Evaluation of apoptosis markers in different cell lines infected with equine arteritis virus

Equine arteritis virus (EAV) induces apoptosis in infected cells. Cell death caused by EAV has been studied mainly using three cell lines, BHK-21, RK-13 and Vero cells. The mechanism of apoptosis varies among cell lines and results cannot be correlated owing to differences in EAV strains used. We evaluated different markers for apoptosis in BHK-21, RK-13 and Vero cell lines using the Bucyrus EAV reference strain. Acridine orange/ethidium bromide staining revealed morphological changes in infected cells, while flow cytometry indicated the extent of apoptosis. We also observed DNA fragmentation, but the DNA ladder was detected at different times post-infection depending on the cell line, i.e., 48, 72 and 96 h post-infection in RK-13, Vero and BHK-21 cells, respectively. Measurement of viral titers obtained with each cell line indicated that apoptosis causes interference with viral replication and therefore decreased viral titers. As an unequivocal marker of apoptosis, we measured the expression of caspase-3 and caspases-8 and -9 as extrinsic and intrinsic markers of apoptosis pathways, respectively. Caspase-8 in BHK-21 cells was the only protease that was not detected at any of the times assayed. We found that Bucyrus EAV strain exhibited a distinctive apoptosis pathway depending on the cell line.     

Potential role of viruses in white plague coral disease

White plague (WP)-like diseases of tropical corals are implicated in reef decline worldwide, although their etiological cause is generally unknown. Studies thus far have focused on bacterial or eukaryotic pathogens as the source of these diseases; no studies have examined the role of viruses. Using a combination of transmission electron microscopy (TEM) and 454 pyrosequencing, we compared 24 viral metagenomes generated from Montastraea annularis corals showing signs of WP-like disease and/or bleaching, control conspecific corals, and adjacent seawater. TEM was used for visual inspection of diseased coral tissue. No bacteria were visually identified within diseased coral tissues, but viral particles and sequence similarities to eukaryotic circular Rep-encoding single-stranded DNA viruses and their associated satellites (SCSDVs) were abundant in WP diseased tissues. In contrast, sequence similarities to SCSDVs were not found in any healthy coral tissues, suggesting SCSDVs might have a role in WP disease. Furthermore, Herpesviridae gene signatures dominated healthy tissues, corroborating reports that herpes-like viruses infect all corals. Nucleocytoplasmic large DNA virus (NCLDV) sequences, similar to those recently identified in cultures of Symbiodinium (the algal symbionts of corals), were most common in bleached corals. This finding further implicates that these NCLDV viruses may have a role in bleaching, as suggested in previous studies. This study determined that a specific group of viruses is associated with diseased Caribbean corals and highlights the potential for viral disease in regional coral reef decline.     

Production of the virus‐like particles of nipah virus matrix protein in Pichia pastoris as diagnostic reagents

The matrix (M) protein of Nipah virus (NiV) is a peripheral protein that plays a vital role in the envelopment of nucleocapsid protein and acts as a bridge between the viral surface and the nucleocapsid proteins. The M protein is also proven to play an important role in production of virus‐like particles (VLPs) and is essential for assembly and budding of NiV particles. The recombinant M protein produced in Escherichia coli assembled into VLPs in the absence of the viral surface proteins. However, the E. coli produced VLPs are smaller than the native virus particles. Therefore, the aims of this study were to produce NiV M protein in Pichia pastoris, to examine the structure of the VLPs formed, and to assess the potential of the VLPs as a diagnostic reagent. The M protein was successfully expressed in P. pastoris and was detected with anti‐myc antibody using Western blotting. The VLPs formed by the recombinant M protein were purified with sucrose density gradient ultracentrifugation, high‐performance liquid chromatography (HPLC), and Immobilized Metal Affinity Chromatography (IMAC). Immunogold staining and transmission electron microscopy confirmed that the M protein assembled into VLPs as large as 200 nm. ELISA revealed that the NiV M protein produced in P. pastoris reacted strongly with positive NiV sera demonstrating its potential as a diagnostic reagent. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1038–1045, 2016     

Effects of Nigella sativa on apoptosis and GABAA receptor density in cerebral cortical and hippocampal neurons in pentylenetetrazol induced kindling in rats

We investigated the effects of Nigella sativa on apoptosis and gamma-aminobutyric acid (GABA_A) receptor density in cerebral cortical and hippocampal neurons in a pentylenetetrazol (PTZ)-induced kindling model in rats. The PTZ kindling model was produced by injecting PTZ in subconvulsive doses to rats on days 1, 3, 5, 8, 10, 12, 15, 17, 19, 22 and 24 of the study into animals of PTZ treated (PTZ) and PTZ + N. sativa treated (PTZ + NS) groups. Clonic and tonic seizures were induced by injecting a convulsive dose of PTZ on day 26 of the study. Rats in the PTZ + NS group were treated also with a 10 mg/kg methanolic extract of N. sativa 2 h before each PTZ injection. Rats in the control group were treated with 4 ml/kg saline. The number of neurons that expressed GABA_A receptors in the hippocampus and cerebral cortex of rats in the PTZ and PTZ + NS groups increased significantly. There was no significant difference in the number of GABA_A receptors between the PTZ and PTZ + NS groups. GABA_A receptor density of the neurons in the cerebral cortex, but not hippocampus, was increased in PTZ group compared to controls. We observed a significant increase in the number of apoptotic neurons in the cerebral cortex of rats of both the PTZ and PTZ + NS groups compared to controls. We observed a significant decrease in the number of the apoptotic neurons in the cerebral cortex of rats in the PTZ + NS group compared to the PTZ group. N. sativa treatment ameliorated the PTZ induced neurodegeneration in the cerebral cortex as reflected by neuronal apoptosis and neuronal GABA_A receptor frequency.     

Functional Analysis of the Helicobacter pylori Flagellar Switch Proteins

Helicobacter pylori uses flagellum-mediated chemotaxis to promote infection. Bacterial flagella change rotational direction by changing the state of the flagellar motor via a subcomplex referred to as the switch. Intriguingly, the H. pylori genome encodes four switch complex proteins, FliM, FliN, FliY, and FliG, instead of the more typical three of Escherichia coli or Bacillus subtilis. Our goal was to examine whether and how all four switch proteins participate in flagellation. Previous work determined that FliG was required for flagellation, and we extend those findings to show that all four switch proteins are necessary for normal numbers of flagellated cells. Furthermore, while fliY and fliN are partially redundant with each other, both are needed for wild-type levels of flagellation. We also report the isolation of an H. pylori strain containing an R54C substitution in fliM, resulting in bacteria that swim constantly and do not change direction. Along with data demonstrating that CheY-phosphate interacts with FliM, these findings suggest that FliM functions in H. pylori much as it does in other organisms.Flagellar motility is important for gastric colonization by the ulcer-causing bacterium Helicobacter pylori and also for suborgan localization within the stomach (16-18, 33, 45). Flagellar motility is regulated by a set of signal transduction proteins, collectively referred to as the chemotaxis pathway, that control the migration of microbes in response to environmental cues. This pathway is well elucidated in organisms such as Escherichia coli, Salmonella enterica serovar Typhimurium (referred to hereinafter as S. Typhimurium), and Bacillus subtilis. Sequence analysis of the genomes of other flagellated bacteria, including H. pylori, has suggested that there is diversity in the set of chemotaxis proteins that a particular microbe contains. Here we analyze the diversity of H. pylori''s flagellar switch proteins, which control flagellar rotational direction.The molecular mechanisms underlying chemotactic signal transduction in E. coli and S. Typhimurium have been extensively studied (7, 50) The overall function of this pathway is to convert the perception of local environmental conditions into a swimming response that drives bacteria toward beneficial conditions and away from harmful ones. Such migration is accomplished by interspersing straight, or smooth, swimming with periods of random reorientations or tumbles. Smooth swimming occurs when the flagella rotate counterclockwise (CCW), while reorienting occurs when the flagella rotate clockwise (CW). The chemotaxis signal transduction system acts to appropriately alter flagellar rotation. The canonical chemotaxis pathway consists of a chemoreceptor bound to the coupling protein CheW, which is in turn bound to the histidine kinase CheA. If a beneficial/attractant ligand is not bound (or a repellant is bound) to the chemoreceptor, CheA autophosphorylates and passes a phosphate to the response regulator CheY. Phosphorylated CheY (CheY-P) interacts with a protein complex called the flagellar switch (discussed at more length below). This interaction causes a switch in the direction of flagellar rotation from CCW to CW, thus reorienting the cells, via an as-yet-unknown mechanism (reviewed in references 23 and 29).Bacterial flagella are complex, multiprotein organelles (reviewed in references 23, 25, and 29). Each flagellum is composed of several parts, including the filament, the hook, and the basal body (listed from outside the cell to inside the cytoplasm). The flagellar basal body spans from the outer membrane to the cytoplasm and is responsible for rotating the flagellum. This part of the flagellum is further made up of several subassemblies that are named for their locations. The innermost is called the switch or C ring, based on its location in the cytoplasm. The switch is comprised of three proteins in E. coli, FliM, FliN, and FliG (reviewed in references 23 and 29). Experimental evidence strongly suggests that these proteins, along with the stator proteins MotA and MotB, drive motor rotation, because one can obtain point mutations in these proteins that disrupt rotation but not flagellation. Null mutations, however, in fliM, fliN, or fliG also result in aflagellated cells, a phenotype that has been proposed to arise because these proteins are needed to complete the flagellar export apparatus (23).There is extensive structural information about each of the switch proteins and their arrangement in the flagellum (reviewed in references 23 and 29, with additional key references added below). There are 26 copies of FliG, 34 copies of FliM, and ∼136 copies of FliN, arranged in a circular structure at the base of each flagellum. FliM is positioned between FliG and FliN and interacts with both. FliM also binds CheY-P via sequences in the first 16 amino acids, and elsewhere (15), to play a key role in switching flagellar rotation direction. FliG, the switch protein closest to the cytoplasmic membrane, interacts with the stator protein MotA, the FliF membrane protein that forms the flagellar basal-body MS ring, and the membrane-bound respiratory protein fumarate reductase (11). FliG has the most direct role in creating flagellar rotation. FliN is the most cytoplasmic component of the switch, and its role is not fully understood. FliN may play a role in switching by possibly binding CheY-P directly (36) and an additional role in flagellar assembly, because it binds to the flagellar export protein FliH and localizes it, along with its interaction partners FliI and FliJ, to the flagellum (20, 28, 36). FliN contains significant sequence similarity to secretion proteins of type III secretion systems of Yersinia pestis and Shigella flexneri. The conserved domain comprises most of FliN and is called a SpoA or PFAM PF01052 domain. Other FliN homologs include YscL and Spa33 (25).The flagellar switch of another well-studied chemotactic microbe, B. subtilis, differs slightly in its protein makeup from that of E. coli. B. subtilis contains FliM and FliG, which function similarly to their E. coli counterparts, but instead of FliN it has a protein called FliY (6, 42). FliY of B. subtilis has two functional domains, one of which is homologous to E. coli FliN, while the other shares similarity with the B. subtilis chemotaxis protein CheC, which functions to dephosphorylate CheY-P. FliY is the most active known phosphatase of CheY-P in B. subtilis (40, 41).H. pylori contains homologs of many of the chemotaxis and flagellar genes found in other organisms (32, 48). Curiously, its genome encodes four predicted flagellar switch proteins, FliG, FliM, and both FliY and FliN, although FliY was not annotated in the original genome analysis. Previous work had determined that H. pylori strain SS1 lacking fliG was aflagellated (1), but the other switch proteins had not been analyzed. As noted above, FliN and FliY share a FliN domain and so could have functional redundancy. fliY and fliM appear to reside in an operon, suggesting that the two encoded proteins function together (see Fig. S1 in the supplemental material).Since having all four flagellar switch proteins in one microbe is unusual, we were curious as to whether all four serve “switch” functions. As noted above, fliM and fliG deletions typically result in an aflagellated phenotype in other organisms. Others had previously shown that fliG mutations have this phenotype in H. pylori (1), and we additionally show here that fliM null mutants are also almost completely aflagellate. In spite of a shared domain that might indicate functional redundancy, we show that fliN and fliY are each necessary for normal numbers of flagellated cells. Finally, we characterize a fliM point mutant that results in a lock-smooth swimming bias and demonstrate physical interaction between CheY-P and FliM, indicating that FliM responds to CheY signaling in H. pylori in a manner similar to that found in E. coli, S. Typhimurium, B. subtilis, and other studied organisms.     

Augmentation of partially regenerated nerves by end-to-side side-to-side grafting neurotization: experience based on eight late obstetric brachial plexus cases

Objective

The effect of end-to-side neurotization of partially regenerated recipient nerves on improving motor power in late obstetric brachial plexus lesions, so-called nerve augmentation, was investigated.

Methods

Eight cases aged 3 – 7 years were operated upon and followed up for 4 years (C5,6 rupture C7,8T1 avulsion: 5; C5,6,7,8 rupture T1 avulsion:1; C5,6,8T1 rupture C7 avulsion:1; C5,6,7 ruptureC8 T1 compression: one 3 year presentation after former neurotization at 3 months). Grade 1–3 muscles were neurotized. Grade0 muscles were neurotized, if the electromyogram showed scattered motor unit action potentials on voluntary contraction without interference pattern. Donor nerves included: the phrenic, accessory, descending and ascending loops of the ansa cervicalis, 3^rd and 4^th intercostals and contralateral C7.

Results

Superior proximal to distal regeneration was observed firstly. Differential regeneration of muscles supplied by the same nerve was observed secondly (superior supraspinatus to infraspinatus regeneration). Differential regeneration of antagonistic muscles was observed thirdly (superior biceps to triceps and pronator teres to supinator recovery). Differential regeneration of fibres within the same muscle was observed fourthly (superior anterior and middle to posterior deltoid regeneration). Differential regeneration of muscles having different preoperative motor powers was noted fifthly; improvement to Grade 3 or more occurred more in Grade2 than in Grade0 or Grade1 muscles. Improvements of cocontractions and of shoulder, forearm and wrist deformities were noted sixthly. The shoulder, elbow and hand scores improved in 4 cases.

Limitations

The sample size is small. Controls are necessary to rule out any natural improvement of the lesion. There is intra- and interobserver variability in testing muscle power and cocontractions.

Conclusion

Nerve augmentation improves cocontractions and muscle power in the biceps, pectoral muscles, supraspinatus, anterior and lateral deltoids, triceps and in Grade2 or more forearm muscles. As it is less expected to improve infraspinatus power, it should be associated with a humeral derotation osteotomy and tendon transfer. Function to non improving Grade 0 or 1 forearm muscles should be restored by muscle transplantation.

Level of evidence

Level IV, prospective case series.     

Comparison of multimarker logistic regression models, with application to a genomewide scan of schizophrenia

Background

Imprinted genes show expression from one parental allele only and are important for development and behaviour. This extreme mode of allelic imbalance has been described for approximately 56 human genes. Imprinting status is often disrupted in cancer and dysmorphic syndromes. More subtle variation of gene expression, that is not parent-of-origin specific, termed 'allele-specific gene expression' (ASE) is more common and may give rise to milder phenotypic differences. Using two allele-specific high-throughput technologies alongside bioinformatics predictions, normal term human placenta was screened to find new imprinted genes and to ascertain the extent of ASE in this tissue.

Results

Twenty-three family trios of placental cDNA, placental genomic DNA (gDNA) and gDNA from both parents were tested for 130 candidate genes with the Sequenom MassArray system. Six genes were found differentially expressed but none imprinted. The Illumina ASE BeadArray platform was then used to test 1536 SNPs in 932 genes. The array was enriched for the human orthologues of 124 mouse candidate genes from bioinformatics predictions and 10 human candidate imprinted genes from EST database mining. After quality control pruning, a total of 261 informative SNPs (214 genes) remained for analysis. Imprinting with maternal expression was demonstrated for the lymphocyte imprinted gene ZNF331 in human placenta. Two potential differentially methylated regions (DMRs) were found in the vicinity of ZNF331. None of the bioinformatically predicted candidates tested showed imprinting except for a skewed allelic expression in a parent-specific manner observed for PHACTR2, a neighbour of the imprinted PLAGL1 gene. ASE was detected for two or more individuals in 39 candidate genes (18%).

Conclusions

Both Sequenom and Illumina assays were sensitive enough to study imprinting and strong allelic bias. Previous bioinformatics approaches were not predictive of new imprinted genes in the human term placenta. ZNF331 is imprinted in human term placenta and might be a new ubiquitously imprinted gene, part of a primate-specific locus. Demonstration of partial imprinting of PHACTR2 calls for re-evaluation of the allelic pattern of expression for the PHACTR2-PLAGL1 locus. ASE was common in human term placenta.     

The mutation rates of di-, tri- and tetranucleotide repeats in Drosophila melanogaster

In a recent study, we reported that the combined average mutation rate of 10 di-, 6 tri-, and 8 tetranucleotide repeats in Drosophila melanogaster was 6.3 x 10(-6) mutations per locus per generation, a rate substantially below that of microsatellite repeat units in mammals studied to date (range = 10(-2)-10(-5) per locus per generation). To obtain a more precise estimate of mutation rate for dinucleotide repeat motifs alone, we assayed 39 new dinucleotide repeat microsatellite loci in the mutation accumulation lines from our earlier study. Our estimate of mutation rate for a total of 49 dinucleotide repeats is 9.3 x 10(-6) per locus per generation, only slightly higher than the estimate from our earlier study. We also estimated the relative difference in microsatellite mutation rate among di-, tri-, and tetranucleotide repeats in the genome of D. melanogaster using a method based on population variation, and we found that tri- and tetranucleotide repeats mutate at rates 6.4 and 8.4 times slower than that of dinucleotide repeats, respectively. The slower mutation rates of tri- and tetranucleotide repeats appear to be associated with a relatively short repeat unit length of these repeat motifs in the genome of D. melanogaster. A positive correlation between repeat unit length and allelic variation suggests that mutation rate increases as the repeat unit lengths of microsatellites increase.