Biology subjects

Bao, J.

Publications and source records attributed to Bao, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Probiotic-Directed Fermentation Reprograms the Metabolic Profile of a Traditional Mongolian Whole-Wheat Diet and Modulates Escherichia coli-Induced Gut Microbiota Dysbiosis

Traditional Mongolian fermented foods have been extensively utilized for dietary regulation and the promotion of gastrointestinal health. However, spontaneous fermentation remains inherently unpredictable, leading to significant variations in microbial community dynamics, metabolite accumulation, and the consistency and quality of the final product. Drawing on the traditional preparation of Mongolian acidic foods, this study established a controlled production strategy for whole-wheat probiotic fermented soup (WWPFS) by combining enzymatic pretreatment with probiotic-directed fermentation. Physicochemical characterization, 16S rRNA gene-based microbial community profiling, LC-MS/MS-based untargeted metabolomics, safety evaluation, and an Escherichia coli-induced gut microbiota dysbiosis model were employed to optimize and comprehensively characterize the fermentation process of WWPFS. The optimized process established a reproducible fermentation system consistently dominated by Lactobacillus and Bacillus across independent fermentation batches. Compared with traditional spontaneous fermentation, probiotic-directed fermentation remodeled the physicochemical properties of the whole-wheat matrix, including carbon, nitrogen, phosphorus, sulfur, and mineral composition, and facilitated the accumulation of putatively annotated LC-MS/MS features, including DL-lactate, 1,4-D-xylobiose, diacetyl, and phenyllactic-acid-related features derivatives. Acute oral and 28-day repeated-dose toxicity evaluations showed no treatment-related adverse effects within the tested dose range and study duration. In the Escherichia coli-induced gut microbiota dysbiosis mouse model, microbial richness, diversity, and community structure differed among the experimental groups, and both low- and high-dose WWPFS groups showed significant shifts in overall gut microbial community composition relative to the model group after multiple-testing correction, together with directional recovery of selected model-responsive bacterial genera. Cross-system integration identified coordinated response patterns between fermentation-derived metabolite features and model-responsive gut bacterial taxa, supporting a potential metabolite-microbiota link in WWPFS-mediated gut microbiota modulation. In summary, probiotic-directed fermentation improved the controllability of the traditional Mongolian fermented food production process, reshaped its metabolic profile, and enhanced its potential to modulate the gut microbiota. These findings provide experimental evidence supporting the modernization of traditional Mongolian fermented foods and the development of probiotic-based functional foods.

microbiology↗

bioRxiv2026

A cinnamyl alcohol dehydrogenase scaffold organizes monoterpenoid indole alkaloid biosynthesis

Biosynthesis of ~3,000 monoterpenoid indole alkaloids (MIAs), including the anticancer drug vinblastine, involves the highly unstable intermediate strictosidine aglycone. Its formation by strictosidine {beta}-glucosidase (SGD) and subsequent conversion by geissoschizine synthase (GS) occur in spatially separated compartments, representing a major biosynthesis bottleneck. Here we discover VinBLAST, a cinnamyl alcohol dehydrogenase-like protein repurposed as a scaffold for efficient processing of this labile intermediate. VinBLAST physically mediates SGD and GS interaction in the nucleus and allosterically enhances GS catalytic efficiency. VinBLAST homologues from diverse plant families enhance biosynthesis of several representative MIAs, with the production of catharanthine increased to ~160 mg L-1 in yeast, nearly 1,000-fold higher than previous studies. Our discovery provides a missing link in organizing MIA biosynthesis and enables scalable bioproduction of geissoschizine-derived therapeutics.

synthetic biology↗

bioRxiv2025

Proteome profiling reveals HES1-driven mitotic catastrophe in ovarian serous carcinoma

Ovarian high-grade serous cancer (HGSC) is an aggressive subtype of epithelial ovarian cancer. Here, we identify BX-912, a phosphoinositide-dependent kinase 1 (PDPK1) inhibitor, as a promising therapeutic agent for HGSC. BX-912 suppressed HGSC growth as a single agent and synergized with olaparib independently of BRCA status. Unexpectedly, BX-912 treatment induced multinucleation, a phenotype not observed with other PDPK1 inhibitors. Proteome Integral Solubility Alteration (PISA) profiling revealed the transcription factor HES1 as a functional target of BX-912. Structural modeling showed that BX-912 binds the Orange domain of HES1, while its WRPW motif mediates interactions with protein partners, including the AP2 endocytic protein complex, coordinating their nuclear accumulation that leads to a mitotic catastrophe. Furthermore, cell cycle analyses showed that BX-912 combined with olaparib synergistically enhanced DNA damage and G2-M arrest. Our study demonstrates the value of proteomics for revealing hidden drug activities. It also identifies potential inhibition strategies for HES1, which is commonly overexpressed in HGSC. Additionally, this study proposes a novel strategy of targeting consecutive cell cycle phases to enhance treatment efficacy in HGSC.

cancer biology↗

bioRxiv2025

Recurrent connectivity supports carbon dioxide sensitivity in Aedes aegypti mosquitoes

The mosquito Aedes aegyptis human host-seeking behavior depends on the integration of multiple sensory cues. One of these cues, carbon dioxide (CO2), gates odorant and heat pathways and activates host-seeking behavior. The neuronal circuits underlying processing of CO2 information remain unclear. We used automated serial-section transmission electron microscopy (EM) to image and reconstruct the circuitry of the glomeruli that are innervated by the Ae. aegypti maxillary palp, including the glomerulus that responds to CO2. Notably, CO2-sensitive olfactory sensory neurons (OSNs) make high levels of recurrent synaptic connections with one another, while making a low density of feedforward synapses. At some of these contacts between CO2 OSNs, we observe ribbon- like presynaptic structures, which may further enhance recurrent signaling. We compared both feedforward and recurrent connectivity with all olfactory glomeruli in Drosophila melanogaster, and we found more recurrent connections between the Ae. aegypti CO2-responsive OSNs than in any D. melanogaster glomeruli. We developed a computational circuit model that demonstrates recurrent synapses are necessary for robust CO2 detection under normal physiological conditions. Together, elevated levels of recurrent connectivity and ribbon-like structures may amplify sensory information detected by CO2-sensitive OSNs to support mosquito activation and sensitization by CO2, even in the presence of high levels of other odorants in the environment. We propose that this circuit organization supports the salience of CO2 as a mosquito host cue. One Sentence SummaryConnectomic analysis of carbon dioxide circuitry in the disease-vector mosquito Aedes aegypti.

neuroscience↗

bioRxiv2024

Uncovering pre-sensitizing agents to FLT3 inhibitors in acute myeloid leukemia with ReSisTrace lineage tracing

While FLT3 inhibitors have significantly improved the treatment of aggressive FLT3-mutated acute myeloid leukemia (AML), the emergence of resistance remains as a major challenge. Here, we applied our recently developed single-cell lineage-tracing method ReSisTrace to identify cells that are pre-resistant or pre-sensitive to FLT3 inhibitors midostaurin and quizartinib in FLT3-ITD-positive AML. By comparing the gene expression profiles of these cells, we unraveled the transcriptional pre-resistance signatures, including G1 to S phase transition 1 (GSPT1) gene. Targeting GSPT1 with the small molecule CC-90009 exhibited strong synergistic effect when combined with FLT3 inhibitors in the FLT3-ITD-mutated MOLM-13 and MV4-11 cell lines and primary AML patient samples. Further, we identified novel compounds that induced transcriptomic changes opposite to the pre-resistance signatures, thereby driving cells to FLT3 inhibitor-sensitive states. Vistusertib (mTOR inhibitor), linsitinib (IGF1R and insulin receptor inhibitor), and meisoindigo (IGF1R and Src family kinase inhibitor), all inhibiting pathways parallel to or downstream of oncogenic FLT3 signaling, were predicted and validated to pre-sensitize the FLT3-ITD-mutated cell lines and primary cells to FLT3 inhibitors. Collectively, these findings demonstrate the validity of our lineage-tracing method in unveiling pre-existing transcriptional features of treatment vulnerability in hematological cancers, and elucidate novel strategies for enhancing FLT3 inhibitor treatment efficacy in FLT3-ITD-positive AML by preventing the emergence of treatment resistance.

cancer biology↗

bioRxiv2024

Microsporidian Nosema bombycis secretes serine protease inhibitor to suppress host cell apoptosis via caspase BmICE

Microsporidia are a group of intracellular pathogens that actively manipulate host cell biological processes to facilitate their intracellular niche. Apoptosis is an important defense mechanism by which host cell control intracellular pathogens. Microsporidia modulating host cell apoptosis has been reported previously, however the molecular mechanism is not yet clear. In this report, we describe that the microsporidia Nosema bombycis inhibits apoptosis of Bombyx mori cells through a secreted protein NbSPN14, which is a serine protease inhibitor (Serpin). An immunofluorescent assay demonstrated that upon infection with N. bombycis, NbSPN14 was initially found in the B. mori cell cytoplasm and then became enriched in the host cell nucleus. Overexpression and RNA-interference (RNAi) of NbSPN14 in B. mori embryo cells confirmed that NbSPN14 inhibited host cell apoptosis. Immunofluorescent and Co-IP assays verified the co-localization and interaction of NbSPN14 with the BmICE, the caspase 3 homolog in B. mori. Knocking out of BmICE or mutating the BmICE-interacting P1 site of NbSPN14, eliminated the localization of NbSPN14 into the host nucleus and prevented the apoptosis-inhibiting effect of NbSPN14, which also proved that the interaction between BmICE and NbSPN14 occurred in host cytoplasm and the NbSPN14 translocation into host cell nucleus is dependent on BmICE. These data elucidate that N. bombycis secretory protein NbSPN14 inhibits host cell apoptosis by directly inhibiting the caspase protease BmICE, which provides an important insight for understanding pathogen-host interactions and a potential therapeutic target for N. bombycis proliferation. Author SummaryMicrosporidia constitute a class of eukaryotic pathogens that exclusively reside within host cells. The species Nosema bombycis is the first microsporidian identified as the pathogen of silkworm Pebrine disease. In our research, we discovered how N. bombycis cleverly evades the hosts defenses. It has developed a strategy to survive inside host cells by manipulating host cell apoptosis, disarming the host cells self-destruct mechanism. In this study, we discovered that the N. bombycis secretes a serine protease inhibitor named NbSPN14, which infiltrates the cytoplasm of the host cell. The NbSPN14 interacts with the executioner Caspase protease BmICE within the silkworms apoptotic pathway, effectively neutralizing its apoptoic activity and thus curbing the apoptosis of the host cells.

microbiology↗

bioRxiv2024

Multi-omics data integration reveals molecular mechanisms of carfilzomib resistance in multiple myeloma

Multiple myeloma represents a complex hematological malignancy, characterized by its wide array of genetic and clinical events. The introduction of proteasome inhibitors, such as carfilzomib or bortezomib, into the therapeutic landscape has notably enhanced the quality of life and survival rates for patients suffering from this disease. Nonetheless, a significant obstacle in the long-term efficacy of this treatment is the inevitable development of resistance to PIs, posing a substantial challenge in managing the disease effectively. Our study investigates the molecular mechanisms behind carfilzomib resistance by analyzing multi-omics profiles from four multiple myeloma cell lines: AMO-1, KMS-12-PE, RPMI-8226 and OPM-2, together with their carfilzomib-resistant variants. We uncovered a significant downregulation of metabolic pathways linked to strong mitochondrial dysfunction in resistant cells. Further examination of patient samples identified key genes - ABCB1, RICTOR, PACSIN1, KMT2D, WEE1 and GATM - potentially crucial for resistance, guiding us towards promising carfilzomib combination therapies to circumvent resistance mechanisms. The response profiles of tested compounds have led to the identification of a network of gene interactions in resistant cells. We identified two already approved drugs, benidipine and tacrolimus, as potential partners for combination therapy with carfilzomib to counteract resistance. This discovery enhances the clinical significance of our findings.

cancer biology↗

bioRxiv2024

Automated Cytometric Gating with Human-Level Performance Using Bivariate Segmentation

Recent advances in cytometry technology have enabled high-throughput data collection with multiple single-cell protein expression measurements. The significant biological and technical variance between samples in cytometry has long posed a formidable challenge during the gating process, especially for the initial gates which deal with unpredictable events, such as debris and technical artifacts. Even with the same experimental machine and protocol, the target population, as well as the cell population that needs to be excluded, may vary across different measurements. To address this challenge and mitigate the labor-intensive manual gating process, we propose a deep learning framework UNITO to rigorously identify the hierarchical cytometric subpopulations. The UNITO framework transformed a cell-level classification task into an image-based semantic segmentation problem. For reproducibility purposes, the framework was applied to three independent cohorts and successfully detected initial gates that were required to identify single cellular events as well as subsequent cell gates. We validated the UNITO framework by comparing its results with previous automated methods and the consensus of at least four experienced immunologists. UNITO outperformed existing automated methods and differed from human consensus by no more than each individual human. Most critically, UNITO framework functions as a fully automated pipeline after training and does not require human hints or prior knowledge. Unlike existing multi-channel classification or clustering pipelines, UNITO can reproduce a similar contour compared to manual gating for each intermediate gating to achieve better interpretability and provide post hoc visual inspection. Beyond acting as a pioneering framework that uses image segmentation to do auto-gating, UNITO gives a fast and interpretable way to assign the cell subtype membership, and the speed of UNITO will not be impacted by the number of cells from each sample. The pre-gating and gating inference takes approximately 2 minutes for each sample using our pre-defined 9 gates system, and it can also adapt to any sequential prediction with different configurations.

immunology↗

bioRxiv2024

MoLst8 regulates autophagy and lipid homeostasis in Magnaporthe oryzae

TOR, a widely conserved eukaryotic protein kinase, forms TORC1 and TORC2 to regulate diverse cell signaling. TORC1 controls protein synthesis, cell cycle, and autophagy, whereas TORC2 manages cell polarity, cytoskeleton, and membrane structure. Our previous research found that MoVast2, along with MoVast1, regulates TOR in rice blast fungus Magnaporthe oryzae, maintaining lipid and autophagy balance. Lst8, a key TOR complex component in yeast and mammalian cells. However, the precise role of MoLst8 in M. oryzae is still unclear. In this study, we obtained the {Delta}Molst8 mutant through high-through gene knockout strategies. The results showed that loss of MoLST8 leading to a series of defects, such as growth and sporulation reduction, abnormal conidia, and loss of virulence. In addition, this mutant is highly sensitive to rapamycin, leading to growth arrest and autophagy impairment, indicated that MoLst8 positively regulates TORC1 for cellular growth, metabolism, and autophagy. Lipidomics analysis in the mutant revealed lipid metabolism dysregulation, sphingolipid reduction, disrupting membrane tension and homeostasis, suggested that TORC2 mediated lipid regulation is disordered in {Delta}Molst8 mutant. Additionally, the study explored TOR-MAPK crosstalk, finding that the mutant shows heightened cell wall stress sensitivity but fails to restore integrity despite MAPK activation. These findings offer insights into MoLst8s role in fungal pathogenesis, contributing to an understanding of fungal biology and disease control strategies.

microbiology↗

bioRxiv2024

Patient-derived functional immuno-oncology platform identifies responders to ATR inhibitor and immunotherapy combinations in ovarian cancer

Responses to single agent immunotherapies have remained modest in high-grade serous ovarian cancer (HGSC), suggesting the need for combination treatments. Identifying clinically effective immunotherapy combinations (IC) requires pre-clinical testing using models representing the patient-specific immune microenvironment. Here, we established a functional immuno-oncology platform for high-throughput and functional testing of IC using HGSC patient-derived immunocompetent cultures (iPDCs) established on patient-derived omentum gel matrix. We employed genomic and single-cell analysis to assess the intricate and functional characteristics of the iPDCs combined with tumor and immune cell-specific cytotoxic responses. Corroborating the clinical response to Poly (ADP-ribose) polymerase inhibitors (PARPi), iPDCs showed homologous recombination deficiency (HRD) - specific response to PARPi. Importantly, drug responses from iPDCs of chemotherapy and PARPi refractory patients corresponded with patient outcomes and aligned with distinct pathway activities from single-cell RNA sequencing analysis. Furthermore, iPDCs from HRD tumors showed response to anti-PD1 antibody as measured by decrease in tumor cells combined with augmented T cell activation. High-throughput drug testing followed by single cell-imaging from iPDCs revealed patient-specific responses to combination of ataxia telangiectasia and Rad3-related inhibitor (ATRi) with DNA damaging agents or immunotherapies. Integration of cytotoxic responses with immune cell states uncovered patient-specific immune activation with the combination of ATRi and a novel immunotherapy targeting Autotaxin (ATX), and this response was significantly associated with a tumor-cell replication stress biomarker in single-cell analysis of tCycIF highly multiplexed imaging. In conclusion, iPDCs provide a platform for high-throughput screening and functional testing of immuno-oncology agents for precision oncology in HGSC.

cancer biology↗

bioRxiv2024

Nature AND Nurture: Enabling formate-dependent growth in Methanosarcina acetivorans

Methanogenic archaea are crucial in global carbon cycling as around 1 Gt of the potent greenhouse gas, methane, is produced annually. Major contributors belong to the order Methanosarcinales, which contain some of the most versatile methanogens that are capable of acetotrophic, methylotrophic and CO2-reducing methanogenesis. The genetically tractable model methanogen, Methanosarcina acetivorans, by its nature shows versatility in substrate utilization and energy conservation pathways but cannot utilize formate. In this study, we expanded the primary metabolism of M. acetivorans to include formate-dependent methanogenesis. By introducing an exogenous formate dehydrogenase, the two metabolically engineered M. acetivorans strains acquired the capacity for formate-dependent methanogenesis pathways with one capable of formate-dependent methyl-reduction and the other capable of formate-dependent CO2-reduction. Through nurturing the strain capable of CO2-reduction with adaptive laboratory evolution, we were able to enable growth and methanogenesis of M. acetivorans solely on formate, a metabolism only reported in methanogens without cytochromes which are limited by their versatility. M. acetivorans also showed acetogenic potential where the formate-dependent CO2-reducing strain was able to divert {approx} 10% of carbon to acetate instead of methane. Our results show that even though M. acetivorans lacks energy converting hydrogenase and cannot use H2, it has yet-uncharacterized capacity to obtain reduced ferredoxins from oxidizing formate. Our work encourages reevaluation of our understanding of formate utilization in Methanosarcinales. By enabling formate-dependent methanogenesis, we have expanded the substrate spectrum of a versatile model methanogen with cytochromes to include formate as well.

microbiology↗

bioRxiv2023

Homological landscape of human brain functional sub-circuits

Human whole-brain functional connectivity networks have been shown to exhibit both local/quasilocal (e.g., set of functional sub-circuits induced by node or edge attributes) and non-local (e.g., higher-order functional coordination patterns) properties. Nonetheless, the non-local properties of topological strata induced by local/quasilocal functional sub-circuits have yet to be addressed. To that end, we proposed a homological formalism that enables the quantification of higher-order characteristics of human brain functional sub-circuits. Our results indicated that each homological order uniquely unravels diverse, complementary properties of human brain functional sub-circuits. Noticeably, the H1 homological distance between rest and motor task were observed at both whole-brain and sub-circuit consolidated level which suggested the self-similarity property of human brain functional connectivity unraveled by homological kernel. Furthermore, at the whole-brain level, the rest-task differentiation was found to be most prominent between rest and different tasks at different homological orders: i) Emotion task (H0), ii) Motor task (H1), and iii) Working memory task (H2). At the functional sub-circuit level, the rest-task functional dichotomy of default mode network is found to be mostly prominent at the first and second homological scaffolds. Also at such scale, we found that the limbic network plays a significant role in homological reconfiguration across both task- and subject-domain which sheds light to subsequent investigations on the complex neuro-physiological role of such network. From a wider perspective, our formalism can be applied, beyond brain connectomics, to study non-localized coordination patterns of localized structures stretching across complex network fibers.

neuroscience↗

bioRxiv2023

Microsporidia Ser/Thr Protein Phosphatase PP1 Targets DCs MAPK Pathway and Impair Immune Functions

Microsporidia are difficult to completely eliminate. Their persistence may disrupt host cell functions. Here in this study, we aimed to elucidate the impairing effects and consequences of microsporidia infection upon dendritic cells (DCs). We used the zoonotic microsporidia species, Enterocytozoon hellem, in our studies. In vivo experiments showed that E. hellem-infected mice were more susceptible to further pathogenic challenges. DCs were identified as the most affected group of cells. In vitro assays revealed that E. hellem infection impaired the immune functions of DCs as reflected by down-regulation of cytokine expression, lower extent of maturation and antigen presentation. E. hellem infection decreased the ability of DCs to prime and stimulate T cells, thereby hampering host immune cell functions. We further demonstrate that E. hellem Ser/Thr protein phosphatase PP1 directly interacts with host p38(MAPK14) to manipulate the p38 (MAPK14)/NFAT-5 axis of the MAPK pathway. Our study is the first to elucidate the molecular mechanisms of the impairing effects of microsporidia on host DCs immune functions. The emerging of microsporidiosis may be great threat to public health. HighlightsO_LIPersistence of Microsporidia within host impairs dendritic cell functions such as phagocytosis, maturation, antigen presentation and T cell priming, thereby disrupting both innate and adaptive immunities and making the host more vulnerable to secondary infections C_LIO_LIMicrosporidia impairs DCs function via Serine/Threonine Protein Phosphatase PP1 directly targeting DCs p38/MAPK pathway C_LIO_LILatent Microsporidia infection and persistence is a great threat to public health when assessing acute and emerging pathogen risk C_LI

microbiology↗

bioRxiv2023

T2T-YAO: a Telomere-to-telomere Assembled Diploid Reference Genome for Han Chinese

Since its initial release in 2001, the human reference genome has been continuously improved in both continuity and accuracy, and the recently-released telomere-to-telomere version--T2T-CHM13--reaches its top quality after 20 years of effort. However, T2T-CHM13 does not represent an authentic diploid human genome, but rather one derived from a simplified, nearly homozygous genome of a hydatidiform mole cell line. To address this limitation and provide an alternative pertinent to the Chinese population, the largest ethnic group in the world, we have assembled a complete diploid human genome of a male Han Chinese, T2T-YAO, which includes telomere-to-telomere assemblies for all the 22+X+M and 22+Y chromosomes in his two haploids inherited separately from his parents. Both haplotypes contain no artificial sequences or model nucleotides and possess a high quality comparable to CHM13, with fewer than one error per [~]14 Mb. Derived from the individual who lives in the aboriginal region of Han Chinese, T2T-YAO shows clear ancestry and potential genetic continuity from the ancient ancestors of the Han population. Each haplotype of T2T-YAO possesses [~]340 Mb exclusive sequences and [~]3100 unique genes as compared to CHM13, and their genome sequences show greater genetic distance to CHM13 than to each other in terms of nucleotide polymorphism and structural variations. The construction of T2T-YAO would serve as a high-quality diploid reference that enables precise delineation of genomic variations in a haplotype-sensitive manner, which could advance our understandings in human evolution, hereditability of diseases and phenotypes, especially within the context of the unique variations of the Chinese population.

genetics↗

bioRxiv2023

Discovery of microRNA-derived RNAs and proteins in human cells

MicroRNAs (miRNAs) are short noncoding RNAs that can regulate gene expression through the binding of their 5-ends to mRNAs. However, the biological effects of miRNA binding via their 3-ends remain unclear. Here, we discover that the exact reverse pairing of the 3-ends of miRNAs or miRNA-like RNAs, collectively termed microsized RNAs (msRNAs), with template RNAs can initiate the production of msRNA-derived RNAs (msdRs), which can subsequently be translated into polypeptides (msdPs). Using 2,632 human msRNAs from miRBase, 11,121 msdRs and 1,239 msdPs were predicted based on a 15-nucleotide pairing threshold, and the presence of representative msdRs and msdPs was confirmed in human cells. Of clinical relevance, msdP0188 is highly expressed in human lung and breast cancers, and its corresponding msRNAs and msdRs represent novel anti-cancer targets. Notably, inhibiting telomerase reverse transcriptase, a putative RNA-dependent RNA polymerase identified by bioinformatic screening, led to reduced levels of msdP0188 in human cancer cells. Our findings reveal a novel "msRNA [->] msdR [->] msdP" axis, expanding the classical central dogma and predicting many previously unrecognized RNAs and polypeptides with potential biological functions in human cells and other systems.

cell biology↗

bioRxiv2023

CRISPR/Cas12a toolbox for genomic manipulation in Methanosarcina acetivorans

Methanogenic archaea play an important role in the global carbon cycle and are regarded as promising host organisms for the biotechnological generation of fuels and chemicals from one-carbon substrates. Methanosarcina acetivorans is extensively studied as a model methanogen due to the availability of genetic tools and its versatile substrate range. Although genome editing in M. acetivorans via CRISPR/Cas9 has already been demonstrated, we now describe a user-friendly CRISPR/Cas12a toolbox that recognizes a T-rich (5'-TTTV) PAM sequence. This new system can manage deletions of 3500 bp (i.e., knockout of the entire frhADGB operon) and heterologous gene insertions with 80% efficiency observed in ten PurR transformants. Our CRISPR/Cas12a system also enables multiplex genome editing at high efficiency, which helps speed up genetic engineering. Deletions of 100 bp generated on two separate sites of the genome yielded 8/8 correctly edited transformants. Simultaneous gene deletion (100 bp) and replacement (100-bp region replaced by the 2400-bp uidA expression cassette) at a separate site was achieved, with 3/6 of transformants being edited correctly. In combination with the Cas9-based system, our CRISPR/Cas12a toolbox enables targeted genome editing at two sites (guanine-rich and thymine-rich, respectively) and, in so doing, hastens the overall genetic engineering of the Methanosarcinales species.

molecular biology↗

bioRxiv2023

Biallelic Variants in MAD2L1BP (p31comet) Cause Female Infertility Characterized by Oocyte Maturation Arrest

Human oocyte maturation arrest represents one of the severe conditions for female patients with primary infertility. However, the genetic factors underlying this human disease remain largely unknown. The spindle assembly checkpoint (SAC) is an intricate surveillance mechanism that ensures accurate segregation of chromosomes throughout cell cycles. Once the kinetochores of chromosomes are correctly attached to bipolar spindles and the SAC is satisfied, the MAD2L1BP, best known as p31comet, binds MAD2 and recruits the AAA+-ATPase TRIP13 to disassemble the mitotic checkpoint complex (MCC), leading to the cell cycle progression. In this study, by whole-exome sequencing (WES), we identified homozygous and compound heterozygous MAD2L1BP variants in three families with female patients diagnosed with primary infertility owing to oocyte metaphase I (MI) arrest. Functional studies revealed that the protein variants resulting from the C-terminal truncation of MAD2L1BP lost their binding ability to MAD2. cRNA microinjection of full-length or truncated MAD2L1BP uncovered their discordant roles in driving the extrusion of polar body 1 (PB1) in mouse oocytes. Furthermore, the patients oocytes carrying the mutated MAD2L1BP variants resumed polar body extrusion (PBE) when rescued by microinjection of full-length MAD2L1BP cRNAs. Together, our studies identified and characterized novel biallelic variants in MAD2L1BP responsible for human oocyte maturation arrest at MI, and thus prompted new therapeutic avenues for curing female primary infertility.

developmental biology↗

bioRxiv2022

Oxytocin enhances the triangular association among behavioral performance, resting state, and task-state functional connectivity

The role of oxytocin (OT) in social behavior and social brain networks has been widely documented. However, the effect of OT on the association between social behavior and brain functional connectivity (FC) is yet to be comprehensively explored. In this study, using a face-perception task and multiple connectome-based predictive (CPM) models, we aimed to: 1) determine whether OT could enhance the association between task behavioral performance, resting-state functional connectivity (rsFC), and task-state functional connectivity (tsFC), and 2) if so, determine the role of OT in enhancing this triangular association. We found that both rsFC and tsFC could independently and significantly predict task performance in the OT group, but not in the placebo (PL) group. In addition, the correlation coefficient between rsFC and tsFC was substantially higher in the OT group than in the PL group. The strength of these associations could be partly explained by OT altering the brains FCs related to social cognition and face-perception in both resting and task states, mainly in brain regions such as the limbic system, prefrontal cortex (PFC), temporal poles (TP), and temporoparietal junction (TPJ). Together, these results suggest that neuropeptides can increase the consistency of individual differences in different modalities (e.g., behavioral and brain level data).

neuroscience↗