N‐cadherin/catenin–based costameres in cultured chicken cardiomyocytes

N‐cadherin is a member of the Ca²⁺‐dependent cell adhesion molecules and plays an important role in the assembly of the adherens junction in chicken cardiomyocytes. In addition to being present at the cell‐cell junction, N‐cadherin is associated with costameres in extrajunctional regions. The significance of the N‐cadherin‐associated costameres and whether catenins are components of costameres in chicken cardiomyocytes are not known. In this study, double‐labeling immunofluorescence microscopy was used to determine the extrajunctional distribution of both N‐cadherin and its cytoplasmic associated proteins, α‐ and β‐catenins, and their relationship to myofibrillar Z‐disc α‐actinin. N‐cadherin, α‐, and β‐catenins were all found to be present at the extrajunctional region and, in some cases, were codistributed with myofibrillar α‐actinin exhibiting a periodic staining pattern. Confocal microscopy confirmed that both N‐cadherin and β‐catenin colocalized with peripheral myofibrillar α‐actinin on the dorsal surface of cardiomyocytes as components of the costameres. Intracellular application of antibodies specific for the cytoplasmic portions of N‐cadherin, α‐, and β‐catenin, either by electroporation or microinjection, resulted in myofibril disorganization and disassembly. These results suggest the existence of N‐cadherin/catenin‐based costameres in the dorsal surface of cultured chicken cardiomyocytes in addition to the integrin/vinculin‐based costameres found in the ventral surface and indicate that the former set of costameres is essential for cardiac myofibrillogenesis. J. Cell. Biochem. 75:93–104, 1999. © 1999 Wiley‐Liss, Inc.     

Binpairs: Utilization of Illumina Paired-End Information for Improving Efficiency of Taxonomic Binning of Metagenomic Sequences

Motivation

Paired-end sequencing protocols, offered by next generation sequencing (NGS) platforms like Illumia, generate a pair of reads for every DNA fragment in a sample. Although this protocol has been utilized for several metagenomics studies, most taxonomic binning approaches classify each of the reads (forming a pair), independently. The present work explores some simple but effective strategies of utilizing pairing-information of Illumina short reads for improving the accuracy of taxonomic binning of metagenomic datasets. The strategies proposed can be used in conjunction with all genres of existing binning methods.

Results

Validation results suggest that employment of these “Binpairs” strategies can provide significant improvements in the binning outcome. The quality of the taxonomic assignments thus obtained are often comparable to those that can only be achieved with relatively longer reads obtained using other NGS platforms (such as Roche).

Availability

An implementation of the proposed strategies of utilizing pairing information is freely available for academic users at https://metagenomics.atc.tcs.com/binning/binpairs.     

Urine proteome analysis reflects atherosclerotic disease in an ApoE-/- mouse model and allows the discovery of new candidate biomarkers in mouse and human atherosclerosis

Noninvasive diagnosis of atherosclerosis via single biomarkers has been attempted but remains elusive. However, a previous polymarker or pattern approach of urine polypeptides in humans reflected coronary artery disease with high accuracy. The aim of the current study is to use urine proteomics in ApoE(-/-) mice to discover proteins with pathophysiological roles in atherogenesis and to identify urinary polypeptide patterns reflecting early stages of atherosclerosis. Urine of ApoE(-/-) mice either on high fat diet (HFD) or chow diet was collected over 12 weeks; urine of wild type mice on HFD was used to exclude diet-related proteome changes. Capillary electrophoresis coupled to mass spectrometry (CE-MS) of samples identified 16 polypeptides specific for ApoE(-/-) mice on HFD. In a blinded test set, these polypeptides allowed identification of atherosclerosis at a sensitivity of 90% and specificity of 100%, as well as monitoring of disease progression. Sequencing of the discovered polypeptides identified fragments of α(1)-antitrypsin, epidermal growth factor (EGF), kidney androgen-regulated protein, and collagen. Using immunohistochemistry, α(1)-antitrypsin, EGF, and collagen type I were shown to be highly expressed in atherosclerotic plaques of ApoE(-/-) mice on HFD. Urinary excretion levels of collagen and α(1)-antitrypsin fragments also significantly correlated with intraplaque collagen and α(1)-antitrypsin content, mirroring plaque protein expression in the urine proteome. To provide further confirmation that the newly identified proteins are relevant in humans, the presence of collagen type I, α(1)-antitrypsin, and EGF was also confirmed in human atherosclerotic disease. Urine proteome analysis in mice exemplifies the potential of a novel multimarker approach for the noninvasive detection of atherosclerosis and monitoring of disease progression. Furthermore, this approach represents a novel discovery tool for the identification of proteins relevant in murine and human atherosclerosis and thus also defines potential novel therapeutic targets.     

Prognostic Implication of Human Papillomavirus Types and Species in Cervical Cancer Patients Undergoing Primary Treatment

High-risk human papillomavirus (HPV) types are associated with cervical cancer. It is well established that individual HPV types vary in oncogenicity, but current data on their prognostic implication remain controversial. We examined the association between HPV types/species and the survival of 236 Chinese women aged 26–87 (mean 54.4) years after receiving primary treatment for cervical cancer. Overall, 45.8% were of FIGO stage I, 41.9% stage II, and 12.3% stage III. The four most prevalent types found were HPV-16 (60.2%), HPV-18 (21.6%), HPV-52 (11.9%), and HPV-58 (9.3%). Overall, 19.5% of patients had multiple-type infections, 78.4% harboured one or more alpha-9 species, and 28.8% harboured one or more alpha-7 species. After a median follow-up of 8.0 years, 156 (66.1%) patients survived. The 3-year overall survival rate was 75.5%. Factors independently associated with a poorer 3-year overall survival were age >60 years, tumour size >4 cm, lymph node involvement and treatment with radiotherapy+/-chemotherapy. Univariate analysis showed HPV-16 single-type infection was associated with a marginally poorer disease-specific survival (71.6% vs. 87.0%, HR: 1.71, 95% CI = 1.01–2.90), whereas non-HPV-16 alpha-9 species was associated with a better disease-specific survival (90.0% vs. 76.2%, HR: 0.36, 95% CI = 0.16–0.79). However, on multivariate analysis, HPV infection status irrespective of different grouping methods, including individual types, species, single-type or co-infection, did not carry any significant prognostic significance. In conclusion, we did not observe any association between infection with a particular HPV type/species and survival. An HPV type-based stratification in treatment and follow-up plan could not be recommended.     

Recent trends and some applications of isothermal titration calorimetry in biotechnology

Isothermal titration calorimeters (ITCs) are thermodynamic instruments used for the determination of enthalpy changes in any physical/chemical reaction. This can be applied in various fields of biotechnology. This review explains ITC applications, especially in bioseparation, drug development and cell metabolism. In liquid chromatography, the separation/purification of specific proteins or polypeptides in a mixture is usually achieved by varying the adsorption affinities of the different proteins/polypeptides for the adsorbent under different mobile-phase conditions and temperatures. Using ITC analysis, the binding mechanism of proteins with adsorbent solid material is derived by elucidating enthalpy and entropy changes, which offer valuable guidelines for designing experimental conditions in chromatographic separation. The binding affinity of a drug with its target is studied by deriving binding enthalpy and binding entropy. To improve the binding affinity, suitable lead compounds for a drug can be identified and their affinity tested by ITC. Recently ITC has also been used in studying cell metabolism. The heat produced by animal cells in culture can be used as a primary indicator of the kinetics of cell metabolism, which provides key information for drug bioactivity and operation parameters for process cell culture.     

Investigating the Antiproliferative Activity of High Affinity DNA Aptamer on Cancer Cells

Vascular endothelial growth factor (VEGF) is an angiogenic mitogen involved in promoting tumor angiogenesis inside the body. VEGF is a key protein required for progression of tumor from benign to malignant phenotype. In this study, we investigated the binding affinity of a previously selected 26-mer DNA aptamer sequence (SL₂-B) against heparin binding domain (HBD) of VEGF₁₆₅ protein. The SL₂-B was first chemically modified by introduction of phosphorothioate linkages (PS-linkages). Subsequently, surface plasmon resonance (SPR) spectroscopy and circular dichroism (CD) were used to determine the binding affinity, specificity and to deduce the conformation of PS-modified SL₂-B sequence. Finally, antiproliferative activity of the modified SL₂-B sequence on Hep G2 cancer cells was investigated. Our results demonstrate a marked enhancement in the biostability of the SL₂-B sequence after PS modification. The modified SL₂-B sequence also exhibits enhanced antiproliferative activity against Hep G2 cancer cells in hypoxia conditions. In addition, modified SL₂-B sequence inhibits the expression of Jagged-1 protein, which is one of the ligands to VEGF linked delta/jagged-notch signaling pathway.     

Cell surface display of Chi92 on Escherichia coli using ice nucleation protein for improved catalytic and antifungal activity

The gene encoding chitinase 92 (Chi92) from Aeromonas hydrophila JP10 has been displayed on the cell surface of Escherichia coli using the N-terminal region of ice nucleation proteins (INPN) as an anchoring motif. Immunofluorescence microscopy confirmed that Chi92 was anchored on the cell surface. Western blot analysis further identified the synthesis of INP derivatives containing the N-terminal domain INPN-Chi92 fusion protein of the expected size (112 kDa). Whole cell enzyme assay indicated that the displayed Chi92 showed enhanced catalytic activity toward colloidal chitin. In addition, the Chi92-displayed cells exhibited inhibitory effects on the mycelial growth of phytopathogenic fungi, including Fusarium decemcellulare, Sclerotium rolfsii, Rhizoctonia solani kuhn, and Fusarium oxysporum f.sp. melonis. This study suggested that the INP-based display systems can be used to express a large protein (90 kDa Chi92) on the cell surface of E. coli without growth inhibition. In addition, the display of chitinase on the cell surface may provide an attractive method for the development of biocontrol agents against phytopathogenic fungi.