Discovery of the First-in-Class Inhibitors of Hypoxia Up-Regulated Protein†1 (HYOU1) Suppressing Pathogenic Fibroblast Activation.

Fibroblasts are key regulators of inflammation, fibrosis, and cancer. Targeting their activation in these complex diseases has emerged as a novel strategy to restore tissue homeostasis. Here, we present a multidisciplinary lead discovery approach to identify and optimize small molecule inhibitors of pathogenic fibroblast activation. The study encompasses medicinal chemistry, molecular phenotyping assays, chemoproteomics, bulk RNA-sequencing analysis, target validation experiments, and chemical absorption, distribution, metabolism, excretion and toxicity (ADMET)/pharmacokinetic (PK)/in vivo evaluation. The parallel synthesis employed for the production of the new benzamide derivatives enabled us to a) pinpoint key structural elements of the scaffold that provide potent fibroblast-deactivating effects in cells, b) discriminate atoms or groups that favor or disfavor a desirable ADMET profile, and c) identify metabolic "hot spots". Furthermore, we report the discovery of the first-in-class inhibitor leads for hypoxia up-regulated protein 1 (HYOU1), a member of the heat shock protein 70 (HSP70) family often associated with cellular stress responses, particularly under hypoxic conditions. Targeting HYOU1 may therefore represent a potentially novel strategy to modulate fibroblast activation and treat chronic inflammatory and fibrotic disorders.

Successful treatment of complex regional pain syndrome type 1 of upper limb with cryoneurolysis of the stellate ganglion: A rare case report.

Complex regional pain syndrome type I (CRPS I) is a multifactorial condition that is related to neural damage, and leads to chronic neuropathic pain. Due to its multifactorial nature, there are many methods that have been recruited for its management. Most of them limit the symptoms to some degree, but the most effective of them so far seems to be blockade of the sympathetic innervation of the region. We present the case of a 71-year-old woman, with an upper limb fracture which was treated conservatively, and later on developed CRPS type I. Analgesic medications and pregabalin were ineffective in controlling her symptoms. Blockade of the stellate ganglion was performed with local anesthetic and dexamethasone. After the stellate ganglion blockade was marked as successful in relieving her symptoms, cryoneurolysis was performed which was very effective. There are not many cases that have been treated with cryoneurolysis, and this is what makes this case interesting.

Combination of subtherapeutic anti-TNF dose with dasatinib restores clinical and molecular arthritogenic profiles better than standard anti-TNF treatment.

BACKGROUND: New medications for Rheumatoid Arthritis (RA) have emerged in the last decades, including Disease Modifying Antirheumatic Drugs (DMARDs) and biologics. However, there is no known cure, since a significant proportion of patients remain or become non-responders to current therapies. The development of new mode-of-action treatment schemes involving combination therapies could prove successful for the treatment of a greater number of RA patients. METHODS: We investigated the effect of the Tyrosine Kinase inhibitors (TKIs) dasatinib and bosutinib, on the human TNF-dependent Tg197 arthritis mouse model. The inhibitors were administered either as a monotherapy or in combination with a subtherapeutic dose of anti-hTNF biologics and their therapeutic effect was assessed clinically, histopathologically as well as via gene expression analysis and was compared to that of an efficient TNF monotherapy. RESULTS: Dasatinib and, to a lesser extent, bosutinib inhibited the production of TNF and proinflammatory chemokines from arthritogenic synovial fibroblasts. Dasatinib, but not bosutinib, also ameliorated significantly and in a dose-dependent manner both the clinical and histopathological signs of Tg197 arthritis. Combination of dasatinib with a subtherapeutic dose of anti-hTNF biologic agents, resulted in a synergistic inhibitory effect abolishing all arthritis symptoms. Gene expression analysis of whole joint tissue of Tg197 mice revealed that the combination of dasatinib with a low subtherapeutic dose of Infliximab most efficiently restores the pathogenic gene expression profile to that of the healthy state compared to either treatment administered as a monotherapy. CONCLUSION: Our findings show that dasatinib exhibits a therapeutic effect in TNF-driven arthritis and can act in synergy with a subtherapeutic anti-hTNF dose to effectively treat the clinical and histopathological signs of the pathology. The combination of dasatinib and anti-hTNF exhibits a distinct mode of action in restoring the arthritogenic gene signature to that of a healthy profile. Potential clinical applications of combination therapies with kinase inhibitors and anti-TNF agents may provide an interesting alternative to high-dose anti-hTNF monotherapy and increase the number of patients responding to treatment.

In Silico Identification and Evaluation of Natural Products as Potential Tumor Necrosis Factor Function Inhibitors Using Advanced Enalos Asclepios KNIME Nodes.

Tumor necrosis factor (TNF) is a regulator of several chronic inflammatory diseases, such as rheumatoid arthritis. Although anti-TNF biologics have been used in clinic, they render several drawbacks, such as patients' progressive immunodeficiency and loss of response, high cost, and intravenous administration. In order to find new potential anti-TNF small molecule inhibitors, we employed an in silico approach, aiming to find natural products, analogs of Ampelopsin H, a compound that blocks the formation of TNF active trimer. Two out of nine commercially available compounds tested, Nepalensinol B and Miyabenol A, efficiently reduced TNF-induced cytotoxicity in L929 cells and production of chemokines in mice joints' synovial fibroblasts, while Nepalensinol B also abolished TNF-TNFR1 binding in non-toxic concentrations. The binding mode of the compounds was further investigated by molecular dynamics and free energy calculation studies, using and advancing the Enalos Asclepios pipeline. Conclusively, we propose that Nepalensinol B, characterized by the lowest free energy of binding and by a higher number of hydrogen bonds with TNF, qualifies as a potential lead compound for TNF inhibitors' drug development. Finally, the upgraded Enalos Asclepios pipeline can be used for improved identification of new therapeutics against TNF-mediated chronic inflammatory diseases, providing state-of-the-art insight on their binding mode.

Repurposing the antipsychotic drug amisulpride for targeting synovial fibroblast activation in arthritis.

Synovial fibroblasts (SFs) are key pathogenic drivers in rheumatoid arthritis (RA). Their in vivo activation by TNF is sufficient to orchestrate full arthritic pathogenesis in animal models, and TNF blockade proved efficacious for a high percentage of patients with RA albeit coinducing rare but serious side effects. Aiming to find new potent therapeutics, we applied the L1000CDS2 search engine, to repurpose drugs that could reverse the pathogenic expression signature of arthritogenic human TNF-transgenic (hTNFtg) SFs. We identified a neuroleptic drug, namely amisulpride, which reduced SFs' inflammatory potential while decreasing the clinical score of hTNFtg polyarthritis. Notably, we found that amisulpride function was neither through its known targets dopamine receptors D2 and D3 and serotonin receptor 7 nor through TNF-TNF receptor I binding inhibition. Through a click chemistry approach, potentially novel targets of amisulpride were identified, which were further validated to repress hTNFtg SFs' inflammatory potential ex vivo (Ascc3 and Sec62), while phosphoproteomics analysis revealed that treatment altered important fibroblast activation pathways, such as adhesion. Thus, amisulpride could prove beneficial to patients experiencing RA and the often-accompanying comorbid dysthymia, reducing SF pathogenicity along with its antidepressive activity, serving further as a "lead" compound for the development of novel therapeutics against fibroblast activation.

Polyclonal Endemicity of Carbapenemase-Producing Klebsiella pneumoniae in ICUs of a Greek Tertiary Care Hospital.

Carbapenemase-producing Klebsiella pneumoniae (CPKP) emerged in Greece in 2002 and became endemic thereafter. Driven by a notable variability in the phenotypic testing results for carbapenemase production in K. pneumoniae isolates from the intensive care units (ICUs) of our hospital, we performed a study to assess the molecular epidemiology of CPKP isolated between 2016 and 2019 using pulse-field gel electrophoresis (PFGE) including isolates recovered from 165 single patients. We investigated the molecular relatedness among strains recovered from rectal surveillance cultures and from respective subsequent infections due to CPKP in the same individual (48/165 cases). For the optimal interpretation of our findings, we carried out a systematic review regarding the clonality of CPKP isolated from clinical samples in ICUs in Europe. In our study, we identified 128 distinguishable pulsotypes and 17 clusters that indicated extended dissemination of CPKP within the hospital ICU setting throughout the study period. Among the clinical isolates, 122 harbored KPC genes (74%), 2 harbored KPC+NDM (1.2%), 38 harbored NDM (23%), 1 harbored NDM+OXA-48 (0.6%), 1 harbored NDM+VIM (0.6%) and 1 harbored the VIM (0.6%) gene. Multiple CPKP strains in our hospital have achieved sustained transmission. The polyclonal endemicity of CPKP presents a further threat for the selection of pathogens resistant to last-resort antimicrobial agents.

Factors underlying denial of and disbelief in COVID-19.

OBJECTIVE: To investigate factors that influence or promote disbelief and negative attitudes toward COVID-19. METHODS: This was cross-sectional study involving 544 males and females ≥ 18 years of age in Greece between December of 2020 and January of 2021. All participants were informed about the purpose of the study, protection of anonymity, and volunteer participation. Participants completed an online anonymous 40-item questionnaire. Analysis of data included the identification of correlations and use of t-tests and ANOVA. RESULTS: The level of knowledge regarding COVID-19 transmission routes, manifestations, and prevention was high in our sample. Women appeared to have a more positive attitude toward COVID-19 prevention and management than did men (p = 0.032 and p = 0.018, respectively). Younger people (18-30 years of age) seemed to deny the validity of scientific data and mass media reports about ways to deal with the pandemic more commonly than did those > 30 years of age (p = 0.003 and p = 0.001, respectively). People who resided in cities more commonly believed in scientific announcements than did those living in villages (p = 0.029). CONCLUSIONS: In order to minimize cases of denial of and disbelief in COVID-19 and to promote vaccination, a series of actions are required. Governments should implement a series of measures to contain the disease, taking into consideration the psychological and social aspects of those policies.

Evaluation of the "AMR Direct Flow Chip Kit" DNA microarray for detecting antimicrobial resistance genes directly from rectal and nasopharyngeal clinical samples upon ICU admission.

INTRODUCTION: Prompt detection of antibiotic resistance genes in healthcare institutions is of utmost importance in tackling the spread of multi-drug resistant micro-organisms. We evaluated the Antimicrobial Resistance (AMR) Direct Flow Chip Kit versus phenotypic screening assays for rectal and nasopharyngeal specimens upon ICU admission. METHODS: A total of 184 dual specimens (92 rectal and 92 nasopharyngeal swabs) from 92 patients were collected from 11/2017 to 8/2018. All swabs were subjected to both AMR and phenotypic tests according to their origin. The degree of agreement of the two methods was assessed by the kappa coefficient. RESULTS: The kappa coefficient showed perfect agreement for MRSA, ESBLs, oxacillinases and vancomycin resistance genes (1.000, p<0.01) and very good agreement for mecA-positive CoNS, KPC-carbapenemases and metallo-beta-lactamases (0.870, p<0.01; 0.864, p<0.01; and 0.912, p<0.01, respectively). CONCLUSION: The AMR Direct Flow Chip Kit is a useful alternative to phenotypic testing for rapid detection of resistance markers.

In Silico Discovery of Plant-Origin Natural Product Inhibitors of Tumor Necrosis Factor (TNF) and Receptor Activator of NF-κB Ligand (RANKL).

An in silico drug discovery pipeline for the virtual screening of plant-origin natural products (NPs) was developed to explore new direct inhibitors of TNF and its close relative receptor activator of nuclear factor kappa-B ligand (RANKL), both representing attractive therapeutic targets for many chronic inflammatory conditions. Direct TNF inhibition through identification of potent small molecules is a highly desired goal; however, it is often hampered by severe limitations. Our approach yielded a priority list of 15 NPs as potential direct TNF inhibitors that were subsequently tested in vitro against TNF and RANKL. We thus identified two potent direct inhibitors of TNF function with low micromolar IC50 values and minimal toxicity even at high concentrations. Most importantly, one of them (A11) was proved to be a dual inhibitor of both TNF and RANKL. Extended molecular dynamics simulations with the fully automated EnalosMD suite rationalized the mode of action of the compounds at the molecular level. To our knowledge, these compounds constitute the first NP TNF inhibitors, one of which being the first NP small-molecule dual inhibitor of TNF and RANKL, and could serve as lead compounds for the development of novel treatments for inflammatory and autoimmune diseases.

Factors underlying denial of and disbelief in COVID-19 / Fatores subjacentes à negação e descrença em relação à COVID-19

ABSTRACT Objective: To investigate factors that influence or promote disbelief and negative attitudes toward COVID-19. Methods: This was cross-sectional study involving 544 males and females ≥ 18 years of age in Greece between December of 2020 and January of 2021. All participants were informed about the purpose of the study, protection of anonymity, and volunteer participation. Participants completed an online anonymous 40-item questionnaire. Analysis of data included the identification of correlations and use of t-tests and ANOVA. Results: The level of knowledge regarding COVID-19 transmission routes, manifestations, and prevention was high in our sample. Women appeared to have a more positive attitude toward COVID-19 prevention and management than did men (p = 0.032 and p = 0.018, respectively). Younger people (18-30 years of age) seemed to deny the validity of scientific data and mass media reports about ways to deal with the pandemic more commonly than did those > 30 years of age (p = 0.003 and p = 0.001, respectively). People who resided in cities more commonly believed in scientific announcements than did those living in villages (p = 0.029). Conclusions: In order to minimize cases of denial of and disbelief in COVID-19 and to promote vaccination, a series of actions are required. Governments should implement a series of measures to contain the disease, taking into consideration the psychological and social aspects of those policies.

RESUMO Objetivo: Investigar fatores que influenciam ou promovem a descrença e atitudes negativas em relação à COVID-19. Métodos: Estudo transversal envolvendo 544 homens e mulheres com ≥ 18 anos de idade na Grécia entre dezembro de 2020 e janeiro de 2021. Todos os participantes foram informados sobre o objetivo do estudo, proteção do anonimato e participação voluntária. Os participantes responderam a um questionário on-line anônimo com 40 itens. A análise dos dados incluiu a identificação de correlações e o uso de testes t e ANOVA. Resultados: O nível de conhecimento sobre as vias de transmissão, manifestações e prevenção da COVID-19 foi alto em nossa amostra. As mulheres pareceram ter uma atitude mais positiva em relação à prevenção e manejo da COVID-19 do que os homens (p = 0,032 e p = 0,018, respectivamente). As pessoas mais jovens (18-30 anos de idade) pareceram negar mais a validade dos dados científicos e reportagens da mídia sobre as formas de lidar com a pandemia do que aquelas com > 30 anos de idade (p = 0,003 e p = 0,001, respectivamente). As pessoas que residiam em cidades grandes acreditavam mais nos pronunciamentos científicos do que as que residiam em vilas (p = 0,029). Conclusões: Para minimizar o número de casos de negação e descrença em relação à COVID-19 e promover a vacinação, é necessária uma série de ações. Os governos devem implementar uma série de medidas para conter a doença, levando em consideração os aspectos psicológicos e sociais dessas políticas.

Evaluation of the “AMR Direct Flow Chip Kit” DNA microarray for detecting antimicrobial resistance genes directly from rectal and nasopharyngeal clinical samples upon ICU admission / Evaluación de la micromatriz de ADN «AMR Direct Flow Chip Kit» para la detección de genes de resistencia antimicrobiana directamente de muestras clínicas rectales y nasofaríngeas en el momento del ingreso en la UCI

Introduction: Prompt detection of antibiotic resistance genes in healthcare institutions is of utmost importance in tackling the spread of multi-drug resistant micro-organisms. We evaluated the Antimicrobial Resistance (AMR) Direct Flow Chip Kit versus phenotypic screening assays for rectal and nasopharyngeal specimens upon ICU admission. Methods: A total of 184 dual specimens (92 rectal and 92 nasopharyngeal swabs) from 92 patients were collected from 11/2017 to 8/2018. All swabs were subjected to both AMR and phenotypic tests according to their origin. The degree of agreement of the two methods was assessed by the kappa coefficient. Results: The kappa coefficient showed perfect agreement for MRSA, ESBLs, oxacillinases and vancomycin resistance genes (1.000, p<0.01) and very good agreement for mecA-positive CoNS, KPC-carbapenemases and metallo-beta-lactamases (0.870, p<0.01; 0.864, p<0.01; and 0.912, p<0.01, respectively). Conclusion: The AMR Direct Flow Chip Kit is a useful alternative to phenotypic testing for rapid detection of resistance markers.(AU)

Introducción: La detección rápida de genes de resistencia a antibióticos en instituciones de salud es de importancia extrema para abordar la propagación de microorganismos multi-resistentes. Evaluamos el Antimicrobial Resistance Direct Flow Chip (AMR) versus los ensayos de detección fenotípica para muestras rectales y nasofaríngeas al ingreso en la UCI. Métodos: Se recogieron 184 muestras duales (92 rectales y 92 nasofaríngeos) de 92 pacientes durante 11/2017-8/2018. Todos los hisopos se sometieron a pruebas de AMR y fenotípicas según su fuente. El grado de acuerdo de los 2 métodos fue evaluado por el coeficiente kappa. Resultados: El coeficiente kappa mostró una concordancia perfecta para MRSA, ESBL, oxacilinasas y para genes de resistencia a vancomicina (1.000, p<0,01) y muy buena concordancia para CoNS mecA positivos, carbapenemasas KPC y metalo-beta-lactamasas (0,870, p<0,01; 0,864, p<0,01 y 0,912, p<0,01, respectivamente). Conclusión: El AMR es una alternativa útil a las pruebas fenotípicas para la detección rápida de marcadores de resistencia.(AU)

Objective definition of rosette shape variation using a combined computer vision and data mining approach.

Computer-vision based measurements of phenotypic variation have implications for crop improvement and food security because they are intrinsically objective. It should be possible therefore to use such approaches to select robust genotypes. However, plants are morphologically complex and identification of meaningful traits from automatically acquired image data is not straightforward. Bespoke algorithms can be designed to capture and/or quantitate specific features but this approach is inflexible and is not generally applicable to a wide range of traits. In this paper, we have used industry-standard computer vision techniques to extract a wide range of features from images of genetically diverse Arabidopsis rosettes growing under non-stimulated conditions, and then used statistical analysis to identify those features that provide good discrimination between ecotypes. This analysis indicates that almost all the observed shape variation can be described by 5 principal components. We describe an easily implemented pipeline including image segmentation, feature extraction and statistical analysis. This pipeline provides a cost-effective and inherently scalable method to parameterise and analyse variation in rosette shape. The acquisition of images does not require any specialised equipment and the computer routines for image processing and data analysis have been implemented using open source software. Source code for data analysis is written using the R package. The equations to calculate image descriptors have been also provided.