An integrated toolkit for human microglia functional genomics.

BACKGROUND: Microglia, the brain's resident immune cells, play vital roles in brain development, and disorders like Alzheimer's disease (AD). Human iPSC-derived microglia (iMG) provide a promising model to study these processes. However, existing iMG generation protocols face challenges, such as prolonged differentiation time, lack of detailed characterization, and limited gene function investigation via CRISPR-Cas9. METHODS: Our integrated toolkit for in-vitro microglia functional genomics optimizes iPSC differentiation into iMG through a streamlined two-step, 20-day process, producing iMG with a normal karyotype. We confirmed the iMG's authenticity and quality through single-cell RNA sequencing, chromatin accessibility profiles (ATAC-Seq), proteomics and functional tests. The toolkit also incorporates a drug-dependent CRISPR-ON/OFF system for temporally controlled gene expression. Further, we facilitate the use of multi-omic data by providing online searchable platform that compares new iMG profiles to human primary microglia: https://sherlab.shinyapps.io/IPSC-derived-Microglia/ . RESULTS: Our method generates iMG that closely align with human primary microglia in terms of transcriptomic, proteomic, and chromatin accessibility profiles. Functionally, these iMG exhibit Ca2 + transients, cytokine driven migration, immune responses to inflammatory signals, and active phagocytosis of CNS related substrates including synaptosomes, amyloid beta and myelin. Significantly, the toolkit facilitates repeated iMG harvesting, essential for large-scale experiments like CRISPR-Cas9 screens. The standalone ATAC-Seq profiles of our iMG closely resemble primary microglia, positioning them as ideal tools to study AD-associated single nucleotide variants (SNV) especially in the genome regulatory regions. CONCLUSIONS: Our advanced two-step protocol rapidly and efficiently produces authentic iMG. With features like the CRISPR-ON/OFF system and a comprehensive multi-omic data platform, our toolkit equips researchers for robust microglial functional genomic studies. By facilitating detailed SNV investigation and offering a sustainable cell harvest mechanism, the toolkit heralds significant progress in neurodegenerative disease drug research and therapeutic advancement.

Single Cell Analysis of Lung Lymphatic Endothelial Cells and Lymphatic Responses during Influenza Infection.

Tissue lymphatic vessels network plays critical roles in immune surveillance and tissue homeostasis in response to pathogen invasion, but how lymphatic system per se is remolded during infection is less understood. Here, we observed that influenza infection induces a significant increase of lymphatic vessel numbers in the lung, accompanied with extensive proliferation of lymphatic endothelial cells (LECs). Single-cell RNA sequencing illustrated the heterogeneity of LECs, identifying a novel PD-L1+ subpopulation that is present during viral infection but not at steady state. Specific deletion of Pd-l1 in LECs elevated the expansion of lymphatic vessel numbers during viral infection. Together these findings elucidate a dramatic expansion of lung lymphatic network in response to viral infection, and reveal a PD-L1+ LEC subpopulation that potentially modulates lymphatic vessel remolding.

Regulation of craving training to support healthy food choices under stress: A randomized control trial employing the hierarchical drift-diffusion model.

Stress increases the likelihood of consuming unhealthy food in some individuals. Previous research has demonstrated that the Regulation of Craving - Training (ROC-T) intervention can reduce unhealthy food intake. However, its effectiveness under stress and the underlying mechanism remained uncertain. This study aimed to assess the efficacy of the ROC-T intervention in improving healthy food choices and to explore the intervention mechanism through computational modeling employing the hierarchical drift-diffusion model (HDDM). This study adopted a 2 (ROC-T intervention vs. control) * 2 (stress vs. no-stress) between-subject experimental design. A total of 118 employees (72 women, Mage  = 28.74) participated in the online experiment. Results show that the ROC-T intervention increases healthy food choices under stress and no-stress conditions. The HDDM results reveal a significant two-way interaction for non-decision time (Bayes factor, BF = 32.722) and initial bias (BF = 27.350). Specifically, in the no-stress condition, the ROC-T intervention resulted in lower non-decision time and higher initial bias compared with the control group. The findings validated the negative impact of stress on healthy food choices, and that the ROC-T intervention promotes healthy food choices both under stress and no-stress conditions.

m6A RNA modification regulates innate lymphoid cell responses in a lineage-specific manner.

Innate lymphoid cells (ILCs) can quickly switch from a quiescent state to an active state and rapidly produce effector molecules that provide critical early immune protection. How the post-transcriptional machinery processes different stimuli and initiates robust gene expression in ILCs is poorly understood. Here, we show that deletion of the N6-methyladenosine (m6A) writer protein METTL3 has little impact on ILC homeostasis or cytokine-induced ILC1 or ILC3 responses but significantly diminishes ILC2 proliferation, migration and effector cytokine production and results in impaired antihelminth immunity. m6A RNA modification supports an increase in cell size and transcriptional activity in activated ILC2s but not in ILC1s or ILC3s. Among other transcripts, the gene encoding the transcription factor GATA3 is highly m6A methylated in ILC2s. Targeted m6A demethylation destabilizes nascent Gata3 mRNA and abolishes the upregulation of GATA3 and ILC2 activation. Our study suggests a lineage-specific requirement of m6A for ILC2 responses.

Noncoding translation mitigation.

Translation is pervasive outside of canonical coding regions, occurring in long noncoding RNAs, canonical untranslated regions and introns1-4, especially in ageing4-6, neurodegeneration5,7 and cancer8-10. Notably, the majority of tumour-specific antigens are results of noncoding translation11-13. Although the resulting polypeptides are often nonfunctional, translation of noncoding regions is nonetheless necessary for the birth of new coding sequences14,15. The mechanisms underlying the surveillance of translation in diverse noncoding regions and how escaped polypeptides evolve new functions remain unclear10,16-19. Functional polypeptides derived from annotated noncoding sequences often localize to membranes20,21. Here we integrate massively parallel analyses of more than 10,000 human genomic sequences and millions of random sequences with genome-wide CRISPR screens, accompanied by in-depth genetic and biochemical characterizations. Our results show that the intrinsic nucleotide bias in the noncoding genome and in the genetic code frequently results in polypeptides with a hydrophobic C-terminal tail, which is captured by the ribosome-associated BAG6 membrane protein triage complex for either proteasomal degradation or membrane targeting. By contrast, canonical proteins have evolved to deplete C-terminal hydrophobic residues. Our results reveal a fail-safe mechanism for the surveillance of unwanted translation from diverse noncoding regions and suggest a possible biochemical route for the preferential membrane localization of newly evolved proteins.

Collateral activity of the CRISPR/RfxCas13d system in human cells.

CRISPR/Cas13 systems are increasingly used for programmable targeting of RNAs. While Cas13 nucleases are capable of degrading both target RNAs and bystander RNAs in vitro and in bacteria, initial studies fail to detect collateral degradation of non-target RNAs in eukaryotic cells. Here we show that RfxCas13d, also known as CasRx, a widely used Cas13 system, can cause collateral transcriptome destruction when targeting abundant reporter RNA and endogenous RNAs, resulting in proliferation defect in target cells. While these results call for caution of using RfxCas13d for targeted RNA knockdown, we demonstrated that the collateral activity can be harnessed for selective depletion of a specific cell population defined by a marker RNA in an in vitro setting.

Clinical characteristics of catheter-related infection in patients with chronic renal failure End Stage Renal failure undergoing semi-permanent catheter placement during maintenance hemodialysis through tunnelled cuffed hemodialysis catheter.

Objective: To analysis the relevant infections and risk factors of patients undergoing hemodialysis semi-permanent catheter (tunneled cuffed) placement during for maintenance hemodialysis. Methods: A total of 158 patients with chronic renal failure (CRF) End stage renal failure (ESRF) treated in our hospital from September 2018 to September 2021 were retrospectively analyzed. All the patients underwent semi-permanent catheter placement during maintenance hemodialysis. The occurrence of catheter-related infections in the patients were recorded. The patients with catheter-related infections were included in the infection group, and the others without infection in the non-infection group. The differences in hypertension, gender, diabetes, age, catheter indwelling time and dialysis time between the two groups were analyzed, and the distribution of pathogens in the patients with infections was analyzed. Results: The patients were followed up for 13 to 36 months, with an average of (22.18 ± 6.09) months. Among the 158 patients who underwent going semi-permanent catheter placement, 42 (26.58%) presented semi-permanent catheter-related infections, including four cases of catheter-related bacteremia, 16 cases of tunnel infection and 22 cases of catheter exit-site infection. Among total of 42 strains of pathogens were isolated from the 42 patients with catheter-related infections, including 243 strains of Gram-positive cocci were identified in 24/42(57.14%), and 163 strains of Gram-negative bacilli were identified 16/42(38.10%) and one starin of fungus was identified in 2/42 patients. Statistically significant differences were found in dialysis duration time, hypoalbuminemia, average mean age, diabetes and catheter indwelling time between patients with and without catheter-related infections (P < 0.05). Hypoalbuminemia, catheter indwelling time and diabetes were risk factors for catheter-related infections (P < 0.05). Conclusions: Patients with ESRF CRF are at risk and prone to catheter-related infections during hemodialysis using catheter, mainly tunnel infection and catheter exit-site infection. Gram-positive cocci are the main pathogens. Hypoalbuminemia, too long catheter indwelling time and diabetes are the risk factors for infections.

Overview of the LAMOST survey in the first decade.

The Large Sky Area Multi-Object Fiber Spectroscopic Telescope (LAMOST), also known as the Guoshoujing Telescope, is a major national scientific facility for astronomical research located in Xinglong, China. Beginning with a pilot survey in 2011, LAMOST has been surveying the night sky for more than 10 years. The LAMOST survey covers various objects in the Universe, from normal stars to peculiar ones, from the Milky Way to other galaxies, and from stellar black holes and their companions to quasars that ignite ancient galaxies. Until the latest data release 8, the LAMOST survey has released spectra for more than 10 million stars, ∼220,000 galaxies, and ∼71,000 quasars. With this largest celestial spectra database ever constructed, LAMOST has helped astronomers to deepen their understanding of the Universe, especially for our Milky Way galaxy and the millions of stars within it. In this article, we briefly review the characteristics, observations, and scientific achievements of LAMOST. In particular, we show how astrophysical knowledge about the Milky Way has been improved by LAMOST data.

Structural Basis for the RNA-Guided Ribonuclease Activity of CRISPR-Cas13d.

CRISPR-Cas endonucleases directed against foreign nucleic acids mediate prokaryotic adaptive immunity and have been tailored for broad genetic engineering applications. Type VI-D CRISPR systems contain the smallest known family of single effector Cas enzymes, and their signature Cas13d ribonuclease employs guide RNAs to cleave matching target RNAs. To understand the molecular basis for Cas13d function and explain its compact molecular architecture, we resolved cryoelectron microscopy structures of Cas13d-guide RNA binary complex and Cas13d-guide-target RNA ternary complex to 3.4 and 3.3 Å resolution, respectively. Furthermore, a 6.5 Å reconstruction of apo Cas13d combined with hydrogen-deuterium exchange revealed conformational dynamics that have implications for RNA scanning. These structures, together with biochemical and cellular characterization, provide insights into its RNA-guided, RNA-targeting mechanism and delineate a blueprint for the rational design of improved transcriptome engineering technologies.

Publisher Correction: Pairwise library screen systematically interrogates Staphylococcus aureus Cas9 specificity in human cells.

The original HTML version of this Article incorrectly listed an affiliation of Josh Tycko as 'Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA', instead of the correct 'Present address: Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA'. It also incorrectly listed an affiliation of this author as 'Present address: Arrakis Therapeutics, 35 Gatehouse Dr., Waltham, MA, 02451, USA'.The original HTML version incorrectly listed an affiliation of Luis A. Barrera as 'Present address: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, 06511, USA', instead of the correct 'Present address: Arrakis Therapeutics, 35 Gatehouse Dr., Waltham, MA 02451, USA'.Finally, the original HTML version incorrectly omitted an affiliation of Nicholas C. Huston: 'Present address: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511, USA'.This has been corrected in the HTML version of the Article. The PDF version was correct from the time of publication.

Combined effects of octreotide and cisplatin on the proliferation of side population cells from anaplastic thyroid cancer cell lines.

Anaplastic thyroid cancer (ATC) represents the most aggressive subtype of thyroid cancer and has a poor prognosis. In addition to surgery, chemotherapy is an important treatment for ATC; however, the therapeutic effects of current chemotherapies for ATC are not particularly promising. There is a high proportion of side population (SP) cells in ATC, which may be a reason for its drug resistance. In the present study, the antitumor activities of combined octreotide (OCT) and cisplatin (DDP) on the proliferation and apoptosis of ATC SP cells were evaluated. First, SP cells from 8305C and BHT101 cell lines were detected and sorted. Following in vitro culture for 1 week, cluster of differentiation (CD)44, CD133, ATP-binding cassette (ABC) subfamily B member 1 (ABCB1), ABC subfamily G member 2 (ABCG2) and somatostatin receptor expression was detected to characterize the SP cells. An MTT assay was performed to investigate the combined effects on 8305C-SP cell proliferation in vitro, and a mouse model was used to investigate the combined effects on 8305C-SP cell proliferation in vivo. Annexin V/propidium iodide staining was used to investigate the combined effects on 8305C-SP cell apoptosis. Chemotherapeutic drug resistance-associated protein expression and apoptosis-associated protein expression were also detected following combined treatment. As a result, SP cells were identified in 8305C and BHT101 cells, and the proportion of 8305C-SP cells was increased compared with that of BTH101-SP cells. SP cells have enhanced proliferation, tumorigenicity and drug resistance compared with main population cells. The combined treatment of OCT with DDP suppressed the proliferation of 8305C-SP cells in vitro and in vivo, and induced 8305C-SP cell apoptosis. Combined treatment decreased the ABCB1 and ABCG2 expression by SP cells and activated mitochondrial apoptotic signaling, resulting in cell apoptosis. In conclusion, these data support the hypothesis that combined treatment with OCT and DDP induces ATC cell apoptosis and suppresses cell proliferation. These data provide a theoretical basis for further combined chemotherapy clinical applications.

Pairwise library screen systematically interrogates Staphylococcus aureus Cas9 specificity in human cells.

Therapeutic genome editing with Staphylococcus aureus Cas9 (SaCas9) requires a rigorous understanding of its potential off-target activity in the human genome. Here we report a high-throughput screening approach to measure SaCas9 genome editing variation in human cells across a large repertoire of 88,692 single guide RNAs (sgRNAs) paired with matched or mismatched target sites in a synthetic cassette. We incorporate randomized barcodes that enable whitelisting of correctly synthesized molecules for further downstream analysis, in order to circumvent the limitation of oligonucleotide synthesis errors. We find SaCas9 sgRNAs with 21-mer or 22-mer spacer sequences are generally more active, although high efficiency 20-mer spacers are markedly less tolerant of mismatches. Using this dataset, we developed an SaCas9 specificity model that performs robustly in ranking off-target sites. The barcoded pairwise library screen enabled high-fidelity recovery of guide-target relationships, providing a scalable framework for the investigation of CRISPR enzyme properties and general nucleic acid interactions.

A genetic program mediates cold-warming response and promotes stress-induced phenoptosis in C. elegans.

How multicellular organisms respond to and are impacted by severe hypothermic stress is largely unknown. From C. elegans screens for mutants abnormally responding to cold-warming stimuli, we identify a molecular genetic pathway comprising ISY-1, a conserved uncharacterized protein, and ZIP-10, a bZIP-type transcription factor. ISY-1 gatekeeps the ZIP-10 transcriptional program by regulating the microRNA mir-60. Downstream of ISY-1 and mir-60, zip-10 levels rapidly and specifically increase upon transient cold-warming exposure. Prolonged zip-10 up-regulation induces several protease-encoding genes and promotes stress-induced organismic death, or phenoptosis, of C. elegans. zip-10 deficiency confers enhanced resistance to prolonged cold-warming stress, more prominently in adults than larvae. We conclude that the ZIP-10 genetic program mediates cold-warming response and may have evolved to promote wild-population kin selection under resource-limiting and thermal stress conditions.

Transcriptional Pause Sites Delineate Stable Nucleosome-Associated Premature Polyadenylation Suppressed by U1 snRNP.

Regulation of RNA polymerase II (Pol II) elongation is a critical step in gene regulation. Here, we report that U1 snRNP recognition and transcription pausing at stable nucleosomes are linked through premature polyadenylation signal (PAS) termination. By generating RNA exosome conditional deletion mouse embryonic stem cells, we identified a large class of polyadenylated short transcripts in the sense direction destabilized by the RNA exosome. These PAS termination events are enriched at the first few stable nucleosomes flanking CpG islands and suppressed by U1 snRNP. Thus, promoter-proximal Pol II pausing consists of two processes: TSS-proximal and +1 stable nucleosome pausing, with PAS termination coinciding with the latter. While pausing factors NELF/DSIF only function in the former step, flavopiridol-sensitive mechanism(s) and Myc modulate both steps. We propose that premature PAS termination near the nucleosome-associated pause site represents a common transcriptional elongation checkpoint regulated by U1 snRNP recognition, nucleosome stability, and Myc activity.

Rescue of Fragile X Syndrome Neurons by DNA Methylation Editing of the FMR1 Gene.

Fragile X syndrome (FXS), the most common genetic form of intellectual disability in males, is caused by silencing of the FMR1 gene associated with hypermethylation of the CGG expansion mutation in the 5' UTR of FMR1 in FXS patients. Here, we applied recently developed DNA methylation editing tools to reverse this hypermethylation event. Targeted demethylation of the CGG expansion by dCas9-Tet1/single guide RNA (sgRNA) switched the heterochromatin status of the upstream FMR1 promoter to an active chromatin state, restoring a persistent expression of FMR1 in FXS iPSCs. Neurons derived from methylation-edited FXS iPSCs rescued the electrophysiological abnormalities and restored a wild-type phenotype upon the mutant neurons. FMR1 expression in edited neurons was maintained in vivo after engrafting into the mouse brain. Finally, demethylation of the CGG repeats in post-mitotic FXS neurons also reactivated FMR1. Our data establish that demethylation of the CGG expansion is sufficient for FMR1 reactivation, suggesting potential therapeutic strategies for FXS.

kpLogo: positional k-mer analysis reveals hidden specificity in biological sequences.

Motifs of only 1-4 letters can play important roles when present at key locations within macromolecules. Because existing motif-discovery tools typically miss these position-specific short motifs, we developed kpLogo, a probability-based logo tool for integrated detection and visualization of position-specific ultra-short motifs from a set of aligned sequences. kpLogo also overcomes the limitations of conventional motif-visualization tools in handling positional interdependencies and utilizing ranked or weighted sequences increasingly available from high-throughput assays. kpLogo can be found at http://kplogo.wi.mit.edu/.

Widespread Influence of 3'-End Structures on Mammalian mRNA Processing and Stability.

The physiological relevance of structures within mammalian mRNAs has been elusive, as these mRNAs are less folded in cells than in vitro and have predicted secondary structures no more stable than those of random sequences. Here, we investigate the possibility that mRNA structures facilitate the 3'-end processing of thousands of human mRNAs by juxtaposing poly(A) signals (PASs) and cleavage sites that are otherwise too far apart. We find that RNA structures are predicted to be more prevalent within these extended 3'-end regions than within PAS-upstream regions and indeed are substantially more folded within cells, as determined by intracellular probing. Analyses of thousands of ectopically expressed variants demonstrate that this folding both enhances processing and increases mRNA metabolic stability. Even folds with predicted stabilities resembling those of random sequences can enhance processing. Structure-controlled processing can also regulate neighboring gene expression. Thus, RNA structure has widespread roles in mammalian mRNA biogenesis and metabolism.

Editing DNA Methylation in the Mammalian Genome.

Mammalian DNA methylation is a critical epigenetic mechanism orchestrating gene expression networks in many biological processes. However, investigation of the functions of specific methylation events remains challenging. Here, we demonstrate that fusion of Tet1 or Dnmt3a with a catalytically inactive Cas9 (dCas9) enables targeted DNA methylation editing. Targeting of the dCas9-Tet1 or -Dnmt3a fusion protein to methylated or unmethylated promoter sequences caused activation or silencing, respectively, of an endogenous reporter. Targeted demethylation of the BDNF promoter IV or the MyoD distal enhancer by dCas9-Tet1 induced BDNF expression in post-mitotic neurons or activated MyoD facilitating reprogramming of fibroblasts into myoblasts, respectively. Targeted de novo methylation of a CTCF loop anchor site by dCas9-Dnmt3a blocked CTCF binding and interfered with DNA looping, causing altered gene expression in the neighboring loop. Finally, we show that these tools can edit DNA methylation in mice, demonstrating their wide utility for functional studies of epigenetic regulation.

Cell-Type-Specific Alternative Splicing Governs Cell Fate in the Developing Cerebral Cortex.

Alternative splicing is prevalent in the mammalian brain. To interrogate the functional role of alternative splicing in neural development, we analyzed purified neural progenitor cells (NPCs) and neurons from developing cerebral cortices, revealing hundreds of differentially spliced exons that preferentially alter key protein domains-especially in cytoskeletal proteins-and can harbor disease-causing mutations. We show that Ptbp1 and Rbfox proteins antagonistically govern the NPC-to-neuron transition by regulating neuron-specific exons. Whereas Ptbp1 maintains apical progenitors partly through suppressing a poison exon of Flna in NPCs, Rbfox proteins promote neuronal differentiation by switching Ninein from a centrosomal splice form in NPCs to a non-centrosomal isoform in neurons. We further uncover an intronic human mutation within a PTBP1-binding site that disrupts normal skipping of the FLNA poison exon in NPCs and causes a brain-specific malformation. Our study indicates that dynamic control of alternative splicing governs cell fate in cerebral cortical development.

In vivo genome editing using Staphylococcus aureus Cas9.

The RNA-guided endonuclease Cas9 has emerged as a versatile genome-editing platform. However, the size of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for basic research and therapeutic applications that use the highly versatile adeno-associated virus (AAV) delivery vehicle. Here, we characterize six smaller Cas9 orthologues and show that Cas9 from Staphylococcus aureus (SaCas9) can edit the genome with efficiencies similar to those of SpCas9, while being more than 1 kilobase shorter. We packaged SaCas9 and its single guide RNA expression cassette into a single AAV vector and targeted the cholesterol regulatory gene Pcsk9 in the mouse liver. Within one week of injection, we observed >40% gene modification, accompanied by significant reductions in serum Pcsk9 and total cholesterol levels. We further assess the genome-wide targeting specificity of SaCas9 and SpCas9 using BLESS, and demonstrate that SaCas9-mediated in vivo genome editing has the potential to be efficient and specific.