Biochemical Validation Research Articles

Abstract 1516 Computational Prediction and Biochemical Validation of CVD-associated SNPs Altering NKX2-5 DNA-binding

Abstract 1516 Computational Prediction and Biochemical Validation of CVD-associated SNPs Altering NKX2-5 DNA-binding

The Impact of Military Tobacco Control Policy: A Systematic Review.

Tobacco use is synonymous with the military. Despite that military personnel are trained to follow commands, opportunities exist to implement various tobacco control strategies. We conducted a systematic review to evaluate the impact of tobacco control policy employed in military settings. We searched for published English articles in Medline, Web of Science, Scopus, and Google Scholar databases using relevant subject headings without year restriction. We included randomized controlled trials, nonrandomized controlled trials, case-control, cohort, controlled before and after, and uncontrolled before and after studies evaluating the impact of tobacco control policy in the military population. Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, three independent reviewers independently screened initially identified articles, reviewed the full text, and extracted the data and any disagreements resolved by consensus after data recheck. Five reviewers used a validated tool to assess the quality of the included studies. The primary outcome was the reduction of any tobacco or nicotine-contained products (TNCPs) use among the troops. The impacts of the tobacco control policy were synthesized and analyzed qualitatively. This study is registered with the International Prospective Register of Systematic Review (CRD42022314117). Fourteen studies were included in the analysis from 5372 studies screened. Most of the studies were from the USA, and fractions were from Thailand, France, and Taiwan. These studies were methodologically heterogeneous. Most studies employed a total ban policy on TNCP use during basic military training or operational deployment as the primary strategy. Other methods utilized were the brief tobacco intervention, targeted treatment, support, and counseling provided through telephone or mailing systems, the adjunctive behavioral intervention, providing free nicotine gum, the "Pharsai clinic", active and regular smoking restriction, and interventions aimed at intrapersonal, interpersonal, and organizational levels. There is a moderate quality of evidence that the tobacco control policies effectively reduced the prevalence of TNCP use, increased the cessation rate, reduced the intake, and lowered the dependency. The adjunctive interventions provided after the total ban on TNCP use may increase its effectiveness. However, findings from this review need to be carefully considered as the definition of TNCP use status was not universal between studies and lacked a biochemical validation procedure. There is reasonable evidence to support that the tobacco control policy employed in the military population has multiple positive impacts in reducing the prevalence of TNCP use, increasing the cessation rates, reducing the intake, and lowering dependency. Other evidence-based strategies need to be fully utilized to materialize the tobacco endgame.

Evaluation of Genetic or Cellular Impairments in Type I IFN Immunity in a Cohort of Young Adults with Critical COVID-19

Several genetic and immunological risk factors for severe COVID-19 have been identified, with monogenic conditions relating to 13 genes of type I interferon (IFN) immunity proposed to explain 4.8% of critical cases. However, previous cohorts have been clinically heterogeneous and were not subjected to thorough genetic and immunological analyses. We therefore aimed to systematically investigate the prevalence of rare genetic variants causing inborn errors of immunity (IEI) and functionally interrogate the type I IFN pathway in young adults that suffered from critical COVID-19 yet lacked comorbidities. We selected and clinically characterized a cohort of 38 previously healthy individuals under 50 years of age who were treated in intensive care units due to critical COVID-19. Blood samples were collected after convalescence. Two patients had IFN-α autoantibodies. Genome sequencing revealed very rare variants in the type I IFN pathway in 31.6% of the patients, which was similar to controls. Analyses of cryopreserved leukocytes did not indicate any defect in plasmacytoid dendritic cell sensing of TLR7 and TLR9 agonists in patients carrying variants in these pathways. However, lymphocyte STAT phosphorylation and protein upregulation upon IFN-α stimulation revealed three possible cases of impaired type I IFN signaling in carriers of rare variants. Together, our results suggest a strategy of functional screening followed by genome analyses and biochemical validation to uncover undiagnosed causes of critical COVID-19.

Structural basis for competitive binding of productive and degradative co-transcriptional effectors to the nuclear cap-binding complex

The nuclear cap-binding complex (CBC) coordinates co-transcriptional maturation, transport, or degradation of nascent RNA polymerase II (Pol II) transcripts. CBC with its partner ARS2 forms mutually exclusive complexes with diverse "effectors" that promote either productive or destructive outcomes. Combining AlphaFold predictions with structural and biochemical validation, we show how effectors NCBP3, NELF-E, ARS2, PHAX, and ZC3H18 form competing binary complexes with CBC and how PHAX, NCBP3, ZC3H18, and other effectors compete for binding to ARS2. In ternary CBC-ARS2 complexes with PHAX, NCBP3, or ZC3H18, ARS2 is responsible for the initial effector recruitment but inhibits their direct binding to the CBC. We show that invivo ZC3H18 binding to both CBC and ARS2 is required for nuclear RNA degradation. We propose that recruitment of PHAX to CBC-ARS2 can lead, with appropriate cues, to competitive displacement of ARS2 and ZC3H18 from the CBC, thus promoting a productive rather than a degradative RNA fate.

Therapeutic potential of earthworm-derived proteins: Targeting NEDD4 for cardiovascular disease intervention

Targeting neuronal precursor cell-expressed developmentally down-regulated 4 (NEDD4) offers a promising strategy for cardiovascular therapies. NEDD4, a ubiquitin ligase enzyme, is crucial in protein degradation and cellular signaling. Earthworms (Lumbricus genus) are noteworthy for their rich biochemical composition and pharmacological properties. This study investigated the interactions between Lumbricus-derived proteins and NEDD4 to identify potential cardiovascular therapeutic candidates. Using advanced computational techniques, including 3D structure modeling, protein-protein docking simulations, 100 ns molecular dynamics (MD) simulations, and Molecular mechanics/Poisson–Boltzmann surface area (MM/PBSA) calculations, we assessed the binding affinities and functional impacts of these proteins on NEDD4 activity. The findings indicated that Lumbricus-derived proteins such as Lumbrokinase, heat shock protein, and elongation factor 1 (EF-1)-alpha showed activities similar to standard antagonists in modulating NEDD4. These results aligned with previous studies showing the inhibitory effects of heat shock protein and EF-1 on NEDD4 ubiquitination and ligase activity. Additionally, MM/PBSA calculations revealed favorable binding free energies for these compounds, underscoring their therapeutic potential in cardiovascular diseases. In conclusion, this study highlighted the potential of Lumbricus-derived compounds in cardiovascular disease therapy via the NEDD4 pathway, warranting further biochemical and preclinical validation and exploring broader therapeutic applications.

Expression and Purification of Mammalian NMDA Receptor Protein for Functional Characterization.

N-methyl-D-aspartate (NMDA) receptors are critical for brain function and serve as drug targets for the treatment of neurological and psychiatric disorders. They typically form thetetrameric assembly of GluN1-GluN2 (2A to 2D) subtypes, with their diverse three-dimensional conformations linked with the physiologically relevant function in vivo. Purified proteins of tetrameric assembled NMDA receptors have broad applications in the structural elucidation, hybridoma technology for antibody production, and high-throughput drug screening. However, obtaining sufficient quantity and monodisperse NMDA receptor protein is still technically challenging. Here, we summarize a paradigm for the expression and purification of diverse NMDA receptor subtypes, with detailed descriptions on screening constructs by fluorescence size-exclusion chromatography (FSEC), generation of recombinant baculovirus, expression in theeukaryotic expression system, protein purification by affinity chromatography and size-exclusion chromatography (SEC), biochemical and functional validation assays.

Comments on: "Comparative evaluation of the efficacy of nicotine chewing gum and nicotine patches as nicotine replacement therapy using salivary cotinine levels as a biochemical validation measure".

Comments on: "Comparative evaluation of the efficacy of nicotine chewing gum and nicotine patches as nicotine replacement therapy using salivary cotinine levels as a biochemical validation measure".

Leveraging Flavonoids as Potential Inhibitors of METTL3 in Combating Cancer: A Combined Structure‐Based Drug Design and DFT Approach

AbstractMethyltransferase‐like 3 (METTL3) serves as a primary catalytic enzyme within the N6‐ methyladenosine (m6 A) methyltransferase system, playing a crucial role in various biological processes. However, its involvement in the pathogenesis of several cancers, have been well documented. The pursuit of small molecule inhibitors to counteract the cancer‐promoting effects of METTL3 has gained considerable momentum; however, no approved drugs are available. In this study, we employed computational techniques, including structure‐based virtual screening, consensus molecular docking, Density Function Theory calculations and MMPBSA energy profiling to identify potential METTL3 inhibitors from flavonoids. ST024026, ST99039 and ST4135876 were identified as promising candidates for METTL3 inhibition, as indicated by their favorable docking scores and robust energy profiles. Notably, their binding interactions were characterized by strong hydrogen bonds and hydrophobic contacts, ensuring high stability within the METTL3 binding site. Furthermore, these compounds exhibited favorable physicochemical properties and ADME characteristics, coupled with minimal toxicity. Notably, their binding induced conformational changes in the protein, underscoring their potential as METLL3 inhibitors. These findings highlight the selected flavonoids as potential novel entry inhibitors subject to modifications and optimizations. To advance this line of research, additional biochemical testing and experimental validation are warranted to facilitate the development of highly selective and effective METTL3 inhibitors.

Pyramided transgenic jute (Corchorus capsularis) with biotic stress resistance and herbicide tolerance

Jute (Corchorus sp.) is a commercially important bast (stem) fibre–producing crop. The natural, long, lustrous, and strong jute fibres are globally popular for their domestic and industrial utility. Unfortunately, jute fibre production is under continuous threat from three major agricultural challenges: insect infestation (predominantly lepidopteran insects), fungal infection, and weed management issues. Creating jute plants that could themselves overcome these three challenges would be spectacular. Hence, multi–trait transgenic lines were developed by incorporating three genes—cry1Ab/Ac (encodes Bt δ–endotoxin for Lepidopteran insect resistance), chi11 (encodes chitinase for fungus resistance), and bar (encodes phosphinothricin acetyltransferase (PAT) for glufosinate herbicide tolerance)—into the white jute (C. capsularis) cultivar JRC–321 through Agrobacterium–mediated co–transformation. A bioassay–based event selection strategy was applied for the selection of the transformed jute lines. Each plant was tested through consecutive bioassays involving herbicide, insects, and fungus, followed by molecular and biochemical validation. The functions of the three genes, incorporated in the greenhouse–grown transgenic progeny plants, were confirmed for every generation (till the T2 generation). The plants were able to detoxify the glufosinate herbicide Basta®, resist the two devastating lepidopteran pests tested: hairy caterpillars (Spilarctia obliqua) and semiloopers (Anomis sabulifera), and recover from the stem rot–causing fungal pathogen Macrophomina phaseolina, without compromising agronomic characteristics or fibre quality. The white jute lines thus developed may be effectively used to increase the production of quality jute fibres, meet local and global demands, and minimise input cultivation costs.

EPCO-28. MULTIPLATFORM MOLECULAR AND FUNCTIONAL GENOMIC ANALYSES OF NF1 MUTANT GLIOBLASTOMA IDENTIFY DISTINCT CO-MUTATION PATTERNS AND DRUGGABLE DEPENDENCIES UNDERLYING SELUMETINIB RESPONSE

Abstract The tumor suppressor NF1 is recurrently mutated in sporadic IDH-wildtype glioblastoma and activates Ras/Raf/MEK signaling, motivating the use of targeted MEK inhibition. Here, we combine multiplatform molecular analysis of NF1 mutant glioblastoma human resection specimens with functional genomic CRISPRi screens to the MEK inhibitor selumetinib. METHODS: We identified 123 consecutive patients treated at the University of California San Francisco between 2016-2022 with NF1 mutant, IDH-wildtype glioblastoma. DNA methylation data was processed using the minfi package, and hierarchical clustering, principal component analysis (PCA), and survival analysis was performed in R. Human NF1 mutant GBM43 and mouse NRAS mutant SB28 GBM cells expressing CRISPRi machinery were used for genome wide selumetinib-resistance screens. Immunoblotting was performed to validate CRISPRi screen hits and selumetinib responses in cell lines. RESULTS: DNA sequencing of human glioblastomas identified 12 recurrent alterations in greater than 10% of cases including cell cycle regulators (CDKN2A/B, TP53, RB1), PI3K signaling (PTEN, PIK3R1, PIK3CA), epigenetic regulators (ATRX, SETD2), and Ras signaling (EGFR, PDGFRA, PTPN11). Methylation data (n=55 tumors) identified three groups marked by differences in MGMT methylation (p=0.04, chi-square test) and CDKN2A/B loss (p=0.01, chi-square test). CRISPRi screens in GBM43 and SB28 cells identified driver phenotypes in cell cycle regulators (TP53, CDKN2A, CDK4), PI3K signaling (PTEN, MTOR), and Ras signaling (RASA2, PTPN11) consistent with co-occurring mutations in the human NF1 mutant glioblastoma cohort. In contrast, mediators of selumetinib response converged on regulators of Ras/Raf/MEK signaling including the Ras effector SHOC2. Biochemical analysis validated SHOC2 repression is sufficient for selumetinib sensitization. CONCLUSIONS: Using a combination of human tumor genomic analysis, functional genetic screens, and biochemical validation, we identify three epigenetic groups of NF1 mutant glioblastoma with distinct co-mutation patterns and validate compensatory Ras/Raf/MEK activation as a conserved mechanism of selumetinib resistance, suggesting combination molecular therapy as a rational therapeutic combination.

Identification of potent inhibitors for Leishmania donovani homoserine kinase: an integrated in silico and kinetic study

Leishmaniasis is caused by ∼20 species of Leishmania that affects millions in endemic areas. Available therapies are not sufficient to effectively control the disease, cause severe side effects and eventually lead to drug resistance, making the discovery of novel therapeutic molecules an immediate need. Molecular target-based drug discovery, where the target is a defined molecular gene, protein or a mechanism, is a rationale driven approach for novel therapeutics. Humans obtain the essential amino acid such as threonine from dietary sources, while Leishmania synthesize it de-novo. Enzymes of the threonine biosynthesis pathway, including the rate limiting Homoserine kinase (HSK) which converts L-homoserine into ortho-phospho homoserine are thus attractive targets for rationale driven therapy. The absence of HSK in humans and its presence in Leishmania donovani enhances the opportunity to exploit HSK as a molecular target for anti-leishmanials therapeutic development. In this study, we utilize structure-based high throughput drug discovery (SBDD), followed by biochemical validation and identified two potential inhibitors (RH00038 and S02587) from Maybridge chemical library that targets L. donovani HSK. These two inhibitors effectively induced the mortality of Leishmania donovani in both amastigote and promastigote stages, with one of them being specific to parasite and twice as effective as the standard therapeutic molecule.

Biochemical test as an efficient system to improve safety in parenteral nutrition for premature infants

ObjectiveThe biochemical test validation process of parenteral nutrition bags is described to verify the correct composition of critical compounds before its administration, as well as its impact on safety after two years since its implantation in a population of premature infants. MethodFor the validation of the technique, 35 samples of parenteral nutrition without lipids were processed by the emergency laboratory, following the routine procedures used to measure the concentrations of glucose and electrolytes in plasma and urine. To analyze its impact on safety, pre-implantation and post-implantation risk analysis was carried out using failure mode, effects and criticality analysis (FMECA). Likewise, all out-of-range results and their potential repercussions on patient safety were evaluated. ResultsRegression analysis showed no systematic measurement error for glucose, calcium and potassium; however, there is a constant systematic error for sodium and magnesium, thus both were discarded for routine analysis. Failure mode, effects and criticality analysis results showed a decrease in the risk of the process of 11% for the biochemical test. We tested 1,734 parenteral nutritions from 218 premature neonates; 58 (3.3%) results were out of the acceptance range, and 7 were considered to be potentially dangerous compounding errors. ConclusionsThe biochemical test of glucose and electrolytes is an efficient and reproducible method that prevents possible compounding errors from reaching the patient.

An unconventional gatekeeper mutation sensitizes inositol hexakisphosphate kinases to an allosteric inhibitor.

Inositol hexakisphosphate kinases (IP6Ks) are emerging as relevant pharmacological targets because a multitude of disease-related phenotypes has been associated with their function. While the development of potent IP6K inhibitors is gaining momentum, a pharmacological tool to distinguish the mammalian isozymes is still lacking. Here, we implemented an analog-sensitive approach for IP6Ks and performed a high-throughput screen to identify suitable lead compounds. The most promising hit, FMP-201300, exhibited high potency and selectivity toward the unique valine gatekeeper mutants of IP6K1 and IP6K2, compared to the respective wild-type (WT) kinases. Biochemical validation experiments revealed an allosteric mechanism of action that was corroborated by hydrogen deuterium exchange mass spectrometry measurements. The latter analysis suggested that displacement of the αC helix, caused by the gatekeeper mutation, facilitates the binding of FMP-201300 to an allosteric pocket adjacent to the ATP-binding site. FMP-201300 therefore serves as a valuable springboard for the further development of compounds that can selectively target the three mammalian IP6Ks; either as analog-sensitive kinase inhibitors or as an allosteric lead compound for the WT kinases.

An unconventional gatekeeper mutation sensitizes inositol hexakisphosphate kinases to an allosteric inhibitor

Inositol hexakisphosphate kinases (IP6Ks) are emerging as relevant pharmacological targets because a multitude of disease-related phenotypes has been associated with their function. While the development of potent IP6K inhibitors is gaining momentum, a pharmacological tool to distinguish the mammalian isozymes is still lacking. Here, we implemented an analog-sensitive approach for IP6Ks and performed a high-throughput screen to identify suitable lead compounds. The most promising hit, FMP-201300, exhibited high potency and selectivity toward the unique valine gatekeeper mutants of IP6K1 and IP6K2, compared to the respective wild-type (WT) kinases. Biochemical validation experiments revealed an allosteric mechanism of action that was corroborated by hydrogen deuterium exchange mass spectrometry measurements. The latter analysis suggested that displacement of the αC helix, caused by the gatekeeper mutation, facilitates the binding of FMP-201300 to an allosteric pocket adjacent to the ATP-binding site. FMP-201300 therefore serves as a valuable springboard for the further development of compounds that can selectively target the three mammalian IP6Ks; either as analog-sensitive kinase inhibitors or as an allosteric lead compound for the WT kinases.

Evaluating the impact of varying expired carbon monoxide thresholds on smoking relapse identification: insights from the E3 trial on e-cigarette efficacy for smoking cessation

ObjectivesExpired carbon monoxide (ECO) is often used in smoking cessation trials to biochemically validate self-reported smoking status. The optimal ECO threshold to distinguish individuals who smoke from those who do...

Clickable Glutathione-Based Identification of Cysteine Glutathionylation.

Clickable glutathione is a glutathione-derived chemical probe designed to identify and analyze protein S-glutathionylation, a major cysteine oxidation in redox signaling. An engineered glutathione synthetase mutant (GS M4) is used to synthesize clickable glutathione in cells or in vitro, which affords utility via click chemistry to detect, identify, and quantify glutathionylation on individual or global proteins in biochemical and mass spectrometric analyses. The clickable glutathione approach is valuable for the unequivocal identification of glutathionylated cysteines, among many reversible cysteine oxoforms, via the direct enrichment and detection of glutathionylated proteins or peptides. Clickable glutathione, in combination with GS M4, has demonstrated utility in the mass-spectrometry-based discovery and profiling of new proteins and cysteines for glutathionylation in cell lines in response to physiologic and oxidative stress. The approach is versatile and applicable to validating the glutathionylation of proteins and cysteines in other biochemical analysis beside mass spectrometry. Here, we describe the applications of clickable glutathione and provide detailed protocols for the identification, profiling, and detection of glutathionylated proteins and cysteines. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Identification of glutathionylated cysteine in individual proteins in vitro Basic Protocol 2: Proteomic identification and quantification of glutathionylation Basic Protocol 3: Biochemical validation of glutathionylation in cells.

Bicc1 ribonucleoprotein complexes specifying organ laterality are licensed by ANKS6-induced structural remodeling of associated ANKS3.

Organ laterality of vertebrates is specified by accelerated asymmetric decay of Dand5 mRNA mediated by Bicaudal-C1 (Bicc1) on the left side, but whether binding of this or any other mRNA to Bicc1 can be regulated is unknown. Here, we found that a CRISPR-engineered truncation in ankyrin and sterile alpha motif (SAM)-containing 3 (ANKS3) leads to symmetric mRNA decay mediated by the Bicc1-interacting Dand5 3' UTR. AlphaFold structure predictions of protein complexes and their biochemical validation by in vitro reconstitution reveal a novel interaction of the C-terminal coiled coil domain of ANKS3 with Bicc1 that inhibits binding of target mRNAs, depending on the conformation of ANKS3 and its regulation by ANKS6. The dual regulation of RNA binding by mutually opposing structured protein domains in this multivalent protein network emerges as a novel mechanism linking associated laterality defects and possibly other ciliopathies to perturbed dynamics in Bicc1 ribonucleoparticle (RNP) formation.

BabyBreathe trial: protocol for a randomised controlled trial of a complex intervention to prevent postpartum return to smoking

IntroductionMany people quit smoking during pregnancy, but postpartum smoking relapse is common. Maintaining smoking abstinence achieved during pregnancy is key to improving maternal and child health. There are no evidence-based...

Systematic analysis of the Frazzled receptor interactome establishes previously unreported regulators of axon guidance.

The Netrin receptor Dcc and its Drosophila homolog Frazzled play crucial roles in diverse developmental process, including axon guidance. In Drosophila, Fra regulates midline axon guidance through a Netrin-dependent and a Netrin-independent pathway. However, what molecules regulate these distinct signaling pathways remain unclear. To identify Fra-interacting proteins, we performed affinity purification mass spectrometry to establish a neuronal-specific Fra interactome. In addition to known interactors of Fra and Dcc, including Netrin and Robo1, our screen identified 85 candidate proteins, the majority of which are conserved in humans. Many of these proteins are expressed in the ventral nerve cord, and gene ontology, pathway analysis and biochemical validation identified several previously unreported pathways, including the receptor tyrosine phosphatase Lar, subunits of the COP9 signalosome and Rho-5, a regulator of the metalloprotease Tace. Finally, genetic analysis demonstrates that these genes regulate axon guidance and may define as yet unknown signaling mechanisms for Fra and its vertebrate homolog Dcc. Thus, the Fra interactome represents a resource to guide future functional studies.

Kinase Suppressor of RAS 1 (KSR1) Maintains the Transformed Phenotype of BRAFV600E Mutant Human Melanoma Cells.

Kinase Suppressor of RAS 1 (KSR1) is a scaffolding protein for the RAS-RAF-MEK-ERK pathway, which is one of the most frequently altered pathways in human cancers. Previous results have shown that KSR1 has a critical role in mutant RAS-mediated transformation. Here, we examined the role of KSR1 in mutant BRAF transformation. We used CRISPR/Cas9 to knock out KSR1 in a BRAFV600E-transformed melanoma cell line. KSR1 loss produced a complex phenotype characterised by impaired proliferation, cell cycle defects, decreased transformation, decreased invasive migration, increased cellular senescence, and increased apoptosis. To decipher this phenotype, we used a combination of proteomic ERK substrate profiling, global protein expression profiling, and biochemical validation assays. The results suggest that KSR1 directs ERK to phosphorylate substrates that have a critical role in ensuring cell survival. The results further indicate that KSR1 loss induces the activation of p38 Mitogen-Activated Protein Kinase (MAPK) and subsequent cell cycle aberrations and senescence. In summary, KSR1 function plays a key role in oncogenic BRAF transformation.