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We describe a rare case of severe primary spondylitis caused by Streptococcus gordonii in a 45-year-old immunocompetent woman with no relevant comorbidities. The surgical site infection arose after a L4-L5 microdiscectomy and resulted in severe clinical disability. Allegations of possible negligence as the cause prompted a forensic review to clarify the original source and transmission of this uncommon pathogen, which dismissed its cause as due to malpractice during treatment., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: The Lung Ultrasound (LUS) is routinely used as a point-of-care imaging tool in Emergency Department (ED) and its role in COVID-19 is being studied. The Lung UltraSound Score (LUSS) is a semi quantitative score of lung damage severity. Alongside instrumental diagnostic, the PaO2/FiO2 (P/F) ratio, obtained from arterial blood gas analysis, is the index used to assess the severity of the acute respiratory distress syndrome (ARDS), according to the Berlin definition., Objectives: The primary objective of the study was to evaluate a possible correlation between the LUSS score and the P/F Ratio, obtained from the arterial sampling in COVID-19 positive patients., Materials and Methods: This was a cross-perspective monocentric observational study and it was carried out in the Emergency Department of the "AOU delle Marche" (Ancona, Italy), from 1 January 2023 to 28 February 2023. The study foresaw, once the patient was admitted to the ED, the execution of the LUS exam and the subsequent calculation of the LUSS score., Results: The sample selected for the study was of 158 patients. The proportion of LUSS ≤4 was statistically higher in those with a P/F >300 (76.2%), compared to those with a P/F ≤300 (13.2%). On the other end, the proportion of LUSS >4 was lower in those who have P/F >300 (23.8%), while it was higher in those who have P/F ≤300 (86.8%). Those patients with a LUSS >4 were 1.76 (95% CI: 1.57 - 1.99) times more likely to have a P/F ≤300, compared to those with LUSS ≤4. The Odds Ratio of having a P/F ≤300 value in those achieving a LUSS >4, compared to those achieving a LUSS ≤4, was 21.0 (95% CI: 8.4 - 52.4). The study identified pO2, Hb and dichotomous LUSS as predictors of the level of P/F ≤300 or P/F >300., Discussion: We found that the LUSS score defined by our study was closely related to the P/F ratio COVID-19 positive patients. Our study presented provides evidence on the potential rule of the LUSS for detecting the stage of lung impairment and the need for oxygen therapy in COVID-19 positive patients.

Low-grade chronic inflammation is a hallmark of ageing, associated with impaired tissue function and disease development. However, how cell-intrinsic and -extrinsic factors collectively establish this phenotype, termed inflammaging, remains poorly understood. We addressed this question in the mouse intestinal epithelium, using mouse organoid cultures to dissect stem cell-intrinsic and -extrinsic sources of inflammaging. At the single-cell level, we found that inflammaging is established differently along the crypt-villus axis, with aged intestinal stem cells (ISCs) strongly upregulating major histocompatibility complex class II (MHC-II) genes. Importantly, the inflammaging phenotype was stably propagated by aged ISCs in organoid cultures and associated with increased chromatin accessibility at inflammation-associated loci in vivo and ex vivo, indicating cell-intrinsic inflammatory memory. Mechanistically, we show that the expression of inflammatory genes is dependent on STAT1 signaling. Together, our data identify that intestinal inflammaging in mice is promoted by a cell-intrinsic mechanism, stably propagated by ISCs, and associated with a disbalance in immune homeostasis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Over the last decades, PCR and molecular cloning have profoundly impacted various biological areas, from basic to pharmaceutical sciences. Presented in this study is a simple and step-by-step protocol that uses PCR to recover a poor-quality ligase product. In fact, a classic step that can be problematic in typical recombinant DNA manipulations can be the recovery of a product from a T4 DNA ligase reaction between two or more suitably prepared DNA fragments (sticky ends, blunt ends, TA cloning, etc.). This reaction can result in poor yields of the ligation product, due to various causes, mainly the preparation of the DNA fragments, and the poor yield can severely invalidate all subsequent steps. To overcome this problem, we designed a pair of PCR primers to amplify the entire ligase product into satisfactory amount. Of course, high-fidelity DNA polymerase must be used to obtain a faithful copy of the DNA of interest. The fragment thus amplified can then be inserted into a suitable vector and propagated by bacterial transformation. We applied this procedure to modify a synthetic gene by adding a His-Tag to its 5' end, and to insert this new construct into an expression cassette. This last step was achieved by employing a PCR cloning system. In our practical example, comprehensive PCR-based protocol with important tips were introduced. This methodological paper can serve as a roadmap for biologists who want to quickly/fully exploit the potential of the PCR-cloning to get desired constructs., (© 2013-2023 The Journal of Biological Methods, All rights reserved.)

Peroxidases are widespread key antioxidant enzymes that catalyse the oxidation of electron donor substrates in parallel with the decomposition of H 2 O 2 . In this work, a novel tomato peroxidase, named SAAP2, was isolated from MicroTom cell cultures, purified, and characterised. The enzyme was identified with 64% sequence coverage as the leprx21 gene product (suberization-associated anionic peroxidase 2-like) from Solanum lycopersicum, 334 amino acids long. Compared to other plant peroxidases, SAAP2 was more active at elevated temperatures, with the optimal temperature and pH at 90 °C and 5.0, respectively. Furthermore, the enzyme retained more than 80% of its maximal activity over the range of 70-80 °C and the presence of NaCl (1.0-4.5 M). It also exhibited broad pH versatility (65% relative activity over the pH range 2.0-7.0), acid-tolerance (80% residual activity after 22 h at pH 2.0-7.0), high thermostability (50% residual activity after 2 h at 80 °C) and proteolytic resistance. SAAP2 exhibited exceptional resistance under thermo-acidic conditions compared to the horseradish peroxidase benchmark, suggesting that it may find potential applications as a supplement or anti-pollution agent in the food industry., (© 2023. Springer Nature Limited.)

Background: Breakthrough SARS-CoV-2 infections following vaccination against COVID-19 are of international concern. Patients with cancer have been observed to have worse outcomes associated with COVID-19 during the pandemic. We sought to evaluate the clinical characteristics and outcomes of patients with cancer who developed breakthrough SARS-CoV-2 infections after 2 or 3 doses of mRNA vaccines., Methods: We evaluated the clinical characteristics of patients with cancer who developed breakthrough infections using data from the multi-institutional COVID-19 and Cancer Consortium (CCC19; NCT04354701). Analysis was restricted to patients with laboratory-confirmed SARS-CoV-2 diagnosed in 2021 or 2022, to allow for a contemporary unvaccinated control population; potential differences were evaluated using a multivariable logistic regression model after inverse probability of treatment weighting to adjust for potential baseline confounding variables. Adjusted odds ratios (aOR) and 95% confidence intervals (CI) are reported. The primary endpoint was 30-day mortality, with key secondary endpoints of hospitalization and ICU and/or mechanical ventilation (ICU/MV)., Findings: The analysis included 2486 patients, of which 564 and 385 had received 2 or 3 doses of an mRNA vaccine prior to infection, respectively. Hematologic malignancies and recent receipt of systemic anti-neoplastic therapy were more frequent among vaccinated patients. Vaccination was associated with improved outcomes: in the primary analysis, 2 doses (aOR: 0.62, 95% CI: 0.44-0.88) and 3 doses (aOR: 0.20, 95% CI: 0.11-0.36) were associated with decreased 30-day mortality. There were similar findings for the key secondary endpoints of ICU/MV (aOR: 0.60, 95% CI: 0.45-0.82 and 0.37, 95% CI: 0.24-0.58) and hospitalization (aOR: 0.60, 95% CI: 0.48-0.75 and 0.35, 95% CI: 0.26-0.46) for 2 and 3 doses, respectively. Importantly, Black patients had higher rates of hospitalization (aOR: 1.47, 95% CI: 1.12-1.92), and Hispanic patients presented with higher rates of ICU/MV (aOR: 1.61, 95% CI: 1.06-2.44)., Interpretation: Vaccination against COVID-19, especially with additional doses, is a fundamental strategy in the prevention of adverse outcomes including death, among patients with cancer., Funding: This study was partly supported by grants from the National Cancer Institute grant number P30 CA068485 to C-YH, YS, SM, JLW; T32-CA236621 and P30-CA046592 to C.R.F; CTSA 2UL1TR001425-05A1 to TMW-D; ACS/FHI Real-World Data Impact Award, P50 MD017341-01, R21 CA242044-01A1, Susan G. Komen Leadership Grant Hunt to MKA. REDCap is developed and supported by Vanderbilt Institute for Clinical and Translational Research grant support (UL1 TR000445 from NCATS/NIH)., Competing Interests: TKC reports grants, personal fees and non-financial support from Merck, BMS, Exelixis, Astra Zeneca, Eli Lilly, Eisai, Novartis, GSK, Pfizer, EMD Serono; stocks in Pionyr, Tempest, outside the submitted work; In addition, TKC reports patent: pending International Patent Application No. PCT/US2018/12209, entitled “PBRM1 Biomarkers Predictive of Anti-Immune Checkpoint Response,” filed January 3, 2018, claiming priority to U.S. Provisional Patent Application No. 62/445,094, filed January 11, 2017; pending International Patent Application No. PCT/US2018/058430, entitled “Biomarkers of Clinical Response and Benefit to Immune Checkpoint Inhibitor Therapy,” filed October 31, 2018, claiming priority to U.S. Provisional Patent Application No. 62/581,175, filed November 3, 2017; TKC sits on National Comprehensive Cancer Network kidney panel. CL reports grants from Genentech/ImCore. ZB reports non-financial support from Bristol Myers Squibb, grants from Genentech/ImCore, personal fees from UpToDate, outside the submitted work. ALS reports non-financial support from Astellas and Pfizer outside the submitted work. GdLL reports personal fees from Boehringer Ingelheim, Pfizer, AstraZeneca; grants from AstraZeneca, Merck Sharp & Dohme, EMD Serono, AstraZeneca, Blueprint Medicines, Tesaro, Bavarian Nordic, Novartis, G1 Therapeutics, Adaptimmune, BMS, GSK, Abbvie, Rgenix, Pfizer, Roche, Genentech, Eli Lilly, Janssen; personal fees from Boehringer Ingelheim, Pfizer, E.R. Squibb Sons, LLC, Janssen; all outside the submitted work. CH reports grants from Merck, Bayer, Genentech, AstraZeneca, Bausch Health; Consulting fees from Tempus, Genzyme, EMD Sorono, payment or honoraria from OncLive/MJH Life Sciences, support for attending meetings and/or travel from Merck, participation on a data safety monitoring or advisory board of Henry Ford Cancer Institute, Hoosier Cancer Research Network; Leadership or fiduciary role in Wayne County Medical Society of Southeast Michigan; Stock or stock options in Johnson and Johnson, all outside the submitted work. EAG reports Consulting fees from Alexion Inc, Picnic Health, AbbVie, CTI Biopharma, Genentech Inc., Novartis, Celgene/Bristol Myers-Squibb, Takeda oncology, Taiho Oncology and Research Funding from Genentech Inc, Astex Pharmaceuticals, and BluePrint Medicines, outside the submitted work. SH reports grants/research supports from ASCO TAPUR, Astellas; honoraria or consultation fees from Sanofi, Aveo Oncology, outside the submitted work. SB reports Consulting fees from BMS, Exelexis, Eisai, Pfizer, Myovant, SeaGen; Payment or honoraria from Exelexis, Eisai, BMS; Participation on a Data Safety Monitoring Board or Advisory Board from SeaGen, Pfizer, Myovant; Stock or stock options in Natera; outside the submitted work. MJ reports grants from AstraZeneca, Pfizer; Eisai, personal fees from Seagen, Sanofi, outside the submitted work. NTNG reports personal fees from Novocure, outside the submitted work. RRM reports Advisory board/consultant—Aveo, AstraZeneca, Bayer, Bristol Myers Squib, Calithera, Caris, Dendreon, Exelixis, Janssen, Merck, Myovant, Novartis, Pfizer, Sanofi, Sorrento therapeutics, Pfizer, Tempus, Vividion, unrelated to this work. SMR reports advisory for Roche, Janssen, Sanofi, and EUSA Pharma, unrelated to this work. PV reports institutional research funding from Sanofi; stocks or stock options in Novavax, Biontech. HAZ acknowledges support from Georgia NCORP. CRF reports grants from Merck Foundation, grants from NCCN/Pfizer, grants from National Cancer Institute, other from National Cancer Institute, other from Patient-Centered Outcomes Research Institute, outside the submitted work. SM reports support from National Cancer Institute, and Intl Assoc. for the Study of Lung Cancer during the conduct of the study; and personal fees from National Geographic outside the submitted work. DF reports Grants or contracts from Merck, Viracor, Astellas; Support for attending meetings and/or travel from Viracor; outside the submitted work. JLW reports grants from NIH during the conduct of the study; personal fees from Roche, Westat, Flatiron Health, Melax Tech, IBM Watson Health, ownership of HemOnc.org LLC, grants from AACR; outside the submitted work. TMW-D reports grants from BMS, Merck & Co, GSK/Tesaro, Janssen; personal fees from Exicure, Shattuck Labs, SITC, Merck & Co, Caris Life Sciences, outside the submitted work. C-YH, SRKS, CJ, LBW, UW, TEO'C, OAP, EJK, HS, RN, TKN, RQ, MP, BHM, SADP, KR, BF, DYR, MKA, HS, DRR, YS, and SB have nothing to disclose. The content is solely the responsibility of the authors and does not necessarily represent the US Food and Drug Administration official views or policies., (© 2023 The Author(s).)

To reduce the mortality of COVID-19 older patients, clear criteria to predict in-hospital mortality are urgently needed. Here, we aimed to evaluate the performance of selected routine laboratory biomarkers in improving the prediction of in-hospital mortality in 641 consecutive COVID-19 geriatric patients (mean age 86.6 ± 6.8) who were hospitalized at the INRCA hospital (Ancona, Italy). Thirty-four percent of the enrolled patients were deceased during the in-hospital stay. The percentage of severely frail patients, assessed with the Clinical Frailty Scale, was significantly increased in deceased patients compared to the survived ones. The age-adjusted Charlson comorbidity index (CCI) score was not significantly associated with an increased risk of death. Among the routine parameters, neutrophilia, eosinopenia, lymphopenia, neutrophil-to-lymphocyte ratio (NLR), C-reactive protein, procalcitonin, IL-6, and NT-proBNP showed the highest predictive values. The fully adjusted Cox regressions models confirmed that high neutrophil %, NLR, derived NLR (dNLR), platelet-to-lymphocyte ratio (PLR), and low lymphocyte count, eosinophil %, and lymphocyte-to-monocyte ratio (LMR) were the best predictors of in-hospital mortality, independently from age, gender, and other potential confounders. Overall, our results strongly support the use of routine parameters, including complete blood count, in geriatric patients to predict COVID-19 in-hospital mortality, independent from baseline comorbidities and frailty., (Copyright © 2022 Elsevier B.V. All rights reserved.)

In this paper, the efficiency of the carbonic anhydrase (CA) enzyme in accelerating the hydration of CO 2 is evaluated using a measurement system which consists of a vessel in which a gaseous flow of mixtures of nitrogen and CO 2 is bubbled into water or water solutions containing a known quantity of CA enzyme. The pH value of the solution and the CO 2 concentration at the measurement system gas exhaust are continuously monitored. The measured CO 2 level allows for assessing the quantity of CO 2 , which, subtracted from the gaseous phase, is dissolved into the liquid phase and/or hydrated to bicarbonate. The measurement procedure consists of inducing a transient and observing and modelling the different kinetics involved in the steady-state recovery with and without CA. The main contribution of this work is exploiting dynamical system theory and chemical kinetics modelling for interpreting measurement results for characterising the activity of CA enzymes. The data for model fitting are obtained from a standard bioreactor, in principle equal to standard two-phase bioreactors described in the literature, in which two different techniques can be used to move the process itself away from the steady-state, inducing transients.

Coumarins are widely diffused secondary metabolites possessing a plethora of biological activities. It has been established that coumarins represent a peculiar class of human carbonic anhydrase (hCA) inhibitors having a distinct mechanism of action involving a non-classical binding with amino acid residues paving the entrance of hCA catalytic site. Herein, we report the synthesis of a small series of new coumarin derivatives 7-11 , 15 , 17 prepared via classical Pechmann condensation starting from resorcinol derivatives and suitable β-ketoesters. The evaluation of inhibitory activity revealed that these compounds possessed nanomolar affinity and high selectivity towards tumour-associated hCA IX and XII over cytosolic hCA I and hCA II isoforms. To investigate the binding mode of these new coumarin-inspired inhibitors, the most active compounds 10 and 17 were docked within hCA XII catalytic cleft.

There is significant interest in increasing the microalgal efficiency for producing high-quality products that are commonly used as food additives in nutraceuticals. Some natural substances that can be extracted from algae include lipids, carbohydrates, proteins, carotenoids, long-chain polyunsaturated fatty acids, and vitamins. Generally, microalgal photoautotrophic growth can be maximised by optimising CO 2 biofixation, and by adding sodium bicarbonate and specific bacteria to the microalgal culture. Recently, to enhance CO 2 biofixation, a thermostable carbonic anhydrase (SspCA) encoded by the genome of the bacterium Sulfurihydrogenibium yellowstonense has been heterologously expressed and immobilised on the surfaces of bacteria. Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous metalloenzymes, which catalyse the physiologically reversible reaction of carbon dioxide hydration to bicarbonate and protons: CO 2 + H 2 O ⇄ HCO 3 - + H + . Herein, we demonstrate for the first time that the fragments of bacterial membranes containing immobilised SspCA (M-SspCA) on their surfaces can be doped into the microalgal culture of the green unicellular alga, Chlorella sorokiniana , to significantly enhance the biomass, photosynthetic activity, carotenoids production, and CA activity by this alga. These results are of biotechnological interest because C. sorokiniana is widely used in many different areas, including photosynthesis research, human pharmaceutical production, aquaculture-based food production, and wastewater treatment.

A primary strategy to combat antimicrobial resistance is the identification of novel therapeutic targets and anti-infectives with alternative mechanisms of action. The inhibition of the metalloenzymes carbonic anhydrases (CAs, EC 4.2.1.1) from pathogens (bacteria, fungi, and protozoa) was shown to produce an impairment of the microorganism growth and virulence. As phosphonamidates have been recently validated as human α-CA inhibitors (CAIs) and no phosphorus-based zinc-binding group have been assessed to date against β-class CAs, herein we report an inhibition study with this class of compounds against β-CAs from pathogenic bacteria, fungi, and protozoa. Our data suggest that phosphonamidates are among the CAIs with the best selectivity for β-class over human isozymes, making them interesting leads for the development of new anti-infectives.

A β-carbonic anhydrase (CA, EC 4.2.1.1) from the widespread bacterium Escherichia coli (EcoCAβ), encoded by the CynT2 gene, has been investigated for its catalytic properties and enzymatic activation by a panel of amino acids and amines. EcoCAβ showed a significant catalytic activity for the hydration of CO 2 to bicarbonate and a proton, with a kinetic constant k cat s 5 s - and a Michaelis-Menten constant K s from 2.76 to 10.7 µM. L-His, 2-pyridyl-methylamine, L-Asn and L-Gln were relatively weak activators (K M s from 36.0 to 49.5 µM). D-His, L- and D-Phe, L- and D-Trp, D-Tyr, histamine, dopamine, 2-(aminoethyl)pyridine/piperazine/morpholine, L-Asp, L- and D-Glu have K s from 11.3 to 23.7 µM. Endogenous CA activators may play a role in bacterial virulence and colonisation of the host.A s from 2.76 to 10.7 µM. L-His, 2-pyridyl-methylamine, L-Asn and L-Gln were relatively weak activators (K A s from 36.0 to 49.5 µM). D-His, L- and D-Phe, L- and D-Trp, D-Tyr, histamine, dopamine, 2-(aminoethyl)pyridine/piperazine/morpholine, L-Asp, L- and D-Glu have K A s from 11.3 to 23.7 µM. Endogenous CA activators may play a role in bacterial virulence and colonisation of the host.

Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous metalloenzymes involved in biosynthetic processes, transport, supply, and balance of CO 2 /HCO 3 - into the cell. In Bacteria, CAs avoid the depletion of the dissolved CO 2 /HCO 3 - from the cell, providing them to the central metabolism that is compromised without the CA activity. The involvement of CAs in the survival, pathogenicity, and virulence of several bacterial pathogenic species is recent. Here, we report the kinetic properties of the recombinant γ -CA (EcoCA γ ) encoded in the genome of Escherichia coli . EcoCA γ is an excellent catalyst for the physiological CO 2  s cat of 5.7 × 10 5  s -1 and k cat /K M s 6  M -1 s -1 . The EcoCA γ inhibition profile with a broad series of known CA inhibitors, the substituted benzene-sulphonamides, and clinically licenced drugs was explored. Benzolamide showed a K I lower than 100 nM. Our study reinforces the hypothesis that the synthesis of new drugs capable of interfering selectively with the bacterial CA activity, avoiding the inhibition of the human α -CAs, is achievable and may lead to novel antibacterials.

The inhibition of δ- and η-class carbonic anhydrases (CAs; EC 4.2.1.1) was poorly investigated so far. Only one δ-CA, TweCA from the diatom Thalassiosira weissflogii, and one η-CA, PfCA, from Plasmodium falciparum , have been cloned and characterised to date. To enrich δ- and η-CAs inhibition profiles, a panel of 22 phenols was investigated for TweCA and PfCA inhibition. Some derivatives showed effective, sub-micromolar inhibition of TweCA (K I s 0.81-65.4 µM) and PfCA (K I s 0.62-78.7 µM). A subset of compounds demonstrated a significant selectivity for the target CAs over the human physiologically relevant ones. This study promotes the identification of new potent and selective inhibitors of TweCA and PfCA , which could be considered as leads for finding molecular probes in the study of carbon fixation processes (in which TweCA and orthologue enzymes are involved) or drug candidates in the treatment of malaria.

The carbonic anhydrases (CAs, EC 4.2.1.1) catalyse a simple but physiologically crucial reversible reaction, the carbon dioxide hydration with the production of bicarbonate and protons. In the last years, and especially, to the rapid emergence of the bacterial antibiotic resistance that is occurring worldwide, the understanding of the function of bacterial CAs has increased significantly. Recently, a new CA-class (ι-CA) was discovered in the marine diatom T. pseudonana . It has been reported that bacterial genomes may contain genes with relevant homology to the diatom ι-class CA. Still, the catalytic activity of the enzyme encoded by the gene was not investigated. Thus, herein, for the first time, we cloned, expressed, and purified the recombinant bacterial ι-CA (acronym BteCAι) identified in the genome of Burkholderia territorii . The recombinant BteCAι resulted in a good catalyst for the hydration of CO 2 to bicarbonate and protons, with a k cat of 3.0 × 10 5  s -1 and k cat /K M of 3.9 × 10 7  M -1 s -1 , and is also sensitive to inhibition by the sulphonamide acetazolamide. Furthermore, with the aid of the protonography, it has been demonstrated that BteCAι can be present as a dimer. This result is corroborated by the construction of a molecular model of BteCAι, which showed that the enzyme is formed by two equivalent monomers having a structure similar to a butterfly.

Acidity, hypoxia and increased release of exosomes are severe phenotypes of tumours. The regulation of pH in tumours involves the interaction of several proteins, including the carbonic anhydrases which catalyze the formation of bicarbonate and protons from carbon dioxide and water. Among CA isoforms, CA IX is over-expressed in a large number of solid tumours, conferring to cancer cells a survival advantage in hypoxic and acidic microenvironment, but there isn't evidence that CA IX expression could have a real clinical impact. Therefore, in this study for the first time the expression and activity of CA IX have been investigated in the plasmatic exosomes obtained from patients with prostate carcinoma (PCa). For this purpose, the study was performed through different methodological approaches, such as NTA, western blot analysis, enzyme activity assay, Nanoscale flow cytometry, ELISA, confocal microscopy. The results showed that PCa exosomes significantly overexpressed CA IX levels and related activity as compared to healthy donors. Furthermore, CA IX expression and activity were correlated to the exosome intraluminal pH, demonstrating for the first time that PCa exosomes are acidic. Our data suggest the possible use of the exosomal CA IX expression and activity as a biomarker of cancer progression in PCa.

A series of urea/thiourea substituted benzoxaboroles was investigated for the inhibition of the three carbonic anhydrases encoded by Vibrio cholerae (VchCAα, VchCAβ, and VchCAγ). In particular, benzoxaborole derivatives were here first assayed for the inhibition of a γ-class CA, extending the panel of CA classes that benzoxaboroles efficiently target beyond α and β. Inhibition profiles demonstrated that VchCAα was significantly more inhibited compared to VchCAγ and, in turn, more efficiently modulated than VchCAβ. Among the many selective benzoxaborole ligands detected against VchCAα over the off-target hCA II, compound 18 , a p -NO 2 -phenylthiourea derivative, even exhibited a fully selective inhibition profile against the three VchCAs over hCA II. A comprehensive ligand/target interaction study was performed in silico for all three VchCA isoforms providing the first molecular modeling investigation with inhibitors of a γ-class CA to the best of our knowledge. The present study reinforces the rationale behind the use of benzoxaboroles as innovative antibacterial agents with a new mechanism of action, furnishing suggestions for the rational design of new potent and selective inhibitors targeting V. cholerae CAs over human off-target ones., Competing Interests: The authors declare no competing financial interest.

Carbonic anhydrases from Vibrio cholerae (VchCAs) play a significant role in bacterial pathophysiological processes. Therefore, their inhibition leads to a reduction of gene expression virulence and bacterial growth impairment. Herein, we report the first ligand-based pharmacophore model as a computational tool to study selective inhibitors of the β-class of VchCA. By a virtual screening on a collection of sulfonamides, we retrieved 9 compounds that were synthesized and evaluated for their inhibitory effects against VchCAβ as well as α- and γ-classes of VchCAs and selectivity over human ubiquitous isoforms hCA I and II. Notably, all tested compounds were active inhibitors of VchCAs. The N -(4-sulfamoylbenzyl)-[1,1'-biphenyl]-4-carboxamide ( 20e ) stood out as the most exciting inhibitor toward the β-class ( K i = 95.6 nM), also showing a low affinity against the tested human isoforms. By applying docking procedures, we described the binding mode of the inhibitor 20e within the catalytic cavity of the modeled open conformation of VchCAβ., Competing Interests: The authors declare no competing financial interest.

Proteins are relevant antimicrobial drug targets, and among them, enzymes represent a significant group, since most of them catalyze reactions essential for supporting the central metabolism, or are necessary for the pathogen vitality. Genomic exploration of pathogenic and non-pathogenic microorganisms has revealed genes encoding for a superfamily of metalloenzymes, known as carbonic anhydrases (CAs, EC 4.2.1.1). CAs catalyze the physiologically crucial reversible reaction of the carbon dioxide hydration to bicarbonate and protons. Herein, we investigated the sulfonamide inhibition profile of the recombinant β -CA (CynT2) identified in the genome of the Gram-negative bacterium Escherichia coli . This biocatalyst is indispensable for the growth of the microbe at atmospheric pCO 2 . Surprisingly, this enzyme has not been investigated for its inhibition with any class of CA inhibitors. Here, we show that CynT2 was strongly inhibited by some substituted benzene-sulfonamides and the clinically used inhibitor sulpiride (K I s in the range of 82-97 nM). This study may be relevant for identifying novel CA inhibitors, as well as for another essential part of the drug discovery pipeline, such as the structure-activity relationship for this class of enzyme inhibitors.

The interconversion of CO 2 and HCO 3 - is catalyzed by a superfamily of metalloenzymes, known as carbonic anhydrases (CAs, EC 4.2.1.1), which maintain the equilibrium between dissolved inorganic CO 2 and HCO 3 - . In the genome of Escherichia coli , a Gram-negative bacterium typically colonizing the lower intestine of warm-blooded organisms, the cyn operon gene includes the CynT gene, encoding for a β-CA, and CynS gene, encoding for the cyanase. CynT (β-CA) prevents the depletion of the cellular bicarbonate, which is further used in the reaction catalyzed by cyanase. A second β-CA (CynT2 or Can or yadF), as well as a γ and ι-CAs were also identified in the E. coli genome. CynT2 is essential for bacterial growth at atmospheric CO 2 concentration. Here, we characterized the kinetic properties and the anion inhibition profiles of recombinant CynT2. The enzyme showed a good activity for the physiological CO 2 s cat = 5.3 × 10 5 s -1 and k cat /K M s 7 M -1 s -1 . Sulfamide, sulfamate, phenylboronic acid, phenylarsonic acid, and diethyldithiocarbamate were the most effective CynT2 inhibitors (K I = 2.5 to 84 µM). The anions allowed for a detailed understanding of the interaction of inhibitors with the amino acid residues surrounding the catalytic pocket of the enzyme and may be used as leads for the design of more efficient and specific inhibitors.

Carbonic anhydrase (CA) is a zinc enzyme that catalyzes the reversible conversion of carbon dioxide to bicarbonate and proton. Currently, CA inhibitors are widely used as antiglaucoma, anticancer, and anti-obesity drugs and for the treatment of neurological disorders. Recently, the potential use of CA inhibitors to fight infections caused by protozoa, fungi, and bacteria has emerged as a new research line. In this article, the X-ray crystal structure of β-CA from Burkholderia pseudomallei was reported. The X-ray crystal structure of this new enzyme was solved at 2.7 Å resolution, revealing a tetrameric type II β-CA with a "closed" active site in which the zinc is tetrahedrally coordinated to Cys46, Asp48, His102, and Cys105. B. pseudomallei is known to encode at least two CAs, a β-CA, and a γ-CA. These proteins, playing a pivotal role in its life cycle and pathogenicity, offer a novel therapeutic opportunity to obtain antibiotics with a different mechanism of action. Furthermore, the new structure can provide a clear view of the β-CA mechanism of action and the possibility to find selective inhibitors for this class of CAs.

A series of 2-thio- and 2-seleno-acetamides bearing the benzenesulfonamide moiety were evaluated as Carbonic Anhydrase (CA, EC 4.2.1.1) inhibitors against different pathogenic bacteria such as the Vibrio cholerae (VchCA-α and VchCA-β), Burkholderia pseudomallei (BpsCA-β and BpsCA-γ), Mycobacterium tuberculosis (Rv3723-β) and the Salmonella enterica serovar Typhimurium (StCA2-β). The molecules represent interesting leads worth developing as innovative antibacterial agents since they possess new mechanism of action and isoform selectivity preferentially against the bacterial expressed CAs. The identification of potent and selective inhibitors of bacterial CAs may lead to tools also useful for deciphering the physiological role(s) of such proteins.

The critical CO 2 hydration reaction to bicarbonate and protons is catalyzed by carbonic anhydrases (CAs, EC 4.2.1.1). Their physiological role is to assist the transport of the CO 2 and HCO 3 - at the cellular level, which will not be ensured by the low velocity of the uncatalyzed reaction. CA inhibition may impair the growth of microorganisms. In the yeasts, Candida albicans and Malassezia globosa , the activity of the unique β-CA identified in their genomes was demonstrated to be essential for growth of the pathogen. Here, we decided to investigate the sulfonamide inhibition profile of the homologous β-CA (MreCA) identified in the genome of Malassezia restricta, an opportunistic pathogen triggering dandruff and seborrheic dermatitis. Among 40 investigated derivatives, the best MreCA sulfonamide inhibitors were dorzolamide, brinzolamide, indisulam, valdecoxib, sulthiam, and acetazolamide (K I < 1.0 μM). The MreCA inhibition profile was different from those of the homologous enzyme from Malassezia globosa (MgCA) and the human isoenzymes (hCA I and hCA II). These results might be useful to for designing CA inhibitor scaffolds that may selectively inhibit the dandruff-producing fungi.

Famotidine, an antiulcer drug belonging to the H 2 antagonists class of pharmacological agents, was recently shown to potently inhibit human (h) and bacterial carbonic anhydrases (CAs, EC 4.2.1.1). We investigated the inhibitory effects of famotidine against all classes of CAs from the pathogenic bacteria Vibrio cholerae, Burkholderia pseudomallei and Mycobacterium tuberculosis Rv3273 β-CA, as well as the CAs from the nonpathogenic bacteria/cyanobacteria Sulfurihydrogenibium yellowstonensis, S. azorense, Pseudoalteromonas haloplanktis, Colwellia psychrerythraea and Nostoc commune. The δ- and ζ-CAs from the diatom Thalassiosira weissflogii, the fungal enzymes from Cryptococcus neoformans, Candida glabrata and Malassezia globosa, as well as the protozoan enzymes from Trypanosoma cruzi and Plasmodium falciparum, were also investigated. Anopheles gambiae β-CA was also investigated. All these enzymes were effectively inhibited by famotidine, with affinities between the low nanomolar to the micromolar range. The best inhibition was observed against C. glabrata β-CA and TweCAζ, with K I s ranging between 13.6 and 22.1 nM.

A series of sixteen benzenesulfonamide derivatives has been synthesised and tested as inhibitors of Vibrio cholerae carbonic anhydrase (CA) enzymes, belonging to α -CA, β -CA, and γ -CA classes (VchCA α , VchCA β , and VchCA γ ). The determined K i values were compared to those of selected human CA isoforms (hCA I and hCA II). Structure-affinity relationship analysis highlighted that all tested compounds proved to be active inhibitors of VchCA α at nanomolar concentration. The VchCA β activity was lower to respect inhibitory efficacy toward VchCA α , whereas, these benzenesulfonamide derivatives failed to inhibit VchCA γ . Interestingly, compound 7e combined the best activity toward VchCA α and VchCA β . In order to obtain a model for binding mode of our inhibitors toward bacterial CAs, we carried out docking simulations by using the available crystal structures of VchCA β .

The genome of Helicobacter pylori encodes for carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the α- and β-CA classes, which together with urease, have a pivotal role in the acid acclimation of the microorganism within the human stomach. Recently, in the exoproteome of H. pylori, a CA with no indication of the corresponding class was identified. Here, using the protonography and the mass spectrometry, a CA belonging to the α-class was detected in the outer membrane vesicles (OMVs) generated by planktonic and biofilm phenotypes of four H. pylori strains. The amount of this metalloenzyme was higher in the planktonic OMVs (pOMVs) than in the biofilm OMVs (bOMVs). Furthermore, the content of α-CA increases over time in the pOMVs. The identification of the α-CA in pOMVs and bOMVs might shed new light on the role of this enzyme in the colonization, survival, persistence, and pathogenesis of H. pylori.

γ-Class carbonic anhydrases (CAs; EC 4.2.1.1) lack of extended inhibition characterization in comparison to α- and β-class isozymes. For this reason, a panel of 22 phenols was investigated here for the inhibition of the γ-CAs from the pathogenic bacteria Burkholderia pseudomallei (BpsCAγ), Porphyromonas gingivalis (PgiCA), Vibrio cholerae (VchCAγ) and from the antarctic bacteria Pseudoalteromonas haloplanktis (PhaCAγ) and Colwellia psychrerythraea (CpsCAγ). The exploration of the chemical space around the main phenolic group led to the discovery of a number of such derivatives showing effective, sometimes sub-micromolar inhibition against BpsCAγ (K I s 0.45-8.6 µM), PgiCA (K I s 0.36-9.8 µM) and VchCAγ (K I s 0.47-9.6 µM). A subset of compounds even demonstrated a significant selectivity for the target γ-CAs over the human physiologically most relevant isoform CA II. This study enriches the inhibitory profiles database for γ-class CAs and promotes the identification of new potent and selective inhibitors against bacterial isoforms over human off-target ones. These agents are of remarkable interest and importance in the search of novel, worldwide required, antibiotic agents possessing alternative mechanisms of action as a strategy to overcome the spread to antimicrobic resistance., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The activation of the β-class carbonic anhydrases (CAs, EC 4.2.1.1) from the bacteria Brucella suis and Francisella tularensis with amine and amino acids was investigated. BsuCA 1 was sensitive to activation with amino acids and amines, whereas FtuCA was not. The most effective BsuCA 1 activators were L-adrenaline and D-Tyr (K A s of 0.70-0.95 µM). L-His, L-/D-Phe, L-/D-DOPA, L-Trp, L-Tyr, 4-amino-L-Phe, dopamine, 2-pyridyl-methylamine, D-Glu and L-Gln showed activation constants in the range of 0.70-3.21 µM. FtuCA was sensitive to activation with L-Glu (K A of 9.13 µM). Most of the investigated compounds showed a weak activating effect against FtuCA (K A s of 30.5-78.3 µM). Many of the investigated amino acid and amines are present in high concentrations in many tissues in vertebrates, and their role in the pathogenicity of the two bacteria is poorly understood. Our study may bring insights in processes connected with invasion and pathogenic effects of intracellular bacteria.

Carbonic anhydrases (CAs) are ubiquitous metalloenzymes, which started to be investigated in detail in pathogenic, as well as non-pathogenic species since their pivotal role is to accelerate the physiological CO 2 hydration/dehydration reaction significantly. Here, we propose the marine unicellular diatom Phaeodactylum tricornutum as a model organism for testing the membrane penetrability of CA inhibitors (CAIs). Seven inhibitors belonging to the sulphonamide type and possessing a diverse scaffold have been explored for their in vitro inhibition of the whole diatom CAs and the in vivo inhibitory effect on the growth of P. tricornutum. Interesting, inhibition of growth was observed, in vivo, demonstrating that this diatom is a good model for testing the cell wall penetrability of this class of pharmacological agents. Considering that many pathogens are difficult and dangerous to grow in the laboratory, the growth inhibition of P. tricornutum with different such CAIs may be subsequently used to design inhibition studies of CAs from pathogenic organisms.

The use of natural systems, such as outer membrane protein A (OmpA), phosphoporin E (PhoE), ice nucleation protein (INP), etc., has been proved very useful for the surface exposure of proteins on the outer membrane of Gram-negative bacteria. These strategies have the clear advantage of unifying in a one-step the production, the purification and the in vivo immobilisation of proteins/biocatalysts onto a specific biological support. Here, we introduce the novel Anchoring-and-Self-Labelling-protein-tag (ASL tag ), which allows the in vivo immobilisation of enzymes on E. coli surface and the labelling of the neosynthesised proteins with the engineered alkylguanine-DNA-alkyl-transferase (H 5 ) from Sulfolobus solfataricus. Our results demonstrated that this tag enhanced the overexpression of thermostable enzymes, such as the carbonic anhydrase (SspCA) from Sulfurihydrogenibium yellowstonense and the β-glycoside hydrolase (SsβGly) from S. solfataricus, without affecting their folding and catalytic activity, proposing a new tool for the improvement in the utilisation of biocatalysts of biotechnological interest.

Acidity and hypoxia are crucial phenotypes of tumour microenvironment both contributing to the selection of malignant cells under a micro evolutionistic pressure. During the tumour progression, nanovesicles, called exosomes and the metalloenzyme carbonic anhydrase IX (CA IX) affect the tumour growth and proliferation. Exosomes are released into the tumour microenvironment and spilt all over the body, while CA IX is a tumour-associated protein overexpressed in many different solid tumours. In the present study, to better understand the relationships between exosomes and CA IX, it has been used an in vitro cellular model of cells cultured in different pH conditions. The results showed that the acidic microenvironment induced upregulation of both expression and activity of CA IX in cancer cells and their exosomes, together with increasing the number of released exosomes. These data strongly support the importance of CA IX as a cancer biomarker and as a valuable target of new anticancer therapies.

Carbonic anhydrases (CAs, EC 4.2.1.1) are a superfamily of ubiquitous metalloenzymes present in all living organisms on the planet. They are classified into seven genetically distinct families and catalyse the hydration reaction of carbon dioxide to bicarbonate and protons, as well as the opposite reaction. CAs were proposed to be used for biotechnological applications, such as the post-combustion carbon capture processes. In this context, there is a great interest in searching CAs with robust chemical and physical properties. Here, we describe the enhancement of thermostability of the α-CA from Sulfurihydrogenibium yellowstonense (SspCA) by using the anchoring-and-self-labelling-protein-tag system (ASL tag ). The anchored chimeric H 5 -SspCA was active for the CO 2 hydration reaction and its thermostability increased when the cells were heated for a prolonged period at high temperatures (e.g. 70 °C). The ASL tag can be considered as a useful method for enhancing the thermostability of a protein useful for biotechnological applications, which often need harsh operating conditions.

A series of benzenesulfonamides incorporating selenazoles with diverse substitution patterns were investigated as inhibitors of six bacterial carbonic anhydrases (CAs, EC 4.2.1.1) from bacterial pathogens, such as Helicobacter pylori (hpCAα was the investigated enzyme), Vibrio cholerae (all the three CAs from this pathogen were considered, VchCAα, VchCAβ and VchCAγ) and Burkholderia pseudomallei (with its two CAs, BpsCAβ and BpsCAγ). All these sulfonamides were effective CA inhibitors, with potencies in the low micromolar or submicromolar range, making them attractive as lead compounds for designing antibacterials with a novel mechanism of action, which could counteract the extensive resistance problem observed with many clinically used antibiotics.

Neoadjuvant chemotherapy (NAC) of breast cancer (BC) improves outcomes, especially in patients with locally advanced and inflammatory cancer. Further insight into clinic-pathological factors influencing outcomes is essential to define the optimal therapeutic strategy for each category of patients and to predict the response to the treatment. In total, 117 patients with BC were treated with NAC with or without trastuzumab between 2010 and 2015. The histologic response to NAC was defined as a pathological complete response (pCR) when there was no evidence of residual invasive tumor in the breast or axillary lymph nodes. Relapse-free survival (RFS) was estimated using the Kaplan-Meier method and compared using log rank analysis. P-value <0.05 was considered statistically significant. The median age of the 117 patients enrolled in the present study was 52 years (age range, 35-85 years). The overall response rate (complete and partial responses) assessed by radiological and pathological evaluation were 76 and 72%, respectively. pCR was achieved in 35 out of 117 patients (~30%). In total, 6 patients (5%) developed progressive disease during chemotherapy. The RFS was 85 months (SE=3; 95% CI 79-91). The median was not reached and the mean follow-up time was 55 months (median 52 months; range 11-100 months). In this time, 20 patients (17%) experienced tumor recurrence. From the univariate analysis, the pathological response was significantly associated with receptor-based subtype, menopausal status and T-stage. From the multivariate analysis by using linear multiple regression and including receptor- menopausal status and T-stage, the model was not significant (P=0.062). However, by using the multiple logistic regression, and including age, pCR was significantly associated with ER+ HER2neg (P=0.006), T2 (P=0.043) and T3 (P=0.018). T-stage, menopausal status and receptor status are significantly associated with the pathological response in patients with inoperable BC treated with NAC.

The cloning, purification, and initial characterization of the β-carbonic anhydrase (CA, EC 4.2.1.1) from the genome of the opportunistic pathogen Malassezia restricta (MreCA), which a fungus involved in dandruff and seborrheic dermatitis (SD), is reported. MreCA is a protein consisting of 230 amino acid residues and shows high catalytic activity for the hydration of CO 2 into bicarbonate and protons, with the following kinetic parameters: k cat of 1.06 × 10 6 s -1 and k cat /K M of 1.07 × 10 8 M -1 s -1 . It is also sensitive to inhibition by the sulfonamide acetazolamide (K I of 50.7 nM). Phylogenetically, MreCA and other CAs from various Malassezia species seem to be on a different branch, distinct from that of other β-CAs found in fungi, such as Candida spp., Saccharomyces cerevisiae , Aspergillus fumigatus , and Sordaria macrospora , with only Cryptococcus neoformans and Ustilago maydis enzymes clustering near MreCA. The further characterization of this enzyme and the identification of inhibitors that may interfere with its life cycle might constitute new strategies for fighting dandruff and SD.

A series of 1,2,3-triazole-bearing benzenesulfonamides was assessed for the inhibition of carbonic anhydrases (CA, EC 4.2.1.1) from bacteria Vibrio cholerae (VchCAα and VchCAβ) and Mycobacterium tuberculosis (β-mtCA3). Growing resistance phenomena against existing antimicrobial drugs are globally spreading and highlight a urgent need of agents endowed with alternative mechanisms of action. Two global WHO strategies aim to reduce cholera deaths by 90% and eradicate the tuberculosis epidemic by 2030. The derivatives here reported represent interesting leads towards the optimization of new antibiotic agents showing excellent inhibitory efficiency and selectivity for the target CAs over the human (h) off-target isoform hCA I. In detail, the first subset of derivatives potently inhibits VchCAα in a low nanomolar range (K I s between 0.72 and 22.6 nM). Compounds of a second subset, differing from the first one for the position of the spacer between benzenesulfonamide and triazole, preferentially inhibit VchCAβ (K I s in the range 54.8-102.4 nM) and β-mtCA3 (K I s in the range 28.2-192.5 nM) even more than the clinically used AAZ, used as the standard., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The γ-carbonic anhydrases (CAs, EC 4.2.1.1) present in the Antarctic marine bacteria Pseudoalteromonas haloplanktis and Colwellia psychrerythraea , herein referred to as PhaCA and CpsCA, respectively, were investigated for their activation with a panel of 24 amino acids and amines. Both bacteria are considered Antarctic models for the investigation of photosynthetic and metabolic pathways in organisms adapted to live in cold seawater. PhaCA was much more sensitive to activation by these compounds compared to the genetically related enzyme CpsCA. The most effective PhaCA activators were d-Phe, l-/d-DOPA, l-Tyr and 2-pyridyl-methylamine, with the activation constant K A values of 0.72-3.27 µM. d-His, l-Trp, d-Tyr, histamine, dopamine, serotonin anddicarboxylic amino acids were also effective activators of PhaCA, with K A values of 6.48-9.85 µM. CpsCA was activated by d-Phe, d-DOPA, l-Trp, l-/d-Tyr, 4-amino-l-Phe, histamine, 2-pyridyl-methylamine and l-/d-Glu with K A values of 11.2-24.4 µM. The most effective CpsCA activator was l-DOPA (K A of 4.79 µM). Given that modulators of CAs from Antarctic bacteria have not been identified and investigated in detail for their metabolic roles to date, this research sheds some light on these poorly understood processes.

Background: The floral transition is a complex developmental event, fine-tuned by various environmental and endogenous cues to ensure the success of offspring production. Leaves are key organs in sensing floral inductive signals, such as a change in light regime, and in the production of the mobile florigen. CONSTANS and FLOWERING LOCUS T are major players in leaves in response to photoperiod. Morphological and molecular events during the floral transition have been intensively studied in the shoot apical meristem. To better understand the concomitant processes in leaves, which are less described, we investigated the nuclear changes in fully developed leaves during the time course of the floral transition., Results: We highlighted new putative regulatory candidates of flowering in leaves. We observed differential expression profiles of genes related to cellular, hormonal and metabolic actions, but also of genes encoding long non-coding RNAs and new natural antisense transcripts. In addition, we detected a significant increase in ploidy level during the floral transition, indicating endoreduplication., Conclusions: Our data indicate that differentiated mature leaves, possess physiological plasticity and undergo extensive nuclear reprogramming during the floral transition. The dynamic events point at functionally related networks of transcription factors and novel regulatory motifs, but also complex hormonal and metabolic changes.

The authors report the study of a clinical case, which presented eosinophilia both in the secretion of the nasal mucosa and in the blood count. After a careful examination of all the pathologies related to hypereosinophilia, through a clinical study, they have documented the presence of an adenocarcinoma located in the ileocecal junction of the colon. From what has been documented it is clear that only a clinical observation of precision, carried out above all through nasal cytology and colonoscopy, is able to diagnose an important pathology, such as oncology. For the literature review we used the Scopus and PubMed search engines to analyze other authors who were interested in the relationship between eosinophilia and colorectal cancer. Much of the studies analyzed reported a close relationship between the presence of tissue eosinophilia and tumor, and the prognosis of colorectal cancer. KEY WORD: Colorectal cancer, Eosinophils, Hypereosinophilia.

Thirty novel sulfonamide derivatives incorporating dipeptide were synthesized by facile acylation through benzotriazole mediated reactions and their structures were identified by 1 H NMR, 13 C NMR, MS and FT-IR spectroscopic techniques and elemental analysis. The carbonic anhydrase (CA, EC 4.2.1.1) inhibitory activity of the new compounds was assessed against four human (h) isoforms, hCA I, hCA II, hCA IV and hCA XII. Most of the synthesized compounds showed excellent in vitro carbonic anhydrase inhibitory properties comparable to those of the clinically used drug acetazolamide (AAZ). The new unprotected dipeptide-sulfonamide conjugates showed very effective inhibitory activity, in the low nanomolar range against II and XII, being less effective as hCA I and IV inhibitors. Four of the thirty compounds also showed strong inhibitory activity against hCA XII compared to AAZ., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The ubiquitous metalloenzymes carbonic anhydrases (CAs, EC 4.2.1.1) are responsible for the reversible hydration of CO₂ to bicarbonate (HCO₃ - ) and protons (H⁺). Bicarbonate may subsequently generate carbonate used in many functional activities by marine organisms. CAs play a crucial role in several physiological processes, e.g., respiration, inorganic carbon transport, intra and extra-cellular pH regulation, and bio-mineralization. Multiple transcript variants and protein isoforms exist in the organisms. Recently, 16 α-CA isoforms have been identified in the coral Stylophora pistillata . Here, we focalized the interest on three coral isoforms: SpiCA1 and SpiCA2, localized in the coral-calcifying cells; and SpiCA3, expressed in the cytoplasm of the coral cell layers. The three recombinant enzymes were heterologously expressed and investigated for their inhibition profiles with sulfonamides and sulfamates. The three coral CA isoforms differ significantly in their susceptibility to inhibition with sulfonamides. This study provides new insights into the coral physiology and the comprehension of molecular mechanisms involved in the bio-mineralization processes, since CAs interact with bicarbonate transporters, accelerating the trans-membrane bicarbonate movement and modulating the pH at both sides of the plasma membranes.

Purpose: Approximately 3% to 10% of EGFR (epidermal growth factor receptor) -mutant non-small cell lung cancers (NSCLCs) undergo transformation to small-cell lung cancer (SCLC), but their clinical course is poorly characterized., Methods: We retrospectively identified patients with EGFR-mutant SCLC and other high-grade neuroendocrine carcinomas seen at our eight institutions. Demographics, disease features, and outcomes were analyzed., Results: We included 67 patients-38 women and 29 men; EGFR mutations included exon 19 deletion (69%), L858R (25%), and other (6%). At the initial lung cancer diagnosis, 58 patients had NSCLC and nine had de novo SCLC or mixed histology. All but these nine patients received one or more EGFR tyrosine kinase inhibitor before SCLC transformation. Median time to transformation was 17.8 months (95% CI, 14.3 to 26.2 months). After transformation, both platinum-etoposide and taxanes yielded high response rates, but none of 17 patients who received immunotherapy experienced a response. Median overall survival since diagnosis was 31.5 months (95% CI, 24.8 to 41.3 months), whereas median survival since the time of SCLC transformation was 10.9 months (95% CI, 8.0 to 13.7 months). Fifty-nine patients had tissue genotyping at first evidence of SCLC. All maintained their founder EGFR mutation, and 15 of 19 with prior EGFR T790M positivity were T790 wild-type at transformation. Other recurrent mutations included TP53, Rb1, and PIK3CA. Re-emergence of NSCLC clones was identified in some cases. CNS metastases were frequent after transformation., Conclusion: There is a growing appreciation that EGFR-mutant NSCLCs can undergo SCLC transformation. We demonstrate that this occurs at an average of 17.8 months after diagnosis and cases are often characterized by Rb1, TP53, and PIK3CA mutations. Responses to platinum-etoposide and taxanes are frequent, but checkpoint inhibitors yielded no responses. Additional investigation is needed to better elucidate optimal strategies for this group.

Introduction: Concurrent platinum-based chemoradiation currently represents the standard treatment for advanced head and neck cancer (HNC), but it induces a significant toxicity, in particular among elderly patients. Elderly and unfit patients have been underrepresented in clinical trials and there is a need for tailored guidelines., Methods: A retrospective review of clinical data of HNC patients treated at the Operative Oncology Unit of the San Giovanni di Dio Hospital in Frattamaggiore (Naples, Italy) was performed. At study entry, a comprehensive assessment including absolute contraindications for cisplatin use, as well as comorbidities, socioeconomic status, BMI, and weight loss, was performed. The treatment included high-dose radiotherapy plus weekly cetuximab (initially at a dose of 400 mg/m2of body surface area and thereafter at 250 mg weekly during the whole radiotherapy). The aim of this study was to evaluate the activity and toxicity of this schedule in a series of patients aged older than 69 years., Results: Between May 30, 2013, and March 30, 2015, sixty-four patients (age range, 69-87 years; median age, 73.7 years; male/female ratio, 46/18) were treated. The overall response rate was 67% in this series of patients. The disease control rate was 76%. Disease progression was recorded in 25% of the patients. The median duration of loco-regional control was 17 months (range, 15.8-17.7 months). PFS was 14.8 months (range, 13.9-15.5 months). The overall survival was 34 months, with a median follow-up of 41.0 months (range, 31.1-36.8 months). The main grade 3/4 adverse events were acne rash in 52% and radiation dermatitis in 32% of the cases., Conclusion: Cetuximab plus radiotherapy appears to be feasible and active in elderly patients unsuitable for cisplatin treatment. The treatment was supported by a favorable toxicity profile., (© 2019 S. Karger AG, Basel.)

Purpose: To measure corneal epithelial thickness (CET) in patients with glaucoma using anterior-segment optical coherence tomography and to evaluate CET changes in relation to corneal epithelial microvilli analyzed by scanning electron microscopy (SEM)., Methods: Twenty-two eyes (16 patients) being treated with preservative-containing topical medications and 12 normal eyes underwent anterior-segment optical coherence tomography imaging using RTVue-100. The CET maps generated corresponded to a 6-mm diameter area of cornea that was divided into 17 sectors. We compared the CETs of each sector obtained in the glaucomatous group with those obtained in the control group., Results: Glaucomatous eyes were divided into 2 groups based on the number of microvilli on SEM: group 1 (6 eyes) = grades 1 and 2 at SEM (range: 500-3000) and group 2 (10 eyes) = grades 3 and 4 at SEM (range: 0-500). Four CET sectors were significantly thinner in group 1 than in normal eyes: central (P = 0.012), superior (P = 0.005), temporal paracentral (P = 0.003), and temporal midperipheral (P = 0.023). No significant differences were observed between group 2 and normal eyes. CET sectors were significantly thinner in group 1 than in group 2 only in the superior (P = 0.024) and superior-temporal paracentral (P = 0.020) sectors. CET progressively increased in patients with glaucoma as the number of corneal epithelial microvilli decreased., Conclusions: CET and corneal epithelial microvilli are new parameters with which to evaluate early stages of corneal epithelial changes during glaucoma therapy. In advanced stages of corneal epithelial damage, SEM evaluation reveals ultrastructural epithelial changes that may not be observed on CET measurements.

The activation of a β-class carbonic anhydrase (CAs, EC 4.2.1.1) from Mycobacterium tuberculosis, encoded by the gene Rv3273 (mtCA 3), was investigated using a panel of natural and non-natural amino acids and amines. mtCA 3 was effectively activated by D-DOPA, L-Trp, dopamine and serotonin, with K A s ranging between 8.98 and 12.1 µM. L-His and D-Tyr showed medium potency activating effects, with K A s in the range of 17.6-18.2 µM, whereas other amines and amino acids were relatively ineffective activators, with K A s in the range of 28.9-52.2 µM. As the physiological roles of the three mtCAs present in this pathogen are currently poorly understood and considering that inhibition of these enzymes has strong antibacterial effects, discovering molecules that modulate their enzymatic activity may lead to a better understanding of the factors related to the invasion and colonisation of the host during Mycobacterium tuberculosis infection.

The activation of the δ-class carbonic anhydrase (CAs, EC 4.2.1.1) from the diatom Thalassiosira weissflogii (TweCAδ) was investigated using a panel of natural and non-natural amino acids and amines. The most effective activator of TweCAδ was d-Tyr (K A of 51 nM), whereas several other amino acids and amines, such as L-His, L-Trp, d-Trp, dopamine and serotonin were submicromolar activators (K A s from 0.51 to 0.93 µM). The most ineffective activator of TweCAδ was 4-amino-l-Phe (18.9 µM), whereas d-His, l-/d-Phe, l-/d-DOPA, l-Tyr, histamine, some pyridyl-alkylamines, l-adrenaline and aminoethyl-piperazine/morpholine were moderately potent activators (K A s from 1.34 to 8.16 µM). For any δ-CA, there are no data on the crystal structure, homology modelling and the amino acid residues that are responsible for proton transfer to the active site are currently unknown making it challenging to provide a detailed rational for these findings. However, these data provide further evidence that this class of underexplored CA deserves more attention.

The inhibition of α-, β-, γ-, and δ-class carbonic anhydrases (CAs, EC 4.2.1.1) from bacteria (Vibrio cholerae and Porphyromonas gingivalis) and diatoms (Thalassiosira weissflogii) with a panel of N'-aryl-N-hydroxy-ureas is reported. The α-/β-CAs from V. cholerae (VchCAα and VchCAβ) were effectively inhibited by some of these derivatives, with K I s in the range of 97.5 nM - 7.26 µM and 52.5 nM - 1.81 µM, respectively, whereas the γ-class enzyme VchCAγ was less sensitive to inhibition (K I s of 4.75 - 8.87 µM). The β-CA from the pathogenic bacterium Porphyromonas gingivalis (PgiCAβ) was not inhibited by these compounds (K I s > 10 µM) whereas the corresponding γ-class enzyme (PgiCAγ) was effectively inhibited (K I s of 59.8 nM - 6.42 µM). The δ-CA from the diatom Thalassiosira weissflogii (TweCAδ) showed effective inhibition with these derivatives (K I s of 33.3 nM - 8.74 µM). As most of these N-hydroxyureas are also ineffective as inhibitors of the human (h) widespread isoforms hCA I and II (K I s > 10 µM), this class of derivatives may lead to the development of CA inhibitors selective for bacterial/diatom enzymes over their human counterparts and thus to anti-infectives or agents with environmental applications.

The synthesis of a new series of sulfamides incorporating ortho-, meta, and para-benzenesulfamide moieties is reported, which were investigated for the inhibition of two human (h) isoforms of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), hCA I and II, and two Vibrio cholerae enzymes, belonging to the α- and β-CA classes (VchCAα, VchCAβ). The compounds were prepared by using the "tail approach", aiming to overcome the scarcity of selective inhibition profiles associated to CA inhibitors belonging to the zinc binders. The built structure-activity relationship showed that the incorporation of benzhydryl piperazine tails on a phenyl sulfamide scaffold determines rather good efficacies against hCA I and VchCAα, with several compounds showing K I s < 100 nM. The activity was lower against hCA II and VchCAβ, probably due to the fact that the incorporated tails are quite bulky. The obtained evidences allow us to continue the investigations of different tails/zinc binding groups, with the purpose to increase the effectiveness/selectivity of such inhibitors against bacterial CAs from pathogens, affording thus potential new anti-infectives.

Infections caused by pathogens resistant to the available antimicrobial treatments represent nowadays a threat to global public health. Recently, it has been demonstrated that carbonic anhydrases (CAs) are essential for the growth of many pathogens and their inhibition leads to growth defects. Principal drawbacks in using CA inhibitors (CAIs) as antimicrobial agents are the side effects due to the lack of selectivity toward human CA isoforms. Herein we report a new class of CAIs, which preferentially interacts with microbial CA active sites over the human ones. The mechanism of action of these inhibitors was investigated against an important fungal pathogen, Cryptococcus neoformans, revealing that they are also able to inhibit CA in microbial cells growing in vitro. At our best knowledge, this is the first report on newly designed synthetic compounds selectively targeting β-CAs and provides a proof of concept of microbial CAs suitability as an antimicrobial drug target.