A sensitive WST-8-based bioassay for PEGylated granulocyte colony stimulating factor using the NFS-60 cell line.

CONTEXT: Granulocyte colony stimulating factor (G-CSF) has been commonly used to treat neutropenia caused by chemotherapy, radiotherapy, and organ transplants. Improved in vitro efficacy of G-CSF has already been observed by conjugating it to polyethylene glycol (PEG). OBJECTIVE: The in vivo bioassay using tetrazolium dye with the NFS-60 cell line has been recommended for G-CSF but no such monographs are available for PEGylated G-CSF in pharmacopeias. In the present study, the assay recommended for G-CSF was evaluated for its suitability to PEGylated G-CSF. MATERIALS AND METHODS: The generally used MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]-based assay was compared with a bioassay employing a water-soluble tetrazolium dye, WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium], using NFS-60 cells at a concentration of 7 × 10(5) cells/ml against 800 IU/ml of PEGylated G-CSF at 24, 48, 72, and 72 h time points to determine the efficacy of PEGylated G-CSF. Further, the optimized WST-8 dye-based assay was used to test the potency of various commercially available PEGylated G-CSF preparations. RESULTS: The results demonstrated enhanced sensitivity of the WST-8-based assay over the conventional MTS-based assay for determining the potency of PEGylated G-CSF using the NFS-60 cell line. CONCLUSION: Our study demonstrates the potential application of WST-8-based bioassays for other biotherapeutic proteins of human and veterinary interest.

The Challenges and Successes of Dealing with the COVID-19 Pandemic in India.

As the infectivity of the SARS-CoV-2 virus is higher compared with other coronaviruses reported so far, so effective therapeutics and vaccines are the best way to control the proliferation of this infection The COVID-19 mortality rate is lower compared with other similar viral diseases such as severe acute respiratory Ssndrome (SARS) and Middle East respiratory syndrome (MERS). However, due to the evolution of SARS-CoV-2 mutants that are responsible for the subsequent waves, mortality due to COVID-19 has increased across the globe. Currently, the magnitude of SARS-CoV-2 infection is highly severe and is leading to a tremendously increased number of deaths globally. Scientists expect that SARS-CoV-2 has the potential to become a seasonal disease like influenza and may persist with humanity in the future. Currently, preventive strategies such as sanitation, social distancing, use of masks, potential chemotherapies (pathogen-centric and host-centric), and vaccines are the only option to fight against COVID-19. Many groups of Indian government-public private consortia had set up different strategies (development of multiple vaccines) for combat of this unique threat through stepssuch as an increase in vaccinations and sample testing per day. In this focused review, we have discussed the challenges faced and success stories employed to manage COVID-19.

Fast and efficient detection of tuberculosis antigens using liposome encapsulated secretory proteins of Mycobacterium tuberculosis.

PURPOSE: A rapid and efficient diagnostic test was developed for the detection of Mycobacterium tuberculosis antigens in serum samples of active tuberculosis (TB) and extrapulmonary TB patients via a liposomal agglutination-based method. METHODS: A rapid card test has been developed to facilitate the recognition of high-affinity binding rabbit raised purified culture filtrate protein antibodies coupled on the surface of activated liposomal preparation. In the presence of TB antigens, the polyclonal antibodies bound to the liposomal particles demonstrate a visible agglutination reaction. RESULTS: The developed assay was simple, rapid, reliable, sensitive, and specific as a diagnostic test for the detection of antigens in serum samples of clinically confirmed cases of TB within 4-5 minutes' duration. The test was evaluated at different hospitals, medical colleges, and pathology centers, and involved 1483 participants. This investigation was conducted to detect the presence of these antigens during the period of active growth of the microorganism in serum samples for pulmonary TB and processed tissue biopsy for other extrapulmonary TB. Results obtained using this test were compared with acid-fast bacilli smear and culture results. CONCLUSION: Our study demonstrated that the newly developed liposome tuberculosis antigen card test detected antigens in our study population with approximately 97.48% sensitivity and 95.79% specificity. This is the first study to report the liposomal encapsulation of culture filtrate proteins from M. tuberculosis for diagnostic application.

Artificial Intelligence vs. Statistical Modeling and Optimization of Continuous Bead Milling Process for Bacterial Cell Lysis.

For a commercially viable recombinant intracellular protein production process, efficient cell lysis and protein release is a major bottleneck. The recovery of recombinant protein, cholesterol oxidase (COD) was studied in a continuous bead milling process. A full factorial response surface methodology (RSM) design was employed and compared to artificial neural networks coupled with genetic algorithm (ANN-GA). Significant process variables, cell slurry feed rate (A), bead load (B), cell load (C), and run time (D), were investigated and optimized for maximizing COD recovery. RSM predicted an optimum of feed rate of 310.73 mL/h, bead loading of 79.9% (v/v), cell loading OD600nm of 74, and run time of 29.9 min with a recovery of ~3.2 g/L. ANN-GA predicted a maximum COD recovery of ~3.5 g/L at an optimum feed rate (mL/h): 258.08, bead loading (%, v/v): 80%, cell loading (OD600nm): 73.99, and run time of 32 min. An overall 3.7-fold increase in productivity is obtained when compared to a batch process. Optimization and comparison of statistical vs. artificial intelligence techniques in continuous bead milling process has been attempted for the very first time in our study. We were able to successfully represent the complex non-linear multivariable dependence of enzyme recovery on bead milling parameters. The quadratic second order response functions are not flexible enough to represent such complex non-linear dependence. ANN being a summation function of multiple layers are capable to represent complex non-linear dependence of variables in this case; enzyme recovery as a function of bead milling parameters. Since GA can even optimize discontinuous functions present study cites a perfect example of using machine learning (ANN) in combination with evolutionary optimization (GA) for representing undefined biological functions which is the case for common industrial processes involving biological moieties.

Efficient Purification of rhG-CSF and its PEGylated Forms and Evaluation for In Vitro Activities.

Granulocyte-colony stimulating factor (G-CSF) has commonly been used to help the patients to recover from neutropenia inflicted due to radiotherapy, organ transplants and chemotherapy. As the number of people undergoing these therapies and procedures are increasing world-wide, the need for more economical ways of G-CSF production and improvement in its efficacy has become increasingly crucial. In the present study, recombinant human G-CSF (rhG-CSF) was expressed in E. coli and its purification process was optimized by demonstrating better efficiency and higher recoveries (upto 54%) in a multi-step chromatographic purification process, which is greater than the existing reports. Additionally, the efficacy of rhG-CSF was increased by derivatizing with polyethylene glycol (PEG; upto 85% PEGylation), which increases the plasma clearance time, reduces the immunogenicity and requires less frequent administration to the patient. Overall, the present study suggests a cost-effective purification process of rhG-CSF and also proposes its efficient conjugation with PEG for enhanced efficacy as compared to the existing commercially available forms.

Maternal Music Exposure during Pregnancy Influences Neonatal Behaviour: An Open-Label Randomized Controlled Trial.

Objective. This study evaluated the effect of antenatal music exposure to primigravida healthy mothers on the behaviour of their term appropriate-for-date newborns assessed using Brazelton Neonatal Behavioral Assessment Scale (BNBAS). Methods. This was a single-centre, randomized, open-label controlled trial. Primigravida mothers aged 19-29 years, free of chronic medical diseases or significant deafness, with singleton pregnancy, with a gestation of 20 weeks or less, were randomized to listen to a pre-recorded music cassette for approximately 1 hour/day in addition to standard antenatal care (intervention arm) or standard care only (control arm). Perinatal factors with adverse effect on neonatal behaviour were deemed as protocol violations. Outcome measure included scores on 7 clusters of BNBAS. Primary analysis was per protocol. The trial is registered with ClinicalTrials.gov (NCT01278329). Results. One hundred and twenty-six newborns in the music group and 134 in the control group were subjected to BNBAS assessment. The infants of mothers exposed to music during pregnancy performed significantly better on 5 of the 7 BNBAS clusters. The maximal beneficial effect was seen with respect to orientation (ES 1.13, 95% CI 0.82-1.44, P < 0.0001) and habituation (ES 1.05, 95% CI 0.53-1.57, P = 0.0001). Conclusion. Prenatal music exposure to mother significantly and favourably influences neonatal behaviour.

Glycolipids of Mycobacterium tuberculosis strain H37Rv are potential serological markers for diagnosis of active tuberculosis.

A simple and cost-effective diagnostic tool (TB Screen Test) for the screening of patients with pulmonary and extrapulmonary tuberculosis and for differentiation of those individuals from individuals without tuberculosis, other common infections, and healthy controls has been developed. The serological responses of purified mycobacterial glycolipid antigens were examined by a liposome agglutination assay. The assay was able to detect very low antiglycolipid antibody concentrations in the infected individuals. The sera from the tuberculosis patient group had significantly higher concentrations of antiglycolipid antibody than the sera from uninfected control subjects, with 94% sensitivity and 98.3% specificity. Glycolipids of Mycobacterium tuberculosis H37Rv antigens were isolated, purified, and characterized. After interchelation with liposome particles, these purified antigens specifically bound to the antiglycolipid antibodies present in the sera of patients with tuberculosis, resulting in the formation of a blue agglutination. This protocol clearly differentiates healthy controls and M. bovis BCG-vaccinated subjects from those with active tuberculosis. The resultant diagnostic tool, the TB Screen Test, is more economical and rapid (4 min) than other currently available products and can be used for the mass screening of a heavily afflicted population.

Diagnosis of tuberculosis: available technologies, limitations, and possibilities.

Rapid diagnosis and treatment are important for preventing transmission of Mycobacterium tuberculosis. However, the diagnosis of tuberculosis continues to pose serious problems, mainly because of difficulties in differentiating between patients with active tuberculosis and those with healed lesions, normal mycobacterium boris BCG (Bacillus Calmette Guerin) vaccinated individuals, and unvaccinated Manteux positives. Physicians still rely on conventional methods such as Ziehl-Neelsen (ZN) staining, fluorochrome staining, sputum culture, gastric lavage, and other non-traditional methods. Although the tuberculin test has aided in the diagnosis of tuberculosis for more than 85 years, its interpretation is difficult because sensitization with nontuberculous mycobacteria leads to false-positive tests. There have been numerous unsuccessful attempts to develop clinically useful serodiagnostic kits for tuberculosis. A number of proteinaceous and nonprotein antigens (such as acyltrehaloses and phenolglycolipids) have been explored from time to time for the development of such assays but they have not proved to be clinically useful. It has been difficult to develop an ELISA utilizing a suitable antigen because M. tuberculosis shares a large number of antigenic proteins with other microorganisms that may or may not be pathogenic. With the advent of molecular biology techniques, there have been significant advances in nucleic acid-based amplification and hybridization, which are helping to rectify existing flaws in the diagnosis of tuberculosis. The detection of mycobacterial DNA in clinical samples by polymerase chain reaction (PCR) is a promising approach for the rapid diagnosis of tuberculous infection. However, the PCR results must be corrected for the presence of inhibitors as well as for DNA contamination. In the modern era of genetics, marked by proteomics and genomics, the day is not far off when DNA chip-based hybridization assays will instantly reveal mycobacterial infections.