Development of a Plant-Expressed Subunit Vaccine against Brucellosis.

Brucellosis is an important bacterial disease of livestock and the most common zoonotic disease. The current vaccines are effective but unsafe, as they result in animal abortions and are pathogenic to humans. Virus-like particles are being investigated as molecular scaffolds for foreign antigen presentation to the immune system. Here, we sought to develop a new-generation vaccine by presenting selected Brucella melitensis T cell epitopes on the surface of Orbivirus core-like particles (CLPs) and transiently expressing these chimeric particles in Nicotiana benthamiana plants. We successfully demonstrated the assembly of five chimeric CLPs in N. benthamiana plants, with each CLP presenting a different T cell epitope. The safety and protective efficacy of three of the highest-yielding CLPs was investigated in a mouse model of brucellosis. All three plant-expressed chimeric CLPs were safe when inoculated into BALB/c mice at specific antigen doses. However, only one chimeric CLP induced protection against the virulent Brucella strain challenge equivalent to the protection induced by the commercial Rev1 vaccine. Here, we have successfully shown the assembly, safety and protective efficacy of plant-expressed chimeric CLPs presenting B. melitensis T cell epitopes. This is the first step in the development of a safe and efficacious subunit vaccine against brucellosis.

Novel strategies for drug repurposing.

Synthetic biology, precision medicine, and nanobiotechnology are the three main emerging areas that drive translational innovation toward commercialization. There are several strategies used in precision medicine and drug repurposing is one of the key approaches as it addresses the challenges in drug discovery (high cost and time). Here, we provide a perspective on various new approaches to drug repurposing for cancer precision medicine. We report here our optimized wound healing methodology that can be used to validate drug sensitivity and drug repurposing. Using HeLa as our benchmark, we demonstrated that the assay can be applied to identify drugs that limit cell proliferation. From a future perspective, this assay can be expanded to ex vivo culturing of solid tumors in 2D culture and leukemia in 3D culture.

Protocol for Testing the Effects of ssDNA Aptamer in HeLa and MCF-7.

Conventional approaches for treating tumors encompass chemotherapy, radiotherapy, and surgery. However, these methods come with their limitations when applied in clinical practice. Aptamers are often referred to as "chemical antibodies" and consist of short DNA or RNA molecules, designed to bind to a wide range of targets, including proteins or nucleic acid structures. They exhibit strong affinities and remarkable specificity for their target molecules, making them capable of functioning as therapeutic agents to directly impede tumor cell proliferation. This approach helps minimize the harm to normal cells, thus reducing toxicity through decreased side effects. Here we report the procedure to develop ssDNA aptamer and investigate its ability to inhibit cancer cell proliferation in HeLa and MCF-7 cancer cell lines.

DirectedCHO: A new miniaturized directed evolution process for phenotype stability trial test of CHO cells before bioreactor scale-up.

Most of the biopharmaceuticals that are currently on the market are expressed using the Chinese Hamster Ovary (CHO) cell lines. However, the production yield of these biopharmaceuticals is affected due to CHO cellular heterogeneity and challenges in adaptability during the bioreactor scale-up stage. In this communication, we report the protocol for the miniaturized directed evolution process for CHO cells. The results of the directed evolution process would guide adapting the CHO cell line before bioreactor scale-up. With our approach, we have established the protocol that can be used to streamline superior CHO cell lines for biopharmaceutical production which would be the first of its kind in Africa. Our directed evolution protocol includes a method for a low-cost multiplex directed evolution process that can be used on CHO cells using 20 stressors in 8 concentrations and provides stable trial results for the scale-up process. Using our process, we can provide a simple consumable kit that manufacturers can use for the CHO cell phenotype stability test before the scale-up process. With our approach, we would further develop a platform that can streamline superior CHO cell lines for biopharmaceutical production. This approach would be the first of its kind in South Africa/ Africa.

Effects of nanoadditives on the performance and emissions of rapeseed biodiesel in an insulated piston diesel engine.

Energy consumption and management have emerged as crucial production functions because of the high cost of energy. Since the total consumption of fossil fuels like diesel has increased proportionally to the expansion in demand for power generation, industry, and transportation services, researchers have long been interested in constructing a more energy-efficient engine. With its improved efficiency, reduced fuel consumption, and fewer emissions, the application of nano-coating technology to engine components has become more popular in recent years. This study involved the application of a thermal barrier coating (TBC) using zirconia on the test engine piston. The aim of this research is to examine the impact of aluminium oxide nano-additives in rapeseed biodiesel blends on the performance of a diesel engine with a thermal barrier-coated piston. The four test fuels were prepared using 20% and 40% blends of rapeseed biodiesel with and without the addition of aluminium oxide at 25 ppm and 50 ppm. The full factorial design methodology was employed to examine the influential factors, specifically the rapeseed blend ratio and aluminium oxide concentration, in order to enhance performance and reduce emissions. The blends of RSB20AO25 and RSB20AO50 showed significant results on energy consumption and emissions. The RSB20AO50 blend performed with a 5.4% increase in brake thermal efficiency and a 6.5% reduction in fuel consumption compared with standard diesel. Similarly, blends of RSB20AO25 and RSB20AO50 show 6% and 11% reductions in carbon monoxide and 5.2% and 9.5% reductions in hydrocarbon emissions.

Recent advances in genome annotation and synthetic biology for the development of microbial chassis.

This article provides an overview of microbial host selection, synthetic biology, genome annotation, metabolic modeling, and computational methods for predicting gene essentiality for developing a microbial chassis. This article focuses on lactic acid bacteria (LAB) as a microbial chassis and strategies for genome annotation of the LAB genome. As a case study, Lactococcus lactis is chosen based on its well-established therapeutic applications such as probiotics and oral vaccine development. In this article, we have delineated the strategies for genome annotations of lactic acid bacteria. These strategies also provide insights into streamlining genome reduction without compromising the functionality of the chassis and the potential for minimal genome chassis development. These insights underscore the potential for the development of efficient and sustainable synthetic biology systems using streamlined microbial chassis with minimal genomes.

Machine learning models for predicting vegetation conditions in Mahanadi River basin.

The vegetation of a river basin is affected by various climate factors, such as precipitation and land surface temperature (LST). This study explores the best machine learning model for the prediction of normalized difference vegetation index (NDVI) with LST and precipitation as input parameters. The study also determines the correlation between NDVI, LST, and precipitation of the Mahanadi basin from 2003 to 2021. Monthly precipitation data was extracted from the Center for Hydrometeorology and Remote Sensing (CHRS) portal. The Moderate Resolution Imaging Spectroradiometer (MODIS) products were used to derive the LST and NDVI using Google Earth Engine (GEE). Four different machine learning models were used to predict the NDVI of the Mahanadi basin: linear regression (LR), random forest (RF), support vector regression (SVR), and k-nearest neighbors (KNN). The coefficient of determination (R2), root mean square error (RMSE), mean square error (MSE), mean absolute error (MAE), and explained variance score (EVS) were calculated to evaluate the performance of the models. The results show that the RF model has the highest R2 value in both the training and testing sets among these models, indicating that it is the most optimal among these models for predicting NDVI. The SVR model has the lowest RMSE value in the training set, but the KNN model has the lowest RMSE value in the testing set. The results also show that there is a positive correlation between precipitation and NDVI, a negative correlation between precipitation and LST, and between NDVI and LST. This study provides insights into the relationship between NDVI, LST, and precipitation, and the best machine-learning model for predicting NDVI. The findings of this study can be used to improve the management of river basins and to predict the effects of climate change on vegetation.

Treatment Strategies for Multiple Myeloma Treatment and the Role of High-Throughput Screening for Precision Cancer Therapy.

In the past few years, development of approved drug candidates has improved the disease management of multiple myeloma (MM). However, due to drug resistance, some of the patients do not respond positively, while some of the patients acquire drug resistance, thereby these patients eventually relapse. Hence, there are no other therapeutic options for multiple myeloma patients. Therefore, this necessitates a precision-based approach to multiple myeloma therapy. The use of patient's samples to test drug sensitivity to increase efficacy and reduce treatment-related toxicities is the goal of functional precision medicine. Platforms such as high-throughput-based drug repurposing technology can be used to select effective single drug and drug combinations based on the efficacy and toxicity studies within a time frame of couple of weeks. In this article, we describe the clinical and cytogenetic features of MM. We highlight the various treatment strategies and elaborate on the role of high-throughput screening platforms in a precision-based approach towards clinical treatment.

Recent advances in genetic tools for engineering probiotic lactic acid bacteria.

Synthetic biology has grown exponentially in the last few years, with a variety of biological applications. One of the emerging applications of synthetic biology is to exploit the link between microorganisms, biologics, and human health. To exploit this link, it is critical to select effective synthetic biology tools for use in appropriate microorganisms that would address unmet needs in human health through the development of new game-changing applications and by complementing existing technological capabilities. Lactic acid bacteria (LAB) are considered appropriate chassis organisms that can be genetically engineered for therapeutic and industrial applications. Here, we have reviewed comprehensively various synthetic biology techniques for engineering probiotic LAB strains, such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 mediated genome editing, homologous recombination, and recombineering. In addition, we also discussed heterologous protein expression systems used in engineering probiotic LAB. By combining computational biology with genetic engineering, there is a lot of potential to develop next-generation synthetic LAB with capabilities to address bottlenecks in industrial scale-up and complex biologics production. Recently, we started working on Lactochassis project where we aim to develop next generation synthetic LAB for biomedical application.

Application of Drug Repurposing-Based Precision Medicine Platform for Leukaemia Patient Treatment.

Drug resistance in leukaemia is a major problem that needs to be addressed. Precision medicine provides an avenue to reduce drug resistance through a personalised treatment plan. It has helped to better stratify patients based on their molecular profile and therefore improved the sensitivity of patients to a given therapeutic regimen. However, therapeutic options are still limited for patients who have already been subjected to many lines of chemotherapy. The process of designing and developing new drugs requires significant resources, including money and time. Drug repurposing has been explored as an alternative to identify effective drug(s) that could be used to target leukaemia and lessen the burden of drug resistance. The drug repurposing process usually includes preclinical studies with drug screening and clinical trials before approval. Although most of the repurposed drugs that have been identified are generally safe for leukaemia treatment, they seem not to be good candidates for monotherapy but could have value in combination with other drugs, especially for patients who have exhausted therapeutic options. In this review, we highlight precision medicine in leukaemia and the role of drug repurposing. Specifically, we discuss the several screening methods via chemoinformatic, in vitro, and ex vivo that have facilitated and accelerated the drug repurposing process.

Aptamer-Based Tumor-Targeted Diagnosis and Drug Delivery.

Early cancer identification is crucial for providing patients with safe and timely therapy. Highly dependable and adaptive technologies will be required to detect the presence of biological markers for cancer at very low levels in the early stages of tumor formation. These techniques have been shown to be beneficial in encouraging patients to develop early intervention plans, which could lead to an increase in the overall survival rate of cancer patients. Targeted drug delivery (TDD) using aptamer is promising due to its favorable properties. Aptamer is suitable for superior TDD system candidates due to its desirable properties including a high binding affinity and specificity, a low immunogenicity, and a chemical composition that can be simply changed.Due to these properties, aptamer-based TDD application has limited drug side effect along with organ damages. The development of aptasensor has been promising in TDD for cancer cell treatment. There are biomarkers and expressed molecules during cancer cell development; however, only few are addressed in aptamer detection study of those molecules. Its great potential of attachment of binding to specific target molecule made aptamer a reliable recognition element. Because of their unique physical, chemical, and biological features, aptamers have a lot of potential in cancer precision medicine.In this review, we summarized aptamer technology and its application in cancer. This includes advantages properties of aptamer technology over other molecules were thoroughly discussed. In addition, we have also elaborated the application of aptamer as a direct therapeutic function and as a targeted drug delivery molecule (aptasensor) in cancer cells with several examples in preclinical and clinical trials.

Ex vivo drug sensitivity screening in multiple myeloma identifies drug combinations that act synergistically.

The management of multiple myeloma (MM) is challenging: An assortment of available drug combinations adds complexity to treatment selection, and treatment resistance frequently develops. Given the heterogeneous nature of MM, personalized testing tools are required to identify drug sensitivities. To identify drug sensitivities in MM cells, we established a drug testing pipeline to examine ex vivo drug responses. MM cells from 44 patients were screened against 30 clinically relevant single agents and 44 double- and triple-drug combinations. We observed variability in responses across samples. The presence of gain(1q21) was associated with low sensitivity to venetoclax, and decreased ex vivo responses to dexamethasone reflected the drug resistance observed in patients. Less heterogeneity and higher efficacy was detected with many combinations compared to the corresponding single agents. We identified new synergistic effects of melflufen plus panobinostat using low concentrations (0.1-10 nm and 8 nm, respectively). In agreement with clinical studies, clinically approved combinations, such as triple combination of selinexor plus bortezomib plus dexamethasone, acted synergistically, and synergies required low drug concentrations (0.1 nm bortezomib, 10 nm selinexor and 4 nm dexamethasone). In summary, our drug screening provided results within a clinically actionable 5-day time frame and identified synergistic drug efficacies in patient-derived MM cells that may aid future therapy choices.

Micro and nanofluidics for high throughput drug screening.

In this book chapter, we elaborate on the state-of-the-art technology developments in high throughput screening, microfluidics and nanofluidics. This book chapter further elaborated on the application of microfluidics and nanofluidics for high throughput drug screening with respect to communicable diseases and non-communicable diseases such as cancer. As a future perspective, there is tremendous potential for microfluidics and nanofluidics to be applied in high throughput drug screening which could be applied for various biotechnology applications such as in cancer precision medicine, point-of-care diagnostics and imaging. With the integration of Fourth industrial revolution (4IR) technologies with micro and nanofluidics technologies, it envisioned that such integration along with digital health would enable next generation technology development in medical field.

Advances in CRISPR-Cas9 for the Baculovirus Vector System: A Systematic Review.

The baculovirus expression vector systems (BEVS) have been widely used for the recombinant production of proteins in insect cells and with high insert capacity. However, baculovirus does not replicate in mammalian cells; thus, the BacMam system, a heterogenous expression system that can infect certain mammalian cells, was developed. Since then, the BacMam system has enabled transgene expression via mammalian-specific promoters in human cells, and later, the MultiBacMam system enabled multi-protein expression in mammalian cells. In this review, we will cover the continual development of the BEVS in combination with CRPISPR-Cas technologies to drive genome-editing in mammalian cells. Additionally, we highlight the use of CRISPR-Cas in glycoengineering to potentially produce a new class of glycoprotein medicines in insect cells. Moreover, we anticipate CRISPR-Cas9 to play a crucial role in the development of protein expression systems, gene therapy, and advancing genome engineering applications in the future.

Frequency of Subclinical Hypothyroidism in Women With Polycystic Ovary Syndrome.

INTRODUCTION: Polycystic ovary syndrome (PCOS) is a heterogeneous disorder characterized by hyperandrogenism and chronic anovulation. It may also influence thyroid hormones. Increasing evidence suggests that PCOS is linked with an increased prevalence of thyroid diseases such as nodular goiter, autoimmune thyroiditis, and subclinical hypothyroidism (SCH). Due to very limited global and regional data related to the prevalence of SCH in women with PCOS, we will determine the association between the two. METHODS: This case-control study was conducted in the endocrinology ward of a tertiary care hospital in Pakistan from March 2020 to April 2021. We enrolled 200 females between the ages of 18 and 30 years, with documented evidence of PCOS in the study. Further 200 females without PCOS were enrolled as the case group. After demographics were noted, blood was drawn from their cubital vein via phlebotomy and sent to the laboratory to assess for thyroid-stimulating hormone, free thyroxine, and free triiodothyronine. RESULTS: SCH was found to be more prevalent in participant with PCOS compared to participants without PCOS (43.5% vs. 20.5%; p-value: <0.00001). Increased weight (65.12 ± 5.62 kg vs. 60.02 ± 4.41 kg; p-value: <0.0001) and BMI (25.12 ± 2.51 kg/m2 vs. 22.51 ± 2.01 kg/m2; p-value: <0.0001) was significantly more in participants with PCOS compared to participants without PCOS. CONCLUSION: Based on our findings, this study demonstrated the strong association of SCH in women with PCOS as compared to their normal counterparts. Therefore, the clinical implication is to maintain a high index of suspicion for signs and symptoms of SCH, and awareness is needed for such women to enhance the reproductive and clinical pregnancy outcomes.

Risk Factors Associated With Clostridium difficile-Associated Diarrhea.

INTRODUCTION: Recent years have been alarming due to the sudden, dramatic rise in the incidence of Clostridium difficile infection (CDI). Identifying and addressing the risk factors associated with CDI will help in reducing the incidence of infection and associated complications. METHODS: This case-control study was conducted in a tertiary care hospital in Pakistan from June 2020 to March 2021, in which 200 patients diagnosed with Clostridium difficile-associated diarrhea (CDAD) were enrolled in the study. CDAD was diagnosed based on clinical symptoms and stool enzyme immunoassay. Another 200 participants without a diagnosis of CDAD were enrolled from the outpatient department as a control group. Participants were enrolled after seeking informed consent. RESULTS:  In patients older than 65, risk of CDI was higher compared to participants lower than 65 years old (15.5% vs. 8.0%; p value: 0.02). Hospitalization (25.5% vs. 6.0%; p value < 0.0001), the use of proton pump inhibitors in last 30 days (23.0% vs. 10.5%; p value: 0.001) , and use of antibiotics in the last 30 days (36.0% vs. 10.5%; p value < 0.0001) were significantly higher in participants with CDI. CONCLUSION: Hospitalization, the usage of proton pump inhibitors, and antibiotics in the last 30 days were significantly associated with CDI. A higher incidence of CDI was associated with risk factors like increased body mass index, diabetes, chronic kidney disease, and malignancy.

Impact of Coronavirus Diseases on Liver Enzymes.

Introduction Coronavirus disease 2019 (COVID-19) affects various organs including lungs, brain, and eyes. Very limited data is available related to the effect of COVID-19 on liver. This study is conducted to determine the impact of COVID-10 on liver by measuring the frequency of participants with deranged liver enzymes in patients diagnosed with COVID-19. Methods This cross-sectional study was conducted in a COVID-19 unit of a tertiary care hospital in Pakistan from February 2021 to June 2021. A total of 900 patients admitted with COVID-19 were enrolled in the study after seeking informed consent. After enrollment, taking history and vitals, 5 mL blood was drawn via phlebotomy and sent to the laboratory to test for C-reactive protein, lactate dehydrogenase, and liver enzymes. Results Overall 141 (28.2%) participants had a minimum of one deranged liver enzyme. The most commonly deranged liver enzyme found was alanine transaminase (ALT), both in males (19.9%) and females (21.3%), followed by aspartate transaminase (male: 18.3% and female: 20.3%). Serum total bilirubin was deranged in both males (8.4%) and females (8.3%). There was no significant difference in the gender-wise prevalence of deranged liver enzymes.  Conclusion Liver enzymes are frequently deranged in patients admitted with COVID-19. Liver enzymes should be regularly monitored during the course of management of COVID-19, as various medications used in the treatment of COVID-19 may further deteriorate liver enzymes and may cause long-term damage.

Patents, ethics, biosafety and regulation using CRISPR technology.

In this review chapter, we provide full comprehensive analysis on the patent, ethics and biosafety regulation with respect to the application of CRISPR technology in mammalian systems. We focused on recent development in CRISPR technology and its patent landscape between countries such as US, European Union, China and Australia. Further, we emphasized on the current scenarios on the ethics regulations with respect to CRISPR research, its applicability in patent and technology transfer. Finally, we elaborated on the biosafety regulation on CRISPR/Cas9 technology application in both mammalian and non-mammalian host system.

SynBac: Enhanced Baculovirus Genomes by Iterative Recombineering.

Baculovirus expression vector systems (BEVS) are widely used to produce heterologous proteins for a wide range of applications. Developed more than 30 years ago, BEVS have been constantly modified to improve product quality and ease-of-use. Plasmid reagents were tailored and engineered to facilitate introduction of heterologous genes into baculoviral genomes. At the same time, detrimental modalities such as genes encoding proteases or apoptotic factors were removed to improve protein yield. Advances in DNA synthesis and manipulation now enable the engineering of part or whole synthetic baculovirus genomes, opening up new avenues to redesign and tailor the system to specific applications. Here, we describe a simple protocol for designing and constructing baculovirus genomes comprising segments of synthetic DNA through the use of iterative Red/ET homologous recombination reactions.

Development of insect cell line using CRISPR technology.

In this chapter, we delineated the methods of CRISPR technology that has been used for the development of engineered insect cell line. We elaborated on how CRISPR/Cas9 genome editing in Drosophila melanogaster, Bombyx mori, Spodoptera frugiperda (Sf9 and Sf21), and Mosquitoes enabled the use of model or non-model insect system in various biological and medical applications. Also, the application of synthetic baculovirus genome along with CRISPR/Cas9 vector system to enable genome editing of insect cell systems for treatment of communicable and non-communicable diseases.