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Objectives: The aim of the study was to determine the clinical features & autoantibody profile of patients having late onset Systemic Lupus Erythematosus (SLE) and to compare with young onset SLE due to its scarce data from India., Methods: All patients who fulfilled the 1997 ACR criteria for SLE were included. Late onset patients were >50 years of age and young onset were <50 years >18 years at the time of first SLE-related symptom. Clinical, laboratory, and autoantibody (ENA 25 & APLA) profiles were compared between the two groups using descriptive statistics and chi square test., Results: Of the 305 patients, 69 had late onset (75.4% females). Mean age was 59.42±6.7 years (Late onset lupus) and 33.13±8.44 years (young onset lupus). The most common symptom was arthritis (60%) followed by oral ulceration (50%), fever (43%), and serositis (37.68%). Most common antibody was SSA/Ro60 (50%) and anti-SSA/Ro52 (46%). Interstitial lung disease (ILD) (14.5%), pancytopenia (13%) and diffuse alveolar haemorrhage (4.3%) were more frequent in late onset group. Statistically significant differences were found between two groups in terms of photosensitivity (p=0.009), malar rash (p=0.005), excessive hair loss (p=0.0006), Raynaud's phenomenon (p=0.001), lymphadenopathy (p=0.01), nephritis (p=0.0007), ILD (p=0.01), anti-dsDNA (p=0.005), anti-nucleosome (p=0.01), anti-Sm (p=0.007), Ribosomes P0 (p=0.0004)., Conclusion: This study suggests that late onset SLE has distinct clinical and serological manifestations when compared with young onset SLE patients., Competing Interests: The authors declare no conflict of interest., (© 2023 The Mediterranean Journal of Rheumatology (MJR).)

Competing Interests: The authors declare no conflict of interest.

Immunoglobulin G4-related mastitis (IgG4-RM) is an uncommon entity in clinical practice which has an evolving spectrum of manifestations and presently is of high clinical interest among rheumatologists. Since its closest differential remains breast carcinoma, the importance of describing this entity is in the fact that early diagnosis, awareness and timely management can save the patient from unnecessary surgical intervention and its complications. Tumefactive lesions are considered hallmark of this disease, but rarely abscess may also be the presenting feature. In this article, we describe a 24-year-old female patient of a very unusual case of IgG4-RM presenting as recurrent breast abscess, its successful management and discussion about novel treatment strategies., Competing Interests: Conflict of Interest: The authors declared no conflicts of interest with respect to the authorship and/or publication of this article., (Copyright © 2020, Turkish League Against Rheumatism.)

We describe protein synthesis, folding and assembly of antibody fragments and full-length aglycosylated antibodies using an Escherichia coli-based open cell-free synthesis (OCFS) system. We use DNA template design and high throughput screening at microliter scale to rapidly optimize production of single-chain Fv (scFv) and Fab antibody fragments that bind to human IL-23 and IL-13α1R, respectively. In addition we demonstrate production of aglycosylated immunoglobulin G (IgG 1) trastuzumab. These antibodies are produced rapidly over several hours in batch mode in standard bioreactors with linear scalable yields of hundreds of milligrams/L over a 1 million-fold change in scales up to pilot scale production. We demonstrate protein expression optimization of translation initiation region (TIR) libraries from gene synthesized linear DNA templates, optimization of the temporal assembly of a Fab from independent heavy chain and light chain plasmids and optimized expression of fully assembled trastuzumab that is equivalent to mammalian expressed material in biophysical and affinity based assays. These results illustrate how the open nature of the cell-free system can be used as a seamless antibody engineering platform from discovery to preclinical development of aglycosylated monoclonal antibodies and antibody fragments as potential therapeutics.

Metabolomics involves qualitative and/or quantitative analysis of hundreds of metabolites in a complex sample. As most of the metabolites are polar in nature (for example, amino acids, nucleotides, carboxylic acids), their chromatographic separation and analysis present a difficult challenge. Recently, hydrophilic interaction chromatography (HILIC) has become a useful tool for analysis of such polar molecules. In this chapter, we present a simple HILIC LC-MS/MS method for targeted multiple reaction monitoring (MRM)-based relative quantitation of hundreds of polar metabolites in a complex sample. The method uses an aminopropyl column with acetonitrile as weak solvent and 20 mM ammonium acetate buffer (pH 9.4) as strong solvent. The method does not use any ion pairing reagent making it suitable for analysis in both positive and negative ionization mode using a simple LC-MS/MS instrument setup. The method has been thoroughly tested, validated, and applied to a variety of samples such as bacteria, yeast, plants, human body fluids, and cell cultures.

The ability of an organism to acclimate to its environment is a key determinant in its global distribution and capacity to compete with other organisms. The heat stress response, a highly conserved environmental and developmental program in eukaryotic and prokaryotic organisms, is an important component of the acclimation response of plants. Previous studies have shown that heat shock transcription factors play an important role in thermotolerance in plants and other organisms, controlling the expression of different heat shock proteins and detoxifying enzymes. In contrast, although several other pathways, involving ethylene, salicylic acid (SA), and trehalose, were recently shown to play a central role in thermotolerance in plants, a key regulator of these responses was not identified. Here we report that the highly conserved transcriptional co-activator, MBF1c (multiprotein bridging factor 1c), is a key regulator of thermotolerance in Arabidopsis thaliana. MBF1c protein accumulates rapidly and is localized to nuclei during heat stress. MBF1c is required for thermotolerance and functions upstream to SA, trehalose, ethylene, and pathogenesis-related protein 1 during heat stress. In contrast, MBF1c is not required for the expression of transcripts encoding HSFA2 and different heat shock proteins. Interestingly, MBF1c interacts with TPS5 (trehalose phosphate synthase 5), which is also heat-inducible, and mutants deficient in TPS5 are thermosensitive. Our results provide evidence for the existence of a tightly coordinated heat stress-response network, involving trehalose-, SA-, and ethylene-signaling pathways, that is under the control of MBF1c.

The 'omics' approaches - genomics, proteomics and metabolomics - are based on high-throughput, high-information-content analysis. Using these approaches, as opposed to targeting one or a few analytes, a holistic understanding of the composition of a sample can be obtained. These approaches have revolutionized sample-analysis and data-processing protocols. In metabolomic studies, hundreds of small molecules are simultaneously analyzed using analytical platforms (e.g., gas chromatography-mass spectrometry (GC-MS) or liquid chromatography coupled to tandem mass spectrometry (LC-MS(2))). This philosophy of holistic analysis and the application of high-throughput, high-information-content analysis offer several advantages. In this article, we compare the conventional analytical approach of one or a few analytes per sample to the LC-MS(2)-based metabolomics-type approach in the context of pharmaceutical and environmental analysis.

The intraerythrocytic malaria parasite constructs an intracellular haem crystal, called haemozoin, within an acidic digestive vacuole where haemoglobin is degraded. Haem crystallization is the target of the widely used antimalarial quinoline drugs. The intracellular mechanism of molecular initiation of haem crystallization, whether by proteins, polar membrane lipids or by neutral lipids, has not been fully substantiated. In the present study, we show neutral lipid predominant nanospheres, which envelop haemozoin inside Plasmodium falciparum digestive vacuoles. Subcellular fractionation of parasite-derived haemozoin through a dense 1.7 M sucrose cushion identifies monoacylglycerol and diacylglycerol neutral lipids as well as some polar lipids in close association with the purified haemozoin. Global MS lipidomics detects monopalmitic glycerol and monostearic glycerol, but not mono-oleic glycerol, closely associated with haemozoin. The complex neutral lipid mixture rapidly initiates haem crystallization, with reversible pH-dependent quinoline inhibition associated with quinoline entry into the neutral lipid microenvironment. Neutral lipid nanospheres both enable haem crystallization in the presence of high globin concentrations and protect haem from H2O2 degradation. Conceptually, the present study shifts the intracellular microenvironment of haem crystallization and quinoline inhibition from a polar aqueous location to a non-polar neutral lipid nanosphere able to exclude water for efficient haem crystallization.

Objective: The objective of these studies was to determine the role of ascorbate deficiency in HIV infection in the defective detoxification of sulfamethoxazole-nitroso, the metabolite thought to mediate sulfonamide hypersensitivity reactions., Methods: Fifty-one HIV-infected patients and 26 healthy volunteers were evaluated. Vitamin supplementation histories were obtained, and blood samples were collected for determination of plasma ascorbate, dehydroascorbate, and cysteine concentrations, erythrocyte glutathione concentrations, and plasma reduction of sulfamethoxazole-nitroso in vitro., Results: Plasma ascorbate concentrations were significantly lower in HIV-positive patients not taking vitamin supplements (29.5 +/- 22.3 microM) than in healthy subjects (54.8 +/- 22.3 microM; P = 0.0005) and patients taking 500-1000 mg of ascorbate daily (82.5 +/- 26.3 microM; P < 0.0001). Plasma ascorbate deficiency was strongly correlated with impaired reduction of sulfamethoxazole-nitroso to its hydroxylamine (r = 0.60, P < 0.0001), and during in vitro reduction, the loss of plasma ascorbate was strongly associated with the amount of nitroso reduced (r = 0.70, P < 0.0001). Ascorbate added ex vivo normalized this reduction pathway. Erythrocyte glutathione concentrations were significantly lower in HIV-positive patients (0.98+/-0.32 mM) than in healthy subjects (1.45+/-0.49 mM; P = 0.001), but this finding was unrelated to ascorbate supplementation. There was trend toward lower plasma cysteine concentrations in patients (8.4+/-3.9 microM) than in controls (10.3+/-4.3 microM), but this trend was similarly unrelated to ascorbate supplementation. Dehydroascorbate concentrations were not significantly higher in HIV-positive patients (7.4+/-10.5%) than in healthy controls (4.0+/-6.2%), even in the subset of patients taking ascorbate (8.4+/-9.4%)., Conclusions: Ascorbate deficiency is common in HIV-positive patients and is associated with impaired detoxification of sulfamethoxazole-nitroso, the suspected proximate toxin in sulfonamide hypersensitivity. Patients taking daily ascorbate supplements (500-1000 mg) achieved high plasma ascorbate concentrations and did not show this detoxification defect. Ascorbate deficiency (or supplementation) was not associated with changes in glutathione or cysteine concentrations. These data suggest that ascorbate deficiency, independent of thiol status, may be an important determinant of impaired drug detoxification in HIV infection.

Potential of piperine, an active alkaloid of black and long peppers, to increase the bioavailability of drugs in humans is of great clinical significance owing to its omnipresence in food. In an attempt to further study the reported differences in its metabolism in rats and humans, a new major urinary metabolite was detected in rat urine and plasma using HPLC. The metabolite was partially purified using reverse phase column chromatography on Sephadex((R))-LH 20 and characterized as 5-(3, 4-methylenedioxy phenyl)-2E,4E-pentadienoic acid-N-(3-yl propionic acid)-amide with the help of LC/NMR/positive ESI-MS studies. Complete mass fragmentation pattern could be assigned with MS/MS studies. The metabolite has a unique structure compared to the previously reported metabolites in that it retains methylenedioxy ring and conjugated double bonds while the piperidine ring is modified to form propionic acid group. Mechanism of formation of the metabolite by oxidation and cleavage of piperidine ring is proposed. Kidney appears to be the major excretion route for piperine metabolites in rats as no metabolite could be detected in feces.

Piperine, a major alkaloid of Piper longum and Piper nigrum has been reported to have several pharmacological/toxicological effects. Though a number of methods for analysis of this omnipresent food component in pepper fruits are available, its analysis in body fluids has been largely neglected. A high-performance liquid chromatography method for the analysis of piperine in rat plasma is presented in this communication. Analysis was performed using a Symmetry C(18) column (250x4.6 mm) by isocratic elution with 25 mM KH(2)PO(4) (pH 4.5)-acetonitrile (35:65) and UV detection at 340 nm. The calibration plot was linear over the range studied (2-2000 ng) with correlation coefficient of 0.9984. Limit of detection and limit of quantitation were 1 ng/ml and 3 ng/ml, respectively. Good overall recovery (85.5+/-6%) was obtained with 4 ml ethyl acetate and extraction time of 3 min. Intra- and inter-assay coefficient of variation was found to be less than 7.5%. Plasma concentration-time profile of piperine in a conscious rat implanted with jugular vein cannula was obtained using this method. The method is simple, sensitive and reproducible., (Copyright 2002 Elsevier Science B.V.)

Piperine, a major alkaloid of black and long peppers has been reported to act as bioavailability enhancer of several drugs by inhibiting drug metabolising enzymes and/or by increasing oral absorption. Ketoconazole is a well established potent inhibitor of CYP 3A4 and P-glycoprotein. A simple and rapid HPLC method has been developed for the simultaneous analysis of ketoconazole and piperine in rat plasma and hepatocyte culture. Analysis was performed using a Symmetry C18 column (150x4.6 mm, 5 microm) and isocratic elution with 25 mM KH2PO4 (pH 4.5)-acetonitrile (50:50) with a flow-rate of 1 ml/min. Photodiode array detection was used to simultaneously monitor piperine at 340 nm and ketoconazole at 231 nm in a single sample. Calibration plots in spiked plasma, hepatocytes and William's medium E were linear over the range studied (10-2000 ng for both drugs). The detection limits for piperine and ketoconazole are 2 and 4 ng, respectively, and the limits of quantitation are 10 and 12 ng, respectively. Intra- and inter-assay variations were less than 8%.