Phosphoinositide-dependent kinase-1 (PDK-1) is a serine/threonine protein kinase that phosphorylates members of the conserved AGC kinase superfamily, including AKT and PKC, and is implicated in important cellular processes including survival, metabolism and tumorigenesis. In large cohorts of nevi and melanoma samples, PDK1 expression was significantly higher in primary melanoma, compared with nevi, and was further increased in metastatic melanoma. PDK1 expression suffices for its activity, due to auto-activation, or elevated phosphorylation by phosphoinositide 3'-OH-kinase (PI 3-K). Selective inactivation of Pdk1 in the melanocytes of BrafV600E::Pten−/− or BrafV600E::Cdkn2a−/−::Pten−/− mice delayed the development of pigmented lesions and melanoma induced by systemic or local administration of 4-HT. Melanoma invasion and metastasis were significantly reduced or completely prevented by Pdk1 deletion. Administration of the PDK1 inhibitor GSK2334470 (PDKi) effectively delayed melanomagenesis and metastasis in BrafV600E::Pten−/− mice. Pdk1−/− melanomas exhibit a marked decrease in the activity of AKT, P70S6K and PKC. Notably, PDKi was as effective in inhibiting AGC kinases and colony forming efficiency of melanoma with Pten WT genotypes. Gene expression analyses identified Pdk1-dependent changes in FOXO3a-regulated genes and inhibition of FOXO3a restored proliferation and colony formation of Pdk1−/− melanoma cells. Our studies provide direct genetic evidence for the importance of PDK1, in part through FOXO3a-dependent pathway, in melanoma development and progression.
PDK1; FOXO3a; melanoma; Braf; Pten; GSK2334470
Radiofrequency ablation (RFA) of atrial flutter (AFL) is a commonly performed procedure with low risk of complications. Several case reports and animal studies cautioned about the risk of right coronary artery (RCA) injury following AFL ablation. This risk is due to the anatomic proximity of the RCA to the cavo-tricuspid isthmus where ablation is performed. We present a case report that demonstrates postmortem evidence of RCA injury following RFA of AFL.
Atrial flutter; Coronary artery; Cardiac arrhythmia; Myocardial infarction; Radiofrequency catheter ablation
DDR2 gene, playing an essential role in regulating osteoblast differentiation and chondrocyte maturation, may influence bone mineral density (BMD) and osteoporosis, but the genetic variations actually leading to the association remain to be elucidated. Therefore, the aim of this study was to investigate whether the genetic variants in DDR2 are associated with BMD and fracture risk. This study was performed in three samples from two ethnicities, including 1,300 Chinese Han subjects, 700 Chinese Han subjects (350 with osteoporotic hip fractures and 350 healthy controls) and 2,286 US white subjects. Twenty-eight SNPs in DDR2 were genotyped and tested for associations with hip BMD and fractures. We identified 3 SNPs in DDR2 significantly associated with hip BMD in the Chinese population after multiple testing adjustments, which were rs7521233 (P = 1.06×10−4, β: −0.018 for allele C), rs7553831 (P = 1.30×10−4, β: −0.018 for allele T), and rs6697469 (P = 1.59×10−3, β: −0.015 for allele C), separately. These three SNPs were in high linkage disequilibrium. Haplotype analyses detected two significantly associated haplotypes, including one haplotype in block 2 (P = 9.54×10−4, β: −0.016) where these three SNPs located. SNP rs6697469 was also associated with hip fractures (P = 0.043, OR: 1.42) in the Chinese population. The effect on fracture risk was consistent with its association with lower BMD. However, in the white population, we didn’t observe significant associations with hip BMD. eQTL analyses revealed that SNPs associated with BMD also affected DDR2 mRNA expression levels in Chinese. Our findings, together with the prior biological evidence, suggest that DDR2 could be a new candidate for osteoporosis in Chinese population. Our results also reveal an ethnic difference, which highlights the need for further genetic studies in each ethnic group.
Osteoporosis is caused by excessive bone resorption (by osteoclasts) over bone formation (by osteoblasts). Monocytes are important to osteoporosis by serving as progenitors of osteoclasts and produce cytokines for osteoclastogenesis.
To identify osteoporosis-related genes, we performed microarray analyses of monocytes using Affymetrix 1.0 ST arrays in 42 (including 16 pre- and 26 postmenopausal) high hip BMD (bone mineral density) vs. 31 (including 15 pre- and 16 postmenopausal) low hip BMD Caucasian female subjects. Here, high vs. low BMD is defined as belonging to top vs. bottom 30% of BMD values in population.
Differential gene expression analysis in high vs. low BMD subjects was conducted in the total cohort as well as pre- and post-menopausal subjects. Focusing on the top differentially expressed genes identified in the total, the pre- and the postmenopausal subjects (with a p <5E-03), we performed replication of the findings in 3 independent datasets of microarray analyses of monocytes (total N = 125).
We identified (in the 73 subjects) and successfully replicated in all the 3 independent datasets 2 genes, DAXX and PLK3. Interestingly, both genes are apoptosis induction genes and both down-regulated in the low BMD subjects. Moreover, using the top 200 genes identified in the meta-analysis across all of the 4 microarray datasets, GO term enrichment analysis identified a number of terms related to induction of apoptosis, for which the majority of component genes are also down-regulated in the low BMD subjects. Overall, our result may suggest that there might be a decreased apoptosis activity of monocytes in the low BMD subjects.
Our study for the first time suggested a decreased apoptosis rate (hence an increased survival) of monocytes, an important osteoclastogenic cell, as a novel mechanism for osteoporosis.
Co-injection of zygotes with Cas9 mRNA and sgRNA has been proven to be an efficient gene-editing strategy for genome modification of different species. Genetic engineering in pigs holds a great promise in biomedical research. By co-injection of one-cell stage embryos with Cas9 mRNA and Npc1l1 sgRNA, we achieved precise Npc1l1 targeting in Chinese Bama miniature pigs at the efficiency as high as 100%. Meanwhile, we carefully analyzed the Npc1l1 sgRNA:Cas9-mediated on- and off-target mutations in various somatic tissues and ovaries, and demonstrated that injection of zygotes with Cas9 mRNA and sgRNA is an efficient and reliable approach for generation of gene-modified pigs.
Glucocorticoids are among the most commonly used anti-inflammatory agents. Despite the enormous efforts in elucidating the glucocorticoid-mediated anti-inflammatory actions, how glucocorticoids tightly control overactive inflammatory response is not fully understood. Here we show that glucocorticoids suppress bacteria-induced inflammation by enhancing IRAK-M, a central negative regulator of Toll-like receptor signalling. The ability of glucocorticoids to suppress pulmonary inflammation induced by non-typeable Haemophilus influenzae is significantly attenuated in IRAK-M-deficient mice. Glucocorticoids improve the survival rate after a lethal non-typeable Haemophilus influenzae infection in wild-type mice, but not in IRAK-M-deficient mice. Moreover, we show that glucocorticoids and non-typeable Haemophilus influenzae synergistically upregulate IRAK-M expression via mutually and synergistically enhancing p65 and glucocorticoid receptor binding to the IRAK-M promoter. Together, our studies unveil a mechanism by which glucocorticoids tightly control the inflammatory response and host defense via the induction of IRAK-M and may lead to further development of anti-inflammatory therapeutic strategies.
Glucocorticoids strongly suppress inflammation. Here the authors show that this suppression is mediated by induction of the negative inflammatory regulator IRAK-M, and demonstrate its important role in host defense against the pneumonia-causative bacterium, non-typeable Haemophilus influenzae.
Serotonin (5-HT) receptors are valuable molecular targets for antipsychotic drug discovery. Current reported methods for detecting 5-HT receptors, such as cAMP accumulation and calcium influx assay, are often demanding specialized instruments and inconvenient. The luciferase reporter gene assay, based on the responsible-element-regulated expression of luciferase, has been widely applied in the high-throughput functional assay for many targets because of its high sensitivity and reliability. However, 5-HT receptors couple to multiple G-proteins regulate respective downstream signalling pathways and are usually detected using different response elements. Hence, finding a suitable response element to fulfil the detection of different 5-HT receptors and make the results of luciferase reporter gene assays generalizable is very useful for active compounds screening. Here, we conducted three luciferase reporter assays using CRE, NFAT, and SRE response elements attached to 5-HT to detect the activation of different 5-HT receptors in CHO-K1 cells. The potencies and efficacies of the reported ligands (agonists and antagonists) were determined and compared. Our results indicate that CRE-luciferase reporter gene is sensitive and reliable to detect the activities of G protein-coupled 5-HT receptors.
Fungi are important soil components as both decomposers and plant symbionts and play a major role in ecological and biogeochemical processes. However, little is known about the richness and structure of fungal communities. DNA sequencing technologies allow for the direct estimation of microbial community diversity, avoiding culture-based biases. We therefore used 454 pyrosequencing to investigate the fungal communities in the rhizosphere of Xinjiang jujube. We obtained no less than 40,488 internal transcribed spacer (ITS) rDNA reads, the number of each sample was 6943, 6647, 6584, 6550, 6860, and 6904, and we used bioinformatics and multivariate statistics to analyze the results. The index of diversity showed greater richness in the rhizosphere fungal community of a 3-year-old jujube than in that of an 8-year-old jujube. Most operational taxonomic units belonged to Ascomycota, and taxonomic analyses identified Hypocreales as the dominant fungal order. Our results demonstrated that the fungal orders are present in different proportions in different sampling areas. Redundancy analysis (RDA) revealed a significant correlation between soil properties and the abundance of fungal phyla. Our results indicated lower fungal diversity in the rhizosphere of Xinjiang jujube than that reported in other studies, and we hope our findings provide a reference for future research.
Nephrotoxicity is the dose-limiting factor for colistin, but the exact mechanism is unknown. This study aimed to investigate the roles of the mitochondrial, death receptor, and endoplasmic reticulum pathways in colistin-induced nephrotoxicity. Mice were intravenously administered 7.5 or 15 mg of colistin/kg of body weight/day (via a 3-min infusion and divided into two doses) for 7 days. Renal function, oxidative stress, and apoptosis were measured. Representative biomarkers involved in the mitochondrial, death receptor, and endoplasmic reticulum pathways were investigated, and the key markers involved in apoptosis and autophagy were examined. After 7-day colistin treatment, significant increase was observed with blood urea nitrogen, serum creatinine, and malondialdehyde, while activities of superoxide dismutase (SOD) and catalase decreased in the kidneys. Acute tubular necrosis and mitochondrial dysfunction were detected, and colistin-induced apoptosis was characterized by DNA fragmentation, cleavage of poly(ADP-ribose) polymerase (PARP-1), increase of 8-hydroxydeoxyguanosine (8-OHdG), and activation of caspases (caspase-8, -9, and -3). It was evident that colistin-induced apoptosis involved the mitochondrial pathway (downregulation of Bcl-2 and upregulation of cytochrome C [cytC] and Bax), death receptor pathway (upregulation of Fas, FasL, and Fas-associated death domain [FADD]), and endoplasmic reticulum pathway (upregulation of Grp78/Bip, ATF6, GADD153/CHOP, and caspase-12). In the 15-mg/kg/day colistin group, expression of the cyclin-dependent kinase 2 (CDK2) and phosphorylated JNK (p-JNK) significantly increased (P < 0.05), while in the 7.5-mg/kg/day colistin group, a large number of autophagolysosomes and classic autophagy were observed. Western blot results of Beclin-1 and LC3B indicated that autophagy may play a protective role in colistin-induced nephrotoxicity. In conclusion, this is the first study to demonstrate that all three major apoptosis pathways and autophagy are involved in colistin-induced nephrotoxicity.
Chemokines produced in distinct tissue microenvironments sustain migration of mature lymphocytes in lymphoglandula. Chemokine receptors expressed on chronic lymphocytic leukemia (CLL) cells regulate the migration of the leukemia cells within the bone marrow (BM), lymphoid organs in collaboration with chemokines. Chemokines form a pro-survival circuitry by regulating leukocyte trafficking, maintaining extended lymphocyte survival. Therefore, chemokines in tumor cell–microenvironment interactions represent a target for treatment of CLL. AMD3100 disrupts the CLL/microenvironment interactions and influences CXCL12/CXCR4 survival signaling. Fostamatinib, ibrutinib, and GS-1101 as B-cell receptor (BCR)-related kinase inhibitors inhibit BCR- and chemokine-receptor-signal-regulated kinase and have a good clinical response in CLL. Lenalidomide, sorafenib, and dasatinib are other additional drugs associated with chemokine in microenvironment. Inhibiting signaling through chemokine and microenvironment associated signaling are emerging as innovative therapeutic targets in CLL. In this article, we reviewed the role of chemokines in CLL microenvironment and novel therapeutics targeting CLL microenvironment.
chronic leukemia lymphoma; microenvironment; chemokines; chemokines receptors; targeted therapy
Chitinases are essential enzymes for crustaceans and participates in several biological processes, such as nutrient digestion, morphogenesis, pathogenesis, and pathogen defense. In the present study, the full-length cDNA of Chi (named EcChi) was cloned from the hemocytes of ridgetail white prawn Exopalaemon carinicauda by rapid amplification of cDNA ends methods. The full-length cDNA of EcChi was 1,319 bp, including contains a 5′-untranslated region (UTR) of 42 bp, 3′-UTR of 101 bp with a poly (A) tail, an open-reading frame of 1,176 bp, encoding a 391-amino acid polypeptide with the predicted molecular weight of 43.71 kDa and estimated isoelectric point of 4.78. Sequence analysis revealed that the conserved chitinases family 18 active site was predicted in the amino acid sequence of EcChi. BLAST analysis revealed that amino acids of EcChi shared high identity (61–77 %) with that of other crustaceans. Quantitative real-time PCR analysis indicated that EcChi could be detected in all the tested tissues, and strongly expressed in hepatopancreas of E. carinicauda. After challenged with Vibrio anguillarum and WSSV, EcChi transcripts both in hemocytes and hepatopancreas increased significantly in the first 3 h, respectively. These results indicated that EcChi might be involved in the innate immune responses to V. anguillarum and WSSV in E. carinicauda.
Exopalaemon carinicauda; Chitinase (Chi); Vibrio anguillarum; White spot syndrome virus (WSSV); Immune response
Insulin-like growth factor-1 (IGF-1) and insulin-like growth factor binding protein-1 (IGFBP-1) play a pivotal role in regulating cellular hypoxic response. In this study, we cloned and characterized the genes encoding IGF-1 and IGFBP-1 to improve the current knowledge on their roles in highland Bos grunniens (Yak). We also compared their expression levels in the liver and kidney tissues between yaks and lowland cattle. We obtained full-length 465 bp IGF-1 and 792 bp IGFBP-1, encoding 154 amino acids (AA) IGF-1, and 263 AA IGFBP-1 protein, respectively using reverse transcriptase-polyerase chain reaction (RT-PCR) technology. Analysis of their corresponding amino acid sequences showed a high identity between B. grunniens and lowland mammals. Moreover, the two genes were proved to be widely distributed in the examined tissues through expression pattern analysis. Real-time PCR results revealed that IGF-1 expression was higher in the liver and kidney tissues in B. grunniens than in Bos taurus (p<0.05). The IGFBP-1 gene was expressed at a higher level in the liver (p<0.05) of B. taurus than B. grunniens, but it has a similar expression level in the kidneys of the two species. These results indicated that upregulated IGF-1 and downregulated IGFBP-1 are associated with hypoxia adaptive response in B. grunniens.
Bos grunniens; Insulin-like Growth Factor-1; Insulin-like Growth Factor Binding Protein-1; Cloning; Gene expression
The Allostatic Load Index (ALI) has been used to establish associations between stress
and health-related outcomes. This review summarizes the measurement and methodological
challenges of allostatic load in occupational settings. Databases of Medline, PubPsych,
and Cochrane were searched to systematically explore studies measuring ALI in working
adults following the PRISMA statement. Study characteristics, biomarkers and methods were
tabulated. Methodological quality was evaluated using a standardized checklist. Sixteen
articles (2003–2013) met the inclusion criteria, with a total of 39 (range 6–17) different
variables used to calculate ALI. Substantial heterogeneity was observed in the number and
type of biomarkers used, the analytic techniques applied and study quality. Particularly,
primary mediators were not regularly included in ALI calculation. Consensus on methods to
measure ALI in working populations is limited. Research should include longitudinal
studies using multi-systemic variables to measure employees at risk for biological wear
Allostasis; Biomarker; Chronic stress; Employee health; Work-related stress
The proprotein convertase subtilisin/kexin type 9 (PCSK9) has been confirmed as a major factor regulating cholesterol homeostasis and has low-density lipoprotein receptor (LDLR) independent effects. In addition, the pathogenesis of acute myocardial infarction (AMI) involves lipids alteration and other acute phase responses. It remains unknown whether the PCSK9 expression is influenced by the impact of AMI. The present study aimed to investigate the changes of PCSK9 concentration using AMI rat model.
AMI (n = 6-8 at each time point) or sham operated (n = 6) adult male rats model were used. Whole blood and liver tissue were collected at 1, 3, 6, 9, 12, 24, 48, and 96 hour (h) post infarction. The plasma PCSK9 concentration was measured by ELISA and lipid profiles were measured by enzymatic assay. The liver mRNA levels of PCSK9, LDLR, sterol response element binding protein-2 (SREBP-2) and hepatocyte nuclear factor 1α (HNF1α) were measured by quantitative real-time PCR.
The plasma PCSK9 concentration was increased from 12 h to 96 h (P < 0.05 vs. control). Paralleled with the enhanced plasma PCSK9 concentration, the hepatic PCSK9 mRNA expression was up-regulated by 2.2-fold at 12 h and 4.1-fold at 24 h. Hepatic mRNA levels of LDLR, SREBP-2 and HNF1α were all increased and lipid profiles underwent great changes at this acute period.
We firstly demonstrated that PCSK9 was transiently up-regulated in the acute period of AMI, which is also driven by transcriptional factors, SREBP-2 and HNF1α, suggesting that the role of PCSK9 in myocardial injury may be needed further study.
Acute myocardial infarction; PCSK9; Rat
The accuracy of dose calculation is crucial to the quality of treatment planning and, consequently, to the dose delivered to patients undergoing radiation therapy. Current general calculation algorithms such as Pencil Beam Convolution (PBC) and Collapsed Cone Convolution (CCC) have shortcomings in regard to severe inhomogeneities, particularly in those regions where charged particle equilibrium does not hold. The aim of this study was to evaluate the accuracy of the PBC and CCC algorithms in lung cancer radiotherapy using Monte Carlo (MC) technology.
Methods and materials
Four treatment plans were designed using Oncentra Masterplan TPS for each patient. Two intensity-modulated radiation therapy (IMRT) plans were developed using the PBC and CCC algorithms, and two three-dimensional conformal therapy (3DCRT) plans were developed using the PBC and CCC algorithms. The DICOM-RT files of the treatment plans were exported to the Monte Carlo system to recalculate. The dose distributions of GTV, PTV and ipsilateral lung calculated by the TPS and MC were compared.
For 3DCRT and IMRT plans, the mean dose differences for GTV between the CCC and MC increased with decreasing of the GTV volume. For IMRT, the mean dose differences were found to be higher than that of 3DCRT. The CCC algorithm overestimated the GTV mean dose by approximately 3% for IMRT. For 3DCRT plans, when the volume of the GTV was greater than 100 cm3, the mean doses calculated by CCC and MC almost have no difference. PBC shows large deviations from the MC algorithm. For the dose to the ipsilateral lung, the CCC algorithm overestimated the dose to the entire lung, and the PBC algorithm overestimated V20 but underestimated V5; the difference in V10 was not statistically significant.
PBC substantially overestimates the dose to the tumour, but the CCC is similar to the MC simulation. It is recommended that the treatment plans for lung cancer be developed using an advanced dose calculation algorithm other than PBC. MC can accurately calculate the dose distribution in lung cancer and can provide a notably effective tool for benchmarking the performance of other dose calculation algorithms within patients.
3-Dimensional conformal radiation therapy; Collapsed cone convolution; Pencil beam convolution; Lung cancer; Monte Carlo; Intensity-modulated radiation therapy
The study was to estimate the likelihood of axillary downstaging and to identify the factors predicting a pathologically node negative status after neoadjuvant chemotherapy (NAC) with or without trastuzumab in HER2-positive breast cancer.
Patients with HER2-positive, stage IIa-IIIc breast cancer were enrolled. Axillary status was evaluated by palpation and fine needle aspiration (FNA) before NAC. All patients received 4–6 cycles of PCrb (paclitaxel 80 mg/m2 and carboplatin AUC = 2 d1, 8, and 15 of a 28-day cycle, or paclitaxel 175 mg/m2 and carboplatin AUC = 6 every-3-week) and were non-randomly administered trastuzumab (2 mg/kg weekly or 6 mg/kg every-3-week) or not. After NAC, each patient underwent standard axillary lymph node dissection and breast-conserving surgery or mastectomy. And some patients received sentinel lymph node biopsy (SLNB) before axillary dissection.
Between November-2007 and June-2013, 255 patients were enrolled. Of them, 157 were confirmed as axillary node positive by FNA (group-A) and 98 as axillary node negative either by FNA or impalpable (group-B). After axillary dissection, the overall pathologically node negative rates (pNNR) were 52.9% in group-A and 69.4% in group-B. The ER-poor/HER2-positive subtype acquired the highest pNNR (79.6% in group-A and 87.9% in group-B, respectively) and the lowest rate of residual with ≥4 nodes involvement (1.9% and 3%, respectively) after PCrb plus trastuzumab. In multivariate analysis, trastuzumab added and ER-poor status were independent factors in predicting a higher pNNR in HER2-positive breast cancer. Forty-six tested patients showed that the ER-poor/HER2-positive subtype acquired a considerable high pNNR and axillary status with SLNB was well macthed with the axillary dissection.
ER-poor/HER2-positive subtype of breast cancer is a potential candidate for undergoing sentinel lymph node biopsy instead of regional node dissection for accurate axillary evaluation after effective downstaging by neoadjuvant chemo-trastuzumab therapy.
This study aimed to establish yak mammary epithelial cells (YMECs) for an in vitro model of yak mammary gland biology. The primary culture of YMECs was obtained from mammary gland tissues of lactating yak and then characterized using immunocytochemistry, RT-PCR, and western blot analysis. Whether foreign genes could be transfected into the YMECs were examined by transfecting the EGFP gene into the cells. Finally, the effect of Staphylococcus aureus infection on YMECs was determined. The established YMECs retained the mammary epithelial cell characteristics. A spontaneously immortalized yak mammary epithelial cell line was established and could be continuously subcultured for more than 60 passages without senescence. The EGFP gene was successfully transferred into the YMECs, and the transfected cells could be maintained for a long duration in the culture by continuous subculturing. The cells expressed more antimicrobial peptides upon S.aureus invasion. Therefore, the established cell line could be considered a model system to understand yak mammary gland biology.
High-quality protein crystals of suitable size are an important prerequisite for applying X-ray crystallography to determine the 3-dimensional structure of proteins. However, it is often difficult to obtain protein crystals of appropriate size and quality because nucleation and growth processes can be unsuccessful. Here, we show that by adsorbing proteins onto porous polystyrene-divinylbenzene microspheres (SDB) floating on the surface of the crystallisation solution, a localised high supersaturation region at the surface of the microspheres and a low supersaturation region below the microspheres can coexist in a single solution. The crystals will easily nucleate in the region of high supersaturation, but when they grow to a certain size, they will sediment to the region of low supersaturation and continue to grow. In this way, the probability of crystallisation and crystal quality can be simultaneously increased in a single solution without changing other crystallisation parameters.
A 47-year-old male presented with a six-month history of fatigue and a four-month history of alanine and aspartate aminopherase elevation. Laboratory examination revealed that the serum α-fetoprotein (AFP) level was 371.51 μg/l (normal range, 0–20 μg/l), and a computed tomography scan revealed a hypodense lesion in the left hepatic lobe. During laparotomy, a dark red-colored soft tumor (1.5×1.7 cm in diameter) was found in segment eight of the liver. Intra-operative pathology and post-operative histopathology examinations revealed that the tumor was a hepatic cavernous hemangioma. The serum AFP level was decreased to 24.45 μg/l by the second post-operative week. The literature was searched and only three similar cases were found. A brief review of this rare disease entity was produced, which attempted to explain this appearance reasonably.
hepatic cavernous hemangioma; α-fetoprotein; cancer stem cell
Next generation sequencing (NGS) is now being used for detecting chromosomal abnormalities in blastocyst trophectoderm (TE) cells from in vitro fertilized embryos. However, few data are available regarding the clinical outcome, which provides vital reference for further application of the methodology. Here, we present a clinical evaluation of NGS-based preimplantation genetic diagnosis/screening (PGD/PGS) compared with single nucleotide polymorphism (SNP) array-based PGD/PGS as a control.
A total of 395 couples participated. They were carriers of either translocation or inversion mutations, or were patients with recurrent miscarriage and/or advanced maternal age. A total of 1,512 blastocysts were biopsied on D5 after fertilization, with 1,058 blastocysts set aside for SNP array testing and 454 blastocysts for NGS testing. In the NGS cycles group, the implantation, clinical pregnancy and miscarriage rates were 52.6% (60/114), 61.3% (49/80) and 14.3% (7/49), respectively. In the SNP array cycles group, the implantation, clinical pregnancy and miscarriage rates were 47.6% (139/292), 56.7% (115/203) and 14.8% (17/115), respectively. The outcome measures of both the NGS and SNP array cycles were the same with insignificant differences. There were 150 blastocysts that underwent both NGS and SNP array analysis, of which seven blastocysts were found with inconsistent signals. All other signals obtained from NGS analysis were confirmed to be accurate by validation with qPCR. The relative copy number of mitochondrial DNA (mtDNA) for each blastocyst that underwent NGS testing was evaluated, and a significant difference was found between the copy number of mtDNA for the euploid and the chromosomally abnormal blastocysts. So far, out of 42 ongoing pregnancies, 24 babies were born in NGS cycles; all of these babies are healthy and free of any developmental problems.
This study provides the first evaluation of the clinical outcomes of NGS-based pre-implantation genetic diagnosis/screening, and shows the reliability of this method in a clinical and array-based laboratory setting. NGS provides an accurate approach to detect embryonic imbalanced segmental rearrangements, to avoid the potential risks of false signals from SNP array in this study.
Electronic supplementary material
The online version of this article (doi:10.1186/2047-217X-3-30) contains supplementary material, which is available to authorized users.
Preimplantation genetic diagnosis/screening; Next generation sequencing; Blastocyst; Cryopreserved embryo transfer; Clinical outcome
An ultrasound-assisted extraction (UAE) method using ethanol was applied for extracting arabinogalactan (AG) and dihydroquercetin (DHQ) simultaneously from larch wood, as a pretreatment for pulping and papermaking. The extraction parameters were optimized by a Box-Behnken experimental design with the yields of AG and DHQ as the response values. Under optimum conditions (three extractions, each using 40% ethanol, for 50 min, 200 W ultrasound power and 1∶18 solid-liquid ratio), the yields of AG and DHQ were 183.4 and 36.76 mg/g, respectively. After UAE pretreated, the wood chips were used for Kraft pulping (KP) and high boiling solvent pulping (HBSP). The pulping yield after pretreatment was higher than that of untreated (the pulping yields of untreated HBSP and KP were 42.37% and 39.60%, and the pulping yields of HBSP and KP after UAE-pretreated were 44.23% and 41.50% respectively), as indicated by a lower kappa number (77.91 and 27.30 for untreated HBSP and KP; 77.01 and 26.83 for UAE-pretreated HBSP and KP). Furthermore, the characteristics of paper produced from pretreated wood chips were superior to those from the untreated chips: the basis weight was lower (85.67 and 82.48 g·cm−2 for paper from untreated KP and HBSP; 79.94 and 80.25 g·cm−2 for paper from UAE-pretreated KP and HBSP), and the tensile strengths, tearing strengths, bursting strengths, and folding strengths were higher than these of paper after UAE-pretreated, respectively.
Arrestins are dynamic proteins which move between cell compartments triggered by stimulation of G-protein-coupled receptors. Even more dynamically in vertebrate photoreceptors, arrestin1 (Arr1) moves between the inner and outer segments according to the lighting conditions. Previous studies have shown that the light-driven translocation of Arr1 in rod photoreceptors is initiated by rhodopsin through a phospholipase C/protein kinase C (PKC) signaling cascade. The purpose of this study is to identify the PKC substrate that regulates the translocation of Arr1.
Mass spectrometry was used to identify the primary phosphorylated proteins in extracts prepared from PKC-stimulated mouse eye cups, confirming the finding with in vitro phosphorylation assays. Our results show that BBS5 is the principal protein phosphorylated either by phorbol ester stimulation or by light stimulation of PKC. Via immunoprecipitation of BBS5 in rod outer segments, Arr1 was pulled down; phosphorylation of BBS5 reduced this co-precipitation of Arr1. Immunofluorescence and immunoelectron microscopy showed that BBS5 principally localizes along the axonemes of rods and cones, but also in photoreceptor inner segments, and synaptic regions.
Our principal findings in this study are three-fold. First, we demonstrate that BBS5 is post-translationally regulated by phosphorylation via PKC, an event that is triggered by light in photoreceptor cells. Second, we find a direct interaction between BBS5 and Arr1, an interaction that is modulated by phosphorylation of BBS5. Finally, we show that BBS5 is distributed along the photoreceptor axoneme, co-localizing with Arr1 in the dark. These findings suggest a role for BBS5 in regulating light-dependent translocation of Arr1 and a model describing its role in Arr1 translocation is proposed.
arrestin; cilia; BBS5; BBSome; translocation
To explore the hypothesis that alterations in cellular membrane lipids are present at the stage of preclinical Alzheimer’s disease (AD) (i.e., cognitively normal at death, but with AD neuropathology), we performed targeted shotgun lipidomics of lipid extracts from postmortem brains of subjects with preclinical AD. We found sulfatide levels were significantly lower in subjects with preclinical AD compared to those without AD neuropathology. We also found that the level of ethanolamine glycerophospholipid was marginally lower at this stage of AD, whereas changes of the ceramide levels were undetectable with the available samples. These results indicate that cellular membrane defects are present at the earliest stages of AD pathogenesis and also suggest that sulfatide loss is among the earliest events of AD development, while alterations in the levels of ethanolamine glycerophospholipid and ceramide occur relatively later in disease.
Ceramide; membrane lipids; plasmalogen; preclinical Alzheimer’s disease; shotgun lipidomics; sulfatide