Exome sequencing and functional analyses revealed CETN1 variants leads to impaired cell division and male fertility.

Human spermatogenesis requires an orchestrated expression of numerous genes in various germ cell subtypes. Therefore, the genetic landscape of male infertility is highly complex. Known genetic factors alone account for at least 15% of male infertility. However, ~40% of infertile men remain undiagnosed and are classified as idiopathic infertile men. We performed exome sequencing in 47 idiopathic infertile men (discovery cohort), followed by replication study (40 variants in 33 genes) in 844 infertile men and 709 controls using Sequenom MassARRAY® based genotyping. We report 17 variants in twelve genes that comprise both previously reported (DNAH8, DNAH17, FISP2 and SPEF2) and novel candidate genes (BRDT, CETN1, CATSPERD, GMCL1, SPATA6, TSSK4, TSKS and ZNF318) for male infertility. The latter have a strong biological nexus to human spermatogenesis and their respective mouse knockouts are concordant with human phenotypes. One candidate gene CETN1, identified in this study, was sequenced in another independent cohort of 840 infertile and 689 fertile men. Further, CETN1 variants were functionally characterized using biophysical and cell biology approaches. We demonstrate that CETN1 variant- p.Met72Thr leads to multipolar cells, fragmented nuclei during mitosis leading to cell death and show significantly perturbed ciliary disassembly dynamics. Whereas CETN1-5' UTR variant; rs367716858 leads to loss of a methylation site and increased reporter gene expression in vitro. We report a total of eight novel candidate genes identified by exome sequencing, which may have diagnostic relevance and can contribute to improved diagnostic workup and clinical management of male infertility.

Truncated variants of thyroid hormone receptor beta display disease-inflicting malfunctioning at cellular level.

Thyroid hormone receptor ß (THRß) is a member of the nuclear receptor superfamily of ligand-modulated transcription factors. Upon ligand binding, THRß sequentially recruits the components of transcriptional machinery to modulate target gene expression. In addition to regulating diverse physiological processes, THRß plays a crucial role in hypothalamus-pituitary-thyroid axis feedback regulation. Anomalies in THRß gene/protein structure are associated with onset of diverse disease states. In this study, we investigated disease-inflicting truncated variants of THRß using in-silico analysis and cell-based assays. We examined the THRß truncated variants on multiple test parameters, including subcellular localization, ligand-receptor interactions, transcriptional functions, interaction with heterodimeric partner RXR, and receptor-chromatin interactions. Moreover, molecular dynamic simulation approaches predicted that shortened THRß-LBD due to point mutations contributes proportionally to the loss of structural integrity and receptor stability. Deviant subcellular localization and compromised transcriptional function were apparent with these truncated variants. Present study shows that 'mitotic bookmarking' property of some THRß variants is also affected. The study highlights that structural and conformational attributes of THRß are necessary for normal receptor functioning, and any deviations may contribute to the underlying cause of the inflicted diseases. We anticipate that insights derived herein may contribute to improved mechanistic understanding to assess disease predisposition.

TEX13B is essential for metabolic reprogramming during germ cell differentiation.

STUDY QUESTION: What is the functional significance of Tex13b in male germ cell development and differentiation? SUMMARY ANSWER: Tex13b regulates male germ cell differentiation by metabolic reprogramming during spermatogenesis. WHAT IS KNOWN ALREADY: Studies in mice and humans suggest that TEX13B is a transcription factor and is exclusively expressed in germ cells. STUDY DESIGN, SIZE, DURATION: We sequenced the coding regions of TEX13B in 628 infertile men and 427 ethnically matched fertile control men. Further, to identify the molecular function of Tex13b, we created a Tex13b knockout and conditional overexpression system in GC-1spg (hereafter, GC-1) cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: Our recent exome sequencing study identified novel candidate genes for male infertility. TEX13B was found to be one of the potential candidates, hence we explored the role of TEX13B in male infertility within a large infertile case-control cohort. We performed functional analyses of Tex13b in a GC-1 cell line using CRISPR-Cas9. We differentially labelled the cell proteins by stable isotope labelling of amino acids in cell culture (SILAC) and performed mass spectrometry-based whole-cell proteomics to identify the differential protein regulation in knockout cells compared to wild-type cells. We found that Tex13b knockout leads to downregulation of the OXPHOS complexes and upregulation of glycolysis genes, which was further validated by western blotting. These results were further confirmed by respirometry analysis in Tex13b knockout cells. Further, we also performed a conditional overexpression of TEX13B in GC-1 cells and studied the expression of OXPHOS complex proteins by western blotting. MAIN RESULTS AND THE ROLE OF CHANCE: We identified a rare variant, rs775429506 (p.Gly237Glu), exclusively in two non-obstructive-azoospermia (NOA) men, that may genetically predispose these men for infertility. Further, we demonstrated that Tex13b functions in the transcription regulation of OXPHOS complexes. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: We examined the function of Tex13b in GC-1 in vitro by knocking out and conditional overexpression, for understanding the function of Tex13b in germ cells. Unfortunately, this could not be replicated in either an animal model or in patient-derived tissue due to the non-availability of an animal model or patient's testis biopsies. WIDER IMPLICATIONS OF THE FINDINGS: This study identified that Tex13b plays an important role in male germ cell development and differentiation. The findings of this study would be useful for screening infertile males with spermatogenic failure and counselling them before the implementation of assisted reproduction technique(s). STUDY FUNDING/COMPETING INTEREST(S): Funding was provided by the Council of Scientific and Industrial Research (CSIR) under the network project (BSC0101 and MLP0113) and SERB, the Department of Science and Technology, Government of India (J C Bose Fellowship: JCB/2019/000027). The authors do not have any competing interest.

Chronic pulmonary aspergillosis incidence in newly detected pulmonary tuberculosis cases during follow-up.

BACKGROUND: Chronic pulmonary aspergillosis (CPA) is known to complicate patients with post-tubercular lung disease. However, some evidence suggests that CPA might co-exist in patients with newly-diagnosed pulmonary tuberculosis (P.TB) at diagnosis and also develop during therapy. The objective of this study was to confirm the presence of CPA in newly diagnosed P.TB at baseline and at the end-of-TB-therapy. MATERIALS AND METHODS: This prospective longitudinal study included newly diagnosed P.TB patients, followed up at third month and end-of-TB-therapy with symptom assessment, anti-Aspergillus IgG antibody and imaging of chest for diagnosing CPA. RESULTS: We recruited 255 patients at baseline out of which 158 (62%) completed their follow-up. Anti-Aspergillus IgG was positive in 11.1% at baseline and 27.8% at end-of-TB-therapy. Overall, proven CPA was diagnosed in 7% at baseline and 14.5% at the end-of-TB-therapy. Around 6% patients had evidence of aspergilloma in CT chest at the end-of-TB-therapy. CONCLUSIONS: CPA can be present in newly diagnosed P.TB patients at diagnosis and also develop during anti-tubercular treatment. Patients with persistent symptoms or developing new symptoms during treatment for P.TB should be evaluated for CPA. Whether patients with concomitant P.TB and CPA, while receiving antitubercular therapy, need additional antifungal therapy, needs to be evaluated in future studies.

Soil-mustard revitalization via rice husk ash, a promising soil amendment material for sustainable management of heavy metal contamination in tropical ecosystem.

Prolonged wastewater irrigation in agriculture has led to the accumulation of heavy metals in soil, endangering both the soil quality and food safety, thereby posing a potential threat to human health through the consumption of contaminated crops. The present study aimed to enhance the yield of mustard (Brassica juncea L. cv. Varuna and NRCHB 101) plants and stabilize heavy metals (Cd, Cr, Ni, Cu, and Zn) in wastewater-irrigated soil using rice husk ash (RHA), rice mill by-product, collected from Chandauli region of Eastern Uttar Pradesh, India. Results demonstrated significant improvements in growth, biomass, physiology, and yield of mustard plant with increasing RHA application in wastewater irrigated soil (p ≤ 0.05). Heavy metal accumulation in different parts of mustard plants decreased as RHA application rate increased. Applying RHA at 2% in soil proved to be most effective in reducing Cd, Cr, Ni, Cu, and Zn accumulation in seeds by 29%, 29.6%, 23.1%, 21.3% and 20.1%, respectively in Varuna and 30.1%, 21.4%, 11.1%, 12.1%, and 28.5%, respectively in NRCHB 101cultivars. The present findings showed that RHA amendment in wastewater irrigated soil had reduced bioaccumulation of Cd, Cr, Ni, Cu, and Zn and consequently their toxicity in cultivated mustard plants. A novel application of RHA is unveiled in this research, offering a promising solution to promote sustainable agriculture and to reduce heavy metal associated health risks within the soil-mustard system.

Screening of mustard cultivars for phytoremediation of heavy metals contamination in wastewater irrigated soil systems.

The mustard (Brassica juncea L.) plant is a well-known and widely accepted hyper-accumulator of heavy metals. The genetic makeup of mustard's cultivars may significantly impact their phytoremediation capabilities. The present study aimed to investigate the growth performance, yield attributes, and heavy metal accumulation potential of B. juncea cv. Varuna, NRCHB 101, RH 749, Giriraj, and Kranti, cultivated in soil irrigated with wastewater (EPS) and bore-well water (MPS). EPS contributed more Cr, Cd, Cu, Zn, and Ni to tested mustard cultivars than the MPS. EPS reduced morphological, biochemical, physiological, and yield attributes of tested mustard cultivars significantly (p < 0.05) than the MPS. Among the tested cultivars of mustard plants, Varuna had the highest heavy metal load with the lowest harvest index (35.8 and 0.21, respectively). Whereas NRCHB 101 showed the lowest heavy metal load with the highest harvest index (26.9 and 0.43, respectively). The present study suggests that B. juncea cv. Varuna and NRCHB 101 could be used for the phytoextraction of heavy metals and reducing their contamination in food chain, respectively in wastewater irrigated areas of peri-urban India. The outcomes of the present study can also be utilized to develop a management strategy for sustainable agriculture in heavy metal polluted areas resulting from long-term wastewater irrigation.

Association of altered metabolic profiles and long non-coding RNAs expression with disease severity in breast cancer patients: analysis by 1H NMR spectroscopy and RT-q-PCR.

INTRODUCTION: Globally, one of the major causes of cancer related deaths in women is breast cancer. Although metabolic pattern is altered in cancer patients, robust metabolic biomarkers with a potential to improve the screening and disease monitoring are lacking. A complete metabolome profiling of breast cancer patients may lead to the identification of diagnostic/prognostic markers and potential targets. OBJECTIVES: The aim of this study was to analyze the metabolic profile in the serum from 43 breast cancer patients and 13 healthy individuals. MATERIALS & METHODS: We used 1H NMR spectroscopy for the identification and quantification of metabolites. q-RT-PCR was used to examine the relative expression of lncRNAs. RESULTS: Metabolites such as amino acids, lipids, membrane metabolites, lipoproteins, and energy metabolites were observed in the serum from both patients and healthy individuals. Using unsupervised PCA, supervised PLS-DA, supervised OPLS-DA, and random forest classification, we observed that more than 25 metabolites were altered in the breast cancer patients. Metabolites with AUC value > 0.9 were selected for further analysis that revealed significant elevation of lactate, LPR and glycerol, while the level of glucose, succinate, and isobutyrate was reduced in breast cancer patients in comparison to healthy control. The level of these metabolites (except LPR) was altered in advanced-stage breast cancer patients in comparison to early-stage breast cancer patients. The altered metabolites were also associated with over 25 signaling pathways related to metabolism. Further, lncRNAs such as H19, MEG3 and GAS5 were dysregulated in the breast tumor tissue in comparison to normal adjacent tissue. CONCLUSION: The study provides insights into metabolic alteration in breast cancer patients. It also provides an avenue to examine the association of lncRNAs with metabolic patterns in patients.

Acute pancreatitis is characterized by generalized intestinal barrier dysfunction in early stage.

BACKGROUND AND AIMS: The role of intestinal-barrier in acute pancreatitis(AP) is poorly understood. We aimed to assess structural and functional changes in the intestinal-barrier in patients with early AP (time from onset<2 weeks) and the effect of enteral nutrition on them. METHODS: In this prospective observational study, patients with early AP not on enteral nutrition were compared with controls for baseline intestinal-permeability(lactulose: mannitol ratio(L:M)), endotoxinemia(serum IgM/IgG anti-endotoxin antibodies), bacterial-translocation(serum bacterial 16S rRNA) and duodenal epithelial tight-junction structure by immunohistochemistry(IHC) for tight-junction proteins(claudin-2,-3,-4, zonula occludens-1(ZO1), junctional adhesion molecule(JAM) and occludin) and electron microscopy. These parameters were reassessed after 2 weeks enteral feeding in a AP patients subset. RESULTS: 96 patients with AP(age: 38.0 ± 14.5 years; etiology: biliary[46.8%]/alcohol[39.6%]; severe:53.2%, mortality:11.4%) and 40 matched controls were recruited. Patients with AP had higher baseline intestinal permeability(median L:M 0.176(IQR 0.073-0.376) vs 0.049(0.024-0.075) in controls; p < 0.001) and more frequent bacteraemia(positive bacterial 16S rRNA in 24/48 AP vs 0/21 controls; p < 0.001) with trend towards higher serum endotoxinemia(median IgG anti-endotoxin 78(51.2-171.6) GMU/ml vs 51.2(26.16-79.2) in controls; p = 0.061). Claudin-2, claudin-3, ZO1 were downregulated in both duodenal crypts and villi while claudin-4 and JAM were downregulated in duodenal villi and crypts respectively. 22 AP patients reassessed after initiation of enteral nutrition showed trend towards improving intestinal permeability, serum endotoxinemia and bacteraemia, with significant improvement in claudin-2,-3 in duodenal villi. CONCLUSION: Patients with AP have significant disturbances in intestinal barrier structure and function in first 2 weeks from onset that persist despite institution of enteral nutrition.

Modulation of macrophage inflammatory function through selective inhibition of the epigenetic reader protein SP140.

BACKGROUND: SP140 is a bromodomain-containing protein expressed predominantly in immune cells. Genetic polymorphisms and epigenetic modifications in the SP140 locus have been linked to Crohn's disease (CD), suggesting a role in inflammation. RESULTS: We report the development of the first small molecule SP140 inhibitor (GSK761) and utilize this to elucidate SP140 function in macrophages. We show that SP140 is highly expressed in CD mucosal macrophages and in in vitro-generated inflammatory macrophages. SP140 inhibition through GSK761 reduced monocyte-to-inflammatory macrophage differentiation and lipopolysaccharide (LPS)-induced inflammatory activation, while inducing the generation of CD206+ regulatory macrophages that were shown to associate with a therapeutic response to anti-TNF in CD patients. SP140 preferentially occupies transcriptional start sites in inflammatory macrophages, with enrichment at gene loci encoding pro-inflammatory cytokines/chemokines and inflammatory pathways. GSK761 specifically reduces SP140 chromatin binding and thereby expression of SP140-regulated genes. GSK761 inhibits the expression of cytokines, including TNF, by CD14+ macrophages isolated from CD intestinal mucosa. CONCLUSIONS: This study identifies SP140 as a druggable epigenetic therapeutic target for CD.

Individualized Homeopathic Medicines for Low Back Pain in Lumbar Spondylosis: Double-Blind, Randomized, Placebo-Controlled Trial.

INTRODUCTION: Lumbar spondylosis (LS) is a degenerative disorder of the lumbar spine. Despite substantial research efforts, no gold-standard treatment for LS has been identified. The efficacy of individualized homeopathic medicines (IHMs) in LS has remained under-researched. In this study, the efficacy of IHMs was compared with identical-looking placebos in the treatment of low back pain associated with LS. METHODS: A double-blind, randomized (1:1), placebo-controlled trial was conducted at the National Institute of Homoeopathy, West Bengal, India. Patients were randomized to receive IHMs or placebos, along with standardized concomitant care for both the groups. The Oswestry low back pain and disability questionnaire (ODQ) was the primary outcome; the Roland-Morris questionnaire (RMQ) and the short form of the McGill pain questionnaire (SF-MPQ) were the secondary outcomes. Each was measured at baseline and every month for 3 months. The intention-to-treat (ITT) sample was analyzed to detect any inter-group differences using two-way repeated measures analysis of variance models overall and by unpaired t-tests at different time points. RESULTS: Enrolment was stopped prematurely because of time restrictions; 55 patients were randomized (verum: 28; control: 27); 49 were analyzed by ITT (verum: 26; control: 23). Inter-group differences in ODQ (F 1, 47 = 0.001, p = 0.977), RMQ (F 1, 47 = 0.190, p = 0.665) and SF-MPQ total score (F 1, 47 = 3.183, p = 0.081) at 3 months were not statistically significant. SF-MPQ total score after 2 months (p = 0.030) revealed inter-group statistical significance, favoring IHMs against placebos. Some of the SF-MPQ sub-scales at different time points were also statistically significant: e.g., the SF-MPQ average pain score after 2 months (p = 0.002) and 3 months (p = 0.007). Rhus toxicodendron, Sulphur and Pulsatilla nigricans were the most frequently indicated medicines. CONCLUSION: Owing to failure in detecting a statistically significant effect for the primary outcome and in recruiting a sufficient number of participants, our trial remained inconclusive. TRIAL REGISTRATION: CTRI/2019/11/021918.

Synthesis and physicochemical characterization of rhamnolipid fabricated fucoxanthin loaded bovine serum albumin nanoparticles supported by simulation studies.

BACKGROUND: Fucoxanthin is a hydrophobic carotenoid with many beneficial biological activities. However, due to low aqueous solubility their clinical efficacy is limited thus leading to poor oral bioavailability. To address this issue, we encapsulated fucoxanthin in rhamnolipid fabricated bovine serum albumin (BSA) loaded nanoparticles (LNPs) for improving solubility dependent bioavailability of fucoxanthin. RESULTS: These synthesized LNPs were characterized by dynamic light scattering (DLS), ultraviolet (UV)-visible spectrophotometry, high-performance liquid chromatography (HPLC), Fourier-transform infrared (FTIR), scanning electron microscopy (SEM), differential scanning calorimetry (DSC). Our results showed that LNPs were spherical in shape with particle size around 180 nm along with positive zeta potential. The encapsulation efficiency and loading efficiency calculated for LNPs were 69.66 ± 1.5% and 14 ± 0.2%, respectively. The antioxidant assay of LNPs indicate high radical scavenging activity compared to pure fucoxanthin. Besides this, our release studies indicates that drug release occur from the matrix of nanocarrier system through diffusion based on concentration. Thus, these findings indicate successful encapsulation of fucoxanthin, with improved solubility thereby leading to increased bioavailability. This nano formulation is derived from components which are FDA approved that could be exploited for encapsulating other vital nutraceutical molecules. CONCLUSION: Overall, our results showed successful synthesis of biodegradable nanocarrier for delivering fucoxanthin supported by molecular docking, molecular dynamics simulation and thermodynamics of free binding energy studies. © 2022 Society of Chemical Industry.

Fabrication of Imatinib Mesylate-Loaded Lactoferrin-Modified PEGylated Liquid Crystalline Nanoparticles for Mitochondrial-Dependent Apoptosis in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a major cause of concern as it has substantial morbidity associated with it. Previous reports have ascertained the antiproliferative activity of imatinib mesylate (IMS) against diverse types of carcinomas, but limited bioavailability has also been reported. The present study envisaged optimized IMS-loaded lactoferrin (LF)-modified PEGylated liquid crystalline nanoparticles (IMS-LF-LCNPs) for effective therapy of IMS to HCC via asialoglycoprotein receptor (ASGPR) targeting. Results displayed that IMS-LF-LCNPs presented an optimum particle size of 120.40 ± 2.75 nm, a zeta potential of +12.5 ± 0.23 mV, and 73.94 ± 2.69% release. High-resolution transmission electron microscopy and atomic force microscopy were used to confirm the surface architecture of IMS-LF-LCNPs. The results of cytotoxicity and 4,6-diamidino-2-phenylindole revealed that IMS-LF-LCNPs had the highest growth inhibition and significant apoptotic effects. Pharmacokinetics and biodistribution studies showed that IMS-LF-LCNPs have superior pharmacokinetic performance and targeted delivery compared to IMS-LCNPs and plain IMS, which was attributed to the targeting action of LF that targets the ASGPR in hepatic cells. Next, our in vivo experiment established that the HCC environment existed due to suppression of BAX, cyt c, BAD, e-NOS, and caspase (3 and 9) genes, which thus owed upstream expression of Bcl-xl, iNOS, and Bcl-2 genes. The excellent therapeutic potential of IMS-LF-LCNPs began the significant stimulation of caspase-mediated apoptotic signals accountable for its anti-HCC prospect. 1H nuclear magnetic resonance (serum) metabolomics revealed that IMS-LF-LCNPs are capable of regulating the disturbed levels of metabolites linked to HCC triggered through N-nitrosodiethylamine. Therefore, IMS-LF-LCNPs are a potentially effective formulation against HCC.

An elaborative NMR based plasma metabolomics study revealed metabolic derangements in patients with mild cognitive impairment: a study on north Indian population.

Mild cognitive impairment (MCI) is transition phase between cognitive decline and dementia. The current study aims to investigate altered metabolic pattern in plasma of MCI for potential biomarkers. MCI (N = 50) and healthy controls (HC, N = 50) age group 55-75 years were screened based on Mini Mental State Examination Test (MMSE) and diffusion tensor imaging (DTI imaging). The MMSE score of MCI was significantly lower (25.74 ± 1.83) compared to healthy control subjects (29 ± 1). The MCI patients exhibit significant changes in white matter integrity in the right frontal lobe, right temporal lobe, left frontal lobe, forcep major, fornix, corpus callosum. Further, the plasma samples of twenty seven MCI patients (N = 27) and twenty HC subjects (N = 20; having no significant differences in any demographics) were analyzed using 1H NMR based metabolomics approach. Consistent with many previous reports, the levels of several plasma metabolites were found to be elevated in MCI patients compared to healthy controls. Further univariate and multivariate ROC curve analyses provided three plasma metabolites as a diagnostic panel of biomarker for MCI; which are lysine, glycine, and glutamine. Overall, the results of this study will help to improve the diagnostic and prognostic strategies of MCI in addition to improving our understanding about disease pathogenesis. We believe that the over-nutritional metabolic phenotype of MCI needs to be targeted for developing future dietary interventions so that the progression of MCI can be limited. Metabolic derangements associated with Mild Cognitive Impairment.

Sagittal Maxillary Fracture: Diagnosis and Management.

Background The sagittal maxillary fracture often coexists with maxillary fractures and warrants a definitive management strategy together with other maxillary fractures. Method This study was conducted on 60 patients suffering from sagittal maxillary fracture. Palatal fractures were classified into six subgroups. During management, patients were divided into three groups. In group A, patients with type I, IV, V, and VI were managed with maxillomandibular fixation and anterior maxillary buttress stabilization. Group B patients included type II, III, and IV palatal fractures. These fractures were undisplaced and were managed with maxillomandibular fixation, anterior alveolar plating, and anterior maxillary buttress stabilization. Group C included type II and III fractures with visible gap in the palate and were managed with maxillomandibular fixation, palatal vault plating, anterior alveolar plating, and anterior maxillary buttress stabilization. Result Sagittal maxillary fracture was more common in young males. Le Fort I and II fractures were more frequently associated with it in isolation or in combination. Parasagittal and sagittal fractures were the most common types. Sixteen patients of group A, twenty patients of group B, and twenty-four patients of group C were managed. Malocclusion (2), plate extrusion (2), and oroantral fistula (2) were the most common complications. Conclusion Sagittal maxillary fracture can be diagnosed with clinical and radiological examination. Palatal vault plating is required in displaced palatal fractures of type II and III. Single plate fixed in posterior half of middle one-third of palate gives sufficient stability to the palatal vault.

Nuclear magnetic resonance-based targeted profiling of urinary acetate and citrate following cyclophosphamide therapy in patients with lupus nephritis.

OBJECTIVE: Metabolomics, the study of global alterations in small metabolites, is a useful tool to look for novel biomarkers. Recently, we reported a reprogramming of the serum metabolomic profile by nuclear magnetic resonance (NMR) spectroscopy following treatment in lupus nephritis (LN). This study aimed to compare the urine excretory levels of citrate and acetate in patients with biopsy-proven LN before and six months after cyclophosphamide induction therapy and to evaluate their correlation with the Systemic Lupus Erythematosus Disease Activity Index 2K (SLEDAI 2K) and renal SLEDAI. METHODS: Urine obtained from LN patients (N = 18, 16 female) at diagnosis and six months following induction therapy with cyclophosphamide and healthy controls (HC; N = 18, median age = 35 years, all female) were stored at -80°C. Metabolomic profiling was done using high resolution 800 MHz 1D 1H NMR spectroscopy. The urinary ratio of metabolites was calculated as (metabolite×1000)/creatinine. Disease activity was measured using the SLEDAI. Metabolomic profiles were compared between groups and correlated with clinical parameters. RESULTS: Compared to HC, LN patients had significantly lower median urinary citrate/creatinine levels (LN = 18.26, range 12.80-27.62; HC = 107.7, range 65.39-138.4; p < 0.0001) which significantly increased after six months of cyclophosphamide treatment (51.05, range 11.51-170.2; p = 0.03). LN patients also differed from HC by having a higher mean urinary acetate/creatinine ratio (LN = 17.44, range 11.6-32.7; HC = 9.61, range 7.97-13.71; p = 0.054) with a non-significant fall in values after six months of treatment. The Area under curve for differentiating LN from HC for urinary citrate was 0.9136, and urinary acetate was 0.6883. The urinary acetate levels correlated with SLEDAI (r = 0.337, p = 0.048). Urinary citrate levels correlated positively with C3 (r = 0.362, p = 0.03) and negatively with urine protein/creatinine (r = -0.346, p = 0.039). CONCLUSIONS: Urinary citrate, which reflects dampened aerobic glycolysis and oxidative phosphorylation, improved significantly and is a potential non-invasive biomarker for diagnosis and monitoring treatment response in LN.

Novel Classification of Posttraumatic Ear Deformities and its Surgical Management.

Background Classification of posttraumatic ear deformities and its reconstruction is an uphill task for a reconstructive surgeon as they present in various combinations. In our study, we have described ear deformity as per a new classification and reconstructed the ear accordingly. Method Posttraumatic ear deformity was described under the following four headings: (a) zone of defect, (b) size of defect, (c) missing components, and (d) condition of surrounding skin. Twenty-six posttraumatic ear deformities were operated using postauricular skin flap (14), temporoparietal fascial (TPF) flaps (8), preauricular skin flap (1), intralesional excision (2), and primary closure with chondrocutaneous advancement in one patient. Costal cartilage was used for reconstruction of framework wherever required. Framework elevation was done 4 to 6 months postoperatively. Results Posttraumatic ear deformity was more common in males. Bite injury and road traffic accidents were the common causes. Zones I, II and III were most frequently involved. Four patients complained about size, contour, and projection of reconstructed ear. Three patients were not satisfied by the appearance of junction between reconstructed and residual ear. Four patients in whom the reconstruction was done with TPF, costal cartilage, and thin (SSG) split skin grafts complained of hyperpigmentation of reconstructed ear. Conclusion Classification of posttraumatic ear deformity and its reconstruction is a surgical challenge. Unscarred postauricular skin and TPF flaps are the workhorse flaps for reconstruction of acquired ear deformities. Our classification helps in describing the defect, documenting it, planning reconstruction, and aiding in assessing postoperative outcomes.

Novel 1,3,4-thiadiazoles inhibit colorectal cancer via blockade of IL-6/COX-2 mediated JAK2/STAT3 signals as evidenced through data-based mathematical modeling.

We attempted a preclinical study using DMH-induced CRC rat model to evaluate the antitumor potential of our recently synthesized 1,3,4-thiadiazoles. The molecular insights were confirmed through ELISA, qRT-PCR and western blot analyses. The CRC condition was produced in response to COX-2 and IL-6 induced activation of JAK2/STAT3 which, in turn, was due to the enhanced phosphorylation of JAK2 and STAT3. The treatment with 1,3,4-thiadiazole derivatives (VR24 and VR27) caused the significant blockade of this signaling pathway. The behavior of STAT3 populations in response to IL-6 and COX-2 stimulations was further confirmed through data-based mathematical modeling using the quantitative western blot data. Finally, VR24 and VR27 restored the perturbed metabolites associated to DMH-induced CRC as evidenced through 1H NMR based serum metabolomics. The tumor protecting ability of VR24 and VR27 was found comparable or to some degree better than the marketed chemotherapeutics, 5-flurouracil.

Antineoplastic properties of zafirlukast against hepatocellular carcinoma via activation of mitochondrial mediated apoptosis.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwideand haslimited treatment options. In view of this, zafirlukast (ZAF) was administered orally to DEN-induced HCC rats to evaluate its antineoplastic properties. ELISA, qRT-PCR and Western blot were used to determine the molecular mechanism associated with ZAF therapy for HCC. We found that HCC developed as a result of lower expression of caspases 3 and 9, but their levels returned to normal when the expression of eNOS, BAX, BAD, and Cyt C was decreased and when the expression of iNOS, Bcl-xl, and Bcl-2 was increased. Again, ZAF (80 mg/kg dose) treatment normalized the expression of caspase-mediated apoptotic factors, i.e. BAX and Bcl-2 proteins, as established through Western blot analysis. Later, 1H NMR-based serum metabolomics study revealed that levels of perturbed metabolites in DEN-induced rat serum returned to normal after ZAF administration. Altogether, the antineoplastic potential of ZAF was found to be comparable, and to some degree better, than the marketed chemotherapeutic 5-flurouracil, which may be beneficial for anti-HCC treatment from a future drug design perspective.

NMR-Based Serum Metabolomics of Patients with Takayasu Arteritis: Relationship with Disease Activity.

Takayasu arteritis (TA) is a large vessel vasculitis of unknown pathogenesis. Assessment of disease activity is a challenge, and there is an unmet need for relevant biomarker(s). In our previous study, NMR based serum metabolomics had revealed distinctive metabolic signatures in TA patients compared with age/sex matched healthy controls and systemic lupus erythematosus (SLE). In this study we investigate whether the metabolites correlate with disease activity. Patients with TA fulfilling American College of Rheumatology (ACR) criteria were enrolled, and disease activity was assessed using Indian Takayasu Clinical Activity Score using acute phase reactant-erythrocyte sedimentation rate [ITAS-A (ESR)]. Sera were analyzed using 800 MHz NMR spectrometer to identify metabolites [based on partial least squares discriminant analysis (PLS-DA) VIP (variable importance in projection) score > 1.0 and permutation test, p-value <0.01]. 45 active and 53 inactive TA patients with median age 27 [(IQR) 22-35 years] and 27 [(IQR) 23-37 years], female to male ratio 3.5:1 and 4.9:1, and median duration of illness 5 [(IQR) 2-9 years] and 3 [(IQR) 1-6 years], respectively, were enrolled. The key metabolites with highest discriminatory potential in active TA (ITAS-A ≥ 4) were glutamate and N-acetyl glycoprotein (NAG), both elevated, with area under the curve 0.775 and 0.769 ( p-value <0.001). On follow up assessment, metabolic spectra started to differ with change in disease activity. This large cohort of patients revealed metabolic profiles discriminating between clinically active and inactive TA patients. It suggests glutamate and NAG have strong potential as biomarkers for disease activity in TA and may serve as a guide to therapy. We are now working to further validate these results in longitudinal studies.

NMR-Based Serum Metabolomics Reveals Reprogramming of Lipid Dysregulation Following Cyclophosphamide-Based Induction Therapy in Lupus Nephritis.

Lupus nephritis (LN) is a major cause of morbidity and mortality in lupus. Renal biopsy is the gold standard for classification of nephritis, but because of its impracticality, new approaches for improving patient prognostication and monitoring treatment efficacy are needed. We aimed to evaluate the potential of metabolic profiling in identifying biomarkers to distinguish disease and monitor treatment efficacy in patients with LN. Serum samples from patients with LN ( n = 18) were profiled on NMR-based metabolomics platforms at diagnosis and after 6 months of treatment. LN patients had a different metabolomic fingerprint as compared with healthy controls, with increased lipoproteins and lipids and reduced acetate and amino acids. Using multivariate statistical analysis, we found that the metabolic changes observed in naïve LN patients at diagnosis displayed a variation in the opposite direction upon responding to treatment. Increased levels of lipid metabolites including low- and very-low-density lipoproteins (LDL/VLDL) in LN patients significantly decreased after 6 months of treatment, whereas the serum levels of acetate increased. These levels correlated significantly with SLE Disease Activity Index (SLEDAI 2K), renal SLEDAI, and serum C3 and C4 levels. The result presented in this pilot longitudinal study revealed the reprogramming of metabolome in LN patients on immunosuppressive therapy using NMR-based metabolomics, and thus this approach may be used to monitor the response to treatment.