Electron tomography visualization of HIV-1 virions trapped by fusion inhibitors to host cells in infected tissues.

HIV-1 delivers its genetic material to infect a cell after fusion of the viral and host cell membranes, which takes place after the viral envelope (Env) binds host receptor and co-receptor proteins. Binding of host receptor CD4 to Env results in conformational changes that allow interaction with a host co-receptor (CCR5 or CXCR4). Further conformational rearrangements result in an elongated pre-hairpin intermediate structure in which Env is anchored to the viral membrane by its transmembrane region and to the host cell membrane by its fusion peptide. Although budding virions can be readily imaged by electron tomography (ET) of HIV-1-infected tissues and cultured cells, virions that are fusing (attached to host cells via pre-hairpin intermediates) are not normally visualized, perhaps because the process of membrane fusion is too fast to capture by EM. To image virions during fusion, we used fusion inhibitors to prevent downstream conformational changes in Env that lead to membrane fusion, thereby trapping HIV-1 virions linked to target cells by prehairpin intermediates. ET of HIV-1 pseudovirions bound to CD4+/CCR5+ TZM-bl cells revealed presumptive pre-hairpin intermediates as 2-4 narrow spokes linking a virion to the cell surface. To extend these results to a more physiological setting, we used ET to image tissues and organs derived from humanized bone marrow, liver, thymus (BLT) mice infected with HIV-1 and then treated with CPT31, a high-affinity D-peptide fusion inhibitor linked to cholesterol. Trapped HIV-1 virions were found in all tissues studied (small intestine, mesenteric lymph nodes, spleen, and bone marrow), and spokes representing pre-hairpin intermediates linking trapped virions to cell surfaces were similar in structure and number to those seen in the previous pseudovirus and cultured cell ET study.

The genomic and cellular basis of biosynthetic innovation in rove beetles.

How evolution at the cellular level potentiates macroevolutionary change is central to understanding biological diversification. The >66,000 rove beetle species (Staphylinidae) form the largest metazoan family. Combining genomic and cell type transcriptomic insights spanning the largest clade, Aleocharinae, we retrace evolution of two cell types comprising a defensive gland-a putative catalyst behind staphylinid megadiversity. We identify molecular evolutionary steps leading to benzoquinone production by one cell type via a mechanism convergent with plant toxin release systems, and synthesis by the second cell type of a solvent that weaponizes the total secretion. This cooperative system has been conserved since the Early Cretaceous as Aleocharinae radiated into tens of thousands of lineages. Reprogramming each cell type yielded biochemical novelties enabling ecological specialization-most dramatically in symbionts that infiltrate social insect colonies via host-manipulating secretions. Our findings uncover cell type evolutionary processes underlying the origin and evolvability of a beetle chemical innovation.

9-fold symmetry is not essential for centriole elongation and formation of new centriole-like structures.

As daughter centrioles assemble during G2, they recruit conserved Ana3/RTTN followed by its partner Rcd4/PPP1R35. Together, this contributes to the subsequent recruitment of Ana1/CEP295, required for the centriole's conversion to a centrosome. Here, we show that Rcd4/PPP1R35 is also required to maintain 9-fold centriole symmetry in the Drosophila male germline; its absence causes microtubule triplets to disperse into a reduced number of doublet or singlet microtubules. rcd4-null mutant spermatocytes display skinny centrioles that elongate normally and localize centriolar components correctly. Mutant spermatocytes also have centrioles of normal girth that splay at their proximal ends when induced to elongate by Ana1 overexpression. Skinny and splayed spermatid centrioles can still recruit a proximal centriole-like (PCL) structure marking a capability to initiate features of centriole duplication in developing sperm. Thus, stable 9-fold symmetry of microtubule triplets is not essential for centriole growth, correct longitudinal association of centriole components, and aspects of centriole duplication.

Variant mutation in SARS-CoV-2 nucleocapsid enhances viral infection via altered genomic encapsidation.

The evolution of SARS-CoV-2 variants and their respective phenotypes represents an important set of tools to understand basic coronavirus biology as well as the public health implications of individual mutations in variants of concern. While mutations outside of Spike are not well studied, the entire viral genome is undergoing evolutionary selection, particularly the central disordered linker region of the nucleocapsid (N) protein. Here, we identify a mutation (G215C), characteristic of the Delta variant, that introduces a novel cysteine into this linker domain, which results in the formation of a disulfide bond and a stable N-N dimer. Using reverse genetics, we determined that this cysteine residue is necessary and sufficient for stable dimer formation in a WA1 SARS-CoV-2 background, where it results in significantly increased viral growth both in vitro and in vivo. Finally, we demonstrate that the N:G215C virus packages more nucleocapsid per virion and that individual virions are larger, with elongated morphologies.

The fatty liver disease-causing protein PNPLA3-I148M alters lipid droplet-Golgi dynamics.

Nonalcoholic fatty liver disease, recently renamed metabolic dysfunction-associated steatotic liver disease (MASLD), is a progressive metabolic disorder that begins with aberrant triglyceride accumulation in the liver and can lead to cirrhosis and cancer. A common variant in the gene PNPLA3, encoding the protein PNPLA3-I148M, is the strongest known genetic risk factor for MASLD. Despite its discovery 20 y ago, the function of PNPLA3, and now the role of PNPLA3-I148M, remain unclear. In this study, we sought to dissect the biogenesis of PNPLA3 and PNPLA3-I148M and characterize changes induced by endogenous expression of the disease-causing variant. Contrary to bioinformatic predictions and prior studies with overexpressed proteins, we demonstrate here that PNPLA3 and PNPLA3-I148M are not endoplasmic reticulum-resident transmembrane proteins. To identify their intracellular associations, we generated a paired set of isogenic human hepatoma cells expressing PNPLA3 and PNPLA3-I148M at endogenous levels. Both proteins were enriched in lipid droplet, Golgi, and endosomal fractions. Purified PNPLA3 and PNPLA3-I148M proteins associated with phosphoinositides commonly found in these compartments. Despite a similar fractionation pattern as the wild-type variant, PNPLA3-I148M induced morphological changes in the Golgi apparatus, including increased lipid droplet-Golgi contact sites, which were also observed in I148M-expressing primary human patient hepatocytes. In addition to lipid droplet accumulation, PNPLA3-I148M expression caused significant proteomic and transcriptomic changes that resembled all stages of liver disease. Cumulatively, we validate an endogenous human cellular system for investigating PNPLA3-I148M biology and identify the Golgi apparatus as a central hub of PNPLA3-I148M-driven cellular change.

The fatty liver disease-causing protein PNPLA3-I148M alters lipid droplet-Golgi dynamics.

Non-alcoholic fatty liver disease (NAFLD), recently renamed metabolic dysfunction-associated steatotic liver disease (MASLD), is a progressive metabolic disorder that begins with aberrant triglyceride accumulation in the liver and can lead to cirrhosis and cancer. A common variant in the gene PNPLA3, encoding the protein PNPLA3-I148M, is the strongest known genetic risk factor for MASLD to date. Despite its discovery twenty years ago, the function of PNPLA3, and now the role of PNPLA3-I148M, remain unclear. In this study, we sought to dissect the biogenesis of PNPLA3 and PNPLA3-I148M and characterize changes induced by endogenous expression of the disease-causing variant. Contrary to bioinformatic predictions and prior studies with overexpressed proteins, we demonstrate here that PNPLA3 and PNPLA3-I148M are not endoplasmic reticulum-resident transmembrane proteins. To identify their intracellular associations, we generated a paired set of isogenic human hepatoma cells expressing PNPLA3 and PNPLA3-I148M at endogenous levels. Both proteins were enriched in lipid droplet, Golgi, and endosomal fractions. Purified PNPLA3 and PNPLA3-I148M proteins associated with phosphoinositides commonly found in these compartments. Despite a similar fractionation pattern as the wild-type variant, PNPLA3-I148M induced morphological changes in the Golgi apparatus, including increased lipid droplet-Golgi contact sites, which were also observed in I148M-expressing primary human patient hepatocytes. In addition to lipid droplet accumulation, PNPLA3-I148M expression caused significant proteomic and transcriptomic changes that resembled all stages of liver disease. Cumulatively, we validate an endogenous human cellular system for investigating PNPLA3-I148M biology and identify the Golgi apparatus as a central hub of PNPLA3-I148M-driven cellular change.

The genomic and cellular basis of biosynthetic innovation in rove beetles.

How evolution at the cellular level potentiates change at the macroevolutionary level is a major question in evolutionary biology. With >66,000 described species, rove beetles (Staphylinidae) comprise the largest metazoan family. Their exceptional radiation has been coupled to pervasive biosynthetic innovation whereby numerous lineages bear defensive glands with diverse chemistries. Here, we combine comparative genomic and single-cell transcriptomic data from across the largest rove beetle clade, Aleocharinae. We retrace the functional evolution of two novel secretory cell types that together comprise the tergal gland-a putative catalyst behind Aleocharinae's megadiversity. We identify key genomic contingencies that were critical to the assembly of each cell type and their organ-level partnership in manufacturing the beetle's defensive secretion. This process hinged on evolving a mechanism for regulated production of noxious benzoquinones that appears convergent with plant toxin release systems, and synthesis of an effective benzoquinone solvent that weaponized the total secretion. We show that this cooperative biosynthetic system arose at the Jurassic-Cretaceous boundary, and that following its establishment, both cell types underwent ∼150 million years of stasis, their chemistry and core molecular architecture maintained almost clade-wide as Aleocharinae radiated globally into tens of thousands of lineages. Despite this deep conservation, we show that the two cell types have acted as substrates for the emergence of adaptive, biochemical novelties-most dramatically in symbiotic lineages that have infiltrated social insect colonies and produce host behavior-manipulating secretions. Our findings uncover genomic and cell type evolutionary processes underlying the origin, functional conservation and evolvability of a chemical innovation in beetles.

An extracellular vesicle targeting ligand that binds to Arc proteins and facilitates Arc transport in vivo.

Communication between distant cells can be mediated by extracellular vesicles (EVs) that deliver proteins and RNAs to recipient cells. Little is known about how EVs are targeted to specific cell types. Here, we identify the Drosophila cell-surface protein Stranded at second (Sas) as a targeting ligand for EVs. Full-length Sas is present in EV preparations from transfected Drosophila Schneider 2 (S2) cells. Sas is a binding partner for the Ptp10D receptor tyrosine phosphatase, and Sas-bearing EVs preferentially target to cells expressing Ptp10D. We used co-immunoprecipitation and peptide binding to show that the cytoplasmic domain (ICD) of Sas binds to dArc1 and mammalian Arc. dArc1 and Arc are related to retrotransposon Gag proteins. They form virus-like capsids which encapsulate Arc and other mRNAs and are transported between cells via EVs. The Sas ICD contains a motif required for dArc1 binding that is shared by the mammalian and Drosophila amyloid precursor protein (APP) orthologs, and the APP ICD also binds to mammalian Arc. Sas facilitates delivery of dArc1 capsids bearing dArc1 mRNA into distant Ptp10D-expressing recipient cells in vivo.

Perilipin-2 promotes lipid droplet-plasma membrane interactions that facilitate apocrine lipid secretion in secretory epithelial cells of the mouse mammary gland.

Secretory epithelial cells (sMEC) in mammary glands of lactating animals secrete lipids by a novel apocrine mechanism in which cytoplasmic lipid droplets (LD) contact and are enveloped by elements of the apical plasma membrane (APM) before being released into the lumen of the gland as membrane bound structures. The molecular properties of LD-APM contacts and the mechanisms regulating LD membrane envelopment and secretion are not fully understood. Perilipin-2 (Plin2) is a constitutive LD protein that has been proposed to tether LD to the APM through formation of a complex with the transmembrane protein, butyrophilin1a1 (BTN) and the redox enzyme, xanthine oxidoreductase (XOR). Using mice lacking Plin2 and physiological inhibition of apocrine lipid secretion, we demonstrate that LD-APM contact and envelopment are mechanistically distinct steps that they are differentially regulated by Plin2 and independent of LD secretion. We find that Plin2 is not required for formation of LD-APM contacts. However, it increases the percentage of LD that contact the APM and mediates enlargement of the LD-APM contact zone as LD undergo membrane envelopment. The effects of Plin2 LD-APM interactions are associated with increased abundances of BTN, XOR and Cidea, which are implicated as mediators of LD-APM contact formation, on membranes surrounding secreted LD, and with promotion of glycocalyx remodeling at LD-APM contact sites. We propose that Plin2 does not directly mediate contact between LD and the APM but acts by enhancing molecular interactions that stabilize LD-APM contacts and govern membrane envelopment of LD during apocrine lipid secretion. Plin2 does not appear to significantly affect the lipid content of milk in fully lactating animals, but it does increase lipid secretion at the onset of lactation in primaparous dams, which suggest a role in facilitating apocrine lipid secretion in sMEC during their initial transition to a secretory phenotype.

Microbiota imbalance induced by dietary sugar disrupts immune-mediated protection from metabolic syndrome.

How intestinal microbes regulate metabolic syndrome is incompletely understood. We show that intestinal microbiota protects against development of obesity, metabolic syndrome, and pre-diabetic phenotypes by inducing commensal-specific Th17 cells. High-fat, high-sugar diet promoted metabolic disease by depleting Th17-inducing microbes, and recovery of commensal Th17 cells restored protection. Microbiota-induced Th17 cells afforded protection by regulating lipid absorption across intestinal epithelium in an IL-17-dependent manner. Diet-induced loss of protective Th17 cells was mediated by the presence of sugar. Eliminating sugar from high-fat diets protected mice from obesity and metabolic syndrome in a manner dependent on commensal-specific Th17 cells. Sugar and ILC3 promoted outgrowth of Faecalibaculum rodentium that displaced Th17-inducing microbiota. These results define dietary and microbiota factors posing risk for metabolic syndrome. They also define a microbiota-dependent mechanism for immuno-pathogenicity of dietary sugar and highlight an elaborate interaction between diet, microbiota, and intestinal immunity in regulation of metabolic disorders.

A gut-derived metabolite alters brain activity and anxiety behaviour in mice.

Integration of sensory and molecular inputs from the environment shapes animal behaviour. A major site of exposure to environmental molecules is the gastrointestinal tract, in which dietary components are chemically transformed by the microbiota1 and gut-derived metabolites are disseminated to all organs, including the brain2. In mice, the gut microbiota impacts behaviour3, modulates neurotransmitter production in the gut and brain4,5, and influences brain development and myelination patterns6,7. The mechanisms that mediate the gut-brain interactions remain poorly defined, although they broadly involve humoral or neuronal connections. We previously reported that the levels of the microbial metabolite 4-ethylphenyl sulfate (4EPS) were increased in a mouse model of atypical neurodevelopment8. Here we identified biosynthetic genes from the gut microbiome that mediate the conversion of dietary tyrosine to 4-ethylphenol (4EP), and bioengineered gut bacteria to selectively produce 4EPS in mice. 4EPS entered the brain and was associated with changes in region-specific activity and functional connectivity. Gene expression signatures revealed altered oligodendrocyte function in the brain, and 4EPS impaired oligodendrocyte maturation in mice and decreased oligodendrocyte-neuron interactions in ex vivo brain cultures. Mice colonized with 4EP-producing bacteria exhibited reduced myelination of neuronal axons. Altered myelination dynamics in the brain have been associated with behavioural outcomes7,9-14. Accordingly, we observed that mice exposed to 4EPS displayed anxiety-like behaviours, and pharmacological treatments that promote oligodendrocyte differentiation prevented the behavioural effects of 4EPS. These findings reveal that a gut-derived molecule influences complex behaviours in mice through effects on oligodendrocyte function and myelin patterning in the brain.

Severe Acute Respiratory Syndrome Coronavirus 2 Viremia Is Associated With Coronavirus Disease 2019 Severity and Predicts Clinical Outcomes.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral RNA (vRNA) is detected in the bloodstream of some patients with coronavirus disease 2019 (COVID-19), but it is not clear whether this RNAemia reflects viremia (ie, virus particles) and how it relates to host immune responses and outcomes. METHODS: SARS-CoV-2 vRNA was quantified in plasma samples from observational cohorts of 51 COVID-19 patients including 9 outpatients, 19 hospitalized (non-intensive care unit [ICU]), and 23 ICU patients. vRNA levels were compared with cross-sectional indices of COVID-19 severity and prospective clinical outcomes. We used multiple imaging methods to visualize virions in plasma. RESULTS: SARS-CoV-2 vRNA was detected in plasma of 100%, 52.6%, and 11.1% of ICU, non-ICU, and outpatients, respectively. Virions were detected in plasma pellets using electron tomography and immunostaining. Plasma vRNA levels were significantly higher in ICU > non-ICU > outpatients (P < .0001); for inpatients, plasma vRNA levels were strongly associated with higher World Health Organization (WHO) score at admission (P = .01), maximum WHO score (P = .002), and discharge disposition (P = .004). A plasma vRNA level >6000 copies/mL was strongly associated with mortality (hazard ratio, 10.7). Levels of vRNA were significantly associated with several inflammatory biomarkers (P < .01) but not with plasma neutralizing antibody titers (P = .8). CONCLUSIONS: Visualization of virus particles in plasma indicates that SARS-CoV-2 RNAemia is due, at least in part, to viremia. The levels of SARS-CoV-2 RNAemia correlate strongly with disease severity, patient outcome, and specific inflammatory biomarkers but not with neutralizing antibody titers.

Fis1 ablation in the male germline disrupts mitochondrial morphology and mitophagy, and arrests spermatid maturation.

Male germline development involves choreographed changes to mitochondrial number, morphology and organization. Mitochondrial reorganization during spermatogenesis was recently shown to require mitochondrial fusion and fission. Mitophagy, the autophagic degradation of mitochondria, is another mechanism for controlling mitochondrial number and physiology, but its role during spermatogenesis is largely unknown. During post-meiotic spermatid development, restructuring of the mitochondrial network results in packing of mitochondria into a tight array in the sperm midpiece to fuel motility. Here, we show that disruption of mouse Fis1 in the male germline results in early spermatid arrest that is associated with increased mitochondrial content. Mutant spermatids coalesce into multinucleated giant cells that accumulate mitochondria of aberrant ultrastructure and numerous mitophagic and autophagic intermediates, suggesting a defect in mitophagy. We conclude that Fis1 regulates mitochondrial morphology and turnover to promote spermatid maturation.

Live imaging of SARS-CoV-2 infection in mice reveals that neutralizing antibodies require Fc function for optimal efficacy.

Neutralizing antibodies (NAbs) are effective in treating COVID-19, but the mechanism of immune protection is not fully understood. Here, we applied live bioluminescence imaging (BLI) to monitor the real-time effects of NAb treatment during prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. Real-time imaging revealed that the virus spread sequentially from the nasal cavity to the lungs in mice and thereafter systemically to various organs including the brain, culminating in death. Highly potent NAbs from a COVID-19 convalescent subject prevented, and also effectively resolved, established infection when administered within three days. In addition to direct neutralization, depletion studies indicated that Fc effector interactions of NAbs with monocytes, neutrophils, and natural killer cells were required to effectively dampen inflammatory responses and limit immunopathology. Our study highlights that both Fab and Fc effector functions of NAbs are essential for optimal in vivo efficacy against SARS-CoV-2.

In vivo imaging of retrovirus infection reveals a role for Siglec-1/CD169 in multiple routes of transmission.

Early events in retrovirus transmission are determined by interactions between incoming viruses and frontline cells near entry sites. Despite their importance for retroviral pathogenesis, very little is known about these events. We developed a bioluminescence imaging (BLI)-guided multiscale imaging approach to study these events in vivo. Engineered murine leukemia reporter viruses allowed us to monitor individual stages of retrovirus life cycle including virus particle flow, virus entry into cells, infection and spread for retroorbital, subcutaneous, and oral routes. BLI permitted temporal tracking of orally administered retroviruses along the gastrointestinal tract as they traversed the lumen through Peyer's patches to reach the draining mesenteric sac. Importantly, capture and acquisition of lymph-, blood-, and milk-borne retroviruses spanning three routes was promoted by a common host factor, the I-type lectin CD169, expressed on sentinel macrophages. These results highlight how retroviruses co-opt the immune surveillance function of tissue-resident sentinel macrophages for establishing infection.

The Evolution of Interdependence in a Four-Way Mealybug Symbiosis.

Mealybugs are insects that maintain intracellular bacterial symbionts to supplement their nutrient-poor plant sap diets. Some mealybugs have a single betaproteobacterial endosymbiont, a Candidatus Tremblaya species (hereafter Tremblaya) that alone provides the insect with its required nutrients. Other mealybugs have two nutritional endosymbionts that together provision these same nutrients, where Tremblaya has gained a gammaproteobacterial partner that resides in its cytoplasm. Previous work had established that Pseudococcus longispinus mealybugs maintain not one but two species of gammaproteobacterial endosymbionts along with Tremblaya. Preliminary genomic analyses suggested that these two gammaproteobacterial endosymbionts have large genomes with features consistent with a relatively recent origin as insect endosymbionts, but the patterns of genomic complementarity between members of the symbiosis and their relative cellular locations were unknown. Here, using long-read sequencing and various types of microscopy, we show that the two gammaproteobacterial symbionts of P. longispinus are mixed together within Tremblaya cells, and that their genomes are somewhat reduced in size compared with their closest nonendosymbiotic relatives. Both gammaproteobacterial genomes contain thousands of pseudogenes, consistent with a relatively recent shift from a free-living to an endosymbiotic lifestyle. Biosynthetic pathways of key metabolites are partitioned in complex interdependent patterns among the two gammaproteobacterial genomes, the Tremblaya genome, and horizontally acquired bacterial genes that are encoded on the mealybug nuclear genome. Although these two gammaproteobacterial endosymbionts have been acquired recently in evolutionary time, they have already evolved codependencies with each other, Tremblaya, and their insect host.

Live Imaging of SARS-CoV-2 Infection in Mice Reveals Neutralizing Antibodies Require Fc Function for Optimal Efficacy.

Neutralizing antibodies (NAbs) are effective in treating COVID-19 but the mechanism of immune protection is not fully understood. Here, we applied live bioluminescence imaging (BLI) to monitor the real-time effects of NAb treatment in prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. We could visualize virus spread sequentially from the nasal cavity to the lungs and thereafter systemically to various organs including the brain, which culminated in death. Highly potent NAbs from a COVID-19 convalescent subject prevented, and also effectively resolved, established infection when administered within three days. In addition to direct Fab-mediated neutralization, Fc effector interactions of NAbs with monocytes, neutrophils and natural killer cells were required to effectively dampen inflammatory responses and limit immunopathology. Our study highlights that both Fab and Fc effector functions of NAbs are essential for optimal in vivo efficacy against SARS-CoV-2.

Intestinal Host Response to SARS-CoV-2 Infection and COVID-19 Outcomes in Patients With Gastrointestinal Symptoms.

BACKGROUND & AIMS: Given that gastrointestinal (GI) symptoms are a prominent extrapulmonary manifestation of COVID-19, we investigated intestinal infection with SARS-CoV-2, its effect on pathogenesis, and clinical significance. METHODS: Human intestinal biopsy tissues were obtained from patients with COVID-19 (n = 19) and uninfected control individuals (n = 10) for microscopic examination, cytometry by time of flight analyses, and RNA sequencing. Additionally, disease severity and mortality were examined in patients with and without GI symptoms in 2 large, independent cohorts of hospitalized patients in the United States (N = 634) and Europe (N = 287) using multivariate logistic regressions. RESULTS: COVID-19 case patients and control individuals in the biopsy cohort were comparable for age, sex, rates of hospitalization, and relevant comorbid conditions. SARS-CoV-2 was detected in small intestinal epithelial cells by immunofluorescence staining or electron microscopy in 15 of 17 patients studied. High-dimensional analyses of GI tissues showed low levels of inflammation, including down-regulation of key inflammatory genes including IFNG, CXCL8, CXCL2, and IL1B and reduced frequencies of proinflammatory dendritic cells compared with control individuals. Consistent with these findings, we found a significant reduction in disease severity and mortality in patients presenting with GI symptoms that was independent of sex, age, and comorbid illnesses and despite similar nasopharyngeal SARS-CoV-2 viral loads. Furthermore, there was reduced levels of key inflammatory proteins in circulation in patients with GI symptoms. CONCLUSIONS: These data highlight the absence of a proinflammatory response in the GI tract despite detection of SARS-CoV-2. In parallel, reduced mortality in patients with COVID-19 presenting with GI symptoms was observed. A potential role of the GI tract in attenuating SARS-CoV-2-associated inflammation needs to be further examined.

Intractable Coronavirus Disease 2019 (COVID-19) and Prolonged Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Replication in a Chimeric Antigen Receptor-Modified T-Cell Therapy Recipient: A Case Study.

A chimeric antigen receptor-modified T-cell therapy recipient developed severe coronavirus disease 2019, intractable RNAemia, and viral replication lasting >2 months. Premortem endotracheal aspirate contained >2 × 1010 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA copies/mL and infectious virus. Deep sequencing revealed multiple sequence variants consistent with intrahost virus evolution. SARS-CoV-2 humoral and cell-mediated immunity were minimal. Prolonged transmission from immunosuppressed patients is possible.

Mitochondrial fission factor (Mff) is required for organization of the mitochondrial sheath in spermatids.

BACKGROUND: Mitochondrial fission counterbalances fusion to maintain organelle morphology, but its role during development remains poorly characterized. Mammalian spermatogenesis is a complex developmental process involving several drastic changes to mitochondrial shape and organization. Mitochondria are generally small and spherical in spermatogonia, elongate during meiosis, and fragment in haploid round spermatids. Near the end of spermatid maturation, small mitochondrial spheres line the axoneme, elongate, and tightly wrap around the midpiece to form the mitochondrial sheath, which is critical for fueling flagellar movements. It remains unclear how these changes in mitochondrial morphology are regulated and how they affect sperm development. METHODS: We used genetic ablation of Mff (mitochondrial fission factor) in mice to investigate the role of mitochondrial fission during mammalian spermatogenesis. RESULTS: Our analysis indicates that Mff is required for mitochondrial fragmentation in haploid round spermatids and for organizing mitochondria in the midpiece in elongating spermatids. In Mff mutant mice, round spermatids have aberrantly elongated mitochondria that often show central constrictions, suggestive of failed fission events. In elongating spermatids and spermatozoa, mitochondrial sheaths are disjointed, containing swollen mitochondria with large gaps between organelles. These mitochondrial abnormalities in Mff mutant sperm are associated with reduced respiratory chain Complex IV activity, aberrant sperm morphology and motility, and reduced fertility. CONCLUSIONS: Mff is required for organization of the mitochondrial sheath in mouse sperm. GENERAL SIGNIFICANCE: Mitochondrial fission plays an important role in regulating mitochondrial organization during a complex developmental process.