DOI: 10.1016/j.cell.2026.05.049

Resource: Zeiss LSM 800 with Airyscan Microscope (RRID:SCR_015963)

Curator: @scibot

SciCrunch record: RRID:SCR_015963

What is this?

DOI: 10.1016/j.cell.2026.05.049

Resource: (Abcam Cat# ab62352, RRID:AB_944418)

Curator: @scibot

SciCrunch record: RRID:AB_944418

What is this?

DOI: 10.1016/j.cell.2026.05.049

Resource: RRID:Addgene_12263

Curator: @scibot

SciCrunch record: RRID:Addgene_12263

What is this?

DOI: 10.1016/j.cell.2026.05.049

Resource: Zeiss LSM 700 microscope (RRID:SCR_017377)

Curator: @scibot

SciCrunch record: RRID:SCR_017377

What is this?

DOI: 10.1016/j.cell.2026.05.049

Resource: RRID:Addgene_15799

Curator: @scibot

SciCrunch record: RRID:Addgene_15799

What is this?

DOI: 10.1016/j.cell.2026.05.049

Resource: Bio-Rad CFX384 Real-Time Detection System (RRID:SCR_018057)

Curator: @scibot

SciCrunch record: RRID:SCR_018057

What is this?

DOI: 10.1016/j.cell.2026.05.049

Resource: RRID:Addgene_62988

Curator: @scibot

SciCrunch record: RRID:Addgene_62988

What is this?

DOI: 10.1016/j.cell.2026.05.049

Resource: NIS-Elements Advanced Research (RRID:SCR_027181)

Curator: @scibot

SciCrunch record: RRID:SCR_027181

What is this?

DOI: 10.1016/j.cell.2026.05.049

Resource: RRID:Addgene_212936

Curator: @scibot

SciCrunch record: RRID:Addgene_212936

What is this?

DOI: 10.1016/j.cell.2026.05.049

Resource: RRID:SCR_027182

Curator: @scibot

SciCrunch record: RRID:SCR_027182

What is this?

DOI: 10.1016/j.cell.2026.05.049

Resource: RRID:SCR_027183

Curator: @scibot

SciCrunch record: RRID:SCR_027183

What is this?

DOI: 10.1016/j.cell.2026.05.049

Resource: (RRID:CVCL_1926)

Curator: @scibot

SciCrunch record: RRID:CVCL_1926

What is this?

DOI: 10.1016/j.cell.2026.05.049

Resource: RRID:SCR_027184

Curator: @scibot

SciCrunch record: RRID:SCR_027184

What is this?

DOI: 10.1016/j.cell.2026.05.049

Resource: (RCB Cat# RCB3676, RRID:CVCL_0126)

Curator: @scibot

SciCrunch record: RRID:CVCL_0126

What is this?

DOI: 10.1016/j.cell.2026.05.049

Resource: BioTek Synergy H1 Hybrid Multi-Mode Microplate Reader (RRID:SCR_019748)

Curator: @scibot

SciCrunch record: RRID:SCR_019748

What is this?

DOI: 10.1016/j.cell.2026.05.049

Resource: LI-COR Odyssey Fc Imaging System (RRID:SCR_023227)

Curator: @scibot

SciCrunch record: RRID:SCR_023227

What is this?

DOI: 10.1016/j.cell.2026.05.049

Resource: (Santa Cruz Biotechnology Cat# sc-33725, RRID:AB_628459)

Curator: @scibot

SciCrunch record: RRID:AB_628459

What is this?

DOI: 10.1016/j.cell.2026.05.049

Resource: (CellNest Cat# CNG004-0005, RRID:AB_3698716)

Curator: @scibot

SciCrunch record: RRID:AB_3698716

What is this?

DOI: 10.1016/j.cell.2026.05.049

Resource: (Thermo Fisher Scientific Cat# PA1-85250, RRID:AB_2259435)

Curator: @scibot

SciCrunch record: RRID:AB_2259435

What is this?

DOI: 10.1016/j.cell.2026.05.049

Resource: (CellNest Cat# CNG005-0005, RRID:AB_3698717)

Curator: @scibot

SciCrunch record: RRID:AB_3698717

What is this?

DOI: 10.1016/j.cell.2026.05.049

Resource: (Cell Signaling Technology Cat# 55920, RRID:AB_2734718)

Curator: @scibot

SciCrunch record: RRID:AB_2734718

What is this?

DOI: 10.1016/j.cell.2026.05.049

Resource: (Jackson ImmunoResearch Labs Cat# 715-096-151, RRID:AB_2340796)

Curator: @scibot

SciCrunch record: RRID:AB_2340796

What is this?

DOI: 10.1016/j.bcp.2026.118202

Resource: (Abcam Cat# 2251-1, RRID:AB_1267174)

Curator: @scibot

SciCrunch record: RRID:AB_1267174

What is this?

DOI: 10.1016/j.bcp.2026.118202

Resource: (ABclonal Cat# A11952, RRID:AB_2861643)

Curator: @scibot

SciCrunch record: RRID:AB_2861643

What is this?

DOI: 10.1016/j.bcp.2026.118202

Resource: RRID:AB_11000297

Curator: @scibot

SciCrunch record: RRID:AB_11000297

What is this?

DOI: 10.1016/j.bcp.2026.118202

Resource: RRID:AB_1861654

Curator: @scibot

SciCrunch record: RRID:AB_1861654

What is this?

DOI: 10.1016/j.bcp.2026.118202

Resource: (ABclonal Cat# A19083, RRID:AB_2862575)

Curator: @scibot

SciCrunch record: RRID:AB_2862575

What is this?

DOI: 10.1016/j.bcp.2026.118202

Resource: RRID:AB_3674771

Curator: @scibot

SciCrunch record: RRID:AB_3674771

What is this?

DOI: 10.1016/j.bcp.2026.118202

Resource: (Abcam Cat# ab151230, RRID:AB_2753144)

Curator: @scibot

SciCrunch record: RRID:AB_2753144

What is this?

DOI: 10.1016/j.bcp.2026.118202

Resource: RRID:AB_2758406

Curator: @scibot

SciCrunch record: RRID:AB_2758406

What is this?

DOI: 10.1016/j.bcp.2026.118198

Resource: (RRID:CVCL_B288)

Curator: @scibot

SciCrunch record: RRID:CVCL_B288

What is this?

DOI: 10.1016/j.bcp.2026.118198

Resource: (ATCC Cat# TIB-71, RRID:CVCL_0493)

Curator: @scibot

SciCrunch record: RRID:CVCL_0493

What is this?

DOI: 10.1007/s00109-026-02695-5

Resource: (RRID:CVCL_C1W7)

Curator: @scibot

SciCrunch record: RRID:CVCL_C1W7

What is this?

DOI: 10.1002/jmv.71034

Resource: (ICLC Cat# HTL98017, RRID:CVCL_0032)

Curator: @scibot

SciCrunch record: RRID:CVCL_0032

What is this?

DOI: 10.1002/jmv.71034

Resource: (BCRC Cat# 60005, RRID:CVCL_0030)

Curator: @scibot

SciCrunch record: RRID:CVCL_0030

What is this?

DOI: 10.1002/jmv.71034

Resource: (ATCC Cat# CRL-10302, RRID:CVCL_1727)

Curator: @scibot

SciCrunch record: RRID:CVCL_1727

What is this?

DOI: 10.1002/jmv.71034

Resource: (ATCC Cat# HTB-31, RRID:CVCL_1094)

Curator: @scibot

SciCrunch record: RRID:CVCL_1094

What is this?

DOI: 10.1002/jmv.71034

Resource: (ATCC Cat# CRL-1550, RRID:CVCL_1100)

Curator: @scibot

SciCrunch record: RRID:CVCL_1100

What is this?

DOI: 10.1002/advs.76340

Resource: Seurat (RRID:SCR_016341)

Curator: @scibot

SciCrunch record: RRID:SCR_016341

What is this?

DOI: 10.1002/advs.76340

Resource: Cell Ranger (RRID:SCR_017344)

Curator: @scibot

SciCrunch record: RRID:SCR_017344

What is this?

DOI: 10.1002/advs.76340

Resource: Creighton University Histology Core Facility (RRID:SCR_025163)

Curator: @scibot

SciCrunch record: RRID:SCR_025163

What is this?

DOI: 10.1002/advs.76277

Resource: (ATCC Cat# CRL-4053, RRID:CVCL_9Q53)

Curator: @scibot

SciCrunch record: RRID:CVCL_9Q53

What is this?

DOI: 10.1002/advs.76277

Resource: (ATCC Cat# CRL-2539, RRID:CVCL_0125)

Curator: @scibot

SciCrunch record: RRID:CVCL_0125

What is this?

DOI: 10.64898/2026.05.13.724991

Resource: RRID:AB_2341193

Curator: @nmaralla

SciCrunch record: RRID:AB_2341193

What is this?

DOI: 10.3390/v18050536

Resource: RRID:Addgene_235668

Curator: @nmaralla

SciCrunch record: RRID:Addgene_235668

What is this?

DOI: 10.21203/rs.3.rs-7437638/v1

Resource: RRID:AB_2333093

Curator: @nmaralla

SciCrunch record: RRID:AB_2333093

What is this?

DOI: 10.1186/s12934-026-02979-y

Resource: RRID:Addgene_249722

Curator: @nmaralla

SciCrunch record: RRID:Addgene_249722

What is this?

DOI: 10.1016/j.neuron.2026.04.046

Resource: RRID:Flybase_FBst0466398

Curator: @nmaralla

SciCrunch record: RRID:Flybase_FBst0466398

What is this?

DOI: 10.1016/j.neuron.2026.04.046

Resource: RRID:DGRC_1298

Curator: @nmaralla

SciCrunch record: RRID:DGRC_1298

What is this?

DOI: 10.1016/j.devcel.2025.05.008

Resource: RRID:IMSR_JAX:006054

Curator: @nmaralla

SciCrunch record: RRID:IMSR_JAX:006054

What is this?

DOI: 10.1016/j.devcel.2025.05.008

Resource: RRID:IMSR_JAX:000664

Curator: @nmaralla

SciCrunch record: RRID:IMSR_JAX:000664

What is this?

DOI: 10.1016/j.celrep.2026.117456

Resource: RRID:IMSR_JAX:000664

Curator: @nmaralla

SciCrunch record: RRID:IMSR_JAX:000664

What is this?

PMID: 24698326

Gene: PIK3CD

HGNC: 8977

Case#: Case 1

DiseaseAssertion: APDS

FamilyInfo: no familial history of PID

CaseHPOFreeText: He was referred to our hospital at the age of 2 years with recurrent bronchopulmonary infections, lymphadenopathy, hepato-splenomegaly, liver disease (elevated transaminases and portal septal fibrosis at liver biopsy). He had increased serum IgM levels (4.25g/L), normal IgG (5.7 g/L) and decreased IgA (0.65g/L) levels, compatible with the diagnosis of CSR-D. The CD40L and CD40 defects were excluded and intravenous IgG substitution was initiated. At 8 years of age, he developed a high grade diffuse large B-cell lymphoma (DLBCL, WHO classification) of biliary tract (Figure 1 a-c). In situ hybridization for Epstein Barr virus (EBV) was negative and Bcl-6 was expressed as shown by immunohistochemistry. The patient recovered after nine courses of chemotherapy (UKCCSG 9002 protocol; “see E3”). At 19 years of age, under IgG substitution, he again developed a high grade EBV(-) DLBCL of the colon, which was found to be Bcl-6 negative (Figure 1 d-f). He received CHOP (Cyclophophamide, vincristine, steroids) plus rituximab. He died from large bowel perforation and bleeding 12 days after the third course of chemotherapy.

CasePreviousTesting: None. Genotyping only done at position c.3061 of PIK3CD

GenotypingMethod: We genotyped the PIK3CD gene at position c.3061G as described previously (1) in a cohort of 139 patients with immunological phenotype of Ig CSR-D. We found 8 new APDS patients with the E1021K heterozygous mutation in the PIK3CD gene

Variant: E1021K

CAID: CA145460

gnomAD: absent in gnomAD v2.1.1

SupplementalData: Clinical features of patients 3-8 in supplementary

Case#: Case 2

DiseaseAssertion: APDS

FamilyInfo: no familial history of PID

CaseHPOFreeText: belongs to a family in which two siblings were reported as suffering from a CSR-D (data from the affected sister P7 “see Tables E1 and E2”). From the age of 5 months, he suffered recurrent upper (recurrent acute otitis media) and lower respiratory tract infections complicated by bronchiectasis, chronic non-infectious diarrhea with malabsorption syndrom and failure to thrive. Other infections were also noticed, including pericarditis caused by Echo virus infection and recurrent synovitis. The diagnosis of CSR-D was made, according to his familial history and IgG substitution was started. At 6 and 8 years of age, he displayed episodes of massive enlargement of lymph nodes (cervical and mesenteric) with no malignant feature at biopsy. Serum Ig levels revealed an increase of IgM (4.5g/L at 5 years and 13g/L at 11 years) and a decrease of IgG (<1.9g/L) and IgA (0.41 g/L). At 11 years of age, he had a new episode of cervical lymph nodes enlargement which led to the diagnosis of Hodgkin disease, histological type nodular sclerosis, stage III with localization to cervical, mediastinum, retroperitoneum and spleen (EBV status was unknown and could not be studied retrospectively) (Figure 1 g-i). Patient received chemotherapy and radiotherapy with irradiation of regions above and below diaphragma, which induced complete remission. He is now well on IgG substitution and prophylactic antibiotherapy with a follow-up of more than 10 years.

CasePreviousTesting: None. Genotyping only done at position c.3061 of PIK3CD

GenotypingMethod: We genotyped the PIK3CD gene at position c.3061G as described previously (1) in a cohort of 139 patients with immunological phenotype of Ig CSR-D. We found 8 new APDS patients with the E1021K heterozygous mutation in the PIK3CD gene

Variant: E1021K

CAID: CA145460

gnomAD: absent in gnomAD v2.1.1

SupplementalData: Clinical features of patients 3-8 in supplementary

PMID: 30619796

Gene: PIK3CD

HGNC: 8977

Case#: 2 year old female

DiseaseAssertion: APDS (She was diagnosed with polymorphous B-cell lymphoproliferative disorder)

FamilyInfo: no family history suggestive of an immunodeficiency

CaseHPOFreeText: referred by pulmonary to clinical immunology for evaluation of her recurrent pneumonias and bronchiectasis. Work-up at that time revealed IgA < 6 (34–305 mg/dL), IgG 30 (572–1,474 mg/dL), IgM 1190 (31–208 mg/dL). In addition to low levels of IgG and IgA but elevated IgM levels, immune evaluation revealed T and B cell lymphopenia −827 (876–3,394 CU MM) and 28 (200–1,259/CU MM), respectively. Her CD4 count was 331 (412–2,095/CU MM) and CD8 count was 481 (236–995/CU MM). She had a normal response to lymphocyte mitogen and antigen stimulation. Due to these results and her clinical history, she was started on intravenous immunoglobulin replacement which helped decrease her incidence of infections. As she grew older she began to have increased hospitalizations for hemolytic anemia and recurrent pneumonias and sinusitis. She also developed lymphadenopathy and splenomegaly. She received rituximab with resultant improvement in her counts and decrease in size of her lymphadenopathy and hepatosplenomegaly. She was also started on trimethoprim/sufamethoxazole prophylaxis to help prevent infections, and though she continued to have intermittent respiratory infections the amount was improved. In addition to asymmetrical cervical lymphadenopathy and parotid gland enlargement Figures 2A,B, a CT neck/chest/abdomen and pelvis done at that time showed mediastinal, hilar, abdominal, and pelvic lymphadenopathy, impressive retroperitoneal lymphadenopathy, hepatosplenomegaly, and cecum and terminal ileum thickening Figures 2C,D.

CasePreviousTesting: Genetic testing revealed no mutation in AID, UNG, CD40, or CD40L but later gene sequencing discovered a dominant activating mutation in PIK3CD–c.3061G > A (p.Glu1021Lys).

GenotypingMethod:

Variant: PIK3CD–c.3061G > A (p.Glu1021Lys).

CAID: CA145460

gnomAD: absent in gnomAD v2.1.1

PMID: 37758162

Gene: PIK3R1

HGNC: 8979

Case#: 15-year-old boy

DiseaseAssertion: SHORT syndrome and Immunodeficiency 36

FamilyInfo: Table2 Father is wild type, mother was unavailable for testing. Consanguinity was reported at Table 4. No affected family members Table4.

CasePresentingHPOs: HP:0001511(Intrauterine growth retardation) HP:0004322(Short stature) HP:0000325(Triangular face) HP:0010751(Dimple chin) HP:0000684(Delayed eruption of teeth) HP:0000347(micrognathia) HP:0100750(Atelectasis) HP:0004469(chronic bronchitis) HP:0002110(bronchiectasis) HP:0002720(Decreased circulating IgA level) HP:0011342(Mild global developmental delay) HP:0004279(short hands) HP:0000954(Single transverse palmar crease) HP:0002205(Recurrent respiratory infections)

CaseHPOFreeText:

CaseNotHPOs: Height -5.5 to -6.1 SDS

CaseNotHPOFreeText: N/A

CasePreviousTesting: CMA and MS-MLPA for chromosomes 6,14,20 was performed.

GenotypingMethod: Whole-exome sequencing was performed on the patient’s whole blood sample.

PreviouslyPublished: No

Variant: NM_001242466.2:c.68G > A p.Arg23Gln

ClinVar: 1361868

CAID: CA3290343

gnomAD: 0.00005439 https://gnomad.broadinstitute.org/variant/5-67589169-G-A

PIK3CG variants p.Arg1021Cys and p.Arg1021Pro described and functionally characterized in experiments.

PMID: 29180244

Gene: PIK3CD

HGNC: 8977

PMID: 28713374

Gene: PIK3CD

HGNC: 8977

PMID: 34115277

Gene: PIK3CD

HGNC: 8977

Case#: 19 year old, Indian, male

DiseaseAssertion: APDS

FamilyInfo: Neither parent carried this mutation, suggesting a de novo origin.

CaseHPOFreeText: referred for the evaluation of chronic active EBV disease. The patient’s medical history revealed congenital stenosis of the left bronchus, recurrent sinopulmonary infections, and autism spectrum disorder with an IQ of 80–86. EBV seroconversion was identified at 7 years of age when the patient presented idiopathic abdominal lymphadenopathy. Furthermore, a previous laboratory examination revealed IgA deficiency and low IgG2 and IgG4 levels without an overarching diagnosis. Four months before presentation, the patient developed fatigue, fever, night sweats, hepatosplenomegaly, anemia, hepatitis, weight loss of 10 kg, and lymphadenopathy (Fig. 1). Serum EBV copy numbers were repeatedly > 1500/mL. Histology of the three lymph nodes did not reveal any evidence of malignant lymphoma, but rather a markedly positive EBV-encoded RNA (EBER). Chronically active EBV was diagnosed, and rituximab therapy of 500 mg weekly during 4 weeks was initiated. A stable condition without any additional immunosuppressive drugs was achieved for 3 months after which he developed high spiking fever, pneumonia, progressive lymphadenopathy, severe unexplained cardiomyopathy with progressive anemia, thrombocytopenia, and hyperferritinemia (Table ​(Table1).1). Malignant lymphoma was excluded and hemophagocytosis was detected in the bone marrow (Fig. 3A, B). The patient therefore fulfilled six out of eight diagnostic HLH-04 criteria (Table ​(Table2)2) [7]. His clinical status quickly deteriorated, and he developed acute respiratory distress syndrome and multiple organ failure. Salvage therapy was initiated with high-dose steroids, sirolimus, and intravenous immunoglobulin. PI3K delta inhibitors were withheld due to heart and respiratory failure. Etoposide was withheld due to leucopenia and severe sepsis with systemic infection (hospital acquired pneumonia with H. influenza, not responding to treatment). Within 1 month, the patient died from cardiac failure with refractory pulmonary edema.

CasePreviousTesting: whole exome sequencing (confirmed by Sanger sequencing)

GenotypingMethod: whole exome sequencing (confirmed by Sanger sequencing)

Variant: heterozygous c.3074A > C, p.Glu1025Gly mutation in the PIK3CD gene (NM_005026.3)

CAID: CA16617216

gnomAD: absent from gnomAD v2.1.1

Case#: P7, diagnosed at age 7, female

DiseaseAssertion: APDS

FamilyInfo:

CaseHPOFreeText: recurrent respiratory tract infections, EBV viremia conjunctivitis, lymphadenopathy, hepatosplenomegaly, short stature, thrombocytopenia

GenotypingMethod: Unclear, possibly WES ("In a previous study (15), we reviewed clinical and routine immunological features of 15 APDS patients diagnosed in our center by next-generation sequencing (NGS). In the present study, we extended our previous study...")

Variant: Y524N

CAID: CA338303802

gnomAD: absent from gnomAD v2.1.1

SupplementalData: Table S1

Case#: P23, Male, diagnosed at age 7

DiseaseAssertion: APDS

FamilyInfo:

CaseHPOFreeText: cytomegalovirus, lymphadenopathy, EBV lymphadenitis, B cell lymphoma, IgA vasculitis

GenotypingMethod: Unclear, possibly WES ("In a previous study (15), we reviewed clinical and routine immunological features of 15 APDS patients diagnosed in our center by next-generation sequencing (NGS). In the present study, we extended our previous study...")

Variant: E81K

CAID: CA338300169

gnomAD: absent from gnomAD v2.1.1

SupplementalData: Table S1

PMID: 19679072

Gene: PIK3CD

HGNC: 8977

PMID: 32641742

Gene: PIK3CD

HGNC: 8977

PMID: 28842185

Gene: PIK3CD

HGNC: 8977

Case#: 2 year old female, Albanian

DiseaseAssertion: APDS

FamilyInfo: The patient was the first of three children from a non-consanguineous family of Albanian origin

CaseHPOFreeText: presented with recurrent otitis media, respiratory infections, persistent splenomegaly and nonmalignant lymphadenopathy. In the first year of life, she had recurrent episodes of wheezing associated with viral infections. In four occasions, she developed otitis media. Clinical evaluation at 17 months of age revealed splenomegaly suggesting Autoimmune lymphoproliferative disease (ALPS), but analysis of CD4 − /CD8 − /TCR alpha/beta + T cells was normal. In addition, bone marrow morphology and karyotype were normal. At the age of 21 months, the patient was hospitalized due to an additional episode of otitis caused by multidrug resistant Pseudomonas aeruginosa . Since then, she suffered of recurrent otorrhea, due to Haemophilus influenzae and Moraxella catarrhalis . Virological testing ( Table 1 ) revealed chronic low-level Epstein–Barr virus (EBV) viraemia characterized by EBV-DNA persistence and elevated anti-VCA IgM (total viral load ranging from negative to 506 copies/ml; VCA IgM ranging from 43 AU/ml to 186 AU/ml).

CasePreviousTesting: No genotyping ot other genes

GenotypingMethod: Genetic analysis of PIK3CD by Sanger sequencing revealed a heterozygous G > A mutation at the position c.3061 resulting in E1021K substitution

Variant: heterozygous G > A mutation at the position c.3061 resulting in E1021K substitution

CAID: CA145460

gnomAD: variant is absent in gnomAD v2.1.1

Case#: Wang_2018_P1, M, 2 y.o. (onset), origin in China

DiseaseAssertion: APDS

FamilyInfo: None reported

CasePresentingHPOs: RRTI (HP:0002205) Tonsillitis (HP:0011110) Diarrhea (HP:0002014) Lymphadenopathy (HP:0002716) Hepatomegaly (HP:0002240) Splenomegaly (HP:0001744) ASD (HP:0001631) Dacrocystitis (HP:0000620) Inguinal hernia (HP:0000023) autoimmune cytopenia (HP:0001973) increased transitional B cells (HP:0030381) increased plasmablasts (HP:0032128) decreased CD3 (HP:0045080) increased CD4 (HP:0032219) decreased CD4 naive (HP:0410378) increased CD4 EM (HP:0025625) decreased CD8 naive (HP:0410377) decreased T cells (HP:0005403) decreased b cells (HP:0010976) decreased CD4/CD8 (HP:0033222) decreased IgG (HP:0004315) increased IgM (HP:0003496)

HP:0002205, HP:0011110, HP:0002014, HP:0002716, HP:0002240, HP:0001744, HP:0001631, HP:0000620, HP:0000023, HP:0001973, HP:0030381, HP:0032128, HP:0045080, HP:0032219, HP:0410378, HP:0025625, HP:0410377, HP:0010976, HP:0004315, HP:0003496

CaseHPOFreeText: EBV DNA, decreased antibodies to Hepatitis-B, presence of autoantibodies, decreased CD19, increased CD8, increased CD8 CM, increased NK cells, increased CD8 EM

CaseNotHPOs: abnormal naive B cells (HP:0030370) abnormal memory B cells (HP:0030373) abnormal CD8 TEMRA (HP:0020177) abnormal wbc counts (HP:0011893) abnormal IgA (HP:0410240)

HP:0030370, HP:0020177, HP:0410240, HP:0011893, HP:0020177

CaseNotHPOFreeText: CMV-IgM negative, Fungus negative, abnormal CD4 CM, abnormal DNT

CasePreviousTesting: None reported

GenotypingMethod: WES + Sanger

PreviouslyPublished: Additional info published in 2022 (PMID:35799777)

Variant: NM_005026.5(PIK3CD):c.3061G>A (p.Glu1021Lys)

ClinVarID: 88675

CAID: CA145460

gnomAD: Not present in gnomAD

SupplementalData: Phenotypic info in table S4

PMID: 37600808

Gene: PIK3CD Gene: PIK3R1

HGNC: 8977 HGNC: 8979

Douglass disagrees with people who think everyone should forget the Civil War.

Frederick believes the country must have liberty, justice and obey the constitution before there can be true peace.

Douglass starts by honoring Abraham Lincoln and explaining why everyone has gathered.

Background info about what the speech is about

Tittle of the book or page

Directed this to the audience to evoke a feeling of injustice and patriotism. Wants others to fight for what they stand for.

starts with this to establish credibility and connection with the audience. Makes him a credible and honored speaker. Ethos

There is a separation from is power and out of power.

Uses an appeal to a specific audience: those of religious background.

Speaks from experience and forgives ex-slaveholders, gets others to sympathize with him and feel from his point of view.

Change of pronouns in these paragraphs is used to emphasize a point and to connect more deeply with the audience.

Repetition of "If" is used to emphasize a point, and the reminder of history is used as a logical argument. Logos

main claim

key term

Key term

Key terms

key term

key term

Key term

Key Term

Main point: reminding others what the Civil War was about, how it came out, and what the difference is between the Union and the Confederacy

Purpose, what Douglass hopes to get out of this speech

Support: those emancipated and those who fought in the war

Support

Background information about what the speech was about: Others forgetting the Civil War, having to bring a reminder of what the Civil War was about. Freeing slaves and fighting treason. Frederick Douglass was a black leader, abolitionist, orator, writer, and reformer. Frederick Douglass wants to remind Americans of why the Civil War occurred, and why they should keep fighting toward liberty, equality, and justice.

Title: Frederick Douglass on Remembering the Civil War, 1877 Author: Frederick Douglass Publisher: The American Yawp Reader Given in: 1877

The title shows the experience of the Civil War after ended. Douglass wants to remember to people the war were important.

what is time? is it time? is time it?

Túm lại, máy Super...có những phương pháp nào để phân biệt phông neutrino siêu tân tinh khuếch tán DSNB giữa tạp âm?

Bằng cách nào ngta có thể truy vết được neutrino

Thí nghiệm Super-Kamiokande là gì và dùng để làm gì?

Làm sao để bắt đợc những hạt neutrio khi chúng không chịu dừng lại

Vì sao lại có cấu trúc này và vì sao lại là Nhật Bản?

Hạt neutrino là gì, có đặc điểm như thế nào?

Vụ nổ siêu tân tinh (supernova) là một sự kiện thiên văn học, đánh dấu sự kết thúc của một ngôi sao khổng lồ --> giải phóng nguồn năng lượng khổng lồ

Mình bị lừa bởi cách đặt tên của bài báo ka ka. Ko dễ hiểu các thuật ngữ mình đã từng nghe khi học cấp 3 môn vật lý và rất lâu rồi không dùng lại ^^

In short, neutrino là một dạng hộp đen của vũ trụ, lưu trữ toàn vẹn thông tin hành tinh

ản dụ nhưng quá khó hiểu với dân KHXH (:

Dùng từ này hay quá nè, rất là địa phương và Việt Nam hóa

Đoạn này mô tả một đặc trưng của khoa học: lý thuyết đến trước và xuất phát từ trí tưỡng tượng của mọi người, sau đó mới được kiểm chứng thực nghiệm.

Khi không chứng minh được một thứ không có nghĩa là nó không tồn tại.

những vụ nổ mà ở đó các ngôi sao lớn nặng ký qua đời

ây bùn quá z

Neutrino có đặc tính là không tương tác với thứ gì. Cho nên khi nghiên cứu về bức xạ vũ trụ, nhà khoa học dựa vào Neutrino đễ đo đạc. Vì nó không tương tác với vật chất hay bụi khác.

Khái niệm hạt ma (cả para)

wowww thật hảa

Cụ Xuân Diệu không còn giục giã mọi người vì neutrino đã tạo ra tiếng thét thay thế

rất wow

1. Khái niệm: highlight khái niệm mới và diễn giải lại bằng lời của mình.
2. Tóm tắt: Tự mình tóm tắt lại một vài đoạn văn liên quan tới nhau.
3. Cảm nhận: GHi lại cảm nhận của mình khi đọc.

ABC

xonic, suffix for axis. "either 1 axis, or 4 axises. yet never 3 axises".

oligotrophic = clear water with low nutrient concentration. Eutrophic = water with high nutrient levels; leading to abundant plant growth and murky water, high BO.

I learned that Native American groups worked together to bring attention to problems like broken treaties, the way Native people were treated, and poor living conditions. They pushed for change and helped people understand what was really going on.

This stood out to me because Native Americans were being talked about by outsiders instead of getting to tell their own stories. It also shows how students fought to make colleges fairer and to get them to include different cultures in what was being taught, and that's amazing.

This is news to me, I never heard of {AIS) or (NAS) it's awesome to read about or hear about American Indian activist. This also awesome to see so many minority groups coming and working together to fight for equality in schools.

• Nasilenie presji inflacyjnej i zmiana perspektywy stóp procentowych

  • Wskaźnik inflacji konsumenckiej (CPI) w USA osiągnął poziom 4,2% rok do roku, notując najwyższy odczyt od trzech lat i znacznie przekraczając rynkowe prognozy [00:01:56, 00:02:33].
  • Inflacja producencka (PPI) wzrosła o 1,1% miesiąc do miesiąca oraz aż o 6,5% rok do roku (najwyżej od listopada 2022). Wzrost PPI wyprzedza CPI, ponieważ odzwierciedla wcześniejsze podwyżki cen surowców i energii u producentów [00:01:27, 00:03:14].
  • Największy wpływ na skok PPI miały koszty energii (napędzane wysokimi cenami ropy naftowej) oraz wzrost cen surowców na początkowych etapach produkcji (o 12% r/r) [00:03:22, 00:03:49].
  • Rynek pracy wysłał mieszane sygnały: liczba nowych miejsc pracy (Nonfarm Payrolls) skoczyła do 172 tys. (wobec prognozy 85 tys.), lecz długoterminowy trend wskazuje na wyhamowywanie dynamiki. Stopa bezrobocia wzrosła do 4,3%, co historycznie bywało sygnałem ostrzegawczym przed korektami rynkowymi [00:04:24, 00:04:59].
  • Te odczyty wymusiły zmianę rynkowego konsensusu dotyczącego stóp procentowych. Zamiast obniżek, rynki zaczęły wyceniać potencjalne podwyżki pod koniec 2026 i w 2027 roku. Wyższy koszt pieniądza uderza bezpośrednio w spółki mocno zadłużone, podnosząc koszty finansowe ich funkcjonowania [00:05:40, 00:06:44].

• Analiza fundamentalna Oracle (Wyniki za Q4 i FY2026)

  • Przychody Oracle za czwarty kwartał urosły o 21% r/r do 19,2 miliarda dolarów, a zysk na akcję (EPS) wyniósł 2,11 $, pokonując konsensus rynkowy o 8,2% [00:08:30]. Całoroczne przychody firmy po raz pierwszy przekroczyły 67 miliardów dolarów [00:08:46].
  • Sektor infrastruktury chmurowej (Cloud Infrastructure) zyskał aż 93% r/r, co potwierdza transformację Oracle w kierunku głównego dostawcy technologii pod projekty AI [00:08:52]. Zakontraktowane, przyszłe przychody (RPO) wzrosły o 363% r/r do gigantycznego poziomu ponad 630 miliardów dolarów, przewyższając obecną kapitalizację giełdową spółki [00:09:26, 00:09:43].
  • Głównym problemem pozostają potężne wydatki inwestycyjne (CapEx), które w FY2026 wyniosły 55 miliardów dolarów (prognoza na FY2027 zakłada wzrost do 70 miliardów). Przekraczają one generowane wolne przepływy pieniężne, spychając wolny cash flow na poziom blisko -24 miliardów dolarów [00:09:50, 00:10:11].
  • Aby finansować te inwestycje, spółka planuje pozyskać 40 miliardów dolarów (20 mld poprzez stopniową emisję akcji w formule at-the-market i 20 mld poprzez nowy dług). Wskaźnik zadłużenia do aktywów (Total Debt to Total Assets) wynosi już około 60%, co przy widmie wyższych stóp niepokoi inwestorów i wywołało kilkunastoprocentową korektę kursu [00:11:56, 00:12:37].

• Spektakularne IPO SpaceX i narodziny akronimu "MANGO"

  • Debiut giełdowy SpaceX okazał się największym IPO w historii rynków finansowych. Cena emisyjna wynosiła 135 $, jednak notowania otworzyły się z luką popytową na poziomie 150 $, zamykając sesję w okolicach 160 $ [00:15:11, 00:15:41].
  • Spółka sprzedała ponad 55,5 miliona akcji, pozyskując 75 miliardów dolarów kapitału (ponad 2,5 raza więcej niż dotychczasowy rekordzista Saudi Aramco). Popyt ze strony inwestorów przekroczył 250 miliardów dolarów, wywołując silną redukcję zapisów [00:15:28, 00:17:25].
  • Dzięki temu debiutowi SpaceX z kapitalizacją rzędu 2 bilionów dolarów stało się z miejsca 7. największą firmą świata, a Elon Musk formalnie został pierwszym na świecie bilionerem (z majątkiem netto na papierze) [00:15:41, 00:16:06].
  • Rynkowi komentatorzy rewidują dotychczasowy akronim największych spółek (FAANG/MAG7) na rzecz MANGO: Meta, Antropic, Nvidia, Alphabet, Open AI oraz SpaceX [00:17:03].
  • Część brokerów wprowadziła dla akcji SpaceX tzw. okres zamrożenia (lock-up) na 15–30 dni, by ograniczyć natychmiastową spekulację i tzw. flipowanie akcji tuż po debiucie [00:18:41].

• Statystyka historyczna debiutów technologicznych vs. wycena SpaceX

  • Przed samym IPO SpaceX widoczna była kilkunastoprocentowa pompka na spółkach satelitarnych (np. Echostar) połączona z natychmiastowym zjazdem po debiucie, co sugeruje, że inwestorzy szukali tam tymczasowej ekspozycji, po czym masowo przesiedli się na właściwy walor [00:17:46, 00:18:33].
  • Krótkoterminowa korekta na indeksie S&P 500 (ok. 5%) przed samym IPO mogła wynikać z faktu, że inwestorzy wyprzedawali inne płynne bigtechy (Google, Nvidia, Palantir), by uwolnić gotówkę na zapisy na SpaceX [00:18:55].
  • Analiza historyczna 12 głośnych debiutów technologicznych (przeprowadzona za pomocą platformy TradingView i Investing Pro) pokazuje, że wejścia na giełdę rzadko przynoszą natychmiastowe zyski. W horyzoncie 6 miesięcy spółki te traciły średnio 2%, po roku traciły 19%, a ich średnie maksymalne obsunięcie (drawdown) wynosiło aż -52% [00:20:34, 00:21:16].
  • Przykłady Mety (Facebooka) oraz Palantira potwierdzają ten schemat – obie spółki po debiucie zaliczyły spektakularne, kilkudziesięcioprocentowe spłuczki cenowe (Meta -60%, Palantir -78%), zanim ich kurs wszedł w wieloletni trend wzrostowy poparty fundamentami [00:21:23, 00:22:08].
  • Aktualny wskaźnik Cena/Sprzedaż (P/S) dla SpaceX przekracza 100. Wycena ta bazuje na dalekosiężnych obietnicach (kolonizacja Marsa, orbitalne centra danych), a nie bieżących zyskach. Po minięciu pierwszych kwartałów i blokad lock-up, cena SpaceX prawdopodobnie ulegnie korekcie i zbliży się do twardych fundamentów, co stworzy lepszy moment do zakupów długoterminowych [00:21:55, 00:23:26].

• Ewolucja finansowa i operacyjna

  • W ciągu ostatnich 5 lat Mercado Libre odnotowało spektakularny wzrost fundamentalny: wartość sprzedanych towarów (GMV) wzrosła o 238%, wolumen płatności o 516%, przychody o 700%, a zysk operacyjny o ponad 2200% [00:00:38].
  • Kurs akcji w tym samym okresie wzrósł zaledwie o kilkanaście procent, tworząc potężny rozjazd między realnymi wynikami biznesowymi a wyceną rynkową [00:00:50].
  • Kluczowym motorem marży jest model Third-Party (3P), czyli pobieranie prowizji od zewnętrznych sprzedawców, kosztem wygaszanego, niezależnego projektu Mercado Shops na rzecz wewnętrznego ekosystemu "Mi Pagina" [00:01:25].

• Logistyka jako bariera wejścia (Moat)

  • Skala rozwoju sieci Mercado Envios jest ogromna: z 1,8 miliona paczek (2,2% udziału) w 2013 roku do 1,5 miliarda przesyłek i obsługi 98% dostaw w 2023 roku [00:04:03].
  • Na koniec 2025 roku firma dysponowała 6 milionami m² powierzchni magazynowej oraz własną flotą samolotów cargo w Brazylii i Meksyku [00:04:30].
  • Głównym celem logistyki nie jest bezpośrednia rentowność, lecz cementowanie przewagi konkurencyjnej, podbijanie konwersji i generowanie ruchu dla zyskowniejszych segmentów [00:04:56].

• Potęga segmentu fintech (Mercado Pago)

  • Wolumen transakcji handlowych (acquiring) w roli agenta rozliczeniowego osiągnął w I kwartale 2026 roku wartość 204 miliardów dolarów [00:05:30].
  • Cyfrowy portfel idealnie zagospodarowuje ponad 100 milionów ludzi funkcjonujących w szarej strefie Ameryki Łacińskiej bez konta w tradycyjnym banku. Liczba aktywnych użytkowników miesięcznych (MAU) wzrosła do prawie 83 milionów [00:05:51].
  • Segment kredytowy rozwija się w tempie blisko 88% rocznie z przychodami rzędu 6,7 miliarda dolarów, bazując na unikalnych danych transakcyjnych użytkowników, co obniża koszty operacyjne w porównaniu do tradycyjnych banków [00:06:48].
  • Całkowity wolumen płatności Pago (306,7 miliarda dolarów) jest ponad czterokrotnie większy niż GMV samego marketplace'u, co czyni z firmy giganta finansowego działającego głównie poza własną platformą [00:07:32].

• Główne czynniki ryzyka

  • Makroekonomia: Prognozy Banku Światowego wskazują na wolny wzrost PKB regionu w 2026 roku (ok. 2,1%) oraz osłabienie dynamiki w Brazylii i Meksyku, co ogranicza siłę nabywczą konsumentów [00:08:00].
  • Geopolityka i waluty: Wysokie ryzyko niestabilności politycznej, przestępczości zorganizowanej oraz drastycznych wahań walutowych (głównie reala brazylijskiego i peso argentyńskiego) [00:08:47].
  • Konkurencja: Silny nacisk ze strony Amazona (8 mld USD inwestycji w Meksyku), agresywne cenowo Shopee (wzrost o 600% od 2020 roku w tańszych kategoriach) oraz dynamicznie wchodzący TikTok Shop [00:09:48]. Na polu fintechu głównym rywalem pozostaje Nubank ze 135 milionami klientów [00:11:29].

• Perspektywy dalszego wzrostu

  • Niska penetracja e-commerce: Udział handlu elektronicznego w Ameryce Łacińskiej wynosi 12–15% wobec średniej światowej wynoszącej 18%, dając przestrzeń do długoterminowego podwojenia wyników [00:12:05].
  • Reklama cyfrowa: Dynamiczny rozwój sieci reklamowej na bazie bazy 121 milionów kupujących przyniósł ponad 10-krotny wzrost przychodów z reklam do poziomu ponad 1,5 miliarda dolarów [00:13:06].
  • Sektor fintech: Szacunki zakładają wzrost wartości branży o ok. 20% rocznie do 2034 roku, gdzie przewagą firmy jest darmowe pozyskiwanie klientów z poziomu lojalnego marketplace'u [00:13:43].

Absolutely true

This is interesting.

General thoughts - this could be like 4 articles! It's depth and breadth are a lot to cover here.

If not executed well, it can destroy companies.

😂

This is something that businesses will need to start to think about. AWS AgentCore does have some interesting concepts around memory/context separation: https://aws.amazon.com/bedrock/agentcore/resources/?trk=9cd2d6e3-79cb-48fa-b04c-0233ea17f395&sc_channel=ps

Mostly agree. AI as a 'colleague' is strong, but I agree with the sentiment.

Testing this app.

Does this mean that many women get pregnant and miscarry without ever realizing it?

Agent pre-plans calls too -- you can share your calendars and it makes suggested times/matches

• Need push notifications.
• Need to be able to synchronize this with your calendar.
• Needs to be very smartphone friendly, or at least be able to switch between modes.

tooltip what the point of htis is let people upvote or downvote responses ,and then sorted by this or in chronological order.

it should automatically highlight the matches, e.g., if you're both up for a drive time call ... Tell you how many minutes you could have overlapping

don't show yourself in the thingbelow

Communicating and listening are 2 different things. To nonverbal and verbal going.

I have found myself in this "action" listening style as an IT Work Study tech at the CWI Help Desk. When I see a pupil bring in a broken laptop, I'm always tempted to ignore the feeling of frustration and just see what steps I can take to fix the problem. This is good for me to get to the root of the issue and close tickets quickly, but I need to keep in mind that sometimes users just want to be heard before we dive into the troubleshooting. If you strike a balance between that and some empathy, you'll have a better user experience.

being social is a performance, and lonely people/socially awkward people are more aware of it than people who are confident.

從#1開始 移至#2 升至第6名 升至第7名 升至第8名 升至第6名 升至第4名 升至第8名 升至第5名 升至第4名 移至#2 升至第3名 升至第5名 升至第7名 升至第3名 最後移至#1

reply to u/Typed_Anew at https://old.reddit.com/r/typewriters/comments/1ug4q1i/any_information_on_thisa_repair_question/

There don't appear to be any examples in the typewriter database, so be sure to create an account and upload the data and photos you've got. https://typewriterdatabase.com/register-today.php (It's not every day someone gets to upload a new make/model!)

Similar machines and details: - Gundka https://typewriterdatabase.com/no_info.414.typewriter-serial-number-database - Frolio https://typewriterdatabase.com/no_info.415.typewriter-serial-number-database

By drilling down into the database and looking at pictures and descriptions of other models as well as looking at the galleries for specific examples, you may be able to troubleshoot what is missing or "off" on your machine (which is obviously missing the spools, though you may be able to fashion something similar to get yours working.)

Other examples via online search may be helpful as well: - https://www.glennsantiques.co.uk/shop/p/02/060/11 - https://www.worthpoint.com/worthopedia/vintage-toy-typewriter-emg-dover-1000802769 - https://www.ebay.com/sch/i.html?_nkw=EMG+Dover+Junior+typewriter&_sacat=0&_from=R40&_trksid=m570.l1313

Documenting your own example will make it much easier for collectors in the future.

“he looks at past things from a distance”

“He could not express what he wanted more forcefully than by appealing to their conscience and, as it were, by confessing shame in their person.”

"over which things you are now ashamed."

Or more idiomatically:

"the things of which you are now ashamed."

"to be fruitless" or "to be without fruit."

“But now, you have your fruit, which leads to sanctification…”

Or more idiomatic:

“But now you are producing fruit that results in sanctification…”

“For the end of those things is death.”

“because of which things you are now ashamed”

“then” / “at that time” / “at that point”

shame,” “disgrace,” or “a sense of dishonor.”

to be ashamed with respect to / because of something

on account of which

directional, intensifying, or affective force

on,” “upon,” “over,” or “in relation to.”

1. With different cases 🔹 With GENITIVE (ἐπί + gen.)

Often means:

“on the basis of” “concerning” “in the time of”

Example sense:

“in the days of…” / “on the authority of…” 🔹 With DATIVE (ἐπί + dat.)

Often means:

“on,” “upon” (location) “at / in connection with” “because of / regarding” (very common in moral or emotional contexts)

Example:

“still,” “yet,” “further,” or “even now.”

“because of these things, which you are now ashamed of”

“to act and to bear/produce the consequences of those actions.”

on account of which

“you will not be ashamed of such imitation” or more naturally, “you will not feel shame in such imitation.”

“lest you be ashamed because of our friendship” or “do not be ashamed of our friendship.”

to be ashamed of something,

“on account of which things” or “because of which (things)”

“of those,” “of those things,” “of those people,” “belonging to those.”

“For the end of those things is death.”

“What things are you ashamed of?” or more naturally, “What are you now ashamed of?”

“For you did not even divide the mode of your slavery between righteousness and sin, but rather you were entirely surrendering yourselves to wickedness.”

Secrets to Remembering Names and Faces (1988 Harry Lorayne) (VHS Rip)<br /> https://www.youtube.com/watch?v=qtrMtoxHGjI

Reminding me a lot of Dave Cormier's work in Learning in a Time of Abundance and elsewhere.

See Patrick H. WIllems - Why is every streaming blockbuster so bad?!

Miroslav Šašek illustrations<br /> https://en.wikipedia.org/wiki/Miroslav_%C5%A0a%C5%A1ek

Use of repetition creates rhythm.

Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

Learn more at Review Commons

Reply to the reviewers

• *

Review Commons: RC-2026-03417

We thank all three reviewers for their thoughtful and constructive evaluations of our manuscript. We are encouraged that the reviewers recognize the significance of identifying a role for the TRAPPIII-Rab1 module in Wingless (Wg) trafficking and secretion. We also appreciate the insightful comments that have helped us identify areas where our interpretations, experimental support, and presentation can be strengthened.

Below, we provide a detailed point-by-point response to all comments and concerns raised by the reviewers.

Reviewer #1

__Evidence, reproducibility and clarity __

Summary

In this manuscript, the authors investigate the role of Transport Protein Particle (TRAPP) complexes in the secretion of Wingless (Wg) in Drosophila. Two TRAPP complexes are known to exist: TRAPPII and TRAPPIII. Through the systematic analysis of individual subunits, the authors identify the requirement of the TRAPPIII subunit TRAPPC8 for Wg trafficking.

They demonstrate that the absence of TRAPPC8 disrupts the retrograde trafficking of both Wg and its carrier Evi, resulting in their intracellular accumulation within Wg- producing cells. Further analyses suggest that TRAPPC8 controls the post-apical internalisation and endosomal trafficking of Wg and Evi. Consistent with the established function of TRAPPIII as a Rab1-specific GEF, they demonstrate that inhibiting Rab1 produces similar effects as depleting TRAPPC8, while constitutively active Rab1 reverses the trafficking defects. Furthermore, depletion of either TRAPPC8 or Rab1 increases the levels of Wg-unbound Evi, suggesting that they act downstream of Evi-Wg dissociation. Taken together, these findings suggest that TRAPPIII and its effector Rab1 are essential regulators of retrograde Wg trafficking, which is necessary for efficient secretion.

Overall, the work is carefully performed and the results are presented clearly. The controls are appropriate and the study expands the functional scope of Rab1-dependent trafficking beyond early secretory pathways. The identification of a previously unrecognised function of TRAPPC8 in Wg trafficking is a valuable contribution.

Major Points

1. It is not entirely clear whether the RNAi lines used in the initial screen were validated for knockdown efficiency. Notably, some core TRAPPIII subunits (e.g., TRAPPC3 and TRAPPC5) do not show a phenotype. This could indicate that the complex retains partial function upon their depletion, or alternatively that the RNAi lines are ineffective. While this point may not critically affect the main conclusion regarding TRAPPC8, it is important for drawing conclusions about the specificity of TRAPPIII versus TRAPPII involvement. For instance, TRAPPC10 (a TRAPPII-specific subunit) was analysed using a single RNAi line, yet no evidence of knockdown efficiency was provided. Validation of these RNAi reagents would strengthen the conclusions regarding complex specificity.

Response: *We thank the reviewer for highlighting this shortcoming in the manuscript. We agree that testing the knockdown efficiency of the TRAPPII complex subunits will strengthen our conclusion on the specificity of TRAPPC8/TRAPPIII towards Wg trafficking. While testing the protein levels would be ideal for checking knockdown efficiency, specific antibodies to these subunits are not commercially available. Therefore, as an alternative approach, we will perform RT-PCR to assess the efficiency of RNAi-mediated depletion of the TRAPPII-specific subunits (TRAPPC9 and TRAPPC10). *

Furthermore, we would also like to highlight that previous studies have reported variable phenotypic outcomes upon loss of TRAPP complex subunits, ranging from no apparent defects to early lethality (Sun and Sui 2023; Riedel et al. 2018). For example, complete deletion of the C9 gene and a frameshift mutation in the C10 gene were found to be homozygously viable, whereas C8 and C11 homozygous mutants showed early larval lethality (Riedel et al., 2018).

*To better contextualize our findings, we will include a more detailed discussion comparing our results with the phenotypes reported for these published mutants, in addition to the RT-PCR analysis of RNAi efficiency. *

Minor Points

In Figure 2C, the clone appears restricted to the apical region, and I do not clearly observe GFP loss in the basolateral domain. Larger clones or additional sections would help clarify the spatial distribution and strengthen the interpretation.

Response - *We thank the reviewer for pointing this out. This issue arises from the pseudostratified nature of the wing epithelium. For smaller clones, such as those observed in the TRAPPC8 mutant, cross-sections can occasionally pass through the tissue at an oblique angle, making it difficult to capture the entire clone within a single section. *

We have repeated the experiment and now provide better images where the apical and basolateral distribution of the mutant clone is visible, and accumulation of exWg can be observed (see updated Figure 2A-C).

The authors should discuss why TRAPPC8 depletion results primarily in apical Wg enrichment, whereas Rab1 inhibition leads to Wg accumulation at both apical and basolateral membranes. This difference may provide insight into whether Rab1 has additional TRAPPIII-independent functions or whether TRAPPC8 affects a more spatially restricted trafficking step.

__ ____Response__: We agree that, while inhibition of either TRAPPC8 or Rab1 led to a strong intracellular accumulation of Wg, subtle differences were observed in the localization of the accumulated Wg. In agreement with your comment and as suggested by previous studies, C8 may direct the Rab1-GEF activity of the core TRAPP complex towards a specific location, while Rab1DN will affect all downstream functions, possibly also including TRAPPIII-independent effects (Riedel et al. 2018; Gyurkovska et al. 2026)*. We will modify the text to incorporate these points more clearly. *

__ ____Significance__

This study broadens our understanding of Rab1-dependent membrane trafficking by identifying a previously unrecognized role for the TRAPPIII complex in retrograde transport during Wingless secretion. ____While the study convincingly establishes the involvement of the TRAPPIII-Rab1 module in Wg trafficking, it does not define the specific retrograde trafficking step that is regulated by this machinery. In addition, the functional relationship between TRAPPIII-Rab1 and established retrograde regulators, such as the retromer complex, is not addressed.

____Response: We thank the reviewer for this comment. This issue was also raised by Reviewer 3. We agree that the precise retrograde trafficking step regulated by the TRAPPC8-Rab1 module remained unclear in the study. To address this, we will perform additional experiments to investigate the functional interactions between TRAPPC8-Rab1 and components of the retrograde trafficking machinery, including the retromer complex, and will incorporate these findings into the revised manuscript. These analyses should help clarify the specific trafficking step controlled by the TRAPPC8-Rab1 module.

Further molecular and mechanistic analyses will be required to position TRAPPIII within the broader retrograde trafficking network and to fully elucidate how Rab1 activity is coordinated with other pathways involved in Wnt secretion. These unresolved issues represent the main limitation of the current study.

__ ____Response__: Wg secretion in polarized epithelial cells involves multiple transport routes and regulatory mechanisms, including exosome-mediated transport, cytonemes, and association with carrier molecules. A systematic analysis of the contribution of TRAPPC8-Rab1 to each of these pathways would require substantial additional investigation and falls outside the scope of the present study.

In this work, we focused on a key, relatively underexplored aspect of Wg transport: the retrograde pathway that mediates the separation and recycling of Wg and Evi/Wls. Our findings identify TRAPPC8-Rab1 as an important component of this process and provide a basis for future studies to define its broader mechanistic integration into intracellular trafficking pathways.

Reviewer #2

__Evidence, reproducibility and clarity __

__Summary Sharma, Sabnis et al. show in this report that TRAPPC8 and Rab1 are important for Wingless (Wg) secretion in developing Drosophila wing discs. - Loss of TRAPPC8 (either knockdown or mutant clones) lead to higher levels of apical Wg in Wg-producing cells (total and extracellular staining). - The levels of the intracellular Wg transporter Evi/Wls are also increased, in particular in its 'unbound' form. - A similar phenotype is observed upon overexpression of Rab1 dominant negative. - Overexpression of constitutively active Rab1 has no effect in otherwise wild type discs but does rescue the effect of TRAPPC8 loss of function.

Major concerns 1) The author's key message is that TRAPPC8 is essential for retrograde transport of Wg and Evi. However, in apparent contradiction, TRAPPC8 loss does not appear to affect the basolateral distribution of exWg (or total Evi) (Fig2B', FigS3). Therefore, while Wg transport may be less efficient in the absence of TRAPPC8, the conclusion that TRAPPC8 is essential should be toned down. It is warranted to suggest that transport of unbound Evi is disrupted in the absence of TRAPPC8, as unbound Evi increases apically and is lost basally (Fig 4A-B).__

Response:

*Thank you for these comments, which prompted us to retest the extracellular Wg levels in these experiments. We repeated the experiments and reanalyzed the basolateral distribution of extracellular Wg in TRAPPC8-depleted cells. While we consistently observed an increase in apical extracellular Wg, the effects on basolateral Wg were more variable. In several samples, basolateral Wg appeared largely unaffected, whereas in others we observed some reduction. We speculate that this variability may arise from differences in RNAi efficiency, with stronger depletion of TRAPPC8 potentially required to reveal basolateral defects. Furthermore, these defects are completely rescued by the expression of Rab1CA, indicating Rab1-dependent effects of TRAPPC8 loss. *

*We apologize for this oversight in our analysis in the original manuscript and would like to correct our conclusions regarding the basolateral Wg. We have included both results in the partially revised manuscript, indicating the variability observed in the basolateral Wg, albeit with consistent apical accumulation of extracellular Wg. *

Furthermore, we have modified the statement (line 235, page 8) "These results suggest that TRAPPC8 functions after the dissociation of the Evi-Wg complex, likely promoting proper sorting of Evi and Wg within maturing endosomes" to "These results suggest that TRAPPC8 functions after the dissociation of the Evi-Wg complex, likely affecting proper retrograde trafficking of unbound-Evi."

Pending revision: *We will further update our discussion section to include these points after completing all additional experiments suggested by the reviewers. *

2) The relative roles of Rab1 and TRAPPC8 are not equally considered. As the authors show, unlike TRAPPC8 loss of function, Rab1 DN causes a decrease in basal exWg. Doesn't this suggest that Rab1 is more important for Wg trafficking than TRAPPC8?

Response: This issue is addressed in the previous comment.

a) Rab1 DN causes also accumulation of total basal Wg. Does this mean that Wg can be trafficked basally but fails to be secreted at the basal surface?

Response: Yes, we agree with this interpretation. In Rab1DN-expressing cells, total Wg accumulates throughout the cell, including at the basal side, while basolateral extracellular Wg is reduced. This suggests that Wg-containing vesicles can reach the basolateral region but are inefficiently secreted at the basal surface. One possibility is that Rab1 function is required for the apical-to-basolateral transcytosis and/or the final delivery of Wg-containing vesicles to sites of basolateral secretion.

b) A similar assay (total basal Wg) is lacking in condition of TRAPPC8 loss (clones or RNAi); only exWg is shown.__

Response: We will also include the basolateral sections of total Wg in TRAPPC8 depletion conditions (mutant clones and RNAi) in the revised manuscript.

c) Also needed is an assessment of total and exWg at the basal surface in the Rab1 CA experiment. Based on the cross-section image shown in Fig6B, it seems that the distribution of Wg might be more confined to the apical plane of the cells compared to other conditions (Fig6F for example), but there is little difference in this regard between the anterior and posterior compartments.__

Response: *We thank the reviewer for this important point, and we will reanalyze both total and extracellular Wg levels in Rab1CA samples, specifically the apical and basolateral distributions and provide images in the revised manuscript. *

3) A significant claim of the paper is "TRAPPC8 regulates retrograde Wg trafficking post-apical internalization". a. The increase in apical extracellular Wg (FigS3), which seems to remain associated with the membrane of Wg-producing cells (i.e. no onward spread) would suggest that apical internalization may be affected, but this is not considered by the authors.

Response: *We agree that the increased apical extracellular Wg observed in Fig. S3 (now Fig 2D-F and S3) could also suggest alterations in apical uptake dynamics. However, based on our internalization assay, we believe that the major defect is likely in the post-endocytic sorting/trafficking of Wg after apical internalization rather than a complete block in internalization itself. In these internalization assays, following the internalization pulse, the tissue was subjected to a brief acid wash to remove extracellular and surface-bound antibody. At the same time, the internalized antibody-antigen complexes remain protected. Therefore, the Wg signal detected after the acid wash represents internalized apical Wg, demonstrating that apical internalization does occur in the TRAPPC8 loss condition. *

4) From the antibody chase experiment, it is not clear if the effect of TRAPPC8 loss affects endocytosis and/or trafficking in general or whether it is specific to Wg and Evi. A fluorescent Dextran control should be included as in Witte et al., 2020 (https://doi.org/10.1242/dev.186833 ). Also, can the authors be sure that they are visualising internalised Wg and not just internalised Wg antibody? Can the author show a field of view where Wg is not expressed?

__ Response: __Thank you for this important suggestion. We would first like to clarify that the Wg internalization assay, using the highly specific monoclonal anti-Wg antibody, is an established approach that has been used in multiple previous studies to monitor Wg internalization and trafficking (Hemalatha et al. 2016; Sharma and Chaudhary 2024)*. Furthermore, as shown in Figure S4C-C′ (now moved to Fig 3D), the internalized Wg signal is detected specifically in cells close to the DV boundary, while more distal receiving cells do not show comparable staining. This spatially restricted pattern strongly supports the specificity of the assay for Wg-producing cells and a limited number of nearby receiving cells that may internalize secreted Wg, rather than nonspecific internalization of the antibody alone. We will revise the text to clarify this point in the revised manuscript. *

To address whether TRAPPC8 loss affects general endocytosis, we will perform a fluorescent Dextran uptake assay, as suggested by the reviewer and similar to the approach described by (Witte et al. 2020)*. *

5) The authors suggest that TRAPPC8 loss leads to increased Rab7 levels. This is taken as evidence for a role of acidification in driving Wg dissociation from Evi, with TRAPPC8 acting downstream to sort Wg from mature endosomes. ____Neither of these claims are supported because in Fig.S3G loss of TRAPPC8 results in an increase in Rab7 everywhere except at the DV boundary where the Wg-producing cells are. This should be acknowledged in text, and these data should appear in the main Fig4.

Response: To address this concern, we have repeated the experiment and reanalyzed the RAb7 and lysotracker levels specifically in cells at the DV boundary. The data is now moved to the updated main Figure 5E-F and 5G-H. A significant increase in both lysotracker and Rab7 can be observed, suggesting that loss of TRAPPC8 affected late endosomal maturation.

a) The increase in lysotracker also does not support the above claims as it could be due to an increase in unbound Evi that cannot be trafficked back to the ER (and hence targeted for degradation in lysosomes).

Response: We respectfully disagree with this interpretation. If TRAPPC8 loss primarily caused increased lysosomal degradation of unbound Evi, we would expect a reduction in total Evi levels, as observed upon loss of retromer function, in which Evi is diverted to lysosomes and specifically depleted in Wg-producing cells (Port et al. 2008). In contrast, we observe an accumulation of both total and unbound Evi in TRAPPC8-depleted cells (Figures 4A-D and 5A-D), arguing against enhanced lysosomal degradation as the primary defect.

b) In fig S3G, the purported Rab7 increase in the posterior compartment is not readily apparent (and not quantified), in contrast to the authors' description of the effect of TRAPPC8 depletion. This suggest that the model proposed by the authors needs to be revised (Fig6G) and the relevant paragraphs from Results and Discussion section must be significantly edited or removed.

Response: We apologize for the lack of clarity in our previous images. As noted above, we now observe a significant increase in Rab7 and Lysotracker signals in Wg-producing cells, indicating an expansion of Rab7-positive acidic late endosomal compartments upon TRAPPC8 depletion. Furthermore, our interpretation is consistent with our previous findings showing that the Evi-Wg complex dissociates within maturing endosomes.

Minor concerns 1) The way data in Fig 2, S3 and S4 is presented and referred to in text could be rearranged slightly to make it easier to follow: first talk about the work using clones (Fig2, FigS4 becomes FigS3), then mention similar results with the knockdown (FigS3 becomes FigS4). This arrangement would link naturally to the effect on Evi.

Response: Thanks for your suggestion, we have revised the supplementary figure order by changing FigS3 (now Fig S4) to FigS4 (now Fig S5) and FigS4 (now Fig S5) to FigS3 (now Fig S4), and the corresponding figure references in the main text have been updated accordingly. We have also moved some of the RNAi data from Fig S3 to main figures (Fig 2D-F and Fig 3D-F) to increase the clarity and make the results easier to follow.

2) How do the authors explain that the knockdown of some core TRAPP subunits does not have a phenotype? Some sort of rationalisation (or experimental follow up) is desirable.

*__Response: __The lack of a detectable phenotype following knockdown of some core TRAPP subunits may reflect several possibilities. Previous studies have suggested that certain core subunits, including TRAPPC2, TRAPPC2L, TRAPPC6A and TRAPPC6B, are not universally required for mammalian cell viability, indicating potential functional redundancy or their context-dependent requirements within the TRAPP core complex (Lipatova and Segev, 2019; Sun and Sui, 2023). It is also possible that residual protein levels after RNAi-mediated depletion are sufficient to support partial TRAPP function. Testing the RNAi-mediated protein depletion of these subunits is beyond the scope of the current study. *

*We would like to highlight that the focus of the study is TRAPPC8, a TRAPPIII-specific subunit, which was validated with a genomic mutation. However, to strengthen our conclusions regarding the TRAPPIII-specific effect, we will validate the known efficiency of the TRAPPII complex subunits with RT-PCR, also addressing the comment from Reviewer 1. *

3) The authors should give more detail about how they quantified normalised intensity profiles and clarify if the profiles correspond to just the representative image shown or the average of multiple discs (possible for the compartment experiments, but presumably impossible for the clone experiments as they would need to normalise by the size of the clone too).

Response: *Thank you. We have updated the figure legend to clearly indicate the representative images corresponding to each plot profile. The plot profiles were generated from representative images only and do not represent averages from multiple discs. The intensity values were normalized by dividing each value by the mean intensity across the quantified region. We have also added this information in the Materials and Methods section (line 447, page 16). *

__ ____Significance__

This report is a valuable report for the Wg secretion subfield, and useful for the Wnt community. It makes some interesting observations and brings the importance of Rab1 back into the conversation after Ching et al., 2008. However, the insight remain limited and the mechanism/trafficking defects remain unclear. There is convincing evidence that TRAPPC8 and Rab1 affect Wg and Evi, but the claims that TRAPPC8 is essential and acts downstream of acidic endosomes is inadequate.

__ ____Response:__ * Please refer to the responses for Reviewer 3. We have addressed these concerns in the next section.*

Reviewer #2 (Significance (Required)):

__This report is a valuable report for the Wg secretion subfield, and useful for the Wnt community. It makes some interesting observations and brings the importance of Rab1 back into the conversation after Ching et al., 2008. However, the insight remain limited and the mechanism/trafficking defects remain unclear. There is convincing evidence that TRAPPC8 and Rab1 affect Wg and Evi, but the claims that TRAPPC8 is essential and acts downstream of acidic endosomes) is inadequate. ____ __

__Reviewer #3 __

This manuscript uncovers a direct or indirect role of RAB1/TRAPPIII in regulating the intracellular fate of Wng in the columnar epithelial cells of the wing imaginal disc of the fruit fly. This observation is interesting, although it should come as no surprise that, given the fact that Wnts are secreted morphogens and considering the involvement of TRAPPIII in the early stages of the secretory pathway, the key TRAPPIII subunit, TRS85/TRAPPC8, is crucial for their normal trafficking. The main finding of this work is that cells deficient in TRAPPIII/RAB1 accumulate the morphogen and its receptor in the apical region of morphogen-producing cells, which is interpreted as a block in the retrograde trafficking of the morphogen.

---

1) While it is unclear to me what the authors consider as retrograde trafficking of Wng (see below), the arguments fall short of being convincing, in part because the intracellular trafficking of Wng is rather intricate, but also because the physiological role of TRAPPIII is insufficiently understood. It is well established that Wng transits through endosomes to reach multivesicular bodies, where it is incorporated into the inwardly budding vesicles that are secreted as exosomes (Gross et al, cited). Do the authors consider this a retrograde pathway? The authors do not delineate further the location at which Wng accumulates, for example using co-localization studies.

Response: *We would like to clarify that, in our manuscript, we use the term "retrograde trafficking" specifically to describe the trafficking route followed by apically internalized Wg/Evi complexes through the endosomal system before their re-secretion from Wg-producing cells. *

*Wg trafficking after internalization is highly complex and can involve multiple intracellular routes. Importantly, besides trafficking of internalized Wg to multivesicular bodies (MVBs) for exosomal secretion, other pathways downstream of apical internalization have also been reported, including Rab4-dependent apical recycling, apical-to-basolateral transcytosis involving HSPGs, and secretion of Wg on lipoprotein particles. Testing the effect of TRAPPC8 in multiple trafficking routes is currently beyond the scope of this study. *

*Our current study does not attempt to distinguish between these individual downstream secretory routes. Rather, our data support a requirement for TRAPPC8/Rab1 in trafficking steps occurring after apical internalization of Wg/Evi and before their redistribution and/or re-secretion. The detailed characterization of the precise endosomal compartments and carrier-specific secretion mechanisms affected by TRAPPC8/Rab1 will require significant additional lines of experiments, which are beyond the scope of the current work. *

__2) They have also not provided a rationalization of their observations with current knowledge of retrograde trafficking between the endosomes and the Golgi. It would have been interesting to address the effects of Trs85 depletion in mutant backgrounds deficient in the master regulator of retrograde pathways, RAB6, its effector, the GARP complex, or RAB7 and the retromer; it would have been important to study TRAPPIII depletion under conditions in which endocytic internalization is blocked, or the biogenesis of multivesicular bodies is prevented. __

__ ____Response: __We agree that understanding the functional relationship between TRAPPIII and other retrograde trafficking regulators would provide important mechanistic insight into Wg/Evi trafficking.

*Among the known regulators of retrograde trafficking, we are particularly interested in testing the functional interaction between TRAPPC8 and the retromer complex, as retromer is one of the best-characterized regulators of Evi recycling in the Drosophila Wg pathway (Port et al., 2008; Franch-Marro et al., 2008; Belenkaya et al., 2008). Therefore, we will analyze the functional interactions between TRAPPC8 and retromer components and provide results in the revised manuscript. *

However, the roles of other retrograde trafficking regulators, such as GARP and Rab6, in retrograde Wg trafficking in Drosophila are not yet well established and would first require independent characterization before meaningful epistasis analyses with TRAPPIII can be performed.

*Regarding the suggestion to block endocytic internalization, our antibody internalization experiments indicate that early Wg internalization, including uptake of Wg in neighboring receiving (non-secreting) cells, is not detectably affected upon TRAPPC8 or Rab1 depletion. These observations suggest that TRAPPC8 and Rab1 are unlikely to play a general role in endocytic uptake. *

We therefore focused our analyses on post-internalization trafficking events affecting Wg and Evi. Furthermore, blocking endocytosis globally would likely introduce strong secondary effects on both Evi-Wg complex internalization in secreting cells and uptake of extracellular Wg in receiving cells, making it difficult to distinguish direct effects on retrograde trafficking from broader defects in Wg trafficking dynamics.

__Specific comments __

3) There are no page or line numbers, which makes very cumbersome to comment on specific sections of the manuscript.

*__Response: __We apologize for this inconvenience and have now added page and line numbers. *

__4) The introduction contains a factual mistake. TRAPPC11, 12, and 13 are not metazoan specific. They are present in fungi but have been lost in Saccharomyces cerevisiae. Pinar M, Arias-Palomo E, de Los Ríos V, Arst HN Jr, Peñalva MA. Characterization of Aspergillus nidulans TRAPPs uncovers unprecedented similarities between fungi and metazoans and reveals the modular assembly of TRAPPII. PLoS Genet. 2019 Dec 23;15(12):e1008557. doi: 10.1371/journal.pgen.1008557. This reference should have been cited. __

Response: *We thank the reviewer for pointing this out and apologize for missing this reference. We have now updated the text and added the reference (see line 83 on page 3). *

__5) Materials and methods are very incomplete, particularly in the section that deals with the antibodies, which are essential tools for understanding the experiments. __

__Is the Wnt antibody a monoclonal antibody? __

__Are there different antibodies specific for extracellular and intracellular Wnt? __

__What is the molecular basis for this differential detection? __

__The transgene expressing GFP under the engrailed driver is not described anywhere. __

__ ____Response:__ We apologize for the lack of sufficient detail in the Materials and Methods section. We have now revised this section to include the missing methodological details and clarifications requested by the reviewer.* *

For all Wg-related stainings, including total, extracellular, and internalization assays, we used the mouse monoclonal anti-Wg antibody obtained from DSHB. Antibody details and working dilutions are provided in the revised Materials and Methods section.

The differential detection of extracellular versus total Wg does not arise from the use of different antibodies, but rather from differences in the staining protocol. Total Wg staining was performed after permeabilization of wing imaginal discs using 0.2% Triton X-100 in 1X PBS, allowing detection of both intracellular and extracellular Wg pools. In contrast, extracellular Wg staining was performed without tissue permeabilization, thereby restricting antibody access to extracellular or cell surface-associated Wg. Similarly, the Wg internalization assay was performed using established protocols already described in the manuscript. We have now updated the Materials and Methods section to include additional details for the extracellular Wg staining procedure (line 409, page 14).

• *The GFP expression used in our study is driven by the Gal4-UAS system, where engrailed-Gal4 (en-Gal) drives UAS-GFP expression. We have provided the details for these two fly lines in the Drosophila stocks section in Materials and Methods. For our experiments, we generated a recombinant fly stock having both en-Gal and UAS-GFP on the second chromosome using Drosophila genetics, and this genotype, along with different combinations of genes, is listed in our Supplementary Information.

• *__6) The labeling of the figures and the figure legends themselves are excessively simple and appear to be accessible for fly experts only. __

Response: We thank the reviewer for this suggestion. We have revised the details for labeling the figure in the legends and expanded the figure legends to improve clarity and accessibility for a broader audience. In particular, we added more detailed descriptions of the specific images with reference to their corresponding quantified graphs (as also suggested by Reviewer 2). We also incorporated additional minor explanatory details (for example, GFP-negative clones mean the homozygous mutant) wherever necessary to help non-fly experts to better understand the figures.

__7) The authors have not considered the possibility that ablating TRAPPC8 of TRAPPIII can have off-target effects in TRAPPII. It would have been very interesting to address the phenotype of down-regulating TRAPPII and of down-regulating one of the core subunits of TRAPPs. __

__ ____ Response: __*If the reviewer is suggesting the off-target effects of TRAPPC8 RNAi on TRAPPII complex member, we would like point out that the observations from the RNAi were validated by the TRAPPC8 mutant. However, if the concern is whether TRAPPC8 loss functionally affected TRAPPII complex besides TRAPPIII, then it we have not directly tested this. However, several past studies have shown that TRAPPC8 is a TRAPPIII-specific subunit and not part of TRAPPII complex. Furthermore, and more importantly, we have rescued the RNAi phenotype with the expression of Rab1CA, indicating the effects TRAPPIII-specific are unlikely to be via the TRAPPII-Rab11 pathway. *

__8) Figure 1, Panel 1i: What is the basis at this point that justifies "likely by altering intra-cellular trafficking"? __

Response: Since loss of TRAPPC8 resulted in increased levels of total Wg, we wanted to determine whether this increase could be due to transcriptional upregulation of wg. To address this, we examined the established wg-LacZ reporter and found that its expression was not altered upon loss of TRAPPC8 (Fig 1i).

Therefore, the increased Wg levels are unlikely to arise from increased wg transcription. In addition, previous studies have shown that loss of Evi/Wls leads to intracellular accumulation of Wg as a consequence of trafficking defects rather than transcriptional regulation. Based on these observations, we concluded that the accumulation of Wg upon TRAPPC8 depletion is more likely due to altered intracellular trafficking.

__9) Several figures: where are the boundaries of the cells in orthogonal views? If GFP labels whole cells, why is there an area at the top of the cross-sections that hasn't got GFP staining? __

__ ____Response:__ In all orthogonal views of the GFP-negative mutant clones, we have used dotted lines to indicate the clone boundaries. The wing disc epithelium consists of two distinct epithelial layers: a squamous epithelium (the peripodial membrane) and a pseudostratified columnar epithelium. Although GFP-negative clones are generated in both layers, our analysis focuses specifically on the columnar epithelium, where Wg is expressed. Therefore, the signal observed from the peripodial membrane can vary in the orthogonal views and does not affect our interpretation of Wg localization in the columnar cells.

__10) I can follow the point that accumulation in the apical side means that retrograde trafficking is impaired. I miss the connection between the observation and the conclusion. __

Response: We apologize for the lack of clarity in explaining the retrograde trafficking defects and would like to clarify this point for Wg.

In our experiments, we performed both total Wg staining (predominantly intracellular) and extracellular Wg staining. The apical accumulation observed in total Wg staining upon TRAPPC8/Rab1 depletion, by itself, does not directly demonstrate impaired retrograde trafficking. However, the increase in extracellular Wg indicates that Wg can still reach the plasma membrane, suggesting that the anterograde delivery pathway remains functional.

Our conclusion regarding defective retrograde trafficking is primarily based on the antibody internalization assays. In these experiments, internalized Wg and Evi accumulate intracellularly upon loss of TRAPPC8/Rab1, consistent with a defect in post-endocytic trafficking and recycling. Since both Wg and Evi normally undergo endocytic recycling through retrograde pathways, the accumulation of internalized Evi and Wg supports the interpretation that retrograde trafficking is impaired in TRAPPC8/Rab1-depleted cells.

__11) VPS34 is an effector of RAB5 and therefore its down-regulation impairs the maturation of early endosomes because their membranes cannot acquire the key component phosphatidylinositol-3-phosphate, which is recognized by the ESCRT machinery to proceed with multivesicular body biogenesis. __

__ ____Response: __We agree with the reviewer that VPS34 acts as an effector of Rab5 and plays an important role in early endosome maturation. However, more recent studies have shown that VPS34 functions within two related but functionally distinct complexes, VPS34 complex I and VPS34 complex II. VPS34 complex II, which contains UVRAG, functions predominantly in the endolysosomal system and is associated with Rab5. In contrast, VPS34 complex I, which contains Atg14, functions in autophagy and has been shown to interact with Rab1. Importantly, Rab1 and Rab5 bind VPS34 in a mutually exclusive manner at overlapping interaction sites (Scott and Burke 2026; Cook et al. 2025; Špokaitė et al. 2026; Tremel et al. 2021)*. *

• *Therefore, while the reviewer's interpretation regarding Rab5-dependent VPS34 function in endosomal maturation is fully valid, these studies also support the possibility that Rab1 can regulate VPS34-dependent trafficking pathways through a distinct VPS34 complex.

__12) The Q70L mutation, widely used as a constitutive activator of RABs, is borrowed from studies in RAS and it might not lead to constitutive activation of RAB1 (Langemeyer, L., Nunes Bastos, R., Cai, Y., Itzen, A., Reinisch, K.M., and Barr, F.A. (2014). Diversity and plasticity in Rab GTPase nucleotide release mechanism has consequences for Rab activation and inactivation. eLife 3, e01623). __

Response: We thank the reviewer for raising this important point. We admit that we have not performed an independent analysis of the GTP-locked status of the Rab1Q70L mutant in flies. Our rationale for using Rab1Q70L as a GTP-locked or functionally hyperactive Rab1 variant is based on its extensive prior use in the field (Tisdale et al. 1992; Levin et al. 2016; Russo et al. 2016; van Vliet et al. 2026). Importantly, a past study in Drosophila has shown functional hyperactivity of the Rab1Q70L compared with WT Rab1 (Sechi et al. 2017)*. ** *

References (response to reviewers):

Cook, Annan S. I., Minghao Chen, Thanh N. Nguyen, et al. 2025. "Structural Pathway for PI3-Kinase Regulation by VPS15 in Autophagy." Science (New York, N.Y.) 388 (6743): eadl3787.

Gyurkovska, Valeriya, Rakhilya Murtazina, Sarah F. Zhao, Christopher B. Huppenbauer, Vadim Gaponenko, and Nava Segev. 2026. "Distinct TRAPP Complexes Activate Ypt/Rab GTPases in Secretion and Autophagy." The Journal of Cell Biology 225 (5). https://doi.org/10.1083/jcb.202507166.

Hemalatha, Anupama, Chaitra Prabhakara, and Satyajit Mayor. 2016. "Endocytosis of Wingless via a Dynamin-Independent Pathway Is Necessary for Signaling in Drosophila Wing Discs." Proceedings of the National Academy of Sciences of the United States of America 113 (45): E6993-E7002.

Levin, Rebecca S., Nicholas T. Hertz, Alma L. Burlingame, Kevan M. Shokat, and Shaeri Mukherjee. 2016. "Innate Immunity Kinase TAK1 Phosphorylates Rab1 on a Hotspot for Posttranslational Modifications by Host and Pathogen." Proceedings of the National Academy of Sciences of the United States of America 113 (33): E4776-83.

Nakajima, Yu-Ichiro. 2021. "Analysis of Epithelial Architecture and Planar Spindle Orientation in the Drosophila Wing Disc." Methods in Molecular Biology (Clifton, N.J.) (New York, NY), Methods in molecular biology (Clifton, N.J.), vol. 2346: 51-62.

Port, Fillip, Marco Kuster, Patrick Herr, et al. 2008. "Wingless Secretion Promotes and Requires Retromer-Dependent Cycling of Wntless." Nature Cell Biology 10 (2): 178-185.

Riedel, Falko, Antonio Galindo, Nadine Muschalik, and Sean Munro. 2018. "The Two TRAPP Complexes of Metazoans Have Distinct Roles and Act on Different Rab GTPases." The Journal of Cell Biology 217 (2): 601-617.

Russo, Ashley J., Alyssa J. Mathiowetz, Steven Hong, Matthew D. Welch, and Kenneth G. Campellone. 2016. "Rab1 Recruits WHAMM during Membrane Remodeling but Limits Actin Nucleation." Molecular Biology of the Cell 27 (6): 967-978.

Scott, Mackenzie K., and John E. Burke. 2026. "Two Binding Sites Are Better than One." eLife 15 (e110917). https://doi.org/10.7554/eLife.110917.

Sechi, Stefano, Anna Frappaolo, Roberta Fraschini, et al. 2017. "Rab1 Interacts with GOLPH3 and Controls Golgi Structure and Contractile Ring Constriction during Cytokinesis in Drosophila Melanogaster." Open Biology 7 (1): 160257.

Sharma, Satyam, and Varun Chaudhary. 2024. "Dissociation of Drosophila Evi-Wg Complex Occurs Post Apical Internalization in the Maturing Acidic Endosomes." Traffic (Copenhagen, Denmark) 25 (9): e12955.

Špokaitė, Saulė, Yohei Ohashi, Maxime Bourguet, Antoine N. Dessus, and Roger L. Williams. 2026. "A Novel RAB5 Binding Site in Human VPS34-CII That Is Likely the Primordial Site in Eukaryotic Evolution." In eLife. ELife, March 31. https://doi.org/10.7554/elife.110040.

Sun, Shan, and Sen-Fang Sui. 2023. "Structural Insights into Assembly of TRAPPII and Its Activation of Rab11/Ypt32." Current Opinion in Structural Biology 80 (102596): 102596.

Tisdale, E. J., J. R. Bourne, R. Khosravi-Far, C. J. Der, and W. E. Balch. 1992. "GTP-Binding Mutants of rab1 and rab2 Are Potent Inhibitors of Vesicular Transport from the Endoplasmic Reticulum to the Golgi Complex." The Journal of Cell Biology 119 (4): 749-761.

Tremel, Shirley, Yohei Ohashi, Dustin R. Morado, et al. 2021. "Structural Basis for VPS34 Kinase Activation by Rab1 and Rab5 on Membranes." Nature Communications 12 (1): 1564.

Vliet, Alexander R. van, Alison K. Gillingham, Tomos E. Morgan, et al. 2026. "A Rab1 Interactome Illuminates a Dual Role in Autophagy and Membrane Trafficking." The Journal of Cell Biology 225 (3): e202507084.

Witte, Leonie, Karen Linnemannstöns, Kevin Schmidt, et al. 2020. "The Kinesin Motor Klp98A Mediates Apical to Basal Wg Transport." Development 147 (15). https://doi.org/10.1242/dev.186833.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Referee #3

Evidence, reproducibility and clarity

This manuscript uncovers a direct or indirect role of RAB1/TRAPPIII in regulating the intracellular fate of Wng in the columnar epithelial cells of the wing imaginal disc of the fruit fly. This observation is interesting, although it should come as no surprise that, given the fact that Wnts are secreted morphogens and considering the involvement of TRAPPIII in the early stages of the secretory pathway, the key TRAPPIII subunit, TRS85/TRAPPC8, is crucial for their normal trafficking. The main finding of this work is that cells deficient in TRAPPIII/RAB1 accumulate the morphogen and its receptor in the apical region of morphogen-producing cells, which is interpreted as a block in the retrograde trafficking of the morphogen. While it is unclear to me what the authors consider as retrograde trafficking of Wng (see below), the arguments fall short of being convincing, in part because the intracellular trafficking of Wng is rather intricate, but also because the physiological role of TRAPPIII is insufficiently understood. It is well established that Wng transits through endosomes to reach multivesicular bodies, where it is incorporated into the inwardly budding vesicles that are secreted as exosomes (Gross et al, cited). Do the authors consider this a retrograde pathway? The authors do not delineate further the location at which Wng accumulates, for example using co-localization studies. They have also not provided a rationalization of their observations with current knowledge of retrograde trafficking between the endosomes and the Golgi. It would have been interesting to address the effects of Trs85 depletion in mutant backgrounds deficient in the master regulator of retrograde pathways, RAB6, its effector, the GARP complex, or RAB7 and the retromer; it would have been important to study TRAPPIII depletion under conditions in which endocytic internalization is blocked, or the biogenesis of multivesicular bodies is prevented.

Specific comments

There are no page or line numbers, which makes very cumbersome to comment on specific sections of the manuscript.

The introduction contains a factual mistake. TRAPPC11, 12, and 13 are not metazoan specific. They are present in fungi but have been lost in Saccharomyces cerevisiae. Pinar M, Arias-Palomo E, de Los Ríos V, Arst HN Jr, Peñalva MA. Characterization of Aspergillus nidulans TRAPPs uncovers unprecedented similarities between fungi and metazoans and reveals the modular assembly of TRAPPII. PLoS Genet. 2019 Dec 23;15(12):e1008557. doi: 10.1371/journal.pgen.1008557. This reference should have been cited.

Materials and methods are very incomplete, particularly in the section that deals with the antibodies, which are essential tools for understanding the experiments. Is the Wnt antibody a monoclonal antibody? Are there different antibodies specific for extracellular and intracellular Wnt? What is the molecular basis for this differential detection? The transgene expressing GFP under the engrailed driver is not described anywhere.

The labeling of the figures and the figure legends themselves are excessively simple and appear to be accessible for fly experts only.

The authors have not considered the possibility that ablating TRAPPC8 of TRAPPIII can have off-target effects in TRAPPII. It would have been very interesting to address the phenotype of down-regulating TRAPPII and of down-regulating one of the core subunits of TRAPPs.

Figure 1, Panel 1i: What is the basis at this point that justifies "likely by altering intra-cellular trafficking"?

Several figures: where are the boundaries of the cells in orthogonal views? If GFP labels whole cells, why is there an area at the top of the cross-sections that hasn't got GFP staining?

I can follow the point that accumulation in the apical side means that retrograde trafficking is impaired. I miss the connection between the observation and the conclusion.

VPS34 is an effector of RAB5 and therefore its down-regulation impairs the maturation of early endosomes because their membranes cannot acquire the key component phosphatidylinositol-3-phosphate, which is recognized by the ESCRT machinery to proceed with multivesicular body biogenesis.

The Q70L mutation, widely used as a constitutive activator of RABs, is borrowed from studies in RAS and it might not lead to constitutive activation of RAB1 (Langemeyer, L., Nunes Bastos, R., Cai, Y., Itzen, A., Reinisch, K.M., and Barr, F.A. (2014). Diversity and plasticity in Rab GTPase nucleotide release mechanism has consequences for Rab activation and inactivation. eLife 3, e01623).

Referee cross-commenting

I also feel that six months is a more realistic estimation

Significance

In summary an interesting observation that deserves a more detailed follow-up to unveil the actual role of TRAPPIII in the Wng pathway.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Referee #2

Evidence, reproducibility and clarity

Summary

Sharma, Sabnis et al. show in this report that TRAPPC8 and Rab1 are important for Wingless (Wg) secretion in developing Drosophila wing discs.

• Loss of TRAPPC8 (either knockdown or mutant clones) lead to higher levels of apical Wg in Wg-producing cells (total and extracellular staining).
• The levels of the intracellular Wg transporter Evi/Wls are also increased, in particular in its 'unbound' form.
• A similar phenotype is observed upon overexpression of Rab1 dominant negative.
• Overexpression of constitutively active Rab1 has no effect in otherwise wild type discs but does rescue the effect of TRAPPC8 loss of function.

Major concerns

1) The authors key message is that TRAPPC8 is essential for retrograde transport of Wg and Evi. However, in apparent contradiction, TRAPPC8 loss does not appear to affect the basolateral distribution of exWg (or total Evi) (Fig2B', FigS3). Therefore, while Wg transport may be less efficient in the absence of TRAPPC8, the conclusion that TRAPPC8 is essential should be toned down. It is warranted to suggest that transport of unbound Evi is disrupted in the absence of TRAPPC8, as unbound Evi increases apically and is lost basally (Fig4A-B).

2) The relative roles of Rab1 and TRAPPC8 are not equally considered. As the authors show, unlike TRAPPC8 loss of function, Rab1 DN causes a decrease in basal exWg. Doesn't this suggest that Rab1 is more important for Wg trafficking than TRAPPC8? a. Rab1 DN causes also accumulation of total basal Wg. Does this mean that Wg can be trafficked basally but fails to be secreted at the basal surface? b. A similar assay (total basal Wg) is lacking in condition of TRAPPC8 loss (clones or RNAi); only exWg is shown. c. Also needed is an assessment of total and exWg at the basal surface in the Rab1 CA experiment. Based on the cross-section image shown in Fig6B, it seems that the distribution of Wg might be more confined to the apical plane of the cells compared to other conditions (Fig6F for example), but there is little difference in this regard between the anterior and posterior compartments.

3) A significant claim of the paper is "TRAPPC8 regulates retrograde Wg trafficking post-apical internalization". a. The increase in apical extracellular Wg (FigS3), which seems to remain associated with the membrane of Wg-producing cells (i.e. no onward spread) would suggest that apical internalization may be affected, but this is not considered by the authors. b. From the antibody chase experiment, it is not clear if the effect of TRAPPC8 loss affects endocytosis and/or trafficking in general or whether it is specific to Wg and Evi. A fluorescent Dextran control should be included as in Witte et al., 2020 (https://doi.org/10.1242/dev.186833 ). Also, can the authors be sure that they are visualising internalised Wg and not just internalised Wg antibody? Can the author show a field of view where Wg is not expressed?

4) The authors suggest that TRAPPC8 loss leads to increased Rab7 levels. This is taken as evidence for a role of acidification in driving Wg dissociation from Evi, with TRAPPC8 acting downstream to sort Wg from mature endosomes. a. Neither of these claims are supported because in Fig.S3G loss of TRAPPC8 results in an increase in Rab7 everywhere except at the DV boundary where the Wg-producing cells are. This should be acknowledged in text, and these data should appear in the main Fig4. b. The increase in lysotracker also does not support the above claims as it could be due to an increase in unbound Evi that cannot be trafficked back to the ER (and hence targeted for degradation in lysosomes). c. In fig S3G, the purported Rab7 increase in the posterior compartment is not readily apparent (and not quantified), in contrast to the authors' description of the effect of TRAPPC8 depletion. This suggest that the model proposed by the authors needs to be revised (Fig6G) and the relevant paragraphs from Results and Discussion section must be significantly edited or removed.

Minor concerns

1) The way data in Fig 2, S3 and S4 is presented and referred to in text could be rearranged slightly to make it easier to follow: first talk about the work using clones (Fig2, FigS4 becomes FigS3), then mention similar results with the knockdown (FigS3 becomes FigS4). This arrangement would link naturally to the effect on Evi.

2) How do the authors explain that the knockdown of some core TRAPP subunits does not have a phenotype? Some sort of rationalisation (or experimental follow up) is desirable.

3) The authors should give more detail about how they quantified normalised intensity profiles and clarify if the profiles correspond to just the representative image shown or the average of multiple discs (possible for the compartment experiments, but presumably impossible for the clone experiments as they would need to normalise by the size of the clone too).

Referee cross-commenting

Reviewer 3 was thorough and makes some good points that I had not considered because of coming from a different research field (e.g. the fact that losing TRAPPC8 can have off-target effects in TRAPPII, or that the figures may not be clear to non-fly people). In my review I identified the lack of testing of other TRAPP subunits as a minor point, but having read R3's comments I would probably increase this to a major issue. Reviewer 1 and I agree on multiple points as well (e.g. the lack of testing of other TRAPP subunits, the difference between the TRAPPC8 and Rab1 phenotypes). I believe that 1 - 3 months to complete revisions is optimistic. 6 months is a more realistic, with the caveat that the results from some of the requested experiments could upend the conclusions of this study.

Significance

This report is a valuable report for the Wg secretion subfield, and useful for the Wnt community. It makes some interesting observations and brings the importance of Rab1 back into the conversation after Ching et al., 2008. However, the insight remain limited and the mechanism/trafficking defects remain unclear. There is convincing evidence that TRAPPC8 and Rab1 affect Wg and Evi, but the claims that TRAPPC8 is essential and acts downstream of acidic endosomes) is inadequate.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Referee #1

Evidence, reproducibility and clarity

Summary

In this manuscript, the authors investigate the role of Transport Protein Particle (TRAPP) complexes in the secretion of Wingless (Wg) in Drosophila. Two TRAPP complexes are known to exist: TRAPPII and TRAPPIII. Through the systematic analysis of individual subunits, the authors identify the requirement of the TRAPPIII subunit TRAPPC8 for Wg trafficking. They demonstrate that the absence of TRAPPC8 disrupts the retrograde trafficking of both Wg and its carrier Evi, resulting in their intracellular accumulation within Wg-producing cells. Further analyses suggest that TRAPPC8 controls the post-apical internalisation and endosomal trafficking of Wg and Evi. Consistent with the established function of TRAPPIII as a Rab1-specific GEF, they demonstrate that inhibiting Rab1 produces similar effects as depleting TRAPPC8, while constitutively active Rab1 reverses the trafficking defects. Furthermore, depletion of either TRAPPC8 or Rab1 increases the levels of Wg-unbound Evi, suggesting that they act downstream of Evi-Wg dissociation. Taken together, these findings suggest that TRAPPIII and its effector Rab1 are essential regulators of retrograde Wg trafficking, which is necessary for efficient secretion. Overall, the work is carefully performed and the results are presented clearly. The controls are appropriate and the study expands the functional scope of Rab1-dependent trafficking beyond early secretory pathways. The identification of a previously unrecognised function of TRAPPC8 in Wg trafficking is a valuable contribution.

Major Points

It is not entirely clear whether the RNAi lines used in the initial screen were validated for knockdown efficiency. Notably, some core TRAPPIII subunits (e.g., TRAPPC3 and TRAPPC5) do not show a phenotype. This could indicate that the complex retains partial function upon their depletion, or alternatively that the RNAi lines are ineffective. While this point may not critically affect the main conclusion regarding TRAPPC8, it is important for drawing conclusions about the specificity of TRAPPIII versus TRAPPII involvement. For instance, TRAPPC10 (a TRAPPII-specific subunit) was analysed using a single RNAi line, yet no evidence of knockdown efficiency was provided. Validation of these RNAi reagents would strengthen the conclusions regarding complex specificity.

Minor Points

• In Figure 2C, the clone appears restricted to the apical region, and I do not clearly observe GFP loss in the basolateral domain. Larger clones or additional sections would help clarify the spatial distribution and strengthen the interpretation.
• The authors should discuss why TRAPPC8 depletion results primarily in apical Wg enrichment, whereas Rab1 inhibition leads to Wg accumulation at both apical and basolateral membranes. This difference may provide insight into whether Rab1 has additional TRAPPIII-independent functions or whether TRAPPC8 affects a more spatially restricted trafficking step.

Significance

This study broadens our understanding of Rab1-dependent membrane trafficking by identifying a previously unrecognized role for the TRAPPIII complex in retrograde transport during Wingless secretion. While the study convincingly establishes the involvement of the TRAPPIII-Rab1 module in Wg trafficking, it does not define the specific retrograde trafficking step that is regulated by this machinery. In addition, the functional relationship between TRAPPIII-Rab1 and established retrograde regulators, such as the retromer complex, is not addressed. Further molecular and mechanistic analyses will be required to position TRAPPIII within the broader retrograde trafficking network and to fully elucidate how Rab1 activity is coordinated with other pathways involved in Wnt secretion. These unresolved issues represent the main limitation of the current study. This work will be of particular interest to researchers in the fields of cell signaling, membrane trafficking, and intercellular communication.

My expertise lies in cell communication and the regulation of cellular proliferation.

Аспарух не е създавал България , той просто е разширил Стара Велика България на юго-изток

https://www.golddustantiques.com/products/antique-german-oak-filing-cabinet-with-drop-down-doors-by-stolzenberg-1930s

Byles, R. B. 1911. The Card Index System; Its Principles, Uses, Operation, and Component Parts. London: Sir Isaac Pitman & Sons, Ltd. http://archive.org/details/cardindexsystemi00bylerich (February 13, 2024).

Annotations<br /> Alternate annotations: urn:x-pdf:9c84c139e7502ca806dd83bfecf80c20

Read: 2026-06-23 to 06-26

R. B. Byles attributes the origin of the card index system to Europe, but provides no direct details or history.

Interesting section here on the state of the art of carbon copies of letters

To guard against an accidental upset of the drawer and its entire contents which in the absence of a rod

Card indexes and filing cabinets should have tray stops to help prevent pulling drawers out too quickly and losing all the file organization by dropping the drawer out on the floor.

There are generally two types of rods for a card index:<br /> - plain rod with round hole that typically needs to be removed to insert cards. These are useful for safety (and not dumping cards out) or losing them (esp. in public settings like a library. - flat rod (various forms) allows cards to be 'locked" or released easily for filing efficiency, especially valuable for business use. - no rod at all; by 1911 there were more easily moved follower blocks which helped to get rid of the necessity of rods at all, at least in Byles' estimation.

He's not using the traditional wording, but he's talking about the need for having "cards of equal size" for use in a card index here.