Revealing novel cytb and nad5 genes-based population diversity and benzimidazole resistance in Echinococcus granulosus of bovine origin.

Cystic echinococcosis (CE) is a neglected zoonotic disease caused by Echinococcus granulosus (sensu stricto). The parasite affects a wide range of livestock and wild animals. In this study, the population diversity of the Echinococcus species was investigated based on mitochondrial cytochrome b (cytb) and NADH dehydrogenase subunit 5 (nad5) genes. In addition to this, ß-tubulin gene isoforms of Echinococcus granulosus were amplified to determine the resistance against benzimidazoles. For this purpose, 40 cyst samples from cattle (n = 20) and buffaloes (n = 20) were collected from the main abattoir of Sialkot. DNA extraction was performed using Qiagen Blood and Tissue Kits. Amplification was performed through PCR. Each amplicon was confirmed by GelRed™ stained agarose gel (2%). Samples were sequenced in a DNA analyzer and viewed for any misread nucleotide by using MEGA (v.11). Corrections in nucleotide sequence and multiple sequence alignment were made through the same software. NCBI-BLAST was used for sample specific sequences to identify them as belonging to a particular species. Diversity indices were estimated using DnaSP (v.6) while phylogenetic analysis was inferred using the Bayesian method using MrBayes (v.1.1). ß-tubulin gene isoforms sequence analysis was performed to find out the candidate gene causing benzimidazole resistance. All 40 isolates were found positive for E. granulosus. BLAST-based searches of sequences of each isolate for each gene (nad5 and cytb) confirmed their maximum similarity with the G1 genotype. Overall, high haplotype diversity (Hd nad5 = 1.00; Hd cytb = 0.833) and low nucleotide diversity (π nad5 = 0.00560; π = cytb = 0.00763) was identified based on diversity indices. For both the genes, non-significant values of Tajima's D (nad5 = -0.81734; cytb = -0.80861) and Fu's Fs (nad5 = -1.012; cytb = 0.731) indicate recent population expansion. Bayesian phylogeny-based results of nad5 and cytb sequences confirmed their genotypic status as distinct from other Echinococcus species. This study shed light on the status of benzimidazole resistance in Echinococcus granulosus for the very first time from Pakistan. The findings of this study will significantly add in the information available on genetic diversity of Echinoccous granulosus based on cytb and nad5 genes sequences.

PI3K inhibitors in trastuzumab-resistant HER2-positive breast cancer cells with PI3K pathway alterations.

The activation of the PI3K signaling pathway resulting from genetic alterations induces carcinogenesis and resistance to anticancer therapies. Breast cancer is a major malignancy that is associated with dysregulation of the PI3K signaling pathway. PIK3CA mutations and PTEN loss occur in every subtype of breast cancer. PI3K inhibitors are being evaluated in breast cancer after the success of an alpha isoform-specific PI3K inhibitor in estrogen receptor (ER)-positive/HER2-negative metastatic breast cancer. Some preclinical data indicate the potential for PI3K/mTOR targeting in combination with trastuzumab for HER2-positive breast cancer with or without expression of the estrogen receptor. However, the role of this therapy in HER2-positive breast cancer with PIK3CA mutations and/or PTEN loss remains unclear. We examined three HER2-positive, ER-negative breast cancer cell lines to determine the efficacy of a novel alpha isoform-specific PI3K inhibitor in combination with trastuzumab. The results indicated that this combination was effective in PIK3CA-mutant or PTEN-deficient breast cancer cells by inducing apoptosis and inhibiting the expression of downstream proteins. PTEN loss by siRNA modulation in parental HER2-positive cancer cells with PI3K signaling pathway alterations could not confer resistance to alpelisib or GDC-0077 plus trastuzumab. We selected the CK-MB-1 cell line without alterations in the PI3K pathway to demonstrate that PI3K inhibitors plus trastuzumab represented a biomarker-specific treatment. In vivo effects of alpelisib plus trastuzumab were tested and confirmed in a mouse model, showing the combination strategy offered the best opportunity to achieve tumor volume reduction. With known safety profiles, this cytotoxic chemotherapy-free regimen warrants further attention as a biomarker-driven strategy for treating HER2-positive breast cancer.

M33 condenses chromatin through nuclear body formation and methylation of both histone H3 lysine 9 and lysine 27.

Heterochromatin, a type of condensed DNA in eukaryotic cells, has two main categories: Constitutive heterochromatin, which contains H3K9 methylation, and facultative heterochromatin, which contains H3K27 methylation. Methylated H3K9 and H3K27 serve as docking sites for chromodomain-containing proteins that compact chromatin. M33 (also known as CBX2) is a chromodomain-containing protein that binds H3K27me3 and compacts chromatin in vitro. However, whether M33 mediates chromatin compaction in cellulo remains unknown. Here we show that M33 compacts chromatin into DAPI-intense heterochromatin domains in cells. The formation of these heterochromatin domains requires H3K27me3, which recruits M33 to form nuclear bodies. G9a and SUV39H1 are sequentially recruited into M33 nuclear bodies to create H3K9 methylated chromatin in a process that is independent of HP1α. Finally, M33 decreases progerin-induced nuclear envelope disruption caused by loss of heterochromatin. Our findings demonstrate that M33 mediates the formation of condensed chromatin by forming nuclear bodies containing both H3K27me3 and H3K9me3. Our model of M33-dependent chromatin condensation suggests H3K27 methylation corroborates with H3K9 methylation during the formation of facultative heterochromatin and provides the theoretical basis for developing novel therapies to treat heterochromatin-related diseases.

Development of the CK-MB-1 trastuzumab-resistant HER2-positive breast cancer cell line and xenograft animal models.

BACKGROUND: Patients with human epidermal growth factor receptor 2 (HER2)-positive breast cancer who fail to respond to anti-HER2 treatments have poor prognoses. Most trastuzumab-resistant breast cancer cell lines available from biobanks feature either phosphoinositide-3-kinase, catalytic, alpha (PIK3CA) mutation or the loss of phosphatase and tensin homolog (PTEN). However, PIK3CA mutations and/or PTEN loss do not account for most trastuzumab-resistant tumors in humans. METHODS: Breast cancer cells were collected from one patient's malignant ascites. These cells were cultured and maintained to develop a stable cell line, which we named CK-MB-1. We used western blotting to evaluate protein expression. The PIK3CA status of CK-MB-1 cells was analyzed using Sanger sequencing and validated using next-generation sequencing. In vivo, CK-MB-1 xenograft tumor models were developed in zebrafish and immunodeficient mice. RESULTS: CK-MB-1 cells maintained the major characteristics of the parental tumor including HER2 positivity and estrogen receptor negativity. The HER2 gene amplification of CK-MB-1 cells was detected by fluorescence in situ hybridization. The integrity of PTEN was confirmed by its positive protein expression and the absence of gene mutations. No common PIK3CA mutation was detected. Compared with the findings in two other HER2-positive trastuzumab-resistant cell lines, CK-MB-1 cells exhibited greater resistance to trastuzumab, chemotherapeutics, and small-molecule drugs. Trastuzumab resistance in CK-MB-1 cells was confirmed in vivo using the NOD SCID mouse model. CONCLUSIONS: CK-MB-1 cells represent a stable HER2-positive trastuzumab-resistant breast cancer cell line. The resistance of CK-MB-1 cells does not originate from the PTEN or phosphoinositide 3-kinase signaling pathway, which can provide an alternative approach for potential drugs.

Correction to: Occurrence of 25 pharmaceuticals in Taihu Lake and their removal from two urban drinking water treatment plants and a constructed wetland.

The article Occurrence of 25 pharmaceuticals in Taihu Lake and their removal from two urban drinking water treatment plants and a constructed wetland, written by Xia-Lin Hu, Yi-Fan Bao, Jun-Jian Hu, You-Yu Liu and Da-Qiang Yin, was originally published electronically on the publisher's internet portal.

Effects and mechanism of STAT3 silencing on the growth and apoptosis of colorectal cancer cells.

Signal transducer and activator of transcription 3 (STAT3) have roles in various cellular processes, including angiogenesis, apoptosis, cell cycle progression, cell migration and drug resistance. To clarify the effects of STAT3 in colorectal cancer (CRC) cells and the underlying molecular mechanisms, STAT3 was directly silenced, and the effects of STAT3 silencing on cell proliferation, apoptosis and growth with phenotype-associated molecules were examined.pSH1-Si-STAT3 was successfully transfected into the CRC HCT-116 and SW480 cell lines, which was verified by GFP tagging under a fluorescence microscope. An MTT assay revealed that the proliferation of both cell lines that were transfected with pSH1-Si-STAT3 was significantly suppressed in comparison with the control and mock (P<0.05). Acridine orange/ethidium bromide staining and flow cytometry indicated that the transfected cell lines had a significantly higher rate of apoptosis than the control- and mock-treated cells (P<0.05). STAT3-silienced cells were also significantly arrested at the G2/M stage compared with the cells that were transfected with control and mock plasmids (P<0.05). At the mRNA level, the expression of STAT3 and survivin was significantly downregulated (P<0.05), but p53 and caspase-3 were significantly upregulated (P<0.05). The significantly different patterns of expression were observed in western blot analysis (P<0.05). The findings of the present study indicate that STAT3 silencing may suppress the proliferation and growth of CRC cells, and induce their apoptosis by upregulating the expression of survivin, p53 and caspase-3. Therefore, STAT3 may be a good candidate for CRC gene therapy.

Simultaneous determination of 29 pharmaceuticals in fish muscle and plasma by ultrasonic extraction followed by SPE-UHPLC-MS/MS.

A confirmatory method for the simultaneous detection of 29 pharmaceuticals in fish muscle and plasma was developed by using solid-phase extraction combined with ultra-high performance liquid chromatography and tandem mass spectrometry. Fish samples were extracted with methanol and enriched using Oasis HLB solid-phase extraction columns in one step. Twenty-nine target pharmaceuticals were quantified by the internal standard method and the calibration curves showed good linearity in a wide range with determination coefficients of greater than 0.913. The detection limits of the pharmaceuticals ranged from 0.01 to 2.00 µg/kg (µg/L). The applicability of the method was checked by precision and recovery experiments. The average recoveries of the 29 pharmaceuticals were between 61 and 111%, and all the relative standard deviations were below 25%. Our reported method has been demonstrated to be sensitive, convenient, rapid, and reliable for the simultaneous determination of 29 pharmaceuticals in fish muscle and plasma. Real sample determination showed that 25 and 9 of the 29 compounds were detected in fish muscle and plasma, respectively.

Occurrence of 25 pharmaceuticals in Taihu Lake and their removal from two urban drinking water treatment plants and a constructed wetland.

Pharmaceuticals in drinking water sources have raised significant concerns due to their persistent input and potential human health risks. The seasonal occurrence of 25 pharmaceuticals including 23 antibiotics, paracetamol (PAR), and carbamazepine (CMZ) in Taihu Lake was investigated; meanwhile, the distribution and removal of these pharmaceuticals in two drinking water treatment plants (DWTPs) and a constructed wetland were evaluated. A high detection frequency (>70%) in the Taihu Lake was observed for nearly all the 25 pharmaceutics. Chlortetracycline (234.7 ng L-1), chloramphenicol (27.1 ng L-1), erythromycin (72.6 ng L-1), PAR (71.7 ng L-1), and CMZP (23.6 ng L-1) are compounds with both a high detection frequency (100%) and the highest concentrations, suggesting their wide use in the Taihu Basin. Higher concentrations of chloramphenicols, macrolides, PAR, and CMZP were observed in dry season than in wet season, probably due to the low flow conditions of the lake in winter and the properties of pharmaceuticals. The overall contamination levels of antibiotic pharmaceutics (0.2-74.9 ng L-1) in the Taihu Lake were lower than or comparable to those reported worldwide. However, for nonantibiotic pharmaceutics, PAR (45.0 ng L-1) and CMZP (14.5 ng L-1), significantly higher concentrations were observed in the Taihu Lake than at a global scale. High detection frequencies of 25 pharmaceuticals were observed in both the two DWTPs (100%) and the wetland (>60%) except for florfenicol and sulfapyridine. The removal efficacies of the studied pharmaceuticals in DWTP B with advanced treatment processes including ozonation and granular activated carbon filtration (16.7-100%) were superior to DWTP A with conventional treatment processes (2.9-100%), except for sulfonamides. Wetland C with the constructed root channel technology was efficient (24.2-100%) for removing most pharmaceuticals. This work suggests that the application of cost-effective technologies such as constructed wetlands should be considered as an efficient alternative for removing pharmaceuticals from water supply sources.

XRCC1 and XPD genetic polymorphisms and susceptibility to age-related cataract: a meta-analysis.

OBJECTIVE: This meta-analysis aimed to determine the relationships between XRCC1 Arg399Gln (rs25487 G>A) and XPD Lys751Gln (rs1052559 A>C) polymorphisms and susceptibility to age-related cataract. METHODS: Medline (1966-2013), the Cochrane Library Database (Issue 12, 2013), EMBASE (1980-2013), CINAHL (1982-2013), Web of Science (1945-2013) and the Chinese Biomedical Database (CBM; 1982-2013) were searched without language restrictions. Various combinations of the keywords and MeSH terms were used to screen for potentially relevant studies, specifically "genetic polymorphisms" or "SNPs" or "variation" or "single nucleotide polymorphism" or "polymorphism" or "mutation" or "variant"; "X-ray repair cross complementing protein 1" or "Xeroderma Pigmentosum Group D Protein" or "X-ray repair cross complementing protein 1" or "Xeroderma Pigmentosum Group D Protein" or "XPD" or "Xeroderma Pigmentosum Complementation Group D Protein" or "ERCC2" or "XRCC1" or "XRCC1 DNA repair protein"; and "Cataract" or " Membranous Cataract" or " Pseudoaphakia." Meta-analyses were conducted using Stata 12.0 software. Crude odds ratios (ORs) and their corresponding 95% confidence intervals (95% CI) were calculated. RESULTS: Six independent case-control studies were included in the meta-analysis. Our results indicated that the association between the genetic polymorphisms of XRCC1 Arg399Gln G>A and XPD Lys751Gln A>C and increased susceptibility to age-related cataracts was statistically significant (XRCC1 Arg399Gln: OR=1.30, 95% CI=1.17-1.44, p<0.001; XPD Lys751Gln: OR=1.25, 95% CI=1.12-1.40, p<0.001, respectively). Ethnicity-stratified analysis indicated that the XRCC1 Arg399Gln G>A polymorphism was correlated with the development and progression of age-related cataract in China, India, and Turkey in the allele model and the dominant model. For the XPD Lys751Gln A>C variant, the association with the pathogenesis of age-related cataract in China and Turkey in the allele model and the dominant model was investigated. CONCLUSIONS: The association of XRCC1 Arg399Gln and XPD Lys751Gln polymorphisms with age-related cataract susceptibility observed in our meta-analyses supports the view that XRCC1 and XPD may play important roles in susceptibility to age-related cataract.

Selective synthesis of Zn(1 - x)Mn(x)Se nanobelts and nanotubes from [Zn(1 - x)Mn(x)Se](DETA)0.5 nanbelts in solution (x = 0-0.15) and their EPR and optical properties.

Zn(1 - x)Mn(x)Se (x = 0-0.15) nanobelts and nanotubes can be synthesized via the removal of diethylenetriamine (DETA) in 1-octadecene (ODE) and ethylene glycol (EG), respectively, using [Zn(1 - x)Mn(x)Se](DETA)(0.5) nanobelts as a template. The as-prepared ZnSe nanobelts are single-crystalline and grown along the [001] direction, and the ZnSe nanotubes consist of nanoparticles assembled along the [001] direction. In addition, Mn(2+)-doped Zn(1 - x)Mn(x)Se (x = 0.05, 0.10, 0.15) nanotubes are prepared for the first time if doped [Zn(1 - x)Mn(x)Se](DETA)(0.5) nanobelts are used as the template. The formation process of Zn(1 - x)Mn(x)Se nanobelts and nanotubes has been studied, and a plausible mechanism is proposed. Photoluminescence (PL) and electron paramagnetic resonance (EPR) spectra of Zn(1 - x)Mn(x)Se nanobelts and Zn(1 - x)Mn(x)Se nanotubes have been investigated.