Bioreactor-produced iPSCs-derived dopaminergic neuron-containing neural microtissues innervate and normalize rotational bias in a dose-dependent manner in a Parkinson rat model.

A breadth of preclinical studies now support the rationale of pluripotent stem cell-derived cell replacement therapies to alleviate motor symptoms in Parkinsonian patients. Replacement of the primary dysfunctional cell population in the disease, i.e. the A9 dopaminergic neurons, is the major focus of these therapies. To achieve this, most therapeutical approaches involve grafting single-cell suspensions of DA progenitors. However, most cells die during the transplantation process, as cells face anoïkis. One potential solution to address this challenge is to graft solid preparations, i.e. adopting a 3D format. Cryopreserving such a format remains a major hurdle and is not exempt from causing delays in the time to effect, as observed with cryopreserved single-cell DA progenitors. Here, we used a high-throughput cell-encapsulation technology coupled with bioreactors to provide a 3D culture environment enabling the directed differentiation of hiPSCs into neural microtissues. The proper patterning of these neural microtissues into a midbrain identity was confirmed using orthogonal methods, including qPCR, RNAseq, flow cytometry and immunofluorescent microscopy. The efficacy of the neural microtissues was demonstrated in a dose-dependent manner using a Parkinsonian rat model. The survival of the cells was confirmed by post-mortem histological analysis, characterised by the presence of human dopaminergic neurons projecting into the host striatum. The work reported here is the first bioproduction of a cell therapy for Parkinson's disease in a scalable bioreactor, leading to a full behavioural recovery 16 weeks after transplantation using cryopreserved 3D format.

Neurogenic Dysphagia: Systematic Review and Proposal of a Classification System.

OBJECTIVE: Introduction and validation of a phenotypic classification of neurogenic dysphagia based on flexible endoscopic evaluation of swallowing (FEES). METHODS: A systematic literature review was conducted, searching MEDLINE from inception to May 2020 for FEES findings in neurologic diseases of interest. Based on a retrospective analysis of FEES videos in neurologic diseases and considering the results from the review, a classification of neurogenic dysphagia was developed distinguishing different phenotypes. The classification was validated using 1,012 randomly selected FEES videos of patients with various neurologic disorders. Chi-square tests were used to compare the distribution of dysphagia phenotypes between the underlying neurologic disorders. RESULTS: A total of 159 articles were identified, of which 59 were included in the qualitative synthesis. Seven dysphagia phenotypes were identified: (1) "premature bolus spillage" and (2) "delayed swallowing reflex" occurred mainly in stroke, (3) "predominance of residue in the valleculae" was most common in Parkinson disease, (4) "predominance of residue in the piriform sinus" occurred only in myositis, motoneuron disease, and brainstem stroke, (5) "pharyngolaryngeal movement disorder" was found in atypical Parkinsonian syndromes and stroke, (6) "fatigable swallowing weakness" was common in myasthenia gravis, and (7) "complex disorder" with a heterogeneous dysphagia pattern was the leading mechanism in amyotrophic lateral sclerosis. The interrater reliability showed a strong agreement (kappa = 0.84). CONCLUSION: Neurogenic dysphagia is not a symptom, but a multietiologic syndrome with different phenotypic patterns depending on the underlying disease. Dysphagia phenotypes can facilitate differential diagnosis in patients with dysphagia of unclear etiology.

Computationally Empowered Workflow Identifies Novel Covalent Allosteric Binders for KRASG12C.

Due to its frequent mutations in multiple lethal cancers, KRAS is one of the most-studied anticancer targets nowadays. Since the discovery of the druggable allosteric binding site containing a G12C mutation, KRASG12C has been the focus of attention in oncology research. We report here a computationally driven approach aimed at identifying novel and selective KRASG12C covalent inhibitors. The workflow involved initial enumeration of virtual molecules tailored for the KRAS allosteric binding site. Tools such as pharmacophore modeling, docking, and free-energy perturbations were deployed to prioritize the compounds with the best profiles. The synthesized naphthyridinone scaffold showed the ability to react with G12C and inhibit KRASG12C . Analogues were prepared to establish structure-activity relationships, while molecular dynamics simulations and crystallization of the inhibitor-KRASG12C complex highlighted an unprecedented binding mode.

FEES-based assessment of pharyngeal hypesthesia-Proposal and validation of a new test procedure.

BACKGROUND: Intact pharyngeal sensation is essential for a physiological swallowing process, and conversely, pharyngeal hypesthesia can cause dysphagia. This study introduces and validates a diagnostic test to quantify pharyngeal hypesthesia. METHODS: A total of 20 healthy volunteers were included in a prospective study. Flexible endoscopic evaluation of swallowing (FEES) and a sensory test were performed both before and after pharyngeal local anesthesia. To test pharyngeal sensation, a small tube was positioned transnasally in the upper third of the oropharynx with contact to the lateral pharyngeal wall. Increasing volumes of blue-dyed water were injected through the tube, and the latency of swallowing response (LSR) was determined by two independent raters from the endoscopic video recording. Three trials were performed for each administered volume starting with 0.1 mL and increased by 0.1 mL up to 0.5 mL. KEY RESULTS: The average LSR without anesthesia was 2.24 ± 0.80 s at 0.1 mL, 1.79 ± 0.84 s at 0.2 mL, 1.29 ± 0.62 s at 0.3 mL, 1.17 ± 0.41 s at 0.4 mL, and 1.19 ± 0.52 s at 0.5 mL. With anesthesia applied, the average LSR was 2.65 ± 0.62 s at 0.1 mL, 2.64 ± 0.49 s at 0.2 mL, 2.44 ± 0.65 s at 0.3 mL, 2.10 ± 0.80 s at 0.4 mL, and 2.18 ± 0.85 s at 0.5 mL. LSR was significantly longer following anesthesia at 0.2 mL (t = -3.82; P = .001), 0.3 mL (t = -4.65; P < .000), 0.4 mL (t = -5.77; P < .000), and 0.5 mL (t = -3.49; P = .005). CONCLUSION AND INFERENCES: Pharyngeal hypesthesia can be quantified with sensory testing using LSR. Suitable volumes to distinguish between normal and impaired pharyngeal sensation are 0.2 mL, 0.3 mL, 0.4 mL and 0.5 mL. Experimentally induced pharyngeal anesthesia represents a valid model of sensory dysphagia.

Extubation Readiness in Critically Ill Stroke Patients.

Background and Purpose- Predicting safe extubation represents a clinical challenge in acute stroke patients. Classical respiratory weaning criteria have not proven reliable. Concerning the paramount relevance of postextubation dysphagia in this population, criteria related to airway safety seem to perform better, but diagnostic standards are lacking. We compare clinical and instrumental swallowing examination tools to assess extubation readiness and propose a simple Determine Extubation Failure in Severe Stroke score for decision making. Methods- Data of 133 orally intubated acute stroke patients were prospectively collected in this observational study. Classical extubation criteria, a modified semiquantitative airway score, and an oral motor function score were assessed before extubation. A 3-ounce water swallow test and validated 6-point fiberoptic endoscopic dysphagia severity scoring were performed thereafter. Association of demographic and clinical parameters with extubation failure (EF) was investigated. Independent predictors of EF were translated into a point scoring system. Ideal cutoff values were determined by receiver operator characteristics analyses. Results- Patients with EF (24.1% after 24±43 hours) performed worse in all swallowing assessments (P<0.001). Fiberoptic endoscopic dysphagia severity scoring was the only independent predictor of EF (adjusted odds ratio, 4.2; P<0.007) with optimal cutoff ≥5 (sensitivity 84.6% and specificity 76.5%). Restricting regression analysis to parameters collected before extubation, a 4-item Determine Extubation Failure in Severe Stroke score (duration of ventilation, the examination of oral motor function, infratentorial lesion, and stroke severity) was derived. The score demonstrated excellent discrimination (area under the curve 0.89; 95% CI, 0.83-0.95) and calibration (Nagelkerkes R2=0.54) with an ideal cutoff ≥4 (sensitivity: 81.3% and specificity: 78.2%). Conclusions- Risk of EF is strongly correlated with postextubation dysphagia severity in stroke. Fiberoptic endoscopic examination of swallowing best predicts necessity of reintubation but requires a trial of extubation. The Determine Extubation Failure In Severe Stroke score is based on easy to collect clinical data and may guide extubation decision making in critically ill stroke patients.

Discovery of potent SOS1 inhibitors that block RAS activation via disruption of the RAS-SOS1 interaction.

Since the late 1980s, mutations in the RAS genes have been recognized as major oncogenes with a high occurrence rate in human cancers. Such mutations reduce the ability of the small GTPase RAS to hydrolyze GTP, keeping this molecular switch in a constitutively active GTP-bound form that drives, unchecked, oncogenic downstream signaling. One strategy to reduce the levels of active RAS is to target guanine nucleotide exchange factors, which allow RAS to cycle from the inactive GDP-bound state to the active GTP-bound form. Here, we describe the identification of potent and cell-active small-molecule inhibitors which efficiently disrupt the interaction between KRAS and its exchange factor SOS1, a mode of action confirmed by a series of biophysical techniques. The binding sites, mode of action, and selectivity were elucidated using crystal structures of KRASG12C-SOS1, SOS1, and SOS2. By preventing formation of the KRAS-SOS1 complex, these inhibitors block reloading of KRAS with GTP, leading to antiproliferative activity. The final compound 23 (BAY-293) selectively inhibits the KRAS-SOS1 interaction with an IC50 of 21 nM and is a valuable chemical probe for future investigations.

Disulphide-reduced psoriasin is a human apoptosis-inducing broad-spectrum fungicide.

The unexpected resistance of psoriasis lesions to fungal infections suggests local production of an antifungal factor. We purified Trichophyton rubrum-inhibiting activity from lesional psoriasis scale extracts and identified the Cys-reduced form of S100A7/psoriasin (redS100A7) as a principal antifungal factor. redS100A7 inhibits various filamentous fungi, including the mold Aspergillus fumigatus, but not Candida albicans. Antifungal activity was inhibited by Zn(2+), suggesting that redS100A7 interferes with fungal zinc homeostasis. Because S100A7-mutants lacking a single cysteine are no longer antifungals, we hypothesized that redS100A7 is acting as a Zn(2+)-chelator. Immunogold electron microscopy studies revealed that it penetrates fungal cells, implicating possible intracellular actions. In support with our hypothesis, the cell-penetrating Zn(2+)-chelator TPEN was found to function as a broad-spectrum antifungal. Ultrastructural analyses of redS100A7-treated T. rubrum revealed marked signs of apoptosis, suggesting that its mode of action is induction of programmed cell death. TUNEL, SYTOX-green analyses, and caspase-inhibition studies supported this for both T. rubrum and A. fumigatus. Whereas redS100A7 can be generated from oxidized S100A7 by action of thioredoxin or glutathione, elevated redS100A7 levels in fungal skin infection indicate induction of both S100A7 and its reducing agent in vivo. To investigate whether redS100A7 and TPEN are antifungals in vivo, we used a guinea pig tinea pedes model for fungal skin infections and a lethal mouse Aspergillus infection model for lung infection and found antifungal activity in both in vivo animal systems. Thus, selective fungal cell-penetrating Zn(2+)-chelators could be useful as an urgently needed novel antifungal therapeutic, which induces programmed cell death in numerous fungi.

SPINK9 stimulates metalloprotease/EGFR-dependent keratinocyte migration via purinergic receptor activation.

Serine protease inhibitors of the Kazal-type 9 (SPINK9) is a keratinocyte-derived cationic peptide that is found most abundantly in the upper layers of the palmar-plantar epidermis. In vitro, the peptide displays the capacity to inhibit specifically kallikrein-related peptidase 5 (KLK5). Here, we report that cells expressing SPINK9 secrete the peptide constitutively. Recombinant SPINK9 (rSPINK9) provoked transactivation of the EGFR in human keratinocytes, resulting in efficient downstream triggering of cell migration. Transactivation occurred via functional upregulation of a disintegrin and metalloproteases (ADAMs), as evidenced by suppression with a metalloproteinase inhibitor and an EGFR-blocking antibody. SPINK9 preparations isolated from human skin also displayed EGFR-transactivating capacity. The classical purinergic receptor antagonists oxidized ATP and pyridoxalphosphate-6-azophenyl-2',4',-disulfonic acid effectively suppressed EGFR transactivation by rSPINK9, indicating that in analogy to what has recently been reported for the cationic antimicrobial peptides cathelicidin LL-37 and bee venom melittin, purinergic receptors have an essential bridging role in promoting the upregulation of ADAM function by the cationic peptide. SPINK9 could represent an example of how a cationic peptide may subserve multiple and interrelated functions that contribute to the maintenance of the physical and immunological barrier of the skin.

Chemerin is an antimicrobial agent in human epidermis.

Chemerin, a chemoattractant ligand for chemokine-like receptor 1 (CMKLR1) is predicted to share similar tertiary structure with antibacterial cathelicidins. Recombinant chemerin has antimicrobial activity. Here we show that endogenous chemerin is abundant in human epidermis, and that inhibition of bacteria growth by exudates from organ cultures of primary human skin keratinocytes is largely chemerin-dependent. Using a panel of overlapping chemerin-derived synthetic peptides, we demonstrate that the antibacterial activity of chemerin is primarily mediated by Val(66)-Pro(85), which causes direct bacterial lysis. Therefore, chemerin is an antimicrobial agent in human skin.

Assessment of styling performance in hair gels and hair sprays by means of a new two-point stiffness test.

A new two-point bending stiffness method on flat hair strands was developed and validated after application of hair styling gels and hair styling sprays. A special mold was used to align single hair fibers after applying the formulations to the hair. The styling gels used contain different commercially available thickeners and styling polymers, e.g., carbomer, acrylates/beheneth-25 methacrylate copolymer, Polyquaternium-86, PVP, VP/VA copolymers, and VP/methacrylamide/vinylimidazole copolymer. Evaluation of hair sprays was performed after spray application on flat hair strands. Commercially available hair styling resins were used, e.g. acrylates/t-butylacrylamide copolymer, octylacrylamide/acrylates/butylaminoethyl methacrylate copolymer, and VP/VA copolymer (30:70). The new stiffness test method provided the best correlation with practically relevant sensory assessments on hair strands and a panel test in which styling gels were evaluated. However, we did not observe a correlation between the new stiffness method on flat hair strands and practical assessments in hair spray application. We postulate that different polymer/hair composites are responsible for these discrepancies. Hairs on model heads for half-side testing are spot-welded after spray application, while hairs are seam-welded in the stiffness test after alignment of single hair fibers. This alignment is necessary to achieve reproducible results.

High-potential human mesenchymal stem cells.

Bone marrow-derived stromal mesenchymal stem cells (MSCs) have been characterized in vitro by their growth characteristics, the expression of a panel of surface antigens, and their potential to differentiate into mesenchymal lineages. They can be separated by physical methods as well as by immunological or chemical separation or cultivation. Different protocols are used in different laboratories, making the comparison of various reported MSC populations difficult. Here we describe a population of bone marrow-derived adult stem cells that has been separated on a Percoll gradient with low density. It is characterized by an extraordinary high proliferative potential and a conserved phenotype characteristic of MSCs that retain their plutipotentiality in culture, as evidenced by their ability to differentiate into osteo-, chondro-, and adipogenic lineages. Separation of these cells provide an effective and convenient method for rapid expansion of pluripotential human MSCs for clinical use where large amounts of stem cells are needed.

Neurotropic action of androgens: principles, mechanisms and novel targets.

The importance of androgen signaling is well recognized for numerous aspects of central nervous system (CNS) function, ranging from sex-specific organization of neuroendocrine and behavioral circuits to adaptive capacity, resistance and repair. Nonetheless, concepts for the therapeutic use of androgens in neurological and mental disorders are far from being established. This review outlines some critical issues which interfere with decisions on the suitability of androgens as therapeutic agents for CNS conditions. Among these, sex-specific organization of neural substrates and resulting differential responsiveness to endogenous gonadal steroids, convergence of steroid hormone actions on common molecular targets, co-presence of different sex steroid receptors in target neuronal populations, and in situ biotransformation of natural androgens apparently pose the principal obstacles for the characterization of specific neurotropic effects of androgens. Additional important, albeit less explored aspects consist in insufficient knowledge about molecular targets in the CNS which are under exclusive or predominant androgen control. Own experimental data illustrate the variability of pharmacological effects of natural and synthetic androgens on CNS functions of adaptive relevance, such as sexual behavior, anxiety and endocrine responsiveness to stress. Finally, we present results from an analysis of the consequences of aging for the rat brain transcriptome and examination of the influence of androgens on differentially expressed genes with presumable significance in neuropathology.

Inducible and constitutive beta-defensins are differentially expressed in Crohn's disease and ulcerative colitis.

Antimicrobial peptides such as defensins provide nonspecific mucosal defense against a multitude of microorganisms. Recently, it has been shown that luminal bacteria may invade the mucosa in inflammatory bowel diseases, suggesting a defect in innate mucosal immunity. The aim of this study was to investigate the expression of human beta-defensins (HBD) in controls, Crohn's disease (CD), ulcerative colitis (UC), and unspecific inflammation. Up to 4 biopsies were taken from 103 patients (33 controls, 24 with Crohn's disease, 36 with ulcerative colitis, 10 with unspecific colitis). Mucosal mRNA was measured using real-time fluorescence temperature cycler reverse-transcription polymerase chain reaction with primers for HBD-1, HBD-2, HBD-3, tumor necrosis factor alpha, and interleukin 8. Mucosal HBD-1 expression was marginally decreased in both CD and UC. HBD-2 was increased exclusively in UC but not in CD. The expression of the novel defensin HBD-3 was strongly correlated with HBD-2 and also raised predominantly in UC. The expression of both inducible beta-defensins was enhanced in the state of inflammation. Expression of HBD-2 showed a weak correlation with interleukin 8 only in inflamed CD biopsies but not with tumor necrosis factor alpha. The missing induction of both inducible beta-defensins in CD as compared with UC may cause a defect in barrier function that predisposes to bacterial invasion.

The primary role in biologic activity of the neutrophil chemokines IL-8 and GRO-alpha in cultured nasal epithelial cells.

Primary nasal epithelial cells were investigated for their ability to synthesize and deliver neutrophil chemotactic factors (chemokines) following tumor necrosis factor-alpha (TNF-alpha) induction. The chemokines interleukin8 (IL-8), growth-related oncogene-alpha (GRO-alpha), epithelial cell-derived neutrophil attractant-78 (ENA-78), and granulocyte chemotactic protein-2 (GCP-2) have been detected and characterized and shown to have different potencies in the chemotaxis of neutrophils. Cultures of primary nasal epithelial cells were treated with TNF-alpha in concentrations of 20 and 200 ng/ml for 2, 8, 24, and 72 h. The chemokine protein concentrations in the supernatants of the incubations were determined by the ELISA technique. Chemokine mRNA expression in epithelial cells was also measured using the reverse transcriptase-polymerase chain reaction (RT-PCR). The biologic activity of the chemokines was identified using a three-step high-performance liquid chromatography (HPLC) technique, a bioassay involving measurement of neutrophil chemotaxis in a single Boyden chamber. Both the IL-8 and GRO-alpha proteins and their respective mRNA appear to be induced by TNF-alpha in epithelial cells. The chemotactic responsiveness of both GRO-alpha and IL-8 appears to predominate after 24 h incubation with TNF-alpha. The chemokines GCP-2 and ENA-78 were only weakly induced by TNF-alpha. The neutrophil chemokines IL-8 and GRO-alpha were synthesized in nasal epithelial cell culture induced by TNF-alpha in biologically active concentrations of 0.8 ng/ml and 1.42 ng/ml, respectively. It appears that both the IL-8 and GRO-alpha chemokines may contribute to neutrophil tissue migration in sinusitis, whereas GCP-2 and ENA-78 are of secondary importance to the chemotaxis of neutrophils in this condition.