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The photoreceptor rhodopsin (Rho) becomes active when a tethered inverse agonist ligand (11CR) is photoconverted to an agonist (ATR). The ligand-binding pocket of inactive rhodopsin is completely enclosed, whereas active rhodopsin displays pores accessible from the lipid bilayer. Stabilization of active rhodopsin impedes 11CR binding and photoreceptor dark adaptation. Here, we used genetic code expansion and bioorthogonal labeling to engineer Rho mutants that serve as FRET sensors for measuring 11CR binding kinetics and energetics. We found that mutations that alter a channel between transmembrane helices 5 and 6 (TM5/6) dramatically affect 11CR binding kinetics but not agonist release kinetics. Our data provide direct experimental evidence for 11CR entry between TM5/6 in Rho that involves dynamic allosteric control of the ligand entry channel. Our findings provide a conceptual framework for understanding the function of G protein-coupled receptors with hydrophobic ligands that are hypothesized to enter their binding pockets through transmembrane pores.

Previously published digital autoradiography of H-3-labeled capecitabine reveals a near-uniform distribution of activity throughout a murine pancreatic model. This is in contrast both to C-14-labeled gemcitabine, and established expectations, as the dense stroma of pancreatic cancer is understood to inhibit drug penetration. Capecitabine is a pro-drug for 5 FU. The positioning of the radiolabel on capecitabine leaves open the possibility that much of the autoradiographic signal is generated by nontoxic compounds. Studies were performed on tumors derived via organoid culture from a murine KPC tumor. As before, we performed autoradiography comparing H-3 capecitabine to the gemcitabine analog F-18-FAC. The metabolism of capecitabine in this model was studied through LC-MS of tumor tissue. The autoradiographs confirmed that the H-3 label from capecitabine was much more uniformly distributed through the tumor than the F-18 from the gemcitabine analog. LC-MS revealed that approximately 75% of the molar mass of capecitabine had been converted into 5 FU or pre-5 FU compounds. The remainder had been converted into nontoxic species. Therapeutically relevant capecitabine metabolites achieve a relatively even distribution in this pancreatic cancer model, in contrast to the gemcitabine analog F-18-FAC. In a human xenograft model, (BxPC3), the H-3 label from capecitabine was also uniformly spread across the tumor autoradiographs. However, at 2 h post-administration the metabolism of capecitabine had proceeded further and the bulk of the agent was in the form of nontoxic species.

These data identify a host pathway by which VPS29 and associated factors control the endosomal environment in a manner that influences susceptibility to viral infection. This pathway could serve as a pharmaceutical target for intervention in zoonotic viral diseases, including those caused by coronaviruses, influenza viruses, and filoviruses, all of which are pandemic threats. Emerging zoonotic viral pathogens threaten global health, and there is an urgent need to discover host and viral determinants influencing infection. We performed a loss-of-function genome-wide CRISPR screen in a human lung cell line using HCoV-OC43, a human betacoronavirus. One candidate gene, VPS29, a component of the retromer complex, was required for infection by HCoV-OC43, SARS-CoV-2, other endemic- and pandemic-threat coronaviruses, as well as ebolavirus. Notably, we observed a heightened requirement for VPS29 by the recently described Omicron variant of SARS-CoV-2 compared to the ancestral variant. However, VPS29 deficiency had no effect on certain other viruses that enter cells via endosomes and had an opposing, enhancing effect on influenza A virus infection. Deficiency in VPS29 or other retromer components caused changes in endosome morphology and acidity and attenuated the activity of endosomal proteases. These changes in endosome properties caused incoming coronavirus, but not influenza virus particles, to become entrapped therein. Overall, these data show how host regulation of endosome characteristics can influence cellular susceptibility to viral infection and identify a host pathway that could serve as a pharmaceutical target for intervention in zoonotic viral diseases. IMPORTANCE These data identify a host pathway by which VPS29 and associated factors control the endosomal environment in a manner that influences susceptibility to viral infection. This pathway could serve as a pharmaceutical target for intervention in zoonotic viral diseases, including those caused by coronaviruses, influenza viruses, and filoviruses, all of which are pandemic threats. Our findings show how host regulation of endosome characteristics can influence viral susceptibility in both a positive and negative manner.

Globally, autosomal recessive IFNAR1 deficiency is a rare inborn error of immunity underlying susceptibility to live attenuated vaccine and wild-type viruses. We report seven children from five unrelated kindreds of western Polynesian ancestry who suffered from severe viral diseases. All the patients are homozygous for the same nonsense IFNAR1 variant (p.GLu386*). This allele encodes a truncated protein that is absent from the cell surface and is loss-of-function. The fibroblasts of the patients do not respond to type I IFNs (IFN-alpha 2, IFN-omega, or IFN-beta). Remarkably, this IFNAR1 variant has a minor allele frequency >1% in Samoa and is also observed in the Cook, Society, Marquesas, and Austral islands, as well as Fiji, whereas it is extremely rare or absent in the other populations tested, including those of the Pacific region. Inherited IFNAR1 deficiency should be considered in individuals of Polynesian ancestry with severe viral illnesses.

Proper segregation of chromosomes during mitosis depends on "amphitelic attachments"-load-bearing connections of sister kinetochores to the opposite spindle poles via bundles of microtubules, termed as the "K-fibers." Current models of spindle assembly assume that K-fibers arise largely from stochastic capture of microtubules, which occurs at random times and locations and independently at sister kinetochores. We test this assumption by following the movements of all kinetochores in human cells and determine that most amphitelic attachments form synchronously at a specific stage of spindle assembly and within a spatially distinct domain. This biorientation domain is enriched in bundles of antiparallel microtubules, and perturbation of microtubule bundling changes the temporal and spatial dynamics of amphitelic attachment formation. Structural analyses indicate that interactions of kinetochores with microtubule bundles are mediated by noncentrosomal short microtubules that emanate from most kinetochores during early prometaphase. Computational analyses suggest that momentous molecular motor-driven interactions with antiparallel bundles rapidly convert these short microtubules into nascent K-fibers. Thus, load-bearing connections to the opposite spindle poles form simultaneously on sister kinetochores. In contrast to the uncoordinated sequential attachments of sister kinetochores expected in stochastic models of spindle assembly, our model envisions the formation of amphitelic attachments as a deterministic process in which the chromosomes connect with the spindle poles synchronously at a specific stage of spindle assembly and at a defined location determined by the spindle architecture. Experimental analyses of changes in the kinetochore behavior in cells with perturbed activity of molecular motors CenpE and dynein confirm the predictive power of the model.

Embryonic cells grow in environments that provide a plethora of physical cues, including mechanical forces that shape the development of the entire embryo. Despite their prevalence, the role of these forces in embryonic development and their integration with chemical signals have been mostly neglected, and scrutiny in modern molecular embryology tilted, instead, towards the dissection of molecular pathways involved in cell fate determination and patterning. It is now possible to investigate how mechanical signals induce downstream genetic regulatory networks to regulate key developmental processes in the embryo. Here, we review the insights into mechanical control of early vertebrate development, including the role of forces in tissue patterning and embryonic axis formation. We also highlight recent in vitro approaches using individual embryonic stem cells and self-organizing multicellular models of human embryos, which have been instrumental in expanding our understanding of how mechanics tune cell fate and cellular rearrangements during human embryonic development. Cells in the embryo are subject to autonomous and external mechanical forces that help steer embryonic tissue patterning. Technical developments, such as in vitro models of early embryos, allow probing of the roles of mechanical forces in animal and human embryonic development.

Coagulopathy is a significant aspect of morbidity in COVID-19 patients. The clotting cascade is propagated by a series of proteases, including factor Xa and thrombin. While certain host proteases, including TMPRSS2 and furin, are known to be important for cleavage activation of SARS-CoV-2 spike to promote viral entry in the respiratory tract, other proteases may also contribute. Using biochemical and cell-based assays, we demonstrate that factor Xa and thrombin can also directly cleave SARS-CoV-2 spike, enhancing infection at the stage of viral entry. Coagulation factors increased SARS-CoV-2 infection in human lung organoids. A drug-repurposing screen identified a subset of protease inhibitors that promiscuously inhibited spike cleavage by both transmembrane serine proteases and coagulation factors. The mechanism of the protease inhibitors nafamostat and camostat may extend beyond inhibition of TMPRSS2 to coagulation-induced spike cleavage. Anticoagulation is critical in the management of COVID-19, and early intervention could provide collateral benefit by suppressing SARS-CoV-2 viral entry. We propose a model of positive feedback whereby infection-induced hypercoagulation exacerbates SARS-CoV-2 infectivity.

Ethical considerations are central to all medicine though, likely, nowhere more essential than in the practice of reproductive endocrinology and infertility. Through in vitro fertilization (IVF), this is the only field in medicine involved in creating human life. IVF has, indeed, so far led to close to 10 million births worldwide. Yet, relating to substantial changes in clinical practice of IVF, the medical literature has remained surprisingly quiet over the last two decades. Major changes especially since 2010, however, call for an updated commentary. Three key changes deserve special notice: Starting out as a strictly medical service, IVF in recent years, in efforts to expand female reproductive lifespans in a process given the term "planned" oocyte cryopreservation, increasingly became more socially motivated. The IVF field also increasingly underwent industrialization and commoditization by outside financial interests. Finally, at least partially driven by industrialization and commoditization, so-called add-ons, the term describing mostly unvalidated tests and procedures added to IVF since 2010, have been held responsible for worldwide declines in fresh, non-donor live birthrates after IVF, to levels not seen since the mid-1990s. We here, therefore, do not offer a review of bioethical considerations regarding IVF as a fertility treatment, but attempt to point out ethical issues that arose because of major recent changes in clinical IVF practice.

Nup358, a protein of the nuclear pore complex, facilitates a nuclear positioning pathway that is essential for many biological processes, including neuromuscular and brain development. Nup358 interacts with the dynein adaptor Bicaudal D2 (BicD2), which in turn recruits the dynein machinery to position the nucleus. However, the molecular mechanisms of the Nup358/BicD2 interaction and the activation of transport remain poorly understood. Here for the first time, we show that a minimal Nup358 domain activates dynein/dynactin/BicD2 for processive motility on microtubules. Using nuclear magnetic resonance titration and chemical exchange saturation transfer, mutagenesis, and circular dichroism spectroscopy, a Nup358 alpha-helix encompassing residues 2162-2184 was identified, which transitioned from a random coil to an alpha-helical conformation upon BicD2 binding and formed the core of the Nup358-BicD2 interface. Mutations in this region of Nup358 decreased the Nup358/BicD2 interaction, resulting in decreased dynein recruitment and impaired motility. BicD2 thus recognizes Nup358 through a 'cargo recognition alpha-helix,' a structural feature that may stabilize BicD2 in its activated state and promote processive dynein motility.