Analysis of EGFR binding hotspots for design of new EGFR inhibitory biologics.

The epidermal growth factor (EGF) receptor (EGFR) is activated by the binding of one of seven EGF-like ligands to its ectodomain. Ligand binding results in EGFR dimerization and stabilization of the active receptor conformation subsequently leading to activation of downstream signaling. Aberrant activation of EGFR contributes to cancer progression through EGFR overexpression/amplification, modulation of its positive and negative regulators, and/or activating mutations within EGFR. EGFR targeted therapeutic antibodies prevent dimerization and interaction with endogenous ligands by binding the ectodomain of EGFR. However, these antibodies have had limited success in the clinic, partially due to EGFR ectodomain resistance mutations, and are only applicable to a subset of patients with EGFR-driven cancers. These limitations suggest that alternative EGFR targeted biologics need to be explored for EGFR-driven cancer therapy. To this end, we analyze the EGFR interfaces of known inhibitory biologics with determined structures in the context of endogenous ligands, using the Rosetta macromolecular modeling software to highlight the most important interactions on a per-residue basis. We use this analysis to identify the structural determinants of EGFR targeted biologics. We suggest that commonly observed binding motifs serve as the basis for rational design of new EGFR targeted biologics, such as peptides, antibodies, and nanobodies.

HER4 is a high-affinity dimerization partner for all EGFR/HER/ErbB family proteins.

Human epidermal growth factor receptors (HER)-also known as EGFR or ErbB receptors-are a subfamily of receptor tyrosine kinases (RTKs) that play crucial roles in cell growth, division, and differentiation. HER4 (ErbB4) is the least studied member of this family, partly because its expression is lower in later stages of development. Recent work has suggested that HER4 can play a role in metastasis by regulating cell migration and invasiveness; however, unlike EGFR and HER2, the precise role that HER4 plays in tumorigenesis is still unresolved. Early work on HER family proteins suggested that there are direct interactions between the four members, but to date, there has been no single study of all four receptors in the same cell line with the same biophysical method. Here, we quantitatively measure the degree of association between HER4 and the other HER family proteins in live cells with a time-resolved fluorescence technique called pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). PIE-FCCS is sensitive to the oligomerization state of membrane proteins in live cells, while simultaneously measuring single-cell protein expression levels and diffusion coefficients. Our PIE-FCCS results demonstrate that HER4 interacts directly with all HER family members in the cell plasma membrane. The interaction between HER4 and other HER family members intensified in the presence of a HER4-specific ligand. Our work suggests that HER4 is a preferred dimerization partner for all HER family proteins, even in the absence of ligands.

Interrogation of a fluoropolymer dispersion manufactured with a non-fluorinated polymerization aid for targeted and non-targeted fluorinated residuals by liquid chromatography high resolution mass spectrometry.

Recent advances in fluoropolymer polymerization have focused on replacing perfluorinated polymerization aids (PAs) with hydrocarbon-based alternatives. Hydrocarbon PAs are vulnerable to fluorinated radicals during polymerization, leading to the creation of hundreds of process-specific polyfluorinated residuals. These residuals, which include low molecular weight extractable or leachable impurities, are challenging to detect at trace levels. This study investigates a polytetrafluoroethylene (PTFE) dispersion prepared with a hydrocarbon-based surfactant (DOSS) to measure these process-specific fluorinated residues. Liquid chromatography high resolution mass spectrometry is one of the few analytical methods that offers the sensitivity and selectivity required to detect these residuals in complex matrices at concentrations as low as parts per billion. The results indicate that using a hydrocarbon PA during emulsion polymerization produces numerous polyfluorinated residuals. These must be identified and monitored to develop effective abatement strategies, ensuring responsible fluoropolymer manufacturing.

Genetic rescue often leads to higher fitness as a result of increased heterozygosity across animal taxa.

Biodiversity loss has reached critical levels partly due to anthropogenic habitat loss and degradation. These landscape changes are damaging as they can fragment species distributions into small, isolated populations, resulting in limited gene flow, population declines and reduced adaptive potential. Genetic rescue, the translocation of individuals to increase genetic diversity and ultimately fitness, has produced promising results for fragmented populations but remains underutilized due to a lack of long-term data and monitoring. To promote a better understanding of genetic rescue and its potential risks and benefits over the short-term, we reviewed and analysed published genetic rescue attempts to identify whether genetic diversity increases following translocation, and if this change is associated with increased fitness. Our review identified 19 studies that provided genetic and fitness data from before and after the translocation; the majority of these were on mammals, and included experimental, natural and conservation-motivated translocations. Using a Bayesian meta-analytical approach, we found that on average, genetic diversity and fitness increased in populations post translocations, although there were some exceptions to this trend. Overall, genetic diversity was a positive predictor of population fitness, and in some cases this relationship extended three generations post-rescue. These data suggest a single translocation can have lasting fitness benefits, and support translocation as another tool to facilitate conservation success. Given the limited number of studies with long-term data, we echo the need for genetic monitoring of populations post-translocation to understand whether genetic rescue can also limit the loss of adaptive potential in the long-term.

Backtracking childhood leukaemia to birth: A battle of addition.

Paediatric leukaemia has a long tail of driver mutations each of which must be 'backtracked' to samples taken at birth to identify the prenatal origin of a subtype. Presently, Bardini et al. describe the first successful backtracking of an NUTM1 rearrangement, which sheds light on the biology of this particular alteration. Continued backtracking of NUTM1 rearrangements, and all leukaemia-typical somatic alterations, is necessary to fully understand the prenatal origin of these diseases. Commentary on: Bardini et al. Prenatal origin of NUTM1 gene rearrangement in infant B-cell precursor acute lymphoblastic leukaemia. Br J Haematol 2024 (Online ahead of print). doi: 10.1111/bjh.19685.

Genome-wide association studies of Down syndrome associated congenital heart defects.

Congenital heart defects (CHDs) are the most common structural birth defect and are present in 40-50% of children born with Down syndrome (DS). To characterize the genetic architecture of DS-associated CHD, we sequenced genomes of a multiethnic group of children with DS and a CHD (n=886: atrioventricular septal defects (AVSD), n=438; atrial septal defects (ASD), n=122; ventricular septal defects (VSD), n=170; other types of CHD, n=156) and DS with a structurally normal heart (DS+NH, n=572). We performed four GWAS for common variants (MAF>0.05) comparing DS with CHD, stratified by CHD-subtype, to DS+NH controls. Although no SNP achieved genome-wide significance, multiple loci in each analysis achieved suggestive significance (p<2×10-6). Of these, the 1p35.1 locus (near RBBP4) was specifically associated with ASD risk and the 5q35.2 locus (near MSX2) was associated with any type of CHD. Each of the suggestive loci contained one or more plausible candidate genes expressed in the developing heart. While no SNP replicated (p<2×10-6) in an independent cohort of DS+CHD (DS+CHD: n=229; DS+NH: n=197), most SNPs that were suggestive in our GWASs remained suggestive when meta-analyzed with the GWASs from the replication cohort. These results build on previous work to identify genetic modifiers of DS-associated CHD.

Membrane Stabilization of Helical Previtamin D Conformers as Possible Enhancement of Vitamin D Photoproduction.

Photoinduced vitamin D formation occurs 10-15-fold faster in phospholipid bilayers (PLB) than in isotropic solution. It has been hypothesized that amphipatic interactions of the PLB with the rotationally flexible previtamin D (Pre) stabilize its helical conformers, enhancing thermal intramolecular [1,7]-hydrogen transfer, forming vitamin D. To test this hypothesis, we carried out molecular dynamics (MD) simulations of Pre in a PLB composed of dipalmitoylphosphatidylcholine (DPPC). We designed a classical force field capable of accurately describing the equilibrium composition of Pre conformers. Using adaptive biasing force MD simulations, we determined the free energy of Pre conformers in isotropic environments (hexane and gas-phase) and in the anisotropic environment of a DPPC PLB. We find a total increase of 25.5% of the population of both helical conformers (+20.5% g+Zg+ and +5% g-Zg-) in DPPC compared to hexane. In view of ab initio simulations, showing that hydrogen transfer occurs in both helical conformers, our study strongly suggests the validity of the initial hypothesis. Regarding the amphipatic interactions of Pre with the PLB, we find that, similar to cholesterol (Chol) and 7-dehydrocholesterol (7-DHC), Pre entertains hydrogen bonds mainly to the carbonyl groups of DPPC and, to a lesser extent, with phosphate oxygen atoms and rarely to water molecules at the interface. We further report order parameters of the Pre/DPPC system, which are slightly smaller than those for Chol/DPPC and 7-DHC/DPPC, but larger than for pure DPPC. This indicates a loss in membrane viscosity upon photochemical ring-opening of 7-DHC to form Pre.

Blood-based multivariate methylation risk score for cognitive impairment and dementia.

INTRODUCTION: The established link between DNA methylation and pathophysiology of dementia, along with its potential role as a molecular mediator of lifestyle and environmental influences, positions blood-derived DNA methylation as a promising tool for early dementia risk detection. METHODS: In conjunction with an extensive array of machine learning techniques, we employed whole blood genome-wide DNA methylation data as a surrogate for 14 modifiable and non-modifiable factors in the assessment of dementia risk in independent dementia cohorts. RESULTS: We established a multivariate methylation risk score (MMRS) for identifying mild cognitive impairment cross-sectionally, independent of age and sex (P = 2.0 × 10-3). This score significantly predicted the prospective development of cognitive impairments in independent studies of Alzheimer's disease (hazard ratio for Rey's Auditory Verbal Learning Test (RAVLT)-Learning = 2.47) and Parkinson's disease (hazard ratio for MCI/dementia = 2.59). DISCUSSION: Our work shows the potential of employing blood-derived DNA methylation data in the assessment of dementia risk. HIGHLIGHTS: We used whole blood DNA methylation as a surrogate for 14 dementia risk factors. Created a multivariate methylation risk score for predicting cognitive impairment. Emphasized the role of machine learning and omics data in predicting dementia. The score predicts cognitive impairment development at the population level.

Validated 3D finite-element model of the Risso's dolphin (Grampus griseus) head anatomy demonstrates gular sound reception and channelling through the mandibular fats.

Like other odontocetes, Risso's dolphins actively emit clicks and passively listen to the echoes during echolocation. However, the head anatomy of Risso's dolphins differs from that of other odontocetes by a unique vertical cleft along the anterior surface of the forehead and a differently-shaped lower jaw. In this study, 3D finite-element sound reception and production models were constructed based on computed tomography (CT) data of a deceased Risso's dolphin. Our results were verified by finding good agreement with experimental measurements of hearing sensitivity. Moreover, the acoustic pathway for sounds to travel from the seawater into the dolphin's tympanoperiotic complexes (TPCs) was computed. The gular reception mechanism, previously discovered inDelphinus delphisandZiphius cavirostris, was also found in this species. The received sound pressure levels and relative displacement at TPC surfaces were compared between the cases with and without the mandibular fats or mandible. The results demonstrate a pronounced wave-guiding role of the mandibular fats and a limited bone-conductor role of the mandible. For sound production modelling, we digitally filled the cleft with neighbouring soft tissues, creating a hypothetical 'cleftless' head. Comparison between sound travelling through a 'cleftless' head vs. an original head indicates that the distinctive cleft plays a limited role in biosonar sound propagation.

Functional Connectivity Favors Aberrant Visual Network c-Fos Expression Accompanied by Cortical Synapse Loss in a Mouse Model of Alzheimer's Disease.

Background: While Alzheimer's disease (AD) has been extensively studied with a focus on cognitive networks, visual network dysfunction has received less attention despite compelling evidence of its significance in AD patients and mouse models. We recently reported c-Fos and synaptic dysregulation in the primary visual cortex of a pre-amyloid plaque AD-model. Objective: We test whether c-Fos expression and presynaptic density/dynamics differ in cortical and subcortical visual areas in an AD-model. We also examine whether aberrant c-Fos expression is inherited through functional connectivity and shaped by light experience. Methods: c-Fos+ cell density, functional connectivity, and their experience-dependent modulation were assessed for visual and whole-brain networks in both sexes of 4-6-month-old J20 (AD-model) and wildtype (WT) mice. Cortical and subcortical differences in presynaptic vulnerability in the AD-model were compared using ex vivo and in vivo imaging. Results: Visual cortical, but not subcortical, networks show aberrant c-Fos expression and impaired experience-dependent modulation. The average functional connectivity of a brain region in WT mice significantly predicts aberrant c-Fos expression, which correlates with impaired experience-dependent modulation in the AD-model. We observed a subtle yet selective weakening of excitatory visual cortical synapses. The size distribution of cortical boutons in the AD-model is downscaled relative to those in WT mice, suggesting a synaptic scaling-like adaptation of bouton size. Conclusions: Visual network structural and functional disruptions are biased toward cortical regions in pre-plaque J20 mice, and the cellular and synaptic dysregulation in the AD-model represents a maladaptive modification of the baseline physiology seen in WT conditions.

Corticotropin-releasing factor and GABA in the ventral tegmental area modulate partner preference formation in male and female prairie voles (Microtus ochrogaster).

Introduction: The mesolimbic reward system is associated with the promotion and rewarding benefits of social relationships. In the socially monogamous prairie vole (Microtus ochrogaster), the establishment of a pair bond can be displayed by a robust preference for a breeding partner and aggressive rejection of unfamiliar conspecifics. Mesolimbic dopamine signaling influences bond-related behaviors within the vole through dopamine transmission and receptor activity in the nucleus accumbens. However, only one experiment has examined how the ventral tegmental area (VTA), a region that produces much of the fore- and mid-brain dopamine, regulates these social behaviors. Specifically, inhibition of either glutamate or GABA neurons in the VTA during a brief courtship promoted a partner preference formation in male prairie voles. The VTA is a heterogeneous structure that contains dopamine, GABA, and glutamate neurons as well as receives a variety of projections including corticotropin-releasing factor (CRF) suggested to modulate dopamine release. Methods: We used pharmacological manipulation to examine how GABA and CRF signaling in the VTA modulate partner preference formation in male and female prairie voles. Specifically, we used a 3 h partner preference test, a social choice test, to assess the formation of a partner preference following an infused bicuculline and CRF during a 1 h cohabitation and muscimol and CP154526, a CRFR1 antagonist, during a 24 h cohabitation with an opposite-sex conspecific. Results: Our study demonstrated that bicuculline, a GABA A receptor antagonist, and CRF in the VTA promoted a partner preference, whereas low-dose muscimol, a GABA A receptor agonist, and CP154526, a CRFR1 antagonist, inhibited a partner preference in both male and female prairie voles. Conclusion: This study demonstrated that GABA and CRF inputs into the VTA is necessary for the formation of a partner preference in male and female prairie voles.

Effects of a range of 6 prefabricated orthotic insole designs on plantar pressure in a healthy population: A randomized, open-label crossover investigation.

BACKGROUND: Prefabricated orthotic insoles are widely commercially available for self-selection to treat foot and lower-body musculoskeletal pain, without requiring advice from health care professionals. Although they are generally designed to mimic traditional design features of custom-made orthotics used in clinical practice, the effects of prefabricated insoles on plantar pressure distribution are poorly understood. OBJECTIVE: This investigation aimed to evaluate and directly compare the effects of a range of 6 different commercially available prefabricated orthotic insole designs on plantar pressure in healthy individuals. METHODS: This was a single-center, randomized, open-label, crossover investigation. In-shoe dynamic pressure (F-scan) was investigated in 24 healthy subjects with normal foot posture, wearing standard shoes alone and in combination with 6 different orthotic insoles, consecutively, measured on a single day. The biomechanical impact of each insole was determined by the statistical significance of changes from baseline measurements (standard shoe alone). RESULTS: Insoles with heel cups and medial arch geometries consistently increased contact area at medial arch and whole-foot regions and reduced both plantar peak pressure (PP) and pressure time integral at medial arch and heel regions. CONCLUSIONS: This investigation has aided in further understanding the mode of action of prefabricated insoles in a healthy population. The insoles in this study redistributed plantar pressure at key regions of the foot, based on design features common to prefabricated insoles. Prefabricated orthotic insoles represent an easily accessible means of reducing lower-body musculoskeletal stress for those who spend prolonged periods of time on their feet.

The validity of single-item measures of health-related quality of life across groups differing in acute respiratory symptom severity.

PURPOSE: Practical considerations precluding health-related quality of life (HRQOL) monitoring in population and clinical research have spawned development of improved items for more brief surveys of frequently measured HRQOL outcomes. The aim of this study was to validate the use of the Quality of Life General (QGEN-8), a shorter 8-item alternative to the longer 36-item short form (SF)-36 Health Survey for measuring the same eight HRQOL domains across groups of adults with varying severity of acute respiratory symptoms, such as cough and sore throat. METHODS: National Opinion Research Center (NORC) representative probability (N = 1,648) and supplemental opt-in (N = 5,915) U.S. adult samples were surveyed cross-sectionally online in 2020. Parallel analyses compared QGEN-8 and SF-36 estimates of group means for each of eight matching profile domains and summary physical and mental scores across groups differing in severity of acute symptoms and chronic respiratory conditions using analysis of covariance (ANCOVAs) controlling for socio-demographics and presence of chronic respiratory conditions. RESULTS: In support of discriminant validity, ANCOVA estimates of QGEN-8 means with SF-36 estimates revealed the same patterns of declining HRQOL with the presence and increasing severity of symptoms and chronic condition severity. CONCLUSION: QGEN-8® shows satisfactory validity and warrants further testing in cross-sectional and longitudinal population and clinical survey research as a more practical method for estimating group differences in SF-36 profile and summary component HRQOL scores.

Upper respiratory tract infections (URTI) with symptoms such as cough and sore throat are highly prevalent and negatively impact on health-related quality of life (HRQOL). Existing instruments that comprehensively measure HRQOL are lengthy, potentially increasing respondent burden and restricting their use in clinical studies and research. The aim of this study was to evaluate whether eight newly constructed survey items, the QGEN-8®, measure the same HRQOL outcomes as the 36-item SF-36 Health Survey well enough to serve as a more practical alternative for purposes of detecting the physical and mental HRQOL effects on differing severity of acute URTI symptoms, specifically cough and sore throat. The results showed that the QGEN-8® was psychometrically sound and able to differentiate between different levels of URTI symptoms, even in cases where respondents had chronic respiratory conditions. This indicates that the briefer QGEN-8® with 75% shorter response time is able to provide HRQOL measurements comparable to those derived from lengthier instruments thereby lending itself more readily to use in clinical studies and research of URTI symptoms, such as cough and sore throat.

Engineering of RNase P Ribozymes for Therapy against Human Cytomegalovirus Infection.

Nucleic acid-based gene interference and editing strategies, such as antisense oligonucleotides, ribozymes, RNA interference (RNAi), and CRISPR/Cas9 coupled with guide RNAs, are exciting research tools and show great promise for clinical applications in treating various illnesses. RNase P ribozymes have been engineered for therapeutic applications against human viruses such as human cytomegalovirus (HCMV). M1 ribozyme, the catalytic RNA subunit of RNase P from Escherichia coli, can be converted into a sequence-specific endonuclease, M1GS ribozyme, which is capable of hydrolyzing an mRNA target base-pairing with the guide sequence. M1GS RNAs have been shown to hydrolyze essential HCMV mRNAs and block viral progeny production in virus-infected cell cultures. Furthermore, RNase P ribozyme variants with enhanced hydrolyzing activity can be generated by employing in vitro selection procedures and exhibit better ability in suppressing HCMV gene expression and replication in cultured cells. Additional studies have also examined the antiviral activity of RNase P ribozymes in mice in vivo. Using cytomegalovirus infection as an example, this review summarizes the principles underlying RNase P ribozyme-mediated gene inactivation, presents recent progress in engineering RNase P ribozymes for applications in vitro and in mice, and discusses the prospects of using M1GS technology for therapeutic applications against HCMV as well as other pathogenic viruses.

Early-life tobacco exposure is causally implicated in aberrant RAG-mediated recombination in childhood acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children, yet few environmental risk factors have been identified. We previously found an association between early-life tobacco smoke exposure and frequency of somatic deletions of 8 leukemia driver genes among childhood ALL patients in the California Childhood Leukemia Study. To expand analysis genome-wide and examine potential mechanisms, we conducted tumor whole-genome sequencing in 35 ALL patients, including 18 with high prenatal tobacco exposure and 17 with low exposure as determined by established epigenetic biomarkers. High tobacco exposure patients had significantly more structural variants (P < .001) and deletions (P = .001) genome-wide than low exposure patients. Investigation of off-target RAG recombination revealed that 41% of deletions in the high tobacco exposure patients were putatively RAG-mediated (full RAG motif identified at one or both breakpoints) compared with only 21% in the low exposure group (P = .001). In a multilevel model, deletions in high tobacco exposure patients were 2.44-fold (95% CI:1.13-5.38) more likely to be putatively RAG-mediated than deletions in low exposure patients. No point mutational signatures were associated with prenatal tobacco exposure. Our findings suggest that early-life tobacco smoke exposure may promote leukemogenesis by driving development of somatic deletions in pre-leukemic lymphocytes via off-target RAG recombination.

Climate change increases flowering duration, driving phenological reassembly and elevated co-flowering richness.

Changes to flowering phenology are a key response of plants to climate change. However, we know little about how these changes alter temporal patterns of reproductive overlap (i.e. phenological reassembly). We combined long-term field (1937-2012) and herbarium records (1850-2017) of 68 species in a flowering plant community in central North America and used a novel application of Bayesian quantile regression to estimate changes to flowering season length, altered richness and composition of co-flowering assemblages, and whether phenological shifts exhibit seasonal trends. Across the past century, phenological shifts increased species' flowering durations by 11.5 d on average, which resulted in 94% of species experiencing greater flowering overlap at the community level. Increases to co-flowering were particularly pronounced in autumn, driven by a greater tendency of late season species to shift the ending of flowering later and to increase flowering duration. Our results demonstrate that species-level phenological shifts can result in considerable phenological reassembly and highlight changes to flowering duration as a prominent, yet underappreciated, effect of climate change. The emergence of an autumn co-flowering mode emphasizes that these effects may be season-dependent.

Family supplemented patient monitoring after surgery (SMARTER): a pilot stepped-wedge cluster-randomised trial.

BACKGROUND: Mortality after surgery in Africa is twice that in high-income countries. Most deaths occur on wards after patients develop postoperative complications. Family members might contribute meaningfully and safely to early recognition of deteriorating patients. METHODS: This was a stepped-wedge cluster-randomised trial of an intervention training family members to support nursing staff to take and record patient vital signs every 4 h after surgery. Adult inpatients across four surgical wards (clusters) in a Ugandan hospital were included. Clusters crossed once from routine care to the SMARTER intervention at monthly intervals. The primary outcome was frequency of vital sign measurements from arrival on the postoperative ward to the end of the third postoperative day (3 days). RESULTS: We enrolled 1395 patients between April and October 2021. Mean age was 28.2 (range 5-89) yr; 85.7% were female. The most common surgical procedure was Caesarean delivery (74.8%). Median (interquartile range) number of sets of vital signs increased from 0 (0-1) in control wards to 3 (1-8) in intervention wards (incident rate ratio 12.4, 95% confidence interval [CI] 8.8-17.5, P<0.001). Mortality was 6/718 (0.84%) patients in the usual care group vs 12/677 (1.77%) in the intervention group (odds ratio 1.32, 95% CI 0.1-14.7, P=0.821). There was no difference in length of hospital stay between groups (usual care: 2 [2-3] days vs intervention: 2 [2-4] days; hazard ratio 1.11, 95% CI 0.84-1.47, P=0.44). CONCLUSIONS: Family member supplemented vital signs monitoring substantially increased the frequency of vital signs after surgery. Care interventions involving family members have the potential to positively impact patient care. CLINICAL TRIAL REGISTRATION: NCT04341558.

Limbic oxytocin receptor expression alters molecular signaling and social avoidance behavior in female prairie voles (Microtus ochrogaster).

Introduction: The social defeat paradigm is the most representative animal model to study social anxiety disorder (SAD) and its underlying neuronal mechanisms. We have previously reported that defeat progressively reduces oxytocin receptors (OXTR) in limbic regions of the brain over an eight-week period in female prairie voles (Microtus ochrogaster). Oxytocin receptors activate the mitogen-activated protein kinase (MAPK) pathway, which has been previously associated with the anxiolytic effects of oxytocin. Here, we assessed the functional significance of OXTR in stress-induced social avoidance and the response of the MAPK signaling pathway in the nucleus accumbens (NAc), anterior cingulate cortex (ACC), and basolateral amygdala (BLA) of female prairie voles. Methods: In experiment 1, Sexually naïve adult female prairie voles were defeated for three consecutive days and tested a week after for social preference/avoidance (SPA) test. Control subjects were similarly handled without defeat conditioning. In experiment 2, sexually and stress naïve adult female prairie voles were bilaterally injected into the NAc, ACC, or the BLA with a CRISPR/Cas9 virus targeting the Oxtr coding sequence to induce OXTR knockdown. Two weeks post-surgery, subjects were tested for SPA behavior. Viral control groups were similarly handled but injected with a control virus. A subgroup of animals from each condition in both experiments were similarly treated and euthanized without being tested for SPA behavior. Brains were harvested for OXTR autoradiography, western blot analysis of MAPK proteins and quantification of local oxytocin content in the NAc, BLA, ACC, and PVN through ELISA. Results: Social defeat reduced OXTR binding in the NAc and affected MAPK pathway activity and oxytocin availability. These results were region-specific and sensitive to exposure to the SPA test. Additionally, OXTR knockdown in the NAc, ACC, and BLA induced social avoidance and decreased basal MAPK activity in the NAc. Finally, we found that OXTR knockdown in these regions was associated with less availability of oxytocin in the PVN. Conclusion: Dysregulation of the oxytocin system and MAPK signaling pathway in the NAc, ACC, and BLA are important in social behavior disruptions in female voles. This dysregulation could, therefore, play an important role in the etiology of SAD in women.