Spatial multi-omics at subcellular resolution via high-throughput in situ pairwise sequencing.

Technology for spatial multi-omics aids the discovery of new insights into cellular functions and disease mechanisms. Here we report the development and applicability of multi-omics in situ pairwise sequencing (MiP-seq), a method for the simultaneous detection of DNAs, RNAs, proteins and biomolecules at subcellular resolution. Compared with other in situ sequencing methods, MiP-seq enhances decoding capacity and reduces sequencing and imaging costs while maintaining the efficacy of detection of gene mutations, allele-specific expression and RNA modifications. MiP-seq can be integrated with in vivo calcium imaging and Raman imaging, which enabled us to generate a spatial multi-omics atlas of mouse brain tissues and to correlate gene expression with neuronal activity and cellular biochemical fingerprints. We also report a sequential dilution strategy for resolving optically crowded signals during in situ sequencing. High-throughput in situ pairwise sequencing may facilitate the multidimensional analysis of molecular and functional maps of tissues.

The potential of antifungal peptides derived from Lactiplantibacillus plantarum WYH for biocontrol of Aspergillus flavus contamination.

Aspergillus flavus is a notorious fungus that contaminates food crops with toxic aflatoxins, posing a serious threat to human health and the agricultural economy. To overcome the inadequacy of traditional control methods and meet consumer preferences for natural-sources additives, there is an urgent demand for novel biocontrol agents that are safe and efficient. This study aims to investigate the antifungal properties of a novel antifungal agent derived from the biologically safe Lactiplantibacillus plantarum WYH. Firstly, antifungal peptides (AFPs) with a molecular weight of less than 3kD, exhibiting remarkable temperature stability and effectively retarding fungal growth in a dose-dependent manner specifically against A. flavus, were concentrated from the fermentation supernatant of L. plantarum WYH and were named as AFPs-WYH. Further analysis demonstrated that AFPs-WYH might exert antifungal effects through the induction of oxidative stress, disruption of mitochondrial function, alteration of membrane permeability, and cell apoptosis in A. flavus. To further validate our findings, a transcriptomics analysis was conducted on A. flavus treated with 2 and 5 mg/mL of AFPs-WYH, which elucidated the potential effect of AFPs-WYH administration on the regulation of genes involved in impairing fungal development and preventing aflatoxin biosynthesis pathways. Overall, AFPs-WYH reduced the A. flavus proliferation and affected the AFB1 biosynthesis, exhibiting a promising potential for food industry applications as a biopreservative and biocontrol agent.

MGA-seq: robust identification of extrachromosomal DNA and genetic variants using multiple genetic abnormality sequencing.

Genomic abnormalities are strongly associated with cancer and infertility. In this study, we develop a simple and efficient method - multiple genetic abnormality sequencing (MGA-Seq) - to simultaneously detect structural variation, copy number variation, single-nucleotide polymorphism, homogeneously staining regions, and extrachromosomal DNA (ecDNA) from a single tube. MGA-Seq directly sequences proximity-ligated genomic fragments, yielding a dataset with concurrent genome three-dimensional and whole-genome sequencing information, enabling approximate localization of genomic structural variations and facilitating breakpoint identification. Additionally, by utilizing MGA-Seq, we map focal amplification and oncogene coamplification, thus facilitating the exploration of ecDNA's transcriptional regulatory function.

Safety Assessment of One Lactiplantibacillus plantarum Isolated from the Traditional Chinese Fermented Vegetables-Jiangshui.

Lactiplantibacillus plantarum is a kind of extensively utilized probiotic species, which plays a critical role in the prevention of pathogenic bacteria and development of functional probiotics. Our group previously isolated one Lactiplantibacillus from Jiang Shui, a traditional Chinese fermented vegetable, which remarkably inhibited the growth of Aspergillus flavus. Herein, the safety of this isolate was assessed to ensure its application feasibility in food industry. Firstly, the phenotypic analyses including tolerance to low pH and bile salt, aggregation ability, and hemolytic activity detection, indicated the isolate could survive and colonize in the gastrointestinal tract, without hemolysin activity. The susceptibilities of the isolate to eight antibiotics and the absence of most resistance genes were demonstrated by agar disk diffusion and PCR, respectively. Furthermore, no mortality or toxicity was observed in mice by in vivo tests using gross autopsy, hematology, serum biochemistry, and HE-staining. Taken together, this study demonstrated the safety of Lactiplantibacillus plantarum WYH as a probiotic strain in terms of phenotypic analyses, absence of antimicrobial resistance and toxin-related genes, as well as mice toxicity test, while supported the prospect of applying isolate in suppression of fungal growth and mycotoxin biosynthesis.

In Silico Infection Analysis (iSFA) Identified Coronavirus Infection and Potential Transmission Risk in Mammals.

Coronaviruses are a great source of threat to public health which could infect various species and cause diverse diseases. However, the epidemic's spreading among different species remains elusive. This study proposed an in silico infection analysis (iSFA) system that includes pathogen genome or transcript mining in transcriptome data of the potential host and performed a comprehensive analysis about the infection of 38 coronaviruses in wild animals, based on 2,257 transcriptome datasets from 89 mammals' lung and intestine, and revealed multiple potential coronavirus infections including porcine epidemic diarrhea virus (PEDV) infection in Equus burchellii. Then, through our transmission network analysis, potential intermediate hosts of five coronaviruses were identified. Notably, iSFA results suggested that the expression of coronavirus receptor genes tended to be downregulated after infection by another virus. Finally, binding affinity and interactive interface analysis of S1 protein and ACE2 from different species demonstrated the potential inter-species transmission barrier and cross-species transmission of SARS-CoV-2. Meanwhile, the iSFA system developed in this study could be further applied to conduct the source tracing and host prediction of other pathogen-induced diseases, thus contributing to the epidemic prevention and control.

Reprogramming Mycobacterium tuberculosis CRISPR System for Gene Editing and Genome-wide RNA Interference Screening.

Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), which is still the leading cause of mortality from a single infectious disease worldwide. The development of novel anti-TB drugs and vaccines is severely hampered by the complicated and time-consuming genetic manipulation techniques for M. tuberculosis. Here, we harnessed an endogenous type III-A CRISPR/Cas10 system of M. tuberculosis for efficient gene editing and RNA interference (RNAi). This simple and easy method only needs to transform a single mini-CRISPR array plasmid, thus avoiding the introduction of exogenous protein and minimizing proteotoxicity. We demonstrated that M. tuberculosis genes can be efficiently and specifically knocked in/out by this system as confirmed by DNA high-throughput sequencing. This system was further applied to single- and multiple-gene RNAi. Moreover, we successfully performed genome-wide RNAi screening to identify M. tuberculosis genes regulating in vitro and intracellular growth. This system can be extensively used for exploring the functional genomics of M. tuberculosis and facilitate the development of novel anti-TB drugs and vaccines.

Screening of Compounds for Anti-tuberculosis Activity, and in vitro and in vivo Evaluation of Potential Candidates.

Tuberculosis (TB) is a debilitating infectious disease responsible for more than one million deaths per year. The emergence of drug-resistant TB poses an urgent need for the development of new anti-TB drugs. In this study, we screened a library of over 4,000 small molecules and found that orbifloxacin and the peptide AK15 possess significant bactericidal activity against Mycobacterium tuberculosis (Mtb) in vitro. Orbifloxacin also showed an effective ability on the clearance of intracellular Mtb and protect mice from a strong inflammatory response but not AK15. Moreover, we identified 17 nucleotide mutations responsible for orbifloxacin resistance by whole-genome sequencing. A critical point mutation (D94G) of the DNA gyrase (gyrA) gene was found to be the key role of resistance to orbifloxacin. The computational docking revealed that GyrA D94G point mutation can disrupt the orbifloxacin-protein gyrase interactions mediated by magnesium ion bridge. Overall, this study indicated the potential ability of orbifloxacin as an anti-tuberculosis drug, which can be used either alone or in combination with first-line antibiotics to achieve more effective therapy on TB.

Bioinformatic analyses of a potential Salmonella-virus-FelixO1 biocontrol phage BPS15S6 and the characterisation and anti-Enterobacteriaceae-pathogen activity of its endolysin LyS15S6.

Foodborne Enterobacteriaceae pathogens, especially Salmonella, still seriously threaten food safety. To establish a foundation for further developing phage- and endolysin-based methods combating these pathogens, in this study, the newly isolated Salmonella-virus-FelixO1 phage BPS15S6 for biocontrol purposes was characterised by genomic bioinformatic analysis, and then its endolysin LyS15S6 was obtained using a prokaryotic expression system, characterised in vitro and evaluated in the antibacterial efficacy. It was shown that BPS15S6 had an 87,609-bp genome with 130 open reading frames and does not appear to carry known lysogeny-associated genes and other damaging genetic determinants and is unlikely to perform generalised transduction. Furthermore, LyS15S6 was determined to possess the high enzymatic activity of 1,001,000 U mg-1 and the broad spectrum of lysing 56 tested Gram-negative strains. The assays of thermostability and optimum pH revealed that LyS15S6 was stable up to 40 °C and more active at pH 7. Notably, we demonstrate that edible ε-poly-L-lysine (EPL) can be used as an outer-membrane permeabiliser to improve the antibacterial performance of endolysins. When combined with 1 µg ml-1 EPL, 2 µM LyS15S6 could cause 3-4 log viable cell reductions of the three tested Enterobacteriaceae pathogens in vitro after 2 h of reaction at 25 °C and 2.56 and 3.14 log reductions of Salmonella ATCC13076 after 15 min of reaction at 25 °C and 2 h of reaction at 8 °C respectively. A new strategy, the combined application of endolysins and edible EPL for combating Enterobacteriaceae pathogens in food, is thus presented in this work.

Differences in neurotropism and neurotoxicity among retrograde viral tracers.

BACKGROUND: Neurotropic virus-based tracers have been extensively applied in mapping and manipulation of neural circuits. However, their neurotropic and neurotoxic properties remain to be fully characterized. METHODS: Through neural circuit tracing, we systematically compared the neurotropism discrepancy among different multi-trans-synaptic and mono-synaptic retrograde viral tracers including pseudorabies virus (PRV), rabies virus (RV), and the newly engineered retro adeno-associated virus (rAAV2-retro) tracers. The (single-cell) RNA sequencing analysis was utilized for seeking possible attribution to neurotropism discrepancy and comparing cell toxicity caused by viral infection between glycoprotein-deleted RV (RV-∆G) and rAAV2-retro. Viral toxicity induced microglia activation and neuronal protein change were evaluated by immunohistochemistry. RESULTS: Multi-trans-synaptic retrograde viral tracers, PRV and RV, exhibit differential neurotropism when they were used for central neural circuit tracing from popliteal lymph nodes. Mono-synaptic retrograde tracers, including RV-∆G and rAAV2-retro, displayed discrepant neurotropic property, when they were applied to trace the inputs of lateral hypothalamic area and medial preoptic nucleus. rAAV2-retro demonstrated preference in cerebral cortex, whereas RV-∆G prefers to label basal ganglia and hypothalamus. Remarkably, we detected a distinct preference for specific cortical layer of rAAV2-retro in layer 5 and RV-∆G in layer 6 when they were injected into dorsal lateral geniculate nucleus to label corticothalamic neurons in primary visual cortex. Complementation of TVA receptor gene in RV-resistant neurons enabled EnvA-pseudotyped RV infection, supporting receptors attribution to viral neurotropism. Furthermore, both RV-∆G and rAAV2-retro exerted neurotoxic influence at the injection sites and retrogradely labeled sites, while the changes were more profound for RV-∆G infection. Finally, we demonstrated a proof-of-concept strategy for more comprehensive high-order circuit tracing of a specific target nucleus by combining rAAV2-retro, RV, and rAAV tracers. CONCLUSIONS: Different multi-trans-synaptic and mono-synaptic retrograde viral tracers exhibited discrepant neurotropism within certain brain regions, even cortical layer preference. More neurotoxicity was observed under RV-∆G infection as compared with rAAV2-retro. By combining rAAV2-retro, RV, and rAAV tracers, high-order circuit tracing can be achieved. Our findings provide important reference for appropriate application of viral tracers to delineate the landscape and dissect the function of neural network.

Simultaneous determination of NOGE-related and BADGE-related compounds in canned food by ultra-performance liquid chromatography-tandem mass spectrometry.

An improved analytical method enabling rapid and accurate determination and identification of bisphenol F diglycidyl ether (novolac glycidyl ether 2-ring), novolac glycidyl ether 3-ring, novolac glycidyl ether 4-ring, novolac glycidyl ether 5-ring, novolac glycidyl ether 6-ring, bisphenol A diglycidyl ether, bisphenol A (2,3-dihydroxypropyl) glycidyl ether, bisphenol A (3-chloro-2-hydroxypropyl) glycidyl ether, bisphenol A bis(3-chloro-2-hydroxypropyl) ether, and bisphenol A (3-chloro-2-hydroxypropyl) (2,3-dihydroxypropyl) ether in canned food and their contact packaging materials has been developed by using, for the first time, ultra-performance liquid chromatography coupled with tandem mass spectrometry. After comparison of electrospray ionization and atmospheric pressure chemical ionization in positive and negative-ion modes, tandem mass spectrometry with positive electrospray ionization was chosen to carry out selective multiple reaction monitoring analysis of novolac glycidyl ethers, bisphenol A diglycidyl ether, and its derivatives. The analysis time is only 5.5 min per run. Limits of detection varied from 0.01 to 0.20 ng g(-1) for the different target compounds on the basis of a signal-to-noise ratio (S/N) = 3; limits of quantitation were from 0.03 to 0.66 ng g(-1). The relative standard deviation for repeatability was <8.01%. Analytical recovery ranged from 87.60 to 108.93%. This method was successfully applied to twenty samples of canned food and their contact packaging materials for determination of migration of NOGE, BADGE, and their derivatives from can coatings into food.

Effects of high dietary fat and cholesterol on expression of PPAR alpha, LXR alpha, and their responsive genes in the liver of apoE and LDLR double deficient mice.

The significance of transcription factors PPAR alpha, LXR alpha, and their responsive/target genes for the pathogenesis of atherosclerosis in apolipoprotein E and low-density lipoprotein receptor double deficient (AL) mice fed with high fat and cholesterol (HF) diet were studied. C57BL/6J wild-type (WT) mice were used as control to the AL mice. Plasma lipid metabolites and morphological atherosclerotic lesions in aortic wall were determined. Semi- and real-time quantitative RT-PCR were used to measure gene expression patterns between AL mice and the controls, which were fed with HF or normal chow diet. The results showed that in AL mice fed with HF diet, plasma lipid levels, hepatic lipid accumulation, and atherogenesis together with upregulated PPAR alpha, LXR alpha, and their target genes, i.e., FAT, SCD1, FAS, Angptl3, and apoB100 significantly increased in a 12-week long feeding period. In contrast, apoAI, apoAIV, apoF, LPL, and SR-BI were decreased compared to chow-fed group. In WT mice, PPAR alpha, LXR alpha, FAS, Angpt13, CPT1, apoF, ACOX1, LPL, and SR-BI were increased with HF treatment, while apoAI and apoAIV were decreased markedly. The different changes of lipid metabolism-related genes between AL and WT mice, fed with HF diet or chow diet indicated that the mechanisms of dietary effects on gene mutant mice are different from those of intact WT mice. Since lipid metabolic system defected genetically in AL mice, we suggest that the changes of PPAR alpha, LXR alpha, and their target genes aggravated lipid metabolic disorder in the liver and further accelerated the development of atherosclerosis on a stress of HF diet feeding in AL mice.

[Expressions of atherosclerosis-related genes in aorta in young apoE/LDLR double knockout mice].

To systematically clarify the effects of apolipoprotein E (aopE) and low-density lipoprotein receptor (LDLR) gene mutant on hyperlipidemia, vascular inflammation impairment and pathogenesis of atherosclerosis (AS), total RNA was isolated from fresh aortas of young apoE/LDLR double knockout (apoE(-/-)/LDLR(-/-)) and wild type (WT) mice using TRIzol reagent. Then RNA was reversely transcribed to first-strand cDNA by reverse transcriptase for reverse transcription polymerase chain reaction (RT-PCR) and real-time RT-PCR. Primer pairs were designed using primer design software according to the gene sequences available in GenBank. ß-actin was used as an internal control. Then RT-PCR assay was used to analyze the expression patterns of interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), nuclear factor-κB (NF-κB), granulocyte-macrophage colony-stimulating factor (GM-CSF), CD36, endothelin-1 (ET-1), toll-like receptor 2 (TLR2), monocyte chemoattractant protein-1 (MCP-1), vascular adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and platelet-derived growth factor-α (PDGF-α). SYBR Green quantitative real-time RT-PCR was used to validate gene expressions identified by RT-PCR. Blood samples were taken from the retro-orbital venous plexus, and serum levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL) and high-density lipoprotein (HDL) were measured by using biochemical techniques. Serum concentrations of circulating TNF-α, IL-1ß and oxidized LDL (ox-LDL) were determined by ELISA. Frozen sections of aortic sinus were stained with Sudan IV to visualize intimal fatty lesions. The results showed that the relative expressions of IL-1ß, GM-CSF, ET-1, TLR2, CD36, MCP-1, ICAM-1 and VCAM-1 in apoE(-/-)/LDLR(-/-) mice at the age of 1 month were higher than those in age-matched WT mice (P<0.05, P<0.01), respectively. The expressions of PDGF-α and TNF-α in apoE(-/-)/LDLR(-/-) mice at the age of 2 months were up-regulated compared to those in age-matched WT mice (P<0.05). All the expressions of target genes continued to be up-regulated (P<0.05, P<0.01) except that ET-1 expression at the age of 2 months, TLR2, VCAM-1 and ICAM-1 expressions at the age of 3 months were down-regulated to that in WT mice. NF-κB expression had no significant changes between two genotype mice at different ages. All the gene expressions kept unchanged in WT mice at different ages, except that IL-1b expressions were slightly up-regulated at the ages of 2 and 3 months. Serum levels of TC, TG, LDL, HDL, TNF-α, IL-1ß and ox-LDL in apoE(-/-)/LDLR(-/-) mice at different ages were higher than those in age-matched WT mice (P<0.05, P<0.01), and were increasing with age. Primary atherosclerotic lesions were observed in 1-month old apoE(-/-)/LDLR(-/-) mice and were progressing with age. There were no lesions observed in all WT mice at different ages. The data suggest that hyperlipidemia due to apoE and LDLR gene mutant may stimulate the temporal expressions of AS-related genes and contribute to primary atherogenetic lesions and vascular inflammation impairment.