Current Landscape of Methods to Evaluate Antimicrobial Activity of Natural Extracts.

Natural extracts have been and continue to be used to treat a wide range of medical conditions, from infectious diseases to cancer, based on their convenience and therapeutic potential. Natural products derived from microbes, plants, and animals offer a broad variety of molecules and chemical compounds. Natural products are not only one of the most important sources for innovative drug development for animal and human health, but they are also an inspiration for synthetic biology and chemistry scientists towards the discovery of new bioactive compounds and pharmaceuticals. This is particularly relevant in the current context, where antimicrobial resistance has risen as a global health problem. Thus, efforts are being directed toward studying natural compounds' chemical composition and bioactive potential to generate drugs with better efficacy and lower toxicity than existing molecules. Currently, a wide range of methodologies are used to analyze the in vitro activity of natural extracts to determine their suitability as antimicrobial agents. Despite traditional technologies being the most employed, technological advances have contributed to the implementation of methods able to circumvent issues related to analysis capacity, time, sensitivity, and reproducibility. This review produces an updated analysis of the conventional and current methods to evaluate the antimicrobial activity of natural compounds.

Chemical Properties and Biological Activity of Bee Pollen.

Pollen, a remarkably versatile natural compound collected by bees for its abundant source of proteins and nutrients, represents a rich reservoir of diverse bioactive compounds with noteworthy chemical and therapeutic potential. Its extensive biological effects have been known and exploited since ancient times. Today, there is an increased interest in finding natural compounds against oxidative stress, a factor that contributes to various diseases. Recent research has unraveled a multitude of biological activities associated with bee pollen, ranging from antioxidant, anti-inflammatory, antimicrobial, and antifungal properties to potential antiviral and anticancer applications. Comprehending the extensive repertoire of biological properties across various pollen sources remains challenging. By investigating a spectrum of pollen types and their chemical composition, this review produces an updated analysis of the bioactive constituents and the therapeutic prospects they offer. This review emphasizes the necessity for further exploration and standardization of diverse pollen sources and bioactive compounds that could contribute to the development of innovative therapies.

Evaluation of Biological Activity of Natural Compounds: Current Trends and Methods.

Natural compounds have diverse structures and are present in different forms of life. Metabolites such as tannins, anthocyanins, and alkaloids, among others, serve as a defense mechanism in live organisms and are undoubtedly compounds of interest for the food, cosmetic, and pharmaceutical industries. Plants, bacteria, and insects represent sources of biomolecules with diverse activities, which are in many cases poorly studied. To use these molecules for different applications, it is essential to know their structure, concentrations, and biological activity potential. In vitro techniques that evaluate the biological activity of the molecules of interest have been developed since the 1950s. Currently, different methodologies have emerged to overcome some of the limitations of these traditional techniques, mainly via reductions in time and costs. These emerging technologies continue to appear due to the urgent need to expand the analysis capacity of a growing number of reported biomolecules. This review presents an updated summary of the conventional and relevant methods to evaluate the natural compounds' biological activity in vitro.

Developing a Cas9-based tool to engineer native plasmids in Synechocystis sp. PCC 6803.

The oxygenic photosynthetic bacterium Synechocystis sp. PCC 6803 (S6803) is a model cyanobacterium widely used for fundamental research and biotechnology applications. Due to its polyploidy, existing methods for genome engineering of S6803 require multiple rounds of selection to modify all genome copies, which is time-consuming and inefficient. In this study, we engineered the Cas9 tool for one-step, segregation-free genome engineering. We further used our Cas9 tool to delete three of seven S6803 native plasmids. Our results show that all three small-size native plasmids, but not the large-size native plasmids, can be deleted with this tool. To further facilitate heterologous gene expression in S6803, a shuttle vector based on the native plasmid pCC5.2 was created. The shuttle vector can be introduced into Cas9-containing S6803 in one step without requiring segregation and can be stably maintained without antibiotic pressure for at least 30 days. Moreover, genes encoded on the shuttle vector remain functional after 30 days of continuous cultivation without selective pressure. Thus, this study provides a set of new tools for rapid modification of the S6803 genome and for stable expression of heterologous genes, potentially facilitating both fundamental research and biotechnology applications using S6803.

Engineering xylose metabolism for production of polyhydroxybutyrate in the non-model bacterium Burkholderia sacchari.

BACKGROUND: Despite its ability to grow and produce high-value molecules using renewable carbon sources, two main factors must be improved to use Burkholderia sacchari as a chassis for bioproduction at an industrial scale: first, the lack of molecular tools to engineer this organism and second, the inherently slow growth rate and poly-3-hydroxybutyrate [P(3HB)] production using xylose. In this work, we have addressed both factors. RESULTS: First, we adapted a set of BglBrick plasmids and showed tunable expression in B. sacchari. Finally, we assessed growth rate and P(3HB) production through overexpression of xylose transporters, catabolic or regulatory genes. Overexpression of xylR significantly improved growth rate (55.5% improvement), polymer yield (77.27% improvement), and resulted in 71% of cell dry weight as P(3HB). CONCLUSIONS: These values are unprecedented for P(3HB) accumulation using xylose as a sole carbon source and highlight the importance of precise expression control for improving utilization of hemicellulosic sugars in B. sacchari.

xylA and xylB overexpression as a successful strategy for improving xylose utilization and poly-3-hydroxybutyrate production in Burkholderia sacchari.

Despite the versatility and many advantages of polyhydroxyalkanoates as petroleum-based plastic substitutes, their higher production cost compared to petroleum-based polymers has historically limited their large-scale production. One appealing approach to reducing production costs is to employ less expensive, renewable feedstocks. Xylose, for example is an abundant and inexpensive carbon source derived from hemicellulosic residues abundant in agro-industrial waste (sugarcane bagasse hemicellulosic hydrolysates). In this work, the production of poly-3-hydroxybutyrate P(3HB) from xylose was studied to develop technologies for conversion of agro-industrial waste into high-value chemicals and biopolymers. Specifically, this work elucidates the organization of the xylose assimilation operon of Burkholderia sacchari, a non-model bacterium with high capacity for P(3HB) accumulation. Overexpression of endogenous xylose isomerase and xylulokinase genes was successfully assessed, improving both specific growth rate and P(3HB) production. Compared to control strain (harboring pBBR1MCS-2), xylose utilization in the engineered strain was substantially improved with 25% increase in specific growth rate, 34% increase in P(3HB) production, and the highest P(3HB) yield from xylose reported to date for B. sacchari (YP3HB/Xil = 0.35 g/g). This study highlights that xylA and xylB overexpression is an effective strategy to improve xylose utilization and P(3HB) production in B. sacchari.

Organization and mode of action of two component system signaling circuits from the various kingdoms of life.

Two-component system (TCS) signaling circuits regulate numerous cellular processes in response to environmental cues in both prokaryotic and eukaryotic cells. These signaling circuits are all based on phosphoryl-group transfers between histidine and aspartate containing modules of sensor kinase and response regulator proteins. Curiously, the architecture and organization of prokaryotic and eukaryotic two-component systems reveal notable variations, raising the question of whether the input-response specificity that governs the majority of prokaryotic TCSs also governs the eukaryotic ones. In this review, we contrast the TCS architecture and signaling circuits of prokaryotes and eukaryotes, and discuss their possible consequences on signaling specificity.

Bacteriophage-mediated approaches for biofilm control.

Biofilms are complex microbial communities in which planktonic and dormant bacteria are enveloped in extracellular polymeric substances (EPS) such as exopolysaccharides, proteins, lipids, and DNA. These multicellular structures present resistance to conventional antimicrobial treatments, including antibiotics. The formation of biofilms raises considerable concern in healthcare settings, biofilms can exacerbate infections in patients and compromise the integrity of medical devices employed during treatment. Similarly, certain bacterial species contribute to bulking, foaming, and biofilm development in water environments such as wastewater treatment plants, water reservoirs, and aquaculture facilities. Additionally, food production facilities provide ideal conditions for establishing bacterial biofilms, which can serve as reservoirs for foodborne pathogens. Efforts to combat antibiotic resistance involve exploring various strategies, including bacteriophage therapy. Research has been conducted on the effects of phages and their individual proteins to assess their potential for biofilm removal. However, challenges persist, prompting the examination of refined approaches such as drug-phage combination therapies, phage cocktails, and genetically modified phages for clinical applications. This review aims to highlight the progress regarding bacteriophage-based approaches for biofilm eradication in different settings.

Exploring the Multifaceted Biological Activities of Anthocyanins Isolated from Two Andean Berries.

Natural pigments extracted from plant species are used in foods, cosmetics, and pharmaceuticals. This study evaluates the comprehensive biological activities of anthocyanins isolated from Andean blueberry (Vaccinium floribundum Kunth) and Andean blackberry (Rubus glaucus Benth), focusing on their antimicrobial, antioxidant, antitumoral, anti-inflammatory, and hemolytic properties. Chemical characterization revealed significant anthocyanin content with complex mass spectrometric profiles indicating diverse glycosylation patterns that may influence their bioactivity. The antimicrobial assays showed that the extracts were particularly effective against Gram-positive bacteria, with minimal inhibitory concentrations (MICs) as low as 1 mg/mL for Rubus glaucus, indicating strong potential for therapeutic use. The antioxidant capacity of the berries was substantial, albeit slightly lower than that of ascorbic acid. The extracts also exhibited notable antitumoral activity in various cancer cell lines, showing promise as adjunctive or preventive treatments. The anti-inflammatory effects were confirmed by inhibiting nitric oxide production in macrophage cells, highlighting their potential in managing inflammatory diseases. In terms of hemolytic activity, Rubus glaucus exhibited dose-dependent effects, potentially attributable to anthocyanins and phenolics, while Vaccinium floribundum demonstrated no significant hemolytic activity, underscoring its safety. These findings suggest that anthocyanins from Andean berries possess potent biological activities, which could be leveraged for health benefits in pharmaceutical and nutraceutical applications. Further studies are needed to isolate specific bioactive compounds and investigate their synergistic effects in clinical and real-world contexts.

Phytosynthesis of Silver Nanoparticles Using Mansoa alliacea (Lam.) A.H. Gentry (Bignoniaceae) Leaf Extract: Characterization and Their Biological Activities.

Background. Mansoa alliacea is a native plant renowned for its medicinal properties in traditional healing in the Amazon Region. This plant is rich in polyphenols, flavonoids, anthocyanins, phenolic acids, tannins, ketones, triterpenes, as well as other bioactive compounds. Objectives. This study aims to develop an innovative, eco-friendly method for synthesizing silver nanoparticles using an aqueous extract of M. alliacea (Ma-AgNPs), enhancing the biological activities of AgNPs by leveraging the therapeutic potential of the plant's bioactive compounds. Methods. Silver nanoparticles were synthesized using the aqueous extract of M. alliacea. The biological activities of Ma-AgNPs were assessed, including antibacterial, anti-inflammatory, antioxidant, antitumor, and anti-biofilm effects, along with evaluating their hemolytic activity. Results. Quantitative analysis revealed that Ma-AgNPs exhibit potent antibacterial activity against multidrug and non-multidrug-resistant bacteria, with MIC values ranging from 1.3 to 10.0 µg/mL. The Ma-AgNPs significantly reduced NO production by 86.9% at 4 µg/mL, indicating strong anti-inflammatory effects. They demonstrated robust antioxidant activity with an IC50 of 5.54 ± 1.48 µg/mL and minimal hemolytic activity, with no hemolysis observed up to 20 µg/mL and only 4.5% at 40 µg/mL. Their antitumor properties were notable, with IC50 values between 2.9 and 5.4 µg/mL across various cell lines, and they achieved over 50% biofilm inhibition at concentrations of 30-40 µg/mL. Conclusions. These findings underscore the potential of Ma-AgNPs for biomedical applications, particularly in developing new antimicrobial agents and bioactive coatings with reduced toxicity. This research highlights a sustainable approach that not only preserves but also amplifies the inherent biological activities of plant extracts, paving the way for innovative therapeutic solutions.

A critical tyrosine residue determines the uncoupling protein-like activity of the yeast mitochondrial oxaloacetate carrier.

The mitochondrial Oac (oxaloacetate carrier) found in some fungi and plants catalyses the uptake of oxaloacetate, malonate and sulfate. Despite their sequence similarity, transport specificity varies considerably between Oacs. Indeed, whereas ScOac (Saccharomyces cerevisiae Oac) is a specific anion-proton symporter, the YlOac (Yarrowia lipolytica Oac) has the added ability to transport protons, behaving as a UCP (uncoupling protein). Significantly, we identified two amino acid changes at the matrix gate of YlOac and ScOac, tyrosine to phenylalanine and methionine to leucine. We studied the role of these amino acids by expressing both wild-type and specifically mutated Oacs in an Oac-null S. cerevisiae strain. No phenotype could be associated with the methionine to leucine substitution, whereas UCP-like activity was dependent on the presence of the tyrosine residue normally expressed in the YlOac, i.e. Tyr-ScOac mediated proton transport, whereas Phe-YlOac lost its protonophoric activity. These findings indicate that the UCP-like activity of YlOac is determined by the tyrosine residue at position 146.

CRISPR-Cas-Based Antimicrobials: Design, Challenges, and Bacterial Mechanisms of Resistance.

The emergence of antibiotic-resistant bacterial strains is a source of public health concern across the globe. As the discovery of new conventional antibiotics has stalled significantly over the past decade, there is an urgency to develop novel approaches to address drug resistance in infectious diseases. The use of a CRISPR-Cas-based system for the precise elimination of targeted bacterial populations holds promise as an innovative approach for new antimicrobial agent design. The CRISPR-Cas targeting system is celebrated for its high versatility and specificity, offering an excellent opportunity to fight antibiotic resistance in pathogens by selectively inactivating genes involved in antibiotic resistance, biofilm formation, pathogenicity, virulence, or bacterial viability. The CRISPR-Cas strategy can enact antimicrobial effects by two approaches: inactivation of chromosomal genes or curing of plasmids encoding antibiotic resistance. In this Review, we provide an overview of the main CRISPR-Cas systems utilized for the creation of these antimicrobials, as well as highlighting promising studies in the field. We also offer a detailed discussion about the most commonly used mechanisms for CRISPR-Cas delivery: bacteriophages, nanoparticles, and conjugative plasmids. Lastly, we address possible mechanisms of interference that should be considered during the intelligent design of these novel approaches.

Iron Oxide Nanoparticles: Green Synthesis and Their Antimicrobial Activity.

The rise of antimicrobial resistance caused by inappropriate use of these agents in various settings has become a global health threat. Nanotechnology offers the potential for the synthesis of nanoparticles (NPs) with antimicrobial activity, such as iron oxide nanoparticles (IONPs). The use of IONPs is a promising way to overcome antimicrobial resistance or pathogenicity because of their ability to interact with several biological molecules and to inhibit microbial growth. In this review, we outline the pivotal findings over the past decade concerning methods for the green synthesis of IONPs using bacteria, fungi, plants, and organic waste. Subsequently, we delve into the primary challenges encountered in green synthesis utilizing diverse organisms and organic materials. Furthermore, we compile the most common methods employed for the characterization of these IONPs. To conclude, we highlight the applications of these IONPs as promising antibacterial, antifungal, antiparasitic, and antiviral agents.

Current landscape and future directions of synthetic biology in South America.

Synthetic biology (SynBio) is a rapidly advancing multidisciplinary field in which South American countries such as Chile, Argentina, and Brazil have made notable contributions and have established leadership positions in the region. In recent years, efforts have strengthened SynBio in the rest of the countries, and although progress is significant, growth has not matched that of the aforementioned countries. Initiatives such as iGEM and TECNOx have introduced students and researchers from various countries to the foundations of SynBio. Several factors have hindered progress in the field, including scarce funding from both public and private sources for synthetic biology projects, an underdeveloped biotech industry, and a lack of policies to promote bio-innovation. However, open science initiatives such as the DIY movement and OSHW have helped to alleviate some of these challenges. Similarly, the abundance of natural resources and biodiversity make South America an attractive location to invest in and develop SynBio projects.

The Neurospora crassa DCC-1 protein, a putative histidine kinase, is required for normal sexual and asexual development and carotenogenesis.

Two-component signaling pathways based on phosphoryl group transfer between histidine kinase and response regulator proteins regulate environmental responses in bacteria, archaea, plants, slime molds, and fungi. Here we characterize a mutant form of DCC-1, a putative histidine kinase encoded by the NCU00939 gene of the filamentous fungus Neurospora crassa. We show that this protein participates in the regulation of processes such as conidiation, perithecial development, and, to a certain degree, carotenogenesis. Furthermore, DCC-1 is suggested to exert its effect by promoting cyclic AMP production, thereby placing this protein within the context of a signaling pathway.

Protective role of butyrate in obesity and diabetes: New insights.

Studies in human microbiota dysbiosis have shown that short-chain fatty acids (SCFAs) like propionate, acetate, and particularly butyrate, positively affect energy homeostasis, behavior, and inflammation. This positive effect can be demonstrated in the reduction of butyrate-producing bacteria observed in the gut microbiota of individuals with type 2 diabetes (T2DM) and other energy-associated metabolic alterations. Butyrate is the major end product of dietary fiber bacterial fermentation in the large intestine and serves as the primary energy source for colonocytes. In addition, it plays a key role in reducing glycemia and improving body weight control and insulin sensitivity. The major mechanisms involved in butyrate regulation include key signaling pathways such as AMPK, p38, HDAC inhibition, and cAMP production/signaling. Treatment strategies using butyrate aim to increase its intestine levels, bioavailability, and improvement in delivery either through direct supplementation or by increasing dietary fiber in the diet, which ultimately generates a higher production of butyrate in the gut. In the final part of this review, we present a summary of the most relevant studies currently being carried out in humans.

High-throughput and automated screening for COVID-19.

The COVID-19 pandemic has become a global challenge for the healthcare systems of many countries with 6 million people having lost their lives and 530 million more having tested positive for the virus. Robust testing and a comprehensive track and trace process for positive patients are essential for effective pandemic control, leading to high demand for diagnostic testing. In order to comply with demand and increase testing capacity worldwide, automated workflows have come into prominence as they enable high-throughput screening, faster processing, exclusion of human error, repeatability, reproducibility and diagnostic precision. The gold standard for COVID-19 testing so far has been RT-qPCR, however, different SARS-CoV-2 testing methods have been developed to be combined with high throughput testing to improve diagnosis. Case studies in China, Spain and the United Kingdom have been reviewed and automation has been proven to be promising for mass testing. Free and Open Source scientific and medical Hardware (FOSH) plays a vital role in this matter but there are some challenges to be overcome before automation can be fully implemented. This review discusses the importance of automated high-throughput testing, the different equipment available, the bottlenecks of its implementation and key selected case studies that due to their high effectiveness are already in use in hospitals and research centres.

Tracking SARS-CoV-2: Novel Trends and Diagnostic Strategies.

The COVID-19 pandemic has had an enormous impact on economies and health systems globally, therefore a top priority is the development of increasingly better diagnostic and surveillance alternatives to slow down the spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In order to establish massive testing and contact tracing policies, it is crucial to have a clear view of the diagnostic options available and their principal advantages and drawbacks. Although classical molecular methods such as RT-qPCR are broadly used, diagnostic alternatives based on technologies such as LAMP, antigen, serological testing, or the application of novel technologies such as CRISPR-Cas for diagnostics, are also discussed. The present review also discusses the most important automation strategies employed to increase testing capability. Several serological-based diagnostic kits are presented, as well as novel nanotechnology-based diagnostic methods. In summary, this review provides a clear diagnostic landscape of the most relevant tools to track COVID-19.

In silico Analyses of Immune System Protein Interactome Network, Single-Cell RNA Sequencing of Human Tissues, and Artificial Neural Networks Reveal Potential Therapeutic Targets for Drug Repurposing Against COVID-19.

Background: There is pressing urgency to identify therapeutic targets and drugs that allow treating COVID-19 patients effectively. Methods: We performed in silico analyses of immune system protein interactome network, single-cell RNA sequencing of human tissues, and artificial neural networks to reveal potential therapeutic targets for drug repurposing against COVID-19. Results: We screened 1,584 high-confidence immune system proteins in ACE2 and TMPRSS2 co-expressing cells, finding 25 potential therapeutic targets significantly overexpressed in nasal goblet secretory cells, lung type II pneumocytes, and ileal absorptive enterocytes of patients with several immunopathologies. Then, we performed fully connected deep neural networks to find the best multitask classification model to predict the activity of 10,672 drugs, obtaining several approved drugs, compounds under investigation, and experimental compounds with the highest area under the receiver operating characteristics. Conclusion: After being effectively analyzed in clinical trials, these drugs can be considered for treatment of severe COVID-19 patients. Scripts can be downloaded at https://github.com/muntisa/immuno-drug-repurposing-COVID-19.

Clinical, molecular, and epidemiological characterization of the SARS-CoV-2 virus and the Coronavirus Disease 2019 (COVID-19), a comprehensive literature review.

Coronaviruses are an extensive family of viruses that can cause disease in both animals and humans. The current classification of coronaviruses recognizes 39 species in 27 subgenera that belong to the family Coronaviridae. From those, at least 7 coronaviruses are known to cause respiratory infections in humans. Four of these viruses can cause common cold-like symptoms. Those that infect animals can evolve and become infectious to humans. Three recent examples of these viral jumps include SARS CoV, MERS-CoV and SARS CoV-2 virus. They are responsible for causing severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS) and the most recently discovered coronavirus disease during 2019 (COVID-19). COVID-19, a respiratory disease caused by the SARS-CoV-2 virus, was declared a pandemic by the World Health Organization (WHO) on 11 March 2020. The rapid spread of the disease has taken the scientific and medical community by surprise. Latest figures from 20 May 2020 show more than 5 million people had been infected with the virus, causing more than 330,000 deaths in over 210 countries worldwide. The large amount of information received daily relating to COVID-19 is so abundant and dynamic that medical staff, health authorities, academics and the media are not able to keep up with this new pandemic. In order to offer a clear insight of the extensive literature available, we have conducted a comprehensive literature review of the SARS CoV-2 Virus and the Coronavirus Diseases 2019 (COVID-19).