Disruption of the acetate kinase (ack) gene of Clostridium acetobutylicum results in delayed acetate production

In microorganisms, the enzyme acetate kinase (AK) catalyses the formation of ATP from ADP by de-phosphorylation of acetyl phosphate into acetic acid. A mutant strain of Clostridium acetobutylicum lacking acetate kinase activity is expected to have reduced acetate and acetone production compared to the wild type. In this work, a C. acetobutylicum mutant strain with a selectively disrupted ack gene, encoding AK, was constructed and genetically and physiologically characterized. The ack− strain showed a reduction in acetate kinase activity of more than 97% compared to the wild type. The fermentation profiles of the ack− and wild-type strain were compared using two different fermentation media, CGM and CM1. The latter contains acetate and has a higher iron and magnesium content than CGM. In general, fermentations by the mutant strain showed a clear shift in the timing of peak acetate production relative to butyrate and had increased acid uptake after the onset of solvent formation. Specifically, in acetate containing CM1 medium, acetate production was reduced by more than 80% compared to the wild type under the same conditions, but both strains produced similar final amounts of solvents. Fermentations in CGM showed similar peak acetate and butyrate levels, but increased acetoin (60%), ethanol (63%) and butanol (16%) production and reduced lactate (−50%) formation by the mutant compared to the wild type. These findings are in agreement with the proposed regulatory function of butyryl phosphate as opposed to acetyl phosphate in the metabolic switch of solventogenic clostridia

Lysine-Specific Demethylase 1 (LSD1) Is Required for the Transcriptional Repression of the Telomerase Reverse Transcriptase (hTERT) Gene

BACKGROUND: Lysine-specific demethylase 1 (LSD1), catalysing demethylation of mono- and di-methylated histone H3-K4 or K9, exhibits diverse transcriptional activities by mediating chromatin reconfiguration. The telomerase reverse transcriptase (hTERT) gene, encoding an essential component for telomerase activity that is involved in cellular immortalization and transformation, is silent in most normal human cells while activated in up to 90% of human cancers. It remains to be defined how exactly the transcriptional activation of the hTERT gene occurs during the oncogenic process. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we determined the effect of LSD1 on hTERT transcription. In normal human fibroblasts with a tight hTERT repression, a pharmacological inhibition of LSD1 led to a weak hTERT expression, and a robust induction of hTERT mRNA was observed when LSD1 and histone deacetylases (HDACs) were both inhibited. Small interference RNA-mediated depletion of both LSD1 and CoREST, a co-repressor in HDAC-containing complexes, synergistically activated hTERT transcription. In cancer cells, inhibition of LSD1 activity or knocking-down of its expression led to significant increases in levels of hTERT mRNA and telomerase activity. Chromatin immunoprecipitation assay showed that LSD1 occupied the hTERT proximal promoter, and its depletion resulted in elevated di-methylation of histone H3-K4 accompanied by increased H3 acetylation locally in cancer cells. Moreover, during the differentiation of leukemic HL60 cells, the decreased hTERT expression was accompanied by the LSD1 recruitment to the hTERT promoter. CONCLUSIONS/SIGNIFICANCE: LSD1 represses hTERT transcription via demethylating H3-K4 in normal and cancerous cells, and together with HDACs, participates in the establishment of a stable repression state of the hTERT gene in normal or differentiated malignant cells. The findings contribute to better understandings of hTERT/telomerase regulation, which may be implicated in the development of therapeutic strategies for telomerase dysregulation-associated human diseases including cancers

The burden and spatial distribution of bovine African trypanosomes in small holder crop-livestock production systems in Tororo District, south-eastern Uganda

African animal trypanosomiasis (AAT) is considered to be one of the greatest constraints to livestock production and livestock-crop integration in most African countries. South-eastern Uganda has suffered for more than two decades from outbreaks of zoonotic Human African Trypanosomiasis (HAT), adding to the burden faced by communities from AAT. There is insufficient AAT and HAT data available (in the animal reservoir) to guide and prioritize AAT control programs that has been generated using contemporary, sensitive and specific molecular techniques. This study was undertaken to evaluate the burden that AAT presents to the small-scale cattle production systems in south-eastern Uganda. Randomised cluster sampling was used to select 14% (57/401) of all cattle containing villages across Tororo District. Blood samples were taken from all cattle in the selected villages between September-December 2011; preserved on FTA cards and analysed for different trypanosomes using a suite of molecular techniques. Generalized estimating equation and Rogen-Gladen estimator models were used to calculate apparent and true prevalences of different trypanosomes while intra cluster correlations were estimated using a 1-way mixed effect analysis of variance (ANOVA) in R statistical software version 3.0.2.ResultsThe prevalence of all trypanosome species in cattle was 15.3% (95% CI; 12.2-19.1) while herd level trypanosome species prevalence varied greatly between 0-43%. Trypanosoma vivax (17.4%, 95% CI; 10.6-16.8) and Trypanosoma brucei rhodesiense (0.03%) were respectively, the most, and least prevalent trypanosome species identified. The prevalence of bovine trypanosomes in this study indicates that AAT remains a significant constraint to livestock health and livestock production. There is need to implement tsetse and trypanosomiasis control efforts across Tororo District by employing effective, cheap and sustainable tsetse and trypanosomiasis control method that could be integrated in the control of other endemic vector borne diseases like tick-borne diseases

Improvements on Restricted Insecticide Application Protocol for Control of Human and Animal African Trypanosomiasis in Eastern Uganda

African trypanosomes constrain livestock and human health in Sub-Saharan Africa, and aggravate poverty and hunger of these otherwise largely livestock-keeping communities. To solve this, there is need to develop and use effective and cheap tsetse control methods. To this end, we aimed at determining the smallest proportion of a cattle herd that needs to be sprayed on the legs, bellies and ears (RAP) for effective Human and Animal African Trypanosomiasis (HAT/AAT) control.; Cattle in 20 villages were ear-tagged and injected with two doses of diminazene diaceturate (DA) forty days apart, and randomly allocated to one of five treatment regimens namely; no treatment, 25%, 50%, 75% monthly RAP and every 3 month Albendazole drench. Cattle trypanosome re-infection rate was determined by molecular techniques. ArcMap V10.3 was used to map apparent tsetse density (FTD) from trap catches. The effect of graded RAP on incidence risk ratios and trypanosome prevalence was determined using Poisson and logistic random effect models in R and STATA V12.1 respectively. Incidence was estimated at 9.8/100 years in RAP regimens, significantly lower compared to 25.7/100 years in the non-RAP regimens (incidence rate ratio: 0.37; 95% CI: 0.22-0.65; P>0.001). Likewise, trypanosome prevalence after one year of follow up was significantly lower in RAP animals than in non-RAP animals (4% vs 15%, OR: 0.20, 95% CI: 0.08-0.44; P>0.001). Contrary to our expectation, level of protection did not increase with increasing proportion of animals treated.; Reduction in RAP coverage did not significantly affect efficacy of treatment. This is envisaged to improve RAP adaptability to low income livestock keepers but needs further evaluation in different tsetse challenge, HAT/AAT transmission rates and management systems before adopting it for routine tsetse control programs

Artificial intelligence and bladder cancer arrays.

Non-muscle invasive bladder cancer is a heterogenous disease whose management is dependent upon the risk of progression to muscle invasion. Although the recurrence rate is high, the majority of tumors are indolent and can be managed by endoscopic means alone. The prognosis of muscle invasion is poor and radical treatment is required if cure is to be obtained. Progression risk in non-invasive tumors is hard to determine at tumor diagnosis using current clinicopathological means. To improve the accuracy of progression prediction various biomarkers have been evaluated. To discover novel biomarkers several authors have used gene expression microarrays. Various statistical methods have been described to interpret array data, but to date no biomarkers have entered clinical practice. Here, we describe a new method of microarray analysis using neurofuzzy modeling (NFM), a form of artificial intelligence, and integrate it with artificial neural networks (ANN) to investigate non-muscle invasive bladder cancer array data (n=66 tumors). We develop a predictive panel of 11 genes, from 2800 expressed genes, that can significantly identify tumor progression (average Logrank p = 0.0288) in the analyzed cancers. In comparison, this panel appears superior to those genes chosen using traditional analyses (average Logrank p = 0.3455) and tumor grade (Logrank, p = 0.2475) in this non-muscle invasive cohort. We then analyze panel members in a new non-muscle invasive bladder cancer cohort (n=199) using immunohistochemistry with six commercially available antibodies. The combination of 6 genes (LIG3, TNFRSF6, KRT18, ICAM1, DSG2 and BRCA2) significantly stratifies tumor progression (Logrank p = 0.0096) in the new cohort. We discuss the benefits of the transparent NFM approach with respect to other reported methods