Expression, purification and characterization of a biologically active and thermally stable human lysyl oxidase

Lysyl oxidase (LOX), a promising therapeutic target for the progression of cancer and fibrosis, has not been well characterized yet. A major difficulty faced in LOX characterization is its lack of solubility in common buffers. In this study, mature LOX (mLOX) was cloned, purified and its purity was ascertained by mass spectroscopy. Through screening various buffers, 0.2 M glycine-NaOH buffer with 10% glycerol pH 8.0 was identified to maintain mLOX in its soluble state. About 67% of the refolded mLOX was found to be in copper bound state after His-tag removal. Catalytic properties Km and kcat were found to be 3.72 × 10−4 M and 7.29 ×103s−1. In addition, collagen cross-linking in ARPE-19 cells was augmented on exposure to mLOX, endorsing its biological activity. Circular Dichroism revealed that mLOX comprises 8.43% of α-helix and 22% of β-strand and it was thermally stable up to 90°C. Disulfide linkage imparts the structural stability in LOX which was experimentally ascertained with intrinsic and extrinsic fluorescence studies

Somatic Mutations Profile of a Young Patient With Metastatic Urothelial Carcinoma Reveals Mutations in Genes Involved in Ion Channels

Background: Urothelial carcinoma is the most common malignancy of the bladder and is primarily considered as a disease of the elderly. Studies that address bladder tumor occurrence in young age groups are rare.Case Presentation: A 19-year-old male presented with a gross total painless hematuria. A histology after biopsy revealed a high-grade transitional cell carcinoma with lymph node metastasis. The patient succumbed to the disease on day 72 of the treatment. Here, we used whole-exome sequencing of a paired tumor-normal sample to identify the somatic mutations and the possible targets of treatment.Result: We predicted eight potential driver mutations (TP53 p.V157L, RB1 c.1498+1G>T, MED23 p.L1127P, CTNND1 p.S713C, NSD1 p.P2212A, MED17 p.G556V, DPYD p.Q814K, and SPEN p.S1078*). In addition, we predicted deleterious mutations in genes involved in the ion channels (CACNA1S p.E1581K, CACNG1 p.P71T, CACNG8 p.G404W, GRIN2B p.A1096T, KCNC1 p.G16V, KCNH4 p.E874K, KCNK9 p.R131S, P2RX7 p.A296D, and SCN8A p.R558H).Conclusions: Most likely, mutations in genes involved in ion channels may be responsible for the aggressive behavior of a tumor. Ion channels are the second largest class of drug targets, and may thus serve as a putative potential therapeutic target in advanced stage urothelial carcinoma

Trace Elements Iron, Copper and Zinc in Vitreous of Patients with Various Vitreoretinal Diseases

<b>Purpose:</b> To measure the concentrations of iron, copper and zinc in human vitreous and to interpret their levels with various vitreoretinal diseases like proliferative diabetic retinopathy, retinal detachment, intraocular foreign body, Eales&#x2032; disease and macular hole. <b> Methods: </b> Undiluted vitreous fluid collected during pars plana vitrectomy was used to measure trace elements using an atomic absorption spectrophotometer. <b> Results:</b> The level of vitreous iron increased threefold in Eales&#x2032; disease (1.85 &#x00B1; 0.36 pg/ml), 2.5-fold in proliferative diabetic retinopathy (1.534 &#x00B1; 0.17 pg/ml) and 2.3-fold in eyes with intraocular foreign body (1.341 &#x00B1; 0.25 pg/ml) when compared with macular hole (0.588 &#x00B1; 0.16 pg/ml). This was statistically significant (P &lt; 0.05). Zinc was found to be low in Eales&#x2032; disease (0.57 &#x00B1; 0.22 pg/ml) when compared with other groups, though the difference was not statistically significant. <b> Conclusion:</b> The increased level of iron with decreased zinc content in Eales&#x2032; disease confirms the earlier reported oxidative stress mechanism for the disease. In proliferative diabetic retinopathy and intraocular foreign body the level of iron increases. This is undesirable as iron can augment glycoxidation, which can lead to increased susceptibility to oxidative damage, in turn causing vitreous liquefaction, posterior vitreous detachment and ultimately retinal detachment and vision los

Determination of Carbonyl Group Content in Plasma Proteins as a Useful Marker to Assess Impairment in Antioxidant Defense in Patients with Eales&#x2032; Disease

Purpose: Formation of protein carbonyl groups is considered an early biomarker for the oxidant/antioxidant barrier impairment in various inflammatory diseases. We evaluated the intensity of free radical reactions in patients with Eales&#x2032; disease, an idiopathic inflammatory condition of the retina. Methods: Twenty patients with Eales&#x2032; disease in active vasculitis stage, 15 patients with Eales&#x2032; disease in healed vasculitis stage and 20 healthy control subjects were recruited for the study. Plasma protein carbonyl groups,plasma glutathione (GSH) superoxide dismutase (SOD) activity and thiobarbituric acid reactive substances (TBARS) were determined in erythrocytes. Results: Plasma protein carbonyl content was elevated by a factor of 3.5 and 1.8 respectively in active and healed vasculitis stages. The increase of carbonyl group content in active and healed stage of patients with Eales&#x2032; disease correlated with diminished SOD activity and GSH content. There was also increased accumulation of TBARS in active and healed vasculitis stages of Eales&#x2032; disease, and this correlated with diminished SOD activity. Conclusion: Our results showed that protein carbonyl group content increases with severity of Eales&#x2032; disease. The increase in carbonyl content correlated with diminished antioxidant status. This confirms an earlier report that free radical mediated tissue damage occurs in Eales&#x2032; disease. The determination of protein carbonyl content may be used as a simple biomarker to monitor the efficacy of antioxidant supplementation in controlling retinal vasculitis in patients with Eales&#x2032; disease

Expression, purification and characterization of a biologically active and thermally stable human lysyl oxidase

105-116Lysyl oxidase (LOX), a promising therapeutic target for the progression of cancer and fibrosis, has not been well characterized yet. A major difficulty faced in LOX characterization is its lack of solubility in common buffers. In this study, mature LOX (mLOX) was cloned, purified and its purity was ascertained by mass spectroscopy. Through screening various buffers, 0.2 M glycine-NaOH buffer with 10% glycerol pH 8.0 was identified to maintain mLOX in its soluble state. About 67% of the refolded mLOX was found to be in copper bound state after His-tag removal. Catalytic properties Km and kcat were found to be 3.72 × 10−4 M and 7.29 ×103s−1. In addition, collagen cross-linking in ARPE-19 cells was augmented on exposure to mLOX, endorsing its biological activity. Circular Dichroism revealed that mLOX comprises 8.43% of α-helix and 22% of β-strand and it was thermally stable up to 90°C. Disulfide linkage imparts the structural stability in LOX which was experimentally ascertained with intrinsic and extrinsic fluorescence studies

Homocysteine & its metabolite homocysteine-thiolactone & deficiency of copper in patients with age related macular degeneration - A pilot study

Background & objectives: Age related macular degeneration (ARMD) is a leading cause of blindness, particularly in persons above 60 yr of age. Homocysteine is implicated in many ocular diseases including ARMD. This study was undertaken to assess the status and relationship between plasma homocysteine, homocysteine - thiolactone, homocysteinylated protein and copper levels in patients with ARMD. Methods: A total of 16 patients with ARMD and 16 age-matched controls were recruited for the study. Plasma glutathione, homocysteine, homocysteine - thiolactone and extent of homocysteine conjugation with proteins, copper and thiobarbituric acid reactive substances were measured. Results: Homocysteine levels were elevated with increase in homocysteine-thiolactone, thiobarbituric acid reactive substances and a decrease of glutathione. The levels of homocysteinylated protein were elevated in ARMD. The elevated homocysteine, homocysteine-thiolactone correlated with the decrease in copper level. Interpretation & conclusions: Elevated homocysteine and its metabolite homocysteine-thiolactone and decreased levels of copper may play an important role in the pathogenesis of ARMD

Hyperhomocysteinemia and Low Methionine Stress Are Risk Factors for Central Retinal Venous Occlusion in an Indian Population

PURPOSE. The underlying cause of disturbed homocysteine metabolism is incompletely understood in young persons with central retinal vein occlusion (CRVO) with mild hyperhomocysteinemia (HHcys) and no other systemic disease in India. A 2-year prospective study was undertaken to determine whether HHcys is a risk factor for CRVO in an Indian population. METHOD. The prevalence of fasting HHcys was evaluated in a consecutive series of 29 patients with CRVO (mean age, 30 Ϯ 6 years) along with 57 age-and sex-matched control subjects (healthy subjects, mean age 27 Ϯ 5 years). Strict inclusion and exclusion criteria were used. Plasma levels of homocysteine (Hcys), methionine, cysteine, glutathione, B 12 , and folate were measured. Multivariate logistic regression analysis was performed to determine the risk factors for CRVO. RESULT. Fifteen of 29 patients with CRVO (51.72%) exhibited HHcys (Ͼ15 M). The mean Hcys level was significantly elevated in the patients with CRVO (19.1 Ϯ 13.1 M) compared with that in the healthy control subjects (14.7 Ϯ 6.2 M) with P ϭ 0.04. The increased Hcys levels in CRVO cases was associated with decreased methionine (P ϭ 0.052) and decreased B 12 (P ϭ 0.001). A multivariate logistic regression analysis revealed an odds ratio of 1.9 (95% CI ϭ 0.50 -7.16) for Hcys and 15.9 for methionine (95%CI ϭ 1.50 -169.62; P ϭ 0.022). CONCLUSION. Elevated Hcys and low methionine were risk factors for CRVO in an Indian population. (Invest Ophthalmol Vis Sci

Common mutations identified in the MLH1 gene in familial Lynch syndrome

Lynch syndrome (Hereditary Non Polyposis Colorectal Cancer, HNPCC) is one of the most common hereditary familial colorectal cancers (CRC) with an autosomal dominant pattern of inheritance. It accounts for 2-5% of the total CRCs reported worldwide. Although a lower incidence for CRCs have been observed in India, the last decade has shown a remarkable increase of CRC incidences (2-4 %). Features of Lynch syndrome associated colorectal cancer include early age of cancer onset, accelerated carcinogenesis of adenomas into carcinomas, and predilection to cancer of the proximal colon (about 70% of Lynch syndrome–related colorectal cancers occur in the right colon, which is not accessed through sigmoidoscopy). Mutations in the DNA mismatch repair genes (MMR) MLH1, MSH2, MSH6 or PMS2 cells are known to be responsible for Lynch syndrome. Almost 70% of the mutations in Lynch syndrome are seen in the MLH1 and MSH2 gene. In this study we identified three families with Lynch syndrome from a rural cancer center in western India (KCHRC, Goraj, Gujarat), where 70-75 CRC patients are seen annually. DNA isolated from the blood of consented family members of all three families (8-10 members/family) was subjected to NGS sequencing methods on an Illumina HiSeq 4000 platform. We identified unique mutations in the MLH1 gene in all three HNPCC family members. Two of the three unrelated families shared a common mutation (154delA and 156delA). Total 8 members of a family were identified as carriers for 156delA mutation of which 5 members were unaffected while 3 were affected (age of onset: 1 member &lt;30yrs &amp; 2 were&gt;40yr). The family with 154delA mutation showed 2 affected members (&gt;40yr) carrying the mutations.LYS618DEL mutation found in 8 members of the third family showed that both affected and unaffected carried the mutation. Thus the common mutations identified in the MLH1 gene in two unrelated families had a high risk for lynch syndrome especially above the age of 40

Integrated analysis of whole exome and RNA sequencing for Neo-epitope peptide prediction in buccal cancer

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy globally, but ranks first in India because of the extensive use of tobacco, betel-quid and alcohol.  Despite, the availability of aggressive treatments, the survival for HNSCC patients remains relatively poor. High-throughput sequencing technologies have identified several mutant genes and pathways in cancer genomes but the transcription patterns of these genetic alterations remain unclear. We performed an integrated analysis of whole exome and RNA sequencing to gain a better understanding of the transcriptional consequences of these genetic mutations, thus paving the way to identifying biomarkers and neo-epitopes. To this end, we conducted a prospective study on patients diagnosed with HNSCC from Gujarat, India. Detailed clinical data was collected from a total of 541 consented patients. Additionally, DNA and RNA was extracted from a subset of nine buccal cancer patients from both tumor and blood (Age 44 ± 9: M: 7, F: 2). Whole exome and RNA sequencing was performed on an Illumina platform followed by data analysis using bioinformatics tools. Normalized expression of all coding variants were compiled and variants with Alt allele depth in RNA-seq &gt;= 1 was prioritized for HLA binding and neo-epitope prediction using our proprietary pipeline OncoPeptVAC. As previously reported, DNA sequencing results revealed recurrent frameshift/nonsense and missense mutations in the TP53, PIK3CA, CASP8, FAT1, TTN, FSIP2 and CDKN2A genes. Additionally, unique mutations were observed in the CA6, FGD3, FLG2, COX 4l1, CD58, MAPK1, HLA-A and HLA-DQB1genes, previously not associated with head and neck cancers. A positive correlation between the mutant allele frequency in Exome and RNA-seq data confirmed the expression of both missense and frameshift mutant genes. Our results show that the majority of the mutation derived peptides were not predicted to be immunogenic for the HLA types considered. Peptides derived from variants in TP53, ANKRD12, EPHB2, ZNF200, TRPM4, CHD4, PLEC, KMT2C, GLTSCR1 were predicted to have neo-epitopes properties in buccal cancer. These findings provide a platform to predict immunogenic neo-epitopes which could be used in the development of personalized vaccines for patients with buccal cancer