ß-Coronaviruses Use Lysosomes for Egress Instead of the Biosynthetic Secretory Pathway.

ß-Coronaviruses are a family of positive-strand enveloped RNA viruses that includes the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Much is known regarding their cellular entry and replication pathways, but their mode of egress remains uncertain. Using imaging methodologies and virus-specific reporters, we demonstrate that ß-coronaviruses utilize lysosomal trafficking for egress rather than the biosynthetic secretory pathway more commonly used by other enveloped viruses. This unconventional egress is regulated by the Arf-like small GTPase Arl8b and can be blocked by the Rab7 GTPase competitive inhibitor CID1067700. Such non-lytic release of ß-coronaviruses results in lysosome deacidification, inactivation of lysosomal degradation enzymes, and disruption of antigen presentation pathways. ß-Coronavirus-induced exploitation of lysosomal organelles for egress provides insights into the cellular and immunological abnormalities observed in patients and suggests new therapeutic modalities.

Virulence and Pathogenicity of Chytrid Fungi Causing Amphibian Extinctions.

Ancient enzootic associations between wildlife and their infections allow evolution to innovate mechanisms of pathogenicity that are counterbalanced by host responses. However, erosion of barriers to pathogen dispersal by globalization leads to the infection of hosts that have not evolved effective resistance and the emergence of highly virulent infections. Global amphibian declines driven by the rise of chytrid fungi and chytridiomycosis are emblematic of emerging infections. Here, we review how modern biological methods have been used to understand the adaptations and counteradaptations that these fungi and their amphibian hosts have evolved. We explore the interplay of biotic and abiotic factors that modify the virulence of these infections and dissect the complexity of this disease system. We highlight progress that has led to insights into how we might in the future lessen the impact of these emerging infections.

Rac1 controls cell turnover and reversibility of the involution process in postpartum mammary glands.

Cell turnover in adult tissues is essential for maintaining tissue homeostasis over a life span and for inducing the morphological changes associated with the reproductive cycle. However, the underlying mechanisms that coordinate the balance of cell death and proliferation remain unsolved. Using the mammary gland, we have discovered that Rac1 acts as a nexus to control cell turnover. Postlactational tissue regression is characterised by the death of milk secreting alveoli, but the process is reversible within the first 48 h if feeding recommences. In mice lacking epithelial Rac1, alveolar regression was delayed. This defect did not result from failed cell death but rather increased cell turnover. Fitter progenitor cells inappropriately divided, regenerating the alveoli, but cell death also concomitantly accelerated. We discovered that progenitor cell hyperproliferation was linked to nonautonomous effects of Rac1 deletion on the macrophageal niche with heightened inflammation. Moreover, loss of Rac1 impaired cell death with autophagy but switched the cell death route to apoptosis. Finally, mammary gland reversibility failed in the absence of Rac1 as the alveoli failed to recommence lactation upon resuckling.

The human fungal pathogen Aspergillus fumigatus can produce the highest known number of meiotic crossovers.

Sexual reproduction involving meiosis is essential in most eukaryotes. This produces offspring with novel genotypes, both by segregation of parental chromosomes as well as crossovers between homologous chromosomes. A sexual cycle for the opportunistic human pathogenic fungus Aspergillus fumigatus is known, but the genetic consequences of meiosis have remained unknown. Among other Aspergilli, it is known that A. flavus has a moderately high recombination rate with an average of 4.2 crossovers per chromosome pair, whereas A. nidulans has in contrast a higher rate with 9.3 crossovers per chromosome pair. Here, we show in a cross between A. fumigatus strains that they produce an average of 29.9 crossovers per chromosome pair and large variation in total map length across additional strain crosses. This rate of crossovers per chromosome is more than twice that seen for any known organism, which we discuss in relation to other genetic model systems. We validate this high rate of crossovers through mapping of resistance to the laboratory antifungal acriflavine by using standing variation in an undescribed ABC efflux transporter. We then demonstrate that this rate of crossovers is sufficient to produce one of the common multidrug resistant haplotypes found in the cyp51A gene (TR34/L98H) in crosses among parents harboring either of 2 nearby genetic variants, possibly explaining the early spread of such haplotypes. Our results suggest that genomic studies in this species should reassess common assumptions about linkage between genetic regions. The finding of an unparalleled crossover rate in A. fumigatus provides opportunities to understand why these rates are not generally higher in other eukaryotes.

Two-speed genome evolution drives pathogenicity in fungal pathogens of animals.

The origins and evolution of virulence in amphibian-infecting chytrids Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal) are largely unknown. Here, we use deep nanopore sequencing of Bsal and comparative genomics against 21 high-quality genome assemblies that span the fungal Chytridiomycota. We discover that Bsal has the most repeat-rich genome of the Chytridiomycota, comprising 40.9% repetitive elements; this genome has expanded to more than 3× the length of its conspecific Bd, with autonomous and fully functional LTR/Gypsy elements contributing significantly to the expansion. The M36 metalloprotease virulence factors are highly expanded (n = 177) in Bsal, most of which (53%) are flanked by transposable elements, suggesting they have a repeat-associated expansion. We find enrichment upstream of M36 metalloprotease genes of three novel repeat families belonging to the repeat superfamily of LINEs that are implicated with gene copy number variations. Additionally, Bsal has a highly compartmentalized genome architecture, with virulence factors enriched in gene-sparse/repeat-rich compartments, while core conserved genes are enriched in gene-rich/repeat-poor compartments. Genes upregulated during infection are primarily found in the gene-sparse/repeat-rich compartment in both Bd and Bsal. Furthermore, genes with signatures of positive selection in Bd are enriched in repeat-rich regions, suggesting these regions are a cradle for the evolution of chytrid pathogenicity. These are the hallmarks of two-speed genome evolution, and this study provides evidence of two-speed genomes in an animal pathogen, shedding light on the evolution of fungal pathogens of vertebrates driving global declines and extinctions.

Phosphates form spectroscopically dark state assemblies in common aqueous solutions.

Phosphates and polyphosphates play ubiquitous roles in biology as integral structural components of cell membranes and bone, or as vehicles of energy storage via adenosine triphosphate and phosphocreatine. The solution phase space of phosphate species appears more complex than previously known. We present nuclear magnetic resonance (NMR) and cryogenic transmission electron microscopy (cryo-TEM) experiments that suggest phosphate species including orthophosphates, pyrophosphates, and adenosine phosphates associate into dynamic assemblies in dilute solutions that are spectroscopically "dark." Cryo-TEM provides visual evidence of the formation of spherical assemblies tens of nanometers in size, while NMR indicates that a majority population of phosphates remain as unassociated ions in exchange with spectroscopically invisible assemblies. The formation of these assemblies is reversibly and entropically driven by the partial dehydration of phosphate groups, as verified by diffusion-ordered spectroscopy (DOSY), indicating a thermodynamic state of assembly held together by multivalent interactions between the phosphates. Molecular dynamics simulations further corroborate that orthophosphates readily cluster in aqueous solutions. This study presents the surprising discovery that phosphate-containing molecules, ubiquitously present in the biological milieu, can readily form dynamic assemblies under a wide range of commonly used solution conditions, highlighting a hitherto unreported property of phosphate's native state in biological solutions.

Archival mitogenomes identify invasion by the Batrachochytrium dendrobatidis CAPE lineage caused an African amphibian extinction in the wild.

Outbreaks of emerging infectious diseases are influenced by local biotic and abiotic factors, with host declines occurring when conditions favour the pathogen. Deterioration in the population of the micro-endemic Tanzanian Kihansi spray toad (Nectophrynoides asperginis) occurred after the construction of a hydropower dam, implicating habitat modification in this species decline. Population recovery followed habitat augmentation; however, a subsequent outbreak of chytridiomycosis caused by Batrachochytrium dendrobatidis (Bd) led to the spray toad's extinction in the wild. We show using spatiotemporal surveillance and mitogenome assembly of Bd from archived toad mortalities that the outbreak was caused by invasion of the BdCAPE lineage and not the panzootic lineage BdGPL. Molecular dating reveals an emergence of BdCAPE across southern Africa overlapping with the timing of the spray toad's extinction. That our post-outbreak surveillance of co-occurring amphibian species in the Udzungwa Mountains shows widespread infection by BdCAPE yet no signs of ill-health or decline suggests these other species can tolerate Bd when environments are stable. We conclude that, despite transient success in mitigating the impact caused by dams' construction, invasion by BdCAPE caused the ultimate die-off that led to the extinction of the Kihansi spray toad.

Stable Measurement-Induced Floquet Enriched Topological Order.

The Floquet code utilizes a periodic sequence of two-qubit measurements to realize the topological order. After each measurement round, the instantaneous stabilizer group can be mapped to a honeycomb toric code, explaining the topological feature. The code also possesses a time-crystal order-the e-m transmutation after every cycle, breaking the Floquet symmetry of the measurement schedule. This behavior is distinct from the stationary topological order realized in either random circuits or time-independent Hamiltonian. Therefore, the resultant phase belongs to the overlap between the classes of Floquet enriched topological orders and measurement-induced phases. In this Letter, we construct a continuous path interpolating between the Floquet and toric codes, focusing on the transition between the time-crystal and stationary topological phases. We show that this transition is characterized by a divergent length scale. We also add single-qubit perturbations to the model and obtain a richer two-dimensional parametric phase diagram of the Floquet code, showing the stability of the Floquet enriched topological order.

Modular and divergent synthesis of 2,N3-disubstituted 4-quinazolinones facilitated by regioselective N-alkylation.

The synthesis of a biologically relevant 2-amino-N3-alkylamido 4-quinazolinone has been accomplished in four steps from commercially available materials using design principles from both modular and divergent synthesis. N3-Alkylation of 2-chloro-4(3H)-quinazolinone using methyl bromoacetate, followed by C2-amination produced a suitable scaffold for introducing molecular diversity. Optimization of alkylation conditions afforded full regioselectivity, enabling exclusive access to the N-alkylated isomer. Subsequent C2-amination using piperidine, pyrrolidine, or diethylamine, followed by amide bond formation using variously substituted phenethylamines, generated fifteen unique 4-quinazolinones bearing C2-amino and N3-alkylamido substituents. These efforts highlight the reciprocal influence of C2 and N3 substitution on functionalization at either position, establish an effective synthetic pathway toward 2,N3-disubstituted 4-quinazolinones, and enable preliminary bioactivity studies while providing an experiential learning opportunity for undergraduate student researchers.

Neo-Natal Castration Leads to Subtle Differences in Porcine Anterior Cruciate Ligament Morphology and Function in Adolescence.

Female adolescent athletes are at a higher risk of tearing their anterior cruciate ligament (ACL) than male counterparts. While most work related to hormones has focused on the effects of estrogen to understand the increased risk of ACL injury, there are other understudied factors, including testosterone. The purpose of this study was to determine how surgical castration in the male porcine model influences ACL size and function across skeletal growth. Thirty-six male Yorkshire crossbreed pigs were raised to 3 (juvenile), 4.5 (early adolescent), and 6 months (adolescent) of age. Animals were either castrated (barrows) within 2 weeks after birth or were left intact (boars). Posteuthanasia, joint and ACL size were assessed via MRI, and biomechanics were assessed via a robotic testing system. Joint size increased throughout age, yet barrows had smaller joints than boars. ACL cross-sectional area (CSA), length, volume, and in situ stiffness increased with age, as did the percent contribution of the ACL anteromedial (AM) bundle to resisting loads. Boar ACL, AM bundle, and PL bundle volumes were 19%, 25%, and 15% larger than barrows across ages. However, ACL CSA, in situ stiffness, and bundle contribution were similar between boars and barrows. The barrows had smaller temporal increases in AM bundle function than boars, but these data were highly variable. Early and sustained loss in testosterone leads to subtle differences in ACL morphology but may not influence measures associated with increased injury risk, such as CSA or bundle forces in response to applied loads.

Controlled Stiffness of Direct-Write, Near-Field Electrospun Gelatin Fibers Generates Differences in Tenocyte Morphology and Gene Expression.

Tendinopathy is a leading cause of mobility issues. Currently, the cell-matrix interactions involved in the development of tendinopathy are not fully understood. In vitro tendon models provide a unique tool for addressing this knowledge gap as they permit fine control over biochemical, micromechanical, and structural aspects of the local environment to explore cell-matrix interactions. In this study, direct-write, near-field electrospinning of gelatin solution was implemented to fabricate micron-scale fibrous scaffolds that mimic native collagen fiber size and orientation. The stiffness of these fibrous scaffolds was found to be controllable between 1 MPa and 8 MPa using different crosslinking methods (EDC, DHT, DHT+EDC) or through altering the duration of crosslinking with EDC (1 h to 24 h). EDC crosslinking provided the greatest fiber stability, surviving up to 3 weeks in vitro. Differences in stiffness resulted in phenotypic changes for equine tenocytes with low stiffness fibers (∼1 MPa) promoting an elongated nuclear aspect ratio while those on high stiffness fibers (∼8 MPa) were rounded. High stiffness fibers resulted in the upregulation of matrix metalloproteinase (MMPs) and proteoglycans (possible indicators for tendinopathy) relative to low stiffness fibers. These results demonstrate the feasibility of direct-written gelatin scaffolds as tendon in vitro models and provide evidence that matrix mechanical properties may be crucial factors in cell-matrix interactions during tendinopathy formation.

A Systems Approach to Biomechanics, Mechanobiology, and Biotransport.

The human body represents a collection of interacting systems that range in scale from nanometers to meters. Investigations from a systems perspective focus on how the parts work together to enact changes across spatial scales, and further our understanding of how systems function and fail. Here, we highlight systems approaches presented at the 2022 Summer Biomechanics, Bio-engineering, and Biotransport Conference in the areas of solid mechanics; fluid mechanics; tissue and cellular engineering; biotransport; and design, dynamics, and rehabilitation; and biomechanics education. Systems approaches are yielding new insights into human biology by leveraging state-of-the-art tools, which could ultimately lead to more informed design of therapies and medical devices for preventing and treating disease as well as rehabilitating patients using strategies that are uniquely optimized for each patient. Educational approaches can also be designed to foster a foundation of systems-level thinking.

Cancer-associated fibroblasts promote cancer stemness by inducing expression of the chromatin-modifying protein CBX4 in squamous cell carcinoma.

The chromobox-containing protein CBX4 is an important regulator of epithelial cell proliferation and differentiation, and has been implicated in several cancer types. The cancer stem cell (CSC) population is a key driver of metastasis and recurrence. The undifferentiated, plastic state characteristic of CSCs relies on cues from the microenvironment. Cancer-associated fibroblasts (CAFs) are a major component of the microenvironment that can influence the CSC population through the secretion of extracellular matrix and a variety of growth factors. Here we show CBX4 is a critical regulator of the CSC phenotype in squamous cell carcinomas of the skin and hypopharynx. Moreover, CAFs can promote the expression of CBX4 in the CSC population through the secretion of interleukin-6 (IL-6). IL-6 activates JAK/STAT3 signaling to increase ∆Np63α-a key transcription factor that is essential for epithelial stem cell function and the maintenance of proliferative potential that is capable of regulating CBX4. Targeting the JAK/STAT3 axis or CBX4 directly suppresses the aggressive phenotype of CSCs and represents a novel opportunity for therapeutic intervention.

Cross Entropy Benchmark for Measurement-Induced Phase Transitions.

We investigate prospects of employing the linear cross entropy to experimentally access measurement-induced phase transitions without requiring any postselection of quantum trajectories. For two random circuits that are identical in the bulk but with different initial states, the linear cross entropy χ between the bulk measurement outcome distributions in the two circuits acts as an order parameter, and can be used to distinguish the volume law from area law phases. In the volume law phase (and in the thermodynamic limit) the bulk measurements cannot distinguish between the two different initial states, and χ=1. In the area law phase χ<1. For circuits with Clifford gates, we provide numerical evidence that χ can be sampled to accuracy ϵ from O(1/ϵ^{2}) trajectories, by running the first circuit on a quantum simulator without postselection, aided by a classical simulation of the second. We also find that for weak depolarizing noise the signature of the measurement-induced phase transitions is still present for intermediate system sizes. In our protocol we have the freedom of choosing initial states such that the "classical" side can be simulated efficiently, while simulating the "quantum" side is still classically hard.

Screening p-Phenylenediamine Antioxidants, Their Transformation Products, and Industrial Chemical Additives in Crumb Rubber and Elastomeric Consumer Products.

Recently, roadway releases of N,N'-substituted p-phenylenediamine (PPD) antioxidants and their transformation products (TPs) received significant attention due to the highly toxic 6PPD-quinone. However, the occurrence of PPDs and TPs in recycled tire rubber products remains uncharacterized. Here, we analyzed tire wear particles (TWPs), recycled rubber doormats, and turf-field crumb rubbers for seven PPD antioxidants, five PPD-quinones (PPDQs), and five other 6PPD TPs using liquid chromatography-tandem mass spectrometry. PPD antioxidants, PPDQs, and other TPs were present in all samples with chemical profiles dominated by 6PPD, DTPD, DPPD, and their corresponding PPDQs. Interestingly, the individual [PPDQ]/[PPD] and [TP]/[PPD] ratios significantly increased as total concentrations of the PPD-derived chemical decreased, indicating that TPs (including PPDQs) dominated the PPD-derived compounds with increased environmental weathering. Furthermore, we quantified 15 other industrial rubber additives (including bonding agents, vulcanization accelerators, benzotriazole and benzothiazole derivatives, and diphenylamine antioxidants), observing that PPD-derived chemical concentrations were 0.5-6 times higher than these often-studied additives. We also screened various other elastomeric consumer products, consistently detecting PPD-derived compounds in lab stoppers, sneaker soles, and rubber garden hose samples. These data emphasize that PPD antioxidants, PPDQs, and related TPs are important, previously overlooked contaminant classes in tire rubbers and elastomeric consumer products.

Long-read Sequencing and de novo Genome Assembly of Three Aspergillus fumigatus Genomes.

Aspergillus fumigatus is a genetically diverse fungal species, which is near ubiquitous in its global distribution and is the major cause of the life-threatening disease invasive aspergillosis. We present 3 de novo genome assemblies that were selected to be representative of the genetic diversity of clinical and environmental A. fumigatus. Sequencing using long-read Oxford Nanopore and subsequent assembly of the genomes yielded 10-23 contigs with an N50 of 4.05 Mbp to 4.93 Mbp.

Multi-sectoral action to promote psychological wellbeing: Theorising the role of place-based policy.

ISSUES ADDRESSED: Several national governments are showing interest in policies to promote wellbeing. One common strategy is to devise systems to measure indictors of wellbeing, presuming that governments act on what they measure. This article will argue instead that formation of multisectoral policies to promote psychological wellbeing requires a different kind of theoretical and evidentiary basis. METHODS: The article integrates ideas from literature on wellbeing, health in all policies, political science, mental health promotion, and social determinants of health to make a case for place-based policy as the central feature of multi-sectoral policy for psychological wellbeing. RESULTS AND DISCUSSION: I argue that the required theoretical foundation for policy action on psychological wellbeing lies with understanding certain basic functions of human social psychology including the role of stress arousal. I then draw on policy theory to propose three steps to translate this theoretical understanding of psychological wellbeing into practicable, multi-sectoral policies. Step one is concerned with adopting a thoroughly revised conception of psychological wellbeing as a policy problem. Step two involves uptake of a theory of change in policy, grounded on recognition of essential social conditions required to promote psychological wellbeing. Proceeding from these, I will argue that a necessary (but not sufficient) third step is to implement place-based strategies involving government-community partnerships, to generate essential conditions for psychological wellbeing on a universal basis. Finally, I examine implications of the proposed approach for current theory and practice in mental health promotion policy. CONCLUSIONS: Place-based policy is foundational for effective multi-sectoral policy to promote psychological wellbeing. SO WHAT?: Governments aiming to promote psychological wellbeing should position place-based policy at the centre of their strategies.

p63-related signaling at a glance.

p63 (also known as TP63) is a transcription factor of the p53 family, along with p73. Multiple isoforms of p63 have been discovered and these have diverse functions encompassing a wide array of cell biology. p63 isoforms are implicated in lineage specification, proliferative potential, differentiation, cell death and survival, DNA damage response and metabolism. Furthermore, p63 is linked to human disease states including cancer. p63 is critical to many aspects of cell signaling, and in this Cell science at a glance article and the accompanying poster, we focus on the signaling cascades regulating TAp63 and ΔNp63 isoforms and those that are regulated by TAp63 and ΔNp63, as well the role of p63 in disease.

Exploring a novel genomic safe-haven site in the human pathogenic mould Aspergillus fumigatus.

Aspergillus fumigatus is the most important airborne fungal pathogen and allergen of humans causing high morbidity and mortality worldwide. The factors that govern pathogenicity of this organism are multi-factorial and are poorly understood. Molecular tools to dissect the mechanisms of pathogenicity in A. fumigatus have improved significantly over the last 20 years however many procedures have not been standardised for A. fumigatus. Here, we present a new genomic safe-haven locus at the site of an inactivated transposon, named SH-aft4, which can be used to insert DNA sequences in the genome of this fungus without impacting its phenotype. We show that we are able to effectively express a transgene construct from the SH-aft4 and that natural regulation of promoter function is conserved at this site. Furthermore, the SH-aft4 locus is highly conserved in the genome of a wide range of clinical and environmental isolates including the isolates commonly used by many laboratories CEA10, Af293 and ATCC46645, allowing a wide range of isolates to be manipulated. Our results show that the aft4 locus can serve as a site for integration of a wide range of genetic constructs to aid functional genomics studies of this important human fungal pathogen.

Citizen Science Surveillance of Triazole-Resistant Aspergillus fumigatus in United Kingdom Residential Garden Soils.

Compost is an ecological niche for Aspergillus fumigatus due to its role as a decomposer of organic matter and its ability to survive the high temperatures associated with the composting process. Subsequently, composting facilities are associated with high levels of A. fumigatus spores that are aerosolized from compost and cause respiratory illness in workers. In the UK, gardening is an activity enjoyed by individuals of all ages, and it is likely that they are being exposed to A. fumigatus spores when handling commercial compost or compost they have produced themselves. In the present study, 246 citizen scientists collected 509 soil samples from locations in their gardens in the UK, from which were cultured 5,174 A. fumigatus isolates. Of these isolates, 736 (14%) were resistant to tebuconazole: the third most-sprayed triazole fungicide in the UK, which confers cross-resistance to the medical triazoles used to treat A. fumigatus lung infections in humans. These isolates were found to contain the common resistance mechanisms in the A. fumigatus cyp51A gene TR34/L98H or TR46/Y121F/T289A, as well as the less common resistance mechanisms TR34, TR53, TR46/Y121F/T289A/S363P/I364V/G448S, and (TR46)2/Y121F/M172I/T289A/G448S. Regression analyses found that soil samples containing compost were significantly more likely to grow tebuconazole-susceptible and tebuconazole-resistant A. fumigatus strains than those that did not and that compost samples grew significantly higher numbers of A. fumigatus than other samples. IMPORTANCE The findings presented here highlight compost as a potential health hazard to individuals with predisposing factors to A. fumigatus lung infections and as a potential health hazard to immunocompetent individuals who could be exposed to sufficiently high numbers of spores to develop infection. Furthermore, we found that 14% of A. fumigatus isolates in garden soils were resistant to an agricultural triazole, which confers cross-resistance to medical triazoles used to treat A. fumigatus lung infections. This raises the question of whether compost bags should carry additional health warnings regarding inhalation of A. fumigatus spores, whether individuals should be advised to wear facemasks while handling compost, or whether commercial producers should be responsible for sterilizing compost before shipping. The findings support increasing public awareness of the hazard posed by compost and investigating measures that can be taken to reduce the exposure risk.