Fitness factor genes conserved within the multi-species core genome of Gram-negative Enterobacterales species contribute to bacteremia pathogenesis.

There is a critical gap in knowledge about how Gram-negative bacterial pathogens, using survival strategies developed for other niches, cause lethal bacteremia. Facultative anaerobic species of the Enterobacterales order are the most common cause of Gram-negative bacteremia, including Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, Citrobacter freundii, and Enterobacter hormaechei. Bacteremia often leads to sepsis, a life-threatening organ dysfunction resulting from unregulated immune responses to infection. Despite a lack of specialization for this host environment, Gram-negative pathogens cause nearly half of bacteremia cases annually. Based on our existing Tn-Seq fitness factor data from a murine model of bacteremia combined with comparative genomics of the five Enterobacterales species above, we prioritized 18 conserved fitness genes or operons for further characterization. Mutants were constructed for all genes in all five species. Each mutant was used to cochallenge C57BL/6 mice via tail vein injection along with each respective wild-type strain to determine competitive indices for each fitness gene. Five fitness factor genes, when mutated, attenuated mutants in four or five species in the spleen and liver (tatC, ruvA, gmhB, wzxE, arcA). Five additional fitness factor genes or operons were validated as outcompeted by wild-type in three, four, or five bacterial species in the spleen (xerC, prc, apaGH, atpG, aroC). Overall, 17 of 18 fitness factor mutants were attenuated in at least one species in the spleen or liver. Together, these findings allow for the development of a model of bacteremia pathogenesis that may include future targets of therapy against bloodstream infections.

Navigating the blurred path of mixed neuroimmune signaling.

Evolution has created complex mechanisms to sense environmental danger and protect tissues, with the nervous and immune systems playing pivotal roles. These systems work together, coordinating local and systemic reflexes to restore homeostasis in response to tissue injury and infection. By sharing receptors and ligands, they influence the pathogenesis of various diseases. Recently, a less-explored aspect of neuroimmune communication has emerged: the release of neuropeptides from immune cells and cytokines/chemokines from sensory neurons. This article reviews evidence of this unique neuroimmune interplay and its impact on the development of allergy, inflammation, itch, and pain. We highlight the effects of this neuroimmune signaling on vital processes such as host defense, tissue repair, and inflammation resolution, providing avenues for exploration of the underlying mechanisms and therapeutic potential of this signaling.

Role of Charge Heterogeneity on Physical Stability of Monoclonal Antibody Biotherapeutic Products.

PURPOSE: Chemical modifications in monoclonal antibodies can change hydrophobicity, charge heterogeneity as well as conformation, which eventually can impact their physical stability. In this study, the effect of the individual charge variants on physical stability and aggregation propensity in two different buffer conditions used during downstream purification was investigated. METHODS: The charge variants were separated using semi-preparative cation exchange chromatography and buffer exchanged in the two buffers with pH 6.0 and 3.8. Subsequently each variant was analysed for size heterogeneity using size exclusion chromatography and dynamic light scattering, conformational stability, colloidal stability, and aggregation behaviour under accelerated stability conditions. RESULTS: Size variants in each charge variant were similar in both pH conditions when analyzed without extended storage. However, conformational stability was lower at pH 3.8 than pH 6.0. All charge variants showed similar apparent melting temperature at pH 6.0. In contrast, at pH 3.8 variants A3, A5, B2, B3 and B4 display lower Tm, suggesting reduced conformational stability. Further, A2, A3 and A5 exhibit reduced colloidal stability at pH 3.8. In general, acidic variants are more prone to aggregation than basic variants. CONCLUSION: Typical industry practice today is to examine in-process intermediate stability with acidic species and basic species taken as a single category each. We suggest that perhaps stability evaluation needs to be performed at specie level as different acidic or basic species have different stability and this knowledge can be used for clever designing of the downstream process to achieve a stable product.

Acaricide resistance status of deltamethrin and coumaphos in Hyalomma anatolicum ticks collected from different districts of Haryana.

To study the acaricide resistance status and possible mechanisms of action in conferring resistance to commonly used acaricides (deltamethrin and coumaphos), Hyalomma anatolicum ticks were collected from 6 dairy farms of Hisar and Charkhi Dadri districts of Haryana. By using standard larval packet test, H. anatolicum tick larvae of Charkhi Dadri isolates were found to be susceptible (100% mortality) to both the acaricides. Level-I resistance against coumaphos was recorded from four isolates, whereas, level-II was observed in only one isolate, collected from Hisar. One isolates (Kaimri) from Hisar also showed level-I resistance against deltamethrin. Biochemically, the ticks having higher values of resistance factor (RF) against coumaphos were found to possess increased enzymatic activity of α-esterase, ß-esterase, glutathione-S-transferase (GST) and mono-oxygenase enzymes, whereas, the monoamine oxidase did not show any constant trend. However, the RF showed a statistical significant correlation with GST only. Native PAGE analysis of H. anatolicum ticks revealed the presence of nine types of esterases (EST-1 h to EST-9 h) by using napthyl acetate as substrate. In the inhibitory assay, esterases were found to be inhibited by PMSF, indicating the presence of serine residue at catalytic triad. The partial cds of carboxylesterase and domain II of sodium channel genes were sequenced to determine any proposed mutations in resistant isolates of H. anatolicum ticks, however, no mutations were observed in either gene, indicating that increased expression of detoxification enzymes as a possible mechanism for resistance development, in the current study.

Metal and catalyst-free strategy to access 1,3-thio-heteroaryl BCP derivatives.

The widespread presence of bicyclo[1.1.1]pentane (BCP) and sulfur motifs in pharmaceutical compounds underscores the significance of synthesizing suitably functionalized BCP thioethers. In response, we have developed a metal-free and photocatalyst-free strategy that harnesses visible light-induced radical cascades. This approach culminates in the synthesis of essential thio-BCP derivatives, which serve as crucial precursors for the formation of the corresponding sulfoxides, sulfones, and sulfoximines. Importantly, this methodology exhibits potential for large-scale applications, displaying commendable tolerance towards various functional groups while operating under mild reaction conditions.

Understanding the resistance mechanisms of Rhipicephalus microplus ticks to synthetic pyrethroids and organophosphates in south-west regions of Haryana, North India.

Chemical control of tick infestation on dairy farms in India strongly relies upon the use of synthetic pyrethroids (deltamethrin) and organophosphate (coumaphos) drugs. Therefore, the present manuscript aims to investigate the resistance status of Rhipicephalus microplus ticks against these acaricides. Fully engorged adult R. microplus ticks were randomly collected from 8 dairy farms in North India and evaluated for acaricide resistance by using the Larval Packet Test (LPT). Of these, ticks collected from one and three farms showed the emergence of Level I acaricide resistance against deltamethrin and coumaphos, respectively. Significant positive correlations were found in the enzymatic activity (α-esterase, ß-esterase, glutathione-S-transferase, and mono-oxygenase) of R. microplus tick resistant against coumaphos. Native electrophoretogram analysis showed six different types of esterase activity in R. microplus (EST-1b to EST-6b), and EST-5b activity was more predominantly expressed in resistant ticks. Further, inhibitor studies using various esterase inhibitors suggested that EST-5b is a putative acetylcholine-esterase (AchE), and increased expression of one of the AchE might be responsible for the emergence of acaricide resistance. Further, no mutations were detected in the carboxylesterase (G1120A) and domain II S4-5 linker region (C190A) of the sodium channel genes of resistant R. microplus ticks, indicating that increased expression of detoxification enzymes was the probable mechanism for the development of acaricide resistance in the resistant ticks.

Multi-Level High-Throughput Screening for Discovery of Ligands That Inhibit Insulin Aggregation.

We have developed a multi-level virtual screening protocol to identify lead molecules from the FDA inactives database that can inhibit insulin aggregation. The method is based on the presence of structural and interaction specificity in non-native aggregation pathway protein-protein interactions. Some key challenges specific to the present problem, when compared with native protein association, include structural heterogeneity of the protein species involved, multiple association pathways, and relatively higher probability of conformational rearrangement of the association complex. In this multi-step method, the inactives database was first screened using the dominant pharmacophore features of previously identified molecules shown to significantly inhibit insulin aggregation nucleation by binding to its aggregation-prone conformers. We then performed ensemble docking of several low-energy ligand conformations on these aggregation-prone conformers followed by molecular dynamics simulations and binding affinity calculations on a subset of docked complexes to identify a final set of five potential lead molecules to inhibit insulin aggregation nucleation. Their effect on aggregation inhibition was extensively investigated by incubating insulin under aggregation-prone aqueous buffer conditions (low pH, high temperature). Aggregation kinetics were characterized using size exclusion chromatography and Thioflavin T fluorescence assay, and the secondary structure was determined using circular dichroism spectroscopy. Riboflavin provided the best aggregation inhibition, with 85% native monomer retention after 48 h incubation under aggregation-prone conditions, whereas the no-ligand formulation showed complete monomer loss after 36 h. Further, insulin incubated with two of the screened inactives (aspartame, riboflavin) had the characteristic α-helical dip in CD spectra, while the no-ligand formulation showed a change to ß-sheet rich conformations.

A Medical Records Review Study Assessing Safety of Zoster Vaccine Recombinant, Adjuvanted in Patients With Rheumatic Disease.

OBJECTIVE: Patients with rheumatic disease are at increased risk for herpes zoster infection. Because of limited safety data in this population and concerns over vaccine-precipitated flares, there are no guidelines for vaccination with the zoster vaccine recombinant, adjuvanted (ZRA). We evaluated self-reported adverse events (AEs) and disease activity after ZRA administration in adults with rheumatic disease. METHODS: In this medical records review study at our large academic center, patients who had received at least 1 dose of ZRA from January 1, 2018 to March 11, 2020 were assessed. Self-reported AEs and disease activity were monitored 3 months after each ZRA administration. Measures of disease activity were reviewed 6 months before ZRA in those who received both doses, or 3 months before ZRA in those who received 1 dose. RESULTS: We identified 65 patients, of whom 34 (52.3%) received both doses of ZRA. Four patients (6.2%) self-reported AEs after receiving ZRA, all of which were minor and systemic. Three patients (9.2%) developed a flare after receiving ZRA, compared with 8 (12.3%) who experienced a flare in the baseline period. There was no significant change in flare incidence or disease activity after vaccination. Subgroup analysis of those on biologic and nonbiologic disease-modifying antirheumatic drugs revealed no differences in frequency of postvaccination AEs, flares, or disease activity. CONCLUSIONS: In our cohort, disease activity seemed stable when comparing disease markers before and after ZRA administration. In addition, ZRA was well-tolerated with minor AEs. Further studies are needed to guide formal vaccination recommendations.

In vitro anti-trypanosomal effect of ivermectin on Trypanosoma evansi by targeting multiple metabolic pathways.

High cytotoxicity and increasing resistance reports of existing chemotherapeutic agents against T. evansi have raised the demand for novel, potent, and high therapeutic index molecules for the treatment of surra in animals. In this regard, repurposing approach of drug discovery has provided an opportunity to explore the therapeutic potential of existing drugs against new organism. With this objective, the macrocyclic lactone representative, ivermectin, has been investigated for the efficacy against T. evansi in the axenic culture medium. To elucidate the potential target of ivermectin in T. evansi, mRNA expression profile of 13 important drug target genes has been studied at 12, 24, and 48 h interval. In the in vitro growth inhibition assay, ivermectin inhibited T. evansi growth and multiplication significantly (p < 0.001) with IC50 values of 13.82 µM, indicating potent trypanocidal activity. Cytotoxicity assays on equine peripheral blood mononuclear cells (PBMCs) and Vero cell line showed that ivermectin affected the viability of cells with a half-maximal cytotoxic concentration (CC50) at 17.48 and 22.05 µM, respectively. Data generated showed there was significant down-regulation of hexokinase (p < 0.001), ESAG8 (p < 0.001), aurora kinase (p < 0.001), casein kinase 1 (p < 0.001), topoisomerase II (p < 0.001), calcium ATPase 1 (p < 0.001), ribonucleotide reductase I (p < 0.05), and ornithine decarboxylase (p < 0.01). The mRNA expression of oligopeptidase B remains refractory to the exposure of the ivermectin. The arginine kinase 1 and ribonucleotide reductase II showed up-regulation on treatment with ivermectin. The ivermectin was found to affect glycolytic pathways, ATP-dependent calcium ATPase, cellular kinases, and other pathway involved in proliferation and maintenance of internal homeostasis of T. evansi. These data imply that intervention with alternate strategies like nano-formulation, nano-carriers, and nano-delivery or identification of ivermectin homologs with low cytotoxicity and high bioavailability can be explored in the future as an alternate treatment for surra in animals.

Leukemia associated RUNX1T1 gene reduced proliferation and invasiveness of glioblastoma cells.

RUNX1T1 has been found to be mutated in different cancers such as prostate, lung, colon, and breast cancer. A recent computational study involving the TCGA database of glioma patients found RUNX1T1 as one of the downregulated driver genes associated with poor overall survival of glioma patients. Hypoxia-inducible factor 1α (HIF1α) is upregulated in glioma and has been associated with the severity and drug resistance of glioma. Previously, we have shown that RUNX1T3 degrades HIF1α affecting the proliferation of leukemia cells. We hypothesize that RUNX1T1 might be associated with the growth and development of glioma through the regulation of HIF1α. We have evaluated the expression level of RUNX1T1 at different stages of glioma and the effect of RUNX1T1 on the proliferation and invasiveness of glioblastoma cells in vitro. We further looked at the effect of RUNX1T1 on the expression and stability of HIF1α in vitro. Expression of RUNX1T1 was significantly downregulated, both at RNA and protein levels in glioma samples as studied by quantitative real-time polymerase chain reaction and immunohistochemistry. While expression of HIF1α was higher in glioma tissues compared with its level in the normal brain. In vitro studies demonstrated that RUNX1T1 interacted with HIF1α and recruited HIF1α modification factor such as PHD2 and GSK3ß causing hydroxylation of HIF1α following ubiquitination by FBW7. RUNX1T1 led to the degradation of HIF1α and decreased proliferation/invasiveness of glioblastoma cell lines. Further, RUNX1T1 increased the effectiveness of temozolomide (TMZ), a conventional glioma drug toward glioblastoma cell lines. This study indicates that downregulation of RUNX1T1 might play an important role in the severity and development of glioma.

First report of molecular and phylogenetic analysis of Physaloptera praeputialis in naturally infected stray cats from India.

Nematodes of the genus Physaloptera are globally distributed and infect a multitude of hosts. Their life cycle involves orthopterans and coleopterans as intermediate hosts. The morphological characters alone are inadequate to detect and differentiate Physaloptera spp. from its congeners. Moreover, molecular studies are limited to compare them precisely. The present communication reports the first molecular phylogenetic characterization of feline Physaloptera spp. from India based on mitochondrial cytochrome c oxidase subunit 1 (COX1) and small subunit ribosomal DNA (18S rDNA). The nematodes were first isolated from the stomach of adult stray cats during necropsy examination. Based on the gross and microscopic characters, the worms were identified as P. praeputialis. Morphological identification was further confirmed through PCR targeting the barcode region of the mitochondrial cytochrome c oxidase subunit I (MT-COI) gene, using nematode-specific primers cocktail followed by species specific primers targeting partial COX1 and 18S rRNA genes. Generated sequences were submitted in NCBI GenBank (MW517846, MW410927, MW411349), and phylogenetic trees were constructed using the maximum likelihood method. When compared with other sequences of Physaloptera species across the globe, the present isolates showed 85.6-97.7% and 97.3-99% nucleotide homology based on COX1 and 18S rRNA gene, respectively. BLASTn analysis revealed a strong identity to other Physaloptera spp., and the phylogenetic tree placed all Physaloptera spp. in the same cluster. This study again indicates the usefulness of molecular techniques to substantiate the identity of species that may lack adequate descriptions and impart new insight for the potentially overlooked significance of P. praeputialis infections in felines.

Fate of the Left Pulmonary Artery and Thoracic Aorta After Transcatheter Patent Ductus Arteriosus Closure in Low Birth Weight Premature Infants.

Transcatheter patent ductus arteriosus closure (TCPC) is an emerging treatment for low birth weight extremely premature neonates (EPNs). Left pulmonary artery (LPA) and descending aorta (DAO) obstruction are described device-related complications, however, data on mid- and long-term vascular outcomes are lacking. A retrospective analysis of EPNs who underwent successful TCPC at our institution from 03/2013 to 12/2018 was performed. Two-dimensional echocardiography and spectral Doppler velocities from various time points before and after TCPC were used to identify LPA and DAO flow disturbances. A total of 44 EPNs underwent successful TCPC at a median (range) procedural weight of 1150 g (755-2500 g). Thirty-two (73%) patients were closed with the AVP II and 12 (27%) with the Amplatzer Piccolo device. LPA and DAO velocities on average remained within normal limits and improved spontaneously in long-term follow up (26.1 months, range 1-75 months). One patient, who had concerning LPA flow characteristics immediately after device implant (peak velocity 2.6 m/s) developed progressive LPA stenosis requiring stent placement 3 months post-procedure. In the remaining infants, including 7 (16%) who developed LPA and 3 (7%) who developed DAO flow disturbances (range 2-2.4 m/s), all had progressive normalization of flow velocities over time. TCPC can be performed safely in EPNs with a low incidence of LPA and DAO obstruction. In the absence of significant progressive vascular obstruction in the early post-procedure period, mild increases in LPA and DAO flow velocities tend to improve spontaneously and normalize in long-term follow-up.

Perioperative risk factors for pulmonary complications after non-cardiac surgery.

BACKGROUND AND AIMS: Postoperative pulmonary complications (PPCs) lead to increased morbidity, mortality, length of hospital stay, and cost to the patient. This study was conducted to determine the risk factors and assess the incidence of PPC after non-cardiac surgery. MATERIAL AND METHODS: This prospective, observational study was conducted on 1,170 patients undergoing non-cardiac surgery. Details of patient, surgical, and anesthetic factors were collected and patients were followed up for the entire duration of hospital stay for the occurrence of PPC. Assess Respiratory Risk in Surgical Patients in Catalonia (ARISCAT) score and the length of hospital stay was noted for all the patients. Regression analysis was used to find the risk factors associated with development of respiratory complications. RESULTS: The incidence of PPC was found to be 59 in 1,170 patients (5%) in our hospital. Multivariate analysis revealed that patients with intermediate and high risk ARISCAT scoring had higher odds of developing PPC. Higher age (>50 years), positive cough test, presence of nasogastric tube, and intraoperative pulmonary complications were identified as independent risk factors associated with the occurrence of PPC. CONCLUSION: We found 5% incidence of PPC in our study. Recognition of the delineated risk factors and routine use of ARISCAT score for preoperative assessment may help identify patients at a higher risk of developing postoperative pulmonary complications.

Analytical Platform for Monitoring Aggregation of Monoclonal Antibody Therapeutics.

PURPOSE: To develop an analytical platform for the estimation as well as characterization of aggregates over the complete size spectrum (from invisible monomer to visible precipitates). METHODS: Two mAb samples were incubated at 30°C in different buffer systems of protein A chromatography for observing degradation due to aggregation. The aggregation in these samples was quantified by size exclusion chromatography (SEC), dynamic light scattering (DLS), and micro flow imaging (MFI). RESULTS: The results obtained from various characterization tools were analysed in various size ranges - size exclusion chromatography (SEC) (1 nm - 25 nm), dynamic light scattering (DLS) (10 nm - 5 µm), and micro flow imaging (MFI) (2 µm - 300 µm). Since each characterization tool covers a particular size range, data from multiple tools was collected in the "handover" regions to demonstrate accuracy of the platform. CONCLUSIONS: Based on the observations from the experiments, an analytical platform has been proposed covering the whole size spectrum that would be of utility to those engaged in formulation development as well as other aspects related to stability of biotherapeutic products.

Efficient production of endotoxin depleted bioactive α-hemolysin of uropathogenic Escherichia coli.

Uropathogenic E. coli (UPEC), especially associated with severe urinary tract infections (UTI) pathologies, harbors an important virulence factor known as α-hemolysin (110 kDa). Hemolytic activity of α-hemolysin (HlyA) requires modification (acylation) of two lysine residues of HlyA by HlyC, part of operon hlyCABD. Most of the previous studies had used whole operon hlyCABD and gene tolC cloning for the production of active α-hemolysin. Studies involving α-hemolysin are limited due to the cumbersome and manual method of purification for this toxin. Here, we report a simple method for production of both active and inactive recombinant α-hemolysin by cloning only hlyA and hlyC genes of operon hlyCABD. Presence of both active and inactive α-hemolysin would be advantageous for functional characterization. After translation, the yield of the purified α-hemolysin was 1 mg/200 ml. Functionality of the recombinant α-hemolysin protein was confirmed using hemolytic assay. This is the first report of the production of active and inactive recombinant α-hemolysin for functional studies.

Neuroprotective Effects of Nicorandil in Chronic Cerebral Hypoperfusion-Induced Vascular Dementia.

BACKGROUND: Ischemia-induced chronic cerebral hypoperfusion (CCH) is associated with reduced cerebral blood flow and vascular dementia (VaD). Brain mitochondrial potassium (adenosine triphosphate-sensitive potassium [KATP]) channels have a beneficial role in various brain conditions. The utility of KATP channels in CCH-induced VaD is still unknown. The aim of this study is to investigate the role of nicorandil, a selective KATP channel opener, in CCH-induced VaD. METHODS: The method of 2-vessel occlusion (2VO) was used to induce CCH in mice. Cognitive impairment was assessed using Morris water maze. Serum nitrosative stress (nitrite/nitrate), brain cholinergic dysfunction (acetylcholinesterase [AChE] activity), brain oxidative stress (thiobarbituric acid reactive substances, glutathione [GSH], catalase [CAT], and superoxide dismutase [SOD]), inflammation (myeloperoxidase [MPO]), and infarct size (2,3,5-triphenyltetrazolium chloride staining) were assessed. RESULTS: 2-vessels-occluded animals have shown significant cognitive impairment, serum nitrosative stress (reduced nitrite/nitrate), cholinergic dysfunction (increased brain AChE activity), and increased brain oxidative stress (reduction in GSH content and SOD and CAT activities with a significant increase in lipid peroxidation), along with a significant increase in MPO activity and infarct size. However, nicorandil treatment has significantly attenuated various CCH-induced behavioral and biochemical impairments. CONCLUSIONS: It may be said that 2VO provoked CCH leading to VaD, which was attenuated by the treatment of nicorandil. So, modulation of KATP channels may provide benefits in CCH-induced VaD.

Effect of stress on academic performance in medical students--a cross sectional study.

INTRODUCTION: Stress, a universal phenomenon, affects an individual's productivity either by increasing it ('eustress') or decreasing it ('distress'). It is widely acknowledged that the medical fraternity is predisposed to enormous stress. The same may be true for the budding medicos- the undergraduate medical students. In our study we attempted to identify situations that predisposed the medical students to stress and their effects on academic performance and to suggest certain coping mechanisms. OBJECTIVES: firstly to explore common sources of stress in medical students, secondly to establish correlation of stress, gender, attendance, and academic performance if any. METHOD: 114 medical undergraduates were assessed for the common sources of stress and the level of stress using semi structured Performa and stress scale. The results were compared and correlated with various variables like attendance, demographic factors, average marks etc. Pearson correlation coefficient was used for statistical correlation amongst different variables. RESULTS & CONCLUSIONS: Stress shows beneficial effects in females when compared to males. High attendance and better day to day performance in female medical students was associated with more amount of stress when compared to male students. Thus, stress among medical students should be acknowledged and attempts should be made to alleviate it.

Montelukast Ameliorates 2K1C-Hypertension Induced Endothelial Dysfunction and Associated Vascular Dementia.

BACKGROUND: Declined kidney function associated with hypertension is a danger for cognitive deficits, dementia, and brain injury. Cognitive decline and vascular dementia (VaD) are serious public health concerns, which highlights the urgent need for study on the risk factors for cognitive decline. Cysteinyl leukotriene (CysLT1) receptors are concerned with regulating cognition, motivation, inflammatory processes, and neurogenesis. OBJECTIVE: This research aims to examine the consequence of montelukast (specific CysLT1 antagonist) in renovascular hypertension 2-kidney-1-clip-2K1C model-triggered VaD in experimental animals. METHODS: 2K1C tactics were made to prompt renovascular hypertension in mature male rats. Morris water maze was employed to measure cognition. Mean arterial pressure (MAP), serum nitrite levels, aortic superoxide content, vascular endothelial activity, brain's oxidative stress (diminished glutathione, raised lipid peroxides), inflammatory markers (IL-10, IL-6, TNF-α), cholinergic activity (raised acetylcholinesterase), and cerebral injury (staining of 2, 3, 5- triphenylterazolium chloride) were also examined. RESULTS: Montelukast in doses of 5.0 and 10.0 mg kg-1 was used intraperitoneally as the treatment drug. Along with cognitive deficits, 2K1C-operated rats showed elevated MAP, endothelial dysfunction, brain oxidative stress, inflammation, and cerebral damage with diminished serum nitrite/nitrate. Montelukast therapy significantly and dose-dependently mitigated the 2K1Chypertension- provoked impaired behaviors, biochemistry, endothelial functions, and cerebral infarction. CONCLUSION: The 2K1C tactic caused renovascular hypertension and associated VaD, which was mitigated via targeted regulation of CysLT1 receptors by montelukast administration. Therefore, montelukast may be taken into consideration for the evaluation of its complete potential in renovascular-hypertension-induced VaD.

Novel purification platform based on multimodal preparative scale separation of mAb fragments and aggregates.

Monoclonal antibodies (mAbs) continue to dominate the biopharmaceutical industry. Certain mAbs are prone to fragmentation and clipping and in these cases, adequate removal of these species is critical during manufacturing. Fragments can be generated during fermentation, purification, storage, formulation, and administration. Their addition to the acidic charge-variant of the purified mAb has been reported to decrease stability and potency of the final product. However, contrary to mAb aggregation, manufacturers have not given much attention to removal of fragments and clipped species and as a result most conventional mAb platforms offer at best limited capabilities for their removal. In this study, we propose a novel purification platform that uses multimodal chromatography and achieves complete removal of a range of mAb fragments and clipped products (25-120 kDa). The utility of the platform has been successfully demonstrated for 2 IgG1s and 2 IgG4s. Further, adequate removal of the various host cell impurities such as host cell proteins (<10 ppm) and host cell DNA (<5 ppb) has been achieved. Finally, the platform was able to deliver adequate removal of high molecular weight impurities (<1 %) and a 30 % clearance of the acidic charge variant. The proposed single step has been shown to deliver what the polishing chromatography and intermediate purification chromatography steps deliver in a traditional mAb platform.

Assessment of change in the basic variants composition of trastuzumab during dilution in saline for administration.

Postproduction handling of drug products during preparation or clinical use may affect the structure and efficacy of the drug and perhaps remain unnoticed. Since chemical modifications can impact the product's structure, stability, and biological activity, this study investigates the impact of elevated temperature and subtle shift in pH on the drug product post-dilution in saline. The mAb sample diluted in saline for administration was stressed at elevated temperature and slightly acidic pH condition. Extended stability studies were performed and monitored for size and charge heterogeneity. Size heterogeneity shows no significant changes, whereas charge heterogeneity shows an increase in basic variants and a reduction in main species. Further, basic variants were isolated and characterized to identify the type and site of chemical modification. Intact mass analysis and peptide mapping identify that the basic variants were attributed mainly to the isomerization of HC Asp102 into iso-Asp or its succinimide intermediate. Four basic variants were found to exhibit similar structural properties as the main and control samples. However, basic variants showed reduced binding affinity to HER2 receptor, while there was no significant difference in FcRn binding. The results indicate that modification in the HC Asp102, which is present in the CDR, affects antigen binding and thus can influence the potency of the drug product. Hence, with the conventional stability studies required to license the drug product, including in-use or extended stability studies to mimic the postproduction handling would be desirable.