Changing Trends in the Prevalence of Shigella Species: Emergence of Multi-Drug Resistant Shigella sonnei Biotype g in Bangladesh

Shigellosis, caused by Shigella species, is a major public health problem in Bangladesh. To determine the prevalence and distribution of different Shigella species, we analyzed 10,827 Shigella isolates from patients between 2001 and 2011. S. flexneri was the predominant species isolated throughout the period. However, the prevalence of S. flexneri decreased from 65.7% in 2001 to 47% in 2011, whereas the prevalence of S. sonnei increased from 7.2% in 2001 to 25% in 2011. S. boydii and S. dysenteriae accounted for 17.3% and 7.7% of the isolates respectively throughout the period. Of 200 randomly selected S. sonnei isolates for extensive characterization, biotype g strains were predominant (95%) followed by biotype a (5%). Resistance to commonly used antibiotics including trimethoprim-sulfamethoxazole, nalidixic acid, ciprofloxacin, mecillinam and ampicillin was 89.5%, 86.5%, 17%, 10.5%, and 9.5%, respectively. All isolates were susceptible to ceftriaxone, cefotaxime, ceftazidime and imipenem. Ninety-eight percent of the strains had integrons belonging to class 1, 2 or both. The class 1 integron contained only dfrA5 gene, whereas among class 2 integron, 16% contained dhfrAI-sat1-aadA1-orfX gene cassettes and 84% harbored dhfrA1-sat2 gene cassettes. Plasmids of ∼5, ∼1.8 and ∼1.4 MDa in size were found in 92% of the strains, whereas only 33% of the strains carried the 120 MDa plasmid. PFGE analysis showed that strains having different integron patterns belonged to different clusters. These results show a changing trend in the prevalence of Shigella species with the emergence of multidrug resistant S. sonnei. Although S. flexneri continues to be the predominant species albeit with reduced prevalence, S. sonnei has emerged as the second most prevalent species replacing the earlier dominance by S. boydii and S. dysenteriae in Bangladesh.     

Computational Design of the Affinity and Specificity of a Therapeutic T Cell Receptor

T cell receptors (TCRs) are key to antigen-specific immunity and are increasingly being explored as therapeutics, most visibly in cancer immunotherapy. As TCRs typically possess only low-to-moderate affinity for their peptide/MHC (pMHC) ligands, there is a recognized need to develop affinity-enhanced TCR variants. Previous in vitro engineering efforts have yielded remarkable improvements in TCR affinity, yet concerns exist about the maintenance of peptide specificity and the biological impacts of ultra-high affinity. As opposed to in vitro engineering, computational design can directly address these issues, in theory permitting the rational control of peptide specificity together with relatively controlled increments in affinity. Here we explored the efficacy of computational design with the clinically relevant TCR DMF5, which recognizes nonameric and decameric epitopes from the melanoma-associated Melan-A/MART-1 protein presented by the class I MHC HLA-A2. We tested multiple mutations selected by flexible and rigid modeling protocols, assessed impacts on affinity and specificity, and utilized the data to examine and improve algorithmic performance. We identified multiple mutations that improved binding affinity, and characterized the structure, affinity, and binding kinetics of a previously reported double mutant that exhibits an impressive 400-fold affinity improvement for the decameric pMHC ligand without detectable binding to non-cognate ligands. The structure of this high affinity mutant indicated very little conformational consequences and emphasized the high fidelity of our modeling procedure. Overall, our work showcases the capability of computational design to generate TCRs with improved pMHC affinities while explicitly accounting for peptide specificity, as well as its potential for generating TCRs with customized antigen targeting capabilities.     

Fabrication and Intracellular Delivery of Doxorubicin/Carbonate Apatite Nanocomposites: Effect on Growth Retardation of Established Colon Tumor

In continuing search for effective treatments of cancer, the emerging model aims at efficient intracellular delivery of therapeutics into tumor cells in order to increase the drug concentration. However, the implementation of this strategy suffers from inefficient cellular uptake and drug resistance. Therefore, pH-sensitive nanosystems have recently been developed to target slightly acidic extracellular pH environment of solid tumors. The pH targeting approach is regarded as a more general strategy than conventional specific tumor cell surface targeting approaches, because the acidic tumor microclimate is most common in solid tumors. When nanosystems are combined with triggered release mechanisms in endosomal or lysosomal acidic pH along with endosomolytic capability, the nanocarriers demonstrated to overcome multidrug resistance of various tumors. Here, novel pH sensitive carbonate apatite has been fabricated to efficiently deliver anticancer drug Doxorubicin (DOX) to cancer cells, by virtue of its pH sensitivity being quite unstable under an acidic condition in endosomes and the desirable size of the resulting apatite-DOX for efficient cellular uptake as revealed by scanning electron microscopy. Florescence microscopy and flow cytometry analyses demonstrated significant uptake of drug (92%) when complexed with apatite nanoparticles. In vitro chemosensitivity assay revealed that apatite-DOX nanoparticles executed high cytotoxicity in several human cancer cell lines compared to free drugs and consequently apatite-DOX-facilitated enhanced tumor inhibitory effect was observed in colorectal tumor model within BALB/cA nude mice, thereby shedding light on their potential applications in cancer therapy.     

Low dose of bisphenol A impairs the reproductive axis of prepuberal male rats

The objective of the present work was to study the effect of a low dose of bisphenol A (BPA), on the reproductive axis of prepuberal male rats exposed to the endocrine disruptor (ED) during gestation and lactation period. Wistar-mated rats were treated with either 0.1 % ethanol or BPA in their drinking water until their offspring were weaned at the age of 21 days. The estimated average dose of exposure to dams was approximately 3 μg/kg/day of BPA. The pups were sacrificed on the 35th day of life. Body weight was measured during the development and at the moment of the sacrifice; testicular and seminal vesicles weight and their respective relative weights were also measured. LH, FSH and testosterone were determined and histological studies of testicular tissue were also performed. Body weight at the moment of the sacrifice was significantly higher in the group exposed to BPA; testicular weight decreased significantly; seminal vesicles weight and relative weights of testes and seminal vesicles were not modified by treatment. LH and FSH serum levels increased significantly after treatment, meanwhile testosterone showed no significant changes. Histological studies showed the lumen of seminal tubes reduced by the presence of immature cells of the spermatic lineage. Our results suggest that pre- and early postnatal exposure to a low dose of BPA disrupts the normal function of the reproductive axis in prepuberal male rats. The effects of the ED may be exerted at different levels of the axis and may be dependent on the dose, manner of administration, and the moment of exposure to the disruptor.     

18O-labeled proteome reference as global internal standards for targeted quantification by selected reaction monitoring-mass spectrometry

Selected reaction monitoring (SRM)-MS is an emerging technology for high throughput targeted protein quantification and verification in biomarker discovery studies; however, the cost associated with the application of stable isotope-labeled synthetic peptides as internal standards can be prohibitive for screening a large number of candidate proteins as often required in the preverification phase of discovery studies. Herein we present a proof of concept study using an (18)O-labeled proteome reference as global internal standards (GIS) for SRM-based relative quantification. The (18)O-labeled proteome reference (or GIS) can be readily prepared and contains a heavy isotope ((18)O)-labeled internal standard for every possible tryptic peptide. Our results showed that the percentage of heavy isotope ((18)O) incorporation applying an improved protocol was >99.5% for most peptides investigated. The accuracy, reproducibility, and linear dynamic range of quantification were further assessed based on known ratios of standard proteins spiked into the labeled mouse plasma reference. Reliable quantification was observed with high reproducibility (i.e. coefficient of variance <10%) for analyte concentrations that were set at 100-fold higher or lower than those of the GIS based on the light ((16)O)/heavy ((18)O) peak area ratios. The utility of (18)O-labeled GIS was further illustrated by accurate relative quantification of 45 major human plasma proteins. Moreover, quantification of the concentrations of C-reactive protein and prostate-specific antigen was illustrated by coupling the GIS with standard additions of purified protein standards. Collectively, our results demonstrated that the use of (18)O-labeled proteome reference as GIS provides a convenient, low cost, and effective strategy for relative quantification of a large number of candidate proteins in biological or clinical samples using SRM.     

Regulation of Osteoblast Gene Expression and Phenotype by Polylactide-fatty Acid Surfaces

Cell function is influenced by surface structure and molecules. Molecules that enhance cellular differentiation can be applied to tissue scaffold surfaces to stimulate endogenous tissue regeneration. The application of this approach to bone implants yields surfaces coated with factors (proteins, peptides, etc...) that promote the differentiation of osteoblasts, the cells that make bone. Increased bone formation leads to increased healing and union of the implant with endogenous bone. To obtain better control over surface coating we developed PLLA copolymers with allyl (PLLA-co-DAG) and 3-hydroxypropyl (PLLA-co-HP) side chains to which we can attach functional groups. Given the potential of fatty acids being able to incorporate into lipid bilayers and/or influence gene expression, we grafted different fatty acid side chains to PLLA-co-HP by esterifying the corresponding fatty acids with the PLLA-co-HP 3-hydroxypropyl side chains. The effects of the polymer modifications on osteoblasts were then evaluated. While cellular morphology differed between surface coatings, they did not reflect changes in cellular phenotype. Changes in gene expression were most evident with arachidonate and 3-hydroxypropyl side-chains which exhibited osteoblast differentiating capabilities. Linoleate, myristate, oleate, and stearate ester side-chains did not have a significant influence on osteoblast phenotype. Growth characteristics of osteoblasts did not differ between the fatty acid copolymer films, although cells grown on PLLA-co-HP exhibited a trend toward increased growth. Taken together our findings demonstrate that surface fatty acid composition can impact osteoblast phenotype.     

Survival of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> O1 on fomites

It is well established that the contamination sources of cholera causing bacteria, Vibrio cholerae, are water and food, but little is known about the transmission role of the fomites (surfaces that can carry pathogens) commonly used in households. In the absence of appropriate nutrients or growth conditions on fomites, bacteria have been known to assume a viable but non-culturable (VBNC) state after a given period of time. To investigate whether and when V. cholerae O1 assumes such a state, this study investigated the survival and viable quantification on a range of fomites such as paper, wood, glass, plastic, cloth and several types of metals under laboratory conditions. The fomites were inoculated with an outbreak strain of V. cholerae and its culturability was examined by drop plate count method at 30 min intervals for up to 6 h. For molecular detection, the viable/dead stain ethidium monoazide (EMA) which inhibits amplification of DNA from dead cells was used in combination with real-time polymerase chain reaction (EMA-qPCR) for direct quantitative analyses of viable V. cholerae at 2, 4, 6, 24 h and 7 day time intervals. Results showed that V. cholerae on glass and aluminum surfaces lost culturability within one hour after inoculation but remained culturable on cloth and wood for up to four hours. VBNC V. cholerae on dry fomite surfaces was detected and quantified by EMA-qPCR even 7 days after inoculation. In conclusion, the prolonged survival of V. cholerae on various household fomites may play vital role in cholera transmission and needs to be further investigated.     

Induction of glucoamylase production by non-starchy carbohydrates inAspergillus terreus

Glucoamylase production inAspergillus terreus was induced, in order, by glucose, cellobiose, sorbitol, sucrose, -methyl mannoside and -methyl glucoside. Optimal induction was at 38°C, pH 4.0 and with 8 mg glucose/ml. Cycloheximide at 10 g/ml completely inhibited induction indicatingde novo protein synthesis was involved in induction of glucoamylase.

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Résumé La production de gluco-amylase est induite chezAspergillus terreus, en ordre décroissant, par le glucose, le celloboise, le sorbitol, le sucrose, l'-methylmannoside et l'-methylglucoside. L'induction est optimum à 38°C, pH 4.0 et en présence de 8 mg de glucose parml. La cycloheximide à 10 g par ml inhibe complètement l'induction, ce qui implique use synthèse de protéinede novo dans l'induction de la gluco-amylase.

     

The astrocyte-expressed integrin αvβ8 governs blood vessel sprouting in the developing retina

The mouse retina is vascularized after birth when angiogenic blood vessels grow and sprout along a pre-formed latticework of astrocytes. How astrocyte-derived cues control patterns of blood vessel growth and sprouting, however, remains enigmatic. Here, we have used molecular genetic strategies in mice to demonstrate that αvβ8 integrin expressed in astrocytes is essential for neovascularization of the developing retina. Selective ablation of αv or β8 integrin gene expression in astrocytes leads to impaired blood vessel sprouting and intraretinal hemorrhage, particularly during formation of the secondary vascular plexus. These pathologies correlate, in part, with diminished αvβ8 integrin-mediated activation of extracellular matrix-bound latent transforming growth factor βs (TGFβs) and defective TGFβ signaling in vascular endothelial cells, but not astrocytes. Collectively, our data demonstrate that αvβ8 integrin is a component of a paracrine signaling axis that links astrocytes to blood vessels and is essential for proper regulation of retinal angiogenesis.     

Flacourtia indica fruit extract modulated antioxidant gene expression,prevented oxidative stress and ameliorated kidney dysfunction in isoprenaline administered rats

This study evaluated the effect of Flacourtia indica fruit extract against isoprenaline (ISO) induced renal damage in rats. This investigation showed that ISO administration in rats increased the level oxidative stress biomarkers such as malondialdehyde (MDA), nitric oxide (NO), advanced protein oxidation product (APOP) in kidneys followed by a decrease in antioxidant enzymes functions. Flacourtia indica fruit extract, which is rich in strong antioxidants, also reduced the MDA, NO and APOP level in kidney of ISO administered rats. Inflammation and necrosis was also visible in kidney section of ISO administered rats which was significantly prevented by atenolol and Flacourtia indica fruit extract. Moreover, atenolol and Flacourtia indica fruit extract also modulated the genes expressions related to inflammation and oxidative stress in kidneys. The beneficial effects could be attributed to the presence of a number of phenolic antioxidants. This study suggests that Flacourtia indica fruit extract may prevent kidney dysfunction in ISO administered rats, probably by preventing oxidative stress and inflammation.