The rising roles of exosomes in the tumor microenvironment reprogramming and cancer immunotherapy.

Exosomes are indispensable for intercellular communications. Tumor microenvironment (TME) is the living environment of tumor cells, which is composed of various components, including immune cells. Based on TME, immunotherapy has been recently developed for eradicating cancer cells by reactivating antitumor effect of immune cells. The communications between tumor cells and TME are crucial for tumor development, metastasis, and drug resistance. Exosomes play an important role in mediating these communications and regulating the reprogramming of TME, which affects the sensitivity of immunotherapy. Therefore, it is imperative to investigate the role of exosomes in TME reprogramming and the impact of exosomes on immunotherapy. Here, we review the communication role of exosomes in regulating TME remodeling and the efficacy of immunotherapy, as well as summarize the underlying mechanisms. Furthermore, we also introduce the potential application of the artificially modified exosomes as the delivery systems of antitumor drugs. Further efforts in this field will provide new insights on the roles of exosomes in intercellular communications of TME and cancer progression, thus helping us to uncover effective strategies for cancer treatment.

Human mutations in SLITRK3 implicated in GABAergic synapse development in mice.

This study reports on biallelic homozygous and monoallelic de novo variants in SLITRK3 in three unrelated families presenting with epileptic encephalopathy associated with a broad neurological involvement characterized by microcephaly, intellectual disability, seizures, and global developmental delay. SLITRK3 encodes for a transmembrane protein that is involved in controlling neurite outgrowth and inhibitory synapse development and that has an important role in brain function and neurological diseases. Using primary cultures of hippocampal neurons carrying patients' SLITRK3 variants and in combination with electrophysiology, we demonstrate that recessive variants are loss-of-function alleles. Immunostaining experiments in HEK-293 cells showed that human variants C566R and E606X change SLITRK3 protein expression patterns on the cell surface, resulting in highly accumulating defective proteins in the Golgi apparatus. By analyzing the development and phenotype of SLITRK3 KO (SLITRK3-/-) mice, the study shows evidence of enhanced susceptibility to pentylenetetrazole-induced seizure with the appearance of spontaneous epileptiform EEG as well as developmental deficits such as higher motor activities and reduced parvalbumin interneurons. Taken together, the results exhibit impaired development of the peripheral and central nervous system and support a conserved role of this transmembrane protein in neurological function. The study delineates an emerging spectrum of human core synaptopathies caused by variants in genes that encode SLITRK proteins and essential regulatory components of the synaptic machinery. The hallmark of these disorders is impaired postsynaptic neurotransmission at nerve terminals; an impaired neurotransmission resulting in a wide array of (often overlapping) clinical features, including neurodevelopmental impairment, weakness, seizures, and abnormal movements. The genetic synaptopathy caused by SLITRK3 mutations highlights the key roles of this gene in human brain development and function.

Effects of Differential Shading on Summer Tea Quality and Tea Garden Microenvironment.

Shading is an effective agronomic technique to protect tea plants from intense sunlight. However, there are currently very few studies on more effective shading methods to improve the quality of summer tea. In this study, 'Longjing43' plants were grown under four different shading treatments for 14 days, with no shading as the control. Among the four shading treatments, double-layer-net shadings had the most positive impact on the tea quality, resulting in higher levels of amino acids but lower levels of tea polyphenols. Additionally, double-layer-net shadings provided more suitable microenvironments for tea plants. The tea leaves in T4 (double nets 50 cm above the plant canopy) contained 16.13 mg∙g-1 of umami and sweet amino acids, which was significantly higher than in other treatments. T4 had the lowest air temperature and the most suitable and stable soil water content. Interestingly, the ratio of red light to far-red light in T4 was only 1.65, much lower than other treatments, which warrants further study. In conclusion, the microenvironment induced by shading can greatly affect the tea quality, and double-layer-net shading is better for improving the quality of summer tea.

Base editing of the HBG promoter induces potent fetal hemoglobin expression with no detectable off-target mutations in human HSCs.

Reactivating silenced γ-globin expression through the disruption of repressive regulatory domains offers a therapeutic strategy for treating ß-hemoglobinopathies. Here, we used transformer base editor (tBE), a recently developed cytosine base editor with no detectable off-target mutations, to disrupt transcription-factor-binding motifs in hematopoietic stem cells. By performing functional screening of six motifs with tBE, we found that directly disrupting the BCL11A-binding motif in HBG1/2 promoters triggered the highest γ-globin expression. Via a side-by-side comparison with other clinical and preclinical strategies using Cas9 nuclease or conventional BEs (ABE8e and hA3A-BE3), we found that tBE-mediated disruption of the BCL11A-binding motif at the HBG1/2 promoters triggered the highest fetal hemoglobin in healthy and ß-thalassemia patient hematopoietic stem/progenitor cells while exhibiting no detectable DNA or RNA off-target mutations. Durable therapeutic editing by tBE persisted in repopulating hematopoietic stem cells, demonstrating that tBE-mediated editing in HBG1/2 promoters is a safe and effective strategy for treating ß-hemoglobinopathies.

Design and application of the transformer base editor in mammalian cells and mice.

Fusing apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like cytidine deaminase with catalytically impaired Cas proteins (e.g., nCas9 or dCas9) provides a novel gene-editing technology, base editing, that grants targeted base substitutions with high efficiency. However, genome-wide and transcriptome-wide off-target mutations are observed in base editing, which raises safety concerns regarding therapeutic applications. Previously, we developed a new base editing system, the transformer base editor (tBE), to induce efficient editing with no observable genome-wide or transcriptome-wide off-target mutations both in mammalian cells and in mice. Here we describe a detailed protocol for the design and application of the tBE. Steps for designing single-guide RNA (sgRNA) and helper sgRNA pairs, making constructs, determining the genome-wide and transcriptome-wide off-target mutations, producing the tBE-containing adeno-associated viruses, delivering adeno-associated viruses into mice and examining the in vivo editing effects are included in this protocol. High-precision base editing by the tBE can be completed within 2-3 weeks (in mammalian cells) or within 6-8 weeks (in mice), with sgRNA-helper sgRNA pairs. The whole process can be collaboratively accomplished by researchers using standard techniques from molecular biology, bioinformatics and mouse husbandry.

Regulation of NLGN3 and the Synaptic Rho-GEF Signaling Pathway by CDK5.

Neuroligins (NLGNs) are postsynaptic cell adhesion molecules that are involved in synapse assembly and function. The NLGN gene family consists of 5 genes (NLGN1-3, 4X, and 4Y). NLGN3 forms heterodimers with other NLGNs and is expressed at both excitatory and inhibitory synapses, although the distinct role at different synapses is not fully understood. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase that targets various neuronal substrates to impact neuronal migration, neurite outgrowth, synaptic transmission, and plasticity. Both NLGNs and their presynaptic binding partners neurexins are highly associated with neurodevelopmental disorders. The NLGN3 gene is on the X chromosome and variants in NLGN3 have been linked to the pathophysiology in neurodevelopmental disorders. To better understand the endogenous modulation of NLGN3, we generated an HA-tagged knock-in mouse. We found that Cdk5 associates with NLGN3 in vivo and phosphorylates NLGN3 on serine 725 (S725) in the knock-in mouse of either sex. The phosphorylation affects the NLGN3 association with Kalirin-7, a postsynaptic guanine nucleotide exchange factors for Rho GTPase family proteins. We further observed that the phosphorylation modulates NLGN3 surface expression and NLGN3-mediated synaptic currents in cultured rat neurons. Thus, we characterized NLGN3 as a novel Cdk5 substrate and revealed the functional consequences of NLGN3 S725 phosphorylation in neurons. Our study provides a novel molecular mechanism underlying Cdk5-mediated regulation of postsynaptic cell adhesion molecules.SIGNIFICANCE STATEMENT NLGN3 is involved in synapse assembly and function at both excitatory and inhibitory synapses and has been associated with the pathophysiology of neurodevelopmental disorders. Cdk5 has brain-specific activity and is involved in neuronal transmission, synapse function, and plasticity. Here, we characterize NLGN3 as a Cdk5 substrate for the first time and show that Cdk5-mediated phosphorylation regulates NLGN3 function. We demonstrate that NLGN3 S725 is a Cdk5 phosphorylation site, and reveal that the site is important for NLGN3 association with Kalirin-7, NLGN3 surface expression, and NLGN3-mediated synaptic transmission.

Genome-wide association studies of human and rat BMI converge on synapse, epigenome, and hormone signaling networks.

A vexing observation in genome-wide association studies (GWASs) is that parallel analyses in different species may not identify orthologous genes. Here, we demonstrate that cross-species translation of GWASs can be greatly improved by an analysis of co-localization within molecular networks. Using body mass index (BMI) as an example, we show that the genes associated with BMI in humans lack significant agreement with those identified in rats. However, the networks interconnecting these genes show substantial overlap, highlighting common mechanisms including synaptic signaling, epigenetic modification, and hormonal regulation. Genetic perturbations within these networks cause abnormal BMI phenotypes in mice, too, supporting their broad conservation across mammals. Other mechanisms appear species specific, including carbohydrate biosynthesis (humans) and glycerolipid metabolism (rodents). Finally, network co-localization also identifies cross-species convergence for height/body length. This study advances a general paradigm for determining whether and how phenotypes measured in model species recapitulate human biology.

Lipids regulate peripheral serotonin release via gut CD1d.

The crosstalk between the immune and neuroendocrine systems is critical for intestinal homeostasis and gut-brain communications. However, it remains unclear how immune cells participate in gut sensation of hormones and neurotransmitters release in response to environmental cues, such as self-lipids and microbial lipids. We show here that lipid-mediated engagement of invariant natural killer T (iNKT) cells with enterochromaffin (EC) cells, a subset of intestinal epithelial cells, promoted peripheral serotonin (5-HT) release via a CD1d-dependent manner, regulating gut motility and hemostasis. We also demonstrated that inhibitory sphingolipids from symbiotic microbe Bacteroides fragilis represses 5-HT release. Mechanistically, CD1d ligation on EC cells transduced a signal and restrained potassium conductance through activation of protein tyrosine kinase Pyk2, leading to calcium influx and 5-HT secretion. Together, our data reveal that by engaging with iNKT cells, gut chemosensory cells selectively perceive lipid antigens via CD1d to control 5-HT release, modulating intestinal and systemic homeostasis.

Automated quantitative trait locus analysis (AutoQTL).

BACKGROUND: Quantitative Trait Locus (QTL) analysis and Genome-Wide Association Studies (GWAS) have the power to identify variants that capture significant levels of phenotypic variance in complex traits. However, effort and time are required to select the best methods and optimize parameters and pre-processing steps. Although machine learning approaches have been shown to greatly assist in optimization and data processing, applying them to QTL analysis and GWAS is challenging due to the complexity of large, heterogenous datasets. Here, we describe proof-of-concept for an automated machine learning approach, AutoQTL, with the ability to automate many complicated decisions related to analysis of complex traits and generate solutions to describe relationships that exist in genetic data. RESULTS: Using a publicly available dataset of 18 putative QTL from a large-scale GWAS of body mass index in the laboratory rat, Rattus norvegicus, AutoQTL captures the phenotypic variance explained under a standard additive model. AutoQTL also detects evidence of non-additive effects including deviations from additivity and 2-way epistatic interactions in simulated data via multiple optimal solutions. Additionally, feature importance metrics provide different insights into the inheritance models and predictive power of multiple GWAS-derived putative QTL. CONCLUSIONS: This proof-of-concept illustrates that automated machine learning techniques can complement standard approaches and have the potential to detect both additive and non-additive effects via various optimal solutions and feature importance metrics. In the future, we aim to expand AutoQTL to accommodate omics-level datasets with intelligent feature selection and feature engineering strategies.

An exponential increase in QTL detection with an increased sample size.

Power analyses are often used to determine the number of animals required for a genome-wide association study (GWAS). These analyses are typically intended to estimate the sample size needed for at least 1 locus to exceed a genome-wide significance threshold. A related question that is less commonly considered is the number of significant loci that will be discovered with a given sample size. We used simulations based on a real data set that consisted of 3,173 male and female adult N/NIH heterogeneous stock rats to explore the relationship between sample size and the number of significant loci discovered. Our simulations examined the number of loci identified in subsamples of the full data set. The subsampling analysis was conducted for 4 traits with low (0.15 ± 0.03), medium (0.31 ± 0.03 and 0.36 ± 0.03), and high (0.46 ± 0.03) SNP-based heritabilities. For each trait, we subsampled the data 100 times at different sample sizes (500, 1,000, 1,500, 2,000, and 2,500). We observed an exponential increase in the number of significant loci with larger sample sizes. Our results are consistent with similar observations in human GWAS and imply that future rodent GWAS should use sample sizes that are significantly larger than those needed to obtain a single significant result.

Differential expression of lysine acetylation proteins in gastric cancer treated with a new antitumor agent bioactive peptide chelate selenium.

The method of anticancer bioactive peptide (ACBP) functionalized selenium particle (Se), which has enhanced anticancer activity, inhibited the growth of gastric cancer (GC) cells, and increased the ability of apoptosis in vitro, has been reported in previous studies. We used tandem mass spectrometry (TMT) labeling to construct a complete atlas of the acetylation-modified proteome in GC MKN-45 cells treated with ACBP-Se. The proteomics data database was searched and analyzed by bioinformatics: Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), functional enrichment, and protein-protein interaction network. Finally, we conducted a quantitative PRM analysis of the selected target-modified peptides. We identified 4,958 acetylation sites from 1,926 proteins in this research. Among these, 4,467 acetylation sites corresponding to 1,777 proteins were quantified. Based on the above data and standards, we found that in the ACBP-Se group vs. the control group, 297 sites were upregulated, and 665 sites were downregulated. We systematically assessed the proteins containing quantitative information sites, including protein annotation, functional classification, and functional enrichment, cluster analysis supported by functional enrichment, domain structures, and protein interaction networks. Finally, we evaluated differentially expressed lysine acetylation sites. We revealed that SHMT2 K200 and PGK1 K97 were the most critical acetylated non-histone proteins, which may have an essential role in ACBP-Se treatment. Here, we identified and quantified the lysine acetylation proteins in GC cells treated with ACBP-Se. The characterization of acetylation indicates that acetylated proteins might be pivotal in the biological process, molecular binding, and metabolic pathways of ACBP-Se treatment progress. Our findings provide a broad understanding of acetylation ACBP-Se treatment of GC, suggesting a potential application for molecular targeted therapy.

Automated quantitative trait locus analysis (AutoQTL).

Background: Quantitative Trait Locus (QTL) analysis and Genome-Wide Association Studies (GWAS) have the power to identify variants that capture significant levels of phenotypic variance in complex traits. However, effort and time are required to select the best methods and optimize parameters and pre-processing steps. Although machine learning approaches have been shown to greatly assist in optimization and data processing, applying them to QTL analysis and GWAS is challenging due to the complexity of large, heterogenous datasets. Here, we describe proof-of-concept for an automated machine learning approach, AutoQTL, with the ability to automate many complex decisions related to analysis of complex traits and generate diverse solutions to describe relationships that exist in genetic data. Results: Using a dataset of 18 putative QTL from a large-scale GWAS of body mass index in the laboratory rat, Rattus norvegicus , AutoQTL captures the phenotypic variance explained under a standard additive model while also providing evidence of non-additive effects including deviations from additivity and 2-way epistatic interactions from simulated data via multiple optimal solutions. Additionally, feature importance metrics provide different insights into the inheritance models and predictive power of multiple GWAS-derived putative QTL. Conclusions: This proof-of-concept illustrates that automated machine learning techniques can be applied to genetic data and has the potential to detect both additive and non-additive effects via various optimal solutions and feature importance metrics. In the future, we aim to expand AutoQTL to accommodate omics-level datasets with intelligent feature selection strategies.

Diversity in rhizospheric microbial communities in tea varieties at different locations and tapping potential beneficial microorganisms.

Soil microenvironments and plant varieties could largely affect rhizosphere microbial community structure and functions. However, their specific effects on the tea rhizosphere microbial community are yet not clear. Beneficial microorganisms are important groups of microbial communities that hold ecological functionalities by playing critical roles in plant disease resistance, and environmental stress tolerance. Longjing43 and Zhongcha108 are two widely planted tea varieties in China. Although Zhongcha108 shows higher disease resistance than Longjing43, the potential role of beneficial tea rhizosphere microbes in disease resistance is largely unknown. In this study, the structure and function of rhizosphere microbial communities of these two tea varieties were compared by using the Illumina MiSeq sequencing (16S rRNA gene and ITS) technologies. Rhizosphere soil was collected from four independent tea gardens distributed at two locations in Hangzhou and Shengzhou cities in eastern China, Longjing43 and Zhongcha108 are planted at both locations in separate gardens. Significant differences in soil physicochemical properties as demonstrated by ANOVA and PCA, and distinct rhizosphere microbial communities by multiple-biotech analyses (PCoA, LEfSe, Co-occurrence network analyses) between both locations and tea varieties (p < 0.01) were found. Functions of bacteria were annotated by the FAPROTAX database, and a higher abundance of Nitrososphaeraceae relating to soil ecological function was found in rhizosphere soil in Hangzhou. LDA effect size showed that the abundance of arbuscular mycorrhizal fungi (AMF) was higher in Zhongcha108 than that in Longjing43. Field experiments further confirmed that the colonization rate of AMF was higher in Zhongcha108. This finding testified that AMF could be the major beneficial tea rhizosphere microbes that potentially function in enhanced disease resistance. Overall, our results confirmed that locations affected the microbial community greater than that of tea varieties, and fungi might be more sensitive to the change in microenvironments. Furthermore, we found several beneficial microorganisms, which are of great significance in improving the ecological environment of tea gardens and the disease resistance of tea plants. These beneficial microbial communities may also help to further reveal the mechanism of disease resistance in tea and potentially be useful for mitigating climate change-associated challenges to tea gardens in the future.

Sleep and wake cycles dynamically modulate hippocampal inhibitory synaptic plasticity.

Sleep is an essential process that consolidates memories by modulating synapses through poorly understood mechanisms. Here, we report that GABAergic synapses in hippocampal CA1 pyramidal neurons undergo daily rhythmic alterations. Specifically, wake inhibits phasic inhibition, whereas it promotes tonic inhibition compared to sleep. We further utilize a model of chemically induced inhibitory long-term potentiation (iLTP) to examine inhibitory plasticity. Intriguingly, while CA1 pyramidal neurons in both wake and sleep mice undergo iLTP, wake mice have a much higher magnitude. We also employ optogenetics and observe that inhibitory inputs from parvalbumin-, but not somatostatin-, expressing interneurons contribute to dynamic iLTP during sleep and wake. Finally, we demonstrate that synaptic insertion of α5-GABAA receptors underlies the wake-specific enhancement of iLTP at parvalbumin-synapses, which is independent of time of the day. These data reveal a previously unappreciated daily oscillation of inhibitory LTP in hippocampal neurons and uncover a dynamic contribution of inhibitory synapses in memory mechanisms across sleep and wake.

Genome-Wide Identification and Expression Analysis of Isopentenyl transferase Family Genes during Development and Resistance to Abiotic Stresses in Tea Plant (Camellia sinensis).

The tea plant is an important economic crop and is widely cultivated. Isopentenyl transferase (IPT) is the first and rate-limiting enzyme of cytokinin (CK) signaling, which plays key roles in plant development and abiotic stress. However, the IPT gene family in tea plants has not been systematically investigated until now. The phylogenetic analyses, gene structures, and conserved domains were predicted here. The results showed that a total of 13 CsIPT members were identified from a tea plant genome database and phylogenetically classified into four groups. Furthermore, 10 CsIPT members belonged to plant ADP/ATP-IPT genes, and 3 CsIPTs were tRNA-IPT genes. There is a conserved putative ATP/GTP-binding site (P-loop motif) in all the CsIPT sequences. Based on publicly available transcriptome data as well as through RNA-seq and qRT-PCR analysis, the CsIPT genes which play key roles in the development of different tissues were identified, respectively. Furthermore, CsIPT6.2 may be involved in the response to different light treatments. CsIPT6.4 may play a key role during the dormancy and flush of the lateral buds. CsIPT5.1 may play important regulatory roles during the development of the lateral bud, leaf, and flower. CsIPT5.2 and CsIPT6.2 may both play key roles for increased resistance to cold-stress, whereas CsIPT3.2 may play a key role in improving resistance to high-temperature stress as well as drought-stress and rewatering. This study could provide a reference for further studies of CsIPT family's functions and could contribute to tea molecular breeding.

Expression and functional analysis of CsA-IPT5 splice variants during shoot branching in Camellia sinensis.

Alternative splicing (AS) is a process by which several functional splice variants are generated from the same precursor mRNA. In our recent study, five CsA-IPT5 splice variants with various numbers of ATTTA motifs in the untranslated regions (UTRs) were cloned. Meanwhile, their transient expression, as well as the expression and functional analysis in the two shoot branching processes were studied. Here, we examined how these splice variants regulate the other three important shoot branching processes, including the spring tea development, the distal branching of new shoots, and the shoot branching induced by 2,3,5-triiodobenzoic acid (TIBA) spraying, and thus unraveling the key CsA-IPT5 transcripts which play the most important roles in the shoot branching of tea plants. The results showed that the increased expression of 5' UTR AS3, 3' UTR AS1 and 3' UTR AS2 could contribute to the increased synthesis of tZ/iP-type cytokinins (CKs), thus promoting the spring tea development. Meanwhile, in the TIBA-induced shoot branching or in the distal branching of the new shoots, CsA-IPT5 transcripts regulated the synthesis of CsA-IPT5 protein and CKs through transcriptional regulation of the ratios of its splice variants. Moreover, 3' UTR AS1 and 3' UTR AS2 both play key roles in these two processes. In summary, it is revealed that 3' UTR AS1 and 3' UTR AS2 of CsA-IPT5 might act as the predominant splice variants in shoot branching of the tea plant, and they both can serve as gene resources for tea plant breeding.

Association of rs1137101 with hypertension and type 2 diabetes mellitus of Mongolian and Han Chinese.

BACKGROUND: Hypertension (HTN) and type 2 diabetes mellitus (T2DM) are often coincident, and each condition is considered a risk factor for the other. Both occur frequently in the Inner Mongolia region of China. The reasons for differences in risk between Han and Mongolian ethnic groups are not known. The LEPR gene and its polymorphism, rs1137101 (Gln223Arg), are both considered risk factors for HTN and T2DM, but any role of rs1137101 in the occurrence of HTN + T2DM remains unclear for Mongolian and Han populations in the Inner Mongolia region. AIM: To investigate the relationship between rs1137101 and the occurrence of HTN with T2DM in Mongolian and Han populations in Inner Mongolia. METHODS: A total of 2652 subjects of Han and Mongolian ethnic origins were enrolled in the current study, including 908 healthy controls, 1061 HTN patients and 683 HTN patients with T2DM. RESULTS: The association between the rs1137101 polymorphism and HTN with T2DM was analyzed, and differences between Han and Mongolian individuals assessed. There was a significant correlation between rs1137101 and HTN (co-dominant, dominant, over-dominant and log-additive models) and HTN + T2DM (co-dominant, dominant, over-dominant and log-additive models) after adjustment for sex and age in individuals of Mongolian origin. rs1137101 was significantly associated with HTN (co-dominant, recessive and log-additive models) and HTN + T2DM (co-dominant, dominant, over-dominant and log-additive models) in the Han Chinese population. CONCLUSION: Mongolian and Han subjects from Inner Mongolia with HTN who had rs1137101 were protected against the development of T2DM. Allele A has the opposite impact on the occurrence of HTN in Mongolian and Han Chinese populations.

Variations in Soil Nutrient Dynamics and Bacterial Communities After the Conversion of Forests to Long-Term Tea Monoculture Systems.

The soil microbial community is a key indicator to evaluate the soil health and productivities in agricultural ecosystems. Monoculture and conversions of forests to tea plantations have been widely applied in tea plantation globally, but long-term monoculture of tea plantation could lead to soil degradation and yield decline. Understanding how long-term monoculture systems influence the soil health and ecosystem functions in tea plantation is of great importance for soil environment management. In this study, through the comparison of three independent tea plantations across eastern China composed of varying stand ages (from 3 to 90 years after conversion from forest), we found that long-term tea monoculture led to significant increases in soil total organic carbon (TOC) and microbial nitrogen (MBN). Additionally, the structure, function, and co-occurrence network of soil bacterial communities were investigated by pyrosequencing 16S rRNA genes. The pyrosequencing analysis revealed that the structures and functions of soil bacterial communities were significantly affected by different stand ages, but sampling sites and land-use conversion (from forest to tea plantation) had stronger effects than stand age on the diversity and structure of soil bacterial communities. Soil bacterial diversity can be improved with increasing stand ages in tea plantation. Further RDA analysis revealed that the C and N availability improvement in tea plantation soils led to the variation of structure and function in soil bacterial communities. Moreover, co-occurrence network analysis of soil bacterial communities also demonstrated that interactions among soil bacteria taxa were strengthened with increasing stand age. Our findings suggest that long-term monoculture with proper managements could be beneficial to soil ecosystems by increasing the C and N content and strengthening bacterial associations in tea plantations. Overall, this study provides a comprehensive understanding of the impact of land-use change and long-term monoculture stand age on soil environments in tea plantation.

Shisa7 phosphorylation regulates GABAergic transmission and neurodevelopmental behaviors.

GABA-A receptors (GABAARs) are crucial for development and function of the brain. Altered GABAergic transmission is hypothesized to be involved in neurodevelopmental disorders. Recently, we identified Shisa7 as a GABAAR auxiliary subunit that modulates GABAAR trafficking and GABAergic transmission. However, the underlying molecular mechanisms remain elusive. Here we generated a knock-in (KI) mouse line that is phospho-deficient at a phosphorylation site in Shisa7 (S405) and combined with electrophysiology, imaging and behavioral assays to illustrate the role of this site in GABAergic transmission and plasticity as well as behaviors. We found that expression of phospho-deficient mutants diminished α2-GABAAR trafficking in heterologous cells. Additionally, α1/α2/α5-GABAAR surface expression and GABAergic inhibition were decreased in hippocampal neurons in KI mice. Moreover, chemically induced inhibitory long-term potentiation was abolished in KI mice. Lastly, KI mice exhibited hyperactivity, increased grooming and impaired sleep homeostasis. Collectively, our study reveals a phosphorylation site critical for Shisa7-dependent GABAARs trafficking which contributes to behavioral endophenotypes displayed in neurodevelopmental disorders.