Photodynamic Inactivation of Bacteria in Ionic Environments Using the Photosensitizer SAPYR and the Chelator Citrate

Many studies show that photodynamic inactivation (PDI) is a powerful tool for the fight against pathogenic, multiresistant bacteria and the closing of hygiene gaps. However, PDI studies have been frequently performed under standardized in vitro conditions comprising artificial laboratory settings. Under real-life conditions, however, PDI encounters substances like ions, proteins, amino acids and fatty acids, potentially hampering the efficacy of PDI to an unpredictable extent. Thus, we investigated PDI with the phenalene-1-one-based photosensitizer SAPYR against Escherichia coli and Staphylococcus aureus in the presence of calcium or magnesium ions, which are ubiquitous in potential fields of PDI applications like in tap water or on tissue surfaces. The addition of citrate should elucidate the potential as a chelator. The results indicate that PDI is clearly affected by such ubiquitous ions depending on its concentration and the type of bacteria. The application of citrate enhanced PDI, especially for Gram-negative bacteria at certain ionic concentrations (e.g. CaCl2 or MgCl2: 7.5 to 75 mmol L−1). Citrate also improved PDI efficacy in tap water (especially for Gram-negative bacteria) and synthetic sweat solution (especially for Gram-positive bacteria). In conclusion, the use of chelating agents like citrate may facilitate the application of PDI under real-life conditions

Photodynamic inactivation of different pathogenic bacteria on human skin using a novel photosensitizer hydrogel

Background The colonization of skin with pathogenic, partially antibiotic-resistant bacteria is frequently a severe problem in dermatological therapies. For instance, skin colonization with Staphylococcus aureus is even a disease-promoting factor in atopic dermatitis. The photodynamic inactivation (PDI) of bacteria could be a new antibacterial procedure. Upon irradiation with visible light, a special photosensitizer exclusively generates singlet oxygen. This reactive oxygen species kills bacteria via oxidation independent of species or strain and their antibiotic resistance profile causing no bacterial resistance on its part. Objective To investigate the antibacterial potential of a photosensitizer, formulated in a new hydrogel, on human skin ex vivo. Methods The photochemical stability of the photosensitizer and its ability to generate singlet oxygen in the hydrogel was studied. Antimicrobial efficacy of this hydrogel was tested step by step, firstly on inanimate surfaces and then on human skin ex vivo against S. aureus and Pseudomonas aeruginosa using standard colony counting. NBTC staining and TUNEL assays were performed on skin biopsies to investigate potential necrosis and apoptosis effects in skin cells possibly caused by PDI. Results None of the hydrogel components affected the photochemical stability and the life time of singlet oxygen. On inanimate surfaces as well as on the human skin, the number of viable bacteria was reduced by up to 4.8 log10 being more effective than most other antibacterial topical agents. Histology and assays showed that PDI against bacteria on the skin surface caused no harmful effects on the underlying skin cells. Conclusion Photodynamic inactivation hydrogel proved to be effective for decolonization of human skin including the potential to act against superficial skin infections. Being a water-based formulation, the hydrogel should be also suitable for the mucosa. The results of the present ex vivo study form a good basis for conducting clinical studies in vivo

Inhibitory effects of calcium or magnesium ions on PDI

Photodynamic inactivation of microorganisms (PDI) finds use in a variety of applications. Several studies report on substances enhancing or inhibiting PDI. In this study, we analyzed the inhibitory potential of ubiquitous salts like CaCl2 and MgCl2 on PDI against Staphylococcus aureus and Pseudomonas aeruginosa cells using five cationic photosensitizers methylene blue, TMPyP, SAPYR, FLASH-02a and FLASH-06a. TMPyP changed its molecular structure when exposed to MgCl2, most likely due to complexation. CaCl2 substantially affected singlet oxygen generation by MB at small concentrations. Elevated concentrations of CaCl2 and MgCl2 impaired PDI up to a total loss of bacterial reduction, whereas CaCl2 is more detrimental for PDI than MgCl2. Binding assays cannot not explain the differences of PDI efficacy. It is assumed that divalent ions tightly bind to bacterial cells hindering close binding of the photosensitizers to the membranes. Consequently, photosensitizer binding might be shifted to outer compartments like teichoic acids in Gram-positives or outer sugar moieties of the LPS in Gram-negatives, attenuating the oxidative damage of susceptible cellular structures. In conclusion, CaCl2 and MgCl2 have an inhibitory potential at different phases in PDI. These effects should be considered when using PDI in an environment that contains such salts like in tap water or different fields of food industry

Survival Data and Predictors of Functional Outcome an Average of 15 Years after the Fontan Procedure: The Pediatric Heart Network Fontan Cohort

ObjectiveMulticenter longitudinal outcome data for Fontan patients surviving into adulthood are lacking. The aim of this study was to better understand contemporary outcomes in Fontan survivors by collecting follow‐up data in a previously well‐characterized cohort.DesignBaseline data from the Fontan Cross‐Sectional Study (Fontan 1) were previously obtained in 546 Fontan survivors aged 11.9 ± 3.4 years. We assessed current transplant‐free survival status in all subjects 6.8 ± 0.4 years after the Fontan 1 study. Anatomic, clinical, and surgical data were collected along with socioeconomic status and access to health care.ResultsThirty subjects (5%) died or underwent transplantation since Fontan 1. Subjects with both an elevated (>21 pg/mL) brain natriuretic peptide and a low Child Health Questionnaire physical summary score (<44) measured at Fontan 1 were significantly more likely to die or undergo transplant than the remainder, with a hazard ratio of 6.2 (2.9–13.5). Among 516 Fontan survivors, 427 (83%) enrolled in this follow‐up study (Fontan 2) at 18.4 ± 3.4 years of age. Although mean scores on functional health status questionnaires were lower than the general population, individual scores were within the normal range in 78% and 88% of subjects for the Child Health Questionnaire physical and psychosocial summary score, and 97% and 91% for the SF‐36 physical and mental aggregate score, respectively. Since Fontan surgery, 119 (28%) had additional cardiac surgery; 55% of these (n = 66) in the interim between Fontan 1 and Fontan 2. A catheter intervention occurred in 242 (57%); 32% of these (n = 78) after Fontan 1. Arrhythmia requiring treatment developed in 118 (28%) after Fontan surgery; 58% of these (n = 68) since Fontan 1.ConclusionsWe found 95% interim transplant‐free survival for Fontan survivors over an average of 7 years of follow‐up. Continued longitudinal investigation into adulthood is necessary to better understand the determinants of long‐term outcomes and to improve functional health status.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/110738/1/chd12193.pd

Micro RNAs of Epstein-Barr Virus Promote Cell Cycle Progression and Prevent Apoptosis of Primary Human B Cells

Cellular and viral microRNAs (miRNAs) are involved in many different processes of key importance and more than 10,000 miRNAs have been identified so far. In general, relatively little is known about their biological functions in mammalian cells because their phenotypic effects are often mild and many of their targets still await identification. The recent discovery that Epstein-Barr virus (EBV) and other herpesviruses produce their own, barely conserved sets of miRNAs suggests that these viruses usurp the host RNA silencing machinery to their advantage in contrast to the antiviral roles of RNA silencing in plants and insects. We have systematically introduced mutations in EBV's precursor miRNA transcripts to prevent their subsequent processing into mature viral miRNAs. Phenotypic analyses of these mutant derivatives of EBV revealed that the viral miRNAs of the BHRF1 locus inhibit apoptosis and favor cell cycle progression and proliferation during the early phase of infected human primary B cells. Our findings also indicate that EBV's miRNAs are not needed to control the exit from latency. The phenotypes of viral miRNAs uncovered by this genetic analysis indicate that they contribute to EBV-associated cellular transformation rather than regulate viral genes of EBV's lytic phase

Repertoire of microRNAs in Epithelial Ovarian Cancer as Determined by Next Generation Sequencing of Small RNA cDNA Libraries

MicroRNAs (miRNAs) are small regulatory RNAs that are implicated in cancer pathogenesis and have recently shown promise as blood-based biomarkers for cancer detection. Epithelial ovarian cancer is a deadly disease for which improved outcomes could be achieved by successful early detection and enhanced understanding of molecular pathogenesis that leads to improved therapies. A critical step toward these goals is to establish a comprehensive view of miRNAs expressed in epithelial ovarian cancer tissues as well as in normal ovarian surface epithelial cells.We used massively parallel pyrosequencing (i.e., "454 sequencing") to discover and characterize novel and known miRNAs expressed in primary cultures of normal human ovarian surface epithelium (HOSE) and in tissue from three of the most common histotypes of ovarian cancer. Deep sequencing of small RNA cDNA libraries derived from normal HOSE and ovarian cancer samples yielded a total of 738,710 high-quality sequence reads, generating comprehensive digital profiles of miRNA expression. Expression profiles for 498 previously annotated miRNAs were delineated and we discovered six novel miRNAs and 39 candidate miRNAs. A set of 124 miRNAs was differentially expressed in normal versus cancer samples and 38 miRNAs were differentially expressed across histologic subtypes of ovarian cancer. Taqman qRT-PCR performed on a subset of miRNAs confirmed results of the sequencing-based study.This report expands the body of miRNAs known to be expressed in epithelial ovarian cancer and provides a useful resource for future studies of the role of miRNAs in the pathogenesis and early detection of ovarian cancer

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

A humanized model to investigate targeted therapeutic strategies against cancer bone metastasis

Bone metastasis represents a common and terminal outcome for patients suffering from advanced breast and prostate cancer. The underlying molecular mechanisms are still not fully understood and to date no curative treatments are available. In this PhD project, a humanized mouse model was, for the first time, explored as drug response tool, using new and already clinically approved targeted treatments against cancer bone metastasis. The outcomes and observations presented, show that further humanization of the model will allow for the in-depth dissection of molecular differences between the murine and human bone-microenvironment in the context of drug response

Homozygous mutation in murine retrovirus integration site 1 gene associated with a non‐syndromic form of isolated familial achalasia

Background Achalasia is a condition characterized by impaired function of esophageal motility and incomplete relaxation of the lower esophagus sphincter, causing dysphagia and regurgitation. Rare cases of early-onset achalasia appear often in combination with further symptoms in a syndromic form as an inherited disease. Methods Whole genome sequencing was used to investigate the genetic basis of isolated achalasia in a family of Tunisian origin. We analyzed the function of the affected protein with immunofluorescence and affinity chromatography study. Key Results A homozygous nonsense mutation was detected in murine retrovirus integration site 1 (MRVI1) gene (Human Genome Organisation Gene Nomenclature Committee (HGNC) approved gene symbol:IRAG1) encoding the inositol 1,4,5-trisphosphate receptor 1 (IP(3)R1)-associated cyclic guanosine monophosphate (cGMP) kinase substrate (IRAG). Sanger sequencing confirmed co-segregation of the mutation with the disease. Sequencing of the entireMRVI1gene in 35 additional patients with a syndromic form of achalasia did not uncover further cases withMRVI1mutations. Immunofluorescence analysis of transfected COS7 cells revealed GFP-IRAG with the truncating mutation p.Arg112* (transcript variant 1) or p.Arg121* (transcript variant 2) to be mislocalized in the cytoplasm and the nucleus. Co-transfection with cGMP-dependent protein kinase 1 isoform beta (cGK1 beta) depicted a partial mislocalization of cGK1 beta due to mislocalized truncated IRAG. Isolation of protein complexes revealed that the truncation of this protein causes the loss of the interaction domain of IRAG with cGK1 beta. Conclusions & Inferences In individuals with an early onset of achalasia without further accompanying symptoms,MRVI1mutations should be considered as the disease-causing defect

Immunogenetic Predisposition to SARS-CoV-2 Infection

Herein, we included 527 individuals from two Hospitals, Chemnitz and University-Hospital Leipzig. In total, 199 were negative for PCR and 328 were positive upon first admission. We used next generation sequencing for HLA-A, B, C, DRB1, DRB345, DQA1, DQB1, DPA1, and DPB1, and in some cases, HLA-E, F, G, and H. Furthermore, we molecularly defined 22 blood group systems comprising 26 genes and 5 platelet antigen genes. We observed a significant enrichment of homozygosity for DQA/DQB in the positive group. Within the negative subjects, HLA-B*57:01, HLA-B*55:01, DRB1*13:01, and DRB1*01:01 were enriched, and in the positive group, homozygosity for DQA/DQB, DRB1*09:01, and DRB1*15:01 was observed. DQA1*01:01, DQA1*02:01, and DQA1*01:03 were enriched in the negative group. HLA-DQB1*06:02 was enriched in the positive group, and HLA-DQB1*05:01 and HLA-DQB1*06:03 were enriched in the negative group. For the blood group systems MNS, RH, LE, FY, JK, YT, DO, and KN, enrichment was seen in both groups, depending on the antigen under observation. Homozygosity for D-positive RHD alleles, as well as the phenotypes M-N+ of the MNS blood group system and Yk(a-) of the KN system, were enriched in the positive group. All of these significances disappeared upon correction. Subjects who carried homozygous HPA-1a were more frequent in the negative group, contrasting with the finding that HPA-1ab was enriched in the positive group