Histological improvements following energy restriction and exercise: The role of insulin resistance in resolution of MASH.

BACKGROUND & AIMS: Metabolic-dysfunction associated steatohepatitis (MASH) is one of the most common liver diseases worldwide and is characterized by multi-tissue insulin resistance. The effects of a 10-month energy restriction and exercise intervention on liver histology, anthropometrics, plasma biochemistries, and insulin sensitivity were compared to standard of care (control) to understand mechanisms that support liver health improvements. METHODS: Following medical diagnosis of MASH, subjects were randomized to treatment (n=16) or control (n=8). Liver fat (MRS), 18-hour plasma biochemical measurements, and isotopically-labeled hyperinsulinemic-euglycemic clamps were completed pre- and post-intervention. Body composition and cardiorespiratory fitness (VO2peak) were also measured mid-intervention. Treatment subjects were counseled to reduce energy intake and completed supervised, high-intensity interval training (3x/week) for 10 months. Control subjects continued physician-directed care. RESULTS: Treatment induced significant (P<0.05) reductions in body weight, fat mass, and liver injury, while VO2peak (P<0.05) and fatty acid (NEFA) suppression (P=0.06) were improved. Both groups exhibited reductions in total energy intake, HbA1c, hepatic insulin resistance, and liver fat (P<0.05). Compared to control, treatment induced a two-fold increase in peripheral insulin sensitivity which was significantly related to higher VO2peak and resolution of liver disease, despite no group differences in peripheral insulin sensitivity. CONCLUSIONS: Exercise and energy-restriction elicited significant and clinically-meaningful treatment effects on liver health, potentially driven by a redistribution of excess nutrients to skeletal muscle, thereby reducing hepatic nutrient toxicity. Clinical guidelines should emphasize the addition of aerobic exercise in lifestyle treatments for the greatest histologic benefit in individuals with advanced MASH. CLINICAL TRIAL NUMBER: NCT03151798.

Isotope Labeling and Biochemical Assessment of Liver-Triacylglycerol in Patients with Different Levels of Histologically-Graded Liver Disease.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) prevalence is rapidly growing, and fatty liver has been found in a quarter of the US population. Increased liver lipids, particularly those derived from the pathway of de novo lipogenesis (DNL), have been identified as a hallmark feature in individuals with high liver fat. This has led to much activity in basic science and drug development in this area. No studies to date have investigated the contribution of DNL across a spectrum of disease, although it is clear that inhibition of DNL has been shown to reduce liver fat. OBJECTIVES: The purpose of this study was to determine whether liver lipid synthesis increases across the continuum of liver injury. METHODS: Individuals (n = 49) consumed deuterated water for 10 d before their scheduled bariatric surgeries to label DNL; blood and liver tissue samples were obtained on the day of the surgery. Liver lipid concentrations were quantitated, and levels of protein and gene expression assessed. RESULTS: Increased liver DNL, measured isotopically, was significantly associated with liver fatty acid synthase protein content (R = 0.470, P = 0.003), total steatosis assessed by histology (R = 0.526, P = 0.0008), and the fraction of DNL fatty acids in plasma very low-density lipoprotein-triacylglycerol (R = 0.747, P < 0.001). Regression analysis revealed a parabolic relationship between fractional liver DNL (percent) and NAFLD activity score (R = 0.538, P = 0.0004). CONCLUSION: These data demonstrate that higher DNL is associated with early to mid stages of liver disease, and this pathway may be an effective target for the treatment of NAFLD and nonalcoholic steatohepatitis. This study was registered at clinicaltrials.gov as NCT03683589.

Labeled breath tests in patients with NASH: Octanoate oxidation relates best to measures of glucose metabolism.

In vivo methods to estimate human liver mitochondrial activity are lacking and this project's goal was to use a non-invasive breath test to quantify complete mitochondrial fat oxidation and determine how test results changed when liver disease state was altered over time. Patients with suspected non-alcoholic fatty liver disease (NAFLD; 9 men, 16 women, 47 ± 10 years, 113 ± 23 kg) underwent a diagnostic liver biopsy and liver tissue was histologically scored by a pathologist using the NAFLD activity score (0-8). To assess liver oxidation activity, a labeled medium chain fatty acid was consumed orally (23.4 mg 13C4-octanoate) and breath samples collected over 135 min. Total CO2 production rates were measured using breath 13CO2 analysis by isotope ratio mass spectrometry. Fasting endogenous glucose production (EGP) was measured using an IV infusion of 13C6-glucose. At baseline, subjects oxidized 23.4 ± 3.9% (14.9%-31.5%) of the octanoate dose and octanoate oxidation (OctOx) was negatively correlated with fasting plasma glucose (r = -0.474, p = 0.017) and EGP (r = -0.441, p = 0.028). Twenty-two subjects returned for repeat tests 10.2 ± 1.0 months later, following lifestyle treatment or standardized care. OctOx (% dose/kg) was significantly greater across all subjects (p = 0.044), negatively related to reductions in EGP (r = -0.401, p = 0.064), and tended to correlate with reduced fasting glucose (r = -0.371, p = 0.090). Subjects exhibited reductions in steatosis (p = 0.007) which tended to correlate with increased OctOx (% of dose/kg, r = -0.411, p = 0.058). Based on our findings, the use of an 13C-octanoate breath test may be an indicator of hepatic steatosis and glucose metabolism, but these relationships require verification through larger studies in NAFLD populations.

The Utility and Diagnostic Accuracy of Transient Elastography in Adults with Morbid Obesity: A Prospective Study.

Patients with morbid obesity are at high risk for nonalcoholic fatty liver disease (NAFLD) complicated by liver fibrosis. The clinical utility of transient elastography (TE) by Fibroscan in patients with morbid obesity (body mass index (BMI) ≥ 40 kg/m2) is not well-defined. We examined the diagnostic accuracy of Fibroscan in predicting significant liver fibrosis (fibrosis stage ≥2) in morbidly obese patients (BMI ≥ 40 kg/m2). Patients scheduled for bariatric surgery were prospectively enrolled. Intraoperative liver biopsy, liver-stiffness measurement (LSM) by Fibroscan (XL probe), and biochemical evaluation were all performed on the same day. The endpoint was significant liver fibrosis defined as fibrosis stage ≥2 based on the Nonalcoholic Steatohepatitis Clinical Research Network. The optimal LSM cutoff value for detecting significant fibrosis was determined by using the Youden Index method. Routine clinical, laboratory, and elastography data were analyzed by stepwise logistic regression analysis to identify predictors of significant liver fibrosis and build a predictive model. An optimal cutoff point of the new model's regression formula for predicting significant fibrosis was determined by using the Youden index method. One hundred sixty-seven patients (mean age, 46.4 years) were included, of whom 83.2% were female. Histological assessment revealed the prevalence of steatohepatitis and significant fibrosis of 40.7% and 11.4%, respectively. The median LSM was found to be significantly higher in the significant fibrosis group compared to those in the no or non-significant fibrosis group (18.2 vs. 7.7 kPa, respectively; p = 0.0004). The optimal LSM cutoff for predicting significant fibrosis was 12.8 kPa, with an accuracy of 71.3%, sensitivity of 73.7%, specificity of 70.9%, positive predictive value of 24.6%, negative predictive value of 95.5%, and ROC area of 0.723 (95% CI: 0.62-0.83). Logistic regression analysis identified three independent predictors of significant fibrosis: LSM, hemoglobin A1c, and alkaline phosphatase. A risk score was developed by using these three variables. At an optimal cutoff value of the regression formula, the risk score had an accuracy of 79.6% for predicting significant fibrosis, sensitivity of 89.5%, specificity of 78.4%, positive predictive value of 34.7%, negative predictive value of 98.3%, and ROC area of 0.855 (95% CI: 0.76-0.95). Fibroscan utility in predicting significant liver fibrosis in morbidly obese subjects is limited with accuracy of 71.3%. A model incorporating hemoglobin A1c and alkaline phosphatase with LSM improves accuracy in detecting significant fibrosis in this patient population.

Compromised hepatic mitochondrial fatty acid oxidation and reduced markers of mitochondrial turnover in human NAFLD.

BACKGROUND AND AIMS: NAFLD and its more-advanced form, steatohepatitis (NASH), is associated with obesity and is an independent risk factor for cardiovascular, liver-related, and all-cause mortality. Available human data examining hepatic mitochondrial fatty acid oxidation (FAO) and hepatic mitochondrial turnover in NAFLD and NASH are scant. APPROACH AND RESULTS: To investigate this relationship, liver biopsies were obtained from patients with obesity undergoing bariatric surgery and data clustered into four groups based on hepatic histopathological classification: Control (CTRL; no disease); NAFL (steatosis only); Borderline-NASH (steatosis with lobular inflammation or hepatocellular ballooning); and Definite-NASH (D-NASH; steatosis, lobular inflammation, and hepatocellular ballooning). Hepatic mitochondrial complete FAO to CO2 and the rate-limiting enzyme in ß-oxidation (ß-hydroxyacyl-CoA dehydrogenase activity) were reduced by ~40%-50% with D-NASH compared with CTRL. This corresponded with increased hepatic mitochondrial reactive oxygen species production, as well as dramatic reductions in markers of mitochondrial biogenesis, autophagy, mitophagy, fission, and fusion in NAFL and NASH. CONCLUSIONS: These findings suggest that compromised hepatic FAO and mitochondrial turnover are intimately linked to increasing NAFLD severity in patients with obesity.

Assessing the levels of intraspecific admixture and interspecific hybridization in Iberian wild goats (Capra pyrenaica).

Iberian wild goats (Capra pyrenaica, also known as Iberian ibex, Spanish ibex, and Spanish wild goat) underwent strong genetic bottlenecks during the 19th and 20th centuries due to overhunting and habitat destruction. From the 1970s to 1990s, augmentation translocations were frequently carried out to restock Iberian wild goat populations (very often with hunting purposes), but they were not systematically planned or recorded. On the other hand, recent data suggest the occurrence of hybridization events between Iberian wild goats and domestic goats (Capra hircus). Augmentation translocations and interspecific hybridization might have contributed to increase the diversity of Iberian wild goats. With the aim of investigating this issue, we have genotyped 118 Iberian wild goats from Tortosa-Beceite, Sierra Nevada, Muela de Cortes, Gredos, Batuecas, and Ordesa and Monte Perdido by using the Goat SNP50 BeadChip (Illumina). The analysis of genotypic data indicated that Iberian wild goat populations are strongly differentiated and display low diversity. Only three Iberian wild goats out from 118 show genomic signatures of mixed ancestry, a result consistent with a scenario in which past augmentation translocations have had a limited impact on the diversity of Iberian wild goats. Besides, we have detected eight Iberian wild goats from Tortosa-Beceite with signs of domestic goat introgression. Although rare, hybridization with domestic goats could become a potential threat to the genetic integrity of Iberian wild goats; hence, measures should be taken to avoid the presence of uncontrolled herds of domestic or feral goats in mountainous areas inhabited by this iconic wild ungulate.

Critical Role for Hepatocyte-Specific eNOS in NAFLD and NASH.

Regulation of endothelial nitric oxide synthase (eNOS) in hepatocytes may be an important target in nonalcoholic fatty liver disease (NAFLD) development and progression to nonalcoholic steatohepatitis (NASH). In this study, we show genetic deletion and viral knockdown of hepatocyte-specific eNOS exacerbated hepatic steatosis and inflammation, decreased hepatic mitochondrial fatty acid oxidation and respiration, increased mitochondrial H2O2 emission, and impaired the hepatic mitophagic (BNIP3 and LC3II) response. Conversely, overexpressing eNOS in hepatocytes in vitro and in vivo increased hepatocyte mitochondrial respiration and attenuated Western diet-induced NASH. Moreover, patients with elevated NAFLD activity score (histology score of worsening steatosis, hepatocyte ballooning, and inflammation) exhibited reduced hepatic eNOS expression, which correlated with reduced hepatic mitochondrial fatty acid oxidation and lower hepatic protein expression of mitophagy protein BNIP3. The current study reveals an important molecular role for hepatocyte-specific eNOS as a key regulator of NAFLD/NASH susceptibility and mitochondrial quality control with direct clinical correlation to patients with NASH.

A Model Incorporating Serum Alkaline Phosphatase for Prediction of Liver Fibrosis in Adults with Obesity and Nonalcoholic Fatty Liver Disease.

We assessed the relationship between serum alkaline phosphatase (ALP) and liver fibrosis by histology, in addition to other noninvasive parameters, in obese patients undergoing metabolic surgery. Patients scheduled for elective bariatric surgery were prospectively recruited from a bariatric clinic. An intraoperative liver biopsy was performed, and liver histology was evaluated by a pathologist blinded to the patients' data. The endpoint was significant fibrosis defined as fibrosis stage ≥ 2. Independent predictors of fibrosis were identified by logistic regression. Two hundred ten patients were recruited. Liver histology revealed steatosis in 87.1%, steatohepatitis in 21.9%, and significant fibrosis in 10%. Independent predictors of significant fibrosis were ALP (Odds Ratio (OR) 1.03; 95% Confidence interval (CI), 1.01-1.05), alanine aminotransferase (OR 1.02; 95% CI, 1.01-1.03), HbA1c (OR 1.58; 95% CI, 1.20-2.09), and body mass index (OR 1.06; 95% CI, 1.00-1.13). A tree-based model was developed to predict significant fibrosis, with a receiver operating characteristic (ROC) area of 0.845, sensitivity of 0.857, specificity of 0.836, and accuracy of 0.931. The applicability of serum ALP as an independent biomarker of liver fibrosis should be considered in obesity surgery patients, and in the broader context of obese patients with nonalcoholic fatty liver disease.

Congenital Appendiceal Diverticulum: An Incidental Finding During an Appendectomy.

Diverticula are small outpouchings that form at weak points in the wall of the digestive tract. They are commonly found in the colon, and while they can occur anywhere in the digestive tract, occurrence in the appendix is rare. Here, we report the case of a young woman presenting with complaints of right lower quadrant abdominal pain. The patient's physical examination, laboratory values, and computed tomography (CT) result supported the presumptive diagnosis of uncomplicated appendicitis. The patient underwent a laparoscopic appendectomy, where an appendiceal diverticulum was appreciated. A postoperative pathology report supported the diagnosis of a true appendiceal diverticulum. The patient's symptoms improved postoperatively, and her recovery has been unremarkable. We conclude that appendiceal diverticula are often incidental findings that should be removed along with the appendix to reduce the risk of malignancy and perforation.

A case report of pseudoaneurysm after a core needle biopsy of breast cancer.

Breast pseudoaneurysms after a core needle biopsy are a rare complication with a low incidence. However, it is important to be aware of the possibility of complications that require treatment.

Mastectomía ahorradora de piel y complejo areola-pezón con reconstrucción inmediata: una técnica en auge en el tratamiento del cáncer de mama / Nipple-sparing mastectomy with immediate reconstruction: An increasing technique for breast cancer treatment

Introducción: La mastectomía ahorradora de piel y pezón (MAP-CAP) es una técnica quirúrgica cada vez más empleada en el tratamiento del cáncer de mama. Nuestro objetivo es describir la técnica quirúrgica y exponer nuestros resultados. Métodos: Se analizaron retrospectivamente 46 MAP-CAP con reconstrucción inmediata con implante definitivo, realizadas en 35 pacientes con cáncer de mama. Resultados: Se realizaron 46 MAP-CAP entre 2010 y 2015. En 9 casos se realizó una mastectomía contralateral reductora de riesgo por mutaciones en los genes BRCA o acúmulo de riesgo familiar. La edad media fue de 48 años. El tipo histológico más frecuente fue el carcinoma ductal infiltrante (58%). El 26% de las neoplasias fueron carcinoma ductal in situ. Se evidenció multicentricidad en el 44,7% de los casos. Se administró neoadyuvancia en el 41% de los casos y radioterapia adyuvante (RT) en 24 casos (63%). En 4 casos aparecieron complicaciones postoperatorias (8,7%). En 13 casos se produjo una contractura capsular, habiéndose administrado RT en todos ellos. En 7 casos (15%) se requirió una segunda intervención quirúrgica para recambio de la prótesis inicial por contractura o alteraciones estéticas significativas. Existió un único caso de recidiva regional en axila (2,2%), con una mediana de seguimiento de 44 meses. Conclusiones: La MAP-CAP con prótesis definitiva es una técnica oncológicamente segura, con una tasa de recidivas similar a la mastectomía clásica. La administración de RT eleva la tasa de contractura capsular, implicando una segunda intervención en el 15% de los casos, por lo que debemos restringir su administración a los casos estrictamente necesarios

Introduction: Nipple-sparing mastectomy (NSM) is a surgical procedure that is increasingly used for the treatment of breast cancer. Our aim was to describe the technique performed at our institution and to present our results. Methods: We retrospectively analysed data from 46 NSM with immediate breast reconstruction with definitive implants performed in 35 patients with breast cancer. Results: We performed 46 NSMs between 2010 and 2015. Nine of these patients received a contralateral prophylactic NSM because of BRCA mutations or high familial cancer risk. The median age was 48 years. The most frequent histologic type was ductal invasive carcinoma (58%). A total of 26% of the neoplasms were ductal carcinoma in situ. Neoadjuvant treatment was administered to 41% of the patients and adjuvant radiotherapy was administered in 24 (63%). Early complications appeared in 4 patients (8.7%). Capsular contracture occurred in 13 patients. All of them had received radiotherapy. In 7 of the 46 patients (15%), a second surgery to change the initial prosthesis was required because of high-grade capsular contracture or to improve cosmetic results. There was only one case of regional recurrence (2.2%) in the axilla, with a median of follow-up of 44 months. Conclusions: NSM is an oncologically safe technique for breast cancer, with a similar recurrence rate to classic mastectomy. Radiotherapy increases the rate of capsular contracture, requiring a second intervention in 15% of patients. Consequently, its use should be restricted to cases in which it is strictly necessary

Freshwater scarcity effects on the aquatic macrofauna of a European Mediterranean-climate estuary.

In the Mediterranean-climate zone, recurrent drought events and increasing water demand generally lead to a decrease in freshwater input to estuaries. This water scarcity may alter the proper function of estuaries as nursery areas for marine species and as permanent habitat for estuarine species. A 12-year data set of the aquatic macrofauna (fish, decapod and mysid crustaceans) in a Mediterranean estuary (Guadalquivir estuary, South Spain) was analysed to test if water scarcity favours the nursery function of regional estuaries to the detriment of permanent estuarine inhabitants. Target species typically displayed a salinity-related distribution and estuarine salinisation in dry years resulted in a general upstream community displacement. However, annual densities of marine species were neither consistently higher in dry years nor estuarine species during wet years. Exceptions included the estuarine mysid Neomysis integer and the marine shrimp Crangon crangon, which were more abundant in wet and dry years, respectively. High and persistent turbidity, a collateral effect of water scarcity, altered both the structural (salinity-related pattern) and functional (key prey species and predator density) community characteristics, chiefly after the second drought period of the analysis. The observed high inter-year environmental variability, as well as species-specific effects of water scarcity, suggests that exhaustive and long-term sampling programmes will be required for rigorously monitoring the estuarine communities of the Mediterranean-climate region.

IL-35 promotes pancreas cancer growth through enhancement of proliferation and inhibition of apoptosis: evidence for a role as an autocrine growth factor.

Interleukin-35 (IL-35), an IL-12 cytokine family member, mediates the immune inhibitory function of regulatory T cells (Treg). We assayed the presence of IL-35 in paraffin-embedded human pancreas cancer (PCAN) and unexpectedly found IL-35 was expressed mainly by epithelial derived PCAN cells, but not by Treg. We further examined the expression and effect of exogenous IL-35 in human PCAN cell lines and found IL-35 promoted growth and inhibited apoptosis in PCAN cell lines. IL-35 induced proliferation correlated with an increase in cyclin B, cyclin D, cdk2, and cdk4 and a decrease in p27 expression, while inhibition of apoptosis was associated with an increase in Bcl-2 and a decrease in TRAILR1. We conclude IL-35 is produced by PCAN in vivo and promotes PCAN cell line growth in vitro. These results might indicate an important new role for IL-35 as an autocrine growth factor in PCAN growth.

Trace element contamination in the Guadalquivir River Estuary ten years after the Aznalcóllar mine spill.

Sediments, clams Scrobicularia plana and worms Hediste diversicolor from the Guadalquivir estuary were collected ten years after the Aznalcóllar mine spill and analyzed for metals. Significant seasonal and spatial effects were detected for most elements, so data from different sampling periods and locations were treated separately. Overall, the most polluted sites were found upstream, although Zn and Cu tended to accumulate at the estuary mouth. A significant decline of Zn in sediments and clams was observed compared to levels reported following the spill, so the estuarine ecosystem has recovered. However, the concentrations of some elements in S. plana were still higher than those of heavily contaminated regions. In this mollusk, Pb and As levels were higher in 2008 than in previous years, suggesting a new source entering into the estuary. Metals in sediments presented low bioavailability for biota, so other sources must account for the concentrations observed in these species.

Obstructive jaundice due to a rare periampullary tumor.

Gangliocytic paraganglioma is a rare neuroendocrine tumor predominantly arising in the second part of the duodenum with rare local recurrence or metastasis to regional lymph nodes. A 92-year-old female presented with obstructive jaundice. On exam she had pale conjunctiva and icteric sclera. Abdominal examination revealed tenderness in the upper abdomen. Laboratory data was consistent with obstructive jaundice. Computed tomography of the abdomen revealed a dilated gall bladder and a common bile duct (CBD) with no evidence of liver lesions or pancreatic head mass. Endoscopic ultrasonography revealed a 1 cm isoechoic submucosal nodule at the periampullary area, dilated CBD (9 mm), a prominent pancreatic duct (4.1 mm) and a hydropic gall bladder with no stones. Endoscopic retrograde cholangiopancreaticography was performed to relieve obstruction and showed a 1 cm periampullary mass which underwent an en-bloc snare resection. Histopathology analyses with immunohistochemical stains were positive for cytokeratin, synaptophysin, S-100 protein, neuron specific enolase and negative for actin and desmin consistent with periampullary gangliocytic paraganglioma. Periampullary gangliocytic paraganglioma is a rare benign tumor of the small bowel. Common presentation includes abdominal pain and obstructive jaundice which should be included in differential diagnosis of obstructive jaundice. Endoscopic resection is a curative therapy in the absence of local invasion or distant metastasis.

Sarcoidosis: an extremely rare cause of granulomatous enterocolitis.

Clinically recognizable gastrointestinal (GI) system involvement with sarcoidosis is extremely rare. We present a case of a 51-year-old Caucasian male who was evaluated for abdominal pain, elevated liver enzymes, leukopenia, thrombocytopenia, severe peripheral arthralgias, and chronic watery diarrhea. He had a history of mediastinal and periaortic lymphadenopathy. Extensive laboratory work up for liver diseases, infections, malabsorption and a bone marrow biopsy was essentially unremarkable. Eso-gastroduodenoscopy was unremarkable. Colonoscopy showed scattered right colon ulcerations and erythema. The terminal ileum appeared normal. Biopsies from the duodenum, terminal ileum, and colon showed intramucosal non-caseating granulomas with focal multinucleate giant cell formation in a background of chronic active duodenitis, ileitis, and colitis. Liver biopsy showed moderate non-specific chronic hepatitis with non-caseating granulomas present within portal and lobular parenchyma. The clinical presentations, along with biopsy results were suggestive of sarcoidosis. The patient was started on prednisone and had a significant improvement in his symptoms including diarrhea.

Osseous Metaplasia in an Inflammatory Polyp of the Rectum: A Case Report and Review of the Literature.

Osseous metaplasia is a rare finding in colonic neoplasms. We report a case osseous metaplasia in a 74 year-old male who underwent surveillance colonoscopy and found to have a 7 mm rectal polyp. Histopathologic examination revealed an inflammatory polyp with osseous metaplasia.

A pathologist-in-the-loop IHC antibody test selection using the entropy-based probabilistic method.

BACKGROUND: Immunohistochemistry (IHC) is an important tool to identify and quantify expression of certain proteins (antigens) to gain insights into the molecular processes in a diseased tissue. However, it is a challenge for pathologists to remember the discriminative characteristics of the growing number of such antigens across multiple diseases. The complexity of their expression patterns, fueled by continuous discoveries in molecular pathology, gives rise to a combinatorial explosion that places an unprecedented burden on a practicing pathologist and therefore increases cost and variability of IHC studies. MATERIALS AND METHODS: To tackle these issues, we have developed antibody test optimized selection method, a novel informatics tool to help pathologists in improving the IHC antibody selection process. The method uses extensions of Shannon's information entropies and Bayesian probabilities to dynamically build an efficient diagnostic tree. RESULTS: A comparative analysis of our method with the expert and World Health Organization classification guidelines showed that the proposed method brings threefold reduction in number of antibody tests required to reach a diagnostic conclusion. CONCLUSION: The developed method can significantly streamline the antibody test selection process, decrease associated costs and reduce inter- and intrapathologist variability in IHC decision-making.

Following the Aznalcóllar toxic spill.

Ten years after the Aznalcóllar mine spill, the decline in metal contamination in the area is evident. However, some elements are still found in elevated concentrations. In Scrobicularia plana, zinc, lead, and arsenic exceeded health advisory levels at several locations although there is no indication of risks associated with consumption of this mollusc. The estuary seems to have enhanced the bioavailability of arsenic and lead. Research should continue to understand the significance of this contamination.

Identification, inheritance, and variation of microsatellite markers in the black scallop Mimachlamys varia.

Five polymorphic microsatellite loci were identified in the black scallop Mimachlamys varia after construction of a genomic library enriched for (GT)n. To examine the transmission pattern of microsatellite alleles, several families were created and genotypes scored for three loci. The expected Mendelian ratios were found in 12 of 14 segregations examined. Unexpected segregations may be explained by a genotyping error (allelic dropout), given that when a specific allele was treated as dominant, the phenotypic ratios conformed to Mendelian expectations. The five loci were also examined in two samples from the Spanish coast. The two localities displayed similar mean values for the number of alleles per locus (7.2-8.4), allelic richness (7.2-7.9), and observed (0.389-0.484) and expected heterozygosity (0.545-0.618). Significant Hardy-Weinberg deviations were observed at three loci, with heterozygote deficiency occurring in all cases. Global multilocus θ value and allele frequencies at one locus revealed significant differentiation between the two localities.