CogiTx1: A novel subtilisin A inhibitor isolated from the sea anemone Condylactis gigantea belonging to the defensin 4 protein family.

Subtilisin-like enzymes are recognized as key players in many infectious agents. In this context, its inhibitors are very valuable molecular lead compounds for structure based drug discovery and design. Marine invertebrates offer a great source of bioactive molecules, including protease inhibitors. In this work, we describe a new subtilisin inhibitor, from the sea anemone Condylactis gigantea (CogiTx1). CogiTx1 was purified using a combination of cation exchange chromatography, size exclusion chromatography and RP-HPLC chromatography. CogiTx1 it is a protein with 46 amino acid residues, with 4970.44 Da and three disulfide bridges. Is also able to inhibit subtilisin-like enzymes and pancreatic elastase. According to the amino acid sequence, it belongs to the defensin 4 family of proteins. The sequencing showed that CogiTx1 has an amidated C-terminal end, which was confirmed by the presence of the typical -XGR signal for amidation in the protein sequence deduced from the cDNA. This modification was described at protein level for the first time in this family of proteins. CogiTx1 is the first subtilisin inhibitor from the defensin 4 family and accordingly it has a folding consisting primarily in beta-strands in agreement with the analysis by CD and 3D modelling. Therefore, future in-depth functional studies may allow a more detailed characterization and will shed light on structure-function properties.

Desempeño de los estudiantes de quinto año de Estomatología en el proceso docente-educativo durante la COVID-19 / Performance of the fifth year Stomatology students in the teaching-educational process during the COVID-19

Introducción: Durante la COVID-19, la labor de los estudiantes de quinto año de estomatología (internos) constituyó una nueva experiencia teórico-práctica en el proceso docente- educativo de pregrado. Objetivo: Describir las principales actividades estomatológicas desempeñadas por los estudiantes de quinto año de estomatología durante la COVID-19. Métodos: Se realizó un estudio descriptivo y transversal de 11 estudiantes de quinto año de Estomatología que laboraron activamente en la Clínica Estomatológica Provincial Docente Mártires del Moncada de Santiago de Cuba, desde enero hasta abril de 2021. Las variables utilizadas fueron: edad, sexo, nacionalidad, actividades estomatológicas y tratamientos realizados. Los datos se procesaron y se vaciaron en tablas estadísticas. Se determinó el promedio de edad y como medidas de resumen se utilizaron las frecuencias relativa y absoluta. Resultados: Predominaron el grupo etario de 22-25 años, el sexo femenino (54,5 %, respectivamente) y los de nacionalidad extranjera, con cifra muy similar a la de los cubanos. En este período se efectuaron 609 consultas, donde las odontalgias y los abscesos dentoalveolares agudos resultaron ser las afecciones de mayor frecuencia; mientras que los tratamientos más realizados en urgencias fueron las obturaciones temporales, los accesos camerales, así como los obturantes de amalgama en los grupos priorizados. Conclusión: En tiempos de COVID-9, los internos de Estomatología, mediante la educación en el trabajo, desempeñaron con responsabilidad y abnegación todas las habilidades profesionales enmarcadas en el programa de la asignatura como estrategia para reforzar el proceso docente- educativo de pregrado.

Introduction: During the COVID-19, the work of the fifth year stomatology students (intern) constituted a new theoretical-practical experience in the pre-degree teaching- educational process. Objective: To describe the main stomatological activities carried out by the fifth year stomatology students during the COVID-19. Methods: A descriptive and cross-sectional study of 11 fifth year Stomatology students was carried out, they actively worked in Mártires del Moncada Teaching Provincial Stomatological Clinic in Santiago de Cuba, from January to April, 2021. The variables used were: age, sex, nationality, stomatological activities and treatments performed. The data were processed and copied in statistical charts. The age average was determined and the relative and absolute frequencies were used as summary measures. Results: There was a prevalence of the 22-25 age group, female sex (54.5 %, respectively) and those of foreign nationality, with very similar values to those of Cubans. In this period 609 visits were made, in which odontalgias and acute dentoalveolar abscesses were the most frequent affections; while the treatments more performed in emergencies were the temporary obturations, cameral accesses, as well as the amalgam obturants in the prioritized groups. Conclusion: In times of COVID-9, the Stomatology interns, by means of the education at work, carried out with responsibility and abnegation all the professional skills within the program of the subject as strategy to reinforce the pre-degree teaching - educational process.

Relación causa - efecto entre manifestaciones bucales y pacientes con la COVID-19 / Cause - effect relationship between the oral manifestations and the patients with COVID-19

Las manifestaciones clínicas en los pacientes con la COVID-19 han sido notificadas en numerosas investigaciones; sin embargo, respecto a la cavidad bucal es escasa la información. En este artículo se abordan las manifestaciones bucales más frecuentes y su localización. Asimismo, se considera que no puede establecerse la relación causa - efecto entre la infección por coronavirus y la aparición de lesiones bucales, puesto que estas lesiones pueden estar relacionadas con la inmunosupresión; además, se presentan como coinfecciones y manifestaciones secundarias con múltiples aspectos clínicos.

The clinical signs in patients with COVID-19 have been notified in numerous investigations; however, there is lack of information regarding the oral cavity. The most frequent oral manifestations and their localization are approached in this work. Also, it is considered that the cause-effect relationship between the infection by coronavirus and the emergence of oral lesions cannot be established, since these lesions can be related to the immunosupression; also, they are presented as coinfections and secondary signs with multiple clinical aspects.

Arginine substitution by alanine at the P1 position increases the selectivity of CmPI-II, a non-classical Kazal inhibitor.

CmPI-II is a Kazal-type tight-binding inhibitor isolated from the Caribbean snail Cenchritis muricatus. This inhibitor has an unusual specificity in the Kazal family, as it can inhibit subtilisin A (SUBTA), elastases and trypsin. An alanine in CmPI-II P1 site could avoid trypsin inhibition while improving/maintaining SUBTA and elastases inhibition. Thus, an alanine mutant of this position (rCmPI-II R12A) was obtained by site-directed mutagenesis. The gene cmpiR12A was expressed in P. pastoris KM71H yeast. The recombinant protein (rCmPI-II R12A) was purified by the combination of two ionic exchange chromatography (1:cationic, 2 anionic) followed by and size exclusion chromatography. The N-terminal sequence obtained as well as the experimental molecular weight allowed verifying the identity of the recombinant protein, while the correct folding was confirmed by CD experiments. rCmPI-II R12A shows a slightly increase in potency against SUBTA and elastases. The alanine substitution at P1 site on CmPI-II abolishes the trypsin inhibition, confirming the relevance of an arginine residue at P1 site in CmPI-II for trypsin inhibition and leading to a molecule with more potentialities in biomedicine.

Evaluación de la proteólisis en células aisladas vivas de Plasmodium falciparum aisladas durante los estadios asexuales sanguíneos / Proteolysis assessment in isolated Plasmodium falciparum living cells at the asexual blood stages

It has been demonstrated that proteases play crucial roles in Plasmodium falciparum infection and therefore have been considered as targets for the development of new therapeutic drugs. The aim of this study was to describe the specific proteolytic activity profile in all blood stages of P. falciparum isolated parasites in order to explore new antimalarial options. For this purpose, we used the fluorogenic substrate Z-Phe-Arg-MCA (Z: carbobenzoxy, MCA: 7-amino-4-methyl coumarine) and classic inhibitors for the different classes of proteolytic enzymes, such as phenylmethylsulfonyl fluoride (PMSF), 1.10-phenantroline, pepstatin A and E64 to study the inhibition profiles. As expected, due to the high metabolic activity in mature stages, the substrate was mostly degraded in the trophozoite and schizont, with specific activities ~ 20 times higher than in early stages (merozoite/rings). The major actors in substrate hydrolysis were cysteine proteases, as confirmed by the complete hydrolysis inhibition with E64 addition. Proteolytic activity was also inhibited in the presence of PMSF in all but the schizont stage. However, PMSF inhibition was the result of unspecific interaction with cysteine proteases as demonstrated by reversion of inhibition by dithiotreitol (DTT), indicating that serine protease activity is very low or null. To our knowledge, this is the first report aiming to describe the proteolytic profile of P. falciparum isolated parasites at all the erythrocytic cycle stages. The results and protocol described herein can be useful in the elucidation of stage specific action of proteolysis-inhibiting drugs and aid in the development of antimalarial compounds with protease inhibitory activity(AU)

e ha demostrado que las proteasas desempeñan funciones vitales en la infección por Plasmodium falciparum, y por lo tanto se consideran dianas en la elaboración de nuevos medicamentos terapéuticos. El objetivo del estudio era describir el perfil de actividad proteolítica específica de todas las etapas sanguíneas de parásitos aislados de P. falciparum con vistas a explorar nuevas opciones antimaláricas. Con ese propósito, utilizamos el sustrato fluorogénico Z-Phe-Arg-AMC (Z: carbobenzoxi, AMC: 7-amino-4-metilcumarina) e inhibidores clásicos para las diferentes clases de enzimas proteolíticas, tales como el fluoruro de fenilmetilsulfonilo (PMSF), 1,10-fenantrolina, pepstatina A y E64 para estudiar los perfiles de inhibición. Como se esperaba, debido a la elevada actividad metabólica de las etapas de madurez, el sustrato fue degradado mayormente en el trofozoíto y el esquizonte, con actividad específica ~ 20 veces superior a la de las etapas tempranas (merozoíto/ anillos). Los principales actores en la hidrólisis del sustrato fueron las cisteínas proteasas, lo que fue confirmado por la inhibición completa de la hidrólisis con la adición de E64. La actividad proteolítica también fue inhibida en presencia de PMSF en todas las etapas excepto el esquizonte. Sin embargo, la inhibición del PMSF fue resultado de una interacción inespecífica con las cisteínas proteasas, según lo demuestra la reversión de la inhibición con el ditiotreitol (DTT), lo que indica que la actividad de la serina proteasa es muy baja o inexistente. Que sepamos, este es el primer informe dirigido a describir el perfil proteolítico de parásitos aislados de P. falciparum en todas las etapas del ciclo eritrocítico. Los resultados y el protocolo que aquí se describen pueden ser útiles para dilucidar la acción específica de los medicamentos inhibidores de proteólisis en cada etapa, así como contribuir al desarrollo de compuestos antimaláricos con actividad inhibidora de la proteasa(AU)

NMR structure of CmPI-II, a non-classical Kazal protease inhibitor: Understanding its conformational dynamics and subtilisin A inhibition.

Subtilisin-like proteases play crucial roles in host-pathogen interactions. Thus, protease inhibitors constitute important tools in the regulation of this interaction. CmPI-II is a Kazal proteinase inhibitor isolated from Cenchritis muricatus that inhibits subtilisin A, trypsin and elastases. Based on sequence analysis it defines a new group of non-classical Kazal inhibitors. Lacking solved 3D structures from this group prevents the straightforward structural comparison with other Kazal inhibitors. The 3D structure of CmPI-II, solved in this work using NMR techniques, shows the typical fold of Kazal inhibitors, but has significant differences in its N-terminal moiety, the disposition of the CysI-CysV disulfide bond and the reactive site loop (RSL) conformation. The high flexibility of its N-terminal region, the RSL, and the α-helix observed in NMR experiments and molecular dynamics simulations, suggest a coupled motion of these regions that could explain CmPI-II broad specificity. The 3D structure of the CmPI-II/subtilisin A complex, obtained by modeling, allows understanding of the energetic basis of the subtilisin A inhibition. The residues at the P2 and P2' positions of the inhibitor RSL were predicted to be major contributors to the binding free energy of the complex, rather than those at the P1 position. Site directed mutagenesis experiments confirmed the Trp14 (P2') contribution to CmPI-II/subtilisin A complex formation. Overall, this work provides the structural determinants for the subtilisin A inhibition by CmPI-II and allows the designing of more specific and potent molecules. In addition, the 3D structure obtained supports the existence of a new group in non-classical Kazal inhibitors.

Heterologous expression of Cenchritis muricatus protease inhibitor II (CmPI-II) in Pichia pastoris system: Purification, isotopic labeling and preliminary characterization.

Cenchritis muricatus protease inhibitor II (CmPI-II) is a tight-binding serine protease inhibitor of the Kazal family with an atypical broad specificity, being active against several proteases such as bovine pancreatic trypsin, human neutrophil elastase and subtilisin A. CmPI-II 3D structures are necessary for understanding the molecular basis of its activity. In the present work, we describe an efficient and straightforward recombinant expression strategy, as well as a cost-effective procedure for isotope labeling for NMR structure determination purposes. The vector pCM101 containing the CmPI-II gene, under the control of Pichia pastoris AOX1 promoter was constructed. Methylotrophic Pichia pastoris strain KM71H was then transformed with the plasmid and the recombinant protein (rCmPI-II) was expressed in benchtop fermenter in unlabeled or (15)N-labeled forms using ammonium chloride ((15)N, 99%) as the sole nitrogen source. Protein purification was accomplished by sequential cation exchange chromatography in STREAMLINE DirectHST, anion exchange chromatography on Hitrap Q-Sepharose FF and gel filtration on Superdex 75 10/30, yielding high quantities of pure rCmPI-II and (15)N rCmPI-II. Recombinant proteins displayed similar functional features as compared to the natural inhibitor and NMR spectra indicated folded and homogeneously labeled samples, suitable for further studies of structure and protease-inhibitor interactions.

1H, 13C and 15N resonance assignments and secondary structure analysis of CmPI-II, a serine protease inhibitor isolated from marine snail Cenchritis muricatus.

A protease inhibitor (CmPI-II) (UNIPROT: IPK2_CENMR) from the marine mollusc Cenchritis muricatus, has been isolated and characterized. It is the first member of a new group (group 3) of non-classical Kazal-type inhibitors. CmPI-II is a tight-binding inhibitor of serine proteases: trypsin, human neutrophil elastase (HNE), subtilisin A and pancreatic elastase. This specificity is exceptional in the members of Kazal-type inhibitor family. Several models of three-dimensional structure of CmPI-II have been constructed by homology with other inhibitors of the family but its structure has not yet been solved experimentally. Here we report the (1)H, (15)N and (13)C chemical shift assignments of CmPI-II as basis for NMR structure determination and interaction studies. Secondary structure analyses deduced from the NMR chemical shift data have identified three ß-strands ß1: residues 14-19, ß2: 23-35 and ß3: 43-45 and one helix α1: 28-37 arranged in the sequential order ß1-ß2-α1-ß3. These secondary structure elements suggest that CmPI-II adopts the typical scaffold of a Kazal-type inhibitor.

Eficacia del tratamiento tópico con Vimang® en pacientes con estomatitis subprótesis / Effectiveness of topical treatment with Vimang® in patients with denture stomatitis

Se efectuó un ensayo clínico terapéutico controlado de fase III en 120 pacientes con estomatitis subprótesis, atendidos en la Clínica Estomatológica Provincial Docente "Mártires del Moncada" de Santiago de Cuba, desde junio de 2010 hasta mayo de 2011, con vistas a evaluar la eficacia del tratamiento tópico con té de Vimang® en dichos pacientes. Se conformaron 2 grupos de 60 integrantes cada uno (de estudio y de control). Los primeros fueron tratados con enjuagatorios fríos de dicho té 4 veces al día y los segundos con el mismo tratamiento más vitaminas. Se utilizaron el porcentaje para variables cualitativas, así como la media aritmética y la desviación estándar para variables cuantitativas. Todos los integrantes del grupo de estudio se curaron y disminuyó el gasto de material, pero el costo-efectividad fue mayor por ser menor el tiempo de curación. El té de Vimang® resultó eficaz para eliminar las manifestaciones clínicas de esa lesión y no hubo reacciones adversas, de manera que se logró la curación de los afectados con ahorro de tiempo y recursos.

A stage III controlled therapeutic clinical trial was performed in 120 patients with denture stomatitis, attended in the Provincial School of Dentistry "Martires del Moncada" of Santiago de Cuba, from June 2010 to May 2011, in order to evaluate the effectiveness of topical treatment with Vimang® tea in these patients. Two groups (study and control) with 60 members each were divided. The first ones were treated with tea cold mouthwashes 4 times a day and the second ones with the same treatment and vitamins. The percentage for qualitative variables and the arithmetic mean and standard deviation for quantitative variables were used. All members of the study group were cured and material usage decreased but the cost-effectiveness was higher because of lower healing time. The Vimang® tea was effective in eliminating the clinical manifestations of that lesion and there were not adverse reactions, so that it was possible to cure those affected, saving time and resources.

Afecciones del raquis cervical y lumbar en estomatólogos de la Clínica Estomatológica Provincial Docente de Santiago de Cuba / Disorders of the cervical and lumbar spine in stomatologists of the Provincial School of Dentistry of Santiago de Cuba

Se realizó un estudio descriptivo y transversal de 56 estomatólogos con 10 años y más dedicados a la asistencia en la Clínica Estomatológica Provincial Docente de Santiago de Cuba, desde enero hasta mayo del 2012, con vistas a identificar en ellos la presencia de enfermedades del raquis. Se aplicó una encuesta que tuvo en cuenta variables de interés, tales como sexo, años de graduados, especialidad, estado físico del raquis y ubicación de la afección del sistema osteomioarticular. Se utilizó el porcentaje como medida resumen. En la casuística primaron los especialistas dedicados a la atención general integral (77,8 %), diagnosticados con una afección del raquis cervical y lumbar, que tenían entre 10 y 30 años de graduados.

A descriptive and cross-sectional study was conducted in 56 stomatologists with 10 years and over dedicated to the care in the Provincial School of Dentistry in Santiago de Cuba, from January to May 2012, with the purpose of identifying the presence of spine disorders in them. A survey with some variates of interest such as sex, years of graduate, specialty, physical condition of the spine and location of the osteomyoarticular system disorder was applied. Percentage as summary measure was used. The specialists between 10 and 30 years of graduates dedicated to comprehensive general care (77.8%), diagnosed with a disorder of cervical and lumbar spine, prevailed in the case material.