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    Celulose bacteriana como penso curativo

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    Tese de doutoramento em Biomedical EngineeringWounds, in particular traumatic (e.g. burns) and chronic ones, are a major cause of morbidity, impaired life quality and high health care costs. They often result in long hospitalization stays, taking up substantial health resources in developed countries. Conventional treatments are often painful, expensive and may increase the infection risk, compromising the treatments‚Äô time and success. In recent years, there have been efforts to develop new advanced methodologies to heal chronic wounds, including the topic use of growth factors or cell-based therapies. However, in many cases, the therapeutic efficacy is low, the therapies are expensive and require application in a clinical facility. Therefore, development of new therapeutics is absolutely necessary and important to satisfy these unmet clinical needs. So, this work comprised the development of a safe, easy-to-use and non expensive novel dressing, aimed at efficiently addressing these issues, by attaining faster and proper wound healing. The use of bacterial nanocellulose (BNC) has already demonstrated positive results in the treatment of different kinds of wounds. Additionally, BNC is considered a promising drug delivery system. In this work, BNC was conceived as a protective barrier against exogenous agents (particles, microorganisms) that can impair wound healing, and as a drug carrier for the controlled release of hydrophobic drugs, namely of vitamin D3 (Vit D3 ), an inducer of the endogenous expression of antimicrobial peptide (AMP) LL37, known for accelerating the wound healing process. In a first part of this project, the optimization of the static BNC production was performed, aiming at making it viable and economic at large scale. First, an experimental design, based on response surface methodology (RSM) - central composite design (CCD) - was used to optimize the culture medium for BNC production by Komagataeibacter xylinus BPR 2001, using a simple culture medium composition based on byproducts from the food industry. The optimal conditions for BNC production were (% (m/v)): molasses 5.38; CSL 1.91; ammonium sulphate 0.63; disodium phosphate 0.270; citric acid 0.115 and ethanol 1.38 % (v/v). The experimental and predicted maximum BNC production yields were 7.5 ¬Ī0.54 g/L and 6.64 ¬Ī0.079 g/L, respectively, after 9 days at 30 ¬ļC. Furthermore, the effect of the surface area and culture medium depth on the BNC production yield and productivity were evaluated. BNC dry mass production increased with the surface area and with the medium volume (depth) and fermentation time. Also, as long as nutrients were still available in the culture media, the BNC mass productivity was maintained overtime. The pre-inoculum preparation (PIP) step was also optimized with regards to the (a) identification of an inexpensive culture medium for pre-inoculum leading to a high cell density; (b) analysis of the effect of the initial cellular concentration on the static production of BNC and (c) kinetics of cell growth throughout the different steps of pre-inoculum preparation, including static and stirred - laboratorial and pilot-scale ‚Äď fermentations. The best composition for PIP medium was (% (m/v)): Glucose and Fructose syrup 1.5- 2.0; Corn Step Liquor (protein basis) 0.7; citric acid 0.115; Na2HPO4 0.27. The analysis of the cell growth kinetics in the different steps of PIP showed that a careful control on the culture time in each stage is advisable. The time required to reach the exponential phase was very different in each stage of PIP, reducing significantly from the static culture to the stirred culture and for large scale stirred culture, in a 75 L Bioreactor. In a second part of this work, the use of BNC as a drug carrier was addressed. Since Vit D3 is poorly water soluble, and thus not easily incorporated in the highly hydrophilic environment of the BNC membrane, Vit D3 was encapsulated in a self-assembled hyaluronic acid (HA)-based amphiphilic nanogel and then incorporated in the BNC membrane. The carrier was obtained by grafting hexadecylamine (Hexa) into the HA backbone (HA-Hexa). Vit D3 was successfully loaded into the nanogel (HA-Vit D3 ) with an encapsulation efficiency between 60-91 %. The loaded system- HA-Vit D3 - was embedded into BNC, conceived as a transdermal delivery system. The release of Vit D3 was monitored over time using a Franz cell device. Around 70 % of the initial Vit D3 available was released from BNC membranes in the first 48 h. Most importantly, we observed that the released Vit D3 still remained within the HA-Hexa nanogel carrier. Vit D3 is known to stimulate the endogenous production of human cathelicidin (LL37), which is known to accelerate wound healing. Thus, formulations of HA-Vit D3 and HA-LLKKK18 (an analogue of LL37) were tested in vivo, using excision and chronic wound in dexamethasone treated C57BL/6 and db+/db+ mice models, as to evaluate and compare their efficiency in wound repairing. However, the results did not confirm any wound healing improvement.As feridas cr√≥nicas e traum√°ticas (e.g. queimaduras) apresentam uma elevada morbilidade, afetando severamente a qualidade de vida dos pacientes. Os tratamentos convencionais implicam longos per√≠odos de interna√ß√£o hospitalar, com significativo consumo de recursos dos sistemas de sa√ļde nos pa√≠ses desenvolvidos. Al√©m disso, s√£o dolorosos, caros e podem aumentar o risco de infe√ß√£o, comprometendo a dura√ß√£o e o sucesso dos tratamentos. Recentemente, t√™m sido desenvolvidos esfor√ßos para o desenvolvimento de novas metodologias avan√ßadas para o tratamento de feridas cr√≥nicas, incluindo a aplica√ß√£o t√≥pica de fatores de crescimento ou terapias baseadas em c√©lulas. Em muitos casos, estas novas abordagens s√£o caras, devendo ser realizadas numa unidade hospitalar, e a sua efic√°cia terap√™utica √© baixa. Assim, o desenvolvimento de novas solu√ß√Ķes para satisfazer esta necessidade cl√≠nica ainda n√£o satisfeita √© absolutamente necess√°rio. Com este trabalho pretende se desenvolver um penso curativo eficiente, inovador, f√°cil de usar e n√£o dispendioso, atrav√©s de uma abordagem segura, visando uma cicatriza√ß√£o mais r√°pida e adequada da ferida. A nanocelulose bacteriana (BNC) demonstrou j√° resultados positivos no tratamento de diferentes tipos de feridas, assim como foi j√° demonstrado tamb√©m o seu potencial como sistema de entrega de f√°rmacos. Neste trabalho, a BNC foi utilizada como ve√≠culo para a liberta√ß√£o controlada de mol√©culas hidrof√≥bicas, nomeadamente a vitamina D3 (Vit D3 ), que √© um indutor da express√£o end√≥gena do pept√≠do antimicrobiano LL37, conhecido por acelerar o processo de cicatriza√ß√£o de feridas. Al√©m disso, a BNC funciona como uma barreira protetora contra agentes ex√≥genos (poeiras, microorganismos) que podem prejudicar a cicatriza√ß√£o de feridas. Numa primeira parte, foram desenvolvidos trabalhos visando tornar a produ√ß√£o em grande escala de BNC em cultura est√°tica econ√≥mica e vi√°vel. Nesse sentido, foi usado um desenho experimental, baseado na metodologia de superf√≠cie de resposta (RSM) - planeamento composto central (CCD) - para otimizar o meio de cultura, usando subprodutos da ind√ļstria alimentar. Foi utilizada a estirpe Komagataeibacter xylinus BPR 2001, a 30 ¬ļC. Foram identificadas as seguintes condi√ß√Ķes √≥timas para a produ√ß√£o de BNC (% (m/v)): mela√ßo 5,38, xarope de milho (CSL) 1,91; sulfato de am√≥nio 0,63; fosfato diss√≥dico 0,270; √°cido c√≠trico 0,115 e etanol 1,38 % (v/v). Os rendimentos m√°ximos experimentais e previstos de produ√ß√£o de BNC foram 7,5 ¬Ī0,54 g/L e 6,64 ¬Ī0,079 g/L, respetivamente, ap√≥s 9 dias. Adicionalmente, foram avaliados o efeito da √°rea superficial e da profundidade/altura do meio de cultura no rendimento e produtividade em BNC. Verificou-se que a produ√ß√£o de BNC aumenta com a √°rea superficial, com o volume de meio de cultura (profundidade) e com o tempo de fermenta√ß√£o. Al√©m disso, observou-se que a produtividade de BNC se mant√©m constante at√© se esgotarem os nutrientes no meio de cultura. Para a etapa de prepara√ß√£o pr√©-in√≥culo (PIP), a otimiza√ß√£o consistiu em diferentes estudos, especificamente: (a) otimiza√ß√£o dum meio de cultura de custos reduzidos, que permita a obten√ß√£o de uma elevada densidade celular; (b) avalia√ß√£o do efeito da concentra√ß√£o celular inicial na produ√ß√£o est√°tica de BNC e (c) estudo da cin√©tica de crescimento celular ao longo das diferentes etapas de PIP. A melhor composi√ß√£o para o PIP foi (% (m/v)): xarope de glucose e frutose 1,5- 2,0; CSL 0,7; √°cido c√≠trico 0,115 e Na2HPO4 0,27. Os estudos de cin√©tica de crescimento celular para as diferentes etapas do PIP evidenciam a necessidade dum controle cuidadoso do tempo de cultura em cada etapa do PIP. O tempo necess√°rio para atingir a fase exponencial foi muito diferente em cada fase do PIP, reduzindo significativamente da cultura est√°tica, para a cultura agitada, e para cultura agitada em larga escala num bioreator de 75 L. A segunda parte do trabalho relaciona-se com o desenvolvimento da BNC como sistema de entrega de f√°rmacos. A Vit D3 √© pouco sol√ļvel em √°gua e, portanto, n√£o √© facilmente incorporada no ambiente altamente hidrof√≠lico como o da membrana de BNC. Para esse efeito foi usado um nanogel anfif√≠lico auto-organizado obtido pela liga√ß√£o de hexadecilamina (Hexa) na cadeia do √°cido hialur√≥nico (HA). A Vit D3 foi ent√£o encapsulada no nanogel de (HA-Hexa) e em seguida impregnada na membrana de BNC, com uma efici√™ncia de encapsula√ß√£o entre 60-91 %. A liberta√ß√£o da Vit D3 foi monitorizada ao longo do tempo, usando uma c√©lula de Franz e realizando estudos de permea√ß√£o. Observou-se a liberta√ß√£o de cerca de 70 % da Vit D3 , ainda dentro do nanogel de HA-Hexa, das membranas de BNC em 48h. Finalmente, foi testada a utiliza√ß√£o de HA-Vit D3 e de HA-LLKKK18 (um p√©ptido an√°logo √† LL37) em modelos de feridas de excis√£o e cr√≥nicas em ratinhos tratados com dexametasona e diab√©ticos tipo II (db + / db +) C57BL/6. No entanto, os resultados n√£o revelaram uma maior efici√™ncia na cicatriza√ß√£o de feridas na presen√ßa das referidas formula√ß√Ķes.Ao Projeto BioTecNorte (NORTE-01-0145-FEDER-000004), n¬ļ 003435: ‚ÄúBUILD ‚Äď Bacterial cellulose Leather‚ÄĚ, financiado pelo Fundo Europeu de Desenvolvimento Regional (FEDER) atrav√©s do Programa Operacional do Regional do Norte (NORTE 2020) e ao projeto SkinChip: Disruptive cellulose-based microfluidic device for 3D skin modelling, PTDC/BBB-BIO/1889/2014 e ainda √† Funda√ß√£o para a Ci√™ncia e Tecnologia no √Ęmbito do financiamento estrat√©gico da unidade UID / BIO / 04469/2019 e pela atribui√ß√£o da bolsa de doutoramento SFRH/BD/89547/2012

    Application of lactic acid bacteria for the biopreservation of meat products: A systematic review

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    .The increasing concern of consumers about food quality and safety and their rejection of chemical additives has promoted the breakthrough of the biopreservation field and the development of studies on the use of beneficial bacteria and their metabolites as potential natural antimicrobials for shelf life extension and enhanced food safety. Control of foodborne pathogens in meat and meat products represents a serious challenge for the food industry which can be addressed through the intelligent use of bio-compounds or biopreservatives. This article aims to systematically review the available knowledge about biological strategies based on the use of lactic acid bacteria to control the proliferation of undesirable microorganisms in different meat products. The outcome of the literature search evidenced the potential of several strains of lactic acid bacteria and their purified or semi-purified antimicrobial metabolites as biopreservatives in meat products for achieving longer shelf life or inhibiting spoilage and pathogenic bacteria, especially when combined with other technologies to achieve a synergistic effect.S

    Metabolic and nutritional triggers associated with increased risk of liver complications in SARS-CoV-2

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    Obesity, diabetes, cardiovascular and respiratory diseases, cancer and smoking are risk factors for negative outcomes in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which can quickly induce severe respiratory failure in 5% of cases. Coronavirus disease-associated liver injury may occur during progression of SARS-CoV-2 in patients with or without pre-existing liver disease, and damage to the liver parenchyma can be caused by infection of hepatocytes. Cirrhosis patients may be particularly vulnerable to SARS-CoV-2 if suffering with cirrhosis-associated immune dysfunction. Furthermore, pharmacotherapies including macrolide or quinolone antibiotics and steroids can also induce liver damage. In this review we addressed nutritional status and nutritional interventions in severe SARS-CoV-2 liver patients. As guidelines for SARS-CoV-2 in intensive care (IC) specifically are not yet available, strategies for management of sepsis and SARS are suggested in SARS-CoV-2. Early enteral nutrition (EN) should be started soon after IC admission, preferably employing iso-osmolar polymeric formula with initial protein content at 0.8 g/kg per day progressively increasing up to 1.3 g/kg per day and enriched with fish oil at 0.1 g/kg per day to 0.2 g/kg per day. Monitoring is necessary to identify signs of intolerance, hemodynamic instability and metabolic disorders, and transition to parenteral nutrition should not be delayed when energy and protein targets cannot be met via EN. Nutrients including vitamins A, C, D, E, B6, B12, folic acid, zinc, selenium and ŌČ-3 fatty acids have in isolation or in combination shown beneficial effects upon immune function and inflammation modulation. Cautious and monitored supplementation up to upper limits may be beneficial in management strategies for SARS-CoV-2 liver patients

    Criação de uma plataforma de diagnóstico precoce para doença de Alzheimer através do uso de bacteriófagos

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    Disserta√ß√£o de Mestrado em BioengenhariaAlzheimer's disease (AD) is a neurodegenerative disorder and the most common form of dementia. It affects mostly people over 65 years and the main symptoms are loss of memory and cognitive functions. Biologically, it is thought that this disease is characterized by the accumulation of a small peptide called amyloid beta (Aő≤). This peptide aggregates into oligomers, the presumed real reason of synaptic disruption and memory loss. Recently, two sequences were described to recognize Aő≤ oligomers, the Aő≤ ő≥0-ő≥őĽ (AIIGLMVGGV) and Aő≤ 33-42 (LMVGGVVIA). So far no accurate diagnosis exists increasing the need to develop tools for early diagnosis. Nevertheless, it is difficult to access the brain due to the presence of the blood brain barrier (BBB). Based on the ability of the bacteriophage M13 to cross the BBB and the existence of sequences that recognize the Aő≤, the main goal of this work was to develop an early diagnosis tool for AD diagnosis through the engineering of the bacteriophage M13 in order to express the Aő≤ sequences on its capsid. Different approaches were used to genetically manipulate the M13 genome in order to clone the AB sequences, and it was possible to insert only the last four amino acids of the Aő≤ ő≥0-39 sequence (VGGV). With this modified phage particle, immunohistochemistry assays were performed using AD model tissue from the hippocampus of a double transgenic mice APPswe/PS1dE9. The results of these assays proved promising, with a significant affinity of the modified phage towards the AD model tissue, and possibly to the Aő≤ oligomers. In short, the overall objectives of this work were accomplished, leaving the opportunity for further work to improve and validate this phage particle as an AD diagnosis tool.A Doen√ßa de Alzheimer (DA) √© uma doen√ßa neurodegenerativa e a forma mais comum de dem√™ncia. Esta doen√ßa afeta maioritariamente pessoas com mais de 65 anos e os seus sintomas s√£o a perda gradual de mem√≥ria e de fun√ß√Ķes cognitivas. Biologicamente, pensa-se que a principal causa desta doen√ßa √© a acumula√ß√£o de um pequeno p√©ptido chamado amiloidebeta (Aő≤) na sua forma oligom√©rica. Recentemente foram descritas duas sequ√™ncias que mostraram uma elevada afinidade aos olig√≥meros de Aő≤, especificamente as sequ√™ncias Aő≤ ő≥0- ő≥őĽ (AIIGLMVGGV) e Aő≤ ő≥ő≥-42 (LMVGGVVIA). At√© ao momento n√£o foi descoberto nenhuma ferramenta de diagn√≥stico para DA. Um dos motivos principais para a falta de diagn√≥stico √© a exist√™ncia da barreira hematoencef√°lica. Esta barreira impede o acesso de mol√©culas, v√≠rus entre outros ao sistema nervoso central. Tendo em conta os estudos que demonstram a capacidade do bacteri√≥fago M13 para ultrapassar a barreira hematoencef√°lica e tamb√©m a exist√™ncia de sequ√™ncias capazes de reconhecer Aő≤ na sua forma oligom√©rica, o objetivo principal deste trabalho foi a cria√ß√£o de uma plataforma de diagn√≥stico precoce para DA atrav√©s da modifica√ß√£o gen√©tica do fago M13 de modo a este expressar √† sua superf√≠cie as sequ√™ncias Aő≤. Durante a elabora√ß√£o deste trabalho diferentes metodologias foram realizadas na tentativa de modificar geneticamente o fago M13. Atrav√©s destas abordagens foi poss√≠vel a obten√ß√£o de uma part√≠cula f√°gica modificada possuindo na sua superf√≠cie os √ļltimos quatro amino√°cidos da sequ√™ncia Aő≤ 30-39 (VGGV). Ensaios de imunohistoqu√≠mica foram realizados de modo a avaliar a afinidade do fago modificado para o tecido do hipocampo de ratos modelo da DA. Os resultados obtidos nestes ensaios mostraramse promissores, sugerindo que o novo fago possui elevada afinidade para os olig√≥meros Aő≤. Resumidamente, pode dizer-se que os objetivos gerais deste trabalho foram alcan√ßados, deixando oportunidade de trabalhos futuros para o desenvolvimento de um modelo de diagn√≥stico para a DA, utilizando a part√≠cula f√°gica obtida

    Exposure to particulate matter: direct and indirect role in the COVID-19 pandemic

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    Knowing the transmission factors and the natural environment that favor the spread of a viral infection is crucial to stop outbreaks and develop effective preventive strategies. This work aims to evaluate the role of Particulate Matter (PM) in the COVID-19 pandemic, focusing especially on that of PM as a vector for SARS-CoV-2. Exposure to PM has been related to new cases and to the clinical severity of people infected by SARS-CoV-2, which can be explained by the oxidative stress and the inflammatory response generated by these particles when entering the respiratory system, as well as by the role of PM in the expression of ACE-2 in respiratory cells in human hosts. In addition, different authors have detected SARS-CoV-2 RNA in PM sampled both in outdoor and indoor environments. The results of various studies lead to the hypothesis that the aerosols emitted by an infected person could be deposited in other suspended particles, sometimes of natural but especially of anthropogenic origin, that form the basal PM. However, the viability of the virus in PM has not yet been demonstrated. Should PM be confirmed as a vector of transmission, prevention strategies ought to be adapted, and PM sampling in outdoor environments could become an indicator of viral load in a specific area.‚ÄúThis work has been carried out within the framework of the project ‚ÄúAir pollution and COVID-19: what can we learn from this pandemic?‚ÄĚ of the Call for Grants from the BBVA Foundation to Scientific Research Teams in SARS-CoV-2 and COVID-19, in the area of Ecology and Veterinary Medicine

    Integrated Proteomic and Metabolomic Profiling of Phytophthora cinnamomi Attack on Sweet Chestnut (Castanea sativa) Reveals Distinct Molecular Reprogramming Proximal to the Infection Site and Away from It

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    Phytophthora cinnamomi is one of the most invasive tree pathogens that devastates wild and cultivated forests. Due to its wide host range, knowledge of the infection process at the molecular level is lacking for most of its tree hosts. To expand the repertoire of studied Phytophthora-woody plant interactions and identify molecular mechanisms that can facilitate discovery of novel ways to control its spread and damaging effects, we focused on the interaction between P. cinnamomi and sweet chestnut (Castanea sativa), an economically important tree for the wood processing industry. By using a combination of proteomics, metabolomics, and targeted hormonal analysis, we mapped the effects of P. cinnamomi attack on stem tissues immediately bordering the infection site and away from it. P. cinnamomi led to a massive reprogramming of the chestnut proteome and accumulation of the stress-related hormones salicylic acid (SA) and jasmonic acid (JA), indicating that stem inoculation can be used as an easily accessible model system to identify novel molecular players in P. cinnamomi pathogenicity.O

    SUPLEMENTA√á√ÉO DE COL√ĀGENO PARA ENVELHECIMENTO CUT√āNEO: UMA REVIS√ÉO

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    No envelhecimento, o organismo inicia um processo degenerativo progressivo, resultante de um decl√≠nio fisiol√≥gico das suas fun√ß√Ķes ao longo do tempo, o que acarreta em altera√ß√Ķes na epiderme e na derme, culminando no aparecimento de rugas e linhas de express√£o. Na pele, essas altera√ß√Ķes s√£o decorrentes da perda da capacidade dos fibroblastos da derme em produzir os componentes da matriz extracelular, como o col√°geno. Com isso, o col√°geno tem sido utilizado como suplemento para evitar a progress√£o do envelhecimento cut√Ęneo. Entre os tipos de col√°geno dispon√≠veis no mercado, t√™m-se o hidrolisado e os pept√≠deos de col√°geno. Este artigo objetivou realizar uma revis√£o sistem√°tica da literatura sobre a suplementa√ß√£o de col√°geno no envelhecimento cut√Ęneo, associado ou n√£o √† vitamina C, zinco e sil√≠cio. A pesquisa foi realizada nas bases de dados Scielo, Google acad√™mico, Scince Direct, e Pub Med. Foram considerados artigos publicados no per√≠odo de 2014 a 2020, nos idiomas Ingl√™s e Portugu√™s. A amostra final foi composta por cinco artigos experimentais com estudos cl√≠nicos que envolveram seres humanos, em que os investigadores designaram pessoas eleg√≠veis para os grupos de interven√ß√£o. Participaram volunt√°rios com idades entre 18 e 75 anos, sendo a maioria mulheres. A partir do levantamento dos estudos, conclui-se, que a suplementa√ß√£o de col√°geno hidrolisado apresenta efetividade na redu√ß√£o de rugas, linhas de express√£o e melhora a hidrata√ß√£o da pele, para os grupos estudados

    Folates: An Introduction

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    Folate is a naturally occurring essential nutrient which is vital for DNA replication and a necessary substrate in various enzymatic reactions which are involved in synthesis of amino acids and vitamin metabolism. The synthetic and oxidized form of folate is folic acid, it is mainly incorporated into fortified foods and dietary supplements for preventive measures against folate deficiency. Folate deficiency has been linked with several abnormalities in both mother (anemia, peripheral neuropathy) and fetus (congenital abnormalities). Folic acid supplementation taken around the time of conception has been known to alleviate the risk of neural tube defects in the off springs. Optimal intake and absorption of folates is required for the maintenance of the human body’s normal functioning and keeping the genomic integrity intact

    Exploring the Versatility of Benzimidazole Scaffolds as Medicinal Agents: A Brief Update

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    Benzimidazole, one of the finest classes of heterocyclic aromatic compounds have the characteristic structure of benzene fused with a five-membered imidazole ring. Despite being made their first appearance in the late 1870s, they are considered as a ‚Äėprivileged molecule‚Äô. The applications of this wonder molecule range from medicinal chemistry to material science. Benzimidazole being a potent inhibitor for various enzymes has got therapeutic effects like anticancer, antimicrobial, anthelmintic, antioxidant, anticonvulsant, antifungal, anti-inflammatory, antiviral, antihistaminic, antipsychotic, etc. It has also made its existence in various branches of medical science viz ophthalmology, neurology, cardiology and more. The applications of benzimidazole are not only limited to the biological field but also expanded to the field of material chemistry as well. This chapter summarizes the pharmacological properties of benzimidazole, illustrated on numerous derivatives since 2016
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