Challenges in Spinal Endoscopy.

The advent of any new technology or technique is fraught with challenges in the early stages of development and adoption. This situation is no different for spinal endoscopy, which has been continuously developing for decades and has only recently gained significant traction in North America. Patient selection can be challenging for even expert endoscopic surgeons, given the limited abilities of current technology for patients with multilevel disease. Anatomic limitations, such as iliac crest location and small foraminal dimensions, can restrict application of the transforaminal approach, considered the "workhorse" of endoscopic techniques. A paucity of dedicated training opportunities has led many to become late adopters or preclude exposure entirely, limiting the next generation of surgeons and expansion of the field. Finally, economic constraints, including capital expenses and issues with insurance reimbursement, have generated difficulties to widespread acquisition. Nonetheless, the future is bright for spinal endoscopy, with potential solutions to these issues already generating progress. In the present report, we have summarized these challenges and discussed some of the current steps underway to help alleviate their impact.

Haploinsufficiency in tumor predisposition syndromes: altered genomic transcription in morphologically normal cells heterozygous for VHL or TSC mutation.

Tumor suppressor genes and their effector pathways have been identified for many dominantly heritable cancers, enabling efforts to intervene early in the course of disease. Our approach on the subject of early intervention was to investigate gene expression patterns of morphologically normal "one-hit" cells before they become hemizygous or homozygous for the inherited mutant gene which is usually required for tumor formation. Here, we studied histologically non-transformed renal epithelial cells from patients with inherited disorders that predispose to renal tumors, including von Hippel-Lindau (VHL) disease and Tuberous Sclerosis (TSC). As controls, we studied histologically normal cells from non-cancerous renal epithelium of patients with sporadic clear cell renal cell carcinoma (ccRCC). Gene expression analyses of VHLmut/wt or TSC1/2mut/wt versus wild-type (WT) cells revealed transcriptomic alterations previously implicated in the transition to precancerous renal lesions. For example, the gene expression changes in VHLmut/wt cells were consistent with activation of the hypoxia response, associated, in part, with the "Warburg effect". Knockdown of any remaining VHL mRNA using shRNA induced secondary expression changes, such as activation of NFκB and interferon pathways, that are fundamentally important in the development of RCC. We posit that this is a general pattern of hereditary cancer predisposition, wherein haploinsufficiency for VHL or TSC1/2, or potentially other tumor susceptibility genes, is sufficient to promote development of early lesions, while cancer results from inactivation of the remaining normal allele. The gene expression changes identified here are related to the metabolic basis of renal cancer and may constitute suitable targets for early intervention.

Potential prognostic biomarkers of pancreatic cancer.

OBJECTIVES: We evaluated whether pancreatic main duct fluid can provide protein biomarkers with prognostic value. METHODS: Mass spectrometry proteomics was applied to as little as 20µL of fluid collected at the time of tumor surgical resection. Biomarker proteins identified for 27 patients were correlated with clinical outcomes. RESULTS: Thirteen patients had pancreatic ductal adenocarcinomas, 4 had intraductal papillary mucinous neoplasm with in situ adenocarcinoma, 5 had ampullary adenocarcinomas, 2 had intraductal papillary mucinous neoplasms, and 3 had benign diseases. In pathologic stage II or higher pancreatic ductal adenocarcinoma, moderate or high expression of S100A8 or S100A9 proteins was associated with a median disease recurrence-free survival of 5.8 months compared with 17.3 months in patients with low expression (P = 0.002). Median overall survival was 12.6 versus 27 months for patients with moderate to high versus low S100A8 and A9 expression (P = 0.02). CONCLUSIONS: This analysis suggests distinct proteomic signatures for pancreatic cancer. Patients in our study with elevated levels of S100A8 or A9 in the ductal fluid, a near absence of pancreatic enzymes, and high levels of mucins were found to have significantly worse prognosis. Although further validation is needed to corroborate these findings, analysis of pancreatic ductal fluid is a promising tool for identifying biomarkers of interest.

Immunodepletion plasma proteomics by tripleTOF 5600 and Orbitrap elite/LTQ-Orbitrap Velos/Q exactive mass spectrometers.

Plasma proteomic experiments performed rapidly and economically using several of the latest high-resolution mass spectrometers were compared. Four quantitative hyperfractionated plasma proteomics experiments were analyzed in replicates by two AB SCIEX TripleTOF 5600 and three Thermo Scientific Orbitrap (Elite/LTQ-Orbitrap Velos/Q Exactive) instruments. Each experiment compared two iTRAQ isobaric-labeled immunodepleted plasma proteomes, provided as 30 labeled peptide fractions, and 480 LC-MS/MS runs delivered >250 GB of data in 2 months. Several analysis algorithms were compared. At 1% false discovery rate, the relative comparative findings concluded that the Thermo Scientific Q Exactive Mass Spectrometer resulted in the highest number of identified proteins and unique sequences with iTRAQ quantitation. The confidence of iTRAQ fold-change for each protein is dependent on the overall ion statistics (Mascot Protein Score) attainable by each instrument. The benchmarking also suggested how to further improve the mass spectrometry parameters and HPLC conditions. Our findings highlight the special challenges presented by the low abundance peptide ions of iTRAQ plasma proteome because the dynamic range of plasma protein abundance is uniquely high compared with cell lysates, necessitating high instrument sensitivity.

Assessment of two immunodepletion methods: off-target effects and variations in immunodepletion efficiency may confound plasma proteomics.

Immunodepletion of abundant plasma proteins increases the depth of proteome penetration by mass spectrometry. However, the nature and extent of immunodepletion and the effect of off-target depletion on the quantitative comparison of the residual proteins have not been critically addressed. We performed mass spectrometry label-free quantitation to determine which proteins were immunodepleted and by how much. Two immunodepletion resins were compared: Qproteome (Qiagen) which removes albumin+immunoglobulins and Seppro IgY14+SuperMix (Sigma-Aldrich) which removes 14 target proteins plus a number of unidentified proteins. Plasma collected by P100 proteomic plasma collection tubes (BD) from 20 human subjects was individually immunodepleted to minimize potential variability, prior to pooling. The abundant proteins were quantified better when using only albumin+immunoglobulins removal (Qproteome), while lower abundance proteins were evaluated better using exhaustive immunodepletion (Seppro IgY14+SuperMix). The latter resin removed at least 155 proteins, 38% of the plasma proteome in protein number and 94% of plasma protein in mass. The depth of immunodepletion likely accounts for the effectiveness of this resin in revealing low abundance proteins. However, the more profound immunodepletion achieved with the IgY14+SuperMix may lead to false-positive fold-changes between comparison groups if the reproducibility and efficiency of the depletion of a given protein are not considered.

Isobaric labeling and data normalization without requiring protein quantitation.

Isobaric multiplexed quantitative proteomics can complement high-resolution sample isolation techniques. Here, we report a simple workflow exponentially modified protein abundance index (emPAI)-MW deconvolution (EMMOL) for normalizing isobaric reporter ratios within and between experiments, where small or unknown amounts of protein are used. EMMOL deconvolutes the isobaric tags for relative and absolute quantification (iTRAQ) data to yield the quantity of each protein of each sample in the pool, a new approach that enables the comparison of many samples without including a channel of reference standard. Moreover, EMMOL allows using a sufficient quantity of control sample to facilitate the peptide fractionation (isoelectric-focusing was used in this report), and mass spectrometry MS/MS sequencing yet relies on the broad dynamic range of iTRAQ quantitation to compare relative protein abundance. We demonstrated EMMOL by comparing four pooled samples with 20-fold range differences in protein abundance and performed data normalization without using prior knowledge of the amounts of proteins in each sample, simulating an iTRAQ experiment without protein quantitation prior to labeling. We used emPAI, the target protein MW, and the iTRAQ reporter ratios to calculate the amount of each protein in each of the four channels. Importantly, the EMMOL-delineated proteomes from separate iTRAQ experiments can be assorted for comparison without using a reference sample. We observed no compression of expression in iTRAQ ratios over a 20-fold range for all protein abundances. To complement this ability to analyze minute samples, we report an optimized iTRAQ labeling protocol for using 5 µg protein as the starting material.

APC +/- alters colonic fibroblast proteome in FAP.

Here we compared the proteomes of primary fibroblast cultures derived from morphologically normal colonic mucosa of familial adenomatous polyposis (FAP) patients with those obtained from unaffected controls. The expression signature of about 19% of total fibroblast proteins separates FAP mutation carriers from unaffected controls (P < 0.01). More than 4,000 protein spots were quantified by 2D PAGE analysis, identifying 368 non-redundant proteins and 400 of their isoforms. Specifically, all three classes of cytoskeletal filaments and their regulatory proteins were altered as were oxidative stress response proteins. Given that FAP fibroblasts showed heightened sensitivity to transformation by KiMSV and SV40 including elevated levels of the p53 protein, events controlled in large measure by the Ras suppressor protein-1 (RSU-1) and oncogenic DJ-1, here we show decreased RSU1 and augmented DJ-1 expression in both fibroblasts and crypt-derived epithelial cells from morphologically normal colonic mucosa of FAP gene-carriers. The results indicate that heterozygosity for a mutant APC tumor suppressor gene alters the proteomes of both colon-derived normal fibroblasts in a gene-specific manner, consistent with a "one-hit" effect.

In-vivo Endoscopic Visualization of Patho-anatomy in Symptomatic Degenerative Conditions of the Lumbar Spine II: Intradiscal, Foraminal, and Central Canal Decompression.

The patho-anatomy in an aging spine is partly defined by Rauschning's anatomic cryosections. Theories of pain generation and principles of minimally invasive spine surgery are suggested by close examination of these specimens. If the visualized patho-anatomy can be studied in vivo in a partially sedated patient by spinal probing, spinal pain can be better understood, and rational endoscopic treatment options may then evolve.1 A 1997 IRB-approved study provided evidence that endoscopic transforaminal surgery was feasible for the treatment of a wide spectrum of degenerative conditions in the lumbar spine. The technique incorporated evocative chromo-discography to correlate reproduction of pain with in-vivo probing of patho-anatomy. Laser and radiofrequency ablation augmented mechanical decompression to obtain pain relief.1-3 Endoscopic visualization of patho-anatomy ranging from annular tears to spondylolisthesis and stenosis provided clinical evidence that foraminal decompression, ablation, and irrigation could effectively treat these visualized painful conditions with minimal morbidity. This resulted in a better understanding of the pain generators in the lumbar spine, opening up options for surgical pain management.1-5 The procedure does not burn any bridges for more traditional surgical techniques. The learning curve may be steep for some and long for others, but results are very good, concomitant with each individual surgeon overcoming his personal learning curve.

Alteration of Differentiation Potentials by Modulating GATA Transcription Factors in Murine Embryonic Stem Cells.

Background. Mouse embryonic stem (ES) cells can be differentiated in vitro by aggregation and/or retinoic acid (RA) treatment. The principal differentiation lineage in vitro is extraembryonic primitive endoderm. Dab2, Laminin, GATA4, GATA5, and GATA6 are expressed in embryonic primitive endoderm and play critical roles in its lineage commitment. Results. We found that in the absence of GATA4 or GATA5, RA-induced primitive endoderm differentiation of ES cells was reduced. GATA4 (-/-) ES cells express higher level of GATA5, GATA6, and hepatocyte nuclear factor 4 alpha marker of visceral endoderm lineage. GATA5 (-/-) ES cells express higher level of alpha fetoprotein marker of early liver development. GATA6 (-/-) ES cells express higher level of GATA5 as well as mesoderm and cardiomyocyte markers which are collagen III alpha-1 and tropomyosin1 alpha. Thus, deletion of GATA6 precluded endoderm differentiation but promoted mesoderm lineages. Conclusions. GATA4, GATA5, and GATA6 each convey a unique gene expression pattern and influences ES cell differentiation. We showed that ES cells can be directed to avoid differentiating into primitive endoderm and to adopt unique lineages in vitro by modulating GATA factors. The finding offers a potential approach to produce desirable cell types from ES cells, useful for regenerative cell therapy.

A Phosphotyrosine Proteomic Screen Identifies Multiple Tyrosine Kinase Signaling Pathways Aberrantly Activated in Malignant Mesothelioma.

Malignant mesothelioma (MM) is a highly aggressive cancer that is refractory to all current chemotherapeutic regimens. Therefore, uncovering new rational therapeutic targets is imperative in the field. Tyrosine kinase signaling pathways are aberrantly activated in many human cancers and are currently being targeted for chemotherapeutic intervention. Thus, we sought to identify tyrosine kinases hyperactivated in MM. An unbiased phosphotyrosine proteomic screen was employed to identify tyrosine kinases activated in human MM cell lines. From this screen, we have identified novel signaling molecules, such as JAK1, STAT1, cortactin (CTTN), FER, p130Cas (BCAR1), SRC and FYN as tyrosine phosphorylated in human MM cell lines. Additionally, STAT1 and SRC family kinases (SFK) were confirmed to be active in primary MM specimens. We also confirmed that known signal transduction pathways previously implicated in MM, such as EGFR and MET signaling axes, are co-activated in the majority of human MM specimens and cell lines tested. EGFR, MET, and SFK appear to be co-activated in a significant proportion of MM cell lines, and dual inhibition of these kinases was demonstrated to be more efficacious for inhibiting MM cell viability and downstream effector signaling than inhibition of a single tyrosine kinase. Consequently, these data suggest that TKI mono-therapy may not represent an efficacious strategy for the treatment of MM, due to multiple tyrosine kinases potentially signaling redundantly to cellular pathways involved in tumor cell survival and proliferation.

Altered gene expression in morphologically normal epithelial cells from heterozygous carriers of BRCA1 or BRCA2 mutations.

We hypothesized that cells bearing a single inherited "hit" in a tumor suppressor gene express an altered mRNA repertoire that may identify targets for measures that could delay or even prevent progression to carcinoma. We report here on the transcriptomes of primary breast and ovarian epithelial cells cultured from BRCA1 and BRCA2 mutation carriers and controls. Our comparison analyses identified multiple changes in gene expression, in both tissues for both mutations, which were validated independently by real-time reverse transcription-PCR analysis. Several of the differentially expressed genes had been previously proposed as cancer markers, including mammaglobin in breast cancer and serum amyloid in ovarian cancer. These findings show that heterozygosity for a mutant tumor suppressor gene can alter the expression profiles of phenotypically normal epithelial cells in a gene-specific manner; these detectable effects of "one hit" represent early molecular changes in tumorigenesis that may serve as novel biomarkers of cancer risk and as targets for chemoprevention.

Proteomic analyses of pancreatic cyst fluids.

OBJECTIVES: There are currently no diagnostic indicators that are consistently reliable, obtainable, and conclusive for diagnosing and risk-stratifying pancreatic cysts. Proteomic analyses were performed to explore pancreatic cyst fluids to yield effective diagnostic biomarkers. METHODS: We have prospectively recruited 20 research participants and prepared their pancreatic cyst fluids specifically for proteomic analyses. Proteomic approaches applied were as follows: (1) matrix-assisted laser-desorption-ionization time-of-flight mass spectrometry peptidomics with LC/MS/MS (HPLC-tandem mass spectrometry) protein identification; (2) 2-dimensional gel electrophoresis; (3) GeLC/MS/MS (tryptic digestion of proteins fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by LC/MS/MS). RESULTS: Sequencing of more than 350 free peptides showed that exopeptidase activities rendered peptidomics of cyst fluids unreliable; protein nicking by proteases in the cyst fluids produced hundreds of protein spots from the major proteins, making 2-dimensional gel proteomics unmanageable; GeLC/MS/MS revealed a panel of potential biomarker proteins that correlated with carcinoembryonic antigen (CEA). CONCLUSIONS: Two homologs of amylase, solubilized molecules of 4 mucins, 4 solubilized CEA-related cell adhesion molecules (CEACAMs), and 4 S100 homologs may be candidate biomarkers to facilitate future pancreatic cyst diagnosis and risk-stratification. This approach required less than 40 microL of cyst fluid per sample, offering the possibility to analyze cysts smaller than 1 cm in diameter.

One-hit effects in cancer: altered proteome of morphologically normal colon crypts in familial adenomatous polyposis.

We studied patients with Familial Adenomatous Polyposis (FAP) because they are virtually certain to develop colon cancer, and because much is known about the causative APC gene. We hypothesized that the inherited heterozygous mutation itself leads to changes in the proteome of morphologically normal crypts and the proteins that changed may represent targets for preventive and therapeutic agents. We determined the differential protein expression of morphologically normal colon crypts of FAP patients versus those of individuals without the mutation, using two-dimensional gel electrophoresis, mass spectrometry, and validation by two-dimensional gel Western blotting. Approximately 13% of 1,695 identified proteins were abnormally expressed in the morphologically normal crypts of APC mutation carriers, indicating that a colon crypt cell under the one-hit state is already abnormal. Many of the expression changes affect pathways consistent with the function of the APC protein, including apoptosis, cell adhesion, cell motility, cytoskeletal organization and biogenesis, mitosis, transcription, and oxidative stress response. Thus, heterozygosity for a mutant APC tumor suppressor gene alters the proteome of normal-appearing crypt cells in a gene-specific manner, consistent with a detectable one-hit event. These changes may represent the earliest biomarkers of colorectal cancer development, potentially leading to the identification of molecular targets for cancer prevention.

Molecular mechanisms of action of imatinib mesylate in human ovarian cancer: a proteomic analysis.

BACKGROUND: Imatinib mesylate (Gleevec, Novartis, Basel, Switzerland) is a small-molecule tyrosine kinase inhibitor with activity against ABL, BCR-ABL, c-KIT, and PDGFR alpha. Several clinical trials have evaluated the efficacy and safety of imatinib in patients with ovarian carcinoma who have persistent or recurrent disease following front-line platinum/taxane based chemotherapy. However, there is limited pre-clinical and clinical data on the molecular targets and action of imatinib in ovarian cancer. MATERIALS AND METHODS: Human ovarian cancer cells (A2780) were treated with imatinib mesylate for either 6 or 24 h. We employed a 2D (two-dimensional) gel electrophoresis and mass spectrometry-based proteomics approach to identify protein expression patterns and signaling pathways that were altered in response to imatinib. Cells were analyzed for PDGFR alpha and AKT expression, which were then correlated with imatinib sensitivity. RESULTS: Using 2D gel electrophoresis of overlapping pH ranges from pH 4 to 11, about 4,000 protein spots could be analyzed reproducibly. Proteins whose levels changed between twofold to 30 fold were grouped according to whether changes were in the same direction at both time points of treatment with respect to the control, or changed their levels only at one of the time points. CONCLUSION: Differentially regulated proteins following imatinib treatment of A2780 cells involved the regulation of actin cytoskeleton, metabolic pathways, cell cycle, cell proliferation, apoptosis, cell junctions, and signal transduction. Thus, exposure of cells to imatinib produces complex changes in the cell that require further investigation.

Minimally invasive techniques for the management of lumbar disc herniation.

Traditionally, minimally invasive techniques for surgical discectomy have been defined as smaller incisions, tubular retractors, microscopically assisted tissue dissection, and conservative removal of only extruded or sequestered nucleus pulposus with preservation of the annulus. The first truly minimally invasive technique was chymopapain dissolution of the nucleus pulposus. Other percutaneous techniques followed; however, none were as efficacious as the gold standard of microlumbar discectomy until endoscopically visualized methods evolved to allow visualized mechanical discectomy through the foramen. In experienced hands, such a technique is as effective as microlumbar discectomy and results in less surgical morbidity for herniations that are appropriate for this minimally invasive endoscopic surgical portal that completely avoids traumatizing the normal anatomy of the dorsal musculature and ligamentous structures.

Recombinant nucleases CEL I from celery and SP I from spinach for mutation detection.

BACKGROUND: The detection of unknown mutations is important in research and medicine. For this purpose, a mismatch-specific endonuclease CEL I from celery has been established as a useful tool in high throughput projects. Previously, CEL I-like activities were described only in a variety of plants and could not be expressed in an active form in bacteria. RESULTS: We describe expression of active recombinant plant mismatch endonucleases and modification of their activities. We also report the cloning of a CEL I ortholog from Spinacia oleracea (spinach) which we termed SP I nuclease. Active CEL I and SP I nucleases were expressed as C-terminal hexahistidine fusions and affinity purified from the cell culture media. Both recombinant enzymes were active in mutation detection in BRCA1 gene of patient-derived DNA. Native SP nuclease purified from spinach is unable to incise at single-nucleotide substitutions and loops containing a guanine nucleotide, but the recombinant SP I nuclease can cut at these sites. CONCLUSION: The insect cell-expressed CEL I orthologs may not be identical to their native counterparts purified from plant tissues. The present expression system should facilitate further development of CEL I-based mutation detection technologies.

Searchable high-resolution 2D gel proteome of the human colon crypt.

We seek alterations in protein patterns at the earliest possible step on the path to cancer, namely, in cells of the target tissue from normal persons versus the corresponding normally appearing cells from persons who are heterozygous for mutation in a tumor suppressor gene that predisposes strongly to carcinoma in that tissue. To begin a systematic comparison of the proteomes of cells from normal and from neoplastic colons, we have undertaken the isolation of human colon crypts that are derived from the normal-appearing mucosa of left (descending) colon of patients with sporadic colorectal cancer. Two-dimensional (2D) gel electrophoresis is a proteomic approach that excels in the resolution of protein isoforms. Here, we document the practicality of this approach with human samples using gels of three overlapping pH ranges. For the first time, about 800 nonredundant proteins and 900 isoforms from purified human colonic crypts were identified, permitting an assessment of the contributions of protein isoforms. These interactive, searchable, hyperlink-enabled proteome maps and gene ontology analyses will facilitate future studies to discover the earliest markers and intervention targets during progression to colon cancer.