Cleaning the Cellular Factory:Deletion of McrA in Aspergillus oryzae NSAR1 and the generation of a novel kojic acid deficient strain for cleaner heterologous production of secondary metabolites

The use of filamentous fungi as cellular factories, where natural product pathways can be refactored and expressed in a host strain, continues to aid the field of natural product discovery. Much work has been done to develop host strains which are genetically tractable, and for which there are multiple selectable markers and controllable expression systems. To fully exploit these strains, it is beneficial to understand their natural metabolic capabilities, as such knowledge can rule out host metabolites from analysis of transgenic lines and highlight any potential interplay between endogenous and exogenous pathways. Additionally, once identified, the deletion of secondary metabolite pathways from host strains can simplify the detection and purification of heterologous compounds. To this end, secondary metabolite production in Aspergillus oryzae strain NSAR1 has been investigated via the deletion of the newly discovered negative regulator of secondary metabolism, mcrA (multicluster regulator A). In all ascomycetes previously studied mcrA deletion led to an increase in secondary metabolite production. Surprisingly, the only detectable phenotypic change in NSAR1 was a doubling in the yields of kojic acid, with no novel secondary metabolites produced. This supports the previous claim that secondary metabolite production has been repressed in A. oryzae and demonstrates that such repression is not McrA-mediated. Strain NSAR1 was then modified by employing CRISPR-Cas9 technology to disrupt the production of kojic acid, generating the novel strain NSARΔK, which combines the various beneficial traits of NSAR1 with a uniquely clean secondary metabolite background

Cladobotric acids: Metabolites from cultures of Cladobotryum sp., semi-synthetic analogues and antibacterial activity

[Image: see text] Three new polyketide-derived natural products, cladobotric acids G–I (1–3), and six known metabolites (4, 5, 8–11) were isolated from fermentation of the fungus Cladobotryum sp. grown on rice. Their structures were elucidated by extensive spectroscopic methods. Two metabolites, cladobotric acid A (4) and pyrenulic acid A (10), were converted to a series of new products (12–20) by semisynthesis. The antibacterial activities of all these compounds were investigated against the Gram-positive pathogen Staphylococcus aureus including methicillin-susceptible (MSSA), methicillin-resistant and vancomycin-intermediate (MRSA/VISA), and heterogeneous vancomycin-intermediate (hVISA) strains. Results of these antibacterial assays revealed structural features of the unsaturated decalins important for biological activity

Identification of the population density of a species model with nonlocal diffusion and nonlinear reaction

The identification of the population density of a logistic equation backwards in time associated with nonlocal diffusion and nonlinear reaction, motivated by biology and ecology fields, is investigated. The diffusion depends on an integral average of the population density whilst the reaction term is a global or local Lipschitz function of the population density. After discussing the ill-posedness of the problem, we apply the quasi-reversibility method to construct stable approximation problems. It is shown that the regularized solutions stemming from such method not only depend continuously on the final data, but also strongly converge to the exact solution in L²-norm. New error estimates together with stability results are obtained. Furthermore, numerical examples are provided to illustrate the theoretical results

HIV-Associated TB in An Giang Province, Vietnam, 2001–2004: Epidemiology and TB Treatment Outcomes

BACKGROUND: Mortality is high in HIV-infected TB patients, but few studies from Southeast Asia have documented the benefits of interventions, such as co-trimoxazole (CTX), in reducing mortality during TB treatment. To help guide policy in Vietnam, we studied the epidemiology of HIV-associated TB in one province and examined factors associated with outcomes, including the impact of CTX use. METHODOLOGY/PRINCIPAL FINDINGS: We retrospectively abstracted data for all HIV-infected persons diagnosed with TB from 2001-2004 in An Giang, a province in southern Vietnam in which TB patients receive HIV counseling and testing. We used standard WHO definitions to classify TB treatment outcomes. We conducted multivariate analysis to identify risk factors for the composite outcome of death, default, or treatment failure during TB treatment. From 2001-2004, 637 HIV-infected TB patients were diagnosed in An Giang. Of these, 501 (79%) were male, 321 (50%) were aged 25-34 years, and the most common self-reported HIV risk factor was sex with a commercial sex worker in 221 (35%). TB was classified as smear-positive in 531 (83%). During TB treatment, 167 (26%) patients died, 9 (1%) defaulted, and 6 (1%) failed treatment. Of 454 patients who took CTX, 116 (26%) had an unsuccessful outcome compared with 33 (70%) of 47 patients who did not take CTX (relative risk, 0.4; 95% confidence interval [CI], 0.3-0.5). Adjusting for male sex, rural residence, TB smear status and disease location, and the occurrence of adverse events during TB treatment in multivariate analysis, the benefit of CTX persisted (adjusted odds ratio for unsuccessful outcome 0.1; CI, 0.1-0.3). CONCLUSIONS/SIGNIFICANCE: In An Giang, Vietnam, HIV-associated TB was associated with poor TB treatment outcomes. Outcomes were significantly better in those taking CTX. This finding suggests that Vietnam should consider applying WHO recommendations to prescribe CTX to all HIV-infected TB patients

Critical Requirements for the Initiation of a Cardiac Arrhythmia in Rat Ventricle: How Many Myocytes?

Cardiovascular disease is the leading cause of death worldwide due in a large part to arrhythmia. In order to understand how calcium dynamics play a role in arrhythmogenesis, normal and dysfunctional Ca2+ signaling in a subcellular, cellular, and tissued level is examined using cardiac ventricular myocytes at a high temporal and spatial resolution using multiscale computational modeling. Ca2+ sparks underlie normal excitation–contraction coupling. However, under pathological conditions, Ca2+ sparks can combine to form Ca2+ waves. These propagating elevations of (Ca2+)i can activate an inward Na+–Ca2+ exchanger current (INCX) that contributes to early after-depolarization (EADs) and delayed after-depolarizations (DADs). However, how cellular currents lead to full depolarization of the myocardium and how they initiate extra systoles is still not fully understood. This study explores how many myocytes must be entrained to initiate arrhythmogenic depolarizations in biophysically detailed computational models. The model presented here suggests that only a small number of myocytes must activate in order to trigger an arrhythmogenic propagating action potential. These conditions were examined in 1-D, 2-D, and 3-D considering heart geometry. The depolarization of only a few hundred ventricular myocytes is required to trigger an ectopic depolarization. The number decreases under disease conditions such as heart failure. Furthermore, in geometrically restricted parts of the heart such as the thin muscle strands found in the trabeculae and papillary muscle, the number of cells needed to trigger a propagating depolarization falls even further to less than ten myocytes

Effects of Launaea sarmentosa Extract on Lipopolysaccharide-Induced Inflammation via Suppression of NF-κB/MAPK Signaling and Nrf2 Activation

Launaea sarmentosa has been extensively used as a nutrient herb in traditional Vietnamese remedies for the treatment of various diseases, especially inflammatory diseases. However, no detailed research has been conducted examining the molecular mechanisms involved in the suppression of inflammatory response. Here, we studied the effects of L. sarmentosa methanol extract on lipopolysaccharide (LPS)-induced inflammation using RAW 264.7 macrophages. The extract demonstrated potent antioxidant activity owing to the presence of polyphenolic and flavonoid components. Pretreatment with the extract inhibited LPS-mediated secretion of nitric oxide, reactive oxygen species, and tumor necrosis factor-α as well as the expression of inflammatory cytokines. Furthermore, the activation of the nuclear factor-kappa B pathway and phosphoinositide-3-kinase/protein kinase B pathways was blocked by the extract by inhibiting Akt phosphorylation. Additionally, the mitogen-activated protein kinase pathway was suppressed, and endoplasmic reticulum stress was attenuated. Furthermore, the extract promoted the activity of nuclear factor erythroid-2-related factor 2 resulting in the up-regulation of heme oxygenase-1 pathway, leading to the suppression of oxidative stress and inflammatory response. Taken together, the results indicate that L. sarmentosa exhibits anti-inflammatory effects, and hence, can be further developed as a novel drug for the treatment of diseases associated with excessive inflammation