The Aspartate-Semialdehyde Dehydrogenase of Edwardsiella ictaluri and Its Use as Balanced-Lethal System in Fish Vaccinology

asdA mutants of Gram-negative bacteria have an obligate requirement for diaminopimelic acid (DAP), which is an essential constituent of the peptidoglycan layer of the cell wall of these organisms. In environments deprived of DAP, i.e., animal tissues, they will undergo lysis. Deletion of the asdA gene has previously been exploited to develop antibiotic-sensitive strains of live attenuated recombinant bacterial vaccines. Introduction of an Asd+ plasmid into a ΔasdA mutant makes the bacterial strain plasmid-dependent. This dependence on the Asd+ plasmid vector creates a balanced-lethal complementation between the bacterial strain and the recombinant plasmid. E. ictaluri is an enteric Gram-negative fish pathogen that causes enteric septicemia in catfish. Because E. ictaluri is a nasal/oral invasive intracellular pathogen, this bacterium is a candidate to develop a bath/oral live recombinant attenuated Edwardsiella vaccine (RAEV) for the catfish aquaculture industry. As a first step to develop an antibiotic-sensitive RAEV strain, we characterized and deleted the E. ictaluri asdA gene. E. ictaluri ΔasdA01 mutants exhibit an absolute requirement for DAP to grow. The asdA gene of E. ictaluri was complemented by the asdA gene from Salmonella. Several Asd+ expression vectors with different origins of replication were transformed into E. ictaluri ΔasdA01. Asd+ vectors were compatible with the pEI1 and pEI2 E. ictaluri native plasmids. The balanced-lethal system was satisfactorily evaluated in vivo. Recombinant GFP, PspA, and LcrV proteins were synthesized by E. ictaluri ΔasdA01 harboring Asd+ plasmids. Here we constructed a balanced-lethal system, which is the first step to develop an antibiotic-sensitive RAEV for the aquaculture industry

Validated stability-indicating spectrofluorimetric methods for the determination of ebastine in pharmaceutical preparations

Two sensitive, selective, economic, and validated spectrofluorimetric methods were developed for the determination of ebastine (EBS) in pharmaceutical preparations depending on reaction with its tertiary amino group. Method I involves condensation of the drug with mixed anhydrides (citric and acetic anhydrides) producing a product with intense fluorescence, which was measured at 496 nm after excitation at 388 nm

A Low Dose of Dietary Resveratrol Partially Mimics Caloric Restriction and Retards Aging Parameters in Mice

Resveratrol in high doses has been shown to extend lifespan in some studies in invertebrates and to prevent early mortality in mice fed a high-fat diet. We fed mice from middle age (14-months) to old age (30-months) either a control diet, a low dose of resveratrol (4.9 mg kg−1 day−1), or a calorie restricted (CR) diet and examined genome-wide transcriptional profiles. We report a striking transcriptional overlap of CR and resveratrol in heart, skeletal muscle and brain. Both dietary interventions inhibit gene expression profiles associated with cardiac and skeletal muscle aging, and prevent age-related cardiac dysfunction. Dietary resveratrol also mimics the effects of CR in insulin mediated glucose uptake in muscle. Gene expression profiling suggests that both CR and resveratrol may retard some aspects of aging through alterations in chromatin structure and transcription. Resveratrol, at doses that can be readily achieved in humans, fulfills the definition of a dietary compound that mimics some aspects of CR

Preface paper to the Semi-Arid Land-Surface-Atmosphere (SALSA) Program special issue

The Semi-Arid Land-Surface-Atmosphere Program (SALSA) is a multi-agency, multi-national research effort that seeks to evaluate the consequences of natural and human-induced environmental change in semi-arid regions. The ultimate goal of SALSA is to advance scientific understanding of the semi-arid portion of the hydrosphere–biosphere interface in order to provide reliable information for environmental decision making. SALSA approaches this goal through a program of long-term, integrated observations, process research, modeling, assessment, and information management that is sustained by cooperation among scientists and information users. In this preface to the SALSA special issue, general program background information and the critical nature of semi-arid regions is presented. A brief description of the Upper San Pedro River Basin, the initial location for focused SALSA research follows. Several overarching research objectives under which much of the interdisciplinary research contained in the special issue was undertaken are discussed. Principal methods, primary research sites and data collection used by numerous investigators during 1997–1999 are then presented. Scientists from about 20 US, five European (four French and one Dutch), and three Mexican agencies and institutions have collaborated closely to make the research leading to this special issue a reality. The SALSA Program has served as a model of interagency cooperation by breaking new ground in the approach to large scale interdisciplinary science with relatively limited resources

Upregulation of α7 Nicotinic Receptors by Acetylcholinesterase C-Terminal Peptides

BACKGROUND: The alpha-7 nicotinic acetylcholine receptor (alpha7-nAChR) is well known as a potent calcium ionophore that, in the brain, has been implicated in excitotoxicity and hence in the underlying mechanisms of neurodegenerative disorders such as Alzheimer's disease. Previous research implied that the activity of this receptor may be modified by exposure to a peptide fragment derived from the C-terminal region of the enzyme acetylcholinesterase. This investigation was undertaken to determine if the functional changes observed could be attributed to peptide binding interaction with the alpha7-nAChR, or peptide modulation of receptor expression. METHODOLOGY/PRINCIPAL FINDINGS: This study provides evidence that two peptides derived from the C-terminus of acetylcholinesterase, not only selectively displace specific bungarotoxin binding at the alpha7-nAChR, but also alter receptor binding properties for its familiar ligands, including the alternative endogenous agonist choline. Of more long-term significance, these peptides also induce upregulation of alpha7-nAChR mRNA and protein expression, as well as enhancing receptor trafficking to the plasma membrane. CONCLUSIONS/SIGNIFICANCE: The results reported here demonstrate a hitherto unknown relationship between the alpha7-nAChR and the non-enzymatic functions of acetylcholinesterase, mediated independently by its C-terminal domain. Such an interaction may prove valuable as a pharmacological tool, prompting new approaches for understanding, and combating, the process of neurodegeneration

Upregulation of α7 Nicotinic Receptors by Acetylcholinesterase C-Terminal Peptides

BACKGROUND: The alpha-7 nicotinic acetylcholine receptor (alpha7-nAChR) is well known as a potent calcium ionophore that, in the brain, has been implicated in excitotoxicity and hence in the underlying mechanisms of neurodegenerative disorders such as Alzheimer's disease. Previous research implied that the activity of this receptor may be modified by exposure to a peptide fragment derived from the C-terminal region of the enzyme acetylcholinesterase. This investigation was undertaken to determine if the functional changes observed could be attributed to peptide binding interaction with the alpha7-nAChR, or peptide modulation of receptor expression. METHODOLOGY/PRINCIPAL FINDINGS: This study provides evidence that two peptides derived from the C-terminus of acetylcholinesterase, not only selectively displace specific bungarotoxin binding at the alpha7-nAChR, but also alter receptor binding properties for its familiar ligands, including the alternative endogenous agonist choline. Of more long-term significance, these peptides also induce upregulation of alpha7-nAChR mRNA and protein expression, as well as enhancing receptor trafficking to the plasma membrane. CONCLUSIONS/SIGNIFICANCE: The results reported here demonstrate a hitherto unknown relationship between the alpha7-nAChR and the non-enzymatic functions of acetylcholinesterase, mediated independently by its C-terminal domain. Such an interaction may prove valuable as a pharmacological tool, prompting new approaches for understanding, and combating, the process of neurodegeneration

Understanding the limitations of radiation-induced cell cycle checkpoints

The DNA damage response pathways involve processes of double-strand break (DSB) repair and cell cycle checkpoint control to prevent or limit entry into S phase or mitosis in the presence of unrepaired damage. Checkpoints can function to permanently remove damaged cells from the actively proliferating population but can also halt the cell cycle temporarily to provide time for the repair of DSBs. Although efficient in their ability to limit genomic instability, checkpoints are not foolproof but carry inherent limitations. Recent work has demonstrated that the G1/S checkpoint is slowly activated and allows cells to enter S phase in the presence of unrepaired DSBs for about 4–6 h post irradiation. During this time, only a slowing but not abolition of S-phase entry is observed. The G2/M checkpoint, in contrast, is quickly activated but only responds to a level of 10–20 DSBs such that cells with a low number of DSBs do not initiate the checkpoint or terminate arrest before repair is complete. Here, we discuss the limitations of these checkpoints in the context of the current knowledge of the factors involved. We suggest that the time needed to fully activate G1/S arrest reflects the existence of a restriction point in G1-phase progression. This point has previously been defined as the point when mitogen starvation fails to prevent cells from entering S phase. However, cells that passed the restriction point can respond to DSBs, albeit with reduced efficiency

Non Mycobacterial Virulence Genes in the Genome of the Emerging Pathogen Mycobacterium abscessus

Mycobacterium abscessus is an emerging rapidly growing mycobacterium (RGM) causing a pseudotuberculous lung disease to which patients with cystic fibrosis (CF) are particularly susceptible. We report here its complete genome sequence. The genome of M. abscessus (CIP 104536T) consists of a 5,067,172-bp circular chromosome including 4920 predicted coding sequences (CDS), an 81-kb full-length prophage and 5 IS elements, and a 23-kb mercury resistance plasmid almost identical to pMM23 from Mycobacterium marinum. The chromosome encodes many virulence proteins and virulence protein families absent or present in only small numbers in the model RGM species Mycobacterium smegmatis. Many of these proteins are encoded by genes belonging to a “mycobacterial” gene pool (e.g. PE and PPE proteins, MCE and YrbE proteins, lipoprotein LpqH precursors). However, many others (e.g. phospholipase C, MgtC, MsrA, ABC Fe(3+) transporter) appear to have been horizontally acquired from distantly related environmental bacteria with a high G+C content, mostly actinobacteria (e.g. Rhodococcus sp., Streptomyces sp.) and pseudomonads. We also identified several metabolic regions acquired from actinobacteria and pseudomonads (relating to phenazine biosynthesis, homogentisate catabolism, phenylacetic acid degradation, DNA degradation) not present in the M. smegmatis genome. Many of the “non mycobacterial” factors detected in M. abscessus are also present in two of the pathogens most frequently isolated from CF patients, Pseudomonas aeruginosa and Burkholderia cepacia. This study elucidates the genetic basis of the unique pathogenicity of M. abscessus among RGM, and raises the question of similar mechanisms of pathogenicity shared by unrelated organisms in CF patients

Is there a role for melatonin in fibromyalgia?

Fibromyalgia, characterised by persistent pain, fatigue, sleep disturbance and cognitive dysfunction, is a central sensitivity syndrome that also involves abnormality in peripheral generators and in the hypothalamic pituitary adrenal axis. Heterogeneity of clinical expression of fibromyalgia with a multifactorial aetiology has made the development of effective therapeutic strategies challenging. Physiological properties of the neurohormone melatonin appear related to the symptom profile exhibited by patients with fibromyalgia and thus disturbance of it’s production would be compatible with the pathophysiology. Altered levels of melatonin have been observed in patients with fibromyalgia which are associated with lower secretion during dark hours and higher secretion during daytime. However, inconsistencies of available clinical evidence limit conclusion of a relationship between levels of melatonin and symptom profiles in patients with fibromyalgia. Administration of melatonin to patients with fibromyalgia has demonstrated suppression of many symptoms and an improved quality of life consistent with benefit as a therapy for the management of this condition. Further studies with larger samples, however, are required to explore the potential role of melatonin in the pathophysiology of fibromyalgia and determine the optimal dosing regimen of melatonin for the management of fibromyalgia

Laparoscopy in management of appendicitis in high-, middle-, and low-income countries: a multicenter, prospective, cohort study.

BACKGROUND: Appendicitis is the most common abdominal surgical emergency worldwide. Differences between high- and low-income settings in the availability of laparoscopic appendectomy, alternative management choices, and outcomes are poorly described. The aim was to identify variation in surgical management and outcomes of appendicitis within low-, middle-, and high-Human Development Index (HDI) countries worldwide. METHODS: This is a multicenter, international prospective cohort study. Consecutive sampling of patients undergoing emergency appendectomy over 6 months was conducted. Follow-up lasted 30 days. RESULTS: 4546 patients from 52 countries underwent appendectomy (2499 high-, 1540 middle-, and 507 low-HDI groups). Surgical site infection (SSI) rates were higher in low-HDI (OR 2.57, 95% CI 1.33-4.99, p = 0.005) but not middle-HDI countries (OR 1.38, 95% CI 0.76-2.52, p = 0.291), compared with high-HDI countries after adjustment. A laparoscopic approach was common in high-HDI countries (1693/2499, 67.7%), but infrequent in low-HDI (41/507, 8.1%) and middle-HDI (132/1540, 8.6%) groups. After accounting for case-mix, laparoscopy was still associated with fewer overall complications (OR 0.55, 95% CI 0.42-0.71, p < 0.001) and SSIs (OR 0.22, 95% CI 0.14-0.33, p < 0.001). In propensity-score matched groups within low-/middle-HDI countries, laparoscopy was still associated with fewer overall complications (OR 0.23 95% CI 0.11-0.44) and SSI (OR 0.21 95% CI 0.09-0.45). CONCLUSION: A laparoscopic approach is associated with better outcomes and availability appears to differ by country HDI. Despite the profound clinical, operational, and financial barriers to its widespread introduction, laparoscopy could significantly improve outcomes for patients in low-resource environments. TRIAL REGISTRATION: NCT02179112