Regulation of metal homoeostasis by two F-group bZIP transcription factors bZIP48 and bZIP50 in rice.

Zinc (Zn) deficiency not only impairs plant growth and development but also has negative effects on human health. Rice (Oryza Sativa L.) is a staple food for over half of the global population, yet the regulation of Zn deficiency response in rice remains largely unknown. In this study, we provide evidence that two F-group bZIP transcription factors, OsbZIP48/50, play a crucial role in Zn deficiency response. Mutations in OsbZIP48/50 result in impaired growth and reduced Zn/Fe/Cu content under Zn deficiency conditions. The N-terminus of OsbZIP48/OsbZIP50 contains two Zn sensor motifs (ZSMs), deletion or mutation of these ZSMs leads to increased nuclear localization. Both OsbZIP48 and OsbZIP50 exhibit transcriptional activation activity, and the upregulation of 1117 genes involved in metal uptake and other processes by Zn deficiency is diminished in the OsbZIP48/50 double mutant. Both OsbZIP48 and OsbZIP50 bind to the promoter of OsZIP10 and activate the ZDRE cis-element. Amino acid substitution mutation of the ZSM domain of OsbZIP48 in OsbZIP50 mutant background increases the content of Zn/Fe/Cu in brown rice seeds and leaves. Therefore, this study demonstrates that OsbZIP48/50 play a crucial role in regulating metal homoeostasis and identifies their downstream genes involved in the Zn deficiency response in rice.

Mn-Co-Ce/biochar based particles electrodes for removal of COD from coking wastewater by 3D/HEFL system: Characteristics, optimization, and mechanism.

In this work, the Mn, Co, Ce co-doped corn cob biochar (MCCBC) as catalytic particle electrodes in a three-dimensional heterogeneous electro-Fenton-like (3D-HEFL) system for the efficient degradation of coking wastewater was investigated. Various characterization methods such as SEM, EDS, XRD, XPS and electrochemical analysis were employed for the prepared materials. The results showed that the MCCBC particle electrodes had excellent electrochemical degradation performances of COD in coking wastewater, and the COD removal and degradation rates of the 3D/HEFL system were 85.35% and 0.0563 min-1 respectively. RSM optimized conditions revealed higher COD removal rate at 89.23% after 31.6 min of electrolysis. The efficient degradability and wide adaptability of the 3D/HEFL system were due to its beneficial coupling mechanism, including the synergistic effect between the system factors (3D and HEFL) as well as the synergistic interactions between the ROS (dominated by •OH and supplemented by O2•-) in the system. Moreover, the COD removal rate of MCCBC could still remain at 81.41% after 5 cycles with a lower ion leaching and a specific energy consumption of 11.28 kWh kg-1 COD. The superior performance of MCCBC, as catalytic particle electrodes showed a great potential for engineering applications for the advanced treatment of coking wastewater.

NFXL1 functions as a transcriptional activator required for thermotolerance at reproductive stage in Arabidopsis.

Plants are highly susceptible to abiotic stresses, particularly heat stress during the reproductive stage. However, the specific molecular mechanisms underlying this sensitivity remain largely unknown. In the current study, we demonstrate that the Nuclear Transcription Factor, X-box Binding Protein 1-Like 1 (NFXL1), directly regulates the expression of DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN 2A (DREB2A), which is crucial for reproductive thermotolerance in Arabidopsis. NFXL1 is upregulated by heat stress, and its mutation leads to a reduction in silique length (seed number) under heat stress conditions. RNA-Seq analysis reveals that NFXL1 has a global impact on the expression of heat stress responsive genes, including DREB2A, Heat Shock Factor A3 (HSFA3) and Heat Shock Protein 17.6 (HSP17.6) in flower buds. Interestingly, NFXL1 is enriched in the promoter region of DREB2A, but not of either HSFA3 or HSP17.6. Further experiments using electrophoretic mobility shift assay have confirmed that NFXL1 directly binds to the DNA fragment derived from the DREB2A promoter. Moreover, effector-reporter assays have shown that NFXL1 activates the DREB2A promoter. The DREB2A mutants are also heat stress sensitive at the reproductive stage, and DEREB2A is epistatic to NFXL1 in regulating thermotolerance in flower buds. It is known that HSFA3, a direct target of DREB2A, regulates the expression of heat shock proteins genes under heat stress conditions. Thus, our findings establish NFXL1 as a critical upstream regulator of DREB2A in the transcriptional cassette responsible for heat stress responses required for reproductive thermotolerance in Arabidopsis.

Calcium influx: An essential process by which α-Synuclein regulates morphology of erythrocytes.

INTRODUCTION: Morphological abnormalities of erythrocytes/red blood cells (RBCs), e.g., increased acanthocytes, in Parkinson's disease (PD) have been reported previously, although the underlying mechanisms remain to be characterized. In this study, the potential roles of α-synuclein (α-syn), a protein critically involved in PD and highly abundant in RBCs, were studied in PD patients as well as in a PD mouse model. METHODS: Transgenic [PAC-Tg (SNCAA53T), A53T] mice overexpressing A53T mutant α-syn and SNCA knockout mice were employed to characterize the effect of α-syn on RBC morphology. In addition to A53T and SNCA knockout mice, the morphology of RBCs of PD patients was also examined using scanning electron microscopy. The potential roles of α-syn were further investigated in cultured RBCs and mice. RESULTS: Morphological abnormalities of RBCs and increased accumulation of aggregated α-syn on the RBC membrane were observed in PD patients. A similar phenomenon was also observed in A53T mice. Furthermore, while mice lacking α-syn expression showed a lower proportion of acanthocytes, treating RBCs derived from SNCA knockout mice with aggregated α-syn resulted in a higher percentage of acanthocytes. In a follow-up proteomic investigation, several major classes of proteins were identified as α-syn-associated proteins on the RBC membrane, seven of which were calcium-binding proteins. Applying aggregated α-syn to the RBC membrane directly induced extracellular calcium influx along with morphological changes; both observations were adequately reversed by blocking calcium influx. CONCLUSIONS: This study demonstrated that α-syn plays a critical role in PD-associated morphological abnormalities of RBCs, at least partially via a process mediated by extracellular calcium influx.

JNK/c-Jun pathway activation is essential for HBx-induced IL-35 elevation to promote persistent HBV infection.

BACKGROUND: Immunoregulation plays pivotal roles during chronic hepatitis B virus (HBV) infection. Studies have shown that Interleukin (IL)-35 is an important molecule associated with inadequate immune response against HBV. However, the mechanisms involved in the up-regulation of IL-35 expression during persistent HBV infection remain unknown. METHODS: In this study, we constructed a plasmid expressing the HBV X protein (pCMV-HBx) to evaluate the relationship between HBx and IL-35. Activation of the JNK/c-Jun pathway was analyzed and chromatin immunoprecipitation followed by sequencing and luciferase reporter assays were performed to determine whether c-Jun could regulate IL-35 transcription. RESULTS: HBx can significantly activate IL-35 promoter in both LO2 and HepG2 cells compared to the control plasmid (pCMV-Tag2) using the dual-luciferase assay. Whereas other viral proteins, such as S, preS1, the core protein, had no significant effect on IL-35 expression. Similarly, WB and qRT-PCR also showed that HBx can significantly promote IL-35 expression at protein and mRNA levels in the aforementioned cells. The relevant pathway mechanism showed that the expression of JNK and c-Jun genes was significantly higher in transfected cells carrying pCMV-HBx than in the pCMV-Tag2-transfected and -untransfected cells. WB analysis revealed that phosphorylated JNK and c-Jun were overexpressed after HBx action. Conversely, the addition of the JNK/c-Jun signaling pathway inhibitor could significantly suppress HBx-induced IL-35 expression in a dose-dependent manner. CONCLUSIONS: A novel molecular mechanism of HBV-induced IL-35 expression was revealed, which involves JNK/c-Jun signaling in up-regulating IL-35 expression via HBx, resulting in transactivation of the IL-35 subunit EBI3 and p35 promoter.

Yeast transcriptional device libraries enable precise synthesis of value-added chemicals from methanol.

Natural methylotrophs are attractive methanol utilization hosts, but lack flexible expression tools. In this study, we developed yeast transcriptional device libraries for precise synthesis of value-added chemicals from methanol. We synthesized transcriptional devices by fusing bacterial DNA-binding proteins (DBPs) with yeast transactivation domains, and linking bacterial binding sequences (BSs) with the yeast core promoter. Three DBP-BS pairs showed good activity when working with transactivation domains and the core promoter of PAOX1 in the methylotrophic yeast, Pichia pastoris. Fine-tuning of the tandem BSs, spacers and differentiated input promoters further enabled a constitutive transcriptional device library (cTRDL) composed of 126 transcriptional devices with an expression strength of 16-520% and an inducible TRDL (iTRDL) composed of 162 methanol-inducible transcriptional devices with an expression strength of 30-500%, compared with PAOX1. Selected devices from iTRDL were adapted to the dihydromonacolin L biosynthetic pathway by orthogonal experimental design, reaching 5.5-fold the production from the PAOX1-driven pathway. The full factorial design of the selected devices from the cTRDL was adapted to the downstream pathway of dihydromonacolin L to monacolin J. Monacolin J production from methanol reached 3.0-fold the production from the PAOX1-driven pathway. Our engineered toolsets ensured multilevel pathway control of chemical synthesis in methylotrophic yeasts.

Determination of oligosaccharide product distributions of PL7 alginate lyases by their structural elements.

Alginate lyases can be used to produce well-defined alginate oligosaccharides (AOSs) because of their specificities for AOS products. A large number of alginate lyases have been recorded in the CAZy database; however, the majority are annotated-only alginate lyases that include little information on their products, thus limiting their applications. Here, we establish a simple and experiment-saving approach to predict product distributions for PL7 alginate lyases through extensive structural biology, bioinformatics and biochemical studies. Structural study on several PL7 alginate lyases reveals that two loops around the substrate binding cleft determine product distribution. Furthermore, a database containing the loop information of all annotated-only single-domain PL7 alginate lyases is constructed, enabling systematic exploration of the association between loop and product distribution. Based on these results, a simplified loop/product distribution relationship is proposed, giving us information on product distribution directly from the amino acid sequence.

A programmable high-expression yeast platform responsive to user-defined signals.

Rapidly growing yeasts with appropriate posttranslational modifications are favored hosts for protein production in the biopharmaceutical industry. However, limited production capacity and intricate transcription regulation restrict their application and adaptability. Here, we describe a programmable high-expression yeast platform, SynPic-X, which responds to defined signals and is broadly applicable. We demonstrated that a synthetic improved transcriptional signal amplification device (iTSAD) with a bacterial-yeast transactivator and bacterial-yeast promoter markedly increased expression capacity in Pichia pastoris. CRISPR activation and interference devices were designed to strictly regulate iTSAD in response to defined signals. Engineered switches were then constructed to exemplify the response of SynPic-X to exogenous signals. Expression of α-amylase by SynPic-R, a specific SynPic-X, in a bioreactor proved a methanol-free high-production process of recombinant protein. Our SynPic-X platform provides opportunities for protein production in customizable yeast hosts with high expression and regulatory flexibility.

Phosphoproteomic Analysis of Thermomorphogenic Responses in Arabidopsis.

Plants rapidly adapt to elevated ambient temperature by adjusting their growth and developmental programs. To date, a number of experiments have been carried out to understand how plants sense and respond to warm temperatures. However, how warm temperature signals are relayed from thermosensors to transcriptional regulators is largely unknown. To identify new early regulators of plant thermo-responsiveness, we performed phosphoproteomic analysis using TMT (Tandem Mass Tags) labeling and phosphopeptide enrichment with Arabidopsis etiolated seedlings treated with or without 3h of warm temperatures (29°C). In total, we identified 13,160 phosphopeptides in 5,125 proteins with 10,700 quantifiable phosphorylation sites. Among them, 200 sites (180 proteins) were upregulated, while 120 sites (87 proteins) were downregulated by elevated temperature. GO (Gene Ontology) analysis indicated that phosphorelay-related molecular function was enriched among the differentially phosphorylated proteins. We selected ATL6 (ARABIDOPSIS TOXICOS EN LEVADURA 6) from them and expressed its native and phosphorylation-site mutated (S343A S357A) forms in Arabidopsis and found that the mutated form of ATL6 was less stable than that of the native form both in vivo and in cell-free degradation assays. Taken together, our data revealed extensive protein phosphorylation during thermo-responsiveness, providing new candidate proteins/genes for studying plant thermomorphogenesis in the future.

Liver DCE-MRI registration based on sparse recovery of contrast agent curves.

PURPOSE: Dynamic contrast-enhanced MRI (DCE-MRI) registration is a challenging task because of the effect of remarkable intensity changes caused by contrast agent injections. Unrealistic deformation usually occurs by using traditional intensity-based algorithms. To alleviate the effect of contrast agent on registration, we proposed a DCE-MRI registration strategy and investigated the registration performance on the clinical DCE-MRI time series of liver. METHOD: We reconstructed the time-intensity curves of the contrast agent through sparse representation with a predefined dictionary whose columns were the time-intensity curves with high correlations with respect to a preselected contrast agent curve. After reshaping 1D-reconstructed contrast agent time-intensity curves into a 4D contrast agent time series, we aligned the original time series to the reconstructed contrast agent time series through traditional free-form deformation (FFD) registration scheme combined with a residual complexity (RC) similarity and an iterative registration strategy. This study included the DCE-MRI time series of 20 patients with liver cancer. RESULTS: Qualitatively, the time-cut images and subtraction images of different registration methods did not obviously differ. Quantitatively, the mean (standard deviation) of temporal intensity smoothness of all the patients achieved 54.910 (18.819), 54.609 (18.859), and 53.391 (19.031) in FFD RC, RDDR, Zhou et al.'s method and the proposed method, respectively. The mean (standard deviation) of changes in the lesion volume were 0.985 (0.041), 0.983 (0.041), 0.981 (0.046), and 0.989 (0.036) in FFD RC, RDDR, Zhou et al.'s method and the proposed method. CONCLUSION: Our proposed method would be an effective registration strategy for DCE-MRI time series, and its performance was comparable with that of three advanced registration methods.

Functional magnetic resonance imaging progressive deformable registration based on a cascaded convolutional neural network.

BACKGROUND: Intersubject registration of functional magnetic resonance imaging (fMRI) is necessary for group analysis. Accurate image registration can significantly improve the results of statistical analysis. Traditional methods are achieved by using high-resolution structural images or manually extracting functional information. However, structural alignment does not necessarily lead to functional alignment, and manually extracting functional features is complicated and time-consuming. Recent studies have shown that deep learning-based methods can be used for deformable image registration. METHODS: We proposed a deep learning framework with a three-cascaded multi-resolution network (MR-Net) to achieve deformable image registration. MR-Net separately extracts the features of moving and fixed images via a two-stream path, predicts a sub-deformation field, and is cascaded three times. The moving and fixed images' deformation field is composed of all sub-deformation fields predicted by the MR-Net. We imposed large smoothness constraints on all sub-deformation fields to ensure their smoothness. Our proposed architecture can complete the progressive registration process to ensure the topology of the deformation field. RESULTS: We implemented our method on the 1000 Functional Connectomes Project (FCP) and Eyes Open Eyes Closed fMRI datasets. Our method increased the peak t values in six brain functional networks to 19.8, 17.8, 15.0, 16.4, 17.0, and 13.2. Compared with traditional methods [i.e., FMRIB Software Library (FSL) and Statistical Parametric Mapping (SPM)] and deep learning networks [i.e., VoxelMorph (VM) and Volume Tweening Network (VTN)], our method improved 47.58%, 11.88%, 18.60%, and 15.16%, respectively. CONCLUSIONS: Our three-cascaded MR-Net can achieve statistically significant improvement in functional consistency across subjects.

Case Report: The First Report of NUP214-ABL1 Fusion Gene in Acute Myeloid Leukemia Patient Detected by Next-Generation Sequencing.

The NUP214-ABL1 fusion gene is a constitutively active tyrosine kinase that can be detected in 6% of T-cell acute lymphoblastic leukemia (T-ALL) patients, and it can also be found in B-cell acute lymphoblastic leukaemia (B-ALL). However the NUP214-ABL1 fusion in acute myeloid leukemia (AML) has not yet been reported. Up to now, the sensitivity of NUP214-ABL1-positive patients to tyrosine kinase inhibitor (TKI) is still controversial. Here we report the first case of an AML patient carrying NUP214-ABL1 fusion gene. The conventional AML chemotherapy regimen for the patient was successful. Identification of additional AML patients with NUP214-ABL1 fusion gene will provide treatment experience and prognostic evaluation.

Minimal residual disease level determined by flow cytometry provides reliable risk stratification in adults with T-cell acute lymphoblastic leukaemia.

Minimal residual disease (MRD) is an important independent prognostic factor for relapse and survival in acute lymphoblastic leukaemia (ALL). Compared with adult B-cell ALL, reports of adult T-cell ALL (T-ALL) MRD have been scarce and mostly based on molecular methods. We evaluated the prognostic value of multiparameter flow cytometry (FCM)-based MRD at the end of induction (EOI-MRD). The present retrospective study included 94 adult patients with T-ALL. MRD was detected by six- to eight-colour FCM. Patients who were EOI-MRD positive had a higher cumulative incidence of relapse (CIR) (87·6% vs. 38·8%, P = 0·0020), and a lower relapse-free survival (RFS) (5·4% vs. 61·0%, P = 0·0005) and overall survival (OS) (32·7% vs. 69·7%, P < 0·0001) than those who were EOI-MRD negative. Moreover, for patients who received allogeneic haematopoietic stem cell transplantation (allo-HSCT) at their first remission, EOI-MRD positivity was predictive of post-transplant relapse (2-year CIR: 68·2% vs. 4·0%, P = 0·0003). Multivariate analysis showed that EOI-MRD was an independent prognostic factor for CIR [hazard ratio (HR) 2·139, P = 0·046], RFS (HR 2·125, P = 0·048) and OS (HR 2·987, P = 0·017). In conclusion, EOI-MRD based on FCM was an independent prognostic factor for relapse and survival in adult T-ALL. For patients who underwent HSCT, EOI-MRD could be used to identify patients with a high risk of relapse after allo-HSCT.

Whole-brain functional MRI registration based on a semi-supervised deep learning model.

PURPOSE: Traditional registration of functional magnetic resonance images (fMRI) is typically achieved through registering their coregistered structural MRI. However, it cannot achieve accurate performance in that functional units which are not necessarily located relative to anatomical structures. In addition, registration methods based on functional information focus on gray matter (GM) information but ignore the importance of white matter (WM). To overcome the limitations of exiting techniques, in this paper, we aim to register resting-state fMRI (rs-fMRI) based directly on rs-fMRI data and make full use of GM and WM information to improve the registration performance. METHODS: We provide a robust representation of WM functional connectivity features using tissue-specific patch-based functional correlation tensors (ts-PFCTs) as auxiliary information to assist registration. Furthermore, we propose a semi-supervised deep learning model that uses GM and WM information (GM ts-PFCTs and WM ts-PFCTs) during training as a fine tweak to improve registration accuracy when such information is not provided in new test image pairs. We implement our method on the 1000 Functional Connectomes Project dataset. To evaluate our method, a group-level analysis was implemented in resting-state brain functional networks after registration, resulting in t maps. RESULTS: Our method increases the peak t values of the t maps of default mode network, visual network, central executive network, and sensorimotor network to 21.4, 20.0, 18.4, and 19.0, respectively. Through comparison with traditional methods (FMRIB Software Library(FSL), Statistical Parametric Mapping _ Echo Planar Image(SPM_EPI), and SPM_T1), our method achieves an average improvement of 67.39%, 12.96%, and 25.14%. CONCLUSION: We propose a semi-supervised deep learning network by adding GM and WM information as auxiliary information for resting-state fMRI registration. GM and WM information is extracted and described as GM ts-PFCTs and WM ts-PFCTs. Experimental results show that our method achieves superior registration performance.

Virulence, Antimicrobial Susceptibility, Molecular and Epidemiological Characteristics of a New Serotype of Vibrio parahaemolyticus From Diarrhea Patients.

Vibrio parahaemolyticus is the main pathogen of food-borne diarrheal in coastal areas. Through the study of pathogen characteristics of 1870 V. parahaemolyticus, the isolation rate of O4:KUT had increased significantly since 2013. In this study, we analyzed virulence, antimicrobial susceptibility, molecular, and epidemiological characteristics of a new serotype named O4:KUT2. O4:KUT2 strains had tlh + tdh + trh - toxRS/new- characteristics and were prevalent during 2013-2015. The 91.5% O4:KUT2 serotype strains were resistant to ampicillin. The growth curves of O4:KUT2 strains were different with O4:K9, O4:K8, and O3:K6 serotype strains. O4:KUT2 strains belonged to ST332 where four strains had a large fragment inserted at recA. Compared the whole genomes of O4:KUT2 strains with O4:K9 strain which also belonged to ST322 isolated from acute diarrhea patients in Zhejiang province in 2012, no different alleles at 2249 loci was found. This finding implied that O4:KUT2 strains might be derived from O4:K9 strains. Clinical presentation of patients positive for V. parahaemolyticus O4:KUT2 were no significant difference with patients positive for O3:K6, although their genetic characteristics were different. The appearance and the increase of proportion of the new serotype O4:KUT2 strains was aware that we should not relax the monitoring of the pathogen spectrum of acute diarrheal patients.

Pathogenic Characteristics of and Variation in Vibrio parahaemolyticus Isolated from Acute Diarrhoeal Patients in Southeastern China from 2013 to 2017.

OBJECTIVE: Vibrio parahaemolyticus is a major diarrhoea-inducing pathogen in coastal areas. In this study, we analysed the pathogenic characteristics of and variation in V. parahaemolyticus isolated from acute diarrhoeal patients in seven hospitals in different areas of southeastern China from 2013 to 2017. METHODS: The fecal specimens of patients with acute diarrhoea were collected. The routine microbiological test procedure combining with MALDI Biotyper microbial identification system was carried out to identify the V. parahaemolyticus. Serum agglutination tests, PCR for the detection of virulence-related genes and the Kirby-Bauer method to test for antimicrobial sensitivity were performed. RESULTS: From 2013 to 2017 in southeastern China, a total of 1220 V. parahaemolyticus strains were isolated from 16,504 stool specimens collected from acute diarrhoeal patients, and the annual isolation rate fluctuated between 6.1% and 8.7%. In total, 96.7% of the V. parahaemolyticus isolates were isolated in summer and autumn, mainly in people aged 18-44. Fifty-nine serotypes were identified, and the agglutination rate of the O antigen was 98.5%. From 2014 to 2016, the dominant serotype was O3:K6, while in 2013 and 2017, it was O4:KUT. The serotypes of O3:K6, O4:KUT, O4:K8, O3:KUT, O10:K60, O1:KUT and O1:K36 appeared every year from 2013 to 2017. O4:K6 and OUT:K6 began to appear after 2014 and 2015, respectively. A total of 49.5% of the strains belonged to the pandemic group, which consisted of 26 serotypes. Most isolates were sensitive to common antibiotics, excluding ampicillin. CONCLUSION: V. parahaemolyticus is still present at a high level in southeastern China. Although the pandemic O3:K6 serotype is predominant, new serotypes continue to emerge, especially the O4:KUT serotype, which exceeded O3:K6 in prevalence in some years. Long-term surveillance is necessary to prevent the outbreak or transmission of this pathogen.

CD4+ T cell exhaustion revealed by high PD-1 and LAG-3 expression and the loss of helper T cell function in chronic hepatitis B.

BACKGROUND: Immune inhibitory receptors play an important role in chronic infections. However, little is known about their role in hepatitis B virus (HBV) infection. Here, we analyzed the relationship between programmed death-1 (PD-1) and lymphocyte activation gene-3 (LAG-3) expression on CD4+ T cells and HBV disease progression. RESULTS: PD-1 and LAG-3 expression was significantly higher on CD4+ T cells from HBV patients than on those from the HCs. In addition, a significant positive correlation was found between the PD-1 and LAG-3 expression levels and the ALT(alanine aminotransferase) level. CD4+ T cell function was inhibited by high PD-1 and LAG-3 levels, and CD4+ T cells with high PD-1 and LAG-3 expression lost the ability to secrete IFN-γ, IL-2 and TNF-α. Furthermore, blockade of the PD-1 and LAG-3 pathways reversed the damage to CD4+ T cell proliferation and cytokine secretion. CONCLUSIONS: CD4+ T cell exhaustion during chronic HBV had high PD-1 and LAG-3 expression and the absence of helper T cell cytokines, including IFN-γ, IL-2 and TNF-α. After blocking PD-L1 and LAG-3, CD4+ T cell function in chronic hepatitis B patients was partially restored.

Serology, virulence and molecular characteristics of Vibrio parahaemolyticus isolated from seafood in Zhejiang province.

Vibrio parahaemolyticus is a leading foodborne pathogen in southeastern China. In this study, 105 strains of V. parahaemolyticus were isolated from fresh seafood in 2013 and 2014. The serotypes, virulence-associated genes and sequence types (STs) of these strains were analyzed. 26 defined serotypes were identified and 69 strains (65.7%) had untypeable O or K antigen. 8 strains (7.6%) had the virulence-associated gene tdh and no strain carried the trh gene. 45.7% (48/105) of isolates contained all four T3SS1 genes and 50% (4/8) tdh+ trh- V. parahaemolyticus isolates lacked at least one of the four tested T3SS2α genes. 105 strains could be categorized into 84 STs and only 3 STs (ST3, ST8, ST675) had appeared in clinical strains. V. parahaemolyticus strains from seafood have more diverse and untypeable serotypes, less virulence-associated genes and more STs than strains from clinical sources.

Genome-Wide Determination of Gene Essentiality by Transposon Insertion Sequencing in Yeast Pichia pastoris.

In many prokaryotes but limited eukaryotic species, the combination of transposon mutagenesis and high-throughput sequencing has greatly accelerated the identification of essential genes. Here we successfully applied this technique to the methylotrophic yeast Pichia pastoris and classified its conditionally essential/non-essential gene sets. Firstly, we showed that two DNA transposons, TcBuster and Sleeping beauty, had high transposition activities in P. pastoris. By merging their insertion libraries and performing Tn-seq, we identified a total of 202,858 unique insertions under glucose supported growth condition. We then developed a machine learning method to classify the 5,040 annotated genes into putatively essential, putatively non-essential, ambig1 and ambig2 groups, and validated the accuracy of this classification model. Besides, Tn-seq was also performed under methanol supported growth condition and methanol specific essential genes were identified. The comparison of conditionally essential genes between glucose and methanol supported growth conditions helped to reveal potential novel targets involved in methanol metabolism and signaling. Our findings suggest that transposon mutagenesis and Tn-seq could be applied in the methylotrophic yeast Pichia pastoris to classify conditionally essential/non-essential gene sets. Our work also shows that determining gene essentiality under different culture conditions could help to screen for novel functional components specifically involved in methanol metabolism.

PiggyBac transposon-mediated mutagenesis and application in yeast Komagataella phaffii.

OBJECTIVE: Around one-fourth of the Komagataella phaffii genes encode hypothetical proteins with unknown functions. However, lack of powerful tools for genetic screening in K. phaffii significantly limits the functional analysis of these unknown genes. Transposon mutagenesis has been utilized as an insertional mutagenesis tool in many other organisms and would be extremely valuable if it could be applied in K. phaffii. RESULTS: In this study, we investigated in K. phaffii the transposition activity and efficiency of piggyBac (PB) transposon, a DNA transposon from the cabbage looper moth Trichoplusia ni through the integrated-plasmid system. We also designed a binary-plasmid system which could generate stable mutants. Finally we evaluated the quality of this mutagenesis system by a simple screening for functional genes involved in K. phaffii carbon catabolite repression. CONCLUSIONS: Our results demonstrate that PB-mediated mutagenesis could be a feasible and useful tool for functional gene screening in K. phaffii.