missionstatement

stakeholders

benefit corporation (B corporation)

gency problem

transparency,

community benefits

Schedule H

Form 990

residual earnings

privately held company

publicly held companies

tockholders (shareholders

hybrid form

S corporation

C corporation.

iquid investment

for-profit corporation

partnership

proprietorship (or sole proprietorship)

professional liability

cost-containment programs

licensure

patient captureThe concept that oncea patient enters thesystem (e.g., a doctor’soffice), all servicesneeded by that patientshould be provided inthe system

Long-term care consists of healthcare (and some personal care) services provided to indi-viduals who lack all or some functional ability, specifically in the activities of daily livingsuch as eating, bathing, and locomotion.

Ambulatory (outpatient) care encompasses services provided to patients who are not admit-ted to a hospital or nursing home

investor-owned hospital, also known as the for-profit hospital

70 percent of all privatehospitals (57 percent of all community hospitals) are not-for-profit entities (AHA 2021).

charitable origins

private not-for-profit hospital is a nongovernmental entity organized for thesole purpose of providing inpatient healthcare services

The category of government hospital, which makes up about 19 percentof all hospitals, is broken down into federal and public (nonfederal) entities (AmericanHospital Association [AHA] 2021).

The Joint Commission (previously calledthe Joint Commission on Accreditation of Healthcare Organizations). Joint Commissionaccreditation is a voluntary process intended to promote high standards of care.

Those that were linked tended to be part of a horizontal system,which controls a single type of healthcare facility, such as a group of hospitals or nursinghomes. Recently, however, many healthcare organizations have created a vertical system,which controls two or more related types of providers, such as medical practices, hospitals,and nursing homes.

comptrolle

costs, cash, capital, and control

Miroand TRAK proteins act as adaptors that link kinesin-1 and dynein, aswell as myosin of class XIX (MYO19), t

Assays were performed with0.6 mM total lipids at a 1:2 labeled/unlabeled ratio. For all assays,proteoliposomes were mixed with A100 buffer containing 5 mMMgCl2, and then pre-incubated at 37°C for 5 min in a 96-wellplate. Following pre-incubation, the plate was placed in a Sparkplate reader (Tecan), two baseline readings were taken, and then2 mM GTP or A100 buffer of equal volume was added with amultichannel pipette. For 60 min NBD dequenching was moni-tored at 10 s intervals (excitation at 460 nm, dequenching at 538nm). Maximum possible dequenching was determined after60 min via the addition of 0.5% Anapoe X-100, with two morecycles read on the plate reader after Anapoe addition. Fmax wascalculated by taking the average of these two post-Anapoe cy-cles. Fusion calculations were performed using the functionFusion  Fluorescence observed−Initial fluorescence observedF max × 100. In instanceswhere initial kinetics were extremely rapid and the first readingpost-GTP addition was not representative of time zero, the aver-age of two readings taken immediately before GTP addition wasused to calculate initial fluorescence observed. This difference incalculation of initial fluorescence was used for ATL3 at 1:300 andATL2 1-547 at 1:500

with ATL3having the lowest rate, but converging to a similar rate athigher enzyme concentrations

However, due to thelower dimer affinity of ATL3 relative to that of ATL2, the GTPaserates diverge when compared at lower protein concentrations.

It should be noted that the lack of C-terminal autoinhibitionin ATL3 does not rule out the possibility of ATL3 regulation byother means

The lack of autoinhibition in ATL3 and a corresponding lackof autoinhibition in the single Drosophila ortholog leads us tospeculate that having at least one constitutive form of ATL mightbe beneficial to cells and to organisms.

Second, an in-depth phylogenetic analysis reveals that C-terminal auto-inhibition in ATL1 and ATL2 are recent innovations that likelyarose independently during the diversification of vertebrates

Our findings herein support a model in which C-terminal au-toinhibition of human ATL1/2 fusion activity is not a core con-served feature of the ATL mechanism but more likely aregulatory adaptation

(model 1) ancestral autoinhibition, followed byindependent losses of autoinhibition in ATL3 and in ATLi;(model 2) lack of autoinhibition in the ancestor with recent in-dependent gains of autoinhibition in ATL1 and ATL2; or (model3) lack of ancestral autoinhibition, gain of autoinhibition in thevertebrate ancestor, and a later loss in ATL3

indicates that ATL1/2/3 arose throughtwo duplications at the base of the vertebrate lineage, with ATL1branching first and a subsequent duplication leading to ATL2and ATL3, in line with the more recently reported phylogeny(Neufeldt et al., 2019). Significantly, ATL3, which altogetherlacks autoinhibition, and ATL2, the most strongly autoinhibitedparalog (Crosby et al., 2022), are sister taxa, while ATL1 andATL2, the two paralogs that exhibit autoinhibition, are not (Figs.5 C and S4 A); thus, the presence or absence of autoinhibitiondoes not appear to correlate with the overall evolutionary rela-tionship between paralogs. Based on the gene tree, the presenceand absence of autoinhibition in the canonical ATL paralogscould be most parsimoniously explained by three distinctmodels

C-terminal autoinhibition seen in ATL1/2 (Crosby et al., 2022)might represent a vertebrate specialization rather than a core-conserved feature of the ATL fusion machinery. Deciphering theevolutionary history of C-terminal autoinhibition would shedlight on this hypothesis but would require an understanding ofthe phylogeny of the ATLs and their C-termini.

When ATL3 was incor-porated at either two- or approximately threefold higher pro-tein/lipid ratios of 1:500 or 1:300, respectively, it catalyzedfusion at dramatically higher initial rates

To further demonstrate that the ATL3 C-terminal extensionlacks an autoinhibitory activity, an ATL2/3 chimera was con-structed. As anticipated, replacing the C-terminal extension ofATL2 with that of ATL3 resulted in an ATL2/3 chimera thatlargely lacks autoinhibiti

trikingly, ATL3truncation had no effect on the fusion rate (Fig. 3 B) and nosignificant effect on GTPase activity when measured under thesefusion conditions

In contrast to ATL1/2, there appear to be nodocumented C-terminal variants of ATL3.

Thus, when introducedexogenously into ATL1/2/3 KO cells as the sole source of ATL,ATL3 can restore and maintain a normal ER network.

Fusion by ATL3 was also disrupted by a I503Dmutation, similar to the block by an equivalent I507D mutation

We purified His-tagged human ATL3 from a transiently trans-fected HEK293-derived suspension cell line

R membrane fusionfunction for ATL3 that, unlike ATL1/2, is most likely constitu-tive.

we find that ATL3 altogether lacks theC-terminal autoinhibition observed for ATL1/2

Despite substantial advances using DATL, reconstitution offusion activity by the human ATLs had proved refractory untilrecently, when our lab demonstrated that ATL1/2 expressed andpurified from HEK cells, rather than E. coli, has robust fusionactivity

Notably, while Drosophila andinvertebrates in general appear to have a single ATL, humanshave three paralogs (ATL1-3) that are broadly important forhuman health, with mutations in ATL3 causing hereditary sen-sory neuropathy

ith the absence of ATL causingdisruptions to ER structure in both insect and mammalian cells

two of the three human ATL paralogs

Homotypic membrane