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Objectives Studies have demonstrated high rates of mortality in people with proximal femoral fracture and SARS-CoV-2, but there is limited published data on the factors that influence mortality for clinicians to make informed treatment decisions. This study aims to report the 30-day mortality associated with perioperative infection of patients undergoing surgery for proximal femoral fractures and to examine the factors that influence mortality in a multivariate analysis.Setting Prospective, international, multicentre, observational cohort study.Participants Patients undergoing any operation for a proximal femoral fracture from 1 February to 30 April 2020 and with perioperative SARS-CoV-2 infection (either 7 days prior or 30-day postoperative).Primary outcome 30-day mortality. Multivariate modelling was performed to identify factors associated with 30-day mortality.Results This study reports included 1063 patients from 174 hospitals in 19 countries. Overall 30-day mortality was 29.4% (313/1063). In an adjusted model, 30-day mortality was associated with male gender (OR 2.29, 95% CI 1.68 to 3.13, p80 years (OR 1.60, 95% CI 1.1 to 2.31, p=0.013), preoperative diagnosis of dementia (OR 1.57, 95% CI 1.15 to 2.16, p=0.005), kidney disease (OR 1.73, 95% CI 1.18 to 2.55, p=0.005) and congestive heart failure (OR 1.62, 95% CI 1.06 to 2.48, p=0.025). Mortality at 30 days was lower in patients with a preoperative diagnosis of SARS-CoV-2 (OR 0.6, 95% CI 0.6 (0.42 to 0.85), p=0.004). There was no difference in mortality in patients with an increase to delay in surgery (p=0.220) or type of anaesthetic given (p=0.787).Conclusions Patients undergoing surgery for a proximal femoral fracture with a perioperative infection of SARS-CoV-2 have a high rate of mortality. This study would support the need for providing these patients with individualised medical and anaesthetic care, including medical optimisation before theatre. Careful preoperative counselling is needed for those with a proximal femoral fracture and SARS-CoV-2, especially those in the highest risk groups.Trial registration number NCT04323644

Robotic gait training may improve overground ambulation for individuals with poor control over pelvic motion. However, there is a need for an overground gait training robotic device that allows full control of pelvic movement and synchronizes applied forces to the user’s gait. This work evaluates an overground robotic gait trainer that applies synchronized forces on the user’s pelvis, the mobile Tethered Pelvic Assist Device. To illustrate one possible control scheme, we apply assistive frontal plane pelvic moments synchronized with the user’s continuous gait in real-time. Ten healthy adults walked with the robotic device, with and without frontal plane moments. The frontal plane moments corresponded to 10% of the user’s body weight with a moment arm of half their pelvic width. The frontal plane moments significantly increased the range of frontal plane pelvic angles from 2.6° to 9.9° and the sagittal and transverse planes from 4.6° to 10.1° and 3.0° to 8.3°, respectively. The frontal plane moments also significantly increased the activation of the left gluteus medius muscle, which assists in regulating pelvic obliquity. The right gluteus medius muscle activation did not significantly differ when frontal plane moments were applied. This work highlights the ability of the mobile Tethered Pelvic Assist Device to apply a continuous pelvic moment that is synchronized with the user’s gait cycle. This capability could change how overground robotic gait training strategies are designed and applied. The potential for gait training interventions that target gait deficits or muscle weakness can now be explored with the mobile Tethered Pelvic Assist Device.

Abstract Identification of proteins is one of the most computationally intensive steps in genomics studies. It usually relies on aligners that do not accommodate rich information on proteins and require additional pipelining steps for protein identification. We introduce kAAmer, a protein database engine based on amino-acid k-mers that provides efficient identification of proteins while supporting the incorporation of flexible annotations on these proteins. Moreover, the database is built to be used as a microservice, to be hosted and queried remotely.

As a part of our study of the syntheses of aryl amides, the crystal structures of two benzamides were determined from single-crystal X-ray data at 173 K. Both crystal structures contain molecular units as asymmetric units with no solvent in the unit cells. Crystal structure I, TFMP, is the result of the crystallization of N-[4-(trifluoromethyl)phenyl]benzamide, C14H10F3NO. Crystal structure II, MOP, is composed of N-(4-methoxyphenyl)benzamide, C14H13NO2, units. TFMP is triclinic, space group P\overline{1}, consisting of two molecules in the unit cell related by the center of symmetry. MOP is monoclinic, space group P21/c, consisting of four molecules in the unit cell. Both types of molecules contain three planar regions; a phenyl ring, an amide planar region, and a para-substituted phenyl ring. The orientations of these planar regions within the asymmetric units are compared to their predicted orientations, in isolation, from DFT calculations. The aryl rings are tilted approximately 60° with respect to each other in both experimentally determined structures, as compared to 30° in the DFT results. These conformational changes result in more favorable environments for N—H...O hydrogen bonding and aryl ring π-stacking in the crystal structures. Intermolecular interactions were examined by Hirshfeld surface analysis and quantified by calculating molecular interaction energies. The results of this study demonstrate that both hydrogen bonding and dispersion are essential to the side-by-side stacking of molecular units in these crystal structures. Weaker dispersion interactions along the axial directions of the molecules reveal insight into the melting mechanisms of these crystals.

The relationship between lameness-related adaptations in equine appendicular motion and muscle activation is poorly understood and has not been studied objectively. The aim of this study was to compare muscle activity of selected fore- and hindlimb muscles, and movement of the joints they act on, between baseline and induced forelimb (iFL) and hindlimb (iHL) lameness. Three-dimensional kinematic data and surface electromyography (sEMG) data from the fore- (triceps brachii, latissimus dorsi) and hindlimbs (superficial gluteal, biceps femoris, semitendinosus) were bilaterally and synchronously collected from clinically non-lame horses (n = 8) trotting over-ground (baseline). Data collections were repeated during iFL and iHL conditions (2–3/5 AAEP), induced on separate days using a modified horseshoe. Motion asymmetry parameters and continuous joint and pro-retraction angles for each limb were calculated from kinematic data. Normalized average rectified value (ARV) and muscle activation onset, offset and activity duration were calculated from sEMG signals. Mixed model analysis and statistical parametric mapping, respectively, compared discrete and continuous variables between conditions (α= 0.05). Asymmetry parameters reflected the degree of iFL and iHL. Increased ARV occurred across muscles following iFL and iHL, except non-lame side forelimb muscles that significantly decreased following iFL. Significant, limb-specific changes in sEMG ARV, and activation timings reflected changes in joint angles and phasic shifts of the limb movement cycle following iFL and iHL. Muscular adaptations during iFL and iHL are detectable using sEMG and primarily involve increased bilateral activity and phasic activation shifts that reflect known compensatory movement patterns for reducing weightbearing on the lame limb. With further research and development, sEMG may provide a valuable diagnostic aid for quantifying the underlying neuromuscular adaptations to equine lameness, which are undetectable through human observation alone.

Locoregional therapies play an important role in the management of hepatocellular carcinoma (HCC). Percutaneous ablation is one of the most commonly employed nonsurgical methods for treating very early and early HCC. For small HCC, ablation is potentially curative and competes with surgical resection. The widespread availability and the spectrum of ablative techniques mandate uniform approach among interventional radiologists. Thus, it is desirable to have a consensus regarding various aspects of the liver ablation. This article represents a consensus document of the experts from the Indian Society of Vascular and Interventional Radiology involved in the care of patients with HCC. The statements are presented in two parts.

The ongoing SARS-CoV-2 (COVID-19) pandemic poses a major risk to health care delivery all around the world and also poses significant risks to the public and health care workers (HCWs). Surgical procedures, particularly elective procedures, have been cancelled or deferred due to increased risks associated with surgery and anesthesia both to the patient and the HCWs as well as to allocate resources to treat infected patients. This is particularly true for patients with comorbidities and cancer. As countries emerge from this pandemic, a phased return to conventional surgical procedures is being proposed, with emphasis on procedures performed in ambulatory settings and avoiding endotracheal intubation, where possible. Interventional radiological (IR) procedures offer a unique advantage in this setting, as these procedures are often performed with local or regional anesthesia with shorter hospital stay. In this brief communication, we outline the current opinion on surgical risks and guidelines and suggest areas where interventional therapies may offer an advantage to the multidisciplinary team and patient.

X-linked moesin associated immunodeficiency (X-MAID) is a primary immunodeficiency disease in which patients suffer from profound lymphopenia leading to recurrent infections. The disease is caused by a single point mutation leading to a R171W amino acid change in the protein moesin (moesinR171W). Moesin is a member of the ERM family of proteins, which reversibly link the cortical actin cytoskeleton to the plasma membrane. Here, we describe a novel mouse model with global expression of moesinR171W that recapitulates multiple facets of patient disease, including severe lymphopenia. Further analysis reveals that these mice have diminished numbers of thymocytes and bone marrow precursors. X-MAID mice also exhibit systemic inflammation that is ameliorated by elimination of mature lymphocytes through breeding to a Rag1-deficient background. The few T cells in the periphery of X-MAID mice are highly activated and have mostly lost moesinR171W expression. In contrast, single-positive (SP) thymocytes do not appear activated and retain high expression levels of moesinR171W. Analysis of ex vivo CD4 SP thymocytes reveals defects in chemotactic responses and reduced migration on integrin ligands. While chemokine signaling appears intact, CD4 SP thymocytes from X-MAID mice are unable to polarize and rearrange cytoskeletal elements. This mouse model will be a valuable tool for teasing apart the complexity of the immunodeficiency caused by moesinR171W, and will provide new insights into how the actin cortex regulates lymphocyte function.

Small surface-release dams are prevalent across North American watersheds and can alter stream flow, thermal regimes, nutrient dynamics, and sediment transport. These dams are often implicated as a cause of negative water quality impacts-including reduced dissolved oxygen (DO)-and dam removal is increasingly employed to restore natural stream processes and improve DO. Published impacts of small dams on DO vary widely across sites, and even less is known about the extent and timescale of DO recovery following removal. Therefore, we sought to quantify the effects of small dams and dam removal on DO and determine the dam, stream, and watershed characteristics driving inter-site variation in responses. We deployed continuous data loggers for 3 weeks during summer months in upstream (reference), impoundment, and downstream reaches at each of 15 dammed sites and collected equivalent data at 10 of those sites following dam removal. Prior to dam removal, most sites (60%) experienced a decrease in DO (an average of 1.15 mg/L lower) within the impoundment relative to upstream, but no consistent impacts on diel ranges or on downstream reaches. Before dam removal, 5 impacted stream reaches experienced minimum DO levels below acceptable water quality standards (

Abstract An article published in Microbiome in July 2018 uses incorrect definitions of integron integrase IntI1 and of class 1 integrons that affect the interpretation of the data.

Abstract Background Low-abundance microorganisms of the gut microbiome are often referred to as a reservoir for antibiotic resistance genes. Unfortunately, these less-abundant bacteria can be overlooked by deep shotgun sequencing. In addition, it is a challenge to associate the presence of resistance genes with their risk of acquisition by pathogens. In this study, we used liquid culture enrichment of stools to assemble the genome of lower-abundance bacteria from fecal samples. We then investigated the gene content recovered from these culture-enriched and culture-independent metagenomes in relation with their taxonomic origin, specifically antibiotic resistance genes. We finally used a pangenome approach to associate resistance genes with the core or accessory genome of Enterobacteriaceae and inferred their propensity to horizontal gene transfer. Results Using culture-enrichment approaches with stools allowed assembly of 187 bacterial species with an assembly size greater than 1 million nucleotides. Of these, 67 were found only in culture-enriched conditions, and 22 only in culture-independent microbiomes. These assembled metagenomes allowed the evaluation of the gene content of specific subcommunities of the gut microbiome. We observed that differentially distributed metabolic enzymes were associated with specific culture conditions and, for the most part, with specific taxa. Gene content differences between microbiomes, for example, antibiotic resistance, were for the most part not associated with metabolic enzymes, but with other functions. We used a pangenome approach to determine if the resistance genes found in Enterobacteriaceae, specifically E. cloacae or E. coli, were part of the core genome or of the accessory genome of this species. In our healthy volunteer cohort, we found that E. cloacae contigs harbored resistance genes that were part of the core genome of the species, while E. coli had a large accessory resistome proximal to mobile elements. Conclusion Liquid culture of stools contributed to an improved functional and comparative genomics study of less-abundant gut bacteria, specifically those associated with antibiotic resistance. Defining whether a gene is part of the core genome of a species helped in interpreting the genomes recovered from culture-independent or culture-enriched microbiomes.

Abstract Annual wintertime water‐level drawdowns are a common management strategy in recreational lakes; however, few studies have estimated their relative impact on lake littoral habitat among a set of typically co‐occurring anthropogenic stressors including lakeshore development and herbicide application. Within 21 Massachusetts, USA lakes that represented a drawdown magnitude gradient (0.07–2.26 m), we assessed depth‐specific littoral habitat (coarse wood, sediment, macrophytes) at two sites adjacent to forested or developed shorelines. Using generalized linear mixed models, we found coarse wood abundance and branching complexity was not correlated with drawdown magnitude but was primarily explained by the presence of lakeshore development. Drawdown magnitude was negatively correlated with silt cover and positively correlated with coarse substrate cover, with effects further varying by depth (0.5 m vs. 1 m). Macrophyte biomass and biovolume were negatively correlated with drawdown magnitude with effects also varying by depth for biomass. Macrophyte taxa with annual longevity strategies (e.g., Najas flexilis) and amphibious growth forms increased in biomass proportions with drawdown magnitude. Distance‐based redundancy analyses suggested macrophyte taxa composition was driven by drawdown magnitude, coarse substrate, alkalinity, water transparency, and herbicide use. These results suggest the importance of water quality and depth on macrophyte assemblage responses to winter drawdowns and the potential to develop drawdown‐tolerant macrophyte assemblages. Altogether, understanding the unique impacts of anthropogenic stressors on littoral zone habitat across heterogeneous environmental lake conditions can help minimize impacts to lake ecological integrity while maintaining recreational value.

Small, surface-release dams are ubiquitous features of the landscape that typically slow water flow and decrease canopy cover through impounded reaches, potentially increasing stream temperatures. However, reported effects of small dams on water temperature are variable, likely due to differences in landscape and dam characteristics. To quantify the range of thermal effects of small dams, we deployed continuous temperature loggers for one to four years at 30 dam sites across a range of environmental settings throughout Massachusetts (USA). Most dams (67%) warmed downstream waters, with August mean temperatures 0.20–5.25 °C higher than upstream. Downstream temperatures cooled with increased distance from the dam at 68% of sites, such that the warmest temperatures were observed closest to the dam. Where there was both a significant downstream warming effect and cooling pattern (seven sites), elevated temperatures persisted for an average of 1.31 km downstream of the dam. Dams with impoundments that caused the greatest relative widening of the stream channel and those on coldwater streams had the most warming, while streams with short dams in forested watersheds cooled most quickly downstream of the dam. Flow had a homogenizing effect on water temperatures at over half of the sites, whereby summer thermal impacts were more pronounced (e.g., more warming, faster cooling rates) under periods of lower flows. Downstream warming may reduce habitat for coldwater fishes and invertebrates, particularly where dams shift coldwater/coolwater habitat to warmwater. These results suggest that dam removal may mitigate elevated stream temperatures and increase ecosystem resilience in the face of a changing climate via restoration of critical coldwater habitats.

The ability of cells to migrate is a fundamental physiological process involved in embryonic development, tissue homeostasis, immune surveillance, and wound healing. Therefore, the mechanisms governing cellular locomotion have been under intense scrutiny over the last 50 years. One of the main tools of this scrutiny is live-cell quantitative imaging, where researchers image cells over time to study their migration and quantitatively analyze their dynamics by tracking them using the recorded images. Despite the availability of computational tools, manual tracking remains widely used among researchers due to the difficulty setting up robust automated cell tracking and large-scale analysis. Here we provide a detailed analysis pipeline illustrating how the deep learning network StarDist can be combined with the popular tracking software TrackMate to perform 2D automated cell tracking and provide fully quantitative readouts. Our proposed protocol is compatible with both fluorescent and widefield images. It only requires freely available and open-source software (ZeroCostDL4Mic and Fiji), and does not require any coding knowledge from the users, making it a versatile and powerful tool for the field. We demonstrate this pipeline's usability by automatically tracking cancer cells and T cells using fluorescent and brightfield images. Importantly, we provide, as supplementary information, a detailed step-by-step protocol to allow researchers to implement it with their images.

The ability of cells to migrate is a fundamental physiological process involved in embryonic development, tissue homeostasis, immune surveillance, and wound healing. Therefore, the mechanisms governing cellular locomotion have been under intense scrutiny over the last 50 years. One of the main tools of this scrutiny is live-cell quantitative imaging, where researchers image cells over time to study their migration and quantitatively analyze their dynamics by tracking them using the recorded images. Despite the availability of computational tools, manual tracking remains widely used among researchers due to the difficulty setting up robust automated cell tracking and large-scale analysis. Here we provide a detailed analysis pipeline illustrating how the deep learning network StarDist can be combined with the popular tracking software TrackMate to perform 2D automated cell tracking and provide fully quantitative readouts. Our proposed protocol is compatible with both fluorescent and widefield images. It only requires freely available and open-source software (ZeroCostDL4Mic and Fiji), and does not require any coding knowledge from the users, making it a versatile and powerful tool for the field. We demonstrate this pipeline's usability by automatically tracking cancer cells and T cells using fluorescent and brightfield images. Importantly, we provide, as supplementary information, a detailed step-by-step protocol to allow researchers to implement it with their images.

Smallpox and monkeypox pose severe threats to human health. Other orthopoxviruses are comparably virulent in their natural hosts, including ectromelia, the cause of mousepox. Disease severity is linked to an array of immunomodulatory proteins including the B22 family, which has homologs in all pathogenic orthopoxviruses but not attenuated vaccine strains. We demonstrate that the ectromelia B22 member, C15, is necessary and sufficient for selective inhibition of CD4+ but not CD8+ T cell activation by immunogenic peptide and superantigen. Inhibition is achieved not by down-regulation of surface MHC- II or co-stimulatory protein surface expression but rather by interference with antigen presentation. The appreciable outcome is interference with CD4+ T cell synapse formation as determined by imaging studies and lipid raft disruption. Consequently, CD4+ T cell activating stimulus shifts to uninfected antigen-presenting cells that have received antigen from infected cells. This work provides insight into the immunomodulatory strategies of orthopoxviruses by elucidating a mechanism for specific targeting of CD4+ T cell activation, reflecting the importance of this cell type in control of the virus.

The immunological synapse (IS) is a specialized structure that serves as a platform for cell-cell communication between a T cell and an antigen-presenting cell (APC). Engagement of the T cell receptor (TCR) with cognate peptide-MHC complexes on the APC activates the T cell and instructs its differentiation. Proper T cell activation also requires engagement of additional receptor-ligand pairs, which promote sustained adhesion and deliver costimulatory signals. These events are orchestrated by T cell actin dynamics, which organize IS components and facilitate their signaling. The actin network flows from the edge of the cell inward, driving the centralization of TCR microclusters and providing the force to activate the integrin LFA-1. We recently showed that engagement of LFA-1 slows actin flow, and that this affects TCR signaling. This study highlights the physical nature of the IS, and contributes to a growing appreciation in the field that mechanosensing and mechanotransduction are essential for IS function. Additionally, it is becoming clear that there are multiple types of actin structures at the IS that promote signaling in distinct ways. How the different actin structures contribute to force production and mechanotransduction is just beginning to be explored. In this Perspective, we will feature recent work from our lab and others, that collectively points toward a model in which actin dynamics drive mechanical signaling and receptor crosstalk during T cell activation.

HIV-1 Env mediates fusion of viral and target cell membranes, but it can also mediate fusion of infected (producer) and target cells, thus triggering the formation of multinucleated cells, so-called syncytia. Large, round, immobile syncytia are readily observable in cultures of HIV-1-infected T cells, but these fast growing “fusion sinks” are largely regarded as cell culture artifacts. In contrast, small HIV-1-induced syncytia were seen in the paracortex of peripheral lymph nodes and other secondary lymphoid tissue of HIV-1-positive individuals. Further, recent intravital imaging of lymph nodes in humanized mice early after their infection with HIV-1 demonstrated that a significant fraction of infected cells were highly mobile, small syncytia, suggesting that these entities contribute to virus dissemination. Here, we report that the formation of small, migratory syncytia, for which we provide further quantification in humanized mice, can be recapitulated in vitro if HIV-1-infected T cells are placed into 3D extracellular matrix (ECM) hydrogels rather than being kept in traditional suspension culture systems. Intriguingly, live-cell imaging in hydrogels revealed that these syncytia, similar to individual infected cells, can transiently interact with uninfected cells, leading to rapid virus transfer without cell-cell fusion. Infected cells were also observed to deposit large amounts of viral particles into the extracellular space. Altogether, these observations suggest the need to further evaluate the biological significance of small, T cell-based syncytia and to consider the possibility that these entities do indeed contribute to virus spread and pathogenesis.

Full T cell activation requires coordination of signals from multiple receptor–ligand pairs that interact in parallel at a specialized cell–cell contact site termed the immunological synapse (IS). Signaling at the IS is intimately associated with actin dynamics; T cell receptor (TCR) engagement induces centripetal flow of the T cell actin network, which in turn enhances the function of ligand-bound integrins by promoting conformational change. Here, we have investigated the effects of integrin engagement on actin flow, and on associated signaling events downstream of the TCR. We show that integrin engagement significantly decelerates centripetal flow of the actin network. In primary CD4+ T cells, engagement of either LFA-1 or VLA-4 by their respective ligands ICAM-1 and VCAM-1 slows actin flow. Slowing is greatest when T cells interact with low mobility integrin ligands, supporting a predominately drag-based mechanism. Using integrin ligands presented on patterned surfaces, we demonstrate that the effects of localized integrin engagement are distributed across the actin network, and that focal adhesion proteins, such as talin, vinculin, and paxillin, are recruited to sites of integrin engagement. Further analysis shows that talin and vinculin are interdependent upon one another for recruitment, and that ongoing actin flow is required. Suppression of vinculin or talin partially relieves integrin-dependent slowing of actin flow, indicating that these proteins serve as molecular clutches that couple engaged integrins to the dynamic actin network. Finally, we found that integrin-dependent slowing of actin flow is associated with reduction in tyrosine phosphorylation downstream of the TCR, and that this modulation of TCR signaling depends on expression of talin and vinculin. More generally, we found that integrin-dependent effects on actin retrograde flow were strongly correlated with effects on TCR signaling. Taken together, these studies support a model in which ligand-bound integrins engage the actin cytoskeletal network via talin and vinculin, and tune TCR signaling events by modulating actin dynamics at the IS.

Human activities create threats that have consequences for freshwater ecosystems and, in most watersheds, observed ecological responses are the result of complex interactions among multiple threats and their associated ecological alterations. Here we discuss the value of considering multiple threats in research and management, offer suggestions for filling knowledge gaps, and provide guidance for addressing the urgent management challenges posed by multiple threats in freshwater ecosystems. There is a growing literature assessing responses to multiple alterations, and we build off this background to identify three areas that require greater attention: linking observed alterations to threats, understanding when and where threats overlap, and choosing metrics that best quantify the effects of multiple threats. Advancing science in these areas will help us understand existing ecosystem conditions and predict future risk from multiple threats. Because addressing the complex issues and novel ecosystems that arise from the interaction of multiple threats in freshwater ecosystems represents a significant management challenge, and the risks of management failure include loss of biodiversity, ecological goods, and ecosystem services, we also identify actions that could improve decision-making and management outcomes. These actions include drawing insights from management of individual threats, using threat attributes (e.g., causes and spatio-temporal dynamics) to identify suitable management approaches, testing management strategies that are likely to be successful despite uncertainties about the nature of interactions among threats, avoiding unintended consequences, and maximizing conservation benefits. We also acknowledge the broadly applicable challenges of decision-making within a socio-political and economic framework, and suggest that multidisciplinary teams will be needed to innovate solutions to meet the current and future challenge of interacting threats in freshwater ecosystems.

River Run High School, located in rural west central Florida, was the site for a case study of student conflict precipitated by the wearing and display of Confederate flags on campus. Following a series of tense student encounters over Confederate and other racist symbols, a conflict resolution team was invited to intervene. Team members created a series of student dialogues involving the principal disputants. Conversations revealed the basis of the conflict emanated from misunderstandings over history, stereotypical views of different ethnic groups, and inadequate lines of communication. While acknowledging the role for creating a climate of respect among students on campus, legal precedents have established students' rights to freedom of speech and expression that protect unpopular ideas. Tension and conflict on campus over free speech can be mitigated through ongoing dialogues among diverse segments of the student population.

Human immunodeficiency virus type 1 (HIV-1) transmission takes place primarily through cell-cell contacts known as virological synapses. Formation of these transient adhesions between infected and uninfected cells can lead to transmission of viral particles followed by separation of the cells. Alternatively, the cells can fuse, thus forming a syncytium. Tetraspanins, small scaffolding proteins that are enriched in HIV-1 virions and actively recruited to viral assembly sites, have been found to negatively regulate HIV-1 Env-induced cell-cell fusion. How these transmembrane proteins inhibit membrane fusion, however, is currently not known. As a first step towards elucidating the mechanism of fusion repression by tetraspanins, e.g., CD9 and CD63, we sought to identify the stage of the fusion process during which they operate. Using a chemical epistasis approach, four fusion inhibitors were employed in tandem with CD9 overexpression. Cells overexpressing CD9 were found to be sensitized to inhibitors targeting the pre-hairpin and hemifusion intermediates, while they were desensitized to an inhibitor of the pore expansion stage. Together with the results of a microscopy-based dye transfer assay, which revealed CD9- and CD63-induced hemifusion arrest, our investigations strongly suggest that tetraspanins block HIV-1-induced cell-cell fusion at the transition from hemifusion to pore opening.