Ischemic cardiac events can cause significant morbidity and mortality postliver transplantation; however, no validated protocols to screen patients before transplantation exist.
To report the introduction of a noninvasive cardiac screening protocol used at the Liver Unit, University of Calgary (Calgary, Alberta); to determine whether the protocol decreases use of coronary angiograms; and to compare cardiac outcomes using the new protocol with an appropriately matched historical control group.
A new cardiac screening protocol was introduced into the program in 2005, which uses perfusion scintigraphy to screen high-risk cardiac patients, reserving coronary angiograms for abnormal results. Transplanted patients screened using this protocol were compared with matched historical controls. Electronic charts were reviewed for cardiac outcomes intra- and postliver transplantation.
A total of 396 patients were screened between April 2005 and February 2009. Eighty-two were transplanted by February 2009 and included in the study. Eighty-one patients were successfully matched according to age, sex, cardiac history and presence of diabetes. Twelve of 82 (14.6%) and 11 of 81 (13.6%) in the study and control groups, respectively, underwent coronary angiograms (P=0.85). Coronary artery disease was found in six of 12 (50.0%) study patients and three of 11 (27.3%) control patients who underwent coronary angiography (P=0.27). The mean (± SD) length of the follow-up period was 1.87±0.91 years and 4.45±1.89 years in the study and control groups, respectively. One of 81 in the control group and zero of 82 in the study group experienced an acute coronary syndrome event postoperatively.
Coronary events are infrequent in liver transplant recipients. The described protocol is an effective method of coronary artery disease screening before liver transplant but does not reduce the number of cardiac investigations performed.
Acute coronary syndrome; Coronary angiogram; Coronary artery disease; Liver transplantation; Myocardial perfusion imaging
Background and Purpose
Surgical management of a renal neoplasm in a solitary kidney is a balance between oncologic control and preservation of renal function. We analyzed patients with a renal mass in a solitary kidney undergoing nephron-sparing procedures to determine perioperative, oncologic, and renal functional outcomes.
Patients and Methods
A multicenter study was performed from 12 institutions. All patients with a functional or anatomic solitary kidney who underwent nephron-sparing surgery for one or more renal masses were included. Tumor size, complications, and recurrence rates were recorded. Renal function was assessed with serum creatinine level and estimated glomerular filtration rate.
Ninety-eight patients underwent 105 ablations, and 100 patients underwent partial nephrectomy (PN). Preoperative estimated glomerular filtration rate (eGFR) was similar between the groups. Tumors managed with PN were significantly larger than those managed with ablation (P<0.001). Ablations were associated with a lower overall complication rate (9.5% vs 24%, P=0.01) and higher local recurrence rate (6.7% vs 3%, P=0.04). Eighty-four patients had a preoperative eGFR ≥60 mL/min/1.73 m2. Among these patients, 19 (23%) fell below this threshold after 3 months and 15 (18%) at 12 months. Postoperatively, there was no significant difference in eGFR between the groups.
Extirpation and ablation are both reasonable options for treatment. Ablation is more minimally invasive, albeit with higher recurrence rates compared with PN. Postoperative renal function is similar in both groups and is not affected by surgical approach.
Low-grade inflammation (LGI) is a central phenomenon in the genesis of obesity and insulin-resistance characterized by IL-6 in human serum. Whereas this LGI was initially thought to be mainly attributed to macrophage activation, it is now known that pre-adipocytes and adipocytes secrete several adipokines including IL-6 and participate to LGI and associated pathologies. In macrophages, HMGB1 is a nuclear yet secreted protein and acts as a cytokine to drive the production of inflammatory molecules through RAGE and TLR2/4. In this paper we tested the secretion of HMGB1 and the auto- and paracrine contribution to fat inflammation using the human preadipocyte cell line SW872 as a model. We showed that 1) human SW872 secreted actively HMGB1, 2) IL-6 production was positively linked to high levels of secreted HMGB1, 3) recombinant HMGB1 boosted IL-6 expression and this effect was mediated by the receptor RAGE and did not involve TLR2 or TLR4. These results suggest that HMGB1 is a major adipokine contributing to LGI implementation and maintenance, and can be considered as a target to develop news therapeutics in LGI associated pathologies such as obesity and type II diabetes.
Antigen-specific immunotherapy may offer a unique approach to fight cancer. We have demonstrated that specific immunotherapeutic regimens involving recombinant melanoma antigen family A3 (MAGE-A3) and different immunostimulants exert clinical anticancer activity. In particular, the combination of recombinant MAGE-A3 and AS15, a multicomponent immunostimulant, was found to elicit robust antigen-specific immune responses.
biomarkers; cancer; immunotherapy; immunization; MAGE-A3
In patients with advanced post-transplant hepatitis C virus (HCV) recurrence, antiviral treatment (AVT) with interferon and ribavirin is indicated to prevent graft failure. The aim of this study was to determine and report Canadian data with respect to the safety, efficacy, and spontaneous virologic response (SVR) predictors of AVT among transplanted patients with HCV recurrence.
Patients and Methods:
A retrospective chart review was performed on patients transplanted in London, Ontario and Edmonton, Alberta from 2002 to 2012 who were treated for HCV. Demographic, medical, and treatment information was collected and analyzed.
A total of 85 patients with HCV received pegylated interferon with ribavirin post-liver transplantation and 28 of the 65 patients (43%) with genotype 1 achieved SVR. Of the patients having genotype 1 HCV who achieved SVR, there was a significantly lower stage of fibrosis (1.37 ± 0.88 vs. 1.89 ± 0.96; P = 0.03), increased ribavirin dose (total daily dose 1057 ± 230 vs. 856 ± 399 mg; P = 0.02), increased rapid virologic response (RVR) (6/27 vs. 0/31; P = 0.05), increased early virologic response (EVR) (28/28 vs. 18/35; P = 0.006), and longer duration of therapy (54.7 ± 13.4 weeks vs. 40.2 ± 18.7; P = 0.001). A logistic regression model using gender, age, RVR, EVR, anemia, duration of therapy, viral load, years’ post-transplant, and type of organ (donation after cardiac death vs. donation after brain death) significantly predicted SVR (P < 0.001), with duration of therapy having a significant odds ratio of 1.078 (P = 0.007).
This study identified factors that predict SVR in HCV-positive patients who received dual therapy post-transplantation. Extending therapy from 48 weeks to 72 weeks of dual therapy is associated with increased SVR rates. Future studies examining the role of extended therapy are needed to confirm these findings, since the current study is a retrospective one.
Hepatitis C virus; liver transplant; retrospective study
Angiotensin receptor (type 1) blockers (ARBs) can reduce both hypertension and insulin resistance induced by local and systemic activation of the renin-angiotensin-aldosterone system. The effectiveness of azilsartan medoxomil (AZIL-M), a novel imidazole-based ARB, to facilitate metabolic improvements in conditions of angiotensin II (Ang II)-associated insulin resistance is currently unknown. The aim of this study was to determine the impact of chronic AZIL-M treatment on glucose transport activity and key insulin signaling elements in red skeletal muscle of Ang II-treated rats. Male Sprague-Dawley rats were treated for 8 weeks with or without Ang II (200 ng/kg/min) combined with either vehicle or AZIL-M (1 mg/kg/day). Ang II induced significant (p < 0.05) increases in blood pressure, which were completely prevented by AZIL-M. Furthermore, Ang II reduced insulin-mediated glucose transport activity in incubated soleus muscle, and AZIL-M co-treatment increased this parameter. Moreover, AZIL-M treatment of Ang II-infused animals increased the absolute phosphorylation of insulin signaling molecules, including Akt [both Ser473 (81%) and Thr308 (23%)] and AS160 Thr642 (42%), in red gastrocnemius muscle frozen in situ. Absolute AMPKα (Thr172) phosphorylation increased (98%) by AZIL-M treatment, and relative Thr389 phosphorylation of p70 S6K1, a negative regulator of insulin signaling, decreased (51%) with AZIL-M treatment. These results indicate that ARB AZIL-M improves the in vitro insulin action on glucose transport in red soleus muscle and the functionality of the Akt/AS160 axis in red gastrocnemius muscle in situ in Ang II-induced insulin-resistant rats, with the latter modification possibly associated with enhanced AMPKα and suppressed p70 S6K1 activation.
Angiotensin II; Glucose transport; Red skeletal muscle; Akt; AS160; AMPK; p70 S6K1
β-adrenergic stimulation is the main trigger for cardiac events in type-1 long QT syndrome (LQT1). We evaluated a possible association between ion channel response to β-adrenergic stimulation and clinical response to β-blocker therapy according to mutation location.
Methods and Results
The study sample comprised 860 patients with genetically-confirmed mutations in the KCNQ1 channel. Patients were categorized into carriers of missense mutations located in the cytoplasmic loops (C-loops), membrane spanning domain, C/N-terminus, and non-missense mutations. There were 27 aborted cardiac arrest [ACA] and 78 sudden cardiac death [SCD] events from birth through age 40 years. After multivariable adjustment for clinical factors, the presence of C-loop mutations was associated with the highest risk for ACA or SCD (hazard ratio [95% confidence interval] vs. non-missense mutations = 2.75 [1.29-5.86, P=0.009]). β-blocker therapy was associated with a significantly greater reduction in the risk of ACA or SCD among patients with C-loop mutations than among all other patients (hazard ratios = 0.12 [0.02-0.73, P=0.02] and 0.82 [0.31-2.13, P=0.68], respectively; P-for interaction = 0.04). Cellular expression studies showed that membrane spanning and C-loop mutations produced a similar decrease in current, but only C-loop mutations showed a pronounced reduction in channel activation in response to β-adrenergic stimulation.
Patients with C-loop missense mutations in the KCNQ1 channel exhibit a high-risk for life-threatening events and derive a pronounced benefit from treatment with β-blockers. Reduced channel activation following sympathetic activation can explain the increased clinical risk and response to therapy in patients with C-loop mutations.
beta-blockers; ion channels; long QT syndrome; mutation
Current clinical diagnosis of long-QT syndrome (LQTS) includes genetic testing of family members of mutation positive patients. The present study was designed to assess the clinical course of individuals who are found negative for the LQTS-causing mutation in their families.
Methods and Results
Multivariate Cox proportional hazards model was used to assess the risk for cardiac events (comprising syncope, aborted cardiac arrest [ACA], or sudden cardiac death [SCD]) from birth through age 40 years among 1828 subjects from the LQTS Registry who were found negative for their family LQTS-causing mutation. The median QTc of study subjects was 423 msec (interquartile-range: 402–442 msec). The cumulative probability of a first syncope through age 40 years was 15%. However, only 2 patients (0.1%) experienced ACA and none died suddenly during follow-up. Independent risk factors for syncope in genotype negative subjects included female gender (HR 1.60, p = 0.002), prolonged QTc (HR = 1.63 per 100 msec increment, p = 0.02), family history of ACA or SCD (HR = 1.89, p = 0.002), and LQT2 vs. LQT1 family mutation (HR = 1.41, p = 0.03). Subgroup analysis showed that the presence of the K897T polymorphism in the LQT2 gene in an affected family was associated with an 11-fold (p = 0.001) increase in the risk of recurrent syncope in genotype negative subjects.
Our findings suggest that cardiac events among genotype-negative family members of LQTS patients are dominated by nonfatal syncopal episodes without occurrence of sudden cardiac death. The risk for nonfatal events in this population may be mediated by the presence of common polymorphisms in LQTS genes.
gene mutation; genetic polymorphisms; long-QT syndrome; sudden cardiac death arrhythmia; syncope
Pax3 and Pax7 regulate stem cell function in skeletal myogenesis. However, molecular insight into their distinct roles has remained elusive. Using gene expression data combined with genome wide binding-site analysis we show that both Pax3 and Pax7 bind identical DNA motifs and jointly activate a large panel of genes involved in muscle stem cell function. Surprisingly, in adult myoblasts Pax3 binds a subset (6.4%) of Pax7 targets. Despite a significant overlap in their transcriptional network, Pax7 regulates distinct panels of genes involved in the promotion of proliferation and inhibition of myogenic differentiation. We show that Pax7 has a higher binding affinity to the homeodomain-binding motif relative to Pax3, suggesting that intrinsic differences in DNA binding contribute to the observed functional difference between Pax3 and Pax7 binding in myogenesis. Together, our data demonstrates distinct attributes of Pax7 function and provides mechanistic insight into the non-redundancy of Pax3 and Pax7 in muscle development.
Molecular profiling of renal cell carcinomas (RCC) may improve the distinction between oncocytoma and malignant RCC subtypes and aid in early detection of metastasis. Hyaluronic acid (HA) family includes HA-synthases (HAS1, HAS2, HAS3), hyaluronidases (HYAL-1, HYAL-2, HYAL-3, HYAL-4, PH20, HYAL-P1), and HA receptors (CD44s, CD44v and RHAMM). HA family members promote tumor growth and metastasis. We evaluated the expression of HA family members in kidney specimens.
Using quantitative PCR, mRNA levels of twelve HA family members were measured in tumor specimens obtained from 86 consecutive patients undergoing nephrectomy; 80 of them also provided normal specimens. Mean and median follow-up: 15.2 ± 8.8 and 13.8 months. RCC specimens included: clear cell RCC (ccRCC): 65; papillary: 10; chromophobe: 5; oncocytoma: 6; metastasis (+): 17.
Median HAS1, CD44s and RHAMM transcript levels were 3–25 elevated in ccRCC, papillary and chromophobe tumors when compared to normal tissues. HYAL-4, CD44s and RHAMM levels were 4–12-fold elevated in ccRCC and papillary tumors when compared to oncocytomas; only HYAL-4 levels distinguished between chromophobe and oncocytoma (P=0.009). CD44s and RHAMM levels were significantly higher in tumors < 4-cm (510±611; 19.6±20.8, respectively) when compared to oncocytoma (46.4±20; 3.8±2.5; P≤0.006). In univariate and multivariate analyses, CD44s (P<0.0001), RHAMM (P<0.0001), stage, tumor size, and/or renal vein involvement significantly associated with metastasis. The combined CD44s+RHAMM marker had 82% sensitivity and 86% specificity to predict metastasis.
CD44s and RHAMM levels distinguish between oncocytoma and RCC subtypes regardless of tumor size and are potential predictors of RCC metastasis.
Prognostic markers; HA-synthase; Hyaluronidase; HA-receptors; Renal cell carcinoma; metastasis; oncocytoma
Georges Bank is a large, shallow feature separating the Gulf of Maine from the Atlantic Ocean. Previous studies demonstrated a strong tidal-mixing front during the warm season on the northern bank margin between thermally stratified water in the Gulf of Maine and mixed water on the bank. Tides transport warm water off the bank during flood tide and cool gulf water onto the bank during ebb tide. During 10 days in August 2009, we mapped frontal temperatures in five study areas along ∼100 km of the bank margin. The seabed “frontal zone”, where temperature changed with frontal movment, experienced semidiurnal temperature maxima and minima. The tidal excursion of the frontal boundary between stratified and mixed water ranged 6 to 10 km. This “frontal boundary zone” was narrower than the frontal zone. Along transects perpendicular to the bank margin, seabed temperature change at individual sites ranged from 7.0°C in the frontal zone to 0.0°C in mixed bank water. At time series in frontal zone stations, changes during tidal cycles ranged from 1.2 to 6.1°C. The greatest rate of change (−2.48°C hr−1) occurred at mid-ebb. Geographic plots of seabed temperature change allowed the mapping of up to 8 subareas in each study area. The magnitude of temperature change in a subarea depended on its location in the frontal zone. Frontal movement had the greatest effect on seabed temperature in the 40 to 80 m depth interval. Subareas experiencing maximum temperature change in the frontal zone were not in the frontal boundary zone, but rather several km gulfward (off-bank) of the frontal boundary zone. These results provide a new ecological framework for examining the effect of tidally-driven temperature variability on the distribution, food resources, and reproductive success of benthic invertebrate and demersal fish species living in tidal front habitats.
The probiotic lactic acid bacterium Lactobacillus plantarum is a potential delivery vehicle for mucosal vaccines because of its generally regarded as safe (GRAS) status and ability to persist at the mucosal surfaces of the human intestine. However, the inherent immunogenicity of vaccine antigens is in many cases insufficient to elicit an efficient immune response, implying that additional adjuvants are needed to enhance the antigen immunogenicity. The goal of the present study was to increase the proinflammatory properties of L. plantarum by expressing a long (D1 to D5 [D1-D5]) and a short (D4-D5) version of the extracellular domain of invasin from the human pathogen Yersinia pseudotuberculosis. To display these proteins on the bacterial surface, four different N-terminal anchoring motifs from L. plantarum were used, comprising two different lipoprotein anchors, a transmembrane signal peptide anchor, and a LysM-type anchor. All these anchors mediated surface display of invasin, and several of the engineered strains were potent activators of NF-κB when interacting with monocytes in cell culture. The most distinct NF-κB responses were obtained with constructs in which the complete invasin extracellular domain was fused to a lipoanchor. The proinflammatory L. plantarum strains constructed here represent promising mucosal delivery vehicles for vaccine antigens.
The mammalian target of rapamycin (mTOR) is a serine kinase that regulates phosphorylation (p) of its target ribosomal S6 kinase (S6K1), whose activation can lead to glomerular and proximal tubular cell (PTC) injury and associated proteinuria. Increased mTOR/S6K1 signaling regulates signaling pathways that target fibrosis through adherens junctions. Recent data indicate aldosterone signaling through the mineralocorticoid receptor (MR) can activate the mTOR pathway. Further, antagonism of the MR has beneficial effects on proteinuria that occur independent of hemodynamics.
Accordingly, hypertensive transgenic TG(mRen2)27 (Ren2) rats, with elevated serum aldosterone and proteinuria, and age-matched Sprague-Dawley rats were treated with either a low dose (1 mg/kg/day) or a conventional dose (30 mg/kg/day) of spironolactone (MR antagonist) or placebo for 3 weeks.
Ren2 rats displayed increases in urine levels of the PTC brush border lysosomal enzyme N-acetyl-β-aminoglycosidase (β-NAG) in conjunction with reductions in PTC megalin, the apical membrane adherens protein T-cadherin and basolateral α-(E)-catenin, and fibrosis. In concert with these abnormalities, Ren2 renal cortical tissue also displayed increased Ser2448 (p)/activation of mTOR and Thr389 (p)-S6K1 and increased 3-nitrotyrosine (3-NT) content, a marker for peroxynitrite. Low-dose spironolactone had no effect on blood pressure but decreased proteinuria and β-NAG comparable to a conventional dose of this MR antagonist. Both doses of spironolactone attenuated ultrastructural maladaptive alterations and led to comparable reductions in (p)-mTOR/(p)-S6K1, 3-NT, fibrosis, and increased expression of α-(E)-catenin, T- and N-cadherin.
Thereby, MR antagonism improves proximal tubule integrity by targeting mTOR/S6K1 signaling and redox status independent of changes in blood pressure.
Cadherin; Megalin; β-NAG; Proteinuria
Epidemiological studies support the notion that arterial stiffness is an independent predictor of adverse cardiovascular events contributing significantly to systolic hypertension, impaired ventricular-arterial coupling and diastolic dysfunction, impairment in myocardial oxygen supply and demand, and progression of kidney disease. Although arterial stiffness is associated with aging, it is accelerated in the presence of obesity and diabetes. The prevalence of arterial stiffness parallels the increase of obesity that is occurring in epidemic proportions and is partly driven by a sedentary life style and consumption of a high fructose, high salt, and high fat western diet. Although the underlying mechanisms and mediators of arterial stiffness are not well understood, accumulating evidence supports the role of insulin resistance and endothelial dysfunction. The local tissue renin-angiotensin-aldosterone system (RAAS) in the vascular tissue and immune cells and perivascular adipose tissue is recognized as an important element involved in endothelial dysfunction which contributes significantly to arterial stiffness. Activation of vascular RAAS is seen in humans and animal models of obesity and diabetes, and associated with enhanced oxidative stress and inflammation in the vascular tissue. The cross talk between angiotensin and aldosterone underscores the importance of mineralocorticoid receptors in modulation of insulin resistance, decreased bioavailability of nitric oxide, endothelial dysfunction, and arterial stiffness. In addition, both innate and adaptive immunity are involved in this local tissue activation of RAAS. In this review we will attempt to present a unifying mechanism of how environmental and immunological factors are involved in this local tissue RAAS activation, and the role of this process in the development of endothelial dysfunction and arterial stiffness and targeting tissue RAAS activation.
renin-angiotensin-aldosterone system; arterial stiffness; insulin resistance; endothelial dysfunction; obesity; diabetes
This study was designed to evaluate the clinical and prognostic aspects of long QT syndrome-related cardiac events that occur in the first year of life (infancy).
The clinical implications for patients with long QT syndrome who experience cardiac events in infancy have not been studied previously.
The study population of 3,323 patients with QTc ≥ 450 ms enrolled in the International LQTS Registry involved 20 patients with sudden cardiac death (SCD), 16 patients with aborted cardiac arrest (ACA), 34 patients with syncope, and 3,253 patients who were asymptomatic during the first year of life.
The risk factors for a cardiac event among 212 patients who had an ECG recorded in the first year of life included QTc≥500ms, heart rate ≤100bpm, and female sex. ACA before age 1 year was associated with a hazard ratio of 23.4 (p<0.01) for ACA or SCD during age 1-10 years. During the 10-year follow-up after infancy, beta-blocker therapy was associated with a significant reduction in ACA/SCD only in those with a syncopal episode within 2 years before ACA/SCD, but not for those who survived ACA in infancy.
Patients with LQTS who experience ACA during the first year of life are at very high-risk for subsequent ACA or death during their next 10 years of life, and beta-blockers may not be effective in preventing fatal or near fatal cardiac events in this small but high-risk subset.
Long QT Syndrome; Genetics; Infants; Risk Stratification
Altered autonomic (ANS) tone in chronic respiratory disease is implicated as a factor in cardiovascular co-morbidities, yet no studies address its impact on cardiovascular function in the presence of murine allergic airway (AW) hyperresponsiveness (AHR). Since antigen (Ag)-induced AHR is used to model allergic asthma (in which ANS alterations have been reported), we performed a pilot study to assess measurement feasibility of, as well as the impact of allergic sensitization to ovalbumin (OVA) on, heart rate variability (HRV) in a murine model. Heart rate (HR), body temperature (TB), and time- and frequency-domain HRV analyses, a reflection of ANS control, were obtained in chronically instrumented mice (telemetry) before, during and for 22 h after OVA or saline aerosolization in sensitized (OVA) or Alum adjuvant control exposed animals. OVA mice diverged significantly from Alum mice with respect to change in HR during aerosol challenge (P < 0.001, Two-Way ANOVA; HR max change Ctrl = +80 ± 10 bpm vs. OVA = +1 ± 23 bpm, mean ± SEM), and displayed elevated HR during the subsequent dark cycle (P = 0.006). Sensitization decreased the TB during aerosol challenge (P < 0.001). Sensitized mice had decreased HRV prior to challenge (SDNN: P = 0.038; Low frequency (LF) power: P = 0.021; Low/high Frequency (HF) power: P = 0.042), and increased HRV during Ag challenge (RMSSD: P = 0.047; pNN6: P = 0.039). Sensitized mice displayed decreased HRV subsequent to OVA challenge, primarily in the dark cycle (RMSSD: P = 0.018; pNN6: P ≤ 0.001; LF: P ≤ 0.001; HF: P = 0.040; LF/HF: P ≤ 0.001). We conclude that implanted telemetry technology is an effective method to assess the ANS impact of allergic sensitization. Preliminary results show mild sensitization is associated with reduced HRV and a suppression of the acute TB-response to OVA challenge. This approach to assess altered ANS control in the acute OVA model may also be beneficial in chronic AHR models.
airway hyperresponsiveness; mouse; ovalbumin; heart rate variability; autonomic control; allergic sensitization; asthma; cardiovascular comorbidity
In this study, antibiofilm activity of coconut husk extract (CHE) was tested by various assays in the laboratory. The effects of CHE on extracellular polymeric substance (EPS) production, hydrophobicity and adhesion ability of Pseudomonas sp., Alteromonas sp. and Gallionella sp. and the antimicrobial activity of the extract against these bacteria were assessed. CHE was found to possess antibacterial activity against all the bacterial strains and affected the EPS production. The CHE affected the growth of the biofilm-forming bacteria in a culture medium. The hydrophobicity of the bacterial cells was also changed due to the CHE treatment. The active compound of the CHE was characterised by thin-layer chromatography (TLC), high performance liquid chromatography (HPLC) and fourier transform infrared (FT-IR) analysis. HPLC spectrum showed a single peak and the FT-IR spectrum indicated the presence of an OH-group-containing compound in the extract. In conclusion the CHE could be used as a source for the isolation of antifouling compounds.
Biofouling; Biofilm; Polyphenol; Extracellular polymeric substance; Adhesion
High-throughput, culture-independent surveys of bacterial and archaeal communities in soil have illuminated the importance of both edaphic and biotic influences on microbial diversity, yet few studies compare the relative importance of these factors. Here, we employ multiplexed pyrosequencing of the 16S rRNA gene to examine soil- and cactus-associated rhizosphere microbial communities of the Sonoran Desert and the artificial desert biome of the Biosphere2 research facility. The results of our replicate sampling approach show that microbial communities are shaped primarily by soil characteristics associated with geographic locations, while rhizosphere associations are secondary factors. We found little difference between rhizosphere communities of the ecologically similar saguaro (Carnegiea gigantea) and cardón (Pachycereus pringlei) cacti. Both rhizosphere and soil communities were dominated by the disproportionately abundant Crenarchaeota class Thermoprotei, which comprised 18.7% of 183,320 total pyrosequencing reads from a comparatively small number (1,337 or 3.7%) of the 36,162 total operational taxonomic units (OTUs). OTUs common to both soil and rhizosphere samples comprised the bulk of raw sequence reads, suggesting that the shared community of soil and rhizosphere microbes constitute common and abundant taxa, particularly in the bacterial phyla Proteobacteria, Actinobacteria, Planctomycetes, Firmicutes, Bacteroidetes, Chloroflexi, and Acidobacteria. The vast majority of OTUs, however, were rare and unique to either soil or rhizosphere communities and differed among locations dozens of kilometers apart. Several soil properties, particularly soil pH and carbon content, were significantly correlated with community diversity measurements. Our results highlight the importance of culture-independent approaches in surveying microbial communities of extreme environments.
Seaweed is a highly attractive marine crop for the production of biofuels, due to its rapid growth rate as well as high polysaccharide and low lignin content. One appealing exploitation route is the production of biogas by anaerobic digestion. Interestingly, despite the compositional differences between seaweed and lignocellulosic biomass, available data indicate that conditions and inocula traditionally used for the latter may work well for seaweed. To gain more insight into the underlying microbial processes, we have generated 16S rRNA gene amplicon pyrosequencing data to comparatively describe microbial communities in biogas digesters containing either the seaweed Saccharina latissima or wheat straw. The seaweed digesters gave better biogas yield and a higher relative abundance of core group Methanosaeta-affiliated Archaea. Conversely, variation in biomass had only minor abundance effects towards dominant bacterial lineages and influenced only low-abundant bacterial OTUs. Affiliations between dominant archaeal and bacterial phylotypes described here and previously identified anaerobic digestion core groups indicate that trends are beginning to emerge within these newly explored microbial ecosystems, the understanding of which is currently impeded by limited published datasets.
Biogas; Anaerobic digestion; Seaweed; Macroalgae; Methane
Previous surveys have shown that adult honeybee (Apis mellifera) workers harbor a characteristic gut microbiota that may play a significant role in bee health. For three major phylotypes within this microbiota, we have characterized distributions and abundances across the life cycle and among gut organs. These distinctive phylotypes, called Beta, Firm-5, and Gamma-1 (BFG), were assayed using quantitative PCR, fluorescent in situ hybridization (FISH) microscopy, and the experimental manipulation of inoculation routes within developing bees. Adult workers (9 to 30 days posteclosion) contained a large BFG microbiota with a characteristic distribution among gut organs. The crop and midgut were nearly devoid of these phylotypes, while the ileum and rectum together contained more than 95% of the total BFG microbiota. The ileum contained a stratified community in which the Beta and Gamma-1 phylotypes dominated, filling the longitudinal folds of this organ. Deep sequencing of 16S rRNA genes showed clear differences among communities in midgut, ileum, and rectum. In contrast with older workers, larvae and newly emerged workers contain few or no bacteria, and their major food source, bee bread, lacks most characteristic phylotypes. In experiments aimed at determining the route of inoculation, newly emerged workers (NEWs) sometimes acquired the typical phylotypes through contact with older workers, contact with the hive, and emergence from the brood cell; however, transmission was patchy in these assays. Our results outline a colonization pattern for the characteristic phylotypes through A. mellifera ontogeny. We propose the names “Candidatus Snodgrassella alvi” and “Candidatus Gilliamella apicola” for the Beta and Gamma-1 phylotypes, respectively.
Chikungunya virus (CHIKV) is an arthritogenic member of the Alphavirus genus (family Togaviridae) transmitted by Aedes mosquitoes. CHIKV is now known to target non hematopoietic cells such as epithelial, endothelial cells, fibroblasts and to less extent monocytes/macrophages. The type I interferon (IFN) response is an early innate immune mechanism that protects cells against viral infection. Cells express different pattern recognition receptors (including TLR7 and RIG-I) to sense viruses and to induce production of type I IFNs which in turn will bind to their receptor. This should result in the phosphorylation and translocation of STAT molecules into the nucleus to promote the transcription of IFN-stimulated antiviral genes (ISGs). We herein tested the capacity of CHIKV clinical isolate to infect two different human fibroblast cell lines HS 633T and HT-1080 and we analyzed the resulting type I IFN innate immune response.
Indirect immunofluorescence and quantitative RT-PCR were used to test for the susceptibility of both fibroblast cell lines to CHIKV.
Interestingly, the two fibroblast cell lines HS 633T and HT-1080 were differently susceptible to CHIKV infection and the former producing at least 30-fold higher viral load at 48 h post-infection (PI). We found that the expression of antiviral genes (RIG-I, IFN-β, ISG54 and ISG56) was more robust in the more susceptible cell line HS 633T at 48 h PI. Moreover, CHIKV was shown to similarly interfere with the nuclear translocation of pSTAT1 in both cell lines.
Critically, CHIKV can control the IFN response by preventing the nuclear translocation of pSTAT1 in both fibroblast cell lines. Counter-intuitively, the relative resistance of HT-1080 cells to CHIKV infection could not be attributed to more robust innate IFN- and ISG-dependent antiviral responses. These cell lines may prove to be valuable models to screen for novel mechanisms mobilized differentially by fibroblasts to control CHIKV infection, replication and spreading from cell to cell.
CHIKV; Type I IFN; HS 633T; HT-1080; RIG-I; TLR7; STAT-1
Nonsense and frameshift mutations are common in congenital long QT syndrome type 2 (LQT2). We previously demonstrated that hERG nonsense mutations cause degradation of mutant mRNA by nonsense-mediated mRNA decay (NMD) and are associated with mild clinical phenotypes. The impact of NMD on the expression of hERG frameshift mutations and their phenotypic severity is not clear.
To examine the role of NMD in the pathogenesis of a hERG frameshift mutation, P926AfsX14, identified in a large LQT2 kindred and characterize genotype-phenotype correlations.
Genetic screening was performed among family members. Phenotyping was performed by assessment of ECGs and LQTS-related cardiac events. The functional effect of P926AfsX14 was studied using hERG cDNA and minigene constructs expressed in HEK293 cells.
Significant cardiac events occurred in carriers of the P926AfsX14 mutation. When expressed from cDNA, the P926AfsX14 mutant channel was only mildly defective. However, when expressed from a minigene, the P926AfsX14 mutation caused a significant reduction in mutant mRNA, protein, and hERG current. Inhibition of NMD by RNA interference knockdown of up-frameshift protein 1 partially restored expression of mutant mRNA and protein, and led to a significant increase in hERG current in the mutant cells. These results suggest that NMD is involved in the pathogenic mechanism of the P926AfsX14 mutation.
Our findings suggest that the hERG frameshift mutation P926AfsX14 primarily results in degradation of mutant mRNA by the NMD pathway rather than production of truncated proteins. When combined with environmental triggers and genetic modifiers, LQT2 frameshift mutations associated with NMD can manifest with a severe clinical phenotype.
Nonsense-mediated mRNA decay; Long QT syndrome; Potassium channel; Patch clamp; Mutation
Unlike conventional β-blockers, nebivolol, a third-generation β-adrenergic receptor blocker with vasodilator properties, promotes insulin sensitivity. Objective: The objective of this study was to determine whether nebivolol regulates overnutrition-induced activation of cardiac nutrient sensor kinases and inflammatory signaling.
Young Zucker obese (ZO) rats, a rodent model for overnutrition, and age-matched Zucker lean rats were treated with nebivolol (10 mg/kg/day; 21 days) and cardiac function was monitored by echocardiography and pressure volume loop analysis. Activation status of nutrient sensor serine/threonine kinases mammalian target for rapamycin (mTOR), and p70 S6kinase (S6K1) and S6K1-substrate RPS6, inflammatory marker Janus kinase 2 (Jak2) and its substrate STAT1, and energy sensor AMP-dependent kinase (AMPK) were monitored by determining phosphorylation status of pSer2448 of mTOR, pThr389 of S6K1, pSer235/236 of RPS6, pTyr1007/1008 of Jak2, pTyr701 of STAT1, and pThr172 of AMPK, respectively.
Nebivolol reduced weight and improved cardiac function of ZO rats as shown by improvements in the myocardial performance index and a decrease in the diastolic parameter tau (τ), the time constant of isovolumic relaxation. Nebivolol also attenuated excessive activation of the nutrient sensor kinases mTOR and S6K1 and their substrate RPS6 as well as the inflammatory marker Jak2 and substrate STAT1 in ZO myocardium (p < 0.05). Moreover, nebivolol reversed suppression of the energy sensor kinase AMPK in ZO hearts (p < 0.05).
We report for the first time that nebivolol regulates overnutrition-induced activation of cardiac mTOR and Jak/STAT signaling and reverses suppression of AMPK. Since it also suppresses weight gain, nebivolol appears effective in the treatment of overnutrition-related cardiac inflammation and diastolic dysfunction.
Nebivolol; Zucker obese; AMP kinase; mTORC1; Jak/STAT
The molecular basis of cell signal-regulated alternative splicing at the 3′ splice site remains largely unknown. We isolated a protein kinase A-responsive ribonucleic acid (RNA) element from a 3′ splice site of the synaptosomal-associated protein 25 (Snap25) gene for forskolin-inhibited splicing during neuronal differentiation of rat pheochromocytoma PC12 cells. The element binds specifically to heterogeneous nuclear ribonucleo protein (hnRNP) K in a phosphatase-sensitive way, which directly competes with the U2 auxiliary factor U2AF65, an essential component of early spliceosomes. Transcripts with similarly localized hnRNP K target motifs upstream of alternative exons are enriched in genes often associated with neurological diseases. We show that such motifs upstream of the Runx1 exon 6 also bind hnRNP K, and importantly, hnRNP K is required for forskolin-induced repression of the exon. Interestingly, this exon encodes the peptide domain that determines the switch of the transcriptional repressor/activator activity of Runx1, a change known to be critical in specifying neuron lineages. Consistent with an important role of the target genes in neurons, knocking down hnRNP K severely disrupts forskolin-induced neurite growth. Thus, through hnRNP K, the neuronal differentiation stimulus forskolin targets a critical 3′ splice site component of the splicing machinery to control alternative splicing of crucial genes. This also provides a regulated direct competitor of U2AF65 for cell signal control of 3′ splice site usage.
Lignocellulosic biomass remains a largely untapped source of renewable energy predominantly due to its recalcitrance and an incomplete understanding of how this is overcome in nature. We present here a compositional and comparative analysis of metagenomic data pertaining to a natural biomass-converting ecosystem adapted to austere arctic nutritional conditions, namely the rumen microbiome of Svalbard reindeer (Rangifer tarandus platyrhynchus). Community analysis showed that deeply-branched cellulolytic lineages affiliated to the Bacteroidetes and Firmicutes are dominant, whilst sequence binning methods facilitated the assemblage of metagenomic sequence for a dominant and novel Bacteroidales clade (SRM-1). Analysis of unassembled metagenomic sequence as well as metabolic reconstruction of SRM-1 revealed the presence of multiple polysaccharide utilization loci-like systems (PULs) as well as members of more than 20 glycoside hydrolase and other carbohydrate-active enzyme families targeting various polysaccharides including cellulose, xylan and pectin. Functional screening of cloned metagenome fragments revealed high cellulolytic activity and an abundance of PULs that are rich in endoglucanases (GH5) but devoid of other common enzymes thought to be involved in cellulose degradation. Combining these results with known and partly re-evaluated metagenomic data strongly indicates that much like the human distal gut, the digestive system of herbivores harbours high numbers of deeply branched and as-yet uncultured members of the Bacteroidetes that depend on PUL-like systems for plant biomass degradation.