Acetazolamide Inhibits the Level of Tyrosinase and Melanin: An Enzyme Kinetic,In Vitro,In Vivo,and In Silico Studies

Melanin is the major factor that determines skin color and protects from ultraviolet radiation. In present study we evaluated the anti‐melanogenesis effect of acetazolamide (ACZ) using four different approaches: enzyme kinetic, in vitro, in vivo and in silico. ACZ demonstrated significant inhibitory activity (IC₅₀ 7.895 ± 0.24 μm ) against tyrosinase as compared to the standard drug kojic acid (IC₅₀ 16.84 ± 0.64 μm ) and kinetic analyses showed that ACZ is a non‐competitive inhibitor without cytotoxic effect. In in vitro experiments, A375 human melanoma cells were treated with 20 or 40 μm of ACZ with or without 50 μm of l ‐DOPA. Western blot results showed that ACZ significantly (P < 0.05) decreased the expression level of tyrosinase at 40 μm . Zebrafish embryos were treated with 10, 20 or 40 μm of ACZ and of positive control kojic acid. At 72 h of treatment with ACZ and kojic acid, ACZ significantly (P < 0.001) decreased the embryos pigmentation to 40.8% of untreated embryos at the dose of 40 μm of ACZ while kojic acid decreased only 25.0% of pigmentation at the same dose of kojic acid. In silico docking were performed against tyrosinase using PyRx tool. Docking studies suggested that His244, Asn260 and His85 are the major interacting residues in the binding site of the protein. In conclusion, our results suggest that ACZ is a good candidate for the inhibition of melanin and it could be used as a lead for developing the drugs for hyperpigmentary disorders and skin whitening.     

Novel mechanism of cardiac protection by valsartan: synergetic roles of TGF‐β1 and HIF‐1α in Ang II‐mediated fibrosis after myocardial infarction

Transforming growth factor (TGF)‐β1 is a known factor in angiotensin II (Ang II)‐mediated cardiac fibrosis after myocardial infarction (MI). Hypoxia inducible factor‐1 (Hif‐1α) was recently demonstrated to involve in the tissue fibrosis and influenced by Ang II. However, whether Hif‐1α contributed to the Ang II‐mediated cardiac fibrosis after MI, and whether interaction or synergetic roles between Hif‐1α and TGF‐β pathways existed in the process was unclear. In vitro, cardiac cells were incubated under hypoxia or Ang II to mimic ischaemia. In vivo, valsartan was intravenously injected into Sprague–Dawley rats with MI daily for 1 week; saline and hydralazine (another anti‐hypertensive agent like valsartan) was used as control. The fibrosis‐related proteins were detected by Western blotting. Cardiac structure and function were assessed with multimodality methods. We demonstrated in vitro that hypoxia would induce the up‐regulation of Ang II, TGF‐β/Smad and Hif‐1α, which further induced collagen accumulation. By blocking with valsartan, a blocker of Ang II type I (AT1) receptor, we confirmed that the up‐regulation of TGF‐β/Smad and Hif‐1α was through the Ang II‐mediated pathway. By administering TGF‐β or dimethyloxalylglycine, we determined that both TGF‐β/Smad and Hif‐1α contributed to Ang II‐mediated collagen accumulation and a synergetic effect between them was observed. Consistent with in vitro results, valsartan significantly attenuated the expression of TGF‐β/Smad, Hif‐1α and fibrosis‐related protein in rats after MI. Heart function, infarcted size, wall thickness as well as myocardial vascularization of ischaemic hearts were also significantly improved by valsartan compared with saline and hydralazine. Our study may provide novel insights into the mechanisms of Ang II‐induced cardiac fibrosis as well as into the cardiac protection of valsartan.     

Neuropilin 1 expression correlates with the Radio‐resistance of human non‐small‐cell lung cancer cells

The purpose of this study was to determine the correlation between over‐expression of the neuropilin 1 (NRP1) gene and growth, survival, and radio‐sensitivity of non‐small cell lung carcinoma (NSCLC) cells. 3‐[4,5‐dimethylthylthiazol‐2‐yl]‐2,5 diphenyltetrazolium broide (MTT) and colony assays were then performed to determine the effect of NRP1 inhibition on the in vitro growth of NSCLC cells. The Annexin V‐Fluorescein Isothiocyanate (FITC) apoptosis detection assay was performed to analyse the effect of NRP1 enhancement on apoptosis of NSCLC cells. Transwell invasion and migration assays were employed to examine the metastatic ability of A549 cells post X‐ray irradiation. In addition, Western blot assays were carried out to detect the protein level of VEGFR2, PI3K and NF‐κB. Finally, to examine the effect of shNRP1 on proliferation and radio‐sensitivity in vivo, a subcutaneous tumour formation assay in nude mice was performed. Microvessel density in tumour tissues was assessed by immunohistochemistry. The stable transfected cell line (shNRP1‐A549) showed a significant reduction in colony‐forming ability and proliferation not only in vitro, but also in vivo. Moreover, shRNA‐mediated NRP1 inhibition also significantly enhanced the radio‐sensitivity of NSCLC cells both in vitro and in vivo. The over‐expression of NRP1 was correlated with growth, survival and radio‐resistance of NSCLC cells via the VEGF‐PI3K‐ NF‐κB pathway, and NRP1 may be a molecular therapeutic target for gene therapy or radio‐sensitization of NSCLC.     

Mobile genetic elements: in silico,in vitro,in vivo

Mobile genetic elements (MGEs), also called transposable elements (TEs), represent universal components of most genomes and are intimately involved in nearly all aspects of genome organization, function and evolution. However, there is currently a gap between the fast pace of TE discovery in silico, driven by the exponential growth of comparative genomic studies, and a limited number of experimental models amenable to more traditional in vitro and in vivo studies of structural, mechanistic and regulatory properties of diverse MGEs. Experimental and computational scientists came together to bridge this gap at a recent conference, ‘Mobile Genetic Elements: in silico, in vitro, in vivo’, held at the Marine Biological Laboratory (MBL) in Woods Hole, MA, USA.     

Beneficial effects of intramyocardial mesenchymal stem cells and VEGF165 plasmid injection in rats with furazolidone induced dilated cardiomyopathy

To explore the impact of myocardial injection of mesenchymal stem cells (MSCs) and specific recombinant human VEGF₁₆₅ (hVEGF₁₆₅) plasmid on collagen remodelling in rats with furazolidone induced dilated cardiomyopathy (DCM). DCM was induced by furazolidone (0.3 mg/bodyweight (g)/day per gavage for 8 weeks). Rats were then divided into four groups: (i) PBS group (n = 18): rats received equal volume myocardial PBS injection; (ii) MSCs group (n = 17): 100 μl culture medium containing 10⁵ MSCs were injected into four sites of left ventricular free wall (25 μl per site); (iii) GENE group (n = 18): pCMVen‐MLC2v‐EGFP‐VEGF₁₆₅ plasmid [5 × 109 pfu (0.2 ml)] were injected into four sites of left ventricular free wall (0.05 ml per site)] and (iv) MSCs+GENE group (n = 17): rats received both myocardial MSCs and pCMVen‐MLC2v‐EGFP‐VEGF₁₆₅ plasmid injections. After 4 weeks, cardiac function was evaluated by echocardiography. Myocardial mRNA expressions of type I, type III collagen and transforming growth factor (TGF)‐β1 were detected by RT‐PCR. The protein expression of hVEGF₁₆₅ was determined by Western blot. Myocardial protein expression of hVEGF₁₆₅ was demonstrated in GENE and MSCs+GENE groups. Cardiac function was improved in MSCs, GENE and MSCs+GENE groups. Collagen volume fraction was significantly reduced and myocardial TGF‐β1 mRNA expression significantly down‐regulated in both GENE and MSCs+GENE groups, collagen type I/III ratio reduction was more significant in MSCs+GENE group than in MSCs or GENE group. Myocardial MSCs and hVEGF₁₆₅ plasmid injection improves cardiac function possibly through down‐regulating myocardial TGF‐β1 expression and reducing the type I/III collagen ratio in this DCM rat model.     

Synthesis and Biological Evaluation of Matijin‐Su Derivatives as Potential Antihepatitis B Virus and Anticancer Agents

A series of Matijin‐Su (MTS, (2S)‐2‐{[(2S)‐2‐benzamido‐3‐phenylpropanoyl]amino}‐3‐phenylpropyl acetate) derivatives were synthesized and evaluated for their anti‐HBV and cytotoxic activities in vitro. Six compounds ( 4g , 4j , 5c , 5g , 5h and 5i ) showed significant inhibition against HBV DNA replication with the IC₅₀ values in range of 2.18 – 8.55 μm , which were much lower than that of positive control lamivudine (IC₅₀ 82.42 μm ). In particular, compounds 5h (IC₅₀ 2.18 μm ; SI 151.59) and 5j (IC₅₀ 5.65 μm ; SI 51.16) displayed relatively low cytotoxicities, resulting in high SI values. Notably, besides the anti‐HBV DNA replication activity, compound 4j also exhibited more potent in vitro cytotoxic activity than 5‐fluorouracil in two hepatocellular carcinoma cell (HCC) lines (QGY‐7701 and SMMC‐7721), indicating that 4j may be a promising lead for the exploration of drugs with dual therapeutic effects on HBV infection and HBV‐induced HCC.     

Blister‐inducing antibodies target multiple epitopes on collagen VII in mice

Epidermolysis bullosa acquisita (EBA) is an autoimmune subepidermal blistering disease of mucous membranes and the skin caused by autoantibodies against collagen VII. In silico and wet laboratory epitope mapping studies revealed numerous distinct epitopes recognized by EBA patients' autoantibodies within the non‐collagenous (NC)1 and NC2 domains of collagen VII. However, the distribution of pathogenic epitopes on collagen VII has not yet been described. In this study, we therefore performed an in vivo functional epitope mapping of pathogenic autoantibodies in experimental EBA. Animals (n = 10/group) immunized against fragments of the NC1 and NC2 domains of collagen VII or injected with antibodies generated against the same fragments developed to different extent experimental EBA. Our results demonstrate that antibodies targeting multiple, distinct epitopes distributed over the entire NC1, but not NC2 domain of collagen VII induce blistering skin disease in vivo. Our present findings have crucial implications for the development of antigen‐specific B‐ and T cell‐targeted therapies in EBA.     

Carbonic Anhydrase and Urease Inhibitory Potential of Various Plant Phenolics Using in vitro and in silico Methods

Plant phenolics are known to display many pharmacological activities. In the current study, eight phenolic compounds, e.g., luteolin 5‐O‐β‐glucoside ( 1 ), methyl rosmarinate ( 2 ), apigenin ( 3 ), vicenin 2 ( 4 ), lithospermic acid ( 5 ), soyasaponin II ( 6 ), rubiadin 3‐O‐β‐primeveroside ( 7 ), and 4‐(β‐d ‐glucopyranosyloxy)benzyl 3,4‐dihydroxybenzoate ( 8 ), isolated from various plant species were tested at 0.2 mm against carbonic anhydrase‐II (CA‐II) and urease using microtiter assays. Urease inhibition rate for compounds 1  –  8 ranged between 5.0 – 41.7%, while only compounds 1 , 2 , and 4 showed a considerable inhibition over 50% against CA‐II with the IC₅₀ values of 73.5 ± 1.05, 39.5 ± 1.14, and 104.5 ± 2.50 μm , respectively, where IC₅₀ of the reference (acetazolamide) was 21.0 ± 0.12 μm . In silico experiments were also performed through two docking softwares (Autodock Vina and i‐GEMDOCK) in order to find out interactions between the compounds and CA‐II. Actually, compounds 6 (30.0%) and 7 (42.0%) possessed a better binding capability toward the active site of CA‐II. According to our results obtained in this study, among the phenolic compounds screened, particularly 1 , 2 , and 4 appear to be the promising inhibitors of CA‐II and may be further investigated as possible leads for diuretic, anti‐glaucoma, and antiepileptic agents.     

Regulatory T cells protected against abdominal aortic aneurysm by suppression of the COX‐2 expression

CD4⁺CD25⁺ regulatory T cells (Tregs) have been shown to protect against the development of abdominal aortic aneurysm (AAA). Cyclooxygenase‐2 (COX‐2), a pro‐inflammatory protein, can convert arachidonic acid into prostaglandins (PGs). The present study was aimed to investigate the effect of Tregs on COX‐2 expression in angiotension II (Ang II)‐induced AAA in ApoE^?/? mice. Tregs were injected via tail vein in every 2 weeks. Ang II was continuously infused by a micropump for 28 days to induce AAA. In vivo, compared with the control group, adoptive transfer of Tregs significantly reduced the incidence of AAA, maximal diameter, and the mRNA and protein expression of COX‐2 in mice. Immunofluorescence showed that Tregs treatment reduced COX‐2 expression both in smooth muscle cells (SMCs) and macrophages in AAA. In vitro, the Western blot analysis showed that Tregs reduced Ang II‐induced COX‐2 expression in macrophages and SMCs. Meanwhile, ELISA showed that Tregs reduced Ang II‐induced prostaglandin E₂ (PGE₂) secretion. Moreover, Tregs increased SMC viability and induced transition of macrophages phenotype from M1 to M2. In conclusion, Tregs treatment dramatically decreased the expression of COX‐2 in vivo and in vitro, suggesting that Tregs could protect against AAA through inhibition of COX‐2. The study may shed light on the immune treatment of AAA.     

Culture of a Gastric Non‐Helicobacter pylori Helicobacter from the Stomach of a 14‐Year‐Old Girl

Helicobacter felis belongs to the fastidious gastric non‐Helicobacter pylori helicobacter species that are typically found in the stomach of cats and dogs. These bacteria have the potential to colonize the human stomach and are then associated with gastritis, gastroduodenal ulcers, and MALT lymphoma. Strains cultured from the human stomach are rare. Here, we present the first isolation of H. felis from a gastric biopsy specimen of a 14‐year‐old girl who presented with persistent epigastric pain. The strain was cultured using our routine protocol for H. pylori and identified by phylogenetic analyses of partial urease AB and gyrB gene sequences.     

Ultrastructure of grey mullet (Mugil cephalus,Linnaeus, 1758) spermatozoa as revealed from light,scanning and transmission electron microscopy

Spermatozoa morphology and fine structure were studied in the grey mullet, Mugil cephalus using light, scanning and transmission electron microscopy. Light microscopy observations indicate a semi‐cystic type of spermatogenesis in the testis. The electron microscopy micrograph showed that the spermatozoon of M. cephalus is uniflagellated (total length 5.78 ± 1.26 μm), differentiated into an ovoid‐shaped head without acrosome (1.80 ± 0.35 μm in length and 1.91 ± 0.30 μm in diameter), with a short midpiece and a long cylindrical flagellar tail (length 3.60 ± 0.50 μm). The midpiece is characterized by the presence of four to five vacuoles, a cytoplasmic canal, two centriole and two spherical mitochondria having a flat type of cristae. Chromatin granules of the nucleus form an electron‐dense homogeneous mass. The flagellum consists of nine peripheral microtubules and a central pair (9 + 2) surrounded by the plasma membrane with side fins. The results confirm that spermatozoa of M. cephalus are perciform or teleostean type II. Information generated from the present study will be useful in taxonomic classification, cryopreservation and breeding work.     

Synthesis,Biological Evaluation and Molecular Docking of Deferasirox and Substituted 1,2,4‐Triazole Derivatives as Novel Potent Urease Inhibitors: Proposing Repositioning Candidate

A series of new deferasirox derivatives were synthesized through the reaction of monosubstituted hydrazides with 2‐(2‐hydroxyphenyl)‐4H‐benzo[e][1,3]oxazin‐4‐one. For the first time, deferasirox and some of its derivatives were evaluated for their in vitro inhibitory activity against Jack bean urease. The potencies of the members of this class of compounds are higher than that of acetohydroxamic acid. Two compounds, bearing tetrazole and hydrazine derivatives (bioisoester of carboxylate group), represented the most potent urease inhibitory activity with IC₅₀ values of 1.268 and 3.254 μm , respectively. In silico docking studies were performed to delineate possible binding modes of the compounds with the enzyme, urease. Docking analysis suggests that the synthesized compounds were anchored well in the catalytic site and extending to the entrance of binding pocket and thus restrict the mobility of the flap by interacting with its crucial amino acid residues, CME592 and His593. The overall results of urease inhibition have shown that these target compounds can be further optimized and developed as a lead skeleton for the discovery of novel urease inhibitors     

The B subunit of Escherichia coli heat‐labile toxin alters the development and antigen‐presenting capacity of dendritic cells

Escherichia coli's heat‐labile enterotoxin (Etx) and its non‐toxic B subunit (EtxB) have been characterized as adjuvants capable of enhancing T cell responses to co‐administered antigen. Here, we investigate the direct effect of intravenously administered EtxB on the size of the dendritic and myeloid cell populations in spleen. EtxB treatment appears to enhance the development and turnover of dendritic and myeloid cells from precursors within the spleen. EtxB treatment also gives a dendritic cell (DC) population with higher viability and lower activation status based on the reduced expression of MHC‐II, CD80 and CD86. In this respect, the in vivo effect of EtxB differs from that of the highly inflammatory mediator lipopolysaccharide. In in vitro bone marrow cultures, EtxB treatment was also found to enhance the development of DC from precursors dependent on Flt3L. In terms of the in vivo effect of EtxB on CD4 and CD8 T cell responses in mice, the interaction of EtxB directly with DC was demonstrated following conditional depletion of CD11c⁺ DC. In summary, all results are consistent with EtxB displaying adjuvant ability by enhancing the turnover of DC in spleen, leading to newly mature myeloid and DC in spleen, thereby increasing DC capacity to perform as antigen‐presenting cells on encounter with T cells.     

Wnt inhibitors enhance chondrogenesis of human mesenchymal stem cells in a long-term pellet culture

The long-term effects (~3 weeks) of two Wnt inhibitors (dickkopf [DKK]-1 and secreted frizzled-related protein [sFRP]-1), on the chondrogenic differentiation of human mesenchymal stem cells (hMSCs) was determined. Wnt inhibitors significantly increased the amount of glycosaminoglycan (GAG) in treated pellets (P < 0.05). The gene expression of COL2A1 increased and COL1A1 decreased while the gene expression of SOX-9 and COL10A1 did not change significantly after three weeks of in vitro culture. The protein expression of type II collagen significantly increased (P < 0.05) and that of type I collagen significantly decreased (P < 0.05) while SOX-9 and type X collagen protein expression was unaffected. These findings suggest that Wnt inhibitors promote the chondrogenic differentiation of hMSCs when treated for three weeks.     

In vivo recombination efficiency of two site‐specific recombination systems,VCre/VloxP and SCre/SloxP,in medaka (Oryzias latipes)

The present study delineates the in vivo efficiency of two site‐specific recombination systems, VCre/VloxP and SCre/SloxP, in medaka (Oryzias latipes). VCre, SCre, and Cre RNA was microinjected into fertilized medaka eggs belonging to three transgenic lines harboring VloxP, SloxP, and loxP cassette. VCre induced site‐specific recombination specifically at VloxP sequence and SCre at SloxP sequence without any cross‐reactivity. These findings provide two novel alternative recombination systems in vivo in addition to the existing Cre/loxP and Flp/FRT systems, thus enabling sophisticated gene expression in model organisms.