1,357 Matching Annotations
  1. Dec 2022
    1. We fractionated a crude cytoplasmic extract (2, 3) (fig. S3A) and verified by immunoblotting that the resulting cytosol was largely devoid of mitochondria, as indicated by the absence of a mitochondrial marker protein, the voltage-dependent anion channel (fig. S3B).

      Here, the authors wanted to test how the speed of apoptotic trigger waves are affected in the absence of the mitochondria. As a result, they obtained the cytosolic extract by fractionation and checked for the absence of the mitochondria using a marker protein by immunoblotting.

      The marker protein resides and functions in the mitochondria and hence the absence of this protein can be used as an indicator for the absence of the mitochondria in the extract.

      Immunoblotting (also called western blotting) is a technique used to detect the presence and levels of a specific protein in biological samples.

      Here is a video illustrating how immunoblotting is performed in the lab: https://www.youtube.com/watch?v=OkH8u84t84M

    2. To further test this possibility, we added recombinant GST–Bcl-2 protein to cytoplasmic extracts to see whether trigger waves were affected. Bcl-2 is a stoichiometric inhibitor of the pro-apoptotic truncated Bid (tBid) protein and of the pore-forming Bak and Bax proteins, and so the expectation was that GST–Bcl-2 would slow the trigger waves

      Here, the authors wanted to know if Bax and Bak contributed to the generation and speed of apoptotic trigger waves.

      To test this they blocked the activity of Bax and Bak by adding Bcl-2 (known inhibitor of Bax and Bak) to the extract and tested the speed of the apoptotic trigger waves as before.

  2. Nov 2022
    1. Fig. 1. RECS1 induces cell death through the mitochondrial pathway of apoptosis. (A) Left: MEFs expressing doxycycline-inducible Flag-RECS1 were stimulated with doxycycline (DOX), and the levels of RECS1 were determined by Western blot. Right: Cell death was determined by PI and SYTO16 costaining followed by automated microscopy (n = 7). Mw, weight-average molecular weight; NT, not treated. (B) MEFs were transiently transfected with the indicated concentrations of MYC-RECS1 followed by Western blot analysis. (C) Left: Bright-field (BF) and PI fluorescent images of MEFs transfected with 0.4 μg of MYC-RECS1 for 24 hours. Right: Cell death was determined by PI staining followed by FACS (n = 3). (D) Left: Images of MEF Flag-RECS1 cells cultured with doxycycline in the presence or absence of the caspase inhibitor QvD-OPh (QvD; 40 μM) for 24 hours (red, PI staining). Right: Corresponding cell death kinetics (n = 3). (E) PI staining and FACS of cells treated as in (D) (n = 4). (F) MEF Flag-RECS1 wild-type (WT) or BAX and BAK (BB) double-knockout (DKO) cells were treated with doxycycline, and indicated proteins were assessed by Western blot. (G) Indicated cells were cultured with doxycycline for 38 hours, and cell death was determined by FACS (n = 3). (H) Images (left) and quantification (right) of RECS1 (Flag) colocalization with indicated organelle markers in Flag-RECS1 MEFs treated with doxycycline. GA, Golgi apparatus. Data represent means ± SD (A, C, and D) or means ± SEM. Statistically significant differences were determined comparing the best individual fit for each curve using the extra-sum-of-squares F test (A and D) or two-way analysis of variance (ANOVA) followed by Holm-Sidak’s multiple comparisons test (C, E, and G). *P < 0.05; **P < 0.01; ****P < 0.0001).


      Through which pathway does RECS1 induce cell death

      Western blot:

      A technique to analyze/detect the levels of a specific protein in the sample.

      Wild type (WT) Cells:

      These are normal cells with all expected qualities and properties.

      BAX and BAK (BB) double-knockout (DKO) cells: Cells in which both BAX and BAK have been artificially made non-functional.


      Doxycycline was used to control (induce) expression of RECS1. (left) RECS1 expression increased over 24 hours as measured by western blot using anti-FLAG antibody. (Right) Induction of expression of RECS1 by doxycycline alone caused about 20% increase in cell death after 28 hours. Cell death was estimated from the number of cells with propidium iodide stain.


      Increased concentration of plasmid result in more RECS1 in cells. Cells were transformed with MYC-RECS1, and the level of this protein was detected by western blot.


      Cell death increased at higher concentration (overexpression) of RECS1. Here, MYC-RECS1 plasmid was introduced into cells and then treated with PI. Cells that stain red are dead and are more when 0.4ug of MYC-RECS1 is applied, compared to when 0.1ug is applied.

      1D & E.

      QvD-OPh (QvD) is an inhibitor of BAX and BAK proteins. These are caspases that mediate cell death through the mitochondria. Inhibiting these proteins reduced apoptosis.


      Overexpressing RECS1 in cells lacking BAX and BAK prevents cell death. Therefore, RECS1 induces cell death through BAK and BAX i.e., though the mitochondrial apoptosis pathway.


      RECS1 colocalizes with lysosome markers LAMP1, LAMP2 and GM130 but not the ER marker, ERp72. Here, it regulates the susceptibility of cells to lysosomal stress.

    2. Fig. 9. RECS1-like/CG9722 modulates viability in response to intracellular stress in D. melanogaster. (A) One copy of dRECS1 was overexpressed in the wing imaginal discs of animals fed with control food or food supplemented with CQ (100 μg/ml) or Tm (10 μg/ml). The phenotype of adult male (right) and female (left) wings was quantified (n = 3 to 7, >80 wings per condition). (B) Control (yw) and mutant (dRECS1KO) male and female flies were treated with Tm (10 μg/ml), and the animal death rate was quantified daily (n = 2 to 3). (C) WT, dRECS1KO, or dRECS1IR pupae were treated with Tm, and the percentage of eclosion was quantified (n = 2 to 4). (D) WT and dRECS1KO female flies were fed a normal diet or starved, and the death rate was daily quantified (n = 3). (E) WT and dRECS1KO knockout male flies were fed a normal diet or starved, and the death rate was daily quantified (n = 2 to 3). Data represent means ± SEM. Statistically significant differences were determined by two-way ANOVA followed by Holm-Sidak’s multiple comparisons test. ****P < 0.0001.

      Figure 9


      In the presence of lysosomal stress inducers, TM and CQ, overexpression of dRECS1 amplified abnormal wing development in flies.

      9B & C

      Flies with reduced dRECS1 expression exhibit longevity in ER stress-inducing conditions

      9D & E

      dRECS1KO female flies (flies without RECS1 expression) are more resistant to nutrient starvation. CG9722 (dRECS1) regulates ER and lysosomal stress-induced apoptosis in flies

    3. Fig. 7. RECS1 is a pH-dependent calcium and sodium channel. (A) Alignment showing the conservation of the critical Arg60 residue from BsYetJ. Right: Comparison of the conserved di-aspartyl sensor. (B) Tridimensional structural model of the human RECS1 in closed and open conformations. Zoom: Key conserved C-terminal residues involved in pH sensing and channel opening. (C) X. laevis oocytes were injected with mRNA for Flag-RECS1 WT or D295Q, and ionic single-channel currents were recorded in the cell-attached mode using patch clamp at pH 7 or 6.5. Inset: Amplified plots depicting three opening states for RECS1 (O1, O2, and O3). (D) Quantification of the probability to find the channel in the open state (NPo) for RECS1 WT and D295Q (left). The NPo for open states O1 (middle) and O2 (right) is also shown [n = 4 (RECS1 WT) or n = 3 (RECS1 D295Q)]. (E) Images of X. laevis oocytes injected with different concentrations of Flag-RECS1 (top) and oocyte survival (bottom). Arrowhead indicates morphological alterations (n = 3). NI, not injected. (F) Recordings of macroscopic currents using the cut-open technique. (G) Permeability ratios with respect to K+ for indicated cations (n = 3 to 7). (H) Top: Indicated MEF Flag-RECS1 cells were treated with doxycycline, and protein levels were monitored by Western blot. Dashed lines indicate Western blot splicing. Bottom: Cells were treated with doxycycline and 25 μM CQ for 24 hours. Cell death was determined by FACS and normalized to WT (100%) (n = 3). Data represent means ± SEM. Statistical differences were determined by two-way (D, E, and G) or one-way ANOVA followed by Holm-Sidak’s multiple comparisons test. *P < 0.05; ***P < 0.001; ****P < 0.0001.

      Figure 7

      Driving question: What is the possible biochemical role or nature of RECS1?

      7A & B

      Multiple sequence alignment of proteins related to RECS1, based on a critically conserved Arg 60 residue of a closely related bacteria BsYetJ (7A). MODELLER platform was used to construct a 3-D structural model of RECS1 in both the open and closed states. Model suggests RECS1 to be an ion channel, particularly Asp 295 as a critical residue in this function (senses pH changes and regulates opening). An equivalent Asp residue is critical in other related proteins.

      7C & D

      Testing the electrophysiological properties of RECS1. Does RECS1 allow ionic movements? Under what conditions? At a neutral pH, 7 and constant voltage, both RECS1 WT and mutant cells exhibited similar spontaneous current spikes, which reduced at lower pH (6.5) in three open states (01-03), suggesting that RECS1 is pH-dependent channel. Probability of finding RECS1 in an open state also reduces with pH, confirming that RECS1 activity is pH-dependent.


      Frog larvae (Oocytes) overexpressing RECS1 die in RECS1-dose dependent manner, consistent with the role of RECS1 a death regulator.

      7F & G

      Ca2+ ions are more permeable that Na+ ions in RECS1-dependent manner (F), while mutation of RECS1 at Asp295 hinders influx of Ca2+ and Na+ but not Ba+ or Cs2+ (G).


      Overexpression of D295Q (RECS1 mutant) blocks lysosomal stress induced MEF cell death, thus functional RECS1 is required for lysosomal stress-induced cell death regulation.

    4. Fig. 5. Inhibition of the endogenous RECS1 protects cells against ER stress. (A) Knockdown of the endogenous mouse RECS1 using two different siRNA pools was confirmed by quantitative polymerase chain reaction (qPCR). (B) Left: Cells transfected with RECS1 siRNAs were treated with Tm (50 ng/ml), and cell death was determined by SYTOX green staining. A representative experiment from four independent experiments is shown. Right: Quantification of cell death at 24 hours (n = 4). (C) Cell death kinetics of cells transfected as in (B) and treated with 25 nM Tg. Left: Representative kinetics. Right: Quantification at 24 hours (n = 3). Data are shown as means ± SD. Statistical differences were determined by one-way ANOVA followed by Holm-Sidak’s multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001.

      What is the function of native RECS1 in cell death regulation and control of ER stress.


      RNA interference was used to transiently reduce expression of RECS1 in MEFs. No effect on basal cell viability was observed.

      5B & C

      Silencing RECS1 reduced the effect of Tm and Tg- induced ER stress on MEF cells.

    5. Fig. 4. RECS1 sensitizes cells to ER stress. (A) Annexin V versus PI FACS density plots from MEF Flag-RECS1 cells cultured with doxycycline for 16 hours and then treated with Tm (100 ng/ml) for 16 hours followed by FACS (n = 9). (B) Experimental design for cell death kinetic experiments. (C) Cell death kinetic analyses of MEFs treated as in (A) (n = 6). (D) Cell death of MEF Flag-RECS1 cells incubated with doxycycline and then treated with 100 nM Tg for 24 hours (n = 3). (E) Corresponding cell death kinetic analysis (n = 3). (F) Clonal assay of MEF Flag-RECS1 cells treated with doxycycline alone or in the presence of Tm (50 ng/ml) or 25 nM Tg. (G) Cell death kinetics of MEF Flag-RECS1 cells cultured with doxycycline and then treated with Tm (100 ng/ml) in the presence or absence of 40 μM QvD (n = 3). (H) MEF Flag-RECS1 WT and BAX and BAK DKO cells were cultured with doxycycline and treated with Tm (100 ng/ml) or 50 nM Tg for 24 hours. Cell death was determined by FACS (n = 2 to 3). Data are shown as means ± SD (C, E, and G) or means ± SEM. Statistical differences were determined by the extra-sum-of-squares F test (C, E, and G) or two-way ANOVA followed by Holm-Sidak’s multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

      What is the effect of RECS1 over expression on ER- induced cell death


      Overexpression of RECS1 in ER stressed cells (TM treatment) triggers cell death. Dead cells are shown by annexin V and PI.

      4B & C

      Cell death correlates with ER stress induction and progression in RECS1 over expressing MEF cells

      4D & E

      Cell death correlates with ER stress induction and progression in RECS1 over expressing cells treated with calcium pump inhibitor, Thapsigargin (Tg).


      Cells over expressing RECS1 have reduced long term survival and reduced growth under ER stress

      4G & H

      QvD and Tm (G), BAX and BAK deficiency inhibited RECS1-triggered cell death under ER stress as expected.

    6. Fig. 3. RECS1 causes LMP in cells undergoing stress. (A) HeLa Flag-RECS1 cells were cultured with doxycycline and then treated with indicated concentrations of CQ, QvD, or both for 16 hours. Immunofluorescence against galectin-1 was performed. (B) Quantification of galectin-1 puncta per field (n = 2; 6 to 27 fields in total). (C) MEF Flag-RECS1 cells were cultured with doxycycline and treated with 50 μM CQ for indicated time points. The enzymatic activities of cathepsin D, acid phosphatase, and hexosaminidase were assessed from cytosolic, lysosomal-free fractions (n = 3). (D) MEF Flag-RECS1 WT and BAX and BAK DKO cells were cultured with doxycycline and then treated with indicated concentrations of CQ for 24 hours. Cell death was determined by PI staining followed by FACS (n = 3). (E) MEF Flag-RECS1 cells were cultured with doxycycline and treated with CQ for 16 hours. Immunofluorescence against LAMP-2 (green), BAX N-terminal domain (red, BAX 6A7 antibody), and Flag-RECS1 (blue, anti-Flag antibody) was performed. The triple colocalization of Flag-RECS1, LAMP-2, and active BAX is visualized as white color. (F) Representative immunofluorescence images of Flag-RECS1 MEF cells treated as in (E). Bottom panels correspond to zoomed white boxes (marked as z) from the top panels. (G) Intensity profiles for LAMP-2, BAX, and Flag-RECS1 signals along the line shown with an L1 (left) or an L2 (right) in (F). Data are shown as means ± SEM. Statistically significant differences were determined by two-way ANOVA followed by Holm-Sidak’s multiple comparisons test. **P < 0.01; ***P < 0.001; ****P < 0.0001.

      Effect of RECS1 expression on Lysosome membrane permeabilization

      3A & B

      After LMP, the cytosolic proteins LGALS1/galectin-1 and LGALS3/galectin 3 relocate to the lysosomal glycocalyx and form punctate structures. Cells overexpressing RECS1 had increased LGALS1/galectin-1 puncta at high chloroquine doses.


      The activity of lysosome enzymes, notably cathepsin D, acid phosphatase and hexosaminidase is increased in RECS1 overexpressing cells under chloroquine treatment.


      The resistance of BAK and BAX DKO cells to lysosomal stress-induced cell death confirms that these caspases are required in this process.

      3E & G

      BAX colocalizes with RECS1 in in LAMP2 containing vesicles and redistributes to larger LAMP2 puncta, specifically in RECS1-positive lysosomes upon CQ treatment in a RECS1-dpemdndent manner.

    7. Fig. 2. RECS1 triggers cell death upon lysosomal stress. (A) Flag-RECS1 MEFs were stimulated with doxycycline for 16 hours and treated with a library of 77 cytotoxic compounds for 24 hours. Cell death was determined by PI staining using an ImageXpress Micro XL automated microscope. ETO, etoposide; NVB, vinorelbine tartrate; DTX, docetaxel. (B) Heatmap of the top 10 sensitizing compounds in (A). For each compound, heatmap colors depict doxycycline-treated PI values after the subtraction of cell death induced by doxycycline alone and by the compound in the absence of doxycycline. *CQ and HCQ were used at 50 μM. **Cisplatin (CDDP) was used at 150 μM. (C) Left: Representative images of MEF Flag-RECS1 cells pretreated with doxycycline and treated with 50 μM CQ for 24 hours (red, PI staining). Right: Cell death kinetic analyses of cells treated as in (B) (n = 3). (D) Cells were cultured as in (C) and then treated with indicated concentrations of bafilomycin A1 for 24 hours. Cell death was determined by FACS (n = 4). (E) Left: Representative images of HeLa Flag-RECS1 cells treated as in (C). Right: Corresponding cell death kinetic analyses (n = 3). (F) HeLa Flag-RECS1 cells were treated as in (E), and cell death was assessed by FACS (n = 3). Data are shown as means ± SD (C and E) or means ± SEM (A, D, and F). Statistically significant differences were determined using the extra-sum-of-squares F test (C and E) or two-way ANOVA followed by Holm-Sidak’s multiple comparisons test. *P < 0.05; **P < 0.01; ****P < 0.0001.

      Role of RECS1 in stress-induced cell death

      2A & B

      Cell death was triggered in RECS1-overexpressing cells treated with a subset of lysosomal stress-inducing drugs but not other drugs. Chloroquine and Hydroxychloroquine treatments were associated with the highest cell death

      2C & D

      Treatment of RECS1 overexpressing MEF cells with Chloroquine (lysosomal stressor) or Vacuolar ATPase inhibitor, bafilomycin resulted in over 80% cell death in 24 hours, suggesting that RESC1 mediates cell death triggered by lysosomal stress.

      2E & F

      Treatment of RECS1 overexpressing Hela cells with Chloroquine or bafilomycin resulted in over 60% cell death in 24 hours.

    8. In the open state, this latch becomes dislodged, and the second transmembrane is displaced out, leading to the opening of the channel (Fig. 7B, magenta). Our model suggests that RECS1, as demonstrated for other proteins of the LFG and BI-1 families (11, 12, 25), could operate as an ion channel. Our bioinformatic analysis also suggests that Asp295 (D295) is a strong candidate to regulate the channel activity of RECS1 and was therefore selected for mutagenesis and functional studies.

      The authors think that Asp295 is very important in regulating the activity of this predicted ion channel. They then make this residue non-functional to study its function in cell death

    9. Fig. 6. RECS1 increases lysosomal acidification and calcium content. (A) Lysosomal pH of HeLa Flag-RECS1 loaded with FuraDx and OGDx and cultured in the absence or presence of doxycycline for 24 hours (n = 5, 15 to 16 total coverslips). (B) Corresponding lysosomal calcium concentration. (C) Left: Cytosolic calcium levels were determined in HeLa Flag-RECS1 cells treated with doxycycline. Cells were treated with 5 μM Tg to stimulate ER calcium release. Right: Maximum ER calcium peak (n = 4, 10 to 11 coverslips). ns, not significant. (D) MEF WT and BAX and BAK DKO Flag-RECS1 cells were loaded as in (A), and lysosomal pH was determined after 24 hours of doxycycline treatment (n = 6, 12 to 17 coverslips per condition). (E) Lysosomal calcium measurements (n = 6). (F) Knockdown of the endogenous mouse RECS1 by siRNA was confirmed by qPCR after 48 hours of transient transfection. (G) Cells transfected as described in (F) were loaded as described in (A), and lysosomal pH was determined after 48 hours of transfection (n = 5, 14 to 15 coverslips). (H) Lysosomal calcium measurements (n = 5, 14 to 15 coverslips). (I) MEF Flag-RECS1 WT and BAX and BAK DKO cells were treated with doxycycline for 16 hours and analyzed by TEM. Image magnification, ×4000 (n = 13 to 17 cells per condition). (J) Diameter of primary and secondary lysosomes (n = 8 to 13 cells per condition). Data represent means ± SEM. Statistically significant differences were detected using ratio-paired two-tailed t test (A, B, D, E, G, and H) or two-way ANOVA followed by Holm-Sidak’s multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

      Role of RECS1 in lysosomal pH regulation and calcium accumulation.

      6A, B & C

      Lysosomal pH was reduced in RECS1 overexpressing HeLa cells (A), while calcium concentration increased about 2-fold under these conditions (B), but no effect on ER calcium concentration (C).

      6D & E

      The reduction in lysosomal pH in MEFs is dependent on BAX and BAK expression and a over three-fold increase in calcium content was observed in cells overexpressing RECS1.

      6F, G & H

      RECS1 silenced cells have increased lysosomal pH (F, G), whereas the luminal pH was only slightly reduced when RECS1 was inhibited (H).

      6 I & J

      Number of secondary lysosomes but not size increased in cells over expressing RECS1 in BAX/BAK expression-dependent manner, suggesting an increased catabolic.

    10. pH (OGDx)

      Fluorescence probe used for measuring lysosomal pH. Find more information about this probe at the following link: https://journals.biologists.com/jcs/article/115/3/599/34970/pH-dependent-regulation-of-lysosomal-calcium-in

    11. (siRecs1#1 and siRecs1#2)

      Two siRNA would degrade/break down RECS1 and prevent its expression

    12. mobility shift

      A technique used in biology to detect proteins bound to DNA based on their rate of movement in a gel. DNA-free protein appear smaller that the same protein bound to DNA.

    13. ip/Grp78 and Chop/Gadd153 (fig. S5H), two sensitive markers of ER stress (13, 15).

      Proteins whose expression changes during ER stress.

    14. During LMP, lysosomal intraluminal cathepsins and hydrolases leak into the cytosol, where they act as cell death proteases

      LMP results in the escape of protein-degrading proteins from the lysosomes to the cytoplasm, leading to initiation of cell death

    15. BAX- and BAK-dependent manne

      These are required for RECS1 to cause cell death

    16. Specific structural and functional criteria are required to validate a BH3 motif, since definitive bioinformatic procedures that rely purely on sequence analyses are currently lacking (28). To this end, we synthesized two peptides: RECS1280–299 and RECS1296–311, based on the N- and C-terminal BH3-like motifs, respectively.

      Two synthetic (non-natural) amino acid sequences were made . These amino acid sequences resemble the BH3-like motifs. These helped in determining the structure and function of RECS1

    17. deletion mutants: RECS1Δ910, lacking the C-terminal BH3 motif, and RECS1Δ868, lacking both

      Sequences of RECS1 with some nucleotides at the C-terminal removed. These targeted the two BH3-like segments.

    18. RECS1 has been traditionally classified within the TMBIM superfamily (8). However, detailed sequence and phylogenetic analyses suggest that RECS1, together with GRINA, LFG, GAAP, and Tmbim1b, forms a distinct and evolutionarily conserved protein family, termed the “LFG family,” characterized by a shared common ancestry and the lack of the signature BI-1 motif (fig. S1A) (7, 26). Previous sequence comparison using the BH3 motif consensus from Prosite (PS01259) identified one putative BH3 motif on the C-terminal domain of the human RECS1 and LFG proteins (22). The BH3-like motif in the C-terminal region of RECS1 contains the characteristic minimal Leu-X3-Gly-Asp/Glu sequence, but no additional BH3-like motifs of this kind were found in the other members of the LFG and BI-1 protein families (fig. S1B). However, an additional conserved BH3-like sequence consisting of Leu-X4-Asp is also present in an adjacent sequence of RECS1, in addition to GRINA and LFG (fig. S1B). These putative “BH3 motifs” are conserved in humans and in several species of non-human primates (fig. S1, C and D). However, only one such motif is present in rodents, suggesting that they represent a late evolutionary event.

      RECS1 sequence contains a short segment on the C-terminal that is thought to be involved in positive regulation of cell death. This short sequence is also found in other related proteins in other organisms.

    19. BAX and BAK double-knockout (DKO) background (Fig. 1F)

      Cells in which both BAX and BAK are non-functional

    20. transient transfection

      Temporary expression of foreign plasmid DNA in cells. The foreign DNA does not get integrated into the cell genome.

    21. Together, these results suggest that the C-terminal region of RECS1 is essential to induce cell death, but this sequence does not bear functional bona fide BH3 motifs.

      Is there a minimum peptide sequence length within the C-terminal below which the C-terminal is irrelevant in RECS1-regulated cell death?

    22. itochondrial potential (ΔΨm)–sensitive dye JC-1

      JC-1 changes when mitochondria charge changes

    23. o investigate the role of RECS1 on the control of cell death in vivo in a vertebrate model, we overexpressed the full-length human RECS1 in zebrafish by injecting in vitro synthetized mRNA to one-cell stage embryos, followed by the analysis of animal viability and morphology 24 hours post-fertilization (hpf)

      The authors expressed a high dose of human RECS1 in Zebrafish embryo. It is expected that the human RECS1 will have similar function in Zebrafish, as this protein function is conserved across different organisms.

  3. Oct 2022
    1. If the MOMP–caspase–BH3 protein loop contributed to the generation of trigger waves, inhibition of the executioner caspases would be expected to slow or block the waves (Fig. 1A). To test this, we added the caspase-3 and -7 inhibitor N-acetyl–Asp-Glu-Val-Asp–aldehyde (Ac-DEVD-CHO) to the reconstituted extracts. Because high concentrations of the inhibitor make it difficult to monitor trigger waves with the fluorogenic caspase substrate Z-DEVD-R110, we used an additional probe, tetramethylrhodamine ethyl ester (TMRE), a red fluorescent dye that responds to changes in mitochondrial membrane potential

      Here, the authors inhibited caspase activity in reconstituted cytoplasmic extracts and tested its effect on the speed of apoptotic trigger waves by monitoring the caspase activity and mitochondrial membrane potential (mark of an active mitochondria).

  4. May 2022
    1. In this case, the asymmetry of the trace of fluorescence intensity as a function of time measured near the end of the channel (green trace inFig. 4B) indicates that the band is still broadening rapidly as it reaches the end of the channel:

      Figure 4B shows the spread of a fluorescent plug introduced into a straight channel. As the plug moves along the channel, it spreads out due to a process called axial dispersion. As a result its length increases and its intensity decreases. Axial dispersion is often an undesired phenomenon observed in microfluidic devices.

    2. we use the leading edge of the fluorescent region to measure the angular displacement of the fluid in the cross section, Δφ. 

      Here, the authors captured optical images of the leading edge of the fluorescent solution to monitor and quantify the rotation of the fluid due to chaotic mixing by the herringbone ridge structures. The images are analyzed to determine the angular displacement Δφ as shown in Figure 1C.

    3. Figure 3D shows the evolution of σ for flows of different Pe in the SHM (open symbols), in a simple channel as in Fig. 3A (▴), and in a channel with straight ridges as in Fig. 3B (•).

      The authors measured the variation in fluorescence intensity across the channel cross-section throughout its length and plotted the standard deviation as a function of the channel length. Minimal variation in fluorescence signal (σ values near zero) represents effective mixing. The staggered herringbone induces the most effective mixing because the σ values reach zero within a short distance along the channel.

    1. The fabrication process for the tactile sensor based on Co AW and the air gap structure is shown in fig. S3.

      The authors chose to use a co-based amorphous wire (Co AW) for its strong magnetic properties. The researchers were able to use a B-H Loop Tracer machine to test the permeability of the wire. This test showed that as increasing frequency was applied, the wire was able to maintain a high permeability and impedance.

      Additionally, the authors determined the correct thickness of the PDMS ring and free standing membrane that would allow a sufficient air gap to be present between the magnetic particles and inductor. The air gap allows for pressure-induced deformations in the membrane which is detected by the changes in the magnetic flux through the inductor.

    2. recorded from the sensor were converted into digital-frequency signals by using an LC oscillation circuit composed of a tactile sensor and a capacitor of 100 nF

      In this experiment, the authors tested the ability to convert the analog signals from the sensor into digital-frequency signals. They loaded the sensor with 50, 113, and 1000 Pa shown in fig. 4B. These are applied pressures which are commonly experienced by humans throughout the day. The authors found that the number of pulse waveforms increased with increased loading, which is similar to human responses to force stimuli.

    1. For a 10-mm-long robot, as shown in fig. S9 (A and B), we observed noticeable motion even under an AC drive voltage as low as 8 V peak to peak (movie S6), which is a relatively low voltage requirement among insect-scale piezoelectric actuators (45).

      The attached video demonstrates the locomotion of a 10 mm-long prototype in the controlled transparent quartz tube environment. Specifically, the relatively low voltage requirement is demonstrated by the noticeable motion at 8V.


    2. We then fabricated prototype robots with different lengths ranging from 10 to 30 mm at an interval of 5 mm using the map of λ/L and β/π of 0.1 and 0.4 for guidance.

      The authors chose to test different lengths of robots to find which length was most efficient for movement. They began at 10mm and moved up 5mm till they reached 30mm. This yielded 5 tests of different length robots. Given the results in Fig. 3, the 10mm robot was deemed most efficient in locomotion.

  5. Apr 2022
    1. The micrographs in Fig. 3, A and B, show that for flows with high Péclet number (Pe = 2 × 105), there is negligible mixing in a simple channel (Fig. 3A) and incomplete mixing in a channel with straight ridges (Fig. 3B) after the flow has proceeded 3 cm—the typical dimension of a microfluidic chip—down the channel.

      Here the authors describe how they compared the mixing performance of 1) a straight microchannel, 2) a microchannel with straight ridges, and 3) a microchannel with staggered herringbone ridges to prove that their design (staggered herringbone ridges) had the best mixing performance.

    2. 26. Recent experimental results confirm that ridges in the floor of a channel do generate transverse components in electroosmotic flows (28).

      "Here, the authors note that the ridge structures result in mixing not only in pressure driven flows but also in electroosmotic flows."

    3. 22. We have evaluated the extent of the chaotic region in the cross section by numerically integrating an approximate two-dimensional representation of the transverse flow to generate a Poincaré map. The map is dense (no islands) everywhere in the cross section except in a narrow band (<10% of height) at the top of the channel. We have not systematically optimized the design of the mixer with respect to these Δφm and p values.

      Here the authors describe the mathematical process of analyzing the data presented in Fig. 2B to determine the extent of the chaotic region.

    4. 29. Labeled polymers were prepared by allowing poly(ethylenimine) (molecular weight ∼500,000) to react with fluorocein isothiocyanate. The product was dialyzed for several days. Diffusivities were calculated based on the broadening of fluorescent streams of the dye in confocal images flows of known speed: D = 4 × 10−6 cm2/s in water and D = 2 × 10−8 cm2/s in 80% glycerol/20% water. Flow speeds were measured by weighing the fluid collected at the outlet of the channel. Viscosity of the glycerol/water solution was estimated to be 0.67 g/cm·s by comparing the flow rate to that of water through the same channel with the same applied pressure.

      Here the authors provide a description of the fluorescent streams used in the first 3 figures and how they were prepared.

    5. Within the limits of our simple model of mixing (13), we estimate from the linear portion of the plot in Fig. 3E that λ is on the order of a few millimeters; the average width of the filaments of unmixed fluid decreases by a factor of ∼3 as the fluid travels this axial distance. This estimate agrees qualitatively with the evolution seen in Fig. 3C.

      When possible it is important to compare the data gathered experimentally with the expected result (determined logically or mathematically) to make sure that the experiments are valid and accurate within an acceptable range of error. Here the authors state that their experimental data qualitatively agrees with their mathematical model.

    1. A major regional change in the doubled CO2 experiment with our three-dimensional model (6, 8) was the creation of hot, dry conditions in much of the western two-thirds of the United States and Canada and in large parts of central Asia.

      Since the authors' model produces estimates across the globe, it is able to reveal stark regional changes in addition to overall averages.

    2. Volcanic eruptions of the size of Krakatoa or Agung may slow the warming, but barring an unusual coincidence of eruptions, the delay will not exceed several years.

      This model predicts that even large changes in the energy balance that counteract warming due to greenhouse gases will only have a partial effect. These predictions generally agree with observations recorded since the time of publication.

    3. Projected global warming for fast growth is 3° to 4.5°C at the end of the next century, depending on the proportion of depleted oil and gas replaced by synfuels (Fig. 6).

      The authors' predictions of warming due to greenhouse gas emissions are generally in line with observed warming since this paper's publication (1981) as well as modern simulations.

    4. The key fuel choice is between coal and alternatives that do not increase atmospheric CO2.

      The authors use the model to compare energy scenarios in which we use varying amounts of coal, oil, and gas (which emit greenhouse gases) to meet future electricity demand. These scenarios include replacement by either nuclear energy or alternative synthetic fuels. The scenarios also vary in how quickly fossil fuels are removed from the energy mix.

    5. Thus we consider fast growth (~3 percent per year, specifically 4 percent per year in 1980 to 2020, 3 percent per year in 2020 to 2060, and 2 percent per year in 2060 to 2100), slow growth (half of fast growth), and no growth as representative energy growth rates.

      It is difficult to predict future trends in energy consumption, so the authors decide on several scenarios to show the range of possible outcomes in the temperature trend.

    6. Prediction of the climate effect of CO2 requires projections of the amount of atmospheric CO2, which we specify by (i) the energy growth rate and (ii) the fossil fuel proportion of energy use.

      Since the authors' model now agrees well with observation, they use it to generate predictions about future temperature trends due to increasing carbon dioxide emissions. To estimate future conditions, they primarily consider emissions due to the energy sector.

    7. Radiative forcing by CO2 plus volcanoes accounts for 75 percent of the variance in the 5-year smoothed global temperature

      Most of the variation in temperature is found to be the result of changes in the energy balance by carbon dioxide concentration and aerosol emissions from volcanic eruptions. This indicates these two factors are the most important in modeling temperature trends.

    8. The main uncertainties in the climate model-that is, its "tuning knobs"-are (i) the equilibrium sensitivity and (ii) the rate of heat exchange with the ocean beneath the mixed layer.

      By varying the unknown parameters and observing when the model best fits the empirical results, the authors find fitted values for those parameters.

    9. Radiative forcing by CO2 plus volcanoes and forcing by CO2 plus volcanoes plus the sun both yield a temperature trend with a strong similarity to the observed trend of the past century (Fig. 5), which we quantify below.

      By including the best possible values for the parameters in their equation for heat flow, the authors model the temperature trend for the previous century and compare it to the reconstructed global temperature history. The agreement between these results is a good indicator of the reliability of the model.

    10. We developed an empirical equation that fits the heat flux into the earth's surface calculated with the l-D RC climate model (model 4):

      The authors combine the model results into a single equation which relates the flow of heat into the earth's surface with dependence on solar radiation output, aerosol concentrations and properties, and temperature change.

    11. Years later (Fig. 4c) the surface temperature has increased 2.8°C.

      The ocean takes several years to come into balance with the atmosphere and ground once carbon dioxide concentrations increase. This causes a substantial time delay in the warming of the air due to the greenhouse effect.

    12. A few months after the CO2 doubling (Fig. 4b) the stratospheric temperature has cooled by ~5°C.

      When carbon dioxide concentration increases, the model predicts that less radiation is emitted to space. Therefore, after a short increment of time, the stratosphere cools. The troposphere remains roughly the same temperature because the ocean is still absorbing much of the warming.

    13. To relate these empirical tests to the CO2 greenhouse effect, we illustrate the flux changes in the I-D RC model when CO2 is doubled.

      The authors look at how each component of the energy balance changes when carbon dioxide concentration changes.

    14. Phenomena that alter the regional radiation balance provide another model test.

      The authors observe the impact of regional phenomena on the global energy balance. They observe the model's response to seasonal changes in temperature and solar irradiation of the surface. This test shows agreement between the model and real observations.

    15. A primary lesson from the Mount Agung test is the damping of temperature change by the mixed layer's heat capacity, without which the cooling would have exceeded 1.1°C (Fig. 2).

      The slow response time of the ocean to changes in the energy balance reduces the impact of short-term phenomena, like cooling due to the emission of aerosols from volcanic eruptions.

    16. We reexamined the Mount Agung case for comparison with the present global temperature record, using our model with sensitivity ~2.8°C.

      The authors look at a specific volcanic eruption to verify that the observed effect of the resulting aerosols (suspended solid particles in air, here composed of ash and soot) is consistent with the effect predicted by their model. They confirm that the expected cooling is consistent between measurements and their predictions.

    17. Another conclusion is that global surface air temperature rose ~0.4°C in the past century, roughly consistent with calculated CO2 warming.

      The reconstructed temperature record agrees with the overall expected warming due to carbon dioxide emissions. However, other factors clearly contribute to the year-to-year variations.

    18. Northern latitudes warmed ~0.8°C between the 1880's and 1940, then cooled ~0.5C between 1940 and 1970, in agreement with other analyses (9, 43).

      The authors verify that their reconstructed temperature record is in agreement with records produced by other methods to show that the chosen formula is reliable.

    19. The temperature trends in Fig. 3 are smoothed with a 5-year running mean to make the trends readily visible.

      There is substantial variation in individual weather records as air fronts move and interact with one another. The authors average the data for each subsequent five-year period so that short-term trends interfere less with the visibility of long-term trends.

    20. We combined these temperature records with a method designed to extract mean temperature trends.

      The authors attempt to reconstruct the global temperature history by taking records from weather stations and applying them to a grid of cells dividing the planet's surface. This helps account for the uneven distribution of weather measurements and provides general trends for the previous century.

    21. The net impact of measured trace gases has thus been an equilibrium warming of 0.1°C or slightly larger.

      The effect of greenhouse gases other than carbon dioxide is significant for warming. Although carbon dioxide is the largest contributor, gases like methane have a much higher effect per amount released.

    22. A global surface albedo change of 0.015, equivalent to a change of 0.05 over land areas, would affect global temperature by 1.3°C.

      Increasing albedo (light reflected by the land) reduces warming by causing solar radiation to be reflected back out to space rather than absorbed by the surface.

    23. Based on model calculations, stratospheric aerosols that persist for 1 to 3 years after large volcanic eruptions can cause substantial cooling of surface air (Fig. 2).

      Stratospheric aerosols can reduce warming by blocking incoming solar radiation.

    24. A 1 percent increase of solar luminosity would warm the earth 1.6°C at equilibrium (Fig. 2) on the basis of model 4, which we employ for all radiative perturbations to provide a uniform comparison.

      The authors introduce a number of factors other than carbon dioxide to the model to observe their impact on warming. These components all affect the earth's energy balance in some way: increases in solar output and other greenhouse gases increase warming, whereas stratospheric aerosols and increased surface reflectance reduce warming.

    25. The mixed-layer model and thermocline model bracket the likely CO2 warming.

      Overall global warming depends on the ability of the ocean to diffuse heat from the surface to deeper water. By varying the ocean's ability to mix, the authors obtain upper and lower bounds for the likely warming as carbon dioxide concentrations increase.

    26. The heat capacity of just the mixed layer reduces this to 0.4°C, a direct effect of the mixed layer's 6-year thermal response time.

      If the ocean has a higher heat capacity (it can store more heat with a lower change in temperature), it can partially buffer overall surface warming due to carbon dioxide.

    27. This increase in thermal response time is readily understandable, because feedbacks come into play only gradually after some warming occurs.

      The feedback mechanisms increase the amount of warming but not the rate of heat flow, so it will take the oceans longer to reach the increased temperature.

    28. Table 1 shows that the initial rate of heat storage in the ocean is independent of feedbacks.

      Because the models all predict the same flow of heat into the ground (column F in Table 1) if the surface temperature is held constant, this means that the heat flow into the ocean must also not change.

    29. This sensitivity (i) refers to perturbations about today's climate and (ii) does not include feedback mechanisms effective only on long time scales, such as changes of ice sheets or ocean chemistry.

      The change in temperature as carbon dioxide levels grow is only calculated for changes relative to the early 1980s (when this paper was published). The authors also note that effects occurring over decades or centuries are not considered, as they are assumed to contribute little to the timescale of the projections in this article.

    30. The sensitivity of the climate model we use is thus ΔTs ~ 2.8°C for doubled CO2, similar to the sensitivity of three-dimensional climate models (6-8).

      The change in temperature with respect to changes in carbon dioxide concentration in the authors' chosen model is judged to be reliable, partially because it is similar to values generated by more complex calculations.

    31. Model 4 has our estimate of appropriate model sensitivity.

      Model 4 is chosen as the best model for estimating temperature sensitivity to carbon dioxide levels, as the moist adiabatic lapse rate introduced in Model 3 underestimates the temperature response at high latitudes. Furthermore, the overestimation of the effect of cloud temperature is judged to roughly balance the effects of underestimating feedback from snow/ice and vegetation.

    32. The vegetation albedo feedback was obtained

      In order to estimate how much temperature and reflection of light by plants feed into one another, the authors compare current vegetation levels to models of a previous ice age. During an ice age, one would expect vegetation to be at a minimum, so the difference between climate then and now should be partially due to the difference in plant cover.

    33. Models 5 and 6 illustrate snow/ice and vegetation albedo feedbacks (19, 20).

      The final two models incorporate important feedback mechanisms: as the climate warms, snow and ice melt, reducing the amount of light that is reflected out to space. This causes warming to increase even further since more light is absorbed by the surface and atmosphere. The amount of the surface covered by vegetation also responds to temperature and ice cover.

    34. Model 4 has the clouds at fixed temperature levels, and thus they move to a higher altitude as the temperature increases (18).

      This model is different from the previous ones, because clouds are now allowed to move between altitudes as their temperature changes instead of remaining at a fixed level. This causes the clouds to always emit the same amount of thermal radiation, limiting the total outgoing radiation from cloudy regions.

    35. Model 3 has a moist adiabatic lapse rate in the convective region rather than a fixed lapse rate.

      This model is distinct from the previous one, in that the decrease in temperature with altitude is not a fixed value. Rather, it depends on temperature more strongly due to the consideration of how water condenses as a parcel of air rises.

    36. Fixed relative humidity is clearly more realistic than fixed absolute humidity, as indicated by physical arguments (13) and three-dimensional model results (7, 8).

      From this point forward, the authors fix relative humidity in the models (warmer air holds more water) because it causes the calculations to have better agreement with more complex models.

    37. Model 2 has fixed relative humidity, but is otherwise the same as model 1.

      The second model fixes relative humidity (the amount of water in the air, relative to how much water the air can hold at most) rather than absolute humidity (the amount of water per unit volume of air). This means that warmer air will hold more water than cooler air.

    38. This case is of special interest because it is the purely radiative-convective result, with no feedback effects.

      The simplest model shows the effects of only radiation and convection in the atmosphere. The difference between these results and a fuller, more complicated model reveals the total impact of feedback mechanisms, including reflectance off of snow and plants.

    39. inserting them individually into the model

      Many factors contribute to the energy balance of the atmosphere. The authors evaluate the effect of each by running the model, inserting the various elements one at a time. The difference in the output after each factor is added gives a measure of its importance to the climate predictions in general.

    40. Our computations include the weak CO2 bands at 8 to 12 μm, but the strong 15-μm CO2 band, which closes one side of the 7- to 20-μm H2O window, causes ≥ 90 percent of the CO2 warming.

      Carbon dioxide absorbs some frequencies more strongly than others. These calculations consider absorption from strong and weak bands of carbon dioxide's absorption spectrum, even though the strongest band is the major contributor to the gas' warming potential.

    41. Multiple scattering and overlap of gaseous absorption bands are included.

      These calculations account for light that is scattered multiple times as it propagates through air. The model also considers frequencies of light where multiple gases are capable of absorption.

    42. The term dFc/dh is the energy transport needed to prevent the temperature gradient from exceeding a preassigned limit, usually 6.5°C km-1. This limit parameterizes effects of vertical mixing and large-scale dynamics.

      Large changes in temperature over small vertical distances are unlikely to occur in the real world, so the model is constrained to avoid too steep a slope in the altitude/temperature curve.

    43. integrated over all frequencies, using the temperature profile of the previous time step and an assumed atmospheric composition.

      The amount of light thatis absorbed, emitted, and scattered by the atmosphere is different for different frequencies of light, so these calculations must be done in parallel for each frequency. The contributions to the energy balance can then be added together.

    44. The l-D RC model uses a time-marching procedure to compute the vertical temperature profile from the net radiative and convective energy fluxes:

      This model considers the dependence of variables on time by only considering time intervals of fixed duration. This permits the modeling of the atmosphere's response to stimuli without requiring more complicated calculus-based methods.

    45. Observed surface temperatures of these planets confirm the existence and order of magnitude of the predicted greenhouse effect (Eq. 3)

      Observing conditions on other planets allows us to generalize conclusions about Earth, as we cannot conduct a true experiment on the entire atmosphere of Earth to see how different factors are related.

    46. The excess, Ts − Te

      The effective radiating temperature would be the temperature of the surface if only the surface were absorbing and emitting radiation. However, gases in the atmosphere also absorb and emit, so the average altitude from which outgoing radiation originates is somewhere above the surface, and T(e) is the temperature at that altitude.

    47. The effective radiating temperature of the earth, Te

      This model assumes that the Earth emits exactly as much radiation as it absorbs over the long term.

    48. Finally, we compute the CO2 warming expected in the coming century and discuss its potential implications.

      Finding equations that accurately describe historical climate change allows the authors to generate predictions about the future. It is important to look at a variety of approaches to see how the predictions of different models vary.

    49. We first describe the greenhouse mechanism and use a simple model to compare potential radiative perturbations of climate.

      By constructing mathematical models of climate change, the authors can simulate how different atmospheric effects are related to one another. They can also observe how different models agree and disagree with each other, which allows for the selection of the best model by comparison with measurements.

  6. Mar 2022
    1. the sensitivity of the tactile sensor was investigated.

      The authors added vertical forces to the sensor and measured the impedance. This was done to show that the sensor is able to distinguish between noise and subtle pressure.

    2. frequency changes under loadings of 50, 113, and 1000 Pa

      This experiment shows the changes in frequency under different loads. The authors tested their tactile sensor under various pressure and frequency conditions that mimic the human response to force stimuli. Here, they can determine the tactile input (pressure) by the frequency (of frequency shift) measured by the sensor. The authors derived an equation relating the frequency to pressure.

    3. The height should be optimized to ensure that the inductive sensing element works best at a biased magnetic field in the range of 0 to ~6.2 Oe.

      This experiment was done in order to find the optimal height at which the inductive sensing element works within a magnetic field range of 0-6.2 Oe. They measured impedance for polymer magnets with different magnetizations approaching the sensing element. For each magnet, there is a height at which the sensor was most sensitive. For the maximum region of sensitivity, they chose the magnet with a magnetization of 0.3 electromagnetic units (emu) at a height of 1.3 mm.

    4. frequency reached 250 kHz

      Magneto-impedance is a function of frequency. In order to optimize the sensing component of the device, the authors wanted to determine which frequency yielded the highest percent change in magneto-impedance. They tested a range of frequencies from 0.5 kHz to 500 kHz. It was determined that the frequency of 250 kHz produced the largest percent change (500%) in magneto-impedance, and therefore the tactile sensor will be most sensitive when operated at this frequency.

    5. Frequency has a significant effect on the GMI ratio response under the loading.

      In this experiment, they wanted to test the sensitivity at different frequencies. The sensitivity of the device increased as the frequency increased, but decreased after the frequency exceeded 250 kHz. That means the optimal frequency is 250 kHz, which is consistent with the optimal frequency of the inductive sensing element.

    6. a subtle force applied to the polymer magnet caused displacement, and impedance was changed due to the variation in magnetic field.

      In this experiment, the authors wanted to determine the change of the magneto-impedance ratio that was caused from a force being applied to the polymer magnet. They tested four different frequencies by applying a downward force (displacement). As displacement increased, the magneto-impedance ratio decreased.

    1. In this case, a 10-mm-long prototype (0.024 g) robot was used to achieve a relative running speed up to 20 BL/s driven near its resonant frequency at 850 Hz. In comparison, under driving frequencies of 800 and 900 Hz, lower relative running speeds of 13 and 3.6 BL/s were recorded, respectively (movie S3).

      The attached video demonstrates the locomotion of a 10 mm-long prototype in the controlled transparent quartz tube environment. Three different driving frequencies were used to help characterize the relationship between driving frequency and robot speed.


    2. Experimentally, the robustness of the prototype soft robot was demonstrated by applying a 100-g mass (1500 times its own body weight) with little change in its speed after the mass was removed, as shown in movie S7. Moreover, the soft robot could continue to function (one-half of the original speed) after being stepped on by an adult human (59.5 kg), a load about 1 million times its own body weight (Fig. 5, A to C, and movie S7).

      The first 20 seconds of the attached video demonstrate the robustness of the soft robot in two different circumstances. We find that the device continues to function after the application of a 100-g mass, as well as after being stepped on.


    3. To further increase the running speed, we added and attached a back leg to a 3 cm–by–5 cm prototype robot to emulate galloping-like gaits (movie S8).

      The attached video demonstrates a new type of movement pattern, galloping, where back legs have been attached to the device.


    4. With the more effective galloping-like gait mechanism, a two-legged robot achieved a running speed about three times that of a one-legged 3 cm–by–1.5 cm robot under similar driving conditions, as shown in movie S9.

      The attached video provides a comparison of the one-legged robot with the new galloping two-legged robot, with the latter traveling at three times the prior.


    5. One simple way to turn would be to assemble two separated electrical domains, as shown in figs. S13 and S14 and movie S10.

      The attached video demonstrates the ability for the soft robot to turn, specifically through the use of two electrical domains.


    6. The slope climbing capability of the robot is demonstrated in movie S7, in which the robot reached 7 BL/s while climbing a slope with an angle of 7.5° (Fig. 5D) and 1 BL/s while climbing a slope with an angle of 15.6° (Fig. 5E). Our soft prototype robot could also carry loads equal to the weight of a peanut (0.406 g) (Fig. 5, F and G).

      The last 10 seconds of the attached video demonstrate device robustness in 2 circumstances. The robot successfully climbs inclined surfaces and carries a peanut.


    7. Using a prototype robot of 10 mm (length) by 15 mm (width) by 3 mm (height) as an example, we first selected 25 combinations (Fig. 3B, gray dots) of the above geometric parameters to fabricate prototypes and conducted experiments to plot the normalized running speed map as a function of relative leg position (λ/L) and relative leg angle (β/π) in Fig. 3B.

      The authors chose to test different angles for the device curvature to optimize speed for efficient movement. Specifically, the relative leg position (λ/L) and angle (β/π) were varied between 0.1-0.5 and 0.1-0.7 respectively.

    8. Hence, we designed the bending angle of the front leg to be less than 90° to enhance the forward movements and reduce backward movements.

      The authors chose a 90º bending angle for the front leg to optimize the forward moving speed of the robot.

    1. These stirring flows will reduce the mixing length by decreasing the average distance, Δr, over which diffusion must act in the transverse direction to homogenize unmixed volumes.

      The implementation of ridges along the microchannel floor disrupts the flow of fluid, allowing diffusion to act more quickly compared to steady flow down a smooth channel.

  7. Feb 2022
    1. The dynamic sensing was demonstrated by placing small live insects of 0.8-mg mass onto the tactile sensor.

      In this experiment, the authors created a way to test the sensor's ability to detect changing tactile information. To do this, they placed an ant on the sensor and let it walk around. The authors show the path of the ant on the sensor in fig. 3C. They found that the sensor was able to detect the ant's movements across the sensor. They were also able to detect the movement of the ant's antenna when it was still. The authors show the data from this experiment in fig. 3D.

    2. The detection limit of the tactile sensor was further investigated by dropping water of different volumes (1 μl, equal to 10 μN) onto the tactile sensor

      In this experiment, the authors created a different way to test the sensor's detection limits. They dropped different volumes of water on the sensor. The authors found that the impedance increased with increasing volumes of water. This means that the device was able to "sense" the weight of the different volumes of water. They found that the device's detection limit was 0.3 Pa. The authors show the data from this experiment in fig. 3B.

    3. The magnetic field sensitivity of the inductive sensing element was investigated.

      They passed a sinusoidal driving current through the inductive sensing element and measured the impedance at different magnetic fields. The magnetic field sensitivity of the inductive sensing element was expressed as equation 3. This experiment concluded that a subtle force can be detected by the tactile sensor with high magnetic field sensitivity and a near linear response to magnetic field.

    1. To quantify the degree of mixing (convection plus diffusion) as a function of the axial distance traveled in the mixer and ofPe, we measure the standard deviation of the intensity distribution in confocal images of the cross section of the flow like those in Fig. 3, A to C: σ = 〈(I − 〈I〉)2〉1/2, whereI is the grayscale value (between 0 and 1) of a pixel, and 〈 〉 means an average over all the pixels in the image. The value of σ is 0.5 for completely segregated streams and 0 for completely mixed streams.

      The standard deviation (of fluorescence intensity) is determined by averaging the grayscale value of images taken and used as a metric for the degree of mixing present in each channel. Lower standard deviation values indicate less difference in the intensity values across an image and signify more complete mixing.

    2. We used soft lithographic methods to make the channels in poly(dimethylsiloxane) (16, 17).

      After designing the master mold, an elastic polymer known as polydimethylsiloxane (PDMS) is prepared and poured over it. This substance cures (becomes solid) over a short period of time and is removed from the mold, where it is bonded to a glass cover slip. The channel itself is comprised of the space between the glass surface and the patterned PDMS.

    3. One way to produce a chaotic flow is to subject volumes of fluid to a repeated sequence of rotational and extensional local flows (21). This sequence of local flows is achieved in the SHM by varying the shape of the grooves as a function of axial position in the channel: The change in the orientation of the herringbones between half cycles exchanges the positions of the centers of rotation (local rotational flow, “c” in the Fig. 2A) and the up- and down-wellings (local extensional flow, “u” and “d” in Fig. 2A) in the transverse flow. Figure 2B shows the evolution of two streams through one cycle of the SHM.

      To create chaotic flow, the authors inserted angled grooves that change orientation after every half cycle, which is defined as ten ridges of the same pattern. This change in orientation results in rotational flow that passively mixes streams entering the channel.

    4. To generate transverse flows in microchannels by using a steady axial pressure gradient, we place ridges on the floor of the channel at an oblique angle, θ, with respect to the long axis (ŷ) of the channel (Fig. 1A).

      The authors implemented ridges along the bottom of microfluidic channels that extend across the length of the channel, which is labeled here as the y-axis. These ridges are set at an angle to the y-axis such that fluid flows in a rotational manner as it passes over them.

      As a result, the fluid circles back on itself as it flows through the ridged channel and promotes steady mixing.

  8. Jan 2022
    1. the 26°C-specific expression of Kdm6b began in T. scripta gonad-mesonephros complexes as early as stage 13, before the gonad was distinct

      The authors tested the abundance of KDM6B RNA (denoted as Kdm6b) in turtle embryos at the earliest stages of sex development (stages 12-14) to see if there was a difference between males and females.

      The gonad-mesonephros complexes are the earliest sites of gonad development.

    2. This sexually dimorphic expression profile was maintained in gonads throughout the temperature-sensitive period (stages 15 to 20)

      The authors compared Kdm6b gene expression between gonads at male and female temperatures throughout the temperature-sensitive stages of development (stages 15-20).

  9. Dec 2021
    1. the feedbacks of those temperature changes to chemical processes

      While the chem-only simulation is intended to determine the effect of chemistry on ozone depletion, it cannot fully capture this effect because the chemical reaction rates also depend on temperature.

      As the amount of ozone changes, the atmospheric temperature also changes.

      If these temperature-driven variations in chemistry were fully accounted for, the effect of chemistry on ozone depletion could be even larger.

    2. a volcanically clean simulation (vol-clean), considering only background sources of stratospheric sulfur

      This approach only includes sulfur-based aerosols that did not originate from a volcanic eruption.

      Thus, the effect of the volcanic eruption on ozone depletion is removed.

    3. a simulation encompassing observed time-varying changes in temperature and winds from meteorological analyses, with calculated background and volcanic stratospheric particles as well as other types of PSCs (chem-dyn-vol)

      In this approach, the primary contributors to ozone depletion are included in the model, and it is expected to be the closest to the actual measured trends.

    4. full chlorine and bromine chemistry

      The human-made substances that are primarily responsible for the hole in the ozone layer are chlorine- and bromine-based chemicals.

      Thus, chemical reactions involving chlorine and bromine are included in the model to simulate the ozone depletion process.

    5. The year 2002 was anomalous in terms of meteorological behavior in the Antarctic (29), and it is excluded from all trend analyses throughout this paper.

      The goal of this paper is to determine if the reduction in human-made ozone-depleting chemicals is healing the hole in the Antarctic ozone layer. It studies ozone-layer trends over the time period of 2000 to 2015. However, in the year 2002, the Antarctic weather conditions were unusual compared to other years within this time period.

      Since ozone depletion in the atmosphere is extremely sensitive to weather conditions, the year 2002 was not included in this analysis.

    6. The modal submodel calculates variations in stratospheric aerosols from volcanic sources

      Here, modal refers to the aerosols in the atmosphere which have a distribution of sizes.

      The size distribution used in this model includes 3 modes corresponding to smaller (Aitken), intermediate (accumulation), and larger (coarse) diameter aerosols. Read more here: https://www.dwd.de/EN/research/observing_atmosphere/composition_atmosphere/aerosol/cont_nav/particle_size_distribution_node.html

    7. We used the specified dynamics option, SD-WACCM

      A specified dynamics model incorporates actual temperature and wind measurement data.

      This approach improves the specified dynamics model's accuracy compared to a model which does not include measured data.

    8. Model calculations were carried out with the Community Earth System Model 1 (CESM1) Whole Atmosphere Community Climate Model (WACCM)

      The CESM1 version of this model includes effects which extend from the Earth's oceans to the second-highest layer of the atmosphere (thermosphere). This allows for a more accurate representation of how different parts of the Earth's climate system influence the ozone layer.

      In addition, the model accounts for how the climate system interacts with chemical reactions in the atmosphere. This is important for modeling how chemicals deplete the ozone layer.

      The model can be used to calculate historical ozone trends and to make predictions about future trends.

    9. balloon ozone data from the Syowa and South Pole stations

      Balloon ozone data is collected by attaching an ozone-measuring device to a balloon that carries the device into the atmosphere. As the device travels through the atmosphere, it transmits data back to the ground station. Read more here: https://gml.noaa.gov/ozwv/ozsondes/

      The Syowa Station is a Japanese research post in the Antarctic which collects data on atmospheric ozone as well as other scientific data. Read more here: https://www.nipr.ac.jp/english/antarctic/center.html

      The Amundsen–Scott South Pole Station is a research site in the Antarctic administered by the United States' National Science Foundation. The station collects a variety of scientific data including data on atmospheric ozone. Read more here: https://www.nsf.gov/geo/opp/support/southp.jsp

      See animations of yearly South Pole Station ozone data here: https://gml.noaa.gov/dv/spo_oz/movies/index.html

  10. Oct 2021
    1. Lentiviral treatment of two different shRNAs led to a 73 to 82% reduction of Kdm6b transcripts in 26°C gonads from early stage 15 onward (fig. S4), as compared with treatment with nonsilencing scrambled virus

      The authors validated the shRNAs using multiple methods.

      The shRNA was infected with a fluorescence tag, called green fluorescent protein or GFP. The infected embryos were then visualized at multiple developmental stages. The treated embryos fully fluoresced green, including the gonads, which indicated that the virus successfully introduced the shRNA to the embryos.

      The authors also checked the levels of transcript between the control shRNA- (a non-specific sequence) and Kdm6b shRNA-treated embryo gonads. In the green, GFP+ gonads, the shRNAs specifically reduced Kdm6b by up to 80% when compared to the controls.

    2. Immunofluorescence

      A widely-used technique used to image proteins on or within cells. It uses antibodies to bind and tag a protein of interest.

      The first antibody specifically binds the protein of interest, for example KDM6B. Then, a second antibody carrying a fluorescent dye attaches to the first antibody.

      These proteins are visualized using a microscope with lasers. The lasers excite the fluorophore, which in turn emits specific light waves. Here, the KDM6B is marked by the emission of light waves in the green spectrum and beta catenin produces waves in the red spectrum.

      Here is a brief introduction to immunofluorescence techniques: https://oni.bio/nanoimager/super-resolution-microscopy/immunofluorescence/

    3. Knockdown of Kdm6b at 26°C led to a significant increase in H3K27me3 levels within the Dmrt1 locus, without altering histone H3 occupancy

      When Kdm6b was lost, histone methylation was higher at the Dmrt1 locus than the control at MPT. This indicates that H3K27me3 may mediate Dmrt1 expression at MPT.

    4. Consistently, the enrichment of H3K27me3 in the promoter region of Dmrt1 was significantly higher at 32°C than at 26°C

      H3K27me3, which is associated with silenced transcription, was more abundant at the Dmrt1 promoter at FPT compared to MPT gonadal cells.

    5. Knockdown of Kdm6b reduced KDM6B binding to the promoter of Dmrt1 in gonadal cells

      Authors saw that KDM6B was no longer binding to the activating sites for Dmrt1 in developing Kdm6b-knockdown gonadal cells at MPT. The reduction almost brought KDM6B association with the Dmrt1 promoter to that observed in gonads at FPT.

    6. KDM6B is strongly recruited to the promoter region of Dmrt1, with higher enrichment at 26°C than at 32°C

      KDM6B protein occupies the key transcriptional activation site at DMRT1 in gonads at MPT compared to FPT.

    7. Knockdown of Kdm6b increased the total level of H3K27me3 in gonadal cells at 26°C

      Depletion of Kdm6b, which encodes a histone lysine demethylation enzyme, causes overall H3K27 trimethylation levels to increase at MPT.

    8. Immunofluorescence analysis revealed that H3K27me3 was more highly enriched in gonadal cells at 32°C than at 26°C.

      Cell imaging shows that the histone tail modification, H3K27 trimethylation, is specifically enriched in gonads at FPT over that of MPT.

    9. Male-specific medullar distribution of germ cells was observed in the rescued gonads, although some germ cells remained in the cortex

      The Kdm6b-depleted + Dmrt1 overexpression gonads demonstrated primarily male germ cell composition and structure at MPT.

    10. SOX9 protein was robustly activated in the primary sex cords of Kdm6b-deficient 26°C gonads overexpressing Dmrt1 (Fig. 3D), and the Kdm6b knockdown–induced reduction of Amh and the up-regulation of the female markers Cyp19a1 and Foxl2 were all reversed (fig. S13)

      Dmrt1 overexpression was able to restore presence of male sex development marker SOX9 in Kdm6b-depleted gonad germ cells.

      Conversely, Dmrt1 overexpression also reduced the presence of female sex fate genes relative to controls.

    11. Overexpression of Dmrt1 rescued the male pathway of 16 of 18 (88.9%) Kdm6b-deficient 26°C gonads

      When authors increased Dmrt1 in the Kdm6b-depleted embryos at MPT, embryos showed male gonad development.

      In molecular biology, this is known as a "rescue" when addition of a molecule can correct a disrupted system.

    12. Dmrt1 could be a critical target of KDM6B

      Kdm6b is required for the expression of Dmrt1 and is active just before Dmrt1 in MPT development. Thus, authors reason that KDM6B could be required for the activation and function of DMRT1.

    13. Dmrt1 mRNA levels were reduced to ~13% in Kdm6b-deficient 26°C gonads from stage 15 onward (Fig. 3A), and DMRT1 protein was also reduced or absent (Fig. 3B).

      Authors observed that during the peak stages of gonad development, Kdm6b-deficient embryos at MPT also had lowered Dmrt1 RNA and protein levels.

    14. we previously demonstrated that the loss of Dmrt1redirected gonads incubating at 26°C toward female fate, whereas the gain of Dmrt1 redirected gonads incubating at 32°C toward male fate

      In a previous paper, these authors knocked down Dmrt1 like they do for Kdm6b in this study.

      They observed several female gonad features for embryos in MPT that lacked Dmrt1. When they enriched Dmrt1 in embryos developing at FPT, they saw masculinization.

      This indicates that Dmrt1 regulates male gonad development, similarly to how the authors characterize Kdm6b in this paper.

      This lead authors to test if Kdm6b regulates Dmrt1.

    15. Dmrt1 was of particular interest because the early male-specific expression pattern is detected at stage 14

      Dmrt1 is another gene enriched in embryos at MPT. The authors show here that Dmrt1 expression is activated right after Kdm6b at MPT.

    16. exhibited a female-like distribution pattern in the developed outer cortex of Kdm6b-knockdown 26°C gonads

      The gonads lacking Kdm6b at MPT resembled the structure and organization of gonads at FPT.

    17. SOX9 protein was expressed specifically in the nuclei of precursor Sertoli cells in control 26°C gonads, whereas it was sharply reduced or absent in Kdm6b-deficient 26°C gonads

      The male sex fate gene SOX9 was depleted in gonads lacking Kdm6b at MPT.

    18. analyzed the expression

      The authors checked gene expression of male and female sex fate markers in gonads after sex determination across treatment conditions.

    19. To confirm the activation of the female pathway in 26°C embryos with Kdm6b knocked down

      Next, authors wanted to see if the male-to-female sex determination shift could be detected by gene expression of additional important sex-related genes.

    20. Kdm6b-deficient 26°C gonads became elongated

      Embryos incubated at MPT treated with the Kdm6b shRNA developed gonads resembling females, with long and thin structure as opposed to the compact and round male gonads of the control.

    21. Control 26°C embryos treated with the scrambled virus exhibited typical cylindrically shaped testes, and control 32°C embryos displayed typical long and flat ovaries

      It is important to have normal or "wild-type" development processes for comparison to the experimental treatment conditions.

      The authors confirmed here that the control MPT and FPT embryos treated with the scrambled virus develop as expected.

    22. Kdm6b responded quickly to estrogen treatment

      Kdm6b gene expression responded to temperature change and was also sensitive to sex hormone treatment.

      The FPT gonads that were treated with a female sex hormone inhibitor showed similar Kdm6b expression to the male temperature gonads.

    23. significant changes in Kdm6b expression were evident by stage 17, preceding gonadal sex differentiation

      Authors checked the response of Kdm6b gene expression to changes in temperature in the gonads during sex development.

      In the MPT and FPT-to-MPT gonads, authors observed high levels of Kdm6b expression, whereas the FPT and MPT-to-FPT incubated gonads maintained low Kdm6b expression.

    24. We next examined the responses of Kdm6b expression to temperature shifts and sex hormone–induced sex reversal during the temperature-sensitive window

      Now that they have seen that KDM6B is specifically abundant in gonads at MPT, the authors will test if changing temperature or sex hormones will change Kdm6b expression.

    25. In contrast to the ChIP signal at the Dmrt1 locus, no occupancy of KDM6B or H3K27me3 was found in other early sex-biased genes Amh, Cyp19a1, Fdxr, Pcsk6, Nov, and Vwa2

      To see if this H3K27me signature was specific to Dmrt1, the authors also checked other sex-related genes that are expressed around the same time.

  11. Sep 2021
    1. table S4

      The authors used candidate models to characterize the association between control measures and the new confirmed cases.

    2. table S1

      The authors used candidate models to study the association between the Wuhan city travel ban and the arrival time of COVID-19 in other cities

    3. we investigated the possible effects of control measures on the trajectory of the epidemic outside Wuhan city

      In the previous sections, the authors have studied the effectiveness of two types of interventions (the Wuhan lockdown and the transmission control measures) individually. In this section, they move on to simulate the transmission patterns if no interventions had been taken, only one of the two types of intervention had been taken, and under the synergetic effect of the two.

    4. By fitting an epidemic model to the time series of cases reported in each province (fig. S3)

      The authors used an SEIR model for the fitting (with more mathematical details presented in the Supplementary Materials). In this model, the entire population is divided into four groups: susceptible (S), exposed (E), infectious (I), and recovered (R). Susceptible people are those who can be infected. People who are infected but have yet to show any symptoms or become infectious themselves are exposed. After the incubation period, the time taken to develop symptoms, people become infectious. Lastly, having beaten the disease and become immune to it, people are recovered.

    5. These results are robust to the choice of statistical regression model

      To confirm the conclusion drawn from the regression, the authors tried a different way of computing the regression coefficient, confidence interval, and P value. The consistency between results derived using two methods indicates that the results present here are robust to the choice of the regression model.

    6. A separate analysis

      With the effectiveness of early interventions in slowing the spread of COVID-19 first demonstrated by the Mann-Whitney U test, the authors further conducted a regression analysis to reveal the association between several factors and the number of laboratory-confirmed cases. You can find more details of the regression in the Supplementary Materials.

    7. P < 0.01

      A P value below 0.01 means less than a 1% chance that the null hypothesis is true. Typically, a P of 0.05 or below suffices to conclude a significant correlation between two variables.

      This probability is derived through a Student's T-test. This test requires the correlation coefficient (r) and the number of samples (n) as inputs.

    8. For comparison

      COVID-19 spread fast. To show this, the authors compared the outbreak data for COVID-19 with those for H1N1, another contagious disease, in China.

    9. We first investigated the role of the Wuhan city travel ban, comparing travel in 2020 with that in previous years and exploring how holiday travel links to the dispersal of infection across China.

      To investigate the impact of the Wuhan travel ban on the spread of COVID-19, the authors first evaluated the effectiveness of this ban on stopping the movement of people.

      The evaluation was realized by comparing the travel data from the year 2020 with those from the previous two years.

      Note that the start and end of the time period of records are fixed with reference to the date of Spring Festival, which, however, varies from year to year.

    10. These data include the numbers of COVID-19 cases reported each day in each city of China, information on 4.3 million human movements from Wuhan city, and data on the timing and type of transmission control measures implemented across cities of China.

      For the quantitative analysis—linear regression in this work— the input data include official reports of the health commission, mobile phone data, and travel information recorded in online databases.

      For details regarding the time span of the data sets and the means of data acquisition, please refer to the first two sections of the Materials and Methods described in the Supplementary Materials.

    11. during the first 50 days of the COVID-19 epidemic in China, from 31 December 2019 to 19 February 2020

      The authors focused on the first 50 days since the detection of the first COVID-19 case in Wuhan. They summarized the timeline for the implementation of key control measures over this period in Fig. 1.

    1. We estimated robustness of animals to the extirpation of plants (assuming bottom-up control) and robustness of plants to the extirpation of animals (top-down control). We simulated two scenarios, one in which order of extirpation was random and another—more extreme—scenario in which order was from the most generalist to the most specialist species. After using a null model correction on each metric to account for variation in sampling intensity and network dimensions across studies (14), we compared the 95% confidence intervals for the O‘ahu networks with the global dataset.

      The authors designed hypothetical scenarios where generalist or specialist species in a network went extinct, then determined the severity of these extinctions by measuring the amount of additional species that would (theoretically) go extinct as a consequence.

      The use of a null model ensures that the results found by these simulations are not a coincidence, that is, not due to chance.

    2. We assembled and analyzed a dataset of 42 avian SDNs encompassing a broad geographical range, with data from islands (n = 17) and continents (n = 25) in tropical (n = 18) and nontropical (n = 24) areas (table S12). Although some of the other SDNs in the analyses included introduced species [e.g., (7, 34)], SDNs on O‘ahu present an extreme case of dominance by introduced species (>50%), coupled with extinction of all native frugivorous birds

      The authors surveyed data from seed dispersal networks across a variety of habitats, noting that O'ahu was unique in that the majority of its population was mostly made up of introduced species, with the original bird species of the island going extinct.

    3. To what extent are introduced species integrated into seed dispersal networks (SDNs), and do introduced dispersers replace extinct native animals? To investigate these questions, we examined interactions based on 3278 fecal samples from 21 bird species [tables S1 to S3 and (13)] collected over 3 years at seven sites encompassing broad environmental variation across Oʻahu (fig. S1 and table S1).

      With the original bird species extinct, how well does the introduced bird species spread the seeds of native plants? Does this newer bird species show a preference toward introduced species of plants?

      To address these questions, the authors collected poop samples over the course of 3 years from 21 different birds found in 7 different locations across the island of O'ahu. The supplemental figure 1 and table 1 describe the 7 locations' average rainfall, coordinates, and elevation to demonstrate the diversity of these areas. Another set of tables listed the different species and plants (introduced and native) found at each site.

    4. The statistical significance of the observed topological patterns was assessed by contrasting observed values for each metric with the confidence interval from null models (13)

      To determine if an observation is a consequence of a measured phenomenon, and not by chance, researchers must test (and reject) the null hypothesis. A null hypothesis states that a result or observation is due to chance, and so should be disregarded as insignificant. When researchers can reject the null hypothesis, that means their results from a study were not due by chance.

      In this case, the authors test the significance of the identified patterns in the network by comparing these values to a null model, a generated collection of values randomized to produce a pattern based on no ecological mechanism (Gotelli and Graves, 1996). If the observed values differ from the range of null values defined by the null model's confidence interval, they are considered significant.

    1. total ozone column measurements from the South Pole station

      Total ozone column data can be obtained by combining balloon data with data measured using satellites.

      Satellites are able to obtain data at higher altitudes than balloons, which are limited to around 30 kilometers.

    2. The historic discovery of the Antarctic ozone hole was based on observations taken in October (1), and healing cannot be considered complete until the ozone hole ceases to occur in that month, which is expected around mid-century (2, 28). However, October need not be the month when the onset of the healing process occurs.

      The earliest October ozone hole measurements are used as a reference for the trends that follow.

      Nevertheless, September and November data can still be used to track the impact human-made chemicals, climate variations, and volcanic eruptions are having on the ozone layer over time.

    3. a chemistry-only simulation

      This approach removes the contributions of climate variation and volcanic eruptions to ozone depletion.

      Thus, only the effects of variations in chemistry would remain.

    4. our modeled post-2005 total stratospheric volcanic aerosol optical depths are estimated to be accurate to within ±40%

      Since aerosols released from volcanic eruptions can also contribute to ozone depletion, the researchers must account for this contribution as accurately as possible.

      To calculate the accuracy of the model, the modeled data is compared to measurement data during the years that volcanic eruptions occurred.

  12. Aug 2021
    1. site-saturation mutagenesis

      M100 is the specific amino acid residue within the protein sequence that has been identified to be critical for the protein’s function. it is very important to determine the ideal amino acid residue for this position. Site saturation mutagenesis is employed.<br> Site-saturation mutagenesis is a form of random mutagenesis, allowing the substitution of a specific amino acid site with one of 20 possible amino acids in a single experiment. In this study, this technique is employed to generate a series of enzymes with enhanced activity and enantiospecificity.

    2. Carbon–silicon bond formation catalyzed by heme and purified heme proteins.

      Heme proteins that were readily available were screened to identify the one that gave the highest enantioselectivity. This served as a starting point for directed evolution. Purified heme protein, silane, diazo ester, thiosulfate, methyl cyanide and M9-N buffer as the medium for microbial growth were stirred at room temperature in anaerobic conditions. Reactions were performed in triplicate. Unreacted starting material was obtained in all cases and no further purification was carried out.

    1. qRT-PCR analysis showed that expression of Amh and Sox9 sharply decreased, whereas expression of Cyp19a1 and Foxl2 significantly increased in Kdm6b-deficient 26°C gonads relative to controls

      Analysis of the sex fate gene expression showed that male markers were significantly decreased, whereas female markers were significantly increased in Kdm6b-RNAi treated gonads compared to controls.

      Kdm6b-depleted gonads showed marker expression similar to the female control.

    2. two independent experiments with different shRNAs showed that 39 of 45 (86.7%) and 45 of 56 (80.4%) Kdm6b-knockdown embryos displayed a complete male-to-female shift in sexual trajectory at 26°C

      A robust ratio of the Kdm6b-depleted embryos demonstrated a shift from male to female development, despite incubation temperature. This structural data suggests that KDM6B plays an important role in sex determination.

    1. r = 0.69

      We need to take r values with caution, though. Calculating r values is still possible even for plots where two variables are not linearly associated. It is always wise to look at the scatter plot an r corresponds to.

      In the present study, the scatter plot in Figure 2C indeed shows a clear linear association.

    2. the effectiveness of travel restrictions and social distancing measures in preventing the spread of infection is uncertain.

      The present work addressed this question by studying how the spread of COVID-19 was impacted by the travel bans and the closure of entertainment venues. The travel bans encompass the one for Wuhan city and the suspension of inter- and intra-city public transport.

    1. Although the majority of RNA fragments were extremely short (<30 nt), the authenticity of the sequences could be validated

      Given the results demonstrated by Smith and colleagues, the authors decided to look at the size distributions and damage profiled of the RNA recovered from the lung tissue. They found fragments of much greater lengths than 30 nucleotides with fragments ranging from 217-233 nucleotides. They also found no evidence of RNA damage indicating formalin-fixed specimens could serve as a source of well preserved RNA molecules.

    2. mapped to a MeV genome

      These authors took the collection of overlapping DNA fragments produced through sequencing and lined them up to a genome of measles that had already been constructed.

  13. Jul 2021
    1. Fig. 2 Scope of Rma cyt c V75T M100D M103E-catalyzed carbon–silicon bond formation.

      Rma cyt c V75T M100D M103E shows excellent enantioselectivity and turnover over a wide range of substrates. Silicon substrates with weakly electron-donating or -activating methyl substituents (4), strongly electron donating -OMe (5), weakly deactivating -Cl (6), strongly deactivating -CF3 (7), and moderately deactivating COOMe and CONMe (9 and 10 respectively) show moderate to excellent turnover and high selectivity. No direct relationship exists between turnover number and substituent effects from this study. Enantioselectivity is excellent in all substrates. All products were identified using GC-MS, and no traditional organic chemistry techniques were used.

    2. In addition, diazo compounds other than Me-EDA could be used for carbon–silicon bond formation

      Additional diazo compounds that were successful were R3 = -CH3, -CH2CH3, -Ph.

    3. silicon and diazo reagents

      General method for the preparation of phenyl dimethyl silanes: In a 100 mL round bottom flask, chlorodimethylsilane in THF was cooled to zero degrees. A solution of the appropriate Grignard reagent was added dropwise over ~15 minutes. The reaction was allowed to reach room temperature and stirred for ~8 hours. The product mixture was quenched with ammonium chloride solution. The product was extracted into ether and then isolated. Purification was done by column chromatography.

    4. Relative to the wild-type protein, the evolved triple mutant catalyzes the reaction more than seven times faster, with turnover frequency (TOF) of 46 min–1 (Fig. 1E).

      Via site-saturation mutagenesis, V75 and M103 positions along the protein sequence were identified as likely beneficial mutations and were randomized, i.e., the amino acids at these positions are replaced by random ones. A large number of random variants, which together constitute a library, are produced and then screened in an attempt to discover a highly active variant among them. The evolved triple mutant fits the bill.

    5. a 12-fold improvement over the wild-type protein (Fig. 1D).

      Recombinant protein is a protein encoded by a gene that has been cloned in a system that supports expression of the gene (in this case, it is M100). Modification of the gene by recombinant DNA technology can lead to expression of a mutant protein. In this study, M100D mutation is more highly activating than the wild-type protein.

    6. Carbon–silicon bond forming rates over four generations of Rma cyt c.

      Turnover frequency for each variant relative to wild-type protein: WT: 1 M100D 2.8 +/- 0.2 V75T M100D 4.6 +/- 0.3 V75T M100D M103E 7.1 +/- 0.4

      From this experimental data, it is clear that directed evolution has resulted from changing the enzyme from unselective wild-type into a highly enantioselective variant.

    7. using lysates of E. coli expressing Rma cyt c

      Experiments were conducted with purified heme protein, silane, diazo ester, sodium dithionate, MeCN, and M9-N buffer at room temperature in anaerobic conditions for 1.5 h. Three trials were conducted. Turnover number reported is the average of the three trials. Unreacted reactants were not isolated.

  14. Jun 2021
    1. We calculate that 275 million metric tons (MT) of plastic waste was generated in 192 coastal countries in 2010, with 4.8 to 12.7 million MT entering the ocean.

      The range of 4.8 to 12.7 million metric tons of waste comes from uncertainty in the total amount of waste generated, the amount of that waste that is plastic, and the amount of mismanaged waste that enters the ocean.

    2. Our framework was designed to compute, from the best-available data, an order-of-magnitude estimate of the amount of mismanaged plastic waste potentially entering the ocean worldwide.

      Here the authors restate the goal of their investigation.

    1. To investigate how such changes affect communities, we performed multiscale analyses of seed dispersal networks on Oʻahu, Hawaiʻi.

      The authors visited multiple areas in Hawai'i to study how seeds from plants (originally from the area or recently introduced) were being distributed by invasive birds.

  15. Apr 2021
    1. we injected groups of mice with 2×1062×106<math xmlns="http://www.w3.org/1998/Math/MathML"><mn>2</mn><mo>×</mo><msup><mn>10</mn><mn>6</mn></msup></math> wild-type 51BLim10 tumor cells and treated them with anti-CTLA-4 beginning on day 0 as before, or beginning 7 days later

      The authors conducted a new set of experiments to check whether administering anti-CTLA-4 after tumors are detected is as effective as administering it at the same time tumors are introduced. If they are able to successfully treat mice after tumors are already established, then maybe this treatment could work for human patients as well!

    2. Mice that had rejected V51BLim10 tumor cells as a result of treatment with anti-CTLA-4 were challenged with 4×1064×106<math xmlns="http://www.w3.org/1998/Math/MathML"><mn>4</mn><mo>×</mo><msup><mn>10</mn><mn>6</mn></msup></math> wild-type 51BLim10 cells 70 days after their initial tumor injections

      The authors injected the mice which were previously treated with unmodified tumor. If they developed an immune memory, they may be able to clear this tumor even though it is not expressing B7 and they have not been given anti-CTLA-4 antibodies.

    3. injected with 4×1064×106<math xmlns="http://www.w3.org/1998/Math/MathML"><mn>4</mn><mo>×</mo><msup><mn>10</mn><mn>6</mn></msup></math> V51BLim10 tumor cells and left untreated, or treated with anti-CD28

      The mice were split into groups that received different treatment regimens. Two of the groups were untreated or were treated only with anti-CD28 antibodies.

    4. injected with 2×1062×106<math xmlns="http://www.w3.org/1998/Math/MathML"><mn>2</mn><mo>×</mo><msup><mn>10</mn><mn>6</mn></msup></math> tumor cells

      The authors decided to check if there is an effect to changing the tumor dose. They halved the dose to 2x10(^6) and had a group of untreated and a group of anti-CTLA-4 treated mice.

    5. the growth of V51BLim10, a vector control tumor cell line that does not express B7

      This set of experiments was conducted with a variant of the same murine colon cancer tumor cells, but this time the tumors did not express B7. Thus the tumors are not able to provide the secondary signal to T cells.

    6. untreated controls

      The authors had a control group of mice which were injected with tumor cells expressing B7-1 molecules but were not treated with any antibodies.

    7. Two groups were treated with a series of intraperitoneal injections of either anti-CTLA-4 or anti-CD28

      The authors injected groups of mice with tumor cells expressing B7-1 molecules. The injections were administered within the membrane lining of the abdominal (peritoneal) cavity. These mice were then treated with two different regimens. One group of mice was injected with antibodies targeting CTLA-4 and another with antibodies targeting CD28.

  16. Mar 2021
    1. Via compression, we connected the insulin tip to a shaft made solely from biodegradable polymers

      Tips are connected to the shaft which consists of very large molecules composed of many repeating subunits.

    2. We also performed a laparotomy followed by a gastrostomy to manually place milliposts into the gastric tissue. These experiments yielded comparable pharmacokinetics and systemic uptake.

      The authors made a surgical incision into the abdominal cavity and performed a surgical operation to make an opening in the stomach in order to manually implant the millliposts into a swine stomach. This shows the drug being taken in at a very similar rate as to when the device was ingested.

    3. To demonstrate that the mass distribution affected self-orientation, we showed that the SOMA oriented in 100% of trials, whereas a device of the same shape made solely of PCL only oriented 50% of the time.

      The weight distribution of the SOMA device out performed devices made solely of PCL showing that weight distribution plays a large role in the ability to self orient.

    4. milliposts were inserted into the submucosa of swine stomach tissue after being ejected from a SOMA with a 5-N spring. The insulin tips reached the same depth as dye injected by a Carr-Locke needle (Fig. 3, D to F and H). To ensure a safety margin on the insertion force, we ejected stainless steel milliposts using 9-N steel springs (k = 1.13 N/mm) into ex vivo swine tissue, and these still did not perforate the tissue

      The authors show that a force of 5 N from the spring allows the milliposts to enter into the tissue of the swine's stomach. They verify that this will be safe by testing a force of 9 N, which is larger than the used 5 N, showing that even a larger force will not cause the tissue to rip or be damaged.

    5. Dissolution profiles in vitro demonstrated complete dissolution within 60 min

      The authors tested in a simulation that the millipost will dissolve within 60 minutes after released, so that it is not staying in the body for too long of a time period after it is released.

    6. Stability studies conducted at 40°C showed that the insulin milliposts remained stable in a desiccated environment for 16 weeks (fig. S6), as compared with 4 weeks of stability for a liquid formulation.

      The authors show that the device can be stored for an extended period of time in a warm, dry environment, as well as a slightly shorter time in a liquid environment. This demonstrates that the device can be safely stored and it will not affect the device.

    7. The optimized SOMA shape outperformed both a sphere and ellipsoid made from the same materials with equivalent masses, volumes, and density distributions in two biologically relevant metrics: orientation time and stability.

      When devices with different shapes but the same equivalent mass, volume and density distribution were compared, the SOMA shape was shown to be better at self orienting. This shows that the SOMA shape performs better than the sphere and ellipsoid shapes.

    8. Raman spectroscopy validated the protein structure of the API after high-pressure exposure

      The authors used a technique to determine vibrational modes of molecules to analyze the chemical structure of the API used after having it put under high pressure.

    9. Additionally, to ensure safety in the case of a millipost misfire or device retention, we dosed six SOMA prototypes with 3-mm-long protruding 32-gauge stainless steel needles at once in swine; in this experiment, we performed x-rays over the course of 9 days and found no evidence of GI obstruction, pneumoperitoneum, or other adverse clinical effects

      In the case that the millipost did not dissolve, the authors used SOMAs with stainless steel needles to test that problem. They concluded that the SOMAs with needles that did not dissolve did not cause blockages in the GI tract, nor pneumoperitoneum, or the presence of air or gas in the abdomen. This means that the SOMAs do not cause GI blockages, or behavioral changes or other adverse effects in the swine model. This is promising news and could mean that the SOMAs would work in a human model.