Structure-Function Analysis of Heterodimer Formation,Oligomerization, and Receptor Binding of the Staphylococcus aureus Bi-component Toxin LukGH

The bi-component leukocidins of Staphylococcus aureus are important virulence factors that lyse human phagocytic cells and contribute to immune evasion. The γ-hemolysins (HlgAB and HlgCB) and Panton-Valentine leukocidin (PVL or LukSF) were shown to assemble from soluble subunits into membrane-bound oligomers on the surface of target cells, creating barrel-like pore structures that lead to cell lysis. LukGH is the most distantly related member of this toxin family, sharing only 30–40% amino acid sequence identity with the others. We observed that, unlike other leukocidin subunits, recombinant LukH and LukG had low solubility and were unable to bind to target cells, unless both components were present. Using biolayer interferometry and intrinsic tryptophan fluorescence we detected binding of LukH to LukG in solution with an affinity in the low nanomolar range and dynamic light scattering measurements confirmed formation of a heterodimer. We elucidated the structure of LukGH by x-ray crystallography at 2.8-Å resolution. This revealed an octameric structure that strongly resembles that reported for HlgAB, but with important structural differences. Structure guided mutagenesis studies demonstrated that three salt bridges, not found in other bi-component leukocidins, are essential for dimer formation in solution and receptor binding. We detected weak binding of LukH, but not LukG, to the cellular receptor CD11b by biolayer interferometry, suggesting that in common with other members of this toxin family, the S-component has the primary contact role with the receptor. These new insights provide the basis for novel strategies to counteract this powerful toxin and Staphylococcus aureus pathogenesis.     

Enterobacteriaceae Isolated from the River Danube: Antibiotic Resistances,with a Focus on the Presence of ESBL and Carbapenemases

In a clinical setting it seems to be normal these days that a relevant proportion or even the majority of different bacterial species has already one or more acquired antibiotic resistances. Unfortunately, the overuse of antibiotics for livestock breeding and medicine has also altered the wild-type resistance profiles of many bacterial species in different environmental settings. As a matter of fact, getting in contact with resistant bacteria is no longer restricted to hospitals. Beside food and food production, the aquatic environment might also play an important role as reservoir and carrier. The aim of this study was the assessment of the resistance patterns of Escherichia coli and Klebsiella spp. out of surface water without prior enrichment and under non-selective culture conditions (for antibiotic resistance). In addition, the presence of clinically important extended spectrum beta lactamase (ESBL) and carbapenmase harboring Enterobacteriaceae should be investigated. During Joint Danube Survey 3 (2013), water samples were taken over the total course of the River Danube. Resistance testing was performed for 21 different antibiotics. Samples were additionally screened for ESBL or carbapenmase harboring Enterobacteriaceae. 39% of all isolated Escherichia coli and 15% of all Klebsiella spp. from the river Danube had at least one acquired resistance. Resistance was found against all tested antibiotics except tigecycline. Taking a look on the whole stretch of the River Danube the proportion of multiresistances did not differ significantly. In total, 35 ESBL harboring Enterobacteriaceae, 17 Escherichia coli, 13 Klebsiella pneumoniae and five Enterobacter spp. were isolated. One Klebsiella pneumoniae harboring NMD-1 carbapenmases and two Enterobacteriaceae with KPC-2 could be identified. Human generated antibiotic resistance is very common in E. coli and Klebsiella spp. in the River Danube. Even isolates with resistance patterns normally associated with intensive care units are present.     

Anti-Hepatitis C Virus T-Cell Immunity in the Context of Multiple Exposures to the Virus

Characterisation of Hepatitis C virus (HCV)-specific CD8⁺ T-cell responses in the context of multiple HCV exposures is critical to identify broadly protective immune responses necessary for an effective HCV vaccine against the different HCV genotypes. However, host and viral genetic diversity complicates vaccine development. To compensate for the observed variation in circulating autologous viruses and host molecules that restrict antigen presentation (human leucocyte antigens; HLA), this study used a reverse genomics approach that identified sites of viral adaptation to HLA-restricted T-cell immune pressure to predict genotype-specific HCV CD8⁺ T-cell targets. Peptides representing these putative HCV CD8⁺ T-cell targets, and their adapted form, were used in individualised IFN-γ ELISpot assays to screen for HCV-specific T-cell responses in 133 HCV-seropositive subjects with high-risk of multiple HCV exposures. The data obtained from this study i) confirmed that genetic studies of viral evolution is an effective approach to detect novel in vivo HCV T-cell targets, ii) showed that HCV-specific T-cell epitopes can be recognised in their adapted form and would not have been detected using wild-type peptides and iii) showed that HCV-specific T-cell (but not antibody) responses against alternate genotypes in chronic HCV-infected subjects are readily found, implying clearance of previous alternate genotype infection. In summary, HCV adaptation to HLA Class I-restricted T-cell responses plays a central role in anti-HCV immunity and multiple HCV genotype exposure is highly prevalent in at-risk exposure populations, which are important considerations for future vaccine design.     

Utilization of Benchtop Next Generation Sequencing Platforms Ion Torrent PGM and MiSeq in Noninvasive Prenatal Testing for Chromosome 21 Trisomy and Testing of Impact of In Silico and Physical Size Selection on Its Analytical Performance

Objectives

The aims of this study were to test the utility of benchtop NGS platforms for NIPT for trisomy 21 using previously published z score calculation methods and to optimize the sample preparation and data analysis with use of in silico and physical size selection methods.

Methods

Samples from 130 pregnant women were analyzed by whole genome sequencing on benchtop NGS systems Ion Torrent PGM and MiSeq. The targeted yield of 3 million raw reads on each platform was used for z score calculation. The impact of in silico and physical size selection on analytical performance of the test was studied.

Results

Using a z score value of 3 as the cut-off, 98.11% - 100% (104-106/106) specificity and 100% (24/24) sensitivity and 99.06% - 100% (105-106/106) specificity and 100% (24/24) sensitivity were observed for Ion Torrent PGM and MiSeq, respectively. After in silico based size selection both platforms reached 100% specificity and sensitivity. Following the physical size selection z scores of tested trisomic samples increased significantly—p = 0.0141 and p = 0.025 for Ion Torrent PGM and MiSeq, respectively.

Conclusions

Noninvasive prenatal testing for chromosome 21 trisomy with the utilization of benchtop NGS systems led to results equivalent to previously published studies performed on high-to-ultrahigh throughput NGS systems. The in silico size selection led to higher specificity of the test. Physical size selection performed on isolated DNA led to significant increase in z scores. The observed results could represent a basis for increasing of cost effectiveness of the test and thus help with its penetration worldwide.     

Effects of NaCl on surface properties,chlorophyll fluorescence and light remission,and cellular compounds of <Emphasis Type="Italic">Grewia tenax</Emphasis> (Forssk.) Fiori and <Emphasis Type="Italic">Tamarindus indica</Emphasis> L. leaves

Seedlings of the salt-tolerant plant grewia [Grewia tenax (Forssk.) Fiori] and the moderately salt-tolerant tamarind (Tamarindus indica L.) were grown under controlled conditions and treated daily with NaCl solutions to investigate mechanisms of tolerance to salinity. Leaf micromorphology, cuticular wax load, chlorophyll fluorescence and light remission, as well as antioxidative potential were evaluated. As confirmed by energy-dispersive X-ray microanalysis in both species, absorption of sodium and chlorine increased with rising NaCl concentration in the treatment solution. In parallel, accumulation of calcium in grewia leaves was strongly reduced, leading to less crystals of calcium oxalate in leaf tissue. In grewia the cuticular wax load, chlorophyll content, and electron transport rate (ETR) were significantly reduced by comparatively low NaCl concentrations. In tamarind, in contrast, wax load and ETR were not significantly affected, while the decrease of chlorophyll content was less pronounced. Measurements of the antioxidative capacity and the imbalance between values of lipophilic and hydrophilic extracts at different NaCl concentrations confirmed that grewia is more salt tolerant than tamarind. This higher tolerance degree seemed to be associated with grewias’ more efficient scavenging of free radicals and the regulation of the antioxidative potential in lipophilic and hydrophilic extracts.     

Detailed Functional and Proteomic Characterization of Fludarabine Resistance in Mantle Cell Lymphoma Cells

Mantle cell lymphoma (MCL) is a chronically relapsing aggressive type of B-cell non-Hodgkin lymphoma considered incurable by currently used treatment approaches. Fludarabine is a purine analog clinically still widely used in the therapy of relapsed MCL. Molecular mechanisms of fludarabine resistance have not, however, been studied in the setting of MCL so far. We therefore derived fludarabine-resistant MCL cells (Mino/FR) and performed their detailed functional and proteomic characterization compared to the original fludarabine sensitive cells (Mino). We demonstrated that Mino/FR were highly cross-resistant to other antinucleosides (cytarabine, cladribine, gemcitabine) and to an inhibitor of Bruton tyrosine kinase (BTK) ibrutinib. Sensitivity to other types of anti-lymphoma agents was altered only mildly (methotrexate, doxorubicin, bortezomib) or remained unaffacted (cisplatin, bendamustine). The detailed proteomic analysis of Mino/FR compared to Mino cells unveiled over 300 differentially expressed proteins. Mino/FR were characterized by the marked downregulation of deoxycytidine kinase (dCK) and BTK (thus explaining the observed crossresistance to antinucleosides and ibrutinib), but also by the upregulation of several enzymes of de novo nucleotide synthesis, as well as the up-regulation of the numerous proteins of DNA repair and replication. The significant upregulation of the key antiapoptotic protein Bcl-2 in Mino/FR cells was associated with the markedly increased sensitivity of the fludarabine-resistant MCL cells to Bcl-2-specific inhibitor ABT199 compared to fludarabine-sensitive cells. Our data thus demonstrate that a detailed molecular analysis of drug-resistant tumor cells can indeed open a way to personalized therapy of resistant malignancies.     

The de-ubiquitylating enzymes USP26 and USP37 regulate homologous recombination by counteracting RAP80

The faithful repair of DNA double-strand breaks (DSBs) is essential to safeguard genome stability. DSBs elicit a signaling cascade involving the E3 ubiquitin ligases RNF8/RNF168 and the ubiquitin-dependent assembly of the BRCA1-Abraxas-RAP80-MERIT40 complex. The association of BRCA1 with ubiquitin conjugates through RAP80 is known to be inhibitory to DSB repair by homologous recombination (HR). However, the precise regulation of this mechanism remains poorly understood. Through genetic screens we identified USP26 and USP37 as key de-ubiquitylating enzymes (DUBs) that limit the repressive impact of RNF8/RNF168 on HR. Both DUBs are recruited to DSBs where they actively remove RNF168-induced ubiquitin conjugates. Depletion of USP26 or USP37 disrupts the execution of HR and this effect is alleviated by the simultaneous depletion of RAP80. We demonstrate that USP26 and USP37 prevent excessive spreading of RAP80-BRCA1 from DSBs. On the other hand, we also found that USP26 and USP37 promote the efficient association of BRCA1 with PALB2. This suggests that these DUBs limit the ubiquitin-dependent sequestration of BRCA1 via the BRCA1-Abraxas-RAP80-MERIT40 complex, while promoting complex formation and cooperation of BRCA1 with PALB2-BRCA2-RAD51 during HR. These findings reveal a novel ubiquitin-dependent mechanism that regulates distinct BRCA1-containing complexes for efficient repair of DSBs by HR.     

Forage quality of leaf-fodder from the main broad-leaved woody species and its possible consequences for the Holocene development of forest vegetation in Central Europe

Leaf-hay was the principal winter feed of livestock from the Neolithic until the first archaeological records of scythes dated to the Iron Age (700–0 b.c.). Despite the use of meadow hay, leaf-fodder remained an important winter supplement until the present. Archaeological evidence lists Quercus, Tilia, Ulmus, Acer, Fraxinus and Corylus as woody species harvested for leaf-fodder, while Fagus, Populus or Carpinus were rarely used. The aim of our study was to test whether the use of listed woody species followed the pattern of their forage quality (syn. nutritive value). In late May 2012, we collected leaf biomass at four localities in the Czech Republic and determined concentrations of N, P, K, Ca, Mg, neutral- and acid-detergent fibre and lignin. Species with leaves of low forage quality were Carpinus betulus, Fagus sylvatica and Quercus robur, species with leaves of intermediate quality were Corylus avellana and Populus tremula and species with leaves of high quality were Ulmus glabra, Fraxinus excelsior, Tilia cordata and Acer platanoides. Selective browsing and harvesting of high quality species Acer, Fraxinus, Tilia and Ulmus thus probably supported their decline in the Bronze and Iron ages and supported the expansion of Carpinus and Fagus. Our results indicate that our ancestors’ practice of exploiting woody species as leaf-hay for winter fodder followed their nutritive value.