Verification of chlorine exposure via LC-MS/MS analysis of base hydrolyzed chlorophenols from chlorotyrosine-protein adducts.

Inhalation of chlorine gas, with subsequent hydrolysis in the airway and lungs to form hydrochloric acid (HCl) and hypochlorous acid (HOCl), can cause pulmonary edema (i.e., fluid build-up in the lungs), pulmonary inflammation (with or without infection), respiratory failure, and death. The HOCl produced from chlorine is known to react with tyrosine to form adducts via electrophilic aromatic substitution, resulting in 3-chlorotyrosine and 3,5-dichlorotyrosine adducts. While several analysis methods are available for determining these adducts, each method has significant disadvantages. Hence, a simple and sensitive ultra-high performance liquid chromatography-tandem mass spectroscopy (UHPLC-MS/MS) method was developed for the determination of chlorotyrosine adducts. The sample preparation involves base hydrolysis of isolated plasma proteins to form 2-chlorophenol (CP) from monochlorotyrosine adducts and 2,6-dichlorophenol (2,6-DCP), from dichlorotyrosine adducts, as markers of chlorine exposure. The chlorophenols are extracted with cyclohexane prior to UHPLC-MS/MS analysis. The method produced excellent sensitivity for 2,6-DCP with a limit of detection of 2.2 µg/kg, calibration curve linearity extending from 0.054-54 mg/kg (R2 ≥ 0.9997 and %RA > 94), and accuracy and precision of 100 ± 14 %, and <15 % relative standard deviation, respectively. The sensitivity of the method for 2-CP was relatively poor, so it was used only as a secondary marker for severe chlorine exposure. The method successfully detected elevated levels of 2,6-DCP from hypochlorite-spiked plasma protein and plasma protein isolated from chlorine-exposed rats.

TLR4 Ligation by eNAMPT, a Novel DAMP, is Essential to Sulfur Mustard- Induced Inflammatory Lung Injury and Fibrosis.

Objective: Human and preclinical studies of sulfur mustard (SM)-induced acute and chronic lung injuries highlight the role of unremitting inflammation. We assessed the utility of targeting the novel DAMP and TLR4 ligand, eNAMPT (extracellular nicotinamide phosphoribosyltransferase), utilizing a humanized mAb (ALT-100) in rat models of SM exposure. Methods: Acute (SM 4.2 mg/kg, 24 hrs), subacute (SM 0.8 mg/kg, day 7), subacute (SM 2.1 mg/kg, day 14), and chronic (SM 1.2 mg/kg, day 29) SM models were utilized. Results: Each SM model exhibited significant increases in eNAMPT expression (lung homogenates) and increased levels of phosphorylated NFkB and NOX4. Lung fibrosis (Trichrome staining) was observed in both sub-acute and chronic SM models in conjunction with elevated smooth muscle actin (SMA), TGFß, and IL-1ß expression. SM-exposed rats receiving ALT-100 (1 or 4 mg/kg, weekly) exhibited increased survival, highly significant reductions in histologic/biochemical evidence of lung inflammation and fibrosis (Trichrome staining, decreased pNFkB, SMA, TGFß, NOX4), decreased airways strictures, and decreased plasma cytokine levels (eNAMPT, IL-6, IL-1ß. TNFα). Conclusion: The highly druggable, eNAMPT/TLR4 signaling pathway is a key contributor to SM-induced ROS production, inflammatory lung injury and fibrosis. The ALT-100 mAb is a potential medical countermeasure to address the unmet need to reduce SM-associated lung pathobiology/mortality.

Overexpression of ameloblastin in secretory ameloblasts results in demarcated, hypomineralized opacities in enamel.

Introduction: Developmental defects of the enamel manifest before tooth eruption and include amelogenesis imperfecta, a rare disease of underlying gene mutations, and molar-incisor hypomineralization (MIH), a prevalent disease in children originating from environmental and epigenetic factors. MIH enamel presents as the abnormal enamel marked by loss of translucency, demarcation between the healthy and affected enamel, and reduced mineral content. The pathophysiology of opaque, demarcated enamel lesions is not understood; however, the retention of enamel proteins in the matrix has been suggested. Ameloblastin (Ambn) is an enamel protein of the secreted calcium-binding phosphoproteins (SCPPs) critical for enamel formation. When the Ambn gene is mutated or deleted, teeth are affected by hypoplastic amelogenesis imperfecta. Methods: In this study, enamel formation in mice was analyzed when transgenic Ambn was overexpressed from the amelogenin promoter encoding full-length Ambn. Ambn was under- and overexpressed at six increasing concentrations in separate mouse lines. Results: Mice overexpressing Ambn displayed opaque enamel at low concentrations and demarcated lesions at high concentrations. The severity of enamel lesions increased starting from the inner enamel close to the dentino-enamel junction (DEJ) to span the entire width of the enamel layer in demarcated areas. Associated with the opaque enamel were 17-kDa Ambn cleavage products, a prolonged secretory stage, and a thin basement membrane in the maturation stage. Ambn accumulations found in the innermost enamel close to the DEJ and the mineralization front correlated with reduced mineral content. Demarcated enamel lesions were associated with Ambn species of 17 kDa and higher, prolonged secretory and transition stages, a thin basement membrane, and shortened maturation stages. Hypomineralized opacities were delineated against the surrounding mineralized enamel and adjacent to ameloblasts detached from the enamel surface. Inefficient Ambn cleavage, loss of contact between ameloblasts, and the altered basement membrane curtailed the endocytic activity; thus, enamel proteins remained unresorbed in the matrix. Ameloblasts have the ability to distinguish between Ambn concentration and Ambn cleavage products through finely tuned feedback mechanisms. The under- or overexpression of Ambn in murine secretory ameloblasts results in either hypoplastic amelogenesis imperfecta or hypomineralization with opaque or sharply demarcated boundaries of lesions, similar to MIH.

Effect of Two Different Rehabilitation Approaches on Pulmonary Functional Tests, Neuromuscular Functions and Quality of Life in Juvenile Myasthenia Gravis: A Randomized Controlled Trial Study.

Background and Objectives: Children with juvenile myasthenia gravis have a variety of symptoms, ranging from isolated intermittent ocular complaints to overall muscle weakness with or without respiratory insufficiency. This study aimed to investigate the efficacy of a specialized physical therapy with or without partial body weight supported treadmill training on pulmonary functional tests, neuromuscular functions, and quality of life. Materials and Methods: Thirty children, ranging in age from 13 to 16 years, were distributed randomly into two study groups (A or B). Both groups underwent a designed physical therapy program. In addition, group A underwent the partial body weight supported treadmill training. The treatment was conducted three times a week for 12 weeks successively. Pulmonary functional tests (FVC, FEV1, PEFR, and MVV), neuromuscular function tests (compound motor action potential, isometric muscle force of biceps brachii and rectus femoris, balance, walking endurance, and fatigue), and quality of life were measured before and after 12 successive weeks. Results: A significant improvement in all investigated variables were recorded in both groups in favor of group A. Conclusions: Both a specialized physical therapy and partial body weight supported treadmill training are effective in terms of enhancing pulmonary functional tests, neuromuscular functions, and quality of life. Partial body weight supported treadmill training is an excellent adjunctive to the physical therapy program.

Laboratory Evaluation of Mechanical Properties of Modified Asphalt and Mixture Using Graphene Platelets (GnPs).

Recently, nanomaterials have attracted attention in the field of pavement construction as modifiers to endure heavy loads and climate changes. In this study, conventional asphalt (bitumen) of penetration grade AC (60/70) was modified with graphene platelets (GnPs) at three different contents: 0.5%, 1.0%, and 1.5% by weight of asphalt content. Kinematic viscosity, softening point, penetration, and dynamic shear rheology tests were performed to evaluate the mechanical properties of modified binder. The results showed that adding GnPs improves the mechanical properties of asphalt binder; the kinematic viscosities, softening points, and rutting parameters increased but penetrations decreased with the contents of GnPs. Hot mix asphalt specimens with GnPs-modified asphalt were prepared and characterized with Marshall tests, thermal stress restrained specimen tests (TSRST), wheel tracking tests, and indirect tensile tests. Similar to the results of asphalt binder, the mechanical properties of asphalt mixture were improved by GnPs. Marshall stability increased by 21% and flow decreased by 24% with accepted value of 2.8 mm in penetration when the mixture was modified with 1.0 wt% of GnPs. At the same GnPs content, modified asphalt mixture led to lower failure temperature by 2 °C in comparison with unmodified asphalt mixture and the cryogenic failure stress was improved by 12%. The wheel tracking tests showed that GnPs-modified asphalt mixture has outstanding deformation resistance in comparison with unmodified asphalt mixtures: after 5000 cycles, 1.0 wt% of GnPs reduced the rut depth of asphalt mixture by 60%-the rut depth of unmodified asphalt mixture was 6.9 mm compared to 2.75 mm for modified asphalt mixture. After 10,000 cycles, the modified asphalt mixture showed rut depth of 3.24 mm in comparison with 8.12 mm in case of unmodified asphalt mixture. Addition of GnPs into asphalt mixture significantly improved the indirect tensile strength: 1.0 wt% of GnPs increased the indirect tensile strength of unmodified asphalt mixture from 0.79 to 1.1 MPa recording ~40% increment. The results of this study can confirm that graphene platelets enhance the mechanical properties of asphalt mixture and its performance.

Application of remote sensing and geographic information systems in irrigation water management under water scarcity conditions in Fayoum, Egypt.

Egypt suffers from severe water scarcity, which affects the sustainability of agricultural production. Therefore, the sustainable use of available water resources under water scarcity requires the adoption of water allocation policies favoring conservative and efficient use. Water management with free satellite data and geographical information system modeling capabilities can be a valuable approach for optimizing the benefits from the available water resources to meet the requirements for agricultural lands. This study aims to (i) detect and evaluate changes in agricultural areas because of urbanization and reclamation activities using Landsat data in 1999, 2009, and 2019 and (ii) update the irrigation water demand by monitoring the seasonal changes of agricultural area based on normalized difference vegetation index. Water management of Fayoum Governorate in Egypt is characterized by a non-uniform distribution flow over its canals; thus, two pilot areas are selected. The first site is the Sinnuris canal, the served areas of which represents the urbanization problem. The other site is the Gharaq canal, the served areas of which represents the urbanization and agricultural expansion situations. The results reveal that changes in agricultural areas considerably affect the uniformity of water management. Urbanization activities reduce the agricultural area by ∼5.0% and 5.7% in Sinnuris and Gharaq served areas, respectively. However, the newly cultivated lands in Gharaq preserve an increase of 5.8% in the total agricultural area. The considerably changed water allocation strategies in these districts since Sinnuris has an excess of 1.5 m3/s of water supply, while the Gharaq area faced an irrigation shortage of 0.26 m3/s in 2019. As per the proposed approach, the decision-makers can readjust the water allocation plan to satisfy the water requirements for other demand areas.

Virulence Determinants and Antimicrobial Profiles of Pasteurella multocida Isolated from Cattle and Humans in Egypt.

Pasteurella multocida is a Gram-negative bacterium that causes drastic infections in cattle and humans. In this study, 55 isolates were recovered from 115 nasal swabs from apparently healthy and diseased cattle and humans in Minufiya and Qalyubia, Egypt. These isolates were confirmed by kmt1 existence, and molecular classification of the capsular types showed that types B, D, and E represented 23/55 (41.8%), 21/55 (38.1%), and 11/55 (20.0%), respectively. The isolates were screened for five virulence genes with hgbA, hgbB, and ptfA detected in 28/55 (50.9%), 30/55 (54.5%), and 25/55 (45.5%), respectively. We detected 17 capsular and virulence gene combinations with a discriminatory power (DI) of 0.9286; the most prevalent profiles were dcbF type D and dcbF type D, hgbA, hgbB, and ptfA, which represented 8/55 (14.5%) each. These strains exhibited high ranges of multiple antimicrobial resistance indices; the lowest resistances were against chloramphenicol, ciprofloxacin, amoxicillin/clavulanic acid, and levofloxacin. The macrolide-lincosamide-streptogramin B methylase gene erm(Q), with erm(42) encoding MLSB monomethyltransferase, mph(E) encoding a macrolide efflux pump, and msr(E) encoding macrolide-inactivating phosphotransferase were present. The class 1 and 2 integrons and extended-spectrum ß-lactamase genes intl1, intl2, blaCTX-M, blaCTX-M-1, and blaTEM were detected. It is obvious to state that co-occurrence of resistance genes resulted in multiple drug-resistant phenotypes. The identified isolates were virulent, genetically diverse, and resistant to antimicrobials, highlighting the potential risk to livestock and humans.

Expression of LRP1 and CHOP genes associated with peripheral neuropathy in type 2 diabetes mellitus: Correlations with nerve conduction studies.

AIM: Diabetic peripheral neuropathy (DPN) is a frequent and debilitating complication of diabetes mellitus. The low-density lipoprotein receptor-related protein-1 (LRP-1) is a multifunctional cell surface receptor playing critical roles in lipoprotein metabolism and several cell signaling processes. C/EBP homologous protein (CHOP) is a main conduit to endoplasmic reticulum stress-induced apoptosis. We aimed to investigate LRP1 and CHOP gene expression in peripheral blood cells of type 2 diabetes mellitus (T2DM) subjects to clarify its possible relation to DPN pathogenesis. METHOD: The study included 20 non-complicated T2DM subjects, 20 subjects with DPN and 20 healthy controls. Quantitative real time PCR was used to study gene expression. RESULTS: There was a significant reduction in LRP1 mRNA expression and a significant increase in CHOP mRNA expression in subjects with DPN compared to non-complicated group and healthy controls. Both LRP1 and CHOP expression levels were inversely correlated, and both showed significant correlation with HbA1c, hyperlipidemia, hs-CRP, and different electrophysiological parameters. Receiver operating characteristics (ROC) analysis suggested that both LRP1 and CHOP mRNA expression and hs-CRP levels had great potential advantages to predict the progression of DPN. CONCLUSION: LRP1 and CHOP might be involved in DPN pathogenesis and progression, thus providing opportunities for early detection and treatment.

Comparative analysis of novel decynium-22 analogs to inhibit transport by the low-affinity, high-capacity monoamine transporters, organic cation transporters 2 and 3, and plasma membrane monoamine transporter.

Growing evidence supports involvement of low-affinity/high-capacity organic cation transporters (OCTs) and plasma membrane monoamine transporter (PMAT) in regulating clearance of monoamines. Currently decynium-22 (D22) is the best pharmacological tool to study these transporters, however it does not readily discriminate among them, underscoring a need to develop compounds with greater selectivity for each of these transporters. We developed seven D22 analogs, and previously reported that some have lower affinity for α1-adrenoceptors than D22 and showed antidepressant-like activity in mice. Here, we extend these findings to determine the affinity of these analogs for OCT2, OCT3 and PMAT, as well as serotonin, norepinephrine and dopamine transporters (SERT, NET and DAT) using a combination of uptake competition with [3H]methyl-4-phenylpyridinium acetate in overexpressed HEK cells and [3H]citalopram, [3H]nisoxetine and [3H]WIN 35428 displacement binding in mouse hippocampal and striatal preparations. Like D22, all analogs showed greater binding affinities for OCT3 than OCT2 and PMAT. However, unlike D22, some analogs also showed modest affinity for SERT and DAT. Dual OCT3/SERT and/or OCT3/DAT actions of certain analogs may help explain their ability to produce antidepressant-like effects in mice and help account for our previous findings that D22 lacks antidepressant-like effects unless SERT function is either genetically or pharmacologically compromised. Though these analogs are not superior than D22 in discriminating among OCTs/PMAT, our findings point to development of compounds with combined ability to inhibit both low-affinity/high-capacity transporters, such as OCT3, and high-affinity/low-capacity transporters, such as SERT, as therapeutics with potentially improved efficacy for treatment of psychiatric disorders.

Chronic Hepatitis C Patients with Obesity: Do we Need two Operators for Accurate Evaluation of Liver Stiffness?

INTRODUCTION AND AIM: Transient elastography is gaining popularity as a non-invasive method for predicting liver fibrosis, but inter observer agreement and factors influencing reproducibility have not been adequately assessed. MATERIAL AND METHODS: This cross-sectional study was conducted at Specialized Medical Hospital and the Egyptian Liver Foundation, Mansoura, Egypt. The inclusion criteria were: age older than 18 years and chronic infection by hepatitis C. The exclusion criteria were the presence of ascites, pacemaker or pregnancy. Three hundred and fifty-six patients participated in the study. Therefore, 356 pairs of exams were done by two operators on the same day. RESULTS: The overall inter observer agreement ICC was 0.921. The correlation the two operators was excellent (Spearman's value q = 0.808, p < 0.001). Inter-observer reliability values were κ = 0.557 (p < 0.001). A not negligible discordance of fibrosis staging between operators was observed (87 cases, 24.4%). Discordance of at least one stage and for two or more stages of fibrosis occurred in 60 (16.9%) and 27 cases (7.6%) respectively. Obesity (BMI ≥ 30 kg/m2) is the main factor associated with discordance (p = 0.002). CONCLUSION: Although liver stiffness measurement has had an excellent correlation between the two operators, TE presented an inter-observer variability that may not be negligible.

Constitutive plasma membrane monoamine transporter (PMAT, Slc29a4) deficiency subtly affects anxiety-like and coping behaviours.

Originally, uptake-mediated termination of monoamine (e.g., serotonin and dopamine) signalling was believed to only occur via high-affinity, low-capacity transporters ("uptake1 ") such as the serotonin or dopamine transporters, respectively. Now, the important contribution of a second low-affinity, high-capacity class of biogenic amine transporters has been recognised, particularly in circumstances when uptake1 transporter function is reduced (e.g., antidepressant treatment). Pharmacologic or genetic reductions in uptake1 function can change locomotor, anxiety-like or stress-coping behaviours. Comparable behavioural investigations into reduced low-affinity, high-capacity transporter function are lacking, in part, due to a current dearth of drugs that selectively target particular low-affinity, high-capacity transporters, such as the plasma membrane monoamine transporter. Therefore, the most direct approach involves constitutive genetic knockout of these transporters. Other groups have reported that knockout of the low-affinity, high-capacity organic cation transporters 2 or 3 alters anxiety-like and stress-coping behaviours, but none have assessed behaviours in plasma membrane monoamine transporter knockout mice. Here, we evaluated adult male and female plasma membrane monoamine transporter wild-type, heterozygous and knockout mice in locomotor, anxiety-like and stress-coping behavioural tests. A mild enhancement of anxiety-related behaviour was noted in heterozygous mice. Active-coping behaviour was modestly and selectively increased in female knockout mice. These subtle behavioural changes support a supplemental role of plasma membrane monoamine transporter in serotonin and dopamine uptake, and suggest sex differences in transporter function should be examined more closely in future investigations.