Characterization of digestive proteases in the gut of a basal deuterostome.

Digestive systems are complex organs that allow organisms to absorb energy from their environment to fuel vital processes such as growth, development and the maintenance of homeostasis. A comprehensive understanding of digestive physiology is therefore essential to fully understand the energetics of an organism. The digestion of proteins is of particular importance because most heterotrophic organisms are not able to synthesize all essential amino acids. While Echinoderms are basal deuterostomes that share a large genetic similarity with vertebrates, their digestion physiology remains largely unexplored. Using a genetic approach, this work demonstrated that several protease genes including an enteropeptidase, aminopeptidase, carboxypeptidase and trypsin involved in mammalian digestive networks are also found in sea urchin larvae. Through characterization including perturbation experiments with different food treatments and pharmacological inhibition of proteases using specific inhibitors, as well as transcriptomic analysis, we conclude that the trypsin-2 gene codes for a crucial enzyme for protein digestion in Strongylocentrotus purpuratus. Measurements of in vivo digestion rates in the transparent sea urchin larva were not altered by pharmacological inhibition of trypsin (using soybean trypsin inhibitor) or serine proteases (aprotinin), suggesting that proteases are not critically involved in the initial step of microalgal breakdown. This work provides new insights into the digestive physiology of a basal deuterostome and allows comparisons from the molecular to the functional level in the digestive systems of vertebrates and mammals. This knowledge will contribute to a better understanding for conserved digestive mechanisms that evolved in close interaction with their biotic and abiotic environment.

Extracellular carbonic anhydrase activity promotes a carbon concentration mechanism in metazoan calcifying cells.

Many calcifying organisms utilize metabolic CO2 to generate CaCO3 minerals to harden their shells and skeletons. Carbonic anhydrases are evolutionary ancient enzymes that have been proposed to play a key role in the calcification process, with the underlying mechanisms being little understood. Here, we used the calcifying primary mesenchyme cells (PMCs) of sea urchin larva to study the role of cytosolic (iCAs) and extracellular carbonic anhydrases (eCAs) in the cellular carbon concentration mechanism (CCM). Molecular analyses identified iCAs and eCAs in PMCs and highlight the prominent expression of a glycosylphosphatidylinositol-anchored membrane-bound CA (Cara7). Intracellular pH recordings in combination with CO2 pulse experiments demonstrated iCA activity in PMCs. iCA activity measurements, together with pharmacological approaches, revealed an opposing contribution of iCAs and eCAs on the CCM. H+-selective electrodes were used to demonstrate eCA-catalyzed CO2 hydration rates at the cell surface. Knockdown of Cara7 reduced extracellular CO2 hydration rates accompanied by impaired formation of specific skeletal segments. Finally, reduced pHi regulatory capacities during inhibition and knockdown of Cara7 underscore a role of this eCA in cellular HCO3- uptake. This work reveals the function of CAs in the cellular CCM of a marine calcifying animal. Extracellular hydration of metabolic CO2 by Cara7 coupled to HCO3- uptake mechanisms mitigates the loss of carbon and reduces the cellular proton load during the mineralization process. The findings of this work provide insights into the cellular mechanisms of an ancient biological process that is capable of utilizing CO2 to generate a versatile construction material.

An otopetrin family proton channel promotes cellular acid efflux critical for biomineralization in a marine calcifier.

Otopetrins comprise a family of proton-selective channels that are critically important for the mineralization of otoliths and statoconia in vertebrates but whose underlying cellular mechanisms remain largely unknown. Here, we demonstrate that otopetrins are critically involved in the calcification process by providing an exit route for protons liberated by the formation of CaCO3 Using the sea urchin larva, we examined the otopetrin ortholog otop2l, which is exclusively expressed in the calcifying primary mesenchymal cells (PMCs) that generate the calcitic larval skeleton. otop2l expression is stimulated during skeletogenesis, and knockdown of otop2l impairs spicule formation. Intracellular pH measurements demonstrated Zn2+-sensitive H+ fluxes in PMCs that regulate intracellular pH in a Na+/HCO3--independent manner, while Otop2l knockdown reduced membrane proton permeability. Furthermore, Otop2l displays unique features, including strong activation by high extracellular pH (>8.0) and check-valve-like outwardly rectifying H+ flux properties, making it into a cellular proton extrusion machine adapted to oceanic living conditions. Our results provide evidence that otopetrin family proton channels are a central component of the cellular pH regulatory machinery in biomineralizing cells. Their ubiquitous occurrence in calcifying systems across the animal kingdom suggest a conserved physiological function by mediating pH at the site of mineralization. This important role of otopetrin family proton channels has strong implications for our view on the cellular mechanisms of biomineralization and their response to changes in oceanic pH.

Na+/H+ exchangers differentially contribute to midgut fluid sodium and proton concentration in the sea urchin larva.

Regulation of ionic composition and pH is a requisite of all digestive systems in the animal kingdom. Larval stages of the marine superphylum Ambulacraria, including echinoderms and hemichordates, were demonstrated to have highly alkaline conditions in their midgut with the underlying epithelial transport mechanisms being largely unknown. Using ion-selective microelectrodes, the present study demonstrated that pluteus larvae of the purple sea urchin have highly alkaline pH (pH âˆ¼9) and low [Na+] (∼120 mmol l-1) in their midgut fluids, compared with the ionic composition of the surrounding seawater. We pharmacologically investigated the role of Na+/H+ exchangers (NHE) in intracellular pH regulation and midgut proton and sodium maintenance using the NHE inhibitor 5-(n-ethyl-n-isopropyl)amiloride (EIPA). Basolateral EIPA application decreased midgut pH while luminal application via micro-injections increased midgut [Na+], without affecting pH. Immunohistochemical analysis demonstrated a luminal localization of NHE-2 (SpSlc9a2) in the midgut epithelium. Specific knockdown of spslc9a2 using Vivo-Morpholinos led to an increase in midgut [Na+] without affecting pH. Acute acidification experiments in combination with quantitative PCR analysis and measurements of midgut pH and [Na+] identified two other NHE isoforms, Spslc9a7 and SpSlc9a8, which potentially contribute to the regulation of [Na+] and pH in midgut fluids. This work provides new insights into ion regulatory mechanisms in the midgut epithelium of sea urchin larvae. The involvement of NHEs in regulating pH and Na+ balance in midgut fluids shows conserved features of insect and vertebrate digestive systems and may contribute to the ability of sea urchin larvae to cope with changes in seawater pH.

Cytomegalovirus-vectored vaccines for HIV and other pathogens.

: The use of cytomegalovirus (CMV) as a vaccine vector to express antigens against multiple infectious diseases, including simian immunodeficiency virus, Ebola virus, plasmodium, and mycobacterium tuberculosis, in rhesus macaques has generated extraordinary levels of protective immunity against subsequent pathogenic challenge. Moreover, the mechanisms of immune protection have altered paradigms about viral vector-mediated immunity against ectopically expressed vaccine antigens. Further optimization of CMV-vectored vaccines, particularly as this approach moves to human clinical trials will be augmented by a more complete understanding of how CMV engenders mechanisms of immune protection. This review summarizes the particulars of the specific CMV vaccine vector that has been used to date (rhesus CMV strain 68-1) in relation to CMV natural history.

Factors influencing long-term weight loss after bariatric surgery.

BACKGROUND: Bariatric surgery provides sustained weight loss and improves comorbidities. However, long term data has shown that patients gradually regain weight after 1 year. Several factors have been associated with poor weight loss after bariatric surgery. OBJECTIVE: Our goal is to investigate factors associated with poor weight loss following laparoscopic sleeve gastrectomy (SG) and Roux-en-Y gastric bypass (RYGB). SETTING: Military academic medical center. METHODS: Retrospective review of 247 patients who underwent laparoscopic SG or RYGB between 2010-2012 at Eisenhower Army Medical Center and followed for 5 years postoperatively. Factors of age, type of surgery, sex, hypertension, depression, and type 2 diabetes (T2D) are analyzed in univariate and multivariate analysis with percent total weight loss (%TWL) and Body Mass Index (BMI) change as primary endpoints measured at 3 and 5 years. RESULTS: Average BMI change are maximized at 1 year and decreased at 3 and 5 years post-surgery. Age, diabetes, hypertension and type of surgery significantly influenced weight loss at 3 and 5 years on univariate analysis. However, patients with diabetes, hypertension and sleeve gastrectomy were significantly older than comparable control group. Multivariable analysis showed that age and type of surgery, not diabetes or hypertension, were associated with poor %TWL and BMI change at 3 and 5 years. CONCLUSION: While presence of hypertension and diabetes initially appeared to be associated with weight recidivism, their impacts were negligible on multivariable analysis. However, age and sleeve gastrectomy are independent risk factors. Our data can be used to counsel patients on expected weight loss after bariatric surgery.

Activation of cyclic adenosine monophosphate-dependent protein kinase a signaling prevents liver ischemia/reperfusion injury in mice.

Hepatic ischemia/reperfusion injury (IRI) occurs in multiple clinical settings, including liver transplantation. The cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) pathway inhibits hepatocellular apoptosis and regulates toll-like receptor 4-triggered inflammation responses in vitro. Here we examined the function and therapeutic potential of cAMP-PKA activation in a murine (C57/BL6) model of liver warm ischemia (90 minutes) followed by reperfusion. Liver IRI triggered cAMP-PKA activation, whereas the administration of its specific inhibitor, H89, exacerbated hepatocellular damage. Conversely, forskolin therapy, which activates PKA by elevating cAMP levels, protected livers from IRI; this was evidenced by diminished serum alanine aminotransferase levels and well-preserved tissue architecture. Liver protection due to cAMP-PKA stimulation was accompanied by diminished neutrophil and macrophage infiltration/activation, reduced hepatocyte necrosis/apoptosis, and increased cAMP response element-binding protein (CREB) expression and augmented interleukin-10 (IL-10) expression. The neutralization of IL-10 restored liver damage in otherwise ischemia/reperfusion-resistant, forskolin-treated mice. In vitro, cAMP-PKA activation diminished macrophage tumor necrosis factor α, IL-6, and IL-12 in an IL-10-dependent manner and prevented necrosis/apoptosis in primary mouse hepatocyte cultures. Our novel findings in a mouse model of liver IRI document the importance of cAMP-PKA signaling in hepatic homeostasis and cytoprotection in vivo. The activation of cAMP-PKA signaling differentially regulates local inflammation and prevents hepatocyte death, and this provides a rationale for novel therapeutic approaches to combating liver IRI in transplant recipients.

Accuracy in quantification of coronary calcification with CT: a cork-dog heart phantom study.

RATIONALE AND OBJECTIVES: Coronary artery calcium is a sensitive risk predictor of cardiac events. However, measurement of calcium foci is affected by partial-volume effects, which ultimately have an effect on accuracy and reproducibility of calcium scores. In this study, we describe the accuracy of quantification of calcium foci of known size and density using cork-dog heart phantoms. MATERIALS AND METHODS: Five study phantoms were constructed from cork chests and dog hearts containing 135 calcium hydroxyapatite (CaHA) foci of known volume, mass, and concentration located in the coronary arteries or the myocardium. Hearts were separated into two groups: (1) three hearts containing large, high-density foci and (2) two hearts containing small, low-density foci. The phantoms were scanned using a standard coronary artery calcium (CAC) protocol and the volume and mean intensity of foci were measured. RESULTS: In group 1, the total volume of 87 CaHA foci measured was 4284 and 3779 mm(3) with electron beam computed tomography (EBCT); multidetector computed tomography (MDCT), respectively (P < .001). Both were significantly larger than the true volume (2713.9 mm(3), P < .001). In Group 2, the total volume of 57 CaHA foci measured was 592.6 and 702.9 mm(3) with EBT and MDCT, respectively (P < .001). Both were significantly smaller than the true volume (1733.2 mm(3), P < .001). We found that EBCT values for volume were approximately generally higher than MDCT values, but strongly correlated (r = 0.95, P < .0001). Agatston scores were found to be nearly equivalent between EBCT and MDCT and were similarly strongly correlated (r = 0.97, P < .0001). CONCLUSIONS: Computed tomography images overestimate the volume of large, dense CaHA foci while underestimating the volume of smaller (<6.6 mm(3)), less dense foci. This may have significant implications on CAC scoring and volume measurement. EBCT overestimated calcium more than MDCT, most likely from increased image noise.

Cost-effectiveness of multidetector computed tomography compared with myocardial perfusion imaging as gatekeeper to invasive coronary angiography in asymptomatic firefighters with positive treadmill tests.

BACKGROUND: In a prospective evaluation of 3950 Los Angeles County firefighters who underwent wellness/fitness examinations, 495 firefighters had abnormal treadmill tests and were referred for cardiology evaluation. Cost of the traditional myocardial perfusion imaging (MPI) followed by invasive coronary angiography (ICA) was compared with a method incorporating 64-slice multidetector computed tomography (MDCT) with coronary calcium score (CCS) followed by computed tomographic angiography (CTA) and ICA as indicated. OBJECTIVE: We compared the costs of 2 methods of predicting coronary artery disease (CAD) by ICA among asymptomatic patients with positive treadmill tests. METHODS: A decision-analytic framework was used to compare the net direct costs of CAD diagnosis associated with MDCT versus MPI. In the MDCT arm, all received CCS followed by CTA for those with calcium scores>10 and ICA for those with > or =50% stenosis on CTA. For the MPI arm, results were estimated from prior years' experience, in which firefighters with abnormal treadmill results were referred to ICA. RESULTS: Of 495 firefighters, 131 (26.9%) had abnormal CCS and went to CTA; 40 (8.1%) had > or =50% stenosis on CTA and went to ICA. According to prior years' experience with MPI, 146 (29.5%) would have shown abnormalities requiring ICA. Average cost was $1376/person for MPI versus $503/person for CCS with or without CTA as gatekeeper. All sensitivity analyses showed lower costs for the MDCT pathway compared with MPI. CONCLUSION: In this firefighter population, the cost of ICA-confirmed diagnosis of CAD is substantially lower with MDCT as gatekeeper than with MPI.

Improved CZE capabilities with new dynamic coatings.

Electroosmotic flow (EOF) is one of the fundamental processes affecting both resolution and separation times in capillary zone electrophoresis (CZE). EOF is a function of pH and buffer composition (zeta potential) in bare-silica capillaries at any pH above 3 and runs in the cathodal direction, decreasing the effective separation length for cationic species. On the other hand, the absence of EOF at low pH can significantly increase separation times, particularly for low pl zwitterionic species. This interdependence of pH and EOF limits the ability to design effective separations by capillary zone electrophoresis. The EOTrol family of dynamic coatings (Target Discovery, Inc., Palo Alto, CA, U.S.A.) allows both the magnitude and direction of EOF to be adjusted independently of the buffer composition and pH. Here we report the use of EOTrol in two challenging CZE separations: (1) inorganic cations with small mobility differences, and (2) the rapid separation of organic zwitterions that are only resolvable at low pH.

Extra copy of 20q deletion, acute myelomonocytic leukemia (M4) and Niemann-Pick disease.

A 59-year-old hypertensive white male was diagnosed with acute myelogenous leukemia (AML), M4. A bone marrow aspirate showed a karyotype of 46,XY,del(20)(q11.2q13.3)[12]/ 47,XY,del(20)(q11.2q13.3)x2[8]. The majority of cases with 20q deletion are associated with myeloid disorders; however, an extra copy of the 20q deletion has rarely been reported. The patient expired seven days after admission to the hospital. At autopsy hepatosplenomegaly was present. Many foamy macrophages with bubbling cytoplasm in the spleen, liver, bone marrow and lymph nodes were suggestive of Niemann-Pick disease, type E. AML has not previously been reported with Niemann-Pick disease.

Rhabdomyosarcoma of tunica vaginalis masquerading as hydrocele.

Paratesticular rhabdomyosarcomas are rare tumors with aggressive growth patterns. Multimodal therapy with surgery, chemotherapy, and radiotherapy provides the patient with an excellent long-term prognosis. These tumors often present in the first two decades after birth. We report on the case of an 18-year-old man with a paratesticular rhabdomyosarcoma.

Rapid separation of protein isoforms by capillary zone electrophoresis with new dynamic coatings.

Many cellular functions are regulated through protein isoforms. Changes in the expression level or regulatory dysfunctions of isoforms often lead to developmental or pathological disorders. Isoforms are traditionally analyzed using techniques such as gel- or capillary-based isoelectric focusing. However, with proper electro-osmotic flow (EOF) control, isoforms with small pI differences can also be analyzed using capillary zone electrophoresis (CZE). Here we demonstrate the ability to quickly resolve isoforms of three model proteins (bovine serum albumin, transferrin, alpha1-antitrypsin) in capillaries coated with novel dynamic coatings. The coatings allow reproducible EOF modulation in the cathodal direction to a level of 10(-9) m2V(-1)s(-1). They also appear to inhibit protein adsorption to the capillary wall, making the isoform separations highly reproducible both in peak areas and apparent mobility. Isoforms of transferrin and alpha1-antitrypsin have been implicated in several human diseases. By coupling the CZE isoform separation with standard affinity capture assays, it may be possible to develop a cost-effective analytical platform for clinical diagnostics.

Identification of a novel alternative splicing variant of RGS5 mRNA in human ocular tissues.

Regulator of G protein signaling (RGS) proteins act as GTPase-activating proteins (GAPs) for Galpha subunits and negatively regulate G protein-coupled receptor signaling. Using RGS5 gene-specific RT-PCR, we have identified a novel alternative splicing variant of RGS5 mRNA in human ocular tissues. The alternative splicing of RGS5 mRNA occurred at position +44 (GenBank NM_003617), spliced out 174 bp (+44 to +218 bp) of the coding region, and encoded an RGS5s protein with a 108 amino acid N-terminal deletion. This study is the first to document alternative splicing of an RGS5 gene. We therefore studied RGS5 and RGS5s mRNA distribution in human tissues. In the eye, RGS5s was found to be highly expressed in the ciliary body and trabecular meshwork. It was also expressed in the kidney, brain, spleen, skeletal muscle and small intestine, but was not detectable in the liver, lung, heart. RGS5s was not found in monkey and rat ocular tissues, indicating species specificity for the eye. Comparing the recombinant RGS5 and RGS5s expression in HEK293/EBNA cells, RGS5s was present almost exclusively in the cytosolic fraction, whereas RGS5 was present in both membrane and cytosolic fractions. The data suggest that the N-terminal of RGS5 may be important for protein translocation to the cell membrane. Both RGS5 and RGS5s antagonized the rapid phosphorylation of p44/42 MAP kinase induced by Galphai coupled cannibinoid receptor-1 activation. RGS5, but not RGS5s, inhibited the Ca2+ signaling initiated by activation of Galphaq coupled angiotensin II receptors (AT1) and prostaglandin FP receptors. Cotransfection of RGS5s with RGS5 resulted in the blockade of RGS5 actions with respect to inhibition of the signal transduction initiated by activation of both AT1 and FP receptor, suggesting that RGS5s may contain functional domains that compete with RGS5 in the regulation of the Galphaq coupled AT1 and FP receptors. The unique expression pattern, cellular localization and functions of RGS5s suggest that RGS5s may play a critical role in the regulation of intracellular signaling pathways.

Axillary lymph node metastasis following resection of abdominal wall laparoscopic port site recurrence of gallbladder cancer.

Abdominal wall port site recurrence of gallbladder cancer is well described in the literature in patients that have undergone laparoscopic cholecystectomy with the incidental finding of a gallbladder cancer. The etiology and consequences of this type of metastatic recurrence are unclear. This report describes two cases with the unique sequelae of the interval development of nodal metastases to the axillary lymph nodes following resection of an abdominal wall laparoscopic port site recurrence of gallbladder cancer. The first case involves a patient who developed an isolated left axillary lymph node metastasis approximately 10 months after undergoing resection of a left-sided abdominal wall port site recurrence for a T2 gallbladder cancer. The original tumor had been found at laparoscopic cholecystectomy and definitively treated surgically approximately 3 years earlier. The second case involves a patient who developed isolated nodal metastases to the right axillary lymph nodes approximately 4 months after undergoing resection of right-sided abdominal wall port site recurrence, segment 4/5 hepatic resection, and portal lymphadenectomy for a T2 gallbladder cancer. This tumor had originally been found at laparoscopic cholecystectomy approximately 1 year earlier. These unique sequelae of the interval development of nodal metastases to the axillary lymph nodes demonstrated in both cases has not been previously reported.

Upregulation of orphan nuclear receptor Nur77 following PGF(2alpha), Bimatoprost, and Butaprost treatments. Essential role of a protein kinase C pathway involved in EP(2) receptor activated Nur77 gene transcription.

1. Using gene chip technology, we first identified that PGF(2alpha) (FP agonist) and Butaprost (EP(2) agonist) induced about a five-fold upregulation of Nur77 mRNA expression in hFP-HEK 293/EBNA and hEP(2)-HEK293/EBNA cells. Northern Blot analysis revealed that PGF(2alpha)- and Butaprost-induced upregulation of Nur77 expression are dose- and time-dependent. 2. Both PGF(2alpha) and Butaprost upregulated Nur77 gene expression through the protein kinase C (PKC) pathway. These data are the first showing a link between EP(2) receptor stimulation and protein kinase C activation. Calcineurin was found to be involved downstream of the PKC pathway in PGF(2alpha)-induced Nur77 expression, but not in Butaprost-induced Nur77 expression. 3. We also used Nur77 as a marker gene to compare the effects of PGF(2alpha), Butaprost, and Bimatoprost (a prostamide) on Nur77 expression in human primary trabecular meshwork and ciliary smooth muscle (SM) cells, which are target cells for antiglaucoma drugs. The results showed that PGF(2alpha) and Butaprost, but not Bimatoprost, induced upregulation of Nur77 expression in human TM cells. PGF(2alpha), but not Bimatoprost, dramatically induced upregulation of Nur77 mRNA expression in human ciliary SM cells, whereas Butaprost slightly upregulated Nur77 mRNA expression in SM cells. 4. Nur77 promoter deletion analysis indicated that PGF(2alpha), but not Bimatoprost, activated Nur77 promoter-luciferase reporter in hFP-HEK 293/EBNA cells. Butaprost was less efficacious in inducing Nur77 promoter-luciferase reporter activity in hEP(2)-HEK293/EBNA cells relative to PGF(2alpha) in the comparable assay. The data for Nur77 promoter functional analysis were matched to the Northern blot analysis. 5. It appears that PGF(2alpha) and Butaprost activate Nur77 transcription mechanisms through the activation of FP and EP(2) receptor-coupled signaling pathways, whereas Bimatoprost stimulates neither FP nor EP(2) receptors.