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Sex steroids such as testosterone (T) and estradiol (E2) establish structural differences between sexes during sensitive periods of development. These structural changes take place in hormone-sensitive tissues such as the gonads, brain, and larynx, and these structural changes lead to differences in sex and behavior depending on the absence or presence of these sex steroid hormones. For example, the presence of E2 during early development of male rodents masculinizes their brain and can lead to sexually dimorphic brain regions such as the SDN-POA. The difference in brain region size indicates the significance of the SDN in sexually dimorphic traits such as male mating behavior.Studying the presence or absence of hormones can be done through exposure to hormone-altering chemical reagents. For example, T levels can be increased by exposure to flutamide (an antiandrogen) or decreased by exposure to DHT. Likewise, E2 levels can be increased through tamoxifen (an E2 antagonist) or increased through atrazine, an aromatase inducer. Altering the hormone exposure of individuals will allow us to then observe differences in brain and behavior. Sex steroid effects of hormones on sex differences can be observed through an environmental context. For example, the species Xenopus laevis is a widely studied model system for development. Male X. laevis treated with atrazine were outcompeted by control males for amplexus with females. However, removing competition allowed atrazine-treated males to engage in amplexus behavior.I observed how altered T and E2 levels affect mating roles in amplexus. In amplexus, the male-typical role is to clasp during mating behavior and the female-typical role is to show receptivity. When treated males were paired with sexually primed females, I tested whether hormone exposure affected the male-typical role to clasp in amplexus. When treated males were sexually primed then paired with males, I tested whether the treated male exhibited the female-typical role (to be receptive in amplexus). Likewise, when treated females were paired with sexually primed females, I tested for alterations to male-typical role in amplexus, and when treated females were sexually primed then paired with males, I tested for alterations to female-typical role in amplexus.Brain neuropeptide differences were explored to link sex steroid effects of hormones on the brain to behavioral sex differences. Sex steroid effects of hormones may impact sex differences in behavior through structural differences within the brain. The neuropeptide of interest, vasotocin (AVT), is involved in amphibian reproductive behaviors and AVT-IR cell size is sexually dimorphic. AVT-IR cell number and individual cell area were compared across sex and exposure groups and correlated with amplexus behavior to link sex steroid effects of hormones on the brain to behavior.Lastly, a study site offered the opportunity to study how environmental factors affect reproductive hormones in X. laevis. Two sources of frogs exist in the same river in San Diego, CA where one source lives treated with run-off downstream of a golf course, and the other source lives untreated upstream of the golf course. Hormone levels of frogs were measured over years to establish hormonal differences between sources. These studies provide insight into year-to-year changes between sites and sexes and emphasize the importance for multi-year studies in research related to studying environmental effects on physiology.The significance of my work on Xenopus laevis will bring attention to the role of sex steroid effects on brain and reproductive behavior in the context of unique study systems through exposure of hormone-altering reagents. Contributions to the fields of endocrinology and reproductive behavior can be made by providing more consideration to sex steroid effects in understudied contexts, such as studying female mating behavior in addition to (and not only) male mating behavior. In addition, careful consideration for multimodal factors of sex that lead to sex differences can encourage studies in research areas that may often overlook or neglect sex differences.

Intracellular transport is the process by which cargoes, such as organelles, travel to their needed locations within the cell in a timely manner via cytoskeletal filaments. This process involves the cargo that needs to be transported, motor proteins such as kinesin, and a filament, such as the microtubule. The process is broken down into several sub-processes, two of which are the focus in this study: the binding process (in which the cargo-motor protein ensemble binds to the microtubule) and processivity (where the cargo-motor ensemble walks on the microtubule). It is known which structures in the cell are used in intracellular transport, such as motor proteins, microtubules, and tubulin C terminal tails (CTTs); however, it is unclear how their properties affect the entire process. In this study, we use mathematical modeling and computational simulations to explain perplexing details of experimental data. Specifically, we show that motor diffusion alone cannot explain binding times measured from optical trap studies. Through computational analysis, we suggest that ADP release from the motor head may be an integral component of the binding process. We also propose the contribution CTTs provide to motor proteins processivity, where we believe CTTs help motor heads that are searching for microtubule binding sites by holding them near the microtubule, thus allowing for quicker steps. By combining modeling and simulations with experimental data, we can tune physical parameters that have the most potential of explaining the data, which in turns allow us a deeper understanding of intracellular transport.

Metabolic dysfunction–associated steatotic liver disease (MASLD) is a prevalent condition characterized by the accumulation of lipids in the liver alongside other metabolic abnormalities. Currently, the intricate pathogenesis of MASLD is not fully understood, and there is no FDA-approved treatment for this disease. One potential therapeutic target for MASLD is the liver X receptors (LXRs) due to their crucial roles in lipid metabolism and inflammation. However, activating hepatic LXRs unfortunately leads to increased hepatic lipid production, making them unsuitable for MASLD treatment in the clinic.Apart from the liver, LXRs are highly expressed in the intestine with critical roles in cholesterol metabolism, which shows promise in MASLD treatment. However, their role in the intestine is not fully elucidated. Thus, this study aimed to investigate the role of intestinal LXRs by assessing mice with intestinal-specific LXRα/β double knockout (iLXR-DKO) under a high-fat/cholesterol diet (HFCD) that induces MASLD. iLXR-DKO mice exhibited reduced HFCD-induced body weight gain and protection against MASLD development, including diminished hepatic steatosis, hepatocyte injury, and body weight gain. Moreover, iLXR-DKO did not alter food intake or fecal energy output but instead increased energy expenditure, thus further preventing body weight gain and alleviating MASLD symptoms. These findings propose a novel therapeutic strategy in targeting intestinal LXRs for the treatment of MASLD.

In many species, caring for young is essential for reproductive success because it increases offspring survival, development, and quality. However, parental care can be costly by increasing resource expenditure and mortality risk and decreasing likelihood of future reproduction. Therefore, animals are predicted to adjust their levels of caregiving based on both intrinsic (e.g., sex, physical ability) and extrinsic factors (e.g., season, social interactions). Within-family dynamics are particularly interesting because an animal’s level of care determined by intrinsic factors may be influenced by responses to the behaviors of mates, offspring, or siblings. In biparental species, an individual’s social environment includes not only offspring, which can manipulate parents to garner greater levels of care, but also its mate. Little is known about how interactions within biparental families affect parental behavior. Thus, I examined intrinsic and social influences on caregiving behavior and the consequences for offspring in a biparental mammal, the California mouse (Peromyscus californicus). The first study investigated sex as a factor by determining whether parent-offspring discrimination differs between mothers and fathers. Throughout the pup-rearing period, I presented individual parents simultaneously with two mesh balls containing their own pup in one and an unrelated pup in the other. Although parents behaved similarly on individual test days, fathers showed significant changes in behaviors directed towards the non-kin ball over time, suggesting that fathers may show increased interest in unrelated young as their offspring become independent. In the second study, I examined parental behavior of both parents and offspring quality are affected by paternal age. Mothers mated to young fathers nursed pups more than those mated to old fathers; however, few other measures differed between families of old and young fathers. In the last study, I explored the effects of overlapping litters, in which subsequent litters are raised concurrently, on parent-offspring and sibling-sibling interactions and on offspring development.

Defensive behaviors in the presence of predator cues are typical innate behaviors in animals. Although predator signals are best detected with the summation of various sensory modalities in nature, olfactory-specific exposure to a predator specimen is sufficient to yield defensive responses in nocturnal prey animals such as mice. Animals display fixed patterns of defensive behaviors such as freezing, flight, and risk assessment in response to olfactory predator cues. These defensive responses varied between mice of varying age and sex. We found that mice freeze robustly when exposed to freshly collected cat saliva, but with lessened intensity when exposed to saliva that aged several hours. The underlying molecular and neural mechanisms for such shift in behavioral decisions are still not well understood. This freezing behavior was observed only when mice had direct contact with the cat odor and abolished in mice lacking functional Trpc2, indicating that the accessory olfactory system is necessary for this behavioral output. Interestingly, neural activation in some brain areas were with behavioral outputs in the fresh saliva-exposed mice but not in the ones exposed to old saliva. These results suggest that fresh cat saliva contains chemical cues that signal imminence of predator threat, and the signal is processed through a specific population of neurons in the accessory olfactory system. In this study, we reveal the candidate proteins in cat saliva that induces freezing and the mouse sensory receptor that detects them. Our research aims to identify the neural mechanism of imminence of threat detection and shed light on the neural basis underlying the defensive behavioral decision making in prey animals.

Flaviviruses such as dengue, Zika, West Nile, and yellow fever are dangerous viruses that pose a significant global health burden and have massive epidemic potential. Annually, these viruses infect over 400 million individuals worldwide and have been responsible for several pandemics in recent years. Flavivirus infection can result in hepatitis, microcephaly, liver cirrhosis, hemorrhagic shock, and even death. Despite these severe consequences, there are no available therapeutics for the majority of these flaviviruses . The initial response to flavivirus infection is characterized by the mass production of pro-inflammatory molecules in the host’s attempt to defend against pathogen invasion, causing widespread tissue damage through a phenomenon known as a cytokine storm. Consequently, it is crucial to investigate the host immune regulators that govern inflammatory multi-protein complexes called inflammasomes. Previous studies have highlighted the role of Nod-Like Receptor Protein 10 (NLRP10) in suppressing inflammation by inhibiting another human protein called pro-caspase-1 (pro-CASP1), a key component of many inflammasome systems. Therefore, we sought to determine what role NLRP10 played in the context of flavivirus-associated inflammation. Our findings reveal that NLRP10 is cleaved by diverse flavivirus proteases at amino acid site 265. However, this cleavage did not result in the restoration of inflammasome activation. Despite this, we also discovered that flaviviruses also independently antagonize inflammasomes. Collectively, our results demonstrate that flaviviruses exert evolutionary pressures on many inflammasome-associated proteins such as NLRP10, highlighting the intricacy of virus-host dynamics on a broader pan-flavivirus scale.

Infectious diseases are a worldwide problem that have been brought into focus by the 2020 SARS-CoV-22020 outbreak. The respiratory coronavirus is responsible for a global pandemic that has claimed over 6 million lives and affected hundreds of millions more. To slow the spread of the infection and ease the strain on healthcare systems, new vaccines and therapeutics were developed that protected against severe diseases. However, multiple waves of new variants emerged as the virus evolved to escape immunity afforded by vaccination attempts. To generate more effective vaccines that would provide life-long, pan-variant protection against infectious pathogens, we must first understand the immune response and the memory cells that confer immunity.Following infection, memory CD8 T cells can be found surveilling the body in circulation or persisting in the tissues. In the circulating compartment, different subsets of memory CD8 T cells have been identified based on localization pattern, phenotype, and function. Effector memory T cells (TEM) can be found traversing the non-lymphoid organs and are of high effector function, low proliferative, and low differentiation potentials. Central memory T cells (TCM), however, home to the secondary lymphoid tissues and are of low effector function but high differentiation potential. The distinction between memory CD8 T cell subsets is important not only for understanding the immune response, but also for predicting T cell protection post infection or vaccination.While TCM cells are homogeneous based of expression of cell surface markers, several studies have reported heterogeneity within the TEM population. We found that the conventional definition of TEM cells is conflated with a terminally-differentiated (t-TEM) subset, which consists of memory T cells with effector characteristics. The t-TEM population is of distinct ontogeny and fate from TEM and TCM. All three subsets differentially rely on key lineage-defining transcription factors and are transcriptionally and functionally distinct. In human blood, we identified analogous subsets with corresponding cell surface molecules and gene expression patterns. The delineation of t-TEM further clarified our understanding of circulating memory CD8 T cell biology.Through the characterization of memory CD8 T cell subsets, we observed a subset- and tissue-specific expression of granzyme A (GzmA), a serine protease that can induce apoptosis as well as mediate cytokine production. We showed that expression of GzmA by t-TEM in the circulation and tissue-resident (TRM) T cells in the intestines conferred heightened protection against viral reinfections. However, CD8 T cell-specific GzmA in the colon can contribute to pathology in the DSS-induced model of colitis. In addition to enhancing cell-death, GzmA can also mediate in inflammatory cytokine response in vitro and in vivo.Together, these studies clarified the definition of memory T cell subsets and provided insights on the role of CD8 T cell-dependent GzmA in conferring immunity against reinfection as well as contributing to inflammation.

Tumor associated macrophages (TAMs) are a major component in the tumor microenvironment. TAMs are associated with poor prognosis in solid tumors and promote an immunosuppressive environment, which can result from persistent inflammation. Preclinical studies suggest boosting existing immunotherapies by reprogramming TAMs from a pro-tumor phenotype towards an anti-tumor one. To better target these TAMs, we must look at the complexes that regulate them. The BAF complex, an ATP-dependent chromatin remodeler, regulates gene expression by altering the position of nucleosomes to promote gene expression. ARID1A, a core protein subunit of cBAF plays a vital role in the epigenetic regulation of inflammatory genes and the differentiation of myeloid cells. Little is known about the effects of Arid1a loss in TAMs and its implications in cancer. We sought to recapitulate the effects of Arid1a loss on macrophages in vitro, investigate the mechanisms driving the associated phenotype, and assess phagocytotic function in Arid1a-deficient (Arid1aKO) TAMs. Using an in vitro system of bone marrow derived macrophages (BMDMs), we observed that Arid1aKO macrophages upregulated the inhibitory ligand PD-L1 (programmed death ligand-1) and CD86. This upregulation is both JAK1/2 and type I interferon signaling independent and operates through a cell intrinsic mechanism. Furthermore, we found that Arid1aKO TAMs displayed similar phagocytic capabilities to Arid1a-intact TAMs. Our results demonstrate that upregulation of PD-L1 and CD86 in macrophages that occurs upon Arid1a loss may explain enhanced response to anti-PD-L1 immunotherapy. In the future, these results can help further direct preclinical models/strategies aimed at reprogramming macrophages as a cancer therapeutic.

Pre-implantation development depends on several processes, including the deposition of maternal factors into the oocyte during oogenesis, the expression of maternal mRNAs, and the degradation of maternal mRNA in tandem with zygotic genome activation during the maternal-to-zygotic transition (MZT). These processes ultimately determine whether an embryo implants into the uterine lining, which is necessary for pregnancy.It is known that mice with whole body knockouts of Bmal1 (hereafter referred to as Bmal1-/- mice) are infertile. Previous studies have indicated that this infertility deficit occurs in pre-implantation development. Furthermore, other studies found the presence of Bmal1 mRNAs in mouse pre-implantation development. With this, I hypothesized that the maternal expression of Bmal1 is necessary for pre-implantation development.To investigate this hypothesis, I performed immunostaining on embryos from the MZT, and whole ovary sections from wildtype (WT) mice to characterize the expression of BMAL1. I also quantified the number of metaphase II oocytes obtained from WT and Bmal1-/- mice, to look for deficits in pre-implantation development. I found a higher percentage of abnormal oocytes from Bmal1-/- mothers. Fluorescent imaging of these embryos reveals fluorescence of BMAL1 in the cytoplasm and nucleus of embryos throughout the MZT. Immunostaining of whole ovary sections revealed the presence of both BMAL1 and CLOCK protein within developing oocytes and the surrounding ovarian tissue. Now that BMAL1 expression has been characterized, further investigation can begin on the molecular roles of BMAL1 in mouse pre-implantation development.

Matters arising from: Mapping the human genetic architecture of COVID-19 Original Article published on 08 July 2021 https://www.nature.com/articles/s41586-021-03767-x The COVID-19 pandemic continues to pose a major public health threat, especially in countries with low vaccination rates. To better understand the biological underpinnings of SARS-CoV-2 infection and COVID-19 severity, we formed the COVID-19 Host Genetics Initiative1. Here we present a genome-wide association study meta-analysis of up to 125,584 cases and over 2.5 million control individuals across 60 studies from 25 countries, adding 11 genome-wide significant loci compared with those previously identified2. Genes at new loci, including SFTPD, MUC5B and ACE2, reveal compelling insights regarding disease susceptibility and severity. Here we present meta-analyses bringing together 60 studies from 25 countries (Fig. 1 and Supplementary Table 1) for three COVID-19-related phenotypes: (1) individuals critically ill with COVID-19 on the basis of requiring respiratory support in hospital or who died as a consequence of the disease (9,376 cases, of which 3,197 are new in this data release, and 1,776,645 control individuals); (2) individuals with moderate or severe COVID-19 defined as those hospitalized due to symptoms associated with the infection (25,027 cases, 11,386 new and 2,836,272 control individuals); and (3) all cases with reported SARS-CoV-2 infection regardless of symptoms (125,584 cases, 76,022 new and 2,575,347 control individuals). Most studies have reported results before the roll out of the COVID-19 vaccination campaign. An overview of the study design is provided in Supplementary Fig. 1. We found a total of 23 genome-wide significant loci (P

The breast epithelial system is a complex network of various cell types that work together to provide milk for the offspring. This complex network requires three distinct cell types to maintain the structure and function of the breast. This system is extremely dynamic and constantly changes in response to hormones secreted during different developmental stages. This constant change requires rapid growth and involution of the network and is where mutations and cellular dysregulation could lead to serious complications such as breast cancer. This dissertation focuses on creating a reference atlas of the normal breast epithelium and explores the pre-malignant changes that occur in human BRCA1 mutation carriers. This work utilizes single-cell RNAseq to profile the breast epithelium, which enable us to identify three main cell types, namely two distinct luminal and one basal cell type. In addition to the known basal and luminal populations, an additional luminal population was identified. This highlights the heterogeneity that exists at the cellular level in the luminal population that has been previously overlooked by bulk profiling approaches. Comparative analysis with BRCA1 mutation carrier samples reveals an expansion of a luminal progenitor population. This population is over-represented by cells from BRCA1 mutation carriers and have higher overall scEnergy, indicating greater dysregulation. Further characterization of this population reveals markers relating to stem and progenitor identities, suggesting its role as the cancer cell of origin. To perform single-cell profiling and functional assays, we needed to establish a robust method for single-cell isolation and purification. Analysis of the current method for single-cell isolation reveals that biological changes arise and these changes have long-term effects on the growth potential, thereby influencing the results of functional assays. Overall this work takes a high-resolution approach to identify cellular heterogeneity in the breast epithelium and elude the changes that occur during early cancer initiation for BRCA1 mutation carriers.

Y chromosomes are fascinating parts of the genome. In XY determination species, they specify the male sex and to do so, they influence a wide variety of molecular and cellular processes. Furthermore, since the Y is restricted to the male lineage, they undergo unique evolutionary trajectories resulting in gene-poor and repeat-rich chromosomes. This begs the question of how Y chromosomes can elicit drastic biological differences such as differences between sexes and differences between individuals. In my dissertation, I leveraged the Y chromosomes of two Drosophila species to address this fundamental question. Males often die earlier than females in XY species and recent studies have shown that the Y chromosome may play a role in this sex-specific phenomenon. Intriguingly, repetitive DNA such as transposable elements (TEs) show increased expression over aging, suggesting that the Y’s repetitive nature can contribute to this observation. I study sex-specific aging by leveraging the well-assembled genome of D. miranda, whose males have a very large young Y chromosome (neo-Y). The neo-Y was recently formed through a fusion between an autosome and a Y chromosome and therefore contains both genes and TEs. Using chromatin immunoprecipitation followed by sequencing (ChIP-Seq) and RNA-Seq, I showed that D. miranda males age faster than females possibly because of gene activity on the Y that also increases overall TE expression. Therefore, ineffective repression of deleterious elements both on and off the Y can explain the differences in aging.While many Y’s are degenerate, they can still vary in size within a population. The D. pseudoobscura Y especially has long been known to harbor dramatic size variation in the wild. I show that the 24Mb size difference between a large Y chromosome variant and a small variant is due to a handful of recently active TEs using cytological and sequencing approaches. These same TEs are also implicated in the size variation of a related species, D. affinis. Consistent with previous work, these Y chromosomes produce subtle changes in male lifespan and gene expression. Interestingly, I find that males bearing the large Y variant have higher expression of large Y-abundant TEs compared to males bearing the small Y. Thus, the very large Y chromosome variant of D. pseudoobscura was recently formed through highly active TEs and that these particular TEs have the propensity to also amplify in other species. Gene content on the Y was previously believed to be sparse but advances in sequencing have countered these old notions. In particular, Y chromosomes have been implicated in intragenomic conflicts with the X chromosome via gene amplification. I inspect the genomes and gene annotations of three D. pseudoobscura Y variants to investigate gene content evolution within a population. I find that the D. pseudoobscura Y is not a gene desert. In fact, it contains many gene families with hundreds of gene copies and not all variants contain the same families or copy numbers. Expression of these Y chromosome genes reveal that select genes are testis-expressed with sperm-related functions while others are involved in chromatin regulation. Therefore, Y chromosome gene content is highly dynamic and Y chromosome genes may play a role in wide-reaching biological processes.

Chronic alcohol consumption is associated with cancer development and progression. Previous reports have demonstrated that cancer cell metabolism is heavily involved in the development of cancer, including a side population know as cancer stem cells (CSCs). Therefore, it is generally accepted targeting cancer cell metabolism may provide a new and effective method for the treatment of tumors. There are emerging reports of the effects of alcohol on glucose metabolism, however the effects of alcohol and cancer metabolism and its underlying mechanisms are not fully understood in cancers including OSCC. In this study, we investigated the local effect of alcohol on cancer cell metabolism and its underlying mechanism by which alcohol promotes oral cancer progression. We report chronic alcohol promotes malignant growth of OSCC. In addition, chronic alcohol treatment increased the rate of aerobic glycolysis. Increased aerobic glycolysis was required for the maintenance of CSC population as glycolysis inhibition suppressed CSC properties. In order to explore the molecular mechanism by which chronic alcohol induces glycolysis and stemness, we investigate the role of NFAT signaling as NFAT is known to be involved in glycolysis and malignant cancer properties. NFATc2 is upregulated in chronic EtOH treatment OSCC and is required for for EtOH mediated glycolysis and maintenance of CSC population. Our study suggests that NFATc2 may be a potential therapeutic target for eradicating CSCs in OSCC by suppressing glycolysis in alcohol related cancer patients.

IntroductionEndoscopy performance is highly variable among fellows and practicing physicians.1,2 The field lacks a standard endoscopy training curriculum. Moreover, the traditional training under the apprenticeship model has proven to be a slow and ineffective method. Trainees who underwent a simulation-based mastery learning (SBML) curriculum accelerated their acquisition of clinical competency, 2.5x faster than trainees who received traditional training under the apprenticeship model.3 During the COVID-19 pandemic, we adapted our SBML curriculum to train upper endoscopy (esophagogastroduodenoscopy [EGD]) to novice gastroenterology trainees through online virtual coaching. Herein, we performed a hypothesis-generating study to evaluate the effectiveness of SBML-based EGD training, comparing virtual to direct in-person coaching. MethodsWe conducted a 7-day virtual SBML course across 7 academic centers in the USA and Asia. A minimum passing standard was set for each topic. Theoretical material was delivered using Canvas, an online learning management system. For technical skills training, a virtual coach supervised hand-on training at scheduled intervals. At the end of training, an independent rater assessed the trainees skills using a validated scoring system. After the course, we assessed the trainees’ clinical performance for the first 30 EGDs using the Assessment of Competency of Endoscopy (ACE) form. We compared the trainees’ scores to that of our historical control cohort trained using in-person SBML training. Our primary outcomes were competence scores on the written exam, endoscope tip control, standard EGD. Our secondary outcome was clinical EGD evaluations. We used non-inferiority t-test statistics and Fisher’s exact test. ResultsThe virtual coaching group received similar scores as the direct coaching group for the written assessment (virtual coaching 81.9%+8.9% vs direct coaching 78.3%+8.2%, p=0.385). For endoscope handling analysis, the trainees reached the MPS for competency after 31.4+29.1 attempts and mastery after 51.9+36.7 attempts, similar to the control cohort that had undergone the training with direct coaching (competency: 32.5+22.8, p=0.93; mastery: 38.2+31.1, p=0.42). For Standard EGD, the mean scores for the general assessment of the UGI tract were similar between the intervention and control groups (4.6+0.6 vs 4.7+0.5, p=1.00). For clinical EGDs, there were no significant differences in scores for EGD 1-5, 11-15, 16-20, 21-25, and 26-30. For EGDs 6-10, the virtual coaching cohort performed significantly better than the direct coaching cohort (2.73+0.59 vs 1.65+0.59, p

Currently, prostate adenocarcinoma (PRAD) is the second most common cancer in males, and the metastatic progression of prostate tumors to the bone accounts for a large majority of prostate cancer-related deaths. As such, identifying potential therapeutic targets to prevent prostate cancer progression proves to be vital. The low-molecular-weight protein tyrosine phosphatase (LMPTP) has been found to be highly expressed in metastatic hormone-naïve prostate cancer patients and is associated with lower survival rates. However, the molecular mechanism by which LMPTP promotes prostate cancer growth and metastatic development remains unclear. In this paper, we confirmed the oncogenic nature of LMPTP in prostate cancer growth in vitro and in vivo using both CRISPR-Cas9-mediated knockout (KO) of LMPTP and inhibition of LMPTP via an orally bio-available LMPTP inhibitor. Additionally, the use of a soft agar colony formation assay, Matrigel invasion chamber assay, and in vivo intraosseous metastasis model further implicated LMPTP in metastatic prostate cancer development. Through phosphoproteomic and metabolomic analyses we uncovered a possible glutathione deficiency and compensatory model in LMPTP KO cells that would explain the role of LMPTP in prostate cancer progression. Our findings implicate LMPTP in prostate cancer progression which taken together with our confirmation that loss of LMPTP responds synergistically with current prostate cancer drugs establishes LMPTP as a potential drug target for prostate cancer patients.

BACKGROUND Directional deep brain stimulation (DBS) allows for steering of the stimulation field, but extensive and time-consuming testing of all segmented contacts is necessary to identify the possible benefit of steering. It is therefore important to determine under which circumstances directional current steering is advantageous. METHODS Fifty two Parkinson's disease patients implanted in the STN with a directional DBS system underwent a standardized monopolar programming session 5 to 9 months after implantation. Individual contacts were tested for a potential advantage of directional stimulation. Results were used to build a prediction model for the selection of ring levels that would benefit from directional stimulation. RESULTS On average, there was no significant difference in therapeutic window between ring-level contact and best directional contact. However, according to our standardized protocol, 35% of the contacts and 66% of patients had a larger therapeutic window under directional stimulation compared to ring-mode. The segmented contacts warranting directional current steering could be predicted with a sensitivity of 79% and a specificity of 57%. CONCLUSION To reduce time required for DBS programming, we recommend additional directional contact testing initially only on ring-level contacts with a therapeutic window of less than 2.0 mA.

Malaria, Zika virus, West Nile virus, Dengue fever, and Lyme disease are common causes of morbidity and mortality around the world. While arthropod bites may cause local inflammation and discomfort, a greater concern is the potential to develop deadly systemic infection. The use of insect repellents (IR) to prevent systemic infections constitutes a fundamental public health effort. Cost-effectiveness, availability, and high-efficacy against arthropod vectors are key characteristics of an ideal IR. Currently, numerous IRs are available on the market, with DEET (N,N-diethyl-3-methylbenzamide) being the most widely used. DEET has an excellent safety profile and remarkable protection against mosquitoes and various other arthropods. Other EPA-registered IR ingredients (permethrin, picaridin, IR3535, oil of lemon eucalyptus, oil of citronella, catnip oil, and 2-undecanone) are alternative IRs of great interest due to some having efficacies comparable to that of DEET. These alternative IRs possess low toxicity and favorable customer experiences in utilization (e.g., cosmetically pleasant, naturally occurring). This review summarizes currently available EPA-registered IRs: the origins, mechanisms of action, side effect profiles, and available formulations will be discussed. This review will enable the clinician to select the best IR option to meet patients' needs and provide the greatest protection from arthropod bites and sequelae.

AbstractAn Examination of the effects Estrogen on Neural and Behavioral Development in African Clawed Frogs (Xenopus laevis)Although there is strong evidence that estrogens (E) play an important role in sexual differentiation of behavior in mammals, birds, and amphibians, where and how they act in the brain is still unclear. Estradiol (E2) may masculinize the brain during development, but in adulthood, exposure to E2 in males, de-masculinizes adult copulatory behavior. Most researchers who examined exogenous compounds and amphibian sexual and reproductive behaviors have focused on the impacts that these environmental compounds have on the animal’s physiology, morphology, and adult sexual behaviors. Few scientists have examined the molecular and neural mechanisms that underlie the hormonal changes that induce adult morphology and sexual behavior in amphibians. To address these gaps in the literature, I used exogenous compounds known to affect E2 synthesis and activity to understand how changes in E2 during development induce morphological changes in the brain that may influence adult sexual behaviors in amphibians.Chapter 1 provides a general review of how same-sex behavior is observed and analyzed in literature and provides an overview of previous research conducted on the effects of E2 during development to affect adult sexual behavior. In Chapter 2, I examined the effects of atrazine (ATZ), a chemical that induces aromatase --the enzyme that converts testosterone (T) into E2-- on sexual behavior and reproductive function in male African clawed frogs (Xenopus laevis). Tadpoles generated from breeding lab-bred X. laevis from two populations (San Diego [SD] and Golden Gate [GG]) were exposed throughout development to ATZ Sex ratios, E2 and T concentrations, and reproductive behavior were examined. Four percent of males exposed ATZ were sex-reversed. Males in the ATZ-treated group that were not sex-reversed displayed same-sex behavior in which males allowed other males to clasp them. Males that allowed other males to copulate with them (males on bottom) had elevated plasma E2 concentration and a higher E2 to T concentration relative to the males on top. In addition, males that displayed female typical behavior (bottom) were larger in size compared to males on top. Same-sex behavior in ATZ-exposed males is likely due to increased plasma E2 during development. Together, these results suggest that changes in E2 concentration during development can induce same-sex behavior in exposed animals. In chapter 3, I examined sexually dimorphic areas in the brain of adult X. laevis. I used immunohistochemistry (IHC) to analyze oxytocin (OT) and vasotocin (AVT) cells in the brains of control males, control females, and ATZ-exposed males. I sought to determine whether changes in E2 concentration due to ATZ-exposure during development could induce sexually dimorphic changes in OT/AVT cell number and/or distribution in the brain. Control males had twice the number of AVT cells throughout the brain compared to control females. ATZ-exposed males did not significantly differ in AVT cell number compared to control males. IHC for OT in the brain was unsuccessful. In addition to examining OT/AVT cells in the brain, I also analyzed the vocalization, contractile function of the laryngeal muscles, and fictive calls in females, control males, and ATZ-treated males. Although click rates and laryngeal fused tension differed between females and males (female laryngeal muscle fused tension at approximately 20 Hz whereas males laryngeal muscle fused tension at 70 Hz), click rates and laryngeal fused tension (both at 70 Hz) did not significantly differ between control males and ATZ-treated males. These results suggest that ATZ does not affect the vocal nuclei in the brain or laryngeal muscles in ATZ-exposed males. In chapter 4, I used compounds that affect E2-synthesis and activity in adult female X. laevis as tools to understand how changes in E2 during develop can affect adult sexual behavior and reproduction. In this chapter I exposed ovariectomized and sham-operated female X. laevis to tamoxifen (TAM), a compound that can act as an E2 antagonist or agonist depending on the tissues, miconazole (MIC), an aromatase inhibitor, ATZ (which induces aromatase), and ethinylestradiol (EE2), a synthetic estrogen. Ovariectomy significantly reduced plasma E2 and vitellogenin (VTG) expression. Treatment with MIC (3 ppb and 300 ppb) did not affect plasma E2 and VTG expression compared to control in females after four weeks of exposure. ATZ-exposed females had increased E2 and VTG compared to control females. EE2-treated (20 ppb) females had decreased plasma E2 and VTG expression compared to control females. TAM significantly increased plasma E2 concentration but significantly decreased VTG expression in exposed females. TAM did not induce significant changes in plasma E2 and VTG expression in OVX TAM-treated females. TAM, ATZ, EE2, nor MIC affected oviductal morphology. Lastly, in chapter 5, I provide a summary of the results and potential future studies for the experiment conducted. Chapter 5 also discusses the potential impact of my results on the field regarding how we examine sexual differentiation of the brain and sexual behaviors. Finally, this chapter concludes with implications and final thoughts on how society should rethink the view that sex is a binary construct, and explore without bias the diversity of sexuality.

Anorexia nervosa (AN) is an eating disorder characterized, in part, by aversion toward high-fat foods. Foods that are high in fats are major sources of fatty acids (FAs). FAs are crucial for brain health and have shown to be altered in psychiatric illnesses including eating disorders. The n-3 and n-6 FAs can undergo auto-oxidation or catalytic conversions in enzymatic pathways such as lipoxygenases (LOX), cyclooxygenases (COX), and cytochromes P450 (CYP) to form oxygenated metabolites termed oxylipins. Oxylipins formed by the Cytochrome P450 (CYP) enzymes are epoxy fatty acids, which are converted into diol fatty acids by soluble epoxide hydrolase (sEH). The genetic variants in the gene that encodes sEH, EPHX2, have been shown to be associated with AN risk. We studied the role of FAs and sEH pathway in AN using 50 AN cases and 47 control women. We have identified abnormal concentrations of several fatty acids at both fasting and postprandial timepoints in AN, a trend of elevated sEH activity and expression, and significant differences in several sEH-associated oxylipins in AN. These results confirm the n-3 PUFA-driven dysregulation in plasma PUFA, elevation of sEH in vivo and ex vivo, and aberrant oxylipins in AN. More studies with larger sample sizes are needed to confirm these findings and examine the mechanisms by which these dysregulations affect AN pathogenesis.

Osteoclasts, the multinucleated bone-resorbing cells, are involved in the destructive breakdown of bones in many diseases such as osteoporosis and rheumatoid arthritis. Designing an efficient and specific therapeutic strategy to these diseases would depend on understanding osteoclasts differentiation. Although gene expression quantification and biochemical techniques have been used extensively to study osteoclast differentiation, they lack the capability to dynamically examine live osteoclasts at the single-cell level. In this thesis, we explored the practicality of the two minimally invasive microscopy techniques, NAD(P)H Fluorescence Lifetime Imaging and LAURDAN spectral imaging, in observing cellular metabolic profiles and membrane dynamics respectively during osteoclast differentiation. In addition to establishing the practicality of these two imaging platforms, our report offered a deeper understanding regarding the roles of metabolism and membrane dynamics in osteoclasts differentiation and pathogenesis of osteoclasts-associated diseases.

Periophthalmus chrysospilos is an amphibious fish and considered candidates of typical aquarium fish in the Mekong Delta (MD), Vietnam. It is a species that is facing overfishing. Unfortunately, the data on its length-weight relationship (LWR), growth pattern, and condition factor are limited. The present study lasted from April 2020 to March 2021 to contribute knowledge of these data. A collection of 1,031 specimens (508 females and 523 males) was obtained from estuarine and coastal regions. The results showed that the fish total length (TL) and weight (W) were significantly different between sexes, seasons, and sites. In order to determine LWRs, weights of males and females in two sizes, seasons, four sites, and twelve months were estimated from the fish given lengths. The species displayed temporal variation in growth patterns as b values ranged from 2.87 ± 0.12 SE to 3.46 ± 0.07 SE. It generally showed positive allometric growth as b (3.22 ± 0.02 SE) was higher than the threshold of three. The fish body conditional factor was close to the well-being value of one and was not influenced by sex, but by season, site and month variations. The findings concluded that the species was well adapted to its habitat.

This study investigated the effects of applications of international quality assurance certification at farm level on the implementation of traceability along the shrimp supply chain with evidence from Vietnam. Descriptive statistics was explored to compare products, information flows, financial efficiency, and willingness to implement traceability between certified and noncertified quality assurance shrimp farms. A binary logistics model was applied to examine the effects of applications of international quality assurance certification and other factors on the willingness to implement traceability of shrimp farmers. Found results showed that the applications of international quality assurance certification of shrimp farmers supported traceability of shrimp products among stakeholders in the supply chain. The certified farmers indicated a higher willingness to implement traceability. However, the profitability of those applications for shrimp farmers was not statistically found. The economics incentives, including farm-gate price differentiation and minimization of production costs, should be carefully considered.

Introduction Legume crops are an important source of protein and oil for human health and in fixing atmospheric N2 for soil enrichment. With an objective to accelerate much-needed genetic analyses and breeding applications, draft genome assemblies were generated in several legume crops; many of them are not high quality because they are mainly based on short reads. However, the superior quality of genome assembly is crucial for a detailed understanding of genomic architecture, genome evolution, and crop improvement. Objectives Present study was undertaken with an objective of developing improved chromosome-length genome assemblies in six different legumes followed by their systematic investigation to unravel different aspects of genome organization and legume evolution. Methods We employed in situ Hi-C data to improve the existing draft genomes and performed different evolutionary and comparative analyses using improved genome assemblies. Results We have developed chromosome-length genome assemblies in chickpea, pigeonpea, soybean, subterranean clover, and two wild progenitor species of cultivated groundnut (A. duranensis and A. ipaensis). A comprehensive comparative analysis of these genome assemblies offered improved insights into various evolutionary events that shaped the present-day legume species. We highlighted the expansion of gene families contributing to unique traits such as nodulation in legumes, gravitropism in groundnut, and oil biosynthesis in oilseed legume crops such as groundnut and soybean. As examples, we have demonstrated the utility of improved genome assemblies for enhancing the resolution of “QTL-hotspot” identification for drought tolerance in chickpea and marker-trait associations for agronomic traits in pigeonpea through genome-wide association study. Genomic resources developed in this study are publicly available through an online repository, ‘Legumepedia’. Conclusion This study reports chromosome-length genome assemblies of six legume species and demonstrates the utility of these assemblies in crop improvement. The genomic resources developed here will have significant role in accelerating genetic improvement applications of legume crops.

Objective Transglutaminase (TGM)2 and peroxisome proliferator-activated receptor (PPAR)γ are thought to participate in the pathogenesis of nasal polyp formation in cystic fibrosis (CF). We herein investigated expressions of cystic fibrosis transmembrane conductance regulator (CFTR), TGM2, PPARγ and isopeptide bonds, a reaction product of TGM, in non-CF nasal polyps. Methods Nasal polyps and inferior turbinates were collected from chronic rhinosinusitis patients without CF during transnasal endoscopic sinonasal surgery. Expressions of CFTR, TGM2, isopeptide bonds and PPARγ were examined by fluorescence immunohistochemistry and quantitative RT-PCR. Expression of CFTR was also analyzed by Western blot. Results Immunohistochemical fluorescence of the nasal polyp was significantly lower for CFTR and PPARγ, and significantly higher for TGM2 and isopeptide bonds than that of the turbinate mucosa. Lower expression of CFTR in the nasal polyp than in the turbinate mucosa was also observed in Western blot. Expression of PPARG mRNA was significantly lower in the nasal polyp than in the turbinate mucosa, whereas expressions of CFTR mRNA or TGM2 mRNA did not differ between the two tissues. Immunohistochemical fluorescence for CFTR showed significant negative correlation with that for TGM2 and isopeptide bonds, and significant positive correlation with that for PPARγ. The fluorescence for TGM2 was positively correlated with that for isopeptide bonds and negatively correlated with that for PPARγ. The fluorescence for isopeptide bonds tended to be negatively correlated with that for PPARγ. Conclusions These results suggest a possible role of the CFTR-TGM2-PPARγ cascade in the pathogenesis of nasal polyp formation in non-CF patients as in CF patients.

Background Gene dosage imbalance caused by copy number variations (CNVs) is a prominent contributor to brain disorders. In particular, 15q11.2 CNV duplications and deletions have been associated with autism spectrum disorder and schizophrenia, respectively. The mechanism underlying these diametric contributions remains unclear. Methods We established both loss-of-function and gain-of-function mouse models of Cyfip1, one of four genes within 15q11.2 CNVs. To assess the functional consequences of altered CYFIP1 levels, we performed systematic investigations on behavioral, electrophysiological, and biochemical phenotypes in both mouse models. In addition, we utilized RNA immunoprecipitation sequencing (RIP-seq) analysis to reveal molecular targets of CYFIP1 in vivo. Results Cyfip1 loss-of-function and gain-of function mouse models exhibited distinct and shared behavioral abnormalities related to autism spectrum disorder and schizophrenia. RIP-seq analysis identified messenger RNA targets of CYFIP1 in vivo, including postsynaptic NMDA receptor (NMDAR) complex components. In addition, these mouse models showed diametric changes in levels of postsynaptic NMDAR complex components at synapses because of dysregulated protein translation, resulting in bidirectional alteration of NMDAR-mediated signaling. Importantly, pharmacological balancing of NMDAR signaling in these mouse models with diametric Cyfip1 dosages rescues behavioral abnormalities. Conclusions CYFIP1 regulates protein translation of NMDAR and associated complex components at synapses to maintain normal synaptic functions and behaviors. Our integrated analyses provide insight into how gene dosage imbalance caused by CNVs may contribute to divergent neuropsychiatric disorders.