Genotypic and phenotypic properties of cattle-associated Campylobacter and their implications to public health in the USA

Since cattle are a major source of food and the cattle industry engages people from farms to processing plants and meat markets, it is conceivable that beef-products contaminated with Campylobacter spp. would pose a significant public health concern. To better understand the epidemiology of cattle-associated Campylobacter spp. in the USA, we characterized the prevalence, genotypic and phenotypic properties of these pathogens. Campylobacter were detected in 181 (19.2%) out of 944 fecal samples. Specifically, 71 C. jejuni, 132 C. coli, and 10 other Campylobacter spp. were identified. The prevalence of Campylobacter varied regionally and was significantly (P<0.05) higher in fecal samples collected from the South (32.8%) as compared to those from the North (14.8%), Midwest (15.83%), and East (12%). Pulsed Field Gel Electrophoresis (PFGE) analysis showed that C. jejuni and C. coli isolates were genotypically diverse and certain genotypes were shared across two or more of the geographic locations. In addition, 13 new C. jejuni and two C. coli sequence types (STs) were detected by Multi Locus Sequence Typing (MLST). C. jejuni associated with clinically human health important sequence type, ST-61 which was not previously reported in the USA, was identified in the present study. Most frequently observed clonal complexes (CC) were CC ST-21, CC ST-42, and CC ST-61, which are also common in humans. Further, the cattle associated C. jejuni strains showed varying invasion and intracellular survival capacity; however, C. coli strains showed a lower invasion and intracellular survival potential compared to C. jejuni strains. Furthermore, many cattle associated Campylobacter isolates showed resistance to several antimicrobials including ciprofloxacin, erythromycin, and gentamicin. Taken together, our results highlight the importance of cattle as a potential reservoir for clinically important Campylobacter.     

Vaccine platforms combining circumsporozoite protein and potent immune modulators, rEA or EAT-2, paradoxically result in opposing immune responses

Background

Malaria greatly impacts the health and wellbeing of over half of the world''s population. Promising malaria vaccine candidates have attempted to induce adaptive immune responses to Circumsporozoite (CS) protein. Despite the inclusion of potent adjuvants, these vaccines have limited protective efficacy. Conventional recombinant adenovirus (rAd) based vaccines expressing CS protein can induce CS protein specific immune responses, but these are essentially equivalent to those generated after use of the CS protein subunit based vaccines. In this study we combined the use of rAds expressing CS protein along with rAds expressing novel innate immune response modulating proteins in an attempt to significantly improve the induction of CS protein specific cell mediated immune (CMI) responses.

Methods and Findings

BALB/cJ mice were co-vaccinated with a rAd vectors expressing CS protein simultaneous with a rAd expressing either TLR agonist (rEA) or SLAM receptors adaptor protein (EAT-2). Paradoxically, expression of the TLR agonist uncovered a potent immunosuppressive activity inherent to the combined expression of the CS protein and rEA. Fortunately, use of the rAd vaccine expressing EAT-2 circumvented CS protein''s suppressive activity, and generated a fivefold increase in the number of CS protein responsive, IFNγ secreting splenocytes, as well as increased the breadth of T cells responsive to peptides present in the CS protein. These improvements were positively correlated with the induction of a fourfold improvement in CS protein specific CTL functional activity in vivo.

Conclusion

Our results emphasize the need for caution when incorporating CS protein into malaria vaccine platforms expressing or containing other immunostimulatory compounds, as the immunological outcomes may be unanticipated and/or counter-productive. However, expressing the SLAM receptors derived signaling adaptor EAT-2 at the same time of vaccination with CS protein can overcome these concerns, as well as significantly improve the induction of malaria antigen specific adaptive immune responses in vivo.     

Performance of diagnostic biomarkers in predicting liver fibrosis among hepatitis C virus-infected Egyptian children

The aim of the present study was to identify specific markers that mirror liver fibrosis progression as an alternative to biopsy when biopsy is contraindicated, especially in children. After liver biopsies were performed, serum samples from 30 hepatitis C virus (HCV) paediatric patients (8-14 years) were analysed and compared with samples from 30 healthy subjects. All subjects were tested for the presence of serum anti-HCV antibodies. Direct biomarkers for liver fibrosis, including transforming growth factor-β1, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), hyaluronic acid (HA), procollagen type III amino-terminal peptide (PIIINP) and osteopontin (OPN), were measured. The indirect biomarkers aspartate and alanine aminotransferases, albumin and bilirubin were also tested. The results revealed a significant increase in the serum marker levels in HCV-infected children compared with the healthy group, whereas albumin levels exhibited a significant decrease. Significantly higher levels of PIIINP, TIMP-1, OPN and HA were detected in HCV-infected children with moderate to severe fibrosis compared with children with mild fibrosis (p < 0.05). The diagnostic accuracy of these direct biomarkers, represented by sensitivity, specificity and positive predictive value, emphasises the utility of PIIINP, TIMP-1, OPN and HA as indicators of liver fibrosis among HCV-infected children.     

Chemical composition,antioxidant and antimicrobial activities of the essential oil and methanolic extract of Ferulago bernardii Tomk. & M. Pimen of Iran

The aim of the study was to investigate chemical composition, antioxidant, antibacterial and antifungal activities of the essential oil (EO), polar and nonpolar sub-fractions of methanolic extract of Ferulago bernardii. The chemical constituent of the EO was identified by means of GC–MS. The antimicrobial activities of the EO, polar and nonpolar extracts were evaluated by micro-dilution and agar disc diffusion assays. The antioxidant activity was measured by 2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH) free radical scavenging activity assay. The main components of the EO were α-pinene (35.03%), z-β-ocimene (14.24%) and bornyl acetate (11.64%). Bacillus cereus and Salmonella typhimurium were the most susceptible and resistant to the antibacterial activity of the essential oil and extract, respectively. The free radical scavenging activities of all extracts and the essential oil were in the order: polar > non-polar > EO. Our findings indicate that F. bernardii essential oil and methanolic extract has a potential to be applied as antimicrobial and antioxidant agent.     

Bioinformatic and biochemical characterization of DCXR and DHRS2/4 from Caenorhabditis elegans

Several reductases belonging to the large enzyme superfamily of the short-chain dehydrogenases/reductases (SDR) are involved in the reductive metabolism of carbonyl containing xenobiotics. In order to characterize the human enzymes dicarbonyl/l-xylulose reductase (DCXR), and dehydrogenase/reductase members 2 and 4 (DHRS2, DHRS4) in terms of metabolism of xenobiotics, orthologues from the model organism Caenorhabditis elegans (C. elegans) were identified by using hidden Markov models that were developed in the present study. Accordingly, we describe the characterization of proteins from C. elegans as orthologous to the human enzymes DCXR and DHRS2/4 using a combined approach of bioinformatic and biochemical methods. With the hidden Markov model based system we identified the C. elegans proteins SDR20C18, SDR25C21 and SDR25C22 as being homologous to the human enzymes DCXR, and DHRS2 or DHRS4, respectively. After cloning and overexpression of these three C. elegans genes in Escherichia coli we could purify SDR20C18 and SDR25C22 as soluble proteins by Ni-affinity chromatography, whereas recombinant SDR25C21 was only found in inclusion bodies. Both SDR20C18 (UniProtAcc: Q21929) and SDR25C22 (UniProtAcc: Q93790) were tested with a variety of xenobotic carbonyl compounds as substrates. A comparison of the catalytic activities of SDR20C18 and SDR25C22 with well-known substrates of the human forms revealed that SDR20C18 is the DCXR-orthologue enzyme to the human enzyme and that SDR25C22 might be a DHRS2/4 homologue. Due to their high sequence identity, it was so far not possible to distinguish between SDR25C22 and the human DHRS2/4 proteins by means of sequence analysis alone. However, the study of homologue genes in the model organism C. elegans can provide valuable information on the putative physiological role of the corresponding human form.     

Is the Cerebellum the Optimal Reference Region for Intensity Normalization of Perfusion MR Studies in Early Alzheimer’s Disease?

The cerebellum is the region most commonly used as a reference when normalizing the intensity of perfusion images acquired using magnetic resonance imaging (MRI) in Alzheimer’s disease (AD) studies. In addition, the cerebellum provides unbiased estimations with nuclear medicine techniques. However, no reports confirm the cerebellum as an optimal reference region in MRI studies or evaluate the consequences of using different normalization regions. In this study, we address the effect of using the cerebellum, whole-brain white matter, and whole-brain cortical gray matter in the normalization of cerebral blood flow (CBF) parametric maps by comparing patients with stable mild cognitive impairment (MCI), patients with AD and healthy controls. According to our results, normalization by whole-brain cortical gray matter enables more sensitive detection of perfusion abnormalities in AD patients and reveals a larger number of affected regions than data normalized by the cerebellum or whole-brain white matter. Therefore, the cerebellum is not the most valid reference region in MRI studies for early stages of AD. After normalization by whole-brain cortical gray matter, we found a significant decrease in CBF in both parietal lobes and an increase in CBF in the right medial temporal lobe. We found no differences in perfusion between patients with stable MCI and healthy controls either before or after normalization.     

Effects of Silver Nanoparticles on Burn Wound Healing in a Mouse Model

Biological Trace Element Research - Healing of injuries caused by exposure to heat has been discussed in many studies, although a few drugs have been shown to produce satisfactory results. In this...     

Thermostable alkaline phytase from Bacillus sp. MD2: effect of divalent metals on activity and stability

Phytate, the major source of phosphorus in seeds, exists as a complex with different metal ions. Alkaline phytases are known to dephosphorylate phytate complexed with calcium ions in contrast to acid phytases that act only on phytic acid. A recombinant alkaline phytase from Bacillus sp. MD2 has been purified and characterized with respect to the effect of divalent metal ions on the enzyme activity and stability. The presence of Ca²⁺ on both the enzyme and the substrate is required for optimal activity and stability. Replacing Ca²⁺ with Ba²⁺, Mn²⁺, Mg²⁺ and Sr²⁺ in the phytase resulted in the expression of > 90% of the maximal activity with calcium-phytate as the substrate, while Fe²⁺ and Zn²⁺ rendered the enzyme inactive. On the other hand, the calcium loaded phytase showed significant activity (60%) with sodium phytate and lower activity (17-20%) with phytate complexed with only Mg²⁺, Sn²⁺ and Sr²⁺, respectively. On replacing Ca²⁺ on both the enzyme and the substrate with other metal ions, about 20% of the maximal phytase activity was obtained only with Mg²⁺ and Sr²⁺, respectively. Only Ca²⁺ resulted in a marked increase in the melting temperature (T_m) of the enzyme by 12-21 °C, while Ba²⁺, Mn²⁺, Sr²⁺ or Cu²⁺ resulted in a modest (2-3.5 °C) increase in T_m. In the presence of 1-5 mM Ca²⁺, the optimum temperature of the phytase activity was increased from 40 °C to 70 °C, while optimum pH of the enzyme shifted by 0.4-1 pH unit towards the acidic region.     

Heterologous expression of Anabaena sp. PCC7120 cyanophycin metabolism genes cphA1 and cphB1 in Sinorhizobium (Ensifer) meliloti 1021

Sinorhizobium meliloti infects leguminous plants resulting in a nitrogen-fixing symbiosis. Free living cells accumulate poly(3-hydroxybutyrate) (PHB) as carbon and energy source under imbalanced growth conditions. The cphA1 ₇₁₂₀ gene encoding a cyanophycin (CGP) synthetase of Anabaena sp. PCC7120 in plasmids pVLT31::cphA1 ₇₁₂₀ and pBBR1MCS-3::cphA1 ₇₁₂₀ was expressed in the wild-type S. meliloti 1021 and in a phbC-negative mutant generated in this study. Expression of cphA1 ₇₁₂₀ and accumulation of CGP in cells were studied in various media. Yeast mannitol broth (YMB) and pBBR1MCS-3::cphA1 ₇₁₂₀ yielded the highest CGP contents in both S. meliloti 1021 strains. Supplying the YMB medium with isopropyl-β-D-thiogalactopyranoside, aspartic acid, and arginine enhanced CGP contents about 2.5- and 2.8-fold in S. meliloti 1021 (pBBR1MCS-3::cphA1 ₇₁₂₀) and S. meliloti 1021 phbCΩKm (pBBR1MCS-3::cphA1 ₇₁₂₀), respectively. Varying the nitrogen-to-carbon ratio in the medium enhanced the CGP content further to 43.8% (w/w) of cell dry weight (CDW) in recombinant cells of S. meliloti 1021 phbCΩKm (pBBR1MCS-3::cphA1 ₇₁₂₀). Cells of S. meliloti 1021 (pBBR1MCS-3::cphA1 ₇₁₂₀) accumulated CGP up to 39.6% in addition to 12.1% PHB (w/w, of CDW). CGP from the S. meliloti strains consisted of equimolar amounts of aspartic acid and arginine and contained no other amino acids even if the medium was supplemented with glutamic acid, citrulline, ornithine, or lysine. CGP isolated from cells of S. meliloti 1021 (pBBR1MCS-3::cphA1 ₇₁₂₀) and S. meliloti 1021 phbCΩKm (pBBR1MCS-3::cphA1 ₇₁₂₀) exhibited average molecular weights between 20 and 25 kDa, whereas CGP isolated from Escherichia coli S17-1 (pBBR1MCS-3::cphA1 ₇₁₂₀) exhibited average molecular weight between 22 and 30 kDa. Co-expression of cyanophycinase from Anabaena sp. PCC7120 encoded by cphB1 ₇₁₂₀ in cphA1 ₇₁₂₀-positive E. coli S17-1, S. meliloti 1021, and its phbC-negative mutant gave cyanophycinase activities in crude extracts, and no CGP granules occurred. A higher PHB content in S. meliloti 1021 (pBBR1MCS-3::cphB1 ₇₁₂₀::cphA1 ₇₁₂₀) in comparison to the control indicated that the cells used CGP degradation product (β-aspartate-arginine dipeptide) to fuel PHB biosynthesis.     

The Neuroprotective Effect of Curcumin and Nigella sativa Oil Against Oxidative Stress in the Pilocarpine Model of Epilepsy: A Comparison with Valproate

Oxidative stress has been implicated to play a role in epileptogenesis and pilocarpine-induced seizures. The present study aims to evaluate the antioxidant effects of curcumin, Nigella sativa oil (NSO) and valproate on the levels of malondialdehyde, nitric oxide, reduced glutathione and the activities of catalase, Na⁺, K⁺-ATPase and acetylcholinesterase in the hippocampus of pilocarpine-treated rats. The animal model of epilepsy was induced by pilocarpine and left for 22 days to establish the chronic phase of epilepsy. These animals were then treated with curcumin, NSO or valproate for 21 days. The data revealed evidence of oxidative stress in the hippocampus of pilocarpinized rats as indicated by the increased nitric oxide levels and the decreased glutathione levels and catalase activity. Moreover, a decrease in Na⁺, K⁺-ATPase activity and an increase in acetylcholinesterase activity occurred in the hippocampus after pilocarpine. Treatment with curcumin, NSO or valproate ameliorated most of the changes induced by pilocarpine and restored Na⁺, K⁺-ATPase activity in the hippocampus to control levels. This study reflects the promising anticonvulsant and potent antioxidant effects of curcumin and NSO in reducing oxidative stress, excitability and the induction of seizures in epileptic animals and improving some of the adverse effects of antiepileptic drugs.