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Background Cystic echinococcosis (CE) is a life-threatening zoonosis caused by the larval form of Echinococcus granulosus tapeworm. Our previous study showed that an approved drug pyronaridine (PND) is highly effective against CE, both in vitro and in an animal model. To identify possible target genes, transcriptome analysis was performed with E. granulosus sensu stricto protoscoleces treated with PND. Results A total of 1,321 genes were differentially expressed in protoscoleces treated with PND, including 541 upregulated and 780 downregulated genes. Gene ontology and KEGG analyses revealed that the spliceosome, mitogen-activated protein kinase (MAPK) pathway and ATP-binding cassette (ABC) transporters were the top three enriched pathways. Western blot analysis showed that PND treatment resulted in a dose-dependent increase in protein expression levels of EgMKK1 (MKK3/6-like) and EgMKK2 (MEK1/2-like), two members of MAPK cascades. Interestingly, several heat shock protein (HSP) genes were greatly downregulated including stress-inducible HSPs and their constitutive cognates, and some of them belong to Echinococcus-specific expansion of HSP70. Conclusions PND has a great impact on the spliceosome, MAPK pathway and ABC transporters, which may underline the mechanisms by which PND kills E. granulosus protoscoleces. In addition, PND downregulates HSPs expression, suggesting a close relationship between the drug and HSPs.

A one-pot approach has been demonstrated for the synthesis of amorphous luminol–gallium (Ga) nanoassemblies, which are formed by small particles with big irregular spheres or network nanoassemblies. Moreover, luminol–Ga nanoassemblies exhibit peroxidase-mimetic activity toward the oxidation of peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H2O2) owing to its stimulating effect on the formation of reactive oxygen groups, such as hydroxyl radicals (OH˙). After the addition of luminol–Ga nanoassemblies to the TMB–H2O2 system, a remarkable color change is observed from colorless TMB to its blue product TMBox accompanied by an absorption peak at around 652 nm. Owing to the presence of reactive oxygen groups by luminol–Ga nanoassemblies, the peroxidase-mimetic activity is inhibited by PPi. Accordingly, the blue color fades away and the absorbance at 652 nm is decreased. Herein a novel sensitive colorimetric detection of PPi from 0.5 to 15 μM using luminol–Ga nanoassemblies is developed with the limit of detection (LOD) of 62.4 nM. The practicability of the assay toward the detection of PPi is also investigated in real water analysis such as lake water and tap water. Recoveries from 97.3% to 102.6% in local lake water and tap water samples are obtained. Therefore this cost-effective peroxidase-based analysis using luminol–Ga nanoassemblies is a potentially powerful tool for the quantitative determination of PPi in environmental samples.

Surface immobilization of enzymes on magnetic-recoverable carriers is of great interest and importance for the biocatalysis of relatively large molecules. In this work, the nanosized amino-rich filamentous M13 virus, a versatile biological scaffold, was applied as the unique soft backbone for lipase immobilization. Based on the structure and capsid proteins of M13 phages, the magnetic-recoverable mimic-free-lipases (MFLs) composed of the M13 hydrogels and magnetic particles were developed in two designs. In the first design, nanosized wild M13 phages were crosslinked into a phage hydrogel through the N-terminals of pVIII peptides while NH2-Fe3O4 magnetic nanoparticles (MNPs) were attached to the M13 virus through glutaraldehyde, forming the M13-(NH2-Fe3O4) magnetic phage hydrogel. In the second design, special M13 with Fe3O4 affinity pIII-peptide (FAP-M13) was biopanned for strongly binding towards bare Fe3O4 with the “hook”-like pIII-peptide (N-LPLSTQH-C). TEM observation confirmed the direct grasp of FAP-M13 on bare Fe3O4, forming the magnetic (FAP-M13)-Fe3O4 virus hydrogel. Lipases were uniformly anchored on the phage surface of nanoscale by crosslinking with the N-terminals of pVIII peptides, and then lipase@M13-(NH2-Fe3O4) and lipase@(FAP-M13)-Fe3O4 MFLs were constructed. For both MFLs, high activity recovery yield (>95%) and efficient magnetic separation were characterized. Significantly reduced MNP-usage-amount and enhanced lipase-loading-amount both by about 40 folds were obtained, compared with the conventional NH2-Fe3O4 carriers. The quantified Km and Vmax/Km values were almost equal to those of the free lipases, verifying free-enzyme-mimicking features of the MFLs. High pH-tolerance, wide temperature adaptability, enhanced thermal stability and stable magnetic separation capability of both MFLs were also observed. In particular, the (FAP-M13)-Fe3O4 magnetic virus hydrogel simply using bare Fe3O4 MNPs would be more convenient and economical in the scaled-up biocatalysis.

In sexually reproducing organisms maintenance of germ stem cell immortality is fundamental for transmitting genetic material to future generations. While previous research has mainly considered intrinsic regulatory mechanisms in the germline, our recent study has found a direct contribution of somatic cells in preserving germline immortality via the somatically expressed endoribonuclease ENDU-2 in Caenorhabditis elegans. We have identified ENDU-2 as a secreted protein that can be taken up by the germline. Here, we discuss how ENDU-2 might uncouple its RNA-binding and RNA-cleavage activities to control gene expression via either an endoribonuclease dependent or an independent way. We also speculate on a possible functional conservation of its mammalian homologs in mediating cell-cell communication as well as its potential significance in understanding human pathogenesis such as cancer development.

Multicellular organisms coordinate tissue specific responses to environmental information via both cell-autonomous and non-autonomous mechanisms. In addition to secreted ligands, recent reports implicated release of small RNAs in regulating gene expression across tissue boundaries. Here, we show that the conserved poly-U specific endoribonuclease ENDU-2 in C. elegans is secreted from the soma and taken-up by the germline to ensure germline immortality at elevated temperature. ENDU-2 binds to mature mRNAs and negatively regulates mRNA abundance both in the soma and the germline. While ENDU-2 promotes RNA decay in the soma directly via its endoribonuclease activity, ENDU-2 prevents misexpression of soma-specific genes in the germline and preserves germline immortality independent of its RNA-cleavage activity. In summary, our results suggest that the secreted RNase ENDU-2 regulates gene expression across tissue boundaries in response to temperature alterations and contributes to maintenance of stem cell immortality, probably via retaining a stem cell specific program of gene expression.

Kvainickas et al. establish that retromer, a coat complex that promotes endosomal recycling, is also required for the activation of the lysosomal mTORC1 complex. Mechanistically, retromer and the retromer-associated protein TBC1D5 separate mTORC1 and RAB7 microdomains on late endosomes, which is necessary for amino acid–mediated mTORC1 activation., Retromer is an evolutionarily conserved multiprotein complex that orchestrates the endocytic recycling of integral membrane proteins. Here, we demonstrate that retromer is also required to maintain lysosomal amino acid signaling through mTORC1 across species. Without retromer, amino acids no longer stimulate mTORC1 translocation to the lysosomal membrane, which leads to a loss of mTORC1 activity and increased induction of autophagy. Mechanistically, we show that its effect on mTORC1 activity is not linked to retromer’s role in the recycling of transmembrane proteins. Instead, retromer cooperates with the RAB7-GAP TBC1D5 to restrict late endosomal RAB7 into microdomains that are spatially separated from the amino acid–sensing domains. Upon loss of retromer, RAB7 expands into the ragulator-decorated amino acid–sensing domains and interferes with RAG-GTPase and mTORC1 recruitment. Depletion of retromer in Caenorhabditis elegans reduces mTORC1 signaling and extends the lifespan of the worms, confirming an evolutionarily conserved and unexpected role for retromer in the regulation of mTORC1 activity and longevity.

Summary Pig has been proved to be a valuable large animal model used for research on diabetic disease. However, their translational value is limited given their distinct anatomy and physiology. For the last 30 years, we have been developing a laboratory Asian miniature pig inbred line (Bama miniature pig [BM]) from the primitive Bama xiang pig via long-term selective inbreeding. Here, we assembled a BM reference genome at full chromosome-scale resolution with a total length of 2.49 Gb. Comparative and evolutionary genomic analyses identified numerous variations between the BM and commercial pig (Duroc), particularly those in the genetic loci associated with the features advantageous to diabetes studies. Resequencing analyses revealed many differentiated gene loci associated with inbreeding and other selective forces. These together with transcriptome analyses of diabetic pig models provide a comprehensive genetic basis for resistance to diabetogenic environment, especially related to energy metabolism., Graphical Abstract, Highlights • Bama miniature pig (BM) is one of the pig lines with the highest inbreeding coefficient • This atlas is a report on the chromosome-level genome assembly of miniature pig • Genomic analyses revealed genetic basis underlying BM's advantages to study diabetes • Some lncRNAs and mRNAs may be linked to resistance to diabetogenic environment, Biological Sciences; Gene Ontology; Genomic Analysis; Genomics; Omics; Sequence Analysis; Transcriptomics

A highly sensitive sandwiched luminol electrochemiluminescence (ECL) immunosensor has been constructed based on the double catalysis effect and multiamplification strategy of platinum-gold alloy hybrid functionalized zinc oxide nanocomposites (Pt-Au@ZnONPs). First, the improved surface area and excellent biocompatibility of Pt-Au@ZnONPs were used for binding the secondary antibody (Ab2) and glucose oxidase (GOD) to obtain the novel bioconjugate Pt-Au@ZnONPs-GOD-Ab2. Pt-Au@ZnONPs and the loaded GOD amplified luminol ECL signals and promoted the luminol ECL response by efficiently catalyzing glucose to produce hydrogen peroxide (H2O2) in situ as a coreactant of luminol. Then, Pt-Au@ZnONPs catalyzed H2O2 to generate various reactive oxygen species (ROSs) that accelerated the ECL reaction of luminol, thus enhanced the ECL intensity of luminol and improved the sensitivity of the immunosensor. Second, gold nanoparticles decorated reduced graphene sheets (AuNPs@GS) with large specific surface area, good biocompatibility, high conductivity, and high catalytic activity were prepared and used as substrate of the immunosensor to capture the primary antibody (Ab1). Experimental results demonstrated that the proposed ECL immunosensor showed a sensitive response for the detection of α-1-fetoprotein (AFP) from 0.1 pg mL−1 to 200 ng mL−1 with detection limit of 0.03 pg mL−1 (S/N = 3). The as-proposed luminol ECL immunosensor has excellent sensitivity, stability, and specificity, which makes it very promising for clinical applications.

Background: Cystic and alveolar echinococcosis caused by the tapeworms Echinococcus granulosus and E. multilocularis, respectively, are important zoonotic diseases. Protease inhibitors are crucial for the survival of both Echinococcus spp. Kunitz-type inhibitors play a regulatory role in the control of protease activity. In this study，we identified all the Kunitz-type protease inhibitors present in the genomes of these two tapeworms and analyzed the gene sequences using computational, structural bioinformatics and phylogenetic approaches to evaluate the evolutionary relationships of these genes. Results: A total of 19 genes from E. multilocularis and 23 genes from E. granulosus contained single or multiple Kunitz-domains. A neighbor-joining phylogenetic tree indicated that the E. granulosus and E. multilocularis Kunitz domain peptides were divided into three branches containing 9 clusters. Based on available transcriptome data, we analyzed the expression of these Kunitz-domain protease inhibitors in four major developmental stages of E. granulosus and found they were differentially expressed.Conclusion: We identified 19 and 23 Kunitz protease inhibitors in E. multilocularis and E. granulosus respectively; the majority of these genes were expressed in one or four stages of E. granulosus with some being highly expressed in adult worms indicating that these genes likely play different roles in the different developmental stages.

Multicellular organisms coordinate tissue specific response to environmental information via both cell-autonomous and non-autonomous mechanisms. In addition to secreted ligands, secreted small RNAs have recently been reported to regulate gene expression across tissue boundaries. Here we show that the conserved poly-U specific endoribonuclease ENDU-2 is secreted from the soma and taken-up by the germline to ensure germline immortality at elevated temperature inC. elegans. ENDU-2 binds to mature mRNAs and negatively regulates mRNA abundance both in the soma and the germline. While ENDU-2 promotes RNA decay in the soma directly via its endoribonuclease activity, ENDU-2 prevents misexpression of soma-specific genes in the germline and preserves germline immortality independent of its RNA-cleavage activity. In summary, our results suggest that the secreted RNase ENDU-2 transmits environmental information across tissue boundaries and contributes to maintenance of stem cell immortality probably via retaining a stem cell specific program of gene expression.

Improving the predication efficiency of porcine production performance at early stage will contribute to reducing the breeding and production costs. The intestinal microbiota had received plenty of attention in recent years due to their influence on host health and performance. The purpose of this study was to investigate the relationship between the fecal microbiota at early growth period and porcine feed efficiency (FE) under a commercial feeding environment. Ninety-one pigs were reordered according to the residual feed intake (RFI) values between day 90 on test and day 160 off test, 9 lowest RFI pigs and 9 highest RFI pigs were selected as the LRFI group and the HRFI group, respectively. Fecal samples from pigs in the early grower phase (day 80) were performed for microbial diversity, composition, and predicted functionality by using 16S rRNA sequencing. The results showed that no significant differences in microbial alpha diversity were observed between two RFI groups, whereas, some RFI-associated compositional differences were revealed. In particular, the microbiota of the LRFI group (more feed-efficient) had significantly higher levels of some members of Clostridiales and Bacteroidales (e.g., g_1_68 and g_norank_f_p_2534_18B5), which may promoted FE through protecting gut barrier function, compared with those of the HRFI pigs. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis found that the LRFI pigs were likely have microbiota with higher levels of amino acid metabolism. Moreover, redundancy analysis (RDA) showed that litter size, parity, and date of birth had significant effects on the bacterial community structure. These results improved our knowledge of the porcine early-life fecal microbiota and its potential link underlying RFI, which would be useful for future development of microbial biomarkers for predicting and improving porcine FE as well as investigation of targets for dietary strategies.

Macroautophagy/autophagy is an evolutionarily conserved cellular degradation and recycling process that is tightly regulated by external stimuli, diet, and stress. Our recent findings suggest that in C. elegans, a nutrient sensing pathway mediated by MTORC2 (mechanistic target of rapamycin kinase complex 2) and its downstream effector kinase SGK-1 (serum- and glucocorticoid-inducible kinase homolog 1) suppresses autophagy, involving mitophagy. Induced autophagy/mitophagy in MTORC2-deficient animals slows down development and impairs reproduction independently of the SGK-1 effectors DAF-16/FOXO and SKN-1/NFE2L2/NRF2. In this punctum, we discuss how TORC2-SGK-1 signaling might regulate autophagic turnover and its impact on mitochondrial homeostasis via linking mitochondria-derived reactive oxygen species (mtROS) production to mitophagic turnover.

Background: Cystic echinococcosis (CE), a condition caused by the larval stage of the dog tapeworm Echinococcus granulosus sensu stricto, is a globally distributed zoonotic disease. Current treatment options for CE are limited, and an effective and safe anti-echinococcal drug is urgently required. Methods: Drug repurposing strategy was employed to identify new therapeutic agents against echinococcal cysts. An in vitro protoscolicidal assay along with in vivo murine models was applied in the drug screening. A microinjection procedure was employed to mimic the clinical PAIR (puncture, aspiration, injection and reaspiration) technique to evaluate the potential application of the candidate drug in clinical practice. Findings: We repurposed pyronaridine, an approved antimalarial drug, for the treatment of CE. Following a three-dose intraperitoneal regimen (57 mg/kg, q.d. for 3 days), pyronaridine caused 100% cyst mortality. Oral administration of pyronaridine at 57 mg/kg, q.d. for 30 days significantly reduced the parasitic burden in the pre-infected mice compared with albendazole group (p < 0.001). Using a microinjection of drug into cysts, pyronaridine (200 μM) showed highly effective in term of inhibition of cyst growth (p < 0.05, compared with saline group). Pharmacokinetic analysis revealed that pyronaridine was highly distributed in the liver and lungs, the most affected organs of CE. Function analysis showed that pyronaridine inhibited the activity of topoisomerase I (IC50 = 209.7 ± 1.1 μM). In addition, classical apoptotic hallmarks, including DNA fragmentation and caspase activation, were triggered. Interpretation: Given its approved clinical safety, the repurposing of pyronaridine offers a rapidly translational option for treating CE including PAIR. Fund: National Natural Science Foundation of China and International Cooperation Project of the Qinghai Science and Technology Department. Keywords: Echinococcus granulosus sensu stricto, Pyronaridine, Cystic echinococcosis, Drug repurposing, Topoisomerase I

Objectives RING finger protein 8 (RNF8) is an E3 ligase that plays an essential role in DSB repair. p53 is a well‐established tumour suppressor and cellular gatekeeper of genome stability. This study aimed at investigating the functional correlations between RNF8 and p53 in DSB damage repair. Materials and methods In this article, wild‐type, knockout and shRNA‐depleted HCT116 and U2OS cells were stressed, and the roles of RNF8 and p53 were examined. RT‐PCR and Western blot were utilized to investigate the expression of related genes in damaged cells. Cell proliferation, apoptosis and neutral cell comet assays were applied to determine the effects of DSB damage on differently treated cells. DR‐GFP, EJ5‐GFP and LacI‐LacO targeting systems, flow cytometry, mass spectrometry, IP, IF, GST pull‐down assay were used to explore the molecular mechanism of RNF8 and p53 in DSB damage repair. Results We found that RNF8 knockdown increased cellular sensitivity to DSB damage and decreased cell proliferation, which was correlated with high expression of the p53 gene. RNF8 improved the efficiency of DSB repair by inhibiting the pro‐apoptotic function of p53. We also found that RNF8 restrains cell apoptosis by inhibiting over‐activation of ATM and subsequently reducing p53 acetylation at K120 through regulating Tip60. Conclusions Taken together, these findings suggested that RNF8 promotes efficient DSB repair by inhibiting the pro‐apoptotic activity of p53 through regulating the function of Tip60.

Recently, we proposed a new strategy to construct artificial plant protein assemblies, which were induced by adding a small molecule, based on dual supramolecular interactions. In this paper, we further explored this method by employing Human Galectin-1 (Gal-1) as a building block to form self-assembled microribbons. Two non-covalent interactions, including lactose-lectin binding and dimerization of Rhodamine B (RhB), induced by the small molecule ligand addition, were involved in the crosslinking of the animal protein, resulting in the formation of assemblies. By using transmission electron microscopy (TEM), cryo-electron microscopy (cryo-EM), and three-dimensional (3D) tomographic analysis, we arrived at a possible mechanistic model for the microribbon formation. Furthermore, the morphology of protein assemblies could be fine-timed by varying the incubation time, the protein/ligand ratio, and the chemical structures of ligands. Interestingly, the formation of protein microribbons successfully inhibited Gal-1 induced T-cell agglutination and apoptosis. This is because the multivalent and dynamic interactions in protein assemblies compete with the binding between Gal-1 and the glycans on cell surfaces, which suppresses the function of Gal-1 in promotion of tumor progression and metastasis.

We proposed the deprotection-induced block copolymer self-assembly (DISA); that is, the deprotection of hydroxyl groups resulted in in situ self-assembly of glycopolymers. In the previous studies, block copolymers soluble in common organic solvents were employed as the starting material. In this paper, by using the protected glyco-block containing preassembled glycovesicles in water as the starting material, we moved forward and made two exceeding achievements. First, we have observed a deprotection-induced morphology transition triggered by alkali in water. The carbohydrate-carbohydrate interactions were considered to contribute to such a morphology transition during deprotection. Second, lipase was found to be an efficient enzymatic trigger in the sugar deprotection, which motivates the immune-application of this morphology transition process. When lipase and a model antigen, ovalbumin (OVA), were encapsulated inside the glycovesicles, the deprotection of sugars by lipase induced the transition of vesicles to micelles and the lipase and OVA were released accordingly. When glycovesicles were internalized by dentritic cells (DCs), the lipase from lysosomes efficiently induced the release of OVA and presentation of antigen to T cells. During the process, lysosomal lipase performed as a trigger on the deprotection of sugars and the release of protein without any other reagents. The significance of this design is that as a delivery vehicle, the protected glycovesicles not only avoided unnecessary immune activation but also worked with the released OVA together; that is, the glycovehicle successfully activated DCs and improved the presentation efficiency of T cells remarkably.

Background Dogs play a pivotal role in the transmission of cystic echinococcosis (CE), a zoonosis caused by the tapeworm Echinococcus granulosus. We showed previously that dogs vaccinated with two E. granulosus adult-worm specific proteins, EgM9 and EgM123, emulsified with Freund’s adjuvants induced significant protective efficacy in terms of reduction in worm burden and egg production after 45 days post-infection. It was not known whether this protection can be sustained using adjuvants suitable for use in dogs. Methods Recombinant EgM9 and EgM123 were mixed with Quil A or ISCOMs for vaccinating dogs. After three vaccine injections, all the dogs were orally challenge-infected with 200 000 protoscoleces of E. granulosus. After 45 days of infection, all the dogs were euthanized and necropsied for collecting and counting E. granulosus worms. Immunoglobins, including the IgG subclasses IgG1 and IgG2, were detected in the sera of vaccinated dogs by ELISA. To determine whether the protection efficacy could be maintained after 45 days post-infection, we implemented a longevity trial to count eggs in dog faeces for 170 days after infection. Results The dogs vaccinated with EgM9 and EgM123 mixed with Quil A and ISCOMs showed similar protective efficacy as the proteins emulsified with Freund’s adjuvants in our previous study in terms of reduction of worms and eggs at 45 days post-infection. The longevity trial showed that EgM9 protein-vaccinated group released lower number of eggs per gram compared with the egg counts in the control dogs during the dog trial study. Conclusion EgM9 and EgM123 are thus suitable vaccine candidates against E. granulosus infection in dogs. Electronic supplementary material The online version of this article (10.1186/s40249-018-0425-4) contains supplementary material, which is available to authorized users.

The follicular variant of peripheral T-cell lymphoma, not otherwise specified, is very rare. Primary epiglottic follicular variant of peripheral T-cell lymphoma is extremely rare in clinical practice. Here, we report the first case of a follicular variant of peripheral T-cell lymphoma not otherwise specified in a 44-year-old Chinese man, who presented with a tumor in the middle of the epiglottis tongue surface. Microscopically, the tumor had a vague nodular growth pattern and the morphology of the nodules was different from each other at low power. Atypical lymphoid cells were medium to large in size and had round nuclei, with an irregular nuclear membrane, distinct nucleoli, and rapid mitotic activity. Plasma cells were found surrounding the nodules. The tumor cells were positive for follicular helper T-cell markers (CD10, PD-1, CXCL13, and BCL-6). The EBER was negative by in situ hybridization. Polymerase chain reaction-based analysis showed monoclonal rearrangements of TCRβ, TCRγ, and polyclonal rearrangements of IgH, IgK, and IgL. The clinical and imaging features and the prognostic factors of FV PTCL-NOS remain poorly understood. Thus, investigation of more cases and longer follow-up is necessary to understand the disease and to identify the best treatment to improve prognosis.

Background/Aims: Recent evidence has indicated the crucial regulatory roles of long non-coding RNAs (lncRNAs) in tumour biology. In hepatocellular carcinoma (HCC), aberrant expression of lncRNAs plays an essential role in HCC tumourigenesis. However, the potential roles and regulatory mechanisms of the novel human lncRNA, Linc-USP16, in HCC are unclear. Methods: To investigate the function of Linc-USP16 in HCC, we first analysed the expression levels of Linc-USP16 in HCC patient tissues and cell lines via q-RT-PCR and established overexpressed or knockdown HCC cell lines. Results: Here, we found that Linc-USP16 expression was significantly down-regulated in HCC patient tissues and cell lines. Further functional experiments suggested that Linc-USP16 could directly increase PTEN expression by acting as a competing endogenous RNA (ceRNA) for miR-21 and miR-590-5p. These interactions led to repression of AKT pathway and inhibition of HCC cell proliferation and migration. Conclusion: Thus, our data showed that Linc-USP16, as a tumour suppressor, plays an important role in HCC pathogenesis and provides a new therapeutic strategy for HCC treatment.

In this research, a new DSA (Distyryl-anthracene) derivative with four carboxyl groups was designed and synthesized. This molecule exhibits aggregation-induced emission property (AIE). With the AIE character, a convenient and sensitive fluorescent probe for the detection of protamine, heparin and heparinase has been developed. The protamine can directly induce the aggregation of probe, which caused by electrostatic attraction. In this way, the turn-on detection of protamine is achieved, and the detection limit is as low as 30 ng mL−1. When heparin appears, the probes will be redisperse in solution, which causes a decrease in fluorescence intensity. Besides, this method also shows good selectivity and sensitivity, and the linear range of heparin is from 0.08 to 8 μg mL−1 with detection limit of 37 ng mL−1. After hydrolyzing heparin by heparinase, the probes rebind with protamine and the fluorescence enhance. The fluorescence enhancement was linearly related to the concentration of heparinase in the range of 0.02–2.0 μg mL−1 and detection limit as low as 143.7 ng mL−1. In addition, the results exhibited that the recovery percentage of heparinase in bovine samples reached to 96–101%.

A sensitive electrochemiluminescent immunoassay for alkaline phosphatase (ALP) using p-nitrophenyl phosphate (PNPP) as substrate based on the electrochemiluminescence resonance energy transfer (ECRET) is developed. Luminol-doped silica nanoparticles (luminol-SiNPs) are prepared by water/oil (W/O) microemulsion method. PNPP convertes to p-nitrophenol (PNP) in the presence of ALP, which results in the absorption peak shifting from 360 nm to 450 nm. Herein the spectral overlap between absorption spectrum of PNP and electrochemiluminescence (ECL) spectrum of luminol-SiNPs (425 nm) makes energy transfer occur from luminol-SiNPs to PNP. In the optimized conditions, a linear relationship was obtained using this ECRET method at the concentration of ALP from 5 to 50 U/L (r = 0.9905) and with the limit of detection (LOD) of 0.8 U/L. This ECRET method exhibits sufficient specificity for ALP over other enzymes such as horseradish peroxidase, trypsin and lysozyme.

Enzyme immobilization is widely used for large-scale industrial applications. However, the weak absorption through physical methods limits the recovery ability. Here, affinity-binding immobilization of enzymes was explored using a silica-specific affinity peptide (SAP) as a fusion tag to intensify the binding force between the enzyme and mesoporous silica (MPS) carrier. d-amino acid oxidase (DAAO) of Rhodosporidium toruloides was used as a model enzyme. The optimal screened SAP (LPHWHPHSHLQP) was selected from a M13 phage display peptide library and fused to the C-terminal of DAAO to obtain fused DAAOs with one, two and three SAP tags, respectively. The activity of DAAO–SAP–MPS was superior comparing with DAAO–2SAP–MPS and DAAO–3SAP–MPS; meanwhile DAAO–SAP–MPS shows 36% higher activity than that of DAAO–MPS. Fusion with one SAP improved the thermal stability with a 10% activity increase for immobilized DAAO–SAP–MPS compared to that of DAAO–MPS at 50 °C for 3 h. Moreover, the activity recovery of immobilized DAAO–SAP–MPS was 25% higher in operation stability assessment after six-batch conversions of cephalosporin to glutaryl-7-amino cephalosporanic acid than that of DAAO–MPS.

Summary: Autophagy is stimulated by stress conditions and needs to be precisely tuned to ensure cellular homeostasis and organismal development and health. The kinase mechanistic target of rapamycin (mTOR) forms the enzymatic core of the highly conserved mTOR complexes mTORC1 and mTORC2. mTORC1 is a key inhibitor of autophagy, yet the function of mTORC2 in autophagy is controversial. We here show that inactivation of mTORC2 and its direct target serum- and glucocorticoid-inducible kinase 1 (SGK-1) potently induces autophagy and the autophagic degradation of mitochondria in C. elegans. Enhanced autophagy in mTORC2- or SGK-1-deficient animals contributes to their developmental and reproductive defects and is independent of the canonical SGK-1 effector DAF-16/FOXO. Importantly, we find that inactivation of mTORC2-SGK-1 signaling impairs mitochondrial homeostasis and triggers an increased release of mitochondria-derived reactive oxygen species (mtROS) to induce autophagy. Thus, mitochondrial stress couples reduced mTORC2 activity to enhanced autophagic turnover. : The multiprotein complex mTORC2 is a central regulator of development and metabolism. Aspernig et al. implicate C. elegans mTORC2 and its effector SGK-1 in mitochondrial homeostasis and report that an increased release of mitochondria-derived reactive oxygen species induces autophagy in mTORC2/SGK-1-deficient animals. Keywords: mammalian target of rapamycin, mTOR, mTORC2, serum glucocorticoid-regulated kinase 1, SGK-1, autophagy, mitophagy, mitochondria, reactive oxygen species, ROS

Pig has been proven to be a valuable large animal model used for research on diabetic disease. However, their translational value is limited given their distinct anatomy and physiology. For the last 30 years, we have been developing a laboratory Asian miniature pig inbred line (Bama miniature pig, BM) from the primitive Bama xiang pig (BX) via long-term selective inbreeding. Here, we assembled a BM reference genome at full chromosome-scale resolution with a total length of 2.49 Gb. Comparative and evolutionary genomic analyses identified numerous variations between the BM and commercial pig (Duroc), particularly those in the genetic loci associated with the features advantageous to diabetes studies. Resequencing analyses revealed many differentiated gene loci associated with inbreeding and other selective forces. These together with transcriptome analyses of diabetic pig models provide a comprehensive genetic basis for resistance to diabetogenic environment, especially related to energy metabolism.

Autophagy is stimulated by stress conditions and needs to be precisely tuned to ensure cellular homeostasis and organismal development and health. The kinase mechanistic target of rapamycin (mTOR) forms the enzymatic core of the highly conserved mTOR complexes mTORC1 and mTORC2. mTORC1 is a key inhibitor of autophagy, yet the function of mTORC2 in autophagy is controversial. We here show that inactivation of mTORC2 and its direct target serum- and glucocorticoid-inducible kinase 1 (SGK-1) potently induces autophagy and the autophagic degradation of mitochondria in C. elegans. Enhanced autophagy in mTORC2 or SGK-1 deficient animals contributes to their developmental and reproductive defects and is independent of the canonical SGK-1 effector DAF-16/FOXO. Importantly, we find that inactivation of mTORC2 – SGK-1 signaling impairs mitochondrial homeostasis and triggers an increased release of mitochondria derived reactive oxygen species (mtROS) to induce autophagy. Thus, mitochondrial stress couples reduced mTORC2 activity to enhanced autophagic turnover.

Cystic echinococcosis (CE) is a chronic infectious disease caused by Echinococcus granulosus. To confirm whether the infection impacts on the gut microbiota, we established a mouse model of E. granulosus infection in this study whereby BALB/c mice were infected with micro-cysts of E. granulosus. After 4 months of infection, fecal samples were collected for high-throughput sequencing of the hypervariable regions of the 16S rRNA gene. Sequence analysis revealed a total of 13,353 operational taxonomic units (OTUs) with only 40.6% of the OTUs having genera reference information and 101 of the OTUs were significantly increased in infected mice. Bioinformatics analysis showed that the common core microbiota were not significantly changed at family level. However, two genera (Eisenbergiella and Parabacteroides) were enriched in the infected mice (PAMOV A < 0.05) at genus level. Functional analysis indicated that seven pathways were altered in the E. granulosus Infection Group compared with the Uninfected Group. Spearman correlation analysis showed strong correlations of IgG, IgG1 and IgG2a with nine major genera. E. granulosus cyst infection may change the gut microbiota which may be associated with metabolic pathways.

In pre-initiation complexes, RNA helicase A interacts with β-actin and acts as a bridging factor linking nuclear actin with RNA polymerase II (Pol II). In addition, β-actin participates in Pol II-dependent transcription elongation by interacting with the positive transcription elongation factor Cdk9. However, many relationships between β-actin and Pol II remain to be identified. In an interleukin 6 (IL-6)-induced p21 expression model, we demonstrated that β-actin knockdown reduced p21 expression. Immunofluorescence analysis showed that the colocalization of β-actin and Pol II increased significantly in cells treated with IL-6. It is known that the Rpb5, Rpb6 and Rpb7 subunits are located at the surface of the enzyme. We next constructed recombinant pcDNA-HA-Rpb5, pcDNA-HA-Rpb6 and pcDNA-HA-Rpb7 plasmids and expressed the three polymerase II subunits in HepG2 cells. We found that β-actin could be immunoprecipitated with HA-Rpb5 and HA-Rpb7. A Glutathione-S-transferase pull-down assay revealed that ...

Background Preclinical studies have found differential effects of isoflurane and propofol on the Alzheimer's disease (AD)-associated markers tau, phosphorylated tau (p-tau) and amyloid-β (Aβ). Objective We asked whether isoflurane and propofol have differential effects on the tau/Aβ ratio (the primary outcome), and individual AD biomarkers. We also examined whether genetic/intraoperative factors influenced perioperative changes in AD biomarkers. Methods Patients undergoing neurosurgical/otolaryngology procedures requiring lumbar cerebrospinal fluid (CSF) drain placement were prospectively randomized to receive isoflurane (n = 21) or propofol (n = 18) for anesthetic maintenance. We measured perioperative CSF sample AD markers, performed genotyping assays, and examined intraoperative data from the electronic anesthesia record. A repeated measures ANOVA was used to examine changes in AD markers by anesthetic type over time. Results The CSF tau/Aβ ratio did not differ between isoflurane- versus propofol-treated patients (p = 1.000). CSF tau/Aβ ratio and tau levels increased 10 and 24 h after drain placement (p = 2.002×10-6 and p = 1.985×10-6, respectively), mean CSF p-tau levels decreased (p = 0.005), and Aβ levels did not change (p = 0.152). There was no interaction between anesthetic treatment and time for any of these biomarkers. None of the examined genetic polymorphisms, including ApoE4, were associated with tau increase (n = 9 polymorphisms, p > 0.05 for all associations). Conclusion Neurosurgery/otolaryngology procedures are associated with an increase in the CSF tau/Aβ ratio, and this increase was not influenced by anesthetic type. The increased CSF tau/Aβ ratio was largely driven by increases in tau levels. Future work should determine the functional/prognostic significance of these perioperative CSF tau elevations.

Introduction Thrombocytopenia after cardiac surgery independently predicts stroke, acute kidney injury and death. To understand the underlying risks and mechanisms, we analysed genetic variations associated with thrombocytopenia in patients undergoing coronary artery bypass grafting (CABG) surgery. Materials and Methods Study subjects underwent isolated on-pump CABG surgery at Duke University Medical Center. Post-operative thrombocytopenia was defined as platelet count Results A total of 63 single-nucleotide polymorphisms met a priori discovery thresholds for replication, but only 1 (rs9574547) in the intergenic region upstream of sprouty 2 (SPRY2) met nominal significance in the replication cohort. The minor allele of rs9574547 was associated with a lower risk for thrombocytopenia (discovery cohort, odds ratio, 0.45, 95% confidence interval, 0.30–0.67, p = 9.76 × 10−5) with the overall association confirmed by meta-analysis (meta-p = 7.88 × 10−6). Immunoblotting demonstrated expression of SPRY2 and its dynamic regulation during platelet activation. Treatment with a functional SPRY2 peptide blunted platelet extracellular signal-regulated kinase (ERK) phosphorylation after agonist stimulation. Conclusion We identified the association of a genetic polymorphism in the intergenic region of SPRY2 with a decreased incidence of thrombocytopenia after CABG surgery. Because SPRY2—an endogenous receptor tyrosine kinase inhibitor—is present in platelets and modulates essential signalling pathways, these findings support a role for SPRY2 as a novel modulator of platelet responses after cardiac surgery.

Chromatin remodeling complexes play important roles in various DNA metabolism processes, including DNA damage repair. BRG1 is the core subunit of the SWI/SNF complex, which plays critical roles in cell cycle regulation, cell development, cell differentiation, and tumorigenesis. In the present study, we report that BRG1 depletion increased the percentage of apoptotic cells in etoposide-treated cells. Moreover, western blotting and immunofluorescence data showed that BRG1 depletion decreased H2AX phosphorylation and caused defective phosphorylated histone H2AX (γH2AX) clearance. Furthermore, we found that in both SW13 and U2OS cells, BRG1 expression could increase the sensitivity of genomic DNA to micrococcal nuclease (MNase) and facilitate chromatin relaxation around DNA damage sites. Thus, the results provide evidence that BRG1 plays an important role in early DNA damage repair by remodeling the chromatin structure near DNA damage sites.

The present study aimed to determine the protein expression, in addition to the clinical value of the expression, of estrogen receptor α (ERα), progesterone receptor (PR) and epidermal growth factor receptor (EGFR) in papillary thyroid carcinoma (PTC). The expression of ERα, PR and EGFR was examined immunohistochemically on paraffin-embedded thyroid tissues obtained from 64 patients with PTC and 14 patients with nodular thyroid goiter (NTG). The expression level of ERα, PR and EGFR was found to be significantly elevated in the PTC tissues compared with the NTG tissues. In addition, the expression of ERα was found to be correlated with the size of PTC tumors. However, there was no significant difference between the expression levels of ERα, PR and EGFR in males and females with PTC. Thus, immunohistochemical evaluation of ERα, PR and EGFR expression in patients with PTC may aid in the prediction of the prognosis of patients with PTC.

Objective The detection of BRAF V600E mutation in papillary thyroid carcinoma (PTC) may be helpful to offer diagnostic confirmation. Additionally, such detection may provide a targeted therapeutic approach for the radioactive iodine resistant patients to predict adverse outcomes. To compare the results of immunohistochemistry (IHC) method using the anti-BRAF V600E (VE1) antibody with the Quantitative real-time polymerase chain reaction (qPCR) approach in examining BRAF V600E mutation in PTC, we investigated the sensitivity and specificity of BRAF V600E (clone VE1) mouse monoclonal antibody in detecting the BRAF V600E mutation and correlated BRAF V600E mutation with clinicopathologic features in PTC. Methods IHC and qPCR were performed in 40 cases of paraffin-embedded PTCs tissues. The association between BRAFV600E mutation and clinicopathologic features of PTC was assessed with the χ 2 test. Results The concordance rate between IHC and qPCR analyses was 95% (38/40). The BRAF V600E (VE1) antibody has a sensitivity of 100% (34/34) and specificity of 66.67% (4/6) for detecting the mutation. Our study showed that there was no significant association of BRAF V600E mutation with the gender, age, tumor size and lymph node metastasis in PTCs. Conclusion We may draw the conclusion that detection of BRAF V600E mutation by immunohistochemistry is highly sensitive and specific. Immunohistochemical detection of the mutated BRAF V600E protein in PTC may facilitate mutational analysis in the clinical setting.

Managing available resources is a key necessity of each organism to cope with the environment. The nematode C. elegans responds to nutritional deprivation or harsh environmental conditions with a multitude of developmental adaptations, among them a starvation-induced quiescence at early larval development (L1). daf-18, the C. elegans homolog of the human tumor suppressor gene PTEN, is essential for the maintenance of survival and germline stem cell arrest during the L1 diapause. We show here that daf-18 mutants, independently to their failure to maintain G2 arrest of the primordial germ cells, develop a gonad phenotype after refeeding. This highly penetrant gonadal phenotype is further enhanced by a mutation in shc-1, encoding a protein homologous to the human adaptor ShcA. Features of this phenotype are a tumor-like phenotype encompassing hyper-proliferation of germ cell nuclei and disruption/invasion of the basement membrane surrounding the gonad. The penetrance of this phenotype is reduced by decreasing starvation temperature. In addition, it is also ameliorated in a dose-dependent way by exposure to the antibiotic doxycyclin either during starvation or during subsequent refeeding. Since, in eukaryotic cells, doxycyclin specifically blocks mitochondrial translation, our results suggest that daf-18 and shc-1;daf-18 mutants fail to adapt mitochondrial activity to reduced nutritional availability during early larval developing.

Fluorescent silver nanoclusters stabilized by DNA (DNA-AgNCs) exhibit distinct response rates to thiol and disulfide. Glutathione reductase can catalyze the reduction of the oxidized glutathione (GSSG) quickly to reduced glutathione (GSH) in the presence of β-nicotinamide adenine dinucleotide 2'-phosphate reduced tetrasodium salt hydrate (NADPH). Consequently, DNA-AgNCs can serve as a new fluorescent platform for assaying the glutathione reductase (GR) activity. This newly proposed assay has a high sensitivity and a good selectivity toward GR. The GR activity can be detected in the range of 0.2-2.0 mU mL(-1) with a minimum detectable concentration of 0.2 mU mL(-1). Pepsin, lysozyme, trypsin, avidin, thrombin, myoglobin, and BSA have little effect on the fluorescence intensity of DNA-AgNCs. The GR activity assay is successfully used to monitor the inhibition of GR activity by a typical inhibitor 1,3-bis(2-chloroethyl)-1-nitrosourea.

Atrial tissue gene expression profiling may help to determine how differentially expressed genes in the human atrium before cardiopulmonary bypass (CPB) are related to subsequent biologic pathway activation patterns, and whether specific expression profiles are associated with an increased risk for postoperative atrial fibrillation (AF) or altered response to β-blocker (BB) therapy after coronary artery bypass grafting (CABG) surgery. Right atrial appendage (RAA) samples were collected from 45 patients who were receiving perioperative BB treatment, and underwent CABG surgery. The isolated RNA samples were used for microarray gene expression analysis, to identify probes that were expressed differently in patients with and without postoperative AF. Gene expression analysis was performed to identify probes that were expressed differently in patients with and without postoperative AF. Gene set enrichment analysis (GSEA) was performed to determine how sets of genes might be systematically altered in patients with postoperative AF. Of the 45 patients studied, genomic DNA from 42 patients was used for target sequencing of 66 candidate genes potentially associated with AF, and 2,144 single-nucleotide polymorphisms (SNPs) were identified. We then performed expression quantitative trait loci (eQTL) analysis to determine the correlation between SNPs identified in the genotyped patients, and RAA expression. Probes that met a false discovery rate < 0.25 were selected for eQTL analysis. Of the 17,678 gene expression probes analyzed, 2 probes met our prespecified significance threshold of false discovery rate < 0.25. The most significant probe corresponded to vesicular overexpressed in cancer – prosurvival protein 1 gene (VOPP1; 1.83 fold change; P = 3.47 × 10−7), and was up-regulated in patients with postoperative AF, whereas the second most significant probe, which corresponded to the LOC389286 gene (0.49 fold change; P = 1.54 × 10−5), was down-regulated in patients with postoperative AF. GSEA highlighted the role of VOPP1 in pathways with biologic relevance to myocardial homeostasis, and oxidative stress and redox modulation. Candidate gene eQTL showed a trans-acting association between variants of G protein-coupled receptor kinase 5 gene, previously linked to altered BB response, and high expression of VOPP1. In patients undergoing CABG surgery, RAA gene expression profiling, and pathway and eQTL analysis suggested that VOPP1 plays a novel etiological role in postoperative AF despite perioperative BB therapy.

Chromatin remodelling is critical for repairing DNA damage and maintaining genomic integrity. Previous studies have reported that histone acetyltransferase p300 and ATP-dependent chromatin remodeler chromodomain helicase DNA-binding protein 4 (CHD4) functions, respectively, in DNA double-strand breaks (DSBs) repair. But the physiological significance of their interaction remains elusive. Here, we showed that p300 and CHD4 were both recruited to the sites of DSBs. Their ablation led to impaired DSBs repair and sensitised cells to laser and the anti-cancer drug, etoposide. Using DR-GFP and EJ5-GFP reporter systems, we found that knockdown of p300 or CHD4 impaired the homologous recombination (HR) repair but no the non-homologous end joining (NHEJ) repair. Furthermore, p300 or CHD4 knockdown respectively suppressed the recruitment of replication protein A (RPA), a key protein for HR, to the DSB sites. In addition, immunofluorescence results showed that knockdown of p300 reduced the recruitment of CHD4 at DSB sites. In turn, CHD4 knockdown also decreased p300 assembly. Moreover, immunoprecipitation and purified protein pull down assay revealed that p300 physically interacted with CHD4 at DNA damage sites, and this interaction was dependent on the chromodomain and ATPase/helicase domain of CHD4 and the CH2, Bd and HAT domains of p300. These results indicate that p300 and CHD4 could function cooperatively at DSB sites and provide a new insight into the detailed crosstalk among the chromatin remodelling proteins.

Actin, one of the most evolutionarily conservative proteins in eukaryotes, is distributed both in the cytoplasm and the nucleus, and its dynamics plays important roles in numerous cellular processes. Previous evidence has shown that actin interacts with p53 and this interaction increases in the process of p53 responding to DNA damage, but the physiological significance of their interaction remains elusive. Here, we show that DNA damage induces both actin polymerization and p53 accumulation. To further understand the implication of actin polymerization in p53 function, cells were treated with actin aggregation agent. We find that the protein level of p53 decrease. The change in p53 is a consequence of the polymeric actin anchoring p53 in the cytoplasm, thus impairing p53 nuclear import. Analysis of phosphorylation and ubiquitination of p53 reveals that actin polymerization promotes the p53 phosphorylation at Ser315 and reduces the stabilization of p53 by recruiting Aurora kinase A. Taken together, our results suggest that the actin polymerization serves as a negative modulator leading to the impairment of nuclear import and destabilization of p53. On the basis of our results, we propose that actin polymerization might be a factor participating in the process of orchestrating p53 function in response to DNA damage.

In Caenorhabditis elegans (C. elegans), the promotion of longevity by the transcription factor DAF-16 requires reduced insulin/IGF receptor (IIR) signaling or the ablation of the germline, although the reason for the negative impact of germ cells is unknown. FOXO/DAF-16 activity inhibits germline proliferation in both daf-2 mutants and gld-1 tumors. In contrast to its function as a germline tumor suppressor, we now provide evidence that somatic DAF-16 in the presence of IIR signaling can also result in tumorigenic activity, which counteracts robust lifespan extension. In contrast to the cell-autonomous IIR signaling, which is required for larval germline proliferation, activation of DAF-16 in the hypodermis results in hyperplasia of the germline and disruption of the surrounding basement membrane. SHC-1 adaptor protein and AKT-1 kinase antagonize, whereas AKT-2 and SGK-1 kinases promote, this cell-nonautonomous DAF-16 function. Our data suggest that a functional balance of DAF-16 activities in different tissues determines longevity and reveals a novel, cell-nonautonomous role of FOXO/DAF-16 to affect stem cells., Author Summary Previous studies have shown that DAF–16/FOXO transcription factor promotes longevity and stress resistance and inhibits tumor progression in the absence of insulin signaling. Here we show that active DAF-16 in the epidermis can shorten lifespan by promoting a tumorous germline phenotype. In contrast to the known inhibitory effect of insulin signaling upon DAF-16, an active insulin and PI3K signaling are required for DAF-16–mediated signaling to the germline. In addition, AKT-1– and SHC-1–mediated JNK signaling antagonize AKT-2 and SGK-1 to affect the reproductive system. This is to our knowledge the first report about a detrimental effect of DAF-16 on lifespan. Furthermore it emphasizes that DAF-16 activity is highly dependent on the cellular context and communication between different tissues.

Correlative evidence links stress, accumulation of oxidative cellular damage, and aging in several species. Genetic studies in species ranging from yeast to mammals revealed several pathways regulating stress response and life span, including caloric intake, mitochondrial respiration, insulin/IGF-1 (IIS), and JNK (c-Jun N-terminal kinase) signaling. How IIS and JNK signaling cross-talk to defend against diverse stressors contributing to aging is of critical importance but, so far, only poorly understood. In this study, we demonstrate that the adaptor protein SHC-1, the Caenorhabditis elegans homolog of human p52Shc, coordinates mechanisms of stress response and aging. Using genetic and biochemical approaches, we discover that SHC-1 not only opposes IIS but also activates JNK signaling. Loss of shc-1 function results in accelerated aging and enhanced sensitivity to heat, oxidative stress, and heavy metals, whereas expression of human p52Shc rescues the shc-1 mutant phenotype. SHC-1 acts upstream of the insulin/IGF receptor DAF-2 and the PI3 kinase AGE-1 and directly interacts with DAF-2. Moreover, SHC-1 activates JNK signaling by binding to MEK-1 kinase. Both aspects converge on controlling the nuclear translocation and activation of the FOXO transcription factor DAF-16. Our findings establish C. elegans SHC-1 as a critical scaffold that directly cross-connects the two parallel JNK and IIS pathways and help to explain how these signaling cascades cooperate to ascertain normal stress response and life span in C. elegans.