Spike-RBD-Fc and trimeric Spike ectodomain , and ACE2-Fc were produced as biotinylated proteins as previously described.Fabs were subcloned from the Fab-phagemid into an E. coli expression vector pBL347. VH-Fc were cloned into a pFUSE vector with a human IgG1 Fc domain as previously described.The heavy chain of the IgG were cloned from the Fab plasmid into a pFUSE vector with a human IgG1 Fc domain. The light chain of the IgG was cloned from the Fab plasmid into the same vector but lacking the Fc domain. All constructs were sequence verified by Sanger sequencing.Fabs were expressed in E. coli C43 Pro + using an optimized autoinduction media and purified by protein A affinity chromatography.VH-Fc, IgGs, and bi-specifics were expressed in Expi293 BirA cells using transient transfection . 3-5 days after transfection, media was harvested, and VH-Fc and IgGs were purified using protein A affinity chromatography. Bi-specific antibodies were purified by Ni-NTA affinity chromatography. The bi-specifics were then buffer exchanged into PBS containing 20% glycerol, concentrated, and flash frozen for storage. All other proteins were buffer exchanged into PBS by spin concentration and stored in aliquots at -80ºC. Purity and integrity of all proteins were assessed by SDS-PAGE.Phage selections were done according to previously established protocols.Selections were performed using biotinylated antigens captured with streptavidin-coated magnetic beads .

In each round,vertical grow rack system the phage pool was first cleared by incubation with beads loaded with Fc domain only. The unbound phage were then incubated with beads loaded with Spike-RBD-Fc. After washing, the bound phage was eluted by the addition of 2 μg/mL of TEV protease. In total, four rounds of selection were performed with decreasing amounts of Spike-RBD-Fc. All steps were done in PBS buffer + 0.02% Tween-20 + 0.2% BSA . Individual phage clones from the third and fourth round of selections were analyzed by phage ELISA. For each phage clone, 4 different conditions were tested—Direct: Spike-RBD-Fc, Competition: Spike-RBD-Fc with equal concentration of Spike-RBD-Fc in solution, Negative selection: ACE2- Fc/Spike-RBD-Fc complex, and Control: Fc. 384-well Nunc Maxisorp flat-bottom clear plates were coated with 0.5 μg/mL of NeutrAvidin in PBS overnight at 4ºC and subsequently blocked with PBS + 0.02% Tween-20 + 2% BSA for 1 hr at room temperature. Plates were washed 3X with PBS containing 0.05% Tween-20 and were washed similarly between each of the steps. 20 nM of biotinylated Spike-RBD-Fc, ACE2-Fc/Spike-RBD-Fc complex, or Fc diluted in PBSTB was captured on the NeutrAvidin-coated wells for 30 min, then blocked with PBSTB + 10 μM biotin for 30 min. Phage supernatant diluted 1:5 in PBSTB were added for 20 min. For the competition samples, the phage supernatant was diluted into PBSTB with 20 nM Spike-RBD-Fc. Bound phage were detected by incubation with anti-M13-HRP conjugate for 30 min, followed by addition of TMB substrate . The reaction was quenched with addition of 1 M phosphoric acid and the absorbance at 450 nm was measured using a Tecan M200 Pro spectrophotometer. HEK293T-ACE2 cells were a gift from Arun Wiita’s laboratory at the University of California, San Francisco. Cells were cultured in D10 media .

Plasmids to generate pseudotyped HIV-1-derived lentivirus were a gift from Peter Kim’s lab at Stanford University and pseudovirus displaying SARS-CoV-2 Spike was prepared as previously described.Briefly, plasmids at the designated concentrations were added to OptiMEM media with FuGENE HD Transfection Reagent at a 3:1 FuGENE:DNA ratio, incubated for 30 minutes, and subsequently transfected into HEK-293T cells. After 24 hrs, the supernatant was removed and replaced with D10 culture media. Virus was propagated for an additional 48 hrs, and the supernatant was harvested and filtered. Virus was stored in flash-frozen aliquots at -80ºC and thawed prior to use.HEK-ACE2 were seeded at 10,000 cells/well on 96-well white plates . After 24 hrs, pseudovirus stocks were titered via a two-fold dilution series in D10 media and 40 μL were added to cells. After 60 hrs, infection and intracellular luciferase signal was determined using Bright-Glo™ Luciferase Assay , and the dilution achieving maximal luminescent signal within the linear range, ~3-5 x 105 luminescence units, was chosen as the working concentration for neutralization assays.HEK-ACE2 were seeded at 10,000 cells/well in 40 μL of D10 on 96-well white plates 24 hrs prior to infection. To determine IC50 for pseudovirus neutralization, dose series of each VH binder were prepared at 3x concentration in D10 media and 50 μL were aliquoted into each well in 96-well plate format. Next, 50 μL of virus were added to each well, except no virus control wells, and the virus and blocker solution was allowed to incubate for 1 hr at 37ºC. Subsequent to pre-incubation, 80 μL of the virus and blocker inoculum were transferred to HEKACE2. After 60 hrs of infection at 37ºC, intracellular luciferase signal was measured using the Bright-Glo™ Luciferase Assay.

Luminescence was normalized to the no binder control and plotted using Prism 8 software. A non-linear 4-parameter regression was used to determine the IC50. Patients with limited English proficiency have among the highest rates of untreated depression.Depression has been associated with poor quality of life, increased health care costs and non-mental health service utilization, and poor management of other chronic conditions , with subsequently increased risk of complications and earlier mortality.Numerous studies have documented lower access to mental health care among patients with LEP compared to English-speaking patients.Challenges and barriers to seeking depression care include difficulties navigating complex medical and mental health care systems, widespread stigma surrounding depression treatment, and reliance on interpreters during sensitive screening, diagnosis, and treatment. Patients with LEP may also contend with cultural factors that make it difficult to seek or engage in depression care.Patients with LEP are more apt to seek and receive mental health services in primary care than in specialty mental health settings.However, the mental health concerns of racial and ethnic minority patients—including patients with LEP—are more likely to go unnoticed by primary care providers,and patients with LEP often receive poor equality mental health care when they do seek care.These challenges also impact clinicians, as care for patients with depression is often difficult when patients and clinicians do not share a common language and cultural heritage. Eliciting symptoms, establishing trust, and achieving treatment goals are usually more difficult across language barriers. More than 80 randomized controlled trials and numerous systematic reviews have reported improved depressive symptoms, as well as positive effects on other comorbid conditions, when patients are treated using a collaborative care model.Key components of the collaborative care model include care coordination and care management with multidisciplinary team-based patient care. The care team includes asocial worker or behavioral health assistant who engages the patient in regular care and monitors depressive symptoms using validated clinical ratings scales and population-based registries to facilitate active management, while communicating regularly with the primary care providers. A psychiatrist consults as needed for patients failing to achieve remission.While there is a broad research base supporting the collaborative care model among English-speaking patients,vertical farming companies less is known about the effectiveness of this model in treating depressive symptoms among patients with LEP, who face additional barriers when seeking and engaging in mental health care. We conducted a systematic review of the literature evaluating the effectiveness of collaborative care over usual care for the treatment of depressive symptoms among patients with LEP, while also gathering information about intervention adaptations to better serve the needs of this population.We searched PubMed, PsycInfo, Web of Science, and Embase for articles published between January 1, 2000 , and June 10, 2017. Search syntaxes were developed in consultation with an experienced university research librarian, taking into account a broad range of terms and phrases used in defining randomized controlled trials , cohort studies, collaborative care, depression, limited English proficiency, immigrants, and language barriers .

We also searched reference lists of retrieved articles and systematic reviews, and spoke to experts in the field. If the components of collaborative care were not clearly described in the publication, we emailed study investigators to obtain that information.Studies that met all of the following criteria were included in this systematic review: 1) participants were adults with depressive symptoms identified either through routine clinical care or through the use of a validated depression screening tool; 2) study setting was outpatient only; 3) collaborative care, as defined by multidisciplinary team-based care, including a social worker or behavioral health assistant maintaining a registry of patients, and availability of a psychiatrist for consultation; 4) care delivered to patients with limited English proficiency participants having language other than English listed as the preferred language, reporting need for interpretive services, or having a focus on first-generation immigrant populations from non-English speaking countries, or having greater than 75% non English-speaking participants in the study even if the primary outcome was not reported by participant preferred language; and 5) outcomes included the impact of collaborative care on depressive symptoms or on depression treatment. Given the small number of RCTs among patients with LEP, we included other experimental designs in this review. We excluded studies on the basis of one or more of the following: 1) inappropriate population , 2) purely qualitative study design, or 3) collaborative care programs focused on the treatment of other medical conditions rather than depression or depressive symptoms. Two investigators reviewed the titles and abstracts for all citations to identify studies that met inclusion criteria. If the reviewers could not determine from the abstract whether a particular study met inclusion criteria, the article advanced to a full text review. Articles that were selected for inclusion on the basis of the title and abstract also advanced to full-text review. We exported all studies obtained from the database searches to Covidence,a web-based program that allows collaborators to screen and review studies, assess quality, and resolve disagreements on inclusion and exclusion.We used a standardized data extraction form to collect the following: first author name, publication year, study design, study location, study period, patient inclusion and exclusion criteria, participant characteristics , sample size , components of Bcollaborative care^ and control conditions, cultural or linguistic tailoring of the model, length and frequency of follow-up, depression baseline and outcome measures, and main results .Two authors independently assessed the methodological quality of RCTs using the Cochrane Collaboration’s tool for assessing risk of bias.The few discrepancies in assessment were discussed until the investigators reached consensus. We used the Newcastle-Ottawa Scale to assess the methodological quality of cohort and case–control studies.The score for this scale is based on three categories: 1) selection of study groups and ascertainment of exposure, 2) comparability of study groups, and 3) standardized assessment of outcome of interest.In addition, while outcome inconsistency is usually assessed with a test for heterogeneity, problems of power can give misleading results .Finally, given that five of the nine studies originated from the same study group, a test of heterogeneity might be misleading. We could not assess for publication bias using a funnel plot, due to the low number of studies and differing outcomes.Our search strategy yielded 71 articles, of which 13 were included. Text, references, and discussion with experts yielded an additional 15 articles, of which only two were included after full-text review. Table 1 summarizes the basic characteristics of the included studies. Fifteen articles, representing nine studies with distinct patient populations , met the study criteria and are included in this review . All five RCTs compared collaborative care to either usual care or enhanced usual care , and all were included for having ≥75% patients with LEP rather than for reporting outcomes by language preference. One article described the intervention as Bcollaborative care,^ but failed to specify the availability of a consulting psychiatrist; this component was confirmed via an email to the study investigators.We observed substantial variation in sample size as well as intervention duration and frequency of follow-up across the studies . The reviewed studies were diverse in methodological approaches . Two of the RCTs did not report on allocation concealment; however, overall, the five RCTs were found to be at low risk for selection bias. None of the RCTs blinded participants or personnel to the intervention . All five RCTs ascertained outcomes by blinded researchers/ care providers and had complete outcome data, with low risk for attrition or reporting bias. The non-RCTs did not have a usual care control group.Depression measures were generally well described.