QM/MM MD studies of polyester synthesis/hydrolysis

The world is suffering the consequences of plastic pollution, however modern societies remain heavily reliant on plastics. More sustainable alternatives are actively being sought-after. Enzymatic synthesis can offer a more sustainable route for polyester synthesis. Nevertheless, there are still limitations, such as limited activity and selectivity for some monomers, unfavorable compatibility in chemoenzymatic reactions and low stability under harsh reactions conditions. We have studied the catalytic mechanisms for polycaprolactone hydrolysis/synthesis by the wildtype enzymes Archaeoglobus fulgidus carboxylesterase (AfEST) and Candida antarctica lipase B (CaLB) and respective enzyme variants by performing Quantum Mechanics/Molecular Mechanics Molecular Dynamics simulations [1-3]. Our results give important insights towards the design of new enzyme variants combining good activity with high thermostability. [1] Almeida, B., Figueiredo, P., Carvalho, A. PCL enzymatic hydrolysis: a mechanistic study. ACS Omega. Doi: 10.1021/acsomega.9b00345. [2] Carvalho, A. T. P., Dourado, D. F. A. R., Skvortsov, T., de Abreu M., Ferguson, L. J., Quinn, D. J., Moody, T. S. Huang, M. Spatial requirement for PAMO for transformation of non-native linear substrates. Phys. Chem. Chem. Phys. 2018. [3] Dourado D. F. A. R., Swart M., Carvalho A. T. P. Why the flavin dinucleotide cofactor needs to be covalently linked to Complex II of the electron transport chain for conversion of FADH2 to FAD. Chem. – Eur J. 2018

ENZYME ENGINEERING AT ALMAC: Case studies of enzyme discovery and engineering

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Development and application of novel engineered transaminase panels assisted by in- silico rational design for the production of chiral amines

There is a high demand for the synthesis of chiral amines as building blocks for a large number of industrially valuable compounds. Transaminases (TAm) offer an enzymatic route for the synthesis of chiral amines that avoids complex chemical synthesis [1]. However, their catalytic efficiency towards bulky ketone substrates is greatly limited by steric hinderance [2]. This poster highlights a rational design strategy of combining in silico and in vitro methods to engineer the transaminase enzyme with a minimal number of mutations, achieving high catalytic activity and high enantioselectivity. The wildtype TAm showed no detectable activity towards the ketone 2-acetylbiphenyl but upon introduction of two mutations detectable enzyme activity was observed. The reaction rate was improved a further 1716-fold with the rationally designed variant, that contained a further 5 mutations, producing the corresponding enantiomeric pure (S)-amine (enantiomeric excess (ee) value of \u3e99%)[3]. In addition, screening of in silico designed (R)-TAm mutant panels in resolution mode offered an attractive and efficient route for the preparation of problematic (S)-amines. A mutant was identified from the panels that gave complete resolution of the racemic amine (high substrate loading) to leave the desired enantiomer at a low enzyme loading fit for process development towards an economically viable scale up process. [1] R. C. Simon, et al, ACS Catal. 2014, 4(1) [2] F. Steffen-Munsberg, et al, ChemCatChem 2013, 5, (1) [3]D.F.A.R.Dourado et al, ACS Catal. 2016, 6 (11

Market research in the Finnish food industry

This study introduces the important factors of market research and its significancewhen aiming at new foreign markets. Understanding the cultural differences, the consumers and the market itself with the competitors’ actions among other factors, the organization has a better chance to succeed in entering new markets. The case company is a Belgian food industry company which is interested in the Finnish market environment with its consumers and competitors. Currently they are operating in Central European countries with a little market share. An interview was conducted in order to better understand their current situation and expectations on new markets. The company’s products are sold in specialty stores and in bigger hypermarkets due to their higher image which they would like to obtain in Finland. As a small country Finland can not offer big markets but this is no obstacle for the case company since they are not looking to challenge the market leader or even the followers. Instead they are looking for a small market share as in other countries that they already operate in. By using selective distribution focused on the biggest city areas the product availability is guaranteed to the majority of the Finnish population. The thesis emphasizes the different business chains - the different types of stores and their product variety as well as competitors and their product pricing. Among this, the importance of product visibility will be shown as the case company wishes to enter the markets with as little marketing as possible. Regulations on labelling are studied as well as there are little differences from organizations’ home markets. The study also introduces a Finnish importing company that could possibly cooperate with the customer when aiming at the Finnish markets.Tutkimus esittelee markkinatutkimuksen tärkeimmät osa-alueet sekä sen merkityksen tavoiteltaessa uusia markkinoita. Kulttuurillisten eroavaisuuksien, kuluttajien sekä itse markkinoiden ymmärtäminen kilpailijoineen edesauttaa yrityksen menestymistä uudella markkina-alueella. Asiakasyritys on Belgialainen elintarvikeyritys joka kiinnostui Suomen markkinaympäristöstä, kuluttajista sekä kilpailijoista. Tällä hetkellä he toimivat Keski-Euroopan markkinoilla pienin markkinaosuuksin. Haastattelu suoritettiin jotta saataisiin selville heidän nykytilanteensa sekä tulevaisuuden näkymät uusista markkinoista. Yrityksen tuotteet ovat myynnissä erikoisliikkeissä sekä suurimmissa marketeissa korkean imagon vuoksi ja näin he toivoisivat myös tapahtuvan Suomessa. Suomessa ei ole tarjolla suuria markkinoita jo pelkästään maan koon vuoksi. Tämä ei ole kuitenkaan este asiakasyritykselle sillä he eivät lähde haastamaan markkinajohtajaa tai seuraajia, vaan tyytyvät pieneen markkinaosuuteen aivan kuten muillakin markkinoilla. Selektiivisellä tuotejakelulla, keskittyen Suomen suurimpiin kaupunkialueisiin, taataan tuotteiden saatavuus suurimmalle osalle väestöstä. Tutkimus painottuu eri liikeketjuihin, Suomen kauppatyyppeihin ja niiden tuotevalikoiman suuruuteen sekä kilpailijoihin ja heidän tuotehinnoitteluun. Tämän lisäksi tuotteiden näkyvyyden tärkeys osoitetaan, sillä asiakasyritys toivoisi markkinoille tuloa vähäisin markkinointitoiminnoin. Pakkausmerkintäsäännökset tulevat myös esille sillä ne eroavat hieman yrityksen kotimarkkinoiden säännöksistä. Tutkimus esittelee myös suomalaisen maahantuontiyrityksen, joka voisi mahdollisesti toimia asiakasyrityksen yhteistyökumppanina Suomen markkinoille pyrittäessä

Atuação da comissão de farmácia e terapêutica em um hospital de ensino

The hospital activities are characterized by a highly dynamism as a result of new health technologies such as medicines. A hospital due its characteristics of teaching, research and high complexity care, has the highest concentration of different types of health technologies. The Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto – USP (HCFMRP-USP) is a hospital, with proven quality, inserted in the SUS as a tertiary/quaternary referral and has the Pharmaceutical Services Division (DAF) for development of actions of health care. To aid resource management, selection and standardizationof drugs, DAF adopted the strategy of Pharmacy and Therapeutics Committee (CFT). The CFT is a collegial, consultative and deliberative body, established by the World Health Organization as a strategy tool to monitor and promote the quality in the use of medicine, but studies of CFTs are incipient in Brazil. Thus, this study aims to present the CFT of HCFMRP–USP. Objectives: To introduce the composition, responsibilities and working methods of CFT, as well as a critical analysis of its current operation. Methods: A descriptive study aimed to describe the current functioning of the CFT of HCFMRP-USP was performed. Ordinances, internal regulations were surveyed and a bibliographic review of the CFT was performed. To the critical analysis of the current operating, was selected by the committee from the standard one that would fit classification as belonging to “A” and “V” items after the crossing of the curves ABC and VEN, whose selected item was the medicine Sevoflurane. Results: The CFT was established in 2010 to replace the defunct standardization committee. Since then, the CFT examined 134 requests and 41 of these were standardized. The Sevoflurane drug was incorporated into the HCFMRP-USP in 2010 and, starting that year, there was a gradual increase in the consumption of the same. However, after analyzing the requirements of the drug in 2012, it was observed that the dispensation of Sevoflurane does not follow the specifications of the patient profile as established in the protocol established at the time of standardization. Conclusions: We concluded that the implementation of CFT was a strategy that provided a rational standardization. However, it is observed that there is no control of dispensing and use of the product according to the protocol established at the time of standardization. We emphasize that control the use of Sevoflurane is not responsibility of the CFT and this assignment should be delegated to the responsible technical area.As atividades hospitalares caracterizam-se por um acentuado dinamismo em consequência do surgimento de novas tecnologias em saúde, tais como medicamentos. Uma unidade hospitalar, devido suas características de ensino, pesquisa e atendimentos de alta complexidade, possui maior concentração de diferentes tipos de tecnologias em saúde. O Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto –USP (HCFMRP-USP) é uma instituição hospitalar, de qualidade comprovada, inserido no SUS como referência terciária/quaternária e que conta com a Divisão de Assistência Farmacêutica (DAF) para desenvolvimento das ações de atenção a saúde. A DAF para auxílio da gestão de recursos, seleção e padronização de medicamentos adotou como estratégia a Comissão de Farmácia e Terapêutica (CFT). A CFT é uma instância colegiada, de caráter consultivo e deliberativo, estabelecida pela Organização Mundial de Saúde como ferramenta de estratégia para monitorar e promover a qualidade no uso do medicamento, porém estudos que sobre a atuação das CFTs no Brasil são incipientes. Desta forma, este estudo pretende apresentar a CFT do HCFMRP-USP. Objetivos: apresentar a composição, atribuições e metodologia de trabalho da CFT, bem como desenvolver uma análise crítica de seu atual funcionamento. Metodologia: Foi realizado estudo descritivo e exploratório com o objetivo de descrever o atual funcionamento da CFT do HCFMRP-USP. Foram buscadas portarias, regulamentações internas e foi realizada revisão bibliográfica sobre a CFT. Para Análise crítica do atual funcionamento, foi selecionado dentre os itens padronizados pela comissão aquele que se enquadrasse como pertencente a classificação A e V, após o cruzamento das curvas ABC e VEN, cujo item selecionado foi o medicamento Sevoflurano. Resultados: A CFT foi instituída no ano de 2010 em substituição a extinta comissão de padronização. Desde então, a CFT analisou 134 solicitações e destas 41 foram padronizadas. O medicamento sevoflurano foi incorporado no HCFMRP-USP em 2010 e, a partir deste ano, observa-se um aumento gradativo do consumo do mesmo. Entretanto, após análise das prescrições do referido medicamento no ano de 2012, foi observado que a dispensação do sevoflurano não segue as especificações do perfil de pacientes conforme estabelecido no protocolo instituído no momento da padronização. Conclusões: Portanto, concluímos que a implantação da CFT foi uma estratégia que proporcionou a padronização racional. Entretanto, observa-se que não há controle da dispensação e utilização do medicamento de acordo com o protocolo estabelecido no momento da padronização. Salientamos que o controle do uso do Sevoflurano não é atribuição da CFT devendo esta atribuição ser delegada à área técnica responsável

A breakthrough on Amanita phalloides poisoning: an effective antidotal effect by polymyxin B

Amanita phalloides is responsible for more than 90 % of mushroom-related fatalities, and no effective antidote is available. a-Amanitin, the main toxin of A. phalloides, inhibits RNA polymerase II (RNAP II), causing hepatic and kidney failure. In silico studies included docking and molecular dynamics simulation coupled to molecular mechanics with generalized Born and surface area method energy decomposition on RNAP II. They were performed with a clinical drug that shares chemical similarities to a-amanitin, polymyxin B. The results show that polymyxin B potentially binds to RNAP II in the same interface of a-amanitin, preventing the toxin from binding to RNAP II. In vivo, the inhibition of the mRNA transcripts elicited by a-amanitin was efficiently reverted by polymyxin B in the kidneys. Moreover, polymyxin B significantly decreased the hepatic and renal a-amanitin-induced injury as seen by the histology and hepatic aminotransferases plasma data. In the survival assay, all animals exposed to a-amanitin died within 5 days, whereas 50 % survived up to 30 days when polymyxin B was administered 4, 8, and 12 h post-a-amanitin. Moreover, a single dose of polymyxin B administered concomitantly with a-amanitin was able to guarantee 100 % survival. Polymyxin B protects RNAP II from inactivation leading to an effective prevention of organ damage and increasing survival in a-amanitin-treated animals. The present use of clinically relevant concentrations of an already human-use-approved drug prompts the use of polymyxin B as an antidote for A. phalloides poisoning in humans.Juliana Garcia, Vera Marisa Costa, Ricardo Dinis-Oliveira and Ricardo Silvestre thank FCT-Foundation for Science and Technology-for their PhD grant (SFRH/BD/74979/2010), Post-doc grants (SFRH/BPD/63746/2009 and SFRH/BPD/110001/2015) and Investigator grants (IF/01147/2013) and (IF/00021/2014), respectively. This work was supported by the Fundacao para a Ciencia e Tecnologia (FCT) - project PTDC/DTPFTO/4973/2014 - and the European Union (FEDER funds through COMPETE) and National Funds (FCT, Fundacao para a Ciencia e Tecnologia) through project Pest-C/EQB/LA0006/2013

Participation of Candida albicans transcription factor Rlm1 in cell wall biogenesis and virulence

Candida albicans cell wall is important for growth and interaction with the environment. RLM1 is one of the putative transcription factors involved in the cell wall integrity pathway, which plays an important role in the maintenance of the cell wall integrity. In this work we investigated the involvement of RLM1 in the cell wall biogenesis and in virulence. Newly constructed C. albicans Δ/Δrlm1 mutants showed typical cell wall weakening phenotypes, such as hypersensitivity to Congo Red, Calcofluor White, and caspofungin (phenotype reverted in the presence of sorbitol), confirming the involvement of RLM1 in the cell wall integrity. Additionally, the cell wall of C. albicans Δ/Δrlm1 showed a significant increase in chitin (213%) and reduction in mannans (60%), in comparison with the wild-type, results that are consistent with cell wall remodelling. Microarray analysis in the absence of any stress showed that deletion of RLM1 in C. albicans significantly down-regulated genes involved in carbohydrate catabolism such as DAK2, GLK4, NHT1 and TPS1, up-regulated genes involved in the utilization of alternative carbon sources, like AGP2, SOU1, SAP6, CIT1 or GAL4, and genes involved in cell adhesion like ECE1, ALS1, ALS3, HWP1 or RBT1. In agreement with the microarray results adhesion assays showed an increased amount of adhering cells and total biomass in the mutant strain, in comparison with the wild-type. C. albicans mutant Δ/Δrlm1 strain was also found to be less virulent than the wild-type and complemented strains in the murine model of disseminated candidiasis. Overall, we showed that in the absence of RLM1 the modifications in the cell wall composition alter yeast interaction with the environment, with consequences in adhesion ability and virulence. The gene expression findings suggest that this gene participates in the cell wall biogenesis, with the mutant rearranging its metabolic pathways to allow the use of alternative carbon sources.This work was supported by CBMA (Centre of Molecular and Environmental Biology) through the FCT (Fundacao para a Ciencia e Tecnologia) project PEst-C/BIA/UI4050/2011. Yolanda Delgado-Silva was supported by an ALbAN scholarship (No E07D400922PE), and Alexandra Correia by SFRH/BD/31354/2006 fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

A de novo paradigm for male infertility

Funding Information: (DFG, CRU326) to C.F. and F.T. This project was also supported in part by funding from the Australian National Health and Medical Research Council (APP1120356) to M.K.O.B., by grants from the National Institutes of Health of the United States of America (R01HD078641 to D.F.C. and K.I.A., P50HD096723 to D.F.C.) and from the Biotechnology and Biological Sciences Research Council (BB/S008039/1) to D.J.E. Funding Information: We are grateful for the participation of all patients and their parents in this study. We thank Laurens van de Wiel (Radboudumc), Sebastian Judd-Mole (Monash University), Arron Scott and Bryan Hepworth (Newcastle University) for technical support, and Margot J Wyrwoll (University of Münster) for help with handling MERGE samples and data. This project was funded by The Netherlands Organization for Scientific Research (918-15-667) to J.A.V. as well as an Investigator Award in Science from the Wellcome Trust (209451) to J.A.V. a grant from the Catherine van Tussenbroek Foundation to M.S.O. a grant from MERCK to R.S. a UUKi Rutherford Fund Fellowship awarded to B.J.H. and the German Research Foundation Clinical Research Unit “Male Germ Cells” Publisher Copyright: © 2022, The Author(s).De novo mutations are known to play a prominent role in sporadic disorders with reduced fitness. We hypothesize that de novo mutations play an important role in severe male infertility and explain a portion of the genetic causes of this understudied disorder. To test this hypothesis, we utilize trio-based exome sequencing in a cohort of 185 infertile males and their unaffected parents. Following a systematic analysis, 29 of 145 rare (MAF < 0.1%) protein-altering de novo mutations are classified as possibly causative of the male infertility phenotype. We observed a significant enrichment of loss-of-function de novo mutations in loss-of-function-intolerant genes (p-value = 1.00 × 10−5) in infertile men compared to controls. Additionally, we detected a significant increase in predicted pathogenic de novo missense mutations affecting missense-intolerant genes (p-value = 5.01 × 10−4) in contrast to predicted benign de novo mutations. One gene we identify, RBM5, is an essential regulator of male germ cell pre-mRNA splicing and has been previously implicated in male infertility in mice. In a follow-up study, 6 rare pathogenic missense mutations affecting this gene are observed in a cohort of 2,506 infertile patients, whilst we find no such mutations in a cohort of 5,784 fertile men (p-value = 0.03). Our results provide evidence for the role of de novo mutations in severe male infertility and point to new candidate genes affecting fertility.publishersversionpublishe

Bypassing cellular EGF receptor dependence through epithelial-to-mesenchymal-like transitions

Over 90% of all cancers are carcinomas, malignancies derived from cells of epithelial origin. As carcinomas progress, these tumors may lose epithelial morphology and acquire mesenchymal characteristics which contribute to metastatic potential. An epithelial-to-mesenchymal transition (EMT) similar to the process critical for embryonic development is thought to be an important mechanism for promoting cancer invasion and metastasis. Epithelial-to-mesenchymal transitions have been induced in vitro by transient or unregulated activation of receptor tyrosine kinase signaling pathways, oncogene signaling and disruption of homotypic cell adhesion. These cellular models attempt to mimic the complexity of human carcinomas which respond to autocrine and paracrine signals from both the tumor and its microenvironment. Activation of the epidermal growth factor receptor (EGFR) has been implicated in the neoplastic transformation of solid tumors and overexpression of EGFR has been shown to correlate with poor survival. Notably, epithelial tumor cells have been shown to be significantly more sensitive to EGFR inhibitors than tumor cells which have undergone an EMT-like transition and acquired mesenchymal characteristics, including non-small cell lung (NSCLC), head and neck (HN), bladder, colorectal, pancreas and breast carcinomas. EGFR blockade has also been shown to inhibit cellular migration, suggesting a role for EGFR inhibitors in the control of metastasis. The interaction between EGFR and the multiple signaling nodes which regulate EMT suggest that the combination of an EGFR inhibitor and other molecular targeted agents may offer a novel approach to controlling metastasis