Ischemic nephropathy is an emerging cause of end stage renal disease, associated with many co-morbidities especially cardiovascular disease risk and derangement in calcium-phosphorus homeostasis resulting in hyperphosphatemia, influencing bones, a characteristic of advancing chronic kidney disease. The management of elevated serum phosphorus has been a challenge in this patient population with compromised kidney performance, as available phosphorus lowering agents possess many undesirable hazardous secondary effects and/or are very expensive. While niacin in different formulation is known to not only correct dyslipidemia but also reduce phosphorus level, but its clinical use restricted owing to side effects. The objective of present study is to evaluate such effect of niacin extended release (NER) in ischemic nephropathy. The chronic kidney disease patients fulfilling the pre-defined criteria were randomly categorized into two groups of equal size (n=60) and prescribed either atorvastatin 20 mg/day or NER 500 mg/day with the same dose of statin for four months. A control of 50 healthy characters matched was also incorporated for local reference range. Baseline and follow up phosphorus concentration was measured and means were compared using t-test at SPSS version 17 with 0.05 chosen alpha. There was no difference in the baseline levels in both groups while significant (p<0.001) hyperphosphatemia was observed in both units as compared with healthy controls. The administration of atorvastatin alone for four weeks showed an insignificant decrease in phosphorus, whereas, NER significantly reduced phosphorus (p<0.001). The mean percent change from baseline to follow up further endorsed the finding as statin alone brought -13.8 % reduction in phosphorus and NER -47 % from baseline. NER, at its lowest prescribed dose once a day was well tolerated by most of the patients and demonstrated significant goal achievement of phosphorus reduction. It is concluded that NER even at low doses in renal compromised dyslipidemic patients may be a promising approach to prevent the harmful vascular, valvular effects caused by hyperphosphatemia in addition to its principal target of HDL-C elevation. Renal ischemia is an outcome of renal arteriosclerosis manifested by kidney hypo-perfusion beyond level of auto-regulation that brings significant decrease in glomerular filtration rate (GFR) by affecting the whole functional parenchyma following renal vessel occlusion  suggests reduction in enhanced concentrations of phosphorus toward normal range. The epidemiological studies are suggestive of the clinical basis to control serum phosphorus as they concluded up that hyperphosphatemia is a significant risk factor for secondary hyperparathyroidism and for cardiovascular disease . LimiteCalcium salts supplements, on the other side, are effective and well tolerable in controlling phosphorus in end stage renal disease, but they favor cardiovascular disease and calcification of aortic valve . SumminNiacin was first reported to decrease plasma cholesterol in 1955 . The maAs niacin is converted largely to niacinamide, it also inhibits intestinal phosphate absorption. Early research experiments are suggestive that niacin intake might be beneficial as prime or adjunctive therapy to treat pronounced hyperphosphatemia, a characteristic feature of end stage renal disease (ESRD) . ThoughOn the basis of clinical picture and Doppler scan report, finally 135 patients were recognized as potential experimental units and were requested for consent to participate in a follow up study after explaining the purpose and satisfying their queries. Fifteen of total identified individuals did not show consent so skipped. While left over were randomly (randomization by computer generated random number sequence) distributed in two treatment groups. Group 1 was prescribed atorvastatin (20 mg/day) and group 2 NER (500 mg/day) in combination with same dose of atorvastatin (20 mg/day) for 16-weeks in a 1:1 ratio. Information regarding personal and family medical history, demographics, current medication, socio-economic status, physical activity, smoking and other required items were taken on a structured questionnaire at day first. Blood samples were collected from antecubital vein after 12-hour fasting and either plasma or serum was separated according to the assay requirement. All processed samples were stored in 0.5 ml small cuvettes at -80 °C unless used for biochemical estimation.Inorganic phosphorus was estimated in non-hemolysed serum samples using research grade RANDOX, UK kit. Working reagent was composed of 28 ml blank reagent  mixed with 12 ml molybdate reagent . Standard solution was comprised of 5 mg/dl potassium phosphate. Working reagent was allowed to react with standard and samples for 10 minutes at 25 °C in separate test tubes then absorbance was measured against reagent blank at 340 nm. Inorganic phosphorus concentration was calculated using unitary method of known concentration (standard) to unknown (sample).Sodium and potassium were measured in serum samples using Ion Selective Electrode Beckman Coulter AU clinical Chemistry Analyzer intended for quantitative measurement of electrolytes. According to manufacturer's instructions, hemolysis free lithium heparin serum samples were used for estimations within 48 hours after drawing. Apparatus were calibrated prior to sample readings using ISE low to high serum standards for sodium (130-160 mEq/L), for potassium (10-100 mEq/L) respectively using the particular electrodes for both ions. Sample was prepared by adding 20 μL serum in ISE buffer triethanolamine and read directly by inserting the specific electrode. Ions concentration was measured in mEq/L.The study was controlled by the directions set in Declaration of Helsinki and Good Clinical Practice standards prescribed by World Medical Association (2002). The pl® at chosen p-value 0.05. Percent protection induced by drug treatments was calculated by dividing the difference of pre and post treatment with control  and multiplying the result with 100.Collected data was tabulated and reported as Mean ± S.D. t-Test was used to compare mean values through statistical software SPSS 1754 patients taking statin while 51 with statin NER combination completed the protocol successfully for four months and assessed for final changes. The basic characteristics of CKD patients with ischemic renal changes assessed for both drug combinations are illustrated in Table 1The effects of NER on lipid profile and endothelial function have been discussed by same authors earlier  and nOn comparison of the reduction in potassium and phosphate from respective baseline to four months follow up in the patients treatment groups either on statin alone or in combination with NER, it was found that both treatment protocols brought a decrease in potassium  respectively while in case of phosphorus a generalized decrease from baseline value was observed though only significant in statin + NER group (p<0.001) Table 3. Thus onThe mean percent change from baseline to follow up further endorsed the finding as statin alone brought -47 % reduction in potassium as compared with NER -56 % in case of phosphorus it was observed with more difference recorded as -14 %, -47 % respectively Figure 1. Derangments in calcium-phosphorus homeostasis particularly markedly raised serum phosphorus levels are considered to play a significant role in arterial stiffening, hypertension, and CKD associated CVD risk  in phosphorus level as compared to the baseline values Table 3 while MuCheng and Young (2008) did a pAnimal model data highlight plausible mechanisms, especially fecal loss . The enThe use of NER even at low doses in renal compromised dyslipidemic patients may be a promising approach to prevent the harmful vascular, valvular effects caused by hyperphosphatemia in addition to its principal target of HDL-C elevation. The study was partially supported by a research grant from Dean, Faculty of Science’s research support program for which authors express their gratitude. The authors declare that they have no conflict of interest.


Sequences most similar to Metopus species predominated, with the majority of sequences most closely related to M. palaeformis, a cilitated protozoan known to harbor methanogenic symbionts. Quantification of mcrA mRNA transcripts in the groundwater suggested that methanogens closely related to Metopus endosymbionts were metabolically active at this time. There was a strong correlation between the number of mcrA transcripts from the putative endosymbiotic methanogen and Metopus ß-tubulin mRNA transcripts during the course of the field experiment, suggesting that the activity of the methanogens was dependent upon the activity of the Metopus species. Addition of the eukaryotic inhibitors cyclohexamide and colchicine to laboratory incubations of acetate-amended subsurface sediments significantly inhibited methane production and there was a direct correlation between methane concentration and Metopus ß-tubulin and putative symbiont mcrA gene copies. These results suggest that, following the stimulation of subsurface microbial growth with acetate, protozoa harboring methanogenic endosymbionts become important members of the microbial community, feeding on moribund biomass and producing methane.Previous studies have suggested that protozoa prey on Fe(III)- and sulfate-reducing bacteria that are enriched when acetate is added to uranium contaminated subsurface sediments to stimulate U(VI) reduction. In order to determine whether protozoa continue to impact subsurface biogeochemistry after these acetate amendments have stopped, 18S rRNA and ß-tubulin sequences from this phase of an  Methanogenic microbial communities exemplify the importance of interspecies interactions. These include not only the various forms of interspecies electron transfer between bacteria and methanogens  to less soluble U(IV)  reduction is stimulated with the addition of organic electron donors  was reductively precipitated from the groundwater, arsenic was also released  during the months of August-October as previously described  mixed with native groundwater was injected into the subsurface to provide approximately 15 mM acetate to the groundwater over the course of 68 days as previously described  and 3 control (no acetate additions) incubations were monitored over the course of 50 days. Fe(III) reduction, sulfate reduction, or methanogenesis was not observed in control incubations.For sediment incubations, 40 g of the background sediments described above, 16 ml groundwater and acetate (~20 mM) were added to 60 ml serum bottles in an anaerobic chamber under an NGroundwater samples for geochemical analyses were collected after purging 12 l of groundwater from the wells with a peristaltic pump. Ferrous iron was measured spectrophotometrically immediately after sampling using the phenanthroline method  for ferrous iron. After filtration through a 0.2 μm pore size polytetrafluoroethylene [PTFE (Teflon)] filter , acetate, bromide, chloride, sulfate, and thiosulfate were measured using an ion chromatograph  equipped with an AS18 column under isocratic elution with 32 mM KOH as the eluent.−3 mol/L/atm.Samples for dissolved gas analysis were collected using a passive gas sampler consisting of a 5 cm length of gas permeable silicone tubing affixed to the end of a 10 mL valve sealable, gas-tight syringe  patterned on that described by Spalding and Watson  was monitored over time with a ferrozine assay in a split-beam dual-detector spectrophotometer  at an absorbance of 562 nm after a 1 h extraction with 0.5 N HCl  contaminated aquifer during the bioremediation field experiments. In order to obtain sufficient biomass from the groundwater, it was necessary to concentrate 50 l of groundwater by impact filtration on 293 mm diameter Supor membrane disc filters with pore sizes of 1.2 and 0.2 μm , which took about 3 min. All filters were placed into whirl-pack bags, flash frozen in a dry ice/ethanol bath, and shipped back to the laboratory where they were stored at −80°C. RNA was extracted from filters as previously described , microfluidic analysis , and gel electrophoresis showed that high quality DNA and RNA were extracted from the groundwater samples. In order to ensure that RNA samples were not contaminated with DNA, PCR amplification with primers targeting the 16S rRNA gene was conducted on RNA samples that had not undergone reverse transcription.A DuraScript enhanced avian RT single strand synthesis kit (Sigma) was used to generate cDNA as previously described , 2.5 U Taq DNA polymerase (QIAGEN) and 10 ng of DNA template. Amplification was performed with a minicycler PTC 200 (MJ Research) starting with 5 min at 94°C, followed by 35 cycles consisting of denaturation (45 s at 94°C), annealing (see Table S1), extension (90 s at 72°C), and a final extension at 72°C for 10 min.A 50 μl PCR reaction consisted of the following solutions: 10 μl Q buffer (Qiagen), 0.4 mM of each dNTP, 1.5 mM MgClAfter PCR amplification of these gene fragments, PCR products were purified with the Gel Extraction Kit (Qiagen), and cloned into the TOPO TA cloning vector, version M . One hundred plasmid inserts from each of these clone libraries were sequenced with the M13F primer at the University of Massachusetts Sequencing Facility.in situ Metopus species and the mcrA gene from in situ Methanomicrobiales species were designed according to the manufacturer's specifications (Applied Biosystems) and had amplicon sizes ranging from 100 to 200 bp (See Supplementary Table Metopus specific primer set (Met-bt_60f/155r) was designed from Metopus ß-tubulin clone 9 (KJ609554) which accounted for 90% of the sequences from the ß-tubulin cDNA clone library assembled with groundwater collected on day 95 of the field experiment and shared 99% of its nucleotides with the ß-tubulin gene from Metopus palaeformis. The Methanomicrobiales specific primer set (Rifle_mcrA_379f/489r) was designed from Rifle mcrA clone 6 (KJ609576) and accounted for 52% of the mcrA cDNA clone library assembled with groundwater collected on day 95.Quantitative PCR primer sets targeting the ß-tubulin gene from Quantitative PCR amplification and detection were performed with the 7500 Real Time PCR System (Applied Biosystems) using genomic DNA and cDNA made by reverse transcription from mRNA extracted from groundwater collected during the bioremediation experiment. All qPCR assays had triplicate biological and technical replicates. Each reaction mixture consisted of a total volume of 25 μL and contained 1.5 μL of the appropriate primers , 5 ng cDNA, and 12.5 μL Power SYBR Green PCR Master Mix (Applied Biosystems). Standard curves covering 8 orders of magnitude were constructed with serial dilutions of known amounts of purified cDNA quantified with a NanoDrop ND-1000 spectrophotometer at an absorbance of 260 nm. Transcript abundances and qPCR efficiencies (90–99%) were calculated from appropriate standard curves and all qPCR experiments followed MIQE guidelines , followed by an accumulation of sulfide, indicated a typical succession   of the mcrA (methyl coenzyme M reductase A), was also being actively transcribed in the subsurface following the sulfate reducing phase of the experiment. When acetate concentrations were high, the majority of mcrA mRNA transcripts (58.3%) clustered with Methanosarcinales  shared 87% of its nucleotides with an endosymbiont of Metopus contortus  and the dominant Metopus species (represented by ß-tubulin clone 9), transcripts from methanogen- and protozoan-related genes were quantified over time  between the number of transcripts from clone 6 mcrA and Metopus ß-tubulin genes during the course of the field experiment. Transcription of both genes was low (2.04 × 101 − 9.6 × 102 mRNA transcripts per gene copies) until day 81, when the number of mRNA transcripts increased by two orders of magnitude , and between methane production and Metopus ß-tubulin  and putative symbiont mcrA  gene copies.Analysis of gene copies for d Figure . After ain situ bioremediation of uranium-contaminated groundwater with the addition of high concentrations of acetate can have the unintended negative consequence of stimulating methanogenesis. Endosymbiotic methanogens harbored in protozoa appeared to be responsible for much of the methane produced after acetate additions stopped. These studies further emphasize the importance of protozoa in influencing microbial community dynamics and subsurface geochemistry during the bioremediation process.These results demonstrate that stimulating Both the field experiment and laboratory incubation studies indicated that the enrichment of protozoa harboring endosymbiotic methanogens took place after acetate injections stopped and acetate concentrations had significantly declined in the aquifer. Previous studies showed that high concentrations of acetate stimulate the growth of Fe(III)-reducing and sulfate-reducing microorganisms, and specific protozoan populations that appear to specialize in predation of those actively growing microbes (Holmes et al., Metopus species are known to feed both on detritus as well as intact bacterial cells (Esteban et al., Metopus species that possess symbiotic methanogens may have a competitive advantage as the growth rate of protozoa is increased when they harbor methanogens (Finlay and Fenchel, 2 that can be consumed by free-living bacteria. This study showed that, without acetate additions to the subsurface, there was not sufficient electron donor supply to deplete sulfate from the groundwater (Figure 2 produced within the hydrogenosome of the host protozoan. A comparable benefit to methanogens has been reported in marine sediments in which methanogen endosymbionts can account for 90% of the methane produced in the subsurface (Fenchel, Fermentative microorganisms are also expected to be actively involved in degrading the biomass that accumulates following acetate injection (N'Guessan et al., r Figure . Free-lir Figure . HoweverFenchel, . In a siFenchel, .in situ uranium bioremediation. It is likely that protozoa have similar influences in other bioremediation strategies that rely on stimulating microbial metabolism with organic electron donors.These results further demonstrate the important impact that protozoa can have on the ecology and biogeochemistry of The Associate Editor declares that despite having collaborated with author Kenneth H. Williams, the review process was handled objectively. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cattleyak are hybrid between cattle and yak, which exhibit equivalent adaptability on the Qinghai-Tibetan Plateau as yak and much higher capability in economic traits. However, the F1 males of cattleyak are infertile due to spermatogenic arrest and this greatly restricts the effective utilization of this hybrid. In this data article, differentially expressed proteins (DEPs) were identified from testis proteome of cattleyak and yak using high-performance liquid chromatography–electrospray tandem mass spectrometry (LC–ESI-MS/MS). All the DEPs were subjected to functional classification by Gene Ontology (GO) analysis and gene-pathway annotation by Kyoto Encyclopedia of Genes and Genomes (KEGG). The comparative testis proteome dataset here can shed new light on the molecular characteristics of male infertility of cattleyak on proteome level, “Comparative iTRAQ proteomics revealed proteins associated with spermatogenic arrest of cattleyak”  Specifications TableValue of the data•This is the first dataset to examine the male infertility of cattleyak by comparative testis proteome.•The dataset lays the basis for a clear presentation of up-regulated proteins in cattleyak associated with various stresses/cell adhesion/germ cell migration and down-regulated proteins associated with defects in various metabolic processes/cellular processes during spermatogenesis.•The DEPs indicate their potential functions in spermatogenic arrest in cattleyak.1This data is related to DEPs from testis proteome of cattleyak and yak using LC–ESI-MS/MS 22.1The sampling of cattleyak and yak, and preparing of testis from each animal was described in Reference 2.2Each testicular sample was ground into powder by successively adding liquid nitrogen in a mortar. Then each sample powder was extracted with lysis buffer and the protein was prepared as described in Reference 2.3Total protein (100 μg) was extracted from each sample solution. After digestion with Trypsin Gold , peptides from each sample were dried and processed according to the manufacture׳s protocol for 8-plex iTRAQ reagent (Applied Biosystems). SCX fractionation was performed as description in Reference 2.4The preparation of each SCX fraction, data acquisition and data analysis were performed as description in Reference p<0.05) by comparing of the testis proteome between cattleyak and yak, in which 206 proteins were up-regulated , calculating protein numbers for every term. Hypergeometric test was employed to screen significantly enriched GO terms from DEPs by using a strict algorithm as follows:GO enrichment analysis firstly maps all DEPs to GO terms in the database  screened from testis proteome of cattleyak and yak were analyzed by GO according to cellular component, molecular function, and biological process. The 28 significantly enriched GO terms with respect to cellular component were mainly associated with extracellular matrix organization, collagen fibril organization and cytoplasmic part 2.6N denotes the number of all proteins that with KEGG annotation and n denotes the number of DEPs in N. M is the number of all proteins annotated to specific pathways and m is the number of DEPs in M.KEGG (the major public pathway-related database) p≤0.05), in which ECM-receptor interaction, Protein digestion and absorption, Alzheimer׳s disease, Ribosome and Pentose phosphate pathway were the top listed pathways In total, 465 DEPs were mapped to the reference pathways in KEGG database and only 15 significantly enriched pathways were obtained (


Clinical Genetics published by John Wiley & Sons A/S. Published by John Wiley & Sons Ltd., 2014Porter L.F., Black G.C.M. Personalized ophthalmology. Clin Genet 2014: 86: 1–11. © 2014 The Authors. Ophthalmology has been an early adopter of personalized medicine. Drawing on genomic advances to improve molecular diagnosis, such as next-generation sequencing, and basic and translational research to develop novel therapies, application of genetic technologies in ophthalmology now heralds development of gene replacement therapies for some inherited monogenic eye diseases. It also promises to alter prediction, diagnosis and management of the complex disease age-related macular degeneration. Personalized ophthalmology is underpinned by an understanding of the molecular basis of eye disease. Two important areas of focus are required for adoption of personalized approaches: disease stratification and individualization. Disease stratification relies on phenotypic and genetic assessment leading to molecular diagnosis; individualization encompasses all aspects of patient management from optimized genetic counseling and conventional therapies to trials of novel DNA-based therapies. This review discusses the clinical implications of these twin strategies. Advantages and implications of genetic testing for patients with inherited eye diseases, choice of molecular diagnostic modality, drivers for adoption of personalized ophthalmology, service planning implications, ethical considerations and future challenges are considered. Indeed, whilst many difficulties remain, personalized ophthalmology truly has the potential to revolutionize the specialty. The term ‘personalized medicine’ is used in a broad sense to encompass all approaches used to tailor healthcare to the needs of individual patients. The medical profession has always practiced personalized medicine; considering factors such as age, sex, and family history in approaching diagnosis and taking into account co-morbidities, lifestyle, family and socio-economic circumstances stratification and individualization , accelerated by novel imaging technologies and relying on so called ‘deep phenotypic’ clinical assessment and molecular diagnosis S14–S16) . Patients with IRD pose a compelling case for personalized ophthalmology and show the need for widespread genetic testing , S25–S26. PatientMYOC, PITX2, FOXC1, PAX6, CYP1B1, and LTBP2) have been shown to cause overlapping phenotypes associated with congenital or juvenile glaucoma (S27), accounting for around 20% of cases of glaucoma with onset before the age of 40. The impact on clinical care and genetic counseling can be significant for those with a known genetic mutation because appropriate surveillance and timely treatment may prevent or limit sight loss MYOC-related glaucoma revealed that 96% of the family members benefited from genetic counseling and wished to know their myocilin status .Mutations in six genes (Complex disorders such as AMD and adult-onset primary open angle glaucoma (POAG) are less amenable to DNA-based diagnostic and predictive testing. While a number of high-risk AMD genotypes are documented , S29–S36AMD is the commonest cause of blindness in the Western world and is classified in two major forms, neovascular, or ‘wet’ AMD, and ‘dry’ or non-neovascular AMD (S37). The end-stage event in ‘wet’ AMD is choroidal neovascularization (CNV), an outgrowth of blood vessels from the choroid into the subretinal and intraretinal spaces leading to rapid loss of vision. In ‘dry’ AMD, geographic atrophy of the macula results from progressive retinal pigment epithelium (RPE) atrophy (S37). Neovascular AMD is responsible for 80% of severe vision loss and current treatments rely on pharmacological inhibition of vascular endothelial growth factor (VEGF)-A activity in those with overt disease (S38–S39).CFH) (Y402H) and the age-related maculopathy susceptibility 2 (ARMS2) and adjacent high-temperature requirement factor (HTRA1/PRSS11) genes  studies and candidate gene approaches identified major common variants conferring AMD risk on chromosomes 1q31 and 10q26 within, respectively, complement factor H (1) genes , S30–S33Disease stratification and provision of molecular diagnoses to our patients will impact positively upon many aspects of clinical management, enabling enhanced surveillance and development of early and anticipatory treatments in selected conditions, and, through genetic counseling, empower patient decision-making.Genetic counseling is an important and integrative part of personalized medicine for genetic eye diseases. Ideally, patients should not undergo genetic testing without accessing counseling The impact of genetic testing on genetic counseling is significant, providing specificity to a diagnosis and clarifying inheritance patterns , S44–S45Molecular diagnosis enables optimization of conventional therapies for monogenic disorders. The number of conditions for which genetic profiling may improve traditional medical and surgical management are slowly increasing, and not only for ocular-related phenotypes. For example, delineation of the role of transforming growth factor-β (TFGβ) signaling in the genesis of aortic dissection in Marfan syndrome created unexpected avenues for treatment, leading to the use of losartan as a preventative agent (S48). Losartan is a selective angiotensin 1-receptor blocker, uniquely inhibiting TGFβ-mediated activation of extracellular signal-regulated kinase (ERK) while allowing continued signaling through the angiotensin 2 receptor, required for full protection against aneurysm progression in the mouse model (S48–S49).CYP27A1 gene develop CTX, a condition of lipid metabolism associated with juvenile cataracts, progressive neurological decline and cardiovascular disease usually diagnosed in mid adult life. Critically, early diagnosis is optimal because progression of cardiac and neurological phenotypes can be arrested by administration of chenodoxycholic acid in combination with statins (S50). Recessive mutations in GALK1 cause galaktokinase deficiency with CC in around 75% of cases. Avoidance of galactose intake by strict dietary management may reduce and sometimes even reverse lenticular opacities (S51). Molecular diagnostics will increasingly achieve anticipatory diagnosis and alter the management of both presenting children and affected family members (S52).Using conventional medicines to treat genetically heterogeneous diseases underlines the importance of a personalized approach. Advantages of molecular diagnosis in cases of CC associated with treatable systemic complications illustrate this point. Traditionally, diagnosis has relied on clinical suspicion (S50), diagnostic prediction in the post-genomic era enables individualized care pathways and early commencement of preventative treatments. Examples include the biochemical disorders cerebrotendinous xanthomatosis (CTX) and galactokinase deficiency, both associated with early-onset cataract. Patients who carry loss of function mutations in the The development of personalized therapeutics based on specific genetic predisposition is also envisaged. Treatments based on an individual (or tumor) genotype have been pioneered in oncology, using single genes or biomarkers to indicate tumors that will respond to specific chemotherapies (S53). Trastuzumab (Herceptin), a humanized monoclonal antibody that binds to the extracellular domain of human epidermal growth factor receptor 2 (HER2) is used for the treatment of breast cancer subtypes overexpressing HER2 and has revolutionized outcomes of patients with HER2-positive breast cancer. Several new HER2-targeted therapies and HER2/HER3 antibodies have now been developed for metastatic disease (S54).CFH rs1061170 might be associated with treatment response of neovascular AMD to anti-VEGF agents (S62). Another recent study by Abedi et al. suggested a pharmacogenetic association between the HTRA1 promoter SNP (rs11200638) and A69S at LOC387715/ARMS2 and anti-VEGF treatment CFH, and ARMS2) with those SNPs identified in the angiogenesis pathway  showed that patients with four high-risk alleles in CFH and ARMS2 were younger at age of onset of neovascular AMD and failed to show a visual improvement after ranibizumab treatment (S59). Although this approach has limited current benefit it may facilitate future treatment stratification Similar approaches have been proposed for complex ophthalmic disorders and insights gained from AMD genetics are providing the rationale for development of complement inhibitors for the treatment of AMD rs1120063 and A69SCurrently, targeted therapies are emerging principally in the context of clinical trials for patients with inherited retinal dystrophies using gene-replacement strategies RPE65-associated Leber congenital amaurosis (LCA), a type of early-onset RP . Dose-escalation trials ensued in younger patients confirming safety and highlighting visual improvements in children (S9–S10).onset RP , has beeonset RP , S11. InREP1, which encodes Rab escort protein 1(REP1). The condition is associated with night blindness, progressive peripheral to central chorioretinal degeneration and ultimately total blindness (S64). Rab proteins are involved in the control of intracellular trafficking and are modified by geranyl-geranyl moieties necessary for membrane association and target–protein recognition (S65–S66). Choroideremia is generally caused by Rep1 null mutations or rare missense mutations affecting REP1 post-translational lipid modifications (S67–S68), suggesting that REP1-mediated Rab lipid modification (prenylation) is essential for RPE and photoreceptor function. Absence of protein or impaired post-translational modification is thought to affect opsin transport to photoreceptor outer segments, apical migration of RPE melanosomes and phagocytosis of photoreceptor outer segments by the RPE (S66–S68). Recent results from a phase 1/2 clinical gene-replacement trial are encouraging and suggest improved rod and cone function, with improvements in visual acuity, microperimetry and increases in retinal sensitivity in the treated eyes (S12).Choroideremia is an X-linked recessive disease caused by mutations in PDE6 AR RP (S69) and Bardet–Biedl syndromes caused by mutations in BBS1 and BBS4 (S70–S71).Theoretical proof-of-principle of the benefits of targeted genetic therapies for inherited conditions is now established and offers promise for wider application to other retinal degenerations (S12) with ongoing pre-clinical studies for gene replacement strategies in other loss-of-function IRD such as However, the development of personalized therapies remains significantly limited by incomplete understanding of disease pathophysiology at the molecular level, highlighting the need for continued collaborative research efforts in this field.Increasing access to NGS and conventional sequencing technologies is providing unprecedented opportunities for improved clinical diagnosis and personalized interventions. At present genetic testing is particularly valuable when a treatment and counseling plan is dictated by the presence of a disease-causing mutation. This is currently the case for IRD, early onset glaucomas, and inherited optic neuropathies in situ hybridization (FISH) and microarrays to detect copy number variations will remain important in the investigation of the dysmorphic or developmentally delayed infant with an ocular phenotype. For monogenic disorders, conventional sequencing will continue to play a role in the context of specific clinical diagnoses where there is accurate phenotypic data. For example in the case of the AR cone dystrophy associated with KCNV2 mutations a characteristic phenotypic appearance is evident with patients showing supra-normal rod responses on electroretinogram (ERG) testing, directing molecular diagnosis (S72). Other retinal conditions with a characteristic phenotype include Doyne syndrome, where mutation screening of the affected gene can be performed seeking common or unique mutations.Molecular diagnosis of genetic eye disease should be performed ideally with the most cost effective and simple modality available, and it is important to note that NGS will not be a stand-alone technology for advancing genomic medicine. Fluorescent de novo mutations are common. The choice of whether to employ NGS on affected individuals, on trios  and how to filter genomic data remains unclear for such disorders.Thus use of NGS platforms in the diagnosis of IRD, characterized by significant genetic and phenotypic heterogeneity discussed above, has been examined closely and pick-up rates and case selection is now well-understood , S18–S19The increasing pace of NGS technologies presents enormous challenges in terms of data processing, storage and management, hindering translation from sequence data into clinical practice Our ability to understand the effects of identified variants on disease causation poses the greatest challenge to integration of NGS into clinical practice , S3. So The gold standard proof of pathogenicity remains labor-intensive and expensive functional laboratory-based experiments. To bypass the need for such onerous validation, international sharing of variant data linked to standardized phenotypic descriptions is required to streamline data analysis for clinical use As knowledge of the number of genes mutated in eye disease increases, ophthalmologists who have training and experience in genetic eye disease and are supported by the necessary counseling infrastructure will increasingly order genetic tests and provide mechanism-specific treatments for their patients www.cafevariome.org), deployed across sets of diagnostic laboratories with a common disease focus, as well as across groups of diagnostic laboratories from whole nations. Teams in this network can instantly find mutation records and rare disease patients recorded by other groups with summary data without releasing data or compromising patient privacy (S3).Increasingly, disease stratification will require accurate phenotyping alongside molecular diagnostics. As mentioned above, databases collecting clinical phenotypic information alongside gene specific variants and functional information are required to enable diagnostic context-specific data interpretation. Widespread interconnectivity between scientists and research-based data systems, diagnostics data systems and clinical patient-data systems will enable meaningful interaction between these three domains (S3). An example of this principle is the Café Variome . For example, many RP patients are still diagnosed when they have mid to late stage disease with peripheral vision loss as a consequence of photoreceptor degeneration. However, the success of clinical trials may depend on identifying patients as early as possible to maximize the efficacy of treatment , S69–S70Patients with IED provide support for the utility and validity of molecular diagnostic testing. While current availability of genetic testing for IED is variable within the publicly funded English National Health Service (NHS) for example (S26) – because of cost and perceived clinical utility (S77) – evidence for demand from service users is growing (S78–S79). Willis et al. showed in a questionnaire study of 200 patients with IRD that over 90% of participants were in favor of genetic testing for their disease, with support for both diagnostic (96.5%) and predictive testing (91.5%) being strong in these respondents (S44). A positive view of genetic testing in this population has been confirmed in other studies (S78–S79).RB1 mutation testing in retinoblastoma-affected families (S80–S81)  ; and useHowever overall costs of delivering both disease stratification and treatment individualization cannot be overlooked. For example, while the cost of sequencing – on a per base level – has fallen rapidly in recent years Accelerated development of targeted therapies to run alongside improved diagnostic platforms is the ultimate hope of a personalized approach to healthcare. However the increasing costs of drug development for a tailored and therefore focused market will remain a huge hurdle reflected by the 75% increase in personalized medicine investment by industry over the past 5 years according to the Personalized Medicine Coalition (S87). The challenges of development – and critically, of equitable global application – of such therapies will be a huge future challenge. Although not a personalized therapy, the remarkable achievements of the FDA approved VEGF inhibitor ranibizumab in achieving disease control in neovascular AMD represents a triumph of molecular medicine, developed from a better understanding of the pathophysiology of AMD. It has provoked considerable escalation in the costs of treatment, with around 50,000 new patients per year becoming eligible for monthly injections in England alone at the cost of approximately 1500$ per injection (S84). Implementation difficulties of medical advances in Western healthcare systems are enormous. However application in developing systems is a further, often less well recognized problem. At this time, individualized therapies commercialized under the incentives introduced by the Orphan Drug Act, with substantial benefits and reductions in research and development costs, are setting precedents in terms of substantial annual costs (S85), generating acceleration in the orphan drug market, and increases in rates of approval of orphan drugs with a global orphan drug market of 84.9$billion in 2009 . The ability of such processes to keep pace with the speed of discovery remains in doubt, as is the sustainability of current pricing. Acceptable norms to control costs of personalized therapies – such as DNA-guided therapies – will be required, however decreasing profitability may result in drug companies developing fewer products for these until recently neglected diseases (S85). Personalized medicine, highly impacted by NGS with its multiple and diverse applications is profoundly influencing the drug development industry (S86). Overcoming these challenges will require input and consensus from governments, patients, charitable funders, scientists, industry and regulatory authorities and new business models for the pharmaceutical industry (S87).Furthermore, service planning and delivery will have a deep impact on how patients and the medical profession respond to the development of personalized ophthalmology. The delivery of complex personalized care will require increased levels of clinical integration and communication. Ophthalmologists will need to be prepared to view genetic testing as an integral component of patient care, and be prepared to communicate effectively with laboratories and colleagues in medical genetics In conclusion, the prospects of personalized ophthalmology look promising, illustrated by the enhanced experiences of some patients with IED. Drawing on advances in new technologies to improve molecular diagnostics, basic and translational research to develop novel therapies, and fresh approaches to consent and data-sharing, personalized ophthalmology has the potential to revolutionize our specialty. Challenges developing individualized therapies in complex diseases such as AMD and glaucoma are significant, but AMD remains one of the finest examples of molecular dissection of a complex disease, positioning ophthalmology at the forefront of personalized therapies for complex diseases as they emerge. From a commercial point of view, AMD offers the promise of both precise and highly actionable personalized medicine, with the prospect of a sizeable market even for targeted therapies. While some ophthalmologists might question the immediate clinical relevance of molecular diagnostics, staying abreast of new technologies and their developing applications, and incorporating such testing into diagnostic, prognostic, and therapeutic algorithms of patient care when appropriate, will enable timely acquisition of sensitive and specific actionable interventions which improve patient care and outcome. Overcoming the challenges of developing personalized ophthalmology will require both global collaborative efforts integrating academic institutions, patient and clinical networks, regulatory authorities and commercial companies including the pharmaceutical industry. Finally, ophthalmologists themselves will consider the application of genomics and personalized therapies for their patients on a case-by-case basis, assessing clinically utility and cost-effectiveness, and will play a role in overseeing and promoting implementation of beneficial and equitable personalized healthcare advances for patients.The authors would also like to acknowledge the support of the Manchester Academic Health Science Centre and the Manchester National Institute for Health Research Biomedical Research Centre. L. F. P. is a National Institute of Health (NIHR) research fellow.


Epilepsy is a common and serious neurological disorder characterized by occurrence of recurrent spontaneous seizures, and emerging evidences support the association of mitochondrial dysfunction with epilepsy. Sirtuin 5 (SIRT5), localized in mitochondrial matrix, has been considered as an important functional modulator of mitochondria that contributes to ageing and neurological diseases. Our data shows that SIRT5 deficiency strikingly increased mortality rate and severity of response to epileptic seizures, dramatically exacerbated hippocampal neuronal loss and degeneration in mice exposed to Kainate (KA), and triggered more severe reactive astrogliosis. We found that the expression of mitochondrial SIRT5 of injured hippocampus was relatively up-regulated, indicating its potential contribution to the comparably increased survival of these cells and its possible neuroprotective role. Unexpectedly, SIRT5 seems not to apparently alter the decline of antioxidant enzymes superoxide dismutase 2 (SOD2) and glutathione peroxidase (GPx) in hippocampus caused by KA exposure in our paradigm, which indicates the protective role of SIRT5 on seizures and cellular degeneration might through different regulatory mechanism that would be explored in the future. In the present study, we provided strong evidences for the first time to demonstrate the association between SIRT5 and epilepsy, which offers a new understanding of the roles of SIRT5 in mitochondrial functional regulation. The neuroprotection of SIRT5 in KA-induced epileptic seizure and neurodegeneration will improve our current knowledge of the nature of SIRT5 in central nervous system (CNS) and neurological diseases. Epilepsy is a common and serious neurological disorder that affects about 50 million people worldwide, characterized by the unpredictable but recurrent occurrence of spontaneous seizures , critically affecting neuronal excitability. Functional defect of mitochondria has been observed in human and animal epilepsy  deficient mice that exhibited spontaneous electrographic and motor seizures -dependent histone deacetylases that modulate distinct metabolic pathways, diverse stress responses and other biological activities in metabolism, aging and other diseases  mice exposed to KA; (2) to assess the effect of SIRT5 deficiency on hippocampal neurodegeneration and associated reactive astrogliosis; (3) to test whether KA exposure can alter SIRT5 expression and thereby affect mitochondrial antioxidant enzyme superoxide dismutase 2 (SOD2) and glutathione peroxidase (GPx) levels, which might be involved in the underlying mechanism of above neuroprotection. The present study will provide the evidence for the important role of SIRT5 on KA-induced seizure and epilepsy by using genetic ablation of SIRT5 in mice. Further investigation is needed to better understand the underlying mechanisms.Given the important role of SIRT5 in the functional regulation of mitochondria and our previous study, we hypothesized that SIRT5 is an important regulator in KA-induced neurodegeneration and the underlying oxidative stress. Accordingly, our study had three goals: (1) to determine whether the lack of SIRT5 can increase the seizure susceptibility and mortality by comparing SIRT5ad libitum access to food and water. SIRT5−/− mice (n = 42) were purchased from Jackson laboratory. Age matched WT male C57BL/6 mice (n = 38) were used as controls. All animal procedures were performed in accordance with National Institute of Health guidelines and approved by Institutional Animal Care and the relevant Institutional Ethical Review Committees.Eight to 12 weeks old male mice were housed on a 12 h light/dark cycle and allowed Systemic injection of KA was used to investigate whether SIRT5 deficiency affected the behavioral seizures and the mortality rate. KA  solutions were freshly prepared before use according to the manufacturer’s instruction. KA was dissolved at a concentration 2 μg/μl in alkalized isotonic saline. Adult mice were injected intraperitoneally with a single dosage (either 20 mg/kg or a higher dose of 25 mg/kg) of KA. KA-treated mice were evaluated every 20 min for 2 h after KA injection by trained experimenter blind to genotype. The behavioral seizures were scored 0–5 according to reference descriptions  or Saline were deeply anesthetized with isoflurane and sacrificed, then perfused transcardially with phosphate buffered saline  followed by freshly prepared 4% paraformaldehyde in 0.1 M sodium phosphate buffer, 0.1 M phosphate buffer (pH 7.4). Brains were removed, postfixed overnight, gradiently dehydrated with 20 and 30% sucrose in PBS and frozen at −80°C. For each animal, six sets of 30-μm-thick coronal brain sections were cut on Leica cryostat and preserved in PBS-buffered 50% glycerol at −20°C until used for subsequent free-floating immunohistochemistry.2O2 for 15 min at room temperature, and rinsed three times with PBS. After blocking in PBS containing 0.3% Triton X-100 and 5% bovine serum albumin for 1 h, the sections were then incubated with primary antibody overnight at 4°C and followed by incubation with appropriate secondary antibodies (Vectastain laboratories) in room temperature. Markers were visualized with 3,3-diaminobenzidine (DAB), sections were dehydrated and cover slipped with DPX mountant. Mouse anti-glial fibrillary acidic protein (GFAP) antibody  was used for staining in this study. The total number of GFAP-positive astrocytes in CA1 or CA3 region was counted. We also counted the percentage of GFAP-positive reactive astrocyte, characterized by hypertrophic cell bodies and intensely stained processes. Three consecutive sections per brain were analyzed and provided a single value for each mouse. The images were captured and analyzed by NIS-Elements AR microscope software and ImageJ software (NIH).Sections were incubated in 3% HNeuronal death and degeneration were detected by Nissl (Cresyl Violet) and Fluoro-Jade C (FJc) staining. Nissl staining was performed in terms of the manufacturer’s protocol (IHC WORLD). The Nissl substance (rough endoplasmic reticulum) appears dark blue due to the staining of ribosomal RNA, giving the cytoplasm a mottled appearance. These features disappear in dead neurons. The surround glia cells  were not counted. Nissl positive pyramidal cells  were counted in a 250 × 250 μm square applied at the center of the CA1 and CA3 regions. Three consecutive sections per brain were analyzed and provided a single value for each mouse. The data were shown as the normalized percentages. FJc staining was performed as described in the literature  or saline, animals were anesthetized and sacrificed by cervical dislocation. The hippocampus tissue was harvested immediately and stored at −80°C. For tissue lysate preparation, it was homogenized in RIPA buffer. The samples were centrifuged at 12,000 rpm for 5 min and supernatant was collected and kept on ice. Protein concentration was determined using the protein assay kit from Bio-Rad. Supernatant was separated on 4–15% gradient SDS-PAGE gels and transferred to nitrocellulose membranes. The integrated optic density of the immunoreactive bands was measured and quantified with ImageJ . Primary antibodies used in this study include rabbit polyclonal anti-SIRT5 antibody , rabbit polyclonal anti-GPx antibody , rabbit polyclonal anti-SOD2 antibody  and mouse anti-actin . Detection was carried out using horseradish peroxidase-conjugated anti-mouse, anti-rabbit antibody  and Thermo Scientific Pierce ECL Western Blotting Substrate.−/− and WT mice. Total RNA was extracted using Trizol (Invitrogen) and purified using a RNeasy kit (Qiagen) according to the Qiagen protocol. After isolation, RNA (20 ng per sample) was reverse transcribed to cDNA using SuperScript III RT Reverse Transcriptase (Invitrogen), which was stored at −20°C until further analysis. The mRNA levels were quantified with a quantitative real-time PCR (qPCR) system (Stratagene Mx4000). Each sample was detected in triplicate and each experiment was repeated three times. The following primers for expression analysis were designed using TaqMan assays (Life Technologies) and NCBI Primer-Blast: (5′→3′): SIRT5, forward, GCAGACGGGTTGTGGTCAT; reverse, CGATGCAACTCGTCAATGTTCT.At 24 h after KA (20 mg/kg) or saline injection, tissue from hippocampus was dissected as described above from SIRT5t tests, while multigroup comparisons were carried out using two-way ANOVA followed by Bonferroni post hoc tests. Results are expressed as group mean ± SEM and statistical significance was set at p < 0.05.All quantitative data were performed using Prism software (GraphPad Software). The number of independent experiments is stated in the figure or figure legend. Survival was assessed using Kaplan–Meier analysis, and the statistically significant difference between two groups was determined with the log-rank test. Data from seizure scoring were analyzed using two-way analysis of variance (ANOVA) for repeated measurements. Unless otherwise noted, the comparison only between the two groups was analyzed using unpaired Student’s −/− and WT mice were injected with different dosages of KA, and the mortality rate and seizure severity were examined. The Kaplan–Meier survival curves showed that SIRT5 deficiency significantly increased the mortality (p < 0.05) in 24 h following KA injection at both lower (20 mg/kg) and higher (25 mg/kg) dosages , in contrast to less than 10% mortality rate of WT mice  during 24 h following KA administration at higher dosage compared to 12.5% for WT control mice , whereas WT mice were less likely to suffer from seizures. In addition to seizure scores, seizure severity was calculated by integrating individual scores of each mouse over the experiment period  and average (p < 0.001) seizure severity  in SIRT5y Figure . Togethe−/− and WT mice at 5 days after KA exposure, including those with similar seizure scores, were examined by Nissl staining and FJc staining of nearby sections  and CA3 regions  of SIRT5−/− mice at 5 days after KA administration, whereas WT mice exhibited comparably much less neuronal damage , which indicated that SIRT5 deficiency caused remarkably more severe hippocampal neuron degeneration compared with WT controls . This pattern of FJc staining is also consistent with that of neuronal death in hippocampus. In both CA1 and CA3, the average numbers of FJc positive cells in SIRT5−/− group are significantly increased than the value for the WT control group (p < 0.05).To further confirm the above assessment of severity of hippocampal neurodegeneration, we stained dying neurons in the hippocampus with FJc. Figure −/− mice. Immunohistochemical analysis showed that KA exposure elicited a dramatic increase of GFAP immunoreactivity in the number and morphology change throughout the hippocampus at 5 days after KA administration, particularly prominent in the CA1 and CA3 areas; most astrocytes showed reactive phenotype characterized by hypertrophic cell bodies and intensely stained processes. SIRT5 deficiency apparently exacerbated KA-induced reactive astrogliosis . Notably, a much higher percentage of reactive astrocytes induced by KA exposure was detected in the hippocampus, especially that was more prominent in the CA1 and CA3 regions in SIR5−/− mice compared to WT controls . In order to avoid potential bias, two independent analyses were performed by two trained observers blinded to genotype and treatment. In summary, these results indicate that SIRT5 deficiency exacerbates KA-induced reactive astrogliosis in the hippocampus.Quantitative analysis showed that there was a slight increase in the total numbers of GFAP positive astrocytes in hippocampal CA1, CA3 and DG regions in both SIRT5SIRT5 expression is essential for maintaining proper mitochondrial function, which is required for metabolic homeostasis and regulation of the activity of multiple metabolic enzymes Wallace, . Next, w−/− and WT mice. However, to our surprise, at this time point in the present study, the declines of both enzymes triggered by KA were not significantly affected by SIRT5 deficiency. These data revealed that SIRT5 might contribute very little to preserve mitochondrial antioxidant capacity via SOD2 and GPx from oxidative injury caused by KA.Mitochondrial oxidative stress is emerging as a key factor that not only results from seizures, but might also contribute to epileptogenesis. To evaluate whether SIRT5 deficiency exacerbating KA-induced behavioral seizures and degeneration is associated with antioxidant capacity, we examined the expression levels of SOD2 and GPx in the hippocampus of mice by Western Blot Figure . Our resIn rodents, systemic administration of KA can elicit sustained epileptic activity in the hippocampus. The animals exhibit recurrent behavioral seizures and progressive neuronal death, which provides a well-characterized model of neurodegenerative diseases  damage through reactive astrogliosis, that involves structural and metabolic changes. Reactive astrogliosis has been considered as a prominent feature of mesial temporal lobe human epilepsy and most animal models of recurrent seizures  exhibited exacerbated KA-induced mitochondrial aconitase inactivation and neuronal loss in hippocampus (Liang and Patel, Brain injury caused by seizures and epilepsy is a dynamic process that comprises multiple factors contributing to neuronal cell death. Mitochondrial dysfunction and oxidative stress contribute to neuronal death, which is the key feature of neurodegeneration (Rowley et al., The effect of mitochondria in acquired epilepsies, which account for about 60% of all epilepsies, is important but less well understood. Although mitochondrial oxidative stress and dysfunction are associated with inherited epilepsies, little is known concerning their contributions in acquired epilepsies. Initial trauma of KA exposure triggers complex cellular and molecular events, leading to functional and structural modification in the brain, which contributes to the pathogenesis of epilepsy and, as a result, recurrent seizures. Both functional and pathological defects of mitochondria have been reported in epileptogenesis (Volmering et al., In conclusion, the present study shows that SIRT5 deficiency strikingly increased mortality rate and seizure severity, exacerbated hippocampal neuronal death and degeneration, and triggered more severe reactive astrogliosis in mice exposed to KA. We found that the expression of mitochondrial SIRT5 of injured hippocampus was relatively up-regulated, indicating its potential contribution to the comparably increased survival of these cells and its possible neuroprotective role. SIRT5, to our surprise, did not seem to apparently alter the decline of antioxidant enzymes SOD2 and GPx in hippocampus caused by KA exposure in our paradigm. The protective mechanism of SIRT5 on seizures and cellular degeneration might through different regulatory mechanism other than antioxidant enzymes SOD2 and GPx, which we would like to explore in the future experiment. For the first time we illustrate the role of SIRT5 in KA-induced mice model, offering a new understanding of roles of SIRT5 in mitochondrial functional regulation and their potential neuroprotection. These results will enrich our acknowledgment of the natural property of SIRT5 in health and neurological diseases.All authors have made substantial, intellectual and direct contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Activating mutations in RAS are present in the majority of pancreatic cancer cases and represent an ideal therapeutic target. Reolysin is a proprietary formulation of oncolytic reovirus that is currently being evaluated in multiple clinical trials due to its ability to selectively replicate in cells harboring an activated RAS pathway. Here we report for the first time the presence of reovirus replication and induction of endoplasmic reticular (ER) stress in a primary tumor specimen collected from a pancreatic cancer patient receiving intravenous Reolysin and gemcitabine.We describe the case of a 54-year old patient diagnosed with pancreatic adenocarcinoma in February 2012. Analysis of a tumor biopsy revealed an activating KRAS mutation (G12D) and the patient was started on first-line treatment with Reolysin in combination with gemcitabine in March 2012. Stable disease was achieved with significant improvement in cancer-related pain. Following 25 cycles of treatment over 23 months, a second biopsy was collected and immunohistochemical analyses revealed the presence of reovirus replication and induction of the ER stress-related gene GRP78/BIP and the pro-apoptotic protein NOXA. Importantly, co-localization of reoviral protein and active caspase-3 was also observed in the biopsy specimen.This is the first report of reoviral protein detection in primary tumor biopsies taken from a pancreatic cancer patient receiving intravenous Reolysin therapy. The accumulation of reoviral protein was associated with ER stress induction and caspase-3 processing suggesting that Reolysin and gemcitabine treatment exhibited direct pro-apoptotic activity against the tumor. Reolysin is a formulation of wild-type oncolytic reovirus that is currently under investigation in multiple randomized phase II clinical trials in solid tumors, including combination with taxane-based regimens for the treatment of patients with head and neck carcinoma, non-small cell lung cancer (NSCLC), prostate cancer, pancreatic cancer, and ovarian cancer –5. ReoviThe patient is a 54-year-old gentleman, who presented with a few months of mid-epigastric pain, nausea and vomiting with associated weight loss in February 2012. CT and MRI scans revealed a 3.3 × 3.1 cm pancreatic head mass encasing superior mesenteric artery and vein with associated mesenteric periportal lymphadenopathy. He also had sub-centimeter lung nodules presumed to be metastatic deposits. He thus had a clinical stage 4 unresectable pancreatic cancer. Genomic analysis of tumor biopsies revealed the presence of KRAS mutation (G12D) and loss of CDKN2A/B.10 TCID50 IV on days 1, 2, 8, and 9 (immediately after gemcitabine on days 1 and 8) in combination with 800 mg/m2 IV gemcitabine on days 1 and 8, with 21-day cycles. The patient displayed a clinical response with improvement in cancer-related pain. The best radiographical response was documented as stable disease by Response Evaluation Criteria in Solid Tumors (RECIST) guidelines  and loss of CDKN2A/B. Immunohistochemistry (IHC) was performed on Reolysin-treated or untreated HCT116 colon cancer cells as a positive and negative control for reovirus staining, respectively Fig. . Viral rD and losToxicities were manageable and included grade 1 fever likely due to Reolysin and grade 3 thrombocytopenia and neutropenia due to gemcitabine. The patient also had a biliary obstruction, which required stenting in November 2013. He completed 27 cycles of treatment with the last one in April 2014. At this time, he presented with disease progression with ascites and jaundice.To our knowledge, this is the first report that identified reoviral protein within a primary pancreatic tumor following systemic Reolysin therapy. These findings demonstrate that Reolysin and gemcitabine treatment may result in reovirus replication, ER stress, and apoptosis in patients treated with this regimen. Preliminary results from the Phase II study at our institution for patients with advanced pancreatic cancer treated with gemcitabine and Reolysin showed a clinical benefit with acceptable tolerability. Although this patient did not have tumor shrinkage, the patient did have prolonged stable disease for more than 2 years as well as symptomatic improvement with decreased cancer-related pain.Since pancreatic cancers are hypovascular tumors surrounded by dense desmoplastic tissue, drug resistance may occur as chemotherapeutic agents fail to penetrate the primary tumor. This case establishes Reolysin’s ability to invade the dense desmoplastic tissue surrounding the hypovascular pancreatic cancer, suggesting that Reolysin may be an effective agent against drug resistant tumors. Since the majority of pancreatic cancers have RAS pathway activation, Reolysin serves as a potential promising active agent to overcome this barrier when combined with conventional chemotherapy .The mechanisms of Reolysin’s anticancer activity are pleiotrophic and have been reported to include tumor lysis, ER stress, apoptosis, and stimulation of an immune response against the tumor in preclinical models –17. HereWe have also shown significant induction of the ER stress associated markers GRP78/BIP and NOXA in the biopsy specimen analyzed following Reolysin and gemcitabine treatment. Our preclinical studies determined that induction of ER stress and the BH3-only pro-apoptotic gene NOXA are key mediators of Reolysin-induced apoptosis , 13. SinTaken together, we are the first to demonstrate reoviral protein accumulation in a primary tumor from a cancer patient treated with systemic Reolysin and gemcitabine. This treatment regimen yielded stable disease for more than 2 years in a patient with advanced pancreatic cancer, which is a significant improvement compared to the historical median survival of 6–12 months in this patient population , 19.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor of this journal.


Addressing the effect of these environmental factors on flowering events such as time of day of anthesis (TOA) and flowering time (duration from germination till flowering) is critical to understand the adaptation of plants/crops to changing climate and is the major aim of this review. Increasing ambient temperature is the major climatic factor that advances flowering time in crops and other plants, with a modest effect of e[CO2].Integrated environmental stimuli such as photoperiod, temperature and e[CO2] regulating flowering time is discussed. The critical role of plant tissue temperature influencing TOA is highlighted and crop models need to substitute ambient air temperature with canopy or floral tissue temperature to improve predictions. A complex signaling network of flowering regulation with change in ambient temperature involving different transcription factors , flowering suppressors  and autonomous pathway  genes, mainly from Arabidopsis, provides a promising avenue to improve our understanding of the dynamics of flowering time under changing climate. Elevated CO2 mediated changes in tissue sugar status and a direct [CO2]-driven regulatory pathway involving a key flowering gene, MOTHER OF FT AND TFL1 (MFT), are emerging evidence for the role of e[CO2] in flowering time regulation.Flowering is a crucial determinant for plant reproductive success and seed-set. Increasing temperature and elevated carbon-dioxide (e[CO The impact of these climate events has already been documented on agricultural crop production, natural species diversity and distribution, and other ecosystem services such as flowering time, pollination etc.  the timing of flowering in ecosystems or plant communities may affect individual species “fitness” in relation to competition with other species; (iv) the synchrony of pollination with insects in ecosystems and hence the distribution of species may be affected. All the above functions can be affected by climate change; thus flowering time is one of the major factors determining the adaptation of plants to changing climate. Environmental factors such as photoperiod and temperature that control the timing of the first bud or flowering from a whole-plant physiological perspective has been described in detail by Craufurd and Wheeler (Triticum aestivum), which may be influenced by changes in crop management practices such as variable sowing dates, have to be interpreted with caution . Flowering time marks the visible transition from the vegetative to reproductive phase and is important because: (i) the duration from emergence to flowering determines the reproductive competency of a plant; (ii) the timing of flowering  Wheeler . In gene Wheeler . HoweverOryza alta flowering after 2100 h at night and O. officinalis as early as 0600 h in the morning  in a day when temperatures are cooler, leading to enhanced seed-set even with exposure to early-afternoon hotter temperature , which is defined as the appearance of the first open flower or anthesis on any particular flowering day, can also vary and provide an escape route to overcome the damage induced by high temperature. For example, in tropical and sub-tropical rice environments ambient air temperature gradually increases from early morning through solar noon and late afternoon, and decreases thereafter. Pollination occurs at or just prior to anthesis and fertilization is generally completed within a few minutes or hours after pollination Cho, . High-tePlants require a certain amount of heat units , synonymous with growing degree-days, to reach and progress to the next developmental stage. Provided the temperature is below a critical threshold, plants achieve these requirements earlier in warmer than cooler temperatures, thus increasing the rate of development  or sorghum (Sorghum bicolor), using controlled-environment conditions, has provided extensive knowledge on different high-temperature tolerance and escape mechanisms  with the variation in time of day at peak anthesis ; White et al., Temperature affects flowering time by both affecting the rate of development directly and influencing vernalization  showed 28 cases (different species within the same study is considered a case) in which flowering time was earlier (average 8.6 days) and 12 cases in which flowering was delayed (average 5.2 days)  on a grassland ecosystem using a FACE facility in California resulted in forbs flowering 2–4 days earlier, while in the dominant grass community flowering time was delayed by 2–6 days  accumulation in plants  with vegetative and shoot-apical meristem tissue sucrose levels in Arabidopsis  it was 47 and 32 days, respectively  accelerated flowering by 2–5 days; in combination elevated [CO2] completely overcame the accelerated flowering time observed with increased temperature, i.e., a zero net change  on the regulation of flowering time pathways is limited.Flowering time is governed by a complex signaling network involving the regulation of environmental stimuli  as well as endogenous genetic traits. Until now, four major flowering pathways have been identified. The vernalization and photoperiod pathways mediate signals from the environment, the autonomous pathway monitors endogenous signals based on developmental state of the plants, and the gibberellic acid (GA) pathway forms the fourth distinctive promotive pathway  via a thermo-sensory pathway that mediates flowering regulation through the autonomous pathway genes FCA and FVE via a FLOWERING LOCUS C (FLC)-independent pathway  function alters the miR172 expression level, suggesting that SVP is an upstream mediator of miR172. Hence, it is hypothesized that SVP-miR172 signaling is ultimately integrated by FLOWERING LOCUS T (FT). This hypothesis is supported by early flowering of the miR172-overexpressing plants at high and optimum temperature  through up-regulation of FT expression independent of SVP. The SVP−miR172 regulatory circuit is proposed as a fine-tuning mechanism in response to changes in ambient temperature, which allows plants to modify their development toward changing temperature conditions  in flowering time regulation has been highlighted with miR399b over-expression and a loss of function allele of PHO2 resulting in early flowering under higher temperature (23°C) and long day conditions in Arabidopsis . However, an indirect consequence of phosphate toxicity in miR399 overexpressing-pho2 mutant plants could also lead to early flowering and warrants further investigation  compared to ambient temperature (12°C)  in Arabidopsis. Both PIF4 and PIF5 physically suppress phyB photoreceptor mediated suppression of CO resulting in early flowering under warm night conditions , constitute a different regulatory pathway that acts in parallel to PIF4 to support the transition from vegetative to reproductive development with a change in ambient temperature  activates perature 7°C compaNarcissus tazetta var. chinensis, extended exposure to constant heat treatment (30°C) compared with low (15°C) and natural (ranging between 19 and 33°C) conditions promoted flower initiation and the Arabidopsis FT homolog FT-like (Narcissus FLOWERING LOCUS T1) NFT1 gene was up-regulated under higher temperature exposure  coincided with delayed flowering as a consequence of high temperature (30°C) compared with control (20°C) treatment  under long days, whereas, under short days, the expression of FT-like genes was not responsive to 25°C heat treatment. A MADS-box floral repressor HvODDSOC2 that is highly expressed at high temperature in short days might contribute to the inhibition of early reproductive development, suggesting close interaction between high temperature and day-length conditions. These studies suggest that, under higher temperature, the expression of FT/FT-like genes might be species-/crop-specific and flowering induction might share common and distinct factors with those of the photoperiodic flowering pathway.In Chinese narcissus, 2] on flowering time has been reviewed by Springer and Ward (2] in controlled chambers and the field (using open top chambers and FACE) conditions. The physiological basis of e[CO2] effect on flowering time was to enhance relative growth rates, increase plant size at flowering and raise tissue sugar status , a homolog of FT and TERMINAL FLOWER 1 (TFL1), has been identified as a candidate gene influencing flowering time with e[CO2]  and shoot apical meristem produce TREHALOSE-6-PHOSPHATE (T-6-P) as a proxy signal for floral transition and initiation under inductive environmental conditions  failed to flower, showing the essential role of sugar signaling (T-6-P) in regulation of flowering time  population between the Arabidopsis cultivars Cvi-0 (no response) and SG (delayed flowering time), was studied and a major QTL contributing 30% of the variation in flowering time was mapped on Chromosome 1  influencing flowering time at the genetic level to accurately estimate phenological shifts or to select crops better adapted to changing climates. To map regions responsible for e[COFRUITFUL, SEPALLATA, and FLC. However, VvFT on chromosome 7 and a CONSTANS-like VvCOL2 gene on chromosome 14 were the most reliable candidates identified from two of the underlying QTLs co-localizing for the flowering time , a genome wide association analysis (GWAS) identified five independent main-effect QTLs for both days to flowering and thermal sensitivity  using crop simulation models can be attained by: (i) employing canopy/floral tissue temperature rather than ambient air temperature, (ii) incorporating the differential impact of day and night temperature amplitude, and (iii) considering the key interactions between environmental factors such as temperature and RH (VPD).Genetic outputs obtained using global core accessions have and will result in identifying genomic regions (QTLs/SNPs/candidate genes) responsible for changes in flowering time using experimental and natural temperature regimes. The applicability of the candidates identified needs validation at smaller spatial scale to demonstrate the advantage in overcoming yield losses caused due to changes in the timing of flowering related events.Traits such as early-morning flowering, as demonstrated in rice, need to be explored in other crop species or plants as well. A more careful examination and documentation of TOA across landraces and wild relatives of crops could provide novel and pragmatic solutions for adapting crop production to rising temperatures.bHLH PIFs  and microRNAs that might be involved in integrating diverse environmental signals  to regulate flower initiation and development is needed. Additionally, novel unknown regulators of flowering and their interconnections or independencies with other already known pathways under changing climates constitute future direction of research on flowering time response to environmental stimuli.Deciphering differential regulation of flowering genes by TFs such as All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A systematic, combinatorial study of these compounds further improved myoblast viability, showing both synergistic and additive effects. Pathway and target analysis revealed CDK5, CDK2, CDC2, WEE1, and GSK3β as the main target kinases. In particular, CDK5 was the center of the target kinase network. Using our recently developed statistical method based on elastic net regression we computationally validated the key role of CDK5 in cell protection against hypoxia. This method provided a list of potential kinase targets with a quantitative measure of their optimal amount of relative inhibition. A modified version of the method was also able to predict the effect of combinations using single-drug response data. This work is the first step towards a broadly applicable system-level strategy for the pharmacology of hypoxic damage.Cell-based therapies to treat skeletal muscle disease are limited by the poor survival of donor myoblasts, due in part to acute hypoxic stress. After confirming that the microenvironment of transplanted myoblasts is hypoxic, we screened a kinase inhibitor library  In vivo, most transplanted myoblasts die in the first few days following transplantation, preventing their participation to tissue regeneration. This is at least in part due to the hypoxic environment, as large number of cells transplanted into a solid organ form a mass in which blood vessels are not present, hence limiting the oxygen supply [Skeletal muscle wasting is characterized by a progressive loss of muscle mass and function, compromising patient quality of life and survival . Muscle n supply –13.Hypoxia activates a complex set of pathways, supporting the development of a system-level therapeutic approach. Although cellular hypoxia promotes cell death, the lack of oxygen supply also activates several adaptive pathways to promote survival. These include a switch to anaerobic metabolism by enhancing glycolysis and inhibiting the Krebs cycle, a switch from anabolic to catabolic pathways to limit energy expenditures and the activation of autophagy, a key adaptive response to cellular stress , 15. Appin vitro. Several candidate kinase inhibitors were identified with potent effects on primary myoblast survival under hypoxia. Fully factorial analysis uncovered kinase inhibitor combinations able to both additively and synergistically improve myoblast survival. Using a pathway analysis and a novel statistical method developed by our group [In this study, we screened for kinase inhibitors that affect hypoxia-resistance ur group , we haveNOD/SCID and EGFP mice were purchased from Jackson Laboratories. Luciferase mice [EGFP mice to generate Luciferase x EGFP mice. All mice used for transplantation experiments were 2–3 months of age. Local hind limb irradiation was performed following ketamine-xylazine administration (75 and 5 mg/kg). Intramuscular transplantation and non-invasive bioluminescence imaging was performed under 1–4% 1L O2/min isoflurane inhalation. Euthanasia was performed under isoflurane inhalation followed by cervical dislocation.All protocols were approved by the Sanford-Burnham Medical Research Institute Animal Care and Use Committee. C57BL/6, ase mice  were kinLuciferase x EGFP mice as described previously [-1 bFGF). To expose cells to normoxic (20% O2) or hypoxic (~1% O2) culture conditions, cultures were placed in an airtight modular hypoxia chamber adjusted to the indicated oxygen concentration.Primary myoblasts were isolated from skeletal muscle of 2 month old C57BL/6 and eviously , plated 2 and 5% of CO2 into an airtight modular hypoxia chamber for 5 days. Two independent screens were performed with duplicates each run.The EMD kinase inhibitor library was screened for their capability to protect cells from hypoxia-induced myoblast cell death/growth arrest. The cells were plated at 1500 cells/well in 384-well plates in growth media. At least 4 hours after cell seeding, 244 kinase inhibitors were dispensed into the cells-seeded plates at 1 μM final concentration using Echo liquid handler (Labcyte). The cells were cultured under hypoxic environment created by the infusion of a gas mixture of 95% of NRelative cell survival was measured by the luciferase-based assay, ATPlite (PerkinElmer), which measures ATP levels in the metabolically active cells according to the manufacturer’s protocol. Luminescence was read with Analyst HT apparatus (Molecular Devices). The counting of viable cells was performed based on brightfield imaging and dye exclusion following the addition of 0.4% Trypan Blue (Invitrogen) and automated cell counting using the Celigo Imaging Cytometer (Nexcelom Bioscience).Fully factorial kinase inhibitor combinations were generated in CSV file format using our lab-developed high throughput screening (HTS) manager software. The input files that contain combinations were imported into the Echo Cherry Pick software and kinase inhibitor droplets were transferred from the source plates to the destination plates in 2.5 nl increments accordingly.Muscle tissues were prepared for histology as previously described . Cells a50 of less than 2000 nM and 5% residual kinase activity have been used to indicate a significant action of an inhibitor to a given target.Pathway analysis was obtained by combining two datasets containing drug-target information, 25, onead hoc measurements. The Bliss model[v is the cell viability measured using the combination, di is the dose of drug i, and vi(di) is the viability measured using only using drug i. The HSA model assumes that if the effects of a combination exceeds those of its components, then they are necessarily helping each other. The synergy can then be quantified looking at deviations from the best of the single agent viabilities at the corresponding doses:We have used three different methods to quantify synergy: excess with respect to the Bliss independence model, excess with respect to the highest single agent (HSA) and excess with respect to the highest pair agents (HPA). In contrast to the widely-used Loewe synergy, these miss model assumes vij is the viability measured using only the pair of drugs i and j at the doses di, dj.Note that in this case the expected action of the drugs is to increase cell survival. Since we are dealing with combinations with up to 5 drugs, we have generalised the HSA synergy measure to take into account deviations from the best pair of drugs. This measure indicates how important is to include more than two drugs in the combinations. This synergy is the Highest Pair Agent (HPA), and we defined it asvi of the formAk,i the residual activity of the kinase k under the effect of drug i. The residual activities were obtained from a published dataset containing catalytic activities of kinase inhibitor targets.[We used the Kinase Inhibitors Elastic Net (KIEN) method to ident targets. P17 was  targets..β0, β1,…, βp) can obtained using a linear regression procedure. The resulting coefficients βk can be interpreted as a measure of the protective impact of kinase k on the cell viability against hypoxia. A higher value of |βk| indicates a higher protective effect of that kinase on the survivability.Eq  can be rβk were then used to predict the viability of four-drug combinations. We assumed that the residual activity Ak,c of the kinase k under the effect of a four-drug combination c with dosage d =  isThe coefficients vc can then be obtained using a modified form of Eq  muscles of hind limb irradiated immunodeficient NOD/SCID mice and harvested after 1–3 days  was administered intraperitoneally 1 day post-transplantation. This small molecule selectively binds to oxygen-starved cells and can be detected by antibody recognition through histological assessment . Indeed,2) or hypoxic (0.1% O2) conditions for five days. As expected, the number of primary myoblasts was significantly reduced in hypoxic as compared to normoxic culture conditions  using the KIEN method, which we have recently developed . A limitoxia see . The coe the top , confirm the top . This coSeveral studies have demonstrated that the majority of donor myoblasts 70–90%) die in the first few days upon intramuscular transplantation and this in part correlated with a local increase in hypoxia in the transplanted area 0% die in, 32. Eviin vitro condition to induce hypoxia-induced cell death/growth arrest. While the culture of myoblasts in vitro may result in adapted nutrient availability and metabolism, these cells are nonetheless able to proliferate more rapidly under normoxic rather than hypoxic conditions  were shown to play key roles and growing evidence suggests that they are linked with hypoxia-induced cell death–38. UponOur fully factorial studies verified the effectiveness of combinatorial approaches while pathway analysis revealed a few important pathways associated with hypoxia-induced myoblast death. In particular, our study shows that CDK5 is at the center of the target network. The KIEN regression model also predicted CDK5 as a key target kinase that has protective impact on the cells in hypoxia when the resulting coefficients were ranked. Interestingly, KIEN analysis also ranked CDK5 highly when performed for the HT22 neuronal cell line, suggesting that CDK inhibition might be a general protective mechanism relevant to hypoxia in more than one tissue. The important role of CDK5 in hypoxia-associated cell death has previously been described, supporting our findings , 55. AlbFinally, our results support the advancement of combinatorial approaches, rather than single drug methods, to the treatment of hypoxia-induced cell death. These interventions are likely much closer to the combinatorial control we find in nature, (for example by transcription factors or microRNAs) but until now, not in pharmacology. We report both additive and synergistic benefits gained by the use of kinase inhibitors to improve cell viability, increasing their efficiency in this context. While the development of precisely regulated controls will be important to solve concerns of increased risk of toxicity with multiple agents, well-modulated combinatorial drug regimens may rather overcome drug side effects by minimizing biological compensations and reducing administered doses of single agents . TherefoWhile potentially more efficient once identified, the validation of combinatorial approaches via fully factorial screenings of even modest drug libraries may prove costly and require extremely large throughput. To alleviate these concerns, our results demonstrate that KIEN  can alsoIn summary, this study successfully identified kinase inhibitors that overcome myoblast cell death/growth arrest induced by hypoxia. Through fully factorial studies, we found synergistic combinations that significantly improve myoblast survival in hypoxia. In addition, pathway and regression analysis identified the key kinases involved in the hypoxia-associated reduction of viability in myoblasts. Future studies might investigate the in vivo effects of these compounds.S1 Fig(a) The standard curve between ATPlite contents and viable cell number measured by Trypan Blue exclusion assay. (b) The comparison of relative cell survivals measured by ATPlite assay and flurescence-based automatic cell counts in primary myoblasts untreated or treated with the indicated inhibitors (n = 4). (c) Detection of activated caspase-3 in primary myoblasts untreated or treated with the indicated inhibitors. (blue = nuclei and red = cCasp3), Scale bar = 50 μM(TIF)Click here for additional data file.S1 Table(XLS)Click here for additional data file.S2 TableH: high dose (1uM) L: low dose (0.25uM)(XLS)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


These results suggest that pituitary TSH release in response to TRH stimulation might be an important factor contributing to high normal serum TSH concentrations, which is a regular finding in children with overweight and obesity. The clinical significance and the intermediate factors contributing to pituitary TSH release need to be elucidated in future studies.Thyroid stimulating hormone (TSH) concentrations in the high normal range are common in children with overweight and obesity, and associated with increased cardiovascular disease risk. Prior studies aiming at unravelling the mechanisms underlying these high TSH concentrations mainly focused on factors promoting thyrotropin releasing hormone (TRH) production as a cause for high TSH concentrations. However, it is unknown whether TSH release of the pituitary in response to TRH is affected in children with overweight and obesity. Here we describe TSH release of the pituitary in response to exogenous TRH in 73 euthyroid children  with overweight or (morbid) obesity. Baseline TSH concentrations (0.9–5.5 mU/L) were not associated with BMI z score, whereas these concentrations were positively associated with TSH concentrations 20 minutes after TRH administration (r In children with overweight and obesity thyroid stimulating hormone (TSH) concentrations are often higher compared to TSH concentrations of lean children112345123456681011131516Seventy-three children  with overweight and obesity, and a mean age of 12.7 ± 3.1 years were enrolled. Baseline serum TSH concentrations were 2.7 (1.5–4.1) mU/L in the children with overweight, 3.4 (1.5–5.0) mU/L in the children with obesity, and 2.5 (0.9–5.5) mU/L in the children with morbid obesity. FT4 concentrations were within normal range in all children (13.3 ± 2.0 pmol/L). All participant characteristics are presented in Baseline serum TSH concentrations were stratified into quartiles to evaluate TSH response of the pituitary in response to exogenous TRH stimulation for children in the higher and lower normal ranges of baseline serum TSH concentration . This is shown in 20)  and the TSH incremental area under the curve (iAUC) during the TRH stimulation test  , while no associations were found between age and the TSH iAUC during the TRH stimulation test. There were no gender differences regarding baseline serum TSH concentrations, serum TSH concentrations at t20, and TSH iAUC during the TRH stimulation test.Baseline serum TSH concentrations were positively associated with both, serum TSH concentrations twenty minutes after TRH administration (t< 0.001) . Further20, or the TSH iAUC during the TRH stimulation test. Significant inverse associations between serum c-reactive protein (CRP) concentrations and serum TSH concentrations at t20 , and the TSH iAUC during the TRH stimulation test  were demonstrated. Plasma interleukin 6 (IL-6) concentrations were also significantly negative associated with serum TSH concentrations at t20  and with the TSH iAUC during the TRH stimulation test .BMI z-score and waist circumference z-score showed no significant associations with baseline serum TSH concentrations, serum TSH concentrations at tThis is the first study investigating pituitary TSH release in response to exogenous TRH stimulation in a large group of euthyroid children with overweight and obesity. A positive association between baseline serum TSH concentrations and TSH release of the pituitary in response to exogenous TRH stimulation was demonstrated. This suggests that TSH release of the pituitary in response to TRH stimulation might be an important factor contributing to the frequently found high normal baseline serum TSH concentrations in children with overweight and obesity , which i12345620 and with the TSH iAUC during the TRH stimulation test, ruling out inflammatory stimulation as a contributing factor to high pituitary TSH release. Interestingly, this study showed that TSH release of the pituitary in response to exogenous TRH stimulation and high normal baseline serum TSH concentrations are not simply the consequence of excess body weight, since BMI z-score and waist circumference z-score were not associated with baseline serum TSH concentrations, serum TSH concentrations at t20, or the TSH iAUC during the TRH stimulation test. This reinforces the findings of Aeberli et al. who demonstrated no associations between baseline TSH concentrations and the amount of excess body weight or fat in children with obesityStudies investigating TSH release of the pituitary in response to exogenous TRH stimulation in children with overweight and obesity are limited to small study populations15161011181921et al. also demonstrated an increased pituitary TSH release in response to exogenous TRH stimulation in young adults during long-term overfeeding as compared to the preoverfeeding TSH releaseThus, the results of the current study showed that neither pro-inflammatory cytokines nor the amount of excess body weight is associated with high TSH release of the pituitary in response to exogenous TRH stimulation. Considering the evidence that in mice leptin contributes to regulation of HPT-axis activityIn conclusion, baseline serum TSH concentrations are associated with TSH release of the pituitary in response to exogenous TRH stimulation in euthyroid children with overweight and obesity. The clinical significance and the intermediate factors contributing to pituitary TSH release need to be elucidated in future studies.This cross-sectional study was designed and conducted within the setting of the Centre for Overweight Adolescent and Children’s Healthcare (COACH) at the Maastricht University Medical Centre (Maastricht UMC+). Within COACH, the health status of children with overweight, obesity, and morbid obesity is evaluated, and they are monitored and guided as described previouslyAnthropometric data were obtained while children were barefoot and wearing only underwear. Body weight was determined using a digital scale (Seca) and body length was measured using a digital stadiometer. BMI was calculated and BMI z-scores were obtained using a growth analyser . Based on the International Obesity Task Force criteria children were classified as overweight, obese, or morbidly obeseVenous blood samples were collected after a minimum of 8 hours overnight fasting for the determination of baseline serum TSH and fT4 concentrations. Serum TSH concentrations were determined with the Cobas 8000 modular analyser (Roche), and serum fT4 concentrations were determined with the Autodelfia fluoroimmunoassay system (PerkinElmer). Serum TSH concentrations were considered within the normal range or above the normal range based on age specific references ranges0) were determined. A bolus of 200 μg TRH was given intravenously, subsequently venous blood samples were obtained to determine serum TSH concentrations at 20 minutes (t20), 40 minutes (t40), 60 minutes (t60), and 90 minutes (t90) after the TRH administration.At the start of the TRH stimulation test non-fasting serum TSH concentrations . Plasma pro-inflammatory cytokines monocyte protein 1 (MCP-1), IL-6, and interleukin 8 (IL-8), were measured with a commercially available Multi Spot ELISA assay .p-value below 0.05 was considered statistically significant. Data are presented as mean with standard deviation or as median with the minimum and maximum.All statistical analyses were performed using SPSS 23.0 for Windows (SPSS Inc). Shapiro-Wilk test was performed to test for normality. Serum baseline TSH concentrations were stratified into quartiles. The TSH iAUC was calculated using the trapezoidal method. A one-way analysis of variance (ANOVA) with Bonferroni as post hoc analysis was used to evaluate differences in iAUC between serum baseline TSH concentration quartiles. Associations between variables were determined by linear regressions models. Since TSH concentrations are age dependentClinical trial registration at ClinicalTrial.gov; Registration Number: NCT02091544.How to cite this article: Rijks, J. et al. Pituitary response to thyrotropin releasing hormone in children with overweight and obesity. Sci. Rep.6, 31032; doi: 10.1038/srep31032 (2016).