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Insulin/IGF-1 Signaling (IIS) is known to constrain longevity by inhibiting the transcription factor FOXO. How phosphorylation mediated by IIS kinases regulates lifespan beyond FOXO remains unclear. Here, we profile IIS-dependent phosphorylation changes in a large-scale quantitative phosphoproteomic analysis of wild-type and three IIS mutant Caenorhabditis elegans strains. We quantify more than 15,000 phosphosites and find that 476 of these are differentially phosphorylated in the long-lived daf-2/insulin receptor mutant. We develop a machine learning-based method to prioritize 25 potential lifespan-related phosphosites. We perform validations to show that AKT-1 pT492 inhibits DAF-16/FOXO and compensates the loss of daf-2 function, that EIF-2α pS49 potently inhibits protein synthesis and daf-2 longevity, and that reduced phosphorylation of multiple germline proteins apparently transmits reduced DAF-2 signaling to the soma. In addition, an analysis of kinases with enriched substrates detects that casein kinase 2 (CK2) subunits negatively regulate lifespan. Our study reveals detailed functional insights into longevity., How phosphorylation mediated by Insulin/IGF-1 Signaling kinases regulates lifespan remains unclear. Here the authors perform a large-scale quantitative phosphoproteomic analysis of wildtype and IIS mutant C. elegans strains to reveal detailed functional insights into longevity.

Aim: To investigate the expression profiles of circRNAs after intracerebral hemorrhage (ICH). Materials & methods: RNA sequencing and qRT-PCR were used to investigate and validate circRNA expression levels. Bioinformatics analysis was performed to explore potential functions of the circRNAs. Results: Expression levels of 15 circRNAs were consistently altered in patients with ICH compared with their expression levels in hypertension. Three circRNAs, hsa_circ_0001240, hsa_circ_0001947 and hsa_circ_0001386, individually or combined, were confirmed as promising biomarkers for predicting and diagnosing ICH. The circRNAs were involved mainly in lysine degradation and the immune system. Conclusion: This is the first study to report expression profiles of circRNAs after ICH and to propose that three circRNAs are potential biomarkers for ICH.

Background: Circular RNAs (circRNAs) have shown promise as biomarkers because of their stability in peripheral blood, and they participate in various pathological processes of ischemic stroke; however, their expression profiles and the potential functions for intracerebral hemorrhage (ICH) stroke remain unclear. Methods: RNA sequencing was used to investigate the expression profiles of circRNA in a discovery sample of 129 subjects (44 patients with ICH, 42 hypertension (HTN) controls, and 43 patients with cerebral infarction (CI)), and an independent validation sample of 54 subjects (20 patients with ICH, 18 HTN controls, and 16 patients with CI). Quantitative real-time polymerase chain reaction was used to validate the circRNA expression levels. Logistic regression models were conducted to assess circRNAs as potential biomarkers for ICH. Bioinformatics analysis was performed to identify potential functions of circRNAs.Results: We found that 15 circRNAs including 5 upregulated circRNAs and 10 downregulated circRNAs were consistently altered only after ICH (fold change >1.5 and FDR < 0.05). We validated that hsa_circ_0001240 and hsa_circ_0001947 were upregulated and that hsa_circ_0001386 was downregulated in ICH compared with HTN and CI. The combination of 3 circRNAs showed an area under the curve of 0.92 with a sensitivity of 86% and a specificity of 88% for diagnosing ICH. Together with ICH risk factors, the circRNAs showed an area under the curve of 0.97 in diagnosing ICH. Functional analysis revealed circRNA target miRNA and mRNA are mainly involved in fatty acid biogenesis, lysine degradation, integrin cell surface interactions and the immune system.Conclusion: This is the first report to study the expression profile of circRNAs in peripheral blood after ICH, and a set of 3 circRNAs could serve as potential biomarkers for predicting and diagnosing ICH.

Insulin/IGF-1 Signaling (IIS) constrains longevity by inhibiting the transcription factor FOXO. Beyond FOXO, little is known about how phosphorylation—as mediated by IIS kinases— regulates lifespan. Here, we profiled IIS-dependent phosphorylation changes in a large-scale quantitative phosphoproteomic analysis of wild-type and three IIS mutant C. elegans strains. Our state-of-the-art analysis experimentally identified more than 15,000 phosphosites, among which 448 were differentially phosphorylated in the long-lived daf-2/insulin receptor mutant. We developed a machine-learning-based tool for systematically ranking the likely functional importance of phosphosites to guide candidate selection for follow-up validation. We show that AKT-1 pT492 inhibits DAF-16/FOXO and compensates the loss of daf-2 function, that EIF-2α pS49 potently regulates protein synthesis and daf-2 longevity, and that reduced phosphorylation of multiple germline proteins (e.g., CDK-1) apparently transmits a signal representing reduced DAF-2 signaling to the soma. Finally, kinase-substrate analysis and subsequent experimental validation confirm that casein kinase 2 negatively regulates lifespan. Our new benchmark data resource and machine-learning tool enables unprecedented access to detailed functional insights for studies of longevity.

The combination approach is now a well-established treatment for cancer. The present study evaluated the potential of curcumin and resveratrol on p53 post-translational modifications during gastric cancer. We segregated rats into five groups that included normal controls, dimethylhydrazine (DMH) treated, DMH + curcumin treated, DMH + resveratrol treated, and DMH + curcumin + resveratrol treated. Morphological analyses of tumor nodules confirmed carcinogenesis in rats after 25 weeks of DMH administration. The DMH treatment significantly induced carcinogenesis, as evidenced by high tumor burden in DMH-treated rats compared with controls. Moreover, DMH treatment caused a significant increase in the protein expressions of p53 as well as p53 phosphorylation in the DMH-treated rats. In addition, a significant rise was observed in 14C glucose uptake and 3H-thymidin uptakes in DMH-treated rats. Furthermore, enzyme activities of lactate dehydrogenase and alkaline phosphatase also showed a significant rise. On the contrary, significant decline was noticed in the p53 acetylation at residue 382 of DMH-treated rats. Conversely, combined treatment with curcumin and resveratrol to DMH-treated rats resulted in significant moderation in the tumor burden. In addition, a significant rise in p53 acetylation was at residue 382 of DMH-treated rats after treatment with phytochemicals. Supplementation with phytochemicals significantly modulated other biophysical and biochemical indices to near normal levels. Therefore, we conclude that curcumin and resveratrol significantly modulated p53 post-translational modifications during gastric cancer.

PtdIns3P signaling is critical for dynamic membrane remodeling during autophagosome formation. Proteins in the Atg18/WIPI family are PtdIns3P-binding effectors which can form complexes with proteins in the Atg2 family, and both families are essential for macroautophagy/autophagy. However, little is known about the biophysical properties and biological functions of the Atg2-Atg18/WIPI complex as a whole. Here, we demonstrate that an ortholog of yeast Atg18, mammalian WDR45/WIPI4 has a stronger binding capacity for mammalian ATG2A or ATG2B than the other 3 WIPIs. We purified the full-length Rattus norvegicus ATG2B and found that it could bind to liposomes independently of PtdIns3P or WDR45. We also purified the ATG2B-WDR45 complex and then performed 3-dimensional reconstruction of the complex by single-particle electron microscopy, which revealed a club-shaped heterodimer with an approximate length of 22 nm. Furthermore, we performed cross-linking mass spectrometry and identified a set of highly cross-linked intermolecular and intramolecular lysine pairs. Finally, based on the cross-linking data followed by bioinformatics and mutagenesis analysis, we determined the conserved aromatic H/YF motif in the C terminus of ATG2A and ATG2B that is crucial for complex formation.

Aims at the difficult to measure and influence serious in hydraulic impact fault, the intelligent diagnosis of hydraulic impact fault is proposed with improved EEMD and SVM in this paper. Three states of normal and sudden stop and suddenly reversing shock are set up on the hydraulic experimental bench. The improve EEMD is proposed by EEMD noise reduction, SVM extension signal, cubic spline interpolation improvement, and related pseudo components eliminating. The intelligent fault diagnosis in hydraulic system is researched by the improve EEMD to extract the IMF energy as feature vector, and the SVM training classification. Because distinguish and clear between normal state samples with the two impact fault samples, classification results are very right under a linear, polynomial kernel function, or a RBF, sigmoid and precomputed kernel function.

Four novel alkylaryl sulfate extended surfactants were synthesized by introducing a phenyl group, together with a polypropylene oxide (PPO) chain, as the intermediate polarity spacer in the extended surfactant molecules. Some characteristic properties of the novel extended surfactants were investigated in view of the effect of spacer structure on the performance of the surfactants. It is found that not only the PPO chain but also the phenyl group exerts a strong influence on the performance since these extended surfactants exhibit low cmc, great effectiveness and efficiency of surface tension reduction, high electrolyte resistance and ultralow interfacial tension comparing with the phenyl-free counterpart. It is notable that using a polarizable phenyl group as a junction unit in the intermediate polarity spacer may produce two potential effects: one is obviously resulting in a more gradual polarity transition intramolecularly than that of the PPO chain itself as a spacer; and the other is possibly forming a denser molecular packing and a thicker interfacial layer when the novel extended surfactants occupy at air/water (brine) surface or decane/water (brine) interface, which is attributed to a well-designed molecular structure in favor of forming a more gradual polarity transition zone in the interfacial layer.

The Support Vector Regression Machines (SVRM) is considered to be an effective method for time series prediction, while it is not suitable for long-term prediction. In order to improve the SVRM application in eliminating the end effect of EMD, we apply the SVRM to achieve an accurate short-term extension in the end of the original signal before EMD, and then extend two extreme with the Self-adaptive Triangular Waveform Matching extension method (STWM) in the every sifting process of EMD. The results show that the new extension method can effectively contain end effect of EMD.

Though the Support Vector Regression Machines (SVRM) is considered to be an effective method for time series prediction, its performance is greatly influenced by its parameters. In order to improve the rationality of parameter setting, the influences of the parameters (the number of support vectorsNSand the prediction lengthNE) and signal characteristics on the SVRM performance were discussed. The results proved that the existence of confliction between prediction accuracy and prediction efficiency, and SVRM may inappropriate to long-term prediction, andNSshould be greater than a threshold which depends on the signal characteristics for an accurate prediction result. The research results may provide a theoretical basis for the improvments of SVRM algorithm.

In order to diagnose bearing fault with less samples,combined improved EEMD with SVM in the bearing fault intelligent diagnosis under low dimensional small sample is researched in this paper.It is applied to the binary classification and identification in bearing normal and fault state.The results show that depend only on less sample data 5d feature vector classification after training, SVM using linear kernel function and polynomial kernel function classification accuracy is still up to 100. classification accuracy under the less sample data in less 5d characteristic vectors by RBF kernel function under Sigmoid kernel function is relatively low.Choose appropriate SVM kernel function completely can realize low dimensional small sample right binary classification.

In order to perform the bearing intelligent fault diagnosis,combined improved EEMD with SVM respectively applied to the binary classification identification of bearing normal and ball fault, normal and inner circle fault,normal and outer ring fault in this paper.Improve EEMD decomposed 9d normalized energy for characteristic vector,the SVM binary classification and recognition of bearings normal and ball fault, normal and inner circle fault, normal and outer ring fault is researched.Compared to the SVM classification accuracy using different kernel functions that is linear kernel function, polynomial kernel function, RBF kernel function and Sigmoid kernel function.In the same parameters,SVM classification accuracy based on linear kernel function and polynomial kernel function is a hundred percent.Bearing normal and ball fault,normal and inner circle fault,normal and outer ring fault is completely correct apart.And there are the classification errors based on RBF kernel function and Sigmoid kernel functions.

Intelligent diagnosis is the development direction of mechahnical condition monitoring and fault diagnosis.Conbined improved EEMD with SVM in fault intelligent diagnosis is researched in this paper.To bearing normal and fault as an example,impove EEMD decomposed 9D normalized energy for characteristic vector applied to the binary classification and identification.Compared to the SVM classification accuracy using different kernel functions that is linear,polynomial,RBF and Sigmoid kernel function.In the same parameters,SVM classification accuracy based on linear and polynomial kernel function is a hundred percent.Bearing normal and fault two kinds of state is completely correct apart. And the normal and fault state of the binary classification and identification using RBF and Sigmoid kernel function appeared wtong points.

In order to extract effectively detection signals in the noise background for non-stationary signal.On the basis of EEMD, improved EEMD is put forward, the improve EEMD threshold noise reduction is researched in this paper.The simulation signal compared the noise reduction effect of the wavelet,EMD,EEMD,and the improved EEMD. The improved EEMD threshold noise reduction have the best noise reduction result , the highest signal-to-noise ratio, the smallest standard deviation error.After the improved EEMD threshold noise reduction , the measurement signal time domain waveform smooth. More high frequency noise was obviously reduced in Hilbert time- frequency spectrum. Signal-to-noise ratio significantly improve, and signal characteristics are very clear.

The trend of the measured signal can not only reflect the influence of the external environment, and also reflect the performance of the machine itself mutations. Therefore, removing and extracting tendency item is the necessary link in signal pretreatment.In order to eliminate endpoint effect and modal aliasing phenomenon in EMD and EEMD, based on EEMD,improved EEMD is put forward and the improved EEMD in the application of the signal trend analysis is researched in this paper.In the measured signals to join in a ramp signals,With the improved EEMD decomposition extracted residual items,and the residual items with the original slope signal similarity analysis,the similarity is 0.975.compared to EMD extracted residual items similarity 0.898, EEMD extracted residual items similarity 0.961,the improved EEMD extracted residual item can more accurately reflect the trend of signal.

To improve chemical cross-linking of proteins coupled with mass spectrometry (CXMS), we developed a lysine-targeted enrichable cross-linker containing a biotin tag for affinity purification, a chemical cleavage site to separate cross-linked peptides away from biotin after enrichment, and a spacer arm that can be labeled with stable isotopes for quantitation. By locating the flexible proteins on the surface of 70S ribosome, we show that this trifunctional cross-linker is effective at attaining structural information not easily attainable by crystallography and electron microscopy. From a crude Rrp46 immunoprecipitate, it helped identify two direct binding partners of Rrp46 and 15 protein-protein interactions (PPIs) among the co-immunoprecipitated exosome subunits. Applying it to E. coli and C. elegans lysates, we identified 3130 and 893 inter-linked lysine pairs, representing 677 and 121 PPIs. Using a quantitative CXMS workflow we demonstrate that it can reveal changes in the reactivity of lysine residues due to protein-nucleic acid interaction. DOI: http://dx.doi.org/10.7554/eLife.12509.001, eLife digest Proteins fold into structures that are determined by the order of the amino acids that they are built from. These structures enable the protein to carry out its role, which often involves interacting with other proteins. Chemical cross-linking coupled with mass spectrometry (CXMS) is a powerful method used to study protein structure and how proteins interact, with a benefit of stabilizing and capturing brief interactions. CXMS uses a chemical compound called a linker that has two arms, each of which can bind specific amino acids in a protein or in multiple proteins. Only when the regions are close to each other can they be “cross-linked” in this way. After cross-linking, the proteins are cut into small pieces known as peptides. The cross-linked peptides are then separated from the non cross-linked ones and characterized. Although CXMS is a popular method, there are aspects about it that limit its use. It does not work well on complex samples that contain lots of different proteins, as it is difficult to separate the cross-linked peptides from the overwhelming amounts of non cross-linked peptides. Also, although it can be used to detect changes in the shape of a protein, which are often crucial to the protein's role, the method has not been smoothed out. Tan, Li et al. have now developed a new cross-linker called Leiker that addresses these limitations. Leiker cross-links the amino acid lysine to another lysine, and contains a molecular tag that allows cross-linked peptides to be efficiently purified away from non cross-linked peptides. As part of a streamlined workflow to detect changes in the shape of a protein, Leiker also contains a region that can be labeled. Analysing a bacterial ribosome, which contains more than 50 proteins, showed that Leiker-based CXMS could detect many more protein interactions than previous studies had. These included interactions that changed too rapidly to be studied by other structural methods. Tan, Li et al. then applied Leiker-based CXMS to the entire contents of bacterial cells at different stages of growth, and identified a protein interaction that is only found in growing cells. In future, Leiker will be useful for analyzing the structure of large protein complexes, probing changes in protein structure, and mapping the interactions between proteins in complex mixtures. DOI: http://dx.doi.org/10.7554/eLife.12509.002

In order to solve the endpoint effect and modal aliasing phenomenon in EMD and EEMD,Improved EEMD is put forward, and the application in signal singularity detection is researched in this paper. The improved EEMD will signal drops down into a series of different IMF to highlight the different local characteristics of original data, and then calculate Hilbert marginal spectrum and time-frequency spectrum to determine the frequency of these mutations and mutations position. To compared with FT, STFT, WVD,WT, EMD and EEMD etc, No cross-terms and no false IMF components are produced in the Hilbert time-frequency spectrum of the improved EEMD. Different frequency components and frequency mutations position are clearly distinguished at the same time. The Hilbert time-frequency spectrum of the improved EEMD has more superior detection signal singularity ability.

In order to correct the endpoint effect and modal aliasing phenomenon in EMD and EEMD, improved EEMD is put forward in this paper. on the basis of the cause of the endpoint effect,the improvement measures are proposed. The pulse interference and noise pollution is suppressed by threshold noise reduction.The overshoot and undershoot phenomenon is controlled by improving 3-spline interpolation to fit envelope. The endpoint effects is lessened by signal SVM prolongation.Compared with EMD and EEMD, not only the IMF false component is reduced, the modal aliasing avoided, and effectively the endpoint effect restrained, the distortion problem in the signal decomposition produces corrected in the results of simulation signal and the measured signal by the improved EEMD in this paper.

Avian pathogenic Escherichia coli (APEC) is an important respiratory pathogen of poultry. A variety of virulence-associated genes and serogroups are associated with avian colibacillosis caused by APEC strains. One hundred forty-eight E. coli isolates recovered from diagnosed cases of avian colibacillosis from Guangdong province between 2005 and 2008 were serotyped, and characterized for virulence-associated genes, phylogenetic backgrounds, antibiotic susceptibility, and genetic relatedness. Associations between virulence-associated genes and antimicrobial resistance were further analyzed. Although 148 APEC isolates belonged to 21 different serogroups, 81% were of one of eight serogroups: O65 (27%), O78 (10%), O8 (9%), O120 (9%), O2 (7%), O92 (6%), O108 (5%), and O26 (5%). Polymerase chain reaction analysis showed that the most prevalent gene was traT (90%), followed by iroN (63%), fimH (58%), hlyF (55%), cvaC (54%), and sitA (51%). The APEC strains mainly belonged to groups A (73%) and D (14%). Multiple antimicrobial-resistant phenotypes (greater than or equal to three antimicrobials) were detected in all E. coli isolates, with the majority of isolates displaying resistance to tetracycline (97%), sulfamethoxazole (93%) and fluoroquinolones (87% for ciprofloxacin and 84% for enrofloxacin), chloramphenicol (74%), and florfenicol (66%). All E. coli isolates were further genetically characterized by pulsed-field gel electrophoresis. A total of 125 different pulsed-field gel electrophoresis profiles were obtained, implying that the multiresistant E. coli isolates carrying virulence-associated genes and belonging to multiple serogroups were not derived from a specific clone, but represented a wide variety of chromosomal backgrounds. Statistical analysis showed that several virulence-associated genes were significantly present in APEC isolates susceptibility to multiple antimicrobials. The findings demonstrate that a wide variety of serogroups and potential virulence genes, multiple-resistances, and the clear association of susceptibility and virulence genes have commonly emerged in APEC strains, and these also suggest that antimicrobials should be prudently used to reduce the emergence and spread of resistant strains carrying virulence-associated genes.

We developed a high-throughput mass spectrometry method, pLink-SS (http://pfind.ict.ac.cn/software/pLink/2014/pLink-SS.html), for precise identification of disulfide-linked peptides. Using pLink-SS, we mapped all native disulfide bonds of a monoclonal antibody and ten standard proteins. We performed disulfide proteome analyses and identified 199 disulfide bonds in Escherichia coli and 568 in proteins secreted by human endothelial cells. We discovered many regulatory disulfide bonds involving catalytic or metal-binding cysteine residues.

Spontaneous DNA damage may occur nonrandomly in the genome, especially when genome maintenance mechanisms are undermined. We developed single-strand DNA (ssDNA)–associated protein immunoprecipitation followed by sequencing (SPI-seq) to map genomic hotspots of DNA damage. We demonstrated this method with Rad52, a homologous recombination repair protein, which binds to ssDNA formed at DNA lesions. SPI-seq faithfully detected, in fission yeast, Rad52 enrichment at artificially induced double-strand breaks (DSBs) as well as endogenously programmed DSBs for mating-type switching. Applying Rad52 SPI-seq to fission yeast mutants defective in DNA helicase Pfh1 or histone H3K56 deacetylase Hst4, led to global views of DNA lesion hotspots emerging in these mutants. We also found serendipitously that histone dosage aberration can activate retrotransposon Tf2 and cause the accumulation of a Tf2 cDNA species bound by Rad52. SPI-seq should be widely applicable for mapping sites of DNA damage and uncovering the causes of genome instability.

Sleep is a ubiquitous, tightly regulated, and evolutionarily conserved behavior observed in almost all animals. Prolonged sleep deprivation can be fatal, indicating that sleep is a physiological necessity. However, little is known about its core function. To gain insight into this mystery, we used advanced quantitative proteomics technology to survey the global changes in brain protein abundance. Aiming to gain a comprehensive profile, our proteomics workflow included filter-aided sample preparation (FASP), which increased the coverage of membrane proteins; tandem mass tag (TMT) labeling, for relative quantitation; and high resolution, high mass accuracy, high throughput mass spectrometry (MS). In total, we obtained the relative abundance ratios of 9888 proteins encoded by 6070 genes. Interestingly, we observed significant enrichment for mitochondrial proteins among the differentially expressed proteins. This finding suggests that sleep deprivation strongly affects signaling pathways that govern either energy metabolism or responses to mitochondrial stress. Additionally, the differentially-expressed proteins are enriched in pathways implicated in age-dependent neurodegenerative diseases, including Parkinson's, Huntington's, and Alzheimer's, hinting at possible connections between sleep loss, mitochondrial stress, and neurodegeneration.

Spermiogenesis is a series of poorly understood morphological, physiological and biochemical processes that occur during the transition of immotile spermatids into motile, fertilization-competent spermatozoa. Here, we identified a Serpin (serine protease inhibitor) family protein (As_SRP-1) that is secreted from spermatids during nematode Ascaris suum spermiogenesis (also called sperm activation) and we showed that As_SRP-1 has two major functions. First, As_SRP-1 functions in cis to support major sperm protein (MSP)-based cytoskeletal assembly in the spermatid that releases it, thereby facilitating sperm motility acquisition. Second, As_SRP-1 released from an activated sperm inhibits, in trans , the activation of surrounding spermatids by inhibiting vas deferens-derived As_TRY-5, a trypsin-like serine protease necessary for sperm activation. Because vesicular exocytosis is necessary to create fertilization-competent sperm in many animal species, components released during this process might be more important modulators of the physiology and behavior of surrounding sperm than was previously appreciated.

Non-homologous end joining (NHEJ) is an important mechanism for repairing DNA double-strand breaks (DSBs). The fission yeast Schizosaccharomyces pombe has a conserved set of NHEJ factors including Ku, DNA ligase IV, Xlf1, and Pol4. Their roles in chromosomal DSB repair have not been directly characterized before. Here we used HO endonuclease to create a specific chromosomal DSB in fission yeast and examined the imprecise end joining events allowing cells to survive the continuous expression of HO. Our analysis showed that cell survival was significantly reduced in mutants defective for Ku, ligase IV, or Xlf1. Using Sanger sequencing and Illumina sequencing, we have characterized in depth the repair junction sequences in HO survivors. In wild type cells the majority of repair events were one-nucleotide insertions dependent on Ku, ligase IV, and Pol4. Our data suggest that fission yeast Pol4 is important for gap filling during NHEJ repair and can extend primers in the absence of terminal base pairing with the templates. In Ku and ligase IV mutants, the survivors mainly resulted from two types of alternative end joining events: one used microhomology flanking the HO site to delete sequences of hundreds to thousands of base pairs, the other rejoined the break using the HO-generated overhangs but also introduced one- or two-nucleotide base substitutions. The chromosomal repair assay we describe here should provide a useful tool for further exploration of the end joining repair mechanisms in fission yeast.

One hundred two pathogenic Escherichia coli isolates from diseased pigs were analyzed for serotypes, virulence genes, antimicrobial susceptibility, and the molecular basis of phenicol resistance. Of these 102 E. coli isolates, 101 were typeable and belonged to 27 different O serogroups. However, 69% of these isolates belonged to one of the following eight serogroups: O8, O54, O64, O65, O92, O108, O119, and O120. Serogroups O8 (23%) and O64 (10%) were the most prevalent among typeable isolates. High-resistance phenotypes were observed in all the isolates, with the majority displaying resistance to chloramphenicol (89%), streptomycin (83%), enrofloxacin (78%), and doxycycline (60%). The chloramphenicol resistance genes cat1, cat2, and cmlA were detected in 58%, 49%, and 65%, respectively, of the chloramphenicol-resistant isolates. The floR gene was detected in 57% of the florfenicol-resistant isolates and in 52% of chloramphenicol-resistant isolates. Pulsed-field gel electrophoresis showed that the 32 floR-positive isolates with florfenicol minimum inhibitory concentration ≥ 8 μg mL⁻¹ belonged to 25 different pulsed-field gel electrophoresis profiles, indicating the spread of floR among swine pathogenic E. coli isolates.

A genome-wide deletion library is a powerful tool for probing gene functions and one has recently become available for the fission yeast Schizosaccharomyces pombe. Here we use deep sequencing to accurately characterize the barcode sequences in the deletion library, thus enabling the quantitative measurement of the fitness of fission yeast deletion strains by barcode sequencing.

During DNA double-strand break repair, two structure-specific DNA nucleases are controlled by the same regulator Pxd1, but in opposite manners., Structure-specific nucleases play crucial roles in many DNA repair pathways. They must be precisely controlled to ensure optimal repair outcomes; however, mechanisms of their regulation are not fully understood. Here, we report a fission yeast protein, Pxd1, that binds to and regulates two structure-specific nucleases: Rad16XPF-Swi10ERCC1 and Dna2-Cdc24. Strikingly, Pxd1 influences the activities of these two nucleases in opposite ways: It activates the 3′ endonuclease activity of Rad16-Swi10 but inhibits the RPA-mediated activation of the 5′ endonuclease activity of Dna2. Pxd1 is required for Rad16-Swi10 to function in single-strand annealing, mating-type switching, and the removal of Top1-DNA adducts. Meanwhile, Pxd1 attenuates DNA end resection mediated by the Rqh1-Dna2 pathway. Disabling the Dna2-inhibitory activity of Pxd1 results in enhanced use of a break-distal repeat sequence in single-strand annealing and a greater loss of genetic information. We propose that Pxd1 promotes proper DNA repair by differentially regulating two structure-specific nucleases., Author Summary Genome stability maintenance relies on DNA repair enzymes, among which are structure-specific nucleases that cleave DNA in a sequence-independent but structure-dependent manner. It is important to understand how the activities of such nucleases are controlled, because either insufficient or excessive cleavage of DNA could jeopardize genome integrity. In this study, we discovered a new regulator of two different structure-specific nucleases in the fission yeast Schizosaccharomyces pombe. The identified protein, which we named Pxd1, promotes the activity of the 3′ endonuclease Rad16, but restrains the activity of the 5′ endonuclease Dna2. In the absence of Pxd1, several Rad16-dependent DNA repair processes become defective. One of these processes is a DNA-repeat–mediated double-strand break repair pathway called single-strand annealing, which causes genomic deletions. When the Dna2-inhibitory activity of Pxd1 is impaired, Dna2-dependent end processing of double-strand breaks is enhanced and a more extensive deletion occurs during single-strand annealing. Thus, Pxd1 facilitates a potentially dangerous DNA repair process, but in the meantime minimizes its deleterious consequences. We propose that a dual-target regulator like Pxd1 is ideally suited for coordinating multiple enzymatic activities during DNA repair.

The TTAA-specific transposon piggyBac (PB), originally isolated from the cabbage looper moth, Trichoplusia ni, has been utilized as an insertional mutagenesis tool in various eukaryotic organisms. Here, we show that PB transposes in the fission yeast Schizosaccharomyces pombe and leaves almost no footprints. We developed a PB-based mutagenesis system for S. pombe by constructing a strain with a selectable transposon excision marker and an integrated transposase gene. PB transposition in this strain has low chromosomal distribution bias as shown by deep sequencing-based insertion site mapping. Using this system, we obtained loss-of-function alleles of klp5 and klp6, and a gain-of-function allele of dam1 from a screen for mutants resistant to the microtubule-destabilizing drug thiabendazole. From another screen for cdc25-22 suppressors, we obtained multiple alleles of wee1 as expected. The success of these two screens demonstrated the usefulness of this PB-mediated mutagenesis tool for fission yeast.