<p>by Fuchun Zhou, Sivakumar Periasamy, Nathaniel D. Jackson, Wan Sze Cheng, Ruben Soto Acosta, Aarti Tripathi, Kritika Kedarinath, Philipp A. Ilinykh, Chengjin Ye, Shailendra Chauhan, German Nudelman, Elena Zaslavsky, Haiping Hao, Steven G. Widen, Luis Martinez-Sobrido, Stuart C. Sealfon, Alexander Bukreyev</p>
Several SARS-CoV-2 proteins have been shown to counteract the host innate immune response, mostly using in vitro protein expression, which may not fully reflect their role in the context of viral infection. In addition, while each viral protein was characterized in a different experimental system, its relative contribution to immunosuppression remains unclear. Here we used a SARS-CoV-2 bacterial artificial chromosome with <i>en passant</i> mutagenesis to recover a panel of 12 infectious recombinant SARS-CoV-2 viruses, each with mutations in either NSP1, NSP2, NSP3, NSP6, NSP12, NSP13, NSP14, NSP15, NSP16, ORF3a, ORF6, or ORF8. We used the interferon-stimulated response element (ISRE)-driven luciferase assay in 293T-ACE2/TMPRSS2 cells to test the panel, demonstrating that mutations in many proteins, especially in NSP1 and NSP15, increased the type I interferon response relative to the parental wild-type virus. RNA-seq analysis of mutant-virus infected Calu-3 cells showed that the mutations in NSP1 or NSP15 lead to higher expression of multiple genes involved in innate immune response, cytokine-mediated signaling, and regulation of lymphocyte proliferation. Furthermore, mutations in either NSP1 or NSP15 resulted in a greater maturation of human monocyte-derived dendritic cells in vitro. Infection of K18 hACE2 transgenic mice with either NSP1 or NSP15 mutated viruses demonstrated attenuated respiratory tract replication. Analysis of lung immune cells from infected mice by single-cell RNA-seq identified 15 populations of major myeloid and lymphoid cells with changes in the pattern of their activation associated with viral infection. The effects of mutations …
Science Journals
<p>by Siqi Liu, Flávio Silva Costa, Dario Riccardo Valenzano</p>
Host-associated microbiomes are compositionally stable across most of the life span, yet undergo consistent and marked deterioration during aging, a phenomenon linked to metabolic dysfunction and disease. What drives this late-life collapse remains poorly understood, in part because the mechanisms by which hosts actively construct and maintain the microbial niche during adulthood remain incompletely characterized. This Unsolved Mystery integrates evidence from immunology and ecosystem ecology to investigate the role of immunosenescence in age-associated dysbiosis, raising the possibility of interventions that restore immune surveillance capacity alongside ecologically informed microbiome management, rather than targeting community composition in isolation.
Ciliogenesis is a highly ordered process that requires membrane trafficking, fusion, and maturation. In this study, we investigated EXOC6A, a component of the exocyst complex known for secretory vesicle trafficking and fusion, and found that it interacts with myosin-Va (Myo-Va) during ciliogenesis. EXOC6A colocalizes with Myo-Va at various stages of ciliogenesis, including preciliary vesicles, ciliary vesicles (CVs), and ciliary sheath membrane during ciliogenesis. We found that EXOC6A vesicles are actively recruited, integrated, and exit from the CVs and the ciliary sheath, implying that EXOC6A vesicles may facilitate continuous cilia membrane remodeling during ciliogenesis. Importantly, EXOC6A knockout impairs ciliogenesis, arresting most cells at the CV stage and preventing recruitment of NPHP and MKS module components to the transition zone. Furthermore, EXOC6A vesicles are transported to the mother centriole via a dynein-, microtubule-, and actin-dependent mechanism. Our results suggest that EXOC6A functions in both early and late stages of ciliogenesis, and is involved in orchestrating vesicle dynamics, cilia membrane remodeling, and formation.
The assembly of tau into amyloid filaments is associated with more than 20 neurodegenerative diseases, collectively termed tauopathies. Electron cryo-microscopy (cryo-EM) structures of brain-derived tau filaments revealed that specific structures define different diseases, triggering a quest for the development of experimental model systems that replicate the structures of disease. Here, we describe 12 phosphomimetic serine/threonine-to-aspartate mutations in tau, which we term PAD12, that collectively induce the in vitro assembly of full-length three-repeat tau into filaments with the same structure as paired helical filaments extracted from the brains of individuals with Alzheimer’s disease. Solution-state nuclear magnetic resonance spectroscopy suggests that phosphomimetic mutations in the carboxy-terminal domain of tau may facilitate filament formation by disrupting an intramolecular interaction between two IVYK motifs. PAD12 tau can be used for both nucleation-dependent and multiple rounds of seeded assembly in vitro, as well as for the seeding of tau biosensor cells. PAD12 tau can be assembled into paired helical filaments under various shaking conditions, with the resulting filaments being stable for extended periods of time. They can be labelled with fluorophores and biotin. Tau filaments extracted from the brains of individuals with Alzheimer’s disease have been known to be made of hyperphosphorylated and abnormally phosphorylated full-length tau, but it was not known if the presence of this post-translational modification is more than a mere correlation. Our findings suggest that hyperphosphorylation of tau may be sufficient for the formation of the Alzheimer tau fold. PAD12 tau will be a useful tool for the study of molecular mechanisms of neurodegeneration.
Electrical synapses containing Connexin 36 (Cx36) represent the main means for direct electrical communication among neurons in the mammalian nervous system. However, little is known about the protein complexes that constitute these synapses. In the present study, we applied different BioID strategies to screen the interactomes of Connexin 36 and its zebrafish orthologue Cx35.1 in retinal neurons. For in vivo proximity labeling in mice, we took advantage of the Cx36-EGFP strain and expressed a GFP-nanobody-TurboID fusion construct selectively in AII amacrine cells. For in vivo BioID in zebrafish, we generated a transgenic line expressing a Cx35.1-TurboID fusion under control of the <i>Cx35.1</i> promoter. Both strategies allowed us to capture a plethora of molecules that were associated with electrical synapses and showed a high degree of evolutionary conservation in the proteomes of both species. Besides known interactors of Cx36 such as ZO-1 and ZO-2, we have identified more than 50 new proteins, such as scaffold proteins, adhesion molecules, and regulators of the cytoskeleton. Moreover, we determined the subcellular localization of these proteins in mouse retina and tested potential binding interactions with Cx36. Among these new interactors, we identified signal-induced proliferation associated 1 like 3 (Sipa1l3), a protein that has been implicated in cell junction formation and cell polarity, as a new scaffold of electrical synapses. Interestingly, Sipa1l3 was able to interact with ZO-1, ZO-2, and Cx36, suggesting a pivotal role in electrical synapse function. In summary, our study provides the first detailed view of the electrical synapse proteome in retinal neurons, which is likely to apply to electrical synapses elsewhere.
Cognitive dysfunction often co-occurs with psychopathology. Advances in neuroimaging and machine learning have led to neural indicators that predict individual differences in cognition with reasonable performance. We examined whether these indicators explain the relationship between cognition and mental health in the UK Biobank (<i>n</i>>14,000). Using machine learning, we quantified the covariation between cognition and 133 mental health indices and derived neural indicators of cognition from 72 neuroimaging phenotypes across diffusion-weighted MRI (dwMRI), resting-state functional MRI (rsMRI), and structural MRI (sMRI). With commonality analyses, we investigated how much of the cognition–mental health covariation is captured by each indicator and neural indicators combined within and across MRI modalities. The predictive association between mental health and cognition was at <i>r</i>=0.3. Neuroimaging captured 2.1 to 25.8% of the cognition-mental health covariation. Combining phenotypes within modalities improved the explanation to 25.5% for dwMRI, 29.8% for rsMRI, and 31.6% for sMRI, and combining them across modalities enhanced the explanation to 48%. We present an integrated approach to derive multimodal MRI markers of cognition that can be transdiagnostically linked to psychopathology, demonstrating that the predictive ability of neural indicators extends beyond the prediction of cognition itself, enabling us to capture cognition-mental health covariation.
MORC2 is a chromatin-associated ATPase essential for transcriptional silencing and genome stability, yet the biophysical principles governing its regulatory activity remain elusive. Here, we demonstrate that full-length MORC2 undergoes biomolecular condensation to form dynamic nuclear assemblies, a process fundamentally required for its repressor function. Endogenous MORC2 forms discrete, dynamic condensates in neurons from <i>Morc2a<sup>EGFP</sup></i> chimeric mice, supporting the physiological relevance of these assemblies in vivo. Mechanistically, a 3.1 Å crystal structure of coiled-coil 3 (CC3) identifies a dimeric scaffold that serves as a structural hub, while multivalent ‘sticker’ interactions between an intrinsically disordered region (IDR) and a newly defined IDR-binding domain (IBD) drive condensation. We show that DNA acts as a molecular scaffold that triggers MORC2 condensation, which in turn allosterically stimulates its ATPase activity. Critically, by employing a ‘killswitch’ strategy to decouple assembly from internal fluidity, we reveal that only dynamic MORC2 condensates, not static aggregates or condensation-deficient mutants, can restore transcriptional regulation in <i>MORC2</i>-knockout cells. Furthermore, pathogenic variants linked to CMT2Z and SMA differentially perturb these material properties and enzymatic turnover, providing a mechanistic link between condensate dysregulation and human neuropathies. Together, our findings establish a DNA-templated condensation mechanism for MORC2 and provide a molecular framework for understanding how the material state of chromatin-associated machinery dictates gene regulation and disease pathogenesis.
In this narrative medicine essay, an obstetrician discusses the way in which it is not only the joy of patients, but also the grief, that keeps her going.
To the Editor In the DESIGNATION randomized clinical trial, individualized driving pressure–guided high PEEP with recruitment maneuvers did not reduce postoperative pulmonary complications compared with standard low PEEP in adults at increased risk for postoperative pulmonary complications (Assess Respiratory Risk in Surgical Patients in Catalonia [ARISCAT] score ≥26) undergoing open abdominal surgery. As anesthesiologists frequently managing the treatment of such patients, we would like to highlight 2 aspects that may further inform bedside practice.
This Medical News article discusses the American Heart Association’s new scientific statement on dietary guidance to improve cardiovascular health.
The US Centers for Disease Control and Prevention (CDC) issued an advisory about growing reports of medetomidine in the illegal fentanyl supply, which has increased the risk of overdose and severe withdrawal syndrome.
The US Food and Drug Administration (FDA) approved the first generics of the sodium-glucose cotransporter 2 (SGLT2) inhibitor dapagliflozin, marketed as Farxiga.
To the Editor The DESIGNATION trial compared driving pressure–guided high PEEP with standard low PEEP. We seek clarification on 2 methodological aspects of the intervention to better interpret its null result.
Synthetic nanobodies—also called sybodies—have proven valuable for stabilizing conformations of purified proteins, advancing structural and functional studies for example of transmembrane proteins. However, their utility in modulating protein function in living cells has remained less well explored. Structural Maintenance of Chromosomes (SMC) complexes facilitate chromosome organization, a fundamental process in all domains of life. In this study, we target the bacterial SMC complex, Smc-ScpAB, in <i>Bacillus subtilis</i> with synthetic nanobodies, aiming to identify key functional regions of the protein complex in a largely unbiased manner. We first isolate sybodies that specifically bind purified Smc-ScpAB and then express them in <i>B. subtilis</i> to select binders capable of disrupting Smc-ScpAB function, leading to chromosome segregation defects and cell death. Mapping and biochemical characterization show that the 14 disruptive sybodies belong to one of three library designs, target the Smc subunit near the same coiled coil arm interface and modulate its ATPase activity in two principal ways, highlighting the mid-region of the Smc coiled coil as critical feature of the SMC-DNA folding process. These findings underscore the potential of sybodies—and, by extension, designed binders—as versatile tools for probing dynamic protein function in living cells.
This randomized clinical trial compares the effects of a theory-based serious game (a purpose-driven video game) vs usual education on emergency physician adherence to trauma triage guidelines for older adults.
The study aimed to determine whether a theory-based serious game could improve emergency physician adherence to trauma triage guidelines for severely injured older adults in nontrauma centers.
A new bill would cap US insulin costs under private insurance.
Poor sense of smell was associated with physical function decline in older adults, according to research published in JAMA Otolaryngology − Head & Neck Surgery.
Oral semaglutide was associated with sustained improvements in multiple cardiovascular disease (CVD) risk factors, according to research published in JAMA Cardiology.
In Reply In their Letters, Dr Boujenah and Dr Hnid detail unmeasured variables that may have influenced the exposures and outcomes in our recent electronic health record–based study of GLP-1RA use and discontinuation before and in early pregnancy.
To the Editor Positive end-expiratory pressure (PEEP) titration has long been proposed as a strategy to reduce postoperative pulmonary complications. Large randomized trials, including PROVHILO and PROBESE, failed to demonstrate benefit from standardized higher intraoperative PEEP, highlighting the limitations of fixed approaches. In the recent Driving Pressure During General Anesthesia for Open Abdominal Surgery (DESIGNATION) trial, the investigators tested an individualized, driving pressure–guided PEEP strategy (driving pressure = plateau pressure − PEEP) during open abdominal surgery. A small but measurable separation of pulmonary mechanics was achieved between groups, although no reduction in postoperative pulmonary complications was observed.
In Reply The Letters from Dr Mietto and colleagues, Drs Qi and Zhang, and Mr Yang and colleagues question our findings from the DESIGNATION trial, arguing that intermittently scheduled PEEP titration may not reflect dynamic intraoperative physiology, high PEEP may impose hemodynamic harm in the highest-risk patients, and the recruitment maneuver and driving pressure measurement may have been methodologically or physiologically inadequate.
<i>Trypanosoma brucei,</i> the causal agent of Human and Animal African trypanosomiasis proliferates in the extracellular milieu of mammals. It acquires host macromolecular nutrients by receptor-mediated endocytosis. The best characterised cell surface receptor is for transferrin (TfR), and it has been reported to be preferentially localised in the flagellar pocket domain of the plasma membrane, the sole site of endocytosis. In this location, the TfR may be inaccessible to adaptive immune system effectors. The <i>T. brucei</i> genome encodes ~15 TfR variants, and here we compared two, the first attached to the plasma membrane by a single glycosylphosphatidylinositol (GPI)-anchor and the other by two. Transferrin uptake kinetics were similar and rapid for both. Unexpectedly, initial binding of transferrin occurred over the whole cell surface suggesting the TfR was not localised solely in the flagellar pocket. This localisation was confirmed by immunofluorescence assays and was independent of the number of GPI-anchors. Two other GPI-anchored receptors were investigated to determine whether localisation to the whole cell surface was a general property of GPI-anchored receptors. Haptoglobin-haemoglobin uptake assays and immunofluorescence localisation of complement factor H receptor showed both were also whole cell surface localised. The mechanisms by which trypanosome receptors are protected from antibody-mediated attack are more complex than hiding in a pocket.
Animals routinely need to make decisions about what to eat and when. These decisions are influenced not only by the availability and quality of food but also by the internal state of the animal, which needs to compute and give weights to these different variables before making a choice. Feeding preferences of female mosquitoes exemplify this behavioural plasticity. Both male and female mosquitoes usually feed on carbohydrate-rich sources of nectar or sap but the female also feeds on blood, which is essential for egg development. This blood-appetite is modulated across the female’s reproductive cycle, yet little is known about the factors that bring it about. We show that mated, but not virgin <i>Anopheles stephensi</i> females, a major vector of urban malaria in the Indian subcontinent and West Africa, suppress blood feeding between a blood meal and oviposition. We identify several candidate genes through transcriptomics of blood-deprived and -sated <i>An. stephensi</i> central brains that could modulate this behaviour. We show that <i>short neuropeptide F (sNPF</i>) and <i>RYamide (RYa</i>) act together to promote blood feeding and identify a cluster of cells in the subesophageal zone that expresses <i>sNPF</i> transcripts only in the blood-hungry state. Such females also have more <i>sNPF</i> transcripts in their midguts. Based on these data, we propose a model where increased <i>sNPF</i> levels in the brain and gut promote a state of blood-hunger, which drives feeding behaviour either by <i>sNPF’s</i> action in the two tissues independently or via a communication between them. This occurs in the context of the action of <i>RYa</i> in the brain.