Biology subjects

Yao, Q.

Publications and source records attributed to Yao, Q..

31 records · Page 2Linked to original sources

bioRxiv2020

Human fecal microbiota is associated with colorectal cancer

BackgroundColorectal cancer (CRC) is one of the most common cancers. In recent studies, the gut microbiota has been reported to be potentially involved in aggravating or favoring CRC development. However, little is known about the microbiota composition in CRC patients after treatment. In this study, we explored the fecal microbiota composition to obtain a periscopic view of gut microbial communities. We analyzed microbial 16S rRNA genes from 107 fecal samples of Chinese individuals from three groups, including 33 healthy individuals (Normal), 38 CRC patients (Fa), and 36 CRC post-surgery patients (Fb). ResultsSpecies richness and diversity were decreased in the Fa and Fb groups compared with that of the Normal group. Partial least squares discrimination analysis showed clustering of samples according to disease with an obvious separation between the Fa and Normal, and Fb and Normal groups, as well as a partial separation between the Fa and Fb groups. Based on linear discriminant analysis effect size analysis and a receiver operating characteristic model, Fusobacterium was suggested as a potential biomarker for CRC screening. Additionally, we found that surgery greatly reduced the bacterial diversity of microbiota in CRC patients. Some commensal beneficial bacteria of the intestinal canal, such as Faecalibacterium and Prevotella, were decreased, whereas the drug-resistant Enterococcus was visibly increased in CRC post-surgery group. Meanwhile, we observed a declining tendency of Fusobacterium in the majority of follow-up CRC patients who were still alive approximately 3 y after surgery. We also observed that beneficial bacteria dramatically decreased in CRC patients that recidivated or died after surgery. This revealed that important bacteria might be associated with prognosis. ConclusionsThe fecal bacterial diversity was diminished in CRC patients compared with that in healthy individuals. Enrichment and depletion of several bacterial strains associated with carcinomas and inflammation were detected in CRC samples. Fusobacterium might be a potential biomarker for early screening of CRC in Chinese or Asian populations. In summary, this study indicated that fecal microbiome-based approaches could be a feasible method for detecting CRC and monitoring prognosis post-surgery.

microbiology↗

bioRxiv2020

Detection of melanin-mediated false-positive for BaxΔ2 immunohistochemical staining in human skin tissue

Bax{Delta}2 is a pro-apoptotic isoform of the Bax family. We have previously shown that Bax{Delta}2 protein expression is low in most healthy human tissues with the protein mainly scattered in the connective tissues. Surprisingly, in skin tissue, the Bax{Delta}2-positive staining was strikingly strong, especially in the basal layer of the epidermis. It has been documented that melanin in the basal cells displays a brown color that could give false-positive staining in the commonly used DAB-based (3-3-diaminobenzidine) immunostaining. To avoid the false-positive staining from DAB-melanin, we performed immunofluorescent staining on the same set of tissues which were positive for Bax{Delta}2 in the DAB immunostaining. The results from co-immunofluorescent staining of Bax{Delta}2 and a basal cell marker CK-14 showed that the previously detected DAB-stained Bax{Delta}2-positive epidermal cells were CK-14 positive but Bax{Delta}2 negative. Consistent with our previous finding, the Bax{Delta}2-positive cells in the dermis connective tissues are positive in both DAB-based and fluorescence-based immunostainings. These data indicate that the DAB-stained Bax{Delta}2 positive cells in the basal layer of skin tissue are false-positive due to a misinterpretation of the melanin color, but the dermal connective tissue still presents real Bax{Delta}2 positive staining.

cell biology↗

bioRxiv2020

Challenges in solving structures from radiation-damaged tomograms of protein nanocrystals assessed by simulation

Structure determination methods are needed to resolve the atomic details that underlie protein function. X-ray crystallography has provided most of our knowledge of protein structure but is constrained by the need for large, well-ordered crystals and the loss of phase information. The rapidly developing methods of serial femtosecond crystallography, micro-electron diffraction, and single-particle reconstruction circumvent the first of these limitations by enabling data collection from nanocrystals or purified proteins. However, the first two methods also suffer from the phase problem, while many proteins fall below the molecular weight threshold required by single-particle reconstruction. Cryo-electron tomography of protein nanocrystals has the potential to overcome these obstacles of mainstream structure determination methods. Here we present a data processing scheme that combines routines from X-ray crystallography and new algorithms we developed to solve structures from tomograms of nanocrystals. This pipeline handles image processing challenges specific to tomographic sampling of periodic specimens and is validated using simulated crystals. We also assess the tolerance of this workflow to the effects of radiation damage. Our simulations indicate a trade-off between a wider tilt-range to facilitate merging data from multiple tomograms and a smaller tilt increment to improve phase accuracy. Since phase errors but not merging errors can be overcome with additional datasets, these results recommend distributing the dose over a wide angular range rather than using a finer sampling interval to solve the protein structure.

biophysics↗

bioRxiv2020

ZNF410 represses fetal globin by devoted control of CHD4/NuRD

Major effectors of adult-stage fetal globin silencing include the transcription factors (TFs) BCL11A and ZBTB7A/LRF and the NuRD chromatin complex, although each has potential on-target liabilities for rational {beta}-hemoglobinopathy therapeutic inhibition. Here through CRISPR screening we discover ZNF410 to be a novel fetal hemoglobin (HbF) repressing TF. ZNF410 does not bind directly to the{gamma} -globin genes but rather its chromatin occupancy is solely concentrated at CHD4, encoding the NuRD nucleosome remodeler, itself required for HbF repression. CHD4 has two ZNF410-bound regulatory elements with 27 combined ZNF410 binding motifs constituting unparalleled genomic clusters. These elements completely account for ZNF410s effects on{gamma} -globin repression. Knockout of ZNF410 reduces CHD4 by 60%, enough to substantially de-repress HbF while avoiding the cellular toxicity of complete CHD4 loss. Mice with constitutive deficiency of the homolog Zfp410 are born at expected Mendelian ratios with unremarkable hematology. ZNF410 is dispensable for human hematopoietic engraftment potential and erythroid maturation unlike known HbF repressors. These studies identify a new rational target for HbF induction for the {beta}-hemoglobin disorders with a wide therapeutic index. More broadly, ZNF410 represents a special class of gene regulator, a conserved transcription factor with singular devotion to regulation of a chromatin subcomplex.

genetics↗

bioRxiv2020

Generation of ordered protein assemblies using rigid three-body fusion

Protein nanomaterial design is an emerging discipline with applications in medicine and beyond. A longstanding design approach uses genetic fusion to join protein homo-oligomer subunits via -helical linkers to form more complex symmetric assemblies, but this method is hampered by linker flexibility and a dearth of geometric solutions. Here, we describe a general computational method that performs rigid three-body fusion of homo-oligomer and spacer building blocks to generate user-defined architectures, while at the same time significantly increasing the number of geometric solutions over typical symmetric fusion. The fusion junctions are then optimized using Rosetta to minimize flexibility. We apply this method to design and test 92 dihedral symmetric protein assemblies from a set of designed homo-dimers and repeat protein building blocks. Experimental validation by native mass spectrometry, small angle X-ray scattering, and negative-stain single-particle electron microscopy confirms the assembly states for 11 designs. Most of these assemblies are constructed from DARPins (designed ankyrin repeat proteins), anchored on one end by -helical fusion and on the other by a designed homo-dimer interface, and we explored their use for cryo-EM structure determination by incorporating DARPin variants selected to bind targets of interest. Although the target resolution was limited by preferred orientation effects, small scaffold size, and the low-order symmetry of these dihedral scaffolds, we found that the dual anchoring strategy reduced the flexibility of the target-DARPIN complex with respect to the overall assembly, suggesting that multipoint anchoring of binding domains could contribute to cryo-EM structure determination of small proteins.

synthetic biology↗

bioRxiv2020

Decoding herbal materials of representative TCM preparations with the multi-barcoding approach

With the rapid development of high-throughput sequencing (HTS) technology, the techniques for the assessment of biological ingredients in Traditional Chinese Medicine (TCM) preparations have also advanced. By using HTS together with the multi-barcoding approach, all biological ingredients could be identified from TCM preparations in theory, as long as their DNA is present. The biological ingredients of a handful of classical TCM preparations were analyzed successfully based on this approach in previous studies. However, the universality, sensitivity and reliability of this approach used on TCM preparations remain unclear. Here, four representative TCM preparations, namely Bazhen Yimu Wan, Da Huoluo Wan, Niuhuang Jiangya Wan and You Gui Wan, were selected for concrete assessment of this approach. We have successfully detected from 77.8% to 100% prescribed herbal materials based on both ITS2 and trnL biomarkers. The results based on ITS2 have also shown a higher level of reliability than those of trnL at species level, and the integration of both biomarkers could provide higher sensitivity and reliability. In the omics big-data era, this study has undoubtedly made one step forward for the multi-barcoding approach for prescribed herbal materials analysis of TCM preparation, towards better digitization and modernization of drug quality control.

bioinformatics↗

bioRxiv2019

Meta-16S rRNA gene phylogenetic reconstruction reveals astonishing diversity of the cosmopolitan myxobacteria

Numerous ecological studies for myxobacteria have been conducted well, but their true diversity hidden in plain sight remains to be explored. To bridge this gap, we here implemented a comprehensive survey of diversity and distribution of myxobacteria by using 4997 publicly available 16S rRNA gene sequences ([≥]1200 bp) collected from several hundreds of sites across multiple countries and regions. In the study, the meta-16S rRNA gene phylogenetic reconstruction clearly revealed that these sequences were classified to 998 species, 445 genera, 58 families, and 20 suborders, highlighting a considerable taxonomic diversity of myxobacteria, the great majority of which belonged to new taxa. Most cultured myxobacteria including the well-described type strains were strongly inclined to locate on the shallow branches of the phylogenetic tree; on the contrary, the majority of uncultured myxobacteria the deep branches. The geographical analysis of sequences based on their environmental categories clearly demonstrated that myxobacteria showed a nearly cosmopolitan distribution, despite the presence of some habitat-specific taxa, especially at genus and species levels. Among all abundant suborders, members of Suborder_4, Suborder_15, and Suborder_17 were more widely distributed in marine environments, the remaining suborders preferred to reside in terrestrial ecosystems, particularly in soils, indicating a potential selectivity of geographical distribution. In conclusion, this study profiles a clear framework of diversity and distribution of the cosmopolitan myxobacteria and sheds light on the isolation of the uncultured myxobacteria.\n\nIMPORTANCEMyxobacteria are an attractive bacterial group ubiquitous in soil and aquatic environments. However, the biodiversity and ecological preferences of myxobacteria remain poorly understood across heterogeneous environments. We analyzed thousands of publicly available and high-quality 16S rRNA gene sequences of myxobacteria by using the phylogenetic reconstruction. The study presented an astonishing diversity than that expected from the previous studies. This study further demonstrated that the culturability of myxobacteria was perfectly comparable to its phylogeny in the phylogenetic tree. The geographical analysis clearly indicated that myxobacteria showed a nearly cosmopolitan distribution, while some taxa exhibited obvious preferences for specific environmental conditions. Together, our study provides novel insights into the diversity, distributions, and ecological preferences of of myxobacteria from diverse environments and lays the foundation for innovation of isolation techniques and the discovery of new secondary metabolites.

microbiology↗

bioRxiv2018

Fusion of DARPin to aldolase enables visualization of small protein by cryoEM

In recent years, solving protein structures by single particle cryogenic electron microscopy (cryoEM) has become a crucial tool in structural biology. While exciting progress is being made towards the visualization of smaller and smaller macromolecules, the median protein size in both eukaryotes and bacteria is still beyond the reach of single particle cryoEM. To overcome this problem, we implemented a platform strategy in which a small protein target was rigidly attached to a large, symmetric base via a selectable adapter. Seven designs were tested. In the best construct, a designed ankyrin repeat protein (DARPin) was rigidly fused to tetrameric rabbit muscle aldolase through a helical linker. The DARPin retained its ability to bind its target, the 27 kDa green fluorescent protein (GFP). We solved the structure of this complex to 3.0 [A] resolution overall, with 5 to 8 [A] resolution in the GFP region. As flexibility in the DARPin limited the overall resolution of the target, we describe strategies to rigidify this element.\n\nAuthor summarySingle particle cryogenic electron microscopy (cryoEM) is a technique that uses images of purified proteins to determine their atomic structure. Unfortunately, the majority of proteins in the human and bacterial proteomes are too small to be analyzed by cryoEM. Over the years, several groups have suggested the use of a platform to increase the size of small protein targets. The platform is composed of a large protein base and a selectable adapter that binds the target protein. Here we report a platform based on tetrameric rabbit muscle aldolase that is fused to a Designed Ankyrin Repeat Protein (DARPin). Phage display libraries can be used to generate DARPins against target proteins. The residues mutated in a phage display library to generate a DARPin against a new target do not overlap with the DARPin-base fusion in the platform, thus changing the DARPin identity will not disrupt the platform design. The DARPin adapter used here is capable of binding Green Fluorescent Protein (GFP). We report the structure of GFP to 5 to 8 [A] local resolution by single particle cryoEM. Our analysis demonstrates that flexibility in the DARPin-aldolase platform prevents us from achieving higher resolution in the GFP region. We suggest changes to the DARPin design to rigidify the DARPin-aldolase platform. This work expands on current platforms and paves a generally applicable way toward structure determination of small proteins by cryoEM.

biophysics↗

bioRxiv2018

Simulations suggest a constrictive force is required for Gram-negative bacterial cell division

To divide, Gram-negative bacterial cells must remodel their peptidoglycan cell wall to a smaller and smaller radius at the division site, but how this process occurs remains debated. While the tubulin homolog FtsZ is thought to generate a constrictive force, it has also been proposed that cell wall remodeling alone is sufficient to drive membrane constriction, possibly via a make-before-break mechanism in which new hoops of cell wall are made inside the existing hoops (make) before bonds in the existing wall are cleaved (break). Previously, we constructed software, REMODELER 1, to simulate cell wall remodeling in rod-shaped bacteria during growth. Here, we used this software as the basis for an expanded simulation system, REMODELER 2, which we used to explore different mechanistic models of cell wall division. We found that simply organizing the cell wall synthesis complexes at the midcell was not sufficient to cause wall invagination, even with the implementation of a make-before-break mechanism. Applying a constrictive force at the midcell could drive division if the force was sufficiently large to initially constrict the midcell into a compressed state before new hoops of relaxed cell wall were incorporated between existing hoops. Adding a make-before-break mechanism could drive division with a smaller constrictive force sufficient to bring the midcell peptidoglycan into a relaxed, but not necessarily compressed, state.

biophysics↗

bioRxiv2018

CRISPRO Identifies Functional Protein Coding Sequences Based on Genome Editing Dense Mutagenesis

CRISPR/Cas9 pooled screening permits parallel evaluation of comprehensive guide RNA libraries to systematically perturb protein coding sequences in situ and correlate with functional readouts. For the analysis and visualization of the resulting datasets we have developed CRISPRO, a computational pipeline that maps functional scores associated with guide RNAs to genome, transcript, and protein coordinates and structure. No available tool has similar functionality. The ensuing genotype-phenotype linear and 3D maps raise hypotheses about structure-function relationships at discrete protein regions. Machine learning based on CRISPRO features improves prediction of guide RNA efficacy. The CRISPRO tool is freely available at gitlab.com/bauerlab/crispro.

bioinformatics↗

bioRxiv2018

Bacterial swarming reduces Proteus mirabilis and Vibrio parahaemolyticus cell stiffness and increases β-lactam susceptibility

Swarmer cells of the gram-negative pathogenic bacteria Proteus mirabilis and Vibrio parahaemolyticus become long (>10-100 m) and multinucleate during their growth and motility on polymer surfaces. We demonstrate increasing cell length is accompanied by a large increase in flexibility. Using a microfluidic assay to measure single-cell mechanics, we identified large differences in swarmer cell stiffness of (bending rigidity of P. mirabilis, 9.6 x 10-22 N m2; V. parahaemolyticus, 9.7 x 10-23 N m2) compared to vegetative cells (1.4 x 10-20 N m2 and 3.2 x 10-22 N m2, respectively). The reduction in bending rigidity (~3-15 fold) was accompanied by a decrease in the average polysaccharide strand length of the peptidoglycan layer of the cell wall from 28-30 to 19-22 disaccharides. Atomic force microscopy revealed a reduction in P. mirabilis peptidoglycan thickness from 1.5 nm (vegetative) to 1.0 nm (swarmer) and electron cryotomography indicated changes in swarmer cell wall morphology. P. mirabilis and V. parahaemolyticus swarmer cells became increasingly sensitive to osmotic pressure and susceptible to cell wall-modifying antibiotics (compared to vegetative cells)--they were ~30% more likely to die after 3 h of treatment with minimum inhibitory concentrations of the {beta}-lactams cephalexin and penicillin G. The adaptive cost of swarming is offset by the increase in cell susceptibility to physical and chemical changes in their environment, thereby suggesting the development of new chemotherapies for bacteria that leverage swarming for colonization of hosts and survival. ImportanceProteus mirabilis and Vibrio parahaemolyticus are bacteria that infect humans. To adapt to environmental changes, these bacteria alter their cell morphology and move collectively to access new sources of nutrients in a process referred to as swarming. We found that a change in the composition and thickness of the peptidoglycan layer of the cell wall makes swarmer cells of P. mirabilis and V. parahaemolyticus more flexible (i.e., reduced cell stiffness) and increases their sensitivity to osmotic pressure and cell-wall targeting antibiotics (e.g., {beta}-lactams). These results highlight the importance of assessing the extracellular environment in determining antibiotic doses and the use of {beta}-lactams antibiotics for treating infections caused by swarmer cells of P. mirabilis and V. parahaemolyticus.

microbiology↗

bioRxiv2018

Agricultural Pollution Risks Influence Microbial Ecology in Honghu Lake

BackgroundAgricultural activities, such as stock-farming, planting industry, and fish aquaculture, can influence the physicochemistry and biology of freshwater lakes. However, the extent to which these agricultural activities, especially those that result in eutrophication and antibiotic pollution, effect water and sediment-associated microbial ecology, remains unclear.\n\nMethodsWe performed a geospatial analysis of water and sediment associated microbial community structure, as well as physicochemical parameters and antibiotic pollution, across 18 sites in Honghu lake, which range from impacted to less-impacted by agricultural pollution. Furthermore, the co-occurrence network of water and sediment were built and compared accorded to the agricultural activities.\n\nResultsPhysicochemical properties including TN, TP, NO3--N, and NO2--N were correlated with microbial compositional differences in water samples. Likewise, in sediment samples, Sed-OM and Sed-TN correlated with microbial diversity. Oxytetracycline and tetracycline concentration described the majority of the variance in taxonomic and predicted functional diversity between impacted and less-impacted sites in water and sediment samples, respectively. Finally, the structure of microbial co-associations was influenced by the eutrophication and antibiotic pollution.\n\nConclusionThese analyses of the composition and structure of water and sediment microbial communities in anthropologically-impacted lakes are imperative for effective environmental pollution monitoring. Likewise, the exploration of the associations between environmental variables (e.g. physicochemical properties, and antibiotics) and community structure is important in the assessment of lake water quality and its ability to sustain agriculture. These results show agricultural practices can negatively influence not only the physicochemical properties, but also the biodiversity of microbial communities associated with the Honghu lake ecosystem. And these results provide compelling evidence that the microbial community can be used as a sentinel of eutrophication and antibiotics pollution risk associated with agricultural activity; and that proper monitoring of this environment is vital to maintain a sustainable environment in Honghu lake.

microbiology↗

bioRxiv2017

FtsEX-Mediated Regulation Of Inner Membrane Fusion And Cell Separation Reveals Morphogenetic Plasticity In Caulobacter crescentus

During its life cycle, Caulobacter crescentus undergoes a series of coordinated shape changes, including generation of a polar stalk and reshaping of the cell envelope to produce new daughter cells through the process of cytokinesis. The mechanisms by which these morphogenetic processes are coordinated in time and space remain largely unknown. Here we demonstrate that the conserved division complex FtsEX controls both the early and late stages of cytokinesis in C. crescentus, namely initiation of constriction and final cell separation. {Delta}ftsE cells display a striking phenotype: cells are chained, with skinny connections between cell bodies resulting from defects in inner membrane fusion and cell separation. Surprisingly, the thin connections in {Delta}ftsE cells share morphological and molecular features with C. crescentus stalks. Our data uncover unanticipated morphogenetic plasticity in C. crescentus, with loss of FtsE causing a stalk-like program to take over at failed division sites and yield novel cell morphology.\n\nAuthor SummaryBacterial cell shape is genetically hardwired and is critical for fitness and, in certain cases, pathogenesis. In most bacteria, a semi-rigid structure called the cell wall surrounds the inner membrane, offering protection against cell lysis while simultaneously maintaining cell shape. A highly dynamic macromolecular structure, the cell wall undergoes extensive remodeling as bacterial cells grow and divide. We demonstrate that a broadly conserved cell division complex, FtsEX, relays signals from the cytoplasm to the cell wall to regulate key developmental shape changes in the -proteobacterium Caulobacter crescentus. Consistent with studies in diverse bacteria, we observe strong synthetic interactions between ftsE and cell wall hydrolytic factors, suggesting that regulation of cell wall remodeling is a conserved function of FtsEX. Loss of FtsE causes morphological defects associated with both the early and late stages of division. Intriguingly, without FtsE, cells frequently fail to separate and instead elaborate a thin, tubular structure between cell bodies, a growth mode observed in other -proteobacteria. Overall, our results highlight the plasticity of bacterial cell shape and demonstrate how altering the activity of one morphogenetic program can produce diverse morphologies resembling those of other bacteria in nature.

microbiology↗