Alternatively, previous methods to recellularize organ scaffolds have relied see more heavily on direct cell injection, that damaged

the scaffold microarchitecture and produced heterogeneous scaffold seeding.13 The perfusion method introduced here supports cell infusion through the vascular network and deposition throughout the thickness of the bioscaffold, achieving greater seeding efficiency without compromising the integrity of the bioscaffold. Furthermore, by accessing different vessels that feed into the liver, we were able to deliver cells selectively to different compartments of the liver tissue. EC delivered through the vena cava selectively seeded larger and smaller blood vessels up to the pericentral area of the liver lobule, without reaching the periportal space of the lobule where the final branching vessels of portal vein are located (Fig. 4A). On the other hand, cells seeded through the portal vein, which delivers blood from the intestine and other organs to the liver,39 reached predominantly the periportal

area of the liver lobule without extensive penetration to its pericentral space. These ECs cover the entire circumference of a vascular channel and maintained cell-cell junctions (Fig. 4E and Supporting Information Fig. 3A). We were able to confirm this “selective” seeding by delivering fluorescently-labeled ECs through the portal vein and fluorescent beads via the vena cava in the same bioscaffold, showing that they reached discrete locations in the liver lobule. Thus, simultaneous utilization AP24534 datasheet of both vascular routes for cell seeding enables complete access to the entire length of the vascular network, which has an essential importance for prevention of blood clotting and ultimately

failure to transplant the bioengineered liver. Accordingly, we showed that endothelialized bioscaffolds exhibited significant reduction Methisazone in the presence and adhesion of platelets, compared with unseeded bioscaffolds (Fig. 4F). Yet, further improvement to complete endothelialization of every blood vessel and capillary of the bioscaffold will require larger numbers of ECs and longer bioreactor pre-conditioning time. Recent studies by Ott et al.13, 40 and Uygun et al.41 documented the decellularization of rat hearts,13 lungs,40 and livers,41 respectively, using the same perfusion method. The authors used young animal (rat) cells or human cell lines for recellularization experiments. These cells can sustain greater physical and chemical (hypoxia, toxic metabolites, etc.) insults than primary human cells that were used in the current study. The use of primary human cells to recellularize the bioscaffolds provides a clinical application of organ bioengineering.

Their locations were categorized as migrating or nonmigrating based on the relative orientation of the track and net speed. An average of 42% of nonmigrating locations were between 37°S and 45°S, and 53% were south of 52°S, possibly associated with the Subtropical Convergence and Antarctic Polar Front, respectively. Whaling data suggest right whales fed largely on copepods at the former and euphausiids at the latter. If the nonmigrating locations represented feeding at these frontal zones, switching between them would seem to have obvious cost-benefit implications. “
“Muscle samples from 105 marine mammals stranded along the Oregon and Washington coasts (2002–2009) were tested for

levels of total mercury (THg) by Cold Vapor Atomic NVP-BGJ398 datasheet Fluorescence Spectrometry. The THg present is in the form of the highly toxic methylmercury. After normalizing tissue to 75% water weight, Steller sea lions and northern elephant seals exhibited EPZ-6438 cell line the highest mean concentrations of THg followed by harbor seals, harbor porpoises, and California sea lions, 0.34 ± 0.278, 0.34 ± 0.485, 0.21 ± 0.216, 0.17 ± 0.169, and 0.15 ± 0.126 mg/kg normalized wet weight (ww), respectively. The mean normalized values demonstrate limited muscle methylmercury accumulation in these species in the Pacific Northwest. However, actual ww concentrations in some of the stranded carcasses may pose a risk to scavengers. Normalizing muscle mercury concentrations

eliminated the variability from desiccation, and allowed for a clearer indication of the amount of mercury the animal accumulated before stranding. “
“The carrying capacity of the

French Frigate Shoals (FFS) region for the endangered Hawaiian monk seal was appraised using an updated version of the original FFS Ecopath model (Polovina 1984). Model parameters were updated using recent literature, and data from surveys of the seal population and its bottom-associated prey. Together they produced a static mass Fossariinae balance model for 1998 when the prey surveys began. The Ecopath-estimated monk seal biomass was 0.0045 t/km2, which was in close agreement with the biomass calculated from monk seal field beach counts (0.0046 t/km2). Model simulations through time were done in Ecosim using the Ecopath balanced model and included fisheries data time series from 1998 to 2008. Monk seal biomass declined concurrently with decreases in benthic bottomfish biomass, which were influenced by large-scale changes in the environment of the North Pacific. This model scenario was extended from 2010, when the last permitted fishery in the Northwestern Hawaiian Islands was closed, through to 2040, assuming a constant environmental signal. Model results for this time period did not show a recovery of monk seals that exceeded the initial 1998 model biomass levels, highlighting the importance of including environmental variability in estimates of monk seals recovery at FFS.

Their locations were categorized as migrating or nonmigrating based on the relative orientation of the track and net speed. An average of 42% of nonmigrating locations were between 37°S and 45°S, and 53% were south of 52°S, possibly associated with the Subtropical Convergence and Antarctic Polar Front, respectively. Whaling data suggest right whales fed largely on copepods at the former and euphausiids at the latter. If the nonmigrating locations represented feeding at these frontal zones, switching between them would seem to have obvious cost-benefit implications. “
“Muscle samples from 105 marine mammals stranded along the Oregon and Washington coasts (2002–2009) were tested for

levels of total mercury (THg) by Cold Vapor Atomic Trichostatin A Fluorescence Spectrometry. The THg present is in the form of the highly toxic methylmercury. After normalizing tissue to 75% water weight, Steller sea lions and northern elephant seals exhibited LBH589 the highest mean concentrations of THg followed by harbor seals, harbor porpoises, and California sea lions, 0.34 ± 0.278, 0.34 ± 0.485, 0.21 ± 0.216, 0.17 ± 0.169, and 0.15 ± 0.126 mg/kg normalized wet weight (ww), respectively. The mean normalized values demonstrate limited muscle methylmercury accumulation in these species in the Pacific Northwest. However, actual ww concentrations in some of the stranded carcasses may pose a risk to scavengers. Normalizing muscle mercury concentrations

eliminated the variability from desiccation, and allowed for a clearer indication of the amount of mercury the animal accumulated before stranding. “
“The carrying capacity of the

French Frigate Shoals (FFS) region for the endangered Hawaiian monk seal was appraised using an updated version of the original FFS Ecopath model (Polovina 1984). Model parameters were updated using recent literature, and data from surveys of the seal population and its bottom-associated prey. Together they produced a static mass very balance model for 1998 when the prey surveys began. The Ecopath-estimated monk seal biomass was 0.0045 t/km2, which was in close agreement with the biomass calculated from monk seal field beach counts (0.0046 t/km2). Model simulations through time were done in Ecosim using the Ecopath balanced model and included fisheries data time series from 1998 to 2008. Monk seal biomass declined concurrently with decreases in benthic bottomfish biomass, which were influenced by large-scale changes in the environment of the North Pacific. This model scenario was extended from 2010, when the last permitted fishery in the Northwestern Hawaiian Islands was closed, through to 2040, assuming a constant environmental signal. Model results for this time period did not show a recovery of monk seals that exceeded the initial 1998 model biomass levels, highlighting the importance of including environmental variability in estimates of monk seals recovery at FFS.

We report the validity of a segmentation pipeline for the detection of MS-related brain atrophy with 3T MRI. Longitudinal studies are warranted to extend these results. “
“PRES is a reversible neurotoxic state presenting with headache, altered mental status, visual BGJ398 cell line loss, and seizures. Delayed diagnosis can be avoided if radiological patterns could distinguish PRES from cerebral ischemia. Clinical and radiological data were collected on all hospitalized patients who had (1) discharge diagnosis

of PRES and (2) acute CTP/CTA. Data were compared with 10 TIA patients with proven cytotoxic edema on MRI. Of the four PRES patients found, three were correlated with acute blood pressure and one with chemotherapy. At the radiological level, quantitative analyses of the CTP parameters showed that 2 out of 4 patients had bilaterally reduced CBF-values (23.2-47.1 ml/100g/min) in occipital regions, as seen in the pathological regions of TIA patients (27.3 ± 13.5 ml/100g/min). When compared with TIA patients, the pathological ROI’s demonstrated decreased CBV-values (3.4-5.6 ml/100g). Vasogenic edema on MRI FLAIR imaging was seen in only one PRES patient, and cytotoxic edema on DWI-imaging was never found. CT angiography showed in one PRES patient a vasospasm-like unilateral posterior cerebral artery. If confirmed by other groups, CTP and CTA imaging in patients with acute

visual loss and confusion may help to distinguish PRES from bi-occipital

ischemia. These radiological Adenosine ABT-263 price parameters may identify PRES patients at risk for additional tissue infarction. “
“We report a patient with abnormal diffusion tensor imaging (DTI) and tractography of the corticospinal tract caused by mass effect from adjacent enlarged Virchow–Robin spaces. DTI was performed using 25 noncollinear directions. Fractional anisotropy (FA) and mean diffusivity (MD) maps were generated. Region-of-interest measurements of the corticospinal tracts were organized in histograms, and comparisons were made between sides. Statistical analysis consisted of a Wilcoxon rank-sum nonparametric test and a two-sample test of proportions to compare the relative percentage of voxels >.8. The patient had no signs or symptoms of motor weakness. The corticospinal tract adjacent to the enlarged Virchow–Robin spaces showed significant changes in the proportion of FA > .8, distribution of FA and distribution of MD (P < .001). Diffusion tensor changes may be caused by enlarged Virchow–Robin spaces in the absence of clinical signs or symptoms. We hypothesize that the DTI changes are due to alterations in the extravascular extracellular space. Tensor changes should be interpreted with caution in patients with space occupying mass lesions such as brain tumors. Enlarged Virchow–Robin spaces are extensions of the subpial cerebrospinal space surrounding small penetrating arteries and veins.

We report the validity of a segmentation pipeline for the detection of MS-related brain atrophy with 3T MRI. Longitudinal studies are warranted to extend these results. “
“PRES is a reversible neurotoxic state presenting with headache, altered mental status, visual EPZ-6438 purchase loss, and seizures. Delayed diagnosis can be avoided if radiological patterns could distinguish PRES from cerebral ischemia. Clinical and radiological data were collected on all hospitalized patients who had (1) discharge diagnosis

of PRES and (2) acute CTP/CTA. Data were compared with 10 TIA patients with proven cytotoxic edema on MRI. Of the four PRES patients found, three were correlated with acute blood pressure and one with chemotherapy. At the radiological level, quantitative analyses of the CTP parameters showed that 2 out of 4 patients had bilaterally reduced CBF-values (23.2-47.1 ml/100g/min) in occipital regions, as seen in the pathological regions of TIA patients (27.3 ± 13.5 ml/100g/min). When compared with TIA patients, the pathological ROI’s demonstrated decreased CBV-values (3.4-5.6 ml/100g). Vasogenic edema on MRI FLAIR imaging was seen in only one PRES patient, and cytotoxic edema on DWI-imaging was never found. CT angiography showed in one PRES patient a vasospasm-like unilateral posterior cerebral artery. If confirmed by other groups, CTP and CTA imaging in patients with acute

visual loss and confusion may help to distinguish PRES from bi-occipital

ischemia. These radiological SDHB BVD-523 cell line parameters may identify PRES patients at risk for additional tissue infarction. “
“We report a patient with abnormal diffusion tensor imaging (DTI) and tractography of the corticospinal tract caused by mass effect from adjacent enlarged Virchow–Robin spaces. DTI was performed using 25 noncollinear directions. Fractional anisotropy (FA) and mean diffusivity (MD) maps were generated. Region-of-interest measurements of the corticospinal tracts were organized in histograms, and comparisons were made between sides. Statistical analysis consisted of a Wilcoxon rank-sum nonparametric test and a two-sample test of proportions to compare the relative percentage of voxels >.8. The patient had no signs or symptoms of motor weakness. The corticospinal tract adjacent to the enlarged Virchow–Robin spaces showed significant changes in the proportion of FA > .8, distribution of FA and distribution of MD (P < .001). Diffusion tensor changes may be caused by enlarged Virchow–Robin spaces in the absence of clinical signs or symptoms. We hypothesize that the DTI changes are due to alterations in the extravascular extracellular space. Tensor changes should be interpreted with caution in patients with space occupying mass lesions such as brain tumors. Enlarged Virchow–Robin spaces are extensions of the subpial cerebrospinal space surrounding small penetrating arteries and veins.

5 versus 33.9 years; P < 0.003). Males with a truncating variant were younger (31 ± 12 years) than those with missense variant (40 ± 13 years); likewise, females with a truncating variant were younger (26 ±6 years) than those with a missense variant (40 ± 13 years) (P < 0.001). There was no significant association between truncating sequence variations and severity of either acute or chronic biliary complications (severe biliary complications defined as

acute pancreatitis, recurrent cholangitis, segmental spindle-shape dilatation of the biliary tree filled with gallstones): odds ratio (OR) = 0.8 (95% confidence interval [CI] 0.3-3.7, P = 0.8). These complications were more frequent in men (71% versus 45%, P = 0.05, OR 2.9, 95% CI 1.0-9.6), that in women, independently of age at onset of symptoms and type of variant. About half of the women who had pregnancy experienced LY2835219 in vitro ICP. The frequency and severity of ICP did not differ in patients with missense (44%) and truncating variant (69%) (P = 0.2). In patients without an

ABCB4 detectable variant the clinical characteristics Rapamycin purchase were similar to those observed in patients with gene variation. Of note, in a subset of patients the phospholipid/bile acid ratio measured in hepatic bile (ratio of control gallbladder bile >25%) did not differ between the two groups: 11%, range 4%-16% (n = 7) versus 12%, range 1.4%-16% (n = 8) in patients with and without the ABCB4 variant, respectively. In the overall cohort, biliary cirrhosis was detected in only two patients. Both patients had a missense heterozygous variant (location Glutathione peroxidase and nucleotide changes: c.523A>G and c.959C>T). Intrahepatic

cholangiocarcinoma leading to death was observed in a noncirrhosis female patient with a heterozygous splicing mutation (c.1005+5 G>A). All the patients received ursodeoxycholic acid (UDCA) (8-10 mg/kg/day) as the mainstay treatment after the diagnosis had been made. All the patients with severe chronic biliary complications had sphincterotomy. Patients with symptomatic intrahepatic bile duct dilatations filled with gallstones had repeated endoscopic procedures to remove the stones. All the patients with or without detectable variant and free of intrahepatic bile duct dilatations with gallstones became asymptomatic up to now. The only exception was a patient who did not tolerate UDCA because of bile acid-induced watery diarrhea. Among patients presenting with symptomatic cholelithiasis, three main features defined the syndromatic phenotype termed LPAC: recurrence of biliary symptoms after cholecystectomy, intrahepatic lithiasis, and age <40 years. The results of the present study may be summarized as follows: (1) half of the patients with the LPAC phenotype have detectable sequence variations of the ABCB4 gene, most of them being heterozygous missense.

41, 95% confidence interval = 1.16–5.00, P = 0.02). Serum FST levels are significantly associated with HCC prognosis and could represent a predictive click here biomarker in this disease. “
“The role of autophagy in disease pathogenesis following viral infection is beginning to be elucidated. We have previously reported that hepatitis C virus (HCV) infection in hepatocytes induces autophagy. However, the biological significance of HCV-induced autophagy has not been clarified. Autophagy has recently been identified as a novel component of the innate immune system against viral infection. In this study, we found that knockdown of autophagy-related protein

beclin 1 (BCN1) or autophagy-related protein 7 (ATG7) in immortalized human hepatocytes (IHHs) inhibited HCV growth. BCN1- or ATG7-knockdown IHHs, when they were infected with HCV, exhibited increased

expression of interferon-β, 2′,5′-oligoadenylate synthetase 1, interferon-α, and interferon-α–inducible protein 27 messenger RNAs of the interferon signaling pathways in comparison with infected control IHHs. A subsequent study www.selleckchem.com/products/BIBW2992.html demonstrated that HCV infection in autophagy-impaired IHHs displayed caspase activation, poly(adenosine diphosphate ribose) polymerase cleavage, and apoptotic cell death. Conclusion: The disruption of autophagy machinery in HCV-infected hepatocytes activates the interferon signaling pathway and induces apoptosis. Together, these results suggest that HCV-induced autophagy impairs the innate immune tuclazepam response. (HEPATOLOGY 2011;53:406-414) Hepatitis C virus (HCV) infection affects nearly 3.3 million people and is the most common cause of cirrhosis and hepatocellular carcinoma in the United States.1 The currently approved therapy for the treatment of HCV is pegylated interferon

in combination with ribavirin.2, 3 Although several advances have shown promise in improving the management of HCV infection, nevertheless, it remains a major health problem.4-6 HCV is a member of the Flaviviridae family, and its genome contains a positive-strand RNA approximately 9.6 kb long. The HCV genome encodes a polyprotein precursor of approximately 3000 amino acids that is cleaved by both viral and host proteases into structural (core, E1, E2, and p7) and nonstructural proteins [nonstructural protein 2 (NS2), NS3, NS4A, NS4B, NS5A, and NS5B]. HCV-infected cells accumulate lipid droplets and play an important role in the assembly of virus particles.7-9 Autophagy is a catabolic process by which cells remove their own damaged organelles and long-lived proteins for the maintenance of cellular homeostasis. During autophagy, the double-membrane vesicles engulf the damaged organelles and eventually fuse with the lysosomes for degradation.

Conclusions: Within the limitations of this in vitro model, the effect of component manufacturer resulted in a significantly higher RTV in the control group (two-way ANOVA, p= 0.0032) indicating greater residual preload; however, there was no significant decrease in post-fatigue RTV for either manufacturer compared to baseline. “
“Debonding of acrylic teeth from the denture base remains a major problem in prosthodontics. The objective of this study was to evaluate the effect of various

surface treatments on the shear bond strength of the two chemically different denture base resins—polymethyl methacrylate (PMMA) and urethane dimethacrylate (UDMA). Two denture base resins, heat-cured PMMA (Meliodent) and light-activated UDMA (Eclipse), were used in this study. A total of 60 molar acrylic denture teeth were randomly separated Fostamatinib into four groups (n = 15),

according to surface treatment: acrylic untreated (group AC), Eclipse untreated (group EC), treated with eclipse bonding agent (group EB), and Er:YAG laser-irradiated eclipse (group EL). Shear bond strength Compound Library in vitro test specimens were prepared according to the manufacturers’ instructions. Specimens were subjected to shear bond strength test by a universal testing machine with a 1 mm/min crosshead speed. The data were analyzed with one-way ANOVA and post hoc Tukey-Kramer multiple comparison tests (α = 0.05). The highest mean bond strength was observed in specimens of group EB, and the lowest was observed in group EC specimens. A statistically significant difference in shear bond strength was found among all groups (p < 0.001), except between groups EC and EL (p = 0.61). The two chemically different denture base polymers showed different shear bond strength values to acrylic denture teeth. Laser-irradiation of the adhesive surface was found to be ineffective on improving bond strength of acrylic denture

teeth to denture base resin. Eclipse bonding agent should be used as a part of denture fabrication with the Eclipse Resin System. “
“Purpose: This study aimed to evaluate stress distribution on peri-implant bone simulating the influence of platform switching in external Selleckchem Idelalisib and internal hexagon implants using three-dimensional finite element analysis. Materials and Methods: Four mathematical models of a central incisor supported by an implant were created: External Regular model (ER) with 5.0 mm × 11.5 mm external hexagon implant and 5.0 mm abutment (0% abutment shifting), Internal Regular model (IR) with 4.5 mm × 11.5 mm internal hexagon implant and 4.5 mm abutment (0% abutment shifting), External Switching model (ES) with 5.0 mm × 11.5 mm external hexagon implant and 4.1 mm abutment (18% abutment shifting), and Internal Switching model (IS) with 4.5 mm × 11.5 mm internal hexagon implant and 3.8 mm abutment (15% abutment shifting). The models were created by SolidWorks software.

Fixed mouse liver

tissues were embedded Selleckchem U0126 in paraffin and 5-μm sections were used for immunohistochemistry. Cell proliferation was assessed by way of KI-67 staining as described.19 KI-67–positive cells were quantified by counting hepatocytes in at least 10 random fields. Sections from 2 to 4 individual animals for each genotype were used, and the data are expressed as the mean ± standard deviation (SD). For Western blot analysis, livers were lysed at the indicated time points after PH in lysis buffer (50 mM HEPES [pH 7.5], 150 mM NaCl, 1 mM ethylene diamine tetraacetic acid, 10% glycerin, 1% Triton X-100, 10 mM Na4P2O7) and protein concentrations were measured with the BCA Assay. Nitrocellulose membranes were blocked in either 1× NET Gelatine or 5% milk/TBS-T and processed for immunoblotting. Primary antibodies against mig-6 (9; cl. PE-16), Tubulin, and β-actin were from Sigma; p-EGFR Y1173, EGFR, p-c-Jun Ser66, and pan-phospho-tyrosine antibodies (cl. Androgen Receptor antagonist 4G10) were from Upstate; p-ERK1/2, p-Rb and p-AKT Ser473 were from Cell Signaling Technologies. Secondary horseradish peroxidase–conjugated anti-mouse (Sigma), anti-rabbit (BioRad), and anti-sheep (Jackson ImmunoLaboratories) antibodies were used at a 1:10,000 dilution and detected using an ECL reagent

(Perkin Elmer). For immunoprecipitation, cell lysates were prepared in HNTG buffer (20 mM Hepes [pH 7.5], 150 mM NaCl, 0.1% Triton X-100, 10% glycerin, and 10 mM Na4P2O7), followed by incubation overnight at 4°C with 40 μL of protein A beads and 2 μg/mL anti-EGFR (homemade; cl.108.1) or anti-mig-6 antibody (homemade; cl. 1575). Total RNA was isolated from fresh liver tissue using Trizol (Sigma), and complementary DNA was synthesized using AMV Reverse Transcriptase (Roche). Reverse-transcription polymerase chain reaction primers were as follows: EGFR, 5′-GGAGGAAAAGAAAGTCTGCC-3′ (forward) and 5′-ATCGCACAGCACCAATCAGG-3′ (reverse) (Tm = 53°C, 28 cycles); HB-EGF, 5′-GCTGCCGTCGGTGATGCTGAAGC-3′ (forward) and 5′-GATGACAAGAAGACAGACG-3′ (reverse) (Tm = 60°C; 28 cycles); TGFα, 5′-CTCTGCTAGCGCTGGGTATCC-3′ PRKACG (forward) and 5′-AGGGCGCTGGGCTTCTCAT-3′

(reverse) (Tm = 58°C; 28 cycles); and cyclophilin A, 5′-GACGCCACTGTCGCTTTTCG-3′ (forward) and 5′-CTTGCCATCCAGCCATTCAGTC-3′ (reverse) (57°C; 24 cycles). All polymerase chain reactions were performed with an Eppendorf thermocycler. Hs 817.T and HepG2 human liver cancer cell lines were purchased from the American Type Culture Collection and cultured according to its guidelines. Primary hepatocytes were cultured on collagen I-coated (BD Biosciences) cell culture dishes in William’s E medium (Invitrogen, CA) containing 10% fetal bovine serum. All cells were starved in medium containing 0.1% fetal bovine serum overnight, stimulated with 50 ng/mL EGF (Gibco), and lysed at the indicated time points, and protein lysates were subjected to western blot analysis or immunoprecipitation. Phase contrast pictures were taken on a Zeiss Axiovert 300 microscope.

However, the news has not been uniformly positive. Several other studies on the subject have failed to detect a significant association between GS and CVD.[10, 15-20] A recent, large meta-analysis of 11 studies with 14 711 cases and 60 324 controls, including eight previously published studies and three Mendelian randomization studies undertaken by

the authors of this meta-analysis, examined the relationship of risk of various CVDs with three polymorphism sites related to the UGT1A1 gene.[21] In this analysis, homozygous carriers of the variants associated with increased bilirubin (UGT1A1*28/UGT1A1*28 genotype, rs887829 AA, or rs6742078 TT) showed no reduction in risk of various ischemic CVDs taken together (odds ratio = 1.01, 95% CI = 0.88–1.16) BAY 73-4506 cell line compared to heterozygotes and noncarriers.[21]

Moreover, in another study, GS genotype has shown no association with the severity of CAD.[22] Interestingly, our search in the database of human genome-wide association studies[23] failed to show any association of coronary heart disease with genetic markers located on the segment of the long Erlotinib research buy arm of chromosome 2, where UGT1A1 gene is located. This provides an important piece of evidence against association of GS with CAD, since the genome-wide studies on the subject have been quite large. Relationship of GS with other diseases besides CVD, such as cancers and chronic diseases, has also been studied. These studies have found a negative association between the presence Protein tyrosine phosphatase of UGT1A1*28 allele or high serum bilirubin and risk of endometrial[24]

and colon cancers.[25] Protective effects of GS against the occurrence of diabetes[26] and rheumatoid arthritis[27] have also been reported. The study by Horsfall et al. in the previous of the Journal, had several positive features, including a “cohort” study design and a large sample size. Further, the outcome assessed was all-cause death rate, whereas most of the previous studies among healthy persons had focused on CVD. All-cause death rate has the advantage of aggregating the effects of the factor under study on several disease processes (e.g. on CVD, malignant diseases, cerebrovascular accidents, infections, etc.). This obviates the possibility of the factor providing benefit against one disease while simultaneously increasing the risk of other diseases, wherein the harms may outweigh the benefits. Also, mortality is a hard endpoint that does not suffer from ascertainment bias. Thus, all-cause mortality provides a clinically relevant, reliable and preferred endpoint. However, it would have been useful if the authors had, in addition, provided data on cause-specific and age-specific mortality rates in the GS and non-GS groups. Such disaggregated data would have helped us identify the causes of death that are most influenced by the presence of GS. The study design did have some limitations.