Light-gated Integrator for Highlighting Kinase Activity in Living Cells.

Protein kinases are key signaling nodes that regulate fundamental biological and disease processes. Illuminating kinase signaling from multiple angles can provide deeper insights into disease mechanisms and improve therapeutic targeting. While fluorescent biosensors are powerful tools for visualizing live-cell kinase activity dynamics in real time, new molecular tools are needed that enable recording of transient signaling activities for post hoc analysis and targeted manipulation. Here, we develop a light-gated kinase activity coupled transcriptional integrator (KINACT) that converts dynamic kinase signals into "permanent" fluorescent marks. KINACT enables robust monitoring of kinase activity across scales, accurately recording subcellular PKA activity, highlighting PKA signaling heterogeneity in 3D cultures, and identifying PKA activators and inhibitors in high-throughput screens. We further leverage the ability of KINACT to drive signaling effector expression to allow feedback manipulation of the balance of GαsR201C-induced PKA and ERK activation and dissect the mechanisms of oncogenic G protein signaling.

Safety, immunogenicity and efficacy of Relcovax®, a dual receptor binding domain (RBD) and nucleocapsid (N) subunit protein vaccine candidate against SARS-CoV-2 virus.

SARS-CoV-2, severe acute respiratory syndrome coronavirus-2, causes coronavirus disease- 2019 (COVID-19). Mostly, COVID-19 causes respiratory symptoms that can resemble those of a cold, the flu, or pneumonia. COVID-19 may harm more than just lungs and respiratory systems. It may also have an impact on other parts of the body and debilitating effects on humans, necessitating the development of vaccines at an unprecedented rate in order to protect humans from infections. In response to SARS-CoV-2 infection, mRNA, viral vector-based carrier and inactivated virus-based vaccines, as well as subunit vaccines, have recently been developed. We developed Relcovax®, a dual antigen (Receptor binding domain (RBD) and Nucleocapsid (N) proteins) subunit protein vaccine candidate. Preliminary mouse preclinical studies revealed that Relcovax® stimulates cell-mediated immunity and provides broader protection against two SARS-CoV-2 variants, including the delta strain. Before conducting human studies, detailed preclinical safety assessments are required, so Relcovax® was tested for safety, and immunogenicity in 28-day repeated dose toxicity studies in rats and rabbits. In the toxicity studies, there were no mortality or morbidity, abnormal clinical signs, abnormalities in a battery of neurobehavioral observations, abnormalities in detailed clinical and ophthalmological examinations, or changes in body weights or feed consumption. In any of the studies, no abnormal changes in organ weights, haematology, clinical chemistry, urinalysis parameters, or pathological findings were observed. Immunogenicity tests on rats and rabbits revealed 100 % seroconversion. Relcovax® was therefore found to be safe in animals, with a No Observed Adverse Effect Level (NOAEL) of 20 µg/protein in rats and rabbits. In efficacy studies, Relcovax® immunised hamsters demonstrated dose-dependent protection against SARS-CoV-2 infection, with a high dose (20 µg/protein) being the most protective, while in cynomolgus macaque monkey study, lowest dose 5 µg/protein had the highest efficacy. In conclusion, Relcovax® was found to be safe, immunogenic, and efficacious in in vivo studies.

RelCoVax®, a two antigen subunit protein vaccine candidate against SARS-CoV-2 induces strong immune responses in mice.

The COVID-19 pandemic has spurred an unprecedented movement to develop safe and effective vaccines against the SARS-CoV-2 virus to immunize the global population. The first set of vaccine candidates that received emergency use authorization targeted the spike (S) glycoprotein of the SARS-CoV-2 virus that enables virus entry into cells via the receptor binding domain (RBD). Recently, multiple variants of SARS-CoV-2 have emerged with mutations in S protein and the ability to evade neutralizing antibodies in vaccinated individuals. We have developed a dual RBD and nucleocapsid (N) subunit protein vaccine candidate named RelCoVax® through heterologous expression in mammalian cells (RBD) and E. coli (N). The RelCoVax® formulation containing a combination of aluminum hydroxide (alum) and a synthetic CpG oligonucleotide as adjuvants elicited high antibody titers against RBD and N proteins in mice after a prime and boost dose regimen administered 2 weeks apart. The vaccine also stimulated cellular immune responses with a potential Th1 bias as evidenced by increased IFN-γ release by splenocytes from immunized mice upon antigen exposure particularly N protein. Finally, the serum of mice immunized with RelCoVax® demonstrated the ability to neutralize two different SARS-CoV-2 viral strains in vitro including the Delta strain that has become dominant in many regions of the world and can evade vaccine induced neutralizing antibodies. These results warrant further evaluation of RelCoVax® through advanced studies and contribute towards enhancing our understanding of multicomponent subunit vaccine candidates against SARS-CoV-2.

The importance of a halotyrosine dehalogenase for Drosophila fertility.

The ability of iodotyrosine deiodinase to salvage iodide from iodotyrosine has long been recognized as critical for iodide homeostasis and proper thyroid function in vertebrates. The significance of its additional ability to dehalogenate bromo- and chlorotyrosine is less apparent, and none of these functions could have been anticipated in invertebrates until recently. Drosophila, as most arthropods, contains a deiodinase homolog encoded by CG6279, now named condet (cdt), with a similar catalytic specificity. However, its physiological role cannot be equivalent because Drosophila lacks a thyroid and its associated hormones, and no requirement for iodide or halotyrosines has been reported for this species. We have now applied CRISPR/Cas9 technology to generate Drosophila strains in which the cdt gene has been either deleted or mutated to identify its biological function. As previously shown in larvae, expression of cdt is primarily limited to the fat body, and we now report that loss of cdt function does not enhance sensitivity of the larvae to the toxic effects of iodotyrosine. In adult flies by contrast, expression is known to occur in testes and is detected at very high levels in this tissue. The importance of cdt is most evident in the decrease in fertility observed when either males or females carry a deletion or mutation of cdt Therefore, dehalogenation of a halotyrosine appears essential for efficient reproduction in Drosophila and likely contributes to a new pathway for controlling viability in arthropods.

Functional analysis of iodotyrosine deiodinase from drosophila melanogaster.

The flavoprotein iodotyrosine deiodinase (IYD) was first discovered in mammals through its ability to salvage iodide from mono- and diiodotyrosine, the by-products of thyroid hormone synthesis. Genomic information indicates that invertebrates contain homologous enzymes although their iodide requirements are unknown. The catalytic domain of IYD from Drosophila melanogaster has now been cloned, expressed and characterized to determine the scope of its potential catalytic function as a model for organisms that are not associated with thyroid hormone production. Little discrimination between iodo-, bromo-, and chlorotyrosine was detected. Their affinity for IYD ranges from 0.46 to 0.62 µM (Kd ) and their efficiency of dehalogenation ranges from 2.4 - 9 x 103 M-1 s-1 (kcat /Km ). These values fall within the variations described for IYDs from other organisms for which a physiological function has been confirmed. The relative contribution of three active site residues that coordinate to the amino acid substrates was subsequently determined by mutagenesis of IYD from Drosophila to refine future annotations of genomic and meta-genomic data for dehalogenation of halotyrosines. Substitution of the active site glutamate to glutamine was most detrimental to catalysis. Alternative substitution of an active site lysine to glutamine affected substrate affinity to the greatest extent but only moderately affected catalytic turnover. Substitution of phenylalanine for an active site tyrosine was least perturbing for binding and catalysis.

Iodotyrosine deiodinase: a unique flavoprotein present in organisms of diverse phyla.

Iodide is required for thyroid hormone synthesis in mammals and other vertebrates. The role of both iodide and iodinated tyrosine derivatives is currently unknown in lower organisms, yet the presence of a key enzyme in iodide conservation, iodotyrosine deiodinase (IYD), is suggested by genomic data from a wide range of multicellular organisms as well as some bacteria. A representative set of these genes has now been expressed, and the resulting enzymes all catalyze reductive deiodination of diiodotyrosine with kcat/Km values within a single order of magnitude. This implies a physiological presence of iodotyrosines (or related halotyrosines) and a physiological role for their turnover. At least for Metazoa, IYD should provide a new marker for tracing the evolutionary development of iodinated amino acids as regulatory signals through the tree of life.

Determination of 1,25-dihydroxyvitamin D2 in rat serum using liquid chromatography with tandem mass spectrometry.

Vitamin D therapy is widely used for the treatment of hyperparathyroidism associated with chronic renal failure in renal disease patients. The vitamin D prodrug, 1α-hydroxyvitamin D(2) (1α(OH)D(2)), is used for the treatment of the end stage renal disease patients who as a result of impaired kidney function cannot convert the naturally occurring vitamin D to the active hormonal form namely 1,25-dihydroxyvitamin D(2) (1,25(OH)(2)D(2)). The systemic circulating levels of this active form are in the pg/mL range and represent a significant bioanalytical challenge for therapeutic monitoring. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) is considered the gold standard for the selective and sensitive determination of small molecule therapeutics in biological matrices. However, the reported LC-MS/MS bioanalytical assays for 1,25(OH)(2)D(2) suffer from extensive sample preparation procedures or derivatization protocols to achieve the requisite sensitivity and selectivity. In this paper, we describe an assay that employs 96-well plate solid phase extraction sample preparation combined with highly sensitive LC-MS/MS instrumentation. The utility of ultra high pressure liquid chromatography to reduce the analytical run time was also demonstrated. Employing this assay a lower limit of quantitation of 25.0 pg/mL using 300 µL sample aliquot of rat serum was achieved with linearity obtained over the range of 25.0-1000 pg/mL. Both intra-day and inter-day coefficients of variation were <15% and accuracy across the assay range was within 100±7.24%. The application of the assay was demonstrated for the analysis of 1,25(OH)(2)D(2) rat serum samples to support pharmacokinetic studies conducted at doses down to sub-microgram per kilogram of 1α(OH)D(2).