A comparative genomic analysis of the alkalitolerant soil bacterium Bacillus lehensis G1

Bacillus lehensis G1 is a Gram-positive, moderately alkalitolerant bacterium isolated from soil samples. B. lehensis produces cyclodextrin glucanotransferase (CGTase), an enzyme that has enabled the extensive use of cyclodextrin in foodstuffs, chemicals, and pharmaceuticals. The genome sequence of B. lehensis G1 consists of a single circular 3.99 Mb chromosome containing 4017 protein-coding sequences (CDSs), of which 2818 (70.15%) have assigned biological roles, 936 (23.30%) have conserved domains with unknown functions, and 263 (6.55%) have no match with any protein database. Bacillus clausii KSM-K16 was established as the closest relative to B. lehensis G1 based on gene content similarity and 16S rRNA phylogenetic analysis. A total of 2820 proteins from B. lehensis G1 were found to have orthologues in B. clausii, including sodium–proton antiporters, transport proteins, and proteins involved in ATP synthesis. A comparative analysis of these proteins and those in B. clausii and other alkaliphilic Bacillus species was carried out to investigate their contributions towards the alkalitolerance of the microorganism. The similarities and differences in alkalitolerance-related genes among alkalitolerant/alkaliphilic Bacillus species highlight the complex mechanism of pH homeostasis. The B. lehensis G1 genome was also mined for proteins and enzymes with potential viability for industrial and commercial purposes.     

Transgenerational inheritance of centromere identity requires the CENP-A N-terminal tail in the C. elegans maternal germ line

Centromere protein A (CENP-A) is a histone H3 variant that defines centromeric chromatin and is essential for centromere function. In most eukaryotes, CENP-A-containing chromatin is epigenetically maintained, and centromere identity is inherited from one cell cycle to the next. In the germ line of the holocentric nematode Caenorhabditis elegans, this inheritance cycle is disrupted. CENP-A is removed at the mitosis-to-meiosis transition and is reestablished on chromatin during diplotene of meiosis I. Here, we show that the N-terminal tail of CENP-A is required for the de novo establishment of centromeres, but then its presence becomes dispensable for centromere maintenance during development. Worms homozygous for a CENP-A tail deletion maintain functional centromeres during development but give rise to inviable offspring because they fail to reestablish centromeres in the maternal germ line. We identify the N-terminal tail of CENP-A as a critical domain for the interaction with the conserved kinetochore protein KNL-2 and argue that this interaction plays an important role in setting centromere identity in the germ line. We conclude that centromere establishment and maintenance are functionally distinct in C. elegans.

This study of the nematode Caenorhabditis elegans shows that centromere identity is set in the maternal germ line and passed on to the progeny via an epigenetic mechanism that requires the N-terminal tail of the centromeric histone H3 variant CENP-A.     

Erythrocyte Phospholipid and Polyunsaturated Fatty Acid Composition in Diabetic Retinopathy

Background

Long chain polyunsaturated fatty acids (LCPUFAs) including docosahexaenoic acid and arachidonic acid are suspected to play a key role in the pathogenesis of diabetes. LCPUFAs are known to be preferentially concentrated in specific phospholipids termed as plasmalogens. This study was aimed to highlight potential changes in the metabolism of phospholipids, and particularly plasmalogens, and LCPUFAs at various stages of diabetic retinopathy in humans.

Methodology and Principal Findings

We performed lipidomic analyses on red blood cell membranes from controls and mainly type 2 diabetes mellitus patients with or without retinopathy. The fatty acid composition of erythrocytes was determined by gas chromatography and the phospholipid structure was determined by liquid chromatography equipped with an electrospray ionisation source and coupled with a tandem mass spectrometer (LC-ESI-MS/MS). A significant decrease in levels of docosahexaenoic acid and arachidonic acid in erythrocytes of diabetic patients with or without retinopathy was observed. The origin of this decrease was a loss of phosphatidyl-ethanolamine phospholipids esterified with these LCPUFAs. In diabetic patients without retinopathy, this change was balanced by an increase in the levels of several phosphatidyl-choline species. No influence of diabetes nor of diabetic retinopathy was observed on the concentrations of plasmalogen-type phospholipids.

Conclusions and Significance

Diabetes and diabetic retinopathy were associated with a reduction of erythrocyte LCPUFAs in phosphatidyl-ethanolamines. The increase of the amounts of phosphatidyl-choline species in erythrocytes of diabetic patients without diabetic retinopathy might be a compensatory mechanism for the loss of LC-PUFA-rich phosphatidyl-ethanolamines.     

Testicular disorders induced by plant growth regulators: cellular protection with proanthocyanidins grape seeds extract

The present study aims to investigate the adverse effects of plant growth regulators : gibberellic acid (GA₃) and indoleacetic acid (IAA) on testicular functions in rats, and extends to investigate the possible protective role of grape seed extract, proanthocyanidin (PAC). Male rats were divided into six groups; control group, PAC, GA₃, IAA, GA₃ + PAC and IAA + PAC groups. The data showed that GA₃ and IAA caused significant increase in total lipids, total cholesterol, triglycerides, phospholipids and low-density-lipoprotein cholesterol in the serum, concomitant with a significant decrease in high-density-lipoprotein cholesterol, total protein, and testosterone levels. In addition, there was significant decrease in the activity of alkaline phosphatase, acid phosphatase, and gamma-glutamyl transferase. A significant decrease was detected also in epididymyal fructose along with a significant reduction in sperm count. Testicular lipid peroxidation product and hydrogen peroxide (H₂O₂) levels were significantly increased. Meanwhile, the total antioxidant capacity, glutathione, sulphahydryl group content, as well as superoxide dismutase, catalase, and glucose-6-phosphate dehydrogenase activity were significantly decreased. Moreover, there were a number of histopathological testicular changes including Leydig’s cell degeneration, reduction in seminiferous tubule and necrotic symptoms and sperm degeneration in both GA₃- and IAA-treated rats. However, an obvious recovery of all the above biochemical and histological testicular disorders was detected when PAC seed extract was supplemented to rats administered with GA₃ or IAA indicating its protective effect. Therefore it was concluded that supplementation with PAC had ameliorative effects on those adverse effects of the mentioned plant growth regulators through its natural antioxidant properties.     

Prediction of Muscle Activities from Electrocorticograms in Primary Motor Cortex of Primates

Electrocorticography (ECoG) has drawn attention as an effective recording approach for brain-machine interfaces (BMI). Previous studies have succeeded in classifying movement intention and predicting hand trajectories from ECoG. Despite such successes, however, there still remains considerable work for the realization of ECoG-based BMIs as neuroprosthetics. We developed a method to predict multiple muscle activities from ECoG measurements. We also verified that ECoG signals are effective for predicting muscle activities in time varying series when performing sequential movements. ECoG signals were band-pass filtered into separate sensorimotor rhythm bands, z-score normalized, and smoothed with a Gaussian filter. We used sparse linear regression to find the best fit between frequency bands of ECoG and electromyographic activity. The best average correlation coefficient and the normalized root-mean-square error were 0.92±0.06 and 0.06±0.10, respectively, in the flexor digitorum profundus finger muscle. The δ (1.5∼4Hz) and γ2 (50∼90Hz) bands contributed significantly more strongly than other frequency bands (P<0.001). These results demonstrate the feasibility of predicting muscle activity from ECoG signals in an online fashion.     

Reserve proteins in the developing seeds of xhaynaldoticum sardoum.

In this study the pattern of storage protein deposition in the developing seeds of xHaynaldoticum sardoum was examined. The accumulation of the various protein classes did not differ greatly between the two lines tested (Culmo Pieno and Culmo Vuoto). Nevertheless, some differences in the deposition pattern were revealed by polyacrylamide gel electrophoresis. In particular, the mature seeds of the two lines presented different electrophoretograms for the prolamin and glutelin proteins. These patterns reflect the differences between the two lines, which could play a role in their ability to adapt to environmental changes. This work constitutes part of the Ph. D. thesis submitted by the first author. The study was supported by a grant (40 %) from the Ministero Pubblica Istruzione.     

Identification of Novel Cytotoxic Peptide KENPVLSLVNGMF from Marine Sponge <Emphasis Type="Italic">Xestospongia testudinaria</Emphasis>, with Characterization of Stability in Human Serum

Resistance and side effects are common problems for anticancer drugs used in chemotherapy. Thus, continued research to discover novel and specific anticancer drugs is obligatory. Marine sponges hold great promise as a source of potent cytotoxic peptides with future applications in cancer treatments. This study aimed to purify and identify cytotoxic peptides from the protein hydrolysates of the giant barrel sponge Xestospongia testudinaria, guided by a cytotoxicity assay based on the human cervical cancer cell line (HeLa). Comparison among trypsin, chymotrypsin, papain and alcalase hydrolysates of X. testudinaria revealed papain hydrolysate (PH) to be the most active. PH was purified consecutively by membrane ultrafiltration, gel filtration chromatography, and reversed-phase high performance liquid chromatography (RP-HPLC). Following liquid chromatography-tandem mass spectrometric analysis, two peptides were identified from the most cytotoxic RP-HPLC fraction: KENPVLSLVNGMF and LLATIPKVGVFSILV. Between the two, only the synthetic peptide KENPVLSLVNGMF showed cytotoxicity toward HeLa cells in a dose-dependent manner. KENPVLSLVNGMF (EC₅₀ 0.67 mM) was 3.8-fold more cytotoxic compared with anticancer drug 5-fluorouracil (EC₅₀ 2.56 mM). Furthermore, KENPVLSLVNGMF show only marginal 5% cytotoxicity to Hek293, a non-cancerous, human embryonic kidney cell line, when tested at 0.67 mM. The half-life of the peptide was 3.2?±?0.5 h in human serum in vitro, as revealed by RP-HPLC analyses. These results suggest that KENPVLSLVNGMF identified from X. testudinaria papain hydrolysate has potential applications as peptide lead in future development of potent and specific anticancer drugs.     

<Emphasis Type="Italic">Cupriavidus malaysiensis</Emphasis> sp. nov., a novel poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-4-hydroxybutyrate) accumulating bacterium isolated from the Malaysian environment

Bacterial classification on the basis of a polyphasic approach was conducted on three poly(3 hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] accumulating bacterial strains that were isolated from samples collected from Malaysian environments; Kulim Lake, Sg. Pinang river and Sg. Manik paddy field. The Gram-negative, rod-shaped, motile, non-sporulating and non-fermenting bacteria were shown to belong to the genus Cupriavidus of the Betaproteobacteria on the basis of their 16S rRNA gene sequence analyses. The sequence similarity value with their near phylogenetic neighbour, Cupriavidus pauculus LMG3413^T, was 98.5%. However, the DNA–DNA hybridization values (8–58%) and ribotyping analysis both enabled these strains to be differentiated from related Cupriavidus species with validly published names. The RiboPrint patterns of the three strains also revealed that the strains were genetically related even though they displayed a clonal diversity. The major cellular fatty acids detected in these strains included C15:0 ISO 2OH/C16:1 ω7c, hexadecanoic (16:0) and cis-11-octadecenoic (C18:1 ω7c). Their G+C contents ranged from 68.0  to 68.6 mol%, and their major isoprenoid quinone was Ubiquinone Q-8. Of these three strains, only strain USMAHM13 (= DSM 25816 = KCTC 32390) was discovered to exhibit yellow pigmentation that is characteristic of the carotenoid family. Their assembled genomes also showed that the three strains were not identical in terms of their genome sizes that were 7.82, 7.95 and 8.70 Mb for strains USMAHM13, USMAA1020 and USMAA2-4, respectively, which are slightly larger than that of Cupriavidus necator H16 (7.42 Mb). The average nucleotide identity (ANI) results indicated that the strains were genetically related and the genome pairs belong to the same species. On the basis of the results obtained in this study, the three strains are considered to represent a novel species for which the name Cupriavidus malaysiensis sp. nov. is proposed. The type strain of the species is USMAA1020^T (= DSM 19416^T = KCTC 32390^T).