Biology subjects

Shang, M.

Publications and source records attributed to Shang, M..

12 recordsLinked to original sources

bioRxiv2026

Splicing-guaranteed artificial introns enable efficient homology-directed repair at internal exons and streamlined cell engineering

Selection-marker-assisted homology-directed repair (HDR) efficiently edits coding sequence termini but is poorly suited for internal exons. Here, we developed artificial reporter intron-based HDR (ARI-HDR), which inserts a selectable intron into target exons to enable precise internal-exon editing. Although designed for scarless removal by splicing, ARIs frequently exhibit inaccurate splicing. Using a random mutagenesis reporter assay, we identified intron upstream sequences (IUS) as key determinants of splicing fidelity and screened degenerate IUS variants to obtain reliably spliced ARI donors. ARI-HDR successfully introduced site-specific mutations into five human genes. Combining long single-stranded DNA donors with conversion-tract optimization increased editing efficiency to over 80%. This efficiency enabled 'Dirty-Editing,' a streamlined workflow that generates precisely edited mixed-cell populations without single-cell cloning. Together, ARI-HDR extends selection-based enrichment to internal exons across protein-coding genes, while our splicing-optimized intron design offers a broadly applicable solution for artificial intron-based technologies.

molecular biology↗

bioRxiv2025

Oops: A Language for Formalizing Fallible Biological Protocols

Formalizing protocols used in wetlab biological research as programs improves reproducibility by making protocols replicable and standardized. However, existing protocol languages have limited capacity for codifying error sources and standardizing error handling. When protocols inevitably go wrong, debugging must still be done by technicians, a process which is challenging for unfamiliar protocols and a scalability constraint in automated facilities. We introduce OO_SCPLOWOPSC_SCPLOW, a programming language for describing biological protocols along with how they can fail. OO_SCPLOWOPSC_SCPLOW makes expression of how errors affect outcomes concise through probabilistic semantics, an extensible abstraction of the lab, and metaprogramming. Given an outcome, OO_SCPLOWOPSC_SCPLOW enables analyses of what errors occurred by mapping lab observations to enforced program conditions and applying existing probabilistic inference algorithms. We formalize a collection of molecular cloning protocols and present case studies demonstrating how OO_SCPLOWOPSC_SCPLOW can explain errors and assess diagnostic capabilities.

bioinformatics↗

bioRxiv2024

A motif preferred adenine base editor with minimal bystander and off-targets editing

48% of hereditary disease are caused by single C-to-T base conversion, which makes efficient A-to-G base editing tools (ABEs) have great potential in the treatment of these diseases. However, the existing efficient ABE, while catalyzing A-to-G conversion, will bring more A and C bystander editing and off-target events, which poses safety concerns for their clinical application. Here, we developed ABE10 (ABE8e with TadA-8e A48E) for efficient and accurate editing of As in YA motifs with YAY>YAR (Y=T or C, R=A or G) hierarchy through structure-oriented rational design. Compared with ABE3.1, currently the only motif (YAC) preference ABE version, ABE10 exhibited A-to-G editing efficiency improvement with an average up to 3.1-fold in indicated YA motif while maintaining reduced bystander Cs editing and minimized DNA or RNA off-targets. Also, we showed ABE10 corrected pathogenic mutation with high efficiency and precision in human cells. Moreover, by ABE10, we efficiently and precisely generated hypocholes-terolemia and tail-loss mouse models mimicking human associated disease and mouse PCSK9 base editing in vivo for hypercholesterolemia gene therapy, indicating their great potential in broad applications for and disease modeling and gene therapy.

molecular biology↗

bioRxiv2024

Phage-Encoded Bismuth Bicycles: Instant Access to Targeted Bioactive Peptides

Genetically encoded libraries play a crucial role in discovering structurally rigid, high-affinity macrocyclic peptide ligands for therapeutic applications. This study represents the first genetic encoding of peptide-bismuth and peptide-arsenic bicyclic peptides in phage display. We introduce bismuth tripotassium dicitrate (gastrodenol) as a water-soluble Bi(III) reagent for phage library modification and in situ bicyclic peptide preparation, eliminating the need for organic co-solvents. Additionally, we explore As(III) as an alternative thiophilic element used analogously to our previously introduced class of peptide-bismuth bicycles. The modification of phage libraries and peptides with these elements is instantaneous and entirely biocompatible, offering an advantage over conventional alkylation-based methods. In a pilot display screening campaign aimed at identifying ligands for the biotin-binding protein streptavidin, we demonstrate the enrichment of bicyclic peptides with dissociation constants two orders of magnitude lower than those of their linear counterparts, underscoring the impact of structural constraint on binding affinity.

biochemistry↗

bioRxiv2022

inMTSCCA: An Integrated Multi-task Sparse Canonical Correlation Analysis for Multi-omics Brain Imaging Genetics

Identifying genetic risk factors for Alzheimers disease (AD) is an important research topic. To date, different endophenotypes such as imaging-derived endophenotypes and proteomic expression-derived endophenotypes have shown the great value in uncovering risk genes compared to case-control studies. Biologically, a co-varying pattern of these different omics derived endophenotypes could result from the shared genetic basis. However, existing methods mainly focus on the effect of endophenotypes, and that of cross-endophenotype associations remains largely unexploited. In this paper, we used both endophenotypes and their cross-associations of multi-omics to identify genetic risk factors, and proposed two integrated multi-task sparse canonical correlation analysis (MTSCCA) methods, i.e., pairwise endophenotype correlation guided MTSCCA (pcMTSCCA) and high-order endophenotype correlation guided MTSCCA (hocMTSCCA). pcMTSCCA employed pairwise correlations between MRI-derived, plasma-derived, and cerebrospinal fluid (CSF) derived endophenotypes as an additional penalty. hocMTSCCA used high-order correlations among these multi-omics for regularization. To figure out genetic risk factors at individual and group levels, as well as altered endophenotypic markers, we introduced sparsity-inducing penalties in both models. We compared pcMTSCCA and hocMTSCCA with three related methods on both simulation data and real neuroimaging, proteomic analytes, and genetic data. The results showed that our methods obtained better or comparable canonical correlation coefficients (CCCs) and feature subsets than benchmarks. Most importantly, the identified genetic loci and heterogeneous endophenotypic markers showed high relevance. Therefore, jointly using multi-omics endophenotypes and their cross-endophenotype associations are promising to reveal genetic risk factors, and both methods are qualified for this complicated task. The source code and manual of inMTSCCA is available at: https://ngdc.cncb.ac.cn/biocode/tools/BT007330.

bioinformatics↗

bioRxiv2022

Deleterious Mechanical Deformation Selects Mechanoresilient Cancer Cells with Enhanced Proliferation and Chemoresistance

Cancer cells derived from secondary tumors can form new distant metastases more efficiently as compared to their primary tumor counterparts. This is partially due to the unfavorable microenvironments encountered by metastasizing cancer cells that result in the survival of a more metastatic phenotype from the original population. However, it is unclear how cancer cells may acquire such metastatic competency after overcoming deleterious mechanical stresses. Here, by forcing cancer cells to flow through small capillary-sized constrictions, we demonstrate that mechanical deformation can select a tumor cell subpopulation that exhibits resilience to mechanical squeezing-induced cell death. Transcriptomic profiling reveals up-regulated proliferation and DNA damage response pathways in this subpopulation, which are further translated into a more proliferative and chemotherapy-resistant phenotype. Our results highlight a potential link between the microenvironmental physical barriers and the enhanced malignancy of metastasizing cancer cells which may potentially be utilized for novel therapeutic strategies in preventing the metastatic spread of cancer cells.

cancer biology↗

bioRxiv2021

Two-color super-resolution localization microscopy via joint encoding of emitter location and color

Multi-color super-resolution localization microscopy (SRLM) provides great opportunities for studying the structural and functional details of biological samples. However, current multi-color SRLM methods either suffer from medium to high crosstalk, or require a dedicated optical system and a complicated image analysis procedure. To address these problems, here we propose a completely different method to realize multi-color SRLM. This method is built upon a customized RGBW camera with a repeated pattern of filtered (Red, Green, Blue and Near-infrared) and unfiltered (White) pixels. With a new insight that RGBW camera is advantageous for color recognition instead of color reproduction, we developed a joint encoding scheme of emitter location and color. By combing this RGBW camera with the joint encoding scheme and a simple optical set-up, we demonstrated two-color SRLM with [~]20 nm resolution and < 2% crosstalk (which is comparable to the best reported values). This study significantly reduces the complexity of two-color SRLM (and potentially multi-color SRLM), and thus offers good opportunities for general biomedical research laboratories to use multi-color SRLM, which is currently mastered only by well-trained researchers.

biophysics↗

bioRxiv2021

Reduced virulence and enhanced host adaption during antibiotics therapy: A story of a within-host carbapenem-resistant Klebsiella pneumoniae sequence type 11 evolution in a fatal scrotal abscess patient

Carbapenem-resistant Klebsiella pneumoniae (CRKP) has disseminated globally and become a major threat to human life. The sequence type (ST) 11 CRKP is a dominant clone in Asia, especially China, but how this clone evolves in vivo, then adapts to host and facilitates dissemination remain largely unknown. We analyzed the genomic dynamics of 4 ST11-CRKP isolates sequencially isolated from the urine of a patient with initial fatal scrotal abscess and finally recovered without effective medication. Genomic differences were identified and their implications for pathogenesis and host adaptation were investigated. The related transcriptional pathways were further explored by RNA-Seq. Genomic analysis identified 4-24 mutations and 94%-100% were synonymous or intergenic. The mutation rate of ST11-CRKP was 2.1x10-6-1.7x10-5 substitutions/site/year over 47 days of antibiotics therapy. During this period, CRKP underwent several adaptive changes including tigecycline resistance and virulence attenuation. Tigecycline resistance was caused by ramR ribosomal binding site (RBS) deletion, which has been described by us previously. In this study, we demonstrated that mutations associated with acyltransferase (act) and ompK26 caused the virulence attenuation of ST11-CRKP. act deletion reduced the production of capsular polysaccharide and enhanced biofilm formation. RNA-Seq analysis revealed that act influenced the expression of ldhA, bglX, mtnK and metE which likely participate in capsular synthesis and biofilm formation. ompK26 affected the virulence by its overexpression caused by the deletion of upstream repressor binding site. Our finding suggested that the broad genomic diversity, high evolutionary capacity and rapid within-host adaptability of ST11-CRKP might contribute to the worldwide dissemination of this clone. IMPORTANCECarbapenem-resistant Klebsiella pneumoniae (CRKP) has disseminated worldwide and can cause life threatening infections, including pneumonia, bloodstream infections, urinary tract infections, intra-abdominal infection, liver abscess and meningitis. CRKP infection is the leading cause of high mortality in clinical. The sequence type (ST) 11 CRKP is a dominant clone and accounts for 60% of CRKP infections in China. Recently, the ST11-CRKP with high transmissibility are increasingly identified. Understanding how this clone evolved is crucial in controlling its further dissemination. The significance of our research is identifying the in vivo genomic dynamics of ST11-CRKP and the genetic basis for ST11-CRKP to facilitate persistence and dissemination, which will has broader biomedical impacts on understanding of ST11-CRKP dissemination. Furthermore, our study also highlights the importance of monitoring the development of variation in antibiotics susceptibility and virulence of bacteria in clinical practice, considering that pathogens can rapidly adapt to host during the treatment.

microbiology↗

bioRxiv2021

The most recent common ancestor for Y chromosome lived about 3.67 million years ago

Investigating the divergence time between different populations has been of fundamental interest in evolution biology. In this study, we reanalysed the mitochondrial and Y chromosome DNA of the 1000 Genomes Project, and revealed that most single nucleotide polymorphisms (SNPs) are specific to minority and would be classified as low-frequency mutations. Using these polymorphisms, we recalculated the divergence time of different populations by direct comparison between individuals and the out-group.

evolutionary biology↗

bioRxiv2021

Pharmacological inhibition of PAI-1 alleviates cardiopulmonary pathologies induced by exposure to air pollutants PM2.5

OBJECTIVEExposure to air pollutants leads to the development of pulmonary and cardiovascular diseases, and thus air pollution is one of the major global threats to human health. Air pollutant particulate matter 2.5 (PM2.5)-induced cellular dysfunction impairs tissue homeostasis and causes vascular and cardiopulmonary damage. To test a hypothesis that elevated plasminogen activator inhibitor-1 (PAI-1) levels play a pivotal role in air pollutant-induced cardiopulmonary pathologies, we examined the efficacy of a drug-like novel inhibitor of PAI-1, TM5614, in treating PM2.5-induced vascular and cardiopulmonary pathologies. APPROACH AND RESULTSResults from biochemical, histological, and immunohistochemical studies revealed that PM2.5 increases the circulating levels of PAI-1 and thrombin and that TM5614 treatment completely abrogates these effects in plasma. PM2.5 significantly augments levels of pro-inflammatory cytokine IL-6 in bronchoalveolar lavage fluid, and this also can be reversed by TM5614, indicating its efficacy in amelioration of PM2.5-induced increases in inflammatory and pro-thrombotic factors. TM5614 reduces PM2.5-induced increased levels of inflammatory markers Mac3 and pSTAT3, adhesion molecule VCAM1, and apoptotic marker cleaved caspase 3. Longer exposure to PM2.5 induces pulmonary and cardiac thrombosis, but TM5614 significantly ameliorates PM2.5-induced vascular thrombosis. TM5614 also reduces PM2.5-induced increased blood pressure and heart weight. In vitro cell culture studies revealed that PM2.5 induces the levels of PAI-1, type I collagen, fibronectin, and SREBP-1/2, a transcription factor that mediates profibrogenic signaling, in cardiac fibroblasts. TM5614 abrogated that stimulation, indicating that it may block PM2.5-induced PAI-1 and profibrogenic signaling through suppression of SREBP-1. Furthermore, TM5614 blocked PM2.5-mediated suppression of Nrf2, a major antioxidant regulator in cardiac fibroblasts. CONCLUSIONSPharmacological inhibition of PAI-1 with TM5614 is a promising therapeutic approach to control air pollutant PM2.5-induced cardiopulmonary and vascular pathologies. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

pharmacology and toxicology↗

bioRxiv2019

Anti-inflammatory properties of chemical probes in human whole blood: focus on prostaglandin E2 production

We screened 57 chemical probes, high-quality tool compounds, and relevant clinically used drugs to investigate their effect on pro-inflammatory prostaglandin E2 (PGE2) production and interleukin-8 (IL-8) secretion in human whole blood. Freshly drawn blood from healthy volunteers and patients with systemic lupus erythematosus (SLE) or dermatomyositis was incubated with compounds at 0.1 or 1 M and treated with lipopolysaccharide (LPS, 10 g/mL) to induce a pro-inflammatory condition. Plasma was collected after 24 hours for lipid profiling using liquid chromatography tandem mass spectrometry (LC-MS/MS) and IL-8 quantification using enzyme-linked immunosorbent assay (ELISA). Each compound was tested in at least four donors at one concentration based on prior knowledge of binding affinities and in vitro activity. Our screening suggested that PD0325901 (MEK-1/2 inhibitor), trametinib (MEK-1/2 inhibitor), and selumetinib (MEK-1 inhibitor) decreased while tofacitinib (JAK inhibitor) increased PGE2 production. These findings were validated by concentration-response experiment in two donors. Moreover, the tested MEK inhibitors decreased thromboxane B2 (TXB2) production and IL-8 secretion. We also investigated the lysophophatidylcholine (LPC) profile in plasma from treated whole blood as these lipids are potentially important mediators in inflammation, and we did not observe any changes in LPC profiles. Collectively, we deployed a semi-high throughput and robust methodology to investigate anti-inflammatory properties of new chemical probes. HighlightsO_LIInhibitors for MEK decreased PGE2 and TXB2 production C_LIO_LIInhibitors for MEK and ERK decreased IL-8 secretion C_LIO_LIJAK inhibitor tofacitinib increased PGE2 and TXB2 production C_LI

immunology↗

bioRxiv2019

Effect of recombinant phycocyanin on photosynthetic system in Chlamydomonas reinhardtii

The phycobilisome is an important photosynthetic antenna in the photosynthetic cyanobacteria, and phycocyanin is one of the main components of phycobilisomes. It helps cells absorb green light that green-lineage photo-synthetic organisms cannot. In this work, phycocyanin, heme oxidase and ferredoxin oxidoreductase from Arthrospira platensis FACHB 314 were successfully expressed in the green algae Chlamydomonas reinhardtii. Then the effects of this expression on the photosynthesis and growth of C. reinhardtii were detected. Transcriptional level analysis showed that the phycocyanin gene was successfully expressed stably in the transgenic strains. The results of low-temperature fluorescence emission spectra and chlorophyll fluorescence showed that recombinant phycocyanin has considerable optical activity. The expression of phycocyanin, heme oxidase and ferredoxin oxidoreductase in low-light conditions is particularly evident in the promotion of photosynthesis in C. reinhardtii. The growth of transgenic strains was significantly promoted in the early growth phase under low-light conditions. However, the final growth and biomass accumulation of transgenic C. reinhardti were inhibited by this expression. In this paper, the possibility of photoenergy transfer between phycocyanin and heterologous host thylakoid membrane was researched, which provided a useful attempt for the construction of a new photosynthetic system using phycobiliprotein from cyanobacteria.\n\nOne-sentence summaryPhycocyanin from Arthrospira platensis FACHB 314 expressed in Chlamydomonas reinhardtii can effect the photosynthetic system of C. reinhardtii.

molecular biology↗