DNA double-strand breaks (DSBs) are highly cytotoxic lesions and pose a major threat to genome stability if not properly repaired. We and others have previously shown that a class of DSB-induced small RNAs (diRNAs) is produced from sequences around DSB sites. DiRNAs are associated with Argonaute (Ago) proteins and play an important role in DSB repair, though the mechanism through which they act remains unclear. Here, we report that the role of diRNAs in DSB repair is restricted to repair by homologous recombination (HR) and that it specifically relies on the effector protein Ago2 in mammalian cells. Interestingly, we show that Ago2 forms a complex with Rad51 and that the interaction is enhanced in cells treated with ionizing radiation. We demonstrate that Rad51 accumulation at DSB sites and HR repair depend on catalytic activity and small RNA-binding capability of Ago2. In contrast, DSB resection as well as RPA and Mre11 loading is unaffected by Ago2 or Dicer depletion, suggesting that Ago2 very likely functions directly in mediating Rad51 accumulation at DSBs. Taken together, our findings suggest that guided by diRNAs, Ago2 can promote Rad51 recruitment and/or retention at DSBs to facilitate repair by HR.

Fear behaviors and fear memories in rodents have been traditionally assessed by the amount of freezing upon the presentation of conditioned cues or unconditioned stimuli. However, many experiences, such as encountering earthquakes or accidental fall from tree branches, may produce long-lasting fear memories but are behaviorally difficult to measure using freezing parameters. Here, we have examined changes in heartbeat interval dynamics as physiological readout for assessing fearful reactions as mice were subjected to sudden air puff, free-fall drop inside a small elevator, and a laboratory-version earthquake. We showed that these fearful events rapidly increased heart rate (HR) with simultaneous reduction of heart rate variability (HRV). Cardiac changes can be further analyzed in details by measuring three distinct phases: namely, the rapid rising phase in HR, the maximum plateau phase during which HRV is greatly decreased, and the recovery phase during which HR gradually recovers to baseline values. We showed that durations of the maximum plateau phase and HR recovery speed were quite sensitive to habituation over repeated trials. Moreover, we have developed the fear resistance index based on specific cardiac response features. We demonstrated that the fear resistance index remained largely consistent across distinct fearful events in a given animal, thereby enabling us to compare and rank individual mouse's fear responsiveness among the group. Therefore, the fear resistance index described here can represent a useful parameter for measuring personality traits or individual differences in stress-susceptibility in both wild-type mice and post-traumatic stress disorder (PTSD) models.

WRKY transcription factors have been extensively characterized in the past 20 years, but in wheat, studies on WRKY genes and their function are lagging behind many other species. To explore the function of wheat WRKY genes, we identified a TaWRKY68 gene from a common wheat cultivar. It encodes a protein comprising 313 amino acids which harbors 19 conserved motifs or active sites. Gene expression patterns were determined by analyzing microarray data of TaWRKY68 in wheat and of orthologous genes from maize, rice and barley using Genevestigator. TaWRKY68 orthologs were identified and clustered using DELTA-BLAST and COBALT programs available at NCBI. The results showed that these genes, which are expressed in all tissues tested, had relatively higher levels in the roots and were up-regulated in response to biotic stresses. Bioinformatics results were confirmed by RT-PCR experiments using wheat plants infected by Agrobacterium tumefaciens and Blumeria graminis, or treated with Deoxynivalenol, a Fusarium graminearum-induced mycotoxin in wheat or barley. In summary, TaWRKY68 functions differ during plant developmental stages and might be representing a hub gene function in wheat responses to various biotic stresses. It was also found that including data from major cereal genes in the bioinformatics analysis gave more accurate and comprehensive predictions of wheat gene functions.

Some cell-adapted strains of foot-and-mouth disease virus (FMDV) can utilize heparan sulfate (HS) as a receptor to facilitate viral infection in cultured cells. A number of independent sites on the capsid that might be involved in FMDV-HS interaction have been studied. However, the previously reported residues do not adequately explain HS-dependent infection of two cell-adapted PanAsia-1 strains (O/Tibet/CHA/6/99tc and O/Fujian/CHA/9/99tc) of FMDV serotype O. To identify the molecular determinant(s) for the interaction of O/Tibet/CHA/6/99tc and O/Fujian/CHA/9/99tc with HS receptor, several chimeric viruses and site-directed mutants were generated by using an infectious cDNA of a non-HS-utilizing rescued virus (Cathay topotype) as the genomic backbone. Phenotypic properties of these viruses were determined by plaque assays and virus adsorption and penetration assays in cultured cells.

Amycolatopsis orientalis is the type species of the genus and its industrial strain HCCB10007, derived from ATCC 43491, has been used for large-scale production of the vital antibiotic vancomycin. However, to date, neither the complete genomic sequence of this species nor a systemic characterization of the vancomycin biosynthesis cluster (vcm) has been reported. With only the whole genome sequence of Amycolatopsis mediterranei available, additional complete genomes of other species may facilitate intra-generic comparative analysis of the genus.

The uncertainties inherent in clinical measures of prostate cancer (CaP) aggressiveness endorse the investigation of clinically validated tissue biomarkers. MUC1 expression has been previously reported to independently predict aggressive localized prostate cancer. We used a large cohort to validate whether MUC1 protein levels measured by immunohistochemistry (IHC) predict aggressive cancer, recurrence and survival outcomes after radical prostatectomy independent of clinical and pathological parameters.

Missense mutation is the most common mutation type in hemophilia. However, the majority of missense mutations remain uncharacterized. Here we characterize how hemophilia mutations near the unused N-glycosylation site of the A2 domain (N582) of FVIII affect protein conformation and intracellular trafficking. N582 is located in the middle of a short 310-helical turn (D580-S584), in which most amino acids have multiple hemophilia mutations. All 14 missense mutations found in this 310-helix reduced secretion levels of the A2 domain and full-length FVIII. Secreted mutants have decreased activities relative to WT FVIII. Selected mutations also lead to partial glycosylation of N582, suggesting that rapid folding of local conformation prevents glycosylation of this site in wild-type FVIII. Protease sensitivity, stability and degradation of the A2 domain vary among mutants, and between non-glycosylated and glycosylated species of the same mutant. Most of the mutants interact with the ER chaperone BiP, while only mutants with aberrant glycosylation interact with calreticulin. Our results show that the short 310-helix from D580 to S584 is critical for proper biogenesis of the A2 domain and FVIII, and reveal a range of molecular mechanisms by which FVIII missense mutations lead to moderate to severe hemophilia A.

Inflatin G (1), a new aphidicolin analogue, together with seven known compounds inflatin A (2), inflatin B (3), aphidicolin (4), aphidicolin-17-monoacetate (5), gulypyrone A (6), pyridoxatin rotamers A (7) and B (8), were isolated from the ascomycete fungus Tolypocladium inflatum. Their structures were determined through NMR analyses and the circular dichroism data of the in situ formed [Rh₂(OCOCF₃)₄] complexes. Compounds 1, 4, 5, 7, and 8 showed modest cytotoxicity against four human cancer cell lines A549, CNE1-MP1, A375, and MCF-7.

The decreasing cost of genomic technologies has enabled the molecular characterization of large-scale clinical disease samples and of molecular changes upon drug treatment in various disease models. Exploring methods to relate diseases to potentially efficacious drugs through various molecular features is critically important in the discovery of new therapeutics. Here we show that the potency of a drug to reverse cancer-associated gene expression changes positively correlates with that drug's efficacy in preclinical models of breast, liver and colon cancers. Using a systems-based approach, we predict four compounds showing high potency to reverse gene expression in liver cancer and validate that all four compounds are effective in five liver cancer cell lines. The in vivo efficacy of pyrvinium pamoate is further confirmed in a subcutaneous xenograft model. In conclusion, this systems-based approach may be complementary to the traditional target-based approach in connecting diseases to potentially efficacious drugs.

Functional dyspepsia (FD) is a non-organic gastrointestinal disorder that has a marked negative impact on quality of life. Compared with conventional pharmacological therapies, the traditional Chinese medicine weikangning (WKN) is a safe and effective treatment for FD. The present study aimed to determine the molecular mechanisms underlying the efficacy of WKN. The effect of different concentrations of WKN on the proliferation of the human gastric mucosal epithelial cell line GES-1 was assessed. The optimal WKN concentration to promote cell proliferation was determined, and this concentration was used to examine the effect of WKN compared with a domperidone-treated positive control group on the antioxidant capacity of GES-1 cells. The effect of WKN treatment on the growth and antioxidant activity of GES-1 cells was also assessed following nuclear factor erythroid 2 like 2 (Nrf2) knockdown. The optimal WKN dose for promoting cell growth was determined to be 0.025 mg/ml; at this concentration the expression of the antioxidant proteins glutathione S-transferase P and superoxide dismutase 2 (SOD2) were significantly elevated (P<0.0001). Furthermore, the amount of reduced glutathione and activity of SOD2 were significantly increased (P<0.0001 and P<0.01, respectively), and malondialdehyde content was significantly decreased, compared with the controls (P<0.001). With WKN treatment, the transcription of Nrf2 and its downstream genes were significantly upregulated (P<0.01), and the level and nuclear distribution of Nrf2 protein was also markedly increased. Following Nrf2 silencing, the protective antioxidant effects of WKN treatment were impaired and GES-1 cell proliferation decreased. The results of the present study suggest that the efficacy of WKN in protecting gastric mucosal epithelial cells in FD is antioxidant-dependent and mediated by Nrf2 activation.

We created a novel inducible mouse line Keratocan-rtTA (KeraRT) that allows specific genetic modification in corneal keratocytes and tenocytes during development and in adults.

Preoperative mapping of language areas using fMRI greatly depends on the paradigms used, as different tasks harness distinct capabilities to activate speech processing areas. In this study, we compared the ability of 3 covert speech paradigms: Silent Sentence Completion (SSC), category naming (CAT) and verbal fluency (FAS), in localizing the Wernicke's area and studied the association between genomic markers and functional activation. Fifteen right-handed healthy volunteers and 35 mixed-handed patients were included. We focused on the anatomical areas of posterosuperior, middle temporal and angular gyri corresponding to Wernicke's area. Activity was deemed significant in a region of interest if P < 0.05. Association between fMRI activation and genomic mutation status was obtained. Results demonstrated SSC's superiority at localizing Wernicke's area. SSC demonstrated functional activity in 100% of cancer patients and healthy volunteers; which was significantly higher than those for FAS and CAT. Patients with 1p/19q non-co-deleted had higher extent of activation on SSC (P < 0.02). Those with IDH-1 wild-type were more likely to show no activity on CAT (P < 0.05). SSC is a robust paradigm for localizing Wernicke's area, making it an important clinical tool for function-preserving surgeries. We also found a correlation between tumor genomics and functional activation, which deserves more comprehensive study.

Aneuploidy including trisomy results in developmental disabilities and is the leading cause of miscarriages in humans. Unlike trisomy 21, pathogenic mechanisms of trisomy 18 remain unclear. Here, we successfully generated induced pluripotent stem cells (iPSCs) from human amniotic fluid cells (AFCs) with trisomy 18 pregnancies. We found that trisomy 18 iPSCs (18T-iPSCs) were prone to differentiate spontaneously. Intriguingly, 18T-iPSCs lost their extra 18 chromosomes and converted to diploid cells after 10 generations. fluorescence in situ hybridization analysis showed chromosome loss was a random event that might happen in any trisomic cells. Selection undifferentiated cells for passage accelerated the recovery of euploid cells. Overall, our findings indicate the genomic instability of trisomy 18 iPSCs bearing an extra chromosome 18.

Background. Contrast-induced acute kidney injury (CI-AKI) is one of the main causes of iatrogenic acute kidney injury (AKI); however, therapeutic strategies for AKI remain limited. This study aims to explore the effect of relaxin (RLX) on contrast-induced HK-2 apoptosis and its underlying mechanisms. Methods. Renal tubular epithelial cells (HK-2) were incubated either with or without ioversol, human H2 relaxin, and LY294002 (the inhibitor of the PI3K/Akt signal pathway). Cell viability was evaluated with a CCK-8 assay. Apoptotic morphologic alterations were observed using the Hoechst 33342 staining method. Apoptosis was detected with Annexin V staining. Western blot analysis was employed to measure the expression of pAkt (S473), Akt, cleaved caspase-3, Bcl-2, Bax, and actin proteins. Results. Ioversol reduced the viability of HK-2 cells. Western blotting results revealed decreased expression of phosphorylated Akt in cells treated with ioversol. The activities of caspase-3 and Bax protein increased, while the expression of Bcl-2 protein decreased. As a result, the Bax/Bcl-2 ratio increased after treatment with ioversol. These effects were reversed when HK-2 cells were cotreated with RLX. However, with preadministration of PI3K/Akt pathway inhibitor LY294002, the effect of RLX was blocked. Conclusion. Our study demonstrates that relaxin attenuates ioversol induced cell apoptosis via activation of the PI3K/Akt signaling pathway, suggesting that RLX might play a protective role in the treatment of CI-AKI.

Cardiac fibroblasts (CFs) phenotypic conversion to myofibroblasts (MFs) represents a crucial event in cardiac fibrosis that leads to impaired cardiac function. However, regulation of this phenotypic transformation remains unclear. Here, we showed that sirtuin-7 (Sirt7) plays an important role in the regulation of MFs differentiation. Sirt7 expression and phosphorylation were upregulated in CFs upon angiotensin-II (Ang-II) stimulation. Sirt7 depletion by siRNA in CFs resulted in decreased cell proliferation and extracellular matrix (ECM) deposition. Further, examination of Sirt7-depleted CFs demonstrated significantly lower expression of α-smooth muscle actin (α-SMA), the classical marker of MFs differentiation, and decreased formation of focal adhesions. Moreover, overexpression of Sirt7 increased α-SMA expression in Ang-II treated CFs and exacerbated Ang-II-induced MFs differentiation. Moreover, Sirt7 depletion could largely reverse Ang-II induced increase of nuclear translocalization and activity of smad2 and extracellular regulated kinases (ERK) in CFs. Importantly, the increased differentiation of CFs to MFs was also abolished by smad2 siRNA or U0126. Our findings reveal a novel role of Sirt7 and its phosphorylation in the phenotypic conversion of CFs to MFs and might lead to the development of new therapeutic and prognostic tools for cardiac fibrosis.

Nitrogen (N) is essential for the growth of algae, and its concentration varies greatly in the ocean, which has been regarded as a limitation for phytoplankton growth. Despite its great importance, most of the existing studies on the mechanisms underlying the effects of N on diatoms have focused on physiology, biochemistry and a few target genes and have rarely involved whole genomic analyses. Therefore, in this study, we integrated physiological data with RNA and protein profiling data to reveal the response strategy of Thalassiosira pseudonana under N-depleted conditions. Physiological measurements indicated that the cell growth capacity and chlorophyll content of the cells decreased, as did the expression of photosynthesis- and chlorophyll biosynthesis-related genes or proteins. The RNA-Seq profile results showed that T. pseudonana responded to N deprivation through increases in glycolysis, the TCA cycle and N metabolism as well as down-regulation in the Calvin cycle, gluconeogenesis, pentose phosphate, oxidative phosphorylation and lipid synthesis. These results provide a basic understanding for further research addressing how N affects phytoplankton in terms of genomics.

Clinical applications of siRNA are being hindered by poor intracellular uptake and enzymatic degradation. To address these problems, we devised an oral delivery system for telomerase reverse transcriptase siRNA using N-((2-hydroxy-3-trimethylammonium) propyl) chitosan chloride (HTCC) nanoparticles (HNP). Both the porous structure and the positive charge of HNP facilitated siRNA encapsulation. The outer coating of HTCC not only protected siRNA from enzymatic degradation, but also improved siRNA permeability in intestine tract. In vivo and in vitro experiments proved that HNP could effectively deliver siRNA to lesion site and further into tumor cells. On the basis of confirming the antitumor activity of HNP:siRNA, we continued to encapsulate a hydrophobic chemotherapeutic drug-paclitaxel (PTX) into HNP to form a "two-in-one" nano-complex (HNP:siRNA/PTX). We demonstrated that HNP:siRNA/PTX could simultaneously ferry siRNA and PTX into tumor cells and increase drug concentration, which, in particular, was much more effective in tumor suppression than that of traditional cocktail therapy. These results suggested that the HNP, as a powerful delivery system for both siRNA and chemotherapeutic drug, would have a far-reaching application in human cancer therapy.

Influence of fish oil supplementation on postoperative atrial fibrillation (POAF) was inconsistent according to published clinical trials. The aim of the meta-analysis was to evaluate the effects of perioperative fish oil supplementation on the incidence of POAF after cardiac surgery.

Maintaining a constant number and ratio of immune cells is one critical aspect of the tight regulation of immune homeostasis. Breakdown of this balance will lead to autoimmune diseases such as multiple sclerosis (MS). The antiepileptic drug valproic acid (VPA) was reported to regulate the growth, survival, and differentiation of many cells. However, its function in T cell homeostasis and MS treatment remains unknown. In this study, VPA was found to reduce spinal cord inflammation, demyelination, and disease scores in experimental autoimmune encephalomyelitis, a mouse model of MS. Further study indicated that VPA induces apoptosis in activated T cells and maintains the immune homeostasis. This effect was found to be mainly mediated by the caspase-8/caspase-3 pathway. Interestingly, this phenomenon was also confirmed in T cells from normal human subjects and MS patients. Considering the long history of clinical use and our new findings, we believe VPA might be a safe and effective therapy for autoimmune diseases, such as multiple sclerosis.

In Escherichia coli, 12 distinct RNA structures within the transcripts encoding ribosomal proteins interact with specific ribosomal proteins to allow autogenous regulation of expression from large multi-gene operons, thus coordinating ribosomal protein biosynthesis across multiple operons. However, these RNA structures are typically not represented in the RNA Families Database or annotated in genomic sequences databases, and their phylogenetic distribution is largely unknown. To investigate the extent to which these RNA structures are conserved across eubacterial phyla, we created multiple sequence alignments representing 10 of these messenger RNA (mRNA) structures in E. coli. We find that while three RNA structures are widely distributed across many phyla of bacteria, seven of the RNAs are narrowly distributed to a few orders of Gammaproteobacteria. To experimentally validate our computational predictions, we biochemically confirmed dual L1-binding sites identified in many Firmicute species. This work reveals that RNA-based regulation of ribosomal protein biosynthesis is used in nearly all eubacterial phyla, but the specific RNA structures that regulate ribosomal protein biosynthesis in E. coli are narrowly distributed. These results highlight the limits of our knowledge regarding ribosomal protein biosynthesis regulation outside of E. coli, and the potential for alternative RNA structures responsible for regulating ribosomal proteins in other eubacteria.