Next-generation sequencing to genetically diagnose a diverse range of inherited eye disorders in 15 consanguineous families from Pakistan.

Inherited retinal dystrophies (IRDs) are characterized by photoreceptor dysfunction or degeneration. Clinical and phenotypic overlap between IRDs makes the genetic diagnosis very challenging and comprehensive genomic approaches for accurate diagnosis are frequently required. While there are previous studies on IRDs in Pakistan, causative genes and variants are still unknown for a significant portion of patients. Therefore, there is a need to expand the knowledge of the genetic spectrum of IRDs in Pakistan. Here, we recruited 52 affected and 53 normal individuals from 15 consanguineous Pakistani families presenting non-syndromic and syndromic forms of IRDs. We employed single molecule Molecular Inversion Probes (smMIPs) based panel sequencing and whole genome sequencing to identify the probable disease-causing variants in these families. Using this approach, we obtained a 93% genetic solve rate and identified 16 (likely) causative variants in 14 families, of which seven novel variants were identified in ATOH7, COL18A1, MERTK, NDP, PROM1, PRPF8 and USH2A while nine recurrent variants were identified in CNGA3, CNGB1, HGSNAT, NMNAT1, SIX6 and TULP1. The novel MERTK variant and one recurrent TULP1 variant explained the intra-familial locus heterogeneity in one of the screened families while two recurrent CNGA3 variants explained compound heterozygosity in another family. The identification of variants in known disease-associated genes emphasizes the utilization of time and cost-effective screening approaches for rapid diagnosis. The timely genetic diagnosis will not only identify any associated systemic issues in case of syndromic IRDs, but will also aid in the acceleration of personalized medicine for patients affected with IRDs.

Pilea umbrosa ameliorate CCl4 induced hepatic injuries by regulating endoplasmic reticulum stress, pro-inflammatory and fibrosis genes in rat.

BACKGROUND: Pilea umbrosa (Urticaceae) is used by local communities (district Abbotabad) for liver disorders, as anticancer, in rheumatism and in skin disorders. METHODS: Methanol extract of P. umbrosa (PUM) was investigated for the presence of polyphenolic constituents by HPLC-DAD analysis. PUM (150 mg/kg and 300 mg/kg) was administered on alternate days for eight weeks in rats exposed with carbon tetrachloride (CCl4). Serum analysis was performed for liver function tests while in liver tissues level of antioxidant enzymes and biochemical markers were also studied. In addition, semi quantitative estimation of antioxidant genes, endoplasmic reticulum (ER) induced stress markers, pro-inflammatory cytokines and fibrosis related genes were carried out on liver tissues by RT-PCR analysis. Liver tissues were also studied for histopathological injuries. RESULTS: Level of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), peroxidase (POD) and glutathione (GSH) decreased (p < 0.05) whereas level of thiobarbituric acid reactive substance (TBARS), H2O2 and nitrite increased in liver tissues of CCl4 treated rat. Likewise increase in the level of serum markers; alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and total bilirubin was observed. Moreover, CCl4 caused many fold increase in expression of ER stress markers; glucose regulated protein (GRP-78), x-box binding protein1-total (XBP-1 t), x-box binding protein1-unspliced (XBP-1 u) and x-box binding protein1-spliced (XBP-1 s). The level of inflammatory mediators such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) was aggregated whereas suppressed the level of antioxidant enzymes; γ-glutamylcysteine ligase (GCLC), protein disulfide isomerase (PDI) and nuclear erythroid 2 p45-related factor 2 (Nrf-2). Additionally, level of fibrosis markers; transforming growth factor-ß (TGF-ß), Smad-3 and collagen type 1 (Col1-α) increased with CCl4 induced liver toxicity. Histopathological scrutiny depicted damaged liver cells, neutrophils infiltration and dilated sinusoids in CCl4 intoxicated rats. PUM was enriched with rutin, catechin, caffeic acid and apigenin as evidenced by HPLC analysis. Simultaneous administration of PUM and CCl4 in rats retrieved the normal expression of these markers and prevented hepatic injuries. CONCLUSION: Collectively these results suggest that PUM constituted of strong antioxidant chemicals and could be a potential therapeutic agent for stress related liver disorders.

Indigofera linifolia ameliorated CCl4 induced endoplasmic reticulum stress in liver of rat.

ETHNOPHARMACOLOGICAL RELEVANCE: Indigofera linifolia (L.f.) Retz. is used in subcontinent for liver disorders, in wounds, febrile eruption and as diuretic. AIM OF STUDY: The current study evaluates the protective effects of the methanol extract of Indigofera linifolia (ILM) on CCl4-induced endoplasmic reticulum (ER) stress in liver of rat. METHODS: ILM was analyzed for phytochemical classes, total phenolic (TPC) and flavonoid content (TFC) as well as multidimensional in vitro antioxidant assays. Male (Sprague Dawley) rats were dispersed into seven groups (6 rats/group) receiving 0.9% saline (1 ml/kg bw), CCl4 (1 ml/kg bw) diluted in olive oil (3:7 v/v), silymarin (200 mg/kg bw) + CCl4 (30% v/v), ILM (150 mg/kg bw) + CCl4 (30% v/v), ILM (300 mg/kg bw) + CCl4 and ILM alone (either 150 mg/kg bw or 300 mg/kg bw). RESULTS: ILM extract was constituted of different phytochemical classes. Co-administration of ILM along with CCl4 to rat revert the level of alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and total bilirubin in blood serum and antioxidant parameters in liver. Further, CCl4 increased the level of ER stress markers and inflammatory mediators while decreased level of GCLC and Nrf-2 in liver tissues of rat. CCl4-induced histopathological variations were reduced with ILM co-administration in liver tissues. CONCLUSION: The results suggest that active phyto-constituents of I. linifolia might be responsible for its antioxidant, anti-inflammatory and gene-regulating activities.

Evaluation of anti-inflammatory potential of the leaves of Wendlandia heynei (Schult.) Santapau & Merchant in Sprague Dawley rat.

ETHNOPHARMACOLOGICAL RELEVANCE: The leaves of W. heynei (family: Rubiaceae) are used by the folklore in swelling, wounds and body aches. In this study anti-inflammatory potential of W. heynei leaves has been evaluated during in vitro studies and in rat. METHODS: Methanol extract of the leaves of W. heynei (WHLM) and its fractions; n-hexane (WHLH), chloroform (WHLC), ethyl acetate (WHLE), n-butanol (WHLB) and residual soluble aqueous (WHLA) were screened for phytochemical analysis and several active constituents (alkaloids, flavonoids, saponins, tannins, terpenoids, ß-carotene and lycopene) were also quantified. Heat induced albumin denaturation assay and in vitro cell cultures was carried out for in vitro anti-inflammatory activity, while various in vivo assays like TPA induced ear edema, croton oil induced anus edema, formalin and carrageenan-induced hind paw edema was investigated in Sprague-Dawley rats. Alterations on levels of tumor necrosis factor (TNF-α), Interleukin-1ß (IL-1ß), IL-6 and prostaglandins (PGE2) induced with WHLE was studied in serum after carrageenan induced paw edema in rat. Meanwhile, the dose dependent WHLE inhibition of NFκB pathway via regulation of the phosphorylation of IKKs, IκBα, and p65 subunit was studied in LPS-induced rat peritoneal macrophages. On account of marked anti-inflammatory activity of WHLE its bioactive components were analyzed by HPLC-DAD analysis. RESULTS: The phytochemical analysis yielded alkaloids, saponins, tannins, coumarins, glycosides, quinones and vitamin C in WHLM and in all fractions. Fraction (WHLE) was enriched with alkaloids (20.20 ±â€¯2.5%), flavonoids (25.26 ±â€¯2.11%) and tannins (307.2 ±â€¯2.03 mg of GAE/g of extract), while terpenoids (21.60 ±â€¯1.65%) were the major constituents of WHLH. Ethyl acetate fraction convincingly protected heat induced albumin denaturation. WHLE exhibited highest edema inhibition in models of TPA-induced ear edema (74.51 ±â€¯2.05) and croton oil-induced anal edema (75.38 ±â€¯2.83). The pretreatment with WHLE significantly (p < 0.05) reduced the paw edema with formalin (78.99 ±â€¯2.26%) assessed after 6 h and in carrageenan (75.71 ±â€¯4.46%) was detected after 4 h. Level of anti-inflammatory markers; IL-1ß, IL-6, TNF-α and PGE2 in carrageenan induced paw edema in serum of rat was significantly (p < 0.001) decreased with WHLE pretreatment to rat. WHLE significantly inhibited the NFκB by reducing the phosphorylation of IKKs, IκBα, and p65 subunit in LPS-induced inflammation in rat peritoneal macrophages. HPLC-DAD analysis of WHLE indicated the presence of rutin, gallic acid, catechin, caffeic acid and myricetin. CONCLUSIONS: It is concluded that WHLM fractions have marked anti-inflammatory activity and this study endorsed the folklore use of W. heynei leaves for swelling related disorders.

Methanol extract of Iphiona aucheri ameliorates CCl4 induced hepatic injuries by regulation of genes in rats.

We have investigated the protective potential of methanol extract of Iphiona aucheri (IAM) on the expression of endoplasmic reticulum (ER) stress associated genes and inflammatory genes on carbon tetrachloride (CCl4) induced hepatic toxicity in rats. Hepatic damage markers: aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and bilirubin were elevated while the content of antioxidants: catalase (CAT), superoxide dismutase (SOD), peroxidase (POD) and reduced glutathione (GSH) were decreased significantly (p < 0.05) in CCl4 treated rats as compared to the control group. The CCl4 intoxication induced a higher expression of glucose-regulated protein 78 kDa (GRP78), X-box-binding protein 1 total (XBP1t), spliced X-box-binding protein 1 (XBP1s), unspliced X-box-binding protein 1 (XBP1u), C/EBP homologous protein (CHOP) and genes involved in inflammation and fibrosis: tumor necrosis factor alpha (TNF-α), transforming growth factor-beta (TGF-ß), mothers against DPP homolog 3 (SMAD3), alpha skeletal muscle actin (αSMA) and collagen type I alpha 1 chain (COL1A1). The intoxicated rats showed a low expression of the glutamate-cysteine ligase catalytic subunit (GCLC), protein disulfide isomerase (PDI) and nuclear factor (erythroid-derived 2) like-2 (Nrf2). The administration of IAM to intoxicated rats restored the expression of ER stress, inflammatory, fibrosis and antioxidant genes in a dose dependent manner. Our results indicated that IAM can impede the ER stress and inflammatory genes and it could be a complementary and alternative therapeutic agent for oxidative stress associated disorders.