Remarkably, divergent PD-related mutations can generate comparable brain region specific pathologies. This indicates that some intrinsic region-specificity respecting differential neuron vulnerability exists, which codetermines the disease progression. To gain insight into the pathomechanism of PD, we investigated protein expression and protein oxidation patterns of three different brain regions in a PD mouse model, the PINK1 knockout mice (PINK1-KO), in comparison to wild type control mice. The dysfunction of PINK1 presumably affects mitochondrial turnover by disturbing mitochondrial autophagic pathways. The three brain regions investigated are the midbrain, which is the location of substantia nigra; striatum, the major efferent region of substantia nigra; and cerebral cortex, which is more distal to PD pathology. In all three regions, mitochondrial proteins responsible for energy metabolism and membrane potential were significantly altered in the PINK1-KO mice, but with very different region specific accents in terms of up/down-regulations. This suggests that disturbed mitophagy presumably induced by PINK1 knockout has heterogeneous impacts on different brain regions. Specifically, the midbrain tissue seems to be most severely hit by defective mitochondrial turnover, whereas cortex and striatum could compensate for mitophagy nonfunction by feedback stimulation of other catabolic programs. In addition, cerebral cortex tissues showed the mildest level of protein oxidation in both PINK1-KO and wild type mice, indicating either a better oxidative protection or less reactive oxygen species (ROS) pressure in this brain region. Ultra-structural histological examination in normal mouse brain revealed higher incidences of mitophagy vacuoles in cerebral cortex than in striatum and substantia nigra. Taken together, the delicate balance between oxidative protection and mitophagy capacity in different brain regions could contribute to brain region-specific pathological patterns in PD.Parkinson's disease (PD) is histologically well defined by its characteristic degeneration of dopaminergic neurons in the  Other brain regions, for instance cerebral cortex, are only affected in much advanced disease stages  on original tissue samples. Unpaired Student's T-test was used to assess the statistical significance of the value in different brain regions.For the electron microscopy of different brain regions, cortex, striatum and substantia nigra (SN) tissue samples of about one cubic mm of size were obtained from normal control mice (n = 3). Notice that we used escribed ,31. The substantia nigra, we detected a total of 62 protein spots that were differentially expressed in PINK1-KO mice. The majority of them (51) were down-regulated, whereas 11 protein isoforms were up-regulated. Out of these 62 protein spots, 57 (92%) could be identified, which yield 30 distinct protein encoding genes. In cortex tissue samples of the PINK1-KO mice, 75 protein spots were found to be up-regulated while seven proteins were down-regulated. Eighty-five percent (70/82) of these altered proteins could be identified and were assigned to 28 non-redundant gene symbols.Protein expression alterations induced by PINK1 knockout were accessed by the quantitative comparison of the 2DE patterns of PINK1-KO mice (n = 6) to normal control mice (n = 6) in three brain regions. In general, over 5000 protein spots could be detected on each of the 2DE protein patterns. However, protein expression alterations of each brain region in the PD mouse model were quite unique respecting their isoelectric point and molecular weight distribution  were co-changed in striatum and midbrain tissues of PINK1-KO mice , whereas five proteins were co-altered in striatum and cortex tissues . When comparing midbrain vs. cortex, three proteins showed concomitant regulations. They are Aldoa, Gapdh and Vdac1 Figure . An overAltered proteins in PINK1-KO mice were clustered according to their functional involvement and cellular localization. Respecting their cellular component identity, the up-regulated proteins in PINK1-KO mouse brain tissues were enriched in the GO-terms "mitochondrial membrane"  and "actin filament" subunits . The protein subsets of down-regulated proteins in PINK1-KO showed enrichments in the GO-terms "mitochondria" .Cellular functional involvement analysis on altered proteins in PINK1-KO mice showed significant overrepresentation of various energy metabolism pathways, with partially divergent regulation directions in different brain regions. KEGG pathways that bear significant overrepresentation in altered proteins in PINK1-KO mice are summarized in table Indications of selective oxidative protection were observed among different brain regions in our mouse bearing PINK1 loss-of-function. This was reflected predominantly by the behavior of the cellular antioxidant synthesizer glutathione synthetase (Gss). This enzyme was 42% up-regulated in the striatum, and 24% up-regulated in the cortex tissue of PINK1-KO mice. Consistent with this finding, peroxiredoxin 1 (Prdx1) protein was slightly up-regulated in cortex tissue of the transgenic mice. In contrast, no change of these antioxidant proteins was observed in midbrain tissues.In order to detect the extent of oxidative damage in mouse proteome, we employed a well established methodology of redox proteomics on the 2DE gels Figure  32]. Co. Co32]. Since the direct mode-of-action of PINK1 dysfunction is the disturbance of cellular mitophagy, we set out to investigate mitophagy-related protein expression alteration as well as other catabolic processes in different brain regions of PINK1-KO mice. In this respect, we observed that the heat shock protein 70 (Hspa8), an important player of chaperone-mediated autophagy, was significantly up-regulated in cortex tissue, but not in striatum or midbrain. Regarding cellular catabolic programs other than autophagy, ubiquitin carboxy-terminal hydrolase L1 (Uchl1) was significantly down-regulated in midbrain, but up-regulated in striatum tissue of the PINK1-KO mice. Two other proteins that are involved in the ubiquitination pathway were also significantly down-regulated in midbrain . Contrary to this, two isoforms of the Ulip3 protein were observed to be up-regulated in the cerebral cortex tissue of PINK1-KO mice. Notably, no up-regulation of such catabolic process related proteins was observed in midbrain tissues of the PD mouse model.Detailed information regarding protein identification and expression pattern is provided in Table In order to in-depth scrutinize the notion of fundamental differential mitophagy performance in different mouse brain regions, we carried out a cellular ultra-structural examination to quantify the number of mitochondria undergoing mitophagy. There is no difference in either the total number of mitochondria Figure , or the substantia nigra pars compacta (SN), a core complex in the midbrain. As dopamine is physiologically transported from the substantia nigra to the striatum by dopaminergic projections, this leads to a lack of dopamine in the striatum, which in turn causes prominent motor function disturbances [substantia nigra could shed light on potential clues for PD therapeutic concept.The manifestation of Parkinson's disease (PD) is initiated by a selective loss of dopaminergic neurons in the urbances . In a seEmerging evidences have been strengthening the link between the integrity of the neuronal mitochondria to PD. Here, mitophagy, a form of autophagy for selective degradation of defective mitochondria represent one of the key aspects ,34. As aAs typical post-mitotic cells, the neurons cannot distribute their damaged components to daughter cells, whereas cell death is unfavorable regarding tissue function . This unAmong the available Parkinson's disease animal models, the mouse model with a loss-of-function mutation in PINK1 offers the most obvious link between mitophagy dysfunction and PD . Unlike Furthermore, our protein functional categorizations showed that there were significant down regulations of diverse mitochondrial energy metabolism pathways in the midbrain upon PINK1-KO . Lowered energy level can be considered as a general correlate to the accumulation of depolarized mitochondria, which is again associated with heightened oxidative stress ,37.We next investigated the oxidative protein damage patterns in three distinct brain regions with and without PINK1-KO. Our redox proteomic analysis showed that the cortex tissue was subjected to less extent of overall protein oxidative damage at both basal (wild type) and dynamic (under PINK1-KO) level: On the one hand, the Oxyblot staining experiments demonstrated minor extent of oxidative damage in the cortex tissue irrespective to the transgene. On the other hand, higher oxidative defense in cerebral cortex region was mirrored by the up-regulation of antioxidant-related proteins in our PD mouse model (glutathione synthetase and peroxiredoxin 1). Gluthatione is considered as the most important intracellular antioxidant. Upon the lack of glutathion, highly toxic hydroxyl radicals (OH·) will be generated though Fenton reaction. In sequence with this, the feedback up-regulation of glutathione synthetase has been considered as one of the earliest detectable changes in presymptomatic PD development . Under tMost intrinsically, the observed drastically reduced Vdac1 protein level in midbrain tissue of the PINK1-KO mice offer us signs of the loss of mitochondrial membrane potential in the midbrain of PINK1-KO mice. Remarkably, this phenomenon was absent in striatum, whereas an up-regulation of Vdac1 proteins was observed in cortex tissue under PINK1-KO, which could indicate certain compensation mechanism in cortex tissues upon PINK1-KO. Drastically reduced Vdac1 protein level in midbrain tissue of the PD mouse model could reflect the brain region specific depolarization of mitochondria in PINK1-KO mice. Although such speculation would need direct investigations on mitochondrial membrane potential, this could suggest that mitophagy dysfunction induced by PINK1-KO led to a severe accumulation of non-potentiated mitochondria predominantly in the midbrain tissue .substantia nigra, whereas the amount of mitophagic vacuoles in striatum was comparable to that of substantia nigra. Even more notably, SN indeed contained intrinsically higher number of defective mitochondria in comparison to striatum. These observations ultimately validate the intrinsic differential mitophagic capacity in different brain regions.To test this hypothesis of "built-in" brain-region specific mitophagy capacity in mouse, we conducted an electron microscopy study on wild type mice. Here, the normal mouse cortex tissue showed a 60% higher occurrence of mitophagic vacuoles compared to The fact that PINK1 knockout induced mitophagy disturbance did not adversely influence the cortex and striatum tissues in terms of mitochondrial energy metabolism suggests some compensatory mechanism regarding cellular maintenance. In this respect, recent reports showed that autophagy and ubiquitination/proteasome pathways are two closely interwired cellular catabolic machineries ,41. We oIn summary, using the PINK-KO mouse as a model, our hypothesis advanced in this work is that selective neuron vulnerability in Parkinson's disease could be co-determined by differential mitophagy capacity, differential oxidative protection, as well as differential feedback stimulation of alternative catabolic mechanisms in distinct brain regions. Our 2D-DIGE based study provides a starting point towards such advanced hypothesis, which will need to be scrutinized by subsequent investigations on mitochondrial membrane potential, metabolism functions and most importantly, direct studies on comprehensive mitotophagy pathway.2DE: two-dimensional protein electrophoresis; 2D-DIGE: two dimensional difference gel electrophoresis; KO: knock out mouse model; PD: Parkinson's disease; SEM: standard error of mean; SN: substantia nigra.The authors declare that they have no competing interests.MD performed the proteomic experiments and wrote the manuscript. TK generated the transgenic mice and prepared the tissue samples. GN and AK performed the mass spectrometric analysis. JS co-designed the experiments. CZ performed the statistical analysis. JK co-designed the experiments. LM co-designed the experiments, coordinated the project and wrote the manuscript. All authors read and approved the final manuscript.


Non-coding small RNAs of the micro-RNA class (miRNA) are conserved regulators of gene function with a broad impact on biological processes. We screened miRNA levels for age-related changes in individual worms and investigated their influence on the lifespan of the nematode C. elegans. We measured the abundance of 69 miRNAs expressed in individual animals at different ages with over thirty five thousand discrete quantitative nano-fluidic polymerase chain reactions. We found that miRNA abundance was highly variable between individual worms raised under identical conditions and that expression variability generally increased with age. To identify expression differences associated with either reproductive or somatic tissues, we analyzed wild type and mutants that lacked germlines. miRNAs from the mir-35-41 cluster increased in abundance with age in wild type animals, but were nearly absent from mutants lacking a germline, suggesting their age-related increase originates from the germline. Most miRNAs with age-dependent levels did not have a major effect on lifespan, as corresponding deletion mutants exhibited wild-type lifespans. The major exception to this was mir-71, which increased in abundance with age and was required for normal longevity. Our genetic characterization indicates that mir-71 acts at least partly in parallel to insulin/IGF like signals to influence lifespan. An understanding of the mechanisms that modulate the lifespan of eukaryotes has great potential to promote human health. Aging is the single largest risk factor for many debilitating diseases, including neurodegenerative diseases such as Alzheimer's and Parkinson's disease, as well as cardiovascular diseases, and cancer. An inherently important problem for aging research lies in understanding why some people age well and remain disease free, while others age poorly, and incur increased risk for age-related disease. It is clear that there are genetic factors which individually or collectively contribute to age-related disease via altered function, however, relative maintenance of health with increased age alone does not equate with the absence of age-related disease. In the round worm Caenorhabditis elegans, genetically identical animals exhibit large differences in their lifespan with associated declines in motor skills, and pathogen resistance -4. We hamiRNAs are conserved and highly abundant post-transcriptional regulators of gene expression -10.Since their relatively recent discovery , 12 miRNC. elegans decreased insulin/IGF like signaling results in extended lifespan and increased stress resistance through activation of DAF-16 and its transcription network.Insulin/IGF like signaling modulates lifespan in many model organisms ranging from worms to vertebrates -27. Recemir-71 which had a major effect on longevity. Interestingly, we determine that at least part of mir-71 effect on wild type lifespan is independent of insulin/IGF like signaling.Here we present expression and functional evidence that miRNA expression levels vary between individuals and that at least one of these miRNAs has a dramatic influence on aging. There is considerable phenotypic variation in aging animals, some of which may be attributable to miRNA-regulated protein synthesis. We have utilized a high throughput nano-fluidic PCR approach to analyze individual worm miRNA expression profiles, in order to assess age-dependent differences and between-worm variance in miRNA abundance. We report that certain miRNAs exhibit strong age-specific miRNA changes in abundance and individual variation in miRNA levels. We find that few of these miRNAs strongly influence lifespan by themselves. The exception to this finding was To examine age-related miRNA expression changes we used the quantitative polymerase chain reaction (qPCR) to assay a panel of 69 miRNAs in RNA samples collected from 48 C. elegans animals. We used individual worms to assay the miRNA levels, an approach which has previously been used in the context of gene expression profiling via microarrays , 30. SucAnalysis of the expression profiles in single worms lead to the identification of miRNAs which had significantly altered levels between young and old animals Figure . Some mimir-38 which included three outlier signals that shifted the overall mean towards a net decrease in levels, while still having a median increase in abundance with age  mutants are temperature sensitive sterile and at the restrictive temperature lack a germline due to the inability of the germline stem cells to remain mitotically active. These animals are also long-lived in a DAF-16 and DAF-12 dependent manner , howevers Figure . Interess Figure . These rmir-51 expression tended to decrease with age  mutant trended towards an extended lifespan  also failed to show lifespan phenotypes when the miRNAs were added back . A heat shock specific promoter driven construct was more stable and exhibited little developmental delay. Transgenic worms over-expressing mir-71 were found to be long-lived . These data suggested that mir-71 does not directly regulate the expression of DAF-2.It has previously been reported that mir-71 mutants are short-lived and that over-expression of mir-71 extended lifespan . It was r, DAF-2 . DAF-2 is Figure . Collectmir-71 depend on a functional insulin/IGF pathway. DAF-16 is the major transcription factor downstream of DAF-2 and is required for the lifespan effects resulting from IIS in C. elegans. If mir-71 acts solely with IIS, then the effects of mir-71 should depend on functional DAF-16. We therefore tested the lifespan phenotype of mir-71; daf-16 double null mutants. We find that the mir-71(n4115); daf-16(mu86) double mutants are significantly shorter lived than either daf-16 or mir-71 mutants alone . To further test whethermir-71 acts independently of IIS we next tested whether DAF-16 is required for the lifespan extending effects of mir-71 over-expression. We found that the mir-71 over-expressor, maIs352 significantly extends the lifespan of daf-16 null mutants  or somatic tissues . Finally, we identified which miRNAs showed increases in abundance in germline ablated animals (such as mir-60). The last case may be of particular interest for aging studies since these miRNA expression differences in glp-1 long-lived mutants correlate well with other genes known to be involved in germline loss mediated lifespan extension. Additional studies will be required to tell whether these miRNAs play a role in this process. We also note the possibility that some of the miRNA expression profiles obtained in the glp-1 background may be influenced by non-germline related effects of glp-1.The identification of genes that modulate healthy lifespan is a major goal of research on aging. Detailed functional analysis of these genes will strengthen our understanding of the mechanisms through which organisms grow old, and maintain health. We reasoned that individual gene expression differences between young and old animals may contribute to age dependent phenotypes. To identify such genes we performed expression profiling and focused specifically on miRNAs, several of which showed altered levels with age Tables -2. With let-7 and mir-70 as decreasing with age, while the more recent study as well as the work presented here identified the mir-35 family members, mir-54 and mir-71 as increasing with age. However, significant differences exist as well, possibly explained by technical differences in both collection and analysis. The qPCR data presented here has the benefit of being quantitative over a wide dynamic range as well as having high resolution, the potential to relate individual phenotypes to gene expression (single worm), and to capture the biological variance in the aging population through virtue of a high biological N. In comparison the previously reported micro-array and next generation sequencing data has the benefit of being much higher throughput in terms of number of targets assayed, although they were more limited in the number of biological replicates. These other methods also possess larger range of coverage for potential involvement of miRNAs in aging, while our study was limited to a smaller number of assays available for qPCR. The next generation sequencing method in particular has discovery based appeal since the probe based bias is not present as it is with microarray and qPCR experiments.Our expression profiling of miRNAs from aging C. elegans can be qualitatively compared with previous reports presenting similar data obtained with different techniques of both collection (whole population samples) and detection (micro-array and next generation sequencing) , 20. A qmir-71) that differed dramatically from wild type. Some mutants were found to have relatively small or inconsistent lifespan phenotypes and we also tested the effect of adding back wild type copies of the miRNA. For two of these; mir-1 and mir-80 we observed lifespan alteration in the same direction, whether the miRNA was absent or over-expressed . mir-71 has now been identified by multiple recent reports as having age dependent expression, being strongly required for normal longevity and being involved in germline mediated lifespan extension respectively ; mir-85(n4117); mir-42-44(nDf49); mir-84(n4037); mir-273(n4438); mir-51(n4473); mir-54-56(nDf58); mir-240(n4541); mir-60(n4947); mir-70(n4109); mir-81-82(nDf54); mir-53(n4113);mir-235(n4504);nDf63; mir-63(n4568);mir-80(nDf53);mir-64c(nDf63);mir-1(n4101); mir-58(n4640);mir-228(n4382); maIs251[Pmir-1::GFP]; maIs276[Pmir-60::GFP]; maIs188[Pmir-228::GFP].Nematodes were maintained as previously described . The folglp-1. Lifespan assays containing glp-1 were shifted to 25°C immediately after the egg lay and maintained at that temperature, all other lifespan assays were performed at 20°C. Comparisons of survival were performed with GraphPad Prism 4™ software and P values were generated by the log-rank Whitney test of significance.Lifespan assays were performed as previously described . In brieTo quantify changes in miRNA abundance during aging, we cultured worms under standard laboratory conditions and aged them by daily transferring the adults away from their progeny. At selected ages, we harvested individual animals and isolated total RNA from each sample using the miRNeasy (Qiagen) collection kit with automated Qiacube processing (Qiagen). cDNA synthesis was accomplished with the high capacity RT kit (Applied Biosciences). miRNA reverse transcription was performed with Applied Biosciences custom made miRNA reverse transcription primers. Reverse transcription products were subjected to a multiplex pre-amplification prior to the qPCR.C. elegans miRNAs. Currently over 170 miRNAs are listed on miRBase (www.mirbase.org) as existing in the C. elegans genome. However, the validity of this estimate as being representative of the actual number of miRNAs is unclear. A recent report using high-throughput sequencing techniques revealed that of the approximately 170 annotated miRNAs (at the time) only 106 carried nucleotide signatures consistent with processing by the miRNA processing enzymes [C. elegans samples.qPCR assays for 69 miRNAs were obtained from Applied Biosystems (ABI), at the time this represented their entire coverage of  enzymes . qPCR on enzymes  with sliAdditional methods are included with the Supplemental information.


The level of HTLV-I expression is low in vivo. We found that recombinant interferon (IFN)-α and β suppressed viral expression in HTLV-I-infected cells. Non-lymphoid stromal cells also suppressed HTLV-I expression through type-I IFNs. The suppression was reversible after isolation of infected cells from the source of IFNs, mimicking the status of viral expression in freshly isolated ATL cells which is rapidly induced in culture. Nevertheless, HTLV-I-infected individuals maintain acquired immune responses against HTLV-I such as antibodies and cytotoxic T lymphocytes (CTLs), indicating the presence of HTLV-I proteins in vivo. Analysis on Tax-specific CTLs revealed that they were activated in HAM/TSP but unresponsive in ATL patients. We found that a subpopulation of HTLV-I carriers at asymptomatic stage exhibited impaired Tax-specific T-cell response and elevated HTLV-I proviral load. This combination is a feature of ATL and likely to be an underlying risk of ATL. Collectively, HTLV-I is doubly controlled by acquired and innate immunity; HTLV-I-specific CTLs eliminate infected cells, and IFNs suppress viral expression. Both would contribute the reduction of viral pathogenesis, while the efficiency of CTLs could be partial because of limited viral expression. An increase in viral expression would activate CTLs but also accelerate inflammation. When the viral expression is well-controlled, viral pathogenesis may not be apparent until infected cell clones with a malignant phenotype finally emerge, which may occur earlier without proper CTL responses. Diversity in innate and acquired immune responses among individuals might be important determinants of disease manifestation in HTLV-I infection.


The present results demonstrated that insulin is a potent regulator of NCC phosphorylation in the kidney, and that WNK4 and SPAK are involved in this mechanism of NCC phosphorylation by insulin.The NaCl cotransporter (NCC) is essential for sodium reabsorption at the distal convoluted tubules (DCT), and its phosphorylation increases its transport activity and apical membrane localization. Although insulin has been reported to increase sodium reabsorption in the kidney, the linkage between insulin and NCC phosphorylation has not yet been investigated. This study examined whether insulin regulates NCC phosphorylation. In cultured mpkDCT cells, insulin increased phosphorylation of STE20/SPS1-related proline-alanine-rich kinase (SPAK) and NCC in a dose-dependent manner. This insulin-induced phosphorylation of NCC was suppressed in WNK4 and SPAK knockdown cells. In addition, Ly294002, a PI3K inhibitor, decreased the insulin effect on SPAK and NCC phosphorylation, indicating that insulin induces phosphorylation of SPAK and NCC through PI3K and WNK4 in mpkDCT cells. Moreover, acute insulin administration to mice increased phosphorylation of oxidative stress-responsive kinase-1 (OSR1), SPAK and NCC in the kidney. Time-course experiments in mpkDCT cells and mice suggested that SPAK is upstream of NCC in this insulin-induced NCC phosphorylation mechanism, which was confirmed by the lack of insulin-induced NCC phosphorylation in SPAK knockout mice. Moreover, insulin administration to WNK4 hypomorphic mice did not increase phosphorylation of OSR1, SPAK and NCC in the kidney, suggesting that WNK4 is also involved in the insulin-induced OSR1, SPAK and NCC phosphorylation mechanism  The NaCl cotransporter (NCC) is expressed in the distal convoluted tubules (DCT) and plays a major role in renal electrolyte balance Insulin is known to regulate renal sodium reabsorption. In fact, insulin infusion increases sodium reabsorption in the kidney D561A/+WNK4 knock-in mice, an ideal mouse model of PHAII, and found that the pathogenesis of PHAII is the constitutive activation of the WNK-OSR1/SPAK kinases-NaCl cotransporter (NCC) cascade, resulting in gain of function of NCC Pseudohypoaldosteronism type II (PHAII) is an autosomal dominant disease characterized by hypertension due to increased renal salt reabsorption, and hyperkalemia in vivo. These results indicate that insulin is a substantial regulator of the WNK-OSR1/SPAK-NCC phosphorylation cascade in vivo in the kidney.In this study, we investigated whether insulin regulates the NCC phosphorylation. Acute insulin stimulation was found to increase SPAK and NCC phosphorylation in cultured mouse distal convoluted mpkDCT cells To determine whether insulin regulates NCC phosphorylation, mpkDCT cells that endogenously express OSR1, SPAK and NCC were used. First, to evaluate the effect of insulin on SPAK and NCC phosphorylation in mpkDCT cells, a time-course experiment after the stimulation by insulin was performed . Five miNext, to confirm that SPAK is upstream of NCC in the phosphorylation cascade in this insulin-induced NCC phosphorylation mechanism, a SPAK knockdown experiment in mpkDCT cells was performed. As shown in As shown in These results indicate that insulin positively regulates phosphorylation of SPAK and NCC through PI3K in mpkDCT cell and that WNK4 is involved in this insulin-induced phosphorylation of SPAK and NCC.in vivo. Insulin was intraperitoneally injected into C57BL/6 mice (5 U/kg), and OSR1, SPAK and NCC phosphorylation in the kidney was checked. As shown in in vivo in mouse kidney, as well as in cultured cells.Next, mouse experiments were performed to investigate whether the NCC phosphorylation by insulin observed in the cultured cells was also functional To evaluate the effect of insulin on the phosphorylation of OSR1, SPAK and NCC in the mouse kidney, a time-course experiment was performed . Thirty The time-course of insulin-induced phosphorylation of NCC in mouse kidney was slightly different from that of OSR1 and SPAK. In mouse kidney, NCC phosphorylation increased to 2.6-fold at 30 min after insulin injection, and the level of NCC phosphorylation was sustained for at least 90 min, when phosphorylation of OSR1 and SPAK had already declined. Unlike SPAK and OSR1, total NCC protein was also increased in insulin-injected mouse kidney.To explore the physiological significance of insulin-induced NCC phosphorylation, NCC phosphorylation levels were compared in mice on high- and low-salt diets with or without insulin administration . Irrespein vivo, the same acute insulin injection experiment was performed on SPAK knockout mice It is well known that SPAK is a major kinase to phosphorylate NCC in vivo, the insulin injection experiment was performed in WNK4 hypomorphic mice in vivo.WNK4 knockdown in mpkDCT cells impaired insulin-induced phosphorylation of SPAK and NCC, as shown in Insulin is well known to regulate renal sodium reabsorption. It has been reported that insulin infusion increases sodium reabsorption in the kidney in vivo. As shown in In this study, there was a linkage between insulin and NCC phosphorylation. To our knowledge, this is the first demonstration of insulin-induced NCC phosphorylation in a cultured cell line and in vitro and in vivo. Involvement of WNK4 in insulin-induced NCC phosphorylation was also confirmed in this study. The insulin effect on phosphorylation of OSR1, SPAK and NCC was not observed in WNK4 knockdown cells and WNK4 hypomorphic mice. Taken together, these findings clearly show that WNK4 and SPAK are involved in the insulin-stimulated NCC phosphorylation mechanism in vitro and in vivo.These findings suggest the involvement of SPAK in the mechanism of NCC phosphorylation by insulin. To confirm this, the same insulin experiment was performed in the SPAK knockdown cells and SPAK knockout mice  and 10. The detailed mechanism of WNK4 activation by insulin remains to be determined. It is clear that PI3K is involved in the insulin-induced NCC phosphorylation mechanism, as shown in in vivo. This aldosterone-dependent activation of NCC is physiologically important for the regulation of sodium excretion by sodium intake To date, various regulators of NCC phosphorylation have been reported. Of these, there is no doubt that aldosterone is the major player for NCC phosphorylation D561A/+WNK4 knock-in mice, a mouse model of PHAII As shown in in vivo. WNK4 and SPAK are involved in this insulin-stimulated NCC phosphorylation in vivo. The present findings might explain increased salt-sensitivity under hyperinsulinemic conditions.In summary, it was found that insulin increased phosphorylation of NCC in cultured cells and in mouse kidney In acute insulin injection experiments, male mice were fed a normal NaCl diet, a high-NaCl diet (4.0% NaCl (w/w)) or a low-NaCl diet (0.01% NaCl (w/w)) for 7 days before insulin administration. All foods were obtained from Oriental Yeast Co., Ltd . Insulin was administered intraperitoneally at a dose of 5 U/kg, as previously reported Semiquantitative immunoblotting was performed, as described previously gca caa agc cca aca gcu uTT; mouse WNK4 siTRIO SMF27A-2153-2 gga aga uga ugg aga gaa gTT; mouse WNK4 siTRIO SMF27A-2153-3 cga cag agu ugu cga gug uTT; mouse SPAK siTRIO SMF27A-2154-1 ggu cag auc cau agggau uTT; mouse SPAK siTRIO SMF27A-2154-2 cgg aau aaa guc aga aaa aTT; mouse SPAK siTRIO SMF27A-2154-3 uga cau acg auu uga guu uTT. As a negative control, siTRIO negative control siRNA (COSMO BIO), which consists of cocktail of 3 duplexes of negative control sequence siRNA, was used. For knockdown, 50 nM of siRNA cocktail were used, and transfections were carried out using Lipofectamine RNAiMAX (Invitrogen). Experiments were performed at forty-eight hours after siRNA transfection.mpkDCT cells were cultured as previously reported t-test. P-values<0.05 were considered significant. When more than three groups were compared, one-way ANOVA was used, followed by Fisher's post hoc test.Statistical significance was evaluated using an unpaired Figure S1Conformation of total- and phospho-specific- SPAK antibody in vivo. Immunoblot of kidney homogenate from wild-type (left lane) and SPAK knockout mouse (right lane) with our total SPAK and phospho-specific SPAK antibody. The disappearance of bands in the sample from a SPAK knockout mouse confirms the specificity of our antibody.(TIF)Click here for additional data file.Figure S2Absorption test of total- and phospho-specific- NCC antibody for mpkDCT cell. The specificity of these antibodies was confirmed previously (TIF)Click here for additional data file.Figure S3Absorption test of total- and phospho-specific- NCC antibody for mouse kidney. A. Confirmation of specificity of anti-pNCC (Ser71) antibody in a sample from wild-type, WNK4 hypomorphic and SPAK knockout mouse kidney. The signals detected by the antibody disappeared when the antibody was pre-incubated with antigen phosphopeptide (right panels), but they did not disappear with the corresponding non-phosphopeptide (middle panels). B. Confirmation of specificity of anti-NCC antibody in a sample from wild-type, WNK4 hypomorphic and SPAK knockout mouse kidney. The signals detected by the antibody disappeared when the antibody was pre-incubated with antigen.(TIF)Click here for additional data file.


To determine if vessel orientation can be estimated retrospectively, and if this information improves measurements of coronary luminal area.Measurements of coronary cross-sectional area are utilized in vasomotor tone and endothelial function studies, which typically employ breath-held, 2-D multi-slice imaging protocols wherein slices are prescribed orthogonally to a linear vessel segment Fig  1],[2].,[2].1],.Cardiac-gated, breath-held spiral coronary angiography with spectral-spatial excitation was performed using a GE Signa 3T scanner . Transverse views of a cylindrical vessel phantom  and a linear segment of the right coronary artery (RCA) in a healthy volunteer  were acquired, along with field maps, for gridding reconstruction and off-resonance deblurring . We applin vivo results. A higher-order model of the vessel may improve estimation performance. Nonetheless, the error performance after correction was equivalent to acquiring with much smaller prescription angles (Tables in vivo improvement in the sensitivity reduction was 16-fold.Non-orthogonal prescription angles lead to slice-dependent elliptical projections of the cylindrical phantom Fig  and the s Tables ,2. The mCross-sectional lumen area can be retrospectively compensated for non-orthogonally prescribed slices, thereby reducing the sensitivity to slice prescription and breath-hold repeatability. This approach should reduce operator dependence and shorten localization time.


An attempt was made to demonstrate the possibility of performing LanthaScreen™ TR-FRET based nuclear receptor coactivator recruitment assay using PHERAstar, a multi-detection HTS microplate reader. LanthaScreen™ nuclear receptor coactivator recruitment assay  was performed using PPAR-gamma receptor preparation in the agonist mode. TR-FRET measurements were done on PHERAstar, a multimode microplate reader . The Lanthascreen PPAR gamma coactivator recruitment assay was successfully performed in the PHERAstar, multimode microplate reader. This was evidenced by an assay robustness score (Z') of 0.71. The current work demonstrates the suitability of using PHERAstar, a multi-detection HTS microplate reader.for performing LanthaScreen™ TR-FRET based nuclear receptor coactivator recruitment assays. Nuclear receptors (NRs) are a superfamily of ligand-activated transcription factors that mediate a wide range of cellular responses. The endogenous ligands for NRs include steroidal hormones, eicosanoids, retinoids, thyronines, bile acids, and other metabolites. NRs inflIn TR-FRET assays, the biomolecular interaction is detected by energy transfer between two fluorophores that are deliberately employed in these assays. The fluorescence energy transfer results in dual emissions, which can be expressed as a ratio and used as the assay signal. This ratiometric measurement is helpful to counteract assay interference like quenching exhibited by test samples. Besides the ratiometric nature of these assays, TR-FRET also utilizes the phenomenon of time-resolved fluorescence (TRF). TRF is a phenomenon exhibited by chelates of the lanthanide group of elements  Upon excitation, the fluorescence that is emitted by the lanthanide chelate does not decay spontaneously like commonly employed fluorophores, instead there is delayed decay of fluorescence. Autofluorescence of test compounds/microwell plates/buffers (background fluorescence) is the bane of bioassays. The decay time of the background fluorescence falls within the decay time of commonly employed fluorophores in assay systems and thus there can be assay interference. By utilizing lanthanide chelates, the signal measurement window can be set at a much longer time interval which will be free from background autofluorescence. Thus, the TR-FRET method combines the benefits of ratiometric measurement and the TRF phenomenon.Invitrogen, USA, offers a TR-FRET-based nuclear receptor coactivator assay platform under the trade name LanthaScreen™. In the current study an attempt is made to demonstrate the possibility of performing the LanthaScreen™ nuclear receptor coregulator assay using the PHERAstar, a multilabel plate reader from BMG LABTECH, Germany. LanthaScreen™ nuclear receptor assays using the PHERAstar have not so far been reported in the literature. This demonstration would validate the compatibility of PHERAstar instrumentation for LanthaScreen™ nuclear receptor coregulator assays. This is required since advanced assay methodologies like TR-FRET require appropriate instrumentation for proper signal measurement. In the current study, the LanthaScreen™ peroxisome proliferator activating receptor-gamma (PPAR-gamma) coactivator recruitment assay (agonist mode) is employed. The assay principle is described in S transferase-tagged PPAR-gamma receptor) 125 nM of coregulator peptide, 5 nM of TB-anti-GST-tagged antibody (terbium-anti-glutathione S transferase tagged), 5 mM DTT (dithiothreitol), and varying concentrations of rosiglitazone (a known agonist for PPAR gamma receptor) in the assay buffer supplied by Invitrogen. The negative control was devoid of the agonist but contained everything else contained in the agonist wells. Following 1-h incubation in the dark, TR-FRET measurements were made in the PHERAstar using the following settings: optical module - LanthaScreen™, delay time - 100 μsec, and integration time - 200 μsec. The ratiometric emission (520/490) was plotted against varying ligand concentrations. The data was analyzed using GraphPad Prism, version 4.00 for Windows  using the sigmoidal curve equation with variable slope to obtain EC50 values [et al. and was found to be 0.71 . From the observed data, the PHERAstar, a multilabel plate reader from BMG Labtech, Germany, is suitable for LanthaScreen™ nuclear receptor coregulator assays.In brief, the assay procedure was as per Invitrogen's kit insert . All the reagents were procured from Invitrogen, except rosiglitazone, which was obtained from Cayman Chemicals, USA. The assay was performed in 384 black microwell plates (Corning # 3676). A 20 μl total assay reaction included 5 nM GST-tagged PPAR-gamma receptor (glutathione 0 values . The ass


The intimate connection between coagulation and inflammation in the pathogenesis of vascular disease has moved more and more into focus of clinical research. This paper focuses on the essential components of this interplay in the settings of cardiovascular disease and acute coronary syndrome. Tissue factor, the main initiator of the extrinsic coagulation pathway, plays a central role via causing a proinflammatory response through activation of coagulation factors and thereby initiating coagulation and downstream cellular signalling pathways. Regarding activated clotting factors II, X, and VII, protease-activated receptors provide the molecular link between coagulation and inflammation. Hereby, PAR-1 displays deleterious as well as beneficial properties. Unravelling these interrelations may help developing new strategies to ameliorate the detrimental reciprocal aggravation of inflammation and coagulation. Systemic and local proinflammatory changes are in focus when investigating the pathophysiology of arteriosclerosis and acute coronary syndromes. In acute myocardial infarction (AMI), proinflammatory markers such as C-reactive protein (CRP), interleukins, or monocyte-chemoattractant protein (MCP)-1 are elevated  and theiSources of inflammatory response are vascular cells such as activated endothelial cells, which release proinflammatory cytokines such as interleukin (IL)-8 . IL-8 isBesides IL-8, many other cytokines such as IL-6 take part in inflammatory responses by inducing B-cell differentiation, T-cell activation, and synthesis of acute phase proteins , but alsThe pathogenesis of proinflammatory changes in acute coronary syndromes as well as the interplay between coagulation and inflammation is poorly understood and is subject to intense research. The mechanisms by which the coagulation system is altered by inflammatory interactions comprise enhanced synthesis and activation of coagulant proteins, decreased synthesis of anticoagulants, and suppression of fibrinolysis . HoweverIn this paper we focus on possible mechanisms of the interplay between coagulation and inflammation in acute coronary syndromes.A strict separation between the intrinsic and extrinsic coagulation cascade surely fails to reflect physiologic conditions. Nevertheless, in the setting of arteriosclerosis and acute coronary syndromes the extrinsic pathway of coagulation is of particular significance.Tissue Factor (TF) is the most important initiator of the extrinsic coagulation cascade. TF, a 47 kDa transmembrane glycoprotein and member of the class II cytokine receptor family, is the cofactor for the activated plasma clotting factor VII (FVIIa). The TF-FVIIa complex catalyzes the activation of factor X and IX, which leads to the generation of thrombin and thus finally of a fibrin clot. Under physiologic conditions TF is abundantly expressed only in the adventitia and is induced by several inflammatory mediators such as IL-6, IL-8, and MCP-1 , 29. AftFurthermore, increased TF expression can be noticed on circulating monocytes and microparticles in acute coronary syndromes and may, thereby, contribute to activation of coagulation –34. A soStimulation of the TF-thrombin pathway does not only occur at the site of the plaque but also within the ischemic myocardium where activated coagulation factors may enhance inflammatory responses and increase infarct size . TF contTF-mediated intracellular signal transduction has not been completely elucidated so far. On one hand TF allows docking and activation of FVII and, therefore, promotes the generation of downstream coagulation factors and activation of protease-activated receptors (PARs) which themselves possibly induce intracellular signal transduction. On the other hand there is evidence for direct signalling through the cytoplasmic domain of TF following TF-FVIIa complex formation , 56.The endogenous Kunitz-type inhibitor Tissue Factor Pathway Inhibitor-1 (TFPI) inhibits initiation of TF-induced blood coagulation and is mainly expressed on vascular endothelial cells. TFPI binds and inactivates FXa. The TFPI-FXa complex then binds and inactivates FVIIa. Increased levels of the TFPI-FXa complex may reflect both increased FXa generation and increased TFPI concentrations . In addiImportant players in the interaction between coagulation and inflammation are protease-activated receptors (PARs). PARs are G-protein coupled receptors that mediate various cellular reactions as cytokine release, expression of adhesion molecules, cell migration, or proliferation. Unlike other receptors, PARs are not activated by a soluble, external ligand. Proteases, such as activated coagulation factors, detach a defined part of the NH2-terminal chain of the receptor, thereby inducing a conformational change of the receptor. This change causes a self-activation by a “tethered ligand.” This activating sequence comprises only few amino acids. PARs can also be activated by synthetic peptides consisting of the sequence of amino acids representing the tethered ligand.In contrast to other receptors, the activation of PARs by enzymatic cleavage is irreversible. After proteolytic activation the receptor must be internalized, degraded, and resynthesized. PARs are mainly expressed in vascular cells, but also in many different other cell types such as gastrointestinal and bronchial epithelial cells. Four different PARs are known: PAR-1, -3, and -4 show responsible for thrombin signaling whereas PAR-2 is activated by trypsin-like serine proteases, FVIIa, and matriptase but not by thrombin. PAR-1 and PAR-2 are expressed on smooth muscle cells and endothelial cells, whereas mainly PAR-1 is expressed on monocytes.PAR-1 agonists or thrombin induce IL-8 and IL-6 in SMC, EC, and mononuclear cells (MNCs)  thereforIn addition, PARs can also be cleaved downstream of the tethered ligand, resulting in receptor inactivation by preventing further proteolytic activation . PAR-1 sPlatelet activation with subsequent thrombus generation plays a major role in the development of acute coronary syndromes. At low concentrations thrombin activates PAR-1 on platelets through a hirudin-like site and at high concentrations additional PAR-4. This induces shape change, P-selectin, and CD40L mobilization to the platelet membrane and promotes the release of platelet agonists ADP, thromboxane A2, chemokines, and growth factors  and, theBinding of the serine protease FVII to TF results in generation of the coagulation protease FXa (FXa) and subsequently thrombin both known to induce cell signaling. FXa shows dose-dependent induction of intracellular calcium transients in endothelial cells that is active-site-dependent, and independent of thrombin . PotentiElevated TFPI-FXa and prothrombin fragments F1+2 plasma levels indicate activation of the coagulation cascade in acute coronary syndromes. Under physiological conditions an inverse relationship between TFPI-FXa and F1+2 suggests that TFPI-FXa regulates prothrombinase activity in vivo . Under cIn vitro experiments revealed that FXa stimulates IL-8 and MCP-1 transcription in endothelial cells and mononuclear leukocytes . GeneticAccording to the expression of PAR-1 and PAR-2, PAR-1 and PAR-2 agonists induce IL-8 and MCP-1 release in endothelial cells, whereas, only PAR-1 agonists stimulated cytokine release in mononuclear cells .Several studies suggest a signaling mechanism of the TF-FVIIa complex via PAR-2 . In thisIn contrast to FXa, FVIIa does not elicit a proinflammatory response in endothelial cells or mononuclear cells . The TF-In recent studies , it has The interplay between coagulation and inflammation is a matter of intense research. Proinflammatory changes in acute coronary syndromes may substantially influence prognosis . Experimα (TNF) has been shown to effectively improve survival in several animal models of sepsis [While inhibiting proinflammatory cytokines such as Tumor-Necrosis Factor-f sepsis –91, antif sepsis –95. In cf sepsis , 96, andInhibiting coagulation could depict a more promising mechanism in fighting overwhelming inflammatory response. Experimental studies that have shown that anticoagulant treatment not only diminishes activation of coagulation but also inhibits inflammation, underline the cross-talk between activation of coagulation and cytokine release in vivo , 97, 98.So far, anti-inflammatory treatments displayed no explicit benefit in patients with acute coronary syndromes . HoweverYet the question remains if and what anticoagulant therapies will prove beneficial to alter systemic inflammatory responses.


HsPCFT using the substituted-cysteine accessibility method, to investigate the boundaries between the water-accessible surface and inaccessible buried protein segments. Single-cysteines, engineered individually at 40 positions in a functional cysteine-less HsPCFT background construct, were probed for plasma-membrane expression in Xenopus oocytes with a bilayer-impermeant primary-amine-reactive biotinylating agent (sulfosuccinimidyl 6-(biotinamido) hexanoate), and additionally for water-accessibility of the respective engineered cysteine with the sulfhydryl-selective biotinylating agent 2-((biotinoyl)amino)ethyl methanethiosulfonate. The ratio between Cys-selective over amine-selective labeling was further used to evaluate three-dimensional models of HsPCFT generated by homology / threading modeling. The closest homologues of HsPCFT with a known experimentally-determined three-dimensional structure are all members of one of the largest membrane protein super-families, the major facilitator superfamily (MFS). The low sequence identity - 14% or less – between HsPCFT and these templates necessitates experiment-based evaluation and model refinement of homology / threading models. With the present set of single-cysteine accessibilities, the models based on GlpT and PepTSt are most promising for further refinement.The proton-coupled folate transporter (PCFT) was recently identified as the major uptake route for dietary folates in humans. The three-dimensional structure of PCFT and its detailed interplay with function remain to be determined. We screened the water-accessible extracellular surface of  Oxidized and polyglutamate forms of folate are dietary active, but must be deconjugated to remove glutamic acid moieties and reduced to dihydrofolate and tetrahydrofolate to confer biological activity. These reduced compounds play important roles in one-carbon metabolism pathways such as biosynthesis of nucleic-acids and also in synthesis and breakdown of amino acids. Common to all vitamins, folates cannot be synthesized by mammals, and are therefore considered vital nutrients. Folates are highly absorbed from dietary sources in the intestine by specialized membrane proteins. Folate deficiency in pregnant women increases the risk for the development of neural tube defects (NTDs) in the offspring folic acid  into X. laevis oocytes was determined in MES buffered saline (MBS) buffer . Transport of folic acid through PCFT is proton-coupled and therefore facilitated by acidic pH. Therefore, uptake was studied at pH 5.5 folic acid. After incubation for 10 min at room temperature, uptake was halted by 5-6 rapid washes with cold MBS buffer (pH 5.5). Oocytes were individually solubilized in 300 μl of 5% SDS for 60 minutes to overnight, and uptake of radiolabeled substrate was determined with a Packard 1900 TR liquid scintillation analyzer or a Beckman LS 6500 Scintillation Counter. To evaluate non-PCFT mediated folic acid uptake in oocytes, control experiments were performed with uninjected oocytes. Comparison of uptake in noninjected vs. water-injected oocytes showed no significant differences (data not shown). Uptake expressed in picomoles of [3H]folic acid per oocyte. PCFT mediated uptake of folic acid uptake between HsPCFT wild-type and HsPCFT-CYS- injected oocytes. Data shown are means ± SEM. One-way ANOVA with Bonferroni’s multiple comparison test or unpaired The HHpred server together with MODELLER (http://toolkit.tuebingen.mpg.de/hhpred) was used to search for remote homologues of PCFT  with experimentally-determined crystal structures in the PDB database and to generate structural models using default parameters . SimilarE. coli proteins, glycerol-3-phosphate transporter [St, PDB # 4aps)[So, PDB # 2xut)[-24 to 1.8x10-38, and sequence identities between 9 and 14 % , multidrB # 1pw4), L-fucosB # 1pw4), D-XylosB # 1pw4), lactoseB # 1pw4), nitriteB # 1pw4). Two morB # 4aps), and proB # 2xut), and oneB # 2xut). The E-vand 14 % . LOMETS and 14 % . For allHsPCFT) construct with a C-terminal hemagglutinin-tag (HA) and with all seven endogenous Cys replaced by Ser has been previously expressed in HeLa cells using the vector pcDNA3.1 [HsPCFT construct with a C-terminal V5 epitope tag using the vector pXOON (ct-V5-HsPCFT)[HsPCFT wild-type construct in the pXOON vector with Ser to obtain a Cys-less PCFT construct  . We subsFT-Cys-) . We exprt pH 5.5 . No statTo probe and optimize the obtained homology models of PCFT based on various X-ray-crystallography-derived templates by utilizing the substituted-cysteine accessibility method (SCAM), we used the functional Cys-less PCFT construct described above as template for generating 40 single-Cys mutants of PCFT . All conX. laevis oocytes, and applied two procedures to each batch of oocytes in parallel: 1) we assessed the plasma-membrane expression level of each single-Cys construct individually, and 2) we probed the external accessibility of the engineered Cys. To investigate the plasma-membrane expression in X. laevis oocytes we used surface-biotinylation with sulfo-NHS-LC-biotin. Intact oocytes were treated with sulfo-NHS-LC-biotin. Due to the low pKa of the sulfo moiety this biotinylation compound is charged at our experimental labeling pH, and therefore it is water soluble and unable to cross biological membranes. The reactive NHS moiety selectively labels primary amine groups as found in Lys sidechains and the N-terminal primary amine group. If labeling is performed on intact cells, only Lys exposed to the surface can be modified. Since modification introduces a biotin, biotinylated surface proteins including labeled PCFT can be isolated after cell lysis with avidin beads. Avidin-bead-purified proteins were separated by SDS-PAGE and PCFT detected with an antibody against the V5-epitope tag engineered at its C-terminus. PCFT wild-type and Cys-less PCFT are efficiently surface-biotinylated by this reagent and detected in Western blots . The first three are located on the extracellular end of TM11, and the latter two on the extracellular end of TM12. The location of S407C and N411C is below the bilayer level; however, these positions face the center of our PCFT model based on GlpT, and are likely lining the extracellular mouth of PCFT in the outward-facing state. A recent report investigated P425 extracellular accessibility with MTSEA-biotin and found that it is not accessible . The neiOverall, the extracellular water-accessibility of specific positions that are part of loop-helix regions on the extracellular surface of PCFT was determined. The loop between TM1 and TM2 is the largest extracellular loop, extending approximately from S42 to T82. Additionally, several residues that we predicted to be below the plasma membrane level based on our initial homology model were also labeled by MTSEA-biotin, likely because their side chains are accessible to potential hydrophilic substrate or proton-translocation pathway(s) at the center of PCFT. St best reflect our current results. Our ratiometric approach allows to rationally investigate homology models generated by server-based structure-prediction methods that identify distant homologues within the PDB database. If we simply color-code the three ratio-bins and apply this color code to the investigated 40 residues in the 9 homology models we generated with HHpred/MODELLER and LOMETS/MODELLER, it is immediately obvious that several models contradict our experimental observation, since the colors seem to be randomly distributed over the entire transmembrane depth of the modeled PCFT . For somFurther experiments are needed for rational improvement of the alignments used for generating the models, for example with MODalign. Even though the sequence identity between PCFT and the template transporters with presently known X-ray structures is 14% or less at present, the core region is expected to be structurally well conserved with an RMSD for backbone atoms within almost atomic resolution, making the predicted structure of this core region more reliable. By experimentally probing the more flexible ECL regions and in the future also ICL regions, and further experimentation to delineate the exact vertical alignment between individual TMs it should be possible to generate a homology model with a precision within the current X-ray structure determination range for membrane proteins. A verified model will be of great use for future studies investigating residues involved in substrate and proton translocation as well as mechanistic studies.


The development of Kaposi’s sarcoma (KS), the most common malignancy in AIDS patients, results from Kaposi’s sarcoma-associated herpesvirus (KSHV) infection of endothelial cells and subsequent induction of proliferation, angiogenesis, and inflammation. Previously, we demonstrated that KSHV infection of human umbilical vein endothelial cells (HUVEC) induced a transcriptional induction of Angiopoietin-2 (Ang-2), a pro-angiogenic and pro-inflammatory cytokine that is highly present in KS tumors. This transcriptional up-regulation of Ang-2 started at 12 h and peaked at 54 h post-infection. In difference to our previous data, here we demonstrate that KSHV infection of HUVEC induces rapid release of Ang-2 within minutes of viral contact. Pre-made Ang-2 is stored in the Weibel-Palade body in endothelial cells and is released through regulated exocytosis. KSHV binding to HUVEC is responsible for rapid Ang-2 release because blockade of viral binding inhibits this cytokine exocytosis. We find that KSHV binding to its integrin receptors on endothelial cells activates the integrin tyrosine kinase receptors signaling pathways, including tyrosine phosphorylation of the kinases FAK and Src, and triggers rapid calcium mobilization. This mobilization likely plays a key role in mediating Ang-2 release, as its inhibition by various calcium chelators and calcium channel blockers substantially reduces Ang-2 release. We also demonstrate a direct interaction and association of the kinase Src with the alpha1C subunit of L-type calcium channel. Indeed, specific inhibitors of protein tyrosine phosphorylation not only disrupt this interaction but also abolish Ang-2 release. Finally, preliminary data from in vitro cell adhesion assays suggest that this rapidly released Ang-2 enhances migration and adhesion of monocytes to the infected endothelial cells. To our knowledge, this is the first demonstration of interaction between KSHV and its integrins receptors in regulating rapid cytokine release. This study also uncovers a novel mechanism of KSHV induction of angiogenesis and inflammation, which is much faster, and could likely play important roles in the early event of KS tumor development.


Adoptive transfer of Tumor Infiltrating Lymphocytes (TIL), T-cell receptor (TCR)-transduced PBL and Chimeric Antigen Receptor (CAR)-transduced PBL have proven to be clinically effective. CARs have evolved by introducing cytoplastic costimulatory domains. Higher cytokine secretion and longer persistence of CAR-transduced T-cells in vivo imply important roles of costimulatory signals. In contrast, costimulation of TIL or TCR-transduced PBL in vivo relies on the expression of costimulatory receptors on T-cells and ligands on tumor cells or antigen presenting cells (APCs). One way to give T-cells their desired costimulation signal independent of the endogenous receptor/ligand expression would be to introduce a fusion molecule that is comprised of a receptor whose ligand is frequently expressed on tumor cells and an intra-cellular costimulatory domain. In a pursuit for a “universal costimulator” that is applicable to various tumors, we produced fusions of CD2 and various costimulators such as CD28, 4-1BB, ICOS, CD27 and OX-40 (Figure


After ST-elevation myocardial infarction (STEMI), microvascular obstruction (MVO) can be assessed using angiographic myocardial ‘blush’ grade (MBG), subject to interoperator variability. Quantitative blush evaluator (QuBE) is a free computer-calculated algorithm, with improved reproducibility. We aimed to compare QuBE in detecting MVO and its severity in comparison to the gold standard, cardiovascular magnetic resonance (CMR).Fifty-two STEMI treated with successful primary-PCI were enrolled. QuBE values were blindly calculated on post-procedure angiogram and patients divided in tertiles (ter) according to QuBE values. All patients underwent CMR (GE 1.5T) 4-7 days after STEMI, for quantitative assessment of infarct size (IS), area-at-risk (AAR), myocardial salvage index (MSI), MVO (both as first pass MVO (FP-MVO) and delayed-enhancement MVO (DE-MVO)), and presence of intramyocardial hemorrhage on T2-weighted sequences. Indices were measured blindly by two expert readers with SMCR level 3, using ReportCard (v4.0) software. LGE infarct size was quantified using threshold >5SD, T2 sequence >2SD as previously described. MVO was manually traced, and hemorrage identified as the dark band within bright signal on T2QuBE values were inversely related to IS, both as percentage  and as mass , to DE-MVO, both as percentage  and as mass , to FP-MVO , and positively related to MSI . Moreover patients with intramyocardial hemorrhage had significantly lower QuBE values . At receiver operating characteristic (ROC) curve analysis, QuBE accounted for an area under the curve of 0.88  for both DE-MVO and haemorrhage detection. Figure This is the first report to compare quantitative computer-based assessment of angiographic perfusion with comprehensive parameters of microvascular function, infarct size and salvage on early CMR in STEMI patients. We demonstrate the important finding that QuBE values correlate strongly with microvascular obstruction, Intramyocardial hemorrage and myocardial salvage by CMR. This validates the use of QuBE for assessment of tissue reperfusion in the clinical setting, and represents and important advancement in post-procedural evaluation of reperfusion in the cathlab. Our data suggest that a quantitative evaluation of myocardial blush after STEMI will accurately risk-stratify patients, and provide useful prognostic information, better informing decisions such as the selection of patients requiring further imaging (eg by CMR).


Circulating microRNAs (miRNAs) are emerging as promising biomarkers for cancer; however, the significance of circulating miRNAs in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) remains largely unknown. Based on our prior observations that miRNA-101 (miR-101) is downregulated by HBV and induces epigenetic modification, we sought to test whether circulating miR-101 may serve as a potential biomarker for HCC. The expression of miR-101 in HCCs and serum was evaluated by real-time polymerase chain reaction. Tissue and serum miR-101 levels were assessed in samples from patients with HBV-related HCC and healthy controls. A potential correlation was also evaluated between miR-101 expression and the clinicopathological features and prognosis of HCC patients. miR-101 was downregulated in HBV-related HCC tissues compared with adjacent noncancerous tissues. Furthermore, the miR-101 levels in these tissues from HCC patients were significantly lower than those in tissues from control subjects. Notably, serum miR-101 levels were found to have an inverse correlation with tissue miR-101 expression levels. The expression of serum miR-101 in patients with HBV-related HCC was significantly higher than that in the healthy controls, and this increase correlated with hepatitis B surface antigen positivity, HBV DNA levels and tumor size. These results indicate that different factors govern the levels of miR-101 in the tissue and serum of HCC patients. Given the marked and consistent increase in serum miR-101 levels in HCC patients, circulating miR-101 may serve as a promising biochemical marker for monitoring the progression of tumor development in HBV-related HCC. Hepatocellular carcinoma (HCC), the most common primary liver cancer, is a highly lethal disease that causes ~700,000 mortalities worldwide each year . It has microRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression, predominantly through imperfect base pairing with the 3′-untranslated region of target mRNAs . miRNAs Serum and tissues (paired tissue specimens from HBV-related HCC tissues and adjacent noncancerous hepatic tissues) were obtained from patients undergoing surgical HCC resection. The specimens were collected at the Hepatobiliary Surgery Department of the First Affiliated Hospital of Chongqing Medical University, Chongqing, China. As a control, serum was also collected from healthy volunteers. Volunteers had not been diagnosed previously with any type of cancer, based on self-reporting. All participants signed informed consent for the use of their blood samples prior to recruitment. This study was approved by the Ethics Committee of The First Affiliated Hospital of Chongqing Medical University.Total RNA was extracted from serum using TRIzol reagent  according to the instructions provided by the manufacturer. The purity of the isolated RNA was determined by OD260/280-reading using a NanodropND-2000 spectrophotometer .Reverse transcription was performed using the M-MLV Reverse Transcription system . U6 RNA was used as an internal control for the miRNA. The primers used for stem-loop reverse-transcription PCR for miR-101 were purchased from RiboBio Co., Ltd. . qPCR was performed using a standard SYBR-Green PCR kit protocol for a StepOne Plus system . Finally, relative expression was calculated using the comparative Ct method and normalized to the U6RNA internal control.Statistical analysis was performed using SPSS 16.0 software . Correlations were analyzed using Pearson’s correlation. The statistical significance of the differences in data was determined using the Student’s t-test, and data are expressed as the mean ± standard deviation from at least three independent experiments. Differences were considered statistically significant when P<0.05.The demographics of the study subjects are summarized in To verify whether miR-101 is differentially expressed in HBV-related HCC tumors, we measured miR-101 expression levels in the 20 human HBV-related HCC tissues and adjacent noncancerous hepatic tissues by qPCR. Among the 20 matched samples, miR-101 expression within the same patient was significantly decreased in 90% of the HCC samples compared with adjacent noncancerous hepatic tissues , verifyiOn average, the miR-101 expression levels in HBV-related HCC tissue were ~50% lower compared with those in normal adjacent tissue (Circulating miRNAs have emerged as candidate diagnostic markers for various types of cancer –19. We hTo verify the disparate results for tissue and serum miR-101 expression trends, we assessed whether serum miR-101 levels have a positive correlation with miR-101 levels in HBV-related HCC tissue. miR-101 mRNA expression levels in HBV-related HCC tumor tissue and serum were compared using the qPCR data for all 20 HCC patients. Our results show that serum miR-101 levels have an inverse correlation with the levels in tumor tissue . This veTo determine whether circulating miR-101 levels are indicative of the state of HCC progression, we studied whether miR-101 serum expression correlated with the clinicopathological characteristics of HCC. miR-101 levels were not statistically associated with patient age or gender, or several of the classic markers for HCC progression, including AFP levels, alanine transaminase, aspartate aminotransferase, cirrhosis and tumor-node-metastasis staging; however, a statistically significant correlation was observed between miR-101 expression and HBsAg, HBV DNA level and tumor size . This siHCC, which is the fifth most common cause of cancer worldwide, has an extremely poor prognosis. The early diagnosis of HCC is of great clinical importance and may improve the prognosis of HCC if patients were to receive surgical treatment early. Despite noteworthy advances in the effort to develop noninvasive serum biomarkers for the diagnosis of HCC, the reliability of biomarkers, such as AFP, remains debatable. Indeed, the specificity of AFP is low, particularly in the context of chronic liver disease . AccordimiRNAs have been identified in many body fluids, including serum and plasma , and stuIn this study, we demonstrated that miR-101 was downregulated in 90% of HBV-positive HCC tumor tissues compared with adjacent noncancerous tissue. Furthermore, these miR-101 levels were decreased compared with those in healthy controls. Apparently contradictory to these findings, we found that the serum miR-101 expression in patients with HBV-related HCC was significantly higher than that in the healthy controls. Serum miR-101 levels correlated with HBsAg, HBV DNA level and tumor size, indicating that serum miR-101 may be used as a potential predictor of HCC prognosis.Although our results gave a high positive predictive value for our HCC cohort, there are several limitations to this study. For example, the post-surgical serum samples were small in size and varied in time point. It is imperative to test more longitudinal samples in order to justify the specific time or period at which the circulating miRNAs return to basal levels.Substantial evidence has implicated that serum-based miRNAs are useful as noninvasive biomarkers for different types of cancer –33; howeIn conclusion, our findings indicate that the fluctuation in circulating miRNAs during HCC provides an innovative approach that offers a sensitive and convenient means for the early detection of HBV-related HCC carcinogenesis. Serum miR-101 expression, which was closely associated with tumoral size in this study, provides a promising biochemical marker of HBV-related HCC.


Although cinchonine's antiplatelet effect has been reported in the previous study, antiobesity effect of cinchonine has never been studied. The main objective of this study was to investigate whether cinchonine reduces high-fat-diet- (HFD-) induced adipogenesis and inflammation in the epididymal fat tissues of mice and to explore the underlying mechanisms involved in these reductions. HFD-fed mice treated with 0.05% dietary cinchonine for 10 weeks had reduced body weight gain (−38%), visceral fat-pad weights (−26%), and plasma levels of triglyceride, free fatty acids, total cholesterol, and glucose compared with mice fed with the HFD. Moreover, cinchonine significantly reversed HFD-induced downregulations of WNT10b and galanin-mediated signaling molecules and key adipogenic genes in the epididymal adipose tissues of mice. Cinchonine also attenuated the HFD-induced upregulation of proinflammatory cytokines by inhibiting toll-like-receptor-2- (TLR2-) and TLR4-mediated signaling cascades in the adipose tissue of mice. Our findings suggest that dietary cinchonine with its effects on adipogenesis and inflammation may have a potential benefit in preventing obesity.Cinchonine (C Ou. Ou34]. ogenesis . AccordiκB [α, and IFNα, which act on the TNF receptor in hypertrophied adipocytes, thereby inducing pro-inflammatory cytokine production via NF-κB-dependent and -independent mechanisms [α and IFNα) in the epididymal adipose tissue of mice fed on the HFD. In the present study, the plasma concentration of FFA decreased by cinchonine could also be a factor in the reduction of obesity-induced inflammation. Accordingly, we presume that cinchonine contributes to lower adipose inflammation in the diet-induced obese model by down-regulating the TLR-mediated signaling pathway and lowering plasma FFA level in mice maintained on the HFD.FFA is also an endogenous ligand for TLR2 and TLR4 which play important roles in the pathogenesis of noninfectious, inflammatory diseases of host deregulation such as obesity –38. MiceκB . In the κB . Both IRchanisms . DietaryIn conclusion, our results indicate that cinchonine has a dramatic suppressive effect on adipogenesis through the down-regulation of WNT and galanin-mediated adipogenesis signaling cascades, and it also attenuates inflammation by repressing TLR2- and TLR4-mediated pro-inflammatory signaling pathways in the adipose tissue of mice fed on the HFD. In the current study, we demonstrated that cinchonine is a useful dietary phytochemical for the prevention of not only obesity, but also adipose inflammation.


Protein kinase G type I (PKGI/cGMP kinase I) plays a critical role in survival signalling of pre- and postconditioning. However, it is unclear whether cGKI exerts its protective effects in the cardiomyocyte or if other cardiac cell types are involved, and whether nitric oxide (NO) has cGKI-independent effects on cardiomyocytes mitochondria.2B receptor (A2BAR), or the mitochondria-targeted S-nitrosothiol (MitoSNO) was affected. MitoSNO accumulates within mitochondria, driven by the membrane potential, where it generates NO• and S-nitrosated thiol proteins [We developed mice with a cardiomyocyte-specific ablation of the cGKI gene (CMG-KO) and tested whether protection against reperfusion injury by ischemic postconditioning (IPost), soluble guanylyl cyclase (sGC) activation, the adenosine Aproteins .Conditional mice with floxed cGKI alleles were crossed to the MLC2a-Cre transgenic mice. Western Blot and immunohistochemistry confirmed that the Cre-mediated recombination produced the cGKI knock-out specifically in atrial and ventricular cardiomyocytes but not in other organs Figure .In situ hearts of underwent 30 min of regional ischemia followed by 2 h of reperfusion. As expected, in the control animals all interventions at early reperfusion lead to profound infarct size reduction: IPost (six cycles of 10 sec reperfusion and 10 sec of coronary occlusion), treatment with the specific sGC activator BAY 58-2667 (BAY58), the selective A2BAR agonist BAY 60-6583 (BAY60), as well as MitoSNO. In contrast, the hearts of CMG-KO animals were not protected by BAY58, whereas the protective effects of IPost, BAY60, and MitoSNO were unaffected by the lack of cGKI.2BAR, as well as direct NO effects via mitochondrial S-nitrosylation does not depend on cGKI in cardiomyocytes.While cardiomyocyte cGKI is important for the protection afforded via cGMP-signalling, beneficial effects of IPost, activation of the A


Heme oxygenase-1 (HO-1) is a highly inducible enzyme that metabolizes heme to generate equimolar amounts of carbon monoxide (CO), biliverdin, and ferrous iron. Subsequently, biliverdin is metabolized to the potent antioxidant bilirubin by biliverdin reductase. Compelling experimental evidence indicates that HO-1 and its end products protect against the development of vascular dysfunction in animal models atherosclerosis, post-angioplasty restenosis, vein graft stenosis, thrombosis, myocardial infarction, and hypertension.Studies from our laboratory and others have identified HO-1 as a critical regulator of vascular remodeling following arterial injury. Pharmacological induction or gene delivery of HO-1 suppresses neointima formation following arterial injury while HO-1 deletion exacerbates lesion development. Similarly, local or systemic administration of CO inhibits intimal thickening following arterial injury. Studies in cultured vascular smooth muscle cells (SMCs) reveal that overexpression of HO-1 or exogenously applied CO blocks SMC proliferation. The anti-proliferative action of CO is dependent on the activation of soluble guanylate cyclase and is associated with the arrest of SMCs in the G0/G1 phase of the cell cycle. Interestingly, the soluble guanylate cyclase stimulator YC-1 potentiates the anti-proliferative actions of CO in cultured SMCs and uniquely stimulates the production of CO by inducing HO-1 gene expression, providing a novel mechanism by which this agent is able to amplify cGMP production. YC-1 also suppresses neointima formation, and this is associated with an increase in cGMP levels in injured arteries. Finally, HO-1 deficiency impairs endothelium-dependent vasorelaxation and alters vasorelaxation responses to soluble guanylate cyclase stimulators and activators. The alteration in vasoreactivity in HO-1-deficient animals is accompanied by a pronounced decline in soluble guanylate cyclase expression.Collectively, these findings demonstrate that HO-1 elicits important protective actions in the vasculature by stimulating soluble guanylate cyclase activity and preserving soluble guanylate cyclase expression.


QuantiFERON-TB Gold In-Tube (QFT-GIT) is considered an alternative to the tuberculin skin test (TST) for the diagnosis of tuberculosis (TB) infection, but the programmatic impact of QFT-GIT implementation is largely unknown. In March, 2010, the Baltimore City Health Department (BCHD) introduced routine QFT-GIT testing for individuals referred to the TB program for suspected latent TB infection (LTBI).Retrospective study comparing LTBI diagnosis and treatment during the 13 months before and after QFT-GIT implementation at the BCHD TB clinic.607 and 750 individuals were referred by community-providers for suspected LTBI in the pre- and post-QFT-GIT periods, respectively. Most individuals in the pre- and post-QFT-GIT periods were referred on the basis of a positive TST  and were foreign-born . BCHD performed QFT-GIT testing for 375/543 (69%) eligible individuals in the post-QFT-GIT period, of which 185 (49%) were positive, 178 (47%) were negative, 1 (0.25%) was indeterminate, and 11 (3%) did not yield results. Concordance of QFT-GIT with TST was low (183/352[52%]). Foreign-born individuals had higher proportions of QFT-GIT positivity (57%) than US-born individuals . Significantly fewer individuals received a final diagnosis of LTBI in the post-QFT-GIT period (397/567 [70%]) compared to the pre-QFT-GIT period . In the post-QFT-GIT period, only 230/399 (58%) of those receiving QFT-GIT testing had a final diagnosis of LTBI, while 167/168 (99%) of those without QFT-GIT testing were diagnosed with LTBI (p<0.001). There was no difference in treatment initiation between those with and without QFT-GIT testing (175/230 [76%]) vs. 133/167 [80%], respectively) in the post-QFT-GIT period.QFT-GIT implementation for LTBI evaluation in a public health clinic significantly reduced the proportion of referred individuals in whom LTBI was diagnosed. QFT-GIT testing had no impact on treatment initiation or completion among those diagnosed with LTBI. M. tuberculosis infection, but this test has important limitations. The TST can cross-react with non-tuberculous mycobacterial (NTM) species or Bacille Calmette Guerin (BCG) vaccine, thereby complicating the interpretation of TST results especially in BCG-vaccinated foreign-born individuals from TB-endemic settings. Additionally, TST results are subject to inter-reader variability and may differ by level of training of those reading the test Testing and treatment of persons at increased risk for latent tuberculosis infection (LTBI) is a core element of the tuberculosis (TB) elimination strategy in the United States (US) M. tuberculosis (MTB) infection by measuring in vitro IFN-γ release following stimulation of lymphocytes with antigens specific to M. tuberculosis, ands has several potential advantages over TST. QFT-GIT retains specificity in BCG-vaccinated populations and has less cross-reactivity than TST with NTM species M. tuberculosis infection, and are the preferred option in some circumstances including testing of BCG-vaccinated populations Interferon-gamma (IFN-γ) release assays (IGRAs), such as the commercially available QuantiFERON-TB Gold In Tube  test, have the potential to overcome some of TST's limitations. QFT-GIT detects While many research studies have been conducted to assess IGRA test performance, there is limited information about the implementation of these tests in the context of public health TB control programs M. tuberculosis infection, a main BCHD TB clinic activity is the evaluation and clinical management of individuals with suspected M. tuberculosis infection referred from community providers. Within Baltimore City, a variety of clinical care providers perform M. tuberculosis infection testing, almost exclusively using TST, for a number of indications including employment testing, immigration/refugee services, homeless services, and as a requirement for attendance in some drug-treatment programs. Individuals identified with possible M. tuberculosis infection  by community sources are referred to the BCHD TB clinic for further evaluation and treatment. Prior to March 2010, the BCHD TB clinic made decisions on LTBI diagnosis and treatment based upon test results (i.e. TST in most cases) available from the referral source. In March 2010, the BCHD TB clinic decided to implement a new testing program in which QFT-GIT was made available as part of normative clinical care during evaluations for individuals referred with suspected M. tuberculosis infection. We sought to formally evaluate the programmatic impact of QFT-GIT implementation in the BCHD TB program. We assessed the uptake of QFT-GIT testing and availability of interpretable results, and compared programmatic rates of LTBI diagnosis and treatment before and after QFT-GIT implementation. Additional analyses attempted to determine factors associated with QFT-GIT test positivity and concordance with TST.The Baltimore City Health Department (BCHD) TB control program clinic (“BCHD TB clinic”) provides TB clinical care services free-of-charge to residents of Baltimore City, population 620,961, with active TB incidence of 5.2/100,000 in 2010This retrospective study was approved by ethics committees at the Johns Hopkins University School of Medicine  and the Baltimore City Health Department. This study received a waiver of informed consent; this research involved no more than minimal risk to subjects, data was collected solely by review of existing laboratory and medical records, and this research could not practicably be carried out without the waiver of informed consent.M. tuberculosis infection who had not had prior QFT-GIT testing from the referral source. Clinical care staff underwent manufacturer-supervised training in filling the tubes during phlebotomy, and in how to label and send tubes to the single off-site laboratory located approximately 3 miles away prior to QFT-GIT implementation into routine practice. The four prescribing clinicians (2 MDs and 2 Nurse Practitioners) were trained in interpretation of QFT-GIT test results and CDC recommendations The BCHD TB clinic initiated QFT-GIT testing on March 1, 2010 as part of their normative care algorithms. Following March 2010, staff routinely obtain blood for QFT-GIT testing on all individuals referred for suspected We conducted a retrospective cohort study to evaluate LTBI services before and after implementation of the QFT-GIT test in the BCHD TB program, and assessed concordance of QFT-GIT with TST during routine clinic conditions. Data was obtained using the BCHD TB Program electronic database (Microsoft Access 2003) and through chart reviews. We compared 13 months of data prior to QFT-GIT introduction (pre-QFT-GIT) with the 13 month period after QFT-GIT implementation (post-QFT-GIT). Patients were assigned to the pre-QFT-GIT period if they were referred or evaluated between Feb 1, 2009 and Feb 28, 2010; patients were assigned to the post-QFT-GIT period if they were referred or evaluated between March 1, 2010 and March 31, 2011.M. tuberculosis infection were included without age restriction. Individuals are referred to BCHD for evaluation on the basis of a positive TST, positive IGRA test, or an immigration B-Waiver . Individuals with active TB and their close contacts were excluded.Individuals referred to the BCHD TB clinic for suspected M. tuberculosis infection evaluation were interviewed by a BCHD TB clinic staff member for demographic information, medical history, and signs and symptoms of active TB; a chest x-ray and liver chemistries were obtained, and HIV testing was offered. TST and QFT-GIT were not repeated for individuals who had one or both tests performed by a community provider. Patients with signs or symptoms of active TB were evaluated further by sputum smear microscopy, culture, and other testing as indicated.Per routine care, all individuals referred for Prior to March 1, 2010, diagnosis of LTBI was based on available TST or IGRA information from the referral source at the time of referral to the BCHD TB clinic. Starting March 1, 2010, BCHD directed QFT-GIT testing was made available as part of the diagnostic evaluation. Individuals with discordant results on TST and QFT-GIT could be diagnosed with LTBI at the discretion of the BCHD TB clinician. Factors considered by clinicians included but were not limited to HIV status, chest x-ray results, age of individual, degree of TST induration, and QGIT antigen and nil results, country of origin, BCG status, and other TB risk factors. Individuals diagnosed with LTBI during both study periods were offered treatment in accordance with published guidelines, with medications dispensed on a monthly basis after monthly follow-up BCHD TB clinic visits including toxicity assessment QFT-GIT testing was performed according to manufacturer's instructions at a single off-site BCHD laboratory 2 tests. Factors associated with QFT-GIT results and TST concordance were assessed using univariate and multivariate logistic regression analysis. Analysis of treatment completion was restricted to those who started a 9 month INH regimen prior to Nov 30, 2010 or a 4 month Rifampin regimen prior to March 30, 2011, to allow time for treatment completion. Data were analyzed using STATA .The primary objectives of this study were to determine the proportions of LTBI diagnosis and subsequent treatment initiation among individuals referred to BCHD for TB evaluation,comparing the pre-QFT-GIT and post-QFT-GIT period. Additional analysis sought to determine proportions of QFT-GIT positivity among those referred to BCHD for LTBI care, to assess the percent agreement between TST and QFT-GIT, and to assess factors associated with QFT-GIT positivity and concordance with TST. Categorical data were compared using χM. tuberculosis infection. There were more referrals in the post-QFT-GIT period (750) compared to the pre-QFT-GIT period . Among referrals, there was no difference in age, sex, or HIV status comparing the two study periods . Characteristics of individuals adhering to an initial BCHD TB appointment are shown in Among the 567 referrals that came to BCHD for evaluation in the post-QFT-GIT period, 525 (93%) were referred with a TST, 19 (3%) for a positive QFT-GIT, and 5 (1%) individuals had both a QFT-GIT and TST performed by referral source; 18 (3%) B-waiver referrals were evaluated without prior LTBI testing. Among these 567 referrals, 543 did not have a prior IGRA test and were eligible for QFT-GIT testing at BCHD. QFT-GIT testing was performed for 375/543 (69%) eligible individuals . IndividAmong the 375 individuals who underwent QFT-GIT testing at BCHD TB clinic, 185 (49%) were positive by QFT-GIT, 178 (47%) were negative, and 1 (0.25%) was indeterminate, for 11 (3%) individuals the QFT-GIT test did not yield results due to specimen processing or transportation errors.Factors associated with QFT-GIT positivity among those receiving QFT-GIT at BCHD are shown in During the post-QFT-GIT period, 352 individuals referred for evaluation had both an interpretable QFT-GIT and an available TST result available in the post-QFT-GIT period . Overall, there was only modest agreement between TST and QFT-GIT  and post-QFT-GIT periods . Moreover, there was no difference in treatment initiation in the post-QFT-GIT period between those diagnosed with LTBI that had QFT-GIT testing performed (174/230 [76%]) and those that did not have QFT-GIT testing performed . Among those given a diagnosis of LTBI and adequate time for treatment, the proportions completing treatment were similar between the pre-QFT-GIT period (251/445 [56%]) and the post-QFT-GIT period . Treatment completion proportions, among those that initiated treatment, were also similar between the pre-QFT-GIT period (251/341[74%]) and post-QFT-GIT period . In the post-QFT-GIT period, there was no difference in treatment completion rates between those with and without QFT-GIT performed (p = 0.101). Overall, discontinuation of therapy for toxicity was similar in the pre-QFT-GIT period (7/341 (2%) and post-QFT-GIT period (8/244 (3%); p = 0.355).M. tuberculosis infection that came for evaluation at BCHD were tested with QFT-GIT by BCHD after testing became available. Children were less likely to be tested with QFT-GIT than were older individuals. Several potential reasons include difficulty drawing blood in young children, and absence of another clinical indication for phlebotomy , and clinician discretion; in young children, clinicians at BCHD may have been more likely to accept a positive TST result in order to maximize sensitivity in this population in whom IGRA results are more difficult to interpret. In addition, individuals with HIV-infection were less likely to be tested with QFT-GIT by BCHD TB clinic, which may have represented clinician discretion in this high risk population in whom QFT-GIT has reduced sensitivity. Logistical challenges associated with QFT-GIT processing were likely the primary obstacle precluding ordering of the test for all referred individuals. QFT-GIT processing requires an initial incubation of blood at 37°C for 16–24 hours shortly after phlebotomy, after which the specimens can be stored before further processing. During the post-QFT-GIT period, this initial incubation step was performed off-site from the BCHD TB clinic. As a result, the clinic could not offer the test to individuals arriving in the late afternoon because of a need for specimen transport via courier before laboratory closure. A potential solution is to obtain an incubator for use in the TB clinic so that incubation can be initiated in the clinic immediately after phlebotomy rather than being delayed until receipt of specimens in the off-site laboratory.We ascertained the clinical impact of QFT-GIT implementation in a city health department TB control program clinic, as well as barriers to its implementation. With respect to QFT-GIT uptake, we found that two-thirds of individuals referred for suspected Despite these challenges, there was a low proportion of test failure, with 97% of drawn QFT-GIT tests yielding interpretable results and a very low proportion of indeterminate tests (<1%). On balance, our experience suggests that implementation of QFT-GIT in local health departments is likely to be feasible, but requires attention to specimen transport schedules and/or in-clinic initiation of sample incubation, and to training of clinic staff in drawing blood from children if QFT-GIT testing of children is considered part of routine care.Importantly, implementation of QFT-GIT testing in the BCHD TB clinic had a significant programmatic impact on LTBI diagnosis of referred individuals. In Baltimore City, individuals are referred to the BCHD TB clinic with suspected LTBI on the basis of community-based testing, usually for a positive TST. Thirty percent fewer LTBI evaluations in the BCHD TB clinic resulted in a final diagnosis of LTBI in the period after QFT-GIT implementation compared to a similar time period prior to test implementation. This reduction was driven by the additional QFT-GIT testing conducted by BCHD on LTBI referrals. In both study periods, nearly all referred individuals not receiving BCHD directed QFT-GIT testing had a final diagnosis of LTBI, with reliance on TST data from the referral source. In contrast, only 58% of individuals referred for LTBI evaluation were given a final diagnosis of LTBI if QFT-GIT was performed by BCHD as part of the diagnostic evaluation. Interestingly, while the proportion of individuals with LTBI diagnosis was reduced in the post-QFT-GIT period, there was no difference in the proportion of individuals that initiated or completed LTBI treatment between the study periods. This finding suggests that choice of diagnostic test did not influence patient behavior with regards to starting or completing treatment in our setting.M. tuberculosis infection and agreed with TST results in only 52% of cases. These results suggest that TST positivity from community-based testing may have suboptimal positive predictive value for M. tuberculosis infection. This finding is similar to that reported in other low-prevalence settings when QFT-GIT was performed on TST positive referrals Our study evaluated factors associated with QFT-GIT positivity and offers important insights into QFT-GIT test performance and concordance with TST under operational conditions. Overall, the QFT-GIT test was positive in only half of the individuals referred to BCHD as having possible Our study has several important limitations. Our study population consisted of individuals who were referred to the health department on the basis of prior suspicion or diagnosis of latent TB. QFT-GIT positivity and concordance with TST may differ when performed in an unselected group from the general population. Nonetheless, the BCHD TB program structure is not dissimilar from many public health TB programs in the US, and our results have relevance for urban local health departments and other facilities providing latent TB services. Without a reference standard for latent TB diagnosis, interpretation of TST and QFT-GIT discordance can be challenging Our study also has several important strengths. In contrast to studies of QFT-GIT diagnostic accuracy, we evaluated the programmatic impact of implementing this new TB diagnostic modality in a large urban public health department under realistic operational conditions. We also report on factors associated with QFT-GIT positivity which may help guide other local health departments considering test implementation. With continued reductions in public health resources, our study has important implications for local TB prevention programs. Overall, we found that QFT-GIT implementation led to significant reductions in LTBI diagnosis and treatment at BCHD. Future studies are now needed to determine the cost-effectiveness of QFT-GIT implementation, and to examine TB reactivation rates in QFT-GIT and TST discordant patients.Table S1Differences between those who did and did not come for an initial BCHD appointment among those referred for LTBI evaluation. Abbreviations: BCHD, Baltimore City Health Department. SD, Standard Deviation *p = 0.358 comparing percentages of men and women adhering to an initial LTBI evaluation between the pre-QFT-GIT and post-QFT-GIT periods **p = 0.621 comparing mean age of individuals adhering to an initial LTBI evaluation between the pre-QFT-GIT and post-QFT-GIT periods †p = 0.917 comparing percentage of foreign-born individuals adhering to an initial LTBI evaluation between the pre-QFT-GIT and post-QFT-GIT periods.(DOC)Click here for additional data file.Table S2Factors associated with QFT-GIT testing by BCHD in the post-QFT-GIT period. †Comparison of equality of proportions receiving QFT-GIT testing among those with known ethnicities †† Comparison of equality of proportions from each referral source receiving QFT-GIT testing.(DOC)Click here for additional data file.Table S3Factors associated with discordant results among those who came to BCHD for evaluation. *excluded due to colinearity with birth country.(DOC)Click here for additional data file.