The Humoral Immune Response to Various Domains of Protective Antigen of Bacillus anthracis in Cutaneous Anthrax Cases in India

Anthrax, caused by Bacillus anthracis is known to occur globally since antiquity. Besides being an important biothreat agent, it is an important public health importance pathogen also in countries like India. B. anthracis secretes three distinct toxins, namely protective antigen (PA), lethal factor (LF) and edema factor (EF). PA is the central moiety of the anthrax toxin complex and therefore has been a molecule of choice for vaccine development. PA has four different domains with different functions. In this study, the major domains of PA were cloned and expressed in bacterial system. The purified recombinant proteins were used to determine the humoral immune response by ELISA using 43 human cutaneous anthrax serum samples. The maximum immunoreactivity was observed with the whole PA protein followed by domain 2, 4 and 1. The study corroborated that in addition to full PA, individual domain 2 and 4 can also be good target for vaccine development as well as for serodiagnostic assays for cutaneous anthra

Enhanced Production of Protective Antigen, a Potent Diagnostic Protein of Bacillus anthracis, the Causative Agent of Anthrax

Protective antigen (PA) produced by Bacillus anthracis is a highly immunogenic protein. Therefore, it has significant importance in serodiagnosis as well as a vaccine candidate for anthrax. In the present study, codons for PA gene were optimised and synthesised for its expression in Escherichia coli. Various expression conditions were optimised for scaled up production of rPA. The final yield of affinity chromatography purified protein was 40.8 mg/l during batch fermentation. For further purification, affinity purified protein was diafiltered and subjected to anion exchange chromatography. SDS-PAGE and Western blot was used to characterise the purified rPA protein. The diagnostic potential of purified rPA was evaluated in Western blot using standards reference serum AVR 801 and cutaneous anthrax clinical sera. The results of the present study established the optimum production of rPA in E. coli after codon optimisation for its subsequent use in diagnosis of anthrax infection

Production and Purification of Protective Antigen of Bacillus anthracis and Development of a Sandwich ELISA for its Detection

Anthrax, a zoonotic disease caused by Bacillus anthracis is important for biowarfare as well as public health point of view. The virulence factors of B. anthracis are encoded by the two plasmids, pXO1 and pXO2. Protective antigen (PA), an 83 kDa protein encoded by pXO1 along with lethal factor (LF, 90 kDa) or edema factor (EF, 89 kDa), makes the anthrax toxin responsible for causing the disease. Current detection and diagnostic systems for anthrax are mostly based on PA, a potential biomarker of B. anthracis. The objective of the present study was to produce and purify the PA for development of a sandwich ELISA for its detection. In this study, pYS5 plasmid containing the full PA gene was transformed into an 8 proteases deficient Bacillus anthracis host BH480. The PA was produced under shake flask conditions and purified using the gel filtration chromatography. The reactivity of PA with rabbit and mouse anti-PA antibodies was confirmed by Western blotting. The antibodies were purified and used for the development of a sandwich ELISA for detection of PA. The detection sensitivity of ELISA was found to be 3.9 ng/ mL PA

A Rapid Flow through Membrane Enzyme Linked Immunosorbent Assay for Bacillus anthracis using Surface Array Protein as a Biomarker

Anthrax, caused by Bacillus anthracis is an important disease of biowarfare and public health importance. It is imperative to develop a simple system which can detect and differentiate B. anthracis from other closely related species. The surface array protein (Sap), which is secreted during the early growth phase of bacteria can be an important biomarker for detection of B. anthracis. In the present study, we have developed a rapid flow through membrane ELISA for detection of B. anthracis. Polyclonal antibodies were used to develop a sandwich plate ELISA, which could detect 3.9 ng/ml of recombinant Sap. B. anthracis bacteria grown in culture broth could be detected after 5 h of growth. Finally, a rapid flow through membrane ELISA was developed which can be accomplished just within 2 minutes, instead of 3-4 h as required in sandwich plate ELISA. The results established that the developed flow through membrane ELISA may be used for detection of B. anthracis. The proposed method is rapid, safe and user friendly for detection of B. anthracis culture

Enhanced Production of Recombinant Extractable Antigen (EA1) an Extracellular Protein and its use in Detection of Spores of Bacillus anthracis the Causative Agent of Anthrax

Detection of spores of Bacillus anthracis, the causative agent of anthrax in human and animals in environment is cumbersome due to the presence of spores of other closely related Bacillus species. The Extractable Antigen 1 (EA1), an extracellular protein is considered as a biomarker for detection of B. anthracis spores. In the present work, we have cloned and expressed the recombinant EA1 protein in soluble form in Escherichia coli. Optimisation of culture conditions and cultivation media was carried out to achieve enhanced soluble expression of recombinant EA1 protein. Further, the batch fermentation process was also developed using optimised conditions for scale up production of recombinant EA1 protein. The final yield of protein purified employing affinity chromatography was 42.64 mg/l of culture during batch fermentation process. The polyclonal antibodies were raised against recombinant EA in rabbit and mice and used to develop an ELISA for detection of B. anthracis spores. The specificity of the developed assay was ascertained with spores of other Bacillus species. The results corroborated that the EA1 could be a suitable biomarker for detection of B. anthracis spores

Codon Usage Is Influenced by Compositional Constraints in Genes Associated With Dementia

Dementia is a clinical syndrome characterized by progressive cognitive decline, and the symptoms could be gradual, persistent, and progressive. In the present study, we investigated 47 genes that have been linked to dementia. Compositional, selectional, and mutational forces were seen to be involved. The influence of these two compositional constraints on codon usage bias (CUB) was positive for nucleotide A and negative for GC. Nucleotide A also experienced the highest mutational force, and GC-ending codons were preferred over AT-ending codons. A high bias towards GC-ending codons enhanced the gene expression level, evidenced by the positive association between CAI and GC-ending codons. The unusual behavior of TTG codon showing an inverse relationship with GC-ending codon and negative influence of gene expression, a behavior contrary to all other GC-ending codons, shows operative selectional force. Furthermore, parity analysis, higher translational selection value, preference of GC-ending codons over AT-ending codons, and the association of gene length with gene expression refer to the dominant role of selection pressure with compositional constraint and mutational force shaping codon usage

Advancements in the Application of Nanomedicine in Alzheimer’s Disease: A Therapeutic Perspective

Alzheimer’s disease (AD) is a progressive neurodegenerative disease that affects most people worldwide. AD is a complex central nervous system disorder. Several drugs have been designed to cure AD, but with low success rates. Because the blood–brain and blood–cerebrospinal fluid barriers are two barriers that protect the central nervous system, their presence has severely restricted the efficacy of many treatments that have been studied for AD diagnosis and/or therapy. The use of nanoparticles for the diagnosis and treatment of AD is the focus of an established and rapidly developing field of nanomedicine. Recent developments in nanomedicine have made it possible to effectively transport drugs to the brain. However, numerous obstacles remain to the successful use of nanomedicines in clinical settings for AD treatment. Furthermore, given the rapid advancement in nanomedicine therapeutics, better outcomes for patients with AD can be anticipated. This article provides an overview of recent developments in nanomedicine using different types of nanoparticles for the management and treatment of AD

Phytochemicals based therapeutic approaches for Breast cancer targeting: Molecular docking study

Abstract Background: Cancer is a prominent cause of disease burden all over the world. Primary cause of the development of cancer is exposure to potentially hazardous environmental factors and unhealthy lifestyle. Among all types of cancer, breast cancer is most common in women and becomes a public health issue on a global scale. There has been considerable interest in the development of drugs which can target cancer-causing hotspot factors such as mTOR, estrogen receptor alpha, and progesterone receptor to cure breast cancer. The key focus of the present work is to search plant-based phytochemical molecules which can efficiently interact with the targeted proteins responsible for the development and progression of breast cancer using molecular docking studies. Methods: We have screened 1064 phytochemical molecules from plant sources against 5 potential hotspot targets: EGFR kinase domain, Human estrogen receptor alpha ligand-binding domain, FRB fragment of mTOR, Progesterone receptor, and NUDT5 of breast cancer. Results: Our results show that among all molecules, 25 phytochemicals exhibit promising therapeutic potential for breast cancer treatment. Our observation is further strengthened by the available literatures showing the promising therapeutic efficacy exhibited by these lead molecules in in vitro and in vivo cancer models. Conclusion: The virtual screening of phytochemicals hold promise to search potential anticancer drug. Among the lead molecules, Delphinidin 3,5-diglucoside, Baicalein, and Morphine have already been known for their anticancer properties, but the efficacy of Vitexin, IsoSkimmiwallin, Nodifloretin, Jaceosidin and Nepetin have not been explored so far and therefore further in-vitro and in-vivo study is warrante

The Ubiquitin System: An Emerging Therapeutic Target for Lung Cancer

The ubiquitin system, present in all eukaryotes, contributes to regulating multiple types of cellular protein processes such as cell signaling, cell cycle, and receptor trafficking, and it affects the immune response. In most types of cancer, unusual events in ubiquitin-mediated signaling pathway modulation can lead to a variety of clinical outcomes, including tumor formation and metastasis. Similarly, ubiquitination acts as a core component, which contributes to the alteration of cell signaling activity, dictating biosignal turnover and protein fates. As lung cancer acquires the most commonly mutated proteins, changes in the ubiquitination of the proteins contribute to the development of lung cancer. Various inhibitors targeting the ubiquitin system have been developed for clinical applications in lung cancer treatment. In this review, we summarize the current research advances in therapeutics for lung cancer by targeting the ubiquitin system

Modulating the Ubiquitin–Proteasome System: A Therapeutic Strategy for Autoimmune Diseases

Multiple sclerosis (MS) is an autoimmune, neurodegenerative disease associated with the central nervous system (CNS). Autoimmunity is caused by an abnormal immune response to self-antigens, which results in chronic inflammation and tissue death. Ubiquitination is a post-translational modification in which ubiquitin molecules are attached to proteins by ubiquitinating enzymes, and then the modified proteins are degraded by the proteasome system. In addition to regulating proteasomal degradation of proteins, ubiquitination also regulates other cellular functions that are independent of proteasomal degradation. It plays a vital role in intracellular protein turnover and immune signaling and responses. The ubiquitin–proteasome system (UPS) is primarily responsible for the nonlysosomal proteolysis of intracellular proteins. The 26S proteasome is a multicatalytic adenosine-triphosphate-dependent protease that recognizes ubiquitin covalently attached to particular proteins and targets them for degradation. Damaged, oxidized, or misfolded proteins, as well as regulatory proteins that govern many essential cellular functions, are removed by this degradation pathway. When this system is affected, cellular homeostasis is altered, resulting in the induction of a range of diseases. This review discusses the biochemistry and molecular biology of the UPS, including its role in the development of MS and proteinopathies. Potential therapies and targets involving the UPS are also addressed