Aromatic residues surrounding the active site tunnel of TfCel48A influence activity, processivity, and synergistic interactions with other cellulases.

Cel48A of Thermobifida fusca (TfCel48A) is a processive exocellulase that contains an active site tunnel and digests lignocellulosic biomass via synergistic interactions between different cellulases. Cel48A possesses a number of aromatic amino acids lining the tunnel entrance, which are highly conserved across a diverse number of microbial species and appear to play a role in the selection and threading of individual strands of cellulose from highly recalcitrant substrates. In this study, we sought to further elucidate the roles of these tunnel entrance aromatic amino acids by creating a series of double mutants and examining their effect on TfCel48A activity, processivity, and synergistic interactions with the well-studied processive endocellulase TfCel9A. Our results provide further insight concerning the mechanism of Cel48A kinetics with soluble and insoluble substrates and could play an influential role in the application of Cel48A and other exocellulases for industrial purposes.

How Computational Epitope Mapping Identifies the Interactions between Nanoparticles Derived from Papaya Mosaic Virus Capsid Proteins and Immune System.

BACKGROUND: Nanoparticles derived from plant viruses possess fascinating structures, versa-tile functions and safe properties, rendering them valuable for a variety of applications. Papaya mosaic Virus-Like Particles (VLPs) are nanoparticles that contain a repetitive number of virus capsid proteins (PMV-CP) and are considered to be promising platforms for vaccine design. Previous studies have re-ported the antigenicity of PMV nanoparticles in mammalian systems. MATERIALS AND METHODS: As experiments that concern vaccine development require careful design and can be time consuming, computational experiments are of particular importance. Therefore, prior to ex-pressing PMV-CP in E. coli and producing nanoparticles, we performed an in silico analysis of the virus particles using software programs based on a series of sophisticated algorithms and modeling networks as useful tools for vaccine design. A computational study of PMV-CP in the context of the immune sys-tem reaction allowed us to clarify particle structure and other unknown features prior to their introduc-tion in vitro. RESULTS: The results illustrated that the produced nanoparticles can trigger an immune response in the absence of fusion with any foreign antigen. CONCLUSION: Based on the in silico analyses, the empty capsid protein was determined to be recognised by different B and T cells, as well as cells which carry MHC epitopes.

Periplasmic Cytophaga hutchinsonii Endoglucanases Are Required for Use of Crystalline Cellulose as the Sole Source of Carbon and Energy.

UNLABELLED: The soil bacterium Cytophaga hutchinsonii actively digests crystalline cellulose by a poorly understood mechanism. Genome analyses identified nine genes predicted to encode endoglucanases with roles in this process. No predicted cellobiohydrolases, which are usually involved in the utilization of crystalline cellulose, were identified. Chromosomal deletions were performed in eight of the endoglucanase-encoding genes: cel5A, cel5B, cel5C, cel9A, cel9B, cel9C, cel9E, and cel9F Each mutant retained the ability to digest crystalline cellulose, although the deletion of cel9C caused a modest decrease in cellulose utilization. Strains with multiple deletions were constructed to identify the critical cellulases. Cells of a mutant lacking both cel5B and cel9C were completely deficient in growth on cellulose. Cell fractionation and biochemical analyses indicate that Cel5B and Cel9C are periplasmic nonprocessive endoglucanases. The requirement of periplasmic endoglucanases for cellulose utilization suggests that cellodextrins are transported across the outer membrane during this process. Bioinformatic analyses predict that Cel5A, Cel9A, Cel9B, Cel9D, and Cel9E are secreted across the outer membrane by the type IX secretion system, which has been linked to cellulose utilization. These secreted endoglucanases may perform the initial digestion within amorphous regions on the cellulose fibers, releasing oligomers that are transported into the periplasm for further digestion by Cel5B and Cel9C. The results suggest that both cell surface and periplasmic endoglucanases are required for the growth of C. hutchinsonii on cellulose and that novel cell surface proteins may solubilize and transport cellodextrins across the outer membrane. IMPORTANCE: The bacterium Cytophaga hutchinsonii digests crystalline cellulose by an unknown mechanism. It lacks processive cellobiohydrolases that are often involved in cellulose digestion. Critical cellulolytic enzymes were identified by genetic analyses. Intracellular (periplasmic) nonprocessive endoglucanases performed an important role in cellulose utilization. The results suggest a model involving partial digestion at the cell surface, solubilization and uptake of cellodextrins across the outer membrane by an unknown mechanism, and further digestion within the periplasm. The ability to sequester cellodextrins and digest them intracellularly may limit losses of soluble cellobiose to other organisms. C. hutchinsonii uses an unusual approach to digest cellulose and is a potential source of novel proteins to increase the efficiency of conversion of cellulose into soluble sugars and biofuels.

Nutritionally enhanced food crops; progress and perspectives.

Great progress has been made over the past decade with respect to the application of biotechnology to generate nutritionally improved food crops. Biofortified staple crops such as rice, maize and wheat harboring essential micronutrients to benefit the world's poor are under development as well as new varieties of crops which have the ability to combat chronic disease. This review discusses the improvement of the nutritional status of crops to make a positive impact on global human health. Several examples of nutritionally enhanced crops which have been developed using biotechnological approaches will be discussed. These range from biofortified crops to crops with novel abilities to fight disease. The review concludes with a discussion of hurdles faced with respect to public perception, as well as directions of future research and development for nutritionally enhanced food crops.

Helping students see bacteria in 3D: cellular models increase student learning about cell size and diffusion.

In the microbial world, cell size and shape impact physiology, but students struggle to visualize spatial relationships between cells and macromolecules. In prokaryotic cells, cell size is limited by reliance on diffusion for nutrient uptake and the transport of nutrients within the cell. Cells must also meet a minimum size threshold to accommodate essential cellular components such as ribosomes and DNA. Using 3D printing allows for the creation of custom models that can be influential teaching tools in the biology classroom. This lesson uses 3D cell models to teach students enrolled in an introductory microbiology course about bacterial cell size and the biological importance of surface-area-to-volume ratio. During the lesson, students interact with 3D cell models and discuss a series of questions in small groups. Student learning was assessed using quantitative and qualitative student response data collected pre- and post-lesson. Student achievement of learning objectives, and their confidence in their knowledge of these concepts, improved post-lesson, and these gains were statistically significant. Our findings suggest that interacting with 3D-printed cell models improves student understanding about bacterial cell size and diffusion.

Novel approaches to circumvent the devastating effects of pests on sugarcane.

Sugarcane (Saccharum officinarum L.) is a cash crop grown commercially for its higher amounts of sucrose, stored within the mature internodes of the stem. Numerous studies have been done for the resistance development against biotic and abiotic stresses to save the sucrose yields. Quality and yield of sugarcane production is always threatened by the damages of cane borers and weeds. In current study two problems were better addressed through the genetic modification of sugarcane for provision of resistance against insects and weedicide via the expression of two modified cane borer resistant CEMB-Cry1Ac (1.8 kb), CEMB-Cry2A (1.9 kb) and one glyphosate tolerant CEMB-GTGene (1.4 kb) genes, driven by maize Ubiquitin Promoter and nos terminator. Insect Bio-toxicity assays were carried out for the assessment of Cry proteins through mortality percent of shoot borer Chilo infuscatellus at 2nd instar larvae stage. During V0, V1 and V2 generations young leaves from the transgenic sugarcane plants were collected at plant age of 20, 40, 60, 80 days and fed to the Chilo infuscatellus larvae. Up to 100% mortality of Chilo infuscatellus from 80 days old transgenic plants of V2 generation indicated that these transgenic plants were highly resistant against shoot borer and the gene expression level is sufficient to provide complete resistance against target pests. Glyphosate spray assay was carried out for complete removal of weeds. In V1-generation, 70-76% transgenic sugarcane plants were found tolerant against glyphosate spray (3000 mL/ha) under field conditions. While in V2-generation, the replicates of five selected lines 4L/2, 5L/5, 6L/5, L8/4, and L9/6 were found 100% tolerant against 3000 mL/ha glyphosate spray. It is evident from current study that CEMB-GTGene, CEMB-Cry1Ac and CEMB-Cry2A genes expression in sugarcane variety CPF-246 showed an efficient resistance against cane borers (Chilo infuscatellus) and was also highly tolerant against glyphosate spray. The selected transgenic sugarcane lines showed sustainable resistance against cane borer and glyphosate spray can be further exploited at farmer's field level after fulfilling the biosafety requirements to boost the sugarcane production in the country.

Flawed scientific studies block progress and sow confusion.

Research in crop science in recent years has advanced at an unprecedented rate, and the intermingling of old and new crop breeding technologies has made the term "genetically modified" - and its variant, Genetically Modified Organism, or "GMO" - virtually obsolete. A kind of pseudo-category, it is primarily used pejoratively to refer to the use of the newest, most precise, most predictable, molecular genetic techniques. Prodigious amounts of time, effort and care have been expended to ensure that crops developed for commercialization using molecular techniques are safe, and that new traits are beneficial. Â Yet, despite these advances, some skepticism persists about them, partly due to the publication of fraudulent, poorly designed, and biased studies by a few "rogue scientists" whose intention is to contaminate the scientific literature and sow mistrust about molecular genetic modification among regulators and the public. We discuss how such flawed studies make it to publication and how the scientific community can combat such disinformation.

The role of plant expression platforms in biopharmaceutical development: possibilities for the future.

Introduction: Plant-made vaccines have been in the pipeline for nearly thirty years. Generated stably in transgenic plants or transiently using virus expression systems, pharmaceuticals have been developed to address global pandemics as well as several emerging One Health Diseases.Areas covered: This review describes the generation of plant-made vaccines to address some of the world's most growing health concerns, including both infectious and non-communicable diseases, such as cancer. The review provides an overview of the research taking place in this field over the past three to five years. The PubMed database was searched under the topic of plant-made vaccine between the periods of 2014 and 2019.Expert opinion: While vaccines and other biologics have been shown to be cheap safe and efficacious, they have not yet entered the marketplace largely due to regulatory constraints. The lack of an appropriate regulatory structure to guide plant-made vaccines through to commercial development has stalled efforts to provide life-saving medicines to low- and middle-income families. In my opinion, it is paramount that regulatory hurdles are mitigated to address emerging infectious diseases such as Ebola and Zika in a timely manner.

Repurposing Plant Virus Nanoparticles.

Plants have been explored for many years as inexpensive and versatile platforms for the generation of vaccines and other biopharmaceuticals. Plant viruses have also been engineered to either express subunit vaccines or act as epitope presentation systems. Both icosahedral and helical, filamentous-shaped plant viruses have been used for these purposes. More recently, plant viruses have been utilized as nanoparticles to transport drugs and active molecules into cancer cells. The following review describes the use of both icosahedral and helical plant viruses in a variety of new functions against cancer. The review illustrates the breadth of variation among different plant virus nanoparticles and how this impacts the immune response.

Virus-Based RNA Silencing Agents and Virus-Derived Expression Vectors as Gene Therapy Vehicles.

BACKGROUND: In consideration of recent developments in understanding the genomics and proteomics of viruses, the use of viral DNA / RNA sequences as well as their gene expression schemes, have found new in-roads towards the prognosis and therapy of diseases. Correspondingly, the sphere of the patenting scenario has expanded significantly. OBJECTIVES: The current review addresses patented inventions concerning the use of virus sequences as gene silencing machineries and inventions concerning the generation and application of viral sequences as expression vectors. Furthermore, this review also discusses the employment of these patents for clinical, agricultural and biotechnological applications. METHOD: Considering these objectives, the Delphion Research Intellectual Property Network database was searched using keywords such as "gene silencing", "engineered viruses" and "expression vectors" and descriptions of recent patents on the said topics were discussed. CONCLUSION: Despite several recent advances in the use of viruses as disease therapy vehicles and biotechnological vectors, these developments have yet to be proven effective in practice, in clinical and field trials.

Recently Patented Viral Nucleotide Sequences and Generation of Virus-Derived Vaccines.

BACKGROUND: With an increase in comprehension of the molecular biology of viruses, there has been a recent surge in the application of virus sequences and viral gene expression strategies towards the diagnosis and treatment of diseases. RESULTS: The scope of the patenting landscape has widened as a result and the current review discusses patents pertaining to live / attenuated viral vaccines. The vaccines addressed here have been developed by both conventional means as well as by the state-of-the-art genetic engineering techniques. CONCLUSION: This review also addresses the applications of these patents for clinical and biotechnological purposes.

Multi-tasking of nonstructural gene products is required for bean yellow dwarf geminivirus transcriptional regulation.

Mastreviridae, of the family geminiviridae, possess a monopartite genome and are transmitted by leafhoppers. Bean yellow dwarf dirus (BeYDV) is a mastrevirus which originated from South Africa and infects dicoyledenous plants, a feature unusual for mastreviridae. Previously, the nonstructural proteins Rep and RepA were examined with respect to their independent roles in BeYDV replication. This was achieved by placing both gene products under independent constitutive promoter control and examining their effects on replication-competent constructs. In the current study, Rep and RepA are examined further for their roles in regulating BeYDV gene expression using a series of replication-incompetent constructs. While both Rep and RepA are found to behave as equally potent inhibitors of complementary-sense gene expression, they differ considerably with respect to their abilities to transactivate virion-sense gene expression. Furthermore, RepA is identified as playing more than one role in this transactivation process. A nuclear localization domain is identified in Rep which is absent in RepA, and Rep-RepA interactions are examined under in vivo conditions. The study concludes with an investigation into the expression strategies of the BeYDV capsid protein.

Recent Patents in Oncolytic Virotherapy.

Recent innovative and advanced developments in the diagnosis and treatment of human diseases as well as enhanced in-depth understanding of virus molecular biology have opened novel avenues with respect to the patent landscape. Included are viruses utilized in the development of anticancer agents, agents that are employed against the spread of infectious viral diseases, RNA silencing agents and virus-derived expression vectors that can be used for over-expression of therapeutic proteins or as gene therapy vehicles. The current review describes several recent patents pertaining to virus sequences and their medical and biotechnological applications.

DNA Virus Vectors for Vaccine Production in Plants: Spotlight on Geminiviruses.

Plants represent a safe, efficacious and inexpensive production platform by which to provide vaccines and other therapeutic proteins to the world's poor. Plant virus expression vector technology has rapidly become one of the most popular methods to express pharmaceutical proteins in plants. This review discusses several of the state-of-the-art plant expression systems based upon geminiviruses that have been engineered for vaccine production. An overview of the advantages of these small, single-stranded DNA viruses is provided and comparisons are made with other virus expression systems. Advances in the design of several different geminivirus vectors are presented in this review, and examples of vaccines and other biologics generated from each are described.

Broadly neutralizing antibodies and the promise of universal vaccines: where are we now?

Recent research has provided strong support for the utility of broadly neutralizing antibodies generated against viruses, which inherently possess a high degree of antigenic variability (such as influenza virus or HIV) as a feasible means to prevent infection. Many of these antibodies share the ability to bind to highly conserved regions within the stem of the virus 'spike' or surface glycoprotein, in such a way that they interfere with virus entry, including membrane fusion. As a result, broadly neutralizing antibodies could be supplied to patients as a form of passive immunotherapy, as well as play a role in the design of new 'universal' vaccines and antiviral agents. The following article describes the most recent innovations in this exciting field.

Virus expression vectors.

For many years now, virus expression vectors have been explored as a mechanism for gene delivery, cancer therapy and vaccine development. More recently, the next generation of virus vectors have been generated that possess greater attributes such as tissue specificity and improved expression levels, while at the same time lack many of the shortcomings of their predecessors, such as issues regarding immunogenicity and safety. This review article describes several of the recent patents that have been issued in the field of virus expression vector development. Innovations in both plant and animal virus expression vectors are covered. The review concludes with a discussion of future prospects of virus expression vectors as tools in medical research.

Recent patents in plant biotechnology: impact on global health.

Agricultural biotechnology offers a robust series of tools by which to address global concerns such as food security, crop protection, and fuel/energy requirements. A number of advances made recently in plant molecular biology also have resulted in applications which largely focus on improving global human health. This review describes some of the recent innovations in plant biotechnology that have come to the forefront over the past year. Included are novel techniques by which plants can be improved as platforms for biopharmaceutical protein production, a growing field also referred to as 'molecular pharming'. The metabolic engineering of plants to produce compounds which have additional nutritional benefits is also outlined. The review concludes with a discussion of the future impact that these innovations may have both on global health and on the development of our future intellectual property landscape.

Recent patents involving virus nucleotide sequences; host defense, RNA silencing and expression vector strategies.

Improved knowledge of the molecular biology of viruses, including recent gains in virus sequence data analysis, has greatly contributed to recent innovations in medical diagnostics, therapeutics, drug development and other related areas. Virus sequences have been used for the development of vaccines and antiviral agents to block the spread of viral infections, as well as to target and battle chronic diseases such as cancer. Virus sequences are now routinely employed in a wide array of RNA silencing technologies. Viruses can also be engineered into expression vectors which in turn can be used as protein production platforms as well as delivery vehicles for gene therapies. This review article outlines a number of patents that have been recently issued with respect to virus sequence data and describes some of their biotechnological applications.