Antimicrobial compounds produced by Lactobacillus sakei subsp. sakei 2a, a bacteriocinogenic strain isolated from a Brazilian meat product

Bacteriocins produced by lactic acid bacteria are gaining increased importance due to their activity against undesirable microorganisms in foods. In this study, a concentrated acid extract of a culture of Lactobacillus sakei subsp. sakei 2a, a bacteriocinogenic strain isolated from a Brazilian pork product, was purified by cation exchange and reversed-phase chromatographic methods. The amino acid sequences of the active antimicrobial compounds determined by Edman degradation were compared to known protein sequences using the BLAST-P software. Three different antimicrobial compounds were obtained, P1, P2 and P3, and mass spectrometry indicated molecular masses of 4.4, 6.8 and 9.5 kDa, respectively. P1 corresponds to classical sakacin P, P2 is identical to the 30S ribosomal protein S21 of L. sakei subsp. sakei 23 K, and P3 is identical to a histone-like DNA-binding protein HV produced by L. sakei subsp. sakei 23 K. Total genomic DNA was extracted and used as target DNA for PCR amplification of the genes sak, lis and his involved in the synthesis of P1, P2 and P3. The fragments were cloned in pET28b expression vector and the resulting plasmids transformed in E. coli KRX competent cells. The transformants were active against Listeria monocytogenes, indicating that the activity of the classical sakacin P produced by L. sakei 2a can be complemented by other antimicrobial proteins.     

CAFs affect the proliferation and treatment response of head and neck cancer spheroids during co-culturing in a unique in vitro model

To investigate the efficacy of a PLGA-based nanobody complex in photodynamic therapy (PDT) and NIR-II imaging in A549 tumor hypoxic model. IR1048-MZ was firstly synthesized by conjugating a nitro imidazole group to IR1048. IR1048-MZ and Cat were then encapsulated in PLGA-SH solution. Anti-EGFR-Nanobody was also expressed and purified, and finally Anti-EGFR-Nanobody@PLGA-IR1048MZ-Cat (Nb@IC-NPs) nanobody complex was obtained based on the formation of desulfide bond between PLGA-SH and Anti-EGFR-Nanobody. Size distribution and morphology were characterized by TEM and DLS. Spectrum of Nb@IC-NPs towards NTR was measured by UV and fluorescence, while the particle’s selective response was studied using fluorescence. The uptake of Nb@IC-NPs in A549 cells was observed by flow cytometry and CLSM. In the meantime, its’ catalytic ability that decomposes H2O2 both extra-and intra-cellular was observed by fluorescence and CLSM. In vitro photodynamic toxicity of Nb@IC-NPs was examined by MTT, Live/Dead Cell Staining, Flow Cytometry and Apoptosis Assay. Tumor-bearing model was constructed to observe a semi-quantitative fluorescent distribution and the possibility of NIR-II fluorescence/photoacoustic (PA) imaging. Effect of Nb@IC-NPs on enhancing A549 tumor hypoxia and expression profile of HIF-1α was investigated in the presence of NIR. An A549 tumor metastasis model was also constructed to confirm the complex’ potential to destroy primary tumor, inhibit lung metastasis, and prolong mice’ survival. Lastly, impact of Nb@IC-NPs on mice’ main organs and blood indices was observed. Nb@IC-NPs was successfully fabricated with good homogeneity. The fluorescent absorbance of Nb@IC-NPs showed a linear relationship with the concentration of NTR, and a higher concentration of NTR corresponded to a stronger photoacoustic signal. In addition, Nb@IC-NPs showed a stable selectivity toward NTR. Our results also suggested a high efficient uptake of Nb@IC-NPs in A549 cells, which was more efficient than IC-NPs and IR1048-MZ alone. In vitro assays confirmed the effects of Nb@IC-NPs on catalytic O2 generation even in hypoxic cells. The cell viability was upregulated with the nanocomplex at the absence of the laser, whereas it was dramatically declined with laser treatment that excited at 980 nm. Nb@IC-NPs achieved tumor hypoxia NIR-II/PA imaging through assisting A549 gathering. When NIR was applied, Nb@IC-NPs can significantly relieve A549 cellular/tumor hypoxia by generating more reactive oxygen species (ROS), which in turn helps lower the expression level of HIF-1α. In summary, Nb@IC-NPs based PDT can efficiently decimate A549 primary tumor, inhibit metastatic lung cancer, and prolong the lifespan of the mice under tolerable dosage. At last, in vivo toxicity tests of the nanocomplex showed its biosafety to the main organs and normal blood indices values. Nb@IC-NPs improves tumor hypoxia through catalytic reaction and lowers the expression level of HIF-1α. It achieves tumor PA imaging through intensified NIR-II fluorescence signal that caused by response of the complex to the lesion’s nitroreductase (NTR). Nb@IC-NPs based PDT can efficiently kill A549 primary tumor, inhibit a lung metastasis, as well as prolong mice’ survival cycle.     

The effects of lycopene on hepatic ischemia/reperfusion injury in rats

There is a very little information about the protective effect of lycopene (LYC) against hepatic ischemia–reperfusion injury. The present study was designed to examine the possible protective effect of the strong antioxidant and anti-inflammatory agent, LYC, on hepatic ischemia/reperfusion injury. For this purpose, rats were subjected to 45 min of hepatic ischemia followed by 60 min of reperfusion period. LYC at the doses of 2.5 and 5 mg/kg body weight (bw) were injected intraperitoneally, 60 min prior to ischemia. Upon sacrification, hepatic tissue samples were used for the measurement of catalase (CAT) activity and malondialdehyde (MDA) levels. Also, aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) were assayed in serum samples. As a result of the use of LYC at the doses of 2.5 and 5 mg/kg bw; while improvements of the ALT, AST, LDH and MDA values were partial and dose-dependent, the improvement of CAT activity was total and dose-independent (p < 0.05). Our findings suggest that LYC has a protective effect against ischemia/reperfusion injury on the liver.     

Voltammetric discrimination of mandelic acid enantiomers

We report a novel electrochemical biosensor for direct discrimination of d- and l-mandelic acid (d- and l-MA) in aqueous medium. The glassy carbon electrode (GCE) surface was modified with reduced graphene oxide (rGO) and γ-globulin (GLOB). Electrochemical characterization of the modified electrodes was investigated by cyclic voltammetry and electrochemical impedance spectroscopy. The modified electrode surfaces were also characterized by scanning electron microscopy. Electrochemical response of the prepared electrode (GCE/rGO/GLOB) for discrimination of d- and l-MA enantiomers was investigated by cyclic voltammetry and was compared with bare GCE in the concentration range of 2 to 10 mM. Whereas the bare GCE showed no electrochemical response for the MA enantiomers, the GCE/rGO/GLOB electrode exhibited direct and selective discrimination with different oxidation potential values of 1.47 and 1.71 V and weak reduction peaks at potential values of −1.37 and −1.48 V, respectively. In addition, electrochemical performance of the modified electrode was investigated in mixed solution of d- and l-MA. The results show that the produced electrode can be used as electrochemical chiral biosensor for MA.     

Influence of dietary vitamin E (DL-alpha-tocopheryl acetate) and diludine (diethyl 1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylate) levels on growth and lipid peroxidation in rainbow trout,Oncorhynchus mykiss

We aimed to investigate the influence of dietary vitamin E and diludine on growth and lipid peroxidation (malondialdehyde; MDA) in rainbow trout. Fish (1.5 g) were fed different dietary levels of vitamin E (0, 50 and 100 mg/kg) and diludine (0, 0.5 and 1 g/kg) for 10 weeks. Growth performance and feed conversion ratio (FCR) were significantly affected by dietary vitamin E (p < .05) but not diludine. Fish fed 50 mg/kg dietary vitamin E with no diludine had significantly better growth and lower FCR than those fed vitamin E free diets. Liver vitamin E content was significantly influenced by dietary vitamin E and diludine (p < .05). The highest hepatic vitamin E was in fish fed the highest dietary vitamin E and diludine levels. Hepatic MDA level was significantly affected by dietary vitamin E and diludine (p < .05), decreasing with the increase in both dietary vitamin E and diludine. According to our results, diludine had no significant effect on growth; however, decreased hepatic lipid peroxidation independent of vitamin E. Our results reveal that 50 mg/kg vitamin E content is suitable for optimal growth and FCR in rainbow trout juveniles. However, dose dependent effects of dietary diludine remain uncertain and need further researches.     

Ratio of Pro-Resolving and Pro-Inflammatory Lipid Mediator Precursors as Potential Markers for Aggressive Periodontitis

Aggressive periodontitis (AgP) is a rapidly progressing type of periodontal disease in otherwise healthy individuals which causes destruction of the supporting tissues of the teeth. The disease is initiated by pathogenic bacteria in the dental biofilm, and the severity of inflammation and attachment loss varies with the host response. Recently, there has been an increased interest in determining the role of lipid mediators in inflammatory events and the concept of pro-inflammatory and pro-resolution lipid mediators has been brought into focus also in periodontal disease. The present study aimed to determine the profile of omega-3 or n3- as well as omega-6 or n6- polyunsaturated fatty acids (PUFAs) and PUFA-metabolites of linoleic acid, arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in gingival crevicular fluid (GCF), saliva and serum in AgP patients and healthy controls. In total, 60 selected n3- and n6-PUFAs and various PUFA metabolites were measured using high performance liquid chromatography-tandem electrospray ionisation mass spectrometry (HPLC-ESI-MS-MS). Of these, 51 could be quantified in this study. The concentrations of the majority were low in saliva samples compared with serum and GCF, but were mainly higher in AgP patients compared with healthy controls in all three kinds of sample. Ratios of n3- to n6-PUFAs (DHA + EPA)/AA were significantly lower in the GCF of AgP patients than in the healthy controls. Furthermore, various ratios of the direct precursors of the pro-resolution lipid mediators (precursors of resolvins and protectins) were calculated against the precursors of mainly pro-inflammatory lipid mediators. These ratios were mainly lower in GCF and saliva of AgP patients, compared with healthy controls, but only reached significance in GCF (P<0.05). To conclude, the ratios of precursors of pro-resolution/pro-inflammatory lipid mediators seem to be more relevant for describing the disease status of AgP than the concentration of specific lipid mediators.     

New Findings in a Global Approach to Dissect the Whole Phenotype of PLA2G6 Gene Mutations

Mutations in PLA2G6 gene have variable phenotypic outcome including infantile neuroaxonal dystrophy, atypical neuroaxonal dystrophy, idiopathic neurodegeneration with brain iron accumulation and Karak syndrome. The cause of this phenotypic variation is so far unknown which impairs both genetic diagnosis and appropriate family counseling. We report detailed clinical, electrophysiological, neuroimaging, histologic, biochemical and genetic characterization of 11 patients, from 6 consanguineous families, who were followed for a period of up to 17 years. Cerebellar atrophy was constant and the earliest feature of the disease preceding brain iron accumulation, leading to the provisional diagnosis of a recessive progressive ataxia in these patients. Ultrastructural characterization of patients’ muscle biopsies revealed focal accumulation of granular and membranous material possibly resulting from defective membrane homeostasis caused by disrupted PLA2G6 function. Enzyme studies in one of these muscle biopsies provided evidence for a relatively low mitochondrial content, which is compatible with the structural mitochondrial alterations seen by electron microscopy. Genetic characterization of 11 patients led to the identification of six underlying PLA2G6 gene mutations, five of which are novel. Importantly, by combining clinical and genetic data we have observed that while the phenotype of neurodegeneration associated with PLA2G6 mutations is variable in this cohort of patients belonging to the same ethnic background, it is partially influenced by the genotype, considering the age at onset and the functional disability criteria. Molecular testing for PLA2G6 mutations is, therefore, indicated in childhood-onset ataxia syndromes, if neuroimaging shows cerebellar atrophy with or without evidence of iron accumulation.     

Effects of Some Antibiotics on Human Erythrocyte 6-Phosphogluconate Dehydrogenase: An in vitro and in vivo Study

The in vitro and in vivo effects of some antibiotics on human erythrocyte 6-phosphogluconate dehydrogenase were investigated. Human erythrocyte 6-phosphogluconate dehydrogenase was purified with ammonium sulphate precipitation, 2′,5′ ADP-Sepharose 4B affinity and gel filtration chromatography. Some antibiotics (netilmicin sulphate, cefepime, amikacin, isepamycin, chloramphenicol, ceftazidim, teicoplanin, ampicillin, ofloxacin, levofloxacin, cefotaxime, penicillin G, gentamicin sulphate, ciprofloxacin) inhibited enzyme activity in vitro but others (cefozin, decefin, streptomycin, combisid, and meronem) were devoid of inhibitory effects. For the drugs having low IC50 values (netilmicin sulphate and cefepime), in vivo studies were performed in rats. Netilmicin sulphate at 15-mg/kg inhibited enzyme activity significantly (p < 0.001) 1h, 2h, and 3h after dosing and cefepime at 200-mg/kg very significantly (p < 0.001) inhibited the enzyme 1 h and 2 h after dosing. Netilmicin sulphate and cefepime inhibited rat erythrocyte 6-phosphogluconate dehydrogenase both in vivo and in-vitro.     

Limitations of Augmentation Index in the Assessment of Wave Reflection in Normotensive Healthy Individuals

Objectives

Augmentation index (AIx) is widely used as a measure of wave reflection. We compared the relationship between AIx and age, height and sex with ‘gold standard’ measures of wave reflection derived from measurements of pressure and flow to establish how well AIx measures wave reflection.

Materials and Methods

Measurements of carotid pressure and flow velocity were made in the carotid artery of 65 healthy normotensive individuals (age 21–78 yr; 43 male) and pulse wave analysis, wave intensity analysis and wave separation was performed; waveforms were classified into type A, B or C. AIx, the time of the first shoulder (T_s), wave reflection index (WRI) and the ratio of backward to forward pressure (P_b/P_f) were calculated.

Results

AIx did not correlate with log WRI or P_b/P_f. When AIx was restricted to positive values AIx and log WRI were positively correlated (r = 0.33; p = 0.04). In contrast log WRI and P_b/P_f were closely correlated (r = 0.66; p<0.001). There was no correlation between the T_s and the timing of P_b or the reflected wave identified by wave intensity analysis. Wave intensity analysis showed that the morphology of type C waveforms (negative AIx) was principally due to a forward travelling (re-reflected) decompression wave in mid-systole. AIx correlated positively with age, inversely with height and was higher in women. In contrast log WRI and P_b/P_f showed negative associations with age, were unrelated to height and did not differ significantly by gender.

Conclusions

AIx has serious limitations as a measure of wave reflection. Negative AIx values derived from Type C waves should not be used as estimates of wave reflection magnitude.     

Virulence variation of Puccinia striiformis Westend. f. sp. tritici in Pakistan

Yellow rust populations of Pakistan were characterised for their virulence pathotypes/races and pathogenetic variation using seedling evaluation of differential genotypes under glasshouse conditions in Murree (6000 feet above sea level). Differential genotypes comprised a world set, an European set, near isogenic lines and the universally susceptible bread wheat cultivar “Morocco”. Over the two-year study a total of 18 race groups were identified. Out of these 18 race groups, several (68E0, 64E0, 66E0, 70E0, 6E0, 71E0, 6E0, 2E0, 67E0, and 68E16) were found previously. The new race group 70E32 found probably evolved because of mutation from the previously existing 70E16. Virulence frequencies of yellow rust (Yr) resistance genes were also determined on near isogenic lines. The highest virulence frequencies (%) were found for Yr7 (88%), Yr9 (57%), Yr18 (70%), and Yr24 (69%). Virulence frequencies were low for Yr 1 (4%), Yr5 (7%), Yr10 (10%) and Yr15 (4%). Our studies indicated that virulence existed for almost all yr genes, necessitating regular monitoring of the yellow rust populations and intensifying efforts to identify new sources of resistance to this pathogen.