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microRNAs (miRNAs) are important posttranscriptional regulators during hematopoietic lineage commitment and lymphocyte development. Mature miRNAs are processed from primary miRNA transcripts in two steps by the microprocessor complex, consisting of Drosha and its partner DiGeorge Critical Region 8 (DGCR8), and the RNAse III enzyme, Dicer. Conditional ablations of Drosha and Dicer have established the importance of both RNAses in B- and T-cell development. Here, we show that a cre-mediated B-cell specific deletion of DGCR8 in mice results in a nearly complete maturation block at the transition from the pro-B to the pre-B cell stage, and a failure to upregulate Ig μ heavy chain expression in pro-B cells. Furthermore, we found that the death of freshly isolated DGCR8-deficient pro-B cells could be partially prevented by enforced Bcl2 expression. We conclude from these findings that the microprocessor component DGCR8 is essential for survival and differentiation of early B-cell progenitors., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Chirped pulse amplification in optical lasers is a revolutionary technique, which allows the generation of extremely powerful femtosecond pulses in the infrared and visible spectral ranges. Such pulses are nowadays an indispensable tool for a myriad of applications, both in fundamental and applied research. In recent years, a strong need emerged for light sources producing ultra-short and intense laser-like X-ray pulses, to be used for experiments in a variety of disciplines, ranging from physics and chemistry to biology and material sciences. This demand was satisfied by the advent of short-wavelength free-electron lasers. However, for any given free-electron laser setup, a limit presently exists in the generation of ultra-short pulses carrying substantial energy. Here we present the experimental implementation of chirped pulse amplification on a seeded free-electron laser in the extreme-ultraviolet, paving the way to the generation of fully coherent sub-femtosecond gigawatt pulses in the water window (2.3-4.4 nm).

Population-based studies on Staphylococcus aureus nasal colonization are scarce. We examined the prevalence, resistance, and molecular diversity of S. aureus in the general population in Northeast Germany. Nasal swabs were obtained from 3,891 adults in the large-scale population-based Study of Health in Pomerania (SHIP-TREND). Isolates were characterized using spa genotyping, as well as antibiotic resistance and virulence gene profiling. We observed an S. aureus prevalence of 27.2%. Nasal S. aureus carriage was associated with male sex and inversely correlated with age. Methicillin-resistant S. aureus (MRSA) accounted for 0.95% of the colonizing S. aureus strains. MRSA carriage was associated with frequent visits to hospitals, nursing homes, or retirement homes within the previous 24 months. All MRSA strains were resistant to multiple antibiotics. Most MRSA isolates belonged to the pandemic European hospital-acquired MRSA sequence type 22 (HA-MRSA-ST22) lineage. We also detected one livestock-associated MRSA ST398 (LA-MRSA-ST398) isolate, as well as six livestock-associated methicillin-susceptible S. aureus (LA-MSSA) isolates (clonal complex 1 [CC1], CC97, and CC398). spa typing revealed a diverse but also highly clonal S. aureus population structure. We identified a total of 357 spa types, which were grouped into 30 CCs or sequence types. The major seven CCs (CC30, CC45, CC15, CC8, CC7, CC22, and CC25) included 75% of all isolates. Virulence gene patterns were strongly linked to the clonal background. In conclusion, MSSA and MRSA prevalences and the molecular diversity of S. aureus in Northeast Germany are consistent with those of other European countries. The detection of HA-MRSA and LA-MRSA within the general population indicates possible transmission from hospitals and livestock, respectively, and should be closely monitored., (Copyright © 2016 Holtfreter et al.)

Cytochrome P450 monooxygenases (P450) are heme-containing enzymes that oxidize a broad range of substrates in the presence of molecular oxygen and NAD(P)H. For their activity, most P450s rely on one or two redox proteins responsible for the transfer of electrons from the cofactor NAD(P)H to the heme. One of the challenges when using P450s in vitro, especially when non-physiological redox proteins are applied, is the inefficient transfer of electrons between the individual proteins resulting in non-productive consumption of NAD(P)H - referred to as uncoupling. Herein, we describe the improvement of the coupling efficiency between a P450 and its redox partner - diflavin reductase - by fusing both enzymes individually to the hydrophobin HFBI - a small self-assembling protein of the fungus Trichoderma reesei. The separated monooxygenase (BMO) and reductase (BMR) domains of P450 BM3 from Bacillus megaterium were chosen as a P450-reductase model system and individually fused to HFBI. The fusion proteins could be expressed in soluble form in Escherichia coli. When HFBI-fused BMO and BMR were mixed in vitro, substantially higher coupling efficiencies were measured as compared with the respective non-fused enzymes. Consequently, myristic acid conversion increased up to 20-fold (after 6 h) and 5-fold (after 24 h). Size exclusion chromatography demonstrated that in vitro the hydrophobin-fused enzymes build multimeric protein assemblies. Thus, the higher activity is hypothesized to be due to HFBI-mediated self-assembly arranging BMO and BMR in close spatial proximity in aqueous solution.

We experimentally demonstrate that the spectral sensitivity of a Mach-Zehnder (MZ) interferometer can be enhanced through structural slow light. We observe a 20-fold resolution enhancement by placing a dispersion-engineered, slow-light, photonic-crystal waveguide in one arm of a fiber-based MZ interferometer. The spectral sensitivity of the interferometer increases roughly linearly with the group index, and we have quantified the resolution in terms of the spectral density of interference fringes. These results show promise for the use of slow-light methods for developing novel tools for optical metrology and, specifically, for compact high-resolution spectrometers.

Unlabelled: Phenotypic variability among bacteria depends on gene expression in response to different environments, and it also reflects differences in genomic structure. In this study, we analyzed transcriptome sequencing (RNA-seq) profiles of 151 Pseudomonas aeruginosa clinical isolates under standard laboratory conditions and of one P. aeruginosa type strain under 14 different environmental conditions. Our approach allowed dissection of the impact of the genetic background versus environmental cues on P. aeruginosa gene expression profiles and revealed that phenotypic variation was larger in response to changing environments than between genomically different isolates. We demonstrate that mutations within the global regulator LasR affect more than one trait (pleiotropy) and that the interaction between mutations (epistasis) shapes the P. aeruginosa phenotypic plasticity landscape. Because of pleiotropic and epistatic effects, average genotype and phenotype measures appeared to be uncorrelated in P. aeruginosa., Importance: This work links experimental data of unprecedented complexity with evolution theory and delineates the transcriptional landscape of the opportunistic pathogen Pseudomonas aeruginosa. We found that gene expression profiles are most strongly influenced by environmental cues, while at the same time the transcriptional profiles were also shaped considerably by genetic variation within global regulators. The comprehensive set of transcriptomic and genomic data of more than 150 clinical P. aeruginosa isolates will be made publically accessible to all researchers via a dedicated web interface. Both Pseudomonas specialists interested in expression and regulation of specific genes and researchers from other fields with more global interest in the phenotypic and genotypic variation of this important model species can access all information on various levels of detail., (Copyright © 2015 Dötsch et al.)

We designed, constructed, and evaluated a mobile medical care vehicle called "Rollende Arztpraxis" (rolling medical practice, RMP) that delivers the full medical care of a general practitioner to increase medical care supply in rural areas. Six communities have been identified, where the RMP has been visited 501 times in 14 months. Two different schedules of stops and treatment times have been tested. We show that the RMP treated mainly elderly and multimorbid patients. An accompanying study showed high acceptance and satisfaction of treated patients and treating doctors. An economic evaluation of three different utilization models with three treatment modes each showed no financial sustainability. We show that ambulatory care in rural areas can be complemented by a mobile care unit, if legal and financial barriers can be overcome.

Bacterial adaptation to given environmental conditions is largely achieved by complex gene regulatory processes. To address the question how and to what extend single transcriptional regulators modulate gene expression, chromatin immunoprecipitation (ChIP) coupled to DNA microarrays (ChIP-chip) or to next-generation sequencing (ChIP-seq) is one of the preferred methods. Both ChIP-chip and ChIP-seq can generate genome-wide maps of protein-DNA interactions and thus identify primary regulons of transcription factors. In combination with transcriptome analyses, the obtained data can be used to compile complex regulatory networks which in terms will advance our understanding of bacterial adaptation processes to specific environmental conditions.

The role of the mRNA-binding protein human antigen R (HuR) in stabilization and translation of AU-rich elements (ARE) containing mRNAs is well established. However, the trafficking of HuR and bound mRNA cargo, which comprises a fundamental requirement for the aforementioned HuR functions is only poorly understood. By administering different cytoskeletal inhibitors, we found that the protein kinase Cδ (PKCδ)-triggered accumulation of cytoplasmic HuR by Angiotensin II (AngII) is an actin-myosin driven process functionally relevant for stabilization of ARE-bearing mRNAs. Furthermore, we show that the AngII-induced recruitment of HuR and its bound mRNA from ribonucleoprotein particles to free and cytoskeleton bound polysomes strongly depended on an intact actomyosin cytoskeleton. In addition, HuR allocation to free and cytoskeletal bound polysomes is highly sensitive toward RNase and PPtase and structurally depends on serine 318 (S318) located within the C-terminal RNA recognition motif (RRM3). Conversely, the trafficking of the phosphomimetic HuRS318D, mimicking HuR phosphorylation at S318 by the PKCδ remained PPtase resistant. Co-immunoprecipitation experiments with truncated HuR proteins revealed that the stimulus-induced association of HuR with myosin IIA is strictly RNA dependent and mediated via the RRM3. Our data implicate a microfilament dependent transport of HuR, which is relevant for stimulus-induced targeting of ARE-bearing mRNAs from translational inactive ribonucleoprotein particles to polysomes.

Most bacteria control oxidative stress through the H(2)O(2)-responsive transactivator OxyR, a member of the LTTR family (LysR Type Transcriptional Regulators), which activates the expression of defensive genes such as those encoding catalases, alkyl hydroperoxide reductases and superoxide dismutases. In the human opportunistic pathogen Pseudomonas aeruginosa, OxyR positively regulates expression of the oxidative stress response genes katA, katB, ahpB and ahpCF. To identify additional targets of OxyR in P. aeruginosa PAO1, we performed chromatin immunoprecipitation in combination with whole genome tiling array analyses (ChIP-chip). We detected 56 genes including all the previously identified defensive genes and a battery of novel direct targets of OxyR. Electrophoretic mobility shift assays (EMSAs) for selected newly identified targets indicated that ∼70% of those were bound by purified oxidized OxyR and their regulation was confirmed by quantitative real-time polymerase chain reaction. Furthermore, a thioredoxin system was identified to enzymatically reduce OxyR under oxidative stress. Functional classification analysis showed that OxyR controls a core regulon of oxidative stress defensive genes, and other genes involved in regulation of iron homeostasis (pvdS), quorum-sensing (rsaL), protein synthesis (rpsL) and oxidative phosphorylation (cyoA and snr1). Collectively, our results indicate that OxyR is involved in oxidative stress defense and regulates other aspects of cellular metabolism as well.

Yersinia outer protein P (YopP) induces cell death in macrophages and dendritic cells (DC). In DC this YopP-dependent cell death coincides with the inhibition of nuclear factor-kappa B (NF-kappaB) activation. However, as shown by measurement of propidium iodide uptake via disrupted cellular membranes, the preincubation of DC with several NF-kappaB inhibitors prior to infection with Yersinia did not restore the death-inducing capacity of a YopP-deficient Yersinia mutant. These results suggest that in contrast to macrophages, in DC the YopP-dependent inhibition of NF-kappaB activation is not causative for the induction of cell death. Instead, in DC, the inhibition of mitogen-activated protein kinases (MAPKs), in particular, p38 and c-Jun N-terminal kinase, prior to infection with a YopP-deficient Yersinia mutant substituted the death-inducing capacity of the Yersinia wild-type strain, indicating that the YopP-dependent inhibition of MAPKs mediates Yersinia-induced DC death. The differences between DC and macrophages in the mechanisms of cell death induction by YopP presented herein might be crucial for the function of these antigen-presenting cells.

Yersinia outer protein P (YopP) is injected by Y. enterocolitica into host cells thereby inducing apoptotic and necrosis-like cell death in dendritic cells (DC). Here we show the pathways involved in DC death caused by the catalytic activity of YopP. Infection with Yersinia enterocolitica, translocating catalytically active YopP into DC, triggered procaspase-8 cleavage and c-FLIPL degradation. YopP-dependent caspase-8 activation was, however, not mediated by tumor necrosis factor (TNF) receptor family members since the expression of both CD95/Fas/APO-1 and TRAIL-R2 on DC was low, and DC were resistant to apoptosis induced by agonistic anti-CD95 antibodies or TNF-related apoptosis-inducing ligand (TRAIL). Moreover, DC from TNF-Rp55-/- mice were not protected against YopP-induced cell death demonstrating that TNF-R1 is also not involved in this process. Activation of caspase-8 was further investigated by coimmunoprecitation of FADD from Yersinia-infected DC. We found that both cleaved caspase-8 and receptor interacting protein 1 (RIP1) were associated with the Fas-associated death domain (FADD) indicating the formation of an atypical death-inducing signaling complex (DISC). Furthermore, degradation of RIP mediated by the Hsp90 inhibitor geldanamycin significantly impaired YopP-induced cell death. Altogether our findings indicate that Yersinia-induced DC death is independent of death domain containing receptors, but mediated by RIP and caspase-8 at the level of DISC.