RNA–Stable-Isotope Probing Shows Utilization of Carbon from Inulin by Specific Bacterial Populations in the Rat Large Bowel

Knowledge of the trophisms that underpin bowel microbiota composition is required in order to understand its complex phylogeny and function. Stable-isotope (¹³C)-labeled inulin was added to the diet of rats on a single occasion in order to detect utilization of inulin-derived substrates by particular members of the cecal microbiota. Cecal digesta from Fibruline-inulin-fed rats was collected prior to (0 h) and at 6, 12, 18 and 24 h following provision of the [¹³C]inulin diet. RNA was extracted from these cecal specimens and fractionated in isopycnic buoyant density gradients in order to detect ¹³C-labeled nucleic acid originating in bacterial cells that had metabolized the labeled dietary constituent. RNA extracted from specimens collected after provision of the labeled diet was more dense than 0-h RNA. Sequencing of 16S rRNA genes amplified from cDNA obtained from these fractions showed that Bacteroides uniformis, Blautia glucerasea, Clostridium indolis, and Bifidobacterium animalis were the main users of the ¹³C-labeled substrate. Culture-based studies of strains of these bacterial species enabled trophisms associated with inulin and its hydrolysis products to be identified. B. uniformis utilized Fibruline-inulin for growth, whereas the other species used fructo-oligosaccharide and monosaccharides. Thus, RNA–stable-isotope probing (RNA-SIP) provided new information about the use of carbon from inulin in microbiota metabolism.     

Murine splenocyte proliferation by porin of Shigella dysenteriae type 1 and inhibition of bacterial invasion of HeLa cell by anti-porin antibody

Abstract A highly sensitive nested polymerase chain reaction method was designed for the detection of a wide spectrum of strains from Borrelia burgdorferi sensu lato. This technique allows the detection of as little as 3 fg of total genomic DNA extracted and purified from pure cultures of the organism, this amount corresponds to less than 10 organisms. Two sets of primers homologous to conserved spots in the coding region of the hbb gene, encoding a conserved histone-like protein, were constructed. These were based on a multiple sequence alignment of 39 strains representing all the genomic groups described in B. burgdorferi sensu lato.     

Mitochondrial Reshaping Accompanies Neural Differentiation in the Developing Spinal Cord

Mitochondria, long known as the cell powerhouses, also regulate redox signaling and arbitrate cell survival. The organelles are now appreciated to exert additional critical roles in cell state transition from a pluripotent to a differentiated state through balancing glycolytic and respiratory metabolism. These metabolic adaptations were recently shown to be concomitant with mitochondrial morphology changes and are thus possibly regulated by contingencies of mitochondrial dynamics. In this context, we examined, for the first time, mitochondrial network plasticity during the transition from proliferating neural progenitors to post-mitotic differentiating neurons. We found that mitochondria underwent morphological reshaping in the developing neural tube of chick and mouse embryos. In the proliferating population, mitochondria in the mitotic cells lying at the apical side were very small and round, while they appeared thick and short in interphase cells. In differentiating neurons, mitochondria were reorganized into a thin, dense network. This reshaping of the mitochondrial network was not specific of a subtype of progenitors or neurons, suggesting that this is a general event accompanying neurogenesis in the spinal cord. Our data shed new light on the various changes occurring in the mitochondrial network during neurogenesis and suggest that mitochondrial dynamics could play a role in the neurogenic process.     

DNA methylation in Neisseria gonorrhoeae and other Neisseriae

It has been reported in the literature that Neisseria gonorrhoeae DNA is modified by the methyltransferases (MTases) M.NgoI, M.NgoII, and M.NgoIII, as well as three other cytosine MTases and one adenine MTase, even if the corresponding restriction endonucleases are not present. We envisioned the possibility of cloning one of the N. gonorrhoeae MTase-encoding genes for use as a species-specific DNA probe. We therefore undertook a survey of methylation patterns of several clinical isolates of N. gonorrhoeae and N. meningitidis as well as ATCC strains of other Neisseriae. We found, from digestion patterns with isoschizomers, one N. gonorrhoeae strain that lacked M.NgoII and two that lacked M.NgoIII. All N. meningitidis strains (save one) were resistant to digestion with NlaIV thus possessing an MTase like NgoV, and one was resistant to SstII, thus having an NgoIII-like MTase. None were resistant to isoschizomers of NgoI, NgoIII and NgoIV. Some other Neisseriae had an MTase with NlaIV (NgoV) specificity, but none had NgoI, II, III or IV specificity, except for the Branhamella-like N. caviae-ovis group and N. lactamica where these specificities were present in at least one strain of this group. Therefore, among the Neisseriae other than N. caviae only M.NgoI is N. gonorrhoeae-specific.     

Flavone-resistant Leishmania donovani Overexpresses LdMRP2 Transporter in the Parasite and Activates Host MRP2 on Macrophages to Circumvent the Flavone-mediated Cell Death

In parasites, ATP-binding cassette (ABC) transporters represent an important family of proteins related to drug resistance and other biological activities. Resistance of leishmanial parasites to therapeutic drugs continues to escalate in developing countries, and in many instances, it is due to overexpressed ABC efflux pumps. Progressively adapted baicalein (BLN)-resistant parasites (pB²⁵R) show overexpression of a novel ABC transporter, which was classified as ABCC2 or Leishmania donovani multidrug resistance protein 2 (LdMRP2). The protein is primarily localized in the flagellar pocket region and in internal vesicles. Overexpressed LdABCC2 confers substantial BLN resistance to the parasites by rapid drug efflux. The BLN-resistant promastigotes when transformed into amastigotes in macrophage cells cannot be cured by treatment of macrophages with BLN. Amastigote resistance is concomitant with the overexpression of macrophage MRP2 transporter. Reporter analysis and site-directed mutagenesis assays demonstrated that antioxidant response element 1 is activated upon infection. The expression of this phase II detoxifying gene is regulated by NFE2-related factor 2 (Nrf2)-mediated antioxidant response element activation. In view of the fact that the signaling pathway of phosphoinositol 3-kinase controls microfilament rearrangement and translocation of actin-associated proteins, the current study correlates with the intricate pathway of phosphoinositol 3-kinase-mediated nuclear translocation of Nrf2, which activates MRP2 expression in macrophages upon infection by the parasites. In contrast, phalloidin, an agent that prevents depolymerization of actin filaments, inhibits Nrf2 translocation and Mrp2 gene activation by pB²⁵R infection. Taken together, these results provide insight into the mechanisms by which resistant clinical isolates of L. donovani induce intracellular events relevant to drug resistance.     

Structural Maintenance of Chromosome (SMC) Proteins Link Microtubule Stability to Genome Integrity

Structural maintenance of chromosome (SMC) proteins are key organizers of chromosome architecture and are essential for genome integrity. They act by binding to chromatin and connecting distinct parts of chromosomes together. Interestingly, their potential role in providing connections between chromatin and the mitotic spindle has not been explored. Here, we show that yeast SMC proteins bind directly to microtubules and can provide a functional link between microtubules and DNA. We mapped the microtubule-binding region of Smc5 and generated a mutant with impaired microtubule binding activity. This mutant is viable in yeast but exhibited a cold-specific conditional lethality associated with mitotic arrest, aberrant spindle structures, and chromosome segregation defects. In an in vitro reconstitution assay, this Smc5 mutant also showed a compromised ability to protect microtubules from cold-induced depolymerization. Collectively, these findings demonstrate that SMC proteins can bind to and stabilize microtubules and that SMC-microtubule interactions are essential to establish a robust system to maintain genome integrity.     

Engineered Silybin Nanoparticles Educe Efficient Control in Experimental Diabetes

Silybin, is one imminent therapeutic for drug induced hepatotoxicity, human prostrate adenocarcinoma and other degenerative organ diseases. Recent evidences suggest that silybin influences gluconeogenesis pathways favorably and is beneficial in the treatment of type 1 and type 2 diabetes. The compound however is constrained due to solubility (0.4 mg/mL) and bioavailabilty limitations. Appropriate nanoparticle design for silybin in biocompatible polymers was thus proposed as a probable solution for therapeutic inadequacy. New surface engineered biopolymeric nanoparticles with high silybin encapsulation efficiency of 92.11% and zeta potential of +21 mV were designed. Both the pure compound and the nanoparticles were evaluated in vivo for the first time in experimental diabetic conditions. Animal health recovered substantially and the blood glucose levels came down to near normal values after 28 days treatment schedule with the engineered nanoparticles. Restoration from hyperglycemic damage condition was traced to serum insulin regeneration. Serum insulin recovered from the streptozotocin induced pancreatic damage levels of 0.17±0.01 µg/lit to 0.57±0.11 µg/lit after nanoparticle treatment. Significant reduction in glycated hemoglobin level, and restoration of liver glycogen content were some of the other interesting observations. Engineered silybin nanoparticle assisted recovery in diabetic conditions was reasoned due to improved silybin dissolution, passive transport in nanoscale, and restoration of antioxidant status.     

Castration Induces Parkinson Disease Pathologies in Young Male Mice via Inducible Nitric-oxide Synthase

Although Parkinson disease (PD) is a progressive neurodegenerative disorder, available animal models do not exhibit irreversible neurodegeneration, and this is a major obstacle in finding out an effective drug against this disease. Here we delineate a new irreversible model to study PD pathogenesis. The model is based on simple castration of young male mice. Levels of inducible nitric-oxide synthase (iNOS), glial markers (glial fibrillary acidic protein and CD11b), and α-synuclein were higher in nigra of castrated male mice than normal male mice. On the other hand, after castration, the level of glial-derived neurotrophic factor (GDNF) markedly decreased in the nigra of male mice. Accordingly, castration also induced the loss of tyrosine hydroxylase-positive neurons in the nigra and decrease in tyrosine hydroxylase-positive fibers and neurotransmitters in the striatum. Reversal of nigrostriatal pathologies in castrated male mice by subcutaneous implantation of 5α-dihydrotestosterone pellets validates an important role of male sex hormone in castration-induced nigrostriatal pathology. Interestingly, castration was unable to cause glial activation, decrease nigral GDNF, augment the death of nigral dopaminergic neurons, induce the loss of striatal fibers, and impair neurotransmitters in iNOS^−/− male mice. Furthermore, we demonstrate that iNOS-derived NO is responsible for decreased expression of GDNF in activated astrocytes. Together, our results suggest that castration induces nigrostriatal pathologies via iNOS-mediated decrease in GDNF. These results are important because castrated young male mice may be used as a simple, toxin-free, and nontransgenic animal model to study PD-related nigrostriatal pathologies, paving the way for easy drug screening against PD.