Biology subjects

Zhang, W.

Publications and source records attributed to Zhang, W..

At least 1,081 records · Page 60Linked to original sources

bioRxiv2018

Soil bacterial biomass and diversity are affected by different furrow-ridge mulched management systems during potato continuous cropping

The soil bacterial composition is vital for sustainable agriculture due to its importance in biogeochemical processes in the soil environment. Multiple management systems, such as different furrow-ridge mulched cropping systems, have been established to reduce the damage caused by continuous cropping of potato (Solanum tuberosum L.). However, little is known about the responses of soil bacterial biomass and diversity to these systems. In this study, six different ridge-furrow film planting patterns were tested in a 2-year continuous cropping potato field: flat plot without mulch (CK), flat plot with mulch (T1), on-ridge planting with full mulch (T2), on-furrow planting with full mulch (T3), on-ridge planting with half mulch (T4), and on-furrow planting with half mulch (T5). The soil physicochemical properties and bacterial composition were significantly affected by the planting pattern. Mulched soils, especially T2, maintained better soil physicochemical properties than controls. Fully mulched soil maintained higher bacterial biomass and diversity. Among the dominant genera, the abundances of Nitrosomonadaceae in T2 and T4 were higher than those in the other treatments. Consequently, compared with the other treatments, on-ridge with mulching patterns resulted in better soil physicochemical properties and high bacterial biomass and diversity, which could reduce the economic losses due to potato production by continuous cropping.

microbiology↗

bioRxiv2018

Unigene-based RNA-seq provides insights on drought stress responses in Marsdenia tenacissima

Marsdenia tenacissima is a well-known anti-cancer medicinal plant used in traditional Chinese medicine. Drought severely affects production and no information on its transcriptional responses to drought stress is available. In this study, cDNA libraries on control (CK), drought stress (T1), and re-watering (T2) treatments were constructed and HiSeq 2000 sequencing was performed using the Illumina platform. There were 43,129,228, 47,116,844, and 42,815,454 clean reads with Q20 values of 98.06, 98.04, and 97.88, respectively. A total of 8672, 6043, and 6537 differentially expressed genes (DEGs) were identified when CK vs. T1, CK vs. T2, and T1 vs. T2, respectively, were analyzed. In addition, 1039, 1016, and 980 transcription factors (TFs) were identified in CK, T1, and T2, respectively. Among them, 363, 267, and 299 TFs were identified as DEGs in CK vs. T1, CK vs. T2, and T1 vs. T2, respectively. These differentially expressed TFs mainly belonged to the bHLH, bZIP, C2H2, ERF, MYB, MYB-related, and NAC families. A comparative analysis of CK vs. T1 and T1 vs. T2 found that 1174 genes were up-regulated and 2344 were down-regulated under drought stress and this pattern was the opposite to that found after re-watering. Among the 1174 genes up-regulated by drought stress, 64 were homologous to known functional genes that directly protect plants against drought stress. Furthermore, 44 protein kinases and 38 TFs with opposite expression patterns under drought stress and re-watering were identified, which are possibly candidate regulators for drought stress resistance in M. tenacissima. Our study is the first to characterize the M. tenacissima transcriptome in response to drought stress, and will serve as a useful resource for future studies on the functions of candidate protein kinases and TFs involved in M. tenacissima drought stress resistance.

molecular biology↗

bioRxiv2018

Synthetic chromosome fusion: effects on genome structure and function

As part of the Synthetic Yeast 2.0 (Sc2.0) project, we designed and synthesized synthetic chromosome I. The total length of synI is [~]21.4% shorter than wild-type chromosome I, the smallest chromosome in Saccharomyces cerevisiae. SynI was designed for attachment to another synthetic chromosome due to concerns of potential instability and karyotype imbalance. We used a variation of a previously developed, robust CRISPR-Cas9 method to fuse chromosome I to other chromosome arms of varying length: chrIXR (84kb), chrIIIR (202kb) and chrIVR (1Mb). All fusion chromosome strains grew like wild-type so we decided to attach synI to synIII. Through the investigation of three-dimensional structures of fusion chromosome strains, unexpected loops and twisted structures were formed in chrIII-I and chrIX-III-I fusion chromosomes, which depend on silencing protein Sir3. These results suggest a previously unappreciated 3D interaction between HMR and the adjacent telomere. We used these fusion chromosomes to show that axial element Red1 binding in meiosis is not strictly chromosome size dependent even though Red1 binding is enriched on the three smallest chromosomes in wild-type yeast, and we discovered an unexpected role for centromeres in Red1 binding patterns.

synthetic biology↗

bioRxiv2018

3’ Branch Ligation: A Novel Method to Ligate Non-Complementary DNA to Recessed or Internal 3’OH Ends in DNA or RNA

Nucleic acid ligases are crucial enzymes that repair breaks in DNA or RNA during synthesis, repair and recombination. Various molecular tools have been developed using the diverse activities of DNA/RNA ligases. Herein, we demonstrate a non-conventional ability of T4 DNA ligase to join 5 phosphorylated blunt-end double-stranded DNA to DNA breaks at 3 recessive ends, gaps, or nicks to form a 3 branch structure. Therefore, this base pairing-independent ligation is termed 3 branch ligation (3BL). In an extensive study of optimal ligation conditions, similar to blunt-end ligation, the presence of 10% PEG-8000 in the ligation buffer significantly increased ligation efficiency. A low level of nucleotide preference was observed at the junction sites using different synthetic DNAs. Furthermore, we discovered that T4 DNA ligase efficiently ligated DNA to the 3 recessed end of RNA, not to that of DNA, in a DNA/RNA hybrid, whereas RNA ligases are less efficient in this reaction. These novel properties of T4 DNA ligase can be utilized as a broad molecular technique in many important applications. We performed a proof-of-concept study of a new directional tagmentation protocol for next generation sequencing (NGS) library construction that eliminates inverted adapters and allows sample barcode insertion adjacent to genomic DNA. 3BL after single transposon tagmentation can theoretically achieve 100% usable template, and our empirical data demonstrate that the new approach produced higher yield compared with traditional double transposon or Y transposon tagmentation. We further explore the potential use of 3BL for preparing targeted RNA NGS libraries with mitigated structure-based bias and adapter dimer problems.

molecular biology↗

bioRxiv2018

NudCL2 is an Hsp90 cochaperone to regulate sister chromatid cohesion by stabilizing cohesin subunits

Sister chromatid cohesion plays a key role in ensuring precise chromosome segregation during mitosis, which is mediated by the multisubunit complex cohesin. However, the molecular regulation of cohesin subunits stability remains unclear. Here, we show that NudCL2 (NudC-like protein 2) is essential for the stability of cohesin subunits by regulating Hsp90 ATPase activity in mammalian cells. Depletion of NudCL2 induces mitotic defects and premature sister chromatid separation and destabilizes cohesin subunits that interact with NudCL2. Similar defects are also observed upon inhibition of Hsp90 ATPase activity. Interestingly, ectopic expression of Hsp90 efficiently rescues the protein instability and functional deficiency of cohesin induced by NudCL2 depletion, but not vice versa. Moreover, NudCL2 not only binds to Hsp90, but also significantly modulates Hsp90 ATPase activity and promotes the chaperone function of Hsp90. Taken together, these data suggest that NudCL2 is a previously undescribed Hsp90 cochaperone to modulate sister chromatid cohesion by stabilizing cohesin subunits, providing a hitherto unrecognized mechanism that is crucial for faithful chromosome segregation during mitosis.

cell biology↗

bioRxiv2018

Dissecting PCNA function with a systematically designed mutation library in yeast

Proliferating cell nuclear antigen (PCNA), encoded by POL30 in Saccharomyces cerevisiae, is a key component of DNA metabolism. Here a library consisting of 308 PCNA mutants was designed and synthesized to probe the contribution of each residue to its biological function. Five regions were identified with elevated sensitivity to DNA damaging reagents using high-throughput phenotype screening. Using a series of genetic and biochemical analyses, we demonstrated that one particular mutant, K168A, which displayed severe DNA damage sensitivity, abolished the DNA damage tolerance (DDT) pathway by disrupting interactions between PCNA and Rad5p. Subsequent domain analysis showed that the PCNA/Rad5p interaction is prerequisite for the function of Rad5p in DDT. Our study not only provides a resource in the form of a library of versatile mutants to study PCNA functions, but also reveals a key regulatory function of Rad5p, which highlights the importance of the PCNA-Rad5p interaction.\n\nAuthor summaryPCNA is regarded as the maestro of DNA replication fork because of the astonishing ability to interact with lots of partner proteins that participate in various DNA metabolism processes. However, it has remained elusive as to how does PCNA orchestrate these functions in harmony. Here, we constructed a systematic mutation library of PCNA, which covers every amino acid to map the functional sites of it. This carefully designed synthetic mutant pool could be generally useful and serve as a flexible resource, such as dissecting the functional mechanism of PCNA by genetic relationship analysis with key proteins through Synthetic genetic array. We further dissected the intrinsic mechanism for damage sensitivity of PCNAK168A, the most severe DNA damage sensitive mutant in our alanine scanning mutation library, this helps us to get better understanding of how PCNA participates in DNA damage tolerance (DDT) pathways. Our findings indicate that K168 site is vital for the interaction between DDT related partner proteins and PCNA, and also highlight the importance of the PCNA-Rad5p interaction.

genetics↗

bioRxiv2018

Genome-wide identification of functional tRNA-derived fragments in Senescence-accelerated mouse prone 8 brain

tRNA-derived fragments (tRFs) have been linked previously to the development of various diseases, such as cancer and viral infection. However, tRFs seem also related to brain aging and related diseases, especially Alzheimer and Parkinson disease. RNA sequencing, a state-of-the-art technology, has allowed for investigation of tRFs in this field. In this study, we investigated the changes of tRFs in the brains of a senescence-accelerated mouse model, senescence-accelerated mouse prone 8 (SAMP8), that show age-dependent deficits in learning and memory; and a control model, senescence-accelerated mouse resistant 1 (SAMR1), with normal aging, both at 7 months of age. A total of 570 tRF transcripts were discovered. Among these transcripts, 8, including 3 upregulated and 5 downregulated transcripts, were differentially expressed in the SAMP8 mice. Then, we obtained 110 potential target genes in a miRNA-like pattern. GO survey implicated these target genes in the function of various aspects, e.g. postsynaptic density (GO: 0014069). Furthermore, we assessed in detail those tRFs whose miRNA-like pattern was most likely to affect the progression of either Alzheimer and Parkinson disease, such as AS-tDR-011775 acting on Mobp and Park2. In fact, we found the tRFs to be involved in the regulation of gene expression by means other than the miRNA-like pattern. Therefore, these 8 dysregulated tRFs may hold consequences far into the future and can be attractive biomarkers and valid targets. In brief, our study is the first to provide a comprehensive analysis on tRFs in SAMP8 mouse brain, and this breakthrough identified promising new targets for preventing the age-related changes of brain and the therapeutic intervention of Alzheimers and Parkinsons.

neuroscience↗

bioRxiv2018

Single-cell Transcriptomic Landscape of Nucleated Cells in Umbilical Cord Blood

Umbilical cord blood (UCB) transplant is a therapeutic option for both pediatric and adult patients with a variety of hematologic diseases such as several types of blood cancers, myeloproliferative disorders, genetic diseases, and metabolic disorders. However, the level of cellular heterogeneity and diversity of nucleated cells in the UCB has not yet been assessed in an unbiased and systemic fashion. In the current study, nucleated cells from UCB were subjected to single-cell RNA sequencing, a technology enabled simultaneous profiling of the gene expression signatures of thousands of cells, generating rich resources for further functional studies. Here, we report the transcriptomic maps of 19,052 UCB cells, covering 11 major cell types. Many of these cell types are comprised of distinct subpopulations, including distinct signatures in NK and NKT cell types in the UCB. Pseudotime ordering of nucleated red blood cells (NRBC) identifies wave-like activation and suppression of transcription regulators, leading to a polarized cellular state, which may reflect the NRBC maturation. Progenitor cells in the UBC also consist two subpopulations with divergent transcription programs activated, leading to specific cell-fate commitment. Collectively, we provide this comprehensive single-cell transcriptomic landscape and show that it can uncover previously unrecognized cell types, pathways and gene expression regulations that may contribute to the efficacy and outcome of UCB transplant, broadening the scope of research and clinical innovations.

genomics↗

bioRxiv2018

Reliable Multiplex Sequencing with Rare Index Mis-Assignment on DNB-Based NGS Platform

BackgroundMassively-parallel-sequencing, coupled with sample multiplexing, has made genetic tests broadly affordable. However, intractable index mis-assignments (commonly exceeds 1%) were repeatedly reported on some widely used sequencing platforms.\n\nResultsHere, we investigated this quality issue on BGI sequencers using three library preparation methods: whole genome sequencing (WGS) with PCR, PCR-free WGS, and two-step targeted PCR. BGIs sequencers utilize a unique DNB technology which uses rolling circle replication for DNA-nanoball preparation; this linear amplification is PCR free and can avoid error accumulation. We demonstrated that single index mis-assignment from free indexed oligos occurs at a rate of one in 36 million reads, suggesting virtually no index hopping during DNB creation and arraying. Furthermore, the DNB-based NGS libraries have achieved an unprecedentedly low sample-to-sample mis-assignment rate of 0.0001% to 0.0004% under recommended procedures.\n\nConclusionsSingle indexing with DNB technology provides a simple but effective method for sensitive genetic assays with large sample numbers.

genomics↗

bioRxiv2018

Identification of genes affecting saturated fat acid content in Elaeis guineensis by genome-wide association analysis

Oil palm is the highest yielding oil crop per unit area worldwide. Unfortunately, palm oil is often considered unhealthy. In particular, palmic acid (C16:0) is a major component of palm oil. In this study a total of 1 261 501 SNP markers were produced in a diversity panel of 200 oil palm individuals. Oil content in this population varied from 29.8% to 70.3%, palmic acid varied from 31.3% to 48.8%, and oleic acid varied from 31.3% to 50.1%. We identified 274 SNP markers significantly associated with fatty acid compositions; 44 candidate genes in the flanking regions of these SNPs were involved in fatty acid biosynthesis and metabolism. Among them, two acyl-ACP thioesterase B genes had differential expression patterns between the mesocarp and kernel, tissues which show different oil profiles in oil palm (high palmic acid and high lauric acid respectively). Overexpression of both genes caused a significant increase in palmic acid content, while overexpression of the EgFatB2 gene also caused an accumulation of lauric acid and myristic acid. Our research provides genome-wide SNPs, a set of markers significantly associated with fatty acid content, and validated candidate genes for future targeted breeding of lower saturated fat content in palm oil.

plant biology↗

bioRxiv2018

Estimating heterogeneous treatment effects by balancing heterogeneity and fitness

Estimating heterogeneous treatment effects is an important problem in many medical and biological applications since treatments may have different effects on the prognoses of different patients. Recently, several recursive partitioning methods have been proposed to identify the subgroups that with different responds to a treatment, and they rely on a fitness criterion to minimize the error between the estimated treatment effects and the unobservable true effects. In this paper, we propose that a heterogeneity criterion, which maximizes the differences of treatment effects among the subgroups, also needs to be considered. Moreover, we show that better performances can be achieved when the fitness and the heterogeneous criteria are considered simultaneously. Selecting the optimal splitting points then becomes a multi-objective problem; however, a solution that achieves optimal in both aspects are often not available. To solve this problem, we propose a multi-objective splitting procedure to balance both criteria. The proposed procedure is computationally efficient and fits naturally into the existing recursive partitioning framework. Experimental results show that the proposed multi-objective approach performs consistently better than existing ones.\n\nAuthor summaryThe effects of a treatment are often not the same for different individuals with different gene expressions. Learning to predict the heterogeneous treatment effects from clinical and expression data is an important step towards personalized medical treatment. Existing computational methods are not ideal for the task because they do not address the interpretability of the model and do not consider the limited sample sizes in biological and medical applications. Our method addresses these issues and achieves superior performance in analyzing the treatment effects of radiotherapy on breast cancer patients.

bioinformatics↗

bioRxiv2018

Single tube bead-based DNA co-barcoding for cost effective and accurate sequencing, haplotyping, and assembly

Obtaining accurate sequences from long DNA molecules is very important for genome assembly and other applications. Here we describe single tube long fragment read (stLFR), a technology that enables this a low cost. It is based on adding the same barcode sequence to sub-fragments of the original long DNA molecule (DNA co-barcoding). To achieve this efficiently, stLFR uses the surface of microbeads to create millions of miniaturized barcoding reactions in a single tube. Using a combinatorial process up to 3.6 billion unique barcode sequences were generated on beads, enabling practically non-redundant co-barcoding with 50 million barcodes per sample. Using stLFR, we demonstrate efficient unique co-barcoding of over 8 million 20-300 kb genomic DNA fragments. Analysis of the genome of the human genome NA12878 with stLFR demonstrated high quality variant calling and phasing into contigs up to N50 34 Mb. We also demonstrate detection of complex structural variants and complete diploid de novo assembly of NA12878. These analyses were all performed using single stLFR libraries and their construction did not significantly add to the time or cost of whole genome sequencing (WGS) library preparation. stLFR represents an easily automatable solution that enables high quality sequencing, phasing, SV detection, scaffolding, cost-effective diploid de novo genome assembly, and other long DNA sequencing applications.

genomics↗

bioRxiv2018

White spot syndrome virus infection induces Caspase 1-mediated antiviral cell death in crustacean

In vertebrates, pyroptosis is an intensely inflammatory form of programmed cell death which is dependent on Caspase 1 activation and release of cytoplasmic cytokines including IL-1{beta}. This death pathway is critical for controlling pathogenic infection by mobilizing immune cells and stimulating the development of adaptive immune response. In invertebrates, however, due to the lack of adaptive immune response, it is still elusive whether Caspase 1-dependent cell death pathway exists. In this study, our data showed that Caspase 1-mediated cell death was activated by white spot syndrome virus (WSSV) infection to control the virus in shrimp. Caspase 1 had a higher expression level in hemocytes and lymphoid-like organ in shrimp and WSSV infection was significantly promoted upon the inhibition of Caspase 1 enzymatic activity. IL-1{beta}-like protein was identified as the substrate of Caspase 1 and its interaction with Caspase 1 was validated ectopically and endogenously. Moreover, IL-1{beta} like protein was released into extracellular contents under WSSV infection and Prophenoloxidase system was activated, resulting in the reduction of WSSV copies in vivo. Our data unraveled a previously unidentified mechanism through which Caspase 1-dependent cell death controlled virus infection in shrimp. Therefore, our study opened the possibility that an invertebrate cytokine network might be operative and regulate host defenses against virus infection as in vertebrates.\n\nAuthor SummaryThe elimination of infected cells by programmed cell death is considered as one of the most important anti-pathogen responses. Pyroptosis is one form of programmed cell death, which is mediated by Caspase 1 activation. Activated Caspase 1 can process the inactive precursors of interleukin 1{beta} (IL-1{beta}) into mature inflammatory cytokines to induce potent immune responses. Meanwhile, pyroptosis also induces morphologic changes, DNA fragmentation and chromatin condensation to destroy infected cells. However, due to the lack of efficient adaptive immune responses, it is still unclear whether Caspase 1-mediated cell death also exits and plays such an important role in defending hosts from pathogen invasion in invertebrates. In our data, Caspase 1 gene was characterized and Caspase 1-mediated cell death pathway was validated in shrimp. The Caspase 1-mediated cell death pathway was enhanced by white spot syndrome virus (WSSV) infection to control the virus in shrimp. IL-1{beta}-like protein was cleaved by Caspase 1 and released into extracellular contents under WSSV infection to activate Prophenoloxidase system, leading to the reduction of WSSV copies in shrimp in vivo. On the other hand, silencing of Caspase 1 or IL-1{beta}-like gene facilitated virus infection in shrimp. Our study indicated that Casapase-1-mediated cell death played an important antiviral role in shrimp against WSSV infection and opened the possibility that an invertebrate cytokine network might be operative.

immunology↗

bioRxiv2018

The landscape and diagnostic potential of T and B cell repertoire in Immunoglobulin A Nephropathy

Immunoglobulin A Nephropathy (IgAN) is the most common glomerulonephritis worldwide. In IgAN, immune complex deposite in glomerular mesangium, which induce inflammation and affect the kidneys normal functions. However, the exact pathogenesis of IgAN is still incompletely understood. Further, in current practice the clinical diagnosis relies on needle biopsy on renal tissue. Therefore, a non-invasive method for clinical diagnosis and prognosis surveillance of the disease is in high demand. In this paper, we investigated both the T cell receptor bata chain (TCRB) and immunoglobulin heavy chain (IGH) repertoire of kidney infiltrating and circulating lymphocytes of IgAN patients by immune repertoire high throughput sequencing. We found that the features of TCRB and IGH in the renal tissues were remarkably different from that in blood, including a decreased repertoire diversity and increased IgA and IgG frequency, and more activated B cells. The CDR3 length of PBMC TCRB and IGH in patients is significantly shorter than that in healthy controls, which is the result of both VDJ rearrangement and clone selection. We also found that the IgA1 frequency in the PBMC of IgAN is significant higher than that in other Nephropathy (NIgAN) and healthy control, which is consistent with the previous reports on the level of IgA1 producing B cells and serum IgA1. Significantly, we identified a set of IgAN disease related TCRB and IGH CDR3s, which can be used to distinguish IgAN from NIgAN and healthy controls from the blood with high accuracy. These results indicated that TCRB and IGH repertoire can potentially serve as non-invasive biomarkers for IgAN diagnosis. The characteristics of kidney infiltrating and circulating lymphocytes repertoire shed light on IgAN detection, treatment and surveillance.

immunology↗

bioRxiv2018

Identification of small regulatory RNAs involved in persister formation

Small regulatory RNA (srRNA) is widely distributed in three kingdoms of life and fulfills functions in many aspects of cellular life, but their role in bacterial persistence remains unknown. In this study, we comprehensively interrogated the expression levels of the known srRNAs on three critical time points, stage 1 (S1) where no persisters are formed, stage 2 (S2) where persisters are beginning to appear, and stage 3 (S3) where persister numbers increase significantly. Three upregulated srRNAs (OmrB, an outer member associated srRNA; RdlB, a swarming motility and curli expression regulator; McaS, a flagellar motility and biofilm formation regulator) overlapping in S2/S1 and S3/S1, together with the other four upregulated srRNAs (MicF, a ribosome binding inhibitor; MicL, an outer membrane associated srRNA; RybB, a cell envelope stress regulator; RydB, regulator of a global regulator RpoS) in S2/S1 are of special interest. By constructing deletion mutants and overexpression strains in uropathogenic E. coli strain UTI89, we tested their persister-formation capabilities in log phase and stationary phase cultures exposed to antibiotics (gentamicin, cefotaxime and levofloxacin) and stresses (heat, hyperosmosis, H2O2, and acid). The results of the deletion mutant studies showed that all the seven identified sRNAs have varying effects on persister formation with different antibiotics or stresses. Moreover, we found all the deletion mutants of these srRNAs have reduced biofilm formation. Additionally, except the McaS and the RydB overexpression strains, all of the srRNAs overexpression strains demonstrated increased persister-formation in antibiotic and stress persister assays, confirming the role of these srRNAs in persistence. Together, we identified seven srRNAs (OmrB, RdlB, McaS, MicF, MicL, RybB, and RydB) that are involved in type II persister formation for the first time. These findings provide convincing evidence for a new level of rapid persistence regulation via srRNA and furnish novel therapeutic targets for intervention.

microbiology↗

bioRxiv2018

Identification of novel genes including rpmF and yjjQ critical for Type II persister formation in Escherichia coli

Persister cells, which are characterized by inactive metabolism and tolerance to antibiotics or stresses, pose a significant challenge to the treatment of many persistent infections. Although multiple genes have been reported to be involved in persister formation through transposon mutant library screens, how persisters are formed during the natural process of persister formation as the culture transitions from log phase to stationary phase is unclear. Here, using E. coli as a model, we performed a comprehensive transcriptome analysis of gene expression profiles of successive cultures of an E. coli culture at different critical time points, starting from persister-free S1-nonexistence phase (3h) to persister appearing S2-emergence phase (4h), and persister abundant stage S3-abundance phase (5h). The differentially expressed genes ([≥]2-fold) in persister appearing stage (S1 to S2 transition) and persister abundant stage (S1 to S3) were compared, and 51 and 29 genes were identified to be up-regulated, respectively. Importantly, 13 genes (gnsA, gnsB, ybfA, yjjQ, ymdF, yhdU, csgD, yncN, rpmF, ydcX, yohJ, ssrA, rbsD) overlap in both persister S2-emergence phase and S3-abundance phase, including a member of the trans-translation pathway (ssrA) as well as an orphan toxin (ydcX), which are two well-known persister genes while the remaining 11 novel genes (gnsA, gnsB, ybfA, yjjQ, ymdF, yhdU, csgD, yncN, rpmF, yohJ, rbsD) have not been reported previously. Persister levels of 7 constructed knockout mutants ({Delta}gnsA, {Delta}ybfA, {Delta}yjjQ, {Delta}yhdU, {Delta}csgD, {Delta}yohJ and{Delta} rpmF) and 10 overexpression strains (gnsA, gnsB, ybfA, yjjQ, ymdF, yhdU, csgD, rpmF, yohJ, rbsD) in E. coli uropathogenic strain UTI89 were determined upon treatment with different cidal antibiotics (ampicillin, levofloxacin and gentamicin). Additionally, ranking of these overlapping genes according to their impact on persister levels were also performed. Two genes (rpmF encoding 50S ribosomal subunit protein L32, and yjjQ encoding a putative LuxR-type transcription factor) showed the most obvious phenotype on persister levels in both knockout and overexpression studies, which suggests they are broad and key factors for persister formation. While previous studies cannot distinguish if a given persister gene is involved in persister formation or persister survival, our findings clearly identify novel persister forming genes and pathways involving a ribosome protein and a LuxR type transcription factor during the bona fide persister formation process and may have implications for developing improved treatment of persistent infections.

microbiology↗

bioRxiv2018

Identification of Polynucleotide Phosphorylase (PNPase) in Escherichia coli Involved in Persister Formation

Despite the identification of many genes and pathways involved in the persistence phenomenon of bacteria, the mechanisms of persistence are not well understood. Here, using Escherichia coli as a model, we identified polynucleotide phosphorylase (PNPase) as a key regulator in persister formation. We successfully constructed pnp knockout mutant strain and its complemented strain, and exposed the pnp knockout mutant and complemented strain to antibiotics and stress conditions. The results showed that, compared with the wild-type W3110, the pnp knockout strain had defect in persistence to antibiotics and stress conditions, and the persistence to antibiotics and stresses was restored upon complementation. RNA-Seq was performed to identify the transcriptome profile in the pnp knockout strain compared with wild-type strain W3110, and the data revealed that 242 (166 up-regulated, and 76 down-regulated) genes were differentially expressed in the pnp knockout mutant strain. KEGG pathway analysis of the up-regulated genes showed that they were mostly mapped to metabolism and virulence pathways, most of which are positively regulated by the global regulator cyclic AMP receptor protein (CRP). Similarly, the transcription level of the crp gene in the pnp-deletion strain increased 3.22-fold in the early stationary phase. We further explored the indicators of cellular metabolism of the pnp-deletion strain, the persistence phenotype of the pnp and crp double-deletion mutant, and the transcriptional activity of crp gene. Our results indicate that PNPase controls cellular metabolism by negatively regulating the crp operon at the post-transcriptional level by targeting the 5- Untranslated Region (UTR) of the crp transcript. This study offers new insight about the persister mechanisms and provides new targets for development of new drugs against persisters for more effective treatment of persistent bacterial infections.

microbiology↗

bioRxiv2018

Cycloserine for Treatment of Multidrug-Resistant Tuberculosis in China: A Retrospective Observational Study

ObjectivesCycloserine is crucial in multidrug-resistant tuberculosis (MDR-TB) treatment. Although extensive research has been carried out on MDR-TB, most researchers have not treated cycloserine in much detail. Therefore, we evaluate the efficacy and safety of cycloserine and seek to clarify the role of cycloserine for treatment of simple MDR-TB, pre-extensively drug-resistant tuberculosis (pre-XDR-TB), and extensively drug-resistant tuberculosis (XDR-TB).\n\nPatients and methodsA retrospective observational study was performed in China. We determined the treatment outcome as the primary outcome for 144 cycloserine-treated and 181 cycloserine-nontreated patients according to the definitions of WHO. The proportion of patients with sputum-culture conversion and the frequency of adverse drug reactions related to cycloserine were assessed as well.\n\nResultsAmong 325 MDR-TB patients, 144 were treated with cycloserine and 100 (69.4%) out of 144 successfully completed treatment. Compared with patients in non-cycloserine group, the hazard ratio of any unfavorable treatment outcome was 0.53 (95%CI: 0.35-0.81, P=0.003). Culture conversion rate at the intensive phase was similar whether cycloserine was administered or not (P=0.703). Of the 144 patients treated with cycloserine, a total of 16 (11.1%) patients experienced side-effects related to cycloserine, including 7 patients who discontinued cycloserine permanently.\n\nConclusionsCycloserine could be an attractive agent to treat MDR-TB. Its safety profile warrants use in the most of MDR-TB cases. Cycloserine significantly improved the chance of favorable outcome for patients with simple MDR-TB but not pre-XDR-TB and XDR-TB. More aggressive regimens might be required for pre-XDR-TB or XDR-TB patients.

microbiology↗