Phosphatase and tensin homolog (Pten) catalyzes the reverse reaction of PI3K by dephosphorylating PIP3 to PIP2. This negatively regulates downstream Akt/mTOR/S6 signaling resulting in decreased cellular growth and proliferation. Co-injection of a lentivirus knocking Pten down with a control lentivirus allows us to compare the effects of Pten knockdown between individual neurons within the same animal. We find that knockdown of Pten results in neuronal hypertrophy by 21 days post-injection. This neuronal hypertrophy is correlated with increased p-S6 and p-mTOR in individual neurons. We used this system to test whether an environmental factor that has been implicated in cellular hypertrophy could influence the severity of the Pten knockdown-induced hypertrophy. Implantation of mini-osmotic pumps delivering fatty acids results in increased neuronal hypertrophy and p-S6/p-mTOR staining. These hypertrophic effects were reversed in response to rapamycin treatment. However, we did not observe a similar increase in hypertrophy in response to dietary manipulations of fatty acids. Thus, we conclude that by driving growth signaling with fatty acids and knocking down a critical regulator of growth, Pten, we are able to observe an additive morphological phenotype of increased soma size mediated by the mTOR pathway.

Differential expression analysis of RNA sequencing (RNA-seq) data typically relies on reconstructing transcripts or counting reads that overlap known gene structures. We previously introduced an intermediate statistical approach called differentially expressed region (DER) finder that seeks to identify contiguous regions of the genome showing differential expression signal at single base resolution without relying on existing annotation or potentially inaccurate transcript assembly.We present the derfinder software that improves our annotation-agnostic approach to RNA-seq analysis by: (i) implementing a computationally efficient bump-hunting approach to identify DERs that permits genome-scale analyses in a large number of samples, (ii) introducing a flexible statistical modeling framework, including multi-group and time-course analyses and (iii) introducing a new set of data visualizations for expressed region analysis. We apply this approach to public RNA-seq data from the Genotype-Tissue Expression (GTEx) project and BrainSpan project to show that derfinder permits the analysis of hundreds of samples at base resolution in R, identifies expression outside of known gene boundaries and can be used to visualize expressed regions at base-resolution. In simulations, our base resolution approaches enable discovery in the presence of incomplete annotation and is nearly as powerful as feature-level methods when the annotation is complete.derfinder analysis using expressed region-level and single base-level approaches provides a compromise between full transcript reconstruction and feature-level analysis. The package is available from Bioconductor at www.bioconductor.org/packages/derfinder.

Although transposable element (TE) derived DNA accounts for more than half of mammalian genomes and initiates a significant proportion of RNA transcripts, high throughput methods are rarely leveraged specifically to detect expression from interspersed repeats.

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique has been widely used to encode fluorescent proteins, opto- or chemo-genetic controllers of cell activity, or heterologous expression of human genes in model organisms. These viruses have also successfully been used to deliver recombinases to relevant target sites in transgenic animals, or even deliver small hairpin or micro RNAs in order to manipulate gene expression. While these techniques have been fruitful, they rely on transgenic animals (recombinases) or frequently lack high efficacy and specificity (shRNA/miRNA). In contrast, the CRISPR/Cas system uses an exogenous Cas nuclease which targets specific sites in an organism's genome via an exogenous guide RNA in order to induce double stranded breaks in DNA. These breaks are then repaired by non-homologous end joining (NHEJ), producing insertion and deletion (indel) mutations that can result in deleterious missense or nonsense mutations. This manuscript provides detailed methods for the design, production, injection, and validation of single lenti/retro virus particles that can stably transduce neurons to express a fluorescent reporter, Cas9, and sgRNAs to knockout genes in a model organism.

Styrene is a versatile commodity petrochemical used as a monomer building-block for the synthesis of many useful polymers. Although achievements have been made on styrene biosynthesis in microorganisms, several bottleneck problems limit factors for further improvement in styrene production.

Increased African-American research participation is critical to the applicability and generalizability of biomedical research, as population diversity continues to increase both domestically and abroad. Yet numerous studies document historical origins of mistrust, as well as other barriers that may contribute to resistance in the African-American community towards participation in biomedical research. However, a growing body of more recent scientific evidence suggests that African-Americans value research and are willing to participate when asked. In the present study, we set out to determine factors associated with research participation of African-American families in postmortem human brain tissue donation for neuropsychiatric disorders as compared with Caucasian families, from same-day medical examiner autopsy referrals. We retrospectively reviewed brain donation rates, as well as demographic and clinical factors associated with donation in 1,421 consecutive referrals to three medical examiner's offices from 2010-2015. Overall, 69.7% of all next-of-kin contacted agreed to brain donation. While Caucasian families consented to donate brain tissue at a significantly higher rate (74.1%) than African-American families (57.0%) (p<0.001), African-American brain donation rates were as high as 60.5% in referrals from Maryland. Neither African-American nor Caucasian donors differed significantly from non-donors on any demographic or clinical factors ascertained, including age, sex, diagnosis of the donor, or in the relationship of the next-of-kin being contacted (p>0.05). However, Caucasian donors were significantly older, had more years of education, were more likely to be referred for study due to a psychiatric diagnosis, more likely to have comorbid substance abuse, and more likely to have died via suicide, as compared with African-American donors (p<0.05). When African-American participants are identified and approached, African-American families as well as Caucasian families are indeed willing to donate brain tissue on the spot for neuropsychiatric research, which supports the belief that African-American attitudes towards biomedical research may be more favorable than previously thought.

Pten, a gene associated with autism spectrum disorder, is an upstream regulator of receptor tyrosine kinase intracellular signaling pathways that mediate extracellular cues to inform cellular development and activity-dependent plasticity. We therefore hypothesized that Pten loss would interfere with activity dependent dendritic growth. We investigated the effects of this interaction on the maturation of retrovirally labeled postnatally generated wild-type and Pten knockout granule neurons in male and female mouse dentate gyrus while using chemogenetics to manipulate the activity of the perforant path afferents. We find that enhancing network activity accelerates the dendritic outgrowth of wild-type, but not Pten knockout, neurons. This was specific to immature neurons during an early developmental window. We also examined synaptic connectivity and physiological measures of neuron maturation. The input resistance, membrane capacitance, dendritic spine morphology, and frequency of spontaneous synaptic events were not differentially altered by activity in wild-type versus Pten knockout neurons. Therefore, Pten and its downstream signaling pathways regulate the activity-dependent sculpting of the dendritic arbor during neuronal maturation.

Brain-derived neurotrophic factor (BDNF) signals through its cognate receptor tropomyosin receptor kinase B (TrkB) to promote the function of several classes of inhibitory interneurons. We previously reported that loss of BDNF-TrkB signaling in cortistatin (Cort)-expressing interneurons leads to behavioral hyperactivity and spontaneous seizures in mice. We performed bulk RNA sequencing (RNA-seq) from the cortex of mice with disruption of BDNF-TrkB signaling in cortistatin interneurons, and identified differential expression of genes important for excitatory neuron function. Using translating ribosome affinity purification and RNA-seq, we define a molecular profile for Cort-expressing inhibitory neurons and subsequently compare the translatome of normal and TrkB-depleted Cort neurons, revealing alterations in calcium signaling and axon development. Several of the genes enriched in Cort neurons and differentially expressed in TrkB-depleted neurons are also implicated in autism and epilepsy. Our findings highlight TrkB-dependent molecular pathways as critical for the maturation of inhibitory interneurons and support the hypothesis that loss of BDNF signaling in Cort interneurons leads to altered excitatory/inhibitory balance.

Brain-derived neurotrophic factor (Bdnf) transcription is controlled by several promoters, which drive expression of multiple transcripts encoding an identical protein. We previously reported that BDNF derived from promoters I and II is highly expressed in hypothalamus and is critical for regulating aggression in male mice. Here we report that BDNF loss from these promoters causes reduced sexual receptivity and impaired maternal care in female mice, which is concomitant with decreased oxytocin (Oxt) expression during development. We identify a novel link between BDNF signaling, oxytocin, and maternal behavior by demonstrating that ablation of TrkB selectively in OXT neurons partially recapitulates maternal care impairments observed in BDNF-deficient females. Using translating ribosome affinity purification and RNA-sequencing we define a molecular profile for OXT neurons and delineate how BDNF signaling impacts gene pathways critical for structural and functional plasticity. Our findings highlight BDNF as a modulator of sexually-dimorphic hypothalamic circuits that govern female-typical behaviors.

Gene co-expression networks capture biological relationships between genes and are important tools in predicting gene function and understanding disease mechanisms. We show that technical and biological artifacts in gene expression data confound commonly used network reconstruction algorithms. We demonstrate theoretically, in simulation, and empirically, that principal component correction of gene expression measurements prior to network inference can reduce false discoveries. Using data from the GTEx project in multiple tissues, we show that this approach reduces false discoveries beyond correcting only for known confounders.

Plant pathogenic oomycetes deliver a troop of effector proteins into the nucleus of host cells to manipulate plant cellular immunity and promote colonization. Recently, researchers have focused on identifying how effectors are transferred into the host cell nucleus, as well as the identity of the nuclear targets. In this study, we found that the RxLR effector PvAVH53 from the grapevine (Vitis vinifera) oomycete pathogen Plasmopara viticola physically interacts with grapevine nuclear import factor importin alphas (VvImpα and VvImpα4), localizes to the nucleus and triggers cell death when transiently expressed in tobacco (Nicotiana benthamiana) cells. Deletion of a nuclear localization signal (NLS) sequence from PvAVH53 or addition of a nuclear export signal (NES) sequence disrupted the nuclear localization of PvAVH53 and attenuated its ability to trigger cell death. Suppression of two tobacco importin-α genes, namely, NbImp-α1 and NbImp-α2, by virus-induced gene silencing (VIGS) also disrupted the nuclear localization and ability of PvAVH53 to induce cell death. Likewise, we transiently silenced the expression of VvImpα/α4 in grape through CRISPR/Cas13a, which has been reported to target RNA in vivo. Finally, we found that attenuating the expression of the Importin-αs genes resulted in increased susceptibility to the oomycete pathogen Phytophthora capsici in N. benthamiana and P. viticola in V. vinifera. Our results demonstrate that importin-αs are required for the nuclear localization and function of PvAVH53 and are essential for host innate immunity. The findings provide insight into the functions of importin-αs in grapevine against downy mildew.

The consequences of aneuploidy have traditionally been studied in cell and animal models in which the extrachromosomal DNA is from the same species. Here, we explore a fundamental question concerning the impact of aneuploidy on systemic metabolism using a non-mosaic transchromosomic mouse model (TcMAC21) carrying a near complete human chromosome 21. Independent of diets and housing temperatures, TcMAC21 mice consume more calories, are hyperactive and hypermetabolic, remain consistently lean and profoundly insulin sensitive, and have a higher body temperature. The hypermetabolism and elevated thermogenesis are due to sarcolipin overexpression in the skeletal muscle, resulting in futile sarco(endo)plasmic reticulum Ca 2+ ATPase (SERCA) activity and energy dissipation. Mitochondrial respiration is also markedly increased in skeletal muscle to meet the high ATP demand created by the futile cycle. This serendipitous discovery provides proof-of-concept that sarcolipin-mediated thermogenesis via uncoupling of the SERCA pump can be harnessed to promote energy expenditure and metabolic health.

There is growing evidence for the role of DNA methylation (DNAm) quantitative trait loci (mQTLs) in the genetics of complex traits, including psychiatric disorders. However, due to extensive linkage disequilibrium (LD) of the genome, it is challenging to identify causal genetic variations that drive DNAm levels by population-based genetic association studies. This limits the utility of mQTLs for fine-mapping risk loci underlying psychiatric disorders identified by genome-wide association studies (GWAS). Here we present INTERACT, a deep learning model that integrates convolutional neural networks with transformer, to predict effects of genetic variations on DNAm levels at CpG sites in the human brain. We show that INTERACT-derived DNAm regulatory variants are not confounded by LD, are concentrated in regulatory genomic regions in the human brain, and are convergent with mQTL evidence from genetic association analysis. We further demonstrate that predicted DNAm regulatory variants are enriched for heritability of brain-related traits and improve polygenic risk prediction for schizophrenia across diverse ancestry samples. Finally, we applied predicted DNAm regulatory variants for fine-mapping schizophrenia GWAS risk loci to identify potential novel risk genes. Our study shows the power of a deep learning approach to identify functional regulatory variants that may elucidate the genetic basis of complex traits.

RNA sequencing offers advantages over other quantification methods for microRNA (miRNA), yet numerous biases make reliable quantification challenging. Previous evaluations of these biases have focused on adapter ligation bias with limited evaluation of reverse transcription bias or amplification bias. Furthermore, evaluations of the quantification of isomiRs (miRNA isoforms) or the influence of starting amount on performance have been very limited. No study had yet evaluated the quantification of isomiRs of altered length or compared the consistency of results derived from multiple moderate starting inputs. We therefore evaluated quantifications of miRNA and isomiRs using four library preparation kits, with various starting amounts, as well as quantifications following removal of duplicate reads using unique molecular identifiers (UMIs) to mitigate reverse transcription and amplification biases.

As an essential platform chemical mostly used for rubber synthesis, isoprene is produced in industry through chemical methods, derived from petroleum. As an alternative, bio-production of isoprene has attracted much attention in recent years. Previous researches were mostly focused on key enzymes to improve isoprene production. In this research, besides screening of key enzymes, we also paid attention to expression intensity of non-key enzymes.

Antibody class switch recombination (CSR) to IgG, IgA, or IgE is a hallmark of adaptive immunity, allowing antibody function diversification beyond IgM. CSR involves a deletion of the IgM/IgD constant region genes placing a new acceptor Constant gene, downstream of the VDJH exon. CSR depends on non-coding (CSRnc) transcription of donor Iμ and acceptor IH exons, located 5' upstream of each CH coding gene. Although, our knowledge of the role of CSRnc transcription has advanced greatly, its extension and importance in healthy and diseased humans is scarce. We analyzed CSRnc transcription in 70,603 publicly available RNA-seq samples, including GTEx, TCGA, and the Sequence Read Archive using recount2, an online resource consisting of normalized RNA-seq gene and exon counts, as well as, coverage BigWig files that can be programmatically accessed through R. CSRnc transcription was validated with a qRT-PCR assay for Iμ, Iγ3, and Iγ1 in humans in response to vaccination. We mapped IH transcription for the human IGH locus, including the less understood IGHD gene. CSRnc transcription was restricted to B cells and is widely distributed in normal adult tissues, but predominant in blood, spleen, MALT-containing tissues, visceral adipose tissue and some so-called "immune privileged" tissues. However, significant Iγ4 expression was found even in non-lymphoid fetal tissues. CSRnc expression in cancer tissues mimicked the expression of their normal counterparts, with notable pattern changes in some common cancer subsets. CSRnc transcription in tumors appears to result from tumor infiltration by B cells, since CSRnc transcription was not detected in corresponding tumor-derived immortal cell lines. Additionally, significantly increased Iδ transcription in ileal mucosa in Crohn's disease with ulceration was found. In conclusion, CSRnc transcription occurs in multiple anatomical locations beyond classical secondary lymphoid organs, representing a potentially useful marker of effector B cell responses in normal and pathological immune responses. The pattern of IH exon expression may reveal clues of the local immune response (i.e., cytokine milieu) in health and disease. This is a great example of how the public recount2 data can be used to further our understanding of transcription, including regions outside the known transcriptome.

We employed Illumina 450 K Infinium microarrays to profile DNA methylation (DNAm) in neuronal nuclei separated by fluorescence-activated sorting from the postmortem orbitofrontal cortex (OFC) of heroin users who died from heroin overdose (N = 37), suicide completers (N = 22) with no evidence of heroin use and from control subjects who did not abuse illicit drugs and died of non-suicide causes (N = 28). We identified 1298 differentially methylated CpG sites (DMSs) between heroin users and controls, and 454 DMSs between suicide completers and controls (p < 0.001). DMSs and corresponding genes (DMGs) in heroin users showed significant differences in the preferential context of hyper and hypo DM. HyperDMSs were enriched in gene bodies and exons but depleted in promoters, whereas hypoDMSs were enriched in promoters and enhancers. In addition, hyperDMGs showed preference for genes expressed specifically by glutamatergic as opposed to GABAergic neurons and enrichment for axonogenesis- and synaptic-related gene ontology categories, whereas hypoDMGs were enriched for transcription factor activity- and gene expression regulation-related terms. Finally, we found that the DNAm-based "epigenetic age" of neurons from heroin users was younger than that in controls. Suicide-related results were more difficult to interpret. Collectively, these findings suggest that the observed DNAm differences could represent functionally significant marks of heroin-associated plasticity in the OFC.

Beyond being one the most widely used psychoactive drugs in the world, cannabis has been identified as an environmental risk factor for psychosis. Though the relationship between cannabis use and psychiatric disorders remains controversial, consistent association between early adolescent cannabis use and the subsequent risk of psychosis suggested adolescence may be a particularly vulnerable period. Previous findings on gene by environment interactions indicated that cannabis use may only increase the risk for psychosis in the subjects who have a specific genetic vulnerability. The type 1 cannabinoid receptor (CB1), encoded by the CNR1 gene, is a key component of the endocannabinoid system. As the primary endocannabinoid receptor in the brain, CB1 is the main molecular target of the endocannabinoid ligand, as well as tetrahydrocannabinol (THC), the principal psychoactive ingredient of cannabis. In this study, we have examined mRNA expression and DNA methylation of CNR1 in human prefrontal cortex (PFC), hippocampus, and caudate samples. The expression of CNR1 is higher in fetal PFC and hippocampus, then drops down dramatically after birth. The lifespan trajectory of CNR1 expression in the DLPFC differentially correlated with age by allelic variation at rs4680, a functional polymorphism in the COMT gene. Compared with COMT methionine158 carriers, Caucasian carriers of the COMT valine158 allele have a stronger negative correlation between the expression of CNR1 in DLPFC and age. In contrast, the methylation level of cg02498983, which is negatively correlated with the expression of CNR1 in PFC, showed the strongest positive correlation with age in PFC of Caucasian carriers of COMT valine158. Additionally, we have observed decreased mRNA expression of CNR1 in the DLPFC of patients with schizophrenia. Further analysis revealed a positive eQTL SNP, rs806368, which predicted the expression of a novel transcript of CNR1 in human DLPFC, hippocampus and caudate. This SNP has been associated with addiction and other psychiatric disorders. THC or ethanol are each significantly associated with dysregulated expression of CNR1 in the PFC of patients with affective disorder, and the expression of CNR1 is significantly upregulated in the PFC of schizophrenia patients who completed suicide. Our results support previous studies that have implicated the endocannabinoid system in the pathology of schizophrenia and provided additional insight into the mechanism of increasing risk for schizophrenia in the adolescent cannabis users.

Grapevine downy mildew, caused by oomycete fungus Plasmopara viticola, is one of the most devastating diseases of grapes across the major production regions of the world. Although many putative effector molecules have been identified from this pathogen, the functions of the majority of these are still unknown. In this study, we analyzed the potential function of 26 P. viticola effectors from the highly virulent strain YL. Using transient expression in leaf cells of the tobacco Nicotiana benthamiana, we found that the majority of the effectors could suppress cell death triggered by BAX and INF1, while seven could induce cell death. The subcellular localization of effectors in N. benthamiana was consistent with their localization in cells of Vitis vinifera. Those effectors that localized to the nucleus (17/26) showed a variety of subnuclear localization. Ten of the effectors localized predominantly to the nucleolus, whereas the remaining seven localized to nucleoplasm. Interestingly, five of the effectors were strongly related in sequence and showed identical subcellular localization, but had different functions in N. benthamiana leaves and expression patterns in grapevine in response to P. viticola. This study highlights the potential functional diversity of P. viticola effectors.

Phosphatase and tensin homolog (PTEN) is a major negative regulator of the phosphatidylinositol-3-kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) pathway. Loss-of-function mutations in PTEN have been found in a subset of patients with macrocephaly and autism spectrum disorder (ASD). PTEN loss in neurons leads to somal hypertrophy, aberrant migration, dendritic overgrowth, increased spine density, and hyperactivity of neuronal circuits. These neuronal overgrowth phenotypes are present on Pten knock-out (KO) and reconstitution with autism-associated point mutations. The mechanism underlying dendritic overgrowth in Pten deficient neurons is unclear. In this study, we examined how Pten loss impacts microtubule (MT) dynamics in both sexes using retroviral infection and transfection strategies to manipulate PTEN expression and tag the plus-end MT binding protein, end-binding protein 3 (EB3). We found Pten KO neurons sprout more new processes over time compared with wild-type (WT) neurons. We also found an increase in MT polymerization rate in Pten KO dendritic growth cones. Reducing MT polymerization rate to the WT level was sufficient to reduce dendritic overgrowth in Pten KO neurons in vitro and in vivo Finally, we found that rescue of dendritic overgrowth via inhibition of MT polymerization was sufficient to improve the performance of Pten KO mice in a spatial memory task. Taken together, our data suggests that one factor underlying PTEN loss dependent dendritic overgrowth is increased MT polymerization. This opens the possibility for an intersectional approach targeting MT polymerization and mTOR with low doses of inhibitors to achieve therapeutic gains with minimal side effects in pathologies associated with loss of neuronal PTEN function.SIGNIFICANCE STATEMENT Loss of Pten function because of genetic deletion or expression of mutations associated with autism spectrum disorder (ASD), results in overgrowth of neurons including increased total dendritic length and branching. We have discovered that this overgrowth is accompanied by increased rate of microtubule (MT) polymerization. The increased polymerization rate is insensitive to acute inhibition of mechanistic target of rapamycin (mTOR)C1 or protein synthesis. Direct pharmacological inhibition of MT polymerization can slow the polymerization rate in Pten knock-out (KO) neurons to rates seen in wild-type (WT) neurons. Correction of the MT polymerization rate rescues increased total dendritic arborization and spatial memory. Our studies suggest that phosphatase and tensin homolog (PTEN) inhibits dendritic growth through parallel regulation of protein synthesis and cytoskeletal polymerization.