A Heterologous Multiepitope DNA Prime/Recombinant Protein Boost Immunisation Strategy for the Development of an Antiserum against Micrurus corallinus (Coral Snake) Venom

BackgroundEnvenoming by coral snakes (Elapidae: Micrurus), although not abundant, represent a serious health threat in the Americas, especially because antivenoms are scarce. The development of adequate amounts of antielapidic serum for the treatment of accidents caused by snakes like Micrurus corallinus is a challenging task due to characteristics such as low venom yield, fossorial habit, relatively small sizes and ophiophagous diet. These features make it difficult to capture and keep these snakes in captivity for venom collection. Furthermore, there are reports of antivenom scarcity in USA, leading to an increase in morbidity and mortality, with patients needing to be intubated and ventilated while the toxin wears off. The development of an alternative method for the production of an antielapidic serum, with no need for snake collection and maintenance in captivity, would be a plausible solution for the antielapidic serum shortage.ConclusionHere we describe that the genetic immunisation with a synthetic multiepitope gene followed by booster doses with recombinant protein is a promising approach to develop an alternative antielapidic serum against M. corallinus venom without the need of collection and the very challenging maintenance of these snakes in captivity.     

Casein kinase 1 regulates connexin-43 gap junction assembly

Phosphorylation of members of the connexin family of gap junction proteins has been correlated with gap junction assembly, but the mechanisms involved remain unclear. We have examined the role of casein kinase 1 (CK1) in connexin-43 (Cx43) gap junction assembly. Cellular co-immunoprecipitation experiments and in vitro CK1 phosphorylation reactions indicate that CK1 interacted with and phosphorylated Cx43, initially on serine(s) 325, 328, or 330. (32)P(i)-Metabolically labeled cells treated with CKI-7, a specific CK1 inhibitor, showed a reduction in Cx43 phosphorylation on site(s) that can be phosphorylated by CK1 in vitro. To examine CK1 function, normal rat kidney cells were treated with CKI-7, and Cx43 content was analyzed by Triton X-100 extraction, cell-surface biotinylation, and immunofluorescence. Western blot analysis indicated a slight increase in total Cx43, whereas gap junctional (Triton-insoluble) Cx43 decreased, and non-junctional plasma membrane Cx43 increased (as detected by cell surface biotinylation). Immunofluorescence experiments in the presence of CK1 inhibitor showed increases in Cx43 plasma membrane localization but not necessarily accumulation at cell-cell interfaces. Decreased gap junctional and phosphorylated Cx43 was also detected when cells were treated with IC261, a CK1 inhibitor specific for delta or epsilon isoforms. These data suggest CK1delta could regulate Cx43 gap junction assembly by directly phosphorylating Cx43.     

Metastatic phenotype of human prostate tumor cells in athymic nude mice: Alteration by exposure to ethyl methanesulfonate and “reversion” by 5-azacytidine

Summary The human prostate tumor subline 1-LN-PC-3-1A (1-LN) is reproducibly metastatic in adult athymic nude mice. Cells surviving a brief in vitro exposure to ethyl methanesulfonate (EMS) exhibited a profound decrease in capacity for experimental lung metastasis in nude mice. Thirty days after EMS treatment, 1×10⁶ uncloned EMS-treated 1-LN cells (1-LN-EMS-10) were injected IV into groups of 6 to 8-week-old male athymic nude mice (BALB/cAnBOM). A median of 8.5 colonies/lung was observed among 20 1-LN-EMS-10-injected mice, which was significantly different from the median of 51 colonies/lung produced among 14 1-LN-injected mice (P=0.0002). This altered phenotype remained stable during 150 days of continuous culture. However, the 1-LN-EMS-10 cells were tumorigenic in 10/10 nude mice injected SC. Single lung tumor colonies recovered from 1-LN-EMS-10-injected mice and reinjected IV into nude mice produced medians of 32–63 colonies/lung. The altered metastatic phenotype resulting from treatment of 1-LN with EMS was reversed by exposure to a noncytotoxic dose of 5-azacytidine, but unaffected by a second exposure to EMS. Collectively these data demonstrate that the metastatic phenotype of these human tumor cells in athymic nude mice can be heritably altered by in vitro exposure to EMS and 5-azacytidine. Analysis of the mechanisms underlying these phenotypic changes may provide insight into parts of the complex process of tumor cell evolution.     

<Emphasis Type="Italic">Pichia pastoris</Emphasis> fermentation with mixed-feeds of glycerol and methanol: growth kinetics and production improvement

Fed-batch fermentation of a methanol utilization plus (Mut⁺) Pichia pastoris strain typically has a growth phase followed by a production phase (induction phase). In the growth phase glycerol is usually used as carbon for cell growth while in the production phase methanol serves as both inducer and carbon source for recombinant protein expression. Some researchers employed a mixed glycerol-methanol feeding strategy during the induction phase to improve production, but growth kinetics on glycerol and methanol and the interaction between them were not reported. The objective of this paper is to optimize the mixed feeding strategy based on growth kinetic studies using a Mut⁺ Pichia strain, which expresses the heavy-chain fragment C of botulinum neurotoxin serotype C [BoNT/C(Hc)] intracellularly, as a model system. Growth models on glycerol and methanol that describe the relationship between specific growth rate (μ) and specific glycerol/methanol consumption rate (ν _gly, ν _MeOH) were established. A mixed feeding strategy with desired μ _gly/μ _MeOH =1, 2, 3, 4 (desired μ _MeOH set at 0.015 h⁻¹) was employed to study growth interactions and their effect on production. The results show that the optimal desired μ _gly/μ _MeOH is around 2 for obtaining the highest BoNT/C(Hc) protein content in cells: about 3 mg/g wet cells. Electronic Publication     

Thyrotropin secretagogues reduce rat pituitary neuromedin B, a local thyrotropin release inhibitor

Neuromedin B (NB), a bombesin-like peptide, highly concentrated in rat pituitary gland, has been shown to act as an autocrine/paracrine inhibitor of thyrotropin (TSH) release. Here it is shown that a single injection of thyrotropin-releasing hormone (TRH, 1.5 microg/animal, ip), the most important stimulator of thyrotropin secretion, induced approximately 35%-45% decrease in pituitary NB content in rats, as well as an important decrease in NB mRNA at 15 and 30 min (P < 0.05). Acute cold exposure, which induced higher serum TSH with a peak at 30 min, was associated with progressive decrease in pituitary NB, starting at 15 min although only reaching statistical significance after 2 hr (P < 0.05). Although not involved in the early peak, the decrease in NB may be contributing to maintenance of higher serum TSH in cold-exposed animals compared with those at room temperature. Fed rats, 2 hr after being subcutaneously injected with mouse recombinant leptin (8 microg /100 g body wt), showed a x2 increase in serum TSH and 38% reduction in pituitary NB (P < 0.05). In conclusion, TRH and leptin rapidly decreased pituitary NB and it is first proposed that the reduction of the inhibitory tonus of NB on TSH release will ultimately contribute to the amplification of TSH secretion elicited by TSH secretagogues.     

Meta-Analysis of Quantification Methods Shows that Archaea and Bacteria Have Similar Abundances in the Subseafloor

There is no universally accepted method to quantify bacteria and archaea in seawater and marine sediments, and different methods have produced conflicting results with the same samples. To identify best practices, we compiled data from 65 studies, plus our own measurements, in which bacteria and archaea were quantified with fluorescent in situ hybridization (FISH), catalyzed reporter deposition FISH (CARD-FISH), polyribonucleotide FISH, or quantitative PCR (qPCR). To estimate efficiency, we defined “yield” to be the sum of bacteria and archaea counted by these techniques divided by the total number of cells. In seawater, the yield was high (median, 71%) and was similar for FISH, CARD-FISH, and polyribonucleotide FISH. In sediments, only measurements by CARD-FISH in which archaeal cells were permeabilized with proteinase K showed high yields (median, 84%). Therefore, the majority of cells in both environments appear to be alive, since they contain intact ribosomes. In sediments, the sum of bacterial and archaeal 16S rRNA gene qPCR counts was not closely related to cell counts, even after accounting for variations in copy numbers per genome. However, qPCR measurements were precise relative to other qPCR measurements made on the same samples. qPCR is therefore a reliable relative quantification method. Inconsistent results for the relative abundance of bacteria versus archaea in deep subsurface sediments were resolved by the removal of CARD-FISH measurements in which lysozyme was used to permeabilize archaeal cells and qPCR measurements which used ARCH516 as an archaeal primer or TaqMan probe. Data from best-practice methods showed that archaea and bacteria decreased as the depth in seawater and marine sediments increased, although archaea decreased more slowly.     

Pyrrolopyrimidine inhibitors of DNA gyrase B (GyrB) and topoisomerase IV (ParE). Part I: Structure guided discovery and optimization of dual targeting agents with potent,broad-spectrum enzymatic activity

The bacterial topoisomerases DNA gyrase (GyrB) and topoisomerase IV (ParE) are essential enzymes that control the topological state of DNA during replication. The high degree of conservation in the ATP-binding pockets of these enzymes make them appealing targets for broad-spectrum inhibitor development. A pyrrolopyrimidine scaffold was identified from a pharmacophore-based fragment screen with optimization potential. Structural characterization of inhibitor complexes conducted using selected GyrB/ParE orthologs aided in the identification of important steric, dynamic and compositional differences in the ATP-binding pockets of the targets, enabling the design of highly potent pyrrolopyrimidine inhibitors with broad enzymatic spectrum and dual targeting activity.     

Atrial Arrhythmia in Ageing Spontaneously Hypertensive Rats: Unraveling the Substrate in Hypertension and Ageing

Background

Both ageing and hypertension are known risk factors for atrial fibrillation (AF) although the pathophysiological contribution or interaction of the individual factors remains poorly understood. Here we aim to delineate the arrhythmogenic atrial substrate in mature spontaneously hypertensive rats (SHR).

Methods

SHR were studied at 12 and 15 months of age (n = 8 per group) together with equal numbers of age-matched normotensive Wistar-Kyoto control rats (WKY). Electrophysiologic study was performed on superfused isolated right and left atrial preparations using a custom built high-density multiple-electrode array to determine effective refractory periods (ERP), atrial conduction and atrial arrhythmia inducibility. Tissue specimens were harvested for structural analysis.

Results

Compared to WKY controls, the SHR demonstrated: Higher systolic blood pressure (p<0.0001), bi-atrial enlargement (p<0.05), bi-ventricular hypertrophy (p<0.05), lower atrial ERP (p = 0.008), increased atrial conduction heterogeneity (p = 0.001) and increased atrial interstitial fibrosis (p = 0.006) & CD68-positive macrophages infiltration (p<0.0001). These changes resulted in higher atrial arrhythmia inducibility (p = 0.01) and longer induced AF episodes (p = 0.02) in 15-month old SHR. Ageing contributed to incremental bi-atrial hypertrophy (p<0.01) and atrial conduction heterogeneity (p<0.01) without affecting atrial ERP, fibrosis and arrhythmia inducibility. The limited effect of ageing on the atrial substrate may be secondary to the reduction in CD68-positive macrophages.

Conclusions

Significant atrial electrical and structural remodeling is evident in the ageing spontaneously hypertensive rat atria. Concomitant hypertension appears to play a greater pathophysiological role than ageing despite their compounding effect on the atrial substrate. Inflammation is pathophysiologically linked to the pro-fibrotic changes in the hypertensive atria.     

Characterization of cell viability during bioprinting processes

Bioprinting is an emerging technology in the field of tissue engineering and regenerative medicine. The process consists of simultaneous deposition of cells, biomaterial and/or growth factors under pressure through a micro-scale nozzle. Cell viability can be controlled by varying the parameters like pressure and nozzle diameter. The process itself can be a very useful tool for evaluating an in vitro cell injury model. It is essential to understand the cell responses to process-induced mechanical disturbances because they alter cell morphology and function. We carried out analysis and quantification of the degree of cell injury induced by bioprinting process. A parametric study with different process parameters was conducted to analyze and quantify cell injury as well as to optimize the parameters for printing viable cells. A phenomenological model was developed correlating the percentage of live, apoptotic and necrotic cells to the process parameters. This study incorporates an analytical formulation to predict the cell viability through the system as a function of the maximum shear stress in the system. The study shows that dispensing pressure has a more significant effect on cell viability than the nozzle diameter. The percentage of live cells is reduced significantly (by 38.75%) when constructs are printed at 40 psi compared to those printed at 5 psi.     

Cell death in leukemia: passenger protein regulation by topoisomerase inhibitors

Etoposide is a potent inducer of mitotic catastrophe; a type of cell death resulting from aberrant mitosis. It is important in p53 negative cells where p53 dependent apoptosis and events at the G1 and G2 cell cycle checkpoints are compromised. Passenger proteins regulate many aspects of mitosis and siRNA interference or direct inhibition of Aurora B kinase results in mitotic catastrophe. However, there is little available data of clinical relevance in leukaemia models. Here, in p53 negative K562 myeloid leukemia cells, etoposide-induced mitotic catastrophe is shown to be time and/or concentration dependent. Survivin and Aurora remained bound to chromosomes. Survivin and Aurora were also associated with Cdk1 and were shown to form complexes, which in pull down experiments, included INCENP. There was no evidence of Aurora B kinase suppression. These data suggests etoposide will complement Aurora B kinase inhibitors currently in clinical trials for cancer.