Quantitative profiling of protease specificity.

Proteases are an important class of enzymes, whose activity is central to many physiologic and pathologic processes. Detailed knowledge of protease specificity is key to understanding their function. Although many methods have been developed to profile specificities of proteases, few have the diversity and quantitative grasp necessary to fully define specificity of a protease, both in terms of substrate numbers and their catalytic efficiencies. We have developed a concept of "selectome"; the set of substrate amino acid sequences that uniquely represent the specificity of a protease. We applied it to two closely related members of the Matrixin family-MMP-2 and MMP-9 by using substrate phage display coupled with Next Generation Sequencing and information theory-based data analysis. We have also derived a quantitative measure of substrate specificity, which accounts for both the number of substrates and their relative catalytic efficiencies. Using these advances greatly facilitates elucidation of substrate selectivity between closely related members of a protease family. The study also provides insight into the degree to which the catalytic cleft defines substrate recognition, thus providing basis for overcoming two of the major challenges in the field of proteolysis: 1) development of highly selective activity probes for studying proteases with overlapping specificities, and 2) distinguishing targeted proteolysis from bystander proteolytic events.

Comparative expression of immunohistochemical biomarkers in cribriform and pattern 4 non-cribriform prostatic adenocarcinoma.

The morphology of Gleason 4 prostate cancer (PCa) can be subdivided into cribriform and non-cribriform patterns. A large body of evidence has shown that pattern 4 cribriform PCa (especially non-glomeruloid type) is associated with adverse pathologic features and clinical outcomes compared with non-cribriform pattern 4 PCa. The underlying mechanisms for the aggressiveness of cribriform PCa are not fully understood. The aim of this study is to compare the immunohistochemical expression of various biomarkers and to determine the potential proteins that may account for their biologic and clinical differences. A total of 14 biomarkers were studied. The number of non-glomeruloid cribriform PCa cases studied for each biomarker ranged from 18 to 74 and the number of non-cribriform pattern 4 PCa studied for each biomarker ranged from 29 to 112. We demonstrated that, compared with non-cribriform Gleason pattern 4 PCa, EGFR was significantly upregulated and standard CD44 (CD44s) was significantly downregulated in cribriform PCa; no significant differences were found in the expression of AR, NKX3.1, ERG, EZH2, p53, Rb, C-Myc, BCL2, p16, CyclinD1, Her2/Neu, and Synaptophysin between these two groups of pattern 4 PCa. The study also showed, compared to non-cribriform PCa, cribriform PCa presented with significantly higher serum PSA and more advanced tumor stage. The significant overexpression of EGFR and downregulation of CD44s in non-glomeruloid cribriform PCa may, at least, partly explain the unfavorable pathology and clinical results for this growth pattern. Given that EGFR targeted inhibitors are now available, the findings may also have significant therapeutic implications.

The Effect of Pulsed Electric Field on Expression of ECM proteins: Collagen, Elastin, and MMP1 in Human Dermal Fibroblasts.

Electrical stimulation of tissues has many uses in pain management, antibacterial treatment, and wound healing. The electric field stimulates epidermal migration and increases fibroblast cell proliferation. Here we show the effects of electrical field (EF) stimulation of human dermal fibroblasts (HDF) on the expression of collagen, elastin, and collagenase (MMP1; matrix metalloproteinase 1). The effects of EF stimulation are evaluated in terms of changes in cell morphology and extracellular matrix (ECM) protein expression, defined as intracellular concentration of collagen, elastin, and MMP1. HDF are stimulated in a bioreactor using square wave voltage pulses for up to 24 h. The pulse voltage (0-10V), pulse bias (0, +), pulse time (10-1000 ms), and rest time (0.1-10 s) were varied. We show that expression of collagen, elastin, and MMP1 increases in response to applied EF. The intracellular concentration of ECM proteins more than doubles depending on stimulation conditions with a threshold of effective stimulation above 3V/cm. The short time voltage pulses used for EF stimulation are more effective, while the rest time between pulses has a small effect on intracellular concentration of collagen, MMP1 and elastin. The previously studied HDF stimulation with chemical factors (i.e. TNF-α, TGF-ß) shows negative correlation between concentration of collagen and MMP1. Contrary to that observation, we show that EF stimulation causes increase in the intracellular concentration of both collagen and MMP1. We also demonstrate that the transdermal stimulation of HDF in subcutaneous tissue is possible, thus it might be utilized in the future to improve the wound healing and tissue regeneration process.

Diagnosis of paraganglioma as a pancreatic mass: A case report.

Paragangliomas are rare neoplasms that arise from the chromaffin cells of the autonomic nervous system. Although paragangliomas can occur anywhere paraganglia are present, they tend to occur in the head, neck, and retroperitoneum. Rarely, paragangliomas can occur in the peripancreatic area and present as a pancreatic mass, creating a diagnostic challenge for the clinician, radiologist, and pathologist. Here, we present a case of a 70-year-old woman with history of breast carcinoma who presented with chronic constipation, early satiety, and an abdominal mass. Her first abdominal CT described a 3.6 cm × 5 cm × 4.5 cm cystic and solid mass involving the pancreatic tail that was suspicious for a pancreatic neoplasm. A subsequent abdominal CT described a 5.9 cm multilobulated solid and cystic lesion close to the pancreatic tail. Endoscopic ultrasound-guided fine-needle aspirate of the mass demonstrated scant to moderate cellularity of a heterogeneous population of atypical cells, some with epithelioid morphology and others appearing neuroendocrine-like. By morphology and immunohistochemical stains, an extra-adrenal paraganglioma or pheochromocytoma was considered as a possible diagnosis. The surgical resection specimen confirmed the diagnosis of paraganglioma. This case demonstrates the importance of awareness of paragangliomas in the differential diagnosis of a fine-needle aspiration of a pancreatic mass to avoid erroneous diagnosis.

An interesting case of profound hypoxemia.

A 58 year old male with a history of cirrhosis (hepatitis B and C), a long smoking history, and a recently diagnosed high-grade transitional cell carcinoma of the bladder wall presented three days after a biopsy procedure with abdominal pain, nausea, and new hypoxemia on room air. The chest radiograph was clear and the CT angiogram showed only a borderline large pulmonary artery, two small nodules (3mm and 4mm) in the right middle lobe of the lung, and emphysematous changes throughout the lung parenchyma. There was no evidence of pulmonary embolism. A wide range of diagnostic possibilities were entertained, including pneumonia (community or aspiration related to the procedure), COPD exacerbation, pulmonary emboli, porto-pulmonary syndrome, pulmonary hypertension with right to left shunt, tumor emboli, allergic reaction to a medication or chemotherapeutic agent, or lymphangitic/hematogenous spread of tumor to the lungs. The diagnosis was only established on a post mortem examination. The progressive hypoxia was due to diffuse spread of tumor within alveolar capillaries.

Lepidic predominant adenocarcinoma with aerogenous spread of mucin in a young patient -- a case report.

We present a unique case of a 26 year-old female non-smoker who expired following treatment for presumed pneumonias. At autopsy, lepidic predominant adenocarcinoma with aerogenous spread of mucin without evidence of invasion, a rare diagnosis that previously would have fallen under the umbrella of "bronchioloalveolar carcinoma," was found. Histopathology showed mucin-secreting neoplastic cells lining the alveolar walls, as well as exfoliated and dense aggregates of mucinous debris filling the alveoli. The immediate cause of death was respiratory failure, most likely due to the significant amount of tumor-produced mucin that filled the alveolar spaces, which literally drowned the patient.