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Background Circulating tumor cell (CTC) analysis is highly promising for liquid biopsy-based molecular diagnostics. We undertook a comprehensive molecular analysis of in vivo isolated CTCs in breast cancer (BrCa). Methods In vivo isolated CTCs from 42 patients with early and 23 patients with metastatic breast cancer (MBC) were prospectively collected and analyzed for gene expression, DNA mutations, and DNA methylation before and after treatment. 19 healthy donor (HD) samples were analyzed as a control group. In identical blood draws, CTCs were enumerated using CellSearch® and characterized by direct IF staining. Results All 19 HD samples were negative for CK8, CK18, CK19, ERBB2, TWIST1, VEGF, ESR1, PR, and EGFR expression, while CD44, CD24, ALDH1, VIM, and CDH2 expression was normalized to B2M (reference gene). At least one gene was expressed in 23/42 (54.8%) and 8/13 (61.5%) CTCs in early BrCa before and after therapy, and in 20/23 (87.0%) and 5/7 (71.4%) MBC before and after the first cycle of therapy. PIK3CA mutations were detected in 11/42 (26.2%) and 3/13 (23.1%) in vivo isolated CTCs in early BrCa before and after therapy, and in 11/23 (47.8%) and 2/7 (28.6%) MBC, respectively. ESR1 methylation was detected in 5/32 (15.7%) and 1/10 (10.0%) CTCs in early BrCa before and after therapy, and in 3/15(20.0%) MBC before the first line of therapy. The comprehensive molecular analysis of CTC revealed a higher sensitivity in relation to CellSearch or IF staining when based on creatine kinase selection. Conclusions In vivo-CTC isolation in combination with a comprehensive molecular analysis at the gene expression, DNA mutation, and DNA methylation level comprises a highly powerful approach for molecular diagnostic applications using CTCs.

The community-driven initiative Quality Assessment and Reproducibility for Instruments & Images in Light Microscopy (QUAREP-LiMi) wants to improve reproducibility for light microscopy image data through quality control (QC) management of instruments and images. It aims for a common set of QC guidelines for hardware calibration and image acquisition, management and analysis.

Background Increased catabolism has recently been recognized as a clinical manifestation of amyotrophic lateral sclerosis (ALS). The hypothalamic systems have been shown to be involved in the metabolic dysfunction in ALS, but the exact extent of hypothalamic circuit alterations in ALS is yet to be determined. Here we explored the integrity of large-scale cortico-hypothalamic circuits involved in energy homeostasis in murine models and in ALS patients. Methods The rAAV2-based large-scale projection mapping and image analysis pipeline based on Wholebrain and Ilastik software suites were used to identify and quantify projections from the forebrain to the lateral hypothalamus in the SOD1(G93A) ALS mouse model (hypermetabolic) and the FusΔNLS ALS mouse model (normo-metabolic). 3 T diffusion tensor imaging (DTI)-magnetic resonance imaging (MRI) was performed on 83 ALS and 65 control cases to investigate cortical projections to the lateral hypothalamus (LHA) in ALS. Results Symptomatic SOD1(G93A) mice displayed an expansion of projections from agranular insula, ventrolateral orbitofrontal and secondary motor cortex to the LHA. These findings were reproduced in an independent cohort by using a different analytic approach. In contrast, in the FusΔNLS ALS mouse model hypothalamic inputs from insula and orbitofrontal cortex were maintained while the projections from motor cortex were lost. The DTI-MRI data confirmed the disruption of the orbitofrontal-hypothalamic tract in ALS patients. Conclusion This study provides converging murine and human data demonstrating the selective structural disruption of hypothalamic inputs in ALS as a promising factor contributing to the origin of the hypermetabolic phenotype.

Knowledge about converging disease mechanisms in the heterogeneous syndrome amyotrophic lateral sclerosis (ALS) is rare, but may lead to therapies effective in most ALS cases. Previously, we identified serum microRNAs downregulated in familial ALS, the majority of sporadic ALS patients, but also in presymptomatic mutation carriers. A 5-nucleotide sequence motif (GDCGG; D = G, A or U) was strongly enriched in these ALS-related microRNAs. We hypothesized that deregulation of protein(s) binding predominantly to this consensus motif was responsible for the ALS-linked microRNA fingerprint. Using microRNA pull-down assays combined with mass spectrometry followed by extensive biochemical validation, all members of the fragile X protein family, FMR1, FXR1 and FXR2, were identified to directly and predominantly interact with GDCGG microRNAs through their structurally disordered RGG/RG domains. Preferential association of this protein family with ALS-related microRNAs was confirmed by in vitro binding studies on a transcriptome-wide scale. Immunohistochemistry of lumbar spinal cord revealed aberrant expression level and aggregation of FXR1 and FXR2 in C9orf72- and FUS-linked familial ALS, but also patients with sporadic ALS. Further analysis of ALS autopsies and induced pluripotent stem cell-derived motor neurons with FUS mutations showed co-aggregation of FXR1 with FUS. Hence, our translational approach was able to take advantage of blood microRNAs to reveal CNS pathology, and suggests an involvement of the fragile X-related proteins in familial and sporadic ALS already at a presymptomatic stage. The findings may uncover disease mechanisms relevant to many patients with ALS. They furthermore underscore the systemic, extra-CNS aspect of ALS., Freischmidt et al. show direct interaction of fragile X proteins with a subset of microRNAs that share a common sequence motif and that are downregulated in the majority of patients with ALS. Neuropathology suggests contribution of the fragile X proteins to a convergent disease mechanism implicated in many cases of ALS.

Background and study aims Due to demographic transition, neurogenic dysphagia has become an increasingly recognized problem. Patients suffering from dysphagia often get caught between different clinical disciplines. In this study, we implemented a defined examination protocol for evaluating the whole swallowing process by functional endoscopy. Special focus was put on the esophageal phase of swallowing. Patients and methods This prospective observational multidisciplinary study evaluated 31 consecutive patients with suspected neurogenic dysphagia by transnasal access applying an ultrathin video endoscope. Thirty-one patients with gastroesophageal reflux symptoms were used as a control group. We applied a modified approach including standardized endoscopic positions to compare our findings with fiberoptic endoscopic evaluation of swallowing and high-resolution manometry. The primary outcome measure was feasibility of functional endoscopy. Secondary outcome measures were adverse events (AEs), tolerability, and pathologic endoscopic findings. Results Functional endoscopy was successfully performed in all patients. No AEs were recorded. A variety of disorders were documented by functional endoscopy: incomplete or delayed closure of the upper esophageal sphincter in retroflex view, clearance disturbance of tubular esophagus, esophageal hyperperistalsis, and hypomotility. Analysis of results obtained with the diagnostic tools showed some discrepancies. Conclusions By interdisciplinary cooperation with additional assessment of the esophageal phase of deglutition using the innovative method of functional endoscopy, the diagnosis of neurogenic disorders including dysphagia may be significantly improved, leading to a better clinical understanding of complex dysfunctional patterns. To the best of our knowledge, this is the first study to show that a retroflex view of the ultrathin video endoscope within the esophagus can be safely performed. [NCT01995929]

Fungal lytic polysaccharide monooxygenases (LPMOs) depolymerise crystalline cellulose and hemicellulose, supporting the utilisation of lignocellulosic biomass as a feedstock for biorefinery and biomanufacturing processes. Recent investigations have shown that H2O2 is the most efficient cosubstrate for LPMOs. Understanding the reaction mechanism of LPMOs with H2O2 is therefore of importance for their use in biotechnological settings. Here, we have employed a variety of spectroscopic and biochemical approaches to probe the reaction of the fungal LPMO9C from N. crassa using H2O2 as a cosubstrate and xyloglucan as a polysaccharide substrate. We show that a single ‘priming’ electron transfer reaction from the cellobiose dehydrogenase partner protein supports up to 20 H2O2‐driven catalytic cycles of a fungal LPMO. Using rapid mixing stopped‐flow spectroscopy, alongside electron paramagnetic resonance and UV‐Vis spectroscopy, we reveal how H2O2 and xyloglucan interact with the enzyme and investigate transient species that form uncoupled pathways of NcLPMO9C. Our study shows how the H2O2 cosubstrate supports fungal LPMO catalysis and leaves the enzyme in the reduced Cu+ state following a single enzyme turnover, thus preventing the need for external protons and electrons from reducing agents or cellobiose dehydrogenase and supporting the binding of H2O2 for further catalytic steps. We observe that the presence of the substrate xyloglucan stabilises the Cu+ state of LPMOs, which may prevent the formation of uncoupled side reactions., Lytic polysaccharide monooxygenases (LPMOs) consume external electrons and an O2‐containing cosubstrate to depolymerise recalcitrant polysaccharides. We show that interactions of reduced LPMO with the H2O2 cosubstrate leaves the enzyme in a reduced Cu+ state after a single turnover. This allows for continued catalysis without the need for external electrons. The presence of substrate stabilises the Cu+ state of LPMOs, which may prevent the formation of uncoupled side reactions.

We investigated in larval and adult Drosophila models whether loss of the mitochondrial chaperone Hsc70-5 is sufficient to cause pathological alterations commonly observed in Parkinson disease. At affected larval neuromuscular junctions, no effects on terminal size, bouton size or number, synapse size, or number were observed, suggesting that we studied an early stage of pathogenesis. At this stage, we noted a loss of synaptic vesicle proteins and active zone components, delayed synapse maturation, reduced evoked and spontaneous excitatory junctional potentials, increased synaptic fatigue, and cytoskeleton rearrangements. The adult model displayed ATP depletion, altered body posture, and susceptibility to heat-induced paralysis. Adult phenotypes could be suppressed by knockdown of dj-1β, Lrrk, DCTN2-p50, DCTN1-p150, Atg1, Atg101, Atg5, Atg7, and Atg12. The knockdown of components of the macroautophagy/autophagy machinery or overexpression of human HSPA9 broadly rescued larval and adult phenotypes, while disease-associated HSPA9 variants did not. Overexpression of Pink1 or promotion of autophagy exacerbated defects.Abbreviations: AEL: after egg laying; AZ: active zone; brp: bruchpilot; Csp: cysteine string protein; dlg: discs large; eEJPs: evoked excitatory junctional potentials; GluR: glutamate receptor; H2O2: hydrogen peroxide; mEJP: miniature excitatory junctional potentials; MT: microtubule; NMJ: neuromuscular junction; PD: Parkinson disease; Pink1: PTEN-induced putative kinase 1; PSD: postsynaptic density; SSR: subsynaptic reticulum; SV: synaptic vesicle; VGlut: vesicular glutamate transporter.

The cyanobacterium Fischerella ambigua is a natural producer of polychlorinated aromatic compounds, the ambigols A-E. The biosynthetic gene cluster (BGC) of these highly halogenated triphenyls has been recently identified by heterologous expression. It consists of 10 genes named ab1-10. Two of the encoded enzymes, i.e. Ab2 and Ab3, were identified by in vitro and in vivo assays as cytochrome P450 enzymes responsible for biaryl and biaryl ether formation. The key substrate for these P450 enzymes is 2,4-dichlorophenol, which in turn is derived from the precursor 3-chloro-4-hydroxybenzoic acid. Here, the biosynthetic steps leading towards 3-chloro-4-hydroxybenzoic acid were investigated by in vitro assays. Ab7, an isoenzyme of a 3-deoxy-7-phosphoheptulonate (DAHP) synthase, is involved in chorismate biosynthesis by the shikimate pathway. Chorismate in turn is further converted by a dedicated chorismate lyase (Ab5) yielding 4-hydroxybenzoic acid (4-HBA). The stand alone adenylation domain Ab6 is necessary to activate 4-HBA, which is subsequently tethered to the acyl carrier protein (ACP) Ab8. The Ab8 bound substrate is chlorinated by Ab10 in meta position yielding 3-Cl-4-HBA, which is then transfered by the condensation (C) domain to the peptidyl carrier protein and released by the thioesterase (TE) domain of Ab9. The released product is then expected to be the dedicated substrate of the halogenase Ab1 producing the monomeric ambigol building block 2,4-dichlorophenol.

Metformin may act renoprotective prior to kidney transplantation by reducing ischemia-reperfusion injury (IRI). This study examined whether metformin preconditioning and postconditioning duringex vivonormothermic machine perfusion (NMP) of rat and porcine kidneys affect IRI. In the rat study, saline or 300 mg/kg metformin was administered orally twice on the day before nephrectomy. After 15 minutes of warm ischemia, kidneys were preserved with static cold storage for 24 hours. Thereafter, 90 minutes of NMP was performed with the addition of saline or metformin (30 or 300 mg/L). In the porcine study, after 30 minutes of warm ischemia, kidneys were preserved for 3 hours with oxygenated hypothermic machine perfusion. Subsequently, increasing doses of metformin were added during 4 hours of NMP. Metformin preconditioning of rat kidneys led to decreased injury perfusate biomarkers and reduced proteinuria. Postconditioning of rat kidneys resulted, dose-dependently, in less tubular cell necrosis and vacuolation. Heat shock protein 70 expression was increased in metformin-treated porcine kidneys. In all studies, creatinine clearance was not affected. In conclusion, both metformin preconditioning and postconditioning can be done safely and improved rat and porcine kidney quality. Because the effects are minor, it is unknown which strategy might result in improved organ quality after transplantation.

Zinc, a trace element, is necessary for the correct structure and function of many proteins. Therefore, Zn2+ has to be taken up by the cells, using specific Zn2+ transporters or Ca2+ channels. In this study, we have focused on two Ca2+ channels, the L-type voltage-gated Cav1.2 channel and the transient receptor potential channel TRPM3. Stimulation of either channel induces an intracellular signaling cascade leading to the activation of the transcription factor AP-1. The influx of Ca2+ ions into the cytoplasm is essential for this activity. We asked whether extracellular Zn2+ ions affect Cav1.2 or TRPM3-induced gene transcription following stimulation of the channels. The results show that extracellular Zn2+ ions reduced the activation of AP-1 by more than 80% following stimulation of either voltage-gated Cav1.2 channels or TRPM3 channels. Experiments performed with cells maintained in Ca2+-free medium revealed that Zn2+ ions cannot replace Ca2+ ions in inducing gene transcription via stimulation of Cav1.2 and TRPM3 channels. Re-addition of Ca2+ ions to the cell culture medium, however, restored the ability of these Ca2+ channels to induce a signaling cascade leading to the activation of AP-1. Secretory cells, including neurons and pancreatic β-cells, release Zn2+ ions during exocytosis. We propose that the released Zn2+ ions function as a negative feedback loop for stimulus-induced exocytosis by inhibiting Ca2+ channel signaling.

Background Kidney injuries that result in chronic inflammation initiate crosstalk between stressed resident cells and infiltrating immune cells. In animal models, whole-body receptor Notch3 deficiency protects from leukocyte infiltration and organ fibrosis. However, the relative contribution of Notch3 expression in tissue versus infiltrating immune cells is unknown. Methods Chimeric mice deficient for Notch3 in hematopoietic cells and/or resident tissue cells were generated, and kidney fibrosis and inflammation after unilateral ureteral obstruction (UUO) were analyzed. Adoptive transfer of labeled bone marrow-derived cells validated the results in a murine Leishmania ear infection model. In vitro adhesion assays, integrin activation, and extracellular matrix production were analyzed. Results Fibrosis follows UUO, but inflammatory cell infiltration mostly depends upon Notch3 expression in hematopoietic cells, which coincides with an enhanced proinflammatory milieu (e.g., CCL2 and CCL5 upregulation). Notch3 expression on CD45+ leukocytes plays a prominent role in efficient cell transmigration. Functionally, leukocyte adhesion and integrin activation are abrogated in the absence of receptor Notch3. Chimeric animal models also reveal that tubulointerstitial fibrosis develops, even in the absence of prominent leukocyte infiltrates after ureteral obstruction. Deleting Notch3 receptors on resident cells blunts kidney fibrosis, ablates NF-κB signaling, and lessens matrix deposition. Conclusions Cell-specific receptor Notch3 signaling independently orchestrates leukocyte infiltration and organ fibrosis. Interference with Notch3 signaling may present a novel therapeutic approach in inflammatory as well as fibrotic diseases.

Compared to bacteria, fungi often exhibit a lower abundance and a higher temporal volatility in the intestinal microbiota. Analysis of fungi in the microbiota (mycobiota) faces technical limitations with tools that were originally developed for analyzing bacteria. Dysbiotic states of the intestinal mycobiota, often associated with disruption of the healthy bacterial microbiota, are characterized by overgrowth (domination) of specific fungal taxa and loss of diversity. Intestinal domination by Candida species has been shown to be a major source of Candida bloodstream infections. Fungal dysbiosis is also linked to the development and treatment response in non-fungal infections, for example Clostridioides difficile colitis and HIV. Further research is needed to define the contribution of intestinal mycobiota to human fungal and non-fungal infections.

BackgroundmTOR signaling is an essential nutrient and energetic sensing pathway. Here we describe AIMTOR, a sensitive genetically encoded BRET (Bioluminescent Resonance Energy Transfer) biosensor to study mTOR activity in living cells.ResultsAs a proof of principle, we show in both cell lines and primary cell cultures that AIMTOR BRET intensities are modified by mTOR activity changes induced by specific inhibitors and activators of mTORC1 including amino acids and insulin. We further engineered several versions of AIMTOR enabling subcellular-specific assessment of mTOR activities. We then used AIMTOR to decipher mTOR signaling in physio-pathological conditions. First, we show that mTORC1 activity increases during muscle cell differentiation and in response to leucine stimulation in different subcellular compartments such as the cytosol and at the surface of the lysosome, the nucleus, and near the mitochondria. Second, in hippocampal neurons, we found that the enhancement of neuronal activity increases mTOR signaling. AIMTOR further reveals mTOR-signaling dysfunctions in neurons from mouse models of autism spectrum disorder.ConclusionsAltogether, our results demonstrate that AIMTOR is a sensitive and specific tool to investigate mTOR-signaling dynamics in living cells and phenotype mTORopathies.

As of 10 April 2020, New York State had 180,458 cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and 9,385 reported deaths. Patients with cancer comprised 8.4% of deceased individuals1. Population-based studies from China and Italy suggested a higher coronavirus disease 2019 (COVID-19) death rate in patients with cancer2,3, although there is a knowledge gap as to which aspects of cancer and its treatment confer risk of severe COVID-194. This information is critical to balance the competing safety considerations of reducing SARS-CoV-2 exposure and cancer treatment continuation. From 10 March to 7 April 2020, 423 cases of symptomatic COVID-19 were diagnosed at Memorial Sloan Kettering Cancer Center (from a total of 2,035 patients with cancer tested). Of these, 40% were hospitalized for COVID-19, 20% developed severe respiratory illness (including 9% who required mechanical ventilation) and 12% died within 30 d. Age older than 65 years and treatment with immune checkpoint inhibitors (ICIs) were predictors for hospitalization and severe disease, whereas receipt of chemotherapy and major surgery were not. Overall, COVID-19 in patients with cancer is marked by substantial rates of hospitalization and severe outcomes. The association observed between ICI and COVID-19 outcomes in our study will need further interrogation in tumor-specific cohorts.

Genetic and environmental disturbances are expected to increase developmental instability, which may result in higher fluctuating asymmetry (FA), i.e. small random deviations from symmetry. Plant leaves often do not show this pattern, possibly due to high phenotypic plasticity of leaf shape and low adaptive significance of leaf symmetry. In contrast, symmetry in many animal traits but also in flower shape is considered to be under selection, and FA in such traits may better reflect developmental instability. Using geometric morphometrics, I analysed the symmetry of flowers of inbred and outbred Mimulus guttatus (Phrymaceae) plants grown under five stress treatments with and without grass competition. Flower FA was not increased by abiotic stress, but by inbreeding and competition. As inbreeding and competition affected different principal components of flower FA, different mechanisms may be involved in their effects on FA. FA decreased with individual biomass particularly in selfed offspring, which suggests that inbreeding increased FA particularly when growth was limited by environmental or genetic constraints. Increased flower FA of inbred offspring may explain increased flower handling time and reduced pollinator preference for inbred plants in other M. guttatus studies, and could thus have important consequences for plant demography and plant–pollinator interactions.

Hereditary angioedema (HAE), an inherited deficiency offunctional C1 esterase inhibitor (C1-INH), is characterized byrecurrent episodes of disabling and often painful swelling insubcutaneous and/or submucosal tissues.1HAE attacks aregenerally unpredictable, but triggers for an attack can includehaving a dental or medical procedure (eg, surgery), other trauma,or stress. A preemptive management plan for patients under-going these types of situations may reduce the risk of HAE at-tacks. Recommendations include administration of short-termprophylaxis in patients with HAE before invasive medical pro-cedures, especially those involving the upper airways or digestivetract, with C1-INH concentrate typically the medication ofchoice.

The intestinal microbiota is a complex community of bacteria, archaea, viruses, protists and fungi1,2. Although the composition of bacterial constituents has been linked to immune homeostasis and infectious susceptibility3-7, the role of non-bacterial constituents and cross-kingdom microbial interactions in these processes is poorly understood2,8. Fungi represent a major cause of infectious morbidity and mortality in immunocompromised individuals, although the relationship of intestinal fungi (that is, the mycobiota) with fungal bloodstream infections remains undefined9. We integrated an optimized bioinformatics pipeline with high-resolution mycobiota sequencing and comparative genomic analyses of fecal and blood specimens from recipients of allogeneic hematopoietic cell transplant. Patients with Candida bloodstream infection experienced a prior marked intestinal expansion of pathogenic Candida species; this expansion consisted of a complex dynamic between multiple species and subspecies with a stochastic translocation pattern into the bloodstream. The intestinal expansion of pathogenic Candida spp. was associated with a substantial loss in bacterial burden and diversity, particularly in the anaerobes. Thus, simultaneous analysis of intestinal fungi and bacteria identifies dysbiosis states across kingdoms that may promote fungal translocation and facilitate invasive disease. These findings support microbiota-driven approaches to identify patients at risk of fungal bloodstream infections for pre-emptive therapeutic intervention.

The terrestrial cyanobacterium Fischerella ambigua 108b produces the three polychlorinated triphenyls ambigol A–C that exhibit interesting antimicrobial, antiviral and cytotoxic activities. They are structurally related to polybrominated diphenylethers synthesized by diverse marine bacteria that are known to be highly toxic and are bioaccumulating in natural food webs. All ambigols display unusual connectivities: Ambigols A and B exhibit chlorination and ambigol C biaryl-ether bonds in the relative meta position at the central phenol unit, which is flanked by two 2,4-dichlorophenol units in all three compounds. Here we report on the identification of the biosynthetic gene cluster (BGC) reponsible for ambigol production in F. ambigua. After bioinformatic discovery of a putative ambigol BGC (ab) containing 10 genes, we cloned and heterologously expressed this cluster in Synechococcus elongatus PCC 7942 using Direct Pathway Cloning (DiPaC). In vivo and in vitro characterization of the two cytochrome P450 enzymes present in the ab BGC revealed complementary selectivity for either biaryl-ether bond (Ab2) or biaryl formation (Ab3) and provided a biosynthetic route to the ambigols.

BACKGROUNDTransrectal ultrasound-guided prostate biopsy (TRUS) is a standard procedure for prostate cancer diagnosis. Because prostate cancer is a multifocal disease in many patients, multiple sampling (n ≥ 10) is required, which may bear the risk of systemic spread of cancer cells.DESIGNUsing the standardized CellSearch® system that allows for the detection of single epithelial cell adhesion molecule-positive circulating tumor cells (CTCs) in blood, we investigated whether prostate biopsy is associated with release of prostatic tumor cells into the circulation. Peripheral blood was obtained before and within 30 min after performing prostate biopsy from 115 men with increased serum prostate-specific antigen.RESULTSThe number of CTCs significantly increased after biopsy in men with histologically confirmed prostate cancer (odds ratio, 7.8; 95% CI, 4.8–12.8), whereas no biopsy-related changes could be detected in men without confirmed prostate cancer. Multivariable analysis showed that biopsy-related increase of CTCs was significantly correlated with a worse progression-free survival (hazard ratio, 12.4; 95% CI, 3.2–48.6) within the median follow-up of 41 months.CONCLUSIONSProstate biopsies may lead to a tumor-associated release of CTCs into the blood circulation. Larger confirmatory trials with longer follow-up periods are required before any change in clinical practice can be recommended.

Die Kombination aus Glenoidverlust und Rotatorenmanschetten-Defektarthropathie (RM-Defektarthropathie) stellt eine Herausforderung dar. Eine 75-jahrige Patientin stellte sich mit einer posttraumatischen Glenoiddestruktion mit RM-Defektarthropathie vor. Anhand von CT-Aufnahmen erfolgte die Anfertigung einer individualisierten Glenoid-Komponente mittels 3D-Printverfahren. 6 Monate nach Implantation der inversen Prothese war die Patientin schmerzfrei bei akzeptabler Beweglichkeit. Die individualisierte Glenoidkomponente aus dem 3D-Druckverfahren stellt bei Glenoidverlust mit ausgepragter RM-Defektarthropathie eine gute Therapiealternative dar.

Summary Background The use of opioid analgesic drugs (OADs) to treat postoperative pain after sports traumatology procedures at the time of discharge may be associated with prolonged OAD use after discharge. Material and methods Between January and February 2017, 195 patients underwent inpatient surgery in a sports traumatology department. In total, 194 patients were available for retrospective analysis. The use of OAD and patient- and therapy-related factors were recorded. Odds ratios (OR; 95% CI) were calculated to detect potential risk factors of OAD use at the time of patient discharge. Results Overall, 50 patients (25.8%) received OADs as continuous medication, 85 patients (43.8%) received OADs as oral demand medication, and 103 patients (53.1%) received OADs as intravenous on-demand medication. Within the entire study population, 40 patients (20.6%) received OADs at the time of discharge. A high patient age (OR 1.04; 1.01–1.06), female gender (OR 2.19; 1.08–4.46), a long hospital stay (OR 1.44; 1.19–1.74), the use of regional and/or spinal anaesthesia (OR 3.18; 1.32–7.64) and the preoperative use of OADs (OR 27.0; 3.15–231.65) were found to increase patient risk of being discharged with OADs. Conclusions In sports traumatology, high patient age, female gender, long hospital stays and the preoperative use of OADs are risk factors for OAD use at the time of discharge. Physicians should be aware of these risk factors of administering prolonged OAD treatment, and they should try to terminate the use of OAD therapy or switch to non-opiate analgesics before discharging patients.

Candida albicans is a pathobiont that colonizes multiple niches in the body including the gastrointestinal (GI) tract, but is also responsible for both mucosal and systemic infections. Despite its prevalence as a human commensal, the murine GI tract is generally refractory to colonization with the C. albicans reference isolate SC5314. Here, we identify two C. albicans isolates, 529L and CHN1, that stably colonize the murine GI tract in three different animal facilities under conditions where SC5314 is lost from this niche. Analysis of the bacterial microbiota did not show notable differences between mice colonized with the three C. albicans strains. We compared the genotypes and phenotypes of these three strains and identified thousands of SNPs and multiple phenotypic differences, including their ability to grow and filament in response to nutritional cues. Despite striking filamentation differences under laboratory conditions, however, analysis of cell morphology in the GI tract revealed that the three isolates exhibited similar filamentation properties in this in vivo niche. Notably, we found that SC5314 is more sensitive to the antimicrobial peptide CRAMP, and the use of CRAMP-deficient mice increased the ability of SC5314 to colonize the GI tract relative to CHN1 and 529L. These studies provide new insights into how strain-specific differences impact C. albicans traits in the host and advance CHN1 and 529L as relevant strains to study C. albicans pathobiology in its natural host niche.IMPORTANCEUnderstanding how fungi colonize the GI tract is increasingly recognized as highly relevant to human health. The animal models used to study Candida albicans commensalism commonly rely on altering the host microbiome (via antibiotic treatment or defined diets) to establish successful GI colonization by the C. albicans reference isolate SC5314. Here, we characterize two C. albicans isolates that can colonize the murine GI tract without antibiotic treatment and can therefore be used as tools for studying fungal commensalism. Importantly, experiments were replicated in three different animal facilities and utilized three different mouse strains. Differential colonization between fungal isolates was not associated with alterations in the bacterial microbiome but rather with distinct responses to CRAMP, a host antimicrobial peptide. This work emphasizes the importance of C. albicans intra-species variation as well as host anti-microbial defense mechanisms in defining commensal interactions.

Chronic viral infections subvert protective B cell immunity. An early type I interferon (IFN-I)-driven bias to short-lived plasmablast differentiation leads to clonal deletion, so-called "decimation," of antiviral memory B cells. Therefore, prophylactic countermeasures against decimation remain an unmet need. We show that vaccination-induced CD4 T cells prevented the decimation of naïve and memory B cells in chronically lymphocytic choriomeningitis virus (LCMV)-infected mice. Although these B cell responses were largely T independent when IFN-I was blocked, preexisting T help assured their sustainability under conditions of IFN-I-driven inflammation by instructing a germinal center B cell transcriptional program. Prevention of decimation depended on T cell-intrinsic Bcl6 and Tfh progeny formation. Antigen presentation by B cells, interactions with antigen-specific T helper cells, and costimulation by CD40 and ICOS were also required. Importantly, B cell-mediated virus control averted Th1-driven immunopathology in LCMV-challenged animals with preexisting CD4 T cell immunity. Our findings show that vaccination-induced Tfh cells represent a cornerstone of effective B cell immunity to chronic virus challenge, pointing the way toward more effective B cell-based vaccination against persistent viral diseases.

Shedding of SARS-CoV-2 after Immunosuppression The prolonged contagious period after viral infection in immunocompromised patients may affect how long precautions will be necessary to reduce furthe...

Synopsis image The lack of an effective treatment for ALS is calling for the development of novel therapeutic strategies. By using hiPSC���derived motoneurons and focusing on synapse���related processes, we provide new molecular targets rescuing the degenerative processes and neuronal loss in ALS. Human C9orf72���mutant motoneurons are characterized by reduced expression of synaptic gene, progressive loss of CREB activity and synapse loss. Similar alterations are observed also in motoneurons harboring TBK1 pathogenic mutations, and in primary neurons upon overexpression of poly(GA) aggregates. The K+ channel blockers Apamin and XE991 revert the CREB���dependent loss of synaptic contacts and rescue the degenerative phenotypes of ALS motoneurons., publishedVersion

Aspergillus fumigatus is the most common cause of mold pneumonia worldwide, and a significant cause of infectious morbidity and mortality in immunocompromised individuals. The oxidative burst, which generates reactive oxidative species (ROS), plays a pivotal role in host defense against aspergillosis and induces regulated cell death in Aspergillus conidia, the infectious propagules. Beyond the well-established role of NADP (NADPH) oxidase in ROS generation by neutrophils and other innate effector cells, mitochondria represent a major ROS production site in many cell types, though it is unclear whether mitochondrial ROS (mtROS) contribute to antifungal activity in the lung. Following A. fumigatus infection, we observed that innate effector cells, including alveolar macrophages (AMs), monocyte-derived dendritic cells (Mo-DCS), and neutrophils, generated mtROS, primarily in fungus-infected cells. To examine the functional role of mtROS, specifically the H2O2 component, in pulmonary host defense against A. fumigatus, we infected transgenic mice that expressed a mitochondrion-targeted catalase. Using a reporter of fungal viability during interactions with leukocytes, mitochondrial H2O2 (mtH2O2) was essential for optimal AM, but not for neutrophil phagocytic and conidiacidal activity in the lung. Catalase-mediated mtH2O2 neutralization did not lead to invasive aspergillosis in otherwise immunocompetent mice and did not shorten survival in mice that lack NADPH oxidase function. Collectively, these studies indicate that mtROS-associated defects in AM antifungal activity can be functionally compensated by the action of NADPH oxidase and by nonoxidative effector mechanisms during murine A. fumigatus lung infection. IMPORTANCE Aspergillus fumigatus is a fungal pathogen that causes invasive disease in humans with defects in immune function. Airborne conidia, the infectious propagules, are ubiquitous and inhaled on a daily basis. In the respiratory tree, conidia are killed by the coordinated actions of phagocytes, including alveolar macrophages, neutrophils, and monocyte-derived dendritic cells. The oxidative burst represents a central killing mechanism and relies on the assembly of the NADPH oxidase complex on the phagosomal membrane. However, NADPH oxidase-deficient leukocytes have significant residual fungicidal activity in vivo, indicating the presence of alternative effector mechanisms. Here, we report that murine innate immune cells produce mitochondrial reactive oxygen species (mtROS) in response to fungal interactions. Neutralizing the mtROS constituent hydrogen peroxide (H2O2) via a catalase expressed in mitochondria of innate immune cells substantially diminished fungicidal properties of alveolar macrophages, but not of other innate immune cells. These data indicate that mtH2O2 represent a novel AM killing mechanism against Aspergillus conidia. mtH2O2 neutralization is compensated by other killing mechanisms in the lung, demonstrating functional redundancy at the level of host defense in the respiratory tree. These findings have important implications for the development of host-directed therapies against invasive aspergillosis in susceptible patient populations.

COVID-19 is a pandemic disease that causes severe pulmonary damage and hyperinflammation. Vitamin A is a crucial factor in the development of immune functions and is known to be reduced in cases of acute inflammation. This prospective, multicenter observational cross-sectional study analyzed vitamin A plasma levels in SARS-CoV-2 infected individuals, and 40 hospitalized patients were included. Of these, 22 developed critical disease (Acute Respiratory Distress Syndrome [ARDS]/Extracorporeal membrane oxygenation [ECMO]), 9 developed severe disease (oxygen supplementation), and 9 developed moderate disease (no oxygen supplementation). A total of 47 age-matched convalescent persons that had been earlier infected with SARS-CoV-2 were included as the control group. Vitamin A plasma levels were determined by high-performance liquid chromatography. Reduced vitamin A plasma levels correlated significantly with increased levels of inflammatory markers (CRP, ferritin) and with markers of acute SARS-CoV-2 infection (reduced lymphocyte count, LDH). Vitamin A levels were significantly lower in hospitalized patients than in convalescent persons (p &lt, 0.01). Of the hospitalized patients, those who were critically ill showed significantly lower vitamin A levels than those who were moderately ill (p &lt, 0.05). Vitamin A plasma levels below 0.2 mg/L were significantly associated with the development of ARDS (OR = 5.54 [1.01–30.26], p = 0.048) and mortality (OR 5.21 [1.06–25.5], p = 0.042). Taken together, we conclude that vitamin A plasma levels in COVID-19 patients are reduced during acute inflammation and that severely reduced plasma levels of vitamin A are significantly associated with ARDS and mortality.

The genus Aspergillus is ubiquitous in the environment and contains a number of species, primarily A. fumigatus, that cause mold-associated disease in humans. Humans inhale several hundred to several thousand Aspergillus conidia (i.e., vegetative spores) daily and typically clear these in an asymptomatic manner. In immunocompromised individuals, Aspergillus conidia can germinate into tissue-invasive hyphae, disseminate, and cause invasive aspergillosis. In this review, we first discuss novel concepts in host defense against Aspergillus infections and emphasize new insights in fungal recognition and signaling, innate immune activation, and fungal killing. Second, the review focuses on novel concepts of Aspergillus pathogenesis and highlights emerging knowledge regarding fungal strain heterogeneity, stress responses, and metabolic adaptations on infectious outcomes. Mechanistic insight into the host–pathogen interplay is thus critical to define novel druggable fungal targets and to exploit novel immune-based strategies to improve clinical outcomes associated with aspergillosis in vulnerable patient populations.

Standards for estimating mouth width and Cupid's bow width in craniofacial approximation and superimposition are limited. Currently, the only guideline for mouth width, using direct skeletal references, is a general rule indicating a 75% inter-canine to mouth width ratio. The philtrum, which closely corresponds to the Cupid's bow, is said to be equal to the inter-superior prominences of the maxillary central incisors. This study tested these guidelines against newly generated regression models and mean values. Cone-beam CT scans of 120 black and 39 white southern African adults were used. Comparative hard and soft tissue measurements were taken using a 3D DICOM viewer. Regression equations accounting population, sex, and approximate age variables (20-39 and 40+ years), utilizing maxillary inter-canine width to estimate mouth width and maxillary central-lateral incisor junction width to estimate Cupid's bow width, performed statistically best. The regression models were more reliable than existing standards in validation tests.

The life expectancy of unresectable hilar cholangiocellular carcinomas (CCCs) is very limited and endoscopic radiofrequency ablation (ERFA) of the biliary tract may prolong survival. Our single-center-study retrospectively analysed all CCC cases, in whom ERFAs of the biliary tract were performed between 2012 and 2017 and compared these to historical control cases who received the standard treatment of sole stent application. ERFA was performed in 32 patients with malignant biliary strictures that were mainly caused by Bismuth III and IV hilar CCCs (66%). 14 of these patients received repeated ERFAs, for an overall performance of 54 ERFAs. Stents were applied after examination of all patients (100%). Adverse events occurred in 18.5% of examinations. Case-control analysis revealed that the survival time of cases with unresectable Bismuth type III and IV hilar CCCs (n = 20) treated with combined ERFA and stent application significantly increased compared to controls (n = 22) treated with sole stent application (342 +/− 57 vs. 221 +/− 26 days; p = 0.046). In conclusion, ERFA therapy significantly prolonged survival in patients with unresectable Bismuth type III and IV hilar CCC. As an effective and safe method, ERFA should be considered as a palliative treatment for all these patients.

Der folgende Artikel prasentiert den Fall eines 53-jahrigen Patienten, geburtig in Sri Lanka, der sich zunachst mit dem Verdacht auf ein Kniegelenkempyem prasentierte. Im Rahmen einer arthroskopischen Kniegelenkspulung konnte eine Ochronose diagnostiziert und spater histopathologisch gesichert werden. Die Alkaptonurie, durch einen Mangel an Homogentisinsaure-1,2-Dioxygenase bedingt, fuhrt zur Akkumulation von Homogentisat, einem Tyrosinabbauprodukt. Hieraus resultiert u. a. das typische klinische Erscheinungsbild einer Ochronose, der blaulich-schwarzen Farbeinlagerung im Gewebe (z. B. im Bindegewebe, in den Skleren und im Ohrknorpel) und einer Schwarzfarbung des Urins.

The risk gene CACNA1C is strongly implicated in the etiology of all major psychiatric disorders, such as depressive disorder, bipolar disorder, autism spectrum disorder, and schizophrenia. These disorders feature high levels of comorbidity and share an overlap of symptoms; in particular, deficits in social functioning are common. Intriguingly, sex-dependent effects of CACNA1C single nucleotide polymorphisms on prevalence, health outcomes, and psychological traits have been reported, typically suggesting that women are more affected by CACNA1C mutations than men. In rodents, genetic modifications specifically targeting Cacna1c have repeatedly been linked to deficits in social behavior in male mice and rats but many studies neglect the sex-dependent effects observed in humans. Our study focused on the role of Cacna1c in regulating social behavior and communication in adult female rats. We compared social and non-social behavior together with concomitant emission of pro-social 50-kHz ultrasonic vocalizations (USV) associated with positive affect in constitutive heterozygous (Cacna1c+/-) rats to wildtype (Cacna1c+/+) littermate controls. Our results indicate that partial Cacna1c depletion leads to strongly reduced emission of 50-kHz USV and mild social deficits during female direct reciprocal social interaction. Detailed temporal analyses revealed most prominent reductions of 50-kHz USV during non-social behavior, suggesting that reduced positive affect occurs in a social context in Cacna1c+/- rats but is not specifically linked to social behavior. Finally, we observed increased self-grooming behavior in Cacna1c+/- rats, consistent with an autism-like phenotype. Our findings in rats thus support a role of Cacna1c in regulating behavioral phenotypes with relevance for several neuropsychiatric disorders.

Background Availability of SARS-CoV-2 testing in the United States (U.S.) has fluctuated through the course of the COVID-19 pandemic, including in the U.S. state of Illinois. Despite substantial ramp-up in test volume, access to SARS-CoV-2 testing remains limited, heterogeneous, and insufficient to control spread. Methods We compared SARS-CoV-2 testing rates across geographic regions, over time, and by demographic characteristics (i.e., age and racial/ethnic groups) in Illinois during March through December 2020. We compared age-matched case fatality ratios and infection fatality ratios through time to estimate the fraction of SARS-CoV-2 infections that have been detected through diagnostic testing. Results By the end of 2020, initial geographic differences in testing rates had closed substantially. Case fatality ratios were higher in non-Hispanic Black and Hispanic/Latino populations in Illinois relative to non-Hispanic White populations, suggesting that tests were insufficient to accurately capture the true burden of COVID-19 disease in the minority populations during the initial epidemic wave. While testing disparities decreased during 2020, Hispanic/Latino populations consistently remained the least tested at 1.87 tests per 1000 population per day compared with 2.58 and 2.87 for non-Hispanic Black and non-Hispanic White populations, respectively, at the end of 2020. Despite a large expansion in testing since the beginning of the first wave of the epidemic, we estimated that over half (50–80%) of all SARS-CoV-2 infections were not detected by diagnostic testing and continued to evade surveillance. Conclusions Systematic methods for identifying relatively under-tested geographic regions and demographic groups may enable policymakers to regularly monitor and evaluate the shifting landscape of diagnostic testing, allowing officials to prioritize allocation of testing resources to reduce disparities in COVID-19 burden and eventually reduce SARS-CoV-2 transmission.

The gastrointestinal tract contains a vast and diverse microbial reservoir composed of bacteria, fungi, and viruses that contribute positively to human health. There is growing evidence that perturbation of the normal microbiota can promote a variety of human disease states that include tumorigenesis. Whether the fungal component of the gut microbiota (i.e., the mycobiota) can influence tumor development has not been investigated in detail. In the recent issue of the Theranostics, Zhong et al (2021) shed light on an association between mycobiota dysbiosis and gastric cancer. These findings implicate the mycobiota in gastric carcinogenesis and set the stage for future mechanistic studies to explore whether fungal dysbiosis is a cause or consequence of gastric carcinogenesis, with important implications for preventative strategies.

Gram-negative bacteria partly rely on efflux pumps to facilitate growth under stressful conditions and to increase resistance to a wide variety of commonly used drugs. In recent years, Escherichia coli sequence type 131 (ST131) has emerged as a major cause of extraintestinal infection frequently exhibiting a multidrug resistance (MDR) phenotype. The contribution of efflux to MDR in emerging E. coli MDR clones, however, is not well studied. We characterized strains from an international collection of clinical MDR E. coli isolates by MIC testing with and without the addition of the AcrAB-TolC efflux inhibitor 1-(1-naphthylmethyl)-piperazine (NMP). MIC data for 6 antimicrobial agents and their reversion by NMP were analyzed by principal-component analysis (PCA). PCA revealed a group of 17 MDR E. coli isolates (n = 34) exhibiting increased susceptibility to treatment with NMP, suggesting an enhanced contribution of efflux pumps to antimicrobial resistance in these strains (termed enhanced efflux phenotype [EEP] strains). Only 1/17 EEP strains versus 12/17 non-EEP MDR strains belonged to the ST131 clonal group. Whole-genome sequencing revealed marked differences in efflux-related genes between EEP and control strains, with the majority of notable amino acid substitutions occurring in AcrR, MarR, and SoxR. Quantitative reverse transcription-PCR (qRT-PCR) of multiple efflux-related genes showed significant overexpression of the AcrAB-TolC system in EEP strains, whereas in the remaining strains, we found enhanced expression of alternative efflux proteins. We conclude that a proportion of MDR E. coli strains exhibit an EEP, which is linked to an overexpression of the AcrAB-TolC efflux pump and a distinct array of genomic variations. Members of ST131, although highly successful, are less likely to exhibit the EEP.

Receptors and ion channels expressed on the cell surface ensure proper communication between the cells and the environment. In multicellular organism, stimulus-regulated gene transcription is the basis for communication with the environment allowing individual cells to respond to stimuli such as nutrients, chemical stressors and signaling molecules released by other cells of the organism. Hormones, cytokines, and mitogens bind to receptors and ion channels and induce intracellular signaling cascades involving second messengers, kinases, phosphatases, and changes in the concentration of particular ions. Ultimately, the signaling cascades reach the nucleus. Transcription factors are activated that respond to cellular stimulation and induce changes in gene transcription. Investigating stimulus-transcription coupling combines cell biology with genetics. In this review, we discuss the molecular biology of stimulus-induced transcriptional activators and their responsiveness to extracellular and intracellular signaling molecules and to epigenetic regulators. Stimulus-induced gene expression is measured by several methods, including detection of nuclear translocation of transcription factors, phosphorylation or DNA binding. In this article, we emphasize that the most reliable method to directly measure transcriptional activation involves the use of chromatin-embedded reporter genes.

Background: The prone position (PP) is increasingly used in mechanically ventilated coronavirus disease 2019 (COVID-19) acute respiratory distress syndrome (ARDS) patients. However, studies investigating the influence of the PP are currently lacking in these patients. This is the first study to investigate the influence of the PP on the oxygenation and decarboxylation in COVID-19 patients. Methods: A prospective bicentric study design was used, and in mechanically ventilated COVID-19 patients, PP was indicated from a partial pressure of oxygen in arterial blood (PaO2)/fraction of inspired oxygen (FIO2) ratio of &lt, 200. Patients were left prone for 16 h each. Pressure levels, FIO2, were adjusted to ensure a PaO2 greater than 60 mmHg. Blood gas analyses were performed before (baseline 0.5 h), during (1/2/5.5/9.5/13 h), and after being in the PP (1 h), the circulatory/ventilation parameters were continuously monitored, and lung compliance (LC) was roughly calculated. Responders were defined compared to the baseline value (PaO2/FIO2 ratio increase of ≥15%, partial pressure of carbon dioxide (PaCO2) decrease of ≥2%). Results: 13 patients were included and 36 PP sessions were conducted. Overall, PaO2/FIO2 increased significantly in the PP (p &lt, 0.001). Most PaO2/FIO2 responders (29/36 PP sessions, 77%) were identified 9.5 h after turning prone (14% slow responders), while most PaCO2 responders (15/36 PP sessions, 42%) were identified 13 h after turning prone. A subgroup of patients (interval intubation to PP ≥3 days) showed less PaO2/FIO2 responders (16% vs. 77%). An increase in PaCO2 and minute ventilation in the PP showed a significant negative correlation (p &lt, 0.001). LC (median before the PP = 38 mL/cm H2O, two patients with LC &gt, 80 mL/cm H2O) showed a significant positive correlation with the 28 day survival of patients (p = 0.01). Conclusion: The PP significantly improves oxygenation in COVID-19 ARDS patients. The data suggest that they also benefit most from an early PP. A decrease in minute ventilation may result in fewer PaCO2 responders. LC may be a predictive outcome parameter in COVID-19 patients. Trial registration: Retrospectively registered.

Aspergillus fumigatus is a filamentous fungus which can cause multiple diseases in humans. Allergic broncho-pulmonary aspergillosis (ABPA) is a disease diagnosed primarily in cystic fibrosis patients caused by a severe allergic response often to long-term A. fumigatus colonization in the lungs. Mice develop an allergic response to repeated inhalation of A. fumigatus spores; however, no strains have been identified that can survive long-term in the mouse lung and cause ABPA-like disease. We characterized A. fumigatus strain W72310, which was isolated from the expectorated sputum of an ABPA patient, by whole-genome sequencing and in vitro and in vivo viability assays in comparison to a common reference strain, CEA10. W72310 was resistant to leukocyte-mediated killing and persisted in the mouse lung longer than CEA10, a phenotype that correlated with greater resistance to oxidative stressors, hydrogen peroxide, and menadione, in vitro. In animals both sensitized and challenged with W72310, conidia, but not hyphae, were viable in the lungs for up to 21 days in association with eosinophilic airway inflammation, airway leakage, serum IgE, and mucus production. W72310-sensitized mice that were recall challenged with conidia had increased inflammation, Th1 and Th2 cytokines, and airway leakage compared to controls. Collectively, our studies demonstrate that a unique strain of A. fumigatus resistant to leukocyte killing can persist in the mouse lung in conidial form and elicit features of ABPA-like disease. IMPORTANCE Allergic broncho-pulmonary aspergillosis (ABPA) patients often present with long-term colonization of Aspergillus fumigatus. Current understanding of ABPA pathogenesis has been complicated by a lack of long-term in vivo fungal persistence models. We have identified a clinical isolate of A. fumigatus, W72310, which persists in the murine lung and causes an ABPA-like disease phenotype. Surprisingly, while viable, W72310 showed little to no growth beyond the conidial stage in the lung. This indicates that it is possible that A. fumigatus can cause allergic disease in the lung without any significant hyphal growth. The identification of this strain of A. fumigatus can be used not only to better understand disease pathogenesis of ABPA and potential antifungal treatments but also to identify features of fungal strains that drive long-term fungal persistence in the lung. Consequently, these observations are a step toward helping resolve the long-standing question of when to utilize antifungal therapies in patients with ABPA and fungal allergic-type diseases.

Background Neutropenia is commonly encountered in cancer patients. Recombinant human granulocyte colony-stimulating factor (G-CSF, filgrastim), a cytokine that initiates proliferation and differentiation of mature granulocytes, is widely given to oncology patients to counteract neutropenia, reducing susceptibility to infection. However, the clinical impact of neutropenia and G-CSF use in cancer patients with coronavirus disease 2019 (COVID-19) remains unknown. Methods An observational cohort of 379 actively treated cancer patients with COVID-19 was assembled to investigate links between concurrent neutropenia and G-CSF administration on COVID-19-associated respiratory failure and death. These factors were encoded as time-dependent predictors in an extended Cox model, controlling for age and underlying cancer diagnosis. To determine whether the degree of granulocyte response to G-CSF affected outcomes, the degree of response to G-CSF, based on rise in absolute neutrophil count (ANC) 24 hours after growth factor administration, was also incorporated into a similar Cox model. Results In the setting of active COVID-19 infection, outpatient receipt of G-CSF led to an increased number of hospitalizations (hazard ratio [HR]: 3.54, 95% confidence interval [CI]: 1.25–10.0, P value: .017). Furthermore, among inpatients, G-CSF administration was associated with increased need for high levels of oxygen supplementation and death (HR: 3.56, 95% CI: 1.19–10.2, P value: .024). This effect was predominantly seen in patients that exhibited a high response to G-CSF based on their ANC increase post-G-CSF administration (HR: 7.78, 95% CI: 2.05–27.9, P value: .004). Conclusions The potential risks versus benefits of G-CSF administration should be considered in neutropenic cancer patients with COVID-19, because G-CSF administration may lead to worsening clinical and respiratory status.

Fluorescence-based techniques enable researchers to monitor physiologic processes, specifically fungal cell viability and death, during cellular encounters with the mammalian immune system with single event resolution. By incorporating two independent fluorescent probes in fungal organisms either prior to, or ensuing experimental infection in mice or in cultured leukocytes, it is possible to distinguish and quantify live and killed fungal cells to interrogate genetic, pharmacologic, and cellular determinants that shape host-fungal cell outcomes. This chapter reviews the techniques and applications of fluorescent fungal reporters of viability, with emphasis on the North American endemic dimorphic fungus, Blastomyces dermatitidis.

Colorectal cancer (CRC) is one of the leading causes of cancer death worldwide. There is an urgent need to identify prognostic markers for patients undergoing curative resection of CRC. The detection of circulating tumor cells in peripheral blood is a promising approach to identify high-risk patients with disseminated disease in colorectal cancer. This study aims to evaluate the prognostic relevance of preoperative CTCs using the Cellsearch® system (CS) in patients, who underwent resection with curative intent of different stages (UICC I-IV) of colorectal cancer. Out of 91 Patients who underwent colorectal resection, 68 patients were included in this study. CTC analysis was performed in patients with CRC UICC stages I-IV immediately before surgery. Data were correlated with clinicopathological parameters and patient outcomes. One or more CTCs/7.5 mL were detected in 45.6% (31/68) of patients. CTCs were detected in all stages of the Union of International Cancer Control (UICC), in stage I (1/4, 25%), in stage II (4/12, 33.3%), in stage III (5/19, 26.3%) and in stage IV (21/33, 63.6%). The detection of ≥ 1 CTCs/ 7.5ml correlated to the presence of distant overt metastases (p = 0.014) as well as with shorter progression-free (p = 0.008) and overall survival (p = 0.008). Multivariate analyses showed that the detection of ≥ 1 CTCs/ 7.5ml is an independent prognostic indicator for overall survival (HR, 3.14; 95% CI, 1.18–8.32; p = 0.021). The detection of CTCs is an independent and strong prognostic factor in CRC, which might improve the identification of high-risk patients in future clinical trials.

The gut microbiota produces metabolites that regulate host immunity, thereby impacting disease resistance and susceptibility. The extent to which commensal bacteria reciprocally respond to immune activation, however, remains largely unexplored. Herein, we colonized mice with four anaerobic symbionts and show that acute immune responses result in dramatic transcriptional reprogramming of these commensals with minimal changes in their relative abundance. Transcriptomic changes include induction of stress-response mediators and downregulation of carbohydrate-degrading factors such as polysaccharide utilization loci (PULs). Flagellin and anti-CD3 antibody, two distinct immune stimuli, induced similar transcriptional profiles, suggesting that commensal bacteria detect common effectors or activate shared pathways when facing different host responses. Immune activation altered the intestinal metabolome within 6 hours, decreasing luminal short-chain fatty acid and increasing aromatic metabolite concentrations. Thus, intestinal bacteria, prior to detectable shifts in community composition, respond to acute host immune activation by rapidly changing gene transcription and immunomodulatory metabolite production.

Increased cell proliferation is a hallmark of acute lymphoblastic leukemia (ALL), and genetic alterations driving clonal proliferation have been identified as prognostic factors. To evaluate replicative history and its potential prognostic value, we determined telomere length (TL) in lymphoblasts, B-, and T-lymphocytes, and measured telomerase activity (TA) in leukocytes of patients with ALL. In addition, we evaluated the potential to suppress the in vitro growth of B-ALL cells by the telomerase inhibitor imetelstat. We found a significantly lower TL in lymphoblasts (4.3 kb in pediatric and 2.3 kb in adult patients with ALL) compared to B- and T-lymphocytes (8.0 kb and 8.2 kb in pediatric, and 6.4 kb and 5.5 kb in adult patients with ALL). TA in leukocytes was 3.2 TA/C for pediatric and 0.7 TA/C for adult patients. Notably, patients with high-risk pediatric ALL had a significantly higher TA of 6.6 TA/C compared to non-high-risk patients with 2.2 TA/C. The inhibition of telomerase with imetelstat ex vivo led to significant dose-dependent apoptosis of B-ALL cells. These results suggest that TL reflects clonal expansion and indicate that elevated TA correlates with high-risk pediatric ALL. In addition, telomerase inhibition induces apoptosis of B-ALL cells cultured in vitro. TL and TA might complement established markers for the identification of patients with high-risk ALL. Moreover, TA seems to be an effective therapeutic target, hence, telomerase inhibitors, such as imetelstat, may augment standard ALL treatment.

Impairments in social relationships and awareness are features observed in autism spectrum disorders (ASDs). However, the underlying mechanisms remain poorly understood. Shank2 is a high���confidence ASD candidate gene and localizes primarily to postsynaptic densities (PSDs) of excitatory synapses in the central nervous system (CNS). We show here that loss of Shank2 in mice leads to a lack of social attachment and bonding behavior towards pubs independent of hormonal, cognitive, or sensitive deficits. Shank2���/��� mice display functional changes in nuclei of the social attachment circuit that were most prominent in the medial preoptic area (MPOA) of the hypothalamus. Selective enhancement of MPOA activity by DREADD technology re���established social bonding behavior in Shank2���/��� mice, providing evidence that the identified circuit might be crucial for explaining how social deficits in ASD can arise., publishedVersion

International audience; The global burden of the endemic mycoses (blastomycosis, coccidioidomycosis, emergomycosis, histoplasmosis, paracoccidioidomycosis, sporotrichosis, and talaromycosis) continues to rise yearly and these infectious diseases remain a leading cause of patient morbidity and mortality worldwide. Management of the associated pathogens requires a thorough understanding of the epidemiology, risk factors, diagnostic methods and performance characteristics in different patient populations, and treatment options unique to each infection. Guidance on the management of these infections has the potential to improve prognosis. The recommendations outlined in this Review are part of the "One World, One Guideline" initiative of the European Confederation of Medical Mycology. Experts from 23 countries contributed to the development of these guidelines. The aim of this Review is to provide an up-to-date consensus and practical guidance in clinical decision making, by engaging physicians and scientists involved in various aspects of clinical management.

Fluorescence-based techniques enable researchers to monitor physiologic processes, specifically fungal cell viability and death, during cellular encounters with the mammalian immune system with single event resolution. By incorporating two independent fluorescent probes in fungal organisms either prior to, or ensuing experimental infection in mice or in cultured leukocytes, it is possible to distinguish and quantify live and killed fungal cells to interrogate genetic, pharmacologic, and cellular determinants that shape host–fungal cell outcomes. This chapter reviews the techniques and applications of fluorescent fungal reporters of viability, with emphasis on the filamentous mold Aspergillus fumigatus.

Fructose utilization in Corynebacterium glutamicum starts with its uptake and concomitant phosphorylation via the phosphotransferase system (PTS) to yield intracellular fructose 1-phosphate, which enters glycolysis upon ATP-dependent phosphorylation to fructose 1,6-bisphosphate by 1-phosphofructokinase. This is known to result in a significantly reduced oxidative pentose phosphate pathway (oxPPP) flux on fructose (∼10%) compared to glucose (∼60%). Consequently, the biosynthesis of NADPH demanding products, e.g., L-lysine, by C. glutamicum is largely decreased when fructose is the only carbon source. Previous works reported that fructose is partially utilized via the glucose-specific PTS presumably generating fructose 6-phosphate. This closer proximity to the entry point of the oxPPP might increase oxPPP flux and, consequently, NADPH availability. Here, we generated deletion strains lacking either the fructose-specific PTS or 1-phosphofructokinase activity. We used these strains in short-term evolution experiments on fructose minimal medium and isolated mutant strains, which regained the ability of fast growth on fructose as a sole carbon source. In these fructose mutants, the deletion of the glucose-specific PTS as well as the 6-phosphofructokinase gene, abolished growth, unequivocally showing fructose phosphorylation via glucose-specific PTS to fructose 6-phosphate. Gene sequencing revealed three independent amino acid substitutions in PtsG (M260V, M260T, and P318S). These three PtsG variants mediated faster fructose uptake and utilization compared to native PtsG. In-depth analysis of the effects of fructose utilization via these PtsG variants revealed significantly increased ODs, reduced side-product accumulation, and increased L-lysine production by 50%.

Human mutations and haploinsufficiency of the SHANK family genes are associated with autism spectrum disorders (ASD) and intellectual disability (ID). Complex phenotypes have been also described in all mouse models of Shank mutations and deletions, consistent with the heterogeneity of the human phenotypes. However, the specific role of Shank proteins in synapse and neuronal functions remain to be elucidated. Here, we generated a new mouse model to investigate how simultaneously deletion of Shank1 and Shank3 affects brain development and behavior in mice. Shank1–Shank3 DKO mice showed a low survival rate, a developmental strong reduction in the activation of intracellular signaling pathways involving Akt, S6, ERK1/2, and eEF2 during development and a severe behavioral impairments. Our study suggests that Shank1 and Shank3 proteins are essential to developmentally regulate the activation of Akt and correlated intracellular pathways crucial for mammalian postnatal brain development and synaptic plasticity. Therefore, Akt function might represent a new therapeutic target for enhancing cognitive abilities of syndromic ASD patients.

In April 2020, the QUality Assessment and REProducibility for Instruments and Images in Light Microscopy (QUAREP-LiMi) initiative was formed. This initiative comprises imaging scientists from academia and industry who share a common interest in achieving a better understanding of the performance and limitations of microscopes and improved quality control (QC) in light microscopy. The ultimate goal of the QUAREP-LiMi initiative is to establish a set of common QC standards, guidelines, metadata models, and tools, including detailed protocols, with the ultimate aim of improving reproducible advances in scientific research. This White Paper 1) summarizes the major obstacles identified in the field that motivated the launch of the QUAREP-LiMi initiative; 2) identifies the urgent need to address these obstacles in a grassroots manner, through a community of stakeholders including, researchers, imaging scientists, bioimage analysts, bioimage informatics developers, corporate partners, funding agencies, standards organizations, scientific publishers, and observers of such; 3) outlines the current actions of the QUAREP-LiMi initiative, and 4) proposes future steps that can be taken to improve the dissemination and acceptance of the proposed guidelines to manage QC. To summarize, the principal goal of the QUAREP-LiMi initiative is to improve the overall quality and reproducibility of light microscope image data by introducing broadly accepted standard practices and accurately captured image data metrics., Comment: 17 pages, 3 figures, shortened abstract, Co-Lead Authors: Glyn Nelson and Ulrike Boehm, Corresponding author: Roland Nitschke

Average facial soft-tissue thickness (FSTT) databanks are continuously developed and applied within craniofacial identification. This study considered and tested a subject-specific regression model alternative for estimating the FSTT values for oral midline landmarks using skeletal projection measurements. Measurements were taken from cone-beam computed tomography scans of 100 South African individuals (60 male, 40 female; Mage = 35 years). Regression equations incorporating sex categories were generated. This significantly improved the goodness-of-fit ( r2-value). Validation tests compared the constructed regression models with mean FSTT data collected from this study, existing South African FSTT data, a universal total weighted mean approach with pooled demographic data and collection techniques and a regression model approach that uses bizygomatic width and maximum cranial breadth dimensions. The generated regression equations demonstrated individualised results, presenting a total mean inaccuracy (TMI) of 1.53 mm using dental projection measurements and 1.55 mm using cemento-enamel junction projection measurements. These slightly outperformed most tested mean models (TMI ranged from 1.42 to 4.43 mm), and substantially outperformed the pre-existing regression model approach (TMI = 5.12 mm). The newly devised regressions offer a subject-specific solution to FSTT estimation within a South African population. A continued development in sample size and validation testing may help substantiate its application within craniofacial identification.