Omics approaches to investigate the pathogenesis of suicide.

Suicide is the second leading cause of death in U.S. adolescents and young adults, and generally associated with a psychiatric disorder. Suicidal behavior has a complex etiology and pathogenesis. Moderate heritability suggests genetic causes. Associations between childhood and recent life adversity indicate contributions from epigenetic factors. Genomic contributions to suicide pathogenesis remain largely unknown. This paper is based on a workshop held to design strategies to identify molecular drivers of suicide neurobiology that would be putative new treatment targets. The panel determined that, while bulk tissue studies provide comprehensive information, single-nucleus approaches identifying cell-type specific changes are needed. While single nuclei techniques lack information on cytoplasm, processes, spines, and synapses, spatial multiomic technologies on intact tissue detect cell alterations specific to brain tissue layers and subregions. Because suicide has genetic and environmental drivers, multiomic approaches combining cell-type specific epigenome, transcriptome, and proteome provide a more complete picture of pathogenesis. To determine the direction of effect of suicide risk gene variants on RNA and protein expression, and how these interact with epigenetic marks, single nuclei and spatial multiomics quantitative trait loci maps should be integrated with whole genome sequencing and genome-wide association databases. The workshop concluded with the recommendation for the formation of an international suicide biology consortium that will bring together brain banks and investigators with expertise in cutting-edge omics technologies to delineate the biology of suicide and identify novel potential treatment targets to be tested in cellular and animal models for drug and biomarkers discovery, to guide suicide prevention.

Electron beam lithography on nonplanar and irregular surfaces.

E-beam lithography is a powerful tool for generating nanostructures and fabricating nanodevices with fine features approaching a few nanometers in size. However, alternative approaches to conventional spin coating and development processes are required to optimize the lithography procedure on irregular surfaces. In this review, we summarize the state of the art in nanofabrication on irregular substrates using e-beam lithography. To overcome these challenges, unconventional methods have been developed. For instance, polymeric and nonpolymeric materials can be sprayed or evaporated to form uniform layers of electron-sensitive materials on irregular substrates. Moreover, chemical bonds can be applied to help form polymer brushes or self-assembled monolayers on these surfaces. In addition, thermal oxides can serve as resists, as the etching rate in solution changes after e-beam exposure. Furthermore, e-beam lithography tools can be combined with cryostages, evaporation systems, and metal deposition chambers for sample development and lift-off while maintaining low temperatures. Metallic nanopyramids can be fabricated on an AFM tip by utilizing ice as a positive resistor. Additionally, Ti/Au caps can be patterned around a carbon nanotube. Moreover, 3D nanostructures can be formed on irregular surfaces by exposing layers of anisole on organic ice surfaces with a focused e-beam. These advances in e-beam lithography on irregular substrates, including uniform film coating, instrumentation improvement, and new pattern transferring method development, substantially extend its capabilities in the fabrication and application of nanoscale structures.

Exploring the Complex Impact of Proteins on Dopamine Polymerization: Mechanisms and Strategies for Modulation.

Polydopamine (pDA) is a valuable material with wide-ranging potential applications. However, the complex and debated nature of dopamine polymerization complicates our understanding. Specifically, the impact of foreign substances, especially proteins, on pDA formation adds an additional layer of subtlety and complexity. This study delves into specific surface features of proteins that predominantly shape their impact on dopamine polymerization. Notably, the biotin-binding site emerges as a critical region responsible for the pronounced inhibitory effect of avidin and neutravidin on the dopamine polymerization process. The binding of biotin successfully mitigates these inhibitory effects. Moreover, several nucleases demonstrated a significant hindrance to pDA formation, with their impact substantially alleviated through the introduction of DNA. It is speculated that hydrogen bonding, electrostatic, cation-π, and/or hydrophobic interactions may underlie the binding between protein surfaces and diverse oligomeric intermediates formed by the oxidation products of dopamine. Additionally, we observed a noteworthy blocking effect on the dopamine polymerization reaction induced by erythropoietin (EPO), a glycoprotein hormone known for its role in stimulating red blood cell production and demonstrating neuroprotective effects. The inhibitory influence of EPO persisted even after deglycosylation. These findings not only advance our comprehension of the mechanisms underlying dopamine polymerization but also provide strategic insights for manipulating the reaction to tailor desired biomaterials.

Simultaneous single-cell analysis of 5mC and 5hmC with SIMPLE-seq.

Dynamic 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) modifications to DNA regulate gene expression in a cell-type-specific manner and are associated with various biological processes, but the two modalities have not yet been measured simultaneously from the same genome at the single-cell level. Here we present SIMPLE-seq, a scalable, base resolution method for joint analysis of 5mC and 5hmC from thousands of single cells. Based on orthogonal labeling and recording of 'C-to-T' mutational signals from 5mC and 5hmC sites, SIMPLE-seq detects these two modifications from the same molecules in single cells and enables unbiased DNA methylation dynamics analysis of heterogeneous biological samples. We applied this method to mouse embryonic stem cells, human peripheral blood mononuclear cells and mouse brain to give joint epigenome maps at single-cell and single-molecule resolution. Integrated analysis of these two cytosine modifications reveals distinct epigenetic patterns associated with divergent regulatory programs in different cell types as well as cell states.

SPSL1 is essential for spermatophore formation and sperm activation in Spodoptera frugiperda.

The reproductive process in various species has undergone evolutionary adaptations at both the physiological and molecular levels, playing a significant role in maintaining their populations. In lepidopteran insects, the spermatophore is a unique structure formed in the female reproductive system, in which sperm storage and activation take place. It is known that the formation of the spermatophore is regulated by seminal fluid proteins derived from males. However, studies investigating the genetic mechanisms behind spermatophore formation in lepidopterans have been limited. In this study, our focus was on SPSL1, a gene that encodes a trypsin-type seminal fluid protein in Spodoptera frugiperda, a pest species with global invasive tendencies. Our findings revealed that SPSL1 expression was predominantly observed in the male reproductive tracts, and the disruption of this gene resulted in male sterility. Surprisingly, fluorescence analysis indicated that the absence of SPSL1 did not affect spermatogenesis or sperm migration within the male reproductive system. However, when females mated with SPSL1-mutant males, several defects were observed. These included disruptions in spermatophore formation, sperm activation in the copulatory bursae, and sperm migration into the spermathecae. Additionally, mass spectrometry analysis highlighted reduced levels of energy-related metabolites, suggesting that SPSL1 plays an essential role in promoting hydrolysis reactions during copulation. Consequently, our study demonstrates that SPSL1 is crucial for male fertility due to its functions in spermatophore formation and sperm activation. This research provides valuable insights into the genetic factors underlying reproductive processes in lepidopteran insects and sheds light on potential strategies for controlling invasive pest populations.

A comparative atlas of single-cell chromatin accessibility in the human brain.

Recent advances in single-cell transcriptomics have illuminated the diverse neuronal and glial cell types within the human brain. However, the regulatory programs governing cell identity and function remain unclear. Using a single-nucleus assay for transposase-accessible chromatin using sequencing (snATAC-seq), we explored open chromatin landscapes across 1.1 million cells in 42 brain regions from three adults. Integrating this data unveiled 107 distinct cell types and their specific utilization of 544,735 candidate cis-regulatory DNA elements (cCREs) in the human genome. Nearly a third of the cCREs demonstrated conservation and chromatin accessibility in the mouse brain cells. We reveal strong links between specific brain cell types and neuropsychiatric disorders including schizophrenia, bipolar disorder, Alzheimer's disease (AD), and major depression, and have developed deep learning models to predict the regulatory roles of noncoding risk variants in these disorders.

SnapHiC-D: a computational pipeline to identify differential chromatin contacts from single-cell Hi-C data.

Single-cell high-throughput chromatin conformation capture technologies (scHi-C) has been used to map chromatin spatial organization in complex tissues. However, computational tools to detect differential chromatin contacts (DCCs) from scHi-C datasets in development and through disease pathogenesis are still lacking. Here, we present SnapHiC-D, a computational pipeline to identify DCCs between two scHi-C datasets. Compared to methods designed for bulk Hi-C data, SnapHiC-D detects DCCs with high sensitivity and accuracy. We used SnapHiC-D to identify cell-type-specific chromatin contacts at 10 Kb resolution in mouse hippocampal and human prefrontal cortical tissues, demonstrating that DCCs detected in the hippocampal and cortical cell types are generally associated with cell-type-specific gene expression patterns and epigenomic features. SnapHiC-D is freely available at https://github.com/HuMingLab/SnapHiC-D.

SnapFISH: a computational pipeline to identify chromatin loops from multiplexed DNA FISH data.

Multiplexed DNA fluorescence in situ hybridization (FISH) imaging technologies have been developed to map the folding of chromatin fibers at tens of nanometers and up to several kilobases in resolution in single cells. However, computational methods to reliably identify chromatin loops from such imaging datasets are still lacking. Here we present a Single-Nucleus Analysis Pipeline for multiplexed DNA FISH (SnapFISH), to process the multiplexed DNA FISH data and identify chromatin loops. SnapFISH can identify known chromatin loops from mouse embryonic stem cells with high sensitivity and accuracy. In addition, SnapFISH obtains comparable results of chromatin loops across datasets generated from diverse imaging technologies. SnapFISH is freely available at https://github.com/HuMingLab/SnapFISH .

Droplet-based single-cell joint profiling of histone modifications and transcriptomes.

We previously reported Paired-Tag, a combinatorial indexing-based method that can simultaneously map histone modifications and gene expression at single-cell resolution at scale. However, the lengthy procedure of Paired-Tag has hindered its general adoption in the community. To address this bottleneck, we developed a droplet-based Paired-Tag protocol that is faster and more accessible than the previous method. Using cultured mammalian cells and primary brain tissues, we demonstrate its superior performance at identifying candidate cis-regulatory elements and associating their dynamic chromatin state to target gene expression in each constituent cell type in a complex tissue.

Integrated analysis of single-cell chromatin state and transcriptome identified common vulnerability despite glioblastoma heterogeneity.

In 2021, the World Health Organization reclassified glioblastoma, the most common form of adult brain cancer, into isocitrate dehydrogenase (IDH)-wild-type glioblastomas and grade IV IDH mutant (G4 IDHm) astrocytomas. For both tumor types, intratumoral heterogeneity is a key contributor to therapeutic failure. To better define this heterogeneity, genome-wide chromatin accessibility and transcription profiles of clinical samples of glioblastomas and G4 IDHm astrocytomas were analyzed at single-cell resolution. These profiles afforded resolution of intratumoral genetic heterogeneity, including delineation of cell-to-cell variations in distinct cell states, focal gene amplifications, as well as extrachromosomal circular DNAs. Despite differences in IDH mutation status and significant intratumoral heterogeneity, the profiled tumor cells shared a common chromatin structure defined by open regions enriched for nuclear factor 1 transcription factors (NFIA and NFIB). Silencing of NFIA or NFIB suppressed in vitro and in vivo growths of patient-derived glioblastomas and G4 IDHm astrocytoma models. These findings suggest that despite distinct genotypes and cell states, glioblastoma/G4 astrocytoma cells share dependency on core transcriptional programs, yielding an attractive platform for addressing therapeutic challenges associated with intratumoral heterogeneity.

Nanovaccines Fostering Tertiary Lymphoid Structure to Attack Mimicry Nasopharyngeal Carcinoma.

Tertiary lymphoid structures (TLSs) are formed in inflamed tissues, and recent studies demonstrated that the appearance of TLSs in tumor sites is associated with a good prognosis for tumor patients. However, the process of natural TLSs' formation was slow and uncontrollable. Herein, we developed a nanovaccine consisting of Epstein-Barr virus nuclear antigen 1 (EBNA1) and a bi-adjuvant of Mn2+ and cytosine-phosphate-guanine (CpG) formulated with tannic acid that significantly inhibited the development of mimicry nasopharyngeal carcinoma by fostering TLS formation. The nanovaccine activated LT-α and LT-ß pathways, subsequently enhancing the expression of downstream chemokines, CCL19/CCL21, CXCL10 and CXCL13, in the tumor microenvironment. In turn, normalized blood and lymph vessels were detected in the tumor tissues of the nanovaccine group, correlated with increased infiltration of lymphocytes. Especially, the proportion of the B220+ CD8+ T, which was produced via trogocytosis between T and B cells during activation of T cells, was increased in tumors of the nanovaccine group. Furthermore, the intratumoral effector memory T cells (Tem), CD45+, CD3+, CD8+, CD44+, and CD62L-, did not decrease after blocking the egress of T cells from tumor-draining lymph nodes by FTY-720. These results demonstrated that the nanovaccine can foster TLS formation, which thus enhances local immune responses significantly, delays tumor outgrowth, and prolongs the median survival time of murine models of mimicry nasopharyngeal carcinoma, demonstrating a promising strategy for nanovaccine development.

Single-Cell Joint Profiling of Open Chromatin and Transcriptome by Paired-Seq.

Simultaneous detection of chromatin accessibility and transcription from the same cells promises to greatly facilitate the dissection of cell-type-specific gene regulatory programs in complex tissues. Paired-seq enables joint analysis of open chromatin and nuclear transcriptome from up to a million cells in parallel. It achieves ultra-high-throughput single-cell multiomics with the use of a combinatorial barcoding strategy involving sequential ligation of multiplexed DNA barcodes to chromatin DNA fragments and reverse transcription products, followed by high-throughput DNA sequencing of the resulting DNA libraries and deconvolution of single-cell multiomic maps based on cell-specific barcodes.

Nanoparticulate Cationic Poly(amino acid)s Block Cancer Metastases by Destructing Neutrophil Extracellular Traps.

Cancer metastasis that is resistant to conventional therapies has become a major cause of patient death. Recent reports indicate that the neutrophil extracellular trap (NET) is closely associated with cancer distant metastases, and the cell-free DNA of NETs has been identified as the ligand of the transmembrane protein CCDC25 of cancer cells, acting as a chemokine to induce cancer cell migration to distant organs. In this work, we present the poly(aspartic acid) based-cationic materials to interfere with the interaction between NET-DNA and CCDC25, and furthermore to inhibit NET-DNA-mediated cancer cell chemotaxis and migration. Because of a stronger binding affinity to DNA and favorable retention in the liver, nanoparticulate poly(aspartic acid) derivatives (cANP) efficiently reduce the level of hepatic NET-DNA infiltration, leading to a significant suppression of cancer metastases in mice and several human metastatic models. Moreover, the cANP exhibits no toxicity to organs of animals during the entire treatment. Thus, this work suggests a strategy for controlling cancer metastases, which will benefit patients in clinics.

BmHen1 is essential for eupyrene sperm development in Bombyx mori but PIWI proteins are not.

In lepidopteran insects, sperm dimorphism is a remarkable feature, in which males exhibit two different types of sperms. Both sperm morphs are essential for fertilization: Eupyrene sperm carry DNA and fertilize eggs, whereas apyrene sperm, which do not have nuclei, are necessary for transport of eupyrene sperm into eggs. In this study, we showed that the gene BmHen1, which encodes a methyltransferase that modifies piRNAs, is necessary for eupyrene sperm development in the lepidopteran model insect, Bombyx mori. Loss-of-function mutants of BmHen1 of both sexes were sterile. BmHen1 female mutants laid fewer eggs than wild-type females, and the eggs laid had morphological defects. Immunofluorescence analysis of BmHen1 male mutants revealed that nuclei formation in the eupyrene sperm was defective, whereas apyrene sperm were normal. In mice, worms, and flies, the components in piRNA biogenesis pathway play an important role in gonad development; therefore, we constructed mutations in genes encoding core elements in the piRNA biogenesis pathway, Siwi, and BmAgo3. To our surprise, no obvious phenotypes were observed in the male reproduction system in the Siwi and BmAgo3 mutants, which demonstrated that sperm development in B. mori does not depend on piRNAs. As the sperm development phenotype in BmHen1 mutants mimics the phenotype of the BmPnldc1 mutants, we then performed RNA sequencing analysis of sperm bundles from both mutants. We found that the defects in eupyrene sperm resulted from dysregulation of the expression of genes involved in energy metabolism. Taken together, our findings demonstrate the crucial functions of BmHen1 in the development of eupyrene sperm and provide evidence that spermatogenesis in B. mori is PIWI-independent. Our results suggest potential targets for lepidopteran pest control and broaden our knowledge of the reproduction in this order of insects.

Single-cell epigenome analysis reveals age-associated decay of heterochromatin domains in excitatory neurons in the mouse brain.

Loss of heterochromatin has been implicated as a cause of pre-mature aging and age-associated decline in organ functions in mammals; however, the specific cell types and gene loci affected by this type of epigenetic change have remained unclear. To address this knowledge gap, we probed chromatin accessibility at single-cell resolution in the brains, hearts, skeletal muscles, and bone marrows from young, middle-aged, and old mice, and assessed age-associated changes at 353,126 candidate cis-regulatory elements (cCREs) across 32 major cell types. Unexpectedly, we detected increased chromatin accessibility within specific heterochromatin domains in old mouse excitatory neurons. The gain of chromatin accessibility at these genomic loci was accompanied by the cell-type-specific loss of heterochromatin and activation of LINE1 elements. Immunostaining further confirmed the loss of the heterochromatin mark H3K9me3 in the excitatory neurons but not in inhibitory neurons or glial cells. Our results reveal the cell-type-specific changes in chromatin landscapes in old mice and shed light on the scope of heterochromatin loss in mammalian aging.

CRISPR/Cas9-Mediated Disruption of the lef8 and lef9 to Inhibit Nucleopolyhedrovirus Replication in Silkworms.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a pathogen that causes severe disease in silkworms. In a previous study, we demonstrated that by using the CRISPR/Cas9 system to disrupt the BmNPV ie-1 and me53 genes, transgenic silkworms showed resistance to BmNPV infection. Here, we used the same strategy to simultaneously target lef8 and lef9, which are essential for BmNPV replication. A PCR assay confirmed that double-stranded breaks were induced in viral DNA at targeted sequences in BmNPV-infected transgenic silkworms that expressed small guide RNAs (sgRNAs) and Cas9. Bioassays and qPCR showed that replication of BmNPV and mortality were significantly reduced in the transgenic silkworms in comparison with the control groups. Microscopy showed degradation of midgut cells in the BmNPV-infected wild type silkworms, but not in the transgenic silkworms. These results demonstrated that transgenic silkworms using the CRISPR/Cas9 system to disrupt BmNPV lef8 and lef9 genes could successfully prevent BmNPV infection. Our research not only provides more alternative targets for the CRISPR antiviral system, but also aims to provide new ideas for the application of virus infection research and the control of insect pests.

APOLLO: An accurate and independently validated prediction model of lower-grade gliomas overall survival and a comparative study of model performance.

BACKGROUND: Virtually few accurate and robust prediction models of lower-grade gliomas (LGG) survival exist that may aid physicians in making clinical decisions. We aimed to develop a prognostic prediction model of LGG by incorporating demographic, clinical and transcriptional biomarkers with either main effects or gene-gene interactions. METHODS: Based on gene expression profiles of 1,420 LGG patients from six independent cohorts comprising both European and Asian populations, we proposed a 3-D analysis strategy to develop and validate an Accurate Prediction mOdel of Lower-grade gLiomas Overall survival (APOLLO). We further conducted decision curve analysis to assess the net benefit (NB) of identifying true positives and the net reduction (NR) of unnecessary interventions. Finally, we compared the performance of APOLLO and the existing prediction models by the first systematic review. FINDINGS: APOLLO possessed an excellent discriminative ability to identify patients at high mortality risk. Compared to those with less than the 20th percentile of APOLLO risk score, patients with more than the 90th percentile of APOLLO risk score had significantly worse overall survival (HR=54·18, 95% CI: 34·73-84·52, P=2·66 × 10-69). Further, APOLLO can accurately predict both 36- and 60-month survival in six independent cohorts with a pooled AUC36-month=0·901 (95% CI: 0·879-0·923), AUC60-month=0·843 (95% CI: 0·815-0·871) and C-index=0·818 (95% CI: 0·800-0·835). Moreover, APOLLO offered an effective screening strategy for detecting LGG patients susceptible to death (NB36-month=0·166, NR36-month=40·1% and NB60-month=0·258, NR60-month=19·2%). The systematic comparisons revealed APOLLO outperformed the existing models in accuracy and robustness. INTERPRETATION: APOLLO has the demonstrated feasibility and utility of predicting LGG survival (http://bigdata.njmu.edu.cn/APOLLO). FUNDING: National Key Research and Development Program of China (2016YFE0204900); Natural Science Foundation of Jiangsu Province (BK20191354); National Natural Science Foundation of China (81973142 and 82103946); China Postdoctoral Science Foundation (2020M681671); National Institutes of Health (CA209414, CA249096, CA092824 and ES000002).

T Lymphocyte-Captured DNA Network for Localized Immunotherapy.

The efficient isolation of immune cells with high purity and low cell damage is important for immunotherapy and remains highly challenging. We herein report a cell capture DNA network containing polyvalent multimodules for the specific isolation and in situ incubation of T lymphocytes (T-cells). Two ultralong DNA chains synthesized by an enzymatic amplification process were rationally designed to include functional multimodules as cell anchors and immune adjuvants. Mutually complementary sequences facilitated the formation of a DNA network and encapsulation of T-cells, as well as offering cutting sites of a restriction enzyme for the responsive release of T-cells and immune adjuvants. The purity of captured tumor-infiltrating T-cells reached 98%, and the viability of T-cells maintained ∼90%. The T-cells-containing DNA network was further administrated to a tumor lesion for localized immunotherapy. Our work provides a robust nanobiotechnology for efficient isolation of immune cells and other biological particles.

Extending the Storage Time of Clanis bilineata tsingtauica (Lepidoptera; Sphingidae) Eggs through Variable-Temperature Cold Storage.

Clanis bilineata tsingtauica Mell, 1922 (Lepidoptera, Sphingidae), also known as "Doudan" in China, is an important pest in legume crops. As an edible insect, it is most commonly consumed in Jiangsu, Shandong, and Henan Provinces. Mass rearing requires access to large numbers of eggs. This stage, however, is of short duration and supplies are frequently not sufficient for insect production. Therefore, we identified the cold storage conditions for C. bilineata tsingtauica that can effectively prolong the storage time of the eggs, to make supplies more readily available. We found that when stored at 4 °C, only 7.5% of the eggs hatched after 7 days, while at 10 °C the hatch rate was 78.3%. At 15 °C, the egg hatch rate remained at this same level (77.8% even after 14-20 days). Considering various combinations, we found that optimal egg hatch occurred if eggs were stored at 15 °C for 11 days, and then held at 15-20 °C under dark conditions. Stored as described above, the egg hatch rate was not significantly different from the control group (at 28 °C). These conditions allow for easier mass rearing of C. bilineata tsingtauica by providing a stable supply of eggs.