'TB or not TB': the conundrum of pre-European contact tuberculosis in the Pacific.

Tuberculosis (TB) is a major global health threat, infecting one-third of the world's population. Despite this prominence, the age, origin and spread of the disease have been topics of contentious debate. Molecular studies suggest that Mycobacterium tuberculosis 'sensu stricto', the most common strain of TB infecting humans today, originated in Africa and from there spread into Europe and Asia. The M. tuberculosis strains most commonly found across the Pacific and the Americas today are most closely related to European strains, supporting a hypothesis that the disease only reached these regions relatively recently via European sailors or settlers. However, this hypothesis is inconsistent with palaeopathological evidence of TB-like lesions in human remains from across the Pacific that predate European contact. Similarly, genetic evidence from pre-European South American mummies challenges the notion of a European introduction of the disease into the Pacific. Here, we review the complex evidence for the age and origin of TB in the Pacific, and discuss key gaps in our knowledge and how these may be addressed. This article is part of the theme issue 'Insights into health and disease from ancient biomolecules'.

Development of Multiwell-Plate Methods Using Pure Cultures of Methanogens To Identify New Inhibitors for Suppressing Ruminant Methane Emissions.

Hydrogenotrophic methanogens typically require strictly anaerobic culturing conditions in glass tubes with overpressures of H2 and CO2 that are both time-consuming and costly. To increase the throughput for screening chemical compound libraries, 96-well microtiter plate methods for the growth of a marine (environmental) methanogen Methanococcus maripaludis strain S2 and the rumen methanogen Methanobrevibacter species AbM4 were developed. A number of key parameters (inoculum size, reducing agents for medium preparation, assay duration, inhibitor solvents, and culture volume) were optimized to achieve robust and reproducible growth in a high-throughput microtiter plate format. The method was validated using published methanogen inhibitors and statistically assessed for sensitivity and reproducibility. The Sigma-Aldrich LOPAC library containing 1,280 pharmacologically active compounds and an in-house natural product library (120 compounds) were screened against M. maripaludis as a proof of utility. This screen identified a number of bioactive compounds, and MIC values were confirmed for some of them against M. maripaludis and M. AbM4. The developed method provides a significant increase in throughput for screening compound libraries and can now be used to screen larger compound libraries to discover novel methanogen-specific inhibitors for the mitigation of ruminant methane emissions.IMPORTANCE Methane emissions from ruminants are a significant contributor to global greenhouse gas emissions, and new technologies are required to control emissions in the agriculture technology (agritech) sector. The discovery of small-molecule inhibitors of methanogens using high-throughput phenotypic (growth) screening against compound libraries (synthetic and natural products) is an attractive avenue. However, phenotypic inhibitor screening is currently hindered by our inability to grow methanogens in a high-throughput format. We have developed, optimized, and validated a high-throughput 96-well microtiter plate assay for growing environmental and rumen methanogens. Using this platform, we identified several new inhibitors of methanogen growth, demonstrating the utility of this approach to fast track the development of methanogen-specific inhibitors for controlling ruminant methane emissions.

Retrospective study of alemtuzumab vs ATG-based conditioning without irradiation for unrelated and matched sibling donor transplants in acquired severe aplastic anemia: a study from the British Society for Blood and Marrow Transplantation.

This retrospective national study compared the use of alemtuzumab-based conditioning regimens for hematopoietic SCT (HSCT) in acquired severe aplastic anemia with antithymocyte globulin (ATG)-based regimens. One hundred patients received alemtuzumab and 55 ATG-based regimens. A matched sibling donor (MSD) was used in 87 (56%), matched unrelated donor (MUD) in 60 (39%) and other related or mismatched unrelated donor (UD) in 8 (5%) patients. Engraftment failure occurred in 9% of the alemtuzumab group and 11% of the ATG group. Five-year OS was 90% for the alemtuzumab and 79% for the ATG groups, P=0.11. For UD HSCT, OS of patients was better when using alemtuzumab (88%) compared with ATG (57%), P=0.026, although smaller numbers of patients received ATG. Similar outcomes for MSD HSCT using alemtuzumab or ATG were seen (91% vs 85%, respectively, P=0.562). A lower risk of chronic GVHD (cGVHD) was observed in the alemtuzumab group (11% vs 26%, P=0.031). On multivariate analysis, use of BM as stem cell source was associated with better OS and EFS, and less acute and cGVHD; young age was associated with better EFS and lower risk of graft failure. This large study confirms successful avoidance of irradiation in the conditioning regimens for MUD HSCT patients.

Lack of Evidence for Seed Transmission of 'Candidatus Liberibacter africanus' Associated with Greening (Huanglongbing) in Citrus in South Africa.

'Candidatus Liberibacter africanus' is associated with citrus greening (huanglongbing [HLB]) in South Africa. Various unpublished reports have suggested that the related bacterium 'Ca. L. asiaticus' associated with HLB in citrus might be seed transmissible based on real-time PCR results. Seed transmission poses a risk of long distance disease spread, especially with the dissemination of rootstock seed. Therefore, it was essential to determine whether 'Ca. L. africanus' is seed transmitted in citrus. Fruit from 26 'Ca. L. africanus'-infected branches of six citrus cultivars showing greening symptoms were collected and the seed was harvested. Cultivars included were Minneola tangelo (Citrus reticulata × C. paradisi); sweet oranges (C. sinensis) Premier midseason, Clanor midseason, and Olinda Valencia; Eureka lemon (C. limon) and Troyer citrange (Poncirus trifoliata × C. sinensis) rootstock variety. Branches bearing each fruit were collected and confirmed to contain 'Ca. L. africanus' by real-time PCR testing using Taqman probe HLBp and HLBaf and HLBr primers as described by Li et al. (3). The seed of each sample was sorted into five categories ranging from healthy looking to totally aborted based on their appearance before planting. Germination was done in seed trays under vector-free conditions at 24 to 28°C. Thereafter the seedlings were planted in small, plastic bags and monitored for greening-like symptoms or other abnormalities for up to 2 years. A slow-release, balanced fertilizer was applied and supplemented with micro-nutrient sprays for plant maintenance. Plants showing abnormal symptoms were potted into larger pots and closely monitored. These samples and a number of other seedlings showing growth abnormalities were tested for 'Ca. L. africanus' by real-time PCR as described above. In total, 1,570 seedlings were obtained. Some abnormal symptoms such as small chlorotic leaves, interveinal chlorosis, yellow veins, and stunting were seen in some seedlings. Most symptoms resembled deficiencies, and no blotchy mottle typical of 'Ca. L. africanus' infection was noted on any of the seedlings. Abnormal seedlings arose from normal and abortive seed. One hundred and eighteen of these seedlings (8 Minneola tangelo; 24 Premier midseason, 42 Clanor midseason, 33 Olinda Valencia, and 11 Troyer citrange seedlings) were individually tested using real-time PCR for 'Ca. L. africanus' detection. These seedlings had germinated from essentially healthy-looking seed (category 1) to seeds with severe abnormalities (category 5) and 33, 24, 23, 30, and 8 seedlings, respectively, were tested from each of the five seed categories. No samples tested positive with real-time PCR based on a positive/negative threshold Cq value of 35. Buds of some seedlings that yielded the lowest Cq values above 35 were grafted onto healthy 'Madam vinous' sweet orange (C. sinensis) seedlings and monitored for symptom development for 3 months. No symptoms developed and all these indicators also tested negative for 'Ca. L. africanus', indicating the absence of a transmissible agent. Just as other researchers (1,2) have recently indicated a lack of evidence for seed transmission of 'Ca. L. asiaticus', no seed transmission of 'Ca. L. africanus' could be demonstrated in this experiment either. References: (1) U. Albrecht and K. D. Bowman. HortScience 44:1967, 2009. (2) J. S. Hartung et al. Plant Dis. 94:1200, 2010. (3) W. Li et al. J. Microbiol. Methods 66:104, 2006.

Decision-making in teams: issues arising from two UK evaluations.

Interagency and interprofessional working has often been operationalised through the development of integrated, multiprofessional teams in the UK. However, there is considerable ambivalence reported about the success of such teams. This paper reports on two evaluations of different types of inter-agency/intra-agency, interdisciplinary/unidisciplinary teams. One study used a soft systems methodology to evaluate a health and social care team for people with enduring mental health needs and the other used a pluralistic framework to examine integrated nursing teams in primary care. In both studies, the team-working arrangements influenced the decisions made by the team members such that client care became increasingly responsive and proactive. These changes were made possible by two processes. First, information transaction was augmented and was instrumental in supporting effective client-related decision-making. Second, there was enhanced support for decision-making, especially in respect of problem solving. However, the increased autonomy of the team members had the potential to marginalise those outside the team from decision-making. It is suggested that working within a team can impact on the decisions made by team members, which exceeds a collection of individual decisions. The strengths of complex multiprofessional teams for service users may be realised if the processes of decision-making are respected.

Biosynthetic origin of conjugated double bonds: production of fatty acid components of high-value drying oils in transgenic soybean embryos.

Vegetable oils that contain fatty acids with conjugated double bonds, such as tung oil, are valuable drying agents in paints, varnishes, and inks. Although several reaction mechanisms have been proposed, little is known of the biosynthetic origin of conjugated double bonds in plant fatty acids. An expressed sequence tag (EST) approach was undertaken to characterize the enzymatic basis for the formation of the conjugated double bonds of alpha-eleostearic (18:3Delta(9cis, 11trans,13trans)) and alpha-parinaric (18:4Delta(9cis,11trans, 13trans,15cis)) acids. Approximately 3,000 ESTs were generated from cDNA libraries prepared from developing seeds of Momordica charantia and Impatiens balsamina, tissues that accumulate large amounts of alpha-eleostearic and alpha-parinaric acids, respectively. From ESTs of both species, a class of cDNAs encoding a diverged form of the Delta(12)-oleic acid desaturase was identified. Expression of full-length cDNAs for the Momordica (MomoFadX) and Impatiens (ImpFadX) enzymes in somatic soybean embryos resulted in the accumulation of alpha-eleostearic and alpha-parinaric acids, neither of which is present in untransformed soybean embryos. alpha-Eleostearic and alpha-parinaric acids together accounted for as much as 17% (wt/wt) of the total fatty acids of embryos expressing MomoFadX. These results demonstrate the ability to produce fatty acid components of high-value drying oils in transgenic plants. These findings also demonstrate a previously uncharacterized activity for Delta(12)-oleic acid desaturase-type enzymes that we have termed "conjugase."

Cloning and characterization of the promoter for the liver isoform of the rat carnitine palmitoyltransferase I (L-CPT I) gene.

Carnitine palmitoyltransferase I (CPTI) catalyses the transfer of long chain fatty acids to carnitine for translocation across the mitochondrial inner membrane. The cDNAs of two isoforms of CPT I, termed the hepatic and muscle isoforms, have been cloned. Expression of the hepatic CPT I gene (L-CPT I) is subject to developmental, hormonal and tissue specific regulation. We have cloned the promoter of the L-CPTI gene from a rat genomic library. In the L-CPTI gene, there are two exons 5' to the exon containing the ATG that initiates translation. Exon 1 and the 5' end of exon 2 contain sequences that were not previously described in the rat L-CPTI cDNA. There is an alternatively spliced form of the L-CPTI mRNA in which exon 2 is skipped. The proximal promoter of the L-CPTI gene is extremely GC rich and does not contain a TATA box. There are several putative Sp1 binding sites near the transcriptional start site. A 190 base pair fragment of the promoter can efficiently drive transcription of luciferase and CAT (chloramphenicol acetyltransferase) reporter genes transiently transfected into HepG2 cells. Sequences in both the first intron and the promoter contribute to basal expression. Our results provide the foundation for further studies into the regulation of L-CPTI gene expression.

Repair of rat gastric mucosa: effect of 16,16-dimethyl prostaglandin E2.

Prostaglandins protect the gastric mucosa against a variety of injurious agents and may accelerate the recovery of the gastric mucosa following damage. In previous studies prostaglandins were given prior to the injurious agent, so it was not possible to distinguish their potential effects on accelerating repair or reducing initial damage. We have investigated the effect of 16,16-dimethyl prostaglandin E2 (dmPGE2) on the repair of the gastric mucosa after injury induced by several injurious agents. dmPGE2 was given orally 15 min prior to aspirin or sodium salicylate, or 30 min after aspirin, sodium salicylate, or ethanol. dmPGE2 delivered prior to injury reduced the aspirin-induced fall in mucosal potential difference (PD), but had no effect on that induced by sodium salicylate. dmPGE2 administered after ASA injury significantly increased recovery of PD (P < 0.05), but did not alter the rate of recovery of PD with other damaging agents. Histological damage was decreased in rats treated with dmPGE2 after aspirin compared to aspirin-only-treated rats (P < 0.02). Exogenous dmPGE2 protects and restores gastric mucosal integrity after aspirin damage but has no effect on the repair of sodium salicylate and ethanol injured mucosa, suggesting that repair of the gastric mucosa after aspirin damage is enhanced by dmPGE2 due to its ability to prevent ongoing damage, rather than directly enhancing repair processes.

Lactase phlorhizin hydrolase turnover in vivo in water-fed and colostrum-fed newborn pigs.

We have estimated the synthesis rates in vivo of precursor and brush-border (BB) polypeptides of lactase phlorhizin hydrolase (LPH) in newborn pigs fed with water or colostrum for 24h post partum. At the end of the feeding period, piglets were anaesthetized and infused intravenously for 3h with L-[4-3H]- phenylalanine. Blood and jejunal samples were collected at timed intervals. The precursor and BB forms of LPH were isolated from jejunal mucosa by immunoprecipitation followed by SDS/PAGE, and their specific radioactivity in Phe determined. The kinetics of precursor and BB LPH labelling were analysed by using a linear compartmental model. Immunoisolated LPH protein consisted of five polypeptides [high-mannose LPH precursor (proLPHh), complex glycosylated LPH precursor (proLPHe), intermediate complex glycosylated LPH precursor (proLPH1i) and two forms of BB LPH]. The fractional synthesis rate (Ks) of proLPHh and proLPHc (approx. 5%/min) were the same in the two groups but the absolute synthesis rate (in arbitrary units, min-1) of proLPHh in the colostrum-fed animals was twice that of the water-fed animals. The Ks values of proLPHi polypeptides were significantly different (water-fed, 3.89%/min; colostrum-fed, 1.6%/min), but the absolute synthesis rates did not differ. The Ks of BB LPH was not different between experimental treatment groups (on average 0.037%/min). However, the proportion of newly synthesized proLPHh processed to BB LPH was 48% lower in colostrum-fed than in water-fed animals. We conclude that in neonatal pigs, the ingestion of colostrum stimulates the synthesis of proLPHh but, at least temporarily, disrupts the processing of proLPH polypeptides to the BB enzyme.

Dietary cholesterol stimulates hepatic biosynthesis of triglyceride and reduces oxidation of fatty acids in the rat.

Experiments were conducted in the intact rat and in the isolated, perfused rat liver to investigate the possibility that the increase in the concentration of hepatic triglyceride and increase in the secretion of the very low density lipoprotein (VLDL)-triglyceride (TG) resulting from addition of cholesterol to the diet are due to stimulation of synthesis of triglyceride, reduced fatty acid oxidation, or both. Male rats were fed for 7 days with either a cholesterol-free diet to which 5% (w/w) corn oil was added, or with the same diet supplemented with 0.5% cholesterol. Fed animals received [1-14C]oleic acid via the tail vein, as a complex with rat serum, and were killed 2 h later. Feeding cholesterol for 7 days increased hepatic triglyceride and cholesteryl ester (CE) concentrations, moderately elevated free cholesterol, but did not affect phospholipid (PL) levels, as we had previously observed after a feeding period of 3 weeks. Incorporation of [1-14C]oleic acid into hepatic and plasma triglyceride increased significantly (60 and 48%, respectively) with cholesterol feeding. Incorporation of [1-14C]oleic acid into hepatic and plasma cholesteryl esters increased by 63 and 79%, respectively, while incorporation into phospholipid was unaffected. Increasing the fat (corn oil) content of the diet to 20% (w/w) did not change these effects of dietary cholesterol. Studies using isolated, perfused rat livers were carried out in vitro after rats were fed the 5% corn oil diet for 3 weeks. [Perfusions lasted 4 h. The perfusion medium contained 3% bovine serum albumin and 30% washed bovine erythrocytes in Krebs-Henseleit-HCO3 buffer.(ABSTRACT TRUNCATED AT 250 WORDS)

Aetiology and pathogenesis of postinfective tropical malabsorption (tropical sprue).

Postinfective tropical malabsorption (TM; tropical sprue) starts with an acute intestinal infection (bacterial, viral, or parasitic) which can affect predominantly the small or the large intestine. Miscellaneous invasive pathogens cause subsequent enterocyte damage affecting the entire small intestine and, to a lesser extent, the colon. Enteroglucagon, a tropic hormone, is then liberated and reaches a high plasma concentration. Small-intestinal stasis results. Further bacterial colonisation (in the lumen and also at the enterocyte surface) is encouraged. Continuing enterocyte damage is worsened by coexistent folate depletion, which is initiated at the onset of disease; body stores of folate reach a low concentration by 3 or 4 months. The cycle continues until the bacterial overgrowth is eliminated with an antibiotic (eg, tetracycline), or mucosal integrity recovers (hastened by oral folic-acid supplements), or both.

Successful management of serious disopyramide poisoning.

A case of deliberate disopyramide overdosage is described. Circulatory collapse was treated by means of a large dose of isoprenaline, and charcoal haemoperfusion was used in an attempt to enhance the elimination of disopyramide. The suitability of this treatment regime is discussed in the light of findings from animal studies and the implications for the management of the disopyramide-poisoned patient are considered.

Tobacco use, occupation, coffee, various nutrients, and bladder cancer.

In a Canadian population-based case-control study of 480 males and 152 female case-control pairs, the relative risk for development of bladder cancer for ever used versus never used cigarettes was 3.9 for males and 2.4 for females, with a dose-response relationship in both sexes. A reduced risk was associated with the use of filter cigarettes compared to nonfilter cigarettes. After control for cigarette usage, a significant risk was noted for male pipe smokers. For male ex-smokers the risk after 15 years of no smoking was less than one-half that of current male smokers. Bladder cancer risk was found for workers in the chemical, rubber, photographic, petroleum, medical, and food processing industries among males and for workers occupationally exposed to dust or fumes among both sexes. Bladder cancer risk was elevated for males consuming all types of coffee, regular coffee, and instant coffee and for females consuming instant coffee, but no dose-response relationship was found. Risk was found for males consuming water from nonpublic supples but not for females. No risk was observed in males or females consuming nitrate-containing foods, beverages other than coffee, or fiddlehead greens. Hair dye usage in females and phenacetin usage in males and females carried no risk. Divergent findings by area for aspirin suggested that an overall association was not causal. Reevaluation of the data on artificial sweeteners confirmed a significant bladder cancer risk in males and a dose-response relationship. The cumulated population attributable risk for bladder cancer was 90% for males from cigarette smoking, industrial exposure, and exposure to nonpublic water supplies and 29% for females from cigarette smoking, industrial exposure, and instant coffee consumption.