Recognition and unfolding of human telomeric G-quadruplex by short peptide binding identified from the HRDC domain of BLM helicase

Research in recent decades has revealed that the guanine (G)-quadruplex secondary structure in DNA modulates a variety of cellular events that are mostly related to serious diseases. Systems capable of regulating DNA G-quadruplex structures would therefore be useful for the modulation of various cellular events to produce biological effects. A high specificity for recognition of telomeric G-quadruplex has been observed for BLM helicase. We identified peptides from the HRDC domain of BLM using a molecular docking approach with various available solutions and crystal structures of human telomeres and recently created a peptide library. Herein, we tested one peptide (BLM HRDC peptide) from the library and examined its interaction with human telomeric variant-1 (HTPu-var-1) to understand the basis of G4-protein interactions. Our circular dichroism (CD) data showed that HTPu-var-1 folded into an anti-parallel G-quadruplex, and the CD intensity significantly decreased upon increasing the peptide concentration. There was a significant decrease in hypochromicity due to the formation of G-quadruplex-peptide complex at 295 nm, which indicated the unfolding of structure due to the decrease in stacking interactions. The fluorescence data showed quenching upon titrating the peptide with HTPu-var-1-G4. Electrophoretic mobility shift assay confirmed the unfolding of the G4 structure. Cell viability was significantly reduced in the presence of the BLM peptide, with IC₅₀ values of 10.71 μM and 11.83 μM after 72 and 96 hours, respectively. These results confirmed that the selected peptide has the ability to bind to human telomeric G-quadruplex and unfold it. This is the first report in which a peptide was identified from the HRDC domain of the BLM G4-binding protein for the exploration of the G4-binding motif, which suggests a novel strategy to target G4 using natural key peptide segments.

Schematic representation of (HTPu–var-1-G4) located at the 3′ end, formation of G-quadruplex, model of the G-quadruplex structure, base stacking between G-quadruplex planes, G-quadruplex structure-peptide complex and twisting of G-quadruplex planes upon peptide binding.     

Incidence and Impact of Documented Eradication of Breast Cancer Axillary Lymph Node Metastases Before Surgery in Patients Treated With Neoadjuvant Chemotherapy

OBJECTIVE: To determine the incidence and prognostic significance of documented eradication of breast cancer axillary lymph node (ALN) metastases after neoadjuvant chemotherapy. SUMMARY BACKGROUND DATA: Neoadjuvant chemotherapy is the standard of care for patients with locally advanced breast cancer and is being evaluated in patients with earlier-stage operable disease. METHODS: One hundred ninety-one patients with locally advanced breast cancer and cytologically documented ALN metastases were treated in two prospective trials of doxorubicin-based neoadjuvant chemotherapy. Patients had breast surgery with level I and II axillary dissection followed by additional chemotherapy and radiation treatment. Nodal sections from 43 patients who were originally identified as having negative ALNs at surgery were reevaluated and histologically confirmed to be without metastases. An additional 1112 sections from these lymph node blocks were obtained; half were stained with an anticytokeratin antibody cocktail and analyzed. Survival was calculated using the Kaplan-Meier method. RESULTS: Of 191 patients with positive ALNs at diagnosis, 23% (43 patients) were converted to a negative axillary nodal status on histologic examination (median number of nodes removed = 16). Of the 43 patients with complete axillary conversion, 26% (n = 11) had N1 disease and 74% (n = 32) had N2 disease. On univariate analysis, patients with complete versus incomplete histologic axillary conversion were more likely to have initial estrogen-receptor-negative tumors, smaller primary tumors, and a complete pathologic response in the primary tumor. The 5-year disease-free survival rates were 87% in patients with preoperative eradication of axillary metastases and 51% for patients with residual nodal disease after neoadjuvant chemotherapy. Of the 39 patients with complete histologic conversion for whom nodal blocks were available, occult nodal metastases were found in additional nodal sections in 4 patients (10%). At a median follow-up of 61 months, the 5-year disease-free survival rates were 87% in patients without occult nodal metastases and 75% in patients with occult nodal metastases. CONCLUSIONS: Neoadjuvant chemotherapy can completely clear the axilla of microscopic disease before surgery, and occult metastases are found in only 10% of patients with a histologically negative axilla. The results of this study have implications for the potential use of sentinel lymph node biopsy as an alternative to axillary dissection in patients treated with neoadjuvant chemotherapy.     

Risk stratification in patients with remote prior myocardial infarction using rest-stress myocardial perfusion spect: prognostic value and impact on referral to early catheterization

BACKGROUND: Little is known about the prognostic value of myocardial perfusion single photon emission computed tomography (SPECT) in patients with remote prior myocardial infarction (MI). METHODS AND RESULTS: We identified 1413 consecutive patients with remote prior MI who underwent rest-stress myocardial perfusion SPECT. Semiquantitative visual analysis of 20 SPECT segments was used to define the summed stress, rest, and difference scores. The number of non-reversible segments was used as an index of infarct size. During follow-up (>or=1 year), 118 hard events occurred: 64 cardiac deaths (CDs) and 54 recurrent MIs. Annual CD and hard event rates increased significantly as a function of SPECT abnormality. For summed stress scores less than 4, 4 to 8, 9 to 13, and more than 13, the annual CD rates were 0.4%, 0.9%, 1.7%, and 3.5%, respectively (P =.002). Patients with small MI (<4 non-reversible segments) and no or mild ischemia (summed difference score or=4 non-reversible segments) had moderate to high annual CD rates (3.7%-6.6%) regardless of the extent of ischemia. Nuclear testing added incremental prognostic information to pre-scan information. Compared with a strategy in which all patients are referred to catheterization, a strategy that referred only those patients with a risk for CD of greater than 1% by myocardial perfusion SPECT resulted in a 41.6% cost savings. CONCLUSIONS: Myocardial perfusion SPECT adds incremental value to pre-scan information and is highly predictive and cost-efficient in the risk stratification of patients with remote prior MI. Patients with normal or mildly abnormal scan results or small MI in combination with absent or mild ischemia have a low risk for CD.     

Reevaluation of bone marrow-derived cells as a source for hepatocyte regeneration

We have investigated the contribution of intrasplenic bone marrow transplants or in vivo mobilized hematopoietic stem cells to the formation of hepatocytes in normal and injured liver. Direct intrasplenic injections of bone marrow mononuclear cells (5 x 10(5) cells), Scal+/lin- hematopoietic stem cells (5 x 10(3)) cells, and highly purified "side population" hematopoietic stem cells (5 x 10(3)) derived from enhanced green fluorescent protein (EGFP)-transgenic mice [C57Bl/6-TgN(ActbEGFP)1Osb] were performed in normal C57Bl/6 mice (n = 6) and in C57Bl/6 mice following two thirds hepatectomy (n = 8). To test the effect of mobilized stem cells on transdifferentiation, C57Bl/6 mice (n = 12) were lethally irradiated and reconstituted with EGFP-positive bone marrow mononuclear cells in a second series of experiments. Eight to 12 weeks after bone marrow transplantation a subset of mice (n = 3 in each group) received either rhG-CSF for hematopoietic stem cell mobilization, rhG-CSF combined with an intraperitoneal application of carbon tetrachloride (CCl4) as hepatocyte regeneration stimulus, or CCl4 alone. All mice were completely perfused with PBS to remove circulating nonorgan cells for analyses 4 weeks later. Liver as well as heart, intestine, spleen, and kidney tissue was analyzed for the presence of EGFP-transgenic cells. In 100 sections (2.3 x 10(7) cells) of any recipient mouse no EGFP-positive hepatocytes were detected either by analysis of native EGFP fluorescence or by immunofluorescence analysis with anti-EGFP and antidipeptidyl peptidase (DPP) IV antibodies. EGFP-transgenic cells resembling heart, kidney, or intestinal cells could also not be proven. The results demonstrate that there is little or no contribution of bone marrow-derived cells to the regeneration of normal and injured liver in the animal models used. Thus, potential therapeutic prospects of hematopoietic stem cell therapy for liver disease have to be critically reassessed.     

胆管癌组织中MMP-9的表达及其意义

目的　探讨基质金属蛋白酶 9(matrixmetalloproteinase 9,MMP 9)在胆管癌发展中的作用及与胆管癌浸润、转移和预后的关系。方法　应用免疫组织化学法 (S P法 )对 5 2例经手术和病理证实的胆管癌及 10例经手术切除的正常胆管壁标本进行MMP 9蛋白检测。结果　 (1)MMP 9表达在胆管癌组织、癌旁组织及正常胆管上皮组织阳性率分别为 84 6 % (4 4 /5 2 )、2 2 6 % (7/31)和 0 %(0 /10 ) ,有显著性差异 (P <0 0 1)。 (2 )在 5 2例胆管癌组织中 ,MMP 9表达分别与肿瘤的病理分级、TNM分期、浸润和转移及手术方式有关 (P <0 0 5 ) ,而与性别、年龄、肿瘤部位及大小无关 (P >0 0 5 )。 (3)MMP 9表达阳性患者一年生存率明显低于阴性者 (P <0 0 1)。结论　MMP 9表达与胆管癌发展、浸润和转移密切相关 ,检测MMP 9的表达有助于对胆管癌转移潜能和预后的判断。     

Novel insights on the bottom–up rise strength transfer: investigating massed vs. distributed exercise training

Sport Sciences for Health - The purpose of this study is to investigate the Bottom–Up Rise Strength Transfer (BURST) induced by massed vs. distributed-rehabilitative exercise training....     

Entropy Stable Scheme on Two-Dimensional Unstructured Grids for Euler Equations

We propose an entropy stable high-resolution finite volume scheme to approximate systems of two-dimensional symmetrizable conservation laws on unstructured grids. In particular we consider Euler equations governing compressible flows. The scheme is constructed using a combination of entropy conservative fluxes and entropy-stable numerical dissipation operators. High resolution is achieved based on a linear reconstruction procedure satisfying a suitable sign property that helps to maintain entropy stability. The proposed scheme is demonstrated to robustly approximate complex flow features by a series of benchmark numerical experiments.     

Comparison of the diagnostic value of MR imaging and ophthalmoscopy for the staging of retinoblastoma

Purpose

To compare the diagnostic value of magnetic resonance (MR) imaging and ophthalmoscopy for staging of retinoblastoma.

Methods

MR and ophthalmoscopic images of 36 patients who underwent enucleation were evaluated retrospectively following institutional review board approval. Histopathology being the standard of reference, the sensitivity and specificity of both diagnostic modalities were compared regarding growth pattern, iris neoangiogenesis, retinal detachment, vitreous seeds and optic nerve invasion. Data were analysed via McNemar’s test.

Results

Both investigations showed no significant difference in accuracy for the detection of different tumour growth patterns (P?=?0.80). Vitreous seeding detection was superior by ophthalmoscopy (P?<?0.001). For prelaminar optic nerve invasion, MR imaging showed similar sensitivity as ophthalmoscopy but increased specificity of 40 % (CI 0.12–0.74) vs. 20 % (0.03–0.56). MR detected optic nerve involvement past the lamina cribrosa with a sensitivity of 80 % (0.28–0.99) and a specificity of 74 % (0.55–0.88). The absence of optic nerve enhancement excluded histopathological infiltration, but the presence of optic nerve enhancement included a high number of false positives (22–24 %).

Conclusions

Ophthalmoscopy remains the method of choice for determining extent within the globe while MR imaging is useful for evaluating extraocular tumour extension. Thus, both have their own strengths and contribute uniquely to the staging of retinoblastoma.

Key Points

? Ophthalmoscopy: method of choice for determining extent of retinoblastoma within the globe. ? MR imaging provides optimal evaluation of extrascleral and extraocular tumour extension. ? Positive enhancement of the optic nerve on MRI does not necessarily indicate involvement.