Five Days Periodic Fasting Elevates Levels of Longevity Related Christensenella and Sirtuin Expression in Humans.

Periodic fasting (PF) is an increasingly popular approach that assists in the management of metabolic and inflammatory diseases as well as in preventing mechanisms involved in aging. However, little is known about the effects of fasting on gut microbiota and its impact on the epigenetic regulation of metabolically relevant enzymes, especially sirtuins (SIRTs). We analyzed the effect of periodic fasting on the human gut microbiota, SIRTs expression, and mitochondrial content in 51 males and females. The participants fasted under supervision for five consecutive days following the Buchinger fasting guidelines. Ketogenesis, selected mRNAs, miRNAs, mitochondrial (mt) DNA, and gut composition were analyzed before and after PF. PF triggered a significant switch in metabolism, as indicated by the increase in ß-hydroxybutyrate (BHB) and pyruvate dehydrogenase kinase isoform 4 (PDK4) expression in the capillary blood. MtDNA, SIRT1, SIRT3, and miRlet7b-5p expression in blood cells were elevated, whereas SIRT6 and miR125b-5p were not affected. Following fasting, gut microbiota diversity increased, and a statistically significant correlation between SIRT1 gene expression and the abundance of Prevotella and Lactobacillus was detected. The abundance of longevity related Christensenella species increased after fasting and inversely correlated with age as well as body mass index (BMI). Thus, this represents the first study that showing that fasting not only changes the composition of the gut microbiota, making it more diverse, but also affects SIRT expression in humans.

Unraveling the gut microbiome of the long-lived naked mole-rat.

The naked mole-rat (Heterocephalus glaber) is a subterranean mouse-sized African mammal that shows astonishingly few age-related degenerative changes and seems to not be affected by cancer. These features make this wild rodent an excellent model to study the biology of healthy aging and longevity. Here we characterize for the first time the intestinal microbial ecosystem of the naked mole-rat in comparison to humans and other mammals, highlighting peculiarities related to the specific living environment, such as the enrichment in bacteria able to utilize soil sulfate as a terminal electron acceptor to sustain an anaerobic oxidative metabolism. Interestingly, some compositional gut microbiota peculiarities were also shared with human gut microbial ecosystems of centenarians and Hadza hunter-gatherers, considered as models of a healthy gut microbiome and of a homeostatic and highly adaptive gut microbiota-host relationship, respectively. In addition, we found an enrichment of short-chain fatty acids and carbohydrate degradation products in naked mole-rat compared to human samples. These data confirm the importance of the gut microbial ecosystem as an adaptive partner for the mammalian biology and health, independently of the host phylogeny.

Modulation of GLO1 Expression Affects Malignant Properties of Cells.

The energy metabolism of most tumor cells relies on aerobic glycolysis (Warburg effect) characterized by an increased glycolytic flux that is accompanied by the increased formation of the cytotoxic metabolite methylglyoxal (MGO). Consequently, the rate of detoxification of this reactive glycolytic byproduct needs to be increased in order to prevent deleterious effects to the cells. This is brought about by an increased expression of glyoxalase 1 (GLO1) that is the rate-limiting enzyme of the MGO-detoxifying glyoxalase system. Here, we overexpressed GLO1 in HEK 293 cells and silenced it in MCF-7 cells using shRNA. Tumor-related properties of wild type and transformed cells were compared and key glycolytic enzyme activities assessed. Furthermore, the cells were subjected to hypoxic conditions to analyze the impact on cell proliferation and enzyme activities. Our results demonstrate that knockdown of GLO1 in the cancer cells significantly reduced tumor-associated properties such as migration and proliferation, whereas no functional alterations where found by overexpression of GLO1 in HEK 293 cells. In contrast, hypoxia caused inhibition of cell growth of all cells except of those overexpressing GLO1. Altogether, we conclude that GLO1 on one hand is crucial to maintaining tumor characteristics of malignant cells, and, on the other hand, supports malignant transformation of cells in a hypoxic environment when overexpressed.

Ethyl Pyruvate Combats Human Leukemia Cells but Spares Normal Blood Cells.

Ethyl pyruvate, a known ROS scavenger and anti-inflammatory drug was found to combat leukemia cells. Tumor cell killing was achieved by concerted action of necrosis/apoptosis induction, ATP depletion, and inhibition of glycolytic and para-glycolytic enzymes. Ethyl lactate was less harmful to leukemia cells but was found to arrest cell cycle in the G0/G1 phase. Both, ethyl pyruvate and ethyl lactate were identified as new inhibitors of GSK-3ß. Despite the strong effect of ethyl pyruvate on leukemia cells, human cognate blood cells were only marginally affected. The data were compiled by immune blotting, flow cytometry, enzyme activity assay and gene array analysis. Our results inform new mechanisms of ethyl pyruvate-induced cell death, offering thereby a new treatment regime with a high therapeutic window for leukemic tumors.

Ethyl Pyruvate: An Anti-Microbial Agent that Selectively Targets Pathobionts and Biofilms.

The microbiota has a strong influence on health and disease in humans. A causative shift favoring pathobionts is strongly linked to diseases. Therefore, anti-microbial agents selectively targeting potential pathogens as well as their biofilms are urgently demanded. Here we demonstrate the impact of ethyl pyruvate, so far known as ROS scavenger and anti-inflammatory agent, on planktonic microbes and biofilms. Ethyl pyruvate combats preferably the growth of pathobionts belonging to bacteria and fungi independent of the genera and prevailing drug resistance. Surprisingly, this anti-microbial agent preserves symbionts like Lactobacillus species. Moreover, ethyl pyruvate prevents the formation of biofilms and promotes matured biofilms dissolution. This potentially new anti-microbial and anti-biofilm agent could have a tremendous positive impact on human, veterinary medicine and technical industry as well.

Analysis of cultivable microbiota and diet intake pattern of the long-lived naked mole-rat.

BACKGROUND: A variety of microbial communities exist throughout the human and animal body. Genetics, environmental factors and long-term dietary habit contribute to shaping the composition of the gut microbiota. For this reason the study of the gut microbiota of a mammal exhibiting an extraordinary life span is of great importance. The naked mole-rat (Heterocephalus glaber) is a eusocial mammal known for its longevity and cancer resistance. METHODS: Here we analyzed its gut microbiota by cultivating the bacteria under aerobic and anaerobic conditions and identifying their species by mass spectrometry. RESULTS: Altogether, 29 species of microbes were identified, predominantly belonging to Firmicutes, and Bacteroidetes. The most frequent species were Bacillus megaterium (45.2 %), followed by Bacteroides thetaiotaomicron (19.4 %), Bacteroides ovatus, Staphylococcus sciuri and Paenibacillus spp., each with a frequency of 16.1 %. CONCLUSION: Overall, the gut of the naked mole-rat is colonized by diverse, but low numbers of cultivable microbes compared with humans and mice. The primary food plants of the rodents are rich in polyphenols and related compounds, possessing anti-microbial, anti-inflammatory, anti-oxidative as well as anti-cancer activity which may contribute to their exceptionally healthy life.

Analysis of Alpha-2 Macroglobulin from the Long-Lived and Cancer-Resistant Naked Mole-Rat and Human Plasma.

BACKGROUND: The naked mole-rat (NMR) is a long-lived and cancer resistant species. Identification of potential anti-cancer and age related mechanisms is of great interest and makes this species eminent to investigate anti-cancer strategies and understand aging mechanisms. Since it is known that the NMR expresses higher liver mRNA-levels of alpha 2-macroglobulin than mice, nothing is known about its structure, functionality or expression level in the NMR compared to the human A2M. RESULTS: Here we show a comprehensive analysis of NMR- and human plasma-A2M, showing a different prediction in glycosylation of NMR-A2M, which results in a higher molecular weight compared to human A2M. Additionally, we found a higher concentration of A2M (8.3±0.44 mg/mL vs. and 4.4±0.20 mg/mL) and a lower total plasma protein content (38.7±1.79 mg/mL vs. 61.7±3.20 mg/mL) in NMR compared to human. NMR-A2M can be transformed by methylamine and trypsin resulting in a conformational change similar to human A2M. NMR-A2M is detectable by a polyclonal antibody against human A2M. Determination of tryptic and anti-tryptic activity of NMR and human plasma revealed a higher anti-tryptic activity of the NMR plasma. On the other hand, less proteolytic activity was found in NMR plasma compared to human plasma. CONCLUSION: We found transformed NMR-A2M binding to its specific receptor LRP1. We could demonstrate lower protein expression of LRP1 in the NMR liver tissue compared to human but higher expression of A2M. This was accompanied by a higher EpCAM protein expression as central adhesion molecule in cancer progression. NMR-plasma was capable to increase the adhesion in human fibroblast in vitro most probably by increasing CD29 protein expression. This is the first report, demonstrating similarities as well as distinct differences between A2M in NMR and human plasma. This might be directly linked to the intriguing phenotype of the NMR and suggests that A2M might probably play an important role in anti-cancer and the anti-aging mechanisms in the NMR.

Molecular characterization of Mycobacterium tuberculosis isolated from pulmonary tuberculosis patients in Felege Hiwot Referral Hospital, northwest Ethiopia.

BACKGROUND: Tuberculosis (TB), caused by the Mycobacterium tuberculosis complex (MTBC), is a serious infection in humans and animals. Ethiopia is one of the countries in Sub-Saharan Africa with the highest burden of TB. However, limited information is available on the genotypic characteristics of M. tuberculosis strains infecting humans. The objective of the present study was to characterize the mycobacterial species isolated from pulmonary TB patients using molecular typing. MATERIALS AND METHODS: A cross-sectional study was conducted on 123 patients with smear-positive pulmonary TB, using Ziehl Neelsen staining and bacteriological culturing. Molecular characterizations of the mycobacterial isolates were performed using region of difference 9 (RD9) deletion typing and spoligotyping methods. RESULTS: The proportion of culture positivity was 95.9% (118/123). All the 118 isolates were confirmed to be M. tuberculosis by polymerase chain reaction-based RD9 deletion typing. Further characterization of all isolates using spoligotyping resulted in the identification of 36 different spoligotype patterns. Out of these, 32 (88.9%) patterns have already been reported in the SpolDB database, whereas the remaining four (11.1%) patterns were new and not registered in the database. The isolates were further grouped into 17 clustered (99 isolates) and 19 nonclustered patterns. The most predominant spoligotypes were SIT25 and SIT53, consisting of 22 isolates and 14 isolates, respectively. Classification of the spoligotype patterns using TB-insight RUN SPOTCLUST showed that the dominant lineages identified in the present study were Euro-American and Central Asian genotypes consisting of 64 isolates and 37 isolates, respectively. CONCLUSION: This study confirmed the presence of known M. tuberculosis strains and revealed new strains circulating in northwest Ethiopia and the distribution of the major phylogenetic families. It thus contributes to a better understanding of the genotypic profile of M. tuberculosis strains circulating in Ethiopia.

Smear positive extra pulmonary tuberculosis disease at University of Gondar Hospital, Northwest Ethiopia.

BACKGROUND: While pulmonary tuberculosis is the most common presentation, extra pulmonary tuberculosis is also an important clinical problem. However, no adequate information had been made available on the prevalence of smear positive extra pulmonary tuberculosis in Gondar. The aim of this study was to assess the prevalence and possible risk factors of smear positive extra pulmonary tuberculosis among suspected patients at University of Gondar Hospital. METHODS: A cross-sectional study on extra pulmonary tuberculosis suspected patients was conducted at University of Gondar Hospital from January 2012 to April, 2012. Specimens of patients suspected of extra pulmonary tuberculosis were obtained from fine needle aspiration and body fluid samples collected by pathologist. Demographic characteristics and other variables were collected using a pretested semi-structured questionnaire. Smears were prepared from each sample and stained by Ziehel Neelson and Wright stain. The result of the study was analyzed with bivariate and multivariate logistic regression. RESULT: A total of 344 extra pulmonary tuberculosis suspected clients were included in the study and specimens were taken from lymph node aspirates and body fluids. The overall prevalence of smear positive extra pulmonary tuberculosis was 34 (9.9%). Of these cases of extra pulmonary tuberculosis, lymph node tuberculosis constituted the largest proportion (82.4%). Among the 34 extra pulmonary tuberculosis patients, over half of them (52.9%) were positive for human immunodeficiency virus. The largest proportion of tuberculosis and human immunodeficiency virus cases occurred among persons with in the age group of 31-40 years. Previous history of tuberculosis (OR = 4.77, 95% CI 1.86-12.24), contact to a known tuberculosis cases (OR = 6.67 95% CI 2.78-16.90), history of underlying diseases (OR = 2.79 95% CI 1.15-6.78) and income (OR = 12.9 95% CI 2.25-68.02) were significantly associated with extra pulmonary tuberculosis infection. CONCLUSION: The prevalence of smear positive extra pulmonary tuberculosis infection in Gondar is high. Screening of lymph node and other body fluid specimens for extra pulmonary tuberculosis could help for treatment, control and prevention of the disease.

Immunogenicity of bacillus Calmette-Guérin (BCG) in bovine neonates under traditional farming in central Ethiopia.

Vaccination is an alternative method of controlling bovine tuberculosis (BTB) particularly in developing countries where the test and slaughter control method is not acceptable socially and economically. The objective of this study was to evaluate the immunogenicity of bacillus Calmette-Guerin (BCG) vaccination in bovine neonates. Twelve BTB free bovine neonates (six vaccinated with 0.5 mL of 2.4 x 10(6) CFU of BCG and six control) with age less than one month were used for this study. Interferon gamma (IFN-gamma) and antibody responses to mycobacterial antigens were determined at 0, 1, 3, 7, and 13 weeks of post-vaccination. The mean IFN-gamma response to bovine purified protein derivative, PPD in vaccinated group (Mean +/- SEM, 0.541 +/- 0.216) was greater than the mean IFN-gamma response to bovine PPD in non-vaccinated group (Mean +/- SEM, 0.253 +/- 0.101). Within the vaccinated group, the mean IFN-gamma response was greater in cross breed (Mean +/- SEM, 0.779 +/- 0.458) than in zebu breeds (0.303 +/- 0.178). No detectable antibody was observed in both vaccinated and non-vaccinated groups for 13 weeks post vaccination. A sharp rise in IFN-gamma response to bovine PPD was observed between at week 3, and then from week 3 to 7 post-vaccination, there was rapid falling of IFN-gamma response after which the response remained more or less constant in the consecutive weeks. This preliminary study showed the immunogenicity of BCG in bovine neonates under traditional cattle farming in Ethiopia.