Production and characteristics of some new β-agarases from a marine bacterium,Vibrio sp. strain JT0107

Vibrio sp. strain JT0107 is one of the marine bacteria that secrete β-agarases which catalyze the hydrolysis of agarose. The optimum culture conditions for the production of some β-agarases have been determined. To increase agarase activity, aeration and a sufficient concentration of agarose are needed. One of the enzymes that the bacteria secreted into the culture medium was isolated and purified 39-fold using a combination of ultrafiltration and subsequent anion exchange column chromatography. The purified protein migrated as a single band (72 kDa) on sodium dodecyl sulfate polyacrylamide gel electrophoresis and its isoelectric point was 4.7. Amino acid sequence analysis revealed a single N-terminal sequence that had no sequence identity to other marine bacterial agarases. This novel enzyme was found to be an endo-type β-agarase (EC 3.2.1.81) that catalyzes the hydrolysis of the β-1,4 linkage of agarose to yield neoagarotetraose [O-3,6-anhydro-α-l-galactopyranosyl(1→3)-O-β-d-galactopyranosyl(1→4)-O-3,6-anhydro-α-l-galactopyranosyl(1→3)-d -galactose] and neoagarobiose [O-3,6-anhydro-α-l-galactopyranosyl(1→3)-d-galactose]. The optimum pH and temperature for obtaining high activity of the enzyme were at around 8 and 30°C, respectively. The enzyme did not degrade sodium alginate, λ-carrageenan, ι-carrageenan or κ-carrageenan.     

Design and development of a new ambr250® bioreactor vessel for improved cell and gene therapy applications

The emergence of cell and gene therapies has generated significant interest in their clinical and commercial potential. However, these therapies are prohibitively expensive to manufacture and can require extensive time for development due to our limited process knowledge and understanding. The automated ambr250® stirred-tank bioreactor platform provides an effective platform for high-throughput process development. However, the original dual pitched-blade 20 mm impeller and baffles proved sub-optimal for cell therapy candidates that require suspension of microcarriers (e.g. for the culture of adherent human mesenchymal stem cells) or other particles such as activating Dynabeads® (e.g. for the culture of human T-cells). We demonstrate the development of a new ambr250® stirred-tank bioreactor vessel which has been designed specifically to improve the suspension of microcarriers/beads and thereby improve the culture of such cellular systems. The new design is unbaffled and has a single, larger elephant ear impeller. We undertook a range of engineering and physical characterizations to determine which vessel and impeller configuration would be most suitable for suspension based on the minimum agitation speed (N_JS) and associated specific power input (P/V)_JS. A vessel (diameter, T, = 60 mm) without baffles and incorporating a single elephant ear impeller (diameter 30 mm and 45° pitch-blade angle) was selected as it had the lowest (P/V)_JS and therefore potentially, based on Kolmogorov concepts, was the most flexible system. These experimentally-based conclusions were further validated firstly with computational fluid dynamic (CFD) simulations and secondly experimental studies involving the culture of both T-cells with Dynabeads® and hMSCs on microcarriers. The new ambr250® stirred-tank bioreactor successfully supported the culture of both cell types, with the T-cell culture demonstrating significant improvements compared to the original ambr250® and the hMSC-microcarrier culture gave significantly higher yields compared with spinner flask cultures. The new ambr250® bioreactor vessel design is an effective process development tool for cell and gene therapy candidates and potentially for autologous manufacture too.

     

A new lignan and a new alkaloid,and α-glucosidase inhibitory compounds from the grains of Echinochloa utilis Ohwi & Yabuno

A new lignan, utilisin (1), and a new alkaloid, echinoutilin (2), together with eleven known compounds 3–13 were isolated from the grains of Echinochloa utilis Ohwi & Yabuno. Their structures were identified through the analysis of spectroscopic data. The absolute configuration of 2 was determined by Mosher’s method. These compounds were evaluated for α-glucosidase inhibitory activity. Among them, compounds 2, 3 and 6 exhibited considerable α-glucosidase inhibitory activity with IC₅₀ values of 42.1 ± 1.3, 58.9 ± 3.7, and 40.9 ± 1.1 μM, respectively. The results indicate that the grains of E. utilis will be useful in the treatment of diabetes control agents.     

Sensitivity of a PER.C6® cell line to bis(2,4-di-tert-butylphenyl)phosphate and evaluation of a new,biocompatible single-use film

Single-use technologies have brought numerous advantages to the biopharmaceutical industry. In particular, single-use bags made from multi-layered polymeric films have been adopted for cell culture and liquid handling operations in place of traditional stainless-steel systems. Despite the advantages, leachable compounds originating from the film's materials of construction present a new challenge. In 2013, bis(2,4-di-tert-butylphenyl)phosphate (bDtBPP) was identified as a common leachable from several single-use bags that is detrimental to the growth and viability of many Chinese hamster ovary (CHO) cell lines. While much work has been completed to characterize CHO cell sensitivity to bDtBPP, little has been done to characterize its impact on other important production cell lines, particularly PER.C6®. This publication investigates inconsistent cell growth observed in a PER.C6® cell line during bioprocess development. The growth inhibition was linked to leachable migration from Bioclear™ 10, a single-use film from Cytiva (formerly GE Healthcare) that was used for cell expansion. It was shown that the PER.C6® cells displayed a sensitivity to bDtBPP, comparable to that observed in sensitive CHO cell lines. Finally, biocompatibility of PER.C6® with Cytiva's new Fortem film was evaluated, demonstrating that Fortem™ film is a suitable single-use technology for culturing PER.C6® cells.     

Cryptohelops menaticus – a new genus and species of the tribe Helopini (Coleoptera: Tenebrionidae) from the Palaeocene of Menat (France)

A new genus and species, Cryptohelops menaticus gen. et sp. n., are described from the Palaeocene of Menat (France). The new genus belongs to the subtribe Helopina of the tribe Helopini (subfamily Tenebrioninae of Tenebrionidae) based on the coarse and dense punctation of the hypomera, shape of the prosternum and metepisterna, a similar appearance to the native genera Helops and Stenohelops and the structure of the epipleura. The new genus also resembles Raiboscelis and Entomogonus by having the protibiae excised along the base of the inner side. The new genus is the oldest representative of the tribe Helopini. Four species of Helops sensu lato previously described from the Oligocene (H. wetteravicus K. Heyden et L. Heyden, 1865) and Miocene (H. atticus Redtenbacher in Ungern, 1867; H. meissneri Heer, 1847 and H. molassicus Heer, 1883) lack important diagnostic characters and should be regarded as members of the tribe Helopini with uncertain generic attribution.     

Facile synthesis,optical and conformational characteristics,and efficient intracellular delivery of a peptide–DNA conjugate

Covalent conjugation of disparate peptide and oligonucleotide biomacromolecular species produces peptide–oligonucleotide conjugates (POCs), which are interesting molecules with great potential for use in diverse bioapplications. However, peptide–oligonucleotide conjugation methods are not well established, and the intracellular delivery efficacy of POCs is debatable. Here, we describe a simple method for the synthesis and purification of POCs. When peptides are carefully designed to have a near-neutral charge state, a relatively hydrophobic polarity, and receptor-targeting ligands, synthesis and purification become highly efficient and straightforward. UV–vis, fluorescence, and circular dichroism studies show that both types of molecules mutually influence each other, changing their optical and conformational characteristics in the context of POCs. The combined effect of peptide design strategy, targeting ligands, and relatively hydrophobic property, enables the efficient cellular delivery of POCs.     

An ITAM in a Nonenveloped Virus Regulates Activation of NF-κB,Induction of Beta Interferon,and Viral Spread

Immunoreceptor tyrosine-based activation motifs (ITAMs) are signaling domains located within the cytoplasmic tails of many transmembrane receptors and associated adaptor proteins that mediate immune cell activation. ITAMs also have been identified in the cytoplasmic tails of some enveloped virus glycoproteins. Here, we identified ITAM sequences in three mammalian reovirus proteins: μ2, σ2, and λ2. We demonstrate for the first time that μ2 is phosphorylated, contains a functional ITAM, and activates NF-κB. Specifically, μ2 and μNS recruit the ITAM-signaling intermediate Syk to cytoplasmic viral factories and this recruitment requires the μ2 ITAM. Moreover, both the μ2 ITAM and Syk are required for maximal μ2 activation of NF-κB. A mutant virus lacking the μ2 ITAM activates NF-κB less efficiently and induces lower levels of the downstream antiviral cytokine beta interferon (IFN-β) than does wild-type virus despite similar replication. Notably, the consequences of these μ2 ITAM effects are cell type specific. In fibroblasts where NF-κB is required for reovirus-induced apoptosis, the μ2 ITAM is advantageous for viral spread and enhances viral fitness. Conversely, in cardiac myocytes where the IFN response is critical for antiviral protection and NF-κB is not required for apoptosis, the μ2 ITAM stimulates cellular defense mechanisms and diminishes viral fitness. Together, these results suggest that the cell type-specific effect of the μ2 ITAM on viral spread reflects the cell type-specific effects of NF-κB and IFN-β. This first demonstration of a functional ITAM in a nonenveloped virus presents a new mechanism for viral ITAM-mediated signaling with likely organ-specific consequences in the host.     

Establishment and application of a microsatellite-based parentage identification method for Oreochromis niloticus,O. aureus,and O. niloticus × O. aureus

In genetic fish breeding research, clear pedigree information is of great significance for breeding and parental management. In order to establish a stable, highly accurate, and widely applicable parentage identification method for tilapia, 13 highly polymorphic microsatellite loci within populations of Oreochromis niloticus, O. aureus, O. niloticus × O. aureus, and their mixed population were screened. Four groups of fluorescent-labeled multiple capillary electrophoresis were established for allelic genotyping. The assignment success rate reached 100% when 7, 9, 8 and 12 loci were used in the population of O. niloticus, O. aureus, O. niloticus × O. aureus, and their mixed population, respectively. All 175 progeny individuals of “Yuemin No. 1” tilapia were exclusively assigned to their parental pairs when the 12 loci for the mixed population were used. This study established a fluorescent-labeled microsatellite-based parentage assignment method for O. niloticus, O. aureus, O. niloticus × O. aureus, and their mixed population with high identification accuracy and efficiency, which lays a foundation for pedigree information and population genetic management in tilapia breeding.     

In vitro induction and characterization of hexaploid Pennisetum × advena,an ornamental grass

Plant Cell, Tissue and Organ Culture (PCTOC) - Pennisetum × advena (purple fountain grass) is an ornamental grass with considerable commercial value. In vitro induction of...     

Survival of patients treated with intra-aortic balloon counterpulsation at a tertiary care center in Pakistan – patient characteristics and predictors of in-hospital mortality

Background

In most western countries 20% of adults have hypertension. Reports in the literature suggest that from 31 to 86% of treated patients are not at recommended target levels. However it is important to consider how we are determining whether targets are unmet and the degree to which they are unmet. Our underlying hypothesis is that white coat effect is partially responsible for the reported low rates of control of hypertension by primary care practitioners.

Methods

The study population consists of 1142 patients who are being assessed for enrolment in two community-based randomized controlled trials. Patients must have essential hypertension, be on antihypertensive medication, and must not have met their blood pressure targets. We are reporting on the proportion of patients who have not achieved target, and the degree to which they have not achieved their target. We also report on the mean daytime blood pressures on 24 hour ABPM and compare these to mean blood pressures found on the patients' charts.

Results

We identified 3284 patient charts of patients with hypertension. Of these, 1142 were determined to be "out of control" (did not achieve target) and 436 agreed to undergo 24 hour ABPM for final determination of eligibility. Overwhelmingly (95.8% of the time) it was the systolic blood pressure that was not under control. However, most of the patients who had not achieved target according to our criteria were within 10 mmHg of the recommended targets. Isolated systolic blood pressure was the best predictor of elevated mean daytime blood pressure on 24 hour ABPM.

Conclusions

At least 35% of patients had not achieved target blood pressure levels and this is primarily due to lack of control of systolic blood pressure. The best predictor of continuing hypertension on 24 hour ABPM was the mean systolic blood pressure on the patients chart. However, only 69% of patients who were uncontrolled according blood pressures recorded in the chart were uncontrolled according to 24 hour ABPM criteria. This suggests that the white coat effect makes blood pressure measurements in the doctor's offices, at least as currently done, not sufficiently accurate for determining treatment endpoint.     

Description of a new Brazilian Paraportanus and key to the species of the genus (Insecta,Hemiptera,?Cicadellidae,Portanini)

Paraportanus longispinus,, a new leafhopper species from Roraima and Amazonas States, North Brazil, is described and illustrated. The new species can be recognized by the male genital features, especially the distal third of ventral margin of the pygofer with a dentiform short process; plates distinctly longer than pygofer, extending posteriorly beyond pygofer by approximately 1/3 of their length and aedeagus with one pair of spiniform process long crossed and directed ventrally. A checklist and key to males of all known Paraportanus species is provided.     

Description of a new species of Distenia (Coleoptera,Disteniidae, Disteniini) from Southeastern China,with?records and diagnoses of similar species

A new species, Distenia orientalis sp. n. is described from Southeastern China. It was misidentified as Distenia gracilis (Blessig, 1872) but can be separated from the latter by the color of antennae and legs, structure differences on scape, maxillary palp, pronotum, tibiae, punctures on elytra, etc. Three related species are carefully diagnosed and treated.