Relationships between conformational and vibrational features of tryptophan characteristic Raman markers.

Tryptophan (Trp) residue provides characteristic vibrational markers to the middle wavenumber spectral region of the Raman spectra recorded from peptides and proteins. In this report, we were particularly interested in eight Trp Raman markers, referred to as Wi (i = 1,…,8). All responsible for pronounced Raman lines, these markers originate from indole moiety, a bicyclic conjugated segment involved in the Trp structure. Numerous investigations have previously attempted to relate the variations observed in the spectral features of these markers to the environmental changes of Trp residues. To emphasize the most important points we can mention (i) the variations in the Raman profile of W4 (∼1360 cm-1) and W5 (∼1340 cm-1), frequently observed as a doublet with variable intensity ratio. These two markers were thought to result from a Fermi-resonance effect between certain planar and nonplanar modes; (ii) the changes observed in the wavenumbers and relative intensities of W4, W7 (∼880 cm-1) and W8 (∼760 cm-1) were supposed to be related to the accessibility of Trp to surrounding water molecules; and (iii) the wavenumber fluctuations of W3 (∼1550 cm-1), taken as a Trp side chain orientational marker. However, some ambiguities still exist regarding the interpretation of these markers, needing further clarification. Herein, upon a joint experimental and theoretical analysis based on a multiconformational approach, attention was paid to the relationships between structural and vibrational features of three indole-containing compounds with increasing structural complexity, i.e., skatole (3-methylindole), tryptophan, and tripeptide Gly-Trp-Gly. This study clearly shows that the existing assignments given to certain Trp Raman markers should be reconsidered, especially those based on the Fermi-resonance origin of W4-W5 (∼1360-1340 cm-1) doublet, as well as the purely environmental dependence of W7 and W8 markers.

Inhibiting the cGAS-STING Pathway in Ulcerative Colitis with Programmable Micelles.

Ulcerative colitis is a chronic condition in which a dysregulated immune response contributes to the acute intestinal inflammation of the colon. Current clinical therapies often exhibit limited efficacy and undesirable side effects. Here, programmable nanomicelles were designed for colitis treatment and loaded with RU.521, an inhibitor of the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway. STING-inhibiting micelles (SIMs) comprise hyaluronic acid-stearic acid conjugates and include a reactive oxygen species (ROS)-responsive thioketal linker. SIMs were designed to selectively accumulate at the site of inflammation and trigger drug release in the presence of ROS. Our in vitro studies in macrophages and in vivo studies in a murine model of colitis demonstrated that SIMs leverage HA-CD44 binding to target sites of inflammation. Oral delivery of SIMs to mice in both preventive and delayed therapeutic models ameliorated colitis's severity by reducing STING expression, suppressing the secretion of proinflammatory cytokines, enabling bodyweight recovery, protecting mice from colon shortening, and restoring colonic epithelium. In vivo end points combined with metabolomics identified key metabolites with a therapeutic role in reducing intestinal and mucosal inflammation. Our findings highlight the significance of programmable delivery platforms that downregulate inflammatory pathways at the intestinal mucosa for managing inflammatory bowel diseases.

Benchmark Outcomes for Distal Pancreatectomy: A Multicenter Prospective Snapshot Study (SPANDISPAN).

BACKGROUND: Improving the quality of care is a priority for health systems to obtain better care and reduce costs. One of the tools for measuring quality is benchmarking (BM). We presented a one-country prospective study of distal pancreatectomies (DP) and determined BM. STUDY DESIGN: Prospective, multicenter, observational snapshot study of DP carried out at Spanish HPB centers for a year (February 22-January 23). HPB centers were defined as high-volume if they performed > 10 DP per year. Inclusion criteria: any scheduled DP for any diagnosis and age > 18 years. The low-risk group was defined following the Durin et al. criteria and major complications as Clavien-Dindo ≥ III. RESULTS: 313 patients from 42 centers and 46.6% from high-volume centers were included. Median DP by center was 7 (IQR: 5-10), median age was 65 years (IQR: 55-74), and 53.4% were female. The surgical approach was minimally invasive (MIS) in 69.3%. Major complications were 21.1%. Postoperative pancreatic fistula (POPF) grade B/C rate was 20.1%, and 90-day mortality was 1.6%. One hundred and forty-three were low-risk group patients (43.8%). Compared with previous BM data, an increasing MIS rate and fewer hospital stay was obtained. CONCLUSION: We present the first determination of DP-BM in a prospective series, obtaining similar results to the previous ones, but our BM values include a shorter hospital stay and a higher percentage of MIS probably related to ERAS protocols and prospective data collection. BM is a multiparameter valuable tool for reporting outcomes, comparing centers, and identifying the points to improve surgical care.

The relationship between the tyrosine residue 850-830 cm-1 Raman doublet intensity ratio and the aromatic side chain χ1 torsion angle.

Tyrosine (Tyr) residue in a peptide chain is characterized by the presence of seven Raman markers, referred to as Yi (i = 1, …, 7), distributed over the middle wavenumber spectral region. Particularly, the changes observed in the relative intensity of Y5 and Y6 markers, appearing as a side by side doublet at ca. 850-830 cm-1, has received a great attention. Primarily assigned to a Fermi-resonance effect between phenol ring planar and nonplanar modes, former density functional theory calculations led us to affiliate the Y5-Y6 doublet to two distinct fundamental modes. Furthermore, despite the previous assumptions, it was evidenced that the reversal of the doublet intensity ratio cannot be solely explained by hydrogen bonding on the phenol hydroxyl group involved in Tyr. Herein, upon analyzing the observed and theoretical data collected from the cationic species of the tripeptide Gly-Tyr-Gly, the crucial effect of the aromatic side chain orientation, especially that of the χ1 torsion angle defined around the CαCß bond, on the Tyr doublet intensity ratio has been evidenced.

Baricitinib or imatinib in hospitalized COVID-19 patients: Results from COVINIB, an exploratory randomized clinical trial.

Baricitinib and imatinib are considered therapies for coronavirus disease 2019 (COVID-19), but their ultimate clinical impact remains to be elucidated, so our objective is to determine whether these kinase inhibitors provide benefit when added to standard care in hospitalized COVID-19 patients. Phase-2, open-label, randomized trial with a pick-the-winner design conducted from September 2020 to June 2021 in a single Spanish center. Hospitalized adults with COVID-19 pneumonia and a symptom duration ≤10 days were assigned to 3 arms: imatinib (400 mg qd, 7 days) plus standard-care, baricitinib (4 mg qd, 7 days) plus standard-care, or standard-care alone. Primary outcome was time to clinical improvement (discharge alive or a reduction of 2 points in an ordinal scale of clinical status) compared on a day-by-day basis to identify differences ≥15% between the most and least favorable groups. Secondary outcomes included oxygenation and ventilatory support requirements, additional therapies administered, all-cause mortality, and safety. One hundred and sixty-five patients analyzed. Predefined criteria for selection of the most advantageous arm were met for baricitinib, but not for imatinib. However, no statistically significant differences were observed in formal analysis, but a trend toward better results in patients receiving baricitinib was found compared to standard care alone (hazard ratio [HR] for clinical improvement: 1.41, 95% confidence intervals [CI]: 0.96-2.06; HR for discontinuing oxygen: 1.46, 95% CI: 0.94-2.28). No differences were found regarding additional therapies administered or safety. Baricitinib plus standard care showed better results for hospitalized COVID-19 patients, being the most advantageous therapeutic strategy among those proposed in this exploratory clinical trial.

Serological response to vaccines against SARS-CoV-2 in patients with inflammatory bowel disease. / Respuesta serológica a las vacunas frente a SARS-CoV-2 en pacientes con enfermedad inflamatoria intestinal.

OBJECTIVE: To study the serological response (SR) and tolerability of COVID-19 vaccine in patients with inflammatory bowel disease (IBD) and its relation with IBD treatment and type of vaccine. METHODS: Observational, cross-sectional study in patients with IBD vaccinated against COVID-19 without known previous infection. SR was analyzed by the determination of IgG antibodies against the S1 subunit. Safety was studied using a questionnaire to identify adverse effects (AE). RESULTS: 280 patients with IBD were included. Type of vaccines: Comirnaty® 68.8%; Spikevax® 10.8%, Vaxzevria® 18.3%, Ad26.COV2-S® 2.2%. 51.3% had AE, being 100% mild. 65% developed IgG antibodies after vaccination. The SR was higher for vaccines with mRNA technology (100% Spikevax®, 68.5% Comirnaty®) compared to those based on adenovirus vector (38.0% Vaxzevria®, 33.3% Ad26.COV2-S®) (P<.001). In the multivariate analysis, SR was related to age (<60 years; OR: 3.8, 95% CI 1.9-7.0; P<.001). The SR in patients with aminosalicylates was 65.4%, 61.4% with immunosuppressants, 65.8% with anti-TNF, and 68.7% with non-anti-TNF biologicals (P=.9). CONCLUSIONS: One third of patients with IBD did not develop antibodies with the initial vaccination against SARS-CoV-2. The SR to vaccines based on mRNA technology was higher, and it was related to age (higher in younger patients). Immunosuppressants and biologicals did not decrease SR. More than half of the patients presented AD, being mild in all cases.

Clostridioides difficile Infection Dysregulates Brain Dopamine Metabolism.

Gastrointestinal illnesses and dysbiosis are among the most common comorbidities reported in patients with neurodevelopmental disorders. The manuscript reports that C. difficile infection (CDI), predisposed by antibiotic-induced gut dysbiosis, causes significant alterations in dopamine metabolism in major dopaminergic brain regions in mice (P < 0.05). In addition, C. difficile infected mice exhibited significantly reduced dopamine beta-hydroxylase (DBH) activity compared to controls (P < 0.01). Moreover, a significantly increased serum concentration of p-cresol, a DBH inhibiting gut metabolite produced by C. difficile, was also observed in C. difficile infected mice (P < 0.05). Therefore, this study suggests a potential mechanistic link between CDI and alterations in the brain dopaminergic axis. Such alterations may plausibly influence the precipitation and aggravation of dopamine dysmetabolism-associated neurologic diseases in infected patients. IMPORTANCE The gut-brain axis is thought to play a significant role in the development and manifestation of neurologic diseases. This study reports significant alterations in the brain dopamine metabolism in mice infected with C. difficile, an important pathogen that overgrows in the gut after prolonged antibiotic therapy. Such alterations in specific brain regions may have an effect on the precipitation or manifestation of neurodevelopmental disorders in humans.

Characterizing the molecular and immune landscape of canine bladder cancer.

Transitional cell carcinoma (TCC), also known as urothelial carcinoma, is the most common bladder cancer in humans and dogs. Approximately one-quarter of human TCCs are muscle-invasive and associated with a high risk of death from metastasis. Canine TCC (cTCC) tumours are typically high-grade and muscle-invasive. Shared similarities in risk factors, histopathology, and clinical presentation suggest that cTCC may serve as a model for the assessment of novel therapeutics that may inform therapies for human muscle-invasive TCC. The goal of this study was to characterize cTCC at the molecular level to identify drivers of oncogenesis and druggable targets. We performed whole exome sequencing (WES) of 11 cTCC tumours and three matched normal samples, identifying 583 variants in protein-coding genes. The most common variant was a V-to-E missense mutation in BRAF, identified in 4 out of 11 samples (36%) via WES. Sanger sequencing identified BRAF variants in 8 out of the same 11 cTCC samples, as well as in 22 out of 32 formalin-fixed paraffin embedded (FFPE) cTCC samples, suggesting an overall prevalence of 70%. RNA-Seq was performed to compare the gene expression profiles of cTCC tumours to normal bladder tissue. cTCC tumours exhibited up-regulation of genes involved in the cell cycle, DNA repair, and antiviral immunity. We also analysed the immune landscape of cTCC using immune gene signatures and immunohistochemical analysis. A subset of tumours had characteristics of a hot tumour microenvironment and exhibited high expression of signatures associated with complete response to PD-1/PD-L1 blockade in human bladder cancer.

Inmunoglobulinas en la Etiopatogenia del Liquen Plano Oral; La Evidencia Carece de Confiabilidad. Revisión Sistemática Exploratoria

RESUMEN: El liquen plano oral es una enfermedad inflamatoria crónica mucocutánea, de etiología desconocida y se asocia principalmente a una respuesta de inmunidad celular. En los años 70, se comienza a involucrar a las inmunoglobulinas en la etiopatogenia de la enfermedad y hasta la fecha la evidencia científica nos ofrece información reducida, muy variada y con resultados discutibles que hacen necesario una detallada evaluación de esta. Hemos encontrado poca investigación (32 artículos desde 1974 hasta el 2020) y la importancia relativa de las revistas donde han sido publicado los estudios es miscelánea (cuartiles 1, 2 ,3, 4 y no indexadas). Los artículos científicos presentan baja calidad metodológica en el 75% (24 artículos). El 25% restante, presenta calidad media (7 artículos) y de estos, solo el 9 % (3 artículos) encontraron diferencia en la presencia de inmunoglobulinas entre los grupos de casos y controles, y un solo artículo presenta calidad metodológica alta (3 %), pero sus resultados indican que no hay información suficiente. Nuestros resultados revelan que la evidencia científica existente en cuanto a la participación de inmunoglobulinas en la etiopatogenia del liquen plano oral carece de confiabilidad por presentar calidad metodológica de baja calidad. Proponemos dejar de pensar en esta posibilidad o bien mejorar sustancialmente la metodología empleada en los futuros estudios.

ABSTRACT: Oral lichen planus is a chronic mucocutaneous inflammatory disease of unknown etiology and is mainly associated with a cellular immune response. In the 1970s, immunoglobulins began to be involved in the etiopathogenesis of the disease and to date the scientific evidence offers us limited and highly varied information with controversial results that require a detailed evaluation of it. We found scarce research (32 articles from 1974 to 2020) and the relative importance of the journals where the studies have been published is miscellaneous (quartile 1, 2, 3, 4 and not indexed). Scientific articles present low methodological quality in 75% (24 articles). The remaining 25% presented medium quality (7 articles) and of these, only 9% (3 articles) found a difference in the presence of immunoglobulins between the groups of cases and controls, and only one article presented high methodological quality (3%), but their results indicate that there is insufficient information. Our results reveal that the existing scientific evidence regarding the participation of immunoglobulins in the etiopathogenesis of oral lichen planus lacks reliability due to its low-quality methodological quality. We propose to stop thinking about this possibility or to substantially improve the methodology used in future studies.

Multiconformational analysis of tripeptides upon consideration of implicit and explicit hydration effects.

During the last two decades, numerous observed data obtained by various physical techniques, also supported by molecular modeling approaches, have highlighted the structuring features of tripeptides, as well as their aggregation properties. Herein, we focus on the structural dynamics of four trimers, i.e., Gly-Gly-Gly, Gly-Ala-Gly, Ala-Ala-Ala and Ala-Phe-Ala, in an aqueous environment. Density functional theory calculations (DFT) were carried out to assess the stability of four types of secondary structures, i.e., ß-strand, polyproline-II (pP-II), α-helix and γ-turn, of which the formation had been described in these tripeptides. Both implicit and explicit hydration effects were analyzed on the conformational and energetic features of trimers. It has been shown that the use of M062X functional (versus B3LYP) improve the stability of intramolecular H-bonds, especially in inverse γ-turn structures, as well as the energetic and conformational equilibrium in all tripeptides. Explicit hydration reflected by the presence of five water molecules around the backbone polar sites (NH3+, N-H, CO and NH2) considerably changes the conformational landscapes of the trimers. Characteristic intramolecular and intermolecular interactions evidenced by the calculations, were emphasized.

Prevalence, Colonization, Epidemiology, and Public Health Significance of Clostridioides difficile in Companion Animals.

Clostridioides difficile, previously Clostrdium difficile, is a major cause of antibiotic-associated enteric disease in humans in hospital settings. Increased incidence of C. difficile infection (CDI) in community settings raises concerns over an alternative source of CDI for humans. The detection of genetically similar and toxigenic C. difficile isolates in companion animals, including asymptomatic pets, suggests the potential role of household pets as a source of community-associated CDI. The close association between companion animals and humans, in addition to the use of similar antibiotics in both species, could provide a selective advantage for the emergence of new C. difficile strains and thus increase the incidental transmission of CDI to humans. Therefore, screening household pets for C. difficile is becoming increasingly important from a public health standpoint and may become a part of routine testing in the future, for the benefit of susceptible or infected individuals within a household. In this review, we analyze available information on prevalence, pathophysiology, epidemiology, and molecular genetics of C. difficile infection, focusing on companion animals and evaluate the risk of pet-borne transmission of CDI as an emerging public health concern. Molecular epidemiological characterization of companion animal C. difficile strains could provide further insights into the interspecies transmission of CDI. The mosaic nature of C. difficile genomes and their susceptibility to horizontal gene transfer may facilitate the inter-mixing of genetic material, which could increase the possibility of the emergence of new community-associated CDI strains. However, detailed genome-wide characterization and comparative genome analysis are warranted to confirm this hypothesis.

Orthodontic Extrusion: Guidelines for Contemporary Clinical Practice.

Orthodontic extrusion (OE) is an orthodontic tooth movement in a coronal direction to modify the tooth position and/or induce changes on the surrounding bone and soft tissue with a therapeutic purpose. Evidence emanating from clinical reports and case series studies indicates that OE is a predictable treatment option to manage a variety of clinical situations. Common indications include traction of impacted teeth, exposure of teeth presenting structural damage to facilitate restorative therapy, treatment of periodontal bony and papillary defects, and implant site development. Unfortunately, there is a paucity of established protocols and guidelines for its application in clinical practice. Controversy exists in regard to the definition of rapid and slow OE, use of circumferential supracrestal fiberotomy, and tooth stabilization protocols during and upon completion of orthodontic movement. This article provides a concise perspective on the topic of OE by discussing key biologic principles and technical aspects that are translated into guidelines for the management of different clinical scenarios.

Structural Analysis of Nonapeptides Derived from Elastin.

Elastin-derived peptides are released from the extracellular matrix remodeling by numerous proteases and seem to regulate many biological processes, notably cancer progression. The canonical elastin peptide is VGVAPG, which harbors the XGXXPG consensus pattern, allowing interaction with the elastin receptor complex located at the surface of cells. Besides these elastokines, another class of peptides has been identified. This group of bioactive elastin peptides presents the XGXPGXGXG consensus sequence, but the reason for their bioactivity remains unexplained. To better understand their nature and structure-function relationships, herein we searched the current databases for this nonapeptide motif and observed that the XGXPGXGXG elastin peptides define a specific group of tandemly repeated patterns. Further, we focused on four tandemly repeated human elastin nonapeptides, i.e., AGIPGLGVG, VGVPGLGVG, AGVPGLGVG, and AGVPGFGAG. These peptides were analyzed by means of optical spectroscopies and molecular dynamics. Ultraviolet-circular dichroism and Raman spectra are consistent with a mixture of ß-turn, ß-strand, and random-chain secondary elements in aqueous media. Quantitative analysis of their conformations suggested that turns corresponded to half of the total population of structural elements, whereas the remaining half were equally distributed between ß-strand and unordered chains. These distributions were confirmed by molecular dynamics simulations. Altogether, our data suggest that these highly dynamic peptides harbor a type II ß-turn located in their central part. We hypothesize that this structural element could explain their specific bioactivity.

Aspartate: An interesting model for analyzing dipole-ion and ion pair interactions through its oppositely charged amine and acid groups.

Anionic species of aspartic acid, Asp- , having a zwitterionic backbone and a deprotonated side chain, appears to be a good example for analyzing dipole-ion and ion pair interactions. Density functional theory calculations were herein performed to investigate the low energy conformers of Asp- embedded in a dielectric continuum modeling an aqueous environment, through a scan of the potential energy as a function of the side chain (χ1 , χ2 ) torsion angles. The most energetically favorable conformers having g+ g- and g- g+ side chain orientations are found to be stabilized by charge-enhanced intramolecular H-bonding involving the positively charged ( NH3+ ) and the two negatively charged (COO- ) groups. These conformers were further used to analyze Asp- + nW clusters (W: water, n = 1 or 3), and Asp- /Asp- pair formation. COO- groups were found to be the most attractive sites for hosting a water molecule (binding energy: -6.0 ± 1.5 kcal/mol), compared to NH3+ groups (binding energy: -4.7 ± 1.1 kcal/mol). Energy separation between g+ g- and g- g+ conformers increases upon explicit hydration. Asp- /Asp- ion pairs, stabilized by the interaction between the NH3+ group of a partner and the COO- group of the other, shows a quite constant binding energy (-8.1 ± 0.2 kcal/mol), whatever the pair type, and the relative orientation of the two interacting partners. This study suggests a first step to achieve a more realistic image of intermolecular interactions in aqueous environment, especially upon increasing concentration. It can also be considered as a preliminary attempt to assess the interactions of the Lys+ …Asp- /Glu- ion pairs stabilizing intra- and interchain interactions in proteins.

Identifying the ErbB/MAPK Signaling Cascade as a Therapeutic Target in Canine Bladder Cancer.

Transitional cell carcinoma (TCC) of the bladder comprises 2% of diagnosed canine cancers. TCC tumors are generally inoperable and unresponsive to traditional chemotherapy, indicating a need for more effective therapies. BRAF, a kinase in the mitogen-activated protein kinase (MAPK) pathway, is mutated in 70% of canine TCCs. In this study, we use BRAF mutant and wild-type TCC cell lines to characterize the role of BRAF mutations in TCC pathogenesis and assess the efficacy of inhibition of the MAPK pathway alone and in combination with other gene targets as a treatment for canine TCC. Analysis of MAPK target gene expression and assessment of extracellular signal-regulated kinase (ERK) 1/2 phosphorylation following serum starvation indicated constitutive MAPK activity in all TCC cell lines. BRAF mutant TCC cell lines were insensitive to the BRAF inhibitor vemurafenib, with IC50 values greater than 5 µM, but exhibited greater sensitivity to a paradox-breaking BRAF inhibitor (IC50: 0.2-1 µM). All TCC cell lines had IC50 values less than 7 nM to the mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor trametinib independent of their BRAF mutation status. ERK1/2 phosphorylation decreased after 6-hour treatments with MAPK inhibitors, but rebounded by 24 hours, suggesting the presence of resistance mechanisms. Microarray analysis identified elevated expression of the ErbB family of receptors and ligands in TCC cell lines. The pan-ErbB inhibitor sapitinib synergized with BRAF inhibition in BRAF mutant Bliley TCC cells and synergized with MEK1/2 inhibition in Bliley and BRAF wild-type Kinsey cells. These findings suggest the potential for combined MAPK and ErbB receptor inhibition as a therapy for canine TCC. SIGNIFICANCE STATEMENT: The results of this study (1) identify a novel combination strategy for canine bladder cancer treatment: targeting the ErbB/MAPK signaling cascade and (2) establish the utility of canine bladder cancer as a naturally-occurring model for human MAPK-driven cancers.

Dynamical Behavior of Somatostatin-14 and Its Cyclic Analogues as Analyzed in Bulk and on Plasmonic Silver Nanoparticles.

Primarily known as the inhibitor of growth hormone release, the role of somatostatin in many other inhibiting activities upon binding to its five G-protein-coupled receptors has been elucidated. Because of the short half-life of somatostatin, a number of synthetic analogues were elaborated for this peptide hormone. Herein, after recalling the main somatostatin therapeutic interests, we present the dynamical behavior of somatostatin-14 and its two currently used synthetic cyclic analogues, octreotide and pasireotide. Physical techniques, such as fluorescence, UV-visible absorption, circular dichroism, Raman spectroscopy, surface-enhanced Raman spectroscopy, and transmission electron microscopy, were jointly used in order to get information on the solution structural features, as well as on the anchoring sites of the three peptides on silver colloids. While somatostatin-14 adopts a rather unordered chain within the submillimolar concentration range, its cyclic analogues were revealed to be ordered, i.e., stabilized either in a type-II' ß-turn (octreotide) or in a face-to-face γ-turn/type-I ß-turn (pasireotide) structure. Nevertheless, a progressive structuring trend was observed in somatostatin-14 upon increasing concentration to the millimolar range. Because of their cationic character, the three peptides have revealed their capability to bind onto negatively charged silver nanoparticles. The high affinity of the peptides toward metallic particles seems to be extremely promising for the elaboration of somatostatin-based functionalized plasmonic nanoparticles that can be used in diagnosis, drug delivery, and therapy.

Naturally Occurring Canine Melanoma as a Predictive Comparative Oncology Model for Human Mucosal and Other Triple Wild-Type Melanomas.

Melanoma remains mostly an untreatable fatal disease despite advances in decoding cancer genomics and developing new therapeutic modalities. Progress in patient care would benefit from additional predictive models germane for human disease mechanisms, tumor heterogeneity, and therapeutic responses. Toward this aim, this review documents comparative aspects of human and naturally occurring canine melanomas. Clinical presentation, pathology, therapies, and genetic alterations are highlighted in the context of current basic and translational research in comparative oncology. Somewhat distinct from sun exposure-related human cutaneous melanomas, there is growing evidence that a variety of gene copy number alterations and protein structure/function mutations play roles in canine melanomas, in circumstances more analogous to human mucosal melanomas and to some extent other melanomas with murine sarcoma viral oncogene homolog B (BRAF), Neuroblastoma RAS Viral (V-Ras) Oncogene Homolog (NRAS), and neurofibromin 1 tumor suppressor NF1 triple wild-type genotype. Gaps in canine genome annotation, as well as an insufficient number and depth of sequences covered, remain considerable barriers to progress and should be collectively addressed. Preclinical approaches can be designed to include canine clinical trials addressing immune modulation as well as combined-targeted inhibition of Rat Sarcoma Superfamily/Mitogen-activated protein kinase (RAS/MAPK) and/or Phosphatidylinositol-3-Kinase/Protein Kinase B/Mammalian target of rapamycin (PI3K/AKT/mTOR) signal transduction, pathways frequently activated in both human and canine melanomas. Future investment should be aimed towards improving understanding of canine melanoma as a predictive preclinical surrogate for human melanoma and for mutually benefiting these uniquely co-dependent species.

Calcium-dependent disorder-to-order transitions are central to the secretion and folding of the CyaA toxin of Bordetella pertussis, the causative agent of whooping cough.

The adenylate cyclase toxin (CyaA) plays an essential role in the early stages of respiratory tract colonization by Bordetella pertussis, the causative agent of whooping cough. Once secreted, CyaA invades eukaryotic cells, leading to cell death. The cell intoxication process involves a unique mechanism of translocation of the CyaA catalytic domain directly across the plasma membrane of the target cell. Herein, we review our recent results describing how calcium is involved in several steps of this intoxication process. In conditions mimicking the low calcium environment of the crowded bacterial cytosol, we show that the C-terminal, calcium-binding Repeat-in-ToXin (RTX) domain of CyaA, RD, is an extended, intrinsically disordered polypeptide chain with a significant level of local, secondary structure elements, appropriately sized for transport through the narrow channel of the secretion system. Upon secretion, the high calcium concentration in the extracellular milieu induces the refolding of RD, which likely acts as a scaffold to favor the refolding of the upstream domains of the full-length protein. Due to the presence of hydrophobic regions, CyaA is prone to aggregate into multimeric forms in vitro, in the absence of a chaotropic agent. We have recently defined the experimental conditions required for CyaA folding, comprising both calcium binding and molecular confinement. These parameters are critical for CyaA folding into a stable, monomeric and functional form. The monomeric, calcium-loaded (holo) toxin exhibits efficient liposome permeabilization and hemolytic activities in vitro, even in a fully calcium-free environment. By contrast, the toxin requires sub-millimolar calcium concentrations in solution to translocate its catalytic domain across the plasma membrane, indicating that free calcium in solution is actively involved in the CyaA toxin translocation process. Overall, this data demonstrates the remarkable adaptation of bacterial RTX toxins to the diversity of calcium concentrations it is exposed to in the successive environments encountered in the course of the intoxication process.

Privileged hydration sites in aromatic side chains: effect on conformational equilibrium.

Water interaction with peptide chains is one of the key structure stabilizing factors in an aqueous environment. Because of its strong polar character, water can bind to both anionic and cationic sites via electrostatic interactions. It can also act as a hydrogen-bond donor or acceptor according to its interactions with different polar groups in the backbone and side chains of peptides and proteins. Based on density functional theory calculations, the present report aims at illustrating the most energetically favorable interaction sites of aromatic side chains of phenylalanine, tyrosine, tryptophan, and histidine (neutral and protonated species) with surrounding water molecules. It was shown that beyond the strong interactions occurring between water and the aromatic ring acceptor/donor sites, such as O-H, N-H and -N[double bond, length as m-dash] groups, weaker interactions with π-electron clouds should also be considered. The latter type of binding, hereafter referred to as Hwπ interaction, involves one of the water hydrogen atoms (Hw) pointing toward the aromatic ring. Upon comparison between the theoretical data obtained from a purely implicit hydration model, i.e. a polarized solvent continuum, and those collected from a mixture of implicit and explicit hydration models, it has been shown that the explicit water molecule binding to aromatic rings affects the relative energies of the rotamers generated by the two side chain torsion angles (χ1 and χ2).