Charakterisierung der molekularen Determinanten zur hochaffinen Bindung der kompatiblen Soluten Glycin Betain, Prolin Betain, Ectoin und Hydroxyectoin durch Substratbindeproteine von bakteriellen ABC-Transportern

Die Akkumulation von kompatiblen Soluten ist ein weit verbreiteter Schutzmechanismus gegen variierende Umweltbedingungen und wird in vielen Spezies der Bacteria und Archaea verwendet. Das einige kompatible Solute nicht nur osmoprotektive Wirkung haben, sondern generell Protein-stabilisierende Substanzen sind, ist durch in vitro Experimente belegt. Der Protein-stabilisierende Effekt wird in dem „preferential exclusion model“ beschrieben und beruht auf dem thermodynamisch begründeten, bevorzugten Ausschluß der Solute von der Oberfläche der Proteine. In der vorliegenden Arbeit wurden die Substratbindeproteine zweier hochaffiner ABC-Transporter für die kompatiblen Solute Glycin Betain und Prolin Betain auf molekularer Ebene untersucht, um die Grundlagen zur Bindung von „bevorzugt ausgeschlossenen Soluten“ zu beschreiben. Dazu wurden die beiden hochaufgelösten Kristallstrukturen der Substratbindeproteine aus dem ProU-System von E. coli (EcProX, 1,6 Å) und dem ProU-System von A. fulgidus (AfProX 1,9 Å) verwendet. In EcProX wird das quartäre Amin des gebundenen Glycin Betains durch eine Box von drei Tryptophanen koordiniert (Trp-65, Trp-140 und Trp-188), die Carboxylgruppe des Glycin Betains wird durch H-Brücken stabilisiert. In einer Mutagenestudie konnte in dieser Arbeit gezeigt werden, dass Kation-pi-Interaktionen eine Haupt-Determinante in der Bindung von Glycin Betain sind. Es konnte gezeigt werden, dass neben Tryptophan auch Phenylalanin und Tyrosin zu starken Interaktionen mit dem Substrat fähig sind. Die Mutagenesestudie demonstrierte eine unterschiedlich starke Beteiligung der Tryptophane an der Substratbindung. Trp-188 ist dabei essentiell für die Bindung von Glycin Betain, Trp-140 zeigte die geringste Beteiligung. Trp-188 benötigt die Tryptophane Trp-140 und Trp-65 aber zur Stabilisierung des quartären Amins von Glycin Betain in der Bindetasche. Das AfProX Protein zeigt in Affinitätsmessungen eine besonders hohe Affinität zu Glycin Betain und Prolin Betain. Verglichen mit dem EcProX Protein ist die KD von AfProX zu Glycin Betain bei 50°C (0,01 µM) um den Faktor 100 niedriger als die des EcProX Proteins bei Raumtemperatur (KD=1µM) und um den Faktor 1000 niedriger verglichen zum OpuAC Protein (KD=17 µM), einem Glycin Betain und Prolin Betain bindenden Protein aus B. subtilis. In der Kristallstruktur von AfProX zeigte sich eine weitere Konstellation einer aromatischen Bindetasche. Das quartäre Amin wird dort von den vier Tyrosinen Tyr-63, Tyr-111, Tyr-190 und Tyr-214 (Tyrosin-Gürtel) und einem Aspartat koordiniert, die Carboxylgruppe wird durch Salz- und H-Brücken stabilisiert. Eine Mutagenesestudie zeigte auch hier die wichtige Beteiligung von Kation-pi-Interaktionen an der hochaffinen Bindung dieser kompatiblen Solute. Dabei ist die besondere Architektur des Tyrosin-Gürtels, zusammen mit den Carboxylgruppen-stabilisierenden Aminosäuren, essentiell für die hohe Bindeaffinität. Je nach Position des Tyrosins kann der Austausch eines Tyrosins gegen ein Alanin das Absinken der Bindekonstante auf einen Wert, vergleichbar zu dem des EcProX- oder des BsOpuAC-Proteins, oder niedriger bewirken. Ein Austausch von zwei Tyrosinen gegen Alanin bedeutet immer einen kompletten Verlust der Bindeaffinität. Datenbankanalysen der Aminosäuresequenzen von EcProX, AfProX und BsOpuAC zeigten eine weite Verbreitung der konservierten Bindemotive der Proteine und im Falle von BsOpuAC eine besonders starke Konservierung der bindungsrelevanen Tryptophane („Trp-Prisma“) sowie einen Domänentausch im Vergleich zu anderen Glycin Betain Bindeproteinen. Die Entdeckung eines Substratbindeprotein-abhängigen ABC-Transporters (Ehu) für Ectoin und Hydroxyectoin in S. meliloti erlaubte die Analyse der Ectoin-Bindung durch ein Substratbindeprotein. Nach der Überproduktion, Reinigung und Charakterisierung des hochaffinen Ectoin-Bindeproteins (EhuB) und dessen Kristallistation mit gebundenem Ectoin (und Hydroxyectoin) zeigte die Struktur (Auflösung 2,1 Å) in der Substratbindestelle eine aromatische Box aus zwei Phenylalaninen und einem Tyrosin, welche die delokalisierte positive Ladung des Moleküls binden. Der Pyrimidinring und die Carboxylgruppe des Ectoins werden durch Salz- und H-Brücken stabilisiert. Kation-pi-Interaktionen sind eine Hauptdeterminate in der hochaffinen Bindung von Glycin Betain und Prolin Betain, aber auch von Ectoin und Hydroxyectoin und finden eine weite Verbreitung in den Bacteria und Archaea

Charakterisierung der molekularen Determinanten zur hochaffinen Bindung der kompatiblen Soluten Glycin Betain, Prolin Betain, Ectoin und Hydroxyectoin durch Substratbindeproteine von bakteriellen ABC-Transportern

Die Akkumulation von kompatiblen Soluten ist ein weit verbreiteter Schutzmechanismus gegen variierende Umweltbedingungen und wird in vielen Spezies der Bacteria und Archaea verwendet. Das einige kompatible Solute nicht nur osmoprotektive Wirkung haben, sondern generell Protein-stabilisierende Substanzen sind, ist durch in vitro Experimente belegt. Der Protein-stabilisierende Effekt wird in dem „preferential exclusion model“ beschrieben und beruht auf dem thermodynamisch begründeten, bevorzugten Ausschluß der Solute von der Oberfläche der Proteine. In der vorliegenden Arbeit wurden die Substratbindeproteine zweier hochaffiner ABC-Transporter für die kompatiblen Solute Glycin Betain und Prolin Betain auf molekularer Ebene untersucht, um die Grundlagen zur Bindung von „bevorzugt ausgeschlossenen Soluten“ zu beschreiben. Dazu wurden die beiden hochaufgelösten Kristallstrukturen der Substratbindeproteine aus dem ProU-System von E. coli (EcProX, 1,6 Å) und dem ProU-System von A. fulgidus (AfProX 1,9 Å) verwendet. In EcProX wird das quartäre Amin des gebundenen Glycin Betains durch eine Box von drei Tryptophanen koordiniert (Trp-65, Trp-140 und Trp-188), die Carboxylgruppe des Glycin Betains wird durch H-Brücken stabilisiert. In einer Mutagenestudie konnte in dieser Arbeit gezeigt werden, dass Kation-pi-Interaktionen eine Haupt-Determinante in der Bindung von Glycin Betain sind. Es konnte gezeigt werden, dass neben Tryptophan auch Phenylalanin und Tyrosin zu starken Interaktionen mit dem Substrat fähig sind. Die Mutagenesestudie demonstrierte eine unterschiedlich starke Beteiligung der Tryptophane an der Substratbindung. Trp-188 ist dabei essentiell für die Bindung von Glycin Betain, Trp-140 zeigte die geringste Beteiligung. Trp-188 benötigt die Tryptophane Trp-140 und Trp-65 aber zur Stabilisierung des quartären Amins von Glycin Betain in der Bindetasche. Das AfProX Protein zeigt in Affinitätsmessungen eine besonders hohe Affinität zu Glycin Betain und Prolin Betain. Verglichen mit dem EcProX Protein ist die KD von AfProX zu Glycin Betain bei 50°C (0,01 µM) um den Faktor 100 niedriger als die des EcProX Proteins bei Raumtemperatur (KD=1µM) und um den Faktor 1000 niedriger verglichen zum OpuAC Protein (KD=17 µM), einem Glycin Betain und Prolin Betain bindenden Protein aus B. subtilis. In der Kristallstruktur von AfProX zeigte sich eine weitere Konstellation einer aromatischen Bindetasche. Das quartäre Amin wird dort von den vier Tyrosinen Tyr-63, Tyr-111, Tyr-190 und Tyr-214 (Tyrosin-Gürtel) und einem Aspartat koordiniert, die Carboxylgruppe wird durch Salz- und H-Brücken stabilisiert. Eine Mutagenesestudie zeigte auch hier die wichtige Beteiligung von Kation-pi-Interaktionen an der hochaffinen Bindung dieser kompatiblen Solute. Dabei ist die besondere Architektur des Tyrosin-Gürtels, zusammen mit den Carboxylgruppen-stabilisierenden Aminosäuren, essentiell für die hohe Bindeaffinität. Je nach Position des Tyrosins kann der Austausch eines Tyrosins gegen ein Alanin das Absinken der Bindekonstante auf einen Wert, vergleichbar zu dem des EcProX- oder des BsOpuAC-Proteins, oder niedriger bewirken. Ein Austausch von zwei Tyrosinen gegen Alanin bedeutet immer einen kompletten Verlust der Bindeaffinität. Datenbankanalysen der Aminosäuresequenzen von EcProX, AfProX und BsOpuAC zeigten eine weite Verbreitung der konservierten Bindemotive der Proteine und im Falle von BsOpuAC eine besonders starke Konservierung der bindungsrelevanen Tryptophane („Trp-Prisma“) sowie einen Domänentausch im Vergleich zu anderen Glycin Betain Bindeproteinen. Die Entdeckung eines Substratbindeprotein-abhängigen ABC-Transporters (Ehu) für Ectoin und Hydroxyectoin in S. meliloti erlaubte die Analyse der Ectoin-Bindung durch ein Substratbindeprotein. Nach der Überproduktion, Reinigung und Charakterisierung des hochaffinen Ectoin-Bindeproteins (EhuB) und dessen Kristallistation mit gebundenem Ectoin (und Hydroxyectoin) zeigte die Struktur (Auflösung 2,1 Å) in der Substratbindestelle eine aromatische Box aus zwei Phenylalaninen und einem Tyrosin, welche die delokalisierte positive Ladung des Moleküls binden. Der Pyrimidinring und die Carboxylgruppe des Ectoins werden durch Salz- und H-Brücken stabilisiert. Kation-pi-Interaktionen sind eine Hauptdeterminate in der hochaffinen Bindung von Glycin Betain und Prolin Betain, aber auch von Ectoin und Hydroxyectoin und finden eine weite Verbreitung in den Bacteria und Archaea

Remote health monitoring services in nursing homes

Aged people are challenged by serious complications from chronic diseases, such as mood disorder, diabetes, heart disease, and infectious diseases, which are also the most common causes of death in older people. Therefore, elderly care facilities are more important than ever. The most common causes of death in elderly care facilities were reported to be diabetes, cardiovascular disease, and pneumonia. Recently, the coronavirus disease 2019 (COVID-19) pandemic have a great impact on blind spots of safety where aged people were isolated from society. Elderly care facilities were one of the blind spots in the midst of the pandemic, where major casualties were reported from COVID-19 complications because most people had one or two mortality risk factors, such as diabetes or cardiovascular disease. Therefore, medical governance of public health center and hospital, and elderly care facility is becoming important issue of priority. Thus, remote health monitoring service by the Internet of Medical Things (IoMT) sensors is more important than ever. Recently, technological breakthroughs have enabled healthcare professionals to have easy access to patients in medical blind spots through the use of IoT sensors. These sensors can detect medically urgent situations in a timely fashion and make medical decisions for aged people in elderly care facilities. Real-time electrocardiogram and blood sugar monitoring sensors are approved by the medical insurance service. Real-time monitoring services in medical blind spots, such as elderly care facilities, has been suggested. Heart rhythm monitoring could play a role in detecting early cardiovascular disease events and monitoring blood glucose levels in the management of chronic diseases, such as diabetes, in aged people in elderly care facilities. This review presents the potential usefulness of remote monitoring with IoMT sensors in medical blind spots and clinical suggestions for applications

Transkingdom Analysis of the Female Reproductive Tract Reveals Bacteriophages form Communities

The female reproductive tract (FRT) microbiome plays a vital role in maintaining vaginal health. Viruses are key regulators of other microbial ecosystems, but little is known about how the FRT viruses (virome), particularly bacteriophages that comprise the phageome, impact FRT health and dysbiosis. We hypothesize that bacterial vaginosis (BV) is associated with altered FRT phageome diversity, transkingdom interplay, and bacteriophage discriminate taxa. Here, we conducted a retrospective, longitudinal analysis of vaginal swabs collected from 54 BV-positive and 46 BV-negative South African women. Bacteriome analysis revealed samples clustered into five distinct bacterial community groups (CGs), and further, bacterial alpha diversity was significantly associated with BV. Virome analysis on a subset of baseline samples showed FRT bacteriophages clustering into novel viral state types (VSTs), a viral community clustering system based on virome composition and abundance. Distinct BV bacteriophage signatures included increased alpha diversity along with discriminant Bacillus, Burkholderia, and Escherichia bacteriophages. Bacteriophage-bacteria transkingdom associations were also identified between Bacillus and Burkholderia viruses and BV-associated bacteria, providing key insights for future studies elucidating the transkingdom interactions driving BV-associated microbiome perturbations. In this cohort, bacteriophage-bacterial associations suggest complex interactions, which may play a role in the establishment and maintenance of BV

Perspectives of San Juan healthcare practitioners on the detection deficit in oral premalignant and early cancers in Puerto Rico: a qualitative research study

<p>Abstract</p> <p>Background</p> <p>In Puerto Rico, relative to the United States, a disparity exists in detecting oral precancers and early cancers. To identify factors leading to the deficit in early detection, we obtained the perspectives of San Juan healthcare practitioners whose practice could be involved in the detection of such oral lesions.</p> <p>Methods</p> <p>Key informant (KI) interviews were conducted with ten clinicians practicing in or around San Juan, Puerto Rico. We then triangulated our KI interview findings with other data sources, including recent literature on oral cancer detection from various geographic areas, current curricula at the University of Puerto Rico Schools of Medicine and Dental Medicine, as well as local health insurance regulations.</p> <p>Results</p> <p>Key informant-identified factors that likely contribute to the detection deficit include: many practitioners are deficient in knowledge regarding oral cancer and precancer; oral cancer screening examinations are limited regarding which patients receive them and the elements included. In Puerto Rico, specialists generally perform oral biopsies, and patient referral can be delayed by various factors, including government-subsidized health insurance, often referred to as Reforma. Reforma-based issues include often inadequate clinician knowledge regarding Reforma requirements/provisions, diagnostic delays related to Reforma bureaucracy, and among primary physicians, a perceived financial disincentive in referring Reforma patients.</p> <p>Conclusions</p> <p>Addressing these issues may be useful in reducing the deficit in detecting oral precancers and early oral cancer in Puerto Rico.</p

Sequential Delivery of Host-Induced Virulence Effectors by Appressoria and Intracellular Hyphae of the Phytopathogen Colletotrichum higginsianum

Phytopathogens secrete effector proteins to manipulate their hosts for effective colonization. Hemibiotrophic fungi must maintain host viability during initial biotrophic growth and elicit host death for subsequent necrotrophic growth. To identify effectors mediating these opposing processes, we deeply sequenced the transcriptome of Colletotrichum higginsianum infecting Arabidopsis. Most effector genes are host-induced and expressed in consecutive waves associated with pathogenic transitions, indicating distinct effector suites are deployed at each stage. Using fluorescent protein tagging and transmission electron microscopy-immunogold labelling, we found effectors localised to stage-specific compartments at the host-pathogen interface. In particular, we show effectors are focally secreted from appressorial penetration pores before host invasion, revealing new levels of functional complexity for this fungal organ. Furthermore, we demonstrate that antagonistic effectors either induce or suppress plant cell death. Based on these results we conclude that hemibiotrophy in Colletotrichum is orchestrated through the coordinated expression of antagonistic effectors supporting either cell viability or cell death

Growth and CD4 patterns of adolescents living with perinatally acquired HIV worldwide, a CIPHER cohort collaboration analysis.

INTRODUCTION Adolescents living with HIV are subject to multiple co-morbidities, including growth retardation and immunodeficiency. We describe growth and CD4 evolution during adolescence using data from the Collaborative Initiative for Paediatric HIV Education and Research (CIPHER) global project. METHODS Data were collected between 1994 and 2015 from 11 CIPHER networks worldwide. Adolescents with perinatally acquired HIV infection (APH) who initiated antiretroviral therapy (ART) before age 10 years, with at least one height or CD4 count measurement while aged 10-17 years, were included. Growth was measured using height-for-age Z-scores (HAZ, stunting if <-2 SD, WHO growth charts). Linear mixed-effects models were used to study the evolution of each outcome between ages 10 and 17. For growth, sex-specific models with fractional polynomials were used to model non-linear relationships for age at ART initiation, HAZ at age 10 and time, defined as current age from 10 to 17 years of age. RESULTS A total of 20,939 and 19,557 APH were included for the growth and CD4 analyses, respectively. Half were females, two-thirds lived in East and Southern Africa, and median age at ART initiation ranged from 7 years in sub-Saharan African regions. At age 10, stunting ranged from 6% in North America and Europe to 39% in the Asia-Pacific; 19% overall had CD4 counts <500 cells/mm3 . Across adolescence, higher HAZ was observed in females and among those in high-income countries. APH with stunting at age 10 and those with late ART initiation (after age 5) had the largest HAZ gains during adolescence, but these gains were insufficient to catch-up with non-stunted, early ART-treated adolescents. From age 10 to 16 years, mean CD4 counts declined from 768 to 607 cells/mm3 . This decline was observed across all regions, in males and females. CONCLUSIONS Growth patterns during adolescence differed substantially by sex and region, while CD4 patterns were similar, with an observed CD4 decline that needs further investigation. Early diagnosis and timely initiation of treatment in early childhood to prevent growth retardation and immunodeficiency are critical to improving APH growth and CD4 outcomes by the time they reach adulthood

The epidemiology of adolescents living with perinatally acquired HIV: A cross-region global cohort analysis

Background Globally, the population of adolescents living with perinatally acquired HIV (APHs) continues to expand. In this study, we pooled data from observational pediatric HIV cohorts and cohort networks, allowing comparisons of adolescents with perinatally acquired HIV in “real-life” settings across multiple regions. We describe the geographic and temporal characteristics and mortality outcomes of APHs across multiple regions, including South America and the Caribbean, North America, Europe, sub-Saharan Africa, and South and Southeast Asia. Methods and findings Through the Collaborative Initiative for Paediatric HIV Education and Research (CIPHER), individual retrospective longitudinal data from 12 cohort networks were pooled. All children infected with HIV who entered care before age 10 years, were not known to have horizontally acquired HIV, and were followed up beyond age 10 years were included in this analysis conducted from May 2016 to January 2017. Our primary analysis describes patient and treatment characteristics of APHs at key time points, including first HIV-associated clinic visit, antiretroviral therapy (ART) start, age 10 years, and last visit, and compares these characteristics by geographic region, country income group (CIG), and birth period. Our secondary analysis describes mortality, transfer out, and lost to follow-up (LTFU) as outcomes at age 15 years, using competing risk analysis. Among the 38,187 APHs included, 51% were female, 79% were from sub-Saharan Africa and 65% lived in low-income countries. APHs from 51 countries were included (Europe: 14 countries and 3,054 APHs; North America: 1 country and 1,032 APHs; South America and the Caribbean: 4 countries and 903 APHs; South and Southeast Asia: 7 countries and 2,902 APHs; sub-Saharan Africa, 25 countries and 30,296 APHs). Observation started as early as 1982 in Europe and 1996 in sub-Saharan Africa, and continued until at least 2014 in all regions. The median (interquartile range [IQR]) duration of adolescent follow-up was 3.1 (1.5–5.2) years for the total cohort and 6.4 (3.6–8.0) years in Europe, 3.7 (2.0–5.4) years in North America, 2.5 (1.2–4.4) years in South and Southeast Asia, 5.0 (2.7–7.5) years in South America and the Caribbean, and 2.1 (0.9–3.8) years in sub-Saharan Africa. Median (IQR) age at first visit differed substantially by region, ranging from 0.7 (0.3–2.1) years in North America to 7.1 (5.3–8.6) years in sub-Saharan Africa. The median age at ART start varied from 0.9 (0.4–2.6) years in North America to 7.9 (6.0–9.3) years in sub-Saharan Africa. The cumulative incidence estimates (95% confidence interval [CI]) at age 15 years for mortality, transfers out, and LTFU for all APHs were 2.6% (2.4%–2.8%), 15.6% (15.1%–16.0%), and 11.3% (10.9%–11.8%), respectively. Mortality was lowest in Europe (0.8% [0.5%–1.1%]) and highest in South America and the Caribbean (4.4% [3.1%–6.1%]). However, LTFU was lowest in South America and the Caribbean (4.8% [3.4%–6.7%]) and highest in sub-Saharan Africa (13.2% [12.6%–13.7%]). Study limitations include the high LTFU rate in sub-Saharan Africa, which could have affected the comparison of mortality across regions; inclusion of data only for APHs receiving ART from some countries; and unavailability of data from high-burden countries such as Nigeria. Conclusion To our knowledge, our study represents the largest multiregional epidemiological analysis of APHs. Despite probable under-ascertained mortality, mortality in APHs remains substantially higher in sub-Saharan Africa, South and Southeast Asia, and South America and the Caribbean than in Europe. Collaborations such as CIPHER enable us to monitor current global temporal trends in outcomes over time to inform appropriate policy responses

The FANCM:p.Arg658* truncating variant is associated with risk of triple-negative breast cancer.

Breast cancer is a common disease partially caused by genetic risk factors. Germline pathogenic variants in DNA repair genes BRCA1, BRCA2, PALB2, ATM, and CHEK2 are associated with breast cancer risk. FANCM, which encodes for a DNA translocase, has been proposed as a breast cancer predisposition gene, with greater effects for the ER-negative and triple-negative breast cancer (TNBC) subtypes. We tested the three recurrent protein-truncating variants FANCM:p.Arg658*, p.Gln1701*, and p.Arg1931* for association with breast cancer risk in 67,112 cases, 53,766 controls, and 26,662 carriers of pathogenic variants of BRCA1 or BRCA2. These three variants were also studied functionally by measuring survival and chromosome fragility in FANCM -/- patient-derived immortalized fibroblasts treated with diepoxybutane or olaparib. We observed that FANCM:p.Arg658* was associated with increased risk of ER-negative disease and TNBC (OR = 2.44, P = 0.034 and OR = 3.79; P = 0.009, respectively). In a country-restricted analysis, we confirmed the associations detected for FANCM:p.Arg658* and found that also FANCM:p.Arg1931* was associated with ER-negative breast cancer risk (OR = 1.96; P = 0.006). The functional results indicated that all three variants were deleterious affecting cell survival and chromosome stability with FANCM:p.Arg658* causing more severe phenotypes. In conclusion, we confirmed that the two rare FANCM deleterious variants p.Arg658* and p.Arg1931* are risk factors for ER-negative and TNBC subtypes. Overall our data suggest that the effect of truncating variants on breast cancer risk may depend on their position in the gene. Cell sensitivity to olaparib exposure, identifies a possible therapeutic option to treat FANCM-associated tumors