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Aim. To examine the association between lifetime alcohol consumption and significant liver disease in type 2 diabetic patients with NAFLD. Methods. A cross-sectional study assessing 151 patients with NAFLD at risk of clinically significant liver disease. NAFLD fibrosis severity was classified by transient elastography; liver stiffness measurements ≥8.2 kPa defined significant fibrosis. Lifetime drinking history classified patients into nondrinkers, light drinkers (always ≤20 g/day), and moderate drinkers (any period with intake >20 g/day). Result. Compared with lifetime nondrinkers, light and moderate drinkers were more likely to be male (p=0.008) and to be Caucasian (p=0.007) and to have a history of cigarette smoking (p=0.000), obstructive sleep apnea (p=0.003), and self-reported depression (p=0.003). Moderate drinkers required ≥3 hypoglycemic agents to maintain diabetic control (p=0.041) and fibrate medication to lower blood triglyceride levels (p=0.044). Compared to lifetime nondrinkers, light drinkers had 1.79 (95% CI: 0.67–4.82; p=0.247) and moderate drinkers had 0.91 (95% CI: 0.27–3.10; p=0.881) times the odds of having liver stiffness measurements ≥8.2 kPa (adjusted for age, gender, and body mass index). Conclusions. In diabetic patients with NAFLD, light or moderate lifetime alcohol consumption was not significantly associated with liver fibrosis. The impact of lifetime alcohol intake on fibrosis progression and diabetic comorbidities, in particular obstructive sleep apnea and hypertriglyceridemia, requires further investigation.

Genetic variation at the 19q13.3 KLK locus is linked with prostate cancer susceptibility in men. The non-synonymous KLK3 single nucleotide polymorphism (SNP), rs17632542 (c.536 T > C; Ile163Thr-substitution in PSA) is associated with reduced prostate cancer risk, however, the functional relevance is unknown. Here, we identify that the SNP variant-induced change in PSA biochemical activity mediates prostate cancer pathogenesis. The 'Thr' PSA variant leads to small subcutaneous tumours, supporting reduced prostate cancer risk. However, 'Thr' PSA also displays higher metastatic potential with pronounced osteolytic activity in an experimental metastasis in-vivo model. Biochemical characterisation of this PSA variant demonstrates markedly reduced proteolytic activity that correlates with differences in in-vivo tumour burden. The SNP is associated with increased risk for aggressive disease and prostate cancer-specific mortality in three independent cohorts, highlighting its critical function in mediating metastasis. Carriers of this SNP allele have reduced serum total PSA and a higher free/total PSA ratio that could contribute to late biopsy decisions and delay in diagnosis. Our results provide a molecular explanation for the prominent 19q13.3 KLK locus, rs17632542 SNP, association with a spectrum of prostate cancer clinical outcomes., (© 2024. The Author(s).)

Genetic variation at the 19q13.3 KLK locus is linked with prostate cancer susceptibility. The non-synonymous KLK3 SNP, rs17632542 (c.536T>C; Ile163Thr-substitution in PSA) is associated with reduced prostate cancer risk, however, the functional relevance is unknown. Here, we identify that the SNP variant-induced change in PSA biochemical activity as a previously undescribed function mediating prostate cancer pathogenesis. The 'Thr' PSA variant led to small subcutaneous tumours, supporting reduced prostate cancer risk. However, 'Thr' PSA also displayed higher metastatic potential with pronounced osteolytic activity in an experimental metastasis in-vivo model. Biochemical characterization of this PSA variant demonstrated markedly reduced proteolytic activity that correlated with differences in in-vivo tumour burden. The SNP is associated with increased risk for aggressive disease and prostate cancer-specific mortality in three independent cohorts, highlighting its critical function in mediating metastasis. Carriers of this SNP allele had reduced serum total PSA and a higher free/total PSA ratio that could contribute to late biopsy decisions and delay in diagnosis. Our results provide a molecular explanation for the prominent 19q13.3 KLK locus, rs17632542 SNP, association with a spectrum of prostate cancer clinical outcomes., Competing Interests: HL holds patents for free PSA, hK2, and intact PSA assays, and is named on a patent for a statistical method to detect prostate cancer. The marker assay patents and the patent for the statistical model has been licensed and commercialized as the 4Kscore by OPKO Diagnostics. HL receives royalties from sales of this test and owns stock in OPKO. All the other authors declare no conflict of interest.

Competing Interests: Competing interests: None declared.

Venous valves (VVs) prevent venous hypertension and ulceration. We report that FOXC2 and GJC2 mutations are associated with reduced VV number and length. In mice, early VV formation is marked by elongation and reorientation ("organization") of Prox1hi endothelial cells by postnatal day 0. The expression of the transcription factors Foxc2 and Nfatc1 and the gap junction proteins Gjc2, Gja1, and Gja4 were temporospatially regulated during this process. Foxc2 and Nfatc1 were coexpressed at P0, and combined Foxc2 deletion with calcineurin-Nfat inhibition disrupted early Prox1hi endothelial organization, suggesting cooperative Foxc2-Nfatc1 patterning of these events. Genetic deletion of Gjc2, Gja4, or Gja1 also disrupted early VV Prox1hi endothelial organization at postnatal day 0, and this likely underlies the VV defects seen in patients with GJC2 mutations. Knockout of Gja4 or Gjc2 resulted in reduced proliferation of Prox1hi valve-forming cells. At later stages of blood flow, Foxc2 and calcineurin-Nfat signaling are each required for growth of the valve leaflets, whereas Foxc2 is not required for VV maintenance., (© 2017 Lyons et al.)

Background: Current commercial tubes have difficulties in producing "true" serum from all blood samples even within the recommended clotting times. Hence, Becton Dickinson (BD) and now Greiner have produced tubes containing thrombin as the procoagulant to reduce the clotting time and increase the possibility of producing serum from anticoagulated blood samples., Methods: The Greiner BCA Fast Clot (GBBCAFC) tube was evaluated in a hospital environment using 40 participants, (30 healthy and 10 undergoing renal dialysis) for 32 analytes against the Greiner lithium heparin tube and the BD Rapid Serum Tubes (BD RST) tube measured on Beckman DxC 800 and DxI 800 analyzers. Clotting strength was also examined using thromboelastography (TEG)., Results: The analytes results showed there was a very close agreement between the BD RST tube and GBBCAFC tube in comparison with lithium heparin plasma. The result comparison data showed equivalent performance with lower levels of hemolysis. The prolonged storage study also showed very similar agreement between the BD RST and the GBBCAFC tubes. Likewise, the TEG data showed there was very little difference in clotting ability between the tubes, and neither was capable of producing true serum from blood spiked with 2 U heparin/mL of blood., Conclusions: The study showed the GBBCAFC tube with the combination of the two procoagulants blood clotting activator and thrombin produced comparable performance with the lithium heparin plasma and the BD RST serum samples.

Natural experiments have been proposed as a way of complementing manipulative experiments to improve ecological understanding and guide management. There is a pressing need for evidence from such studies to inform a shift to landscape-scale conservation, including the design of ecological networks. Although this shift has been widely embraced by conservation communities worldwide, the empirical evidence is limited and equivocal, and may be limiting effective conservation. We present principles for well-designed natural experiments to inform landscape-scale conservation and outline how they are being applied in the WrEN project, which is studying the effects of 160 years of woodland creation on biodiversity in UK landscapes. We describe the study areas and outline the systematic process used to select suitable historical woodland creation sites based on key site- and landscape-scale variables - including size, age, and proximity to other woodland. We present the results of an analysis to explore variation in these variables across sites to test their suitability as a basis for a natural experiment. Our results confirm that this landscape satisfies the principles we have identified and provides an ideal study system for a long-term, large-scale natural experiment to explore how woodland biodiversity is affected by different site and landscape attributes. The WrEN sites are now being surveyed for a wide selection of species that are likely to respond differently to site- and landscape-scale attributes and at different spatial and temporal scales. The results from WrEN will help develop detailed recommendations to guide landscape-scale conservation, including the design of ecological networks. We also believe that the approach presented demonstrates the wider utility of well-designed natural experiments to improve our understanding of ecological systems and inform policy and practice.

Introduction: Gentamicin due to its low level of resistance and rapid bactericidal activity is commonly used to treat gram-negative bacteria. However, due to its toxic effects it needs to be monitored. To date, no interference has been reported with gentamicin assays., Materials and Methods: A patient with leg cellulitis and sepsis received a single dose of gentamicin and a sample was sent for gentamicin analysis. The sample showed high blank absorbance readings on Beckman DxC800 and DC800 analysers with various dilutions. A second sample was received and analysed on a Roche Cobas system to obtain a result. A third sample was received 107 hours later with the same results and this sample was then analysed neat and post ethanol precipitation on all the turbidimetric assays available on the DxC800 analyser., Results: The high blank absorbance was observed upon addition of the reactive reagents due to protein precipitation. Although not obvious from the patient protein results, it was shown the presence of high IgM paraprotein, 18.9 g/L (reference range 0.4-2.3 g/L) was the cause of precipitation, giving high blank readings. Of all the other turbidimetric assays, only vancomicin and valproate showed similar high blank absorbance readings. To be able to provide more rapid results it was shown ethanol could be used as a precipitant of proteins in both calibrators and patient samples with acceptable recovery., Conclusion: IgM paraprotein was identified as the cause of interference with the gentamicin, vancomicin and valproate assays. Protein interference in these assays can be overcome by precipitation with ethanol.

Background: Glomerular filtration rate (GFR) and mortality is more accurately determined by gold standard measures than serum creatinine-based estimates in cirrhosis. No formal validation of any gold standard method has been reported., Methods: An isocratic methanol-based method incorporating the reference standard iohexol-related compound C was developed and validated in 12 patients with cirrhosis by simultaneously determining GFR using iohexol and chromium-51 labelled ethylenediamine tetraacetic acid ((51)Cr-EDTA) clearance. Iohexol pharmacokinetics was also studied with the collection of blood and ascitic fluid at intervals following an iohexol bolus., Results: Triplicate assays produced a linear calibration curve (R (2)=0.99, N=5) over an iohexol concentration range of 23.6-755 µg/L. Mean (range) extraction recovery of iohexol from serum was greater than 95% (94-97%), with an intra-day coefficient of variation less than 3%. Twelve patients with cirrhosis with mean Child-Pugh score of 9 displayed a mean difference (bias) -1.3 mL/min/1.73 m(2) (-18 to +16) comparing iohexol with (51)Cr-EDTA. Iohexol equilibrated between blood and ascitic compartments after 4 h., Conclusion: A simple, cheap, and accurate isocratic, methanol-based method for the determination of iohexol concentrations is described, validated according to Food and Drug Administration guidance. Iohexol demonstrated comparable performance with (51)Cr-EDTA in determining GFR. Delayed equilibrium of iohexol between blood and ascitic compartments suggests sampling beyond 4 h would improve accuracy of GFR determinations in patients with cirrhosis.

We have investigated the inflammatory infiltrate in post-transplant dn-AIH, a form of late insidious graft rejection, focusing on transcription factors defining effector and T-regs, using an antigen retrieval immunohistochemical method on archived liver tissue, and compared it with ACR and classical AIH. Paraffin-embedded liver biopsies from pediatric patients with dn-AIH (n = 10), ACR (n = 10), and AIH (n = 13) were selected randomly and stained using antibodies directed to CD4, CD8, T-bet (marker of Th1 polarization), GATA-3 (marker of Th2 polarization), FOXP3 (marker for T regulatory cells), IL-17, CD56 (NK cells), and perforin. Portal and lobular lymphocytic infiltrate was assessed semi-quantitatively. Prominent CD4, CD8, and T-bet positivity were present in both the lobular and portal infiltrate of all three conditions. Overall T-bet score of lobular inflammation in the dn-AIH group was lower than in the ACR and AIH groups (p = 0.02). In contrast, most samples showed absent or minimal GATA-3 positivity. FOXP3, CD56, IL-17, and perforin staining of mild to moderate severity were present in all three groups in both the portal and lobular infiltrate. A Th1 polarization of the inflammatory infiltrate characterizes dn-AIH, but also ACR and AIH., (© 2012 John Wiley & Sons A/S.)

Background: Acute and chronic Q fever/Coxiella burnetii infection is diagnosed principally by serology. The management of patients who have serological evidence of chronic Q fever but no other manifestation of chronic infection is challenging., Methods: This paper describes a follow-up study of individuals 6 years after a point source outbreak. The study compares serological and polymerase chain reaction (PCR) results between 3 international reference laboratories in a well-defined cohort of Q fever patients., Results: Concordance in microimmunofluorescence result interpretation from the 3 centers was only 35%. Australian and UK results had the greatest concordance and French and UK results the lowest. Serological testing revealed no chronic serological profiles when tested in either France or Australia but 10 when tested in the UK. Serological results from a patient with treated Q fever endocarditis suggested treated (France), chronic (UK), and borderline chronic (Australia) infection. PCR results on blood were universally negative., Conclusions: This study has shown that the results from Q fever micro-immunofluorescence vary according to the center in which they are carried out. This has implications for the interpretation of such tests, raises questions regarding the validity of using serological criteria alone as a means of diagnosing chronic Q fever, and affects the interpretation of epidemiological studies. We recommend that all results are interpreted according to the clinical picture and particular caution is applied in the interpretation of chronic serological profiles. In order to further our understanding of Q fever infection we propose that an international standard of Q fever serological investigation be developed.

Tachycardia induced cardiomyopathy (TIC) is a rare but potentially reversible cause of heart failure. The case of a patient with severe tachycardiomyopathy who had a favorable outcome following treatment of tachyarrythmia is presented here.

Sonic hedgehog (Shh) is a secreted morphogen necessary for the production of sidedness in the developing embryo. In this study, we describe the morphology of the atrial chambers and atrioventricular junctions of the Shh null mouse heart. We demonstrate that the essential phenotypic feature is isomerism of the left atrial appendages, in combination with an atrioventricular septal defect and a common atrioventricular junction. These malformations are known to be frequent in humans with left isomerism. To confirm the presence of left isomerism, we show that Pitx2c, a recognized determinant of morphological leftness, is expressed in the Shh null mutants on both the right and left sides of the inflow region, and on both sides of the solitary arterial trunk exiting from the heart. It has been established that derivatives of the second heart field expressing Isl1 are asymmetrically distributed in the developing normal heart. We now show that this population is reduced in the hearts from the Shh null mutants, likely contributing to the defects. To distinguish the consequences of reduced contributions from the second heart field from those of left-right patterning disturbance, we disrupted the movement of second heart field cells into the heart by expressing dominant-negative Rho kinase in the population of cells expressing Isl1. This resulted in absence of the vestibular spine, and presence of atrioventricular septal defects closely resembling those seen in the hearts from the Shh null mutants. The primary atrial septum, however, was well formed, and there was no evidence of isomerism of the atrial appendages, suggesting that these features do not relate to disruption of the contributions made by the second heart field. We demonstrate, therefore, that the Shh null mouse is a model of isomerism of the left atrial appendages, and show that the recognized associated malformations found at the venous pole of the heart in the setting of left isomerism are likely to arise from the loss of the effects of Shh in the establishment of laterality, combined with a reduced contribution made by cells derived from the second heart field.

Rebaudioside A was administered via the diet to male and female Han Wistar rats at 0, 7500, 12,500, and 25,000ppm for two generations. Rebaudioside A treatment was not associated with any signs of clinical toxicity or adverse effects on body weight, body weight gain, or food consumption. No treatment-related effects of rebaudioside A were observed in either the F0 or F1 generations on reproductive performance parameters including mating performance, fertility, gestation lengths, oestrous cycles, or sperm motility, concentration, or morphology. The survival and general condition of the F1 and F2 offspring, their pre-weaning reflex development, overall body weight gains, and the timing of sexual maturation, were not adversely affected by rebaudioside A treatment. The NOAEL for reproductive effects was 25,000ppm and the NOAEL for the survival, development, and general condition of the offspring also was considered to be 25,000ppm or 2048-2273mg/kg body weight/day.

Background: High rates of postpartum relapse occur in women with histories of bipolar or schizoaffective disorder. These relapses may be triggered by the postdelivery fall in circulating estrogen through alteration of central neurotransmitter (especially dopaminergic) systems. This study tested the hypothesis that estrogen administration after childbirth would prevent postpartum relapse and would alter dopamine receptor sensitivity., Method: Twenty-nine pregnant women with a Research Diagnostic Criteria diagnosis of hypomania (bipolar II), mania (bipolar I), or schizoaffective disorder participated in an open clinical trial. Three transdermal dose regimens of estrogen (17beta-estradiol) were tested. Starting doses were 200 (N = 13), 400 (N = 3), and 800 (N = 13) micro g/day, beginning within 48 hours after delivery and reduced by one half every 4 days for a total of 12 days. On the fourth day after starting estradiol therapy (before relapse occurred), subjects participated in a neuroendocrine challenge test that measured the sensitivity of the central nervous system (tubero-infundibular) dopaminergic system (plasma prolactin and growth hormone responses to apomorphine)., Results: Estradiol at all dose regimens did not reduce the rate of relapse. However, of the 12 women who relapsed, those who had taken the highest dose of estradiol (800 micro g/day) needed less subsequent psychotropic medication (fewer chlorpromazine equivalents) and were discharged sooner than those who had taken either of the 2 lower doses. No differences in neuroendocrine responses to apomorphine were detected between women receiving the high-dose and the lower-dose regimens., Conclusion: The results do not support the hypothesis that a fall in circulating concentrations of estrogens precipitates relapse in subjects at risk of postpartum affective psychosis. The use of prophylactic estrogen in such circumstances is therefore highly questionable.

The heart is one of the first organs to form during embryogenesis since its circulatory function is critical from early stages for embryo survival. In man, morphological events are affected by molecular perturbations, which can lead to a congenital heart defect. It is important therefore to understand not only the molecular signals, but also the morphological events, which govern myocardial cell identity. The study of transgenic mouse lines, Mlc1v-nlacZ-24 and Mlc3f-nlacZ-2, has led to the identification of a new precardiac territory, the anterior heart field, which has also been described recently in birds, and which contributes to the myocardium of the arterial pole of the heart. The use of explant cultures also indicates that pharyngeal mesoderm participates in the formation of the outflow tract and right ventricle and shows that the primitive heart tube has a predominantly left ventricular identity. We have also shown that Fgf-10 is expressed in the anterior heart field, where a role for FGF signaling in arterial pole morphogenesis is suggested by inhibitor experiments. Finally explant cultures have been employed to examine the acquisition of left-right atrial identity. The Mlc3f-nlacZ-2 line, which marks the right atrium, allowed us to determine the time window during which left-right signaling confers left-right atrial identity.

From 1996 to 2000, ZEBET (Centre for Documentation and Evaluation of Alternative Methods to Animal Experiments at the BgVV, Berlin, Germany) coordinated the European Centre for the Validation of Alternative Methods (ECVAM) prevalidation and validation study on three embryotoxicity tests: a) a test employing embryonic stem cell lines (EST); b) the micromass (MM) test; and c) the postimplantation rat whole-embryo culture assay (WEC test). The main objectives of the study were to assess the performance of these three in vitro tests in discriminating between non- embryotoxic, weakly embryotoxic and strongly embryotoxic compounds. Phase I of the study (1997) was designed as a prevalidation phase, for test protocol optimisation, and for the establishment of a comprehensive database of in vivo and in vitro data on embryotoxic compounds. Phase II (1998-2000) involved a formal validation trial, conducted under blind conditions on 20 test compounds selected from the database, which were coded and distributed to the participating laboratories. In the preliminary phase of the validation study, six chemicals out of the 20, which showed embryotoxic potential, were tested. These results were used to define new biostatistically based prediction models (PMs) for the MM and WEC tests, and to evaluate those developed previously for the EST. As a next step, the PMs were evaluated by using the results for the remaining 14 chemicals of the definitive phase of the validation study. The three in vitro embryotoxicity tests proved to be applicable to testing a diverse group of chemicals with different embryotoxic potentials (non-embryotoxic, weakly embryotoxic, and strongly embryotoxic). The reproducibility of the three in vitro embryotoxicity tests were acceptable according to the acceptance criteria defined by the Management Team. The concordances between the embryotoxic potentials derived from the in vitro data and from the in vivo data were good for the EST and the WEC (PM2) test, and sufficient for the MM test and the WEC (PM1) tests according to the performance criteria defined by the Management Team before the formal validation study. When applying the PM of the EST to the in vitro data obtained in the definitive phase of the formal validation study, chemicals were classified correctly in 78% of the experiments. For the MM and the WEC tests, the PMs provided 70% and 80% (PM2) correct classifications, respectively. And, very importantly, an excellent predictivity (100%, except for PM1 of the WEC test, with 79%, considered as good) was obtained with strong embryotoxic chemicals in each of the three in vitro tests.