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Guanylate-binding protein (GBP) 5 is an interferon-inducible cellular factor with broad anti-viral activity. Recently, GBP5 has been shown to antagonize the glycoproteins of a number of enveloped viruses, in part by disrupting the host enzyme furin. Here we show that GBP5 strongly impairs the infectivity of virus particles bearing not only viral glycoproteins that depend on furin cleavage for infectivity-the envelope (Env) glycoproteins of HIV-1 and murine leukemia virus and the spike (S) glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-but also viral glycoproteins that do not depend on furin cleavage: vesicular stomatitis virus glycoprotein and SARS-CoV S. We observe that GBP5 disrupts proper N -linked protein glycosylation and reduces the incorporation of viral glycoproteins into virus particles. The glycosylation of the cellular protein CD4 is also altered by GBP5 expression. Flow cytometry analysis shows that GBP5 expression reduces the cell-surface levels of HIV-1 Env and the S glycoproteins of SARS-CoV and SARS-CoV-2. Our data demonstrate that, under the experimental conditions used, inhibition of furin-mediated glycoprotein cleavage is not the primary anti-viral mechanism of action of GBP5. Rather, the antagonism appears to be related to impaired trafficking of glycoproteins to the plasma membrane. These results provide novel insights into the broad antagonism of viral glycoprotein function by the cellular host innate immune response., Importance: The surface of enveloped viruses contains viral envelope glycoproteins, an important structural component facilitating virus attachment and entry while also acting as targets for the host adaptive immune system. In this study, we show that expression of GBP5 in virus-producer cells alters the glycosylation, cell-surface expression, and virion incorporation of viral glycoproteins across several virus families. This research provides novel insights into the broad impact of the host cell anti-viral factor GBP5 on protein glycosylation and trafficking., Competing Interests: The authors declare no conflict of interest.

Background: Sex disparities have previously been identified in surgical academia. This study examines sex differences in the top-cited contemporary general surgery articles and compares Altimetric Attention Score (AAS) and other impact metrics between male and female corresponding authors (CAs)., Methods: We conducted a bibliometric analysis of the 100 most cited articles published between 2019 and 2021 in each of the top 10 general surgery journals based on the 2021 Journal Impact Factor. Impact metrics included AAS, citation count, and H-index of the CA. We used multivariable regression analyses to investigate whether the sex of the CA or first author (FA) was independently associated with AAS and citation count., Results: Among 1000 articles, 23.1% had female CAs and 27.4% female FAs. Female CA articles had higher AAS (13.0 [2.0-63.0] vs. 8.0 [1.0-28.5]; p < 0.001) and lower H-indices (24.0 [11.0-45.0] vs. 31.0 [17.0-50.0]; p = 0.015). Although median citation count did not differ by CA sex, articles with Level 1 evidence and a female CA were cited more often (35.5 [24.0-85.0] vs. 25.0 [16.0 vs. 46.0]; p < 0.05). In multivariable regression, female CA articles had higher AAS (OR: 1.002 [95% CI: 1.001-1.004]) and lower H-index (OR: 0.987 [95% CI: 0.977-0.997])., Conclusion: Despite having similar citation counts, articles authored by female CAs exhibit higher AAS scores compared to those authored by their male counterparts. While it is heartening that research authored by female surgeons achieves significant visibility, it remains to be understood how this translates into academic impact and scholarly recognition., (© 2024 International Society of Surgery/Société Internationale de Chirurgie (ISS/SIC).)

Low-grade gliomas (LGG) are brain tumors of glial cells origin. They are grade 1 and grade 2 tumors according to the WHO classification. Diagnosis of LGG is made through imaging, histopathological analysis, and use of molecular markers. Imaging alone does not establish the grade of the tumor and thus a histopathological examination of tissue is crucial in establishing the definite histopathological diagnosis. Clinical presentation varies according to the location and size of the tumor. Surgical resection is strongly recommended in LGG over observation to improve overall survival as surgery leads to greater benefit due to progression-free survival. Radiation has shown benefits in LGG patients in randomized controlled trials and chemotherapy with temozolomide has also shown good results. This paper covers the principles of low-grade gliomas management and summarizes the recommendations for the LMICs.

Objectives: To assess the visual improvement and mean residual astigmatism in patients who underwent cataract surgery with toric intraocular lens., Methods: The retrospective, observational study was conducted at the Department of Ophthalmology, Aga Khan University Hospital, Karachi, and comprised data from January 1, 2018, to December 31, 2020, related to adult patients who had regular astigmatism of at least 0.75D and underwent cataract surgery with toric intraocular lens implantation using a digital marker. The patients were followed up on post-operative days 1, 7, 30, 90 and 180. Along with age, the degree of astigmatism was noted. The visual acuity was calculated pre- and post-operatively. The mean residual astigmatism was then noted for all patients post-operatively. Data was analysed using SPSS 22., Results: The sample comprised 240 eyes of 177 patients; 99(55.9%) males and 78(44.1%) females. The mean age of the sample was 62.5±10.6 years. The mean unaided visual acuity improved post-operatively from 0.57±0.38 to 0.07±0.22 at 90 days. At the 30-day follow-up, mean residual astigmatism had reduced from 1.52±0.84 to 0.01±0.09 (p<0.001). The mean intraocular lens rotation from the intended axis was 0.73°±0.92° on day 30., Conclusions: Toric intraocular lens implantation using a digital marker could effectively reduce the post-operative cylinder, and improve the unaided visual acuity following cataract surgery.

Objective . This paper presents a novel approach for addressing the intricate task of diagnosing aortic valve regurgitation (AR), a valvular disease characterized by blood leakage due to incompetence of the valve closure. Conventional diagnostic techniques require detailed evaluations of multi-modal clinical data, frequently resulting in labor-intensive and time-consuming procedures that are vulnerable to varying subjective assessment of regurgitation severity. Approach . In our research, we introduce the multi-view video contrastive network, designed to leverage multiple color Doppler imaging inputs for multi-view video processing. We leverage supervised contrastive learning as a strategic approach to tackle class imbalance and enhance the effectiveness of our feature representation learning. Specifically, we introduce a contrastive learning framework to enhance representation learning within the embedding space through inter-patient and intra-patient contrastive loss terms. Main results . We conducted extensive experiments using an in-house dataset comprising 250 echocardiography video series. Our results exhibit a substantial improvement in diagnostic accuracy for AR compared to state-of-the-art methods in terms of accuracy by 9.60%, precision by 8.67%, recall by 9.01%, and F 1 -score by 8.92%. These results emphasize the capacity of our approach to provide a more precise and efficient method for evaluating the severity of AR. Significance . The proposed model could quickly and accurately make decisions about the severity of AR, potentially serving as a useful prescreening tool., (© 2024 Institute of Physics and Engineering in Medicine.)

HIV-1 assembly occurs at the inner leaflet of the plasma membrane (PM) in highly ordered membrane microdomains. The size and stability of membrane microdomains is regulated by activity of the sphingomyelin hydrolase neutral sphingomyelinase 2 (nSMase2) that is localized primarily to the inner leaflet of the PM. In this study, we demonstrate that pharmacological inhibition or depletion of nSMase2 in HIV-1-producer cells results in a block in the processing of the major viral structural polyprotein Gag and the production of morphologically aberrant, immature HIV-1 particles with severely impaired infectivity. We find that disruption of nSMase2 also severely inhibits the maturation and infectivity of other primate lentiviruses HIV-2 and simian immunodeficiency virus, has a modest or no effect on nonprimate lentiviruses equine infectious anemia virus and feline immunodeficiency virus, and has no effect on the gammaretrovirus murine leukemia virus. These studies demonstrate a key role for nSMase2 in HIV-1 particle morphogenesis and maturation.

Although HIV-1 Gag is known to drive viral assembly and budding, the precise mechanisms by which the lipid composition of the plasma membrane is remodeled during assembly are incompletely understood. Here, we provide evidence that the sphingomyelin hydrolase neutral sphingomyelinase 2 (nSMase2) interacts with HIV-1 Gag and through the hydrolysis of sphingomyelin creates ceramide that is necessary for proper formation of the viral envelope and viral maturation. Inhibition or depletion of nSMase2 resulted in the production of noninfectious HIV-1 virions with incomplete Gag lattices lacking condensed conical cores. Inhibition of nSMase2 in HIV-1-infected humanized mouse models with a potent and selective inhibitor of nSMase2 termed PDDC [phenyl(R)-(1-(3-(3,4-dimethoxyphenyl)-2, 6-dimethylimidazo[1,2-b]pyridazin-8-yl) pyrrolidin-3-yl)-carbamate] produced a linear reduction in levels of HIV-1 in plasma. If undetectable plasma levels of HIV-1 were achieved with PDDC treatment, viral rebound did not occur for up to 4 wk when PDDC was discontinued. In vivo and tissue culture results suggest that PDDC selectively kills cells with actively replicating HIV-1. Collectively, this work demonstrates that nSMase2 is a critical regulator of HIV-1 replication and suggests that nSMase2 could be an important therapeutic target with the potential to kill HIV-1-infected cells.

The SERINC (serine incorporator) proteins are host restriction factors that inhibit infection by HIV through their incorporation into virions. Here, we found that SERINC3 and SERINC5 exhibited additional antiviral activities by enhancing the expression of genes encoding type I interferons (IFNs) and nuclear factor κB (NF-κB) signaling. SERINC5 interacted with the outer mitochondrial membrane protein MAVS (mitochondrial antiviral signaling) and the E3 ubiquitin ligase and adaptor protein TRAF6, resulting in MAVS aggregation and polyubiquitylation of TRAF6. Knockdown of SERINC5 in target cells increased single-round HIV-1 infectivity, as well as infection by recombinant vesicular stomatitis virus (rVSV) bearing VSV-G or Ebola virus (EBOV) glycoproteins. Infection by an endemic Asian strain of Zika virus (ZIKV), FSS13025, was also enhanced by SERINC5 knockdown, suggesting that SERINC5 has direct antiviral activities in host cells in addition to the indirect inhibition mediated by its incorporation into virions. Further experiments suggested that the antiviral activity of SERINC5 was type I IFN–dependent. Together, these results highlight a previously uncharacterized function of SERINC proteins in promoting NF-κB inflammatory signaling and type I IFN production, thus contributing to its antiviral activities.

An emerging class of cellular inhibitory proteins has been identified that targets viral glycoproteins. These include the membrane-associated RING-CH (MARCH) family of E3 ubiquitin ligases that, among other functions, downregulate cell surface proteins involved in adaptive immunity. The RING-CH domain of MARCH proteins is thought to function by catalyzing the ubiquitination of the cytoplasmic tails (CTs) of target proteins, leading to their degradation. MARCH proteins have recently been reported to target retroviral envelope glycoproteins (Env) and vesicular stomatitis virus G glycoprotein (VSV-G). However, the mechanism of antiviral activity remains poorly defined. Here we show that MARCH8 antagonizes the full-length forms of HIV-1 Env, VSV-G, Ebola virus glycoprotein (EboV-GP), and the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), thereby impairing the infectivity of virions pseudotyped with these viral glycoproteins. This MARCH8-mediated targeting of viral glycoproteins requires the E3 ubiquitin ligase activity of the RING-CH domain. We observe that MARCH8 protein antagonism of VSV-G is CT dependent. In contrast, MARCH8-mediated targeting of HIV-1 Env, EboV-GP, and SARS-CoV-2 S protein by MARCH8 does not require the CT, suggesting a novel mechanism of MARCH-mediated antagonism of these viral glycoproteins. Confocal microscopy data demonstrate that MARCH8 traps the viral glycoproteins in an intracellular compartment. We observe that the endogenous expression of MARCH8 in several relevant human cell types is rapidly inducible by type I interferon. These results help to inform the mechanism by which MARCH proteins exert their antiviral activity and provide insights into the role of cellular inhibitory factors in antagonizing the biogenesis, trafficking, and virion incorporation of viral glycoproteins. IMPORTANCE Viral envelope glycoproteins are an important structural component on the surfaces of enveloped viruses that direct virus binding and entry and also serve as targets for the host adaptive immune response. In this study, we investigate the mechanism of action of the MARCH family of cellular proteins that disrupt the trafficking and virion incorporation of viral glycoproteins across several virus families. This research provides novel insights into how host cell factors antagonize viral replication, perhaps opening new avenues for therapeutic intervention in the replication of a diverse group of highly pathogenic enveloped viruses.

P-selectin glycoprotein ligand-1 (PSGL-1) is a cell surface glycoprotein that binds to P-, E-, and L-selectins to mediate the tethering and rolling of immune cells on the surface of the endothelium for cell migration into inflamed tissues. PSGL-1 has been identified as an interferon-γ (INF-γ)-regulated factor that restricts HIV-1 infectivity, and has recently been found to possess broad-spectrum antiviral activities. Here we report that the expression of PSGL-1 in virus-producing cells impairs the incorporation of SARS-CoV and SARS-CoV-2 spike (S) glycoproteins into pseudovirions and blocks pseudovirus attachment and infection of target cells. These findings suggest that PSGL-1 may potentially inhibit coronavirus replication in PSGL-1 + cells.

Host proteins with antiviral activity have evolved as first-line defenses to suppress viral replication. The HIV-1 accessory protein viral protein U (Vpu) enhances release of the virus from host cells by down-regulating the cell-surface expression of the host restriction factor tetherin. However, the exact mechanism of Vpu-mediated suppression of antiviral host responses is unclear. To further understand the role of host proteins in Vpu's function, here we carried out yeast two-hybrid screening and identified the V0 subunit C of vacuolar ATPase (ATP6V0C) as a Vpu-binding protein. To examine the role of ATP6V0C in Vpu-mediated tetherin degradation and HIV-1 release, we knocked down ATP6V0C expression in HeLa cells and observed that ATP6V0C depletion impairs Vpu-mediated tetherin degradation, resulting in defective HIV-1 release. We also observed that ATP6V0C overexpression stabilizes tetherin expression. This stabilization effect was specific to ATP6V0C, as overexpression of another subunit of the vacuolar ATPase, ATP6V0C″, had no effect on tetherin expression. ATP6V0C overexpression did not stabilize CD4, another target of Vpu-mediated degradation. Immunofluorescence localization experiments revealed that the ATP6V0C-stabilized tetherin is sequestered in a CD63- and lysosome-associated membrane protein 1 (LAMP1)-positive intracellular compartment. These results indicate that the Vpu-interacting protein ATP6V0C plays a role in down-regulating cell-surface expression of tetherin and thereby contributes to HIV-1 assembly and release.

P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric, mucin-like, 120-kDa glycoprotein that binds to P-, E-, and L-selectins. PSGL-1 is expressed primarily on the surface of lymphoid and myeloid cells and is up-regulated during inflammation to mediate leukocyte tethering and rolling on the surface of endothelium for migration into inflamed tissues. Although it has been reported that PSGL-1 expression inhibits HIV-1 replication, the mechanism of PSGL-1-mediated anti-HIV activity remains to be elucidated. Here we report that PSGL-1 in virions blocks the infectivity of HIV-1 particles by preventing the binding of particles to target cells. This inhibitory activity is independent of the viral glycoprotein present on the virus particle; the binding of particles bearing the HIV-1 envelope glycoprotein or vesicular stomatitis virus G glycoprotein or even lacking a viral glycoprotein is impaired by PSGL-1. Mapping studies show that the extracellular N-terminal domain of PSGL-1 is necessary for its anti-HIV-1 activity, and that the PSGL-1 cytoplasmic tail contributes to inhibition. In addition, we demonstrate that the PSGL-1-related monomeric E-selectin-binding glycoprotein CD43 also effectively blocks HIV-1 infectivity. HIV-1 infection, or expression of either Vpu or Nef, down-regulates PSGL-1 from the cell surface; expression of Vpu appears to be primarily responsible for enabling the virus to partially escape PSGL-1-mediated restriction. Finally, we show that PSGL-1 inhibits the infectivity of other viruses, such as murine leukemia virus and influenza A virus. These findings demonstrate that PSGL-1 is a broad-spectrum antiviral host factor with a unique mechanism of action., Competing Interests: Competing interest statement: Two provisional patent applications pertaining to the results presented in this paper have been filed by George Mason University., (Copyright © 2020 the Author(s). Published by PNAS.)

Background: Nef is a multifunctional accessory protein encoded by HIV-1, HIV-2 and SIV that plays critical roles in viral pathogenesis, contributing to viral replication, assembly, budding, infectivity and immune evasion, through engagement of various host cell pathways., Results: To gain a better understanding of the role of host proteins in the functions of Nef, we carried out tandem affinity purification-mass spectrometry analysis, and identified over 70 HIV-1 Nef-interacting proteins, including the autophagy-related 9A (ATG9A) protein. ATG9A is a transmembrane component of the machinery for autophagy, a catabolic process in which cytoplasmic components are degraded in lysosomal compartments. Pulldown experiments demonstrated that ATG9A interacts with Nef from not only HIV-1 and but also SIV (cpz, smm and mac). However, expression of HIV-1 Nef had no effect on the levels and localization of ATG9A, and on autophagy, in the host cells. To investigate a possible role for ATG9A in virus replication, we knocked out ATG9A in HeLa cervical carcinoma and Jurkat T cells, and analyzed virus release and infectivity. We observed that ATG9A knockout (KO) had no effect on the release of wild-type (WT) or Nef-defective HIV-1 in these cells. However, the infectivity of WT virus produced from ATG9A-KO HeLa and Jurkat cells was reduced by ~ fourfold and eightfold, respectively, relative to virus produced from WT cells. This reduction in infectivity was independent of the interaction of Nef with ATG9A, and was not due to reduced incorporation of the viral envelope (Env) glycoprotein into the virus. The loss of HIV-1 infectivity was rescued by pseudotyping HIV-1 virions with the vesicular stomatitis virus G glycoprotein., Conclusions: These studies indicate that ATG9A promotes HIV-1 infectivity in an Env-dependent manner. The interaction of Nef with ATG9A, however, is not required for Nef to enhance HIV-1 infectivity. We speculate that ATG9A could promote infectivity by participating in either the removal of a factor that inhibits infectivity or the incorporation of a factor that enhances infectivity of the viral particles. These studies thus identify a novel host cell factor implicated in HIV-1 infectivity, which may be amenable to pharmacologic manipulation for treatment of HIV-1 infection.

Tunneling nanotubes (TNTs) are intercellular structures that allow for the passage of vesicles, organelles, genomic material, pathogenic proteins and pathogens. The unconventional actin molecular motor protein Myosin-X (Myo10) is a known inducer of TNTs in neuronal cells, yet its role in other cell types has not been examined. The Nef HIV-1 accessory protein is critical for HIV-1 pathogenesis and can self-disseminate in culture via TNTs. Understanding its intercellular spreading mechanism could reveal ways to control its damaging effects during HIV-1 infection. Our goal in this study was to characterize the intercellular transport mechanism of Nef from macrophages to T cells. We demonstrate that Nef increases TNTs in a Myo10-dependent manner in macrophages and observed the transfer of Nef via TNTs from macrophages to T cells. To quantify this transfer mechanism, we established an indirect flow cytometry assay. Since Nef expression in T cells down-regulates the surface receptor CD4, we correlated the decrease in CD4 to the transfer of Nef between these cells. Thus, we co-cultured macrophages expressing varying levels of Nef with a T cell line expressing high levels of CD4 and quantified the changes in CD4 surface expression resulting from Nef transfer. We demonstrate that Nef transfer occurs via a cell-to-cell dependent mechanism that directly correlates with the presence of Myo10-dependent TNTs. Thus, we show that Nef can regulate Myo10 expression, thereby inducing TNT formation, resulting in its own transfer from macrophages to T cells. In addition, we demonstrate that up-regulation of Myo10 induced by Nef also occurs in human monocyte derived macrophages during HIV-1 infection.

The T cell Ig and mucin domain (TIM) proteins inhibit release of HIV-1 and other enveloped viruses by interacting with cell- and virion-associated phosphatidylserine (PS). Here, we show that the Nef proteins of HIV-1 and other lentiviruses antagonize TIM-mediated restriction. TIM-1 more potently inhibits the release of Nef-deficient relative to Nef-expressing HIV-1, and ectopic expression of Nef relieves restriction. HIV-1 Nef does not down-regulate the overall level of TIM-1 expression, but promotes its internalization from the plasma membrane and sequesters its expression in intracellular compartments. Notably, Nef mutants defective in modulating membrane protein endocytic trafficking are incapable of antagonizing TIM-mediated inhibition of HIV-1 release. Intriguingly, depletion of SERINC3 or SERINC5 proteins in human peripheral blood mononuclear cells (PBMCs) attenuates TIM-1 restriction of HIV-1 release, in particular that of Nef-deficient viruses. In contrast, coexpression of SERINC3 or SERINC5 increases the expression of TIM-1 on the plasma membrane and potentiates TIM-mediated inhibition of HIV-1 production. Pulse-chase metabolic labeling reveals that the half-life of TIM-1 is extended by SERINC5 from <2 to ∼6 hours, suggesting that SERINC5 stabilizes the expression of TIM-1. Consistent with a role for SERINC protein in potentiating TIM-1 restriction, we find that MLV glycoGag and EIAV S2 proteins, which, like Nef, antagonize SERINC-mediated diminishment of HIV-1 infectivity, also effectively counteract TIM-mediated inhibition of HIV-1 release. Collectively, our work reveals a role of Nef in antagonizing TIM-1 and highlights the complex interplay between Nef and HIV-1 restriction by TIMs and SERINCs., Competing Interests: The authors declare no conflict of interest.

Introduction Trauma constitutes a major public health problem. Ninty percent of world's fatalities on road occur in low and middle-income countries. Focused assessment with sonography in trauma (FAST) has a key role in trauma investigation, altering subsequent management in a significant number of patients. There is a rising trend of introducing FAST examination to non-radiologists and junior members of healthcare team to hasten triage of trauma patients. Objective To determine interobserver agreement on focused assessment with sonography for trauma in blunt abdominal injury between senior and junior residents. Methods This cross-sectional study was conducted at Aga Khan University Hospital. Three hundred patients with blunt abdominal trauma meeting inclusion criteria were enrolled. FAST was performed using standard curvilinear 3.5-5 MHz transducer. Agreement between junior and senior residents was measured and Kappa statistic was calculated. Results Mean age of the patients included in the study was 30.04 ± 18.09 years. Among these 237 (79%) were male and 63 (21%) were female. Sixteen (5.3%) were positive for intraperitoneal free fluid while the remaining 284 (94.7%) were negative. A total of 19 FAST examinations were reported positive by junior residents, of which 15 (78.9%) were confirmed by a senior resident to be correct, while four (21.05%) were falsely labeled positive by a junior resident. A total of 281 negative FAST examinations were reported by junior residents, of which 280 (99.6%) were confirmed by a senior resident, while only one (0.003%) was falsely labeled negative. Kappa statistic was calculated for inter-observer agreement on FAST examination findings, which showed a kappa value of 0.84 (very good agreement), with a p-value of <0.001. Conclusion Our study suggests very good interobserver agreement on FAST examination between senior and junior resident in patients with blunt trauma to the abdomen. Results suggest that FAST can be easily learnt with minimal radiology training and may have greater applicability in trauma., Competing Interests: The authors have declared that no competing interests exist.

Background: Transcatheter closure of patent foramen ovale (PFO) after cryptogenic stroke has long been a contentious issue. Herein, we pool aggregate data examining safety and efficacy of transcatheter closure of PFO compared with medical therapy following initial cryptogenic stroke., Methods: We searched for randomized clinical trials (RCT) that compared device closure with medical management and reported on subsequent stroke and adverse events. Stroke was considered as the primary efficacy endpoint, whereas bleeding and atrial fibrillation were considered primary safety endpoints. Data were pooled by the random effects model and I2 was used to assess heterogeneity., Results: A total of 5 RCT investigating 3630 patients met inclusion criteria. Pooled analysis revealed that device closure compared to medical management was associated with a significant reduction in stroke (RR=0.3, 95% CI=0.02-0.57). There was, however, a significant increase in atrial arrhythmias with device therapy (RR=4.8, 95% CI=2.2-10.7). We found no increase in bleeding (RR=0.80, 95% CI=0.5-1.4), death (RR=0.76, 95% CI=0.3-1.99) or "any adverse events" (RR=1.02, 95% CI=0.85-1.23) with device therapy. Sub-group analysis revealed that device closure significantly reduced the incidence of the composite primary endpoint among patients who had moderate to large shunt sizes (RR=0.22, 95% CI=0.02-0.42)., Conclusions: Transcatheter closure is associated with a significant reduction in the risk of stroke compared to medical management at the expense of an increased risk of atrial arrhythmias., (Copyright © 2018. Published by Elsevier Inc.)

Tetherin is an interferon-inducible antiviral protein that inhibits the release of a broad spectrum of enveloped viruses by retaining virions at the surface of infected cells. While the role of specific tetherin domains in antiviral activity is clearly established, the role of glycosylation in tetherin function is not clear. In this study, we carried out a detailed investigation of this question by using tetherin variants in which one or both sites of N -linked glycosylation were mutated (N65A, N92A, and N65,92A), and chemical inhibitors that prevent glycosylation at specific stages of oligosaccharide were added or modified. The single N -linked glycosylation mutants, N65A and N92A, efficiently inhibited the release of Vpu-defective human immunodeficiency virus type 1 (HIV-1). In contrast, the non-glycosylated double mutant, N65,92A, lost its ability to block HIV-1 release. The inability of the N65,92A mutant to inhibit HIV-1 release is associated with a lack of cell-surface expression. A role for glycosylation in cell-surface tetherin expression is supported by tunicamycin treatment, which inhibits the first step of N -linked glycosylation and impairs both cell-surface expression and antiviral activity. Inhibition of complex-type glycosylation with kifunensine, an inhibitor of the oligosaccharide processing enzyme mannosidase 1, had no effect on either the cell-surface expression or antiviral activity of tetherin. These results demonstrate that high-mannose modification of a single asparagine residue is necessary and sufficient, while complex-type glycosylation is dispensable, for cell-surface tetherin expression and antiviral activity., Competing Interests: The authors declare no conflict of interest.

The HIV-1 accessory protein Vpu enhances virus release by counteracting the host restriction factor tetherin. To further understand the role of host cell proteins in Vpu function, we carried out yeast two-hybrid screening and identified a previously reported Vpu-interacting host factor, small glutamine-rich tetratricopeptide repeat-containing protein (SGTA). While RNAi-mediated depletion of SGTA did not significantly affect levels of tetherin or virus release efficiency, we observed that overexpression of SGTA inhibited HIV-1 release in a Vpu- and tetherin-independent manner. Overexpression of SGTA in the presence of Vpu, but not in its absence, resulted in a marked stabilization and cytosolic relocalization of a 23-kDa, non-glycosylated tetherin species. Coimmunoprecipitation studies indicated that non-glycosylated tetherin is stabilized through the formation of a ternary SGTA/Vpu/tetherin complex. This accumulation of non-glycosylated tetherin is due to inhibition of its degradation, independent of the ER-associated degradation (ERAD) pathway. Because the SGTA-stabilized tetherin species is partially localized to the cytosol, we propose that overexpression of SGTA in the presence of Vpu blocks the translocation of tetherin across the ER membrane, resulting in cytosolic accumulation of a non-glycosylated tetherin species. Although our results do not provide support for a physiological function of SGTA in HIV-1 replication, they demonstrate that SGTA overexpression regulates tetherin expression and stability, thus providing insights into the function of SGTA in ER translocation and protein degradation.

The biomedical intervention that has had a major impact on the natural history of HIV and on the global HIV epidemic is antiretroviral therapy (ART). However, the emergence of drug-resistant HIV, an inevitable consequence of increasing use of antiretroviral drugs, poses a major threat to ART success. At the turn of this century, access to life-saving ART was accelerated in low and middle-income countries with the Millennium Development Goal of 15 million individuals receiving ART by 2015 expected to be achieved. However, ART access needs to continue to expand to help bring HIV under control by 2030. The standard of care for people living with HIV in resource- limited settings differs dramatically compared to high-income countries, and not unexpectedly, ART rollout in these settings has resulted in an increase in acquired and transmitted drug resistance. Also of concern, the same drug classes used for ART have been approved or are being progressed for HIV prevention and drug resistance could mitigate their effectiveness for treatment and prevention. In the absence of an effective HIV vaccine and cure, it is imperative that the antiretroviral drug pipeline contains new classes of HIV inhibitors that are active against circulating drug-resistant strains. Studies to advance our fundamental understanding of HIV replication needs to continue, including the interplay between virus and host cell factors, to identify and characterize new drug targets for chemotherapeutic intervention.

Unlabelled: HIV-1 uses cellular machinery to bud from infected cells. This cellular machinery is comprised of several multiprotein complexes known as endosomal sorting complexes required for transport (ESCRTs). A conserved late domain motif, Pro-Thr-Ala-Pro (PTAP), located in the p6 region of Gag (p6(Gag)), plays a central role in ESCRT recruitment to the site of virus budding. Previous studies have demonstrated that PTAP duplications are selected in HIV-1-infected patients during antiretroviral therapy; however, the consequences of these duplications for HIV-1 biology and drug resistance are unclear. To address these questions, we constructed viruses carrying a patient-derived PTAP duplication with and without drug resistance mutations in the viral protease. We evaluated the effect of the PTAP duplication on viral release efficiency, viral infectivity, replication capacity, drug susceptibility, and Gag processing. In the presence of protease inhibitors, we observed that the PTAP duplication in p6(Gag) significantly increased the infectivity and replication capacity of the virus compared to those of viruses bearing only resistance mutations in protease. Our biochemical analysis showed that the PTAP duplication, in combination with mutations in protease, enhances processing between the nucleocapsid and p6 domains of Gag, resulting in more complete Gag cleavage in the presence of protease inhibitors. These results demonstrate that duplication of the PTAP motif in p6(Gag) confers a selective advantage in viral replication by increasing Gag processing efficiency in the context of protease inhibitor treatment, thereby enhancing the drug resistance of the virus. These findings highlight the interconnected role of PTAP duplications and protease mutations in the development of resistance to antiretroviral therapy., Importance: Resistance to current drug therapy limits treatment options in many HIV-1-infected patients. Duplications in a Pro-Thr-Ala-Pro (PTAP) motif in the p6 domain of Gag are frequently observed in viruses derived from patients on protease inhibitor (PI) therapy. However, the reason that these duplications arise and their consequences for virus replication remain to be established. In this study, we examined the effect of PTAP duplication on PI resistance in the context of wild-type protease or protease bearing PI resistance mutations. We observe that PTAP duplication markedly enhances resistance to a panel of PIs. Biochemical analysis reveals that the PTAP duplication reverses a Gag processing defect imposed by the PI resistance mutations in the context of PI treatment. The results provide a long-sought explanation for why PTAP duplications arise in PI-treated patients., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Beta-adrenergic receptor antagonists (β-blockers) have been well established for use in portal hypertension for more than three decades. Different Non-selective β-blockers like propranolol, nadolol, timolol, atenolol, metoprolol and carvedilol have been in clinical practice in patients with cirrhosis. Carvedilol has proven 2-4 times more potent than propranolol as a beta-receptor blocker in trials conducted testing its efficacy for heart failure. Whether the same effect extends to its potency in the reduction of portal venous pressures is a topic of on-going debate. The aim of this review is to compare the hemodynamic and clinical effects of carvedilol with propranolol, and attempt assess whether carvedilol can be used instead of propranolol in patients with cirrhosis. Carvedilol is a promising agent among the beta blockers of recent time that has shown significant effects in portal hypertension hemodynamics. It has also demonstrated an effective profile in its clinical application specifically for the prevention of variceal bleeding. Carvedilol has more potent desired physiological effects when compared to Propranolol. However, it is uncertain at the present juncture whether the improvement in hemodynamics also translates into a decreased rate of disease progression and complications when compared to propranolol. Currently Carvedilol shows promise as a therapy for portal hypertension but more clinical trials need to be carried out before we can consider it as a superior option and a replacement for propranolol.

In Pakistan, Plasmodium vivax contributes to major malaria burden. In this case, a pregnant woman presented with P. vivax infection and which was not cleared by chloroquine, despite adequate treatment. This is probably the first confirmed case of chloroquine-resistant vivax from Pakistan, where severe malaria due to P. vivax is already an emerging problem.

Vpu is an accessory protein encoded by HIV-1 that interferes with multiple host-cell functions. Herein we report that expression of Vpu by transfection into 293T cells causes partial proteolytic cleavage of interferon regulatory factor 3 (IRF3), a key transcription factor in the innate anti-viral response. Vpu-induced IRF3 cleavage is mediated by caspases and occurs mainly at Asp-121. Cleavage produces a C-terminal fragment of ∼37 kDa that comprises the IRF dimerization and transactivation domains but lacks the DNA-binding domain. A similar cleavage is observed upon infection of the Jurkat T-cell line with vesicular stomatitis virus G glycoprotein (VSV-G)-pseudotyped HIV-1. Two other HIV-1 accessory proteins, Vif and Vpr, also contribute to the induction of IRF3 cleavage in both the transfection and the infection systems. The C-terminal IRF3 fragment interferes with the transcriptional activity of full-length IRF3. Cleavage of IRF3 under all of these conditions correlates with cleavage of poly(ADP-ribose) polymerase, an indicator of apoptosis. We conclude that Vpu contributes to the attenuation of the anti-viral response by partial inactivation of IRF3 while host cells undergo apoptosis., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

The interferon-inducible cellular protein tetherin (CD317/BST-2) inhibits the release of a broad range of enveloped viruses. The HIV-1 accessory protein Vpu enhances virus particle release by counteracting this host restriction factor. While the antagonism of human tetherin by Vpu has been associated with both proteasomal and lysosomal degradation, the link between Vpu-mediated tetherin degradation and the ability of Vpu to counteract the antiviral activity of tetherin remains poorly understood. Here, we show that human tetherin is expressed at low levels in African green monkey kidney (COS) cells. However, Vpu markedly increases tetherin expression in this cell line, apparently by sequestering it in an internal compartment that bears lysosomal markers. This stabilization of tetherin by Vpu requires the transmembrane sequence of human tetherin. Although Vpu stabilizes human tetherin in COS cells, it still counteracts the ability of tetherin to suppress virus release. The enhancement of virus release by Vpu in COS cells is associated with a modest reduction in cell-surface tetherin expression, even though the overall expression of tetherin is higher in the presence of Vpu. This study demonstrates that COS cells provide a model system in which Vpu-mediated enhancement of HIV-1 release is uncoupled from Vpu-mediated tetherin degradation.

A 27-aa peptide (P27) was previously shown to decrease the accumulation of human immunodeficiency virus type 1 (HIV-1) in the supernatant of chronically infected cells; however, the mechanism was not understood. Here, we show that P27 prevents virus accumulation by inducing macropinocytosis (MPC). Treatment of HIV-1- and human T-cell lymphotropic virus type 1 (HTLV-1)-infected cells with 2-10 μM P27 caused cell membrane ruffling and uptake of virus and polymerized forms of the peptide into large vacuoles. As demonstrated by electron microscopy, activation of MPC did not require virus or cells infected with virus, as P27 initiated its own uptake in the absence of virus. Inhibitors of MPC, Cytochalasin D and amiloride, decreased P27-mediated uptake of soluble dextran and inhibited P27-induced virus uptake by >60%, which provides further evidence that P27 induces MPC. In CD4(+) HeLa cells, HIV-1 infection was enhanced by P27 up to 4-fold, and P27 increased infection at concentrations as low as 20 nM. The 5-aa C-terminal domain of P27 was necessary for virus uptake and may be responsible for the polymerization of P27 into fibrils. These forms of P27 may play a key role in triggering MPC, making this peptide a useful tool for studying virus uptake and infection, as well as MPC of other macromolecules.

Hyperphosphatemia is a nearly universal complication of end-stage renal disease that is widely recognized as one of the most important and most challenging clinical targets to meet in the care of dialysis patients. Left untreated, it can lead to bone pain, pruritus and worsening secondary hyperparathyroidism. Data from observational studies demonstrate that an elevated serum phosphorus level is an independent risk factor for mortality, and that treatment with phosphate binders is independently associated with improved survival. Experimental studies provide support for the epidemiologic findings: phosphate excess promotes vascular calcification, induces endothelial dysfunction and may contribute to other emerging chronic kidney disease-specific mechanisms of cardiovascular toxicity. On the basis of this evidence, clinical practice guidelines recommend specific targets for serum phosphorus levels in the dialysis population. The purpose of this review is to summarize common challenges in meeting these targets and to identify potential opportunities for improvement.

Background: Previously, we reported the conversion of the 12-mer linear and cell-impermeable peptide CAI to a cell-penetrating peptide NYAD-1 by using an i,i + 4 hydrocarbon stapling technique and confirmed its binding to the C-terminal domain (CTD) of the HIV-1 capsid (CA) protein with an improved affinity (K(d) ~ 1 μM) compared to CAI (K(d) ~ 15 μM). NYAD-1 disrupts the formation of both immature- and mature-like virus particles in in vitro and cell-based assembly assays. In addition, it displays potent anti-HIV-1 activity in cell culture against a range of laboratory-adapted and primary HIV-1 isolates., Results: In this report, we expanded the study to i,i + 7 hydrocarbon-stapled peptides to delineate their mechanism of action and antiviral activity. We identified three potent inhibitors, NYAD-36, -66 and -67, which showed strong binding to CA in NMR and isothermal titration calorimetry (ITC) studies and disrupted the formation of mature-like particles. They showed typical α-helical structures and penetrated cells; however, the cell penetration was not as efficient as observed with the i,i + 4 peptides. Unlike NYAD-1, the i,i + 7 peptides did not have any effect on virus release; however, they impaired Gag precursor processing. HIV-1 particles produced in the presence of these peptides displayed impaired infectivity. Consistent with an effect on virus entry, selection for viral resistance led to the emergence of two mutations in the gp120 subunit of the viral envelope (Env) glycoprotein, V120Q and A327P, located in the conserved region 1 (C1) and the base of the V3 loop, respectively., Conclusion: The i,i + 7 stapled peptides derived from CAI unexpectedly target both CA and the V3 loop of gp120. This dual-targeted activity is dependent on their ability to penetrate cells as well as their net charge. This mechanistic revelation will be useful in further modifying these peptides as potent anti-HIV-1 agents.

A retrospective study evaluating the pattern of blood pressure and its related complications before, during, and after percutaneous hemodialysis interventions was performed in patients presenting with asymptomatic hypertension. Hemodialysis patients undergoing percutaneous interventions including tunneled hemodialysis catheter insertion, percutaneous balloon angioplasty and thrombectomy procedure, and stage II hypertension (systolic blood pressure ≥160 mmHg) were included in this analysis. Blood pressure medications were not used while midazolam and fentanyl were routinely administered. Patients were followed for up to 4 weeks to monitor any complications. The mean blood pressure before, during, and after the procedures were 185 ± 18/96 ± 14, 172 ± 22/92 ± 15, and 153 ± 25/87 ± 14, respectively. There was a statistically significant difference between the blood pressure readings before and after the procedure (before = 185 ± 18/96 ± 14, after = 153 ± 25/87 ± 14; p = 0.001). None of the patients had a stroke, myocardial infarction, or acute pulmonary edema before, during, or after the procedure or during the 4-week follow-up period. A significant reduction in blood pressure was observed after the procedure without the administration of any antihypertensive medication. These results suggest that the reduction in blood pressure observed after percutaneous dialysis access interventions (particularly in the presence of midazolam and fentanyl) may make it unnecessary to treat asymptomatic hypertension prior to these procedures., (© 2013 Wiley Periodicals, Inc.)

Lupus nephritis (LN) increases the morbidity and mortality of patients with SLE. This review compares the randomized, controlled trials that examined various maintenance regimens available to treat LN. Currently, mycophenolate mofetil (MMF) and azathioprine (AZA) are the most popular therapeutic agents used for long-term maintenance of LN. Long-term maintenance with MMF is recommended as the first choice after achieving remission with cyclophosphamide or MMF induction. MMF is effective in consolidating remission and preventing relapse and CKD in patients of diverse races and ethnicities. Long-term maintenance with AZA is the recommended second choice, especially when patients develop intolerance of or contraindications to MMF. Azathioprine is particularly effective in consolidating remission and preventing relapse and CKD in patients who receive an induction regimen of cyclophosphamide. To date, there are no data on how rapidly maintenance therapies can be withdrawn; however, it is recommended that the immunosuppressive therapy be maintained indefinitely, unless it is contraindicated, in patients at high risk for progression to CKD.

The Gag proteins of HIV-1 are central players in virus particle assembly, release, and maturation, and also function in the establishment of a productive infection. Despite their importance throughout the replication cycle, there are currently no approved antiretroviral therapies that target the Gag precursor protein or any of the mature Gag proteins. Recent progress in understanding the structural and cell biology of HIV-1 Gag function has revealed a number of potential Gag-related targets for possible therapeutic intervention. In this review, we summarize our current understanding of HIV-1 Gag and suggest some approaches for the development of novel antiretroviral agents that target Gag.

Targeting protein-protein interactions is gaining greater recognition as an attractive approach to therapeutic development. An example of this may be found with the human cellular protein encoded by the tumor susceptibility gene 101 (Tsg101), where interaction with the p6 C-terminal domain of the nascent viral Gag protein is required for HIV-1 particle budding and release. This association of Gag with Tsg101 is highly dependent on a "Pro-Thr-Ala-Pro" ("PTAP") peptide sequence within the p6 protein. Although p6-derived peptides offer potential starting points for developing Tsg101-binding inhibitors, the affinities of canonical peptides are outside the useful range (K(d) values greater than 50 μM). Reported herein are crystal structures of Tsg101 in complex with two structurally-modified PTAP-derived peptides. This data define new regions of ligand interaction not previously identified with canonical peptide sequences. This information could be highly useful in the design of Tsg101-binding antagonists.

Background: The C-terminal domain (CTD) of HIV-1 capsid (CA), like full-length CA, forms dimers in solution and CTD dimerization is a major driving force in Gag assembly and maturation. Mutations of the residues at the CTD dimer interface impair virus assembly and render the virus non-infectious. Therefore, the CTD represents a potential target for designing anti-HIV-1 drugs., Results: Due to the pivotal role of the dimer interface, we reasoned that peptides from the α-helical region of the dimer interface might be effective as decoys to prevent CTD dimer formation. However, these small peptides do not have any structure in solution and they do not penetrate cells. Therefore, we used the hydrocarbon stapling technique to stabilize the α-helical structure and confirmed by confocal microscopy that this modification also made these peptides cell-penetrating. We also confirmed by using isothermal titration calorimetry (ITC), sedimentation equilibrium and NMR that these peptides indeed disrupt dimer formation. In in vitro assembly assays, the peptides inhibited mature-like virus particle formation and specifically inhibited HIV-1 production in cell-based assays. These peptides also showed potent antiviral activity against a large panel of laboratory-adapted and primary isolates, including viral strains resistant to inhibitors of reverse transcriptase and protease., Conclusions: These preliminary data serve as the foundation for designing small, stable, α-helical peptides and small-molecule inhibitors targeted against the CTD dimer interface. The observation that relatively weak CA binders, such as NYAD-201 and NYAD-202, showed specificity and are able to disrupt the CTD dimer is encouraging for further exploration of a much broader class of antiviral compounds targeting CA. We cannot exclude the possibility that the CA-based peptides described here could elicit additional effects on virus replication not directly linked to their ability to bind CA-CTD.

Photodynamic therapy (PDT) has been used as a cancer therapy for forty years but has not advanced to a mainstream cancer treatment. Although it has been shown to be an efficient way to destroy local tumors by a combination of non-toxic dyes and harmless visible light, it is its additional effects in mediating the stimulation of the host immune system that gives PDT great potential to become more widely used. Although the stimulation of tumor-specific cytotoxic T-cells that can destroy distant tumor deposits after PDT has been reported in some animal models, it remains the exception rather than the rule. This realization has prompted several investigators to test various combination approaches that could potentiate the immune recognition of tumor antigens that have been released after PDT. This review will cover these combination approaches using immunostimulants including various microbial preparations that activate Toll-like receptors and other receptors for pathogen-associated molecular patterns, cytokines growth factors, and approaches that target regulatory T-cells. We believe that by understanding the methods employed by tumors to evade immune response and neutralizing them, more precise ways of potentiating PDT-induced immunity can be devised.

Retroviruses undergo several critical steps to complete a replication cycle. These include the complex processes of virus entry, assembly, and budding that often take place at the plasma membrane of the host cell. Both virus entry and release involve membrane fusion/fission reactions between the viral envelopes and host cell membranes. Accumulating evidence indicates important roles for lipids and lipid microdomains in virus entry and egress. In this review, we outline the current understanding of the role of lipids and membrane microdomains in retroviral replication.

We previously reported that human immunodeficiency virus type 1 (HIV-1) develops resistance to the cholesterol-binding compound amphotericin B methyl ester (AME) by acquiring mutations (P203L and S205L) in the cytoplasmic tail of the transmembrane envelope glycoprotein gp41 that create cleavage sites for the viral protease (PR). In the present study, we observed that a PR inhibitor-resistant (PIR) HIV-1 mutant is unable to efficiently cleave the gp41 cytoplasmic tail in P203L and S205L virions, resulting in loss of AME resistance. To define the pathway to AME resistance in the context of the PIR PR, we selected for resistance with an HIV-1 isolate expressing the mutant enzyme. We identified a new gp41 mutation, R236L, that results in cleavage of the gp41 tail by the PIR PR. These results highlight the central role of gp41 cleavage as the primary mechanism of AME resistance.

HIV-1 viral budding involves binding of the viral Gag(p6) protein to the ubiquitin E2 variant domain of the human tumor susceptibility gene 101 protein (Tsg101). Recognition of p6 by Tsg101 is mediated in part by a proline-rich motif that contains the sequence 'Pro-Thr-Ala-Pro' ('PTAP'). Using the p6-derived 9-mer sequence 'PEPTAPPEE', we had previously improved peptide binding affinity by employing N-alkylglycine ('peptoid') residues. The current study applies ring-closing metathesis macrocyclization strategies to Tsg101-binding peptide-peptoid hybrids as an approach to stabilize binding conformations and to observe the effects of such macrocyclization on Tsg101-binding affinity and bioavailability., (Published by Elsevier Ltd.)

Several critical steps in the replication cycle of human immunodeficiency virus type 1 (HIV-1) - entry, assembly and budding - are complex processes that take place at the plasma membrane of the host cell. A growing body of data indicates that these early and late steps in HIV-1 replication take place in specialized plasma membrane microdomains, and that many of the viral and cellular components required for entry, assembly, and budding are concentrated in these microdomains. In particular, a number of studies have shown that cholesterol- and sphingolipid-enriched microdomains known as lipid rafts play important roles in multiple steps in the virus replication cycle. In this review, we provide an overview of what is currently known about the involvement of lipids and membrane microdomains in HIV-1 replication.

Virus assembly constitutes a key phase of the HIV-1 replication cycle. The assembly process is initiated by the synthesis of the Gag precursor protein, Pr55(Gag), in the cytosol of the infected cell. After its synthesis, Pr55(Gag) is rapidly transported in most cell types to the plasma membrane (PM) where it associates with the inner leaflet of the lipid bilayer. Gag-Gag interactions lead to the assembly of an electron-dense patch of Gag proteins at the membrane. The viral envelope (Env) glycoproteins associate with Gag during the assembly process. The highly multimerized Gag complex begins to bud outwardly from the PM and eventually pinches off from the cell surface. Concomitant with release, the viral protease cleaves Pr55(Gag) to the mature Gag proteins matrix, capsid, nucleocapsid and p6, leading to core condensation. The mature infectious virus particle is now able to initiate a new round of infection in a fresh target cell. Techniques have been developed in many laboratories to study each of the distinct phases of the HIV-1 assembly and release pathway. A number of these techniques are described in detail in this chapter.