Relationship between thrombus vWF and NETs with clinical severity and peripheral blood immunocytes' indicators in patients with acute ischemic stroke.

OBJECTIVE: To investigate the association between von Willebrand factor (vWF) and neutrophil extracellular traps (NETs) in thrombus with clinical severity and peripheral blood immunocytes' indicators in patients with early-stage acute ischemic stroke (AIS). METHODS: A retrospective study was conducted using the clinical data of 66 patients with AIS who underwent endovascular mechanical thrombectomy and had their thrombus samples collected. The concentrations of vWF and NETs in the thrombus samples were quantitatively assessed. Peripheral blood samples taken in the early stages of the disease were analyzed for total white blood cell counts (WBC), ratios of neutrophils (NEU%), lymphocytes (LYM%), eosinophils (EOS%), and monocytes (MONO%). The severity of clinical symptoms in these patients was evaluated using the modified Rankin Scale (mRS), Essen Stroke Risk Score (ESRS), Barthel Index (BI), and National Institute of Health Stroke Scale (NIHSS). RESULTS: Higher vWF levels in thrombus were associated with lower NIHSS scores, while higher NETs levels were associated with higher initial NIHSS scores. In the early stages of AIS, WBC count and vWF levels were negatively correlated, as well as NEU%. LYM% was positively correlated with vWF level; however, it was negatively correlated with NETs. EOS% was positively correlated with vWF levels. CONCLUSION: In the early stages of AIS, a higher peripheral WBC count and NEU%, combined with decreased EOS% and LYM%, were significantly correlated with a lower vWF level in the thrombus, potentially indicating more severe symptoms. Consequently, the timely administration of vWF-targeted medications is recommended for such patients. Reduced LYM% is indicative of elevated NETs levels and correlated with more severe clinical symptoms. Therefore, the prompt initiation of NETs-targeted medication is warranted for these patients.

Copper homeostasis in chronic kidney disease and its crosstalk with ferroptosis.

Chronic kidney disease (CKD) has become a global public health problem with high morbidity and mortality. Renal fibrosis can lead to end-stage renal disease (ESRD). However, there is still no effective treatment to prevent or delay the progression of CKD into ESRD. Therefore, exploring the pathogenesis of CKD is essential for preventing and treating CKD. There are a variety of trace elements in the human body that interact with each other within a complex regulatory network. Iron and copper are both vital trace elements in the body. They are critical for maintaining bodily functions, and the dysregulation of their metabolism can cause many diseases, including kidney disease. Ferroptosis is a new form of cell death characterized by iron accumulation and lipid peroxidation. Studies have shown that ferroptosis is closely related to kidney disease. However, the role of abnormal copper metabolism in kidney disease and its relationship with ferroptosis remains unclear. Here, our current knowledge regarding copper metabolism, its regulatory mechanism, and the role of abnormal copper metabolism in kidney diseases is summarized. In addition, we discuss the relationship between abnormal copper metabolism and ferroptosis to explore the possible pathogenesis and provide a potential therapeutic target for CKD.

Stimulation of lipopolysaccharide from Pseudomonas aeruginosa following H9N2 IAV infection exacerbates inflammatory responses of alveolar macrophages and decreases virus replication.

H9N2 IAV infection contributed to P. aeruginosa coinfection, causing severe hemorrhagic pneumonia in mink. In this study, the in vitro alveolar macrophage models were developed to investigate the innate immune responses to P. aeruginosa LPS stimulation following H9N2 IAV infection, using MH-S cells. The cytokine levels, apoptosis levels and the viral nucleic acid levels were detected and analyzed. As a result, the levels of IFN-α, IL-1ß, TNF-α, and IL-10 in MH-S cells with P. aeruginosa LPS stimulation following H9N2 IAV infection were significantly higher than those in MH-S cells with single H9N2 IAV infection and single LPS stimulation (P < 0.05), exacerbating inflammatory responses. LPS stimulation aggravated the apoptosis of MH-S cells with H9N2 IAV infection. Interestingly, LPS stimulation influences H9N2 IAV replication and indirectly reduced H9N2 IAV replications in in vitro AMs. It implied that LPS should play an important role in the pathogenesis of H9N2 IAV and P. aeruginosa coinfection.

Identifying circRNA-associated-ceRNA networks in juvenile spondyloarthropathies patients.

BACKGROUND: Juvenile spondyloarthropathies (JSpA) are defined as a heterogeneous group of diseases that start before the age of 16. The study aimed to identify key genes and pathways that are influenced by circRNAs and to screen potential therapeutic agents for JSpA. The study involved the analysis of circRNA expression profiles, identification of circRNA-miRNA-mRNA regulatory networks, and functional annotation of differentially expressed genes. The results of the study may have provided insights into the molecular mechanisms underlying JSpA and potential therapeutic targets for this disease. METHODS: In this study, sequencing data of circRNA, miRNA, and mRNA were obtained from the GEO datasets. The data were then analyzed to identify candidates for constructing a circRNA-miRNA-mRNA network based on circRNA-miRNA interactions and miRNA-mRNA interactions. Functional enrichments of genes were performed using the DAVID database. A PPI network was constructed using the STRING database and visualized using Cytoscape software. The MCODE plugin app was used to explore hub genes in the PPI network. The expression changes in immune cells were assessed using the online CIBERSORT algorithm to obtain the proportion of various types of immune cells. Finally, the Connectivity Map L1000 platform was used to identify potential agents for JSpA treatment. Overall, this study aimed to provide a comprehensive understanding of the molecular mechanisms underlying JSpA and to identify potential therapeutic agents for this disease. RESULTS: A total of 225 differentially expressed circRNAs (DEcircRNAs), 23 differentially expressed miRNAs (DEmiRNAs) and 1324 differentially expressed mRNAs (DEmRNAs) were identified. We integrated 5 overlapped circRNAs, 7 miRNAs and 299 target mRNAs into a circRNA-miRNA-mRNA network. We next identified 10 hub genes based on the PPI network. KEGG pathway analysis revealed that the DEGs were mainly associated with JAK-STAT signal pathway. We found that neutrophils accounted for the majority of all enriched cells. In addition, we discovered several chemicals as potential treatment options for JSpA. CONCLUSIONS: Through this bioinformatics analysis, we suggest a regulatory role for circRNAs in the pathogenesis and treatment of JSpA from the view of a competitive endogenous RNA (ceRNA) network.

TBK1-medicated DRP1 phosphorylation orchestrates mitochondrial dynamics and autophagy activation in osteoarthritis.

Mitochondrial dynamics, including mitochondrial fission and fusion, are critical for maintaining mitochondrial functions. Evidence shows that TANK-binding kinase 1 (TBK1) regulates mitochondrial fusion and fission and then mitophagy. Since a previous study demonstrates a strong correlation between mitophagy and osteoarthritis (OA), we herein investigated the potential role of TBK1 in OA process and mitochondrial functions. We demonstrated a strong correlation between TBK1 and OA, evidenced by significantly downregulated expression of TBK1 in cartilage tissue samples of OA patients and in the chondrocytes of aged mice, as well as TNF-α-stimulated phosphorylation of TBK1 in primary mouse chondrocytes. TBK1 overexpression significantly attenuated TNF-α-induced apoptosis and abnormal mitochondrial function in primary mouse chondrocytes. Furthermore, TBK1 overexpression induced remodeling of mitochondrial morphology by directly phosphorylating dynamin-related protein 1 (DRP1) at Ser637, abolishing the fission of DRP1 and preventing its fragmentation function. Moreover, TBK1 recruitment and DRP1 phosphorylation at Ser637 was necessary for engulfing damaged mitochondria by autophagosomal membranes during mitophagy. Moreover, we demonstrated that APMK/ULK1 signaling contributed to TBK1 activation. In OA mouse models established by surgical destabilization of the medial meniscus, intraarticular injection of lentivirus-TBK1 significantly ameliorated cartilage degradation via regulation of autophagy and alleviation of cell apoptosis. In conclusion, our results suggest that the TBK1/DRP1 pathway is involved in OA and pharmacological targeting of the TBK1-DRP1 cascade provides prospective therapeutic benefits for the treatment of OA.

Evaluation of Alcohol Markers in Urine and Oral Fluid after Regular and Hard Kombucha Consumption.

Although kombucha is a popular fermented beverage, the presence of alcohol markers has not been well studied despite being potential indicators of unintentional impairment. Ethyl glucuronide (EtG) and ethyl sulfate (EtS) were measured in oral fluid and urine collected after consumption of regular or hard kombucha. Participants drank within 20 min and provided all urine voids for 12 h, the first urine voids on days 2 and 3 and oral fluid specimens at fixed time points for 48 h. Screening employed liquid chromatography-tandem mass spectrometry (LC-MS-MS; EtS, 25 ng/mL cutoff [oral]; 100 ng/mL cutoff [urine]; EtG, 500 ng/mL cutoff [urine] and immunoassay (IA; EtG, 500 ng/mL cutoff [urine]). After consuming regular kombucha (n = 12 participants), EtS was not detected in oral fluid but both markers were detected by LC-MS-MS in urine specimens within the first five voids from 83% of participants with median (range) concentrations of 240 (100-3,700) ng/mL for EtS and 830 (530-2,200) ng/mL for EtG. Neither marker was positive by IA nor LC-MS-MS after day 1. After consuming hard kombucha (n = 7 participants), 2 (2.8%) of the 70 collected oral fluid specimens tested positive for EtS 3 h after consumption; however, 21 (30%) had EtS levels above the limit of detection (LOD, 10 ng/mL) after 0.5-8 h. Both markers were detected in urine specimens from all participants with median (range) concentrations of 3,381 (559-70,250) ng/mL for EtS and 763 (104-12,864) ng/mL For EtG. Urine specimens were negative for EtG and EtS by the end of the 48-hour study.

The relationship between serum vitamin K concentration and coronary artery calcification in middle-aged and elderly people.

BACKGROUND: Vitamin K is involved in the formation of coronary artery calcification which is an independent predictor of coronary heart disease. This study aims to explore the association between coronary artery calcification score and serum concentrations of vitamin K1, menaquinone-4 (MK-4) and menaquinone-7 (MK-7) in middle-aged and elderly Chinese population. METHODS: A total of 116 patients who underwent CT coronary angiography were consecutively enrolled. Serum concentrations of vitamin K1, MK-4 and MK-7 were determined by high performance liquid chromatography tandem mass spectrometry. The relationships between coronary artery calcification score and serum vitamin K concentrations were analyzed. RESULTS: Significantly lower serum vitamin K1 concentration was found in the patients with CACS > 400, comparing with the other CACS categories, respectively. Log (CACS + 1) was significantly higher in MK-4 < 0.05 ng/ml group compared with MK-4 ≥ 0.05 ng/ml group [2.03(0.21, 2.58) vs 1.31(0.00, 2.19), P < 0.05]. In subjects with established coronary calcification (defined as CACS > 10), vitamin K1 was found to be an independent factor contributing to higher CACS (r = -0.288, P = 0.013). CONCLUSIONS: In this retrospective analysis, serum vitamin K1 and MK-4 concentrations were significantly lower in middle-aged and elderly cohorts with increasing calcification scores. The significant effect of vitamin K1 on CACS was only found in individuals who already had calcification. Whether the detection of circulating vitamin K in patients with preexisting coronary calcification could guide vitamin K supplementation needs further exploration.

Peri-treatment adverse events of primary mediastinal non-seminomatous germ cell tumors.

Primary mediastinal non-seminomatous germ cell tumors (PMNSGCT) are rare but life-threatening thoracic cancers. We report our experience from eight patients with peri-treatment adverse events. By analyzing changes in tumor extent, serum tumor markers, and pathologies between diagnosis and transfer, those events could be attributed to postbiopsy respiratory insufficiency, growing teratoma syndrome, secondary histiocytic malignancy, and PMNSGCT progression. Subjecting patients to respiratory therapy, conventional or high-dose chemotherapy, and surgery controlled the disease, with five of the eight patients surviving disease free. These outcomes indicate that integrated appropriate and timely approaches are important in tackling peri-treatment adverse events.

Discharge of treated Fukushima nuclear accident contaminated water: macroscopic and microscopic simulations.

The diffusion process of the treated Fukushima nuclear accident contaminated water to be discharged into the Pacific Ocean from 2023 is analyzed by two analysis models from macroscopic and microscopic perspectives. Results show that the tritium will spread to the whole North Pacific in 1200 days, which is important to formulate global coping strategies.

Determination of morphine, codeine, and thebaine concentrations from poppy seed tea using magnetic carbon nanotubes facilitated dispersive micro-solid phase extraction and GC-MS analysis.

With tightening enforcement and restrictions amid the opioid epidemic, poppy seed tea is consumed as an alternative to mitigate the withdrawal symptoms or as a home remedy to relieve pain and stress. Previously published studies suggested the potential danger of consuming tea brewed with a moderate to a large amount of poppy seed. In this study, the effects of small quantity and repeat brewing on opiate concentrations were evaluated. A dispersive-micro solid phase extraction facilitated by magnetic carbon nanotubes (Mag-CNTs/d-µSPE) was developed, optimized, successfully validated, and applied to ten poppy seed tea samples using gas chromatography-mass spectrometry (GC-MS) analysis. A total of ten poppy seed samples were evaluated in this work. Two grams of bulk poppy seeds were brewed with 6 mL of heated and acidified DI water three times. The brewed tea samples were subjected to the validated Mag-CNTs/d-µSPE/GC-MS analysis. The total mean opiate concentrations obtained from three brews were 1.1-1926, 20.2-311, and 9.0-100 mg/kg for morphine, codeine, and thebaine, respectively. The total opiate yields obtained from the small quantity brewing, i.e., 6 g seed in 18 mL tea, in this study may provide minimal analgesic and euphoric effects. Over 80% of the total opiate yield was extracted in the first brew with acidified deionized water from the 10 min brewing period, and opiate yields from the second and third brew were minimal. However, potential overdose could occur for some tea samples when scaled up to the starter quantity of seed suggested for new users.

Targeting integrin αvß3 by a rationally designed protein for chronic liver disease treatment.

Chronic Liver Diseases (CLD) are characterized by abnormal accumulation of collagen fibrils, neo-angiogenesis, and sinusoidal remodeling. Collagen deposition along with intrahepatic angiogenesis and sinusoidal remodeling alters sinusoid structure resulting in portal hypertension, liver failure, and other complications. Efforts were made to develop treatments for CLDs. However, the success of such treatments is limited and unpredictable. We report a strategy for CLD treatment by induction of integrin αvß3 mediated cell apoptosis using a rationally designed protein (ProAgio). ProAgio is designed to target integrin αvß3 at a novel site. Integrin αvß3 is highly expressed in activated Hepatic Stellate Cells (HSC), angiogenic endothelium, and capillarized Liver Sinusoidal Endothelial Cells (LSEC). ProAgio induces apoptosis of these disease causative cells. Tests with liver fibrosis mouse models demonstrate that ProAgio reverses liver fibrosis and relieves blood flow resistance by depleting activated HSC and capillarized LSEC. Our studies demonstrate an effective approach for CLD treatment.

Potential impacts of 3D modeling and 3D printing in firearm toolmark examinations.

In this work, two gun barrels were 3D-printed using a metal alloy material from the same 3D digital model created in silico. After assembling the barrels in a reference Colt 45 Auto semi-automatic pistol, a total of 100 rounds of ammunition were successfully test-fired through each barrel. Heavy and gross striations were observed on all fired bullets. The striations on bullets discharged from the corresponding 3D-printed gun barrel were found to be identifiable. Moreover, bullets fired from one 3D-printed gun barrel were easily excluded from those fired from the other 3D-printed gun barrel by visual examination under a comparison microscope. The resulting unconventional striations that were observed were apparent to an experienced firearm and toolmark examiner. These features could provide valuable investigative leads related to the 3D printing process. Since 3D printing has become an option for firearm manufacturing, the forensic science community should establish a knowledge base associated with the toolmark features generated by the 3D-printed products.

The response of benthic foraminifera to heavy metals and grain sizes: A case study from Hainan Island, China.

Benthic foraminifera, heavy metals, and sediment grain sizes were studied in three bays of Hainan Island, and canonical correspondence analysis (CCA) and nonparametric regression were used to reveal the relationship between foraminifera and their environment. According to our survey, the three bays were moderately contaminated by Mo and As and uncontaminated to moderately contaminated by Pb, Zn, Cr, Sb, and Hg. The spatial pattern of heavy metals was comparable to sediment transport trends, indicating that their distribution was determined by sediment transport. Both living and dead foraminiferal assemblages were analyzed, and their compositions were similar, although the latter had a higher density and diversity. Based on the CCA method, species were divided into three groups, each of which responded differently to heavy metals and grain sizes. The response curves of individual species to heavy metals and grain sizes were obtained by using the Loess (locally weighted regression) method.

Analysis of Development Mechanism of Giant Cell Arteritis in Nude Mouse Model through Color Duplex Sonography and Computerized Tomography Nanocontrast Agent.

To explore the application value of color duplex sonography and enhanced computerized tomography (CT) inspection based on a nanocontrast agent in diagnosis and pathogenesis in giant cell arteritis (GCA), the GCA nude mouse model was constructed. In this study, 40 healthy male BalB/c nude mice aged 6-8 weeks were randomly divided into a control group (no model) and an experimental group (model), with 20 mice in each group, and the temporal artery tissue of GCA patients diagnosed as positive by temporal artery biopsy was implanted into nude mice to construct a GCA nude mouse model. Abdominal aortic biopsy and immunohistochemistry were used to verify the success of the GCA nude mouse model. All nude mice were subjected to color duplex sonography and enhanced CT examination based on a nanocontrast agent. At the same time, the basic indicators such as body weight, temperature, white blood cell (WBC), lymphocytes (LYM), hemoglobin (HGB), and platelet (PLT) were measured, and the protein expression levels of interleukin-6 (IL-6) and signal transducer and activator of transcription 3 (STAT3) were detected by immunohistochemistry. The results showed that the temporal artery wall of the nude mice in the experimental group thickened and the lumen was significantly narrowed, indicating that the cell arteritis model of nude mice was successfully constructed; ultrasound examination showed that the right superficial temporal artery vascular cavity narrowed, the blood flow signal changed like a filling defect around the periphery, and there was a low echo halo. CT examination showed that the left superficial temporal artery narrowed, and the inner diameter of the narrow segment of blood vessels changed like a bead. The body weight of nude mice in the experimental group decreased significantly after the modeling was completed (P < 0.05); after modeling, the body temperature of the nude mice in the experimental group increased significantly (P < 0.05); LYM and HGB values of nude mice in the experimental group were significantly lower than those in the control group (P < 0.05); the content of IL-6, STAT3, IL-6, and STAT3 proteins in the arterial tissue of nude mice in the experimental group was lower than that of the control group (P < 0.05), indicating that color duplex sonography and CT contrast agent technology can be used in the diagnosis and development mechanism research of GC.

Aiding diagnosis of childhood attention-deficit/hyperactivity disorder of the inattentive presentation: Discriminant function analysis of multi-domain measures including EEG.

INTRODUCTION: We developed a neurocognitive assessment tool (NCAT) in consultation with mental health professionals working with children with AD/HD as a diagnostic aid and screening tool. This study examines the predictive utility of NCAT in the classification of children with AD/HD Inattentive presentation. METHOD: Fifty three children with AD/HD Inattentive presentation and 161 typically-developing children completed an NCAT assessment. Discriminant function analyses examined group membership prediction for separate components of NCAT and for the components combined. RESULTS: The combined model correctly classified 93.4 % of participants, with 91.4 % sensitivity and 93.9 % specificity. Contributions to classification were from SNAP-IV, psychological needs satisfaction, self-regulation, executive function performance, and EEG. The combined model resulted in a 9.3 % increase in specificity and 5.9 % increase in sensitivity compared to SNAP-IV alone. CONCLUSIONS: NCAT provides good discrimination between children with and without AD/HD of the Inattentive presentation, and further investigation including other subtypes and comorbidities is warranted.

CDCA3 Is a Novel Prognostic Biomarker Associated with Immune Infiltration in Hepatocellular Carcinoma.

Cell division cycle-associated protein-3 (CDCA3) contributes to the regulation of the cell cycle. CDCA3 plays an important role in the carcinogenesis of various cancers; however, the association between CDCA3 expression, prognosis of patients, and immune infiltration in the tumor microenvironment is still unknown. Here, we demonstrated that CDCA3 was differentially expressed between the tumor tissues and corresponding normal tissues using in silico analysis in the ONCOMINE and Tumor Immune Estimation Resource (TIMER) databases. We analyzed the relationship between the expression of CDCA3 and prognosis of patients with hepatocellular carcinoma (HCC) using the Kaplan-Meier plotter database and Gene Expression Profiling Interactive Analysis (GEPIA). Furthermore, we determined the prognostic value of CDCA3 expression using univariate and multivariate analyses. We observed that CDCA3 expression closely correlated with immune infiltration and gene markers of infiltrating immune cells in the TIMER database. CDCA3 was highly expressed in the tumor tissues than in the adjacent normal tissues in various cancers, including HCC. Increased expression of CDCA3 was accompanied by poorer overall survival (OS), relapse-free survival (RFS), progression-free survival (PFS), and disease-specific survival (DSS). The correlation between CDCA3 expression and OS and disease-free survival (DFS) was also studied using GEPIA. CDCA3 expression was associated with the levels of immune cell infiltration and was positively correlated with tumor purity. Moreover, CDCA3 expression was associated with gene markers such as PD-1, CTLA4, LAG3, and TIM-3 from exhausted T cells, CD3D, CD3E, and CD2 from T cells, and TGFB1 and CCR8 located on the surface of Tregs. Thus, we demonstrated that CDCA3 may be a potential target and biomarker for the management and diagnosis of HCC.

Non-numerical strategies used by bees to solve numerical cognition tasks.

We examined how bees solve a visual discrimination task with stimuli commonly used in numerical cognition studies. Bees performed well on the task, but additional tests showed that they had learned continuous (non-numerical) cues. A network model using biologically plausible visual feature filtering and a simple associative rule was capable of learning the task using only continuous cues inherent in the training stimuli, with no numerical processing. This model was also able to reproduce behaviours that have been considered in other studies indicative of numerical cognition. Our results support the idea that a sense of magnitude may be more primitive and basic than a sense of number. Our findings highlight how problematic inadvertent continuous cues can be for studies of numerical cognition. This remains a deep issue within the field that requires increased vigilance and cleverness from the experimenter. We suggest ways of better assessing numerical cognition in non-speaking animals, including assessing the use of all alternative cues in one test, using cross-modal cues, analysing behavioural responses to detect underlying strategies, and finding the neural substrate.

Modulation of Cancer-Associated Fibrotic Stroma by An Integrin αvß3 Targeting Protein for Pancreatic Cancer Treatment.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is resistant to most therapeutics owing to dense fibrotic stroma orchestrated by cancer-associated pancreatic stellate cells (CAPaSC). CAPaSC also support cancer cell growth, metastasis, and resistance to apoptosis. Currently, there is no effective therapy for PDAC that specifically targets CAPaSC. We previously reported a rationally designed protein, ProAgio, that targets integrin αvß3 at a novel site and induces apoptosis in integrin αvß3-expressing cells. Because both CAPaSC and angiogenic endothelial cells express high levels of integrin αvß3, we aimed to analyze the effects of ProAgio in PDAC tumor. METHODS: Expression of integrin αvß3 was examined in both patient tissue and cultured cells. The effects of ProAgio on CAPaSC were analyzed using an apoptosis assay kit. The effects of ProAgio in PDAC tumor were studied in 3 murine tumor models: subcutaneous xenograft, genetic engineered (KrasG12D; p53R172H; Pdx1-Cre, GEM-KPC) mice, and an orthotopic KrasG12D; p53R172H; Pdx1-Cre (KPC) model. RESULTS: ProAgio induces apoptosis in CAPaSC. ProAgio treatment significantly prolonged survival of a genetically engineered mouse-KPC and orthotopic KPC mice alone or in combination with gemcitabine (Gem). ProAgio specifically induced apoptosis in CAPaSC, resorbed collagen, and opened collapsed tumor vessels without an increase in angiogenesis in PDAC tumor, enabling drug delivery into the tumor. ProAgio decreased intratumoral insulin-like growth factor 1 levels as a result of depletion of CAPaSC and consequently decreased cytidine deaminase, a Gem metabolism enzyme in cancer cells, and thereby reduced resistance to Gem-induced apoptosis. CONCLUSIONS: Our study suggests that ProAgio is an effective PDAC treatment agent because it specifically depletes CAPaSC and eliminates tumor angiogenesis, thereby enhancing drug delivery and Gem efficacy in PDAC tumors.

Evaluation of the Long-Term Storage Stability of the Cyanide Antidote: Dimethyl Trisulfide and Degradation Product Identification.

This study reports the long-term storage stability of a formulation of the cyanide (CN) antidote dimethyl trisulfide (DMTS). The F3-formulated DMTS was stored in glass ampules at 4, 22, and 37 °C. Over a period of one year, nine ampules (n = 3 at each temperature) were analyzed by high-performance liquid chromatography (HPLC)-UV/vis at daily time intervals in the first week, weekly time intervals in the first month, and monthly thereafter for a period of one year to determine the DMTS content. No measurable loss of DMTS was found at 4 and 22 °C, and good stability was noted up to five months for samples stored at 37 °C. At 37 °C, a 10% (M/M) decrease of DMTS was discovered at the sixth month and only 30% (M/M) of DMTS remained by the end of the study; discoloration of the formulation and the growth of new peaks in the HPLC chromatogram were also observed. To identify the unknown peaks at 37 °C, controlled oxidation studies were performed on DMTS using two strong oxidizing agents: meta-chloroperoxybenzoic acid (mCPBA) and hydrogen peroxide (H2O2). Dimethyl tetrasulfide and dimethyl pentasulfide were observed as products using both of the oxidizing agents. Dimethyl disulfide was also observed as a product of degradation, which was further oxidized to S-methyl methanethiosulfonate only when mCPBA was used. HPLC-UV/vis and gas chromatography-mass spectrometry/solid phase microextraction analysis revealed good agreement between the degradation products of the stability study at 37 °C and those of disproportionation reactions. Furthermore, at 4 and 22 °C, chromatograms were remarkably stable over the one-year study period, indicating that the F3-formulated DMTS shows excellent long-term storage stability at T ≤ 22 °C.

Development of Different Methods for Preparing Acinetobacter baumannii Outer Membrane Vesicles Vaccine: Impact of Preparation Method on Protective Efficacy.

Acinetobacter baumannii (A. baumannii) is becoming a common global concern due to the emergence of multi-drug or pan-drug resistant strains. Confronting the issue of antimicrobial resistance by developing vaccines against the resistant pathogen is becoming a common strategy. In this study, different methods for preparing A. baumannii outer membrane vesicles (AbOMVs) vaccines were developed. sOMV (spontaneously released AbOMV) was extracted from the culture supernatant, while SuOMV (sucrose-extracted AbOMV) and nOMV (native AbOMV) were prepared from the bacterial cells. Three AbOMVs exhibited significant differences in yield, particle size, protein composition, and LPS/DNA content. To compare the protective efficacy of the three AbOMVs, groups of mice were immunized either intramuscularly or intranasally with each AbOMV. Vaccination via both routes conferred significant protection against lethal and sub-lethal A. baumannii challenge. Moreover, intranasal vaccination provided more robust protection, which may be attributed to the induction of significant sIgA response in mucosal sites. Among the three AbOMVs, SuOMV elicited the highest level of protective immunity against A. baumannii infection, whether intramuscular or intranasal immunization, which was characterized by the expression of the most profound specific serum IgG or mucosal sIgA. Taken together, the preparation method had a significant effect on the yield, morphology, and composition of AbOMVs, that further influenced the protective effect against A. baumannii infection.