Spectrometric Analysis Research Articles

Development and characterization of dexamethasone nanodispersion for the effective treatment of diabetic retinopathy

Diabetic retinopathy is the most common complication of diabetes and remains the leading cause of vision loss worldwide. The potent glucocorticoid based anti-inflammatory drug dexamethasone (DEX) chosen for the present work is long-acting with half-life of 37–72 h extensively metabolized in to 6-hydroxy-dexamethasone (6-OH-DEX). The present work aimed to develop dexamethasone loaded nanodispersion (DEX-ND) by improving its solubility, half-life, and bioavailability in biological system thereby this formulation may be afforded economically. With the concept that polymeric surfactant improves the solubility of challenging water insoluble drug the present work has been hypothesized to improve its solubility using polyvinyl pyrrolidone (PVP-K30) polymer and polysorbate 80 (Tween 80) hydrophilic non-ionic surfactant, thereby the bioavailability is expected to get enhanced. By varying the PVP K30 and Tween 80 concentrations three different formulations were developed. The developed DEX-ND was further characterized for its compatibility, particle size, zeta potential, morphology, and in vitro release behavior. The results of Fourier transform infrared spectrometric analysis indicate the compatibility nature of DEX-ND along with other excipients/drug used in the formulation. The developed formulations showed spherical morphology, particle size ranging from 550.46 ± 107.82 to 3560 ± 284.29 nm, zeta potential ranging from −29.23 ± 1.09 to −7.793 ± 1.01 mV. The amount of DEX present in the DEX-NDs was found to be 0.95–3.93 mg. The in vitro release studies as performed by dialysis bag method showed a sustained release pattern. The results of ex vivo corneal permeation studies indicates that the developed DEX- NDs has some exposure to the posterior segment of the eye.

Multifold nanotwin-enhanced catalytic activity of Ni-Zn-Cu for hydrazine oxidation

The hydrazine oxidation reaction (HzOR) plays a critical role in direct hydrazine fuel cells and presents a promising alternative to the kinetics-sluggish oxygen evolution reaction in water reduction. Engineering catalysts with high-density multifold nanotwin structures has emerged as a compelling strategy to enhance catalytic activity. However, the exploration of multifold nanotwins for HzOR applications remains absent. Herein, we report the successful synthesis of a porous Ni-Zn-Cu catalyst featuring high-density twofold and fivefold nanotwins. The Ni-Zn-Cu catalyst supported on Ni foam (Ni-Zn-Cu/Ni foam) exhibits exceptionally high catalytic activity and stability toward the HzOR, achieving a current density of 204.3 mA cm−2 at 0.0 V vs. RHE and retaining up to 89.1 % after 20 h of continuous electrolysis. Online mass spectrometer (MS) analysis indicates that the Ni-Zn-Cu coating facilitates NN bond cleavage of N2H4. This study offers valuable insights into the development of multifold nanotwin catalysts for advancing HzOR applications.

Zein/fucoidan-coated phytol nanoliposome: preparation, characterization, physicochemical stability, in vitro release, and antioxidant activity.

To improve phytol bioavailability, a novel method of magnetic stirring and high-pressure homogenization (HPH) combination was used to prepare zein/fucoidan-coated phytol nanoliposomes (P-NL-ZF). The characterization, the simulated in vitro digestion, and the antioxidant activity of these phytol nanoliposomes from the different processes have been studied. Based on the results of dynamic light scattering (DLS) and gas chromatography-mass spectrometer (GC-MS) analysis, P-NL-ZF prepared through the combination of magnetic stirring and HPH exhibited superior encapsulation efficiency at 76.19% and demonstrated exceptional physicochemical stability under a series of conditions, including storage, pH, and ionic in comparison to single method. It was further confirmed that P-NL-ZF by magnetic stirring and HPH displayed a uniform distribution and regular shape through transmission electron microscopy (TEM). Fourier-transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC) analysis showed that electrostatic interactions and hydrogen bonding were the primary driving forces for the formation of composite nanoliposomes. Additionally, an in vitro digestion study revealed that multilayer composite nanoliposomes displayed significant and favorable slow-release properties (58.21%) under gastrointestinal conditions compared with traditional nanoliposomes (82.36%) and free phytol (89.73%). The assessments of chemical and cell-based antioxidant activities demonstrated that the coating of zein/fucoidan on phytol nanoliposomes resulted in enhanced effectiveness in scavenging activity of ABTS free radical and hydroxyl radical and mitigating oxidative damage to HepG2 cells. Based on our studies, the promising delivery carrier of zein/fucoidan-coated nanoliposomes is contributed to the encapsulation of hydrophobic natural products and enhancement of their biological activity. © 2024 Society of Chemical Industry.

Exploring structure/property relationships to health and environmental hazards of polymeric polyisocyanate prepolymer substances-3. Aquatic exposure and hazard of aliphatic diisocyanate-based prepolymers.

The water extractability and acute aquatic toxicity of seven aliphatic diisocyanate-based prepolymer substances were investigated to determine if lesser reactivity of the aliphatic isocyanate groups, as well as increased ionization potential of the expected (aliphatic amine-terminated) polymeric hydrolysis products, would influence their aquatic behavior compared to that of previously investigated aromatic diisocyanate-based prepolymers. At loading rates of 100 and 1,000mg/L, only the substances having log Kow ≤9 exhibited more than 1% extractability in water, and a maximum of 66% water extractability was determined for a prepolymer having log Kow = 2.2. For the more hydrophobic prepolymer substances (log Kow values from 18-37), water extractability was negligible. High-resolution mass spectrometric analyses were performed on the water-accommodated fractions (WAF) of the prepolymers, which indicated the occurrence of primary aliphatic amine-terminated polymer species having backbones and functional group equivalent weights aligned to those of the parent prepolymers. Measurements of reduced surface tension and presence of suspended micelles in the WAFs further supported the occurrence of these surface-active cationic polymer species as hydrolysis products of the prepolymers. Despite these characteristics, the water-extractable hydrolysis products were practically non-toxic to Daphnia magna. All of the substances tested exhibited 48-h EL50 values of >1,000mg/L, with one exception of EL50 = 157mg/L. The results from this investigation support a grouping of the aliphatic diisocyanate-based prepolymers as a class of water-reactive polymer substances having predictable aquatic exposure and a uniformly low hazard potential, consistent with that previously demonstrated for the aromatic diisocyanate-based prepolymers.

Identification of Secondary Metabolites from Actinomycetes Isolated from the Hilly Region of Nepal

Actinomycetes are a unique class of Gram-positive bacteria with a fungus-like filamentous mycelium. They are often called ‘antibiotics factories’ because they can produce many potent secondary metabolites. At present, multidrug-resistant (MDR) pathogens are the main problem for humans and animals and to overcome this problem, more potent antibiotics are urgently required. Fortunately, Nepal has great geographical and climatic diversity serving as a home for various actinomycetes. This study identified several actinomycetes using morphological and biochemical assays from seven soil samples collected from different elevations in Nepal. The primary objective of this study was to screen the potent metabolites-producing actinomycetes. For this purpose, antimicrobial assays of seven isolates were carried out against Escherichia coli ATCC 25922, Salmonella typhi ATCC 14028, Shigella sonnei ATCC 25931, and Staphylococcus aureus ATCC 43300. The sample BT36 demonstrated the highest zone of inhibition against the tested microorganisms among others. Similarly, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of all isolates were carried out. Among them, the MIC of samples BT36, PT7, and AB1 were found to be 1.952 mg/mL, 0.012 mg/mL, and 0.029 mg/mL, respectively. Ethyl acetate extracts of resulting potent isolates PT7 and AB1 were subjected to mass spectrometric analysis. A total of 22 bioactive compounds were annotated from liquid chromatography-high resolution mass spectroscopy (LC-HRMS), including epopromycin A, myxopyronin B, gilvocarcin HE, and okilactomycin A. It is concluded that this study will be useful in setting up a strategy to isolate useful secondary metabolites from soil microbes from the hilly region of Nepal.

Enhanced Surface Accessibility of SARS-CoV-2 Omicron Spike Protein Due to an Altered Glycosylation Profile.

SARS-CoV-2 spike (S) proteins undergo extensive glycosylation, aiding in proper folding, enhancing stability, and evading host immune surveillance. In this study, we used mass spectrometric analysis to elucidate the N-glycosylation characteristics and disulfide bonding of recombinant spike proteins derived from the SARS-CoV-2 Omicron variant (B.1.1.529) in comparison with the D614G spike variant. Furthermore, we conducted microsecond-long molecular dynamics simulations on spike proteins to resolve how the different N-glycans impact spike conformational sampling in the two variants. Our findings reveal that the Omicron spike protein maintains an overall resemblance to the D614G spike variant in terms of site-specific glycan processing and disulfide bond formation. Nonetheless, alterations in glycans were observed at certain N-glycosylation sites. These changes, in synergy with mutations within the Omicron spike protein, result in increased surface accessibility of the macromolecule, including the ectodomain, receptor-binding domain, and N-terminal domain. Additionally, mutagenesis and pull-down assays reveal the role of glycosylation of a specific sequon (N149); furthermore, the correlation of MD simulation and HDX-MS identified several high-dynamic areas of the spike proteins. These insights contribute to our understanding of the interplay between structure and function, thereby advancing effective vaccination and therapeutic strategies.

Mass Spectrometric Analysis of Purine Intermediary Metabolism Indicates Cyanide Induces Purine Catabolism in Rabbits.

Purines are the building blocks of DNA/RNA, energy substrates, and cofactors. Purine metabolites, including ATP, GTP, NADH, and coenzyme A, are essential molecules in diverse biological processes such as energy metabolism, signal transduction, and enzyme activity. When purine levels increase, excess purines are either recycled to synthesize purine metabolites or catabolized to the end product uric acid. Purine catabolism increases during states of low oxygen tension (hypoxia and ischemia), but this metabolic pathway is incompletely understood in the context of histotoxic hypoxia (i.e., inhibition of oxygen utilization despite normal oxygen tension). In rabbits exposed to cyanide-a classical histotoxic hypoxia agent-we demonstrated significant increases in several concordant metabolites in the purine catabolic pathway (including plasma levels of uric acid, xanthosine, xanthine, hypoxanthine, and inosine) via mass spectrometry-based metabolite profiling. Pharmacological inhibition of the purine catabolic pathway with oxypurinol mitigated the deleterious effects of cyanide on skeletal muscle cytochrome c oxidase redox state, measured by non-invasive diffuse optical spectroscopy. Finally, plasma uric acid levels correlated strongly with those of lactic acid, an established clinical biomarker of cyanide exposure, in addition to a tissue biomarker of cyanide exposure (skeletal muscle cytochrome c oxidase redox state). Cumulatively, these findings not only shed light on the in vivo role(s) of cyanide but also have implications in the field of medical countermeasure (MCM) development.

DNA-Binding Proteins and Passenger Proteins in Plasma DNA-Protein Complexes: Imprint of Parental Cells or Key Mediators of Carcinogenesis Processes?

Knowledge of the composition of proteins that interact with plasma DNA will provide a better understanding of the homeostasis of circulating nucleic acids and the various modes of interaction with target cells, which may be useful in the development of gene targeted therapy approaches. The goal of the present study is to shed light on the composition and architecture of histone-containing nucleoprotein complexes (NPCs) from the blood plasma of healthy females (HFs) and breast cancer patients (BCPs) and to explore the relationship of proteins with crucial steps of tumor progression: epithelial-mesenchymal transition (EMT), cell proliferation, invasion, cell migration, stimulation of angiogenesis, and immune response. MALDI-TOF mass spectrometric analysis of NPCs isolated from blood samples using affine chromatography was performed. Bioinformatics analysis showed that the shares of DNA-binding proteins in the compositions of NPCs in normal and cancer patients are comparable and amount to 40% and 33%, respectively; in total, we identified 38 types of DNA-binding motifs. Functional enrichment analysis using FunRich 3.13 showed that, in BCP blood, the share of DNA-binding proteins involved in nucleic acid metabolism increased, while the proportion of proteins involved in intercellular communication and signal transduction decreased. The representation of NPC passenger proteins in breast cancer also changes: the proportion of proteins involved in transport increases and the share of proteins involved in energy biological pathways decreases. Moreover, in the HF blood, proteins involved in the processes of apoptosis were more represented in the composition of NPCs and in the BCP blood-in the processes of active secretion. For the first time, bioinformatics approaches were used to visualize the architecture of circulating NPCs in the blood and to show that breast cancer has an increased representation of passenger proteins involved in EMT, cell proliferation, invasion, cell migration, and immune response. Using breast cancer protein data from the Human Protein Atlas (HPA) and DEPC, we found that 86% of NPC proteins in the blood of BCPs were not previously annotated in these databases. The obtained data may indirectly indicate directed protein sorting in NPCs, which, along with extracellular vesicles, can not only be diagnostically significant molecules for liquid biopsy, but can also carry out the directed transfer of genetic material from donor cells to recipient cells.

Neocarzilin Inhibits Cancer Cell Proliferation via BST-2 Degradation, Resulting in Lipid Raft-Trapped EGFR.

Neocarzilin (NCA) is a natural product exhibiting potent antimigratory as well as antiproliferative effects. While vesicle amine transport protein 1 (VAT-1) was previously shown to inhibit migration upon NCA binding, the molecular mechanisms responsible for impaired proliferation remained elusive. We here introduce a chemical probe closely resembling the structural and stereochemical features of NCA and unravel bone marrow stromal antigen 2 (BST-2) as one of the targets responsible for the antiproliferative effect of NCA in cancer cells. The antiproliferative mechanism of NCA was confirmed in corresponding BST-2 knockout (KO) HeLa cells, which were less sensitive to compound treatment. Vice versa, reconstitution of BST-2 in the KO cells again reduced proliferation upon NCA addition, comparable to that of wild-type (wt) HeLa cells. Whole proteome mass spectrometric (MS) analysis of NCA-treated wt and KO cancer cells revealed regulated pathways and showed reduced levels of BST-2 upon NCA treatment. In-depth analysis of BST-2 levels in response to proteasome and lysosome inhibitors unraveled a lysosomal degradation path upon NCA treatment. As BST-2 mediates the release of epidermal growth factor receptor (EGFR) from lipid rafts to turn on proliferation signaling pathways, reduced BST-2 levels led to attenuated phosphorylation of STAT3. Furthermore, fluorescence microscopy confirmed increased colocalization of EGFR and lipid rafts in the presence of NCA. Overall, NCA represents a versatile anticancer natural product with a unique dual mode of action and unconventional inhibition of proliferation via BST-2 degradation.

Interfacing High-Throughput Electrosynthesis and Mass Spectrometric Analysis of Azines.

Combinatorial electrochemistry has great promise for accelerated reaction screening, organic synthesis, and catalysis. Recently, we described a new high-throughput electrochemistry platform, colloquially named "Legion". Legion fits the footprint of a 96-well microtiter plate with simultaneous individual control over all 96 electrochemical cells. Here, we demonstrate the versatility of Legion when coupled with high-throughput mass spectrometry (MS) for electrosynthetic product screening and quantitation. Electrosynthesis of benzophenone azine was selected as a model reaction and was arrayed and optimized using a combination of Legion and nanoelectrospray ionization MS. The combination of high-throughput synthesis with Legion and analysis via MS proves a compelling strategy for accelerating reaction discovery and optimization in electro-organic synthesis.

Effect of the 35 nm and 70 nm Size Exclusion Chromatography (SEC) Column and Plasma Storage Time on Separated Extracellular Vesicles.

The technical difficulty of separating extracellular vesicles (EVs) from plasma proteins in human blood presents a significant hurdle in EV research, particularly during nano ultra-high-performance liquid chromatography-tandem mass spectrometric (UHPLC-MS/MS) analysis, where detecting "vesicular" proteins among abundant plasma proteins is challenging. Standardisation is a pressing issue in EV research, prompting collaborative global efforts to address it. While the MISEV guidelines offer valuable recommendations, unanswered questions remain, particularly regarding sample storage. We compared size exclusion chromatography (SEC) columns with pore sizes of 35 nm and 70 nm to identify fractions with minimal contaminating proteins and the highest concentration of small EVs (sEVs). Following column selection, we explored potential differences in the quality and quantity of sEVs isolated from platelet-free plasma (PFP) after long-term storage at -80 °C (>2.5 years) compared to freshly drawn blood. Our methodologically rigorous study indicates that prolonged storage, under correct storage and processing conditions, does not compromise sEV quality. Both columns effectively isolated vesicles, with the 70 nm column exhibiting a higher abundance of "vesicular" proteins. We propose a relatively rapid and moderately efficient protocol for obtaining a comparatively pure sEV fraction from plasma, facilitating sEV processing in clinical trials.

Mass spectrometric analysis of Odonthobuthus Doriae scorpion venom and its non-neutralized fractions after interaction with commercial antivenom

It is believed that antivenoms play a crucial role in neutralizing venoms. However, uncontrolled clinical effects appear in patients stung by scorpions after the injection of antivenom. In this research, non-neutralized components of the venom of the Iranian scorpion Odonthobuthus doriae were analyzed after interacting with the commercial antivenom available in the market. The venom and antivenom interaction was performed, then centrifuged, and the supernatant was analyzed by high-performance liquid chromatography (HPLC). Two peaks of Odonthobuthus doriae venom were observed in the chromatogram of the supernatant. Two components were isolated by HPLC and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) instruments. Peptide sequencing was done by Liquid Chromatography Quadrupole Time-of-Flight Tandem Mass Spectrometry (LC-Q-TOF MS/MS). Results indicate that the components of scorpion venom mainly have a molecular weight below 10 kDa, consisting of toxic peptides that disrupt the function of sodium and potassium channels. The MALDI-TOF MS results show that two toxic peptides with molecular masses of 6941 Da and 6396 Da were not neutralized by the antivenom. According to the MS/MS sequencing data, the components have been related to peptides A0A5P8U2Q6_MESEU and A0A0U4FP89_ODODO, which belong to the sodium and potassium channels toxins family, respectively.

Verbascoside and rare flavone glucosides from Citharexylum spinosum L. flowers as antihyperglycemic agents: Isolation, α-amylase inhibition, molecular docking and drug-likeness prediction

Human concern for its public health has been a cornerstone since ancient times, with humans exploring natural resources in their environment for medicinal purposes. Plants have long served as the primary source of medicinal compounds, reflecting centuries of exploration and experimentation. Today, leveraging cognitive and technological advancements, researchers continue to deepen their studies towards achieving optimal health conditions. In pursuit of antihyperglycemic phyto-alternatives, a phytochemical investigation of EtOAc and n-BuOH flower extracts of Citharexylum spinosum L. yielded four rare flavone glucosides viz. cirsilineol 4′-glucoside, jaceoside, 5,4′-dihydroxy-7,3′-dimethoxyflavone 6-glucoside and pedaliin. Additionally, the flavone aglycone apigenin, and the phenylethanoid glycoside verbascoside were isolated. The flavonoid compounds were isolated for the first time from C. spinosum tree. The extracts underwent silica gel column chromatography for simplification, followed by compound purification using preparative HPLC. Molecular structures were elucidated through NMR spectroscopic experiments, spectrometric ESI-HRMS analysis, and comparisons with existing literature data. Subsequently, extracts and isolated compounds were evaluated for their antihyperglycemic activities by assessing their ability to inhibit α-amylase enzymatic activity. EtOAc and n-BuOH extracts displayed important anti-α-amylase properties with IC50 values of 52.5 ± 1.2 and 36.3 ± 1.1 µg/mL, respectively. The results demonstrated promising inhibitory properties of the α-amylase enzyme by the polyphenols isolated in this study. IC50 values ranged from 12.8 ± 0.1 to 23.2 ± 0.1 µM for the isolated compounds, comparable to the reference standard acarbose (IC50= 12.3 ± 0.1 µM). Furthermore, a structure-activity relationship study was conducted, with all isolated molecules docked to the α-amylase enzyme (PDB: 7TAA) and their drug-likeness properties predicted.

Albumin Oxidation and Albumin Glycation Discordance During Type 2 Diabetes Therapy: Biological and Clinical Implications.

Aims: Cys34 albumin redox modifications (reversible "cysteinylation" and irreversible "di/trioxidation"), besides being just oxidative stress biomarkers, may have primary pathogenetic roles to initiate and/or aggravate cell, tissue, and vascular damage in diabetes. In an exploratory "proof-of-concept" pilot study, we examined longitudinal changes in albumin oxidation during diabetes therapy. Methods: Mass spectrometric analysis was utilized to monitor changes in human serum albumin (HSA) post-translational modifications {glycation [glycated albumin (GA)], cysteinylation [cysteinylated albumin (CA) or human non-mercaptalbumin-1; reversible], di/trioxidation (di/trioxidized albumin or human non-mercaptalbumin-2; irreversible), and truncation (truncated albumin)} during ongoing therapy. Four informative groups of subjects were evaluated [type 1 diabetes (T1DM), type 2 diabetes (T2DM), prediabetes-obesity, and healthy controls] at baseline, and subjects with diabetes were followed for a period up to 280 days. Results: At baseline, T2DM was associated with relatively enhanced albumin cysteinylation (CA% total) compared with T1DM (P = 0.004), despite comparable mean hyperglycemia (P values: hemoglobin A1c = 0.09; GA = 0.09). T2DM, compared with T1DM, exhibited selectively and significantly higher elevations of all the "individual" glycated cum cysteinylated ("multimodified") albumin isoforms (P values: CysHSA+1G = 0.003; CysHSA+2G = 0.007; and CysHSA+3G = 0.001). Improvements in glycemic control and decreases in albumin glycation during diabetes therapy in T2DM were not always associated with concurrent reductions of albumin cysteinylation, and in some therapeutic situations, albumin cysteinylation worsened (glycation-cysteinylation discordance). Important differences were observed between the effects of sulfonylureas and metformin on albumin molecular modifications. Conclusions: T2DM was associated with higher oxidative (cysteinylation) and combined (cysteinylation plus glycation) albumin molecular modifications, which are not ameliorated by improved glucose control alone. Further studies are required to establish the clinical significance and optimal therapeutic strategies to address oxidative protein damage and resulting consequences in diabetes.

LncRNA PCED1B-AS1 mediates miR-3681-3p/MAP2K7 axis to promote metastasis, invasion and EMT in gastric cancer

BackgroundLncRNA PCED1B-AS1 is abnormally expressed in multiple cancers and has been confirmed as an oncogene. Our study aimed to investigate the regulatory mechanism of lncRNA PCED1B-AS1 in gastric cancer.MethodsTCGA database was used to analyze the abnormal expression of lncRNA PCED1B-AS1 in gastric cancer. By database prediction and mass spectrometric analysis, miR-3681-3p and MAP2K7 are potential downstream target molecules of lncRNA PCED1B-AS1 and verified by dual-luciferase report assay. RT-qPCR analysis and western blot were performed to detect the expressions of PCED1B-AS1 and MAP2K7 in gastric cancer cell lines and tissues. CCK-8 kit was applied to measure the cell viability. Wound healing and Transwell experiment were used to detect the migration and invasion. Western blot and immunohistochemical staining were performed to detect the expressions of EMT-related proteins in tissues. The changes of tumor proliferation were detected by xenograft experiment in nude mice.ResultsPCED1B-AS1 expression was higher but miR-3681-3 expression was lower in gastric cancer cell lines or tissues, compared to normal group. Function analysis verified PCED1B-AS1 promoted cell proliferation and inhibited cell apoptosis in gastric cancer cells in vitro and in vivo. LncRNA PCED1B-AS1 could bind directly to miR-3681-3p, and MAP2K7 was found to be a downstream target of miR-3681-3p. MiR-3681-3p mimics or si-MAP2K7 could partly reverse the effect of PCED1B-AS1 on gastric cancer cells.ConclusionPCED1B-AS1 accelerated cell proliferation and inhibited cell apoptosis through sponging miR-3681-3p to upregulate MAP2K7 expression in gastric cancer, which indicated PCED1B-AS1/miR-3681-3p/MAP2K7 axis may serve as a potential therapeutic target for gastric cancer.

Characterizations and solubility profile of novel senary SiO2–CaO–Na2O–P2O5–CaF2–SrO glass structure modified with Nb2O5

The solubility/degradability of bioactive glasses play crucial role in bone tissue engineering applications owing to the release of ions from their structure, which exert therapeutic effects on tissues. The present study evaluates the findings of a detailed investigation on the main features and solubility behaviors of newly formulated Nb incorporated glasses (Nb-BGs). New glass formulation in SiO2–CaO–Na2O–P2O5–CaF2–SrO–xNb2O5 (x: 0.0, 0.5, 1.0, and 1.5 mol. %) system was successfully synthesized by melt-quenching technique. Glass composition was verified with Energy Dispersive X-ray Fluorescence spectrometer analysis and its amorphous nature, functional groups of the matrix and morphological features were identified via X-Ray diffraction analyses, Fourier Transform Infrared Spectroscopy and Scanning Electron Microscope analysis, respectively. Network connectivity (NC) of the Nb-BGs was assessed with respect to the role of Nb2O5 in the glass matrix as network former, modifier, or both. In order to evaluate the in vitro solubility of Nb-BGs, released concentrations of particular ions from the glass compositions were determined at certain time points and comprehensively discussed with several mathematical models such as zero-order, first-order, second-order, Higuchi, and Korsmeyer-Peppas for the first time. Results showed that the incorporation of Nb2O5 (up to 1.5 mol. %) increased the density, oxygen density as well as NC values of newly formulated glasses. Si, Na and Ca ions which play a main role in well-defined Hench's mechanism followed mostly second-order release kinetics for all glasses in the mathematical models studied. When the Nb content increase (to 1.5 mol. %) in glass composition, the release mechanism of Si ion according to Korsmeyer-Peppas model shifts from the diffusion mechanism to an erosion-dominant release mechanism.

Low Hepatic Cardiolipin Levels Promote Increased Mitochondrial Electron Leak and Metabolic Dysfunction-associated Steatohepatitis

Mitochondria adapt to changing energetic demands of the cell. We recently reported that mitochondrial membrane lipid composition represents a regulatory mechanism by which effciency of oxidative phosphorylation becomes altered. Metabolic-associated fatty liver disease (MAFLD) is a progressive disorder characterized by oxidative stress and accumulation of fat in hepatocytes that can lead to metabolic dysfunction-associated steatohepatitis (MASH). We hypothesized that MAFLD alters mitochondrial membrane lipids to induce mitochondrial electron transfer ineffciency and promotes oxidative stress. Lipid mass spectrometric analyses in four models of MAFLD (high-fat diet, ob/ob, carbon tetrachloride administration (CCI4), and Gubra-Amylin-NASH® diet), revealed that low mitochondrial cardiolipin (CL) is a common signature associated with all MAFLD models. In hepa1-6 cells, lentivirus-mediated knockdown of cardiolipin synthase (CLS, generates CL in mitochondria) was suffcient to reduce respiration and increase electron leak. Liver mitochondria from mice with a hepatocyte-specific knockout of CLS exhibited defects in mitochondrial bioenergetics including increased electron leak, concomitant to pathogenesis of MASH. Together, these findings provide evidence that a reduction in hepatic cardiolipin may drive the progression of MAFLD to induce MASH pathology. R01GM144613-03R01AG074535-02S2R01DK107397 07. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Remodeled Extracellular Matrix Guides Endothelial Cell Phenotype and Transcriptome to Fibrotic Niche

Impairment of endothelial barrier integrity causes vascular injury leading to acute lung injury (ALI), which, if not resolved, can compromise a patient’s lung functions and mortality. While it is known that both endothelial cells (EC) and their underlying extracellular matrix (ECM) are required for preserving lung homeostasis and functions, much of the efforts to understand mechanisms regulating these functions have focused mainly on EC. A fundamental unresolved question is whether lung-remodeled ECM formed due to leaky endothelium impacts EC functions, and if so, how? Herein, using lung ECM derived from a mouse lacking focal adhesion kinase (FAK), specifically in the endothelium (remodeled ECM), we assessed ECM components and their role in regulating EC functions and their relevance in vivo. Previously, we have shown that conditional deletion of FAK in endothelium increases EC permeability and induces vascular injury at homeostasis. Mass spectrometric analysis showed 805 proteins in control ECM (FAKfl/fl mice lung derived ECM) consisting of glycoproteins, matrisome-associated proteins, and secreted factors. However, the remodeled ECM had 722 proteins and was highly enriched with fibrillin 1, fibrinogen, and collagen I. Moreover, the remodeled ECM was two-fold stiffer than the control ECM. Remodeled ECM with its inherent composition recapitulates the 3D microenvironment, encouraging EC morphological alteration, increased cell stiffness, and enhanced focal adhesion (FA) formation compared to control ECM. In addition, RNA sequencing identified increased expression of genes linked with fibrosis, including integrin b8, TGFb2, GDF15, SMAD7, COL1A1, IL33, ID3, BMPR1A, and ADAMTS4 in EC seeded on remodeled ECM. Interestingly, fibrotic signaling observed in EC plated on remodeled ECM mimicked in vivo in EC-FAK−/− mice as they showed increased hydroxyproline content and collagen deposition in the lungs, indicating loss of EC-FAK as the crucial initiator of fibrotic signaling. In summary, studies for the first time show disrupted EC signaling as the instructor of fibrotic niche in the lungs for targeting pulmonary fibrosis. NIH. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Isolation and characterization of a Mannheimiahaemolytica secreted serine protease that degrades sheep and bovine fibrinogen

Mannheimiahaemolytica is an opportunistic agent of the respiratory tract of bovines, a member of the Pasteurellaceae family, and the causal agent of fibrinous pleuropneumonia. This bacterium possesses different virulence factors, allowing it to colonize and infect its host. The present work describes the isolation and characterization of a serine protease secreted by M. haemolytica serotype 1. This protease was isolated from M. haemolytica cultured media by precipitation with 50 % methanol and ion exchange chromatography on DEAE-cellulose. It is a 70-kDa protease able to degrade sheep and bovine fibrinogen or porcine gelatin but not bovine IgG, hemoglobin, or casein. Mass spectrometric analysis indicates its identity with protease IV of M. haemolytica. The proteolytic activity was active between pH 5 and 9, with an optimal pH of 8. It was stable at 50 °C for 10 min but inactivated at 60 °C. The sera of bovines with chronic or acute pneumonia recognized this protease. Still, it showed no cross-reactivity with rabbit hyperimmune serum against the secreted metalloprotease from Actinobacilluspleuropneumoniae, another member of the Pasteurellaceae family. M. haemolytica secreted proteases could contribute to the pathogenesis of this bacterium through fibrinogen degradation, a characteristic of this fibrinous pleuropneumonia.

Investigating the quality of ethiopian steel reinforcing bars through comprehensive analysis

The fast economic growth, urbanization, and industrialization of Ethiopia led to the requirement for the establishment of various infrastructures and many residential building constructions consuming different types of materials. Reinforced steel bar is one of the most consumed construction materials in Ethiopia. The demand for reinforced steel bars in Ethiopia is satisfied by the local production and international market. However, most people lack trust to the use local products without justified reasons. This investigation work was done to evaluate the performance of the locally produced rebar concerning the Compulsory Ethiopian Standard (CES) to develop trust in contractors and consultants of different projects. A Compulsory Ethiopian Standard (CES) is an Ethiopian standard that has been prepared under the direction of the Technical Committee for Concrete and Concrete Products and published by the Ethiopian Standards Agency (ESA). Experimental works were done on rebar of grades B400BWR and B500BWR with different diameters collected from four different factories. The collected samples were investigated based on their yield strength, ultimate tensile strength, elongation, hardness, chemical composition, and microstructure formation of the product. The yield strength, ultimate tensile strength, and elongation of the rebar were analyzed based on the tension test results obtained from the 2000 KN Universal Testing Machine (UTM), and the chemical composition analysis was done using the Spark Emission Spectrometer Analyzer. The micro-hardness test was done using the Vickers Hardness Tester with 3kgf and 15-s dwell time at a 0.5 mm gap along the cross-section of the rebar. Microstructure analysis is done using EDX scanning electron microscope (SEM) and optical microscopy with different magnifications and resolutions on the cross-section of the rebar. From the tested samples one sample failed from B400BWR to achieve the minimum required yield strength of 400 MPa stated by CES 101, 2017. Samples of B500BWR satisfy all the requirements stated by the standard even if there is an increment up to 41 % beyond the minimum requirement.