Recombinant monoclonal antibody production in yeasts: Challenges and considerations.

Monoclonal antibodies (mAbs) are laboratory-based engineered protein molecules with a monovalent affinity or multivalent avidity towards a specific target or antigen, which can mimic natural antibodies that are produced in the human immune systems to fight against detrimental pathogens. The recombinant mAb is one of the most effective classes of biopharmaceuticals produced in vitro by cloning and expressing synthetic antibody genes in a suitable host. Yeast is one of the potential hosts among others for the successful production of recombinant mAbs. However, there are very few yeast-derived mAbs that got the approval of the regulatory agencies for direct use for treatment purposes. Certain challenges encountered by yeasts for recombinant antibody productions need to be overcome and a few considerations related to antibody structure, host engineering, and culturing strategies should be followed for the improved production of mAbs in yeasts. In this review, the drawbacks related to the metabolic burden of the host, culturing conditions including induction mechanism and secretion efficiency, solubility and stability, downstream processing, and the pharmacokinetic behavior of the antibody are discussed, which will help in developing the yeast hosts for the efficient production of recombinant mAbs.

Structural and molecular dynamics simulation studies of CBL-interacting protein kinase CIPK and its complexes related to plant salinity stress.

CONTEXT: Calcium-dependent signaling in plants is responsible for several major cellular events, including the activation of the salinity-responsive pathways. Calcium binds to calcineurin B-like protein (CBL), and the resulting CBL-Ca2+ complex binds to CBL-interacting protein kinase (CIPK). The CBL-CIPK complex enhances the CIPK interaction with an upstream kinase. The upstream kinase phosphorylates CIPK that, in turn, phosphorylates membrane transporters. Phosphorylation influences transporter activity to kick-start many downstream functions, such as balancing the cytosolic Na+-to-K+ ratio. The CBL-CIPK interaction is pivotal for Ca2+-dependent salinity stress signaling. METHODS: Computational methods are used to model the entire Arabidopsis thaliana CIPK24 protein structure in its autoinhibited and open-activated states. Arabidopsis thaliana CIPK24-CBL4 complex is predicted based on the protein-protein docking methods. The available structural and functional data support the CIPK24 and the CIPK24-CBL4 complex models. Models are energy-minimized and subjected to molecular dynamics (MD) simulations. MD simulations for 500 ns and 300 ns enabled us to predict the importance of conserved residues of the proteins. Finally, the work is extended to predict the CIPK24-CBL4 complex with the upstream kinase GRIK2. MD simulation for 300 ns on the ternary complex structure enabled us to identify the critical CIPK24-GRIK2 interactions. Together, these data could be used to engineer the CBL-CIPK interaction network for developing salt tolerance in crops.

Insulin evolution: A holistic view of recombinant production advancements.

The increased prevalence of diabetes and the growing popularity of non-invasive methods of recombinant human insulin uptake, such as oral insulin, have increased insulin demand, further limiting the affordability of insulin. Over 40 years have passed since the development of engineered microorganisms that replaced the animal pancreas as the primary source of insulin. To stay ahead of the need for insulin in the present and the future, a few drawbacks with the existing expression systems need to be alleviated, including the inclusion body formation, the use of toxic inducers, and high process costs. To address these bottlenecks and improve insulin production, a variety of techniques are being used in bacteria, yeasts, transgenic plants and animals, mammalian cell lines, and cell-free expression systems. Different approaches for the production of insulin, including two-chain, proinsulin or mini-proinsulin, preproinsulin coupled with fusion protein, chaperone, signal peptide, and purification tags, are explored in upstream, whereas downstream processing takes into account the recovery of intact protein in its bioactive form and purity. This article focuses on the strategies used in the upstream and downstream phases of the bioprocess to produce recombinant human insulin. This review also covers a range of analytical methods and tools employed in investigating the genuity of recombinant human insulin.

Providing optimal care in the neonatal care units in India: How Covid-19 exacerbated existing barriers.

Nearly one quarter (600,000) of all neonatal deaths worldwide per year occur in India. To reduce neonatal mortality, the Indian Ministry of Health and Family Welfare established neonatal care units, including neonatal intensive care units and specialized neonatal care units to provide immediate care at birth, resuscitation for asphyxiation, postnatal care, follow up for high-risk newborns, immunization, and referral for additional or complex healthcare services. Despite these efforts, neonatal mortality remains high, and measures taken to reduce mortality have been severely challenged by multiple problems caused by the Covid-19 pandemic. In this qualitative study, we conducted seven focus group discussions with newborn care unit nurses and pediatric residents and 35 key informant interviews with pediatricians, residents, nurses, annual equipment maintenance contractors, equipment manufacturers, and Ministry personnel in the Vidarbha region of Maharashtra between December 2019 and November 2020. The goal of the study was to understand barriers and facilitators to providing optimal care to neonates, including the challenges imposed by the Covid-19 pandemic. Covid-19 exacerbated existing barriers to providing optimal care to neonates in these newborn care units. As a result of Covid-19, we found the units were even more short-staffed than usual, with trained pediatric nurses and essential equipment diverted from newborn care to attend to patients with Covid-19. Regular training of neonatal nursing staff was also disrupted due to Covid-19, leaving many staff without the skills to provide optimate care to neonates. Infection control was also exacerbated by Covid-19. This study highlights the barriers to providing optimal care for neonates were made even more challenging during Covid-19 because of the diversion of critically important neonatal equipment and staff trained to use that equipment to Covid-19 wards. The barriers at the individual, facility, and systems levels will remain challenging as the Covid-19 pandemic continues.

Neurodevelopment of Children Whose Mothers Were Randomized to Low-Dose Aspirin During Pregnancy.

OBJECTIVE: Because low-dose aspirin is now commonly prescribed in pregnancy, we sought to assess the association between early antenatal exposure and child neurodevelopment. METHODS: We performed a noninferiority, masked, neurodevelopmental follow-up study of children between age 33 and 39 months whose mothers had been randomized to daily low-dose aspirin (81 mg) or placebo between 6 0/7 and 13 6/7 weeks of gestation through 37 weeks. Neurodevelopment was assessed with the Bayley-III (Bayley Scales of Infant and Toddler Development, 3rd Edition) and the ASQ-3 (Ages and Stages Questionnaire, 3rd Edition). The primary outcome was the Bayley-III cognitive composite score with a difference within 4 points demonstrating noninferiority. RESULTS: A total of 640 children (329 in the low-dose aspirin group, 311 in the placebo group) were evaluated between September 2021 and June 2022. The Bayley-III cognitive composite score was noninferior between the two groups (-1, adjusted mean -0.8, 95% CI, -2.2 to 0.60). Significant differences were not seen in the language composite score (difference 0.7, 95% CI, -0.8 to 2.1) or the motor composite score (difference -0.6, 95% CI, -2.5 to 1.2). The proportion of children who had any component of the Bayley-III score lower than 70 did not differ between the two groups. Similarly, the communication, gross motor, fine motor, problem-solving, and personal-social components of the ASQ-3 did not differ between groups. Maternal characteristics, delivery outcomes, breastfeeding rates, breastfeeding duration, and home environment as measured by the Family Care Indicators were similar. CONCLUSION: Antenatal low-dose aspirin exposure was not associated with altered neurodevelopmental outcomes at age 3 years. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov , NCT04888377.