Metabolic, neuroendocrine and behavioral effects of social defeat in male and female mice using the chronic non-discriminatory social defeat stress model.

Stress-related disorders predominately affect females, yet preclinical models of chronic stress exclusively use males especially in models where social stressors are studied. Here, we implemented a 21-day novel social defeat paradigm in which a female and male C57 intruder are simultaneously placed in the cage of a territorial, resident CD-1 male mouse, and the resident proceeds to attack both intruders. Mice were given access to a regular laboratory diet, high in carbohydrates, and a palatable diet, high in fat. Chronic social defeat stress using this paradigm resulted in increased caloric intake in male and female mice, with the effects being more pronounced in females. We observed sex differences in high fat diet intake in response to stress, which was correlated with higher levels of plasma ghrelin observed in female mice but not male mice. Furthermore, females exposed to chronic stress displayed changes in growth hormone secretatogue receptor (ghsr) and neuropeptide-y (npy) expression in the arcuate nucleus of the hypothalamus, potentially increasing ghrelin sensitivity and inducing changes in diet choice and caloric intake. Behavioral results show that females tended to spend more time interacting during the social interaction test, compared to males who displayed higher vigilance towards the stranger mouse. Overall, our results highlight unique neurometabolic alterations in female mice in response to stress that is not present in male mice and may be important for coping with chronic stress and sustaining reproductive function.

Impact of the Dog-Human Bond on Canine Social Evaluation: Attachment Predicts Preference toward Prosocial Actors.

Scholars have argued that social evaluation, the capacity to evaluate different potential social partners, is an important capacity not just for humans but for all cooperative species. Recent work has explored whether domesticated dogs share a human-like ability to evaluate others based on prosocial and antisocial actions toward third parties. To date, this work has shown mixed results, suggesting that individual differences may play a role in dogs' capacity to evaluate others. In the present study, we test whether attachment-an individual difference that affects human social evaluation performance-can explain the mixed pattern of social evaluation results observed in dogs. We first tested dogs on a social evaluation task in which an experimenter either helped or refused to help the dog's owner open a container. We then assessed dogs' attachment strength using a subset of the C-BARQ. We found that attachment was a statistically significant predictor of dogs' preference toward the prosocial actor but was not a predictor in antisocial or control conditions. This finding provides early evidence that attachment may drive positivity biases in dogs and that attachment might explain mixed results within canine social evaluation literature.

Maternal glucose intolerance during pregnancy affects offspring POMC expression and results in adult metabolic alterations in a sex-dependent manner.

Introduction: Gestational diabetes (GDM) is associated with negative outcomes in mothers and their offspring, including greater risks of macrosomia at birth and the development of metabolic disorders. While these outcomes are well-established, the mechanisms by which this increased metabolic vulnerability is conferred on the offspring are comparatively lacking. One proposed mechanism is that maternal glycemic dysregulation alters the development of the hypothalamic regions related to metabolism and energy balance. Methods: To investigate this possibility, in this study, we first examined the effects of STZ-induced maternal glucose intolerance on the offspring on pregnancy day (PD) 19, and, in a second experiment, in early adulthood (postnatal day (PND) 60). Whether effects would be influenced by sex, or exposure of offspring to a high-fat diet was also investigated. The impact of maternal STZ treatment on POMC neuron number in the ARC of offspring at both time points was also examined. Results: As expected, STZ administration on PD 7 decreased maternal glucose tolerance, and increased risk for macrosomia, and loss of pups at birth. Offspring of STZ-treated mothers were also more vulnerable to developing metabolic impairments in adulthood. These were accompanied by sex-specific effects of maternal STZ treatment in the offspring, including fewer POMC neurons in the ARC of female but not male infants in late pregnancy and a higher number of POMC neurons in the ARC of both male and female adult offspring of STZ-treated dams, which was exacerbated in females exposed to a high-fat diet after weaning. Discussion: This work suggests that maternal hyperglycemia induced by STZ treatment, in combination with early-life exposure to an obesogenic diet, leads to adult metabolic alterations that correlate with the increased hypothalamic expression of POMC, showing that maternal glycemic dysregulation can impact the development of hypothalamic circuits regulating energy state with a stronger impact on female offspring.

Prevalence of mental health symptoms in children and adolescents during the COVID-19 pandemic: A meta-analysis.

The COVID-19 pandemic and its accompanying infection control measures introduced sudden and significant disruptions to the lives of children and adolescents around the world. Given the potential for negative impacts on the mental health of youths as a result of these changes, we conducted a systematic review and meta-analysis to examine the prevalence of depressive symptoms, anxiety symptoms, and sleep disturbances in children and adolescents during the pandemic. We searched major literature databases for relevant cross-sectional or longitudinal studies that included primary and secondary school students or children and adolescents ≤18 years of age. Prevalence values were extracted, logit-transformed, and pooled. Based on 191 included studies with 1,389,447 children and adolescents, we found the pooled prevalence of depressive symptoms, anxiety symptoms, and sleep disturbances to be 31%, 31%, and 42%, respectively. Age, grade levels, education levels, gender, geographical regions, and electronics use were correlated with the prevalence of mental health symptoms. The prevalence of mental health symptoms also increased with time, although signs of recovery and stabilization were also observed. Overall, the results from this review demonstrate the need for increased mental health research, monitoring, and intervention for children and adolescents during the current and future pandemics.

Efficacy and safety of corticosteroid regimens for the treatment of hospitalized COVID-19 patients: a meta-analysis.

Aim: To evaluate the efficacy and safety of corticosteroids for treating hospitalized COVID-19 patients. Materials & methods: Efficacy outcomes included time to negative SARS-CoV-2 tests, length of stay, duration and incidence of intensive unit care stay, incidence of mortality and duration and incidence of mechanical ventilation. Safety outcomes included the incidence of adverse events and severe adverse events, incidence of hyperglycemia and incidence of nosocomial infections. Results: Ninety-five randomized controlled trials (RCTs) and observational studies (n = 42,205) were included. Corticosteroids were associated with increased length of stay (based on RCT only), increased time to negative tests, decreased length of mechanical ventilation and increased odds of hyperglycemia. Conclusion: Corticosteroids should be considered in patients requiring mechanical ventilation, and glycemic monitoring may be needed when administering corticosteroids.

Efficacy of chloroquine and hydroxychloroquine for the treatment of hospitalized COVID-19 patients: a meta-analysis.

Aims: To evaluate the efficacy and safety of hydroxychloroquine/chloroquine, with or without azithromycin, in treating hospitalized COVID-19 patients. Materials & methods: Data from randomized and observational studies were included in a random-effects meta-analysis. Primary outcomes included time to negative conversion of SARS-CoV-2 tests, length of stay, mortality, incidence of mechanical ventilation, time to normalization of body temperature, incidence of adverse events and incidence of QT prolongations. Results: Fifty-one studies (n = 61,221) were included. Hydroxychloroquine/chloroquine showed no efficacy in all primary efficacy outcomes, but was associated with increased odds of QT prolongations. Conclusion: Due to a lack of efficacy and increased odds of cardiac adverse events, hydroxychloroquine/chloroquine should not be used for treating hospitalized COVID-19 patients.

Ghrelin receptor signaling is not required for glucocorticoid-induced obesity in female mice.

Chronic exposure to high circulating glucocorticoid or ghrelin concentrations increases food intake, weight gain and adiposity, suggesting that ghrelin could contribute to the metabolic effects of chronic glucocorticoids. In male mice, however, blocking ghrelin receptor (GHSR) signaling increased the weight gain and adiposity induced by chronic corticosterone (CORT), rather than attenuating them. In the current study, we investigated the role of GHSR signaling in the metabolic effects of chronic exposure to high circulating CORT in female mice. To do this, female WT and GHSR KO mice were treated with either CORT in a 1% ethanol (EtOH) solution or 1% EtOH alone in their drinking water for 32 days (n = 5-8/group). Body weight, food, and water intake as well as vaginal cyclicity were assessed daily. As expected, CORT treatment-induced significant increases in body weight, food intake, adiposity and also impaired glucose tolerance. In contrast to results observed in male mice, WT and GHSR KO female mice did not differ on any of these parameters. Neither plasma levels of ghrelin, LEAP-2, the endogenous GHSR antagonist produced by the liver, nor their ratio were altered by chronic glucocorticoid exposure. In addition, CORT treatment disrupted vaginal cyclicity, produced a reduction in sucrose consumption and increased locomotor activity regardless of genotype. Chronic CORT also decreased exploration in WT but not GHSR KO mice. Collectively, these data suggest that most metabolic, endocrine, reproductive and behavioral effects of chronic CORT exposure are independent of GHSR signaling in female mice.

The prevalence of depressive symptoms, anxiety symptoms and sleep disturbance in higher education students during the COVID-19 pandemic: A systematic review and meta-analysis.

The COVID-19 pandemic and its accompanying infection control measures introduced significant disruptions to the routines of many higher education students around the world. It also deprived them of in-person counselling services and social support. These changes have put students at a greater risk of developing mental illness. The objective of this review is to assess the prevalence of depressive symptoms, anxiety symptoms and sleep disturbances in higher education students during the pandemic. A systematic search of English and Chinese databases was conducted current to January 1st, 2021. The quality of included studies was evaluated using a modified Newcastle-Ottawa scale. Prevalence of depressive symptoms, anxiety symptoms and sleep disturbances were pooled using random-effects meta-analysis. Eighty-nine studies (n=1,441,828) were included. The pooled prevalence of depressive symptoms, anxiety symptoms, and sleep disturbances was 34%, 32% and 33%, respectively. The prevalence values differ based on geographical regions, diagnostic criteria, education level, undergraduate year of study, financial situation, living arrangements and gender. Overall, the prevalence of depressive symptoms and anxiety symptoms synthesized in this study was higher compared to pre-pandemic prevalence in similar populations. Evidently, mental health screening and intervention should be a top priority for universities and colleges during the pandemic.

The effect of calcium and vitamin D compounds on bone mineral density in patients undergoing glucocorticoid therapies: a network meta-analysis.

INTRODUCTION/OBJECTIVES: The objective of our systematic review and network meta-analysis (NMA) is to investigate which vitamin D and/or calcium regimen would yield the greatest increase in lumbar spine, femoral neck, and total hip bone mineral density (BMD) in adult patients undergoing glucocorticoid therapy. METHOD: We performed NMAs based on a prospectively developed protocol. A database search of MEDLINE, EMBASE, Web of Science, CINAHL, CENTRAL and Chinese databases were conducted for relevant randomized controlled trials (RCTs). Outcomes were percentage change in lumbar spine, femoral neck, and total hip BMD from baseline. RESULTS: We included 16 RCTs containing 1073 eligible patients in our analysis. We found alfacalcidol+calcium to yield the greatest percentage increase in lumbar spine BMD (MD 6.05, 95% credible interval [CrI] - 4.18 to 16.18) compared to no treatment, and calcitriol+calcium to yield the greatest percentage increase in femoral neck BMD (MD 8.46, 95% CrI - 4.74 to 21.51) compared to no treatment. Cholecalciferol+calcium ranked first in terms of its ability to increase total hip BMD; however this finding needs to be interpreted with caution due to low sample sizes in the cholecalciferol+calcium treatment arm. None of the treatment arms ruled out the possibility of no effect for any outcome. CONCLUSIONS: Alfacalcidol and calcitriol were the most efficacious treatment arms for increasing lumbar spine and femoral neck BMD, respectively. Our findings need to be validated by further investigations using larger, better-designed RCTs. Key Points •The efficacy of calcium/vitamin D compounds was examined using network meta-analyses. •Alfacalcidol + calcium yielded the greatest increase in lumbar spine BMD, calcitriol + calcium yielded the greatest increase in femoral neck BMD. •Future guidelines should place greater emphasis on the efficacy of different vitamin D compounds.

Efficacy of lopinavir-ritonavir combination therapy for the treatment of hospitalized COVID-19 patients: a meta-analysis.

Aim: To evaluate the efficacy and safety of lopinavir-ritonavir (LPV/r) therapy in treating hospitalized COVID-19 patients. Materials & methods: Data from randomized and observational studies were included in meta-analyses. Primary outcomes were length of stay, time for SARS-CoV-2 test conversion, mortality, incidence of mechanical ventilation, time to body temperature normalization and incidence of adverse events. Results: Twenty-four studies (n = 10,718) were included. LPV/r demonstrated no significant benefit over the control groups in all efficacy outcomes. The use of LPV/r was associated with a significant increase in the odds of adverse events. Conclusion: Given the lack of efficacy and increased incidence of adverse events, the clinical use of LPV/r in hospitalized COVID-19 patients is not recommended.

The prevalence of depression, anxiety, and sleep disturbances in COVID-19 patients: a meta-analysis.

Evidence from previous coronavirus outbreaks has shown that infected patients are at risk for developing psychiatric and mental health disorders, such as depression, anxiety, and sleep disturbances. To construct a comprehensive picture of the mental health status in COVID-19 patients, we conducted a systematic review and random-effects meta-analysis to assess the prevalence of depression, anxiety, and sleep disturbances in this population. We searched MEDLINE, EMBASE, PubMed, Web of Science, CINAHL, Wanfang Data, Wangfang Med Online, CNKI, and CQVIP for relevant articles, and we included 31 studies (n = 5153) in our analyses. We found that the pooled prevalence of depression was 45% (95% CI: 37-54%, I2  = 96%), the pooled prevalence of anxiety was 47% (95% CI: 37-57%, I2  = 97%), and the pooled prevalence of sleeping disturbances was 34% (95% CI: 19-50%, I2  = 98%). We did not find any significant differences in the prevalence estimates between different genders; however, the depression and anxiety prevalence estimates varied based on different screening tools. More observational studies assessing the mental wellness of COVID-19 outpatients and COVID-19 patients from countries other than China are needed to further examine the psychological implications of COVID-19 infections.

Pharmacological prevention of fractures in patients undergoing glucocorticoid therapies: a systematic review and network meta-analysis.

OBJECTIVE: To perform a network meta-analysis (NMA) on the efficacy of antiosteoporotic interventions in the prevention of vertebral and non-vertebral fractures in adult patients taking glucocorticoids (GCs). METHODS: We performed NMAs based on a prospectively developed protocol. A librarian-assisted database search of MEDLINE, EMBASE, Web of Science, Cumulative Index of Nursing and Allied Health Literature (CINAHL), the Cochrane Central Register of Controlled Trials (CENTRAL) and Chinese databases was conducted for randomized controlled trials (RCTs) comparing antiosteoporotic interventions in adult patients taking GCs. Outcomes were vertebral and non-vertebral fracture incidences. RESULTS: We included 56 RCTs containing 6479 eligible patients in our analysis. We found that alendronate and teriparatide were associated with decreased odds of both vertebral and non-vertebral fractures. Denosumab and risedronate were associated with decreased odds of vertebral fractures, while etidronate, ibandronate and alfacalcidol were associated with decreased odds of non-vertebral fractures. We observed low network heterogeneity as indicated by the I2 statistic, and we did not detect evidence of publication bias. All outcomes were based on a moderate quality of evidence according to GRADE. CONCLUSION: Bisphosphonates, teriparatide and denosumab are associated with decreased odds of fracture in patients undergoing GC therapy. Vitamin D metabolites and analogues (e.g. alfacalcidol) may have greater anti-fracture efficacy compared with plain vitamin D. SYSTEMATIC REVIEW REGISTRATION: The International Prospective Register of Systematic Reviews (PROSPERO)-CRD42019127073.

Training differences predict dogs' (Canis lupus familiaris) preferences for prosocial others.

Humans evaluate other agents' behavior on a variety of different dimensions, including morally, from a very early age. For example, human infants as young as 6-months old prefer prosocial over antisocial others and demonstrate negative evaluations of antisocial others in a variety of paradigms (Hamlin et al. in Nature 450(7169):557, 2007; Dev Sci 13(6):923-929, 2010; Proc Natl Acad Sci 108(50):19931-19936, 2011). While these tendencies are well documented in the human species, less is known about whether similar preference emerge in non-human animals. Here, we explore this question by testing prosocial preferences in one non-human species: the domestic dog (Canis lupus familiaris). Given the ubiquity of dog-human social interactions, it is possible that dogs display human-like social evaluation tendencies. Unfortunately, prior research examining social evaluation in dogs has produced mixed results. To assess whether differences in methodology or training differences account for these contrasting results, we tested two samples of dogs with different training histories on an identical social evaluation task. Trained agility dogs approached a prosocial actor significantly more often than an antisocial actor, while untrained pet dogs showed no preference for either actor. These differences across dogs with different training histories suggest that while dogs may demonstrate preferences for prosocial others in some contexts, their social evaluation abilities are less flexible and less robust compared to those of humans.

Discovery of O-Linked Carbohydrate on HIV-1 Envelope and Its Role in Shielding against One Category of Broadly Neutralizing Antibodies.

Approximately 50% of the mass of the Envelope (Env) glycoprotein surface subunit (gp120) of human immunodeficiency virus type 1 (HIV-1) is composed of N-linked carbohydrate. Until now, the dogma has been that HIV-1 lacks O-linked carbohydrate on Env. Here we show that a subset of patient-derived HIV-1 isolates contain O-linked carbohydrate on the variable 1 (V1) domain of Env gp120. We demonstrate the presence of this O-glycosylation both on virions and on gp120 expressed as a secreted protein. Further, we establish that these O-linked glycans can confer a more than 1,000-fold decrease in neutralization sensitivity (IC50) to V3-glycan broadly neutralizing antibodies. These findings uncover a structural modification to the HIV-1 Env and suggest a functional role in promoting viral escape from one category of broadly neutralizing antibodies.

A Highly Unusual V1 Region of Env in an Elite Controller of HIV Infection.

HIV elite controllers represent a remarkable minority of patients who maintain normal CD4+ T-cell counts and low or undetectable viral loads for decades in the absence of antiretroviral therapy. To examine the possible contribution of virus attenuation to elite control, we obtained a primary HIV-1 isolate from an elite controller who had been infected for 19 years, the last 10 of which were in the absence of antiretroviral therapy. Full-length sequencing of this isolate revealed a highly unusual V1 domain in Envelope (Env). The V1 domain in this HIV-1 strain was 49 amino acids, placing it in the top 1% of lengths among the 6,112 Env sequences in the Los Alamos National Laboratory online database. Furthermore, it included two additional N-glycosylation sites and a pair of cysteines suggestive of an extra disulfide loop. Virus with this Env retained good infectivity and replicative capacity; however, analysis of recombinant viruses suggested that other sequences in Env were adapted to accommodate the unusual V1 domain. While the long V1 domain did not confer resistance to neutralization by monoclonal antibodies of the V1/V2-glycan-dependent class, it did confer resistance to neutralization by monoclonal antibodies of the V3-glycan-dependent class. Our findings support results in the literature that suggest a role for long V1 regions in shielding HIV-1 from recognition by V3-directed broadly neutralizing antibodies. In the case of the elite controller described here, it seems likely that selective pressures from the humoral immune system were responsible for driving the highly unusual polymorphisms present in this HIV-1 Envelope.IMPORTANCE Elite controllers have long provided an avenue for researchers to reveal mechanisms underlying control of HIV-1. While the role of host genetic factors in facilitating elite control is well known, the possibility of infection by attenuated strains of HIV-1 has been much less studied. Here we describe an unusual viral feature found in an elite controller of HIV-1 infection and demonstrate its role in conferring escape from monoclonal antibodies of the V3-glycan class. Our results suggest that extreme variation may be needed by HIV-1 to escape neutralization by some antibody specificities.

Geographical distribution of soil transmitted helminths and the effects of community type in South Asia and South East Asia - A systematic review.

BACKGROUND: Soil-transmitted helminth (STH) infections are among the most prevalent neglected tropical diseases (NTD) worldwide. Since the publication of the WHO road map to combat NTD in 2012, there has been a renewed commitment to control STH. In this study, we analysed the geographical distribution and effect of community type on prevalence of hookworm, Trichuris and Ascaris in south Asia and south east Asia. METHODOLOGY: We conducted a systematic review of open-access literature published in PubMed Central and the Global Atlas of Helminth Infection. A total of 4182 articles were available and after applying selection criteria, 174 studies from the region were retained for analysis. PRINCIPAL FINDINGS: Ascaris was the commonest STH identified with an overall prevalence of 18% (95% CI, 14-23%) followed by Trichuris (14%, 9-19%) and hookworm (12%, 9-15%). Hookworm prevalence was highest in Laos, Vietnam and Cambodia. We found a geographical overlap in countries with high prevalence rates for Trichuris and Ascaris (Malaysia, Philippines, Myanmar, Vietnam and Bangladesh). When the effect of community type was examined, prevalence rates of hookworm was comparable in rural (19%, 14-24%) and tribal communities (14%, 10-19%). Tribal communities, however, showed higher prevalence of Trichuris (38%, 18-63%) and Ascaris (32%, 23-43%) than rural communities (13%, 9-20% and 14%, 9-20% respectively). Considerable between and within country heterogeneity in the distribution of STH (I2 >90%) was also noted. When available data from school aged children (SAC) were analysed, prevalence of Ascaris (25% 16-31%) and Trichuris (22%, 14-34%) were higher than among the general population while that of hookworm (10%, 7-16%) was comparable. CONCLUSIONS/SIGNIFICANCE: Our analysis showed significant variation in prevalence rates between and within countries in the region. Highlighting the importance of community type in prevalence and species mix, we showed that tribal and rural communities had higher hookworm infections than urban communities and for ascariasis and trichuriasis, tribal populations had higher levels of infection than rural populations. We also found a higher prevalence of ascariasis and trichuriasis in SAC compared to the general population but comparable levels of hookworm infections. These key findings need to be taken into account in planning future MDA and other interventions.

Human Immunodeficiency Virus and Simian Immunodeficiency Virus Maintain High Levels of Infectivity in the Complete Absence of Mucin-Type O-Glycosylation.

A highly conserved threonine near the C terminus of gp120 of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) was investigated for its contributions to envelope protein function and virion infectivity. When this highly conserved Thr residue was substituted with anything other than serine (the other amino acid that can accept O-glycosylation), the resulting virus was noninfectious. We found that this Thr was critical for the association of gp120 with the virion and that amino acid substitution increased the amount of dissociated gp120 in the cell culture supernatant. When HIV virions were generated in cells overexpressing polypeptide N-acetylgalactosaminyltransferase 1 (GalNAcT1), viral infectivity was increased 2.5-fold compared to that of virus produced in wild-type HEK293T cells; infectivity was increased 8-fold when the Thr499Ser mutant was used. These infectivity enhancements were not observed when GalNAcT3 was used. Using HEK293T knockout cell lines totally devoid of the ability to perform O-linked glycosylation, we demonstrated production of normal levels of virions and normal levels of infectivity in the complete absence of O-linked carbohydrate. Our data indicate that O-glycosylation is not necessary for the natural replication cycle of HIV and SIV. Nonetheless, it remains theoretically possible that the repertoire of GalNAc transferase isoforms in natural target cells for HIV and SIV in vivo could result in O-glycosylation of the threonine residue in question and that this could boost the infectivity of virions beyond the levels seen in the absence of such O-glycosylation.IMPORTANCE Approximately 50% of the mass of the gp120 envelope glycoprotein of both HIV and SIV is N-linked carbohydrate. One of the contributions of this N-linked carbohydrate is to shield conserved peptide sequences from recognition by humoral immunity. This N-linked glycosylation is one of the reasons that primary isolates of HIV and SIV are so heavily resistant to antibody-mediated neutralization. Much less studied is any potential contribution from O-linked glycosylation. The literature on this topic to date is somewhat confusing and ambiguous. Our studies described in this report demonstrate unambiguously that O-linked glycosylation is not necessary for the natural replication cycle of HIV and SIV. However, the door is not totally closed because of the diversity of numerous GalNAc transferase enzymes that initiate O-linked carbohydrate attachment and the theoretical possibility that natural target cells for HIV and SIV in vivo could potentially complete such O-linked carbohydrate attachment to further increase infectivity.

The role of MHC class I gene products in SIV infection of macaques.

Human immunodeficiency virus (HIV) remains among the most significant public health threats worldwide. Despite three decades of research following the discovery of HIV, a preventive vaccine remains elusive. The study of HIV elite controllers has been crucial to elaborate the genetic and immunologic determinants that underlie control of HIV replication. Coordinated studies of elite control in humans have, however, been limited by variability among infecting viral strains, host genotype, and the uncertainty of the timing and route of infection. In this review, we discuss the role of nonhuman primate (NHP) models for the elucidation of the immunologic correlates that underlie control of AIDS virus replication. We discuss the importance of major histocompatibility complex class I (MHC-I) alleles in activating CD8+ T-cell populations that promote control of both HIV and simian immunodeficiency virus (SIV) replication. Provocatively, we make the argument that T-cell subsets recognizing the HIV/SIV viral infectivity factor (Vif) protein may be crucial for control of viral replication. We hope that this review demonstrates how an in-depth understanding of the MHC-I gene products associated with elite control of HIV/SIV, and the epitopes that they present, can provide researchers with a glimpse into the protective immune responses that underlie AIDS nonprogression.

HEK293T cell lines defective for O-linked glycosylation.

Here we describe derivatives of the HEK293T cell line that are defective in their ability to generate mucin-type O-linked glycosylation. Using CRISPR/Cas9 and a single-cell GFP-sorting procedure, the UDP-galactose-4-epimerase (GALE), galactokinase 1 (GALK1), and galactokinase 2 (GALK2) genes were knocked out individually and in combinations with greater than 90% of recovered clones having the desired mutations. Although HEK293T cells are tetraploid, we found this approach to be an efficient method to target and disrupt all 4 copies of the target gene. Deficient glycosylation in the GALE knockout cell line could be rescued by the addition of galactose and N-acetylgalactosamine (GalNAc) to the cell culture media. However, when key enzymes of the galactose/GalNAc salvage pathways were disrupted in tandem (GALE+GALK1 or GALE+GALK2), O-glycosylation was eliminated and could not be rescued by the addition of either galactose plus GalNAc or UDP-galactose plus UDP-GalNAc. GALK1 and GALK2 are key enzymes of the galactose/GalNAc salvage pathways. Mass spectrometry was performed on whole cell lysate of the knockout cell lines to verify the glycosylation phenotype. As expected, the GALE knockout was almost completely devoid of all O-glycosylation, with minimal glycosylation as a result of functional salvage pathways. However, the GALE+GALK1 and GALE+GALK2 knockout lines were devoid of all O-glycans. Mass spectrometry analysis revealed that the disruption of GALE, GALK1, and GALE+GALK2 had little effect on the N-glycome. But when GALE was knocked out in tandem with GALK1, N-glycans were exclusively of the high mannose type. Due to the well-characterized nature of these five knockout cell lines, they will likely prove useful for a wide variety of applications.