Investigating the interaction between dietary polyphenols, the SARS CoV-2 spike protein and the ACE-2 receptor.

The global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has called for an urgent need for the identification of compounds able to control, prevent or slow down the global pandemic. Several dietary polyphenols were assayed against binding to the SARS CoV-2 S1 spike protein and the human ACE-2 receptor, the target of the SARS CoV-2 virus using nano differential scanning fluorimetry, suggesting interaction of dietary polyphenols with both proteins. Following this initial screening the two dietary polyphenols with the strongest affinity were evaluated in a second functional binding assay. The assay was based on the thermophoresis of a fluorescently labelled spike protein and the ACE-2 receptor in the presence of dietary concentrations of the polyphenol in question. It could be experimentally shown that 5-caffeoyl quinic acid and epicatechin reduce the binding constant between SARS CoV-2 spike protein of the alpha variant and the ACE-2 receptor by a factor of ten. The finding could as well be applied to black tea and a coffee beverage with dietary 5-CQA concentrations for the alpha variant Spike protein. Hence it can be speculated that a cup of coffee reduces binding of the virus to its human target, therefore reducing the likelihood of infection with SARS CoV-2, acting as a virus entry-inhibitor.

Is insertion of a plastic stent better and safer than epinephrine injection in post sphincterotomy bleeding?

OBJECTIVES: Epinephrine injection is the therapy of first choice in post sphincterotomy bleeding (PSB), but may not be efficient in all cases and can cause postprocedural myocardial infarction. Plastic stent insertion (PSI) may be a better treatment. The aim of this retrospective study was to compare epinephrine injection with PSI with respect to efficacy and safety. METHODS: Clinical success, number of reinterventions and hospital stays after therapy, postprocedural myocardial infarction, bilirubin increase, and pancreatitis as well as factors influencing PSB were analyzed. RESULTS: Seventy-nine PSBs in 5798 endoscopic retrograde cholangiopancreaticographies (ERCPs) from August 2002 through October 2018 were treated by epinephrine injection, PSI or both (n = 34, 30, 15). Clinical success of PSB therapy showed no difference: 33/34 (97%), 30/30 (100%), 14/15 (93%). Reinterventions were more frequent (n = 30 versus n = 1; P ≤ 0.0001) and hospital stay was longer [median: 3 (2-10) versus 2 (1-3) days; P = 0.0357] in patients who received PSI (versus epinephrine injection). Postprocedural adverse events were very rare: bilirubin increase (1/2/0) and pancreatitis (0/2/1). Intraprocedural episodes of hypertension (≥180 mmHg) were documented in 45-54%. CONCLUSIONS: Epinephrine injection is better than PSI in PSB. PSI may be an adequate treatment in patients with otherwise indicated stent insertion. Intraprocedural episodes of hypertension may be a risk factor for PSB.

68Ga-PSMA PET/CT Detects the Location and Extent of Primary Prostate Cancer.

We evaluated the accuracy of PET/CT with 68Ga-PSMA-HBED-CC-a 68Ga-conjugated ligand of human prostate-specific membrane antigen (PSMA)-to localize cancer in the prostate and surrounding tissue at initial diagnosis. METHODS: Twenty-one patients with biopsy-proven prostate cancer underwent 68Ga-PSMA-HBED-CC (68Ga-PSMA) PET/CT at a median of 4 d (range, 0-47 d) before radical prostatectomy. Based on a 6-segment model, the Gleason score and proportion of tumor tissue within each segment (segmental tumor burden, or STB) as determined by histopathology (STBHP) were correlated with SUVmax and STB as determined by different SUV cutoffs for 68Ga-PSMA PET (STBPET1-6). Furthermore, the involvement of seminal vesicles and other extracapsular extension were assessed by histopathology and PET/CT. RESULTS: Histopathology-positive segments (n = 100 of 126; 79%) demonstrated a significantly higher mean ± SD SUVmax (11.8 ± 7.6) than histopathology-negative segments (4.9 ± 2.9; P < 0.001). Receiver-operating-characteristic analysis revealed an optimal SUVmax cutoff of 6.5 for discrimination of histopathology-positive segments from histopathology-negative segments (area under the curve, 0.84; P < 0.001), which gave 67% sensitivity, 92% specificity, a 97% positive predictive value, a 42% negative predictive value, and 72% accuracy. STBPET3 as determined by (2 × blood SUV) + (2 × SD) correlated best with STBHP (Pearson ρ = 0.68; P < 0.001; mean difference ± SD, 19% ± 15%). PET/CT correctly detected invasion of seminal vesicles (n = 11 of 21 patients; 52%) with 86% accuracy and tumor spread through the capsule (n = 12; 57%) with 71% accuracy. CONCLUSION: 68Ga-PSMA PET/CT accurately detected the location and extent of primary prostate cancer. Our preliminary findings warrant further investigation of 68Ga-PSMA PET/CT in conjunction with needle biopsy.

IRSp53 links the enterohemorrhagic E. coli effectors Tir and EspFU for actin pedestal formation.

Actin pedestal formation by pathogenic E. coli requires signaling by the bacterial intimin receptor Tir, which induces host cell actin polymerization mediated by N-WASP and the Arp2/3 complex. Whereas canonical enteropathogenic E. coli (EPEC) recruit these actin regulators through tyrosine kinase signaling cascades, enterohemorrhagic E. coli (EHEC) O157:H7 employ the bacterial effector EspF(U) (TccP), a potent N-WASP activator. Here, we show that IRSp53 family members, key regulators of membrane and actin dynamics, directly interact with both Tir and EspF(U). IRSp53 colocalizes with EspF(U) and N-WASP in actin pedestals. In addition, targeting of IRSp53 is independent of EspF(U) and N-WASP but requires Tir residues 454-463, previously shown to be essential for EspF(U)-dependent actin assembly. Genetic and functional loss of IRSp53 abrogates actin assembly mediated by EHEC. Collectively, these data indentify IRSp53 family proteins as the missing host cell factors linking bacterial Tir and EspF(U) in EHEC pedestal formation.

Impact of the rpoS genotype for acid resistance patterns of pathogenic and probiotic Escherichia coli.

BACKGROUND: Enterohemorrhagic E. coli (EHEC), a subgroup of Shiga toxin (Stx) producing E. coli (STEC), may cause severe enteritis and hemolytic uremic syndrome (HUS) and is transmitted orally via contaminated foods or from person to person. The infectious dose is known to be very low, which requires most of the bacteria to survive the gastric acid barrier. Acid resistance therefore is an important mechanism of EHEC virulence. It should also be a relevant characteristic of E. coli strains used for therapeutic purposes such as the probiotic E. coli Nissle 1917 (EcN). In E. coli and related enteric bacteria it has been extensively demonstrated, that the alternative sigma factor sigmaS, encoded by the rpoS gene, acts as a master regulator mediating resistance to various environmental stress factors. METHODS: Using rpoS deletion mutants of a highly virulent EHEC O26:H11 patient isolate and the sequenced prototype EHEC EDL933 (ATCC 700927) of serotype O157:H7 we investigated the impact of a functional rpoS gene for orchestrating a satisfactory response to acid stress in these strains. We then functionally characterized rpoS of probiotic EcN and five rpoS genes selected from STEC isolates pre-investigated for acid resistance. RESULTS: First, we found out that ATCC isolate 700927 of EHEC EDL933 has a point mutation in rpoS, not present in the published sequence, leading to a premature stop codon. Moreover, to our surprise, one STEC strain as well as EcN was acid sensitive in our test environment, although their cloned rpoS genes could effectively complement acid sensitivity of an rpoS deletion mutant. CONCLUSION: The attenuation of sequenced EHEC EDL933 might be of importance for anyone planning to do either in vitro or in vivo studies with this prototype strain. Furthermore our data supports recently published observations, that individual E. coli isolates are able to significantly modulate their acid resistance phenotype independent of their rpoS genotype.

Effect of the increased stability of the penicillin amidase mRNA on the protein expression levels.

Several factors at transcriptional, post-transcriptional or post-translational level determine the fate of a target protein and can severely restrict its yield. Here, we focus on the post-transcriptional regulation of the biosynthesis of the periplasmic protein, penicillin amidase (PA). The PA mRNA stability was determined under depleted RNase conditions in strains carrying single or multiple RNase deletions. Single deletion of the endonuclease RNase E yielded, as the highest, a fourfold stabilization of the PA mRNA. This effect, however, was reduced twice at post-translational level. The RNase II, generating secondary exonucleolytic cleavages in the mRNA, although not significantly influencing the PA mRNA decay, led also to an increase of the amount of mature PA. The non-proportional correlation between increased mRNA longevity and amount of active enzyme propose that the rational strategies for yield improvement must be based on a simultaneous tuning of more than one yield restricting factor.

Ca2+ is a cofactor required for membrane transport and maturation and is a yield-determining factor in high cell density penicillin amidase production.

Penicillin amidases (PAs) from E. coli and A. faecalis are periplasmic enzymes that contain one tightly bound Ca(2+) per molecule that does not directly participate in the enzymatic function. This ion may, however, be required for the maturation of the pre-pro-enzyme. The pro-enzyme of homologous PAs are translocated through the Tat- (E. coli PA(EC)) and Sec- (A. faecalis PA(AF)) transport systems, respectively. Cell fractionation, electrophoresis, immunoblotting, and activity staining demonstrated that Ca(2+) binding is required for the membrane transport and maturation of the pro-enzyme to active enzyme. Pro-enzyme without Ca(2+) was targeted to the membrane but not translocated. Influence of Ca(2+) in medium and feed was studied for high cell density cultivations of E. coli expressing these enzymes. Without Ca(2+) in the feed the synthesis of the pre-pro-enzyme was hardly influenced. At optimal Ca(2+) content in the feed the active enzyme amount could be increased by 2 orders of magnitude up to 0.9 g/L (PA(EC)) and 2.3 g/L (PA(AF)) or 4% (PA(EC)) and 8% (PA(AF)) of the cell dry weight. The corresponding specific activities are 1700 U (PA(EC)) and 14000 U (PA(AF)) per gram cell dry weight, respectively. These values are higher than those published previously. Thus, for optimal yields of the studied and other extra- and periplasmic enzymes that require Ca(2+) or other ions as cofactors for membrane transport and maturation, sufficient cofactor must be added in the feed.