Development Of Biotechnology Research Articles

An improved CRISPR and CRISPR interference (CRISPRi) toolkit for engineering the model methanogenic archaeon Methanococcus maripaludis

BackgroundThe type II based CRISPR-Cas system remains restrictedly utilized in archaea, a featured domain of life that ranks parallelly with Bacteria and Eukaryotes. Methanococcus maripaludis, known for rapid growth and genetic tractability, serves as an exemplary model for studying archaeal biology and exploring CO2−based biotechnological applications. However, tools for controlled gene regulation remain deficient and CRISPR-Cas tools still need improved in this archaeon, limiting its application as an archaeal model cellular factory.ResultsThis study not only improved the CRISPR-Cas9 system for optimizing multiplex genome editing and CRISPR plasmid construction efficiencies but also pioneered an effective CRISPR interference (CRISPRi) system for controlled gene regulation in M. maripaludis. We developed two novel strategies for balanced expression of multiple sgRNAs, facilitating efficient multiplex genome editing. We also engineered a strain expressing Cas9 genomically, which simplified the CRISPR plasmid construction and facilitated more efficient genome modifications, including markerless and scarless gene knock-in. Importantly, we established a CRISPRi system using catalytic inactive dCas9, achieving up to 100-fold repression on target gene. Here, sgRNAs targeting near and downstream regions of the transcription start site and the 5′end ORF achieved the highest repression efficacy. Furthermore, we developed an inducible CRISPRi-dCas9 system based on TetR/tetO platform. This facilitated the inducible gene repression, especially for essential genes.ConclusionsTherefore, these advancements not only expand the toolkit for genetic manipulation but also bridge methodological gaps for controlled gene regulation, especially for essential genes, in M. maripaludis. The robust toolkit developed here paves the way for applying M. maripaludis as a vital model archaeal cell factory, facilitating fundamental biological studies and applied biotechnology development of archaea.

A Study on Mutation Breeding for Crop Improvement

Despite the tremendous changes happening in the world of agriculture, food insecurity is still an issue. To meet the 70% increase in food consumption by 2050 predicted by the global population, it is essential to improve crop varieties in order to make them more resistant to climate change, efficient with inputs, provide more nutrient-dense food, and operate better in different agro-ecosystems without negatively impacting the environment. A total of 3,362 mutant plant kinds have been published from more than 75 different nations as a result of the very popular breeding method known as mutation breeding, sometimes known as induced mutation. Genetic diversity in crops has been enhanced by the application of physical, chemical, and hybrid mutagens. Of these, 82 are indigenous to Africa and 959 are native to Asia, the Pacific, Australia, Europe, Latin America, and North America. Other notable crop types that have resulted from mutation breeding and forced mutation include 1602 cereals, 501 legumes, and 86 oil seed mutant. Raising agricultural productivity and ending global hunger, this method improves quantitative and qualitative characteristics across a wide range of crops. Developments in biotechnology, genetics, and plant breeding have enabled this improvement.

CFCN: An HLA-peptide Prediction Model based on Taylor Extension Theory and Multi-view Learning

Background: With the increasing development of biotechnology, many cancer solutions have been proposed nowadays. In recent years, Neo-peptides-based methods have made significant contributions, with an essential prerequisite of bindings between peptides and HLA molecules. However, the binding is hard to predict, and the accuracy is expected to improve further. Methods: Therefore, we propose the Crossed Feature Correction Network (CFCN) with deep learning method, which can automatically extract and adaptively learn the discriminative features in HLA-peptide binding, in order to make more accurate predictions on HLA-peptide binding tasks. With the fancy structure of encoding and feature extracting process for peptides, as well as the feature fusion process between fine-grained and coarse-grained level, it shows many advantages on given tasks. Results: The experiment illustrates that CFCN achieves better performances overall, compared with other fancy models in many aspects. Conclusion: In addition, we also consider to use multi-view learning methods for the feature fusion process, in order to find out further relations among binding features. Eventually, we encapsulate our model as a useful tool for further research on binding tasks.

Progress on CRISPR-Cas gene editing technology in sheep production.

Gene editing is a kind of genetic engineering technology that can modify the genome. In recent years, with the rapid development of molecular biotechnology, the clustered regularly interspaced short palindromic repeats associated protein system has been widely used as a powerful gene editing tool due to its high efficiency, accuracy and flexibility. The CRISPR-Cas system makes a significant contribution to different aspects of livestock production by introducing site-specific modifications such as insertions, deletions or single base replacements at specific genomic sites. In terms of sheep production applications, by establishing animal models that improve production economic traits and disease resistance, the function of key genes can be studied to accelerate the improvement of traits, thereby accelerating the improvement of traits. In this review, we summarize the mechanism and function of CRISPR-Cas system and its application in the production of reproductive traits, meat use traits, wool production traits, lactation traits and disease resistance traits of sheep and the establishment of sheep animal models.

Data Integration and Management in Bioinformatics

Bioinformatics is an interdisciplinary field that combines biology, computer science, and information technology to understand and analyze biological data. With the development of high-throughput sequencing technology and other biotechnologies, the speed and scale of biological data generation are unprecedented. Effectively integrating and managing these data has become a significant challenge. This paper explores the current state of bioinformatics data integration and management, the challenges faced, commonly used methods and tools, and future development directions.

Engineering microalgae for robust glycolate biosynthesis: Targeted knockout of hydroxypyruvate reductase 1 combined with optimized culture conditions enhance glycolate production in Chlamydomonas reinhardtii

Microalgae-based glycolate production through the photorespiratory pathway is considered an environmentally friendly approach. However, the potential for glycolate production is limited by photoautotrophic cultivation with low cell density and existing strains. In this study, a targeted knockout approach was used to disrupt the key photorespiration enzyme, Chlamydomonas reinhardtii hydroxypyruvate reductase 1 (CrHPR1), leading to a significant increase in glycolate production of 280.1 mg/L/OD750. The highest potency yield reached 2.1 g/L under optimized mixotrophic conditions, demonstrating the possibility of synchronizing cell growth with glycolate biosynthesis in microalgae. Furthermore, the hypothesis that the cell wall-deficient mutant facilitates glycolate excretion was proposed and validated by comparing the glycolate accumulation trends of various Chlamydomonas reinhardtii strains. This study will facilitate the development of microalgae-based biotechnology and shed lights on the continuous advancement of green biomanufacturing for industrial application.

Enzyme metabolism and functions in vitamin biosynthesis pathways

Vitamins, as indispensable organic compounds in life activities, demonstrate a complex and refined metabolic network in organisms. This network involves the coordination of multiple enzymes and the integration of various metabolic pathways. Despite the achievements in metabolic engineering and catalytic mechanism research, the lack of studies regarding detailed enzymatic properties for a large number of key enzymes limits the enhancement of vitamin production efficiency and hinders the in-depth understanding and optimization of vitamin synthesis mechanisms. Such limitations not only restrict the industrial application of vitamins but also impede the development of related bio-technologies. This study comprehensively reviews the research progress in the enzymes involved in vitamin biosynthesis and details the current status of research on the enzymes of 13 vitamin synthesis pathways, including their catalytic mechanisms, kinetic properties, and applications in biology. In addition, this study compares the properties of enzymes involved in vitamin metabolic pathways and the glycolysis pathway, and reveals the characteristics of catalytic efficiency and substrate affinity of enzymes in vitamin synthesis pathways. Furthermore, this study discusses the potential and prospects of applying deep learning methods to the research on properties of enzymes associated with vitamin biosynthesis, giving new insights into the production and optimization of vitamins.

Single domain antibody: Development and application in biotechnology and biopharma.

Heavy-chain antibodies (HCAbs) are a unique type of antibodies devoid of light chains, and comprised of two heavy chains-only that recognize their cognate antigen by virtue of a single variable domain also referred to as VHH, single domain antibody (sdAb), or nanobody (Nb). These functional HCAbs, serendipitous discovered about three decades ago, are exclusively found in camelids, comprising dromedaries, camels, llamas, and vicugnas. Nanobodies have become an essential tool in biomedical research and medicine, both in diagnostics and therapeutics due to their beneficial properties: small size, high stability, strong antigen-binding affinity, low immunogenicity, low production cost, and straightforward engineering into more potent affinity reagents. The occurrence of HCAbs in camelids remains intriguing. It is believed to be an evolutionary adaptation, equipping camelids with a robust adaptive immune defense suitable to respond to the pressure from a pathogenic invasion necessitating a more profound antigen recognition and neutralization. This evolutionary innovation led to a simplified HCAb structure, possibly supported by genetic mutations and drift, allowing adaptive mutation and diversification in the heavy chain variable gene and constant gene regions. Beyond understanding their origins, the application of nanobodies has significantly advanced over the past 30 years. Alongside expanding laboratory research, there has been a rapid increase in patent application for nanobodies. The introduction of commercial nanobody drugs such as Cablivi, Nanozora, Envafolimab, and Carvykti has boosted confidence among in their potential. This review explores the evolutionary history of HCAbs, their ontogeny, and applications in biotechnology and pharmaceuticals, focusing on approved and ongoing medical research pipelines.

Wild rice: unlocking the future of rice breeding.

Germplasm resources serve as the foundations of advancements in breeding and are crucial for maintaining food security. Wild rice species of the genus Oryza include rich sources of genetic diversity and high adaptability, making them a substantial resource for rice breeding. The discovery of wild-type cytoplasmic male sterility resources enabled the achievement of the 'three lines' goal in hybrid rice, significantly increasing rice yields. The application of resistance alleles from wild rice enables rice production to withstand losses caused by stress. Reduced genetic diversity due to rice breeding poses a significant limitation to further advances and can be alleviated through a systematic use of wild genetic resources that integrate geographic, climatic and environmental data of the original habitat, along with extensive germplasm collection and identification using advanced methods. Leveraging technological advancements in plant genomics, the understanding of genetic mechanisms and the application of artificial intelligence and gene editing can further enhance the efficiency and accuracy of this process. These advancements facilitate rapid isolation and functional studies of genes, and precise genome manipulation. This review systematically summarizes the utilization of superior genes and germplasm resources derived from wild rice sources, while also exploring the collection, conservation, identification and utilization of further wild rice germplasm resources. A focus on genome sequencing and biotechnology developments is leading to new breeding and biotechnology opportunities. These new opportunities will not only promote the development of rice varieties that exhibit high yields, superior stress resistance and high quality but also expand the genetic diversity among rice cultivars.

Experimental uncoupling of hosts and endosymbionts.

Many organisms harbor heritable bacterial symbionts that offer context-specific benefits to their hosts. In some of these symbioses, symbionts live inside host cells as endosymbionts. Studying the biology of endosymbiosis is challenging because it is hard to independently cultivate hosts and endosymbionts. A recent study, using a simple defined growth medium at ambient temperature, established an axenic culture of the pea aphid's heritable bacterial endosymbiont, Candidatus Fukatsuia symbiotica (G. P. Maeda, M. K. Kelly, A. Sundar, and N. A. Moran, mBio 15:e03253-23, 2024, https://doi.org/10.1128/mbio.03253-23). Notably, the monoculture was capable of host recolonization, was stably transmitted, and returned similar host phenotypes to those observed in native infections. This advance in uncoupling the cultivation of an endosymbiont and its host opens avenues for genetic manipulation of the endosymbiont that will facilitate hypothesis-driven work to explore the mechanisms of host-endosymbiont biology and potentially facilitate the development of symbiont-mediated practical-application biotechnologies.

Biotechnology and crisis management developments during the COVID-19 pandemic: Comprehensive analysis

Over the period from 2020 to 2023, a four-year non-military battle drew to a close, marking the end of the COVID-19 pandemic that will be etched into history. During this time frame, policies and technologies aimed at combating the virus were developed, ultimately saving numerous lives and enhancing people's efficacy in battling the virus while demonstrating unwavering determination. However, some less mature biological nucleic acid technologies and some controversial policy bases are still being improved. Through the development of biotechnology levels and policies, the research and development of three mRNA vaccines, health codes and RT-PCR nucleic acid detection methods have greatly improved the efficiency of epidemic prevention and control. The government's policy of controlling speech and behavior at the initial stage of the outbreak brought people together to fight against the virus. These technologies are being researched to reduce the number of casualties and minimize losses. For example, circRNA or saRNA vaccines are still in the research stage. If these more advanced techniques are put into use, it will undoubtedly greatly increase the means of fighting against the virus. This is also the direction and key for future research and development.

Efficient cytosolic delivery of proteins enabled by modular fusion protein

The development of protein-based biotechnology and drugs holds tremendous potential for various applications. However, the effective intracellular delivery of natural proteins remains a challenge due to their inability to spontaneously penetrate the cell membrane. In this study, we have developed a modular fusion protein sequence that overcomes this limitation and enables efficient cytoplasmic protein delivery . This modular fusion protein we designed has demonstrated efficient lysosomal escape ability, allowing for successful delivery of various protein cargoes into the cytoplasm of different cell types. Importantly, the fusion with the mCherry tag does not affect the biological activity of the protein. The fusion protein can still exert its catalytic activity when delivered into cells. We also designed a fluorescent protein probe capable of precisely targeting lysosomes, providing powerful fluorescent probe tools for intracellular localization and tracking in biomedical research. This approach contributes to the advancement of protein drug development and opens up new possibilities for therapeutic interventions.

Learning from Protein Engineering by Deconvolution of Multi-Mutational Variants.

This review analyzes a development in biochemistry, enzymology and biotechnology that originally came as a surprise. Following the establishment of directed evolution of stereoselective enzymes in organic chemistry, the concept of partial or complete deconvolution of selective multi-mutational variants was introduced. Early deconvolution experiments of stereoselective variants led to the finding that mutations can interact cooperatively or antagonistically with one another, not just additively. During the past decade, this phenomenon was shown to be general. In some studies, molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) computations were performed in order to shed light on the origin of non-additivity at all stages of an evolutionary upward climb. Data of complete deconvolution can be used to construct unique multi-dimensional rugged fitness pathway landscapes, which provide mechanistic insights different from traditional fitness landscapes. Along a related line, biochemists have long tested the result of introducing two point mutations in an enzyme for mechanistic reasons, followed by a comparison of the respective double mutant in so-called double mutant cycles, which originally showed only additive effects, but more recently also uncovered cooperative and antagonistic non-additive effects. We conclude with suggestions for future work, and call for a unified overall picture of non-additivity and epistasis.

Lernen aus Protein‐Engineering durch Dekonvolution von Multi‐Mutationalen Enzymvarianten**

AbstractDiese Übersicht analysiert eine Entwicklung in der Biochemie, Enzymologie und Biotechnologie, die ursprünglich als Überraschung betrachtet wurde. Nach der Etablierung der gerichteten Evolution stereoselektiver Enzyme in der organischen Chemie wurde das Konzept der partiellen oder vollständigen Dekonvolution selektiver multi‐mutationaler Varianten eingeführt. Frühe Dekonvolutionsexperimente von stereoselektiven Enzymvarianten führten zu der Erkenntnis, dass Mutationen kooperativ oder antagonistisch miteinander interagieren können, nicht nur additiv. Im letzten Jahrzehnt wurde dieses Phänomen als allgemeingültig nachgewiesen. In einigen Studien wurden molekulardynamische (MD) und quantenmechanische/ molekülmechanische (QM/MM) Berechnungen durchgeführt, um Licht auf den Ursprung der Nicht‐Additivität in allen Phasen eines evolutionären Aufstiegs zu werfen. Daten der vollständigen Dekonvolution können verwendet werden, um einzigartige mehrdimensionale, zerklüftete („rugged“) Fitness‐Pfad‐Landschaften experimentell zu konstruieren. Sie bieten mechanistische Einblicke, die sich von traditionellen Fitnesslandschaften erheblich unterscheiden. In einem verwandten Bereich haben Biochemiker lange das Ergebnis der Einführung von zwei Punktmutationen in einem Enzym aus mechanistischen Gründen getestet, gefolgt von einem Vergleich mit der entsprechenden Doppelmutante in sogenannten Doppelmutanten‐Zyklen, die ursprünglich nur additive Effekte zeigten. In jüngerer Zeit wurden auch hier kooperative und antagonistische nicht‐additive Effekte aufgedeckt. Wir schließen mit Vorschlägen für zukünftige Arbeiten und fordern ein einheitliches Gesamtbild von Nicht‐Additivität und Epistasis.

<i>Spongia officinalis</i> -Associated <i>Pseudomonas fluorescens</i> as a Reservoir of Bioactive Compounds: A Novel Source of Natural Anticancer Compounds

Marine sponges are an important source of chemical variety and a repository of biodiversity. In this study, the microbial communities found in the marine sponge Spongia officinalis that was taken from the Kanyakumari coast in India were explored. We identified, characterized, and evaluated the bioactive potential of the sponge-associated bacteria. A total of 12 bacterial isolates were obtained, primarily consisting of gram-positive rods and some gram-negative rods and cocci. These bacterial isolates showed a variety of bioactive substances when their secondary metabolites were extracted, including phenols, flavonoids, alkaloids, saponins, terpenoids, and steroids. These chemical compounds are well-known for their numerous biological activity and health benefits. The antioxidant and anticancer effects of these metabolites were also investigated. Isolate 6 showed significant antioxidant activity, suggesting it may be able to treat cancer. With an IC50 value of 55.32 g/ml, this isolate also displayed impressive anticancer activity against HeLa cells. The screened isolate was identified as Pseudomonas fluorescens strain ATCC 13525 using 16S rRNA sequencing. This discovery emphasizes the importance of bacteria associated with marine sponges as a source of bioactive compounds with medicinal potential. The conclusion of this research highlights the diverse microbial communities found in marine sponges and their potential for use in biotechnology and medication development. Pseudomonas fluorescens was found to be a prolific generator of bioactive byproducts, including strong antioxidants and anticancer agents, which emphasizes the significance of additional marine resource exploitation for pharmacologically relevant chemicals.

Accurate prediction of protein function using statistics-informed graph networks

Understanding protein function is pivotal in comprehending the intricate mechanisms that underlie many crucial biological activities, with far-reaching implications in the fields of medicine, biotechnology, and drug development. However, more than 200 million proteins remain uncharacterized, and computational efforts heavily rely on protein structural information to predict annotations of varying quality. Here, we present a method that utilizes statistics-informed graph networks to predict protein functions solely from its sequence. Our method inherently characterizes evolutionary signatures, allowing for a quantitative assessment of the significance of residues that carry out specific functions. PhiGnet not only demonstrates superior performance compared to alternative approaches but also narrows the sequence-function gap, even in the absence of structural information. Our findings indicate that applying deep learning to evolutionary data can highlight functional sites at the residue level, providing valuable support for interpreting both existing properties and new functionalities of proteins in research and biomedicine.

A Combination of Transcriptome and Enzyme Activity Analysis Unveils Key Genes and Patterns of Corncob Lignocellulose Degradation by Auricularia heimuer under Cultivation Conditions.

The cultivation of Auricularia heimuer, a species of edible mushroom, heavily relies on the availability of wood resources serving as substrate for the growth of the species. To ensure the sustainable development of the A. heimuer industry and optimize the utilization of corncob as a substrate, this study sought to investigate the potential use of corncob as a substrate for the cultivation of A. heimuer. The purpose of this study was to explore the utilization of corncob lignocellulose by A. heimuer at the mycelium, primordium, and fruiting stages, by specifically examining the expression profiles of both carbohydrate-active enzymes (CAZymes) and the transcriptome of differentially expressed genes (DEGs) relevant to corncob biomass degradation. The results revealed 10,979, 10,630, and 11,061 DEGs at the mycelium, primordium, and fruiting stages, respectively, while 639 DGEs were identified as carbohydrate-active enzymes. Of particular interest were 46 differentially expressed CAZymes genes that were associated directly with lignocellulose degradation. Furthermore, the study found that A. heimuer exhibited adaptive changes that enabled it to effectively utilize the cellulose present in the corncob. These changes were observed primarily at the primordium and fruiting stages. Key genes involved in lignocellulose degradation were also identified, including g6952, g8349, g12487, and g2976 at the mycelium stage, g5775, g2857, g3018, and g11016 at the primordium stage, and g10290, g2857, g12385, g7656, and g8953 at the fruiting stage. This study found that lytic polysaccharide monooxygenase (LPMO) played a crucial role in the degradation of corncob cellulose, further highlighting the complexity of the molecular mechanisms involved in the degradation of lignocellulose biomass by A. heimuer. The study sheds light on the molecular mechanisms underlying the ability of A. heimuer to degrade corncob biomass, with implications for the efficient utilization of lignocellulose resources. The findings from this study may facilitate the development of innovative biotechnologies for the transformation of corncob biomass into useful products.

A New Era of Discovery: How Artificial Intelligence has Revolutionized the Biotechnology

In the growing field of biotechnology, artificial intelligence (AI) has emerged as a pivotal force of innovation, unveiling a new era of discovery and advancement. The convergence of AI with biotechnology has revolutionized the landscape of scientific research and development. The dynamic interplay between AI and biotechnology highlights the transformative power of AI techniques in accelerating advancements in drug discovery, development, personalized medicine, biomolecular engineering, bioprocessing, CRISPR technology, genome editing, genomics, proteomics, metabolomics, transcriptomic, and AI-enabled robotics in biotechnology. This integration of AI with biotechnology helps us combat global challenges and offers environmentally friendly and sustainable solutions like bioremediation, bioplastics, biodiesel, and biofiltration. Current examples of these problems include waste management, air pollution, healthcare, clean water, energy access, sustainable practices, conservation of biodiversity, and ecosystems. This review article provides a comprehensive analysis, drawing on current literature, case studies, and emerging trends, to highlight the transformative potential of AI in reshaping the biotechnological landscape. It also addresses the challenges and opportunities associated with this AI-powered transformation, discussing future directions, ethical considerations, and the need for human-AI collaboration to ensure responsible and sustainable progress for a brighter future.

MRNA therapeutics in diseases: Light inspect into a new horizon

An in-depth analysis of messenger RNA (mRNA) therapeutics and their potential to treat a wide range of diseases is provided in this thorough review paper. mRNA therapy has a great deal of potential for treating various terminal illnesses. By introducing mRNA as a vaccine or therapeutic agent, recent developments in biotechnology and molecular medicine have made it possible to produce almost any functional protein or peptide in the human body. This offers a unique chance to prevent and treat various challenging illnesses, many of which have genetic roots. Moreover, the utilization of in vitro transcribed mRNA has brought about a revolution in the production process, rendering it more efficient, adaptable, customizable, and cost-effective when compared to traditional methods. For a long time, researchers have aspired to enhance various aspects of mRNA, including its stability, immunogenicity, translation efficiency, and delivery systems, with the aim of achieving an efficient and secure means of delivering mRNA. In this comprehensive review, we will delve into the essence of mRNA and mRNA-based therapies, encompassing their fundamental principles, methods of production, diverse applications, outcomes, and constraints. We will underscore the importance of optimizing mRNA and its delivery systems to ensure the success of mRNA therapy, all while addressing the pivotal challenges and prospects in the battle against cancer and COVID-19.

IN MEMORY OF DOCTOR OF BIOLOGICAL SCIENCES, PROFESSOR SVITLANA IGNATOVA

Problem. Doctor of Biological Sciences, Professor Svitlana Oleksandrivna Ignatova dedicated her entire life to several scientific institutions in Odesa: V. Ye. Tairov Institute of Viticulture and Winemaking (1964–1970), All-Union Plant Breeding and Genetics Institute (1970–1991), the Plant Breeding and Genetics Institute – National Center of Seed and Cultivar Investigation (1991–1999 and 2012–2018), and the South Plant Biotechnological Center of the Ukrainian Academy of Agrarian Sciences (2000–2012). Her life and work were inextricably linked to the development of agricultural biotechnology in Ukraine. Aim. The purpose of our research was to honor the memory of Professor S. O. Ignatova, to highlight the main biographical data and the results of her scientific and pedagogical activities. Results. S. O. Ignatova fruitfully worked on theoretical and applied problems of agricultural crops biotechnology and was a pioneer in the use of achievements in Ukraine regarding the successful application in the selection process of the source material obtained by in vitro methods of tissue, organ and cell culture. Under the leadership of Svitlana Oleksandrivna, the principles and complete technology of accelerated production of linear varieties of barley and wheat with the use of haploproducers were developed. The barley varieties – Odessky 115th and Prairie, which were sown on a large area in Ukraine, were created using this technology. The great attention of S. O. Ignatova was devoted to the training of scientific personnel (graduate students, interns and students). Conclusions. S. O. Ignatova’s scientific legacy was a long-term fundamental research in the biotechnology of major agricultural crops, which was a significant contribution to the development of advanced areas of Ukrainian and world science. Svitlana Oleksandrivna was the author of more than 300 scientific works, including monographs, patents, scientific and methodological manuals, methodological recommendations, author’s certificates and publications in leading scientific journals. The life story of S. O. Ignatova is continued by her students, remembering the bright image of their professor – an energetic, determined and inspired woman.