The prestalk region of the Dictyostelium slug is comprised of an anterior population of pstA cells and a posterior population of pstO cells. They are distinguished by their ability to utilize different parts of the promoter of the ecmA gene. We identify, by mutational analysis and DNA transformation, CA-rich sequence elements within the ecmA promoter that are essential for pstA-specific expression and sufficient to direct pstA-specific expression when multimerised. The CA-rich region was used in affinity chromatography with nuclear extracts and bound proteins were identified by mass spectrometry. The CA-rich elements purify MrfA, a protein with extensive sequence similarity to animal Myelin-gene Regulatory Factor (MRF)-like proteins. The MRF-like proteins and MrfA also display more spatially limited but significant sequence similarity with the DNA binding domain of the yeast Ndt80 sporulation-specific transcription factor. Furthermore, the ecmA CA-rich elements show sequence similarity to the core consensus Ndt80 binding site (the MSE) and point mutation of highly conserved arginine residues in MrfA, that in Ndt80 make critical contacts with the MSE, ablate binding of MrfA to its sites within the ecmA promoter. MrfA null strains are delayed in multicellular development and highly defective in pstA-specific gene expression. These results provide a first insight into the intracellular signaling pathway that directs pstA differentiation and identify a non-metazoan orthologue of a family of molecularly uncharacterised transcription factors.

CudA, a nuclear protein required for Dictyostelium prespore-specific gene expression, binds in vivo to the promoter of the cotC prespore gene. A 14 nucleotide region of the cotC promoter binds CudA in vitro and ECudA, an Entamoeba CudA homologue, also binds to this site. The CudA and ECudA DNA-binding sites contain a dyad and, consistent with a symmetrical binding site, CudA forms a homodimer in the yeast two-hybrid system. Mutation of CudA binding sites within the cotC promoter reduces expression from cotC in prespore cells. The CudA and ECudA proteins share a 120 amino acid core of homology, and clustered point mutations introduced into two highly conserved motifs within the ECudA core region decrease its specific DNA binding in vitro. This region, the presumptive DNA-binding domain, is similar in sequence to domains in two Arabidopsis proteins and one Oryza protein. Significantly, these are the only proteins in the two plant species that contain an SH2 domain. Such a structure, with a DNA-binding domain located upstream of an SH2 domain, suggests that the plant proteins are orthologous to metazoan STATs. Consistent with this notion, the DNA sequence of the CudA half site, GAA, is identical to metazoan STAT half sites, although the relative positions of the two halves of the dyad are reversed. These results define a hitherto unrecognised class of transcription factors and suggest a model for the evolution of STATs and their DNA-binding sites.

AIM: To estimate hepatitis B virus (HBV) infection testing rate in cancer patients before chemotherapy with a focus on HBV reactivation.

METHODS: A retrospective study was conducted from January 1, 2009 to June 30, 2010. Inclusion required that patients be naïve to cancer chemotherapy but have indications for it. Patients who did not receive chemotherapy for any reason were excluded. Important clinical information, such as the levels of HBV DNA and serological markers were collected. HBV reactivation was defined as an increase in serum HBV DNA to > 1 log higher than that of the pre-exacerbation baseline, or serum HBV DNA conversion from negative to positive. HBV DNA levels > 1000 copies/mL were defined as HBV DNA positive. The χ2 or Fisher’s exact test was used for analysis of categorized data. Multiple logistic regression analysis was used to estimate the odd ratio and 95%CI of the HBV screening rate.

RESULTS: Of 6646 patients, 5616 (84.5%) received chemotherapy. Only 17.1% of the cancer patients received pre-chemotherapy HBV testing (43.2% for hematological malignancies and 14.9% for solid tumors). Patients who had received rituximab therapy, had elevated aminotransferase levels, or had hematological malignancies were more likely to receive HBV testing. The prevalence of hepatitis B surface antigen (HBsAg) positivity was 13.4%. HBV reactivation (appearance of HBV DNA or an increase in HBV DNA levels by 1 log10) was observed in 33.1% (53/160) of the patients after chemotherapy. Among patients without prophylactic antiviral therapy, the reactivation rate was 43.9% (43/98) in the solid tumor group. Two reactivation cases occurred in patients who were HBsAg negative, but positive for hepatitis B core antibody. HBV reactivation was more likely to occur in patients with lymphoma, high levels of HBV DNA, or hepatitis B e antigen, and in men.

CONCLUSION: Less than 20% of patients received HBV testing before chemotherapy. HBV reactivation would have occurred in about 50% of infected patients with solid tumors without antiviral prophylaxis.

Dynamin-2 (DNM2) is a large GTPase involved in clathrin-mediated endocytosis and related trafficking pathways. Mutations in human DNM2 cause two distinct neuromuscular disorders: centronuclear myopathy and Charcot-Marie-Tooth disease. Zebrafish have been shown to be an excellent animal model for many neurologic disorders, and this system has the potential to inform our understanding of DNM2-related disease. Currently, little is known about the endogenous zebrafish orthologs to human DNM2. In this study, we characterize two zebrafish dynamin-2 genes, dnm2 and dnm2-like. Both orthologs are structurally similar to human DNM2 at the gene and protein levels. They are expressed throughout early development and in all adult tissues examined. Knockdown of dnm2 and dnm2-like gene products resulted in extensive morphological abnormalities during development, and expression of human DNM2 RNA rescued these phenotypes. Our findings suggest that dnm2 and dnm2-like are orthologs to human DNM2, and that they are required for normal zebrafish development.

Mechanisms for cooperation between the cytosolic Hsp70 system and the ubiquitin proteasome system during protein triage are not clear. Herein, we identify new mechanisms for selection of misfolded cytosolic proteins for degradation via defining functional interactions between specific cytosolic Hsp70/Hsp40 pairs and quality control ubiquitin ligases. These studies revolved around the use of S. cerevisiae to elucidate the degradation pathway of a terminally misfolded reporter protein, short-lived GFP (slGFP). The Type I Hsp40 Ydj1 acts with Hsp70 to suppress slGFP aggregation. In contrast, the Type II Hsp40 Sis1 is required for proteasomal degradation of slGFP. Sis1 and Hsp70 operate sequentially with the quality control E3 ubiquitin ligase Ubr1 to target slGFP for degradation. Compromise of Sis1 or Ubr1 function leads slGFP to accumulate in a Triton X-100-soluble state with slGFP degradation intermediates being concentrated into perinuclear and peripheral puncta. Interestingly, when Sis1 activity is low the slGFP that is concentrated into puncta can be liberated from puncta and subsequently degraded. Conversely, in the absence of Ubr1, slGFP and the puncta that contain slGFP are relatively stable. Ubr1 mediates proteasomal degradation of slGFP that is released from cytosolic protein handling centers. Pathways for proteasomal degradation of misfolded cytosolic proteins involve functional interplay between Type II Hsp40/Hsp70 chaperone pairs, PQC E3 ligases, and storage depots for misfolded proteins.

The mammalian target of rapamycin (mTOR), a serine/threonine protein kinase, acts as a “master switch” for cellular anabolic and catabolic processes, regulating the rate of cell growth and proliferation. Dysregulation of the mTOR signaling pathway occurs frequently in a variety of human tumors, and thus, mTOR has emerged as an important target for the design of anticancer agents. mTOR is found in two distinct multiprotein complexes within cells, mTORC1 and mTORC2. These two complexes consist of unique mTOR-in teracting proteins and are regulated by different mechanisms. Enormous advances have been made in the development of drugs known as mTOR inhibitors. Rapamycin, the first defined inhibitor of mTOR, showed effectiveness as an anticancer agent in various preclinical models. Rapamycin analogues (rapalogs) with better pharmacologic properties have been developed. However, the clinical success of rapalogs has been limited to a few types of cancer. The discovery that mTORC2 directly phosphorylates Akt, an important survival kinase, adds new insight into the role of mTORC2 in cancer. This novel finding prompted efforts to develop the second generation of mTOR inhibitors that are able to target both mTORC1 and mTORC2. Here, we review the recent advances in the mTOR field and focus specifically on the current development of the second generation of mTOR inhibitors as anticancer agents.

ADP-ribosylation is the post translational modification of proteins catalyzed by ADP-ribosyltransferases (ARTs). ADP-ribosylation has been implicated in a wide variety of cellular processes, including cell growth and differentiation, apoptosis and transcriptional regulation. Perhaps the best-characterized role, however, is in DNA repair and genome stability, where ADP-ribosylation promotes resolution of DNA single-strand breaks. Although ADP-ribosylation also occurs at DNA double-strand breaks (DSBs), which ARTs catalyze this reaction and the molecular basis of how this modification regulates their repair remains a matter of debate. Here, we review recent advances in our understanding of how ADP-ribosylation regulates DSB repair. Specifically, we highlight studies using the genetic model organism Dictyostelium in addition to vertebrate cells that identify a third ART that accelerates DSB repair by non-homologous end joining through promoting the interaction of repair factors with DNA lesions. The implications of these data with regards to how ADP-ribosylation regulates DNA repair and genome stability are discussed.

Background

Interferon-γ (IFN-γ) is regarded as a potent antitumor agent, but its clinical application is limited by its short half-life and significant side effects. In this paper, we tried to develop IFN-γ gene therapy by a replication defective adenovirus encoding the human IFN-γ (Ad-IFNγ), and evaluate the antitumoral effects of Ad-IFNγ on nasopharyngeal carcinoma (NPC) cell lines in vitro and in xenografts model.

Methods

The mRNA levels of human IFN-γ in Ad-IFNγ-infected NPC cells were detected by reverse transcription-polymerase chain reaction (RT-PCR), and IFN-γ protein concentrations were measured by enzyme-linked immunosorbent assay (ELISA) in the culture supernatants of NPC cells and tumor tissues and bloods of nude mice treated with Ad-IFNγ. The effects of Ad-IFNγ on NPC cell proliferation was determined using MTT assay, cell cycle distribution was determined by flow cytometry analysis for DNA content, and cells apoptosis were analyzed by Annexin V-FITC/7-AAD binding assay and hoechst 33342/PI double staining. The anti-tumor effects and toxicity of Ad-IFNγ were evaluated in BALB/c nude mice carrying NPC xenografts.

Results

The results demonstrated that Ad-IFNγ efficiently expressed human IFN-γ protein in NPC cell lines in vitro and in vivo. Ad-IFNγ infection resulted in antiproliferative effects on NPC cells by inducing G1 phase arrest and cell apoptosis. Intratumoral administration of Ad-IFNγ significantly inhibited the growth of CNE-2 and C666-1 cell xenografts in nude mice, while no significant toxicity was observed.

Conclusions

These findings indicate IFN-γ gene therapy mediated by replication defective adenoviral vector is likely a promising approach in the treatment of nasopharyngeal carcinoma.

Ulcerative colitis (UC) is a major form of chronic inflammation that can frequently progress to colon cancer. Several studies have demonstrated massive infiltration of neutrophils and macrophages into the lamina propria and submucosa in the progression of UC-associated colon carcinogenesis. Macrophages contribute to the development of colitis-associated colon cancer (CAC). However, the role of neutrophils is not well understood. To better understand the involvement of tumor-associated neutrophils (TANs) in the regulation of CAC, we used a mouse CAC model produced by administering azoxymethane (AOM), followed by repeated dextran sulfate sodium (DSS) ingestion. This causes severe colonic inflammation and subsequent development of multiple tumors in mice colon. We observed that colorectal mucosal inflammation became increasingly severe with AOM and DSS treatment. Macrophages infiltrated the lamina propria and submucosa, together with a marked increase in neutrophil infiltration. The chemokine CXCL2 increased in the lamina propria and submucosal regions of the colons of the treated mice, together with the infiltration of neutrophils expressing CXCR2, a specific receptor for CXCL2. This process was followed by neoplastic transformation. After AOM and DSS treatment, the mice showed enhanced production of metalloproteinase (MMP)-9 and neutrophil elastase (NE), accompanied by excessive vessel generation and cell proliferation. Moreover, CXCL2 promoted neutrophil recruitment and induced neutrophils to express MMP-9 and NE in vitro. Furthermore, administration of neutrophil-neutralizing antibodies after the last DSS cycle markedly reduced the number and size of tumors and decreased the expression of CXCR2, CXCL2, MMP-9, and NE. These observations indicate a crucial role for TANs in the initiation and progression of CAC and suggest that the CXCL2–CXCR2 axis might be useful in reducing the risk of UC-associated colon cancer.

Background/Aims

Chronic hepatitis B infection is a common cause of secondary membranous nephropathy (MN) in endemic areas. Lamivudine treatment improves renal outcome in patients with hepatitis B virus-associated MN (HBV-MN), but prolonged use leads to the emergence of lamivudine-resistant variants. We describe our experience treating lamivudine-resistant and other strains of HBV-MN with new antiviral drugs.

Methods

Of the 89 patients biopsied and diagnosed with MN from 1996 to 2011, 10 positive for hepatitis B surface antigen were recruited for this study. We investigated the clinical courses, therapeutic responses, and prognoses of patients with HBV-MN.

Results

The incidence of HBV-MN among the original 89 patients was 11.2%. Of these patients, four were treated with supportive care and six with antiviral drugs. One of the four patients treated with supportive care had a spontaneous remission. Four of the six patients treated with antiviral drugs were given lamivudine, and the other two were given entecavir. Two of the four patients treated with lamivudine achieved complete remission with seroconversion (i.e., development of anti-hepatitis B e antigen antibodies), whereas the other two had lamivudine-resistant strains, which were detected at 22 and 23 months after lamivudine treatment, respectively. We added adefovir to the treatment regimen for one of these patients, and for the other patient we substituted clevudine for lamivudine. Both of these patients experienced complete remission, as did the two patients initially treated with entecavir, neither of whom showed resistance to the drug.

Conclusions

New nucleoside analogues, such as entecavir, adefovir, and clevudine, can be effective for treatment of HBV-MN, including lamivudine-resistant strains.

SecReT4 (http://db-mml.sjtu.edu.cn/SecReT4/) is an integrated database providing comprehensive information of type IV secretion systems (T4SSs) in bacteria. T4SSs are versatile assemblages that promote genetic exchange and/or effector translocation with consequent impacts on pathogenesis and genome plasticity. T4SSs have been implicated in conjugation, DNA uptake and release and effector translocation. The effectors injected into eukaryotic target cells can lead to alteration of host cellular processes during infection. SecReT4 offers a unique, highly organized, readily exploreable archive of known and putative T4SSs and cognate effectors in bacteria. It currently contains details of 10 752 core components mapping to 808 T4SSs and 1884 T4SS effectors found in representatives of 289 bacterial species, as well as a collection of more than 900 directly related references. A broad range of similarity search, sequence alignment, phylogenetic, primer design and other functional analysis tools are readily accessible via SecReT4. We propose that SecReT4 will facilitate efficient investigation of large numbers of these systems, recognition of diverse patterns of sequence-, gene- and/or functional conservation and an improved understanding of the biological roles and significance of these versatile molecular machines. SecReT4 will be regularly updated to ensure its ongoing maximum utility to the research community.

Diabetic neuropathy is a common complication of diabetes. While multiple pathways are implicated in the pathophysiology of diabetic neuropathy, there are no specific treatments and no means to predict diabetic neuropathy onset or progression. Here, we identify gene expression signatures related to diabetic neuropathy and develop computational classification models of diabetic neuropathy progression. Microarray experiments were performed on 50 samples of human sural nerves collected during a 52-week clinical trial. A series of bioinformatics analyses identified differentially expressed genes and their networks and biological pathways potentially responsible for the progression of diabetic neuropathy. We identified 532 differentially expressed genes between patient samples with progressing or non-progressing diabetic neuropathy, and found these were functionally enriched in pathways involving inflammatory responses and lipid metabolism. A literature-derived co-citation network of the differentially expressed genes revealed gene subnetworks centred on apolipoprotein E, jun, leptin, serpin peptidase inhibitor E type 1 and peroxisome proliferator-activated receptor gamma. The differentially expressed genes were used to classify a test set of patients with regard to diabetic neuropathy progression. Ridge regression models containing 14 differentially expressed genes correctly classified the progression status of 92% of patients (P < 0.001). To our knowledge, this is the first study to identify transcriptional changes associated with diabetic neuropathy progression in human sural nerve biopsies and describe their potential utility in classifying diabetic neuropathy. Our results identifying the unique gene signature of patients with progressive diabetic neuropathy will facilitate the development of new mechanism-based diagnostics and therapies.

A 27-year-old man presented at the emergency room with hemoptysis. His blood pressure was 180/100 mm Hg, and he had no history of hypertension. Chest radiographs showed bilateral infiltration, suggestive of alveolar hemorrhage. His laboratory data were consistent with acute kidney injury. His serum creatinine level increased abruptly; therefore, renal biopsy was performed. Steroid pulse therapy was administered because of a strong suspicion of immune-mediated pulmonary renal syndrome. Renal biopsy showed proliferative endarteritis, fibrinoid necrosis, and intraluminal thrombi in the vessels without crescent formation or necrotizing lesions. Steroid pulse therapy rapidly tapered and stopped. His serum creatinine level gradually decreased with strict blood pressure control. Ten months after discharge, his blood pressure was approximately 120/80 mm Hg with a serum creatinine level of 1.98 mg/dL. Pulmonary renal syndrome is generally caused by an immune-mediated mechanism. However, malignant hypertension accompanying renal insufficiency and heart dysfunction causing end-organ damage can create a pulmonary hemorrhage, similar to pulmonary renal syndrome caused by an immune-mediated mechanism. The present case shows that hypertension, a common disease, can possibly cause pulmonary renal syndrome, a rare condition.

Klebsiella pneumoniae is an important pathogen commonly associated with opportunistic infections. Here we report the genome sequence of a strain, HS11286, isolated from human sputum in 2011 in Shanghai, China. It contains one chromosome (5.3 Mb), three multidrug resistance plasmids (∼110 kb), including a carbapenemase producer, and three small plasmids (∼3 kb).

Since the 2006 discovery of the Acinetobacter baumannii strain AYE AbaR1 resistance island, similar elements have been reported in numerous members of this species. As AbaR1 is distantly related to Tn7, we have renamed it TnAbaR1. TnAbaR transposons are known to carry multiple antibiotic resistance- and efflux-associated genes, although none have been experimentally studied en bloc. We deleted the TnAbaR transposon in A. baumannii A424, which we have designated TnAbaR23, and characterized independent deletion mutants DCO163 and DCO174. The NotI pulsed-field gel electrophoresis (PFGE) profile of strain DCO174 was consistent with targeted deletion of TnAbaR23 alone, but strain DCO163 apparently harbored a second large genomic deletion. Nevertheless, “subtractive amplification” targeting 52 TnAbaR and/or resistance-associated loci yielded identical results for both mutants and highlighted genes lost relative to strain A424. PCR mapping and genome sequencing revealed the entire 48.3-kb sequence of TnAbaR23. Consistent with TnAbaR23 carrying two copies of sul1, both mutants exhibited markedly increased susceptibility to sulfamethoxazole. In contrast, loss of tetAR(A) resulted in only a minor and variable increase in tetracycline susceptibility. Despite not exhibiting a growth handicap, strain DCO163 was more susceptible than strain DCO174 to 9 of 10 antibiotics associated with mutant-to-mutant variation in susceptibility, suggesting impairment of an undefined resistance-associated function. Remarkably, despite all three strains sharing identical gyrA and parC sequences, the ciprofloxacin MIC of DCO174 was >8-fold that of DCO163 and A424, suggesting a possible paradoxical role for TnAbaR23 in promoting sensitivity to ciprofloxacin. This study highlights the importance of experimental scrutiny and challenges the assumption that resistance phenotypes can reliably be predicted from genotypes alone.

Thiopeptides are a growing class of sulfur-rich, highly modified heterocyclic peptides that are mainly active against Gram-positive bacteria including various drug-resistant pathogens. Recent studies also reveal that many thiopeptides inhibit the proliferation of human cancer cells, further expanding their application potentials for clinical use. Thiopeptide biosynthesis shares a common paradigm, featuring a ribosomally synthesized precursor peptide and conserved posttranslational modifications, to afford a characteristic core system, but differs in tailoring to furnish individual members. Identification of new thiopeptide gene clusters, by taking advantage of increasing information of DNA sequences from bacteria, may facilitate new thiopeptide discovery and enrichment of the unique biosynthetic elements to produce novel drug leads by applying the principle of combinatorial biosynthesis. In this study, we have developed a web-based tool ThioFinder to rapidly identify thiopeptide biosynthetic gene cluster from DNA sequence using a profile Hidden Markov Model approach. Fifty-four new putative thiopeptide biosynthetic gene clusters were found in the sequenced bacterial genomes of previously unknown producing microorganisms. ThioFinder is fully supported by an open-access database ThioBase, which contains the sufficient information of the 99 known thiopeptides regarding the chemical structure, biological activity, producing organism, and biosynthetic gene (cluster) along with the associated genome if available. The ThioFinder website offers researchers a unique resource and great flexibility for sequence analysis of thiopeptide biosynthetic gene clusters. ThioFinder is freely available at http://db-mml.sjtu.edu.cn/ThioFinder/.

Background

The expression pattern and function of miRNAs in the rat model of temporal lobe epilepsy have not been well defined. Profiling miRNA expression in the rat model of temporal lobe epilepsy and investigating the function of specific miRNAs in epilepsy offers the prospect of a deeper understanding of the mechanisms of epilepsy.

Methods

The lithium-pilocarpine-induced status epilepticus model and the temporal lobe epilepsy model were established in Sprague–Dawley rats. Samples were analysed to detect deregulated miRNAs in the hippocampal temporal lobe, and several of these deregulated miRNAs were confirmed by qPCR. The expression of the pro-apoptotic miR-34a was detected at 1 day, 7 days and 2 weeks post-status epilepticus and at 2 months after temporal lobe epilepsy. The antagomir of miR-34a was then utilised. The expression of miR-34a after targeting and the expression change of activated caspase-3 protein were examined. The effects of altering the expression of miR-34a and activated caspase-3 protein on neuronal survival and neuronal death or apoptosis post-status epilepticus were assessed.

Results

The miRNA microarray detected 9 up-regulated miRNAs (miR-146a, -211, -203, -210, -152, -31, -23a, -34a, -27a) and 15 down-regulated miRNAs (miR-138*, -301a, -136, -153, -19a, -135b, -325-5p, -380, -190, -542-3p, -33, -144, -542-5p, -543, -296*). Some of the deregulated miRNAs (miR-146a, miR-210, miR-27a, miR-135b and miR-33) were confirmed using qPCR. Furthermore, an increase in expression of the pro-apoptotic miR-34a was demonstrated in the post-status epilepticus rat hippocampus. miR-34a was significantly up-regulated at 1 day, 7 days and 2 weeks post-status epilepticus and at 2 months after temporal lobe epilepsy. Experiments with the miR-34a antagomir revealed that targeting miR-34a led to an inhibition of activated caspase-3 protein expression, which may contribute to increased neuronal survival and reduced neuronal death or apoptosis.

Conclusions

Our study showed the expression profile of miRNAs in the hippocampus in a rat model of temporal lobe epilepsy and an increase in the expression of the pro-apoptotic miR-34a in post-status epilepticus rats. The results show that miR-34a is up-regulated during seizure-induced neuronal death or apoptosis, and targeting miR-34a is neuroprotective and is associated with an inhibition of an increase in activated caspase-3 protein.

Objective: To define the roles of gray-scale, color-Doppler ultrasound, and sonoelastography for the assessment of thyroid nodule to determine whether nodule size affects the differential diagnosis of benign and malignant. Methods: A total of 243 consecutive subjects (214 women, 29 men) with 329 thyroid nodules were examined by gray-scale, color-Doppler ultrasound, and sonoelastography in this prospective study. All patients underwent surgery and the final diagnosis was obtained from histopathological examination. Results: Three hundred and twenty-nine nodules (208 benign, 121 malignant) were divided into small (SNs, 5–10 mm, n=137) and large (LNs, >10 mm, n=192) nodules. Microcalcifications were more frequent in malignant LNs than in malignant SNs, but showed no significant difference between benign LNs and SNs. Poorly-circumscribed margins were not significantly different between malignant SNs and LNs, but were less frequent in benign LNs than in benign SNs. Among all nodules, marked intranodular vascularity was more frequent in LNs than in SNs. By comparison, shape ratio of anteroposterior to transverse dimensions (A/T) ≥1 was less frequent in LNs than in SNs. Otherwise, among all nodules, marked hypoechogenicity and elasticity score of 4–6 showed no significant difference between LNs and SNs. Conclusions: The predictive values of microcalcifications, nodular margins, A/T ratio, and marked intranodular vascularity depend on nodule size, but the predictive values of echogenicity and elastography do not.

The mol­ecule of the title compound, C12H14Cl2O2, lies about an inversion center. The six-membered ring is almost planar, with the largest deviation from the least-squares plane being 0.014 (4) Å. The mol­ecular conformation is stabilized by a weak intra­molecular C—H⋯O hydrogen bond. In the crystal, mol­ecules are packed into stacks along the c-axis direction, with an inter­centroid separation of 4.811 (2) Å. Neighboring mol­ecules within the stack are related by the c-glide plane.

The age-related decline of signal joint T-cell receptor rearrangement excision circles (sjTRECs) in human peripheral blood has been demonstrated in our previous study and other reports. Until now, only a few studies on sjTREC detection in bloodstain samples were reported, which were based on a small sample of subjects of a limited age range, although bloodstains are much more frequently encountered in forensic practice. In this present study, we adopted the sensitive Taqman real-time quantitative polymerase chain reaction (qPCR) method to perform sjTREC quantification in bloodstains from individuals ranging from 0–86 years old (n = 264). The results revealed that sjTREC contents in human bloodstains were declined in an age-dependent manner (r = −0.8712). The formula of age estimation was Age  = −7.1815Y−42.458±9.42 (Y dCtTBP-sjTREC; 9.42 standard error). Furthermore, we tested for the influence of short- or long- storage time by analyzing fresh and stored bloodstains from the same individuals. Remarkably, no statistically significant difference in sjTREC contents was found between the fresh and old DNA samples over a 4-week of storage time. However, significant loss (0.16–1.93 dCt) in sjTREC contents was detected after 1.5 years of storage in 31 samples. Moreover, preliminary sjTREC quantification from up to 20-year-old bloodstains showed that though the sjTREC contents were detectable in all samples and highly correlated with donor age, a time-dependent decrease in the correlation coefficient r was found, suggesting the predicting accuracy of this described assay would be deteriorated in aged samples. Our findings show that sjTREC quantification might be also suitable for age prediction in bloodstains, and future researches into the time-dependent or other potential impacts on sjTREC quantification might allow further improvement of the predicting accuracy.

The balance of putative pro- and anti-inflammatory lipoxygenase (LOX)-derived S-hydroxyeicosatetraenoic acids (S-HETEs) in colon mucosa is a potential target for modulating colon cancer risk and progression. The biological effects of S-HETEs and R-HETEs (produced by distinct pathways) may differ, but levels of these compounds in colon are unknown. The objective of this study was to develop chiral methods to characterize HETE enantiomers in colonic mucosa and evaluate the effects of fish oil on HETE formation. C57BL/6 mice (COX-1 null, COX-2 null, wild-type) were fed a diet supplemented with either olive oil or menhaden oil for 11 weeks, and R/S-HETEs in colonic mucosa were quantified by chiral LC-MS/MS. The R-enantiomer comprised 60-72% of 5-HETE, 18-58% of 15-HETE and 1-16% of 12-HETE in colonic mucosa, suggesting that non-LOX sources contribute to HETE profiles. Fish oil reduced levels of both R- and S-HETEs, and increased the preponderance of the R-enantiomers (particularly 12- and 15-HETEs). There was apparent shunting of arachidonic acid to12/15-LOX in the COX-1 null animals. This is the first report of the enantiomeric composition of HETEs in the colon in vivo.

CHIP, the carboxyl-terminus of Hsp70 interacting protein, is both an E3 ubiquitin ligase and an Hsp70 co-chaperone and is implicated in the degradation of cytosolic quality control and numerous disease substrates. CHIP has been shown to monitor the folding status of the CFTR protein, and we have successfully reconstituted this activity using a recombinant CFTR fragment consisting of the cytosolic NBD1 and R domains. We have found that efficient ubiquitination of substrates requires chaperone activity to either deliver the substrate to CHIP or to maintain the substrate in a ubiquitination-competent conformation. This chaperone activity can be provided by the Hsp70/Hsp40 molecular chaperone system as seen in the NBD1–R ubiquitination assay. Alternatively, heat treatment of CHIP can activate its own innate substrate-binding activity and allow for efficient ubiquitination of model substrates, such as denatured luciferase. Here, we describe methods for purifying the recombinant proteins necessary for in vitro reconstitution of CHIP ubiquitin ligase activity, as well as two methods used to monitor CHIP ligase activity. One method allows for the measurement of the Hsp70- and Hsp40-dependent CHIP activity while the other measures the Hsp40- and Hsp70-independent activity of heat-activated CHIP.

OBJECTIVE

A better understanding of the molecular mechanisms underlying the development and progression of diabetic neuropathy (DN) is essential for the design of mechanism-based therapies. We examined changes in global gene expression to define pathways regulated by diabetes in peripheral nerve.

RESEARCH DESIGN AND METHODS

Microarray data for 24-week-old BKS db/db and db/+ mouse sciatic nerve were analyzed to define significantly differentially expressed genes (DEGs); DEGs were further analyzed to identify regulated biological processes and pathways. Expression profile clustering was performed to identify coexpressed DEGs. A set of coexpressed lipid metabolism genes was used for promoter sequence analysis.

RESULTS

Gene expression changes are consistent with structural changes of axonal degeneration. Pathways regulated in the db/db nerve include lipid metabolism, carbohydrate metabolism, energy metabolism, peroxisome proliferator–activated receptor signaling, apoptosis, and axon guidance. Promoter sequences of lipid metabolism–related genes exhibit evidence of coregulation of lipid metabolism and nervous system development genes.

CONCLUSIONS

Our data support existing hypotheses regarding hyperglycemia-mediated nerve damage in DN. Moreover, our analyses revealed a possible coregulation mechanism connecting hyperlipidemia and axonal degeneration.

Experimental and in silico PCR analysis targeting ISAba11 and TnAbaR islands in 196 epidemiologically unrelated Acinetobacter strains representative of ≥19 species were performed. The first two Acinetobacter baumannii ISAba11 elements identified had been found to map to the same site on TnAbaR transposons. However, no further evidence of physical linkage between the two elements was demonstrated. Indeed, examination of 25 definite or putative insertion sites suggested limited sequence specificity. Importantly, an aacC1-tagged version of ISAba11 was shown to actively transpose in A. baumannii. Similarity searches identified nine iso-ISAba11 elements in Acinetobacter and one in Enhydrobacter and single representatives of four distant homologs in bacteria belonging to the phyla “Cyanobacteria” and Proteobacteria. Phylogenetic, sequence, and structural analyses of ISAba11 and/or its associated transposase (TnpISAba11) suggested that these elements be assigned to a new family. All five homologs encode transposases with a shared extended signature comprising 16 invariant residues within the N2, N3, and C1 regions, four of which constituted the cardinal ISAba11 family HHEK motif that is substituted for the YREK DNA binding motif conserved in the IS4 family. Additionally, ISAba11 family members were associated with either no flanking direct repeat (DR) or an ISAba11-typical 5-bp DR and possessed variable-length terminal inverted repeats that exhibited extensive intrafamily sequence identity. Given the limited pairwise identity among TnpISAba11 homologs and the observed restricted distribution of ISAba11, we propose that substantial gaps persist in the evolutionary record of ISAba11 and that this element represents a recent though potentially highly significant entrant into the A. baumannii gene pool.

In the title compound, C12H9N2 +·Br−, the protonated tricyclic ring system is slightly twisted, with a dihedral angle of 3.9 (1)° between the two outer benzene rings. In the crystal, N—H⋯Br and C—H⋯Br hydrogen bonds link two cations and two bromide anions into centrosymmetric assemblies, which are further packed into stacks along [010] via π–π inter­actions between the aromatic rings [centroid–centroid distance = 3.725 (4) Å].