Highly Heterogeneous Probiotic Lactobacillus Species in Healthy Iranians with Low Functional Activities

Background

Lactic acid bacteria (LAB) have been considered as potentially probiotic organisms due to their potential human health properties. This study aimed to evaluate both in vitro and in vivo, the potential probiotic properties of Lactobacillus species isolated from fecal samples of healthy humans in Iran.

Methods and Results

A total of 470 LAB were initially isolated from 53 healthy individual and characterized to species level. Of these, 88 (86%) were Lactobacillus species. Biochemical and genetic fingerprinting with Phene-Plate system (PhP-LB) and RAPD-PCR showed that the isolates were highly diverse consisted of 67(76.1%) and 75 (85.2%) single types (STs) and a diversity indices of 0.994 and 0.997, respectively. These strains were tested for production of adhesion to Caco-2 cells, antibacterial activity, production of B12, anti-proliferative effect and interleukin-8 induction on gut epithelial cell lines and antibiotic resistance against 9 commonly used antibiotics. Strains showing the characteristics consistent with probiotic strains, were further tested for their anti-inflammatory effect in mouse colitis model. Only one L. brevis; one L. rhamnosus and two L. plantarum were shown to have significant probiotic properties. These strains showed shortening the length of colon compared to dextran sulfate sodium and disease activity index (DAI) was also significantly reduced in mouse.

Conclusion

Low number of LAB with potential probiotic activity as well as high diversity of lactobacilli species was evident in Iranian population. It also suggest that specific strains of L. plantarum, L. brevis and L. rhamnosus with anti-inflammatory effect in mouse model of colitis could be used as a potential probiotic candidate in inflammatory bowel disease to decrease the disease activity index.     

A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2

The development of safe and effective vaccines to prevent SARS-CoV-2 infections remains an urgent priority worldwide. We have used a recombinant vesicular stomatitis virus (rVSV)-based prime-boost immunization strategy to develop an effective COVID-19 vaccine candidate. We have constructed VSV genomes carrying exogenous genes resulting in the production of avirulent rVSV carrying the full-length spike protein (S_F), the S1 subunit, or the receptor-binding domain (RBD) plus envelope (E) protein of SARS-CoV-2. Adding the honeybee melittin signal peptide (msp) to the N-terminus enhanced the protein expression, and adding the VSV G protein transmembrane domain and the cytoplasmic tail (Gtc) enhanced protein incorporation into pseudotype VSV. All rVSVs expressed three different forms of SARS-CoV-2 spike proteins, but chimeras with VSV-Gtc demonstrated the highest rVSV-associated expression. In immunized mice, rVSV with chimeric S protein-Gtc derivatives induced the highest level of potent neutralizing antibodies and T cell responses, and rVSV harboring the full-length msp-S_F-Gtc proved to be the superior immunogen. More importantly, rVSV-msp-S_F-Gtc vaccinated animals were completely protected from a subsequent SARS-CoV-2 challenge. Overall, we have developed an efficient strategy to induce a protective response in SARS-CoV-2 challenged immunized mice. Vaccination with our rVSV-based vector may be an effective solution in the global fight against COVID-19.     

Expression of mouse tyrosine hydroxylase in Escherichia coli.

Enzymatically active mouse tyrosine hydroxylase (TH) was successfully expressed at a high level in Escherichia coli using a T7 RNA polymerase directed expression system. The specific activity of mouse TH in E. coli cell lysate was 7.5 nmol/mg protein/min. Kinetic characteristics of recombinant TH were examined. Km for tyrosine and (6R)-tetrahydrobiopterin (6R-BH4) cofactor were determined to be 7.2 microM (420 microM 6R-BH4), 19 microM [( 6R-BH4] less than 55 microM, 20 microM tyrosine) and 54 microM [( 6R-BH4] greater than 55 microM, 20 microM tyrosine), respectively. These were in good agreement with previously reported values for this enzyme.     

Role of tumor suppressor p53 and micro-RNA interplay in multiple myeloma pathogenesis

The molecular mechanisms underlying dysregulated wild type (wt) p53 in multiple myeloma (MM) have been subjects of intense investigation for years. Indeed, correlation of rarely occurring TP53 gene mutations or deletions with adverse clinical outcomes in MM patients is strongly established, while in majority of cases wtp53 seems to be non-functional or dysregulated bearing a high clinical impact. Interestingly, findings from recent investigations show that micro-RNAs (miRNAs) may contribute to suppression of wtp53 in MM, as they are now known to function as key regulatory elements in the p53 network. This area is shedding new light on understanding the biologic effects of dysregulated p53 in MM pathogenesis especially drug resistance. miRNAs such as miR-125b (oncomiR) or miR-34a (tumor suppressor-miR) can be negative or positive regulators of wtp53 function, respectively, with specific effects on MM cell viability. On the other hand, our knowledge of miRNA interaction with mutant (mt) p53 in MM, which is rather related to disease progression and resistance to therapy, is limited which demands in-depth exploration. Here, we will put forward the current knowledge on miRNA-p53 interaction in MM and its role in MM pathogenesis including drug resistance. We will also highlight the pre-clinical approaches for therapeutic application of miRNAs targeting p53 pathway.     

Effects of some flavonoids on the susceptibility of low-density lipoprotein to oxidative modification

The effects of six flavonoids viz., apigenin, genistein, morin, naringin, pelargonidin and quercetin on the susceptibility of low-density lipoprotein (LDL) to oxidative modification were investigated. Flavonoids were added to plasma and incubated for 3 hr at 37 degrees C, and the LDL fraction was separated by ultracentrifugation. Oxidizability of LDL was estimated by measuring conjugated diene (CD), lipid peroxides and thiobarbituric acid-reactive substances (TBARS), after cupric sulfate solution was added. Quercetin and morin significantly (P<0.01 by ANOVA) prolonged the lag time before initiation of oxidation reaction in dose-dependent manner. They also suppressed the formation of lipid peroxides and TBARS more markedly than other flavonoids. The ability to prolong lag time and suppression of lipid peroxides and TBARS formation was in the following order: quercetin >morin >pelargonidin >genistein >naringin >apigenin. LDL exposed to flavonoids reduced oxidizability. These findings suggest that flavonoids may have a role in ameliorating atherosclerosis.     

Gene transfection of Toxoplasma gondii using PEI/DNA polyplexes

The purpose of this study was to explore the potential of using cationic polyethylenimine (PEI) to deliver green fluorescent protein (GFP) to protozoan parasite Toxoplasma gondii. PEI/DNA polyplexes were formed using branched PEI and pEGFP-N1 plasmid with various N/P ratios that ranged from 5 to 50. With the increment of N/P ratio, the average size of formed PEI/DNA polyplexes determined by dynamic light scattering analysis decreased from 306 to 203nm, while the surface charge of polyplexes obtained by zeta potential measurements increased from 20.2 to 36.7mV. Gene transfection efficiency modulated by N/P ratio was determined, indicating PEI/DNA polyplexes were capable of transfecting parasites. The maximal GFP expression was observed 8h post-transfection using N/P ratio of 30. To demonstrate the infectivity and potential use of GFP-expressing T. gondii, transfected parasites were inoculated to the monolayer of human foreskin fibroblast (HFF) cells. GFP-expressing tachyzoites were observed in intracellular milieu of the infected HFF cells one day after the infection. After 12-day culture, the bradyzoites expressing GFP within cysts were clearly visualized extracellularly. Our results revealed that PEI can be harnessed as an effective and inexpensive reagent to construct GFP-expressing T. gondii which has potential uses such as the study of interconversion stages and antimicrobial drug screening.     

The Interplay of Tau Protein and β-Amyloid: While Tauopathy Spreads More Profoundly Than Amyloidopathy,Both Processes Are Almost Equally Pathogenic

Cellular and Molecular Neurobiology - Alzheimer’s disease (AD) is a neurodegenerative disorder, in which amyloid precursor protein (APP) misprocessing and tau protein hyperphosphorylation are...     

Purification of CD47‐streptavidin fusion protein from bacterial lysate using biotin–agarose affinity chromatography

CD47 is a widely expressed transmembrane glycoprotein that modulates the activity of a plethora of immune cells via its extracellular domain. Therefore, CD47 plays important roles in the regulation of immune responses and may serve as targets for the development of immunotherapeutic agents. To make sure CD47 functionality is intact under the process of protein conjugation, CD47‐streptavidin fusion protein was expressed and purified because it can easily bind to biotin‐tagged materials via the unique biotin–streptavidin affinity. In this study, gene sequences of CD47 extracellular domain (CD47ECD) and core streptavidin (coreSA) with a total 834 bp were inserted into pET20b plasmid to construct recombinant plasmid encoding CD47‐SA fusion gene. After bacteria transformation, the CD47‐SA fusion protein was expressed by isopropyl‐β‐d ‐thiogalactopyranoside (IPTG) induction. The collected bacteria lysate was loaded on biotinylated agarose to proceed the purification of CD47‐SA fusion protein. Due to the unexpected high affinity between biotin and coreSA, standard washing and elution approaches (e.g., varying pH, using biotin, and applying guanidine hydrochloride) reported for biotin–streptavidin affinity chromatography were not able to separate the target fusion protein. Instead, using low concentration of the non‐ionic detergent Triton X‐100 followed with alkaline buffer could efficiently weaken the binding between biotin and coreSA, thereby eluting out CD47‐SA fusion protein from the biotin agarose column. The purified CD47‐SA fusion protein was further characterized by molecular biology methods and its antiphagocytic functionality was confirmed by the phagocytosis assay. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:949–958, 2016