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Background. At the end of a long definition and legislative process (Law No.3 of 11 January 2018 and DPR No 131 of 2021), started under the thrust of the European directives, with the Decree of the Ministry of Universities and Research of 29 November 2023, in agreement with the Minister of Health, osteopathy in Italy has become a healthcare profession in all respects. Materials and Methods. In order to understand the current legislative and professional position of the Osteopath, research of the original definitions and the history of the profession has been carried out, assessing an overview of the current situations among EU countries. Therefore, an analysis of the current Italian legislation has been carried out in a medical-legal key, with a critical eye aimed above all at assessing the current shortcomings. Conclusions. the inclusion of osteopathy as a healthcare profession in Italy is a significant step towards the regulation and recognition of this practice, implying considerable innovations both in terms of access to the profession, both in the field of the professional health responsibility. Even if with the Decree of 29 November 2023, a significant step forward has been made, further regulatory and control measures are needed to ensure the quality, safety, and effectiveness of osteopathic treatments, as well as the protection of patients and the professionalism of operators. [ABSTRACT FROM AUTHOR]

OBJECTIVE: The present observational study has the aim to describe the nutritional habits and adherence to Mediterranean diet within a dietary intervention performed by North Naples 2 Local Health Unit in some areas of Campania region. PATIENTS AND METHODS: A semi-quantitative food frequency questionnaire which takes in consideration several kinds of food and the related daily or weekly portions has been administered to people evaluated in the study. An increased score reflects an increased adherence to Mediterranean diet. Patients have been grouped by age, body mass index, education, socio-economic level, income, and score reported to the administered survey. Nutritional intervention has also been evaluated as concerns weight reduction during time. RESULTS: Surveys were administered to 200 patients aged from 12 to 79 years from 21 November 2018 to 27 November 2019. Obese patients were 61.5% in this population. 67.7% of obese people participating to this study had primary/lower secondary school education. 61.5% of study population have been categorized as having a low or low-medium socio-economic level and 68% of them were obese. An intermediate adherence to Mediterranean Diet has been the most represented (76.5%), a significant difference has been found among the groups normal weight, overweight and obese for the variables age, education and income. Obese patients in the present study had metabolic diseases more frequently than normal-weight patients. CONCLUSIONS: A high rate of obese people requesting nutritional counseling showed intermediate/bad adherence to Mediterranean Diet, reflecting the diffuse change from Mediterranean Diet to Western habits in nutrition. The nutritional intervention was found to be effective, especially for overweight patients. These data underline the need for further larger epidemiological analysis and public health interventions. [ABSTRACT FROM AUTHOR]

The frequency of gastroesophageal reflux disease (GERD) is increasing, in part through easy inspection of the upper digestive tract, but especially for a real spread of the disease as a consequence of modernity, lifestyle, incorrect dietary rules, and stress arising from social norms. It is a common chronic gastrointestinal disorder in Europe and the United States.

Background: At the end of a long definition and legislative process (Law No.3 of 11 January 2018 and DPR No 131 of 2021), started under the thrust of the European directives, with the Decree of the Ministry of Universities and Research of 29 November 2023, in agreement with the Minister of Health, osteopathy in Italy has become a healthcare profession in all respects., Materials and Methods: In order to understand the current legislative and professional position of the Osteopath, research of the original definitions and the history of the profession has been carried out, assessing an overview of the current situations among EU countries. Therefore, an analysis of the current Italian legislation has been carried out in a medical-legal key, with a critical eye aimed above all at assessing the current shortcomings., Conclusions: The inclusion of osteopathy as a healthcare profession in Italy is a significant step towards the regulation and recognition of this practice, implying considerable innovations both in terms of access to the profession, both in the field of the professional health responsibility. Even if with the Decree of 29 November 2023, a significant step forward has been made, further regulatory and control measures are needed to ensure the quality, safety, and effectiveness of osteopathic treatments, as well as the protection of patients and the professionalism of operators.

The application of sound wave technology to different plant species has revealed that variations in the Hz, sound pressure intensity, treatment duration, and type of setup of the sound source significantly impact the plant performance. A study conducted on cotton plants treated with Plant Acoustic Frequency Technology (PAFT) highlighted improvements across various growth metrics. In particular, the treated samples showed increases in the height, size of the fourth expanded leaf from the final one, count of branches carrying bolls, quantity of bolls, and weight of individual bolls. Another study showed how the impact of a 4 kHz sound stimulus positively promoted plant drought tolerance. In other cases, such as in transgenic rice plants, GUS expression was upregulated at 250 Hz but downregulated at 50 Hz. In the same way, sound frequencies have been found to enhance the osmotic potential, with the highest observed in samples treated with frequencies of 0.5 and 0.8 kHz compared to the control. Furthermore, a sound treatment with a frequency of 0.4 kHz and a sound pressure level (SPL) of 106 dB significantly increased the paddy rice germination index, as evidenced by an increase in the stem height and relative fresh weight. This paper presents a complete, rationalized and updated review of the literature on the effects of sound waves on the physiology and growth parameters of sound-treated plants.

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.