A highly sensitive nanostructure-based electrochemical sensor for electrocatalytic determination of norepinephrine in the presence of acetaminophen and tryptophan

A new electrochemical sensor for the determination of norepinephrine (NE), acetaminophen (AC) and tryptophan (Trp) is described. The sensor is based on carbon paste electrode (CPE) modified with 3,4-dihydroxybenzaldehyde-2,4-dinitrophenylhydrazone (DDP) and takes the advantages of carbon nanotubes (CNTs), which makes the modified electrode highly sensitive for the electrochemical detection of these compounds. Cyclic voltammetry (CV) at various scan rates was used to investigate the redox properties of the modified electrode. The apparent charge transfer rate constant, k(s), and transfer coefficient, α, for electron transfer between DDP and CNT paste electrode were calculated. The mediated oxidation of NE at the modified electrode was investigated by CV and the values of k, α and diffusion coefficient (D) were calculated. Under the optimum pH of 7.0, the oxidation of NE occurs at a potential about 215 mV less positive than that of the unmodified CPE. Differential pulse voltammetry (DPV) of NE at the modified electrode exhibited two linear dynamic ranges with a detection limit (3σ) of 77±2 nM. DPV was used for simultaneous determination of NE, AC and Trp at the modified electrode, and quantitation of NE in some real samples by the standard addition method.     

Combined evidence annotation of transposable elements in genome sequences

Transposable elements (TEs) are mobile, repetitive sequences that make up significant fractions of metazoan genomes. Despite their near ubiquity and importance in genome and chromosome biology, most efforts to annotate TEs in genome sequences rely on the results of a single computational program, RepeatMasker. In contrast, recent advances in gene annotation indicate that high-quality gene models can be produced from combining multiple independent sources of computational evidence. To elevate the quality of TE annotations to a level comparable to that of gene models, we have developed a combined evidence-model TE annotation pipeline, analogous to systems used for gene annotation, by integrating results from multiple homology-based and de novo TE identification methods. As proof of principle, we have annotated "TE models" in Drosophila melanogaster Release 4 genomic sequences using the combined computational evidence derived from RepeatMasker, BLASTER, TBLASTX, all-by-all BLASTN, RECON, TE-HMM and the previous Release 3.1 annotation. Our system is designed for use with the Apollo genome annotation tool, allowing automatic results to be curated manually to produce reliable annotations. The euchromatic TE fraction of D. melanogaster is now estimated at 5.3% (cf. 3.86% in Release 3.1), and we found a substantially higher number of TEs (n = 6,013) than previously identified (n = 1,572). Most of the new TEs derive from small fragments of a few hundred nucleotides long and highly abundant families not previously annotated (e.g., INE-1). We also estimated that 518 TE copies (8.6%) are inserted into at least one other TE, forming a nest of elements. The pipeline allows rapid and thorough annotation of even the most complex TE models, including highly deleted and/or nested elements such as those often found in heterochromatic sequences. Our pipeline can be easily adapted to other genome sequences, such as those of the D. melanogaster heterochromatin or other species in the genus Drosophila.     

Peptide YY and neuropeptide Y induce villin expression, reduce adhesion, and enhance migration in small intestinal cells through the regulation of CD63, matrix metalloproteinase-3, and Cdc42 activity

Peptide YY (PYY) and neuropeptide Y (NPY) are regulatory peptides synthesized in the intestine and brain, respectively, that modify physiological functions affecting nutrient assimilation and feeding behavior. Because PYY and NPY also alter the expression of intestine-specific differentiation marker proteins and the tetraspanin CD63, which is involved in cell adhesion, we investigated whether intestinal cell differentiation could be linked to mucosal cell adhesion and migration through these peptides. PYY and NPY significantly decreased cell adhesion and increased cell migration in a dose-dependent manner prior to cell confluency in our model system, non-tumorigenic small intestinal hBRIE 380i cells. Both peptides reduced CD63 expression and CD63-dependent cell adhesion. CD63 overexpression increased and antisense CD63 cDNA decreased intestinal cell adhesion. In parallel, both PYY and NPY increased expression of matrix metalloproteinase-3 (MMP-3) to a level sufficient to induce cell migration by activating the Rho GTPase Cdc42. The effects of both peptides on cell migration were blocked in cells constitutively overexpressing dominant-negative Cdc42. PYY and NPY also significantly induced the expression of the differentiation marker villin, which could be eliminated by an MMP inhibitor at a concentration that inhibits cell migration. Increased MMP-3 activity, which enhanced cell migration, also induced villin mRNA levels. Therefore, these data indicate that the alteration of adhesion and migration by PYY and NPY occurs in part by synchronous modulation of three proteins that are involved in extracellular matrix-basolateral membrane interactions, CD63, MMP-3 and Cdc42, and that PYY/NPY regulation of expression of mucosal proteins such as villin is linked to the process of cell migration and adhesion.     

Leishmanin skin test in guinea pig with a single purified protein of Leishmania major

A series of hybridomas was produced by fusion of SP2/0 myeloma cells with spleen cells of mice immunized with Leishmania major (L. major). The reactivity of secreted monoclonal antibodies (mAbs) was evaluated against available leishmanin antigen by enzyme linked immunosorbent assay. Only one hybridoma designated as 7F9 secreted IgG1 mAb which was shown to be reactive with leishmanin. This mAb was further tested against four species of Leishmania (L. donovani, L. tropica, L. infantum, L. major) and a recombinant gp63. Among the four species tested it was shown to be only reactive with promastigotes of L. major. The antigen recognized by this mAb was purified and analyzed from both sonicated and supernatant cultures of L. major by immunoaffinity chromatography and reverse phase high performance liquid chromatography. The purified antigen, which gave a single band of 56kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis elicited a strong delayed-type hypersensitivity (DTH) reaction in guinea pigs sensitized with L. major. It was almost of the same degree as that produced by leishmanin. These results suggest that an L. major-specific antigen is an alternative as a specific diagnostic skin test reagent, which could lead to a better understanding of the mechanism of DTH in L. major.     

A comparison of disease burden and symptoms with age among CoVid-19 patients from data in a Florida clinic

Our knowledge of the disease burden and symptoms with age in COVID-19 patients is limited. Therefore, it is of interest to document the clinical aspect of this association with respect to the disease. We used the data of 3363 patients enrolled with an urgent care clinic in Volusia county, Florida for this study. Data shows difference in age among COVID-19 antibody (Ab) - positive patients (48.3 years, 95% CI = 46.9,49.7 years) and Ab-negative patients (46.1 years, 95% CI = 45.4, 46.8 years). However, disease burden by age is not significant on average. Nonetheless, COVID-19 positive patients between 40-69-years of age experienced the highest burden of disease and highest average number of symptoms. Thus, COVID-19 disease burden and number of symptoms experienced were highest among the 40-69-year-old patients. Those above the populations mean age of 46.4 years old were more likely to test positive for COVID-19.     

Identification of amyloidogenic proteins in the microbiomes of a rat Parkinson's disease model and wild‐type rats

Cross seeding between amyloidogenic proteins in the gut is receiving increasing attention as a possible mechanism for initiation or acceleration of amyloid formation by aggregation‐prone proteins such as αSN, which is central in the development of Parkinson''s disease (PD). This is particularly pertinent in view of the growing number of functional (i.e., benign and useful) amyloid proteins discovered in bacteria. Here we identify two amyloidogenic proteins, Pr12 and Pr17, in fecal matter from PD transgenic rats and their wild type counterparts, based on their stability against dissolution by formic acid (FA). Both proteins show robust aggregation into ThT‐positive aggregates that contain higher‐order β‐sheets and have a fibrillar morphology, indicative of amyloid proteins. In addition, Pr17 aggregates formed in vitro showed significant resistance against FA, suggesting an ability to form highly stable amyloid. Treatment with proteinase K revealed a protected core of approx. 9 kDa. Neither Pr12 nor Pr17, however, affected αSN aggregation in vitro. Thus, amyloidogenicity does not per se lead to an ability to cross‐seed fibrillation of αSN. Our results support the use of proteomics and FA to identify amyloidogenic protein in complex mixtures and suggests that there may be numerous functional amyloid proteins in microbiomes.     

The evolutionary path to terminal differentiation and division of labor in cyanobacteria

A common trait often associated with multicellularity is cellular differentiation, which is a spatial separation of tasks through the division of labor. In principle, the division of labor does not necessarily have to be constrained to a multicellular setting. In this study, we focus on the possible evolutionary paths leading to terminal differentiation in cyanobacteria. We develop mathematical models for two developmental strategies. First, of populations of terminally differentiated single cells surviving by the exchange of common goods. Second, of populations exhibiting terminal differentiation in a multicellular setting. After testing the two strategies against the effect of disruptive mutations (i.e. “cheater” mutants), we assess the effects of selection on the optimization of the ratio of vegetative (carbon fixing) to heterocystous (nitrogen fixing) cells, which in turn leads to the maximization of the carrying capacity for the population density. In addition, we compare the performance of differentiated populations to undifferentiated ones that temporally separate tasks in accordance to a day/night cycle. We then compare some predictions of our model with phylogenetic relationships derived from analyzing 16S rRNA sequences of different cyanobacterial strains. In line with studies indicating that group or spatial structure are ways to evolve cooperation and protect against the spread of cheaters, our work shows that compartmentalization afforded by multicellularity is required to maintain the vegetative/heterocyst division in cyanobacteria. We find that multicellularity allows for selection to optimize the carrying capacity. These results and the phylogenetic analysis indicates that terminally differentiated cyanobacteria evolved after undifferentiated species. In addition, we show that, in regimes of short daylight periods, terminally differentiated species perform worse than undifferentiated species that follow the day/night cycle; indicating that undifferentiated species have an evolutionary advantage in regimes of short daylight periods.     

Ascorbic acid mobilizes endogenous copper in human peripheral lymphocytes leading to oxidative DNA breakage: a putative mechanism for anticancer properties

Several decades back ascorbic acid was proposed as an effective anticancer agent. However, this idea remained controversial and the mechanism of action unclear. In this paper, we show that ascorbic acid at a concentration reported to be achievable through high doses of oral consumption is capable of cytotoxic action against normal cells. Several antioxidants of both animal as well as plant origin including ascorbic acid also possess prooxidant properties. Copper is an essential component of chromatin and can take part in redox reactions. Previously we have proposed a mechanism for the cytotoxic action of plant antioxidants against cancer cells that involves mobilization of endogenous copper ions and the consequent generation of reactive oxygen species. Using human peripheral lymphocytes and Comet assay we show here that ascorbic acid is able to cause oxidatative DNA breakage in normal cells at a concentration of 100–200 μM. Neocuproine, a Cu(I) specific sequestering agent inhibited DNA breakage in a dose dependent manner indicating that Cu(I) is an intermediate in the DNA cleavage reaction. The results are in support of our above hypothesis that involves events that lead to a prooxidant action by antioxidants. The results would support the idea that even a plasma concentration of around 200 μM would be sufficient to cause pharmacological tumor cell death particularly when copper levels are elevated. This would account for the observation of several decades back by Pauling and co-workers where oral doses of ascorbic acid in gram quantities were found to be effective in treating some cancers.     

Plasma exosomes can deliver exogenous short interfering RNA to monocytes and lymphocytes

Despite the promise of RNA interference (RNAi) and its potential, e.g. for use in cancer therapy, several technical obstacles must first be overcome. The major hurdle of RNAi-based therapeutics is to deliver nucleic acids across the cell's plasma membrane. This study demonstrates that exosome vesicles derived from humans can deliver short interfering RNA (siRNA) to human mononuclear blood cells. Exosomes are nano-sized vesicles of endocytic origin that are involved in cell-to-cell communication, i.e. antigen presentation, tolerance development and shuttle RNA (mainly mRNA and microRNA). Having tested different strategies, an optimized method (electroporation) was used to introduce siRNA into human exosomes of various origins. Plasma exosomes (exosomes from peripheral blood) were used as gene delivery vector (GDV) to transport exogenous siRNA to human blood cells. The vesicles effectively delivered the administered siRNA into monocytes and lymphocytes, causing selective gene silencing of mitogen-activated protein kinase 1. These data suggest that human exosomes can be used as a GDV to provide cells with heterologous nucleic acids such as therapeutic siRNAs.