Produção do fator de crescimento epidermal humano (hEGF) em Komagataella phaffii

Dissertação (mestrado)—Universidade de Brasília, Departamento de Biologia Celular, Programa de Pós-Graduação em Tecnologias Química e Biológica, 2018.O sistema de expressão baseado na utilização da levedura Komagataella phaffii tem sido utilizado com sucesso na produção de uma grande variedade de proteínas heterólogas. Esta levedura reúne características como fácil manipulação molecular, crescimento celular rápido, capacidade de realizar modificações pós-traducionais e secreção eficiente de proteínas, além de atingir altas densidades celulares com grande produção da proteína heteróloga. Uma das proteínas de grande interesse na indústria biofarmacêutica e cosmética é o fator de crescimento epidermal humano (hEGF). O objetivo desse estudo foi desenvolver um sistema para a produção de hEGF na levedura K. phaffii. Para isso foram utilizados os peptídeos sinais dos genes αF, SUC2 e PHO1. Três vetores foram construídos, cada um deles contendo um dos peptídeos sinal. Foram construídos os vetores de expressão sob o controle do promotor do PGK1 e utilizados para transformação de K. phaffii M12-K, uma linhagem mutante para o gene KEX1. Os clones recombinantes foram confirmados por PCR de colônia e foram crescidos em meio mínimo para avaliar a cinética de crescimento. Os clones positivos foram selecionados e utilizados na expressão em frasco empregando meio complexo. A presença do hEGF foi avaliada por SDS-PAGE e confirmada por Western blot na presença de um anticorpo específico. Com isso, um clone de cada sistema foi escolhido para a purificação do hEGF por cromatografia de exclusão molecular e RP-HPLC. A partir desses experimentos foi confirmada a presença do hEGF e o polipeptídeo foi parcialmente purificado. Finalmente, foi escolhido o clone 1αF para otimizar o processo de purificação, adicionando uma etapa em HILIC. O hEGF foi satisfatoriamente purificado. O tamanho correto e a sequência do N-terminal igual ao EGF presente em humanos foram confirmados por espectrometria de massas e sequenciamento automático. Os resultados obtidos mostram que o hEGF foi produzido com sucesso em K. phaffii com a sequência primária correta.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).The expression system based on the utilization of the yeast Komagataella phaffii has been used successfully used in the production of a large variety of heterologous proteins. This yeast combines several important features such as easy molecular manipulation, rapid cell growth, ability to perform post-translational modifications and efficient secretion of proteins, in addition to large heterologous protein production at high cell densities. One of the proteins of great interest in the biopharmaceutical and cosmetic industry is the human epidermal growth factor (hEGF). The aim of this study was to develop a system for hEGF production in K. phaffii. For this, the 3 different signal peptides sequences from genes αF, SUC2 and PHO1 were tested. Three vectors were constructed, each containing one of the signal peptides. Expression vectors were constructed under the control of the PGK1 promoter and used for transformation of K. phaffii M12-K, a strain mutant for the KEX1 gene. Recombinant clones were confirmed by colony PCR and grown in minimal medium to evaluate growth kinetics. Positive clones were selected and used in flask expression using complex media. The presence of hEGF was assessed by SDS-PAGE and confirmed by western blot with a specific antibody. One clone from each system was chosen for the production and purification of hEGF by molecular-exchange chromatography and RP-HPLC. From these experiments the presence of hEGF was confirmed and the polypeptide was partially purified. Finally, clone 1αF was chosen to optimize the purification process by adding one step in HILIC. The hEGF was successfully purified. Correct size and N-terminal sequence equal to EGF present in humans were confirmed by mass spectrometry and Edman sequencing. Together, our results show that hEGF was successfully produced in K. phaffii with the proper primary structure.El sistema de expresión basado en la utilización de la levadura Komagataella phaffii ha sido utilizado con éxito en la producción de una gran variedad de proteínas heterólogas. Esta levadura reúne características como fácil manipulación molecular, rápido crecimiento celular, capacidad de realizar modificaciones pos-traduccionales y eficiente secreción de proteínas, además de llegar hasta altas densidades celulares con elevada producción heteróloga. Una de las proteínas de gran interés en la industria biofarmacéutica y cosmétic es el factor de crecimiento epidérmico humano (hEGF). El objetivo de este estudio foi desarrollar un sistema para la producción de hEGF en la levadura K. phaffii. Para eso fueron utilizadas los péptidos señales de los genes αF, SUC2 e PHO1. Fueron construidos tres vectores, cada uno de ellos conteniendo una de las sequencias señales. Los vectores fueron construidos sobre el control del promotor del gen PGK1 y fueron utilizados para la transformación de la cepa M12-K de K. phaffii, que es mutante para el gen KEX1. Los clones recombinantes fueron confirmados mediante PCR de colonias y fueron crecidos en medio mínimo para analizar la cinética de crecimiento. Los clones positivos fueron seleccionados y utilizados para expresión en frasco utilizando medio complejo. La presencia de hEGF fue analizada por SDS-PAGE e confirmada por western blot en presencia de un anticuerpo específico. A partir de ahí, una muestra de cada sistema fue escogida para la purificación de hEGF utilizando cromatografía de exclusión molecular y RP-HPLC. A partir de esos experimentos fue confirmada la presencia de hEGF y el polipeptídeo fue parcialmente purificado. Finalmente, fue escogido el clon 1αF para optimizar el proceso de purificación, adicionando una cromatografía en HILIC. El hEGF fue satisfactoriamente purificado. El tamaño correcto y la secuencia del N-terminal igual al EGF presente en humanos fueron confirmados por espectrometría de masas y sequenciamiento automático. Los resultados obtenidos muestran que fue producido hEGF en K. phaffii, con la sequencia primária correcta

Increased Prevalence of Symptomatic Human Intestinal Spirochetosis in MSM with High-Risk Sexual Behavior in a Cohort of 165 Individuals

Human intestinal spirochetosis (HIS) can cause gastrointestinal symptoms, although asymptomatic infections have been described. Individuals from low-income countries, people living with HIV, and men who have sex with men (MSM) show increased risk. A retrospective review of all patients diagnosed with HIS (n = 165) between January 2013 and October 2020 at a tertiary hospital in Madrid, Spain, was performed to assess risk factors for symptomatic HIS, symptoms, and response to treatment. Most patients were male (n = 156; 94.5%), 86.7% were MSM, and 23.5% practiced chemsex, of whom most were symptomatic (p = 0.039). Most patients (78.4%) reported unprotected oral-anal intercourse. A total of 124 (81.1%) were symptomatic; diarrhea was the most common complaint (68.3%). Multivariable regression showed increased odds of symptoms associated with age under 41 (odds ratio 5.44, 95% CI 1.87-15.88; p = 0.002). Colonoscopy was normal in 153 (92.7%). Furthermore, 66.7% presented previous or concomitant sexually transmitted diseases (STDs). Among the patients, 102 underwent testing for other gastrointestinal pathogens, with positive results in 20 (19.6%). All symptomatic patients without concomitant gastrointestinal infection presenting improvement on follow-up (42 of 53) had received either metronidazole or doxycycline (p = 0.049). HIS should be considered as a cause of chronic diarrhea in MSM with high-risk sexual behavior after other causes have been ruled out; treatment with metronidazole is recommended. Coinfection with other STDs is common.S

Novel indolic AMPK modulators induce vasodilatation through activation of the AMPK-eNOS-NO pathway

Endothelial adenosine monophosphate-activated protein kinase (AMPK) plays a critical role in the regulation of vascular tone through stimulating nitric oxide (NO) release in endothelial cells. Since obesity leads to endothelial dysfunction and AMPK dysregulation, AMPK activation might be an important strategy to restore vascular function in cardiometabolic alterations. Here, we report the identification of a novel AMPK modulator, the indolic derivative IND6, which shows affinity for AMPKα1β1γ1, the primary AMPK isoform in human EA.Hy926 endothelial cells. IND6 shows inhibitory action of the enzymatic activity in vitro, but increases the levels of p-Thr174AMPK, p-Ser1177eNOS and p-Ser79ACC in EA.Hy926. This paradoxical finding might be explained by the ability of IND6 to act as a mixed-type inhibitor, but also to promote the enzyme activation by adopting two distinct binding modes at the ADaM site. Moreover, functional assays reveal that IND6 increased the eNOS-dependent production of NO and elicited a concentration-dependent vasodilation of endothelium-intact rat aorta due to AMPK and eNOS activation, demonstrating a functional activation of the AMPK-eNOS-NO endothelial pathway. This kinase inhibition profile, combined with the paradoxical AMPK activation in cells and arteries, suggests that these new chemical entities may constitute a valuable starting point for the development of new AMPK modulators with therapeutic potential for the treatment of vascular complications associated with obesity

Risk Factors for Gestational Diabetes Mellitus in a Large Population of Women Living in Spain: Implications for Preventative Strategies

The aim of this study is to establish a risk appraisal model for GDM by identifying modifiable factors that can help predict the risk of GDM in a large population of 2194 women living in Spain. They were recruited between 2009-2010 when screening for GDM was performed. Participants completed a questionnaire on socio-demographic, anthropomorphic and behavioral characteristics, and reproductive and medical history. A total of 213 (9.7%) women were diagnosed as having GDM. Age, pregestational body weight (BW) and body mass index (BMI), and number of events of medical, obstetric and family history were significantly associated with GDM. After logistic regression model, biscuits and pastries intake 30 minutes/day, and 30 minutes/day of sports at least 2 days/week, compared with opposite consumption, was associated with less GDM risk. Our study identified several pregestational modifiable lifestyle risk factors associated with an increase in the risk of developing GDM. This may represent a promising approach for the prevention of GDM and subsequent complications. Further intervention studies are needed to evaluate if this appraisal model of risk calculation can be useful for prevention and treatment of GDM

Intracranial atherosclerotic plaque enhancement and long-term risk of future strokes: A prospective, longitudinal study

Background and Purpose The prognostic significance of postcontrast enhancement of intracranial atheromatous plaque is uncertain. Prospective, long-term follow-up studies in Caucasians, using a multicenter design, are lacking. We aimed to evaluate whether this radiological sign predicts long-term new stroke in symptomatic and asymptomatic intracranial atherosclerotic disease (ICAD) patients. Methods This was a prospective, observational, longitudinal, multicenter study. We included a symptomatic and an asymptomatic cohort of ICAD patients that underwent 3T MRI including high-resolution sequences focused on the atheromatous plaque. We evaluated grade of stenosis, plaque characteristics, and gadolinium enhancement ratio (postcontrast plaque signal/postcontrast corpus callosum signal). The occurrence of new events was evaluated at 3, 6, 9, and 12 months and annually thereafter. The association between plaque characteristics and new stroke was studied using Cox multiple regression survival analysis and Kaplan-Meier curves. Results Forty-eight symptomatic and 13 asymptomatic patients were included. During 56.3 ± 16.9 months, 11 patients (18%) suffered a new event (seven ischemic, two hemorrhagic, and two transient ischemic attacks). A receiver operating characteristic curve identified an enhancement ratio of >1.77 to predict a new event. In a multivariable Cox regression, postcontrast enhancement ratio >1.77 (hazard ratio [HR]= 3.632; 95% confidence interval [CI], 1.082-12.101) and cerebral microbleeds (HR = 5.244; 95% CI, 1.476-18.629) were independent predictors of future strokes. Patients with a plaque enhancement ratio >1.77 had a lower survival free of events (p < .05). Conclusions High intracranial postcontrast enhancement is a long-term predictor of new stroke in ICAD patients. Further studies are needed to elucidate whether postcontrast enhancement reflects inflammatory activity of intracranial atheromatous plaque.This study has been funded by the Spanish Ministry of Science, via FIS project PI13/02544, PI16/01396, and PI19/01398 and through the INVICTUS PLUS research network RD16/0019. Beatriz Gómez-Vicente received a research contract from the Junta de Castilla y León and European Social Fund, Spain. María Hernandez-Perez was funded by The Instituto de Salud Carlos III, Spain (JR17/00006)

Comprehensive cross-platform comparison of methods for non-invasive EGFR mutation testing : results of the RING observational trial.

Abstract Several platforms for noninvasive EGFR testing are currently used in the clinical setting with sensitivities ranging from 30% to 100%. Prospective studies evaluating agreement and sources for discordant results remain lacking. Herein, seven methodologies including two next-generation sequencing (NGS)-based methods, three high-sensitivity PCR-based platforms, and two FDA-approved methods were compared using 72 plasma samples, from EGFR-mutant non-small-cell lung cancer (NSCLC) patients progressing on a first-line tyrosine kinase inhibitor (TKI). NGS platforms as well as high-sensitivity PCR-based methodologies showed excellent agreement for EGFR-sensitizing mutations (K = 0.80-0.89) and substantial agreement for T790M testing (K = 0.77 and 0.68, respectively). Mutant allele frequencies (MAFs) obtained by different quantitative methods showed an excellent reproducibility (intraclass correlation coefficients 0.86-0.98). Among other technical factors, discordant calls mostly occurred at mutant allele frequencies (MAFs) ≤ 0.5%. Agreement significantly improved when discarding samples with MAF ≤ 0.5%. EGFR mutations were detected at significantly lower MAFs in patients with brain metastases, suggesting that these patients risk for a false-positive result. Our results support the use of liquid biopsies for noninvasive EGFR testing and highlight the need to systematically report MAFs. Keywords: NGS; circulating free DNA; epidermal growth factor receptor; non-small-cell lung cancer; osimertinib; tyrosine kinase inhibitor

External validity of clinical trials with diverse trastuzumab-based chemotherapy regimens in advanced gastroesophageal adenocarcinoma: data from the AGAMENON-SEOM registry

Background: Trastuzumab combined with cisplatin and fluoropyrimidines, either capecitabine or 5-fluorouracile (XP/FP), is the standard first-line treatment for advanced, HER2-positive, gastric cancer patients based on the ToGA trial. Despite the lack of phase III trials, many clinicians administer trastuzumab with alternative regimens. One meta-analysis suggests that substituting cisplatin for oxaliplatin might lead to greater efficacy and less toxicity. Methods: 594 patients with HER2-positive gastroesophageal adenocarcinoma were recruited from the AGAMENON-SEOM registry. The objective was to evaluate the external validity of clinical trials with chemotherapy and trastuzumab. Results: The regimens used in at least 5% of the patients were XP (27%), oxaliplatin and capecitabine (CAPOX) (26%), oxaliplatin and 5-fluorouracil (FOLFOX) (14%), FP (14%), triplet with anthracycline/docetaxel (7%), and carboplatin-FU (5%). Median exposure to trastuzumab was longer with FOLFOX (11.4 months, 95% CI, 9.1-21.0) versus ToGA regimens (7.5, 6.4-8.5), p < 0.001. Patients with HER2-IHC 3+ cancers had higher response rates than those with IHC 2+/FISH+, odds-ratio 1.97 (95% CI, 1.25-3.09). The results achieved with CAPOX-trastuzumab were comparable to those attained with ToGA regimens. FOLFOX-trastuzumab was superior to ToGA schemes in terms of overall survival (OS), with a greater magnitude of effect in IHC 2+/FISH+ tumors (HR 0.47, 0.24-0.92) compared with IHC 3+ (HR 0.69, 0.49-0.96), and in diffuse (HR 0.37, 0.20-0.69) versus intestinal-type tumors (HR 0.76, 0.54-1.06). Conclusion: We have updated the external validity of clinical trials with trastuzumab in first-line treatment of gastric cancer. Our data confirm the comparable outcomes of ToGA regimens and CAPOX-trastuzumab in clinical practice and point toward a possible benefit of FOLFOX-trastuzumab, contingent on the subtypes typically less sensitive to trastuzumab, to be confirmed in clinical trials

Engineered fluorescent strains of cryptococcus neoformans : a versatile toolbox for studies of host-pathogen interactions and fungal biology, including the viable but nonculturable state

Cryptococcus neoformans is an opportunistic fungal pathogen known for its remarkable ability to infect and subvert phagocytes. This ability provides survival and persistence within the host and relies on phenotypic plasticity. The viable but nonculturable (VBNC) phenotype was recently described in C. neoformans, whose study is promising in understanding the pathophysiology of cryptococcosis. The use of fluorescent strains is improving host interaction research, but it is still underexploited. Here, we fused histone H3 or the poly(A) binding protein (Pab) to enhanced green fluorescent protein (eGFP) or mCherry, obtaining a set of C. neoformans transformants with different colors, patterns of fluorescence, and selective markers (hygromycin B resistance [Hygr ] or neomycin resistance [Neor]). We validated their similarity to the parental strain in the stress response, the expression of virulence-related phenotypes, mating, virulence in Galleria mellonella, and survival within murine macrophages. PAB-GFP, the brightest transformant, was successfully applied for the analysis of phagocytosis by flow cytometry and fluorescence microscopy. Moreover, we demonstrated that an engineered fluorescent strain of C. neoformans was able to generate VBNC cells. GFP-tagged Pab1, a key regulator of the stress response, evidenced nuclear retention of Pab1 and the assembly of cytoplasmic stress granules, unveiling posttranscriptional mechanisms associated with dormant C. neoformans cells. Our results support that the PAB-GFP strain is a useful tool for research on C. neoformans

Guía de práctica clínica SENPE/SEGHNP/SEFH sobre nutrición parenteral pediátrica

Introduction: Parenteral nutrition (PN) in childhood is a treatment whose characteristics are highly variable depending on the age and pathology of the patient. Material and methods: The Standardization and Protocols Group of the Spanish Society for Parenteral and Enteral Nutrition (SENPE) is an interdisciplinary group formed by members of the SENPE, the Spanish Society of Gastroenterology, Hepatology and Pediatric Nutrition (SEGHNP) and the Spanish Society of Hospital Pharmacy (SEFH) that intends to update this issue. For this, a detailed review of the literature has been carried out, looking for the evidences that allow us to elaborate a Clinical Practice Guide following the criteria of the Oxford Center for Evidence-Based Medicine. Results: This manuscript summarizes the recommendations regarding indications, access routes, requirements, modifications in special situations, components of the mixtures, prescription and standardization, preparation, administration, monitoring, complications and home NP. The complete document is published as a monographic number. Conclusions: This guide is intended to support the prescription of pediatric PN. It provides the basis for rational decisions in the context of the existing evidence. No guidelines can take into account all of the often compelling individual clinical circumstances.Introducción: la nutrición parenteral (NP) en la infancia es un tratamiento cuyas características son muy variables en función de la edad y la patología que presente el paciente. Material y métodos: el grupo de Estandarización y Protocolos de la Sociedad Española de Nutrición Parenteral y Enteral (SENPE) es un grupo interdisciplinar formado por miembros de la SENPE, Sociedad Española de Gastroenterología, Hepatología y Nutrición Pediátrica (SEGHNP) y Sociedad Española de Farmacia Hospitalaria (SEFH) que pretende poner al día este tema. Para ello, se ha realizado una revisión pormenorizada de la literatura buscando las evidencias que nos permiten elaborar una Guía de Práctica Clínica siguiendo los criterios del Oxford Centre for Evidence-Based Medicine. Resultados: este manuscrito expone de forma resumida las recomendaciones en cuanto a indicaciones, vías de acceso, requerimientos, modificaciones en situaciones especiales, componentes de las mezclas, prescripción y estandarización, preparación, administración, monitorización, complicaciones y NP domiciliaria. El documento completo se publica como número monográfico. Conclusiones: esta guía pretende servir de apoyo para la prescripción de la NP pediátrica. Constituye la base para tomar decisiones en el contexto de la evidencia existente. Ninguna guía puede tener en cuenta todas las circunstancias clínicas individuale

Modelling the spatial risk of malaria through probability distribution of Anopheles maculipennis s.l. and imported cases

Malaria remains one of the most important infectious diseases globally due to its high incidence and mortality rates. The influx of infected cases from endemic to non-endemic malaria regions like Europe has resulted in a public health concern over sporadic local outbreaks. This is facilitated by the continued presence of competent Anopheles vectors in non-endemic countries. We modelled the potential distribution of the main malaria vector across Spain using the ensemble of eight modelling techniques based on environmental parameters and the Anopheles maculipennis s.l. presence/absence data collected from 2000 to 2020. We then combined this map with the number of imported malaria cases in each municipality to detect the geographic hot spots with a higher risk of local malaria transmission. The malaria vector occurred preferentially in irrigated lands characterized by warm climate conditions and moderate annual precipitation. Some areas surrounding irrigated lands in northern Spain (e.g. Zaragoza, Logroño), mainland areas (e.g. Madrid, Toledo) and in the South (e.g. Huelva), presented a significant likelihood of A. maculipennis s.l. occurrence, with a large overlap with the presence of imported cases of malaria. While the risk of malaria re-emergence in Spain is low, it is not evenly distributed throughout the country. The four recorded local cases of mosquito-borne transmission occurred in areas with a high overlap of imported cases and mosquito presence. Integrating mosquito distribution with human incidence cases provides an effective tool for the quantification of large-scale geographic variation in transmission risk and pinpointing priority areas for targeted surveillance and prevention