Biotechnological Tools Research Articles

Microbial Cellulase Production: Current Technologies and Future Prospects

Enzymes are biocatalysts, that facilitate chemical reactions by lowering their activation energy. Among these, cellulase emerges as a significant enzyme, consisting of a triad of components that work in synergy to degrade cellulosic biomass. Its significance is mostly pronounced in agricultural contexts, where there is an abundance of lignocellulosic biomass making it pivotal for utilization and conversion of biomass. Utilizing the biomass as a substrate for cellulase production offers dual advantages. Firstly, it simplifies the enzymatic synthesis process by the utilization of naturally occurring precursors. Secondly, it contributes to cost reduction by leveraging readily available resources thereby making it economically viable. Microbial cellulases, sourced from diverse microbes found globally, can aid in efficient enzymatic production. Advances in fermentation processes, coupled with the application of biotechnological tools, have significant impacts in production scalability and cost-effectiveness. Optimizing production strategies is crucial to meet the increasing demands of industrial applications while ensuring sustainability. Emphasizing the utilization of biomass substrates and harnessing the potential of emerging biotechnological advancements are key aspects of enzyme production. This review shall aim to provide an in-depth exploration of current cellulase production technologies and future prospects. By elucidating the underlying principles of cellulase catalysis and the intricacies of production methodologies.

Biotechnological Tool for Metal(loid)s as Cd, Cu, Ni, and P Management with Multiple Approaches: Bioremediation, Recovery of Raw Materials, and Food Safety

Contaminated soils are a challenge for implementing biotechnology in bioremediation, the recovery of Critical and Strategic Raw Materials (CRMs and SRMs), and food security. European Union (EU) Governments have established strict limits on As, Pb, Cd, and Hg in foods (Document 32023R0915) and requested the recovery of 34 CRMs within a circular economy (CE) (5th CRMs list). This study proposed a biotechnological tool for the decontamination of soil with heavy metal(loid)s by arbuscular mycorrhizal (AM)-assisted phytoextraction and the subsequent recovery of CRMs or by phytostabilization to prevent their entry into the food chain. It consisted of placing Baccharis salicifolia plants, inoculated or non-inoculated with AM fungi, into bioreactors (BRs) containing mining soil with Cd, Ni, and Cu, according to the Argentinian Patent (AR090183B1). The bioextractive potential (BP) was also estimated at the highest Technological Readiness Level (TRL) using a vegetable depuration module (VDM, TRL 6). Inoculated plants showed significantly higher aerial bioaccumulation coefficients (Cd: 68.62; P: 2.99; Ni: 2.51; Cu: 0.18) in BRs, and the BP values reached 1.16 g, 9.75 g, 2.40 g, and 213.1 g for Ni, Cd, Cu, and P, respectively. Finally, these CRMs and SRMs could be recovered from biomass through hydrometallurgy within a CE framework.

The Multifunctional Anaphase Promoting Complex 7 (APC7) Gene Is Associated With Increased Plant Growth and Improved Resistance to DNA and RNA Viruses.

The anaphase promoting complex 7 (AtAPC7) is an APC/C subunit expressed in different organs of Arabidopsis thaliana and conserved among eukaryotes. A variant of the complete APC7 protein, containing its C-terminal region (named APC-CT), shows a high homology with a tobacco viral replication inhibitor (IVR-like) protein that reduces plant susceptibility to RNA viruses. Here, the role of the AtAPC7 gene was investigated by characterizing Arabidopsis plants overexpressing the full-length AtAPC7 (APC7OE) and the C-terminal portion (APC7-CTOE), by phenotypical, physiological and molecular approaches. APC7OE plants showed improved growth of vegetative organs, earlier flowering and increased photosynthetic efficiency, CO2 assimilation and productivity, compared with Col-0 control plants. Conversely, APC7-CTOE plants showed reduced susceptibility to both RNA and DNA viruses, along with an improvement in plant growth, although not surpassing APC7OE plants. Altogether, the data provide evidence for the role of the AtAPC7 in regulating cell division, expansion and differentiation, accompanied by an increase in photosynthetic capacity, resulting in enhanced plant biomass and seed yield. AtAPC7-CT might reduce growth-defence trade-offs, enabling plants to simultaneously defend themselves while promoting better growth. Our findings highlight the multifunctional role of AtAPC7, unveiling the potential of its orthologous genes as valuable biotechnological tools in important crops.

Rice Yellow Mottle Virus (RYMV): A Review

Rice (Oryza spp.) is mostly grown directly from seed and sown on wet or dry seed beds or usually used as transplants on nursery beds. Among all the economically important viral diseases in the world, rice yellow mottle virus (RYMV) is only prevalent in rice-growing countries in Africa. RYMV has become the main rice production constraint in Africa over the last 20–25 years, causing yield losses of 10 to 100% depending on the age of the plant at the time of infection, degree of varietal susceptibility and the existing climatic conditions. Good agricultural practices and biotechnological tools in the development of improved resistant cultivars have been extensively utilized in controlling the disease. This review focuses on RYMV, its epidemiology, serological and molecular typing, disease management and the way forward for sustainable rice production.

Reproductive Biotechnology Tools as a Game Changer for the Dairy Sector: The Case of Sexed Semen and Estrus Synchronization to Produce Seedstock Dairy Heifers

Human curiosity about the sexual orientation of living things has a long history. For thousands of years, livestock owners wanted to find a suitable way to predict the sex of the progeny to be born for their herds on their farm. Thanks to the development of semen sexing technology, animal breeding has undergone a revolution, which allows farmers to control the sex of their offspring. Sexed semen enhances overall productivity and also assists farmers in meeting the rising demand for high-quality dairy animals by enabling them to selectively generate seedstock female calves. The technology is a one-stop solution to enhance breed improvement especially in a country like Ethiopia where 97 percent of the cattle population is indigenous, which is not selected for milk production and results in undernourishment for animal source origin diet, including milk and meat. The benefits of using sexed semen include sex selection, improved genetic selection, enhanced breeding effectiveness, better control over herd dynamics, greater financial gains, environmental sustainability, and improved animal welfare. Hence, with the finding of this study, an overall result of a 98% response rate 95% female skewness, and 79% conception rate was recorded after being inseminated with sexed semen. The finding of the response rate is closer to the results reported by another researcher, while the average conception rate is comparatively higher. This could be due to careful animal selection, the use of fertile quality semen, proper heat detection, and inseminating at an optimum window of time. In general, from the piece of this study, it is possible to foresee and conclude that the adoption and application of sexed semen technology is a groundbreaking and game-changer technology for the dairy industry in a country like Ethiopia, which overcomes the limitations of improved crossbred dairy cattle, replacement heifers, and high milk prices on the one hand, a shortage of grazing land, ...

Revolutionizing livestock sustainability: Pioneering breeding strategies for superior forage biomass and quality

Livestock primarily rely on forage crops as a source of feed and nutrition. The milk productivity of a cow or meat production in goat/sheep could directly be associated with the availability of a sufficient quantity of quality green fodders with essential nutrients in a balanced ratio. Feeding the cereal/grass: legume fodders in the required proportion will not only improve productivity but also the reproductive capacity of animals. However, many countries of the world experience a huge gap between demand and availability of green fodder. In this context, emphasis should be placed on developing efficient forage genotypes with increased biomass and quality as per the requirements of animals, duly considering their digestibility. Breeding approaches encompassing required classical approaches, including wide hybridization to exploit natural genetic variability, biotechnological tools such as transgenic technology, marker-assisted selection, genomic selection, and various omics techniques alongside high-throughput phenotyping using multispectral cameras, would help to sustain livestock productivity by meeting out the present and future fodder requirements coupled with enhanced nutrients

The digital evolution in toxicology: pioneering computational education for emerging challenges

The educational landscape of toxicology is increasingly integrating computational methodologies due to ethical concerns about animal testing and advancements in biotechnological and data analysis tools. This paper examines the evolution and significance of the Toxicology in the 21st century (Tox21) initiative and its impact on computational toxicology education. It contrasts computational toxicology with traditional methods, highlighting the limitations of conventional approaches and the new perspectives offered by computational techniques. The study emphasizes the importance of incorporating computational toxicology into curricula, including case studies that demonstrate how this integration enhances students’ problem-solving abilities, real-time data analysis skills, and innovation capabilities. Furthermore, it outlines effective teaching content and methods, including software tools, online resources, and academic literature. The paper also addresses the challenges and limitations faced in this educational shift and explores prospects for advancing computational toxicology education. By documenting these developments, the study aims to clarify the current advancements in toxicology education and the preparedness of students to address global chemical safety challenges with innovative solutions.

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC): A COMPREHENSIVE REVIEW

High-Performance Liquid Chromatography (HPLC) is a powerful analytical technique used to separate, identify, and quantify the components of a mixture. It has become an indispensable tool in various fields, including pharmaceuticals, biotechnology, food safety, and environmental monitoring. This review article provides an overview of HPLC principles, instrumentation, applications, advantages, and limitations.

Introduction pages, Notulae Scientia Biologicae 16(3), 2024

Notulae Scientia Biologicae (http://www.notulaebiologicae.ro), Issue 3, Volume 16, 2024: The papers published in this issue represent interesting novelties in different topics of life science. Among the exciting research or reviews, we invite readers to find news about: Exogenous nitric oxide and hydrogen sulfide as biotechnological tools for enhancing plant adaptation to cold; Optimization of the production of a new milk clotting enzyme by Aspergillus flavus thermophile on agro-industrial residues medium and characterization of the enzyme; Chemical composition and potential pharmacological applications of Centaurea acaulis L.; Evaluation of antimalarial compounds in Prosopis africana stem bark fractions against Plasmodium berghei; Bioactive potential of Algerian Citrullus colocynthis resin: Antioxidant and anti-inflammatory effects; Analysis of chemical composition, antimicrobial, antioxidant activities of pomegranate fruit thick and green tea leaf extracts; In silico analysis of Cucumis pubescens Willd. fruit extract phytocompounds and its activity against anti-diabetic targets; In vitro and in vivo antifungal activity of different bacterial isolates against Botrytis gray mold of tomato; Integrated characterization and in vitro biocontrol of Fusarium sp. using Trichoderma asperellum; Morpho-physiological and biochemical responses of radish (Raphanus sativus L.) under cadmium stress; Delineating drought-induced antioxidative traits as potential mechanisms for climate resilience in Argania spinosa.

Ampullaviruses: From Extreme Environments to Biotechnological Innovation.

Ampullaviruses are unique among viruses. They live in extreme environments and have special bottle-shaped architecture. These features make them useful tools for biotechnology. These viruses have compact genomes. They encode a range of enzymes and proteins. Their natural environment highlights their suitability for industrial applications. Ongoing research explores ways in which these viruses can improve enzyme stability. They are also employed in the creation of new biosensors and the development of new bioremediation techniques. High coinfection rates and the ecology of ampullaviruses at larger scales can also reveal new viral vectors. They can also help improve phage therapy. Here, we have explored the structure and function of ampullaviruses. We have focused on their use in biotechnology. We have also identified their characteristics that could prove to be useful. We have also pointed out key knowledge gaps and bridging them could further extend the biotechnological uses.

Use of Bacterial Toxin-Antitoxin Systems as Biotechnological Tools in Plants.

Toxin-antitoxin (TA) systems in bacteria are key regulators of the cell cycle and can activate a death response under stress conditions. Like other bacterial elements, TA modules have been widely exploited for biotechnological purposes in diverse applications, such as molecular cloning and anti-cancer therapies. However, their use in plants has been limited, leaving room for the development of new approaches. In this study, we examined two TA systems previously tested in plants, MazEF and YefM-YoeB, and identified interesting differences between them, likely related to their modes of action. We engineered modifications to these specific modules to transform them into molecular switches that can be activated by a protease, inducing necrosis in the plant cells where they are expressed. Finally, we demonstrated the antiviral potential of the modified TA modules by using, as a proof-of-concept, the potyvirus plum pox virus as an activator of the death phenotype.

Exogenous nitric oxide and hydrogen sulfide as biotechnological tools for enhancing plant adaptation to cold

The challenge of increasing plant tolerance to low temperatures is still relevant despite the upward trend in the average temperatures on Earth. Sharp temperature fluctuations during growing periods and cultivation of thermophilic crops in more northern latitudes increase the likelihood of plants being damaged by cold. Physiologically active substances, primarily hormones and signalling molecules, are considered to be effective for increasing plant tolerance to extreme temperatures. In the last decade, among such compounds, researchers' and practitioners' considerable attention has been paid to gasotransmitters – gaseous substances that are synthesized by living organisms and perform signalling functions. The review analyses the molecular mechanisms of stress protective effects of two major gasotransmitters – nitric oxide (NO) and hydrogen sulfide (H2S). In particular, the physiological effects of gasotransmitters that are attributed to their ability to cause post-translational modifications of numerous target proteins as well as those related to their functional interactions with other signaling intermediaries, primarily ROS and calcium ions, are discussed. The available data on the actions of donors of these gasotransmitters on thermophilic and cold-tolerant plants are summarized. Particular attention is paid to the influence of exogenous NO and H2S on the antioxidant system in plants, their ability to accumulate osmolytes and other low-molecular compounds with multifunctional stress protective effects. Data on the influence of gasotransmitters on fruits during low-temperature storage are analysed separately. It is emphasized that increased storability of fruits under the influence of NO and H2S donors is largely controlled by the ability of both gasotransmitters to inhibit ethylene synthesis. It was concluded that exogenous nitric oxide and hydrogen sulfide could be effective tools for increasing tolerance of different plant taxon’s to low temperatures and used in crop production and storage technologies for fruits and vegetables.

Mechanism of Nucleic Acid Phosphodiester Bond Cleavage by Human Endonuclease V: MD and QM/MM Calculations Reveal a Versatile Metal Dependence.

Human endonuclease V (EndoV) catalytically removes deaminated nucleobases by cleaving the phosphodiester bond as part of RNA metabolism. Despite being implicated in several diseases (cancers, cardiovascular diseases, and neurological disorders) and potentially being a useful tool in biotechnology, details of the human EndoV catalytic pathway remain unclear due to limited experimental information beyond a crystal structure of the apoenzyme and select mutational data. Since a mechanistic understanding is critical for further deciphering the central roles and expanding applications of human EndoV in medicine and biotechnology, molecular dynamics (MD) simulations and quantum mechanics/molecular mechanics (QM/MM) calculations were used to unveil the atomistic details of the catalytic pathway. Due to controversies surrounding the number of metals required for nuclease activity, enzyme-substrate models with different numbers of active site metals and various metal-substrate binding configurations were built based on structural data for other nucleases. Subsequent MD simulations revealed the structure and stability of the human EndoV-substrate complex for a range of active site metal binding architectures. Four unique pathways were then characterized using QM/MM that vary in metal number (one versus two) and modes of substrate coordination [direct versus indirect (water-mediated)], with several mechanisms being fully consistent with experimental structural, kinetic, and mutational data for related nucleases, including members of the EndoV family. Beyond uncovering key roles for several active site amino acids (D240 and K155), our calculations highlight that while one metal is essential for human EndoV activity, the enzyme can benefit from using two metals due to the presence of two suitable metal binding sites. By directly comparing one- versus two-metal-mediated P-O bond cleavage reactions within the confines of the same active site, our work brings a fresh perspective to the "number of metals" controversy.

Biotechnological approaches to reduce the phytic acid content in millets to improve nutritional quality.

The review article summarizes the approaches and potential targets to address the challenges of anti-nutrient like phytic acid in millet grains for nutritional improvement. Millets are a diverse group of minor cereal grains that are agriculturally important, nutritionally rich, and the oldest cereals in the human diet. The grains are important for protein, vitamins, macro and micronutrients, fibre, and energy sources. Despite a high amount of nutrients, millet grains also contain anti-nutrients that limit the proper utilization of nutrients and finally affect their dietary quality. Our study aims to outline the genomic information to identify the target areas of research for the exploration of candidate genes for nutritional importance and show the possibilities to address the presence of anti-nutrient (phytic acid) in millets. So, the physicochemical accessibility of micronutrients increases and the agronomic traits can do better. Several strategies have been adopted to minimize the phytic acid, a predominant anti-nutrient in cereal grains. In the present review, we highlight the potential of biotechnological tools and genome editing approaches to address phytic acid in millets. It also highlights the biosynthetic pathway of phytic acid and potential targets for knockout or silencing to achieve low phytic acid content in millets.

Clear zone formation in microdroplets for high-throughput screening for lactic acid bacteria.

Droplet microfluidic-based technology is a powerful tool for biotechnology, and it is also expected that it will be applied to culturing and screening methods. Using this technology, a new high-throughput screening method for lactic acid bacteria was developed. In this study, the conventional culture of lactic acid bacteria that form clear zones on an agar medium was reproduced in water-in-oil droplets, and only the droplets in which lactic acid bacteria grew were collected one by one. Using this method, the specific recovery of Lactiplantibacillus plantarum from a mixture of L. plantarum and Escherichia coli and the acquirement of lactic acid bacteria from an environmental sample were successful. This method could be applied to various conventional screening methods using the clear zone as a microbial growth indicator. This has expanded the possibilities of applying droplet microfluidic-based technology to microbial cultivations.

Cassava breeding: Classical to recent breeding approaches for food, industry and climate resilience

Cassava ranks as the fourth-most significant starchy root crop in underdeveloped countries in terms of future food and acting as a key source of income for small and marginal farmers. To meet the growing demands for food security and economic development, it is imperative to develop improved cassava varieties that offer higher yields, enhanced nutritional content, safer for consumption, greater resistance to diseases and climate change. The development of these improved varieties necessitates advancements in breeding techniques, leveraging both traditional methods and modern biotechnological tools. However, a major challenge in cassava breeding is heterozygous nature and the crop’s sparse flowering, which limits the potential for sexual reproduction, thereby constraining breeding efforts for predominantly clonal selection. The continuous clonal propagation impedes genetic diversity and the introduction of novel traits, narrowing the overall progress of breeding programs. Integrating genomic tools and accelerating the adoption of biotechnological advancements can overcome these limitations and expedite the development of superior cassava varieties. This review highlights the need of cassava breeding for addressing these challenges with conventional as well as with new breeding techniques with the aim to provide a comprehensive understanding of the current scenario and future directions of cassava breeding research. Key words: Climate change, CRISPR/Cas 9, New breeding techniques, PPD, speed breeding, Waxy cassava

Biotechnology and Genomic Approaches to Mitigating Disease Impacts on Forest Health

Outbreaks of insects and diseases are part of the natural disturbance regime of all forests. However, introduced pathogens have had outsized impacts on many dominant forest tree species over the past century. Mitigating these impacts and restoring these species are dilemmas of the modern era. Here, we review the ecological and economic impact of introduced pathogens, focusing on examples in North America. We then synthesize the successes and challenges of past biotechnological approaches and discuss the integration of genomics and biotechnology to help mitigate the effects of past and future pathogen invasions. These questions are considered in the context of the transgenic American chestnut, which is the most comprehensive example to date of how biotechnological tools have been used to address the impacts of introduced pathogens on naïve forest ecosystems.

Advancements in Biotechnology and Stem Cell Therapies for Breast Cancer Patients.

This comprehensive review article examines the integration of biotechnology and stem cell therapy in breast cancer diagnosis and treatment. It discusses the use of biotechnological tools such as liquid biopsies, genomic profiling, and imaging technologies for accurate diagnosis and monitoring of treatment response. Stem cell-based approaches, their role in modeling breast cancer progression, and their potential for breast reconstruction post-mastectomy are explored. The review highlights the importance of personalized treatment strategies that combine biotechnological tools and stem cell therapies. Ethical considerations, challenges in clinical translation, and regulatory frameworks are also addressed. The article concludes by emphasizing the potential of integrating biotechnology and stem cell therapy to improve breast cancer outcomes, highlighting the need for continued research and collaboration in this field.

Measuring the capacity of yeast for surface display of cell wall-anchored protein isoforms by using β-lactamase as a reporter enzyme.

Yeast surface display is a promising biotechnological tool that uses genetically modified yeast cell wall proteins as anchors for enzymes of interest, thereby transforming yeast cell wall into a living catalytic material. Here, we present a comprehensive protocol for quantifying surface-displayed β-lactamase on the cell wall of model yeast Saccharomyces cerevisiae. We use β-lactamase as a reporter enzyme, which we tagged to be anchored to the cell wall closer to its N or C terminus, through the portion of the Pir2 or Ccw12 cell wall proteins, respectively. The catalytic activity of surface-displayed β-lactamase is assessed by its ability to hydrolyze nitrocefin, which produces a colorimetric change that is quantitatively measured by spectrophotometric analysis at 482 nm. This system enables precise quantification of the potential of S. cerevisiae strains for surface display, continuous real-time monitoring of enzyme activity, and facilitates the study of enzyme kinetics and interactions with inhibitors within the cell's native environment. In addition, the system provides a platform for high-throughput screening of potential β-lactamase inhibitors and can be adapted for the visualization of other enzymes, making it a versatile tool for drug discovery and bioprocess development.

Fusion of FokI and catalytically inactive prokaryotic Argonautes enables site-specific programmable DNA cleavage

Site-specific nucleases are crucial for genome engineering applications in medicine and agriculture. The ideal site-specific nucleases are easily reprogrammable, highly specific in target site recognition, robust in nuclease activities. Prokaryotic Argonaute (pAgo) proteins have received much attention as biotechnological tools due to their ability to recognize specific target sequences without a protospacer adjacent motif (PAM), but their lack of intrinsic double-stranded DNA (dsDNA) unwinding activity limits their utility in key applications such as gene editing. Recently, we developed a pAgo-based system for site-specific DNA cleavage at physiological temperatures independently of the DNA form, using peptide nucleic acids (PNAs) to facilitate unwinding dsDNA targets. Here, we fused catalytically dead pAgos with the nuclease domain of the restriction endonuclease FokI and named this modified platform PNA-assisted FokI-(d)pAgo (PNFP) editors. In the PNFP system, catalytically inactive pAgo recognizes and binds to a specific target DNA sequence based on a programmable guide DNA sequence; upon binding to the target site, the FokI domains dimerize and introduce precise dsDNA breaks. We explored key parameters of the PNFP system including the requirements of PNA and guide DNAs, the specificity of PNA and guide DNA on target cleavage, the optimal concentration of different components, reaction time for invasion and cleavage and ideal temperature and reaction buffer, to ensure efficient DNA editing in vitro. The results demonstrated robust site-specific target cleavage by PNFP system at optimal conditions in vitro. We envision that the PNFP system will provide higher editing efficiency and specificity with fewer off-target effects in vivo.