Discovery of Free Glycated Amines and Glycated Urea in Diabetic Plasma: Potential Implications in Diabetes.

The role of protein glycation in the pathogenesis of diabetes has been well established. Akin to proteins, free amino acids and other small-molecule amines are also susceptible to glycation in hyperglycemic conditions and may have a role in the pathogenesis of the disease. However, information about glycation of free amino acids and other small-molecule amines is relatively obscure. In the quest to discover small-molecule glycated amines in the plasma, we have synthesized glycated amino acids, glycated creatine, and glycated urea, and by using a high-resolution accurate mass spectrometer, a mass spectral library was developed comprising the precursor and predominant fragment masses of glycated amines. Using this information, we report the discovery of the glycation of free lysine, arginine, and leucine/isoleucine from the plasma of diabetic patients. This has great physiological significance as glycation of these amino acids may create their deficiency and affect vital physiological processes such as protein synthesis, cell signaling, and insulin secretion. Also, these glycated amino acids could serve as potential markers of diabetes and its complications. While other amines, such as creatinine and urea, accumulate in the plasma and act as biomarkers of diabetic nephropathy. For the first time, we report the detection of glycated urea in diabetic plasma, which is confirmed by matching the precursor and fragment masses with the in vitro synthesized glycated urea by using 12C6 and 13C6-glucose. Further, we quantified glycated urea detected in two forms, monoglycated urea (MGU) and diglycated urea (DGU), by a targeted mass spectrometric approach in the plasma of healthy, diabetic, and diabetic nephropathy subjects. Both MGU and DGU showed a positive correlation with clinical parameters, such as blood glucose and HbA1c. Given that urea gets converted to glycated urea in hyperglycemic conditions, it is crucial to quantify MGU and DGU along with the urea for the diagnosis of diabetic nephropathy and study their physiological role in diabetes.

The multi-dimensional impact of captopril modification on human serum albumin.

Captopril is a thiol drug, widely used for the management of hypertension and cardiovascular diseases. Reactive thiols are found to covalently modify the cysteines of plasma proteins and affect their structure and function. Human serum albumin (HSA) is prone to undergo modification by various low molecular weight compounds, including drugs. Cysteine34 (Cys34) in HSA has a free thiol group with antioxidant properties, considered to be the most redox-sensitive amino acid in plasma. Through mass-spectrometric analysis, we demonstrate for the first time that captopril forms a disulfide adduct at Cys34 residue and increases the protease susceptibility of HSA to trypsin. As evidenced by our biophysical and electron microscopy studies, HSA undergoes structural alteration, aggregation and morphological changes when treated with different captopril concentrations. Molecular dynamics studies further revealed the regions of secondary structural changes in HSA due to disulfide adduct formation by captopril at Cys34. It also elucidated the residues involved in the noncovalent interactions with captopril. It is envisaged that structural change in HSA may influence the efficacy of drug delivery as well as its own biological function. These findings may thus provide significant insights into the field of pharmacology intriguing further investigation into the effects of long-term captopril treatment.

Surface modification of a commercial bone plate (Ti6Al4V) implant for improved antibacterial and cytocompatibility via thermal dewetting of a silver thin film.

The high demand for bone grafts has motivated the development of implants with excellent osteogenic activity, whereas the risk of implant-associated infection, particularly given the rise of antimicrobial resistance, has compelled the development of implants with innovative antimicrobial strategies in which a small amount of bactericidal agent can effectively kill a wide range of bacteria. To induce antibacterial property, the surface of Grade-5 bone plate titanium implants used in clinical applications was modified using direct current (DC) sputter coating followed by thermal annealing. The 15 nm silver film-coated implants were thermally annealed in the furnace for 15 min at 750 °C. The modified implant surface's antibacterial efficacy againstEscherichia coli(E. coli),Staphylococcus aureus(S. aureus),Salmonella typhi, andMethicillin-resistant staphylococcus aureusbacteria has been assessed using a colony-forming assay. On the modified implant surface, the growth ofE. coliandS. aureusbacteria is reduced by 99.72%, while highly drug-resistant bacteria are inhibited by 96.59%. The MTT assay was used to assess the cytotoxicity of the modified bone-implant surface against NIH3T3 mouse fibroblast cells. The modified bone-implant surface promoted fibroblast growth and demonstrated good cytocompatibility. Furthermore, the mechanical properties of the implant were not harmed by this novel surface modification method. This method is simple and provides new insight into surface modification of commercial metallic implants to have effective antibacterial properties against various classes of bacteria.

SWATH-MS reveals that bisphenol A and its analogs regulate pathways leading to disruption in insulin signaling and fatty acid metabolism.

Bisphenol A (BPA) is an endocrine-disrupting chemical (EDC), associated with obesity and insulin resistance. The FDA prohibited the use of BPA-based polycarbonate resins in infant formula packaging; thus, its analogs, viz. Bisphenol S (BPS) and Bisphenol F (BPF) were considered alternatives in epoxy resins, plastics, and food cans. As these analogs might evoke a similar response, we investigated the role of Bisphenols (BPA, BPF, and BPS), on insulin signaling in CHO-HIRc-myc-GLUT4eGFP cells at environmentally relevant concentrations of 2 nM and 200 nM. Insulin signaling demonstrated that Bisphenols reduced phosphorylation of IR and AKT2, GLUT4 translocation, and glucose uptake. This was accompanied by increased oxidative stress. Furthermore, SWATH-MS-based proteomics of 3T3-L1 cells demonstrated that Bisphenol-treated cells regulate proteins in insulin resistance, adipogenesis, and fatty acid metabolism pathways differently. All three Bisphenols induced differentially expressed proteins enriched similar pathways, although their abundance differed for each Bisphenol. This might be due to their varying toxicity level, structural differences, and estrogen-mimetic activity. This study has important implications in addressing health concerns related to EDCs. Given that the analogs of BPA are considered alternatives to BPA, the findings of this study suggest they are equally potent in altering fatty acid metabolism and inducing insulin resistance.

Enhanced control over size, areal density, and shape of substrate-supported Au and Ag nanoparticles by solid-state dewetting and alloying.

The ability to manipulate the dimensions, areal density, and form of substrate-supported Au and Ag nanoparticles (NPs) is highly desirable for utilizing their plasmonic properties in biosensing, photovoltaics, and nanophotonic applications. The transformation of thin films into the substrate-supported nanostructures by solid-state dewetting (SSD), provides an avenue to manipulate the dimensional aspects of nanostructures simply and cost-effectively on a large scale. However, spontaneous agglomeration of the film produces randomly distributed and non-uniform nanostructures that must be controlled. Here, we have systematically studied the effect of annealing temperature, between 200 °C and 750 °C, on the dewetting morphology evolution of Au, Ag, and Au-Ag bilayer ultrathin films sputter deposited on thec-plane (0001) sapphire substrates. Regardless of the film thickness, Ag films dewet faster than Au films and produce spherical NPs, compared to faceted Au NPs, with broader size distribution. Whereas, by the SSD of Au-Ag bilayer ultrathin films, highly spherical and monodisperse AuAg bimetallic NPs can be fabricated. Furthermore, we have shown the possibility of fabricating the AuAg bimetallic NPs of varying compositions by adjusting the thickness of individual layers, thus enabling us to smoothly tune the spectral location of plasmonic resonance within the visible range.

Prevalence of and Eligibility for Surveillance Without Anticoagulation Among Adults With Lower-Risk Acute Subsegmental Pulmonary Embolism.

Importance: Approximately 8% of acute pulmonary emboli are confined to the subsegmental arteries. The 2016 and 2021 American College of Chest Physicians (CHEST) guidelines and expert panel reports suggest the use of structured surveillance without anticoagulation for select ambulatory patients with subsegmental pulmonary embolism who do not have active cancer, deep vein thrombosis, impaired cardiopulmonary reserve, marked symptoms, or increased risk of recurrent venous thromboembolism; however, guideline uptake in community practice is unknown, as is the proportion of outpatients eligible for surveillance. Objective: To describe the prevalence of surveillance among outpatients with acute subsegmental pulmonary embolism and to estimate the proportion of patients eligible for structured surveillance using modified CHEST criteria. Design, Setting, and Participants: This retrospective cohort study was conducted across 21 US community hospitals in the Kaiser Permanente Northern California integrated health system from January 1, 2017, to December 31, 2021. Adult outpatients with acute subsegmental pulmonary embolism were included. Patients with the following higher-risk characteristics were excluded: codiagnoses requiring hospitalization, non-low-risk vital signs (ie, systolic blood pressure <90 mm Hg, pulse ≥110 bpm, or peripheral cutaneous pulse oximetry ≤92%), prediagnosis anticoagulant use, or hospice care. Data analysis was performed from November 2022 to February 2023. Main Outcomes and Measures: The main outcomes were the (1) prevalence of surveillance and (2) eligibility for surveillance using 2 sets of criteria: the CHEST criteria modified by excluding patients with higher-risk characteristics or right ventricular dysfunction and a stricter set of criteria requiring age younger than 65 years and no more than 1 embolus. The prevalence of structured surveillance was calculated and the proportion of patients eligible for surveillance was estimated. Results: Of the 666 outpatients with acute subsegmental pulmonary embolism included in this study, 229 with lower-risk characteristics were examined. Their median age was 58 (IQR, 42-68) years; more than half were men (120 [52.4%]) and self-identified as non-Hispanic White (128 [55.9%]). Six patients (2.6%) were initially not treated with anticoagulants. Among the lower-risk cohort, only 1 patient (0.4% [95% CI, 0.01%-2.4%]) underwent structured surveillance, without 90-day sequelae. Thirty-five patients (15.3% of the lower-risk group and 5.3% of the full cohort) were surveillance eligible using modified CHEST criteria. Fifteen patients (6.6% of the lower-risk group and 2.3% of the full cohort) were surveillance eligible using stricter criteria. Conclusions and Relevance: In this cohort study of lower-risk outpatients with subsegmental pulmonary embolism, few were eligible for structured surveillance, and only a small proportion of eligible patients underwent surveillance despite the CHEST guideline. If forthcoming trials find surveillance safe and effective, substantial uptake into clinical practice may require more than passive diffusion.

Nanopore Sequencing of RAGE Gene Polymorphisms and Their Association with Type 2 Diabetes.

The receptor for advanced glycation end products (RAGE) is a transmembrane protein that interacts with its ligands, advanced glycation end products (AGEs). AGEs are elevated in diabetes and diabetic complications, leading to increased oxidative stress and activation of pro-inflammatory pathways facilitated by AGE-RAGE signaling. Polymorphisms in the RAGE gene can potentially affect AGE-RAGE interaction and its downstream signaling, which plays a crucial role in the progression of diabetes and its complications. In this study, we used nanopore sequencing for genotyping of RAGE polymorphism and identified a maximum number of 33 polymorphisms, including two previously unreported novel mutations in a cohort of healthy, type 2 diabetics without nephropathy and type 2 diabetics with nephropathy in order to identify associations. Two novel RAGE polymorphisms in the intron 8 and 3'UTR region at genomic locations 32181834 and 32181132, respectively, were detected with a low frequency. For four previously reported polymorphisms, cross-validation by PCR-RFLP showed 99.75% concordance with nanopore sequencing. Analysis of genotype distribution and allele frequencies revealed that five single nucleotide polymorphisms, i.e., rs1800625, rs3131300, rs3134940, rs2070600, and rs9391855, were associated with an increased risk for type 2 diabetes.

Elevated Level of Glycated KQTALVELVK Peptide of Albumin Is Associated with the Risk of Diabetic Nephropathy.

Diabetic nephropathy is a leading cause of end-stage renal disease. Hence, early detection of diabetic nephropathy is essential to mitigate the disease burden. Microalbuminuria, the currently used diagnostic marker of diabetic nephropathy, is not efficient in detecting it at an early stage. Therefore, we explored the utility of glycated human serum albumin (HSA) peptides for risk prediction of diabetic nephropathy. Three glycation-sensitive HSA peptides, namely, FKDLGEENFK, KQTALVELVK, and KVPQVSTPTLVEVSR, with deoxyfructosyllysine (DFL) modification were quantified by targeted mass spectrometry (MS) in a study population comprising healthy and type II diabetes subjects with and without nephropathy. Mass spectrometry, receiver operating characteristic (ROC) curve, and correlation analysis revealed that the DFL-modified KQTALVELVK peptide was better than other glycated HSA peptides and HbA1c for identifying diabetic nephropathy. DFL-modified KQTALVELVK could be a potential marker for risk prediction of diabetic nephropathy.

De novo structure prediction of meteorin and meteorin-like protein for identification of domains, functional receptor binding regions, and their high-risk missense variants.

Meteorin (Metrn) and Meteorin-like (Metrnl) are homologous secreted proteins involved in neural development and metabolic regulation. In this study, we have performed de novo structure prediction and analysis of both Metrn and Metrnl using Alphafold2 (AF2) and RoseTTAfold (RF). Based on the domain and structural homology analysis of the predicted structures, we have identified that these proteins are composed of two functional domains, a CUB domain and an NTR domain, connected by a hinge/loop region. We have identified the receptor binding regions of Metrn and Metrnl using the machine-learning tools ScanNet and Masif. These were further validated by docking Metrnl with its reported KIT receptor, thus establishing the role of each domain in the receptor interaction. Also, we have studied the effect of non-synonymous SNPs on the structure and function of these proteins using an array of bioinformatics tools and selected 16 missense variants in Metrn and 10 in Metrnl that can affect the protein stability. This is the first study to comprehensively characterize the functional domains of Metrn and Metrnl at their structural level and identify the functional domains, and protein binding regions. This study also highlights the interaction mechanism of the KIT receptor and Metrnl. The predicted deleterious SNPs will allow further understanding of the role of these variants in modulating the plasma levels of these proteins in disease conditions such as diabetes.Communicated by Ramaswamy H. Sarma.

Classification of Heart Failure Events by Severity: Insights From the VICTORIA Trial.

BACKGROUND: Hospitalization due to heart failure (HFH) is a major source of morbidity, consumes significant economic resources and is a key endpoint in HF clinical trials. HFH events vary in severity and implications, but they are typically considered equivalent when analyzing clinical trial outcomes. OBJECTIVES: We aimed to evaluate the frequency and severity of HF events, assess treatment effects and describe differences in outcomes by type of HF event in VICTORIA (Vericiguat Global Study in Subjects with Heart Failure with Reduced Ejection Fraction). METHODS: VICTORIA compared vericiguat with placebo in patients with HF with reduced ejection fraction (< 45%) and a recent worsening HF event. All HFHs were prospectively adjudicated by an independent clinical events committee (CEC) whose members were blinded to treatment assignment. We evaluated the frequency and clinical impact of HF events by severity, categorized by highest intensity of HF treatment (urgent outpatient visit or hospitalization treated with oral diuretics, intravenous diuretics, intravenous vasodilators, intravenous inotropes, or mechanical support) and treatment effect by event categories. RESULTS: In VICTORIA, 2948 HF events occurred in 5050 enrolled patients. Overall total CEC HF events for vericiguat vs placebo were 43.9 vs 49.1 events/100 patient-years (P = 0.01). Hospitalization for intravenous diuretics was the most common type of HFH event (54%). HF event types differed markedly in their clinical implications for both in-hospital and post-discharge events. We observed no difference in the distribution of HF events between randomized treatment groups (P = 0.78). CONCLUSION: HF events in large global trials vary significantly in severity and clinical implications, which may have implications for more nuanced trial design and interpretation. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov (NCT02861534).

Effect of vericiguat on left ventricular structure and function in patients with heart failure with reduced ejection fraction: The VICTORIA echocardiographic substudy.

AIM: Vericiguat significantly reduced the primary composite outcome of heart failure (HF) hospitalization or cardiovascular death in the VICTORIA trial. It is unknown if these outcome benefits are related to reverse left ventricular (LV) remodelling with vericiguat in patients with HF with reduced ejection fraction (HFrEF). The aim of this study was to compare the effects of vericiguat versus placebo on LV structure and function after 8 months of therapy in patients with HFrEF. METHODS AND RESULTS: Standardized transthoracic echocardiography (TTE) was performed at baseline and after 8 months of therapy in a subset of HFrEF patients in VICTORIA. The co-primary endpoints were changes in LV end-systolic volume index (LVESVI) and LV ejection fraction (LVEF). Quality assurance and central reading were performed by an echocardiographic core laboratory blinded to treatment assignment. A total of 419 patients (208 vericiguat, 211 placebo) with high-quality paired TTE at baseline and 8 months were included. Baseline clinical characteristics were well balanced between treatment groups and echocardiographic characteristics were representative of patients with HFrEF. LVESVI significantly declined (60.7 ± 26.8 to 56.8 ± 30.4 ml/m2 ; p < 0.01) and LVEF significantly increased (33.0 ± 9.4% to 36.1 ± 10.2%; p < 0.01) in the vericiguat group, but similarly in the placebo group (absolute changes for vericiguat vs. placebo: LVESVI -3.8 ± 15.4 vs. -7.1 ± 20.5 ml/m2 ; p = 0.07 and LVEF +3.2 ± 8.0% vs. +2.4 ± 7.6%; p = 0.31). The absolute rate per 100 patient-years of the primary composite endpoint at 8 months tended to be lower in the vericiguat group (19.8) than the placebo group (29.6) (p = 0.07). CONCLUSIONS: In this pre-specified echocardiographic study, significant improvements in LV structure and function occurred over 8 months in both vericiguat and placebo in a high-risk HFrEF population with recent worsening HF. Further studies are warranted to define the mechanisms of vericiguat's benefit in HFrEF.

Protective Effects of Rifampicin and Its Analog Rifampicin Quinone in a Mouse Model of Obesity-Induced Type 2 Diabetes.

Advanced glycation end-products (AGEs) form when glucose reacts non-enzymatically with proteins, leading to abnormal protein function, oxidative stress, and inflammation. AGEs are associated with aging and age-related diseases; their formation is aggravated during diabetes. Therefore, drugs preventing AGE formation can potentially treat diabetic complications, positively affecting health. Earlier, we demonstrated that rifampicin and its analogs have potent anti-glycating activities and increase the life span of Caenorhabditis elegans. This study aimed to investigate the effects of rifampicin during hyperglycemia in C. elegans and in a mouse model of obesity-induced type 2 diabetes. The effects of rifampicin were assessed by determining the life span of C. elegans cultured in the presence of glucose and by measuring HbA1c, AGE levels, and glucose excursions in the diabetic mouse model. Our results show that rifampicin protects C. elegans from glucose-induced toxicity and increases life span. In mice, rifampicin reduces HbA1c and AGEs, improves insulin sensitivity, and reduces indications of diabetic nephropathy without inducing hepatotoxicity. Rifampicin quinone, an analog with lower anti-microbial activity, also reduces HbA1c levels, improves glucose homeostasis and insulin sensitivity, and lowers indications of diabetic nephropathy, without adversely affecting the liver of the diabetic mice. Altogether, our results indicate that rifampicin and its analog have protective roles during diabetes without inflicting hepatic damage and may potentially be considered for repositioning to treat hyperglycemia-related complications in patients.

Mechanistic insights on anserine hydrolyzing activities of human carnosinases.

Anserine and carnosine represent histidine-containing dipeptides that exert a pluripotent protective effect on human physiology. Anserine is known to protect against oxidative stress in diabetes and cardiovascular diseases. Human carnosinases (CN1 and CN2) are dipeptidases involved in the homeostasis of carnosine. In poikilothermic vertebrates, the anserinase enzyme is responsible for hydrolyzing anserine. However, there is no specific anserine hydrolyzing enzyme present in humans. In this study, we have systematically investigated the anserine hydrolyzing activity of human CN1 and CN2. A targeted multiple reaction monitoring (MRM) based approach was employed for studying the enzyme kinetics of CN1 and CN2 using carnosine and anserine as substrates. Surprisingly, both CN1 and CN2 can hydrolyze anserine effectively. The observed catalytic turnover rate (Vmax/[E]t) was 21.6 s-1 and 2.8 s-1 for CN1 and CN2, respectively. CN1 is almost eight-fold more efficient in hydrolyzing anserine compared to CN2, which is comparable to the efficiency of the carnosine hydrolyzing activity of CN2. The Michaelis constant (Km) value for CN1 (1.96 mM) is almost three-fold lower compared to CN2 (6.33 mM), representing higher substrate affinity for anserine-CN1 interactions. Molecular docking studies showed that anserine binds at the catalytic site of the carnosinases with an affinity similar to carnosine. Overall, the present study elucidated the inherent promiscuity of human carnosinases in hydrolyzing anserine using a sensitive LC-MS/MS approach.

Medication use by US patients with pulmonary hypertension associated with chronic obstructive pulmonary disease: a retrospective study of administrative data.

BACKGROUND: Pulmonary hypertension (PH) is a serious complication of chronic obstructive pulmonary disease (COPD). While clinical guidelines recommend specific drug therapies for pulmonary arterial hypertension (PAH), these drug therapies are not recommended for PH due to lung disease. METHODS: This was a retrospective cohort study using the Optum® Clinformatics® Data Mart from January 2009-September 2019. An algorithm was designed to identify adults with ≥ 2 ICD-9-CM or ICD-10-CM diagnosis codes for PH and with ≥ 2 diagnosis codes for COPD. Sensitivity analyses were conducted among subgroups of patients with evidence of a right heart catheterization (RHC) or pulmonary function test (PFT). Patient characteristics, medications used, and durations of use of PAH and COPD medications were analyzed. RESULTS: A total of 25,975 patients met the study inclusion criteria. Their mean age was 73.5 (SD 10.0) years and 63.8% were female. Medications targeting PAH were prescribed to 643 (2.5%) patients, most frequently a phosphodiesterase-5 inhibitor (2.1%) or an endothelin receptor antagonist (0.75%). Medications for COPD were prescribed to 17,765 (68.4%) patients, most frequently an inhaled corticosteroid (57.4%) or short-acting beta agonist (50.4%). The median durations of use ranged from 4.9 to 12.8 months for PAH medications, and from 0.4 to 5.9 months for COPD medications. Of the subgroup of patients with RHC (N = 2325), 257 (11.1%) were prescribed a PAH medication and 1670 (71.8%) used a COPD medication. Of the subgroup with a PFT (N = 2995), 58 (1.9%) were prescribed a PAH medication and 2100 (70.1%) a COPD medication. CONCLUSIONS: Patients with PH associated with COPD were identified in a US administrative claims database. Very few of these patients received any of the medications recommended for PAH, and only about two thirds received medications for COPD.

"Dentist as Smile Weavers" An Online Survey on Infection Surveillance, Emergency Treatment, Economic Setbacks, and Stress Endured by Periodontists of South India during COVID-19.

Introduction: The SARS-CoV-2 virus introduction and subsequent COVID-19 pandemic have had a profound impact on normal dentistry, particularly periodontal treatment, around the world. This cross-sectional study aimed to evaluate the infection surveillance, emergency treatment, economic setbacks, and stress endured by periodontists of South India during COVID-19. Methods: A cross-sectional study was conducted from March to July 2021 among periodontists in South India. A total of 300 periodontists were chosen through the randomized sampling technique. The data were collected using a self-reported closed-end questionnaire consisting of 38 questions through Google Forms. The information was entered into a database and analyzed using SPSS program version 16.0. Results: A total of 300 people took part in the survey, 277 (92.3%) dentists responded to the survey in its entirety. Periodontists and postgraduates made up 24.18% and 75.9% of those who responded, respectively. For all of the questions asked, there was a statistically significant difference among the responses. The majority of respondents agreed on questions about periodontists' knowledge, practice, mentality, and financial setbacks concerning the COVID-19 epidemic. Conclusion: Most of the periodontists that we surveyed have adequate knowledge about SARS-CoV-2 and have been taking adequate measures in preventing the spread of the disease. This pandemic has a negative impact on the periodontists. This study indicates a high level of depression among a significant number of periodontists. It is important to monitor and address the mental health needs of practitioners during the pandemic. The knowledge, awareness, and attitudes regarding teledentistry were found to be satisfactory among the periodontists.

Stacking herbicide detoxification and resistant genes improves glyphosate tolerance and reduces phytotoxicity in tobacco (Nicotiana tabacum L.) and rice (Oryza sativa L.).

Glyphosate residues retained in the growing meristematic tissues or in grains of glyphosate-resistant crops affect the plants physiological functions and crop yield. Removing glyphosate residues in the plants is desirable with no penalty on crop yield and quality. We report a new combination of scientific strategy to detoxify glyphosate that reduces the residual levels and improve crop resistance. The glyphosate detoxifying enzymes Aldo-keto reductase (AKR1) and mutated glycine oxidase (mGO) with different modes of action were co-expressed with modified EPSPS, which is insensitive to glyphosate in tobacco (Nicotiana tabacum L.) and rice (Oryza sativa L.). The transgenic tobacco plants expressing individual PsAKR1, mGO, CP4-EPSPS, combinations of PsAKR1:CP4EPSPS, PsAKR1:mGO, and multigene with PsAKR1: mGO: CP4EPSPS genes were developed. The bio-efficacy studies of in-vitro leaf regeneration on different concentrations of glyphosate, seedling bioassay, and spray on transgenic tobacco plants demonstrate that glyphosate detoxification with enhanced resistance. Comparative analysis of the transgenic tobacco plants reveals that double and multigene expressing transgenics had reduced accumulation of shikimic acid, glyphosate, and its primary residue AMPA, and increased levels of sarcosine were observed in all PsAKR1 expressing transgenics. The multigene expressing rice transgenics showed improved glyphosate resistance with yield maintenance. In summary, results suggest that stacking genes with two different detoxification mechanisms and insensitive EPSPS is a potential approach for developing glyphosate-resistant plants with less residual content.

Investigation of the Captopril-Insulin Interaction by Mass Spectrometry and Computational Approaches Reveals that Captopril Induces Structural Changes in Insulin.

Post-translational modifications remarkably regulate proteins' biological function. Small molecules such as reactive thiols, metabolites, and drugs may covalently modify the proteins and cause structural changes. This study reports the covalent modification and noncovalent interaction of insulin and captopril, an FDA-approved antihypertensive drug, through mass spectrometric and computation-based approaches. Mass spectrometric analysis shows that captopril modifies intact insulin, reduces it into its "A" and "B" chains, and covalently modifies them by forming adducts. Since captopril has a reactive thiol group, it might reduce the insulin dimer or modify it by reacting with cysteine residues. This was proven with dithiothreitol treatment, which reduced the abundance of captopril adducts of insulin A and B chains and intact Insulin. Liquid chromatography tandem mass spectrometric analysis identified the modification of a total of four cysteine residues, two in each of the A and B chains of insulin. These modifications were identified to be Cys6 and Cys7 of the A chain and Cys7 and Cys19 of the B chain. Mass spectrometric analysis indicated that captopril may simultaneously modify the cysteine residues of intact insulin or its subunits A and B chains. Biophysical studies involving light scattering and thioflavin T assay suggested that the binding of captopril to the protein leads to the formation of aggregates. Docking and molecular dynamics studies provided insights into the noncovalent interactions and associated structural changes in insulin. This work is a maiden attempt to understand the detailed molecular interactions between captopril and insulin. These findings suggest that further investigations are required to understand the long-term effect of drugs like captopril.

Blood Pressure and Safety Events With Vericiguat in the VICTORIA Trial.

Background Although safety and tolerability of vericiguat were established in the VICTORIA (Vericiguat Global Study in Subjects With Heart Failure With Reduced Ejection Fraction) trial in patients with heart failure with reduced ejection fraction, some subgroups may be more susceptible to symptomatic hypotension, such as older patients, those with lower baseline systolic blood pressure (SBP), or those concurrently taking angiotensin receptor neprilysin inhibitors. We described the SBP trajectories over time and compared the occurrence of symptomatic hypotension or syncope by treatment arm in potentially vulnerable subgroups in VICTORIA. We also evaluated the relation between the efficacy of vericiguat and baseline SBP. Methods and Results Among patients receiving at least 1 dose of the study drug (n=5034), potentially vulnerable subgroups were those >75 years old (n=1395), those with baseline SBP 100-110 mm Hg (n=1344), and those taking angiotensin receptor neprilysin inhibitors (n=730). SBP trajectory was plotted as mean change from baseline over time. The treatment effect on time to symptomatic hypotension or syncope was evaluated overall and by subgroup, and the primary efficacy composite outcome (heart failure hospitalization or cardiovascular death) across baseline SBP was examined using Cox proportional hazards models. SBP trajectories showed a small initial decline in SBP with vericiguat in those >75 years old (versus younger patients), as well as those receiving angiotensin receptor neprilysin inhibitors (versus none), with SBP returning to baseline thereafter. Patients with SBP <110 mm Hg at baseline showed a trend to increasing SBP over time, which was similar in both treatment arms. Safety event rates were generally low and similar between treatment arms within each subgroup. In Cox proportional hazards analysis, there were similar numbers of safety events with vericiguat versus placebo (adjusted hazard ratio [HR], 1.18; 95% CI, 0.99-1.39; P=0.059). No difference existed between treatment arms in landmark analysis beginning after the titration phase (ie, post 4 weeks) (adjusted HR, 1.14; 95% CI, 0.93-1.38; P=0.20). The benefit of vericiguat compared with placebo on the primary composite efficacy outcome was similar across the spectrum of baseline SBP (P for interaction=0.32). Conclusions These data demonstrate the safety of vericiguat in a broad population of patients with worsening heart failure with reduced ejection fraction, even among those predisposed to hypotension. Vericiguat's efficacy persisted regardless of baseline SBP. Registration URL: https://www.clinicaltrials.gov; Unique identifier: NCT02861534.