Toll-like receptor (TLR) signaling pathways need to be tightly controlled to avoid excessive inflammation and unwanted damage to the host. Myeloid differentiation primary response gene 88 (MyD88) is a critical adaptor of TLR signaling. Here, we identified the speckle-type POZ protein (SPOP) as a MyD88-associated protein. SPOP was recruited to MyD88 following TLR4 activation. TLR4 activation also caused the translocation of SPOP from the nucleus to the cytoplasm. SPOP depletion promoted the aggregation of MyD88 and recruitment of the downstream signaling kinases IRAK4, IRAK1 and IRAK2. Consistently, overexpression of SPOP inhibited the TLR4-mediated activation of NF-κB and production of inflammatory cytokines, whereas SPOP depletion had the opposite effects. Furthermore, knockdown of SPOP increased MyD88 aggregation and inflammatory cytokine production upon TLR2, TLR7 and TLR9 activation. Our findings reveal a mechanism by which MyD88 is regulated and highlight a role for SPOP in limiting inflammatory responses.

Induction of broadly neutralizing monoclonal antibodies (bNAbs) that bind to the viral envelope glycoproteins is a major goal of hepatitis C virus (HCV) vaccine research. The study of bNAbs arising in natural infection is essential in this endeavor. We generated a human antibody, 8D6, recognizing the E2 protein of HCV isolated from a chronic hepatitis C patient. This antibody shows broadly neutralizing activity, which covers a pan-genotypic panel of cell culture-derived HCV virions (HCVcc). Functional and epitope analyses demonstrated that 8D6 can block the interaction between E2 and CD81 by targeting a highly conserved epitope on E2. We describe how the 8D6 lineage evolved via somatic hypermutation to achieve broad neutralization. We found that the V(D)J recombination-generated junctional and somatic hypermutation-induced disulfide bridge (C-C) motif in the CDRH3 is critical for the broad neutralization and binding activity of 8D6. This motif is conserved among a series of broadly neutralizing HCV antibodies, indicating a common binding model. Next, the 8D6 inferred germline (iGL) was reconstructed and tested for its binding affinity and neutralization activity. Interestingly, 8D6 iGL-mediated relatively strong inhibition of the 1b genotype PR79L9 strain, suggesting that PR79L9 may serve as a potential natural viral strain that provides E2 sequences that induce bNAbs. Overall, our detailed epitope mapping and genetic studies of the HCV E2-specific mAb 8D6 have allowed for further refinement of antigenic sites on E2 and reveal a new mechanism to generate a functional CDRH3, while its iGL can serve as a probe to identify potential HCV vaccine strains.

Beyond classical roles in thrombosis and haemostasis, it becomes increasingly clear that platelets contribute as key players to inflammatory processes. The involvement of platelets in these processes is often mediated through a variety of platelet-derived chemokines which are released upon activation and act as paracrine and autocrine factors. In this study, we investigate CXCL14, a newly described platelet chemokine and its role in thrombus formation as well as monocyte and platelet migration. In addition, we examine the chemokine receptor CXCR4 as a possible receptor for CXCL14 on platelets. Furthermore, with the use of artificially generated platelets derived from induced pluripotent stem cells (iPSC), we investigate the importance of CXCR4 for CXCL14-mediated platelet functions.

Nilotinib is used for treating patients with imatinib-sensitive or -resistant chronic myeloid leukemia (CML); however, nilotinib-resistant cases have been observed in recent years. In addition, a considerable number of patients receiving nilotinib developed diabetes. Metformin is a front-line drug for the treatment of type 2 diabetes, and several studies have shown that diabetes patients treated with metformin have reduced incidence of cancer. This study aimed to define the effect of metformin on CML cells to determine whether metformin overcomes nilotinib resistance, and to identify novel targets for the treatment of nilotinib resistance.

Spreading depolarization (SD) and seizures are pathophysiological events associated with cerebral ischemia. Here, we investigated their role for injury progression in the cerebral cortex. Cerebral ischemia was induced in anesthetized male Wistar rats using the photothrombosis (PT) stroke model. SD and spontaneous neuronal activity were recorded in the presence of either urethane or ketamine/xylazine anesthesia. Blood-brain barrier (BBB) permeability, cerebral perfusion, and cellular damage were assessed through a cranial window and repeated intravenous injection of fluorescein sodium salt and propidium iodide until 4 h after PT. Neuronal injury and early lesion volume were quantified by stereological cell counting and manual and automated assessment of ex vivo T2-weighted magnetic resonance imaging. Onset SDs originated at the thrombotic core and invaded neighboring cortex, whereas delayed SDs often showed opposite propagation patterns. Seizure induction by 4-aminopyridine caused no increase in lesion volume or neuronal injury in urethane-anesthetized animals. Ketamine/xylazine anesthesia was associated with a lower number of onset SDs, reduced lesion volume, and neuronal injury despite a longer duration of seizures. BBB permeability increase inversely correlated with the number of SDs at 3 and 4 h after PT. Our results provide further evidence that ketamine may counteract the early progression of ischemic injury.

Next-generation sequencing (NGS) is used to investigate the presence of somatic mutations. The utility of incorporating routine sequencing to guide diagnosis and therapeutic decisions remains unclear. We report the findings of an observational, multicenter study that aimed to assess the impact of somatic mutation testing by NGS in a reallife setting of chronic myeloid malignancies. A total of 177 patients were enrolled, partitioned into two overlapping groups. In group A (n=94), the indication was to search for clonal hematopoiesis, in a context of suspected myelodysplastic syndrome or myeloproliferative neoplasia. In group B (n=95), the theranostic impact of somatic mutations was studied. A panel of 34 genes was used on DNA extracted from blood or bone marrow samples. Within group A, the detection of clonal hematopoiesis supported the diagnosis of chronic myeloid malignancies for 31 patients while the absence of clonal hematopoiesis ruled out the suspected diagnosis in 47 patients. Within group B, NGS identified prognostically relevant somatic mutations in 32 patients, which had a therapeutic impact in 18 cases. By determining the presence or absence of somatic mutations, the application of NGS in daily practice was found to be useful for an integrated final diagnosis in 83% of the patients. Moreover, the search for somatic mutations had a prognostic impact that led to treatment modification in 19% of the cases. This study outlines the fact that adequate implementation of new investigations may have a significant positive medico-economic impact by enabling appropriate management of patients.

Transcranial direct current stimulation (tDCS) over prefrontal cortex (PFC) regions is currently proposed as therapeutic intervention for major depression and other psychiatric disorders. The in-depth mechanistic understanding of this bipolar and non-focal stimulation technique is still incomplete. In a pilot study, we investigated the effects of bifrontal stimulation on brain metabolite levels and resting state connectivity under the cathode using multiparametric MRI techniques and computational tDCS modeling. Within a double-blind cross-over design, 20 subjects (12 women, 23.7 ± 2 years) were randomized to active tDCS with standard bifrontal montage with the anode over the left dorsolateral prefrontal cortex (DLPFC) and the cathode over the right DLPFC. Magnetic resonance spectroscopy (MRS) was acquired before, during, and after prefrontal tDCS to quantify glutamate (Glu), Glu + glutamine (Glx) and gamma aminobutyric acid (GABA) concentration in these areas. Resting-state functional connectivity MRI (rsfcMRI) was acquired before and after the stimulation. The individual distribution of tDCS induced electric fields (efields) within the MRS voxel was computationally modelled using SimNIBS 2.0. There were no significant changes of Glu, Glx and GABA levels across conditions but marked differences in the course of Glu levels between female and male participants were observed. Further investigation yielded a significantly stronger Glu reduction after active compared to sham stimulation in female participants, but not in male participants. For rsfcMRI neither significant changes nor correlations with MRS data were observed. Exploratory analyses of the effect of efield intensity distribution on Glu changes showed distinct effects in different efield groups. Our findings are limited by the small sample size, but correspond to previously published results of cathodal tDCS. Future studies should address gender and efield intensity as moderators of tDCS induced effects.

Generating publication ready plots to display multiple genomic tracks can pose a serious challenge. Making desirable and accurate figures requires considerable effort. This is usually done by hand or using a vector graphic software.

In single-molecule localization microscopy (SMLM), the use of engineered point spread functions (PSFs) provides access to three-dimensional localization information. The conventional approach of fitting PSFs with a single 2-dimensional Gaussian profile, however, often falls short in analyzing complex PSFs created by placing phase masks, deformable mirrors or spatial light modulators in the optical detection pathway. Here, we describe the integration of PSF modalities known as double-helix, saddle-point or tetra-pod into the phasor-based SMLM (pSMLM) framework enabling fast CPU based localization of single-molecule emitters with sub-pixel accuracy in three dimensions. For the double-helix PSF, pSMLM identifies the two individual lobes and uses their relative rotation for obtaining z-resolved localizations. For the analysis of saddle-point or tetra-pod PSFs, we present a novel phasor-based deconvolution approach entitled circular-tangent pSMLM. Saddle-point PSFs were experimentally realized by placing a deformable mirror in the Fourier plane and modulating the incoming wavefront with specific Zernike modes. Our pSMLM software package delivers similar precision and recall rates to the best-in-class software package (SMAP) at signal-to-noise ratios typical for organic fluorophores and achieves localization rates of up to 15 kHz (double-helix) and 250 kHz (saddle-point/tetra-pod) on a standard CPU. We further integrated pSMLM into an existing software package (SMALL-LABS) suitable for single-particle imaging and tracking in environments with obscuring backgrounds. Taken together, we provide a powerful hardware and software environment for advanced single-molecule studies.

Gluten challenge studies are instrumental in understanding the pathophysiology of celiac disease. Our aims in this study were to reveal early gluten-induced transcriptomic changes in duodenal biopsies and to find tools for clinics.

Patients with heart failure have comparable illness burden and palliative care needs to those with cancer. However, few of them are offered timely palliative care. One main barrier is the difficulty in identifying those who require palliative care. Several palliative care needs-assessment/measurement tools were used to help identify these patients and assess/measure their needs, but it is not known which one is the most appropriate for this population. This review aimed to identify the most appropriate palliative care needs-assessment/measurement tools for patients with heart failure. Cochrane Library, MEDLINE Complete, AMED, PsycINFO, CINAHL Complete, EMBASE, EThOS, websites of the identified tools, and references and citations of the included studies were searched from inception to 25 June 2020. Studies were included if they evaluated palliative care needs-assessment/measurement tools for heart failure populations in terms of development, psychometrics, or palliative care patient/needs identification. Twenty-seven papers were included regarding nineteen studies, most of which were quantitative and observational. Six tools were identified and compared according to their content and context of use, development, psychometrics, and clinical applications in identifying patients with palliative care needs. Despite limited evidence, the Needs Assessment Tool: Progressive Disease - Heart Failure (NAT:PD-HF) is the most appropriate palliative care needs-assessment tool for use in heart failure populations. It covers most of the patient needs and has the best psychometric properties and evidence of identification ability and appropriateness. Psychometric testing of the tools in patients with heart failure and evaluating the tools to identify those with palliative care needs require more investigation.

To assess visceral (VAT), subcutaneous (SAT), and total adipose tissue (TAT) estimates at abdominopelvic CT in COVID-19 patients with different severity, and analyze Body Mass Index (BMI) and CT estimates of fat content in patients requiring hospitalization.

Systematic review.

The COVID-19 infection adversely affects the cardiovascular system. Transthoracic echocardiography has demonstrated diagnostic, prognostic and therapeutic utility. We report biventricular myocardial strain in COVID-19. Methods: Biventricular strain measurements were performed for 12 patients. Patients who were discharged were compared with those who needed intubation and/or died. Results: Seven patients were discharged and five died or needed intubation. Right ventricular strain parameters were decreased in patients with poor outcomes compared with those discharged. Left ventricular strain was decreased in both groups but was not statistically significant. Conclusion: Right ventricular strain was decreased in patients with poor outcomes and left ventricular strain was decreased regardless of outcome. Right ventricular strain measurements may be important for risk stratification and prognosis. Further studies are needed to confirm these findings.

To investigate knowledge levels concerning COVID-19 in elderly patients with T2DM.

Pulse pressure variation (PPV) and cardiac output (CO) can guide perioperative fluid management. Capstesia (Galenic App, Vitoria-Gasteiz, Spain) is a mobile application for snapshot pulse wave analysis (PWAsnap) and estimates PPV and CO using pulse wave analysis of a snapshot of the arterial blood pressure waveform displayed on any patient monitor. We evaluated the PPV and CO measurement performance of PWAsnap in adults having major abdominal surgery. In a prospective study, we simultaneously measured PPV and CO using PWAsnap installed on a tablet computer (PPVPWAsnap, COPWAsnap) and using invasive internally calibrated pulse wave analysis (ProAQT; Pulsion Medical Systems, Feldkirchen, Germany; PPVProAQT, COProAQT). We determined the diagnostic accuracy of PPVPWAsnap in comparison to PPVProAQT according to three predefined PPV categories and by computing Cohen's kappa coefficient. We compared COProAQT and COPWAsnap using Bland-Altman analysis, the percentage error, and four quadrant plot/concordance rate analysis to determine trending ability. We analyzed 190 paired PPV and CO measurements from 38 patients. The overall diagnostic agreement between PPVPWAsnap and PPVProAQT across the three predefined PPV categories was 64.7% with a Cohen's kappa coefficient of 0.45. The mean (± standard deviation) of the differences between COPWAsnap and COProAQT was 0.6 ± 1.3 L min- 1 (95% limits of agreement 3.1 to - 1.9 L min- 1) with a percentage error of 48.7% and a concordance rate of 45.1%. In adults having major abdominal surgery, PPVPWAsnap moderately agrees with PPVProAQT. The absolute and trending agreement between COPWAsnap with COProAQT is poor. Technical improvements are needed before PWAsnap can be recommended for hemodynamic monitoring.

Exosome selectivity mechanisms underlying exosome-target cell interactions and the specific traits affecting their capability to communicate still remain unclear. Moreover, the capacity of exosomes to efficiently deliver their molecular cargos intracellularly needs precise investigation towards establishing functional exosome-based delivery platforms exploitable in the clinical practice. The current study focuses on: (a) exosome production from normal MRC-5 and Vero cells growing in culture, (b) physicochemical characterization by dynamic light scattering (DLS) and cryo-transmission electron microscopy; (c) cellular uptake studies of rhodamine-labeled exosomes in normal and cancer cells, providing to exosomes either "autologous" or "heterologous" cellular delivery environments; and (d) loading exogenous Alexa Fluor 488-labeled siRNA into exosomes for the assessment of their delivering capacity by immunofluorescence in a panel of recipient cells. The data obtained thus far indicate that MRC-5 and Vero exosomes, indeed exhibit an interesting delivering profile, as promising "bio-shuttles," being pharmacologically exploitable in the context of theranostic applications.

Clarifying the coordination of leaf hydraulic traits with gas exchange across closely-related species adapted to varying rainfall can provide insights into plant habitat distribution and drought adaptation. The leaf hydraulic conductance (Kleaf ), stomatal conductance (gs ), net assimilation (A), vein embolism and abscisic acid (ABA) concentration during dehydration were quantified, as well as pressure-volume curve traits and vein anatomy in 10 Caragana species adapted to a range of mean annual precipitation (MAP) conditions and growing in a common garden. We found a positive correlation between Ψleaf at 50% loss of Kleaf (Kleaf P50 ) and maximum Kleaf (Kleaf-max ) across species. Species from low-MAP environments exhibited more negative Kleaf P50 and turgor loss point, and higher Kleaf-max and leaf-specific capacity at full turgor, along with higher vein density and midrib xylem per leaf area, and a higher ratio of Kleaf-max : maximum gs . Tighter stomatal control mediated by higher ABA accumulation during dehydration in these species resulted in an increase in hydraulic safety and intrinsic water use efficiency (WUEi ) during drought. Our results suggest that high hydraulic safety and efficiency combined with greater stomatal sensitivity triggered by ABA production and leading to greater WUEi provides drought tolerance in Caragana species adapted to low-MAP environments.

The postsynaptic density extends across the postsynaptic dendritic spine with discs large (DLG) as the most abundant scaffolding protein. DLG dynamically alters the structure of the postsynaptic density, thus controlling the function and distribution of specific receptors at the synapse. DLG contains three PDZ domains and one important interaction governing postsynaptic architecture is that between the PDZ3 domain from DLG and a protein called cysteine-rich interactor of PDZ3 (CRIPT). However, little is known regarding functional evolution of the PDZ3:CRIPT interaction. Here, we subjected PDZ3 and CRIPT to ancestral sequence reconstruction, resurrection, and biophysical experiments. We show that the PDZ3:CRIPT interaction is an ancient interaction, which was likely present in the last common ancestor of Eukaryotes, and that high affinity is maintained in most extant animal phyla. However, affinity is low in nematodes and insects, raising questions about the physiological function of the interaction in species from these animal groups. Our findings demonstrate how an apparently established protein-protein interaction involved in cellular scaffolding in bilaterians can suddenly be subject to dynamic evolution including possible loss of function.

Rhynchosia nulubilis (black soybean) has many applications in oriental medicine. It is traditionally used to treat disease related with high blood pressure, diabetes, inflammation, and osteoporosis. Furthermore, fermented soybean foods have traditionally been used for immunity enhancement in East Asia. However, the anti-inflammatory effects of germinated R. nulubilis (GR) against delayed-type hypersensitivity (DTH) are not fully understood.