Human Oral Buccal Microbiomes Are Associated with Farmworker Status and Azinphos-Methyl Agricultural Pesticide Exposure.

In a longitudinal agricultural community cohort sampling of 65 adult farmworkers and 52 adult nonfarmworkers, we investigated agricultural pesticide exposure-associated changes in the oral buccal microbiota. We found a seasonally persistent association between the detected blood concentration of the insecticide azinphos-methyl and the taxonomic composition of the buccal swab oral microbiome. Blood and buccal samples were collected concurrently from individual subjects in two seasons, spring/summer 2005 and winter 2006. Mass spectrometry quantified blood concentrations of the organophosphate insecticide azinphos-methyl. Buccal oral microbiome samples were 16S rRNA gene DNA sequenced, assigned to the bacterial taxonomy, and analyzed after "centered-log-ratio" transformation to handle the compositional nature of the proportional abundances of bacteria per sample. Nonparametric analysis of the transformed microbiome data for individuals with and without azinphos-methyl blood detection showed significant perturbations in seven common bacterial taxa (>0.5% of sample mean read depth), including significant reductions in members of the common oral bacterial genus Streptococcus Diversity in centered-log-ratio composition between individuals' microbiomes was also investigated using principal-component analysis (PCA) to reveal two primary PCA clusters of microbiome types. The spring/summer "exposed" microbiome cluster with significantly less bacterial diversity was enriched for farmworkers and contained 27 of the 30 individuals who also had azinphos-methyl agricultural pesticide exposure detected in the blood. IMPORTANCE: In this study, we show in human subjects that organophosphate pesticide exposure is associated with large-scale significant alterations of the oral buccal microbiota composition, with extinctions of whole taxa suggested in some individuals. The persistence of this association from the spring/summer to the winter also suggests that long-lasting effects on the commensal microbiota have occurred. The important health-related outcomes of these agricultural community individuals' pesticide-associated microbiome perturbations are not understood at this time. Future investigations should index medical and dental records for common and chronic diseases that may be interactively caused by this association between pesticide exposure and microbiome alteration.

Neural circuit-wide analysis of changes to gene expression during deafening-induced birdsong destabilization.

Sensory feedback is required for the stable execution of learned motor skills, and its loss can severely disrupt motor performance. The neural mechanisms that mediate sensorimotor stability have been extensively studied at systems and physiological levels, yet relatively little is known about how disruptions to sensory input alter the molecular properties of associated motor systems. Songbird courtship song, a model for skilled behavior, is a learned and highly structured vocalization that is destabilized following deafening. Here, we sought to determine how the loss of auditory feedback modifies gene expression and its coordination across the birdsong sensorimotor circuit. To facilitate this system-wide analysis of transcriptional responses, we developed a gene expression profiling approach that enables the construction of hundreds of spatially-defined RNA-sequencing libraries. Using this method, we found that deafening preferentially alters gene expression across birdsong neural circuitry relative to surrounding areas, particularly in premotor and striatal regions. Genes with altered expression are associated with synaptic transmission, neuronal spines, and neuromodulation and show a bias toward expression in glutamatergic neurons and Pvalb/Sst-class GABAergic interneurons. We also found that connected song regions exhibit correlations in gene expression that were reduced in deafened birds relative to hearing birds, suggesting that song destabilization alters the inter-region coordination of transcriptional states. Finally, lesioning LMAN, a forebrain afferent of RA required for deafening-induced song plasticity, had the largest effect on groups of genes that were also most affected by deafening. Combined, this integrated transcriptomics analysis demonstrates that the loss of peripheral sensory input drives a distributed gene expression response throughout associated sensorimotor neural circuitry and identifies specific candidate molecular and cellular mechanisms that support the stability and plasticity of learned motor skills.

Alteration of oral microbiome composition in children living with pesticide-exposed farm workers.

Our prior work shows that azinphos-methyl pesticide exposure is associated with altered oral microbiomes in exposed farmworkers. Here we extend this analysis to show the same association pattern is also evident in their children. Oral buccal swab samples were analyzed at two time points, the apple thinning season in spring-summer 2005 for 78 children and 101 adults and the non-spray season in winter 2006 for 62 children and 82 adults. The pesticide exposure for the children were defined by the farmworker occupation of the cohabitating household adult and the blood azinphos-methyl detection of the cohabitating adult. Oral buccal swab 16S rRNA sequencing determined taxonomic microbiota proportional composition from concurrent samples from both adults and children. Analysis of the identified bacteria showed significant proportional changes for 12 of 23 common oral microbiome genera in association with azinphos-methyl detection and farmworker occupation. The most common significantly altered genera had reductions in the abundance of Streptococcus, suggesting an anti-microbial effect of the pesticide. Principal component analysis of the microbiome identified two primary clusters, with association of principal component 1 to azinphos-methyl blood detection and farmworker occupational status of the household. The children's buccal microbiota composition clustered with their household adult in ∼95% of the households. Household adult farmworker occupation and household pesticide exposure is associated with significant alterations in their children's oral microbiome composition. This suggests that parental occupational exposure and pesticide take-home exposure pathways elicit alteration of their children's microbiomes.

Ageing compromises mouse thymus function and remodels epithelial cell differentiation.

Ageing is characterised by cellular senescence, leading to imbalanced tissue maintenance, cell death and compromised organ function. This is first observed in the thymus, the primary lymphoid organ that generates and selects T cells. However, the molecular and cellular mechanisms underpinning these ageing processes remain unclear. Here, we show that mouse ageing leads to less efficient T cell selection, decreased self-antigen representation and increased T cell receptor repertoire diversity. Using a combination of single-cell RNA-seq and lineage-tracing, we find that progenitor cells are the principal targets of ageing, whereas the function of individual mature thymic epithelial cells is compromised only modestly. Specifically, an early-life precursor cell population, retained in the mouse cortex postnatally, is virtually extinguished at puberty. Concomitantly, a medullary precursor cell quiesces, thereby impairing maintenance of the medullary epithelium. Thus, ageing disrupts thymic progenitor differentiation and impairs the core immunological functions of the thymus.

Non-BRCA DNA Damage Repair Gene Alterations and Response to the PARP Inhibitor Rucaparib in Metastatic Castration-Resistant Prostate Cancer: Analysis From the Phase II TRITON2 Study.

PURPOSE: Genomic alterations in DNA damage repair (DDR) genes other than BRCA may confer synthetic lethality with PARP inhibition in metastatic castration-resistant prostate cancer (mCRPC). To test this hypothesis, the phase II TRITON2 study of rucaparib included patients with mCRPC and deleterious non-BRCA DDR gene alterations. PATIENTS AND METHODS: TRITON2 enrolled patients who had progressed on one or two lines of next-generation androgen receptor-directed therapy and one taxane-based chemotherapy for mCRPC. Key endpoints were investigator-assessed radiographic response per modified RECIST/PCWG3 and PSA response (≥50% decrease from baseline). RESULTS: TRITON2 enrolled 78 patients with a non-BRCA DDR gene alteration [ATM (n = 49), CDK12 (n = 15), CHEK2 (n = 12), and other DDR genes (n = 14)]. Among patients evaluable for each endpoint, radiographic and PSA responses were observed in a limited number of patients with an alteration in ATM [2/19 (10.5%) and 2/49 (4.1%), respectively], CDK12 [0/10 (0%) and 1/15 (6.7%), respectively], or CHEK2 [1/9 (11.1%) and 2/12 (16.7%), respectively], including no radiographic or PSA responses in 11 patients with confirmed biallelic ATM loss or 11 patients with ATM germline mutations. Responses were observed in patients with alterations in the DDR genes PALB2, FANCA, BRIP1, and RAD51B. CONCLUSIONS: In this prospective, genomics-driven study of rucaparib in mCRPC, we found limited radiographic/PSA responses to PARP inhibition in men with alterations in ATM, CDK12, or CHEK2. However, patients with alterations in other DDR-associated genes (e.g., PALB2) may benefit from PARP inhibition.See related commentary by Sokolova et al., p. 2439.

The presence of macrophages and inflammatory responses in an in vitro testicular co-culture model of male reproductive development enhance relevance to in vivo conditions.

Our 3-dimensional testis co-culture system (3D-TCS) represents a promising model of male reproductive toxicity which captures sensitive processes of male reproductive development and contains the main testes cell types (germ, Leydig and Sertoli cells). Macrophages are another cell type important for testicular function and help to modulate immuno-endocrine processes during testes development. Chemicals such as phthalate esters (PE's) affect macrophage function and testosterone production in the testes in vivo. The aim of this study was to determine whether macrophages were present in the 3D-TCS and investigate responses in our model that may be related to immuno-endocrine functions. We observed consistent expression of the resident macrophage marker ED2 as well as increases in inflammatory cytokines produced by macrophages and testes cells (IL-6, TNF-α and KC/GRO) after exposure to toxic PE's. Pathway analysis of gene expression changes after exposure to PE's showed that IL-6 and TNF-α signaling pathways were enriched after treatment with reproductively toxic, but not non-reproductively toxic phthalates. These results indicate that macrophages and inflammatory processes are captured in the 3D-TCS and that these processes are impacted by exposure to reproductive toxicants. These processes represent a major mode of action for in vivo testis toxicity for a variety of compounds and our novel in vitro model is able to capture toxicant perturbation of immune function.

SAFETY AND TOLERABILITY OF MRI-GUIDED INFUSION OF AAV2-hAADC INTO THE MID-BRAIN OF NON-HUMAN PRIMATE.

Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare, autosomal-recessive neurological disorder caused by mutations in the DDC gene that leads to an inability to synthesize catecholamines and serotonin. As a result, patients suffer compromised development, particularly in motor function. A recent gene replacement clinical trial explored putaminal delivery of recombinant adeno-associated virus serotype 2 vector encoding human AADC (AAV2-hAADC) in AADC-deficient children. Unfortunately, patients presented only modest amelioration of motor symptoms, which authors acknowledged could be due to insufficient transduction of putamen. We hypothesize that, with the development of a highly accurate MRI-guided cannula placement technology, a more effective approach might be to target the affected mid-brain neurons directly. Transduction of AADC-deficient dopaminergic neurons in the substantia nigra and ventral tegmental area with locally infused AAV2-hAADC would be expected to lead to restoration of normal dopamine levels in affected children. The objective of this study was to assess the long-term safety and tolerability of bilateral AAV2-hAADC MRI-guided pressurized infusion into the mid-brain of non-human primates. Animals received either vehicle, low or high AAV2-hAADC vector dose and were euthanized 1, 3 or 9 months after surgery. Our data indicate that effective mid-brain transduction was achieved without untoward effects.