The PRC2.1 Subcomplex Opposes G1 Progression through Regulation of CCND1 and CCND2.

Progression through the G1 phase of the cell cycle is the most highly regulated step in cellular division. We employed a chemogenomics approach to discover novel cellular networks that regulate cell cycle progression. This approach uncovered functional clusters of genes that altered sensitivity of cells to inhibitors of the G1/S transition. Mutation of components of the Polycomb Repressor Complex 2 rescued growth inhibition caused by the CDK4/6 inhibitor palbociclib, but not to inhibitors of S phase or mitosis. In addition to its core catalytic subunits, mutation of the PRC2.1 accessory protein MTF2, but not the PRC2.2 protein JARID2, rendered cells resistant to palbociclib treatment. We found that PRC2.1 (MTF2), but not PRC2.2 (JARID2), was critical for promoting H3K27me3 deposition at CpG islands genome-wide and in promoters. This included the CpG islands in the promoter of the CDK4/6 cyclins CCND1 and CCND2, and loss of MTF2 lead to upregulation of both CCND1 and CCND2. Our results demonstrate a role for PRC2.1, but not PRC2.2, in promoting G1 progression.

Transfluthrin diffusers do not protect two-person US military tents from mosquitoes in open field and canopy warm-temperate habitats.

Spatial repellents are volatile or volatilized chemicals that may repel arthropod vectors in free space, preventing bites and reducing the potential for pathogen transmission. In a 21-week field study, we investigated the efficacy of passive transfluthrin-impregnated diffusers placed in two-person United States (US) military tents located in canopy and open field habitats in north Florida to prevent mosquitoes from entering. Mosquito collections with US Centers for Disease Control and Prevention traps baited with light and carbon dioxide were conducted weekly for weeks 0-4, every two weeks for weeks 5-10, and monthly for weeks 11-21. Our results demonstrated that these transfluthrin-impregnated devices did not function as spatial repellents as expected and did not create a mosquito-free zone of protection. Instead, we observed consistently higher collections of mosquitoes from tents with transfluthrin-impregnated diffusers, and higher rates of mosquito mortality in collections from tents with transfluthrin diffusers, compared to untreated control tents. Based on these findings we do not recommend the use of passive transfluthrin-impregnated diffusers for mosquito protection in two-person US military tents in warm-temperate environments similar to north Florida.

How can we apply decision-making theories to wild animal behavior? Predictions arising from dual process theory and Bayesian decision theory.

Our understanding of decision-making processes and cognitive biases is ever increasing, thanks to an accumulation of testable models and a large body of research over the last several decades. The vast majority of this work has been done in humans and laboratory animals because these study subjects and situations allow for tightly controlled experiments. However, it raises questions about how this knowledge can be applied to wild animals in their complex environments. Here, we review two prominent decision-making theories, dual process theory and Bayesian decision theory, to assess the similarities in these approaches and consider how they may apply to wild animals living in heterogenous environments within complicated social groupings. In particular, we wanted to assess when wild animals are likely to respond to a situation with a quick heuristic decision and when they are likely to spend more time and energy on the decision-making process. Based on the literature and evidence from our multi-destination routing experiments on primates, we find that individuals are likely to make quick, heuristic decisions when they encounter routine situations, or signals/cues that accurately predict a certain outcome, or easy problems that experience or evolutionary history has prepared them for. Conversely, effortful decision-making is likely in novel or surprising situations, when signals and cues have unpredictable or uncertain relationships to an outcome, and when problems are computationally complex. Though if problems are overly complex, satisficing via heuristics is likely, to avoid costly mental effort. We present hypotheses for how animals with different socio-ecologies may have to distribute their cognitive effort. Finally, we examine the conservation implications and potential cognitive overload for animals experiencing increasingly novel situations caused by current human-induced rapid environmental change.

Relating white matter microstructure in theoretically defined addiction networks to relapse in alcohol use disorder.

Theoretical models group maladaptive behaviors in addiction into neurocognitive domains such as incentive salience (IS), negative emotionality (NE), and executive functioning (EF). Alterations in these domains lead to relapse in alcohol use disorder (AUD). We examine whether microstructural measures in the white matter pathways supporting these domains are associated with relapse in AUD. Diffusion kurtosis imaging data were collected from 53 individuals with AUD during early abstinence. We used probabilistic tractography to delineate the fornix (IS), uncinate fasciculus (NE), and anterior thalamic radiation (EF) in each participant and extracted mean fractional anisotropy (FA) and kurtosis fractional anisotropy (KFA) within each tract. Binary (abstained vs. relapsed) and continuous (number of days abstinent) relapse measures were collected over a 4-month period. Across tracts, anisotropy measures were typically (i) lower in those that relapsed during the follow-up period and (ii) positively associated with the duration of sustained abstinence during the follow-up period. However, only KFA in the right fornix reached significance in our sample. The association between microstructural measures in these fiber tracts and treatment outcome in a small sample highlights the potential utility of the three-factor model of addiction and the role of white matter alterations in AUD.

Exploring the Influence of Summer Temperature on Human Mobility During the COVID-19 Pandemic in the San Francisco Bay Area.

Studies on the relationship between temperature and local, small scale mobility are limited, and sensitive to the region and time period of interest. We contribute to the growing mobility literature through a detailed characterization of the observed temperature-mobility relationship in the San Francisco Bay Area at fine spatial and temporal scale across two summers (2020-2021). We used anonymized cellphone data from SafeGraph's neighborhood patterns data set and gridded temperature data from gridMET, and analyzed the influence of incremental changes in temperature on mobility rate (i.e., visits per capita) using a panel regression with fixed effects. This strategy enabled us to control for spatial and temporal variability across the studied region. Our analysis suggested that all areas exhibited lower mobility rate in response to higher summer temperatures. We then explored how several additional variables altered these results. Extremely hot days resulted in faster mobility declines with increasing temperatures. Weekdays were often more resistant to temperature changes when compared to the weekend. In addition, the rate of decrease in mobility in response to high temperature was significantly greater among the wealthiest census block groups compared with the least wealthy. Further, the least mobile locations experienced significant differences in mobility response compared to the rest of the data set. Given the fundamental differences in the mobility response to temperature across most of our additive variables, our results are relevant for future mobility studies in the region.

LIN-35 is necessary in both the soma and germline for preserving fertility in Caenorhabditis elegans under moderate temperature stress.

Maintenance of germline function under stress conditions is crucial for species survival. The germ line in many species is especially sensitive to elevated temperature. We have investigated the role of the pocket protein LIN-35 in preserving fertility in Caenorhabditis elegans under moderate temperature stress. We show that lin-35 mutants display several temperature sensitive germline defects, and more severe reductions in brood size at elevated temperatures compared to wild type. This loss of fertility under temperature stress is primarily due to loss of zygotic, but not maternal, LIN-35. Additionally, we have found that expression of LIN-35 is necessary in both the germ line and soma for the preserving fertility under moderate temperature stress. Specifically, while LIN-35 function in the germ line is required for maintaining fertility in hermaphrodites, broad somatic expression of LIN-35 is also necessary for oocyte formation and/or function under moderate temperature stress. Together, our data add to the emerging understanding of the critical role that LIN-35 plays in preserving tissues against stress.

Lethal neonatal respiratory failure due to biallelic variants in BBS1 and monoallelic variant in TTC21B.

BACKGROUND: Bardet-Biedl syndrome (BBS) is a rare, autosomal recessive ciliopathy characterized by early onset retinal dystrophy, renal anomalies, postaxial polydactyly, and cognitive impairment with considerable phenotypic heterogeneity. BBS results from biallelic pathogenic variants in over 20 genes that encode key proteins required for the assembly or primary ciliary functions of the BBSome, a heterooctameric protein complex critical for homeostasis of primary cilia. While variants in BBS1 are most frequently identified in affected individuals, the renal and pulmonary phenotypes associated with BBS1 variants are reportedly less severe than those seen in affected individuals with pathogenic variants in the other BBS-associated genes. CASE-DIAGNOSIS: We report an infant with severe renal dysplasia and lethal pulmonary hypoplasia who was homozygous for the most common BBS1 pathogenic variant (c.1169 T > G; p.M390R) and also carried a predicted pathogenic variant in TTC21B (c.1846C > T; p.R616C), a genetic modifier of disease severity of ciliopathies associated with renal dysplasia and pulmonary hypoplasia. CONCLUSIONS: This report expands the phenotypic spectrum of BBS with the first infant with lethal neonatal respiratory failure associated with biallelic, pathogenic variants in BBS1 and a monoallelic, predicted pathogenic variant in TTC21B. BBS should be considered among the ciliopathies in the differential diagnosis of neonates with renal dysplasia and severe respiratory failure.

Reduced T Cell Priming in Microbially Experienced "Dirty" Mice Results from Limited IL-27 Production by XCR1+ Dendritic Cells.

Successful vaccination strategies offer the potential for lifelong immunity against infectious diseases and cancer. There has been increased attention regarding the limited translation of some preclinical findings generated using specific pathogen-free (SPF) laboratory mice to humans. One potential reason for the difference between preclinical and clinical findings lies in maturation status of the immune system at the time of challenge. In this study, we used a "dirty" mouse model, where SPF laboratory mice were cohoused (CoH) with pet store mice to permit microbe transfer and immune system maturation, to investigate the priming of a naive T cell response after vaccination with a peptide subunit mixed with polyinosinic-polycytidylic acid and agonistic anti-CD40 mAb. Although this vaccination platform induced robust antitumor immunity in SPF mice, it failed to do so in microbially experienced CoH mice. Subsequent investigation revealed that despite similar numbers of Ag-specific naive CD4 and CD8 T cell precursors, the expansion, differentiation, and recall responses of these CD4 and CD8 T cell populations in CoH mice were significantly reduced compared with SPF mice after vaccination. Evaluation of the dendritic cell compartment revealed reduced IL-27p28 expression by XCR1+ dendritic cells from CoH mice after vaccination, correlating with reduced T cell expansion. Importantly, administration of recombinant IL-27:EBI3 complex to CoH mice shortly after vaccination significantly boosted Ag-specific CD8 and CD4 T cell expansion, further implicating the defect to be T cell extrinsic. Collectively, our data show the potential limitation of exclusive use of SPF mice when testing vaccine efficacy.

Spatial snapshots of amyloid precursor protein intramembrane processing via early endosome proteomics.

Degradation and recycling of plasma membrane proteins occurs via the endolysosomal system, wherein endosomes bud into the cytosol from the plasma membrane and subsequently mature into degradative lysosomal compartments. While methods have been developed for rapid selective capture of lysosomes (Lyso-IP), analogous methods for isolation of early endosome intermediates are lacking. Here, we develop an approach for rapid isolation of early/sorting endosomes through affinity capture of the early endosome-associated protein EEA1 (Endo-IP) and provide proteomic and lipidomic snapshots of EEA1-positive endosomes in action. We identify recycling, regulatory and membrane fusion complexes, as well as candidate cargo, providing a proteomic landscape of early/sorting endosomes. To demonstrate the utility of the method, we combined Endo- and Lyso-IP with multiplexed targeted proteomics to provide a spatial digital snapshot of amyloid precursor protein (APP) processing by ß and γ-Secretases, which produce amyloidogenic Aß species, and quantify small molecule modulation of Secretase action on endosomes. We anticipate that the Endo-IP approach will facilitate systematic interrogation of processes that are coordinated on EEA1-positive endosomes.

SH3-domain mutations selectively disrupt Csk homodimerization or PTPN22 binding.

The kinase Csk is the primary negative regulator of the Src-family kinases (SFKs, e.g., Lck, Fyn, Lyn, Hck, Fgr, Blk, Yes), phosphorylating a tyrosine on the SFK C-terminal tail that mediates autoinhibition. Csk also binds phosphatases, including PTPN12 (PTP-PEST) and immune-cell PTPN22 (LYP/Pep), which dephosphorylate the SFK activation loop to promote autoinhibition. Csk-binding proteins (e.g., CBP/PAG1) oligomerize within membrane microdomains, and high local concentration promotes Csk function. Purified Csk homodimerizes in solution through an interface that overlaps the phosphatase binding footprint. Here we demonstrate that Csk can homodimerize in Jurkat T cells, in competition with PTPN22 binding. We designed SH3-domain mutations in Csk that selectively impair homodimerization (H21I) or PTPN22 binding (K43D) and verified their kinase activity in solution. Disruption of either interaction in cells, however, decreased the negative-regulatory function of Csk. Csk W47A, a substitution previously reported to block PTPN22 binding, had a secondary effect of impairing homodimerization. Csk H21I and K43D will be useful tools for dissecting the protein-specific drivers of autoimmunity mediated by the human polymorphism PTPN22 R620W, which impairs interaction with Csk and with the E3 ubiquitin ligase TRAF3. Future investigations of Csk homodimer activity and phosphatase interactions may reveal new facets of SFK regulation in hematopoietic and non-hematopoietic cells.

Lysoptosis is an evolutionarily conserved cell death pathway moderated by intracellular serpins.

Lysosomal membrane permeabilization (LMP) and cathepsin release typifies lysosome-dependent cell death (LDCD). However, LMP occurs in most regulated cell death programs suggesting LDCD is not an independent cell death pathway, but is conscripted to facilitate the final cellular demise by other cell death routines. Previously, we demonstrated that Caenorhabditis elegans (C. elegans) null for a cysteine protease inhibitor, srp-6, undergo a specific LDCD pathway characterized by LMP and cathepsin-dependent cytoplasmic proteolysis. We designated this cell death routine, lysoptosis, to distinguish it from other pathways employing LMP. In this study, mouse and human epithelial cells lacking srp-6 homologues, mSerpinb3a and SERPINB3, respectively, demonstrated a lysoptosis phenotype distinct from other cell death pathways. Like in C. elegans, this pathway depended on LMP and released cathepsins, predominantly cathepsin L. These studies suggested that lysoptosis is an evolutionarily-conserved eukaryotic LDCD that predominates in the absence of neutralizing endogenous inhibitors.

Assessing transfluthrin mortality against Aedes aegypti and Culex quinquefasciatus inside and outside US military tents in a northern Florida environment.

Mortality caused by passive resin transfluthrin diffusers (∼5 mg AI per 24 h release rate) suspended in small 2-person tents was measured for colony-reared sentinel pyrethroid susceptible Aedes aegypti and Culex quinquefasciatus female mosquitoes, as well as a pyrethroid-resistant strain of Aedes aegypti, in a USA military field camp scenario. Mortality effects were investigated for impact by factors such as sentinel cage location (inside tent, tent doorway and outside tent), exposure time (15, 30, 45 and 60 min), and environmental temperature (°C), all of which were examined over an 8-week period. Analyses determined there was a significant interaction between mosquito strain and transfluthrin susceptibility, with the two susceptible strains experiencing significantly greater mean mortality than the resistant Ae. aegypti strain. Significant differences were likewise observed between the mosquito strains over the 8-week study period, where study week and temperature were both positively correlated with an increase in observed mean mosquito mortality. Mosquito proximity to the transfluthrin diffusers was also influenced by week and showed that sentinel cage placement in the environment demonstrates different mortality measurements, depending on the environmental conditions. The length of exposure to transfluthrin, however, was determined to not significantly impact transfluthrin efficacy on the examined mosquito strains, although increased exposure did result in increased susceptible strain mortality. These results suggest that transfluthrin is highly effective in causing mortality against susceptible Ae. aegypti and Cx. quinquefasciatus mosquitoes under field conditions but is minimally effective against pyrethroid-resistant Ae. aegypti mosquitoes. Transfluthrin-infused devices are influenced by environmental factors that can combine to impact mosquito mortality in the field.

Medicinal Plants in Traditional Herbal Wines and Liquors in the East of Spain and the Balearic Islands.

Homemade herbal preparations from the East of Spain are the witness of traditional medicine inherited from the ancient complex formulas of herbal teas and medicinal wines. In this study, we document the use of traditional alcoholic beverages, identify their ingredients, almost exclusively botanical, record the local medicinal uses of these mixtures, and discuss patterns of distribution of this knowledge in regions of eastern Spain, the Balearic Islands and Andorra. We determine marker species and relevant patterns of herbal formulas in the different regions of the territory. Homemade liquors and liqueurs are consumed for their digestive and tonic-restorative properties but they also play in some cases an important social role. The elderly remember other medicinal uses such as aperitif, emmenagogue, or antidiarrheal, for some of the most popular preparations. The herbal liqueur formulas include predominantly Lamiaceae, Asteraceae, Rosaceae, Rutaceae, and Apiaceae species. Herbs (58%), fruits (28%), and mixtures of both (12%) are ingredients of liquors and wines, being the aerial parts the most frequent in terms of species (30%) and records (49%). Dictamnus hispanicus, Santolina villosa, Salvia blancoana subsp. mariolensis, Rosmarinus officinalis, Thymus vulgaris, and Clinopodium serpyllifolium subsp. fruticosum are the species most frequently used. Others species used to a lesser extent as Polygonatum odoratum, Thymus moroderi, and Saxifraga longifolia are restricted to locally homemade preparations because their collection and uses require special knowledge of the rare or endemic flora. Sustainability of these practices is strongly limited by the overall loss of local traditional knowledge and by the limited availability of most of the wild species; some of them are endangered or threatened mainly by the loss of their natural habitats. Cultivation and domestication are a promising alternative to collecting from wild populations. The cultivation of Thymus moroderi in the province of Alicante and Polygonatum odoratum in the province of Teruel are good examples. There is a notable decrease in the complexity of the formulas registered throughout the nearly 15 years of the study. This is interpreted as a consequence of a loss of knowledge, less accessibility to wild resources, and changes in traditions and preferences.

Severity of Sepsis Determines the Degree of Impairment Observed in Circulatory and Tissue-Resident Memory CD8 T Cell Populations.

Sepsis reduces the number and function of memory CD8 T cells within the host, contributing to the long-lasting state of immunoparalysis. Interestingly, the relative susceptibility of memory CD8 T cell subsets to quantitative/qualitative changes differ after cecal ligation and puncture (CLP)-induced sepsis. Compared with circulatory memory CD8 T cells (TCIRCM), moderate sepsis (0-10% mortality) does not result in numerical decline of CD8 tissue-resident memory T cells (TRM), which retain their "sensing and alarm" IFN-γ-mediated effector function. To interrogate this biologically important dichotomy, vaccinia virus-immune C57BL/6 (B6) mice containing CD8 TCIRCM and skin TRM underwent moderate or severe (∼50% mortality) sepsis. Severe sepsis led to increased morbidity and mortality characterized by increased inflammation compared with moderate CLP or sham controls. Severe CLP mice also displayed increased vascular permeability in the ears. Interestingly, skin CD103+ CD8 TRM, detected by i.v. exclusion or two-photon microscopy, underwent apoptosis and subsequent numerical loss following severe sepsis, which was not observed in mice that experienced moderate CLP or sham surgeries. Consequently, severe septic mice showed diminished CD8 T cell-mediated protection to localized skin reinfection. Finally, the relationship between severity of sepsis and demise in circulatory versus tissue-embedded memory CD8 T cell populations was confirmed by examining tumor-infiltrating and nonspecific CD8 T cells in B16 melanoma tumors. Thus, sepsis can differentially affect the presence and function of Ag-specific CD8 T cells that reside inside tissues/tumors depending on the severity of the insult, a notion with direct relevance to sepsis survivors and their ability to mount protective memory CD8 T cell-dependent responses to localized Ag re-encounter.

Chemical-genetic CRISPR-Cas9 screens in human cells using a pathway-specific library.

The development of CRISPR-Cas9 screening techniques coupled with chemical inhibition of specific biological processes enables high-throughput investigation into many areas of molecular biology. We present a protocol to conduct ubiquitin proteasome system-specific chemical-genetic CRISPR-Cas9 screens in the human HAP1 cell line. This protocol can be adapted for use in other cell lines, with other compounds and types of treatments, and with any other sgRNA library. For complete details on the use and execution of this protocol, please refer to Hundley et al. (2021).

The common ABCA3E292V variant disrupts AT2 cell quality control and increases susceptibility to lung injury and aberrant remodeling.

ATP-binding cassette class A3 (ABCA3) is a lipid transporter that plays a critical role in pulmonary surfactant function. The substitution of valine for glutamic acid at codon 292 (E292V) produces a hypomorphic variant that accounts for a significant portion of ABCA3 mutations associated with lung disorders spanning from neonatal respiratory distress syndrome and childhood interstitial lung disease to diffuse parenchymal lung disease (DPLD) in adults including pulmonary fibrosis. The mechanisms by which this and similar ABCA3 mutations disrupt alveolar type 2 (AT2) cell homeostasis and cause DPLD are largely unclear. The present study, informed by a patient homozygous for the E292V variant, used an in vitro and a preclinical murine model to evaluate the mechanisms by which E292V expression promotes aberrant lung injury and parenchymal remodeling. Cell lines stably expressing enhanced green fluorescent protein (EGFP)-tagged ABCA3 isoforms show a functional deficiency of the ABCA3E292V variant as a lipid transporter. AT2 cells isolated from mice constitutively homozygous for ABCA3E292V demonstrate the presence of small electron-dense lamellar bodies, time-dependent alterations in macroautophagy, and induction of apoptosis. These changes in AT2 cell homeostasis are accompanied by a spontaneous lung phenotype consisting of both age-dependent inflammation and fibrillary collagen deposition in alveolar septa. Older ABCA3E292V mice exhibit increased vulnerability to exogenous lung injury by bleomycin. Collectively, these findings support the hypothesis that the ABCA3E292V variant is a susceptibility factor for lung injury through effects on surfactant deficiency and impaired AT2 cell autophagy.

Biallelic ASCC1 variants including a novel intronic variant result in expanded phenotypic spectrum of spinal muscular atrophy with congenital bone fractures 2 (SMABF2).

Spinal muscular atrophy with congenital bone fractures 2 (SMABF2), a type of arthrogryposis multiplex congenita (AMC), is characterized by congenital joint contractures, prenatal fractures of long bones, and respiratory distress and results from biallelic variants in ASCC1. Here, we describe an infant with severe, diffuse hypotonia, congenital contractures, and pulmonary hypoplasia characteristic of SMABF2, with the unique features of cleft palate, small spleen, transverse liver, and pulmonary thromboemboli with chondroid appearance. This infant also had impaired coagulation with diffuse petechiae and ecchymoses which has only been reported in one other infant with AMC. Using trio whole genome sequencing, our proband was identified to have biallelic variants in ASCC1. Using deep next generation sequencing of parental cDNA, we characterized alteration of splicing encoded by the novel, maternally inherited ASCC1 variant (c.297-8 T > G) which provides a mechanism for functional pathogenicity. The paternally inherited ASCC1 variant is a rare nonsense variant (c.466C > T; p.Arg156*) that has been previously identified in one other infant with AMC. This report extends the phenotypic characteristics of ASCC1-associated AMC (SMABF2) and describes a novel intronic variant that partially disrupts RNA splicing.

Temporal patterns in the social network of core units in Rwenzori Angolan colobus monkeys: Effects of food availability and interunit dispersal.

Multi-level societies are complex, nested social systems where basic social groups (i.e., core units) associate in a hierarchical manner, allowing animals to adjust their group sizes in response to variables such as food availability, predation, or conspecific threat. These pressures fluctuate over time and examining the extent to which this variation affects the clustering of core units into different tiers may be instrumental in understanding the evolution of multi-level societies.The goal of our study was to determine the degree of temporal variability in interunit associations in a multi-level society of Rwenzori Angolan colobus monkey (Colobus angolensis ruwenzorii), and to determine the social and ecological factors that underlie association patterns. The C. a. ruwenzorii multi-level society consists of at least three tiers, with core units clustering into clans that share a home range in a band tier.We performed social network analyses on 21 months of association data from 13 core units (totaling 139 identifiable individuals) at Lake Nabugabo, Uganda. We described the patterns of variation in core-unit associations over time and investigated how changes in rainfall, food availability, and interunit dispersals were correlated with these associations over the short-term (month to month) and long-term (year to year).Although clans were relatively stable, larger-scale changes in association patterns included the formation of an all-male unit and the transfer of one core unit between clans (within the band tier). Seasonally, core units associated significantly more when fruit, their preferred food source, was abundant (i.e., social networks were denser and more clustered) and there was no direct effect of rainfall seasonality or young leaf availability. Male dispersals also occurred more during periods of high fruit availability, suggesting that greater band cohesion allowed males to prospect and transfer between core units. Once males transferred, their previous and new units associated significantly more with one another than with other core units for 1-2 months postdispersal. The dispersal of five males from one core unit to another in a different clan co-occurred with this core unit switching its clan affiliation.By examining temporal shifts in social network structure among core units, this study shows the interconnected roles that food availability and dispersal have in shaping the C. a. ruwenzorii multi-level social system. Our findings highlight how ecological conditions can drive association patterns, impact interunit relationships, and influence social organization.