Individual and Community level Developmental Adversities: Associations with Marijuana and Alcohol Use in Late-Adolescents and Young Adults.

Exposure to community and individual level stressors during adolescence has been reported to be associated with increased substance use. However, it remains unclear what the relative contribution of different community- and individual-level factors play when alcohol and marijuana use become more prevalent during late adolescence. The present study uses a large longitudinal sample of adolescents (Wave 1: N = 2017; 55% Female; 54.5% White, 22.3% Black, 8% Hispanic, 15% other) to evaluate the association and potential interactions between community- and individual-level factors and substance use from adolescence to young adulthood (Wave 1 to Wave 3 Age Mean [SD]: 16.7 [1.1], 18.3 [1.2], 19.3 [1.2]). Across three waves of data, multilevel modeling (MLM) is used to evaluate the association between community affluence and disadvantage, individual household socioeconomic status (SES, measured as parental level of education and self-reported public assistance) and self-reported childhood maltreatment with self-reported 12-month alcohol and 12-month marijuana use occasions. Sample-selection weights and attrition-adjusted weights are accounted for in the models to evaluate the robustness of the estimated effects. Across the MLMs, there is a significant positive association between community affluence and parental education with self-reported alcohol use but not self-reported marijuana use. In post hoc analyses, higher neighborhood affluence in older adolescents is associated with higher alcohol use and lower use in younger adolescents; the opposite association is found for neighborhood disadvantage. Consistent with past literature, there is a significant positive association between self-reported childhood maltreatment and self-reported 12-month alcohol and 12-month marijuana use. Results are largely consistent across weighted and unweighted analyses, however, in weighted analyses there is a significant negative association between community disadvantage and self-reported 12-month alcohol use. This study demonstrates a nuanced relationship between community- and individual-level factors and substance use during the transitional window of adolescence which should be considered when contextualizing and interpreting normative substance use during adolescence.

A conserved isoleucine in the binding pocket of RIG-I controls immune tolerance to mitochondrial RNA.

RIG-I is a cytosolic receptor of viral RNA essential for the immune response to numerous RNA viruses. Accordingly, RIG-I must sensitively detect viral RNA yet tolerate abundant self-RNA species. The basic binding cleft and an aromatic amino acid of the RIG-I C-terminal domain(CTD) mediate high-affinity recognition of 5'triphosphorylated and 5'base-paired RNA(dsRNA). Here, we found that, while 5'unmodified hydroxyl(OH)-dsRNA demonstrated residual activation potential, 5'-monophosphate(5'p)-termini, present on most cellular RNAs, prevented RIG-I activation. Determination of CTD/dsRNA co-crystal structures and mutant activation studies revealed that the evolutionarily conserved I875 within the CTD sterically inhibits 5'p-dsRNA binding. RIG-I(I875A) was activated by both synthetic 5'p-dsRNA and endogenous long dsRNA within the polyA-rich fraction of total cellular RNA. RIG-I(I875A) specifically interacted with long, polyA-bearing, mitochondrial(mt) RNA, and depletion of mtRNA from total RNA abolished its activation. Altogether, our study demonstrates that avoidance of 5'p-RNA recognition is crucial to prevent mtRNA-triggered RIG-I-mediated autoinflammation.

Tumor-derived GDF-15 blocks LFA-1 dependent T cell recruitment and suppresses responses to anti-PD-1 treatment.

Immune checkpoint blockade therapy is beneficial and even curative for some cancer patients. However, the majority don't respond to immune therapy. Across different tumor types, pre-existing T cell infiltrates predict response to checkpoint-based immunotherapy. Based on in vitro pharmacological studies, mouse models and analyses of human melanoma patients, we show that the cytokine GDF-15 impairs LFA-1/ß2-integrin-mediated adhesion of T cells to activated endothelial cells, which is a pre-requisite of T cell extravasation. In melanoma patients, GDF-15 serum levels strongly correlate with failure of PD-1-based immune checkpoint blockade therapy. Neutralization of GDF-15 improves both T cell trafficking and therapy efficiency in murine tumor models. Thus GDF-15, beside its known role in cancer-related anorexia and cachexia, emerges as a regulator of T cell extravasation into the tumor microenvironment, which provides an even stronger rationale for therapeutic anti-GDF-15 antibody development.

Organization and replicon interactions within the highly segmented genome of Borrelia burgdorferi.

Borrelia burgdorferi, a causative agent of Lyme disease, contains the most segmented bacterial genome known to date, with one linear chromosome and over twenty plasmids. How this unusually complex genome is organized, and whether and how the different replicons interact are unclear. We recently demonstrated that B. burgdorferi is polyploid and that the copies of the chromosome and plasmids are regularly spaced in each cell, which is critical for faithful segregation of the genome to daughter cells. Regular spacing of the chromosome is controlled by two separate partitioning systems that involve the protein pairs ParA/ParZ and ParB/Smc. Here, using chromosome conformation capture (Hi-C), we characterized the organization of the B. burgdorferi genome and the interactions between the replicons. We uncovered that although the linear chromosome lacks contacts between the two replication arms, the two telomeres are in frequent contact. Moreover, several plasmids specifically interact with the chromosome oriC region, and a subset of plasmids interact with each other more than with others. We found that Smc and the Smc-like MksB protein mediate long-range interactions on the chromosome, but they minimally affect plasmid-chromosome or plasmid-plasmid interactions. Finally, we found that disruption of the two partition systems leads to chromosome restructuring, correlating with the mis-positioning of chromosome oriC. Altogether, this study revealed the conformation of a complex genome and analyzed the contribution of the partition systems and SMC family proteins to this organization. This work expands the understanding of the organization and maintenance of multipartite bacterial genomes.

Organization and replicon interactions within the highly segmented genome of Borrelia burgdorferi.

Borrelia burgdorferi , a causative agent of Lyme disease, contains the most segmented bacterial genome known to date, with one linear chromosome and over twenty plasmids. How this unusually complex genome is organized, and whether and how the different replicons interact are unclear. We recently demonstrated that B. burgdorferi is polyploid and that the copies of the chromosome and plasmids are regularly spaced in each cell, which is critical for faithful segregation of the genome to daughter cells. Regular spacing of the chromosome is controlled by two separate partitioning systems that involve the protein pairs ParA/ParZ and ParB/SMC. Here, using chromosome conformation capture (Hi-C), we characterized the organization of the B. burgdorferi genome and the interactions between the replicons. We uncovered that although the linear chromosome lacks contacts between the two replication arms, the two telomeres are in frequent contact. Moreover, several plasmids specifically interact with the chromosome oriC region, and a subset of plasmids interact with each other more than with others. We found that SMC and the SMC-like MksB protein mediate long-range interactions on the chromosome, but they minimally affect plasmid-chromosome or plasmid-plasmid interactions. Finally, we found that disruption of the two partition systems leads to chromosome restructuring, correlating with the mis-positioning of chromosome oriC . Altogether, this study revealed the conformation of a complex genome and analyzed the contribution of the partition systems and SMC family proteins to this organization. This work expands the understanding of the organization and maintenance of multipartite bacterial genomes.

Perceptual Grouping in Autism Spectrum Disorder: An Exploratory Magnetoencephalography Study.

Visual information is organised according to visual grouping principles. In visual grouping tasks individuals with ASD have shown equivocal performance. We explored neural correlates of Gestalt grouping in individuals with and without ASD. Neuromagnetic activity of individuals with (15) and without (18) ASD was compared during a visual grouping task testing grouping by proximity versus similarity. Individuals without ASD showed stronger evoked responses with earlier peaks in response to both grouping types indicating an earlier neuronal differentiation between grouping principles in individuals without ASD. In contrast, individuals with ASD showed particularly prolonged processing of grouping by similarity suggesting a high demand of neural resources. The neuronal processing differences found could explain less efficient grouping performance observed behaviourally in ASD.

Polyploidy, regular patterning of genome copies, and unusual control of DNA partitioning in the Lyme disease spirochete.

Borrelia burgdorferi, the tick-transmitted spirochete agent of Lyme disease, has a highly segmented genome with a linear chromosome and various linear or circular plasmids. Here, by imaging several chromosomal loci and 16 distinct plasmids, we show that B. burgdorferi is polyploid during growth in culture and that the number of genome copies decreases during stationary phase. B. burgdorferi is also polyploid inside fed ticks and chromosome copies are regularly spaced along the spirochete's length in both growing cultures and ticks. This patterning involves the conserved DNA partitioning protein ParA whose localization is controlled by a potentially phage-derived protein, ParZ, instead of its usual partner ParB. ParZ binds its own coding region and acts as a centromere-binding protein. While ParA works with ParZ, ParB controls the localization of the condensin, SMC. Together, the ParA/ParZ and ParB/SMC pairs ensure faithful chromosome inheritance. Our findings underscore the plasticity of cellular functions, even those as fundamental as chromosome segregation.

Connecting single-cell ATP dynamics to overflow metabolism, cell growth, and the cell cycle in Escherichia coli.

Adenosine triphosphate (ATP) is an abundant and essential metabolite that cells consume and regenerate in large amounts to support growth. Although numerous studies have inferred the intracellular concentration of ATP in bacterial cultures, what happens in individual bacterial cells under stable growth conditions is less clear. Here, we use the QUEEN-2m biosensor to quantify ATP dynamics in single Escherichia coli cells in relation to their growth rate, metabolism, cell cycle, and cell lineage. We find that ATP dynamics are more complex than expected from population studies and are associated with growth-rate variability. Under stable nutrient-rich condition, cells can display large fluctuations in ATP level that are partially coordinated with the cell cycle. Abrogation of aerobic acetate fermentation (overflow metabolism) through genetic deletion considerably reduces both the amplitude of ATP level fluctuations and the cell-cycle trend. Similarly, growth in media in which acetate fermentation is lower or absent results in the reduction of ATP level fluctuation and cell-cycle trend. This suggests that overflow metabolism exhibits temporal dynamics, which contributes to fluctuating ATP levels during growth. Remarkably, at the single-cell level, growth rate negatively correlates with the amplitude of ATP fluctuation for each tested condition, linking ATP dynamics to growth-rate heterogeneity in clonal populations. Our work highlights the importance of single-cell analysis in studying metabolism and its implication to phenotypic diversity and cell growth.

Brief Report: Specificity of Interpersonal Synchrony Deficits to Autism Spectrum Disorder and Its Potential for Digitally Assisted Diagnostics.

Reliably diagnosing autism spectrum disorders (ASD) in adulthood poses a challenge to clinicians due to the absence of specific diagnostic markers. This study investigated the potential of interpersonal synchrony (IPS), which has been found to be reduced in ASD, to augment the diagnostic process. IPS was objectively assessed in videos of diagnostic interviews in a representative referral population from two specialized autism outpatient clinics. In contrast to the current screening tools that could not reliably differentiate, we found a significant reduction of IPS in interactions with individuals later diagnosed with ASD (n = 16) as opposed to those not receiving a diagnosis (n = 23). While these findings need to be validated in larger samples, they nevertheless underline the potential of digitally-enhanced diagnostic processes for ASD.

Loss of Lymphotoxin Alpha-Expressing Memory B Cells Correlates with Metastasis of Human Primary Melanoma.

Activated antigen-experienced B cells play an unexpected complex role in anti-tumor immunity in human melanoma patients. However, correlative studies between B cell infiltration and tumor progression are limited by the lack of distinction between functional B cell subtypes. In this study, we examined a series of 59 primary and metastatic human cutaneous melanoma specimens with B cell infiltration. Using seven-color multiplex immunohistochemistry and automated tissue imaging and analysis, we analyzed the spatiotemporal dynamics of three major antigen-experienced B cell subpopulations expressing lymphotoxin alpha (LTA/TNFSF1) or interleukin-10 (IL-10) outside tertiary lymphoid structures. The expression of both LTA and IL-10 was not restricted to a particular B cell subtype. In primary melanomas, these cells were predominantly found at the invasive tumor-stroma front and, in metastatic melanomas, they were also found in the intratumoral stroma. In primary melanomas, decreased densities of LTA+ memory-like and, to a lesser extent, activated B cells were associated with metastasis. Compared with metastatic primary tumors, B cell infiltrates in melanoma metastases were enriched in both LTA+ memory-like and LTA+ activated B cells, but not in any of the IL-10+ B cell subpopulations. Melanoma disease progression shows distinct dynamics of functional B cell subpopulations, with the regulation of LTA+ B cell numbers being more significant than IL-10+ B cell subpopulations.

A Standardized Analysis of Tertiary Lymphoid Structures in Human Melanoma: Disease Progression- and Tumor Site-Associated Changes With Germinal Center Alteration.

There is increasing evidence that tertiary lymphoid structures (TLS) control not only local adaptive B cell responses at melanoma tumor sites but also the cellular composition and function of other immune cells. In human melanoma, however, a comprehensive analysis of TLS phenotypes, density and spatial distribution at different disease stages is lacking. Here we used 7-color multiplex immunostaining of whole tissue sections from 103 human melanoma samples to characterize TLS phenotypes along the expression of established TLS-defining molecular and cellular components. TLS density and spatial distribution were determined by referring TLS counts to the tissue area within defined intra- and extratumoral perimeters around the invasive tumor front. We show that only a subgroup of primary human melanomas contains TLS. These TLS rarely formed germinal centers and mostly located intratumorally within 1 mm distance to the invasive tumor front. In contrast, melanoma metastases had a significantly increased density of secondary follicular TLS. They appeared preferentially in stromal areas within an extratumoral 1 mm distance to the invasive tumor front and their density varied over time and site of metastasis. Interestingly, secondary follicular TLS in melanoma often lacked BCL6+ lymphatic cells and canonical germinal center polarity with the formation of dark and light zone areas. Our work provides an integrated qualitative, quantitative and spatial analysis of TLS in human melanoma and shows disease progression- and site-associated changes in TLS phenotypes, density and spatial distribution. The frequent absence of canonical germinal center polarity in melanoma TLS highlights the induction of TLS maturation as a potential additive to future immunotherapy studies. Given the variable evaluation strategies used in previous TLS studies of human tumors, an important asset of this study is the standardized quantitative evaluation approach that provides a high degree of reproducibility.

Spatiotemporal Analysis of B Cell- and Antibody Secreting Cell-Subsets in Human Melanoma Reveals Metastasis-, Tumor Stage-, and Age-Associated Dynamics.

Background: The role of tumor-associated B cells in human cancer is only starting to emerge. B cells typically undergo a series of developmental changes in phenotype and function, however, data on the composition of the B cell population in human melanoma are largely absent including changes during tumor progression and their potential clinical significance. Methods: In this study, we compared the number and distribution of six major B cell and antibody secreting cell subpopulations outside tertiary lymphoid structures in whole tumor sections of 154 human cutaneous melanoma samples (53 primary tumors without subsequent metastasis, 44 primary tumors with metastasis, 57 metastatic samples) obtained by seven color multiplex immunohistochemistry and automated tissue imaging and analysis. Results: In primary melanomas, we observed the highest numbers for plasmablast-like, memory-like, and activated B cell subtypes. These cells showed a patchy, predominant paratumoral distribution at the invasive tumor-stroma margin. Plasma cell-like cells were hardly detected, germinal center- and transitional/regulatory-like B cells not at all. Of the major clinicopathologic prognostic factors for primary melanomas, metastasis was associated with decreased memory-like B cell numbers and a higher age associated with higher plasmablast-like cell numbers. When we compared the composition of B cell subpopulations in primary melanomas and metastatic samples, we found a significantly higher proportion of plasma cell-like cells at distant metastatic sites and a higher proportion of memory-like B cells at locoregional than distant metastatic sites. Both cell types were detected mainly in the para- and intratumoral stroma. Conclusion: These data provide a first comprehensive and comparative spatiotemporal analysis of major B cell and antibody secreting cell subpopulations in human melanoma and describe metastasis-, tumor stage-, and age-associated dynamics, an important premise for B cell-related biomarker and therapy studies.

Cancer-associated cells release citrate to support tumour metastatic progression.

Citrate is important for lipid synthesis and epigenetic regulation in addition to ATP production. We have previously reported that cancer cells import extracellular citrate via the pmCiC transporter to support their metabolism. Here, we show for the first time that citrate is supplied to cancer by cancer-associated stroma (CAS) and also that citrate synthesis and release is one of the latter's major metabolic tasks. Citrate release from CAS is controlled by cancer cells through cross-cellular communication. The availability of citrate from CAS regulated the cytokine profile, metabolism and features of cellular invasion. Moreover, citrate released by CAS is involved in inducing cancer progression especially enhancing invasiveness and organ colonisation. In line with the in vitro observations, we show that depriving cancer cells of citrate using gluconate, a specific inhibitor of pmCiC, significantly reduced the growth and metastatic spread of human pancreatic cancer cells in vivo and muted stromal activation and angiogenesis. We conclude that citrate is supplied to tumour cells by CAS and citrate uptake plays a significant role in cancer metastatic progression.

Conceptualizing protective family context and its effect on substance use: Comparisons across diverse ethnic-racial youth.

Background: Although family behaviors are known to be important for buffering youth against substance use, research in this area often evaluates a particular type of family interaction and how it shapes adolescents' behaviors, when it is likely that youth experience the co-occurrence of multiple types of family behaviors that may be protective. Methods: The current study (N = 1716, 10th and 12th graders, 55% female) examined associations between protective family context, a latent variable comprised of five different measures of family behaviors, and past 12 months substance use: alcohol, cigarettes, marijuana, and e-cigarettes. Results: A multi-group measurement invariance assessment supported protective family context as a coherent latent construct with partial (metric) measurement invariance among Black, Latinx, and White youth. A multi-group path model indicated that protective family context was significantly associated with less substance use for all youth, but of varying magnitudes across ethnic-racial groups. Conclusion: These results emphasize the importance of evaluating psychometric properties of family-relevant latent variables on the basis of group membership in order to draw appropriate inferences on how such family variables relate to substance use among diverse samples.

Inhibition of progesterone receptor activity during development increases reelin-immunoreactivity in Cajal-Retzius cells, alters synaptic innervation in neonatal dentate gyrus, and impairs episodic-like memory in adulthood.

Progesterone receptor (PR) is expressed in Cajal-Retzius (CR) cells of the dentate gyrus (DG) molecular layer during the postnatal period (P1-28), a critical stage of development for the dentate gyrus and its circuitry. CR cells secrete the glycoprotein, reelin, which is required for typical development of the DG and its connections, particularly afferent input from the perforant path. This pathway regulates the processing of sensory information arriving from entorhinal cortex and integrates this information to form episodic memories. To assess the potential role of PR activity on the development of these connections and associated behavior, rats were treated daily from P1 to 7 with the PR antagonist, RU486. RU486 treatment increased the number of reelin-ir cells, suggesting an accumulation of reelin, and implicating PR in the regulation of a principle developmental function of CR cells. RU486 also altered the synaptic bouton marker, synaptophysin-ir, in a sex-specific manner, suggesting a role for PR activity in the development of perforant path innervation of the molecular layer (MOL). Finally, both control and RU486 treated rats spent significantly more time with a temporally distant object in the Relative Recency task, suggesting an intact associative memory for object identity and temporal order in both groups. In contrast, the same RU486 treated rats were impaired in an episodic-like memory task compared to controls, failing to integrate object identity ('what'), time ('when'), and object position ('where'). These findings reveal a novel role for PR in regulating CR cell function within the MOL, thereby altering development of DG connectivity and behavioral function.

Bisphenol A (BPA) induces progesterone receptor expression in an estrogen receptor α-dependent manner in perinatal brain.

Bisphenol A (BPA) is a xenoestrogen that is prevalent in the environment of industrialized nations due its use in the production of many plastic household items. Virtually all adults in the U.S. have detectable levels of BPA in urine and it can be measured in fetal serum and in breastmilk, making developmental exposure a particular concern. The present study utilizes a progesterone receptor (PR) expression bioassay to assess the estrogen receptor α (ERα)-dependent effects of BPA in fetal rodent brain following maternal exposure. Maternal ingestion of 10 µg/kg/day, but not 50 µg/kg/day, BPA from gestational day 14-22 significantly increased levels of PR immunoreactivity (PRir) in the medial preoptic nucleus (MPN) of female offspring. PR expression in the perinatal MPN is highly dependent on the activation of ERα, but not ERß, by estrogens. Indeed, injections of BPA (5 µg/kg) to neonates from postnatal day 2-4 (P2-4) significantly increased PR expression in the MPN of postnatal day 5 females compared to the MPN of females administered the oil vehicle. However, pretreatment with the ER antagonist, ICI 182,780 from P1-4 significantly attenuated the effects of BPA on PR expression, indicating an ERα-dependent mechanism. The present results also demonstrate a non-monotonic effect of BPA on the direct expression of a transcription factor in developing brain.

B cells sustain inflammation and predict response to immune checkpoint blockade in human melanoma.

Tumor associated inflammation predicts response to immune checkpoint blockade in human melanoma. Current theories on regulation of inflammation center on anti-tumor T cell responses. Here we show that tumor associated B cells are vital to melanoma associated inflammation. Human B cells express pro- and anti-inflammatory factors and differentiate into plasmablast-like cells when exposed to autologous melanoma secretomes in vitro. This plasmablast-like phenotype can be reconciled in human melanomas where plasmablast-like cells also express T cell-recruiting chemokines CCL3, CCL4, CCL5. Depletion of B cells in melanoma patients by anti-CD20 immunotherapy decreases tumor associated inflammation and CD8+ T cell numbers. Plasmablast-like cells also increase PD-1+ T cell activation through anti-PD-1 blockade in vitro and their frequency in pretherapy melanomas predicts response and survival to immune checkpoint blockade. Tumor associated B cells therefore orchestrate and sustain melanoma inflammation and may represent a predictor for survival and response to immune checkpoint blockade therapy.

Hazard assessment of air pollutants: The transforming ability of complex pollutant mixtures in the Bhas 42 cell model.

The use of in vitro alternative methods is a promising approach to characterize the hazardous properties of environmental chemical mixtures, including urban airborne particulate matter (PM). The aim of this study was to examine seasonal differences in the toxic and transforming potential of PM samples, by using the in vitro cell transformation assay in Bhas 42 cells for the prediction of potential carcinogenic effects. Bhas 42 cells are already initiated, and the v-Ha-ras transfection, together with genetic modification following the immortalization process, makes them a valuable model to study the late steps of cellular transformation leading to the acquisition of the malignant phenotype. Exposure to organic extracts of PM1 and PM2.5 induced dose-related effects. The transforming and cytotoxic properties are related to the amount of PM collected during the sampling campaign and associated with the concentrations of polycyclic aromatic hydrocarbons (PAHs) in the samples. All the samples induced cell transformation following prolonged exposure of 2 weeks. Our results support the utility of the in vitro top-down approach to characterise the toxicity of real mixtures, thereby supporting regulators in the decision-making process. The results also identify the need for appropriate assay selection within the in vitro testing strategy to address the complexity of the final adverse outcomes.

Loss of Ing3 Expression Results in Growth Retardation and Embryonic Death.

The ING3 candidate tumour suppressor belongs to a family of histone modifying proteins involved in regulating cell proliferation, senescence, apoptosis, chromatin remodeling, and DNA repair. It is a stoichiometric member of the minimal NuA4 histone acetyl transferase (HAT) complex consisting of EAF6, EPC1, ING3, and TIP60. This complex is responsible for the transcription of an essential cascade of genes involved in embryonic development and in tumour suppression. ING3 has been linked to head and neck and hepatocellular cancers, although its status as a tumour suppressor has not been well established. Recent studies suggest a pro-metastasis role in prostate cancer progression. Here, we describe a transgenic mouse strain with insertional mutation of an UbC-mCherry expression cassette into the endogenous Ing3 locus, resulting in the disruption of ING3 protein expression. Homozygous mutants are embryonically lethal, display growth retardation, and severe developmental disorders. At embryonic day (E) 10.5, the last time point viable homozygous embryos were found, they were approximately half the size of heterozygous mice that develop normally. µCT analysis revealed a developmental defect in neural tube closure, resulting in the failure of formation of closed primary brain vesicles in homozygous mid-gestation embryos. This is consistent with high ING3 expression levels in the embryonic brains of heterozygous and wild type mice and its lack in homozygous mutant embryos that show a lack of ectodermal differentiation. Our data provide direct evidence that ING3 is an essential factor for normal embryonic development and that it plays a fundamental role in prenatal brain formation.