Bifunctional glycosphingolipid (GSL) probes to investigate GSL-interacting proteins in cell membranes.

Glycosphingolipids (GSLs) are abundant glycolipids on cells and essential for cell recognition, adhesion, signal transduction, and so on. However, their lipid anchors are not long enough to cross the membrane bilayer. To transduce transmembrane signals, GSLs must interact with other membrane components, whereas such interactions are difficult to investigate. To overcome this difficulty, bifunctional derivatives of II3-ß-N-acetyl-D-galactosamine-GA2 (GalNAc-GA2) and ß-N-acetyl-D-glucosamine-ceramide (GlcNAc-Cer) were synthesized as probes to explore GSL-interacting membrane proteins in live cells. Both probes contain photoreactive diazirine in the lipid moiety, which can crosslink with proximal membrane proteins upon photoactivation, and clickable alkyne in the glycan to facilitate affinity tag addition for crosslinked protein pull-down and characterization. The synthesis is highlighted by the efficient assembly of simple glycolipid precursors followed by on-site lipid remodeling. These probes were employed to profile GSL-interacting membrane proteins in HEK293 cells. The GalNAc-GA2 probe revealed 312 distinct proteins, with GlcNAc-Cer probe-crosslinked proteins as controls, suggesting the potential influence of the glycan on GSL functions. Many of the proteins identified with the GalNAc-GA2 probe are associated with GSLs, and some have been validated as being specific to this probe. The versatile probe design and experimental protocols are anticipated to be widely applicable to GSL research.

Sensitive Method To Analyze Cell Surface GPI-Anchored Proteins Using DNA Hybridization Chain Reaction-Mediated Signal Amplification.

GPI-anchored proteins (GPI-APs) are ubiquitous and essential but exist in low abundances on the cell surface, making their analysis and investigation especially challenging. To tackle the problem, a new method to detect and study GPI-APs based upon GPI metabolic engineering and DNA-facilitated fluorescence signal amplification was developed. In this context, cell surface GPI-APs were metabolically engineered using azido-inositol derivatives to introduce an azido group. This allowed GPI-AP coupling with alkyne-functionalized multifluorophore DNA assemblies generated by hybridization chain reaction (HCR). It was demonstrated that this approach could significantly improve the detection limit and sensitivity of GPI-APs, thereby enabling various biological studies, including the investigation of live cells. This new, enhanced GPI-AP detection method has been utilized to successfully explore GPI-AP engineering, analyze GPI-APs, and profile GPI-AP expression in different cells.

Investigation of Glycosylphosphatidylinositol (GPI)-Plasma Membrane Interaction in Live Cells and the Influence of GPI Glycan Structure on the Interaction.

Glycosylphosphatidylinositols (GPIs) need to interact with other components in the cell membrane to transduce transmembrane signals. A bifunctional GPI probe was employed for photoaffinity-based proximity labelling and identification of GPI-interacting proteins in the cell membrane. This probe contained the entire core structure of GPIs and was functionalized with photoreactive diazirine and clickable alkyne to facilitate its crosslinking with proteins and attachment of an affinity tag. It was disclosed that this probe was more selective than our previously reported probe containing only a part structure of the GPI core for cell membrane incorporation and an improved probe for studying GPI-cell membrane interaction. Eighty-eight unique membrane proteins, many of which are related to GPIs/GPI-anchored proteins, were identified utilizing this probe. The proteomics dataset is a valuable resource for further analyses and data mining to find new GPI-related proteins and signalling pathways. A comparison of these results with those of our previous probe provided direct evidence for the profound impact of GPI glycan structure on its interaction with the cell membrane.

A Biotinylated Glycosylphosphatidylinositol (GPI) as the Universal Platform To Access GPI-Anchored Protein Analogues.

A glycosylphosphatidylinositol (GPI) derivative with biotin linked to its mannose III 6-O-position was prepared by a convergent strategy. This biotinylated GPI was demonstrated to bind avidinated proteins readily through biotin-avidin interaction and, therefore, can serve as a universal platform to access various biologically significant GPI-anchored protein analogues.

Spin-labeling Insights into How Chemical Fixation Impacts Glycan Organization on Cells.

As new methods to interrogate glycan organization on cells develop, it is important to have a molecular level understanding of how chemical fixation can impact results and interpretations. Site-directed spin labeling technologies are well suited to study how the spin label mobility is impacted by local environmental conditions, such as those imposed by cross-linking effects of paraformaldehyde cell fixation methods. Here, we utilize three different azide-containing sugars for metabolic glycan engineering with HeLa cells to incorporate azido glycans that are modified with a DBCO-based nitroxide moiety via click reaction. Continuous wave X-band electron paramagnetic resonance spectroscopy is employed to characterize how the chronological sequence of chemical fixation and spin labeling impacts the local mobility and accessibility of the nitroxide-labeled glycans in the glycocalyx of HeLa cells. Results demonstrate that chemical fixation with paraformaldehyde can alter local glycan mobility and care should be taken in the analysis of data in any study where chemical fixation and cellular labeling occur.

Profiling Glycosylphosphatidylinositol (GPI)-Interacting Proteins in the Cell Membrane Using a Bifunctional GPI Analogue as the Probe.

Glycosylphosphatidylinositol (GPI) anchorage of cell surface proteins to the membrane is biologically important and ubiquitous in eukaryotes. However, GPIs do not contain long enough lipids to span the entire membrane bilayer. To transduce binding signals, GPIs must interact with other membrane components, but such interactions are difficult to define. Here, a new method was developed to explore GPI-interacting membrane proteins in live cell with a bifunctional analogue of the glucosaminylphosphatidylinositol motif conserved in all GPIs as a probe. This probe contained a diazirine functionality in the lipid and an alkynyl group on the glucosamine residue to respectively facilitate the cross-linkage of GPI-binding membrane proteins with the probe upon photoactivation and then the installation of biotin to the cross-linked proteins via a click reaction for affinity-based protein isolation and analysis. Profiling the proteins pulled down from the Hela cells revealed 94 unique and 18 overrepresented proteins compared to the control, and most of them are membrane proteins and many are GPI-related. The results have proved not only the concept of using the new bifunctional GPI probe to investigate GPI-binding membrane proteins but also the important role of inositol in the biological functions of GPI anchors and GPI-anchored proteins.

Labeling cell surface glycosylphosphatidylinositol-anchored proteins through metabolic engineering using an azide-modified phosphatidylinositol.

Glycosylphosphatidylinositol (GPI) anchorage is one of the most common mechanisms to attach proteins to the plasma membrane of eukaryotic cells. GPI-anchored proteins (GPI-APs) play a critical role in many biological processes but are difficult to study. Here, a new method was developed for the effective and selective metabolic engineering and labeling of cell surface GPI-APs with an azide-modified phosphatidylinositol (PI) as the biosynthetic precursor of GPIs. It was demonstrated that this azido-PI derivative was taken up by HeLa cells and incorporated into the biosynthetic pathway of GPIs to present azide-labeled GPI-APs on the live cell surface. The azido group was used as a molecular handle to install other labels through a biocompatible click reaction to enable various biological studies, e.g., fluorescent imaging and protein pull-down, which can help explore the functions of GPI-APs and discover new GPI-APs.

Recent research progress in glycosylphosphatidylinositol-anchored protein biosynthesis, chemical/chemoenzymatic synthesis, and interaction with the cell membrane.

Glycosylphosphatidylinositol (GPI) attachment to the C-terminus of proteins is a prevalent posttranslational modification in eukaryotic species, and GPIs help anchor proteins to the cell surface. GPI-anchored proteins (GPI-APs) play a key role in various biological events. However, GPI-APs are difficult to access and investigate. To tackle the problem, chemical and chemoenzymatic methods have been explored for the preparation of GPI-APs, as well as GPI probes that facilitate the study of GPIs on live cells. Substantial progress has also been made regarding GPI-AP biosynthesis, which is helpful for developing new synthetic methods for GPI-APs. This article reviews the recent advancements in the study of GPI-AP biosynthesis, GPI-AP synthesis, and GPI interaction with the cell membrane utilizing synthetic probes.

Different Biophysical Properties of Cell Surface α2,3- and α2,6-Sialoglycans Revealed by Electron Paramagnetic Resonance Spectroscopic Studies.

Sialoglycans on HeLa cells were labeled with a nitroxide spin radical through enzymatic glycoengineering (EGE)-mediated installation of azide-modified sialic acid (Neu5Ac9N3) and then click reaction-based attachment of a nitroxide spin radical. α2,6-Sialyltransferase (ST) Pd2,6ST and α2,3-ST CSTII were used for EGE to install α2,6- and α2,3-linked Neu5Ac9N3, respectively. The spin-labeled cells were analyzed by X-band continuous wave (CW) electron paramagnetic resonance (EPR) spectroscopy to gain insights into the dynamics and organizations of cell surface α2,6- and α2,3-sialoglycans. Simulations of the EPR spectra revealed average fast- and intermediate-motion components for the spin radicals in both sialoglycans. However, α2,6- and α2,3-sialoglycans in HeLa cells possess different distributions of the two components, e.g., a higher average population of the intermediate-motion component for α2,6-sialoglycans (78%) than that for α2,3-sialoglycans (53%). Thus, the average mobility of spin radicals in α2,3-sialoglycans was higher than that in α2,6-sialoglycans. Given the fact that a spin-labeled sialic acid residue attached to the 6-O-position of galactose/N-acetyl-galactosamine would experience less steric hindrance and show more flexibility than that attached to the 3-O-position, these results may reflect the differences in local crowding/packing that restrict the spin-label and sialic acid motion for α2,6-linked sialoglycans. The studies further suggest that Pd2,6ST and CSTII may have different preferences for glycan substrates in the complex environment of the extracellular matrix. The discoveries of this work are biologically important as they are useful for interpreting the different functions of α2,6- and α2,3-sialoglycans and indicate the possibility of using Pd2,6ST and CSTII to target different glycoconjugates on cells.

Primary intramedullary spinal gliosarcoma: An unusual presentation.

Gliosarcoma is a biphasic central nervous system malignancy composed of glial and mesenchymal components. It is recognized as a rare variant of glioblastoma with unique histology, immunostaining properties, and natural history. Although usually described to primarily involve the brain, a search of the published literature revealed four reported cases of gliosarcoma arising in the spinal cord. This report describes the case of a 35-year-old woman with progressive back pain with loss of sensation and power of both lower limbs. Magnetic resonance imaging showed an intramedullary unifocal space-occupying lesion in her thoracolumbar spinal cord. Subtotal resection and subsequent histopathological and immunohistochemical studies confirmed the diagnosis of gliosarcoma of the spinal cord. This report further establishes the clinical entity of primary spinal gliosarcoma and proposes the need to consider this possibility among the differential diagnoses of a space-occupying spinal lesion.

Poly-lysinated nanoscale carbon probe for low power two-photon bioimaging.

Effective outcome from dynamic live-cell-imaging requires utilization of a probe with high emission intensity and low photobleaching. It would be preferable to achieve such properties at a low power of the applied laser to avoid any probable damage to biological cells or tissue. Most of the used small-molecule fluorophores have been reported to show significant photobleaching in a time-dependent manner and require high laser power to gain significant intensity for bioimaging. Carbon nanoparticles have recently been successfully used for cell imaging with low bleaching characteristics but require high laser power and lack optical nonlinearity at low power levels. Here, we report the preparation, characterization, and application of a Nanoscale Carbon (NC) which, on being surface decorated with crescent-shaped poly-lysine (PLNC), provides two-photon fluorescence (TPF) and low bleaching properties. PLNC was found to stain the cytoplasm of C2C12 muscle cells in the first four-hours of incubation with high TPF in the infrared range and can be useful for deep tissue imaging with further improvements.

Significance of HER2 and Ki-67 in Preneoplastic Lesions and Carcinoma of Gallbladder.

BACKGROUND: HER2 is an oncoprotein which is overexpressed in several cancers including breast and stomach. Several studies have shown that HER2 is overexpressed in gallbladder cancer and in precancerous lesions. The present study was undertaken to assess pattern and level of expression of HER2 in metaplasia, dysplasia, and different stages of gallbladder carcinoma, which would determine its suitability as a prognostic biomarker in neoplastic transformation of gallbladder epithelium. The study was also aimed at to find the significance of Ki-67 index in these lesions. METHODS AND MATERIALS: One hundred and twenty-eight patients who underwent cholecystectomy comprised the study group. Among them, 108 (84.4%) specimens showing metaplasia, dysplasia, and carcinoma on routine histopathology were considered as cases and 20 (15.6%) specimens of chronic cholecystitis having non-metaplastic mucosa were considered as control. Immunohistochemistry (IHC) was performed for HER2 and Ki-67. For HER2 interpretation ASCO/CAP guideline for breast cancer was followed. Chi-square test was used to find out the significance of HER2 expression in dysplasia/metaplasia/carcinoma. The ANOVA and Tukey-Kramer Multiple Comparisons Test were used for determining the association of Ki-67 with malignant transformation. RESULTS AND CONCLUSIONS: Overexpression of HER2 was observed in 48% (n = 12) of adenocarcinomas, 58% (n = 7) of high-grade dysplasia, 47% (n = 8) of low-grade dysplasia, and 74% (n = 25) of intestinal metaplasia. Ki-67 index increases in a non-linear fashion as the precursor lesions progress toward malignancy. In the future, these markers might be used as a prognostic biomarker for gallbladder carcinoma and its precursor lesions and it might become a valid indication for targeted therapies for gallbladder cancer.

Functional magnetic resonance imaging in cervical cancer: current evidence and future directions.

Carcinoma cervix is one of the most common cancers amongst Indian women. Though treatment strategies continue to evolve, there are no established predictive biomarkers of prognosis or therapeutic response. Novel imaging techniques using magnetic resonance (MR) and positron emission tomography (PET) can facilitate time resolved spatial evaluation of biological characteristics (perfusion, permeability, cellularity, proliferation, oxygenation, and apoptosis) thereby serving as early surrogate biomarkers for prognosis and therapeutic response. Several of these imaging modalities such as dynamic contrast-enhanced MRI (DCE-MRI), diffusion-weighted MRI (DW-MRI), MR spectroscopy (MRS) and fluorine-18 fluorodeoxyglucose positron emission tomography (FDG-PET) are now being evaluated for gynecological oncology, with the majority of work being performed on cervical tumors. PUBMED database was searched for this review from January 1966 till February 2011. This review examines the basic principles of functional MR imaging for cervical cancer and its current status as a diagnostic and predictive biomarker for cervical cancer.

Evaluation of diffusion-weighted imaging as a predictive marker for tumor response in patients undergoing chemoradiation for postoperative recurrences of cervical cancer.

PURPOSE: To investigate diffusion-weighted imaging (DWI) as a response biomarker in patients undergoing chemoradiation for postoperative recurrences of cervical cancer. MATERIALS AND METHODS: From October 2008 to March 2011, 20 patients were included. All underwent T2-weighted (T2W) and DWI before and after chemoradiation. Gross tumor volume (GTV), lateral extent, apparent diffusion coefficient (ADC), and presence of regions of focally restricted diffusion were determined at baseline. Response to chemoradiation was categorized as either partial or complete. Receiver operator characteristics (ROC) curve identified thresholds of GTV and ADC that best predict for partial response. Univariate and multivariate analysis were performed on SPSS version 15. RESULTS: The median GTV was 24.5 cc (4.1-110 cc). Central and lateral disease was present in 8 and 12 patients, respectively. The median ADC was 1 × 10 -3 mm² /s (0.8-1.3 × 10⁻³ mm² /s) and 12/20 (60%) patients had focal restricted diffusion. Overall 10/20 patients had partial response. ROC analysis identified volume of 25 cc or higher [sensitivity = 80%, specificity = 80%, area under curve (AUC) = 0.76, P = 0.04] and ADC more than 1 × 10⁻³ mm² /s (sensitivity = 70%, specificity = 50%, AUC = 0.62; P = 0.34) to best predict for partial response. On univariate analysis bulky disease (77.7% vs. 27%; P = 0.03), lateral disease (66.6% vs. 25%; P = 0.08), and focal regions of restricted diffusion (66.6% vs. 25%; P = 0.06) predicted for partial response to chemoradiation. All factors continued to be significant on multivariate analysis. On restricting analysis to bulky tumors ADC greater than 0.95 × 10⁻³ mm² /s predicted partial response with high sensitivity (85.7%) and specificity (100%) (AUC 0.96; P = 0.05). On univariate analysis lateral disease (P = 0.04), high baseline ADC (P = 0.07) predicted for partial response. CONCLUSIONS: Baseline ADC and focal regions of ADC restriction predict for partial response with moderate sensitivity and specificity in patients with postoperative recurrences of cervical cancer and need to be validated in larger cohort.

Carcinoma in situ of true vocal cord in a nonsmoker adolescent female.

Carcinoma in situ (precancerous lesion) of true vocal cord in a nonsmoker adolescent female without any history of prior neck irradiation is rare. A 16-year-old female patient without any of the known risk factors presented with history of gradual-onset hoarseness of voice unrelieved by symptomatic treatments for 1 year. Contrast-enhanced CT scan of neck and laryngoscopy and histopathology of the tissue from irregular lesions along the medial margin of the left vocal cord diagnosed it as a case of carcinoma in situ of vocal cord. Absence of known risk factors and very young age of the patient made this case a rarity and hence the case is being reported.