Varied active-site constraints in the klenow fragment of E. coli DNA polymerase I and the lesion-bypass Dbh DNA polymerase

We report on comparative pre-steady-state kinetic analyses of exonuclease-deficient Escherichia coli DNA polymerase I (Klenow fragment, KF-) and the archaeal Y-family DinB homologue (Dbh) of Sulfolobus solfataricus. We used size-augmented sugar-modified thymidine-5'-triphosphate (T(R)TP) analogues to test the effects of steric constraints in the active sites of the polymerases. These nucleotides serve as models for study of DNA polymerases exhibiting both relatively high and low intrinsic selectivity. Substitution of a hydrogen atom at the 4'-position in the nucleotide analogue by a methyl group reduces the maximum rate of nucleotide incorporation by about 40-fold for KF- and about twelve fold for Dbh. Increasing the size to an ethyl group leads to a further twofold reduction in the rates of incorporation for both enzymes. Interestingly, the affinity of KF- for the modified nucleotides is only marginally affected, which would indicate no discrimination during the binding step. Dbh even has a higher affinity for the modified analogues than it does for the natural substrate. Misincorporation of either TTP or T(Me)TP opposite a G template causes a drastic decline in incorporation rates for both enzymes. At the same time, the binding affinities of KF- for these nucleotides drop by about 16- and fourfold, respectively, whereas Dbh shows only a twofold reduction. Available structural data for ternary complexes of relevant DNA polymerases indicate that both enzymes make close contacts with the sugar moiety of the dNTP. Thus, the varied proficiencies of the two enzymes in processing the size-augmented probes indicate varied flexibility of the enzymes' active sites and support the notion of active site tightness being a criterion for DNA polymerase selectivity.     

Physical and ecological controls on freshwater floc trace metal dynamics

Significantly higher concentrations of Ag, As, Cu, Co, Ni, and Pb are found in suspended floc compared to surficial bed sediments for a freshwater beach in Lake Ontario. Contrasting observed element-specific bed sediment metal partitioning patterns, floc sequestration for all elements is dominated by one substrate: amorphous oxyhydroxides. More specifically, floc metal scavenging is controlled by floc biogeochemical architecture. Floc organics, largely living microbial cells and associated exopolymeric substances (EPS), act as scaffolds for the collection and/or templating of amorphous Fe oxyhydroxides. While interactions between floc organics and amorphous Fe oxyhydroxides affected floc sorption behavior, specific element affinities and competition for these limited substrates was important for overall floc partitioning. Further, assessment of metal dynamics during stormy conditions indicated energy-regime driven shifts in floc and bed sediment partitioning that were specifically linked to the exchange of floc and bed sedimentary materials. These novel results demonstrate that the microbial nature of floc formation exerts an important control on floc metal dynamics distinguishable from surficial bed sediments and that hydrologic energy-regime is an important factor to consider in overall floc metal behavior, especially in beach environments.     

Enhanced cellular uptake of a glutathione selective fluorogenic probe encapsulated in nanoparticles

Selective fluorogenic probes for the labelling of intracellular reduced glutathione (GSH), i.e.?ortho-phthaldialdehyde (OPA) and naphthalene-2,3-dicarboxaldehyde (NDA), have been encapsulated in polymeric nanoparticles (NPs) and the ability of the NPs to enhance uptake of the probe by microbial cells has been evaluated. Preparation of the probe-loaded NPs composed of Eudragit(?) E was based on an oil-in-water emulsification solvent evaporation method using an ultrasonic probe and polyvinyl alcohol as the surfactant. The encapsulation efficiency of the probes in lyophilized NPs was determined using high performance liquid chromatography (HPLC). A higher encapsulation rate of NDA than OPA was found: 47.6 ± 9.9?(n = 6) and 2.1 ± 0.2%?(n = 3), respectively. The NDA-loaded particle diameter and zeta potential were 224.6 ± 14.7?nm and +40.9 ± 6.5?mV, respectively. After 20?min incubation of cultured Candida albicans yeast cells with either free NDA or NDA-loaded NPs (final NDA concentration 100?μM), cells were harvested and corresponding lysates were analysed using HPLC coupled with spectrofluorimetric detection. Incubation of cells with NDA-loaded NPs increased intracellular levels of NDA-GSH adduct by about nine-fold in comparison with the free probe. Adhesion on the cells and the penetration behaviour of NPs loaded with either NDA or fluorescent label (Nile Red) were characterized qualitatively by confocal laser scanning microscopy.     

Surface-enhanced Raman optical activity on adenine in silver colloidal solution

We report the collection of Raman optical activity (ROA) spectra of adenine in silver colloidal solution, that is, surface-enhanced Raman optical activity (SEROA) using considerably shorter data acquisition times, reduced excitation power, and lower concentration, as compared to classical ROA measurements on molecules of biological interest so far reported in the literature. These improvements in experimental parameters for ROA measurements can be explained by enhanced Raman signals in the local optical fields of the silver nanoparticles and by at least 1 order of magnitude higher values for circular intensity differences (CIDs), as compared to classical ROA that has been suggested before and theoretically discussed in terms of large field gradients near a metal surface. The measured ROA effect for adenine can be understood in terms of adsorption-induced chirality in the prochiral molecules on the silver nanoparticles. Surface-enhanced Raman optical activity offers potential capabilities for sensitive, rapid, stereochemical characterization of basic building blocks of biopolymers, such as amino acids and nucleosides, as well as biologically active molecules, in particular, also for probing organization and self-assembling of such molecules on metal surfaces.     

Neutrophils’ Extracellular Trap Mechanisms: From Physiology to Pathology

Neutrophils are an essential part of the innate immune system and the first line of defense against invading pathogens. They phagocytose, release granular contents, produce reactive oxygen species, and form neutrophil extracellular traps (NETs) to fight pathogens. With the characterization of NETs and their components, neutrophils were identified as players of the innate adaptive crosstalk. This has placed NETs at the center not only of physiological but also pathological processes. Aside from their role in pathogen uptake and clearance, NETs have been demonstrated to contribute to the resolution of inflammation by forming aggregated NETs able to degrade inflammatory mediators. On the other hand, NETs have the potential to foster severe pathological conditions. When homeostasis is disrupted, they occlude vessels and ducts, serve as sources of autoantigens and danger or damage associated molecular patterns, directly damage tissues, and exaggerate complement activity and inflammation. This review focusses on the understanding of NETs from their formation to their functions in both physiological and pathological processes.     

Glomerular Endothelial Cell-Derived miR-200c Impairs Glomerular Homeostasis by Targeting Podocyte VEGF-A

Deciphering the pathophysiological mechanisms of primary podocytopathies that can lead to end-stage renal disease and increased mortality is an unmet need. Studying how microRNAs (miRs) interfere with various signaling pathways enables identification of pathomechanisms, novel biomarkers and potential therapeutic options. We investigated the expression of miR-200c in urine from patients with different renal diseases as a potential candidate involved in podocytopathies. The role of miR-200c for the glomerulus and its potential targets were studied in cultured human podocytes, human glomerular endothelial cells and in the zebrafish model. miR-200c was upregulated in urine from patients with minimal change disease, membranous glomerulonephritis and focal segmental glomerulosclerosis and also in transforming growth factor beta (TGF-β) stressed glomerular endothelial cells, but not in podocytes. In zebrafish, miR-200c overexpression caused proteinuria, edema, podocyte foot process effacement and glomerular endotheliosis. Although zinc finger E-Box binding homeobox 1/2 (ZEB1/2), important in epithelial to mesenchymal transition (EMT), are prominent targets of miR-200c, their downregulation did not explain our zebrafish phenotype. We detected decreased vegfaa/bb in zebrafish overexpressing miR-200c and could further prove that miR-200c decreased VEGF-A expression and secretion in cultured human podocytes. We hypothesize that miR-200c is released from glomerular endothelial cells during cell stress and acts in a paracrine, autocrine, as well as context-dependent manner in the glomerulus. MiR-200c can cause glomerular damage most likely due to the reduction of podocyte VEGF-A. In contrast, miR-200c might also influence ZEB expression and therefore EMT, which might be important in other conditions. Therefore, we propose that miR-200c-mediated effects in the glomerulus are context-sensitive.     

Long-Lived Individuals Show a Lower Burden of Variants Predisposing to Age-Related Diseases and a Higher Polygenic Longevity Score

Longevity is a complex phenotype influenced by both environmental and genetic factors. The genetic contribution is estimated at about 25%. Despite extensive research efforts, only a few longevity genes have been validated across populations. Long-lived individuals (LLI) reach extreme ages with a relative low prevalence of chronic disability and major age-related diseases (ARDs). We tested whether the protection from ARDs in LLI can partly be attributed to genetic factors by calculating polygenic risk scores (PRSs) for seven common late-life diseases (Alzheimer’s disease (AD), atrial fibrillation (AF), coronary artery disease (CAD), colorectal cancer (CRC), ischemic stroke (ISS), Parkinson’s disease (PD) and type 2 diabetes (T2D)). The examined sample comprised 1351 German LLI (≥94 years, including 643 centenarians) and 4680 German younger controls. For all ARD-PRSs tested, the LLI had significantly lower scores than the younger control individuals (areas under the curve (AUCs): ISS = 0.59, p = 2.84 × 10⁻³⁵; AD = 0.59, p = 3.16 × 10⁻²⁵; AF = 0.57, p = 1.07 × 10⁻¹⁶; CAD = 0.56, p = 1.88 × 10⁻¹²; CRC = 0.52, p = 5.85 × 10⁻³; PD = 0.52, p = 1.91 × 10⁻³; T2D = 0.51, p = 2.61 × 10⁻³). We combined the individual ARD-PRSs into a meta-PRS (AUC = 0.64, p = 6.45 × 10⁻¹⁵). We also generated two genome-wide polygenic scores for longevity, one with and one without the TOMM40/APOE/APOC1 gene region (AUC (incl. TOMM40/APOE/APOC1) = 0.56, p = 1.45 × 10⁻⁵, seven variants; AUC (excl. TOMM40/APOE/APOC1) = 0.55, p = 9.85 × 10⁻³, 10,361 variants). Furthermore, the inclusion of nine markers from the excluded region (not in LD with each other) plus the APOE haplotype into the model raised the AUC from 0.55 to 0.61. Thus, our results highlight the importance of TOMM40/APOE/APOC1 as a longevity hub.