A Ligation-PCR Approach for Generating Gene Replacement Constructs in Magnaporthe grisea

The conventional approach for generating gene replacement constructs involves several sequence-specific cloning steps and is time-consuming. A ligation-PCR approach was developed to efficiently generate gene replacement constructs. Two vectors useful for this ligation-PCR approach and another vector suitable for improving the efficiency of knockout mutant screens were constructed

Early Clinical and Subclinical Visual Evoked Potential and Humphrey's Visual Field Defects in Cryptococcal Meningitis.

Cryptococcal induced visual loss is a devastating complication in survivors of cryptococcal meningitis (CM). Early detection is paramount in prevention and treatment. Subclinical optic nerve dysfunction in CM has not hitherto been investigated by electrophysiological means. We undertook a prospective study on 90 HIV sero-positive patients with culture confirmed CM. Seventy-four patients underwent visual evoked potential (VEP) testing and 47 patients underwent Humphrey's visual field (HVF) testing. Decreased best corrected visual acuity (BCVA) was detected in 46.5% of patients. VEP was abnormal in 51/74 (68.9%) right eyes and 50/74 (67.6%) left eyes. VEP P100 latency was the main abnormality with mean latency values of 118.9 (±16.5) ms and 119.8 (±15.7) ms for the right and left eyes respectively, mildly prolonged when compared to our laboratory references of 104 (±10) ms (p<0.001). Subclinical VEP abnormality was detected in 56.5% of normal eyes and constituted mostly latency abnormality. VEP amplitude was also significantly reduced in this cohort but minimally so in the visually unimpaired. HVF was abnormal in 36/47 (76.6%) right eyes and 32/45 (71.1%) left eyes. The predominant field defect was peripheral constriction with an enlarged blind spot suggesting the greater impact by raised intracranial pressure over that of optic neuritis. Whether this was due to papilloedema or a compartment syndrome is open to further investigation. Subclinical HVF abnormalities were minimal and therefore a poor screening test for early optic nerve dysfunction. However, early optic nerve dysfunction can be detected by testing of VEP P100 latency, which may precede the onset of visual loss in CM

Nutrient and Stress Sensing in Pathogenic Yeasts

More than 1.5 million fungal species are estimated to live in vastly different environmental niches. Despite each unique host environment, fungal cells sense certain fundamentally conserved elements, such as nutrients, pheromones and stress, for adaptation to their niches. Sensing these extracellular signals is critical for pathogens to adapt to the hostile host environment and cause disease. Hence, dissecting the complex extracellular signal-sensing mechanisms that aid in this is pivotal and may facilitate the development of new therapeutic approaches to control fungal infections. In this review, we summarize the current knowledge on how two important pathogenic yeasts, Candida albicans and Cryptococcus neoformans, sense nutrient availability, such as carbon sources, amino acids, and ammonium, and different stress signals to regulate their morphogenesis and pathogenicity in comparison with the non-pathogenic model yeast Saccharomyces cerevisiae. The molecular interactions between extracellular signals and their respective sensory systems are described in detail. The potential implication of analyzing nutrient and stress-sensing systems in antifungal drug development is also discussed

Investigation of Haemophilus parasuis from healthy pigs in China

Haemophilus parasuis is a common colonizer of the upper respiratory tract of swine and frequently causes disease, especially in weaner pigs. To date, limited epidemiological data was available for H. parasuis from healthy pigs, which might be carriers of potential pathogenic strains. In this study, from September 2016 to October 2017, we investigated the prevalence and characteristics of H. parasuis from healthy pigs in China. Totally, we obtained 244 isolates from 1675 nasal samples from 6 provinces. H. parasuis isolation was more successful in weaner pigs (22.6%, 192/849), followed by finisher pigs (9.3%, 43/463), and sows (2.5%, 9/363). The most prevalent serovars were 7 (20.1%, 49/244), followed by 3 (14.8%, 36/244), 2 (14.3%, 35/244), 11 (12.7%, 31/244), 5/12 (5.7%, 14/244) and 4 (2.5%, 6/244). Bimodal or multimodal distributions of MICs were observed for most of the tested drugs, which suggested the presence of non-wild type populations. It was noted that the MIC90 values of tilmicosin (64 μg/ml) was relatively higher than that reported in previous studies. Our results suggest that: 1) potentially pathogenic serovars of H. parasuis are identified in healthy pigs, and 2) elevated MICs and presence of mechanisms of resistance not yet described for clinically important antimicrobial agents would increase the burden of disease caused by H. parasuis.info:eu-repo/semantics/acceptedVersio

Genome-wide identification of the GT7 and GH16 genes and their correlations with agar metabolism in the agarophyte Gracilariopsis lemaneiformis

The economically important seaweed Gracilariopsis lemaneiformis (Gp. lemaneiformis) is the main resource for agar production and abalone bait. Few studies have focused on the functions of glycosyltransferases (GTs) and glycoside hydrolases (GHs) involved in saccharometabolism, including that of agar. To investigate the possible functions of glycosyltransferase family 7 (GT7) and glycoside hydrolase family 16 (GH16) members in agar metabolism, analyses of these gene families were conducted using bioinformatics-based and physiological methods. In total, five GlGT7 genes and four GlGH16 genes from the Gp. lemaneiformis genome were identified and analyzed. These GT7 and GH16 members are absent in higher plants, and the majority of GlGT7s and GlGH16s have no introns. Catalytic pocket residue analysis revealed that GlGT7s may function as β-1,4-galactosyltransferases and that GlGH16s play roles as agarases. Promoter prediction and qRT-PCR experiments verified that these genes can be regulated by light and phytohormones. GlGT7s were predicted to interact with carbohydrate sulfotransferases (STs), whereas yeast two-hybrid (Y2H) assays revealed no interactions between these proteins. Under heat stress, no significant difference was found in agar content; however, the expression of GlGT7s fluctuated and that of GlGH16 increased. Low nitrogen stress significantly increased the agar content, and the expression of GlGT7s increased, whereas that of GlGH16s decreased. In three cultivars of Gp. lemaneiformis, the expression of GlGT7 genes in cultivars 981 and Lulong No. 1 was upregulated relative to that in the wild type, whereas GlGH16 expression levels were significantly decreased. Fluorescence microscopy further showed that β-1,4-galactose accumulation was consistent with increases in agar content and GlGT7 expression. Pearson correlation analysis confirmed that the expression levels of GlGT7s and GlGH16s were positively and negatively correlated with agar accumulation, respectively. Taken together, these results demonstrated that GlGT7 and GlGH16 are intimately correlated with agar metabolism; in particular, GlGT7-2, GlGT7-5, and GlGH16-4 could act as molecular markers to indicate agar yield. This study will provide a valuable basis for breeding new cultivars with high agar content

Identification of metabolic pathways influenced by the G-protein coupled receptors GprB and GprD in Aspergillus nidulans

Heterotrimeric G-protein-mediated signaling pathways play a pivotal role in transmembrane signaling in eukaryotes. Our main aim was to identify signaling pathways regulated by A. nidulans GprB and GprD G-protein coupled receptors (GPCRs). When these two null mutant strains were compared to the wild-type strain, the DeltagprB mutant showed an increased protein kinase A (PKA) activity while growing in glucose 1% and during starvation. In contrast, the DeltagprD has a much lower PKA activity upon starvation. Transcriptomics and (1)H NMR-based metabolomics were performed on two single null mutants grown on glucose. We noted modulation in the expression of 11 secondary metabolism gene clusters when the DeltagprB and DeltagprD mutant strains were grown in 1% glucose. Several members of the sterigmatocystin-aflatoxin gene cluster presented down-regulation in both mutant strains. The genes of the NR-PKS monodictyphenone biosynthesis cluster had overall increased mRNA accumulation in DeltagprB, while in the DeltagprD mutant strain the genes had decreased mRNA accumulation. Principal component analysis of the metabolomic data demonstrated that there was a significant metabolite shift in the DeltagprD strain. The (1)H NMR analysis revealed significant expression of essential amino acids with elevated levels in the DeltagprD strain, compared to the wild-type and DeltagprB strains. With the results, we demonstrated the differential expression of a variety of genes related mainly to secondary metabolism, sexual development, stress signaling, and amino acid metabolism. We propose that the absence of GPCRs triggered stress responses at the genetic level. The data suggested an intimate relationship among different G-protein coupled receptors, fine-tune regulation of secondary and amino acid metabolisms, and fungal development

Stealthy data poisoning attack method on offline reinforcement learning in unmanned systems

Aiming at the limitations in effectiveness and stealth of existing offline reinforcement learning(RL) data poisoning attacks, a critical time-step dynamic poisoning attack was proposed, perturbing important samples to achieve efficient and covert attacks. Temporal difference errors, identified through theoretical analysis as crucial for model learning, were used to guide poisoning target selection. A bi-objective optimization approach was introduced to minimize perturbation magnitude while maximizing the negative impact on performance. Experimental results show that with only a 1% poisoning rate, the method reduces agent performance by 84%, revealing the sensitivity and vulnerability of offline RL models in unmanned systems

Genetic variation in the tissue factor gene is associated with clinical outcome in severe sepsis patients

INTRODUCTION: Activation of inflammation and coagulation was closely related and mutually interdependent in sepsis. Tissue factor (TF) and its endogenous inhibitor, tissue factor pathway inhibitor (TFPI) was the main regulators of the initiation of coagulation process. Altered plasma levels of TF and TFPI have been related to worse outcome in sepsis. The objective of this study was to investigate whether single nucleotide polymorphisms (SNPs) in the TF and TFPI genes were associated with risk and outcome for patients with severe sepsis. METHODS: Seventeen SNPs in TF and TFPI were genotyped in samples of sepsis (n =577) and severe sepsis patients (n =476), and tested for association in this case–control collection. We then investigated correlation between the associated SNPs and the mRNA expression, and protein level of the corresponding gene. The mRNA levels of TF were determined using real-time quantitative reverse transcription-polymerase chain reaction and the soluble plasma levels of TF were measured using enzyme linked immunosorbent assay (ELISA) method. RESULTS: Association analysis revealed that three TF SNPs in perfect linkage disequilibrium, rs1361600, rs3917615 and rs958587, were significantly associated with outcome of severe sepsis. G allele frequency of rs1361600 in survivor patients was significantly higher than that in nonsurvivor severe sepsis patients (P =4.91 × 10(-5), odds ratio (OR) =0.48, 95% confidence interval (CI) 0.33 to 0.69). The association remained significant after adjustment for covariates in multiple logistic regression analysis and for multiple comparisons. Lipopolysaccharide-induced TF-mRNA expression levels in peripheral blood mononuclear cells from subjects carrying rs1361600 AG and GG genotypes, were significantly lower than those subjects carrying AA genotype (P =0.0012). Moreover, severe sepsis patients of GG and GA genotypes showed lower serum levels of TF than patients with AA genotype (P(adj) =0.02). The plasma levels of TF were also associated with outcome of severe sepsis patients (P(adj) =0.01). However, genotype and allele analyses did not show any significant difference between sepsis and severe sepsis patients. CONCLUSIONS: Our findings indicate that common genetic variation in TF was significantly associated with outcome of severe sepsis in Chinese Han population. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13054-014-0631-9) contains supplementary material, which is available to authorized users

Predictors of Bacterial Meningitis in Resource-Limited Contexts: An Angolan Case

BACKGROUND: Despite the great morbidity and mortality that childhood bacterial meningitis (BM) is experiencing in Africa, diagnosis of BM in resource-limited contexts is still a challenge. Several algorithms and clinical predictors have been proposed to help physicians in decision-making but a lot of these markers used variables that are calculable only in well-equipped laboratories. Predictors or algorithm based on parameters that can be easily performed in basic laboratories can help significantly in BM diagnosis, even in resource-limited settings, rural hospitals or health centers. RESULTS: This retrospective study examined 145 cerebral-spinal fluid (CSF) specimens from children from 2 months to 14 years. CSF specimens were divided into two groups, according to the presence or not of a clinical diagnosis of BM. For each specimen, CSF aspect, CSF white blood cells (WBC) count, CSF glucose and protein concentration were analyzed and statistical analysis were performed. CSF WBC count ≥10/µl is no more a valuable predictor of BM. CSF protein concentration ≥50 mg/dl has a better sensitivity for BM diagnosis and when used with CSF glucose concentration ≤40 mg/dl, can help to diagnose correctly almost all the BM cases. An algorithm including CSF protein concentration, glucose concentration and WBC count has been proposed to rule out BM and to correctly diagnose it. CONCLUSIONS: In resource-limited health centers, the availability of a combination of easy-to-obtain parameters can significantly help physicians in BM diagnosis. The prompt identification of a BM case can be rapid treated or transferred to adequate structures and can modify the outcome in the patient