Synergistic Effects of Clostridium perfringens Enterotoxin and Beta Toxin in Rabbit Small Intestinal Loops

The ability of Clostridium perfringens type C to cause human enteritis necroticans (EN) is attributed to beta toxin (CPB). However, many EN strains also express C. perfringens enterotoxin (CPE), suggesting that CPE could be another contributor to EN. Supporting this possibility, lysate supernatants from modified Duncan-Strong sporulation (MDS) medium cultures of three CPE-positive type C EN strains caused enteropathogenic effects in rabbit small intestinal loops, which is significant since CPE is produced only during sporulation and since C. perfringens can sporulate in the intestines. Consequently, CPE and CPB contributions to the enteropathogenic effects of MDS lysate supernatants of CPE-positive type C EN strain CN3758 were evaluated using isogenic cpb and cpe null mutants. While supernatants of wild-type CN3758 MDS lysates induced significant hemorrhagic lesions and luminal fluid accumulation, MDS lysate supernatants of the cpb and cpe mutants caused neither significant damage nor fluid accumulation. This attenuation was attributable to inactivating these toxin genes since complementing the cpe mutant or reversing the cpb mutation restored the enteropathogenic effects of MDS lysate supernatants. Confirming that both CPB and CPE are needed for the enteropathogenic effects of CN3758 MDS lysate supernatants, purified CPB and CPE at the same concentrations found in CN3758 MDS lysates also acted together synergistically in rabbit small intestinal loops; however, only higher doses of either purified toxin independently caused enteropathogenic effects. These findings provide the first evidence for potential synergistic toxin interactions during C. perfringens intestinal infections and support a possible role for CPE, as well as CPB, in some EN cases.     

Symptoms of psychological distress reported by women from indigenous communities in South India: implications for methodology and future studies

Archives of Women's Mental Health - ‘Indigenous peoples’ across the globe suffer a disproportionate burden of mental illness. However, this burden is not fully explored in India...     

High-resolution voxelation mapping of human and rodent brain gene expression

Voxelation allows high-throughput acquisition of multiple volumetric images of brain gene expression, similar to those obtained from biomedical imaging systems. To obtain these images, the method employs analysis of spatially registered voxels (cubes). For creation of high-resolution maps using voxelation, relatively small voxel sizes are necessary and instruments will be required for semiautomated harvesting of such voxels. Here, we describe two devices that allow spatially registered harvesting of voxels from the human and rodent brain, giving linear resolutions of 3.3 and 1 mm, respectively. Gene expression patterns obtained using these devices showed good agreement with known expression patterns. The voxelation instruments and their future iterations represent a valuable approach to the genome scale acquisition of gene expression patterns in the human and rodent brain.     

Expression of CD105 and CD34 receptors controls BMP‐induced in vitro mineralization of mouse adipose‐derived stem cells but does not predict their in vivo bone‐forming potential

Adipose‐derived stem cells (ADSCs) can be excellent alternative to bone marrow derived stem cells for enhancing fracture repair since ADSCs can be isolated comparatively in large numbers from discarded lipoaspirates. However, osteogenic potential of ADSCs in vivo is very controversial. We hypothesized that adipose‐derived stem cells (ADSCs) that respond maximally to bone morphogenetic proteins (BMPs) in vitro would possess maximum bone‐forming potential. Four purified populations of mouse ADSCs: CD105⁺CD34⁺, CD105^?CD34^?, CD105⁺CD34^? and CD105^?CD34⁺ were obtained using fluorescence‐activated cell sorting (FACS) and their BMP‐responsiveness was determined in vitro. CD105⁺CD34^? population showed the strongest response to BMPs in terms of robust increase in mineralization. Expression of CD105 correlated with high BMP‐responsive phenotype and larger cell size while expression of CD34 correlated with low BMP‐responsive phenotype and smaller cell size. CD105⁺CD34^? population displayed higher gene expression of Alk1 or Alk6 receptors in comparison with other populations. However, CD105⁺CD34^? ADSCs failed to induce ectopic bone formation in vivo after they were transplanted into syngeneic mice, indicating that in vitro BMP‐responsiveness is not a good indicator to predict in vivo bone forming potential of ADSCs. Therefore greater precautions should be executed during selection of competent ADSCs for bone repair. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:625–632, 2015.

     

Recognition, co-internalization, and recycling of an avian riboflavin carrier protein in human placental trophoblasts

Absorption of riboflavin (RF) across membrane barriers is essential to cellular oxidation reduction processes. Riboflavin carrier protein (RCP), a 37-kDa secretory protein, is proposed to play an important role in RF absorption, although information on the mammalian ortholog remains unclear. This study alludes to the existence of a mammalian RF carrier protein and further characterizes its carrier role and fate using avian RCP in human placental trophoblast (BeWo), another mammalian cell line, monkey kidney (COS-1), and the avian control, chicken hepatic (LMH/2A) cells. The presence of RCP and its involvement in RF internalization was analyzed by immunofluorescence and immunobinding assays using chicken RCP (cRCP) antibodies. In the presence of anti-cRCP, cellular RF uptake is significantly decreased (5% of control) in BeWo cells. Kinetic analyses of intracellular accumulation of (125)I-cRCP revealed a J(max) and K(m) of 28.56 +/- 2.70 pmol/mg protein/min and 142.43 +/- 82.16 nM, respectively, in BeWo cells and 75.14 +/- 7.6 pmol/mg protein/min and 104.37 +/- 23.96 nM in the species-specific control, LMH/2A cells. Subcellular fractionation studies revealed colocalization of both radiolabeled RF and cRCP within endosomal and lysosomal fractions, further elucidating RCP's role in trafficking RF through the cell. Following intracellular release of RF from the carrier complex, the protein is either subject to lysosomal breakdown or is conserved via recycling mechanisms for continued RF sequestration and uptake. In summary, mammalian placental trophoblasts exhibit specific carrier protein dependence that sequesters and essentially mediates RF internalization via the proposed receptor-mediated endocytic pathway.     

Anti-Helicobacter pylori therapy in India: differences in eradication efficiency associated with particular alleles of vacuolating cytotoxin (vacA) gene

BACKGROUND AND AIMS: The efficiency of Helicobacter pylori eradication varies geographically, as do many parameters that might affect therapeutic efficiency, including bacterial genotype. The aim of the present study was to determine the efficiency of H. pylori eradication using a 10-day proton pump inhibitor-based triple-therapy regimen (omeprazole, clarithromycin and amoxycillin) in an eastern Indian patient population, and to find out the relationship, if any, of the success or failure of the therapy to known features of bacterial genotype. METHODS: Helicobacter pylori infections were analyzed in 66 duodenal ulcer patients by upper gastrointestinal endoscopy, rapid urease tests, histology and culture. The cytotoxin-associated gene (cagA) and vacuolating cytotoxin (vacA) gene status of cultured strains were studied by polymerase chain reaction. Treatment was given for 10 days and endoscopy was repeated at 4 and 12 weeks post therapy to monitor ulcer healing and H. pylori eradication. RESULTS: Ulcer healing was observed in 60 patients (96.77%). Helicobacter pylori was eradicated in 41 (62.12% intention to treat, 66.13% per protocol) of the 66 duodenal ulcer patients, but not in the other 25. The bacteria from 47 patients were genotyped. The only significant disease-associated difference in patterns observed was that the vacA m1 allele was represented more disproportionately among patients with eradication failures (68%) than in those with successful eradication (39%) (P < 0.05) No significant association of vacAs1 (signal sequence allele) or cag pathogenicity island status with persistence was detected. CONCLUSIONS: This study highlights the public health need for cheaper, more cost-effective anti-H. pylori therapies for developing countries, and suggests that subtle features of bacterial genotype can influence therapeutic efficiency. The possibility that particular vacA mid region alleles affect persistence, perhaps through toxin action on particular gastric cell types, merits further study.     

Interaction of erythroid spectrin with hemoglobin variants: implications in beta-thalassemia

Among the few studies, producing contradictory results, done on the interaction of erythroid membrane skeletal spectrin with hemoglobin (Hb), none has been able to provide a quantitative estimate of the association of spectrin with Hb. In this work, studies on the interactions of erythroid spectrin with Hb have been elaborated upon using a novel fluorescence technique. The concentration-dependent change in the fluorescence intensity of fluorescein-conjugated spectrin (F-spectrin) in presence of oxy-Hb indicated binding with a dissociation constant of approximately 20 microM that has been directly evaluated from the increase in the extent of quenching of the fluorescein fluorescence of F-spectrin by reverse titration with the increasing concentrations of different Hb samples isolated from both normal and beta-thalassemic patients. The Hb compositions, with major components of the normal HbA, the fetal HbF, and the variant HbA2, of each individual were estimated using the Variant HPLC device of Bio-Rad. Results of the present study indicated that the dissociation constant, K(d), of spectrin binding to Hb decreased from 19.5 +/- 2 microM in normal individuals to of 6.5 +/- 0.5 microM in the presence of 73% HbA2 along with coeluted variants in the blood samples of patients suffering from beta-thalassemia, indicating differential interactions of the Hb variants with spectrin.     

Death-associated protein kinase 1 promotes growth of p53-mutant cancers

Estrogen receptor–negative (ER-negative) breast cancers are extremely aggressive and associated with poor prognosis. In particular, effective treatment strategies are limited for patients diagnosed with triple receptor–negative breast cancer (TNBC), which also carries the worst prognosis of all forms of breast cancer; therefore, extensive studies have focused on the identification of molecularly targeted therapies for this tumor subtype. Here, we sought to identify molecular targets that are capable of suppressing tumorigenesis in TNBCs. Specifically, we found that death-associated protein kinase 1 (DAPK1) is essential for growth of p53-mutant cancers, which account for over 80% of TNBCs. Depletion or inhibition of DAPK1 suppressed growth of p53-mutant but not p53-WT breast cancer cells. Moreover, DAPK1 inhibition limited growth of other p53-mutant cancers, including pancreatic and ovarian cancers. DAPK1 mediated the disruption of the TSC1/TSC2 complex, resulting in activation of the mTOR pathway. Our studies demonstrated that high DAPK1 expression causes increased cancer cell growth and enhanced signaling through the mTOR/S6K pathway; evaluation of multiple breast cancer patient data sets revealed that high DAPK1 expression associates with worse outcomes in individuals with p53-mutant cancers. Together, our data support targeting DAPK1 as a potential therapeutic strategy for p53-mutant cancers.