Gamete ultrastructure in some species of the family Mullidae from the South China Sea

In tropical representatives of the family Mullidae, the ultrastructure of oocyte envelopes (Parupeneus multifasciatus and Upeneus tragula) and spermatozoa (P. multifasciatus and P. barberinoides) is studied. The egg envelopes include the single-layered zona radiata and weakly expressed chorion. The spermatozoa are characterized by the presence of the oval head flattened on a side and deep nuclear fossa reaching approximately 90% of the head length. The centriolar complex and basal part of the flagellum are located inside the nuclear fossa.     

Methylenetetrahydrofolate reductase gene polymorphisms in patients with nonalcoholic steatohepatitis (NASH)

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of abnormal hepatic steatosis in the absence of a history of alcohol use. Nonalcoholic steatohepatitis (NASH) is the progressive form of NAFLD. Hyperhomocysteinemia causes steatosis, and the methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C polymorphisms result in hyperhomocysteinemia. To examine whether the C677T and A1298C polymorphisms of the MTHFR gene were associated with NASH, we analysed the allele and genotype distribution of the MTHFR C677T and A1298C polymorphisms in 57 well-diagnosed NASH patients, 324 healthy controls in a case-control study of Turkish subjects of Caucasian origin. The diagnosis of the NASH patients was based on liver biopsy. The method used in the analysis of genotypes was PCR-RFLP. The MTHFR A1298C polymorphism was significantly associated with NASH (chi(2) = 8.439; p = 0.015) in the total NASH patients compared with healthy controls. The MTHFR 1298C allele (odds ratio (OR) = 2.480; 95%CI = 1.286-4.782; chi(2) = 7.703; df = 1; p = 0.006) was significantly associated with NASH in the total NASH patients. The MTHFR C677C/A1298C compound genotype (OR = 2.218; 95%CI = 1.003-4.906; chi(2) = 3.998; df = 1; p = 0.046) in men patients was also significantly associated with NASH. Likewise the MTHFR C1298C genotype was significantly associated with NASH in women patients with NASH (OR = 2.979; 95%CI = 1.027-8.641; chi(2) = 4.343; df = 1; p = 0.037). In conclusion, the MTHFR 1298C allele in all NASH patients, C1298C genotype, C677C/C1298C compound genotype in women NASH patients and C677C/A1298C compound genotype in men NASH patients were genetic risk factors for NASH.     

Sperm motility in three coral reef fish species

A method of computer assisted sperm analysis (CASA) is used to determine the parameters of sperm motility in three fish species from the coral reefs of the Nha Trang Bay, South China Sea, Vietnam: Zebrasoma scopas (Acanthuridae), Abudefduf sexfasciatus, and Dascyllus trimaculatus (Pomacentridae). The representatives of the families are characterized by different reproductive tactics and possess pelagic and demersal eggs, respectively. The main morphological parameters of spermatozoa have been measured. The average curvilinear velocity of spermatozoa movement along the real trajectory (VCL) after 1 min of sperm activation ranges from 15.3 to 74.5 μm/s in Z. scopas, it is comparatively low (12.7–21.6 μm/s) in A. sexfasciatus, and high (58.4–92.2 μm/s) in D. trimaculatus. The duration of progressive movements in more than 50% of spermatozoa at 25°C is 3–20, 5–11, and 6–9 min after sperm activation, respectively. Following storage of the sperm of three species at 4.5°C for 7, 20, and 28 h, respectively, spermatozoa retain the ability of progressive movements. The results are discussed based on the available information on the activity of sperm in fish.     

Rapid and high quality DNA isolation from Origanum onites for RAPD and ISSR analysis

Origanum onites is an economically important medicinal plant with high essential oil content. Lack of an appropriate DNA isolation procedure is a limiting factor for any molecular study of this plant. We have used a protocol for genomic DNA isolation based on a hexadecyltrimethylammonium bromide (CTAB) method described for other plant species. The method involves mortar grinding of leaf tissue, modified CTAB extraction using high salt concentrations and polyvinyl pyrrolidone, and successive isoamyl alcohol/chloroform extractions. The yield was approx. 20 microg DNA per 200 mg of initial fresh plant material. The genomic DNA obtained by this method was suitable to be used in restriction digests, inter simple sequence repeat (ISSR) and randomly amplified polymorphic DNA (RAPD) reactions. This extraction method should facilitate the molecular analysis of Origanum chemotypes.     

Purification of CYP2B-like protein from feral leaping mullet (Liza saliens) liver microsomes and its biocatalytic, molecular, and immunological characterization

In this study, CYP2B-immunoreactive protein was purified to electrophoretic homogeneity from the liver microsomes of leaping mullet. The purified cytochrome P450 (CYP) gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis having a M(r) of 49,300 Da. Absolute absorption spectrum of the purified CYP showed a maximum at 417 nm and CO-difference spectrum of dithionite-reduced cytochrome P450 gave a peak at 450 nm. The purified CYP was found to be active in N-demethylation of benzphetamine, erythromycin, and ethylmorphine, and O-dealkylation of pentoxyresorufin in the reconstituted system. However, it was unable to catalyze O-dealkylation of ethoxyresorufin, methoxyresorufin, benzyloxyresorufin, and hydroxylation of lauric acid and aniline. The purified CYP showed strong cross-reactivity with anti-sheep lung CYP2B, a homologue of CYP2B4. N-terminal amino acid sequence of the mullet P450 had the highest degree of homology with CYP2Bs among the known CYPs. Spectral, electrophoretic, immunochemical, N-terminal amino acid sequence, and biocatalytic properties of the purified CYP are most similar to those of mammalian cytochrome P4502B. All these data indicate that the purified CYP is certainly 2B-like. In this study, we not only purified biocatalytically active CYP2B-like protein from fish, but also demonstrated detailed functional properties of CYP2B-like protein for the first time.     

Transient trade‐off between climate benefit and biodiversity loss of harvesting stumps for bioenergy

To replace fossil fuel and thereby mitigate climate change, harvesting of wood such as stumps for bioenergy will likely increase. Coarse deadwood is an important resource for biodiversity and stumps comprise the main part of the coarse deadwood in managed forests. We provide the first integrated analysis of the long‐term climate and biodiversity impacts of a whole landscape. We simultaneously project climate and biodiversity impacts of harvesting stumps to substitute for fossil coal, assuming scenarios with different proportions of the landscape with stump harvest (10, 50, 80%) the coming 50 years. A life cycle approach was used to calculate future global temperature changes and future metapopulation changes in six epixylic lichens. Metapopulation dynamics were projected using colonization and extinction models based on times series data. Harvesting stumps from ≥50% of the clear‐cut forest land benefits climate with a net global temperature reduction >0.5·10^?9 K ha^?1 after 50 years if assuming substitution of fossil coal. For all scenarios, using stump bioenergy leads to immediate (within 1 year) reductions in temperature of 50% compared to using fossil coal, increasing to 70% reduction after 50 years. However, large‐scale stump harvest inflicted substantial metapopulation declines for five of six lichens. High stump harvest levels (≥50%) put common lichens at risk of becoming red‐listed following the IUCN criteria. The net temperature reduction (cooling effect) from substituting fossil coal with stumps harvested for bioenergy increased over time, while lichen metapopulations stabilized at lower equilibria after two to three decades. This indicates that trade‐offs between climate and metapopulations of commons species are transient, where climate benefits become more prevalent in the long term. As both objectives are important for meeting (inter‐)national climate and biodiversity targets, integrated analyses such as this should be encouraged and urged to guide policymaking about large‐scale implementation of stump harvest.     

Proteomic profiling of HBV infected liver biopsies with different fibrotic stages

Background

Hepatitis B virus (HBV) is a global health problem, and infected patients if left untreated may develop cirrhosis and eventually hepatocellular carcinoma. This study aims to enlighten pathways associated with HBV related liver fibrosis for delineation of potential new therapeutic targets and biomarkers.

Methods

Tissue samples from 47 HBV infected patients with different fibrotic stages (F1 to F6) were enrolled for 2D-DIGE proteomic screening. Differentially expressed proteins were identified by mass spectrometry and verified by western blotting. Functional proteomic associations were analyzed by EnrichNet application.

Results

Fibrotic stage variations were observed for apolipoprotein A1 (APOA1), pyruvate kinase PKM (KPYM), glyceraldehyde 3-phospahate dehydrogenase (GAPDH), glutamate dehydrogenase (DHE3), aldehyde dehydrogenase (ALDH2), alcohol dehydrogenase (ALDH1A1), transferrin (TRFE), peroxiredoxin 3 (PRDX3), phenazine biosynthesis-like domain-containing protein (PBLD), immuglobulin kappa chain C region (IGKC), annexin A4 (ANXA4), keratin 5 (KRT5). Enrichment analysis with Reactome and Kegg databases highlighted the possible involvement of platelet release, glycolysis and HDL mediated lipid transport pathways. Moreover, string analysis revealed that HIF-1α (Hypoxia-inducible factor 1-alpha), one of the interacting partners of HBx (Hepatitis B X protein), may play a role in the altered glycolytic response and oxidative stress observed in liver fibrosis.

Conclusions

To our knowledge, this is the first protomic research that studies HBV infected fibrotic human liver tissues to investigate alterations in protein levels and affected pathways among different fibrotic stages. Observed changes in the glycolytic pathway caused by HBx presence and therefore its interactions with HIF-1α can be a target pathway for novel therapeutic purposes.

     

Poly(hydroxyethyl methacrylate) based affinity cryogel for plasmid DNA purification

The aim of this study is to prepare supermacroporous pseudospecific cryogel which can be used for the purification of plasmid DNA (pDNA) from bacterial lysate. N-methacryloyl-(l)-histidine methyl ester (MAH) was chosen as the pseudospecific ligand and/or comonomer. Poly(hydroxyethyl methacrylate-N-methacryloyl-(l)-histidine methyl ester) [PHEMAH] cryogel was produced by free radical polymerization initiated by N,N,N',N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) pair in an ice bath. Compared with the PHEMA cryogel (50 μg/g polymer), the pDNA adsorption capacity of the PHEMAH cryogel (13,350 μg/g polymer) was improved significantly due to the MAH incorporation into the polymeric matrix. The amount of pDNA bound onto the PHEMAH cryogel disks first increased and then reached a saturation value (i.e., 13,350μg/g) at around 300 μg/ml pDNA concentration. pDNA adsorption amount decreased from 1137 μg/g to 160 μg/g with the increasing NaCl concentration. The maximum pDNA adsorption was achieved at 25 °C. The overall recovery of pDNA was calculated as 90%. The PHEMAH cryogel could be used 3 times without decreasing the pDNA adsorption capacity significantly. The results indicate that the PHEMAH cryogel disks promise high selectivity for pDNA.     

Physiological roles of the angiogenic factors during posthatching development period and adults in the quail lung

The bronchus and vasculature form an intrinsic functional component of the avian lung, and its growth must be tightly regulated and coordinated by lung epithelial and endothelial development. Vascular endothelial growth inhibitor (VEGI), vascular endothelial growth factor (VEGF) and its receptors (flk1/KDR, flt1/fms, flt4) are required for epithelial and endothelial cell survival and apoptosis. Especially, VEGF and its receptors are critical for the development of the lung and serve as a maintenance factor during adult life. To determine the function of VEGI, VEGF and its receptors in the posthatching lung development, we revealed its expression and localization using by immunohistochemical procedure. VEGI, VEGF and its receptors were observed in the structural components of the bronchi, atria and air capillaries, as well as in the pulmonary blood vessels throughout the posthatching development period. On the other hand, immunostaining for VEGI, VEGF and its receptors was faintly detected in the glands of the secondary bronchi. Furthermore, it was determined that the secondary bronchial and atrial muscles did not display VEGF immunoreactions. Our results showed that VEGF and its receptors (flt1/fms, flk1/KDR and flt4) and VEGI were expressed at varying intensity by different cell groups. Therefore, they are also required for the development of the lung component during posthatching period.     

Development of an Optimized Method to Obtain a Limonene-Rich Concentrate from the Discarded Lemon Peels

In this study, lemon peels were used as volatile component source. Automatic solvent extraction has been used for the recovery of limonene rich citrus volatile extract for the first time. The process parameters (amount of raw material, immersion time and washing time) were analyzed to optimize the process by means of Box-Behnken design via response surface methodology. The optimum conditions were achieved by ~10 g fresh lemon peel, and ~15 min immersion time and ~13 min washing time. The difference between the actual (89.37 mg/g limonene) and predicted (90.85 mg/g limonene) results was satisfactory (<2 %). α-Terpinene, β-pinene, citral, ɣ-terpinene and linalool were determined as other major volatiles in the peel extract. FT-IR and ¹H- and ¹³C-NMR spectroscopies were applied to verify the identified volatile compounds.