Protection from Clostridium difficile toxin B-catalysed Rac1/Cdc42 glucosylation by tauroursodeoxycholic acid-induced Rac1/Cdc42 phosphorylation

Toxin A (TcdA) and toxin B (TcdB) are the major virulence factors of Clostridium difficile-associated diarrhoea (CDAD). TcdA and TcdB mono-glucosylate small GTPases of the Rho family, thereby causing actin re-organisation in colonocytes, resulting in the loss of colonic barrier function. The hydrophilic bile acid tauroursodeoxycholic acid (TUDCA) is an approved drug for the treatment of cholestasis and biliary cirrhosis. In this study, TUDCA-induced activation of Akt1 is presented to increase cellular levels of pS71-Rac1/Cdc42 in human hepatocarcinoma (HepG2) cells, showing for the first time that bile acid signalling affects the activity of Rho proteins. Rac1/Cdc42 phosphorylation, in turn, protects Rac1/Cdc42 from TcdB-catalysed glucosylation and reduces the TcdB-induced cytopathic effects in HepG2 cells. The results of this study indicate that TUDCA may prove useful as a therapeutic agent for the treatment of CDAD.     

Intracranial aneurysm risk locus 5q23.2 is associated with elevated systolic blood pressure

Although genome-wide association studies (GWAS) have identified hundreds of complex trait loci, the pathomechanisms of most remain elusive. Studying the genetics of risk factors predisposing to disease is an attractive approach to identify targets for functional studies. Intracranial aneurysms (IA) are rupture-prone pouches at cerebral artery branching sites. IA is a complex disease for which GWAS have identified five loci with strong association and a further 14 loci with suggestive association. To decipher potential underlying disease mechanisms, we tested whether there are IA loci that convey their effect through elevating blood pressure (BP), a strong risk factor of IA. We performed a meta-analysis of four population-based Finnish cohorts (n(FIN) = 11 266) not selected for IA, to assess the association of previously identified IA candidate loci (n = 19) with BP. We defined systolic BP (SBP), diastolic BP, mean arterial pressure, and pulse pressure as quantitative outcome variables. The most significant result was further tested for association in the ICBP-GWAS cohort of 200 000 individuals. We found that the suggestive IA locus at 5q23.2 in PRDM6 was significantly associated with SBP in individuals of European descent (p(FIN) = 3.01E-05, p(ICBP-GWAS) = 0.0007, p(ALL) = 8.13E-07). The risk allele of IA was associated with higher SBP. PRDM6 encodes a protein predominantly expressed in vascular smooth muscle cells. Our study connects a complex disease (IA) locus with a common risk factor for the disease (SBP). We hypothesize that common variants in PRDM6 can contribute to altered vascular wall structure, hence increasing SBP and predisposing to IA. True positive associations often fail to reach genome-wide significance in GWAS. Our findings show that analysis of traditional risk factors as intermediate phenotypes is an effective tool for deciphering hidden heritability. Further, we demonstrate that common disease loci identified in a population isolate may bear wider significance.     

Irrelevance of Microsatellite Instability in the Epidemiology of Sporadic Pancreatic Ductal Adenocarcinoma

Background and Aims

Pancreatic cancer risk is increased in Lynch syndrome (LS) patients with mismatch repair gene defects predisposing to colonic and extracolonic cancers with microsatellite instability (MSI). However, the frequency of MSI pancreatic cancers has never been ascertained in consecutive, unselected clinical series, and their contribution to the sporadic and inherited burden of pancreatic cancer remains to be established. Aims of the study were to determine the prevalence of MSI in surgically resected pancreatic cancers in a multicentric, retrospective study, and to assess the occurrence of pancreatic cancer in LS.

Methods

MS-status was screened by a panel of 5 mononucleotide repeats (Bat26, Bat25, NR-21, NR-24 and NR-27) in 338 consecutive pancreatic ductal adenocarcinoma (PDAC), resected at two Italian and one German referral centres. The personal history of pancreatic cancer was assessed in an independent set of 58 probands with LS and in 138 first degree relatives who had cancers.

Results

Only one PDAC (0.3%) showed MSI. This was a medullary type cancer, with hMLH1-deficiency, and no identified germ-line mutation but methylation of hMLH1. Pancreatic cancer occurred in 5 (2.5%) LS patients. Histological sampling was available for 2 cases, revealing PDAC in one case and an ampullary cancer in the other one.

Conclusions

MSI prevalence is negligible in sporadic, resected PDAC. Differently, the prevalence of pancreatic cancer is 2.5% in LS patients, and cancers other than PDAC may be encountered in this setting. Surveillance for pancreatic cancer should be advised in LS mutation carriers at referral centers.     

Morphofunctional alterations in testicular cells of deslorelin-treated boars: an immunohistochemical study

In this study we thoroughly scrutinized testes morphology and investigated whether treatment of recipient boars with gonadotropin-releasing hormone (GnRH)-agonist deslorelin could alter the expression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), luteinizing hormone receptors (LHRs), and androgen receptors (ARs) in testicular cells. An implant containing 4.7 mg of the GnRH-agonist deslorelin was subcutaneously inserted into crossbred male pigs at 91 and 147 days of age. Testicular traits, morphology of the testes, the proteins' expression, and testosterone concentration in blood plasma were analyzed in all boars after slaughter at 175 days of age. Histological analysis revealed significant alterations in both the interstitial tissue and seminiferous tubules of experimental animals after 28 and 84 days of deslorelin treatment. The intensity of the AR immunostaining within the testis appeared as a function of the severity of testicular dysgenesis. Time-dependent action of deslorelin on the expression of LHR and 3beta-HSD in Leydig cells was also detected. Staining for LHR and 3beta-HSD was very weak or the Leydig cells were immunonegative. Concomitantly, a significant decrease in plasma testosterone level was found in both groups of deslorelin-treated boars when compared with the control group. This is the first report showing the cellular distribution of AR, LHR, and 3beta-HSD in testicular cells of deslorelin-treated boars. It is concluded that morphological and immunohistochemical studies are important for the evaluation of testicular histoarchitecture and steroidogenic function. Subsequently, the endocrine control of reproduction in the GnRH-agonist deslorelin-treated males will be better understood.     

Prospects of androgenetic induction in <Emphasis Type="Italic">Lupinus</Emphasis> spp.

Anthers cultures of six Polish cultivars of pasture lupin (Lupinus L.) were examined for their androgenic response. Anthers with microspores at the uninucleate stage were isolated from flower buds and cultured in liquid media. Better viability of androgenetic structures was obtained when donor plants had grown under field as opposed to greenhouse conditions. A density of five anthers per 0.5 ml medium was more conducive to androgenetic induction than 25 anthers per 0.5 ml medium. Addition of 5% maltose to the induction medium and culture at 25°C without pre-treatment of flowers, buds or anthers promoted microspore release and division. The greatest frequency of androgenic callus, ~70% was developed from cvs. Katon, Wat (white lupin), in contrast to cvs. Legat, Juno (yellow lupin), Polonez and Sonet (narrow-leafed lupin) with callus induction ~30–40%. Despite various combinations of media tested, plant regeneration was not obtained from anther derived callus.     

Overdiagnosis of malaria in patients with severe febrile illness in Tanzania: a prospective study

Objective To study the diagnosis and outcomes in people admitted to hospital with a diagnosis of severe malaria in areas with differing intensities of malaria transmission.Design Prospective observational study of children and adults over the course a year.Setting 10 hospitals in north east Tanzania.Participants 17 313 patients were admitted to hospital; of these 4474 (2851 children aged under 5 years) fulfilled criteria for severe disease.Main outcome measure Details of the treatment given and outcome. Altitudes of residence (a proxy for transmission intensity) measured with a global positioning system.Results Blood film microscopy showed that 2062 (46.1%) of people treated for malaria had Plasmodium falciparum (slide positive). The proportion of slide positive cases fell with increasing age and increasing altitude of residence. Among 1086 patients aged ≥ 5 years who lived above 600 metres, only 338 (31.1%) were slide positive, while in children < 5 years living in areas of intense transmission (< 600 metres) most (958/1392, 68.8%) were slide positive. Among 2375 people who were slide negative, 1571 (66.1%) were not treated with antibiotics and of those, 120 (7.6%) died. The case fatality in slide negative patients was higher (292/2412, 12.1%) than for slide positive patients (142/2062, 6.9%) (P < 0.001). Respiratory distress and altered consciousness were the strongest predictors of mortality in slide positive and slide negative patients and in adults as well as children.Conclusions In Tanzania, malaria is commonly overdiagnosed in people presenting with severe febrile illness, especially in those living in areas with low to moderate transmission and in adults. This is associated with a failure to treat alternative causes of severe infection. Diagnosis needs to be improved and syndromic treatment considered. Routine hospital data may overestimate mortality from malaria by over twofold.     

Effects of Furfural on the Respiratory Metabolism of Saccharomyces cerevisiae in Glucose-Limited Chemostats

Effects of furfural on the aerobic metabolism of the yeast Saccharomyces cerevisiae were studied by performing chemostat experiments, and the kinetics of furfural conversion was analyzed by performing dynamic experiments. Furfural, an important inhibitor present in lignocellulosic hydrolysates, was shown to have an inhibitory effect on yeast cells growing respiratively which was much greater than the inhibitory effect previously observed for anaerobically growing yeast cells. The residual furfural concentration in the bioreactor was close to zero at all steady states obtained, and it was found that furfural was exclusively converted to furoic acid during respiratory growth. A metabolic flux analysis showed that furfural affected fluxes involved in energy metabolism. There was a 50% increase in the specific respiratory activity at the highest steady-state furfural conversion rate. Higher furfural conversion rates, obtained during pulse additions of furfural, resulted in respirofermentative metabolism, a decrease in the biomass yield, and formation of furfuryl alcohol in addition to furoic acid. Under anaerobic conditions, reduction of furfural partially replaced glycerol formation as a way to regenerate NAD⁺. At concentrations above the inlet concentration of furfural, which resulted in complete replacement of glycerol formation by furfuryl alcohol production, washout occurred. Similarly, when the maximum rate of oxidative conversion of furfural to furoic acid was exceeded aerobically, washout occurred. Thus, during both aerobic growth and anaerobic growth, the ability to tolerate furfural appears to be directly coupled to the ability to convert furfural to less inhibitory compounds.