High-throughput screening of SARS-CoV-2 main and papain-like protease inhibitors

The global COVID-19 coronavirus pandemic has infected over 109 million people, leading to over 2 million deaths up to date and still lacking of effective drugs for patient treatment. Here, we screened about 1.8 million small molecules against the main protease (Mpro) and papain like protease (PLpro), two major proteases in severe acute respiratory syndrome-coronavirus 2 genome, and identified 1851Mpro inhibitors and 205 PLpro inhibitors with low nmol/l activity of the best hits. Among these inhibitors, eight small molecules showed dual inhibition effects on both Mpro and PLpro, exhibiting potential as better candidates for COVID-19 treatment. The best inhibitors of each protease were tested in antiviral assay, with over 40% of Mpro inhibitors and over 20% of PLpro inhibitors showing high potency in viral inhibition with low cytotoxicity. The X-ray crystal structure of SARS-CoV-2 Mpro in complex with its potent inhibitor 4a was determined at 1.8 Å resolution. Together with docking assays, our results provide a comprehensive resource for future research on anti-SARS-CoV-2 drug development.

Development and clinical application of hydrogel formulations containing papain and urea for wound healing

Abstract Hydrogels are used for wound treatment, as they may contain one or more active components and protect the wound bed. Papain is one of the active substances that have been used with this purpose, alongside urea. In this paper, carboxypolymethylene hydrogels containing papain (2% and 10% concentrations) and urea (5% concentration) were produced. Physical-chemical stability was performed at 0, 7, 15 and 30 days at 2-8ºC, 25ºC and 40ºC, as well as the rheological aspects and proteolytic activity of papain by gel electrophoresis. Clinical efficacy of the formulations in patients with lower limb ulcers was also evaluated in a prospective, single-center, randomized, double-blind and comparative clinical trial. The results showed 7-day stability for the formulations under 25ºC, in addition to approximately 100% and 15% of protein activity for 10% and 2% papain hydrogel, respectively. The rheological profile was non-Newtonian for the 10% papain hydrogel tested. There were no significant differences regarding the mean time for healing of the lesions, although 10% papain presented a better approach to be used in all types of tissue present in the wound bed.

Multi-level scoring approach to discover multi-targeting potency of medicinal plant phytochemicals against protein targets in SARS-CoV-2 and human ACE-2 receptor

SARS-CoV-2 pandemic has become a major threat to human healthcare and world economy. Due to the rapid spreading and deadly nature of infection, we are in a situation to develop quick therapeutics to combat SARS-CoV-2. In this study, we have adopted a multi-level scoring approach to identify multi-targeting potency of bioactive compounds in selected medicinal plants and compared its efficacy with two reference drugs, Nafamostat and Acalabrutinib which are under clinical trials to treat SARS-CoV-2. In particular, we employ molecular docking and implicit solvent free energy calculations (as implemented in the Molecular Mechanics -Generalized Born Surface Area approach) and QM fragmentation approach for validating the potency of bioactive compounds from the selected medicinal plants against four di?erent viral targets and one human receptor (Angiotensin-converting enzyme 2 -ACE-2) which facilitates the SARS-CoV-2entry into the cell. The protein targets considered for the study are viral 3CL main protease (3CLpro), papain-like protease (PLpro), RNA dependent RNA polymerase (RdRp), and viral spike protein-human hACE-2 complex (Spike:hACE2)including human protein target (hACE-2). Herein, thereliable multi-level scoring approach was used to validate the mechanism behind the multi-targeting potency of selected phytochemicals from medicinal plants. The present study evidenced that the phytochemicals Chebulagic acid, Stigmosterol, Repandusinic acid and Geranin exhibited efficient inhibitory activity against PLpro while Chebulagic acid was highly active against 3CLpro. Chebulagic acid andGeranin also showed excellent target specific activity against RdRp.Luteolin, Quercetin, Chrysoeriol and Repandusinic acid inhibited the interaction of viral spike protein with human ACE-2 receptor. Moreover Piperlonguminine and Piperine displayed significant inhibitory activity against human ACE-2 receptor. Therefore, the identified compounds namely Chebulagic acid, Geranin and Repandusinic acid can serve as potent multi-targeting phytomedicine for treating COVID-19

Brix-3000 as a Preventive Measure in Aerosol Production against Covid-19: Case Report / Utilização do gel Enzimático Brix-3000 Como Medida Preventiva na Produção de Aerossóis Frente à Covid-19: Relato de Caso

In 2020, the World Health Organization declared a pandemic due to a new virus, known as SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2). Faced with exposure to cross-contamination due to the use of aerosols from rotary instruments, saliva, and nasopharyngeal secretions, health professionals had to explore alternative methods for removing decayed tissue, such as BRIX-3000, a papain-based enzymatic gel used for the chemical-mechanical removal of infected decayed tissue using manual instruments, with the aim of reducing the risk of contamination for the dental team. Thus, this paper aims to report a clinical case, using the BRIX-3000 in the removal of carious tissue. An 8 year-old patient complained of sensitivity in teeth 55 and 65, and was proposed removal of decayed tissue by means of chemical-mechanical removal. The procedure was performed without anesthesia, under relative isolation. The enzymatic gel was taken to the cavity following the manufacturer's directions, and after 1 minute the infected tissue was removed by curettage. Then the cavity was prepared, using cotton for washing and drying, avoiding the triple syringe and the formation of aerosols. Glass ionomer was used as a provisional material. It can be concluded that the papain-based enzymatic gel had great efficacy in removing decayed tissue, besides reducing aerosolization and, thus, contamination, making clinical care safer against the spread of infectious and contagious diseases, before the Covid-19 pandemic. (AU)

Em 2020, a Organização Mundial da Saúde declarou uma pandemia devido a um novo vírus, conhecido como SARS-CoV-2 (Síndrome Respiratória Aguda Grave Coronavírus 2). Frente à exposição de uma contaminação cruzada, devido à utilização de aerossóis advindos dos instrumentos rotatórios, salivas e secreções da nasofaringe, os profissionais da saúde precisaram explorar métodos alternativos para remoção do tecido cariado, como no caso o BRIX-3000, um gel enzimático a base de papaína, utilizado na remoção químico-mecânica do tecido cariado infectado, por meio de instrumentos manuais, com o objetivo de diminuir o risco de contaminação da equipe odontológica. Posto isto, esse trabalho visa relatar um caso clínico, utilizando o BRIX-3000 na remoção do tecido cariado. Paciente, 8 anos, procurou atendimento queixando-se de sensibilidade nos elementos dentais 55 e 65. Foi proposto a remoção do tecido cariado por meio da remoção químico-mecânica. O procedimento foi realizado sem anestesia, sob isolamento relativo. O gel enzimático foi levado a cavidade seguindo as orientações do fabricante, decorridos 1 minuto iniciou-se a remoção do tecido infectado por meio de curetagem. Em seguida a cavidade foi preparada, utilizando algodão para lavagem e secagem, evitando a seringa tríplice e a formação de aerossóis. Como material provisório utilizou-se o ionômero de vidro. Diante do caso, pode-se concluir-se que o gel enzimático a base de papaína teve grande eficácia na retirada do tecido cariado, além de reduzir a aerossolização e, assim, a contaminação, tornando o atendimento clínico mais seguro contra a disseminação de doenças infectocontagiosas, frente a pandemia da Covid-19. (AU)

Implementação da teoria do conforto no cuidado ao paciente com ferida neoplásica a partir do desbridamento com papaína: um estudo misto / Implementation of the comfort theory in the care of patients with a neoplastic wound after papain debridement: a mixed study

As feridas neoplásicas são formadas a partir de uma proliferação descontrolada das células tumorais e sua infiltração nas estruturas da pele, sendo a meta principal do cuidado o conforto do paciente a partir do manejo dos sintomas físicos e psicossociais. Este estudo inovador teve como objetivo analisar a implementação da Teoria do Conforto a partir da intervenção clínica de desbridamento no cuidado de enfermagem ao paciente com ferida neoplásica. Trata-se de uma pesquisa de método misto onde as abordagens quantitativa e qualitativa foram contempladas nas diferentes fases da pesquisa passando pela coleta, análise de dados e interpretação dos resultados. No primeiro momento foi desenvolvido um estudo piloto de um ensaio clínico randomizado e no segundo momento foram analisados os dados coletados durante o cuidado e registrados em um diário de campo. Após a aprovação pelo Comitê de Ética em Pesquisa sob o número de parecer 4.292.777, iniciou-se a coleta de dados em novembro de 2020 até novembro de 2021. Os participantes foram distribuídos de forma aleatória em dois grupos, Intervenção (Papaína 6%) e Controle (Gel de Carboximetilcelulose a 2%) por um tempo de seguimento de 12 semanas. Foi realizada uma análise descritiva dos dados baseada em distribuições de frequências e gráficos sendo a mediana a medida síntese principal utilizada. Os resultados apontam que nenhuma participante apresentou evolução positiva de melhora na quantidade de exsudato. No que se refere a variável dor, em termos medianos, houve melhora da dor ao longo das 12 avaliações. Quanto ao odor, pode-se afirmar que no global, houve evolução positiva de melhora do grau de odor das feridas das participantes. Os dados referentes à análise do desconforto mostram um aumento tanto da mediana, de 9,0 na primeira avaliação para 10,0 na segunda avaliação, quanto na média, que passou de 8,0 na primeira avaliação para 8,5 na segunda avaliação. Conclui-se que quanto ao desconforto, houve melhora do escore em apenas 20% das participantes. Com relação aos eventos adversos, o mais incidente e recorrente foi a hiperemia perilesional, que ocorreu para 60,0% das participantes em 64,3% das avaliações. O prurido tem um índice de recorrência igual a 54,2% e a maceração de 20,8%, estando presentes para 40,0% das participantes. O sangramento apresentou um índice de recorrência de 16,7%, estando presente em 20,0% das participantes, sendo induzido pelas remoções das gazes. A identificação das variáveis intervenientes, a partir dos dados coletados durante o cuidado, subsidiou a construção da Estrutura Taxonômica da Teoria de Conforto para pacientes com feridas neoplásicas. Esse produto técnico pode contribuir para a prática profissional, na orientação do enfermeiro para a elaboração do plano de cuidados baseado nas necessidades relatadas pelo paciente. A partir da construção da Estrutura Taxonômica foi possível elaborar um diagrama da Teoria do Conforto de Kolcaba aplicada ao paciente com ferida neoplásica. Na primeira etapa do estudo, apesar dos resultados da análise descritiva terem apontado indícios de benefícios no quesito dor e odor para as participantes no geral, os dados obtidos não mostraram significância suficiente para refutar ou aceitar as hipóteses formuladas. Cabe ressaltar que o número reduzido de participantes, imposto pela pandemia COVID-19, período em que ocorreu a coleta de dados, foi um fator de limitação para o desenvolvimento do estudo. Já na segunda etapa, a partir da análise das diferentes formas de conforto em seus diversos contextos, há indícios favoráveis de que a intervenção clínica de desbridamento é efetiva para o alivio, relaxamento ou transcendência do paciente com ferida neoplásica, à luz da Teoria do Conforto. Este estudo mostra-se relevante ao contribuir com evidencias científicas que podem apoiar um cuidado de enfermagem de qualidade ao paciente com ferida neoplásica tendo em vista a expectativa de aumento no número destas lesões concomitante a um aumento na estimativa do número de casos de câncer. Ao contribuir na formação de profissionais qualificados, integra-se às ações de controle do câncer que propõe um cuidado integral a este paciente, impactando ainda nos custos da assistência de enfermagem. A complexidade desta situação associado ao sofrimento imposto a esta parcela da população demanda uma urgência na implementação de intervenções efetivas e seguras e neste sentido este estudo mostra-se relevante em seu aspecto inovador ao buscar associar uma tecnologia técnica de enfermagem ao conforto do paciente com ferida neoplásica.

Neoplastic wounds develop from an uncontrolled proliferation of tumor cells with infiltration into the skin structures. The main goal of wound care is patient comfort by managing physical and psychosocial symptoms. The objective of this innovative study was to analyze the implementation of the Comfort Theory, drawing on clinical intervention of debridement in nursing care to neoplastic wound patient. This was a mixed method research with quantitative and qualitative approaches contemplated in the various phases of collection, data analysis and interpretation of outcomes. In the first stage, a pilot study of a randomized clinical trial was developed; in the second stage, there was the analysis of data collected during care, which were registered in a field diary. The research was approved by the Research Ethics Committee, under the Opinion Nº 4.292.777. Data collection was conducted from November 2020 to November 2021. The participants were randomly distributed in two groups, Intervention (papain 6%) and Control (Carboxymethyl Cellulose gel 2%), for care continuity of 12 weeks. The descriptive analysis of data was performed based on distribution of frequencies and graphics, with the median used as main synthesis measure. The outcomes point that no participant presented a positive improvement evolution of exudate amount. Regarding the variable pain, in median terms, there was pain improvement during the 12 assessments. Regarding odor, it can be stated that globally there was a positive improvement evolution of the degree of odor of the participants' wounds. Data referring to the analysis of discomfort show an increase of the median, of 9.0 in the first assessment to 10.0 in the second assessment, and of the average, of 8.0 in the first assessment to 8.5 in the second assessment. The conclusion was that in terms of discomfort, there was score improvement in only 20% of participants. In relation to adverse events, the most incident and recurrent was perilesional hyperemia, which occurred for 60.0% of participants in 64.3% of assessments. Itching had a recurrence rate of 54.2% and necrosis of 20.8%, being present in 40.0% of participants. Bleeding presented a recurrence rate of 16.7%, being present in 20.0% of participants, induced by gauze removal. The identification of intervening variables drawing on data collected during care subsidized the construction of the Taxonomic Structure of the Comfort Theory for patients with neoplastic wounds. This technical product can contribute to the professional practice, in guiding the nurse on the elaboration of a plan of care based on the needs reported by the patient. From the construction of the Taxonomic Structure, it was possible to develop a diagram of the Kolcaba's Comfort Theory applied to the patient with neoplastic wound. In the first stage of the study, although the results of the descriptive analysis had pointed indication of benefits in the aspects of pain and odor for participants in general, the data obtained did not show sufficient significance to refute or accept the formulated hypotheses. A limiting factor for the development of the study was the reduced amount of participants due to the Covid-19 pandemic, period when data collection took place. In the second stage, drawing on the analysis of different forms of discomfort in several contexts, there are favorable indications that clinical debridement intervention is effective for relief, ease or transcendence of the patient with neoplastic wound, in the light of the Comfort Theory. This study is relevant by contributing with scientific evidence that can give support to quality nursing care to patients with neoplastic wound, considering the probability of the increase in the number of lesions, concomitant with an estimated increase in the number of cancer cases. By contributing to the training of qualified professionals, this study is integrated to the actions of cancer control that propose patient integral care, with an impact on nursing care costs. The complexity of this situation, associated to the suffering imposed to this segment of the population, requires urgency in the implementation of effective and safe interventions. In this sense, this study is relevant in its innovative aspect by seeking to associate nursing technical technology to the comfort of the patient with neoplastic wound.

High-throughput screening identifies established drugs as SARS-CoV-2 PLpro inhibitors

A new coronavirus (SARS-CoV-2) has been identified as the etiologic agent for the COVID-19 outbreak. Currently, effective treatment options remain very limited for this disease; therefore, there is an urgent need to identify new anti-COVID-19 agents. In this study, we screened over 6,000 compounds that included approved drugs, drug candidates in clinical trials, and pharmacologically active compounds to identify leads that target the SARS-CoV-2 papain-like protease (PLpro). Together with main protease (M

Preprocesamiento de los fármacos antivirales candidatos contra COVID-19 en modelos de SARS cov2. Objetivos / Preprocessing of the candidate antiviral drugs against COVID-19 in models of SARS cov2 targets

Although many viral infections are self-limiting, other are real health challenges like COVID-19 since many viruses possess just few drug gable targets to be treated with small drug molecules. Corona virus genome encodes for up to 17 main proteins. Orf1ab encodes for polyprotein. COVID-19 structural proteins are the spike S, membrane M, envelope E and the nucleocapsid N protein while other are non-structural proteins designated as NSP1-13 for non-structural proteins. Among NSP the most important corona virus targets for developing antiviral drugs are the papain-like protease, PDB ID: 6m03 and RNA polymerase NSP12, PDB ID: 6nur. NCBI, NIH Genbank, Uniprot, PDB, DrugBank, ChemSpider databases and bioinformatics editor softwares like ICM Mol soft pro and Swiss Dock were used in addition to the in vitro lab model of viral protease were integrated to retrieve and analyze corona virus targets and to select the candidate ligands in an attempt to evaluate the inhibitory efficacy of different experimental and approved drugs which were further optimized and searched for the highly similar approved drug. This step aims to adopt drug repurposing to speed the development of antiviral drugs and recommend rational in vivo and clinical studies. After COVID-19 targets had been analyzed the drugs that shared > 70% similarity to the binding sites of those targets were reversin, pentagastrin, remdesivir, norfloxacin and nitazoxanide against COVID-19 papain-like protease whereas benzyl glutathione, lopinavir and hydroxymethylglutathione against RNA polymerase. The anti-resistance reversin showed the highest inhibitory efficacy against COVID-19 papain-like protease as indicated by the ligand-protease binding energy with Mol soft pro analysis. The calculated inhibitory binding was -137.30 kJ/mol z > 1.9 as compared with the tetrazapentadecanoate -129.57 kJ/mol z = 4.0, whereas remdesivir, pentagastrin, nitazoxanide and norfloxacin had a moderate antiprotease activity (>- 100 kJ/mol). Norfloxacin shoresults showed a slight consistency between in vitro and in silico models. Although benzyl glutathione is an experimental compound, however it had the highest RNA polymerase inhibiting efficacy with -129 kJ/mol binding energy which is even higher than lopinavir and Favinavir. From the overall results, reversin, oligopeptides, quinolones and antiviral drugs may widen the treatment options for COVID-19 if further evaluated in clinical studies

Effect of catalpol from the root of rehmannia glutinosa on the expression of inflammatory proteins in synovium of the knee joint in rats with early osteoarthritis / 中国组织工程研究

BACKGROUND: Inflammatory transmitters secreted from the synovium may be one of the important factors inducing the onset of knee osteoarthritis, and further exacerbate knee osteoarthritis. OBJECTIVE: To observe the effect of different concentrations of catalpol from the root of Rehmannia glutinosa on the expression of interleukin-1β, galectin 3 and S100A12 in the synovium of the knee joint in rats with early knee osteoarthritis, and to explore the mechanism of catalpol from the root of Rehmannia glutinosa in the treatment of early knee osteoarthritis. METHODS: The 14 of 48 adult male Wistar rats were randomly selected as a normal control group, and the remaining 34 rats were injected with 4% papain and 0.03 mol/L cysteine solution (0.2 mL) into the right knee joint cavity at 1, 4, 7 days after the initialization of the experiment to duplicate the model of early knee osteoarthritis. At 10 days after the initialization of the experiment, 4 rats in each group were randomly selected for histological observation using hematoxylin-eosin staining and validation of the model using Mankin and OARSI grading evaluation. After the model was successfully verified, the remaining 30 rats in the model group were randomly divided into model control group, low-dose group and high-dose group. At 2 days after successful modeling, each treatment group was infused with corresponding drugs at a dose of 0.2 mL/kg, and normal control group and model control group were infused with normal saline, once a day for 28 consecutive days. The synovium of the right knee joint was then taken from rats in each group, and the expression of interleukin-1β, galectin 3 and S100A12 in the rat synovium was detected by ELISA. RESULTS AND CONCLUSION: Mankin score and OARSI grading were significantly higher in the model control group than the normal control group (P < 0.05). The levels of S100A12, interleukin-1β and galectin 3 in the synovium were significantly higher in the model control group than the normal control group (P < 0.01). Compared with the model control group, the levels of S100A12, interleukin-1β and galectin 3 in the synovium was decreased somewhat in the low-dose group (P < 0.05), but significantly decreased in the high-dose group (P < 0.01). Therefore, catalpol from the root of Rehmannia glutinosa can delay the progression of knee osteoarthritis by reducing the level of inflammatory factors in the synovium.

Discovery of human coronaviruses pan-papain-like protease inhibitors using computational approaches / 药物分析学报

The papain-like protease (PLpro) is vital for the replication of coronaviruses (CoVs), as well as for escaping innate-immune responses of the host. Hence, it has emerged as an attractive antiviral drug-target. In this study, computational approaches were employed, mainly the structure-based virtual screening coupled with all-atom molecular dynamics (MD) simulations to computationally identify specific inhibitors of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PLpro, which can be further developed as potential pan-PLpro based broad-spectrum antiviral drugs. The sequence, structure, and functional con-serveness of most deadly human CoVs PLpro were explored, and it was revealed that functionally important catalytic triad residues are well conserved among SARS-CoV, SARS-CoV-2, and middle east respiratory syndrome coronavirus (MERS-CoV). The subsequent screening of a focused protease in-hibitors database composed of ～7,000 compounds resulted in the identification of three candidate compounds, ADM_13083841, LMG_15521745, and SYN_15517940. These three compounds established conserved interactions which were further explored through MD simulations, free energy calculations, and residual energy contribution estimated by MM-PB(GB)SA method. All these compounds showed stable conformation and interacted well with the active residues of SARS-CoV-2 PLpro, and showed consistent interaction profile with SARS-CoV PLpro and MERS-CoV PLpro as well. Conclusively, the re-ported SARS-CoV-2 PLpro specific compounds could serve as seeds for developing potent pan-PLpro based broad-spectrum antiviral drugs against deadly human coronaviruses. Moreover, the presented infor-mation related to binding site residual energy contribution could lead to further optimization of these compounds.

Development of a new Colombian product, effective for the treatment of carious lesions: a case study / Desarrollo de un nuevo producto colombiano, efectivo para el tratamiento de las lesiones cariosas: un caso de estudio

SUMMARY Purpose: We present a new Colombian product researched and developed by Seven Scientific Foundation, which has been very useful for the removal of the carious tissue in patients who have used this innovative product. Methods: We used a chemical-mechanical removal of dental caries. This product is made with 10% papain which is the active principle and works as a debriding agent, as well as being a bacteriostatic, bactericidal and anti-inflammatory agent; and also contain dragon's blood (Croton lechleri) sap which is an effective as analgesic/anti-inflammatory and especially as a healing agent. In this article, we present a case study of a patient, whose dental carious tissue was removed, using the dental new product. Description of the case report: In this article, we present a case study of a patient, whose dental carious tissue has been removed, using the new dental product, therefore this innovative technique of removal of the atraumatic carious tissue. Conclusions: This method is useful as it is a minimally invasive technique, without requiring the use of local anesthesia, or the additional use of dental instruments. It is used in the Colombian population of all ages and does not generate side effects in people who have used it so far.

RESUMEN Propósito: se investigó y desarrolló un nuevo producto dental el cual ha sido muy útil para la remoción del tejido carioso en los pacientes colombianos que han utilizado este innovador producto para la remoción químico-mecánica de las caries dentales. Métodos: Este producto está hecho a base de papaína al 10 % el cual es el principio activo y funciona como un agente debridante lo cual cicatriza el tejido removido, además de ser un agente bacteriostático, bactericida y antiinflamatorio. Descripción de reporte de caso: en este artículo se presenta un estudio de caso de un paciente al cual se le removió el tejido carioso dental. Conclusiones: Por lo tanto, esta técnica innovadora de remoción del tejido carioso atraumático resulta útil por ser una técnica mínimamente invasiva, sin requerir el uso de anestesia local al tejido afectado ni el uso adicional de instrumentos dentales. Se utiliza en la población colombiana de todas las edades y no genera efectos colaterales en las personas que lo han utilizado hasta el momento.

Effects of papain-based gel used for caries removal on macrophages and dental pulp cells

Abstract Papain-based gel is used for chemical-mechanical caries removal and present antimicrobial and anti-inflammatory activities. However, its effects on dental pulp cells and on macrophages remains largely unknown. Therefore, the aim of this study was to investigate whether the papain-based gel Papacárie Duo® acts as an immunomodulator in lipopolysaccharide (LPS)-activated macrophages and its effects on dental pulp cells . J774.1 macrophage and OD-21 dental pulp cells were stimulated with 0.5% and 5% of Papacárie Duo®, following pre-treatment or not with LPS. After 24 h, a lactate dehydrogenase assay was used to measure cytotoxicity, a tetrazolium-based colorimetric assay (MTT) was used to measure cell viability, and qRT-PCR was used to analyze relative gene expression of Ptgs2, Il10, Tnf, Mmp9, Runx2, Ibsp and Spp1. Papacárie Duo® was cytotoxic and reduced cell viability at 5% but not at 0.5% in both cultures. In macrophages, Papacárie Duo® increased the expression Il10 and LPS-induced Ptgs2, but it did not affect Tnf or Mmp9. In OD-21 cells, Papacárie Duo® inhibited Runx2 and Ibsp expression, but stimulated Spp1 expression. Papain-based gel presented a concentration dependent cytotoxicity, without affecting cell viability, for dental pulp cells and macrophages. Interestingly, the gel presented an inhibitory effect on pulp cell differentiation but modulated the activation of macrophages stimulated with LPS. We speculate that in dental pulp tissue, Papacárie Duo® would impair reparative dentinogenesis but could activate macrophages to perform their role in defense and inflammation.

Resumo O gel à base de papaína é utilizando para remoção químico-mecânica do tecido cariado e apresenta propriedades antimicrobianas e anti-inflamatórias Entretanto, seu efeito sobre as células da polpa dentárias e macrófagos é desconhecido. Portanto, o objetivo deste estudo foi investigar o efeito de um gel de papaína (Papacárie Duo®) em células indiferenciadas da polpa dentária e a capacidade de induzir a ativação e síntese de mediadores inflamatórios por macrófagos estimulados com lipopolissacarídeo bacteriano (LPS). O gel de papaína foi diluído nas concentrações de 0,5 e 5%. Células indiferenciadas da polpa dentária OD-21 e macrófagos J774.1 foram mantidos em cultura com os diferentes estímulos por um período de estimulação de 24 h para realização do teste de citotoxicidade (Ensaio LDH) e para avaliação da viabilidade celular (Ensaio Colorimétrico MTT). A seguir foi realizada avaliação da expressão gênica relativa dos genes Ibsp, Runx2 e Spp1 em células OD-21; e dos genes Il10, Mmp9, Ptgs2 e Tnf em células J774.1, pelo método de transcrição reversa e reação em cadeia de polimerase em tempo real (qRT-PCR), após estimulação pelo período de 24 h. O extrato do gel diluído a 5% foi citotóxico às células da polpa dental, reduziu a viabilidade celular, inibiu a expressão de Runx2 e Ibsp e estimulou a expressão de Spp1. Em macrófagos, o extrato do gel foi citotóxico e reduziu a viabilidade celular na concentração de 5%. O LPS inibiu a viabilidade celular na presença ou não do extrato do gel, sem apresentar citotoxicidade. O extrato do gel induziu a expressão de Ptgs2 e Il10, sem alterar Tnf e Mmp9. O extrato do gel de papaína foi citotóxico, dependente da concentração, tanto em células da polpa dentária como em macrófagos, sem alterar a viabilidade celular. Interessantemente, apresentou efeito inibitório na diferenciação de células da polpa dentária e modulou a ativação de macrófagos estimulados com LPS. No tecido pulpar, o Papacárie Duo® poderia impedir a dentinogênese de reparação, porém ativar macrófagos para desempenhar seu papel na inflamação e defesa.

Papain-mediated reduction of MTT is an artifact but not an indicator of cell apoptosis in Saccharomyces cerevisiae

Papain is a proteolytic enzyme of the cysteine protease family used for tissue dissociation and cell separation. Papain’snonspecific proteolysis of the plasma membrane enzymes plays a crucial role in the homeostasis by disrupting theintracellular pH of the affected cells which might lead to cell death. When the Saccharomyces cerevisiae cellswere treated with different concentrations of papain (1.0, 5.0, 10.0, 20.0, and 30.0 µg/ml), we found no alterationin the trans-plasma membrane electron transport (TPMET) activity and the intracellular pH of the cells, while itsignificantly decreased the mitochondrial dehydrogenase activity when measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay. Additional verification of cell viability by trypan blue assay showed98%–99% cell viability, contrary to the higher cell death observed with MTT assay. To better understand the decreasein cell viability with MTT assay, we tested the cell-free system that demonstrated a significant decrease of MTTconcentration but the trypan blue assay showed more number of viable cells. This study shows that papain interfereswith the MTT assay.

Applicability of papain solutions in immunohematology / Aplicabilidade de soluções de papaína em imuno-hematologia

ABSTRACT Objective: To compare the enzyme activity of different presentations of papain solution to validate in-house preparations. Methods: Two papain solutions were prepared, and the third presentation was a commercial solution. Tests were carried out with samples of red cells typed as weak RhD. Results: In-house prepared papain solutions showed similar enzyme reactivity, and statistically no differences compared to the enzyme activity of the commercial solution. Conclusion: Evaluating the cost-benefit ratio, the in-house prepared papain solutions present more economic advantages, and can be incorporated into immunohematological routines as a way to cope with periods of financial crisis and cost-containment policies.

RESUMO Objetivo: Comparar a atividade enzimática de diferentes apresentações de solução de papaína para validação de preparados in-house. Métodos: Foram preparadas duas soluções de papaína, e a terceira apresentação tratou-se de uma solução comercial. Os testes comparativos das reações enzimáticas foram realizados com amostras de hemácias tipadas como RhD fraco. Resultados: As soluções de papaína preparadas in-house apresentaram reatividade enzimática semelhante e estatisticamente sem diferenças em comparação com a atividade enzimática da solução comercial. Conclusão: Avaliando-se a relação entre custo e benefício, as soluções de papaína preparadas in-house são economicamente vantajosas, podendo ser incorporadas às rotinas imuno-hematológicas como forma de enfrentamento em períodos de crise financeira e em políticas de retenção de gastos.

CBCT analysis of rabbit temporomandibular joints with osteoarthrosis induced by monoiodoacetate and papain / Análisis por TCCB de la articulación temporomandibular de conejos con osteoartrosis inducida por monoiodoacetato y papaina

Osteoarthrosis (OA) is a degenerative disease characterized by loss of joint cartilage, remodelling of the subchondral bone, narrowing of joint spaces and the formation of osteophytes. Animal models are used to study the mechanisms of OA, as well as to test the effects of anti-osteoarthrosis drugs. The objective of the present study was to determine the changes identifiable by imaging techniques occurring in rabbit temporomandibular joints (TMJ) at 15, 25 and 45 days after OA inducement by monoiodoacetate (MIA) and papain. The imaging technology used was cone-beam computerised tomography (CBCT). The model animals were 22 young adult male New Zealand rabbits, divided randomly into three study groups: Four rabbits in the control group, nine in the papain experimental group and nine in the monoiodoacetate (MIA) experimental group. OA was induced by arthrocentesis in the lower compartment of both TMJs. The rabbits were examined by CBCT at 15, 25 and 45 days after the injection of MIA and papain. The mandibular condyles presented loss of their rounded shape, deformation of the condyle or mandibular fossa, cortical irregularity, cortical wear and changes in the dimensions of the condyle. OA induction by MIA and papain generates changes observable by CBCT in the dimensions of the mandibular condyle in rabbits. Both inducers promote signs compatible with OA on the joint surfaces of the TMJ; MIA promotes more expressive changes.

La osteoartrosis (OA) es una enfermedad degenerativa caracterizada por la pérdida de cartílago articular, remodelación ósea subcondral, estrechamiento del espacio articular y formación de osteofitos. El modelo animal es utilizado para estudiar los mecanismos de la OA, así como testar el efecto de drogas anti-osteoartrosis. El objetivo de este estudio fue determinar los cambios imagenológicos, mediante tomografía computarizada cone-beam (TCCB), que se generan en 15, 25 y 45 días, luego de la inducción de OA por medio de Monoiodoacetato (MIA) y Papaína sobre la ATM de conejos. Fueron utilizados 22 conejos machos, adultos jóvenes, de raza New Zealand divididos aleatoriamente en 3 grupos de estudio: 4 conejos para un grupo control, 9 conejos para el grupo experimental con Papaína y 9 conejos para el grupo experimental con monoiodoacetato (MIA). Se realizó la inducción de OA por la técnica de artrocentesis en el compartimiento inferior de ambas ATMs. Se les realizó examen de TCCB en los días 15, 25 y 45 tras la inyección de MIA y de papaina. Los cóndilos mandibulares presentaron pérdida de forma redondeada de cóndilo, deformidad de cóndilo o fosa mandibular, irregularidad de corticales, desgaste de corticales, cambio de dimensiones de cóndilo. La inducción de OA por medio de MIA y papaína genera cambios en la dimensión del cóndilo mandibular de conejos observables a través de TCCB. Además, ambos inductores promueven signos compatibles con OA en las superficies articulares de la ATM, siendo que la MIA promueve cambios más expresivos.

Fruit Enzymes And Their Application: A Review

The pulp and periodontium have embryonic, anatomic and functional inter-relationships. The simultaneous exist-ence of pulpal problems and inflammatory periodontal disease can complicate diagnosis and treatment planning. This case report evaluates the efficacy of G-Bone graft in the management of vertical bone loss associated with an endo-perio lesion in a left mandibular first molar and second molar. A 40 year-old male patient with an endo-perio lesion in the left mandibular first and second molars was initially treated with endodontic therapy. Following the en-dodontic treatment, the defect was treated using G-Bone graft. At the end of 6 months, there was a gain in the clini-cal attachment level and reduction in probing depth. Radiographic evidence showed that there was a significant bony fill.

Purification and biochemical function of Astragalus membtanaceus pathogenesis-related protein 10 / 中国中药杂志

Astragalus membranaceus pathogenesis-related protein 10 (AmPR-10) is largely expressed in case of environmental pressure and pathogen invasion. This study aims to explore the biochemical functions of AmPR-10. The dried root of Astragalus membranaceus was mechanically homogenized and extracted by Tris-HCl buffer to obtain its crude extract, which was then purified by anion exchange chromatography and gel filtration chromatography to obtain electrophoretically pure AmPR-10. The nuclease activity of AmPR-10 was tested with different RNAs by detecting the absorption value at 260 nm. The results demonstrated potent nuclease activity toward yeast tRNA, yeast RNA, Poly (A) and Poly (C). The optimum reaction temperature was 50 °C and pH was 7-8. EDTA showed no effect on its activity, while Mg²⁺ exhibited potent activation effect on the activity, and Co²⁺, Ca²⁺ and Zn²⁺ manifested moderately inhibition of the activity. Since AmPR-10 had no sequence homology with other known nucleases, AmPR-10 was probably a novel nuclease. The inhibition kinetic data against papain was analyzed by Lineweaver-Burk plots, and the results showed that the inhibition of papain followed noncompetitive-type kinetics. AmPR-10 played an important role in Astragalus membranaceus defense mechanism against environmental pressure and pathogen invasion, which may be achieved by inhibiting cycteine enzymes activity.

Úlcera crônica tratada com gel de papaína 10% na Estratégia Saúde da Família: relato de experiência / Chronic ulcer treated with 10% papain gel by Family Health Strategy: experience report / Ulcera crónica tratada con papaína gel al 10% en Estrategia Salud de la Familia: informe de experiencia

As úlceras em membros inferiores são um problema de saúde pública em razão da significativa incidência, do elevado custo e de frequente recidiva. Frequentemente, a equipe da Estratégia Saúde da Família é o primeiro contato do portador de úlcera, necessitando estar atualizada acerca das opções terapêuticas. Um produto tópico utilizado no tratamento das úlceras cutâneas é o gel de papaína a 10%, devido às suas propriedades bactericida, bacteriostática e desbridante de tecidos necrosados, bem como acelerador de crescimento tecidual. Descreve-se a experiência da equipe da Estratégia Saúde da Família com a utilização de gel de papaína a 10% no tratamento de úlcera cutânea, que regrediu após o uso do gel, destacando-se o matriciamento pelo especialista para a resolutividade do caso.

Lower body ulcers are a public health problem due to the significant incidence, high cost and frequent recurrence. Often, the Family Strategy Unit team provides first contact care with the ulcer carrier, so is a requirement for the team to be updated about treatment options. A topical product used for treatment of cutaneous ulcers is 10% papain gel, due to its antibacterial and debridant characteristics of necrotic tissues and the promotion of tissue renovation. This report describes the experience of Family Strategy Unit team using 10% papain gel in the treatment of cutaneous ulcers, which regressed after thirty days of this use, with emphasis on the support by the specialist for the resolution of the case.

Las úlceras en las piernas son un problema de salud pública debido a las repercusiones significativas del alto costo y la recurrencia frecuente. A menudo, el personal de la Estrategia Salud de la Familia es el contacto inicial. El equipo necesita saber sobre los nuevos tratamientos. El producto tópico utilizado en el tratamiento de úlceras de la piel es el gel de papaína 10% debido a sus propiedades bactericidas, bacteriostáticos y desbridamiento de tejido necrótico y acelerador de crecimiento del tejido. Se describe la experiencia del equipo de Salud de la Familia con el utilización de gel de papaína 10% en el tratamiento de úlceras de la piel, que se resolvió después de usar el gel, la matriz destacando el experto para la resolución del caso.

Effect of different combinations of papain-based gels on dissolving pulp tissue / Efeito de diferentes combinações de géis à base de papaína na dissolução do tecido pulpar

The focus of this study was to evaluate the capacity to dissolve pulp tissue of various combinations of papain-based gels and other antimicrobial agents. 105 bovine pulps were used, of standardized sizes, fragmented into 15mm-sized portions and weighed on an analytical balance, divided into 7 groups (n=15): 1 - 0.9% Saline Solution (negative control); 2- 8% Papain gel; 3- 8% Papain gel + 0.5% Chloramine; 4- 0.5% Chloramine gel; 5- 8% Papain gel + 2% Chlorhexidine; 6- 2% Chlorhexidine gel; and 7- 5.25% Sodium Hypochlorite solution (positive control). After initial weighing, the pulp fragments were inserted in test tubes for dissolution for time intervals of 30, 60, 90 and 120 minutes, and then weighed again. The data were analyzed using the Kruskal­Wallis and Mann­Whitney tests (p<0.05). In the time interval of 120 minutes the 0.5% chloramine gel demonstrated 64.9% ability to pulp dissolve, followed by 8% papain gel with 61.3%; papain associated with 0.5% chloramine, 58%; and papain associated with 2% chlorhexidine, 55.4%; which showed statistically significant difference with 5.25% Sodium Hypochlorite (p<0.05). All the gels that contained papain and the 0.5% chloramine gel promoted pulp tissue dissolution, however on a significantly lower scale than 5.25% sodium hypochlorite. The 2% chlorhexidine demonstrated no capacity to dissolve pulp, as did the control.

O foco deste estudo foi avaliar a capacidade de dissolução do tecido pulpar de várias combinações de géis à base de papaína e outros agentes antimicrobianos. Foram utilizadas 105 polpas bovinas, de tamanhos padronizados, fragmentadas em porções de 15mm e pesadas em balança analítica, divididas em 7 grupos (n = 15): 1 - 0,9% de Solução Salina (controle negativo); 2- 8% de gel de papaína; 3- 8% de gel de papaína + 0,5% de cloramina; 4- 0,5% de gel de cloramina; 5- 8% de gel de papaína + 2% de clorhexidina; 6- 2% de gel de clorexidina e 7- 5,25% de solução de hipoclorito de sódio (controlo positivo). Após pesagem inicial, os fragmentos de polpa foram inseridos em tubos de ensaio para dissolução durante intervalos de tempo de 30, 60, 90 e 120 minutos e depois pesados novamente. Os dados foram analisados pelos testes de Kruskal-Wallis e Mann-Whitney (p <0,05). No intervalo de tempo de 120 minutos o gel de cloramina a 0,5% demonstrou 64,9% de capacidade de dissolver polpa, seguido pelo gel de papaína a 8% com 61,3%; papaína associada a 0,5% de cloramina, 58%; e papaína associada a clorexidina a 2%, 55,4%; que apresentaram diferença estatisticamente significativa com hipoclorito de sódio a 5,25% (p <0,05). Todos os géis que continham papaína e o gel de cloramina a 0,5% promoveram a dissolução do tecido de polpa, contudo em uma escala significativamente inferior a 5,25% de hipoclorito de sódio. A clorexidina a 2% não demonstrou capacidade para dissolver a polpa, assim como o controle.

Remoción quimica-mecánica del tejido cariado en dientes permanentes: reporte de caso clínico / Chemo-mechanical caries removal in permanent tooth: case report

En odontopediatría, una de las principales dificultades que se presenta en la remoción del tejido dental cariado, es el manejo del paciente niño debido al miedo y a la ansiedad por el uso de anestesia e instrumentos rotatorios. A fin de resolver este problema, el método químico-mecánico para la eliminación del tejido cariado, es una alternativa conservadora y eficaz ya que evita el uso de anestésicos e instrumental rotatorio. Según estudios, este método es más aceptado por los pacientes comparado con el método tradicional. Este artículo tiene como objetivo presentar un caso clínico del uso del gel a base de papaína (Papacárie ®) para la eliminación químico-mecánica del tejido cariado en dientes permanentes.

anesthesia and rotating instruments. In order to solve this problem, the chemical-mechanical method for the elimination of the carious tissue, is a conservative and effective alternative since it avoids the use of anesthetics and rotary instruments. According to studies, this method is more accepted by patients compared to the traditional method. This paper aims to present a clinical case of the use of papain gel (Papacárie ®) for the chemical-mechanical removal of carious tissue in permanent teeth.

Investigation of irregular antibodies in blood samples from donors in Shanghai area / 中国输血杂志

Objective With the purpose of further improving blood safety and providing the reference basis for a strategy suitable for large-scale routine screening of irregular antibodies in blood service,the irregular antibodies in blood samples from donors in Shanghai area were investigated.Method Irregular blood antibodies were screened in 824 072 blood donors through combined method of saline medium and microplate papain.Result 1 246 samples of blood donors were detected with irregular antibodies among the total 824 072 samples which indicate the positive rate of 1.51‰.Conclusion Screening of irregular antibodies in blood samples from donors can effectively reduce or avoid hemolytic transfusion reactions.The combined method of saline medium and microplate papain is an effective method for large-scale routine screening of irregular antibodies in blood service.