Expression, Purification, and Mass Spectrometric Analysis of (15)N, (13)C-Labeled RGD-Hirudin, Expressed in Pichia pastoris, for NMR Studies

A novel recombinant hirudin, RGD-hirudin, inhibits the activity of thrombin and the aggregation of platelets. Here, we successfully expressed (15)N, (13)C-labeled RGD-hirudin in Pichia pastoris in a fermenter. The protein was subsequently purified to yield sufficient quantities for structural and functional studies. The purified protein was characterized by HPLC and MALDI-TOF mass spectroscopy. Analysis revealed that the protein was pure and uniformly labeled with (15)N and (13)C. A bioassay showed that the anti-thrombin activity and the anti-platelet aggregation ability of the labeled protein were the same as those of unlabeled RGD-hirudin. Multidimensional heteronuclear NMR spectroscopy has been used to determine almost complete backbone (15)N, (13)C and (1)H resonance assignments of the r-RGD-Hirudin. The (15)N-(1)H HSQC spectrum of uniformly (15)N, (13)C-labeled RGD-hirudin allowed successful assignment of the signals. Examples of the quality of the data are provided for the (15)N-(l)H correlation spectrum, and by selected planes of the CBCA(CO)NH, CBCANH, and HNCO experiments. These results provide a basis for further studies on the structure-function relationship of RGD-hirudin with thrombin and platelets.     

Functional and Anatomical Connectivity Abnormalities in Cognitive Division of Anterior Cingulate Cortex in Schizophrenia

Introduction

Current pathophysiological theories of schizophrenia highlight the role of altered brain functional and anatomical connectivity. The cognitive division of anterior cingulate cortex (ACC-cd) is a commonly reported abnormal brain region in schizophrenia for its importance in cognitive control process. The aim of this study was to investigate the functional and anatomical connectivity of ACC-cd and its cognitive and clinical manifestation significance in schizophrenia by using the resting-state functional magnetic resonance imaging (fMRI) and the diffusion tensor imaging (DTI).

Methods

Thirty-three medicated schizophrenics and 30 well-matched health controls were recruited. Region-of-interest (ROI)-based resting-state functional connectivity analysis and Tract-Based Spatial Statistics (TBSS) were performed on 30 patients and 30 controls, and 24 patients and 29 controls, respectively. The Pearson correlation was performed between the imaging measures and the Stroop performance and scores of the Positive and Negative Syndrome Scale (PANSS), respectively.

Results

Patients with schizophrenia showed significantly abnormal in the functional connectivity and its hemispheric asymmetry of the ACC-cd with multiple brain areas, e.g., decreased positive connectivity with the bilateral putamen and caudate, increased negative connectivity with the left posterior cingulated cortex (PCC), increased asymmetry of connectivity strength with the contralateral inferior frontal gyrus (IFG). The FA of the right anterior cingulum was significantly decreased in patients group (p = 0.014). The abnormal functional and structural connectivity of ACC-cd were correlated with Stroop performance and the severity of the symptoms in patients.

Conclusions

Our results suggested that the abnormal connectivity of the ACC-cd might play a role in the cognitive impairment and clinical symptoms in schizophrenia.     

Identification and expansion of cancer stem cells in tumor tissues and peripheral blood derived from gastric adenocarcinoma patients

Gastric cancer is the fourth most common cancer worldwide, with a high rate of death and low 5-year survival rate. To date, there is a lack of efficient therapeutic protocols for gastric cancer. Recent studies suggest that cancer stem cells (CSCs) are responsible for tumor initiation, invasion, metastasis, and resistance to anticancer therapies. Thus, therapies that target gastric CSCs are attractive. However, CSCs in human gastric adenocarcinoma (GAC) have not been described. Here, we identify CSCs in tumor tissues and peripheral blood from GAC patients. CSCs of human GAC (GCSCs) that are isolated from tumor tissues and peripheral blood of patients carried CD44 and CD54 surface markers, generated tumors that highly resemble the original human tumors when injected into immunodeficient mice, differentiated into gastric epithelial cells in vitro, and self-renewed in vivo and in vitro. Our findings suggest that effective therapeutic protocols must target GCSCs. The capture of GCSCs from the circulation of GAC patients also shows great potential for identification of a critical cell population potentially responsible for tumor metastasis, and provides an effective protocol for early diagnosis and longitudinal monitoring of gastric cancer.     

Human Stn1 protects telomere integrity by promoting efficient lagging-strand synthesis at telomeres and mediating C-strand fill-in

Telomere maintenance is critical for genome stability. The newly-identified Ctc1/Stn1/Ten1 complex is important for telomere maintenance, though its precise role is unclear. We report here that depletion of hStn1 induces catastrophic telomere shortening, DNA damage response, and early senescence in human somatic cells. These phenotypes are likely due to the essential role of hStn1 in promoting efficient replication of lagging-strand telomeric DNA. Downregulation of hStn1 accumulates single-stranded G-rich DNA specifically at lagging-strand telomeres, increases telomere fragility, hinders telomere DNA synthesis, as well as delays and compromises telomeric C-strand synthesis. We further show that hStn1 deficiency leads to persistent and elevated association of DNA polymerase α (polα) to telomeres, suggesting that hStn1 may modulate the DNA synthesis activity of polα rather than controlling the loading of polα to telomeres. Additionally, our data suggest that hStn1 is unlikely to be part of the telomere capping complex. We propose that the hStn1 assists DNA polymerases to efficiently duplicate lagging-strand telomeres in order to achieve complete synthesis of telomeric DNA, therefore preventing rapid telomere loss.     

Microsolvation effect and hydrogen-bonding pattern of taurine-water TA-(H2O)n (n = 1-3) complexes

The microsolvation of taurine (TA) with one, two or three water molecules was investigated by a density functional theory (DFT) approach. Quantum theory of atoms in molecules (QTAIM) analyses were employed to elucidate the hydrogen bond (H-bond) interaction characteristics in TA-(H₂O)_n (n = 1–3) complexes. The results showed that the intramolecular H-bond formed between the hydroxyl and the N atom of TA are retained in most TA-(H₂O)_n (n = 1–3) complexes, and are strengthened via cooperative effects among multiple H-bonds from n = 1–3. A trend of proton transformation exists from the hydroxyl to the N atom, which finally results in the cleavage of the origin intramolecular H-bond and the formation of a new intramolecular H-bond between the amino and the O atom of TA. Therefore, the most stable TA-(H₂O)₃ complex becomes a zwitterionic complex rather than a neutral type. A many-body interaction analysis showed that the major contributors to the binding energies for complexes are the two-body energies, while three-body energies and relaxation energies make significant contributions to the binding energies for some complexes, whereas the four-body energies are too small to be significant.     

Shugoshins function as a guardian for chromosomal stability in nuclear division

Accurate chromosome segregation during mitosis and meiosis is regulated and secured by several distinctly different yet intricately connected regulatory mechanisms. As chromosomal instability is a hallmark of a majority of tumors as well as a cause of infertility for germ cells, extensive research in the past has focused on the identification and characterization of molecular components that are crucial for faithful chromosome segregation during cell division. Shugoshins, including Sgo1 and Sgo2, are evolutionarily conserved proteins that function to protect sister chromatid cohesion, thus ensuring chromosomal stability during mitosis and meiosis in eukaryotes. Recent studies reveal that Shugoshins in higher animals play an essential role not only in protecting centromeric cohesion of sister chromatids and assisting bi-orientation attachment at the kinetochores, but also in safeguarding centriole cohesion/engagement during early mitosis. Many molecular components have been identified that play essential roles in modulating/mediating Sgo functions. This review primarily summarizes recent advances on the mechanisms of action of Shugoshins in suppressing chromosomal instability during nuclear division in eukaryotic organisms.     

Thymine DNA glycosylase specifically recognizes 5-carboxylcytosine-modified DNA

Human thymine DNA glycosylase (hTDG) efficiently excises 5-carboxylcytosine (5caC), a key oxidation product of 5-methylcytosine in genomic DNA, in a recently discovered cytosine demethylation pathway. We present here the crystal structures of the hTDG catalytic domain in complex with duplex DNA containing either 5caC or a fluorinated analog. These structures, together with biochemical and computational analyses, reveal that 5caC is specifically recognized in the active site of hTDG, supporting the role of TDG in mammalian 5-methylcytosine demethylation.