ResearchPad - enzyme-assays https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Decyl caffeic acid inhibits the proliferation of colorectal cancer cells in an autophagy-dependent manner <i>in vitro</i> and <i>in vivo</i>]]> https://www.researchpad.co/article/elastic_article_13874 The treatment of human colorectal cancer (CRC) cells through suppressing the abnormal survival signaling pathways has recently become a significant area of focus. In this study, our results demonstrated that decyl caffeic acid (DC), one of the novel caffeic acid derivatives, remarkedly suppressed the growth of CRC cells both in vitro and in vivo. The inhibitory effects of DC on CRC cells were investigated in an in vitro cell model and in vivo using a xenograft mouse model. CRC cells were treated with DC at various dosages (0, 10, 20 and 40 μM), and cell survival, the apoptotic index and the autophagy level were measured using an MTT assay and flow cytometry analysis, respectively. The signaling cascades in CRC were examined by Western blot assay. The anti-cancer effects of DC on tumor growth were examined by using CRC HCT-116 cells implanted in an animal model. Our results indicated that DC differentially suppressed the growth of CRC HT-29 and HCT-116 cells through an enhancement of cell-cycle arrest at the S phase. DC inhibited the expression of cell-cycle regulators, which include cyclin E and cyclin A proteins. The molecular mechanisms of action were correlated to the blockade of the STAT3 and Akt signaling cascades. Strikingly, a high dosage of DC prompted a self-protection action through inducing cell-dependent autophagy in HCT-116 cells. Suppression of autophagy induced cell death in the treatment of DC in HCT-116 cells. DC seemed to inhibit cell proliferation of CRC differentially, and the therapeutic advantage appeared to be autophagy dependent. Moreover, consumption of DC blocked the tumor growth of colorectal adenocarcinoma in an experimental animal model. In conclusion, our results suggested that DC could act as a therapeutic agent through the significant suppression of tumor growth of human CRC cells.

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<![CDATA[Abrogation of pathogenic attributes in drug resistant <i>Candida auris</i> strains by farnesol]]> https://www.researchpad.co/article/elastic_article_7651 Candida auris, a decade old Candida species, has been identified globally as a significant nosocomial multidrug resistant (MDR) pathogen responsible for causing invasive outbreaks. Biofilms and overexpression of efflux pumps such as Major Facilitator Superfamily and ATP Binding Cassette are known to cause multidrug resistance in Candida species, including C. auris. Therefore, targeting these factors may prove an effective approach to combat MDR in C. auris. In this study, 25 clinical isolates of C. auris from different hospitals of South Africa were used. All the isolates were found capable enough to form biofilms on 96-well flat bottom microtiter plate that was further confirmed by MTT reduction assay. In addition, these strains have active drug efflux mechanism which was supported by rhodamine-6-G extracellular efflux and intracellular accumulation assays. Antifungal susceptibility profile of all the isolates against commonly used drugs was determined following CLSI recommended guidelines. We further studied the role of farnesol, an endogenous quorum sensing molecule, in modulating development of biofilms and drug efflux in C. auris. The MIC for planktonic cells ranged from 62.5–125 mM, and for sessile cells was 125 mM (4h biofilm) and 500 mM (12h and 24h biofilm). Furthermore, farnesol (125 mM) also suppresses adherence and biofilm formation by C. auris. Farnesol inhibited biofilm formation, blocked efflux pumps and downregulated biofilm- and efflux pump- associated genes. Modulation of C. auris biofilm formation and efflux pump activity by farnesol represent a promising approach for controlling life threatening infections caused by this pathogen.

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<![CDATA[NAP (davunetide) preferential interaction with dynamic 3-repeat Tau explains differential protection in selected tauopathies]]> https://www.researchpad.co/article/5c92b379d5eed0c4843a4107

The microtubule (MT) associated protein Tau is instrumental for the regulation of MT assembly and dynamic instability, orchestrating MT-dependent cellular processes. Aberration in Tau post-translational modifications ratio deviation of spliced Tau isoforms 3 or 4 MT binding repeats (3R/4R) have been implicated in neurodegenerative tauopathies. Activity-dependent neuroprotective protein (ADNP) is vital for brain formation and cognitive function. ADNP deficiency in mice causes pathological Tau hyperphosphorylation and aggregation, correlated with impaired cognitive functions. It has been previously shown that the ADNP-derived peptide NAP protects against ADNP deficiency, exhibiting neuroprotection, MT interaction and memory protection. NAP prevents MT degradation by recruitment of Tau and end-binding proteins to MTs and expression of these proteins is required for NAP activity. Clinically, NAP (davunetide, CP201) exhibited efficacy in prodromal Alzheimer’s disease patients (Tau3R/4R tauopathy) but not in progressive supranuclear palsy (increased Tau4R tauopathy). Here, we examined the potential preferential interaction of NAP with 3R vs. 4R Tau, toward personalized treatment of tauopathies. Affinity-chromatography showed that NAP preferentially interacted with Tau3R protein from rat brain extracts and fluorescence recovery after photobleaching assay indicated that NAP induced increased recruitment of human Tau3R to MTs under zinc intoxication, in comparison to Tau4R. Furthermore, we showed that NAP interaction with tubulin (MTs) was inhibited by obstruction of Tau-binding sites on MTs, confirming the requirement of Tau-MT interaction for NAP activity. The preferential interaction of NAP with Tau3R may explain clinical efficacy in mixed vs. Tau4R pathologies, and suggest effectiveness in Tau3R neurodevelopmental disorders.

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<![CDATA[Ectopic Otoconin 90 expression in triple negative breast cancer cell lines is associated with metastasis functions]]> https://www.researchpad.co/article/5c6f1491d5eed0c48467a304

Triple negative breast cancer (TNBC) is an aggressive tumor with propensity to metastasize and poor treatment options. Improving treatment options would be impactful; thus, finding a tumor-specific cell surface protein with metastasis promoting functions that could be knocked out was the goal of this study. The Otoconin 90 gene (OC90), frequently amplified in tumors on chromosome 8q24.22, was identified as a potential therapeutic candidate. Normally OC90 is expressed in the cochlea with no known function in other normal tissues. In silico analysis of The Cancer Genome Atlas (TCGA) multi-tumor RNAseq cohorts revealed that OC90 is expressed in many tumor types at high prevalence and genomic amplification is associated with the elevated mRNA expression. In vitro assays in TNBC cell lines revealed OC90 expression with control over cell viability, apoptosis and invasion. RNA-seq analysis of OC90-siRNA knockdown and OC90-overexpression in BT20, BT549, HCC38 cell lines identified co-expressed transcripts, HMGA2, POLE2 and TRIB3. Altered expression of HMGA2, POLE2 and TRIB3 was predictive of survival among members of the Metabric breast cancer cohort. Thus, OC90 represents a potential therapeutic target whose knockdown could improve the treatment of TNBC.

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<![CDATA[Expression optimization, purification, and functional characterization of cholesterol oxidase from Chromobacterium sp. DS1]]> https://www.researchpad.co/article/5c6dc9e1d5eed0c48452a3da

Cholesterol oxidase is a bifunctional bacterial flavoenzyme which catalyzes oxidation and isomerization of cholesterol. This valuable enzyme has attracted a great deal of attention because of its wide application in the clinical laboratory, synthesis of steroid derived drugs, food industries, and its potentially insecticidal activity. Therefore, development of an efficient protocol for overproduction of cholesterol oxidase could be valuable and beneficial in this regard. The present study examined the role of various parameters (host strain, culture media, induction time, isopropyl ß-D-1-thiogalactopyranoside concentration, as well as post-induction incubation time and temperature) on over-expression of cholesterol oxidase from Chromobacterium sp. DS1. Applying the optimized protocol, the yield of recombinant cholesterol oxidase significantly increased from 92 U/L to 2115 U/L. Under the optimized conditions, the enzyme was produced on a large-scale, and overexpressed cholesterol oxidase was purified from cell lysate by column nickel affinity chromatography. Km and Vmax values of the purified enzyme for cholesterol were estimated using Lineweaver-Burk plot. Further, the optimum pH and optimum temperature for the enzyme activity were determined. This study reports a straightforward protocol for cholesterol oxidase production which can be performed in any laboratory.

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<![CDATA[Prognostic role of early D-dimer level in patients with acute ischemic stroke]]> https://www.researchpad.co/article/5c5df327d5eed0c484580dc4

Object

The purpose of our study was to assess the prognostic role of early D-dimer level in patients with acute ischemic stroke (AIS).

Methods

The included patients’ D-dimer levels have to be tested within 24 hours from stroke onset. Poor functional outcome was defined as modified Rankin Scale (mRS) ≥3. The endpoints included recurrence on 5-day diffusion-weighted imaging, 30-day mRS ≥3, 30-day mortality and 90-day mRS ≥3. Regarding to each endpoint, odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to assess the prognostic role of D-dimer in patients with AIS.

Results

A total of 2,479 patients were included. The results showed that elevated D-dimer levels were associated with recurrence on 5-day diffusion-weighted imaging (OR = 2.28, 95% CI = 1.32–3.95), 30-day mRS≥3 (OR = 1.59, 95% CI = 1.37–1.85), 30-day mortality (OR = 1.92, 95% CI = 1.27–2.90) and 90-day mRS≥3 (OR = 1.61, 95% CI = 1.05–2.46).

Conclusions

In conclusion, for patients with AIS, higher D-dimer level within 24 hours from stroke onset was associated with recurrence on 5-day diffusion-weighted imaging, mortality at 30 days, and poor functional outcome at both 30 days and 90 days. However, more studies are warranted to clarify this issue.

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<![CDATA[Screening of brain-derived neurotrophic factor (BDNF) single nucleotide polymorphisms and plasma BDNF levels among Malaysian major depressive disorder patients]]> https://www.researchpad.co/article/5c536c08d5eed0c484a497c8

Background

Brain-derived neurotrophic factor (BDNF) is a neurotrophin found in abundance in brain regions such as the hippocampus, cortex, cerebellum and basal forebrain. It has been associated with the risk of susceptibility to major depressive disorder (MDD). This study aimed to determine the association of three BDNF variants (rs6265, rs1048218 and rs1048220) with Malaysian MDD patients.

Methods

The correlation of these variants to the plasma BDNF level among Malaysian MDD patients was assessed. A total of 300 cases and 300 matched controls recruited from four public hospitals within the Klang Valley of Selangor State, Malaysia and matched for age, sex and ethnicity were screened for BDNF rs6265, rs1048218 and rs1048220 using high resolution melting (HRM).

Findings

BDNF rs1048218 and BDNF rs1048220 were monomorphic and were excluded from further analysis. The distribution of the alleles and genotypes for BDNF rs6265 was in Hardy-Weinberg equilibrium for the controls (p = 0.13) but was in Hardy Weinberg disequilibrium for the cases (p = 0.011). Findings from this study indicated that having BDNF rs6265 in the Malaysian population increase the odds of developing MDD by 2.05 folds (95% CI = 1.48–3.65). Plasma from 206 cases and 206 controls were randomly selected to measure the BDNF level using enzyme-linked immunosorbent assay (ELISA). A significant decrease in the plasma BDNF level of the cases as compared to controls (p<0.0001) was observed. However, there was no evidence of the effect of the rs6265 genotypes on the BDNF level indicating a possible role of other factors in modulating the BDNF level that warrants further investigation.

Conclusion

The study indicated that having the BDNF rs6265 allele (A) increase the risk of developing MDD in the Malaysian population suggesting a possible role of BDNF in the etiology of the disorder.

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<![CDATA[Phorbol ester-induced angiogenesis of endothelial progenitor cells: The role of NADPH oxidase-mediated, redox-related matrix metalloproteinase pathways]]> https://www.researchpad.co/article/5c478cb0d5eed0c484bd3eb3

Endothelial progenitor cells (EPCs) may contribute to ischemia-induced angiogenesis in atherosclerotic diseases. The protein kinase C (PKC) family is involved in the regulation of angiogenesis, however the role of PKCα in EPCs during angiogenesis is unclear. The aim of this study was to evaluate the role of PKCα in EPCs during angiogenesis. Phorbol-12-myristate-13-acetate (PMA), a PKCα activator, significantly increased the activity and expression of matrix metalloproteinases (MMP) -2 and -9 in human (late outgrowth) EPCs in vitro. The MMPs promoted the migratory function and vascular formation of EPCs, which then contributed to neovascularization in a mouse hindlimb-ischemia model. Reactive oxygen species derived from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enhanced the expression of MMPs to increase the bioactivity of EPCs during angiogenesis. The mitogen-activated protein kinase (MAPK) signal pathway was associated with the activation of NADPH oxidase. PMA extensively activated the extracellular signal–regulated kinase (Erk) signal pathway to increase the expression of MMP-9. PMA also activated the p38, Erk, and c-Jun N-terminal kinase signal pathways to increase the expression of MMP-2. PMA-stimulated EPCs enhanced neovascularization in a mouse model of hindlimb ischemia via nuclear factor-κB translocation to up-regulation of the expression of MMP-2 and MMP-9. PMA could activate PKCα and promote the angiogenesis capacity of human EPCs via NADPH oxidase-mediated, redox-related, MMP-2 and MMP-9 pathways. The PKCα-activated, NADPH oxidase-mediated, redox-related MMP pathways could contribute to the function of human EPCs for ischemia-induced neovascularization, which may provide novel insights into the potential modification of EPCs for therapeutic angiogenesis.

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<![CDATA[Epstein-Barr virus microRNAs regulate B cell receptor signal transduction and lytic reactivation]]> https://www.researchpad.co/article/5c3d00f8d5eed0c48403729d

MicroRNAs (miRNAs) are post-transcriptional regulatory RNAs that can modulate cell signaling and play key roles in cell state transitions. Epstein-Barr virus (EBV) expresses >40 viral miRNAs that manipulate both viral and cellular gene expression patterns and contribute to reprogramming of the host environment during infection. Here, we identified a subset of EBV miRNAs that desensitize cells to B cell receptor (BCR) stimuli, and attenuate the downstream activation of NF-kappaB or AP1-dependent transcription. Bioinformatics and pathway analysis of Ago PAR-CLIP datasets identified multiple EBV miRNA targets related to BCR signal transduction, including GRB2, SOS1, MALT1, RAC1, and INPP5D, which we validated in reporter assays. BCR signaling is critical for B cell activation, proliferation, and differentiation, and for EBV, is linked to reactivation. In functional assays, we demonstrate that EBV miR-BHRF1-2-5p contributes to the growth of latently infected B cells through GRB2 regulation. We further determined that activities of EBV miR-BHRF1-2-5p, EBV miR-BART2-5p, and a cellular miRNA, miR-17-5p, directly regulate virus reactivation triggered by BCR engagement. Our findings provide mechanistic insight into some of the key miRNA interactions impacting the proliferation of latently infected B cells and importantly, governing the latent to lytic switch.

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<![CDATA[Optimization of extraction of antioxidant polysaccharide from Pleurotus ostreatus (Jacq.) P. Kumm and its cytotoxic activity against murine lymphoid cancer cell line]]> https://www.researchpad.co/article/5c37b784d5eed0c48449027e

The purpose of this study was to optimize the extraction method for polysaccharide from the fruiting bodies of Pleurotus ostreatus (Jacq.) P. Kumm and to assess the antioxidant and cytotoxic potentials of polysaccharide. In this investigation, polysaccharides from Pleurotus ostreatus (Jacq.) P. Kumm were extricated by utilizing the hot water. One-single factor and response surface methodology was established to optimize the extraction conditions for polysaccharide from Pleurotus ostreatus (Jacq.) P. Kumm. Examination of antioxidant activity of Pleurotus ostreatus polysaccharide (POP) was directed by utilizing 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and 2, 2-azino-bis-3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) techniques. Cytotoxicity of POP was evaluated using an MTT assay. The experimental data were fitted to a quadratic equation utilizing multiple regression investigations, and the ideal conditions were as per the following: water/crude material proportion, 26.04 mL/g; an extraction time of 62.08 minutes; and an extraction temperature 70.5°C. Under such conditions, the polysaccharide yield was 5.32 ± 0.12% with the anticipated yield. POP showed good scavenging activity against DPPH radical (p<0.001, EC50 = 1036.38 μg/mL, R2 = 0.8313) and ABTS radicals (p<0.001, EC50 = 824.37 μg/mL, R2 = 0.8223), with a dose (p<0.001)-and-time (p<0.001) dependent cytotoxic potential on Ehrlich ascites carcinoma cell line in vitro. This demonstrated that polysaccharides (POP) had certain cancer prevention agent exercises. In this manner, these examinations give reference to additionally research and reasonable improvement of Pleurotus ostreatus (Jacq.) P. Kumm polysaccharide and POP may prove a useful therapeutic agent, due to its robust antioxidant and cytotoxic activity.

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<![CDATA[Conformational fingerprint of blood and tissue ACEs: Personalized approach]]> https://www.researchpad.co/article/5c2e7fe0d5eed0c48451be45

Background

The pattern of binding of monoclonal antibodies (mAbs) to 18 epitopes on human angiotensin I-converting enzyme (ACE)–“conformational fingerprint of ACE”–is a sensitive marker of subtle conformational changes of ACE due to mutations, different glycosylation in various cells, the presence of ACE inhibitors and specific effectors, etc.

Methodology/Principal findings

We described in detail the methodology of the conformational fingerprinting of human blood and tissue ACEs that allows detecting differences in surface topography of ACE from different tissues, as well detecting inter-individual differences. Besides, we compared the sensitivity of the detection of ACE inhibitors in the patient’s plasma using conformational fingerprinting of ACE (with only 2 mAbs to ACE, 1G12 and 9B9) and already accepted kinetic assay and demonstrated that the mAbs-based assay is an order of magnitude more sensitive. This approach is also very effective in detection of known (like bilirubin and lysozyme) and still unknown ACE effectors/inhibitors which nature and set could vary in different tissues or different patients.

Conclusions/Significance

Phenotyping of ACE (and conformational fingerprinting of ACE as a part of this novel approach for characterization of ACE) in individuals really became informative and clinically relevant. Appreciation (and counting on) of inter-individual differences in ACE conformation and accompanying effectors make the application of this approach for future personalized medicine with ACE inhibitors more accurate. This (or similar) methodology can be applied to any enzyme/protein for which there is a number of mAbs to its different epitopes.

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<![CDATA[Serum concentration of anti-Cytomegalovirus IgG and ischaemic stroke in patients with advanced HIV infection in Malawi]]> https://www.researchpad.co/article/5c06f041d5eed0c484c6d537

Background

Studies in high-income settings have shown association between Cytomegalovirus (CMV) infection and adverse cardiovascular outcome, especially in HIV infection. We aimed to study the association between serum concentration of anti-CMV IgG and ischaemic stroke in HIV-infected Malawians.

Methods

Our sample was derived from a case-control stroke study in Malawi. Serum concentration of anti-CMV IgG was measured using enzyme-linked immunosorbent assay. Multivariable logistic regression was used to study the association between high concentrations of anti-CMV IgG (above the third tertile) and ischaemic stroke while adjusting for cardiovascular risk factors.

Results

Overall, 139 HIV-positive adults (48.2% women; 48 ischaemic stroke cases and 91 controls; median age: 45 years) were included. The median CD4+ count was 136 and 401 cell/mm3 (IQR: [75–278] and [230–533]) in cases and controls, respectively. High concentration of anti-CMV IgG was associated with ischaemic stroke in the univariable model (OR = 2.56 [1.23–5.34]) but not after adjusting for duration of antiretroviral therapy (ART), CD4+ count, and other cardiovascular risk factors (OR = 0.94 [0.29–3.08]). Low CD4+ count was an independent predictor of stroke. There was a negative correlation between serum concentration of anti-CMV IgG and CD4+ count (rho = -0.30, p < 0.001).

Conclusions

High concentration of anti-CMV IgG is not independently associated with ischaemic stroke in HIV-infected Malawians. Larger cohort studies are needed to further investigate the role of humoral response to CMV in the pathophysiology of HIV-associated stroke.

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<![CDATA[Autophagic cell death associated to Sorafenib in renal cell carcinoma is mediated through Akt inhibition in an ERK1/2 independent fashion]]> https://www.researchpad.co/article/5b694661463d7e3867f4ad07

Objectives

To fully clarify the role of Mitogen Activated Protein Kinase in the therapeutic response to Sorafenib in Renal Cell Carcinoma as well as the cell death mechanism associated to this kinase inhibitor, we have evaluated the implication of several Mitogen Activated Protein Kinases in Renal Cell Carcinoma-derived cell lines.

Materials and methods

An experimental model of Renal Cell Carcinoma-derived cell lines (ACHN and 786-O cells) was evaluated in terms of viability by MTT assay, induction of apoptosis by caspase 3/7 activity, autophagy induction by LC3 lipidation, and p62 degradation and kinase activity using phospho-targeted antibodies. Knock down of ATG5 and ERK5 was performed using lentiviral vector coding specific shRNA

Results

Our data discard Extracellular Regulated Kinase 1/2 and 5 as well as p38 Mitogen Activated Protein Kinase pathways as mediators of Sorafenib toxic effect but instead indicate that the inhibitory effect is exerted through the PI3K/Akt signalling pathway. Furthermore, we demonstrate that inhibition of Akt mediates cell death associated to Sorafenib without caspase activation, and this is consistent with the induction of autophagy, as indicated by the use of pharmacological and genetic approaches.

Conclusion

The present report demonstrates that Sorafenib exerts its toxic effect through the induction of autophagy in an Akt-dependent fashion without the implication of Mitogen Activated Protein Kinase. Therefore, our data discard the use of inhibitors of the RAF-MEK-ERK1/2 signalling pathway in RCC and support the use of pro-autophagic compounds, opening new therapeutic opportunities for Renal Cell Carcinoma.

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<![CDATA[Utilizing ExAC to assess the hidden contribution of variants of unknown significance to Sanfilippo Type B incidence]]> https://www.researchpad.co/article/5b4a2889463d7e4513b897ff

Given the large and expanding quantity of publicly available sequencing data, it should be possible to extract incidence information for monogenic diseases from allele frequencies, provided one knows which mutations are causal. We tested this idea on a rare, monogenic, lysosomal storage disorder, Sanfilippo Type B (Mucopolysaccharidosis type IIIB). Sanfilippo Type B is caused by mutations in the gene encoding α-N-acetylglucosaminidase (NAGLU). There were 189 NAGLU missense variants found in the ExAC dataset that comprises roughly 60,000 individual exomes. Only 24 of the 189 missense variants were known to be pathogenic; the remaining 165 variants were of unknown significance (VUS), and their potential contribution to disease is unknown. To address this problem, we measured enzymatic activities of 164 NAGLU missense VUS in the ExAC dataset and developed a statistical framework for estimating disease incidence with associated confidence intervals. We found that 25% of VUS decreased the activity of NAGLU to levels consistent with Sanfilippo Type B pathogenic alleles. We found that a substantial fraction of Sanfilippo Type B incidence (67%) could be accounted for by novel mutations not previously identified in patients, illustrating the utility of combining functional activity data for VUS with population-wide allele frequency data in estimating disease incidence.

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<![CDATA[Leishmanicidal and fungicidal activity of lipases obtained from endophytic fungi extracts]]> https://www.researchpad.co/article/5b498fc7463d7e0897c6e027

This work describes the production of lipases from endophytic fungi: Vermisporium-like, Emericella nidulans, Dichotomophtora portulacae and D. boerhaaviae and the biological activity against the dermatophyte fungi Malassezia sp and Microsporum canis and the parasite Leishmania amazonensis. All fungal enzymes extract showed lipolysis action in the media that contains long carbon chain lipids. The proteomic analysis of lipases exhibits several molecules mostly ranging in size from 220 to 20 kDa, with clear differences in protein profile’s yield. All fungal enzymes were competent to eliminate promastigote forms of Leishmania amazonensis at 5 mg.mL-1. The antileishmanial activity of lipases from Vermisporium-like, E. nidulans, D. portulacae and D. boerhaaviae in amastigote forms, promoted the reduction in viability of 78.88, 39.65, 63.17 and 98.13%, with selectivity index of 19.56, 30.68, 18.09 and 20.99. In relation to antifungal activity, Dichothomophtora enzymes demonstrate best action with MFC of 14.65 μg.mL-1 against Malassezia sp and Microsporum canis, respectively. These results allow us to infer that lipases from entophytic fungi displays activity against dermatophyte fungi (Malassezia sp. and Microsporum canis) as well as Leishmania.

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<![CDATA[Parkinson-Related LRRK2 Mutation R1628P Enables Cdk5 Phosphorylation of LRRK2 and Upregulates Its Kinase Activity]]> https://www.researchpad.co/article/5989dabcab0ee8fa60baf0d8

Background

Recent studies have linked certain single nucleotide polymorphisms in the leucine-rich repeat kinase 2 (LRRK2) gene with Parkinson’s disease (PD). Among the mutations, LRRK2 c.4883G>C (R1628P) variant was identified to have a significant association with the risk of PD in ethnic Han-Chinese populations. But the molecular pathological mechanisms of R1628P mutation in PD is still unknown.

Principle Findings

Unlike other LRRK2 mutants in the Roc-COR-Kinase domain, the R1628P mutation didn’t alter the LRRK2 kinase activity and promote neuronal death directly. LRRK2 R1628P mutation increased the binding affinity of LRRK2 with Cyclin-dependent kinase 5 (Cdk5). Interestingly, R1628P mutation turned its adjacent amino acid residue S1627 on LRRK2 protein to a novel phosphorylation site of Cdk5, which could be defined as a typical type II (+) phosphorylation-related single nucleotide polymorphism. Importantly, we showed that the phosphorylation of S1627 by Cdk5 could activate the LRRK2 kinase, and neurons ectopically expressing R1628P displayed a higher sensitivity to 1-methyl-4-phenylpyridinium, a bioactive metabolite of environmental toxin MPTP, in a Cdk5-dependent manner.

Conclusion

Our data indicate that Parkinson-related LRRK2 mutation R1628P leads to Cdk5 phosphorylation of LRRK2 at S1627, which would upregulate the kinase activity of LRRK2 and consequently cause neuronal death.

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<![CDATA[Determining the Biochemical Properties of the Oxalate Biosynthetic Component (Obc)1 from Burkholderia mallei]]> https://www.researchpad.co/article/5989da8bab0ee8fa60b9e004

Oxalic acid is produced by a variety of organisms ranging from simple microbes to complex animals. This acid has been proposed to fulfill various physiological and pathological functions which vary between organisms. In bacteria from the Burkholderia genus, oxalate secretion has been shown to be quorum sensing dependent and to support pathogenicity and cell viability. In light of the critical roles of oxalate in Burkholderia as well as other organisms, it is surprising that our understanding of how this simple dicarboxylate is biosynthesized remains incomplete. Here we report the expression, purification, and partial characterization of the first intact bacterial oxalate biosynthetic enzyme, Obc1, from B. mallei. An N-terminal His-tagged Bmobc1 was cloned into pDUET, expressed in E. coli BLR (DE3), and the recombinant enzyme purified by affinity chromatography. Oxalate biosynthetic enzyme assays coupled with HPLC analysis revealed that BmObc1 catalyzed the biosynthesis of oxalate, acetoacetate, and free CoA from oxaloacetate and a short chain acyl-CoA following Michaelis-Menten kinetics. Optimal enzyme activity was measured at pH 8.0 and a temperature around 44°C. Kinetic analysis conducted under conditions of saturating acetyl-CoA and varying oxaloacetate concentrations resulted in a calculated Km value for oxaloacetate of 94.3± 9.2 μM (mean ± SE). Under conditions of saturating oxaloacetate concentration and varying acyl-CoA (acetyl- or propionyl-CoA) concentrations kinetic analysis generated a calculated Km value of 26.8 ± 2.3 μM (mean ± SE) for acetyl-CoA and 104.4 ± 12.7 μM for propionyl-CoA. The significantly lower Km for acetyl-CoA suggests that it is strongly favored as a substrate over propionyl-CoA.

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<![CDATA[Prevalence of Chagas Disease in a U.S. Population of Latin American Immigrants with Conduction Abnormalities on Electrocardiogram]]> https://www.researchpad.co/article/5989db53ab0ee8fa60bdcdff

Chagas disease (CD) affects over six million people and is a leading cause of cardiomyopathy in Latin America. Given recent migration trends, there is a large population at risk in the United States (US). Early stage cardiac involvement from CD usually presents with conduction abnormalities on electrocardiogram (ECG) including right bundle branch block (RBBB), left anterior or posterior fascicular block (LAFB or LPFB, respectively), and rarely, left bundle branch block (LBBB). Identification of disease at this stage may lead to early treatment and potentially delay the progression to impaired systolic function. All ECGs performed in a Los Angeles County hospital and clinic system were screened for the presence of RBBB, LAFB, LPFB, or LBBB. Patients were contacted and enrolled in the study if they had previously resided in Latin America for at least 12 months and had no history of cardiac disease. Enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA) tests were utilized to screen for Trypanosoma cruzi seropositivity. A total of 327 consecutive patients were screened for CD from January 2007 to December 2010. The mean age was 46.3 years and the mean length of stay in the US was 21.2 years. Conduction abnormalities were as follows: RBBB 40.4%, LAFB 40.1%, LPFB 2.8%, LBBB 5.5%, RBBB and LAFB 8.6%, and RBBB and LPFB 2.8%. Seventeen patients were positive by both ELISA and IFA (5.2%). The highest prevalence rate was among those with RBBB and LAFB (17.9%). There is a significant prevalence of CD in Latin American immigrants residing in Los Angeles with conduction abnormalities on ECG. Clinicians should consider evaluating all Latin American immigrant patients with unexplained conduction disease for CD.

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<![CDATA[Transforming growth factor β1 enhances heme oxygenase 1 expression in human synovial fibroblasts by inhibiting microRNA 519b synthesis]]> https://www.researchpad.co/article/5989db52ab0ee8fa60bdc906

Background

Osteoarthritis (OA) is manifested by synovial inflammation and cartilage destruction that is directly linked to synovitis, joint swelling and pain. In the light of the role of synovium in the pathogenesis and the symptoms of OA, synovium-targeted therapy is a promising strategy to mitigate the symptoms and progression of OA. Transforming growth factor beta 1 (TGF-β1), a secreted homodimeric protein, possesses unique and potent anti-inflammatory and immune-regulatory properties in many cell types. Heme oxygenase 1 (HO-1) is an inducible anti-inflammatory and stress responsive enzyme that has been proven to prevent injuries caused by many diseases. Despite the similar anti-inflammatory profile and their involvement in the pathogenesis of arthritic diseases, no studies have as yet explored the possibility of any association between the expression of TGF-β1 and HO-1.

Methodology/Principal findings

TGF-β1-induced HO-1 expression was examined by HO-1 promoter assay, qPCR, and Western blotting. The siRNAs and enzyme inhibitors were utilized to determine the intermediate involved in the signal transduction pathway. We showed that TGF-β1 stimulated the synthesis of HO-1 in a concentration- and time-dependent manner, which can be mitigated by blockade of the phospholipase (PLC)γ/protein kinase C alpha (PKC)α pathway. We also showed that the expression of miRNA-519b, which blocks HO-1 transcription, is inhibited by TGF-β1, and the suppression of miRNA 519b could be reversed via blockade of the PLCγ/PKCα pathway.

Conclusions/Significance

TGF-β1 stimulated the expression of HO-1 via activating the PLCγ/PKCα pathway and suppressing the downstream expression of miRNA-519b. These results may shed light on the pathogenesis and treatment of OA.

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<![CDATA[Ion channel expression patterns in glioblastoma stem cells with functional and therapeutic implications for malignancy]]> https://www.researchpad.co/article/5989db4fab0ee8fa60bdbc83

Ion channels and transporters have increasingly recognized roles in cancer progression through the regulation of cell proliferation, migration, and death. Glioblastoma stem-like cells (GSCs) are a source of tumor formation and recurrence in glioblastoma multiforme, a highly aggressive brain cancer, suggesting that ion channel expression may be perturbed in this population. However, little is known about the expression and functional relevance of ion channels that may contribute to GSC malignancy. Using RNA sequencing, we assessed the enrichment of ion channels in GSC isolates and non-tumor neural cell types. We identified a unique set of GSC-enriched ion channels using differential expression analysis that is also associated with distinct gene mutation signatures. In support of potential clinical relevance, expression of selected GSC-enriched ion channels evaluated in human glioblastoma databases of The Cancer Genome Atlas and Ivy Glioblastoma Atlas Project correlated with patient survival times. Finally, genetic knockdown as well as pharmacological inhibition of individual or classes of GSC-enriched ion channels constrained growth of GSCs compared to normal neural stem cells. This first-in-kind global examination characterizes ion channels enriched in GSCs and explores their potential clinical relevance to glioblastoma molecular subtypes, gene mutations, survival outcomes, regional tumor expression, and experimental responses to loss-of-function. Together, the data support the potential biological and therapeutic impact of ion channels on GSC malignancy and provide strong rationale for further examination of their mechanistic and therapeutic importance.

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