Contribution of human hematopoietic stem cells to liver repair

Immune-deficient mouse models of liver damage allow examination of human stem cell migration to sites of damage and subsequent contribution to repair and survival. In our studies, in the absence of a selective advantage, transplanted human stem cells from adult sources did not robustly become hepatocytes, although some level of fusion or hepatic differentiation was documented. However, injected stem cells did home to the injured liver tissue and release paracrine factors that hastened endogenous repair and enhanced survival. There were significantly higher levels of survival in mice with a toxic liver insult that had been transplanted with human stem cells but not in those transplanted with committed progenitors. Transplantation of autologous adult stem cells without conditioning is a relatively safe therapy. Adult stem cells are known to secrete bioactive factors that suppress the local immune system, inhibit fibrosis (scar formation) and apoptosis, enhance angiogenesis, and stimulate recruitment, retention, mitosis, and differentiation of tissue-residing stem cells. These paracrine effects are distinct from the direct differentiation of stem cells to repair tissue. In patients at high risk while waiting for a liver transplant, autologous stem cell therapy could be considered, as it could delay the decline in liver function

Auswirkungen von kommunalen Wasser- Sanitär- und Hygieneaktivitäten auf das Hygieneverhalten unterschiedlicher ethnischen Gruppen im Nordwesten der Demokratischen Volksrepublik Laos - gemessen vor und nach einer Projektintervention

Worldwide, around 780 million people lack access to improved drinking water sources and 2.5 billion lack improved sanitation (WHO, 2013c). The United Nations (UN) declared access to drinking water and sanitation a fundamental human right in July 2010 (UN, 2010). Each year there are approximately 1.7 billion cases of diarrhoea worldwide. It kills around 2.2 million people globally each year (4.0% of all death), of which 760,000 are children under five years old, mostly in developing countries. Diarrhoeal disease is the second leading cause of death in children under five years old (WHO, 2013c). The lack of drinking water, sanitation facilities and good hygiene practises are considered the main causes for diarrhoea (WHO, 2013d). For this dissertation, a specific health intervention (CBHFA - Community based Health and First Aid) was evaluated which had taken place during three years in 20 target communities in Bokeo Province in Lao PDR. The activities were in the field of water supply, sanitation facilities and hygiene education against diarrhoea. Objective: The objective of this survey study is to show that hygiene behaviour changes in a certain target population through the implementation of CBHFA, if a) individuals have access to improved drinking water sources; b) have access to a HH latrine and c) receive hygiene information. An improvement by 50.0% for each of the hygiene indicator: water use and treatment, hand washing, sanitation, food handling, waste disposal and cleanliness was considered a behavioural change and thus an illustration of the effectiveness of CBHFA. Methods: Hygiene behaviour was measured before and after the CBHFA intervention through above mentioned hygiene indicators. Quantitative figures were collected at two different points in time, in a pre- and post-survey, then analysed and evaluated. Interviews and observations were done through community assessment/evaluation with 20 focus groups in all target communities. 488/487 (pre-survey/post-survey) households (HHs) were interviewed and observed. Knowledge about diarrhoea (definition/signs, prevention, danger, treatment and modes of transmission) was tested. The change in hygiene behaviour as well as the water and sanitation situation was measured through a comparison of the pre- with the post-results. The Relative Risk (RR) and Odds Ratio (OR) were calculated for different variables, such as ethnicity, gender, education, age and income and their changes before and after the implementation of the health intervention project. Results: Water use and sanitation This research clearly shows that the availability of a public water system (gravity fed water system) in the target communities has increased (from 45.0% to 85.0%) as well as the availability and use of hand flush pit latrines (from 20.2% to 63.7%). 95.2% of HHs that owned a latrine used them. The water treatment through “boiling” (from 70.1% to 75.0%) in HHs has improved, but not significantly. Open defecation (OD) decreased from 78.9% to 34.7% (by 56.0%). However it is still practised by more than a third of HHs. OD is one of the highest transmission risks of diarrhoea and can only be eliminated by 100.0% sanitation within the community (WHO, 2008). Hand washing There has been a significant increase of interviewees washing their hands with water and soap (from 8.0% to 38.0%). This result was confirmed by observing the existence of soap in kitchens with 33.0% and in latrines with 20.3% of HHs. The ORs calculated for hand washing “with water and soap” and compared for each stratum of the variables gender, formal education and age group show no statistical difference, but there is a statistical difference regarding income and washing hands “with water and soap”. Relevant occasions for hand washing, such as “after defecation” and “before food preparing”, that could reduce the risk of the transmission of diarrhoeal diseases have not achieved a meaningful improvement. Waste disposal Waste disposal, such as collecting and burning has increased from 23.4% to 42.5% (by 81.6%) according to the HH responses. A matter of concern is the increased HHs (from 26.8% to 29.0%) that disposed their waste by “throwing the waste outside the village”. This waste disposal method is statistically different regarding formal education. The number of HHs with non-educated interviewees where this method was practised has increased over the intervention time in contrast to the number of HHs with educated interviewees. Knowledge about diarrhoea Knowledge was tested. The definition/signs of diarrhoea, its prevention, danger, treatment and modes of transmission were not known by more than 50.0% of the interviewees. It seems that hygiene indicators can improve without this awareness, if improved water and sanitation facilities are provided. However, further investigations are required. The results show that knowledge about diarrhoea is not statistically different regarding formal education and age, but regarding gender. Giving at least one right answer was higher in women than in men. Results and income The results of the pre-survey show that income is significantly associated with owning a latrine and using water vessels, such as pots and jars for drinking, but not associated with using soap for hand washing. However after the implementation of the health interventions, the comparison of the ORs of the HHs with low income per capita (≤200,000 LAK, exchange rate: 1 EUR=10,000 LAK) versus the HHs with high income per capita (>200,000 LAK) shows a statistical difference regarding hand washing “with water and soap”. The OR of the high income per capita group is stronger than the OR of the low income per capita group. Owning a latrine does not show a statistical difference regarding income due to the provision of subsidized latrines by the health intervention project. Occurrence of diarrhoeal diseases The pre-survey results clearly show that diarrhoeal diseases ranked at the top of all recorded cases of diseases. The post-survey results show fewer villages with diarrhoeal outbreak and reduced numbers of reported cases. Summary: An improvement by 50.0% has been reached in hand washing with water and soap, reduction of OD, safe food (by covering), waste disposal (by collecting and burning) and cleanliness but no improvement has been reached in the hygiene indicators “water use and treatment” and in crucial “occasions for hand washing (after defecation, before food preparing)”. Therefore the CBHFA intervention is considered only partially effective in the field of hygiene behaviour change

Evaluation of the effects of PHAST training and community health interventions on the hygiene behaviour of children and women to prevent diarrhoeal diseases- in the Locality of Rashad/ South Kordofan/ Republic of Sudan

Access to drinking water and improved sanitation are a fundamental need for health of all people. Diarrhoea due to infection is widespread throughout the developing world. It is estimated that 90.0% of all cases of diarrhoea can be attributed to three major causes: inadequate sanitation, poor hygiene and unclean water. Each year approximately 2.2 million people die from diarrhoeal diseases worldwide, 90.0% are children under 5 years of age. (WHO, 2000) Besides the mentioned causes of diarrhoeal diseases and its abolishment, knowledge and attitude about hygiene and sanitation and the practice of good hygiene behaviour in children and women is needed to reduce diarrhoeal infections and moreover to prevent getting it. The effects of the undertaken PHAST training related to gained knowledge, attitude and practice (KAP) were investigated by using a quantitative approach in order to prove change in hygiene behaviour according to KAP model. Health indicators in terms of quantitative figures were analyzed. Interviews, observations and tests were done with 118 participants whereby the main target group of the study was based on 70 children and 30 women from three villages in Rashad locality. The tests examined their knowledge in good/bad behaviour related to sanitation, hand washing, water, food preparing and handling as well as in modes of transmission and preventive activities, whereas the passive observation their attitude and practice investigated. It was found a significant better knowledge in all items in children and women who attended the PHAST training (cases) compared to those who have never attended (controls). However changes in their attitude towards better hygiene and sanitation and their performances could not be evidence based confirmed at the end, neither in cases nor in controls. However during the time of observation it could be found that health facilities and staff in the villages and compounds tend to practice good hygienic activities. In summary an association between the undertaken PHAST training and knowledge could be found but the change in hygiene behaviour according to the KAP model could not be proven. The PHAST training and other health interventions tend to prevent people from getting diarrhoeal diseases however valid data to prove it were not available in this region

Structural basis for membrane binding and catalytic activation of the peripheral membrane enzyme pyruvate oxidase from Escherichia coli

The thiamin- and flavin-dependent peripheral membrane enzyme pyruvate oxidase from E. coli catalyzes the oxidative decarboxylation of the central metabolite pyruvate to CO2 and acetate. Concomitant reduction of the enzyme-bound flavin triggers membrane binding of the C terminus and shuttling of 2 electrons to ubiquinone 8, a membrane-bound mobile carrier of the electron transport chain. Binding to the membrane in vivo or limited proteolysis in vitro stimulate the catalytic proficiency by 2 orders of magnitude. The molecular mechanisms by which membrane binding and activation are governed have remained enigmatic. Here, we present the X-ray crystal structures of the full-length enzyme and a proteolytically activated truncation variant lacking the last 23 C-terminal residues inferred as important in membrane binding. In conjunction with spectroscopic results, the structural data pinpoint a conformational rearrangement upon activation that exposes the autoinhibitory C terminus, thereby freeing the active site. In the activated enzyme, Phe-465 swings into the active site and wires both cofactors for efficient electron transfer. The isolated C terminus, which has no intrinsic helix propensity, folds into a helical structure in the presence of micelles

Radical Phosphate Transfer Mechanism for the Thiamin Diphosphate- and FAD-Dependent Pyruvate Oxidase from Lactobacillus plantarum : Kinetic Coupling of Intercofactor Electron Transfer with Phosphate Transfer to Acetyl-thiamin Diphosphate via a Transient FAD Semiquinone/Hydroxyethyl-ThDP Radical Pair

The thiamin diphosphate (ThDP)- and flavin adenine dinucleotide (FAD)-dependent pyruvate oxidase from Lactobacillus plantarum catalyses the conversion of pyruvate, inorganic phosphate, and oxygen to acetyl-phosphate, carbon dioxide, and hydrogen peroxide. Central to the catalytic sequence, two reducing equivalents are transferred from the resonant carbanion/enamine forms of -hydroxyethyl-ThDP to the adjacent flavin cofactor over a distance of approximately 7 Å, followed by the phosphorolysis of the thereby formed acetyl-ThDP. Pre-steady-state and steady-state kinetics using time-resolved spectroscopy and a 1H NMR-based intermediate analysis indicate that both processes are kinetically coupled. In the presence of phosphate, intercofactor electron-transfer (ET) proceeds with an apparent first-order rate constant of 78 s-1 and is kinetically gated by the preceding formation of the tetrahedral substrate-ThDP adduct 2-lactyl-ThDP and its decarboxylation. No transient flavin radicals are detectable in the reductive half-reaction. In contrast, when phosphate is absent, ET occurs in two discrete steps with apparent rate constants of 81 and 3 s-1 and transient formation of a flavin semiquinone/hydroxyethyl-ThDP radical pair. Temperature dependence analysis according to the Marcus theory identifies the second step, the slow radical decay to be a true ET reaction. The redox potentials of the FADox/FADsq (E1 = -37 mV) and FADsq/FADred (E2 = -87 mV) redox couples in the absence and presence of phosphate are identical. Both the Marcus analysis and fluorescence resonance energy-transfer studies using the fluorescent N3'-pyridyl-ThDP indicate the same cofactor distance in the presence or absence of phosphate. We deduce that the exclusive 102-103-fold rate enhancement of the second ET step is rather due to the nucleophilic attack of phosphate on the kinetically stabilized hydroxyethyl-ThDP radical resulting in a low-potential anion radical adduct than phosphate in a docking site being part of a through-bonded ET pathway in a stepwise mechanism of ET and phosporolysis. Thus, LpPOX would constitute the first example of a radical-based phosphorolysis mechanism in biochemistry

Crystallization and preliminary X-ray diffraction analysis of full-length and proteolytically activated pyruvate oxidase from Escherichia coli

The peripheral membrane flavoprotein pyruvate oxidase from E. coli has been crystallized in the full-length form and as a proteolytically activated truncation variant lacking the last 23 amino acids at the C-terminus

Pooled RT-qPCR testing for SARS-CoV-2 surveillance in schools-a cluster randomised trial

Background: The extent to which children and adolescents contribute to SARS-CoV-2 transmission remains not fully understood. Novel high-capacity testing methods may provide real-time epidemiological data in educational settings helping to establish a rational approach to prevent and minimize SARS-CoV-2 transmission. We investigated whether pooling of samples for SARS-CoV-2 detection by RT-qPCR is a sensitive and feasible high-capacity diagnostic strategy for surveillance of SARS-CoV-2 infections in schools. Methods: In this study, students and school staff of 14 educational facilities in Germany were tested sequentially between November 9 and December 23, 2020, two or three times per week for at least three consecutive weeks. Participants were randomized for evaluation of two different age adjusted swab sampling methods (oropharyngeal swabs or buccal swabs compared to saliva swabs using a 'lolli method'). Swabs were collected and pooled for SARS-CoV-2 RT-qPCR. Individuals of positive pooled tests were retested by RTqPCR the same or the following day. Positive individuals were quarantined while the SARS-CoV-2 negative individuals remained in class with continued pooled RT-qPCR surveillance. The study is registered with the German Clinical Trials register (registration number: DRKS00023911). Findings: 5,537 individuals were eligible and 3970 participants were enroled and included in the analysis. In students, a total of 21,978 swabs were taken and combined in 2218 pooled RT-qPCR tests. We detected 41 positive pooled tests (1 cent 8%) leading to 36 SARS-CoV-2 cases among students which could be identified by individual re-testing. The cumulative 3-week incidence for primary schools was 564/100,000 (6/1064, additionally 1 infection detected in week 4) and 1249/100,000 (29/2322) for secondary schools. In secondary schools, there was no difference in the number of SARS-CoV-2 positive students identified from pooled oropharyngeal swabs compared to those identified from pooled saliva samples (lolli method) (14 vs. 15 cases; 1.3% vs. 1.3%; OR 1.1; 95%-CI 0.5-2.5). A single secondary school accounted for 17 of 36 cases (47%) indicating a high burden of asymptomatic prevalent SARS-CoV-2 cases in the respective school and community. Interpretation: In educational settings, SARS-CoV-2 screening by RT-qPCR-based pooled testing with easily obtainable saliva samples is a feasible method to detect incident cases and observe transmission dynamics. Funding: Federal Ministry of education and research (BMBF; Project B-FAST in NaFoUniMedCovid19; registration number: 01KX2021). (c) 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/

Pooled RT-qPCR testing for SARS-CoV-2 surveillance in schools - a cluster randomised trial

Background: The extent to which children and adolescents contribute to SARS-CoV-2 transmission remains not fully understood. Novel high-capacity testing methods may provide real-time epidemiological data in educational settings helping to establish a rational approach to prevent and minimize SARS-CoV-2 transmission. We investigated whether pooling of samples for SARS-CoV-2 detection by RT-qPCR is a sensitive and feasible high-capacity diagnostic strategy for surveillance of SARS-CoV-2 infections in schools. Methods: In this study, students and school staff of 14 educational facilities in Germany were tested sequentially between November 9 and December 23, 2020, two or three times per week for at least three consecutive weeks. Participants were randomized for evaluation of two different age adjusted swab sampling methods (oropharyngeal swabs or buccal swabs compared to saliva swabs using a ‘lolli method’). Swabs were collected and pooled for SARS-CoV-2 RT-qPCR. Individuals of positive pooled tests were retested by RT-qPCR the same or the following day. Positive individuals were quarantined while the SARS-CoV-2 negative individuals remained in class with continued pooled RT-qPCR surveillance. The study is registered with the German Clinical Trials register (registration number: DRKS00023911). Findings: 5,537 individuals were eligible and 3970 participants were enroled and included in the analysis. In students, a total of 21,978 swabs were taken and combined in 2218 pooled RT-qPCR tests. We detected 41 positive pooled tests (1·8%) leading to 36 SARS-CoV-2 cases among students which could be identified by individual re-testing. The cumulative 3-week incidence for primary schools was 564/100,000 (6/1064, additionally 1 infection detected in week 4) and 1249/100,000 (29/2322) for secondary schools. In secondary schools, there was no difference in the number of SARS-CoV-2 positive students identified from pooled oropharyngeal swabs compared to those identified from pooled saliva samples (lolli method) (14 vs. 15 cases; 1·3% vs. 1·3%; OR 1.1; 95%-CI 0·5–2·5). A single secondary school accounted for 17 of 36 cases (47%) indicating a high burden of asymptomatic prevalent SARS-CoV-2 cases in the respective school and community. Interpretation: In educational settings, SARS-CoV-2 screening by RT-qPCR-based pooled testing with easily obtainable saliva samples is a feasible method to detect incident cases and observe transmission dynamics. Funding: Federal Ministry of education and research (BMBF; Project B-FAST in “NaFoUniMedCovid19”; registration number: 01KX2021)