Telomeres play important roles in genome stability and cell proliferation. High risk neuroblastoma (HRNB), an aggressive childhood cancer, is especially reliant on telomere maintenance. Three recurrent genetic aberrations in HRNB (MYCN amplification, TERT re-arrangements, and ATRX mutations) are mutually exclusive and each capable of promoting telomere maintenance mechanisms (i.e., through telomerase or ALT). We analyzed a panel of 5 representative HRNB cell lines and 30 HRNB surgical samples using assays that assess average telomere lengths, length distribution patterns, single-stranded DNA on the G- and C-strand, as well as extra-chromosomal circular telomeres. Our analysis pointed to substantial and variable degrees of telomere DNA damage in HRNB, including pervasive oxidative lesions. Moreover, unlike other cancers, neuroblastoma consistently harbored high levels of C-strand ssDNA overhangs and t-circles, which are consistent with active "telomere trimming". This feature is observed in both telomerase- and ALT-positive tumors and irrespective of telomere length distribution. Moreover, evidence for telomere trimming was detected in normal neural tissues, raising the possibility that TMMs in HRNB evolved in the face of a canonical developmental program of telomere shortening. Telomere trimming by itself appears to distinguish neuroectodermal derived tumors from other human cancers, a distinguishing characteristic with both biologic and therapeutic implications.

To identify an uncommon genetic cause of tooth agenesis (TA) by utilizing whole exome sequencing (WES) and targeted Sanger sequencing in a cohort of 120 patients with isolated TA.

No abstract available

Emerging porcine epidemic diarrhea viruses (PEDVs) have caused large economic losses since 2010, and G2b is the prevalent globally epidemic genotype. Given the fastidious isolation of emerging PEDV in cell culture and difficulties in retaining the isolate infectivity upon further in vitro passage, highly attenuated recombinant vesicular stomatitis virus (rVSVMT) was used as a vector to express the PEDV spike (S) protein, aiming to develop a subunit vaccine against G2b viruses. An S protein with 19 of its cytoplasmic domain amino acids deleted could be incorporated into VSV particles, generating rVSVMT (VSVMT-SΔ19) with high efficiency. Our results suggest that VSVMT-SΔ19 could effectively induce PEDV-specific immunity in pigs via intramuscular, but not intranasal, immunization. Notably, immunizations of sows with VSV MT-SΔ19 provided protective lactogenic immunity against a virulent G2b PEDV challenge in piglets. Consequently, recombinant VSVMT may be a promising platform for preparing a subunit vaccine against PEDV.

The current study evaluated the synergistic role of Plant growth promoting rhizobacteria (PGPR), Pseudomonas aeruginosa and Burkholderia gladioli on different physiological, biochemical and molecular activities of 10-days old Solanum lycopersicum seedlings under Cd stress. Cd toxicity altered the levels of phenolic compounds (total phenols (30.2%), flavonoids (92.7%), anthocyanin (59.5%), polyphenols (368.7%)), osmolytes (total osmolytes (10.3%), total carbohydrates (94%), reducing sugars (64.5%), trehalose (112.5%), glycine betaine (59%), proline (54.8%), and free amino acids (63%)), and organic acids in S. lycopersicum seedlings. Inoculation of P. aeruginosa and B. gladioli alleviated Cd-induced toxicity, which was manifested through enhanced phenolic compound levels and osmolytes. Additionally, the levels of low molecular weight organic acids (fumaric acid, malic acid, succinic acid, and citric acid) were also elevated. The expression of genes encoding enzymes for phenols and organic acid metabolism were also studied to be modulated that included CHS (chalcone synthase; 138.4%), PAL (phenylalanine ammonia lyase; 206.7%), CS (citrate synthase; 61.3%), SUCLG1 (succinyl Co-A ligase; 33.6%), SDH (succinate dehydrogenase; 23.2%), FH (fumarate hydratase; 12.4%), and MS (malate synthase; 41.2%) and found to be upregulated in seedlings inoculated independently with P. aeruginosa and B. gladioli. The results provide insights into the role of micro-organisms in alleviating Cd-induced physiological damage by altering levels of different metabolites.

Mild cognitive impairment (MCI) is a common neurological disorder. This study aims to investigate the modulation effect of Baduanjin (a popular mind-body exercise) on MCI. 69 patients were randomized to Baduanjin, brisk walking, or an education control group for 24 weeks. The Montreal Cognitive Assessment (MoCA) and Magnetic Resonance Imaging scans were applied at baseline and at the end of the experiment. Compared to the brisk walking and control groups, the Baduanjin group experienced significantly increased MoCA scores. Amplitude of low-frequency fluctuations (ALFF) analysis showed significantly decreased ALFF values in the right hippocampus (classic low-freqency band, 0.01-0.08 Hz) in the Baduanjin group compared to the brisk walking group and increased ALFF values in the bilateral anterior cingulate cortex (ACC, slow-5 band, 0.01-0.027 Hz) in the Baduanjin group compared to the control group. Further, ALFF value changes in the right hippocampus and bilateral ACC were significantly associated with corresponding MoCA score changes across all groups. We also found increased gray matter volume in the Baduanjin group in the right hippocampus compared to the brisk walking group and in the bilateral ACC compared to the control group. In addition, there was an increased resting state functional connectivity between the hippocampus and right angular gyrus in the Baduanjin group compared to the control group. Our results demonstrate the potential of Baduanjin for the treatment of MCI.

Protein tyrosine kinases (PTKs) are key signaling molecules and important drug targets. Although the efficient recombinant production of active PTKs is important for both pharmaceutical industry and academic research, most PTKs are still obtained from conventional, expensive and time-consuming insect-cell based expression. Host toxicity, kinase inactivity, insolubility and heterogeneity are among the reasons thought to preclude PTK expression in Escherichia coli. Herein we review these presumed roadblocks and their possible solutions for bacterial expression of PTKs, and give an overview on kinase activity assays. Finally, we report our experiences and observations with the kinases Src, Lyn and FAK as examples to illustrate implementation, effects and pitfalls of E. coli expression and in vitro assaying of PTKs.

Cellular functions are often controlled by small molecular weight molecules such as metabolites. Microorganisms, mainly prokaryotes, have evolved sensing and regulatory mechanisms based on transcriptional regulators (TRs) that are able to activate gene expression in response to changes in intra- and extracellular metabolite (ligand) concentrations. To understanding control mechanisms and cell factory development in synthetic biology applications, high throughput analytical procedures are required. In this chapter, we outline a methodological pipeline to design and build reporter constructs enabling the characterization of metabolite-responsive inducible gene expression systems. As an example, we present the design, cloning and characterization of the itaconate-inducible system which is composed of the LysR-type transcriptional regulator ItcR and the promoter Pccl from Yersinia pseudotuberculosis. Fluorescence-based plate reader and flow cytometry assays are described and the steps for performing data analysis are provided.

Heterologous expression of natural product biosynthetic gene clusters (BGCs) is a robust approach not only to decipher biosynthetic logic behind natural product (NP) biosynthesis, but also to discover new chemicals from uncharacterized BGCs. This approach largely relies on techniques used for cloning large BGCs into suitable expression vectors. Recently, several whole-pathway direct cloning approaches, including full-length RecE-mediated recombination in Escherichia coli, Cas9-assisted in vitro assembly, and transformation-associated recombination (TAR) in Saccharomyces cerevisiae, have been developed to accelerate BGC isolation. In this chapter, we summarize a protocol for TAR cloning large NP BGCs, detailing the process of choosing TAR plasmids, designing pathway-specific TAR vectors, generating yeast spheroplasts, performing yeast transformation, and heterologously expressing BGCs in various host strains. We believe that the established platforms can accelerate the process of discovering new NPs, understanding NP biosynthetic logic, and engineering biosynthetic pathways.

In a variety of malignomas, the acquisition of a mesenchymal phenotype has been linked with anchorage-independent growth and invasiveness. To some extent, glioma cells are able to survive a loss of cell-matrix contact. We here describe that non-adherent culture of glioma cells was accompanied by an increase in hypoxia-inducible factor (HIF)-dependent, but not β-catenin/TCF-induced transcription. Levels of reactive oxygen species decreased in suspension and knockdown of HIF-1α enhanced cell death following detachment. By promoting the adaptation to non-adherent conditions, mechanisms driven by HIF-1α may considerably contribute to the biology and aggressiveness of glioblastoma.

A recently discovered lectin-like protein from mushroom tyrosinase designated as orf239342 inhibits proliferation of the MCF-7 breast cancer cells. This characteristic is likely derived from its ability to recognize sugar entity on the cell surface. Thereby, the binding specificity of orf239342 to sugars was studied. Orf239342 was found to bind specifically to mannose upon analysis with the surface plasmon resonance technique. Finally, our in vitro study showed that mannose impeded orf239342 ability to inhibit proliferation of the MCF-7 breast cancer cells, providing further evidence for the mannose binding onto the protein. Our finding is a breakthrough to characterise orf239342 i.e. to define its functioning in the mushroom, association to the tyrosinase, or even possible application in breast cancer therapy. In addition, the finding allows the more appropriate designation of the protein as Agaricus bisporus mannose binding-protein (AbMb).

Innate lymphoid cells (ILCs) play strategic roles in tissue homeostasis and immunity. ILCs arise from lymphoid progenitors undergoing lineage restriction and the development of specialized ILC subsets. We generated "5x polychromILC" transcription factor reporter mice to delineate ILC precursor states by revealing the multifaceted expression of key ILC-associated transcription factors (Id2, Bcl11b, Gata3, RORγt, and RORα) during ILC development in the bone marrow. This approach allowed previously unattained enrichment of rare progenitor subsets and revealed hitherto unappreciated ILC precursor heterogeneity. In vivo and in vitro assays identified precursors with potential to generate all ILC subsets and natural killer (NK) cells, and also permitted discrimination of elusive ILC3 bone marrow antecedents. Single-cell gene expression analysis identified a discrete ILC2-committed population and delineated transition states between early progenitors and a highly heterogeneous ILC1, ILC3, and NK precursor cell cluster. This diversity might facilitate greater lineage potential upon progenitor recruitment to peripheral tissues.

Brain metastases are a major cause of death in patients with metastatic breast cancer. While surgical resection and radiation therapy are effective treatment modalities, the majority of patients will succumb from disease progression. We have developed a novel therapy for brain metastases that delivers athermal radiofrequency electromagnetic fields that are amplitude-modulated at breast cancer specific frequencies (BCF).

The ability to either erase or update the memories of a previously learned spatial task is an essential process that is required to modify behaviour in a changing environment. Current evidence suggests that the neural representation of such cognitive flexibility involves the balancing of synaptic potentiation (acquisition of memories) with synaptic depression (modulation and updating previously acquired memories). Here we demonstrate that the p38 MAPK/MAPK-activated protein kinase 2 (MK2) cascade is required to maintain the precise tuning of long-term potentiation and long-term depression at CA1 synapses of the hippocampus which is correlated with efficient reversal learning. Using the MK2 knockout (KO) mouse, we show that mGluR-LTD, but not NMDAR-LTD, is markedly impaired in mice aged between 4 and 5 weeks (juvenile) to 7 months (mature adult). Although the amplitude of LTP was the same as in wildtype mice, priming of LTP by the activation of group I metabotropic receptors was impaired in MK2 KO mice. Consistent with unaltered LTP amplitude and compromised mGluR-LTD, MK2 KO mice had intact spatial learning when performing the Barnes maze task, but showed specific deficits in selecting the most efficient combination of search strategies to perform the task reversal. Findings from this study suggest that the mGluR-p38-MK2 cascade is important for cognitive flexibility by regulating LTD amplitude and the priming of LTP.

Croton is an extensive flowering plant genus in the spurge family, Euphorbiaceae. Three croton compounds with the common ent-kaurane skeleton were purified from Croton tonkinensis. By using patch-clamp recording technique, we thoroughly examined the effect of a group of croton compounds, croton-01 (ent-18-acetoxy-7α-hydroxykaur-16-en-15-one), croton-02 (ent-7α,14β-dihydroxykaur-16-en-15-one), and croton-03 (ent-1β-acetoxy-7α,14β-dihydroxykaur-16-en-15-one), on the membrane current in SM826 and BV2 microglial cells. Although neither voltage-gated Na+ nor Ca2+ currents were present in these cells, both delayed-rectifier K+ outward (IK(DR)) and inwardly rectifying K+ currents (IK(IR)) were readily detected. Croton-03 differentially caused inhibition of IK(DR) or IK(IR) in a concentration-dependent manner. According to a minimal scheme, the shortening of the time constant in either the IK(DR)-related block or IK(IR) caused by different concentrations of croton-03 was quantitatively estimated with a dissociation constant of 6.45 and 29.5 μM, respectively. In SM826 cells differentiated with β-amyloid, inhibitory action on these K+ currents remained unaltered. In ultraviolet C-irradiated cells, the magnitude of IK(IR) was still decreased by addition of croton-03. Therefore, our study suggests that these ent-kaurane diterpenoids ought to somehow act on the cellular mechanisms by which they influence the functional activities of microglial cells.

Obstructive sleep apnea hypopnea syndrome (OSAHS) is associated with multiple system diseases. Neurocognitive dysfunction resulting from central nervous system complications has been reported, especially in children with OSAHS. Chronic intermittent hypoxia is accepted to be the major pathophysiological mechanism of OSAHS. Adenosine plays an important role in cellular function via interactions with its receptors. A2a receptor has been recognized as a factor involved in neuroprotection. However, the role of adenosine A2a receptor in intermittent hypoxia induced cellular injury is not completely understood. In this study, we aim to investigate the underlying mechanisms of A2a receptor mediated cellular damage caused by intermittent hypoxia in PC12 cells. We found that activated A2a receptor by CGS21680 decreased cellular viability, increased PKC as well as ATP-sensitive potassium channel (KATP) subunits expression Kir6.2 and SUR1. Inhibition of A2a receptor by SCH58261 increased cellular viability, suppressed PKC and SUR1 expression level, ultimately showing a protective role in PC12 cells. Moreover, we observed that CHE, which is an antagonist of PKC, downregulated Kir6.2 and SUR1 expression and increased cellular viability. Additionally, we found that A2a receptor activation induced cell injury was associated with increased Cleaved-Caspase 3 expression, which can be decreased by inhibition of A2a receptor or PKC. In conclusion, our findings indicate that A2a receptor induced KATP expression by PKC activation and plays a role in accelerating PC12 cells injury induced by intermittent hypoxia exposure via A2a-PKC-KATP signal pathway mediated apoptosis.

Peste des petits ruminants virus (PPRV), a member of the genus Morbillivirus, in the family Paramyxoviridae expresses two membrane glycoproteins, the fusion (F) and haemagglutinin (H) glycoproteins which mediate virus-to-cell fusion and cell-to-cell fusion leading to the induction of syncytia in PPRV infected cells. In the context of the characterization of the virulent lineage IV strain PPRV Kurdistan 2011, isolated from wild goats from the Kurdistan region in Iraq, we observed that both PPRV Kurdistan 2011 and the PPRV Nigeria 75/1 vaccine strain led to induction of large syncytia in Vero-dogSLAM cells within 48 h whereas both failed to induce detectable cell-cell fusion events in two Vero cell lines of differing passage histories. We were unable to detect syncytium formation in transiently transfected cells expressing PPRV F or H alone whereas co-expression of F and H induced large syncytia - in Vero-dogSLAM cells only. In VeroMontpellier cells expressing PPRV F and H, fused cells were rarely detectable indicating that PPRV mediated cell fusion activity is impaired in this cell line. Surprisingly, on Vero-dogSLAM cells the vaccine strain grew to titers of 105.25 TCID50/ml, whereas infectious virus yield was about 200-fold higher on VeroMontpellier and Vero-76 cells. In contrast, the virulent Kurdistan 2011 strain grew to a maximum titer of 107.0 TCID50/ml on Vero-dogSLAM cells and only 104.5 TCID50/ml on normal Vero cells. This was as expected since Vero cells lacking the SLAM receptor for PPRV are regarded as not so permissive for infection. To elucidate the divergent productive replication behaviour of PPRV Nigeria 75/1 vaccine strain on Vero vs Vero-dogSLAM cells, we examined whether intracellular transport and/or maturation of the viral envelope glycoproteins F and H might be implicated with this phenomenon. The results indicate that F in contrast to the H glycoprotein matures inefficiently during intracellular transport in VeroMontpellier cells, thus leading to an absence of detectable syncytia formation. However, in the case of the PPRV Nigeria 75/1 vaccine strain this did not impair efficient virus assembly and release.

Fibroblast growth factor 21 (FGF-21) is an important regulator in glycolipid metabolism that is a promising drug candidate for treatment of diabetes and obesity. However, the productivity of recombinant hFGF-21 (rhFGF-21) in Escherichia coli (E. coli) is relatively low, which limits its clinical application. To meet the clinical demand and control the production cost, rhFGF-21 proteins were expressed in inclusion bodies (IBs) form in Rosetta (DE3) by high cell density fermentation in 50-L scale. Hollow fiber membrane filtration technology was used to enrich the bacteria, wash, denature and refold the IBs in the current report. The renatured proteins were purified by two-step affinity chromatography. Authenticity of the purified rhFGF-21 was confirmed by the N-and C-terminal sequence, disulfide bond composition and molecular weight analyses. Results showed that the average target protein and recovery of rhFGF-21 expressed in IBs form of three batches were more than those of the soluble form. Both the rhFGF-21 proteins from the two forms showed equal potency in improving the glucose uptake in HepG2 cells and anti-diabetic effect in db/db mice. In this study, an efficient method for preparation of FGF-21 was established. This novel process provides an important technical basis for the large-scale production of rhFGF-21.

The effect of Micrurus mipartitus snake venom as a therapeutic alternative for T-acute lymphoblastic leukemia (ALL) is still unknown. This study was aimed to evaluate the cytotoxic effect of M. mipartitus snake venom and a new L-amino acid oxidase (LAAO), named MipLAAO, on human peripheral blood lymphocytes (PBL) and on T-ALL cells (Jurkat), and its mechanism of action. PBL and Jurkat cells were treated with venom and MipLAAO, and morphological changes in the cell nucleus/DNA, mitochondrial membrane potential, levels of intracellular reactive oxygen species and cellular apoptosis markers were determined by fluorescence microscopy, flow cytometry and pharmacological inhibition. Venom and MipLAAO induced apoptotic cell death in Jurkat cells, but not in PBL, in a dose-response manner. Additionally, venom and MipLAAO increased dichlorofluorescein fluorescence intensity, indicative of H2O2 production, increased DJ-1 Cys106-sulfonate, as a marker of intracellular stress and induced the up-regulation of PUMA, p53 and phosphorylation of c-JUN. Additionally, it increased the expression of apoptotic CASPASE-3. In conclusion, M. mipartitus venom and MipLAAO selectively induces apoptosis in Jurkat cells through a H2O2-mediated signaling pathway dependent mostly on CASPASE-3 pathway. Our findings support the potential use of M. mipartitus snake venom compounds as a potential treatment for T-ALL.

Pattern recognition receptors (PRRs) play a crucial role in the host's innate immune system. Among the PPRs, the RIG-I-like (RLRs) and NOD-like receptors (NLRs) are two important subgroups. To understand the role of RLRs (RIG-I, MDA5, LGP2) and NLRs (NOD1, NLRC3, NLRX1, NOD2) in reptilian evolution, we identified six genes from reptilian genomes. A total of 168 putative genes were identified from 28 reptile species, including 141 intact genes, 25 partial genes and two pseudogenes. Interestingly, the NOD2 gene was absent in all reptile species. Phylogenetic results showed that all genes were divided into four major clades corresponding to their traditional taxonomic groups, indicating that these genes are conserved in reptiles. Evolutionary analyses detected positive selection in six genes. Most of the positively selected sites (50/76) were located in known functional domains, reflecting their critical and particular contributions to host defense during reptilian evolution. Branch model analysis showed that NLRs were under different evolutionary forces, while the RLRs were not, suggesting that semiaquatic species and terrestrial species faced different environmental pathogens, leading to different adaptations. Moreover, the positively selected sites identified in MDA5 using the branch-site model among semiaquatic reptiles, suggested their involvement in adaptation to semiaquatic environments.