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A major challenge in coronavirus vaccination and treatment is to counteract rapid viral evolution and mutations. Here the authors show that CRISPR-Cas13d can be used as a broad-spectrum antiviral to inhibit human coronaviruses, including new SARS-CoV-2 variants, combined with small molecule drugs for an enhanced antiviral effect in human primary cells.

Purpose: To retrospectively assess the outcomes of Inferior Vena Cava (IVC) filters placed in critically ill patients in the ICU at bedside using digital radiograph (DR) guidance with previous cross-sectional imaging for planning, compared to IVC filters placed by conventional fluoroscopy (CF). Method and materials: The cohort consisted of 129 IVC filter placements; 48 placed at bedside and 81 placed conventionally from July 2015 to September 2016. Patient demographics, indication, radiation exposures, access site, procedural duration, dwell time, and complications were identified by the EMR. IVC Filter positioning with measurements of tip to renal vein distance and lateral filter tilt were performed when cavograms or post placement CTs were available for review. Statistical analysis was performed using Stata IC 11.2. Results: Technical success of the procedure was 100% in both groups. Procedural duration was longer at the bedside lasting 14.5 +/- 10.2 versus 6.7 +/- 6.0 ​min (p

Abstract Background Most single nucleotide variants (SNVs) occur in noncoding sequence where millions of transcription factor binding sites (TFBS) reside. Here, a comparative analysis of CRISPR-mediated homology-directed repair (HDR) versus the recently reported prime editing 2 (PE2) system was carried out in mice over a TFBS called a CArG box in the Tspan2 promoter. Results Quantitative RT-PCR showed loss of Tspan2 mRNA in aorta and bladder, but not heart or brain, of mice homozygous for an HDR-mediated three base pair substitution in the Tspan2 CArG box. Using the same protospacer, mice homozygous for a PE2-mediated single-base substitution in the Tspan2 CArG box displayed similar cell-specific loss of Tspan2 mRNA; expression of an overlapping long noncoding RNA was also nearly abolished in aorta and bladder. Immuno-RNA fluorescence in situ hybridization validated loss of Tspan2 in vascular smooth muscle cells of HDR and PE2 CArG box mutant mice. Targeted sequencing demonstrated variable frequencies of on-target editing in all PE2 and HDR founders. However, whereas no on-target indels were detected in any of the PE2 founders, all HDR founders showed varying levels of on-target indels. Off-target analysis by targeted sequencing revealed mutations in many HDR founders, but none in PE2 founders. Conclusions PE2 directs high-fidelity editing of a single base in a TFBS leading to cell-specific loss in expression of an mRNA/long noncoding RNA gene pair. The PE2 platform expands the genome editing toolbox for modeling and correcting relevant noncoding SNVs in the mouse.

Objective: Citrin, the mitochondrial aspartate/glutamate carrier isoform 2 (AGC2), is structurally and mechanistically the most complex SLC25 family member, because it consists of three domains and forms a homo-dimer. Each protomer has an N-terminal calcium-binding domain with EF-hands, followed by a substrate-transporting carrier domain and a C-terminal domain with an amphipathic helix. The absence or dysfunction of citrin leads to citrin deficiency, a highly prevalent pan-ethnic mitochondrial disease. Here, we aim to understand the role of different citrin domains and how they contribute to pathogenic mechanisms in citrin deficiency. Methods: We have employed structural modeling and functional reconstitution of purified proteins in proteoliposomes to assess the transport activity and calcium regulation of wild-type citrin and pathogenic variants associated with citrin deficiency. We have also developed a double knockout of citrin and aralar (AGC1), the two paralogs of the mitochondrial aspartate/glutamate carrier, in HAP1 cells to perform mitochondrial imaging and to investigate mitochondrial localisation. Results: Using 33 pathogenic variants of citrin we clarify determinants of subcellular localization and transport mechanism. We identify crucial elements of the carrier domain that are required for transport, including those involved in substrate binding, network formation and dynamics. We show that the N-terminal domain is not involved in calcium regulation of transport, as previously thought, but when mutated causes a mitochondrial import defect. Conclusions: Our work introduces a new role for the N-terminal domain of citrin and demonstrates that dysfunction of the different domains contributes to distinct pathogenic mechanisms in citrin deficiency.

In the first use of the event related optical signal (EROS) as a brain imaging tool for the study of long-term memory, we examined relational or associative aspects of memory, widely presumed to involve the interplay among multiple brain regions in representing and reactivating different elements of a given event. Here, we found that a brain region known to be involved in face processing (the posterior superior temporal sulcus, STS) was active not only when viewing faces during the study phase but also when viewing scenes at test that, through prior learning, were associated with specific faces. These findings, demonstrating the activation of stimulus-specific cortical regions in the absence of stimuli of that type, based on learned relations, reveal cortical substrates of the reactivation of relational memories.

Abstract The ability Gram-negative pathogens have at adapting and protecting themselves against antibiotics has increasingly become a public health threat. Data-driven models identifying molecular properties that correlate with outer membrane (OM) permeation and growth inhibition while avoiding efflux could guide the discovery of novel classes of antibiotics. Here we evaluate 174 molecular descriptors in 1260 antimicrobial compounds and study their correlations with antibacterial activity in Gram-negative Pseudomonas aeruginosa. The descriptors are derived from traditional approaches quantifying the compounds’ intrinsic physicochemical properties, together with, bacterium-specific from ensemble docking of compounds targeting specific MexB binding pockets, and all-atom molecular dynamics simulations in different subregions of the OM model. Using these descriptors and the measured inhibitory concentrations, we design a statistical protocol to identify predictors of OM permeation/inhibition. We find consistent rules across most of our data highlighting the role of the interaction between the compounds and the OM. An implementation of the rules uncovered in our study is shown, and it demonstrates the accuracy of our approach in a set of previously unseen compounds. Our analysis sheds new light on the key properties drug candidates need to effectively permeate/inhibit P. aeruginosa, and opens the gate to similar data-driven studies in other Gram-negative pathogens.

Environmental archeology of the Amazon has conventionally focused on terrestrial spaces, investigating crop domestication and production, agroforestry and anthropogenic soils. Aquatic environments, however, have been given less attention by archeologists and paleoenvironmental scientists alike. The Llanos de Mojos, a 135,000 km2 basin of the Amazon and mosaic landscape of savannas, wetlands and forests, has long been recognized for its archeological record. A variety of anthropogenic earthworks across the region show significant landscape modifications from Pre-Columbian times used for transportation, resource production and control of floodwaters. Here, we investigate the use and management of wetlands, and their interaction in the wider Mojos mosaic landscape, by integrating extensive archeological, paleoenvironmental and GIS datasets. We demonstrate that past human modifications of the landscape enabled the creation of complex wetland-terrestrial system of resource production, in which wetlands were managed, complementary to terrestrial crop production and agroforestry. Across short distances (40 km), land use strategies were heterogeneous despite being connected in time and space. Additionally, mixed wetland-terrestrial resource production was relatively stable during periods of lower rainfall from 1200–1500 CE, implying that this strategy provided flexibility under conditions of variable climate. The wetlands of the Llanos de Mojos are the result of millennia of human intervention and recognition of this deep heritage is required to make informed management and conservation decisions to ensure the future of this culturally and biologically diverse region.

Abstract Background Junipers (Juniperus spp.) are woody native, invasive plants that have caused encroachment problems in the U.S. western rangelands, decreasing forage productivity and biodiversity. A potential solution to this issue is using goats in targeted grazing programs. However, junipers, which grow in dry and harsh environmental conditions, use chemical defense mechanisms to deter herbivores. Therefore, genetically selecting goats for increased juniper consumption is of great interest for regenerative rangeland management. In this context, the primary objectives of this study were to: 1) estimate variance components and genetic parameters for predicted juniper consumption in divergently selected Angora (ANG) and composite Boer x Spanish (BS) goat populations grazing on Western U.S. rangelands; and 2) to identify genomic regions, candidate genes, and biological pathways associated with juniper consumption in these goat populations. Results The average juniper consumption was 22.4% (± 18.7%) and 7.01% (± 12.1%) in the BS and ANG populations, respectively. The heritability estimates (realized heritability within parenthesis) for juniper consumption were 0.43 ± 0.02 (0.34 ± 0.06) and 0.19 ± 0.03 (0.13 ± 0.03) in BS and ANG, respectively, indicating that juniper consumption can be increased through genetic selection. The repeatability values of predicted juniper consumption were 0.45 for BS and 0.28 for ANG. A total of 571 significant SNP located within or close to 231 genes in BS, and 116 SNP related to 183 genes in ANG were identified based on the genome-wide association analyses. These genes are primarily associated with biological pathways and gene ontology terms related to olfactory receptors, intestinal absorption, and immunity response. Conclusions These findings suggest that juniper consumption is a heritable trait of polygenic inheritance influenced by multiple genes of small effects. The genetic parameters calculated indicate that juniper consumption can be genetically improved in both goat populations.

Mechanobiology is a rapidly advancing field, with growing evidence that mechanical signaling plays key roles in health and disease. To accelerate mechanobiology-based drug discovery, novel in vitro systems are needed that enable mechanical perturbation of cells in a format amenable to high throughput screening. Here, both a mechanical stretch device and 192-well silicone flexible linear stretch plate were designed and fabricated to meet high throughput technology needs for cell stretch-based applications. To demonstrate the utility of the stretch plate in automation and screening, cell dispensing, liquid handling, high content imaging, and high throughput sequencing platforms were employed. Using this system, an assay was developed as a biological validation and proof-of-concept readout for screening. A mechano-transcriptional stretch response was characterized using focused gene expression profiling measured by RNA-mediated oligonucleotide Annealing, Selection, and Ligation with Next-Gen sequencing. Using articular chondrocytes, a gene expression signature containing stretch responsive genes relevant to cartilage homeostasis and disease was identified. The possibility for integration of other stretch sensitive cell types (e.g., cardiovascular, airway, bladder, gut, and musculoskeletal), in combination with alternative phenotypic readouts (e.g., protein expression, proliferation, or spatial alignment), broadens the scope of high throughput stretch and allows for wider adoption by the research community. This high throughput mechanical stress device fills an unmet need in phenotypic screening technology to support drug discovery in mechanobiology-based disease areas.

Abstract Little is known about the effects of woody plant encroachment—a recent but pervasive phenomenon—on the hydraulic properties of bedrock substrates. Recent work using stream solute concentrations paired with weathering models suggests that woody plant encroachment accelerates limestone weathering. In this field study, we evaluate this hypothesis by examining bedrock in the Edwards Plateau, an extensive karst landscape in Central Texas. We compared a site that has been heavily encroached by woody plants (mainly Quercus fusiformis and Juniperus ashei), with an adjacent site that has been maintained free of encroachment for the past eight decades. Both sites share the same bedrock, as confirmed by trenching, and originally had very few trees, which enabled us to evaluate how encroachment impacted the evolution of hydraulic properties over a period of no more than 80 years. Using in situ permeability tests in boreholes drilled into the weathered bedrock, we found that the mean saturated hydraulic conductivity of the bedrock was higher—by an order of magnitude—beneath woody plants than in the areas where woody plants have been continuously suppressed. Additionally, woody plant encroachment was associated with greater regolith thickness, greater plant rooting depths, significantly lower rock hardness, and a 24–44% increase in limestone matrix porosity. These findings are strong indicators that woody plant encroachment enhances bedrock weathering, thereby amplifying its permeability—a cycle of mutual reinforcement with the potential for substantial changes within a few decades. Given the importance of shallow bedrock for ecohydrological and biogeochemical processes, the broader impacts of woody plant encroachment on weathering rates and permeability warrant further investigation.

Postgraduate students often find work and thesis preparation demands compelling which in turn affect their thesis completion. The authors conducted a descriptive survey with a sample of 161 graduate thesis candidates (representing a 57% data collection return rate) using the census of the target population. Three scales were used to measure academic stress, academic burnout, and thesis completion, respectively; the questionnaires were administered via Google Forms. The data were analysed using Pearson's correlation coefficient and multiple linear regression. We found that lack of or inadequate sponsorship from beneficent organisations, several administrative bureaucracies, and inaccessibility to research databases, among others, place academic stress on graduate candidates, which connects with burnout, affecting thesis completion. The study also found that academic stress and burnout are essential determinants and predictors of thesis completion. The authors thus recommend that postgraduate schools design and implement thesis preparation schemes that consider this study's findings, especially those that recognise the elements of academic stress and its related burnout concerns for thesis candidates.

IntroductionImmune-mediated inflammatory diseases (IMID), such as multiple sclerosis (MS), inflammatory bowel disease (IBD) or rheumatoid arthritis (RA) have high rates of elevated anxiety symptoms. This can may worsen functioning and increase IMID disease burden. The rate of and factors associated with elevated anxiety symptoms may differ between males and females, which, in turn can affect diagnosis and disease management. We evaluated whether the frequency and factors associated with comorbid elevated anxiety symptoms in those with an IMID differed by sex.MethodsParticipants with an IMID (MS, IBD or RA) completed two anxiety measures (HADS, GAD-7). We used logistic regression to investigate whether sex differences exist in the presence of comorbid elevated anxiety symptoms or in the endorsement of individual anxiety items in those with an IMID.ResultsOf 656 participants, females with an IMID were more likely to have elevated anxiety symptoms compared to males (adjusted odds ratio [aOR] 2.05; 95%CI: 1.2, 3.6). Younger age, higher depressive symptoms and income were also associated with elevated anxiety symptoms in IMID. Lower income in males with an IMID, but not females, was associated with elevated anxiety symptoms (aOR: 4.8; 95%CI: 1.5, 15.6). No other factors demonstrated a sex difference. Males had nearly twice the odds of endorsing restlessness on the GAD-7 (OR = 1.8, 95%CI: 1.07, 3.15) compared to females.DiscussionWe found evidence for sex differences in the factors associated with experiencing elevated anxiety symptoms in those with an IMID. These findings could be helpful to sensitize clinicians to monitor for comorbid anxiety symptoms in males with an IMID.

Agriculture generates ~83% of total US ammonia (NH3) emissions, potentially adversely impacting sensitive ecosystems through wet and dry deposition. Regions with intense livestock production, such as the dairy region of south-central Idaho, generate hotspots of NH3 emissions. Our objective was to measure the spatial and temporal variability of NH3 across this region and estimate its dry deposition. Ambient NH3 was measured using diffusive passive samplers at 8 sites in two transects across the region from 2018–2020. NH3 fluxes were estimated using the Surface Tiled Aerosol and Gaseous Exchange (STAGE) model. Peak NH3 concentrations were 4–5 times greater at a high-density dairy site compared to mixed agriculture/dairy or agricultural sites, and 26 times greater than non-agricultural sites with prominent seasonal trends driven by temperature. Annual estimated dry deposition rates in areas of intensive dairy production can approach 45 kg N ha−1 y−1, compared to −1 y−1 in natural landscapes. Our results suggest that the natural sagebrush steppe landscapes interspersed within and surrounding agricultural areas in southern Idaho receive NH3 dry deposition rates within and above the range of nitrogen critical loads for North American deserts. Finally, our results highlight a need for improved understanding of the role of soil processes in NH3 dry deposition to arid and sparsely vegetated natural ecosystems across the western US.

Neural tube defects (NTDs), including anencephaly and spina bifida, are common major malformations of fetal development resulting from incomplete closure of the neural tube. These conditions lead to either universal death (anencephaly) or severe lifelong complications (spina bifida). Despite hundreds of genetic mouse models of neural tube defect phenotypes, the genetics of human NTDs are poorly understood. Furthermore, pharmaceuticals, such as antiseizure medications, have been found clinically to increase the risk of NTDs when administered during pregnancy. Therefore, a model that recapitulates human neurodevelopment would be of immense benefit to understand the genetics underlying NTDs and identify teratogenic mechanisms. Using our self-organizing single rosette cortical organoid (SOSR-COs) system, we have developed a high-throughput image analysis pipeline for evaluating the SOSR-CO structure for NTD-like phenotypes. Similar to small molecule inhibition of apical constriction, the antiseizure medication valproic acid (VPA), a known cause of NTDs, increases the apical lumen size and apical cell surface area in a dose-responsive manner. GSK3β and HDAC inhibitors caused similar lumen expansion; however, RNA sequencing suggests VPA does not inhibit GSK3β at these concentrations. The knockout of SHROOM3, a well-known NTD-related gene, also caused expansion of the lumen, as well as reduced f-actin polarization. The increased lumen sizes were caused by reduced cell apical constriction, suggesting that impingement of this process is a shared mechanism for VPA treatment and SHROOM3-KO, two well-known causes of NTDs. Our system allows the rapid identification of NTD-like phenotypes for both compounds and genetic variants and should prove useful for understanding specific NTD mechanisms and predicting drug teratogenicity.

Background We have previously reported that the upregulation of galectin-9 (Gal-9) on CD4+ and CD8+ T cells in HIV patients was associated with impaired T cell effector functions. Gal-9 is a ligand for T cell immunoglobulin and mucin domain-3, and its expression on T cells in cancer has not been investigated. Therefore, we aimed to investigate the expression level and effects of Gal-9 on T cell functions in patients with virus-associated solid tumors (VASTs).Methods 40 patients with VASTs through a non-randomized and biomarker-driven phase II LATENT trial were investigated. Peripheral blood mononuclear cells and tumor biopsies were obtained and subjected to immunophenotyping. In this trial, the effects of oral valproate and avelumab (anti-PD-L1) was investigated in regards to the expression of Gal-9 on T cells.Results We report the upregulation of Gal-9 expression by peripheral and tumor-infiltrating CD4+ and CD8+ T lymphocytes in patients with VASTs. Our results indicate that Gal-9 expression is associated with dysfunctional T cell effector functions in the periphery and tumor microenvironment (TME). Coexpression of Gal-9 with PD-1 or T cell immunoglobulin and ITIM domain (TIGIT) exhibited a synergistic inhibitory effect and enhanced an exhausted T cell phenotype. Besides, responding patients to treatment had lower Gal-9 mRNA expression in the TME. Translocation of Gal-9 from the cytosol to the cell membrane of T cells following stimulation suggests persistent T cell receptor (TCR) stimulation as a potential contributing factor in Gal-9 upregulation in patients with VASTs. Moreover, partial colocalization of Gal-9 with CD3 on T cells likely impacts the initiation of signal transduction via TCR as shown by the upregulation of ZAP70 in Gal-9+ T cells. Also, we found an expansion of Gal-9+ but not TIGIT+ NK cells in patients with VASTs; however, dichotomous to TIGIT+ NK cells, Gal-9+ NK cells exhibited impaired cytotoxic molecules but higher Interferon gamma (IFN-γ) expression.Conclusion Our data indicate that higher Gal-9-expressing CD8+ T cells were associated with poor prognosis following immunotherapy with anti-Programmed death-ligand 1 (PD-L1) (avelumab) in our patients’ cohort. Therefore, for the very first time to our knowledge, we report Gal-9 as a novel marker of T cell exhaustion and the potential target of immunotherapy in patients with VASTs.

The Llanos de Mojos of the Bolivian Amazon is a domesticated landscape with a long history of management by pre-Columbian communities. This paper uses a landscape approach to interpret the settlement patterns of pre-Columbian raised-field farmers in west central Mojos. The pre-Columbian landscape was reconstructed by mapping the distribution of three types of landscape features: forest islands, raised agricultural fields, and water systems (rivers, streams and wetlands). Previous research has identified four types of patterned clustering or ‘constellations’ of these landscape features in west central Mojos. These constellations and the immediate area of the landscape that surrounds them afforded Mojos farmers a specific set of tasks or activities to take part in as part of harnessing resources from the landscape. The mapping of landscape features and their associated tasks onto the landscape provides insight into the organization of the communities that constructed and managed them. It was found that the landscape of west central Mojos is organized into two distinct regional patterns. In the northern part of the region, evidence of large farming communities is dispersed along the banks of the permanent rivers with networks of landscape features extending off into remote areas of the savanna. In the southern part of the region, evidence for large farming communities is clustered closer together in remote areas of the savanna with networks of landscape features extending back towards the permanent rivers. The two regions are melded together by a transitional zone that implies a type of interaction between the regions rather than a distinct separation.

To investigate whether oral administration of maize-produced S antigen can provide passive immunity to piglets against porcine epidemic diarrhea virus (PEDV), 16 pregnant sows were randomly assigned to one of four treatments: (1) injection of PEDV vaccine (INJ), (2) maize grain without S protein (CON), (3) maize grain containing low dose of S antigen (LOV) and (4) maize grain containing a high dose of S antigen (HOV). Vaccines were administered on days 57, 85 and 110 of gestation. Sows’ serum and colostrum were collected at farrowing and milk on day 6 post-challenge to quantify neutralizing antibodies (NABs) and cytokines. Piglets were challenged with PEDV 3–5 d after farrowing, and severity of disease and mortality assessed on day 11 post-challenge. Disease severity was lower in LOV and INJ compared with HOV and CON, whereas the survival rate increased in piglets from LOV sows compared with HOV and CON (p ≤ 0.001). Higher titers of NABs and lower levels of cytokine granulocyte-macrophage colony-stimulating factor in sows’ milk were positively correlated with piglet survivability (p ≤ 0.05). These data suggest that feeding S protein in corn to pregnant sows protects nursing piglets against PEDV.

Neuroplasticity is a robust mechanism by which the central nervous system attempts to adapt to a structural or chemical disruption of functional connections between neurons. Mechanical damage from spinal cord injury potentiates via neuroinflammation and can cause aberrant changes in neural circuitry known as maladaptive plasticity. Together, these alterations greatly diminish function and quality of life. This review discusses contemporary efforts to harness neuroplasticity through rehabilitation and neuromodulation to restore function with a focus on motor recovery following cervical spinal cord injury. Background information on the general mechanisms of plasticity and long-term potentiation of the nervous system, most well studied in the learning and memory fields, will be reviewed. Spontaneous plasticity of the nervous system, both maladaptive and during natural recovery following spinal cord injury is outlined to provide a baseline from which rehabilitation builds. Previous research has focused on the impact of descending motor commands in driving spinal plasticity. However, this review focuses on the influence of physical therapy and primary afferent input and interneuron modulation in driving plasticity within the spinal cord. Finally, future directions into previously untargeted primary afferent populations are presented.