Tissue resident cells differentiate S. aureus from S. epidermidis via IL-1ß following barrier disruption in healthy human skin.

The Staphylococcus sp. are a dominant part of the human skin microbiome and present across the body. Staphylococcus epidermidis is a ubiquitous skin commensal, while S. aureus is thought to colonize at least 30% of the population. S. aureus are not only colonizers but a leading cause of skin and soft tissue infections and a critical healthcare concern. To understand how healthy human skin may differentiate commensal bacteria, such as S. epidermidis, from the potential pathogen methicillin-resistant S. aureus (MRSA), we use ex vivo human skin models that allow us to study this host-bacterial interaction in the most clinically relevant environment. Our work highlights the role of the outer stratum corneum as a protective physical barrier against invasion by colonizing Staphylococci. We show how the structural cells of the skin can internalize and respond to different Staphylococci with increasing sensitivity. In intact human skin, a discriminatory IL-1ß response was identified, while disruption of the protective stratum corneum triggered an increased and more diverse immune response. We identified and localized tissue resident Langerhans cells (LCs) as a potential source of IL-1ß and go on to show a dose-dependent response of MUTZ-LCs to S. aureus but not S. epidermidis. This suggests an important role of LCs in sensing and discriminating between bacteria in healthy human skin, particularly in intact skin and provides a detailed snapshot of how human skin differentiates between friend and potential foe. With the rise in antibiotic resistance, understanding the innate immune response of healthy skin may help us find ways to enhance or manipulate these natural defenses to prevent invasive infection.

Complexome profiling on the Chlamydomonas lpa2 mutant reveals insights into PSII biogenesis and new PSII associated proteins.

While the composition and function of the major thylakoid membrane complexes are well understood, comparatively little is known about their biogenesis. The goal of this work was to shed more light on the role of auxiliary factors in the biogenesis of photosystem II (PSII). Here we have identified the homolog of LOW PSII ACCUMULATION 2 (LPA2) in Chlamydomonas. A Chlamydomonas reinhardtii lpa2 mutant grew slower in low light, was hypersensitive to high light, and exhibited aberrant structures in thylakoid membrane stacks. Chlorophyll fluorescence (Fv/Fm) was reduced by 38%. Synthesis and stability of newly made PSII core subunits D1, D2, CP43, and CP47 were not impaired. However, complexome profiling revealed that in the mutant CP43 was reduced to ~23% and D1, D2, and CP47 to ~30% of wild type levels. Levels of PSI and the cytochrome b6f complex were unchanged, while levels of the ATP synthase were increased by ~29%. PSII supercomplexes, dimers, and monomers were reduced to ~7%, ~26%, and ~60% of wild type levels, while RC47 was increased ~6-fold and LHCII by ~27%. We propose that LPA2 catalyses a step during PSII assembly without which PSII monomers and further assemblies become unstable and prone to degradation. The LHCI antenna was more disconnected from PSI in the lpa2 mutant, presumably as an adaptive response to reduce excitation of PSI. From the co-migration profiles of 1734 membrane-associated proteins, we identified three novel putative PSII associated proteins with potential roles in regulating PSII complex dynamics, assembly, and chlorophyll breakdown.

Comparative study of hyperpure chlorine dioxide with two other irrigants regarding the viability of periodontal ligament stem cells.

OBJECTIVES: Periodontal ligament stem cells (PDLSCs) have an underlined significance as their high proliferative capacity and multipotent differentiation provide an important therapeutic potential. The integrity of these cells is frequently disturbed by the routinely used irrigative compounds applied as periodontal or endodontic disinfectants (e.g., hydrogen peroxide (H2O2) and chlorhexidine (CHX)). Our objectives were (i) to monitor the cytotoxic effect of a novel dental irrigative compound, chlorine dioxide (ClO2), compared to two traditional agents (H2O2, CHX) on PDLSCs and (ii) to test whether the aging factor of PDLSC cultures determines cellular responsiveness to the chemicals tested. METHODS: Impedimetry (concentration-response study), WST-1 assays (WST = water soluble tetrazolium salt), and morphology analysis were performed to measure changes in cell viability induced by the 3 disinfectants; immunocytochemistry of stem cell markers (STRO-1, CD90, and CD105) measured the induced mesenchymal characteristics. RESULTS: Cell viability experiments demonstrated that the application of ClO2 does not lead to a significant decrease in viability of PLDSCs in concentrations used to kill microbes. On the contrary, traditional irrigants, H2O2, and CHX are highly toxic on PDLSCs. Aging of PLDSC cultures (passages 3 vs. 7) has characteristic effects on their responsiveness to these agents as the increased expression of mesenchymal stem cell markers turns to decreased. CONCLUSIONS AND CLINICAL RELEVANCE: While the active ingredients of mouthwash (H2O2, CHX) applied in endodontic or periodontitis management have a serious toxic effect on PDLSCs, the novel hyperpure ClO2 is less toxic providing an environment favoring dental structure regenerations during disinfectant interventions.

The Chlamydomonas deg1c Mutant Accumulates Proteins Involved in High Light Acclimation.

Degradation of periplasmic proteins (Deg)/high temperature requirement A (HtrA) proteases are ATP-independent Ser endopeptidases that perform key aspects of protein quality control in all domains of life. Here, we characterized Chlamydomonas reinhardtii DEG1C, which together with DEG1A and DEG1B is orthologous to Arabidopsis (Arabidopsis thaliana) Deg1 in the thylakoid lumen. We show that DEG1C is localized to the stroma and the periphery of thylakoid membranes. Purified DEG1C exhibited high proteolytic activity against unfolded model substrates and its activity increased with temperature and pH. DEG1C forms monomers, trimers, and hexamers that are in dynamic equilibrium. DEG1C protein levels increased upon nitrogen, sulfur, and phosphorus starvation; under heat, oxidative, and high light stress; and when Sec-mediated protein translocation was impaired. DEG1C depletion was not associated with any obvious aberrant phenotypes under nonstress conditions, high light exposure, or heat stress. However, quantitative shotgun proteomics revealed differences in the abundance of 307 proteins between a deg1c knock-out mutant and the wild type under nonstress conditions. Among the 115 upregulated proteins are PSII biogenesis factors, FtsH proteases, and proteins normally involved in high light responses, including the carbon dioxide concentrating mechanism, photorespiration, antioxidant defense, and photoprotection. We propose that the lack of DEG1C activity leads to a physiological state of the cells resembling that induced by high light intensities and therefore triggers high light protection responses.

In vitro functional characterization of biosimilar therapeutic antibodies.

The key factor in successful development and marketing of biosimilar antibodies is a deep understanding of their critical quality attributes and the ability to control them. Comprehensive functional characterization is therefore at the heart of the process and is a crucial part of regulatory requirements. Establishment of a scientifically sound molecule-specific functional in vitro assay panel requires diligent planning and high flexibility in order to respond to both regulatory requirements and the ever-changing demands relevant to the different stages of the development and production process. Relevance of the chosen assays to the in vivo mechanism of action is of key importance to the stepwise evidence-based demonstration of biosimilarity. Use of a sound interdisciplinary approach and orthogonal state-of-the-art techniques is also unavoidable for gaining in-depth understanding of the biosimilar candidate. The aim of the present review is to give a snapshot on the methodic landscape as depicted by the available literature discussing the in vitro techniques used for the functional characterization of approved biosimilar therapeutic antibodies. Emerging hot topics of the field and relevant structure-function relationships are also highlighted.

OX40 stimulation and PD-L1 blockade synergistically augment HBV-specific CD4 T cells in patients with HBeAg-negative infection.

BACKGROUND & AIMS: Current antiviral therapies lack the potential to eliminate persistent hepatitis B virus (HBV) infection. HBV-specific T cells are crucial for HBV control and have recently been shown to be protective in patients following discontinuation of antiviral therapy. Thus, T cell-based approaches may greatly improve the therapeutic landscape of HBV infection. We aimed to augment HBV-specific CD4 T cells from chronically infected patients by targeting different immunological pathways. METHODS: Expression of various co-stimulatory and inhibitory receptors on HBV- and influenza-specific CD4 T cells was analyzed directly ex vivo by MHC class II-tetramers. Patients infected with HBV genotype D were screened for CD4 T cell responses by IFN-γ ELISpot and intracellular cytokine staining following stimulation with overlapping peptides (OLPs) spanning the HBV-polyprotein. Stimulation with recombinant IL-7, an agonistic OX40-antibody or blockade of PD-L1 was performed in antigen-specific in vitro cultures. Cytokine secretion and expression of transcription factors were analyzed by flow cytometry. Responses targeting influenza, Epstein-Barr virus and tetanus toxoid served as controls. RESULTS: Tetramer-staining revealed that the IL-7 receptor-alpha (CD127), OX40 and PD-1 constitute possible therapeutic targets as they were all strongly expressed on HBV-specific CD4 T cells ex vivo. The HBV-specific CD4 T cell responses identified by OLP screening targeted predominantly the HBV-polymerase and core proteins. Combined OX40 stimulation and PD-L1 blockade significantly augmented IFN-γ and IL-21 producing HBV-specific CD4 T cells in vitro, suggesting active T helper type 1 cell and follicular T helper cell programs. Indeed, transcription factors T-bet and Bcl6 were strongly expressed in cytokine-producing cells. CONCLUSIONS: Combined OX40 stimulation and PD-L1 blockade augmented secretion of the helper T cell signature cytokines IFN-γ and IL-21, suggesting that immunotherapeutic approaches can improve HBV-specific CD4 T cell responses. LAY SUMMARY: CD4 T cells are important in controlling viral infections but are impaired in the context of chronic hepatitis B virus (HBV) infection. Therapeutic approaches to cure chronic HBV infection are highly likely to require an immune-stimulatory component. This study demonstrates that HBV-specific CD4 T cells can be functionally augmented by combined stimulation of the co-stimulatory molecule OX40 and blockade of the inhibitory PD-1 pathway.

Can the unemployed be trained to care for the elderly? The effects of subsidized training in elderly care.

Demographic change has increased the need for elderly care. Training unemployed workers might be one way to increase the supply of elderly care nurses. This study analyzes the effectiveness of subsidized training for unemployed individuals in the elderly care professions in Germany over 11.5 years. We find that short further training and long retraining courses significantly increase workers' long-term employment. As approximately 25% to 50% of trained nurses have permanent jobs in the care sector, we estimate that approximately 5% of all employed nurses are formerly trained unemployed workers.

Infants' Processing of Prosodic Cues: Electrophysiological Evidence for Boundary Perception beyond Pause Detection.

Infants as young as six months are sensitive to prosodic phrase boundaries marked by three acoustic cues: pitch change, final lengthening, and pause. Behavioral studies suggest that a language-specific weighting of these cues develops during the first year of life; recent work on German revealed that eight-month-olds, unlike six-month-olds, are capable of perceiving a prosodic boundary on the basis of pitch change and final lengthening only. The present study uses Event-Related Potentials (ERPs) to investigate the neuro-cognitive development of prosodic cue perception in German-learning infants. In adults' ERPs, prosodic boundary perception is clearly reflected by the so-called Closure Positive Shift (CPS). To date, there is mixed evidence on whether an infant CPS exists that signals early prosodic cue perception, or whether the CPS emerges only later-the latter implying that infantile brain responses to prosodic boundaries reflect acoustic, low-level pause detection. We presented six- and eight-month-olds with stimuli containing either no boundary cues, only a pitch cue, or a combination of both pitch change and final lengthening. For both age groups, responses to the former two conditions did not differ, while brain responses to prosodic boundaries cued by pitch change and final lengthening showed a positivity that we interpret as a CPS-like infant ERP component. This hints at an early sensitivity to prosodic boundaries that cannot exclusively be based on pause detection. Instead, infants' brain responses indicate an early ability to exploit subtle, relational prosodic cues in speech perception-presumably even earlier than could be concluded from previous behavioral results.

Effect of SXWS/WSXWS peptides on chemotaxis and adhesion of the macrophage-like cell line J774.

WSXWS motif is a conserved amino acid sequence that is present in type I cytokine receptors. This motif that can be found both in the ligand binding chains and signal transducer molecule of the receptors with different amino acids at the position "X" plays a role in the receptor folding, ligand binding and signal transduction as well. Structural analysis proved that WSEWS motif of IL-6R is located in a highly accessible location in the protein. Structural properties and chemotaxis of a tetrapeptide library with SXWS sequence, where X was the 19 proteinogenic amino acids except cystein were systematically studied earlier. It has been proved that C-terminal amidation and the identity of amino acid X had a pronounced influence on the chemotactic properties but less of the structure of the peptides. Here, we present our findings on the effect of a tetrapeptide and a pentapeptide library with the sequence of SXWS and WSXWS on the chemotaxis and adhesion of J774 murine macrophage cell line. We studied the effect of the presence/absence of N-terminal tryptophan and the different amino acids at the X position on these physiological responses. Results indicated that amino acid X had a marked influence on chemotaxis, adhesion as well as on proliferation induced by (W)SXWS peptides. Elongation of SXWS sequence with a tryptophan at the N terminus also altered pronouncedly all the physiological responses of the cells studied. A good correlation could be observed between the chemotaxis and the proliferation and physicochemical parameters of the amino acid X.

Controlling intracavity dual-comb soliton motion in a single-fiber laser.

Ultrafast science builds on dynamic compositions of precisely timed light pulses, and evolving groups of pulses are observed in almost every mode-locked laser. However, the underlying physics has rarely been controlled or used until now. Here, we demonstrate a general approach to control soliton motion inside a dual-comb laser and the programmable synthesis of ultrashort pulse patterns. Introducing single-pulse modulation inside an Er:fiber laser, we rapidly shift the timing between two temporally separated soliton combs. Their superposition outside the cavity yields ultrashort soliton sequences. On the basis of real-time spectral interferometry, we observe the deterministic switching of intersoliton separation arising from the interplay of attracting and repulsing forces via ultrafast nonlinearity and laser gain dynamics. Harnessing these insights, we demonstrate the high-speed all-optical synthesis of nano- to picosecond pump-probe delays and programmable free-form soliton trajectories. This concept may pave the way to a new class of all-optical delay generators for ultrafast measurements at unprecedented high tuning, cycling, and acquisition speeds.

Chemotaxis induced by SXWS tetrapeptides in Tetrahymena--overlapping chemotactic effects of SXWS sequences and their identical amino acids.

The chemotactic potential of SXWS peptides and the components of the extracellular domain of cytokine receptors were investigated in Tetrahymena as a functional index of substitution with different amino acids in the position 'X' of the tetrapeptide. Data obtained demonstrate that position X plays a special determining role in the ligand, SEWS and STWS possess extremely strong chemoattractant ability, and aromatic amino acids result in chemorepellent ligands. Diverse effects of structurally related molecules, for example, SNWS-SDWS, demonstrate a highly sensitive discrimination potential in the applied model system. Physicochemical characteristics (hydropathy, residue size, and solvent-exposed area) of the amino acids were correlated with the chemotactic activity. Data obtained by computer-assisted conformation analysis of SXWS peptides and the highly overlapping chemotactic effects of the investigated SXWS peptides as well as the presence of the amino acids in the 'X' position indicate that member 'X' of the SXWS sequence performs a special role in interactions with the chemotaxis receptors in the membrane.

Ultrafast imaging of terahertz electric waveforms using quantum dots.

Microscopic electric fields govern the majority of elementary excitations in condensed matter and drive electronics at frequencies approaching the Terahertz (THz) regime. However, only few imaging schemes are able to resolve sub-wavelength fields in the THz range, such as scanning-probe techniques, electro-optic sampling, and ultrafast electron microscopy. Still, intrinsic constraints on sample geometry, acquisition speed and field strength limit their applicability. Here, we harness the quantum-confined Stark-effect to encode ultrafast electric near-fields into colloidal quantum dot luminescence. Our approach, termed Quantum-probe Field Microscopy (QFIM), combines far-field imaging of visible photons with phase-resolved sampling of electric waveforms. By capturing ultrafast movies, we spatio-temporally resolve a Terahertz resonance inside a bowtie antenna and unveil the propagation of a Terahertz waveguide excitation deeply in the sub-wavelength regime. The demonstrated QFIM approach is compatible with strong-field excitation and sub-micrometer resolution-introducing a direct route towards ultrafast field imaging of complex nanodevices in-operando.

A Photoconvertible Reporter System for Bacterial Metabolic Activity Reveals That Staphylococcus aureus Enters a Dormant-Like State to Persist within Macrophages.

Staphylococcus aureus is a leading cause of difficult-to-treat infections. The capacity of S. aureus to survive and persist within phagocytic cells is an important factor contributing to therapy failures and infection recurrence. Therefore, interfering with S. aureus intracellular persistence is key to treatment success. In this study, we used a S. aureus strain carrying the reporter mKikumeGR that enables the monitoring of the metabolic status of intracellular bacteria to achieve a better understanding of the molecular mechanisms facilitating S. aureus survival and persistence within macrophages. We found that shortly after bacteria internalization, a large fraction of macrophages harbored mainly S. aureus with high metabolic activity. This population decreased gradually over time with the concomitant increase of a macrophage subpopulation harboring S. aureus with low metabolic activity, which prevailed at later times. A dual RNA-seq analysis performed in each macrophage subpopulation showed that the host transcriptional response was similar between both subpopulations. However, intracellular S. aureus exhibited disparate gene expression profiles depending on its metabolic state. Whereas S. aureus with high metabolic activity exhibited a greater expression of genes involved in protein synthesis and proliferation, bacteria with low metabolic activity displayed a higher expression of oxidative stress response-related genes, silenced genes involved in energy-consuming processes, and exhibited a dormant-like state. Consequently, we propose that reducing metabolic activity and entering into a dormant-like state constitute a survival strategy used by S. aureus to overcome the adverse environment encountered within macrophages and to persist in the intracellular niche. IMPORTANCE The capacity of Staphylococcus aureus to survive and persist within phagocytic cells has been associated with antibiotic treatment failure and recurrent infections. Here, we investigated the molecular mechanisms leading to S. aureus persistence within macrophages using a reporter system that enables to distinguish between intracellular bacteria with high and low metabolic activity in combinstion with a dual RNA-seq approach. We found that with the progression of infection, intracellular S. aureus transitions from a high metabolic state to a low metabolic dormant-like state by turning off major energy-consuming processes while remaining viable. This process seems to be driven by the level of stress encountered in the intracellular niche. Our study indicates that effective therapies by which to treat S. aureus infections should be able to target not only high metabolic bacteria but also intracellular dormant-like S. aureus.

Chemotactic effect of odorants and tastants on the ciliate Tetrahymena pyriformis.

Naturally occurring aroma compounds are able to elicit physiological and migratory responses such as chemotaxis even at nano to femtomolar concentrations in organisms at different levels of phylogeny. Despite the amazing chemical variety of these substances the apparatus by which they can be detected i.e. the chemosensory receptors and the signaling pathways seem to be rather uniform and evolutionary well-conserved. The intracellular signaling process is supposed to be mediated by either cAMP or inositol 1,4,5-trisphosphate. The present work aimed to investigate the chemotactic behavior of 11 odorants that occur naturally in foods and are also used by the industry as additives, on the eukaryotic ciliate Tetrahymena pyriformis. Intracellular signaling pathways that might be activated by these compounds were also investigated. Activation of the phospholipase C (PLC) was measured by FACS and the stimulation of inositol-1,4,5-trisphosphate 3-kinases (IP3K) was measured using two specific inhibitors, wortmannin and LY294002. The strongest chemoattractant character was observed for isoamyl acetate (10(⁻6) M), propyl isobutyrate (10(⁻8) M), isobutyl propionate (10(⁻6) M). The strongest repellent action was exerted by benzyl acetate (10(⁻8) M), furfuryl thioacetate (10(⁻12) M). Our results suggest that Tetrahymena responds in a very sensitive way to slight changes in the molecular structure. According to our study, tracer amounts of solvents do not contribute significantly to the chemotactic profile of the respective odorants. No significant activation of PLC or PI3K could be observed following stimulation with attractant odorants which implies that some other pathways may be involved, hence further investigation is needed.

Peripheral neuropathy due to neuroborreliosis: Insensitivity for CXCL13 as early diagnostic marker.

The case of a 69-year-old woman with peripheral neuropathy caused by Lyme neuroborreliosis (LNB) in an endemic region in Eastern Austria is reported. The patient had noticed transient numbness of her left leg. On initial examination, she had patchy sensory disturbances of the left lower leg, but ancillary examinations of nerve conduction and cerebrospinal fluid (CSF), including the B-cell chemokine CXCL13, were normal. A re-tap performed 54 days later, following clinical progression with foot drop, widespread lower leg paresthesia, and pain, revealed an increased cell count, autochthonous IgM production, synthesis of Borrelia-specific IgM, and elevated CXCL13. Neurophysiological examinations disclosed an incomplete conduction block, mixed axonal and demyelinating sensorimotor neuropathy, and subacute neurogenic damage of muscles innervated by the peroneal nerve. This case study presents rare evidence of very early diagnostic findings in peripheral neuropathy caused by LNB. These are characterized by insensitivity of CXCL13 in CSF to aid earlier diagnosis and the development of an intrathecal immune response against Borrelia at a later stage. These findings reinforce the need for a re-tap to confirm the diagnosis and facilitate appropriate treatment in this rare manifestation of LNB.

Immunological cure of HBV infection.

Chronic hepatitis B virus (HBV) infection is a major global health burden and cure is rarely achieved by current antiviral therapies. Therefore, there is an urgent need for new therapeutic options. Immune modulation with the goal to restore dysfunctional HBV-specific immunity is an interesting target for new therapeutic strategies. Based on the current evidence on immunology in resolving and persistent HBV infection, we will review the growing field of immunotherapeutic approaches for treatment of chronic HBV infection. We will also discuss the challenge of a heterogeneous patient population and personalized treatment as a possible key factor of success.