Development and Characterization of an Antibody-Labeled Super-Paramagnetic Iron Oxide Contrast Agent Targeting Prostate Cancer Cells for Magnetic Resonance Imaging

In this study we developed, characterized and validated in vitro a functional superparagmagnetic iron-oxide based magnetic resonance contrast agent by conjugating a commercially available iron oxide nanoparticle, Molday ION Rhodamine-B Carboxyl (MIRB), with a deimmunized mouse monoclonal antibody (muJ591) targeting prostate-specific membrane antigen (PSMA). This functional contrast agent is intended for the specific and non-invasive detection of prostate cancer cells that are PSMA positive, a marker implicated in prostate tumor progression and metastasis. The two-step carbodiimide reaction used to conjugate the antibody to the nanoparticle was efficient and we obtained an elemental iron content of 1958±611 per antibody. Immunofluorescence microscopy and flow cytometry showed that the conjugated muJ591:MIRB complex specifically binds to PSMA-positive (LNCaP) cells. The muJ591:MIRB complex reduced cell adhesion and cell proliferation on LNCaP cells and caused apoptosis as tested by Annexin V assay, suggesting anti-tumorigenic characteristics. Measurements of the T2 relaxation time of the muJ591:MIRB complex using a 400 MHz Innova NMR and a multi-echo spin-echo sequence on a 3T MRI (Achieva, Philips) showed a significant T2 relaxation time reduction for the muJ591:MIRB complex, with a reduced T2 relaxation time as a function of the iron concentration. PSMA-positive cells treated with muJ591:MIRB showed a significantly shorter T2 relaxation time as obtained using a 3T MRI scanner. The reduction in T2 relaxation time for muJ591:MIRB, combined with its specificity against PSMA+LNCaP cells, suggest its potential as a biologically-specific MR contrast agent.     

Structural Characteristic of the Initial Unfolded State on Refolding Determines Catalytic Efficiency of the Folded Protein in Presence of Osmolytes

Osmolytes are low molecular weight organic molecules accumulated by organisms to assist proper protein folding, and to provide protection to the structural integrity of proteins under denaturing stress conditions. It is known that osmolyte-induced protein folding is brought by unfavorable interaction of osmolytes with the denatured/unfolded states. The interaction of osmolyte with the native state does not significantly contribute to the osmolyte-induced protein folding. We have therefore investigated if different denatured states of a protein (generated by different denaturing agents) interact differently with the osmolytes to induce protein folding. We observed that osmolyte-assisted refolding of protein obtained from heat-induced denatured state produces native molecules with higher enzyme activity than those initiated from GdmCl- or urea-induced denatured state indicating that the structural property of the initial denatured state during refolding by osmolytes determines the catalytic efficiency of the folded protein molecule. These conclusions have been reached from the systematic measurements of enzymatic kinetic parameters (K _m and k _cat), thermodynamic stability (T _m and ΔH _m) and secondary and tertiary structures of the folded native proteins obtained from refolding of various denatured states (due to heat-, urea- and GdmCl-induced denaturation) of RNase-A in the presence of various osmolytes.     

Association of Common Genetic Variants with Lipid Traits in the Indian Population

Genome-wide association studies (GWAS) have been instrumental in identifying novel genetic variants associated with altered plasma lipid levels. However, these quantitative trait loci have not been tested in the Indian population, where there is a poorly understood and growing burden of cardiometabolic disorders. We present the association of six single nucleotide polymorphisms in 1671 sib pairs (3342 subjects) with four lipid traits: total cholesterol, triglycerides, high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C). We also investigated the interaction effects of gender, location, fat intake and physical activity. Each copy of the risk allele of rs964184 at APOA1 was associated with 1.06 mmol/l increase in triglycerides (SE = 0.049; p = 0.006), rs3764261 at CETP with 1.02 mmol/l increase in both total cholesterol (SE = 0.042; p = 0.017) and HDL-C (SE = 0.041; p = 0.008), rs646776 at CELSR2-PSRC1-SORT1 with 0.96 mmol/l decrease in cholesterol (SE = 0.043; p = 0.0003) and 0.15 mmol/l decrease in LDL-C levels (SE = 0.043; p = 0.0003) and rs2954029 at TRIB1 with 1.02 mmol/l increase in HDL-C (SE = 0.039; p = 0.047). A combined risk score of APOA1 and CETP loci predicted an increase of 1.25 mmol/l in HDL-C level (SE = 0.312; p = 0.0007). Urban location and sex had strong interaction effects on the genetic association of most of the studied loci with lipid traits. To conclude, we validated four genetic variants (identified by GWAS in western populations) associated with lipid traits in the Indian population. The interaction effects found here may explain the sex-specific differences in lipid levels and their heritability. Urbanization appears to influence the nature of the association with GWAS lipid loci in this population. However, these findings will require replication in other Indian populations.     

Fibronectin binding and cell-surface hydrophobicity of attaching effacing enteropathogenic Escherichia coli strains isolated from newborn and weanling rabbits with diarrhoea

Abstract 32 different strains of Escherichia coli isolated from rabbits with diarrhoea were studied for cell-surface properties which may be involved in intestinal colonisation. Strains isolated from diarrhoeic suckling (6 strains) and weaning (26 strains) rabbits which were shown to attach to brush borders in vivo, showed high relative cell-surface hydrophobicity as determined by the Salt Aggregation Test (SAT) when grown on Colonisation Factor Antigen (CFA) agar at 33°C. Cells of these strains grown to express surface hydrophobicity were also defined as high, moderate or low binders of ¹²⁵I-fibronectin or its ¹²⁵I-29-kDa fragment in a standard binding assay. Based on these findings, we propose that binding to intestinal cell surface (mucus)-associated fibronectin may be an early important step in intestinal colonisation of the small bowel in enteropathogenic E. coli (EPEC) diarrhoea in rabbits and other animal species.     

Crystal structure of ferrioxamine B: a comparative analysis and implications for molecular recognition

Ferrioxamine B was successfully co-crystallized with ethanolpentaaquomagnesium(II) and perchlorate ions as counter ions, C27H62Cl3FeMgN6O26, and the crystal structure has been determined by single-crystal X-ray diffraction. The crystals are monoclinic, space group P2(1)/n, four molecules per unit cell with dimensions a=21.1945(7) A, b=10.0034(3) A, c=106.560(1) A, and beta=106.560(1) degrees. The crystal structure contains a racemic mixture of Lambda-N-cis,cis and Delta-N-cis,cis coordination isomers. The structural parameters and the conformational features of ferrioxamine B compare very well with those of ferrioxamines D1 and E, with an exception of the orientation of the pendant protonated amine, which is pointing away from the connecting amide chains and towards the carbonyl face of the inner coordination shell distorted octahedron. This pendant protonated amine, in conjunction with the carbonyl face of the Fe(III) coordination shell, is proposed to play an important role in the recognition and membrane transport processes.     

Maintenance of Sex-Related Genes and the Co-Occurrence of Both Mating Types in Verticillium dahliae

Verticillium dahliae is a cosmopolitan, soilborne fungus that causes a significant wilt disease on a wide variety of plant hosts including economically important crops, ornamentals, and timber species. Clonal expansion through asexual reproduction plays a vital role in recurring plant epidemics caused by this pathogen. The recent discovery of recombination between clonal lineages and preliminary investigations of the meiotic gene inventory of V. dahliae suggest that cryptic sex appears to be rare in this species. Here we expanded on previous findings on the sexual nature of V. dahliae. Only 1% of isolates in a global collection of 1120 phytopathogenic V. dahliae isolates contained the MAT1-1 idiomorph, whereas 99% contained MAT1-2. Nine unique multilocus microsatellite types comprised isolates of both mating types, eight of which were collected from the same substrate at the same time. Orthologs of 88 previously characterized sex-related genes from fungal model systems in the Ascoymycota were identified in the genome of V. dahliae, out of 93 genes investigated. Results of RT-PCR experiments using both mating types revealed that 10 arbitrarily chosen sex-related genes, including MAT1-1-1 and MAT1-2-1, were constitutively expressed in V. dahliae cultures grown under laboratory conditions. Ratios of non-synonymous (amino-acid altering) to synonymous (silent) substitutions in V. dahliae MAT1-1-1 and MAT1-2-1 sequences were indistinguishable from the ratios observed in the MAT genes of sexual fungi in the Pezizomycotina. Patterns consistent with strong purifying selection were also observed in 18 other arbitrarily chosen V. dahliae sex-related genes, relative to the patterns in orthologs from fungi with known sexual stages. This study builds upon recent findings from other laboratories and mounts further evidence for an ancestral or cryptic sexual stage in V. dahliae.     

The Role of CHI3L1 (Chitinase-3-Like-1) in the Pathogenesis of Infections in Burns in a Mouse Model

In severe burn injury the unique setting of a depleted, dysfunctional immune system along with a loss of barrier function commonly results in opportunistic infections that eventually proof fatal. Unfortunately, the dynamic sequence of bacterial contamination, colonization and eventually septic invasion with bacteria such as Pseudomonas species is still poorly understood although a limiting factor in clinical decision making. Increasing evidence supports the notion that inhibition of bacterial translocation into the wound site may be an effective alternative to prevent infection. In this context we investigated the role of the mammalian Chitinase-3-Like-1 (CHI3L1) non-enyzmatic protein predominately expressed on epithelial as well as innate immune cells as a potential bacterial-translocation-mediating factor. We show a strong trend that a modulation of chitinase expression is likely to be effective in reducing mortality rates in a mouse model of burn injury with superinfection with the opportunistic PA14 Pseudomonas strain, thus demonstrating possible clinical leverage.     

Protective and therapeutic efficacy study of divalent fusion protein rL7/L12-Omp25 against B. abortus 544 in presence of IFNγ

Brucella as intracellular pathogen requires a coordinate interaction between Th1 subset of gamma interferon-secreting CD4 T cells and CD8 T cells for optimal protective immunity. It was previously recognized that L7/L12 as T cell-reactive antigen from the pathogen. On other hand, Omp25 was found as another antigen to provide protection against the Brucella infection by eliciting both Th1 and Th2 type of immune responses in mice. Here, we analyzed the prophylactic and therapeutic efficacy of a divalent fusion protein (rL7/L12-Omp25) comprising these two promising immunogens of Brucella in the presence of murine IFN-gamma in mice against B. abortus 544 challenge. rIFN-gamma with rL7/L12-Omp25 resulted in superior immune response when compared to the animal vaccine strain B. abortus S19. The vaccine candidate caused dominance of IgG1 over IgG2a and upregulated cytokine secretion (IFN-gamma, TNF-α, and IL-10) among immunized mice. Moreover, the antigen in combination with murine IFN-gamma elicited stronger cell-mediated immune response among the immunized animals when compared to standard vaccine (S19). The registered log protection unit among challenged mice with B. abortus 544 pathogen was 2.16, p = 0.0001 when rL7/L12-Omp25 was administered alone and 2.4, p = 0.0001 when it was administered along with rIFN-gamma. However, the molecule upon administration with murine IFN-gamma imparted very minimal or no therapeutic effect against brucellosis. To conclude, our study demonstrates the potential of rL7/L12-Omp25 as an immunogen of prospective and efficient prophylaxis as it is capable of eliciting both cell-mediated and humoral immune responses against brucellosis.

     

An exploration of microbial and associated functional diversity in the OMZ and non-OMZ areas in the Bay of Bengal

Depletion of oxygen in certain marine areas creates oxygen minimum zones (OMZs), which can alter the species composition and abundance. We have carried out high-throughput 16S rRNA gene amplicon profiling from the Bay of Bengal (BOB) OMZ and non-OMZ areas. Typically, a total of 35 families of micro-organisms were identified as biomarkers for OMZ and non-OMZ regions in the BOB. Our analysis has identified families Pseudoalteromonadaceae, OM60 and Synechococcaceae to be abundant in oxygenated water, whereas organisms belonging to families Pelagibacteraceae and Caulobacteraceae, which are involved in sulphur and nitrogen metabolism, were prominent in the OMZ areas. Predictive functional analysis for these identified bacteria clearly that suggested an abundance of microbes with assimilatory sulphur-reducing genes (cysl and csH) in the non-OMZ, while bacteria involved in dissimilatory sulphate reduction (known to carry aprA and aprB genes) were enriched in the OMZ areas. Comparative analysis with OMZ areas from Peru and Chile revealed that OMZ areas in the BOB are characterized by specific and distinctive bacterial diversity. Overall, the current analysis provides valuable documentation about the bacterial populations and their characteristics, which can generate pointers for their functional significance in the BOB.     

Isolation,and Virulence Profiles,of Aeromonas hydrophila Implicated in an Outbreak of Food Poisoning in Sweden

A case of food poisoning outbreak involving Aeromonas hydrophila is reported in this study. A group of 27 people consumed a typical Swedish food “landgång” which is a type of smörgåsbord containing shrimps with mayonnaise, liver paté, ham, sausage, and legume salad which was purchased from a food store. Twenty-two of the 27 persons became ill within 20-34 hr of consumption of the food and reported the symptoms ranging from severe acute diarrhea, abdominal pain, headache, fever and vomiting. One person also fainted. The symptoms lasted for a couple of days. Of the remaining 5 healthy persons who consumed the left-over food the next day, 2 became ill with similar symptoms. The bacteriological examination of left-over food samples resulted in the isolation of A. hydrophila from shrimps with mayonnaise, smoked sausage, liver paté and boiled ham. The total number of A. hydrophila in these foods were log 10⁶ to log >10⁷ organisms per gram of food sample. A. hydrophila was however, not isolated from legume/mayonnaise salad samples. All the food samples tested showed low numbers of other expected food contaminating organisms such as coliforms at 37 C and 44 C, fecal streptococci, Staphylococcus aureus, fungi and yeast etc., while Bacillus cereus, Clostridium perfringens and Salmonella spp. were not detected in the food samples. Investigations of the virulence profiles of the A. hydrophila isolates showed their capacity to produce β-hemolysin, cytotoxins, cytotonic toxins, enterotoxins, and adhesion to and invasion of human intestinal (Henle 407) cells in culture.