HNF4A were identified as the cause of Maturity Onset Diabetes of the Young 1 (MODY1) two decades ago, the mechanisms by which this nuclear receptor regulates glucose homeostasis remain unclear. Here we report that loss of Drosophila HNF4 recapitulates hallmark symptoms of MODY1, including adult-onset hyperglycemia, glucose intolerance and impaired glucose-stimulated insulin secretion (GSIS). These defects are linked to a role for dHNF4 in promoting mitochondrial function as well as the expression of Hex-C, a homolog of the MODY2 gene Glucokinase. dHNF4 is required in the fat body and insulin-producing cells to maintain glucose homeostasis by supporting a developmental switch toward oxidative phosphorylation and GSIS at the transition to adulthood. These findings establish an animal model for MODY1 and define a developmental reprogramming of metabolism to support the energetic needs of the mature animal.Although mutations in DOI:http://dx.doi.org/10.7554/eLife.11183.001 HNF4 is mutated or deleted. The symptoms of MODY1 usually appear during early adulthood and include abnormally high levels of sugar in the blood, as well as the pancreas not being able to release the hormone insulin properly in response to these sugars.Diabetes is a complex disease that is caused by a combination of factors, including the person’s habits and environment, as well as their genetic make-up. However, there are some rare forms of diabetes that are caused simply by mutations in single genes and are directly inherited. For example, it has been known for twenty years that a type of diabetes called “Maturity Onset Diabetes of the Young type 1” (or MODY1 for short) occurs when a gene called Previous studies in mice have tried to understand how losing the HNF4 gene leads to MODY1. However, these mouse models did not fully recreate the symptoms of this disorder and the precise role of HNF4 in preventing diabetes remains unclear. Barry and Thummel have now used the fruit fly, because it is a model organism with simple genetics, to help shed light on this question. Furthermore, flies and mammals use many of the same pathways to control metabolism, making the fly a good model for the disease in humans.HNF4 gene in fruit flies and observed that the flies had all the symptoms that are typical in people with MODY1. These symptoms included high sugar levels and decreased production of insulin-like hormones. The experiments also showed that HNF4 normally supports the proper expression of another gene called Hex-C; this gene encodes a protein that senses how much sugar is available and helps to keep the amount of sugar circulating the body within normal levels. Barry and Thummel went on to discover that the HNF4 gene is required for the expression of some genes in structures called mitochondria, which provide most of the energy used by animal cells. Lastly, the HNF4 gene became more active as the flies matured, and appeared to help the metabolism of a developing fruit fly transition towards that of an adult.Barry and Thummel deleted the HNF4 protects against MODY1 by influencing several components of sugar metabolism in fruit flies. In the future, more studies are needed to understand how exactly HNF4 acts in mitochondria and to explore if similar results are seen in mammals.Together these findings show that DOI:http://dx.doi.org/10.7554/eLife.11183.002 Glucokinase (GCK/MODY2), and Insulin (INS/MODY10). Mechanistic insight into the anti-diabetic roles of other MODY genes, however, remains limited.The global rise in the prevalence of diabetes has prompted increased efforts to advance our understanding of metabolic systems and how they become disrupted in the diseased state. Although genetics and environment have a significant impact on diabetes susceptibility, severity, and care, the causal factors are often complex and unclear. Several cases of familial diabetes have been identified, however, that show clear patterns of heritability due to monogenic disease alleles, highlighting these genes as critical factors for glycemic control. To date, mutations in 13 genes have been shown to cause autosomal dominant inheritance of Maturity Onset Diabetes of the Young (MODY1-13), representing the most common forms of monogenic diabetes. MODY patients typically present with hyperglycemia and impaired glucose-stimulated insulin secretion (GSIS) by young adulthood, while having normal body weight and lacking β-cell autoimmunity . ConsistHepatocyte Nuclear Factor 4A (HNF4A) as responsible for MODY1  from neuroendocrine cells in the fly brain  with the goal of defining its roles in maintaining carbohydrate homeostasis. dHNF4 is a close ortholog of human HNF4A, with 89% amino acid identity in the DNA-binding domain and 61% identity in the ligand-binding domain. The spatial expression patterns of the fly and mammalian receptors are also conserved through evolution, raising the possibility that they share regulatory activities . A homolog of mammalian GCK, Hex-C, is also under-expressed in mutants. dHNF4 appears to act through these pathways to promote GSIS in the IPCs and glucose clearance by the fat body. In addition, we show that dHNF4 expression increases dramatically at the onset of adulthood, along with its downstream transcriptional programs. These studies suggest that dHNF4 triggers a developmental transition that establishes the metabolic state of the adult fly, promoting GSIS and OXPHOS to support the energetic needs of the mature animal.Here we describe our functional studies of tivities . In supp mammals . Here wedHNF4 null alleles (Δ17/dHNF4Δ33dHNF4) and genetically-matched controls that were transheterozygous for precise excisions of the EP2449 and KG08976 P-elements, as described previously , less than 30% of mutant animals survive though eclosion, and the rest die primarily during the first day of adulthood , and serine, which is produced from 3-phosphoglycerate, although the increased DHAP was only observed on the 15% sugar diet  metabolomic analysis was used to further characterize the metabolic state of gar diet . This stgar diet . Several pathway . This pa pathway . dHNF4 mhropathy  , with just over half of these genes showing reduced abundance   that contribute to oxidative phosphorylation (OXPHOS). Further analysis of these transcripts by northern blot hybridization confirmed their reduced expression in dHNF4 mutants, corresponding to mtDNA genes involved in Complex I , Complex IV  and Complex V/ATP synthase , along with reduced levels of the mitochondrial large ribosomal RNA (mt:lrRNA)  . Gene on 644 up) . In cont mutants . Interes mutants . In Drost:lrRNA) . Importa mutants . In addi animals .10.7554/dHNF4 for their maximal expression, including genes that encode the alpha and beta subunits of the electron transfer flavoprotein (ETFA and ETFB), ETF-ubiquinone oxidoreductase (ETF-QO), and the Complex II  assembly factor dSdhaf4 , CG5599 (which encodes a protein with homology to the E2 subunit of the α-ketoglutarate dehydrogenase complex (α-KGDHC) as well as the E2 subunit of the branched-chain alpha-ketoacid dehydrogenase complex), CG1544 (which encodes a homolog of α-KGDHC E1), as well as Isocitrate dehydrogenase   . These chenotype .dHNF4 mutants also have decreased expression of the GCK homolog Hexokinase-C (Hex-C) , which is involved in glycogen metabolism, and transaldolase and CG17333, which are involved in the pentose phosphate shunt. Additionally, the gluconeogenesis genes Pyruvate carboxylase (CG1514) and Phosphoenolpyruvate carboxykinase  show reduced expression in mutant animals, similar to their dependence on Hnf4A for expression in the mammalian liver  . GCK is an liver   was performed to identify direct transcriptional targets of the receptor. Through this analysis, forty-seven genes were identified as high confidence targets by fitting the criteria of showing proximal dHNF4 binding along with reduced transcript abundance in mutant animals  . These id CG5599 . We alsod CG5599 . Taken tnt cells . In contstaining . This mi and SOD . Taken tdHNF4 expression in the IPCs, fat body, and intestine to examine the contributions of dHNF4 in these tissues to systemic glucose homeostasis. This revealed a requirement in both the IPCs and fat body for glucose homeostasis, consistent with the well-established roles of these tissues in insulin signaling and the regulation of circulating sugar levels    in eithe 30 kDa) . Fat bodfunction  (Cho et function . In contrphology .10.7554/ATPsynβ RNAi) in the fat body caused lethality prior to eclosion, IPC-specific RNAi produced viable adults that appeared normal but displayed significant hyperglycemia (Cox5a RNAi) failed to produce hyperglycemia, while RNAi in the fat body caused lethality prior to adulthood, similar to ATPsynβ. This premature lethality was accompanied by severe developmental delay and more than 50% of the animals dying prior to puparium formation when raised on the 15% sugar diet. Interestingly, we discovered that these animals are sugar intolerant, similar to dHNF4 mutants, such that rearing them on the 3% sugar diet allowed for 100% survival to puparium formation while also alleviating the developmental delay  for GSIS, fat body (liver) for lipid metabolism and glucose clearance, and intestine to suppress inflammation, there are likely to be divergent roles as well. One example of this is the embryonic lethality of Hnf4A mutant mice, which is clearly distinct from the early adult lethality reported here for dHNF4 mutants. Further studies are required to dissect the degree to which the regulatory functions of this receptor have been conserved through evolution.Interestingly, gene ontology analysis indicates that the up-regulated genes in osophila . This re disease . Disrupt mutants . Moreovevolution . Taken tHnf4A plays a role in hepatocyte differentiation and proliferation in addition to its roles in metabolism   . Fourth,studies) . Some ofl OXPHOS , and stul OXPHOS .dHNF4, dHNF4-B, encodes a predicted mitochondrial localization signal in its 5’-specific exon, providing a molecular mechanism to explain the targeting of this nuclear receptor to this organelle. Efforts are currently underway to conduct a detailed functional analysis of dHNF4-B by using the CRISPR/Cas9 system to delete its unique 5’ exon, as well as establishing transgenic lines that express a tagged version of dHNF4-B under UAS control. Future studies using these reagents, along with our dHNF4 mutants, should allow us to dissect the nuclear and mitochondrial functions of this nuclear receptor and their respective contributions to systemic physiology.Although unexpected, our proposal that dHNF4 may directly regulate mitochondrial gene expression is not unprecedented. A number of nuclear transcription factors have been localized to mitochondria, including ATFS-1, MEF2D, CREB, p53, STAT3, along with several nuclear receptors, including the estrogen receptor, glucocorticoid receptor, and the p43 isoform of the thyroid hormone receptor . The sigHnf4A by immunohistochemistry in tissue sections, including in postnatal pancreatic islets  acts in mid-embryogenesis to directly induce genes that function in biosynthetic pathways related to the Warburg effect, by which cancer cells use glucose to support rapid proliferation  and genetically-matched controls that were transheterozygous for precise excisions of the EP2449 and KG08976 P-elements, as described previously . Fasting was achieved by using 1% agar as a medium. For adult studies, 8–10 day old males were selected for all studies unless otherwise indicated.All genetic studies used a transheterozygous combination of eviously . Sugar dr4-GAL4 , RNAiUAS-Cox5a (TRiP 27548)RNAi, UAS-CIA30 (TRiP 55660), RNAiUAS-ATPsynβ (TRiP 28056), RNAiUAS-Scs-α (TRiP 51807), RNAiUAS-CG5599 (TRiP 32876), and RNAiUAS-HexC  . Animals collected for developmental RNA and glycemia measurements  for 4 day pupae, and males at mid-eclosion from the pupal case  and/or Glucose Oxidase (GO) assay kits  as previously described, with approximately six biological replicates  assayed per condition . Total pMature adult males were placed on 10% sucrose + 1% agar overnight prior to analysis. Six biological replicates per genotype  were analyzed for ATP levels by using a luminescence assay kit  as described .Animals were raised on the 3% sugar diet until five days of adulthood and transferred to a fresh vial with either the 3% or 15% sugar diet for three days prior to collection. Twenty adult males per sample were snap-frozen in liquid nitrogen and prepared for analysis by gas chromatography-mass spectrometry (GC/MS) as described . Data isTissues were dissected in cold 1xPBS, fixed in 4% paraformaldehyde for 20-30 min at room temperature, and washed once quickly followed by three washes for 20 min each in PBS, 0.2% Triton-X100. Samples were blocked using 5% normal goat serum for 1–2 hr at room temperature and incubated with primary antibodies for a minimum of 24 hr at 4˚C, washed, and incubated with secondary antibody at 4˚C for 24 hr. Images were acquired using an Olympus FV1000 confocal microscope and analyzed by Volocity software to generate Z-stack projections and overlay images. The following antibodies were used for immunofluorescence studies: rat anti-dHNF4 , rat antDissected brains stained for DILP2 peptide were mounted in SlowFade® Gold (Life Technologies) and imaged using an Olympus FV1000 confocal microscope and 60X water-immersion objective. Scan settings were fixed to be identical for all captured images, with Z-stack limits set to encompass the entire depth of DILP2 fluorescence for each brain. Maximum projection Z-stacks were analyzed using Volocity software to calculate mean pixel intensity of DILP2 fluorescence within the IPCs (selected by ROI tool) for each set of IPCs analyzed.20, 1 ml 10% SDS, 100 μl sheared herring sperm ssDNA) for two hours at 42˚C prior to the addition of radiolabeled probe. Probes were generated by Klenow-mediated random primer amplification of purified template DNA corresponding to approximately 100–500 bp of the transcript of interest, allowing incorporation of 32P radiolabeled-nucleotide . Probes were incubated with membranes overnight, and the hybridized membranes were washed and exposed to X-ray film at -80˚C using two intensifying screens.Total RNA was extracted from groups of 10–20 animals using TriPure isolation reagent (Roche). RNA was fractionated by 1% formaldehyde gel electrophoresis at a constant voltage (70V) for ~3.5 hr prior to transfer to a nylon membrane overnight. After transfer, RNA was UV cross-linked to the membrane and placed in hybridization buffer  were collected using TriPure isolation reagent (Roche). Pairs of biological replicates were pooled to obtain four biological replicates for further purification using an RNeasy kit (QIAGEN). RNA quality was subsequently analyzed using an Agilent Bioanalyzer RNA 6000. PolyA-selected RNAs from each biological replicate were then assembled into barcoded libraries and pooled into a single-flow cell lane for Illumina HighSeq2000 50-cycle single-read sequencing, which produced ≥21.9 million reads per sample. Standard replicate RNA-seq analysis was performed using USeq and DESeq analysis packages with alignment to the Drosophila melanogaster dm3 genome assembly. Transcripts meeting a cutoff of ≥1.5 fold difference in mRNA abundance and FDR ≤1% were considered as differentially expressed genes. RNA quality control, library preparation, sequencing, and data analysis were performed at the University of Utah High Throughput Genomics and Bioinformatics Core Facilities. Although mt:Cyt-b and mt:ND6 are included among the down-regulated genes in our RNA-Seq dataset  for 1.5 min using a Brinkmann Homogenizer Polytron PT 10/35. The homogenate was filtered through a layer of 100 μm-pore nylon mesh into a pre-chilled glass-Teflon homogenizer, stroked on ice 35 times using a B pestle, and filtered through two layers of 40-μm-pore nylon mesh prior to adding one volume of warm cross-linking buffer  to bring the sample to room temperature for crosslinking . 2.5 M glycine was added to a final concentration of 125 mM to stop the crosslinking after 3 min. The mixture was pelleted for resuspension into 10 ml of RIPA buffer  for sonication. The sonicated material was centrifuged at 20,000 g, and the supernatant was distributed into 500 μl aliquots that were snap frozen in liquid nitrogen. To each aliquot, 1 ml of cold RIPA buffer (without SDS) was added prior to removing 150 μl for an input control, and then 5 µl of polyclonal affinity-purified anti-dHNF4 antibody was added to each sample for immunoprecipitation overnight at 4˚C , transferred to PVDF membrane overnight at 4˚C, and blocked with 5% BSA prior to immunoblotting. Antibodies used for western blots include rabbit anti-pAKT , rabbit anti-AKT , mouse anti-SDHA (Abcam 137756), rabbit anti-SDHB , mouse anti-ATP5A (Abcam 14748), and mouse anti-βTubulin (Chemicon MAB380).UAS-RNAi line or 1118w controls  peptide were used for ELISA assays, essentially as described . The indcontrols  were cro libitum  or faste libitum . The pos40A;tub-GAL4)yw,hsFLP,UAS-GFP; tub-GAL80,FRT were crossed to ∆33/CyO twi>GFPw;HNF4 males for MARCM analysis. Flies were reared on the 3% sugar diet and heat treated at 37°C for one hour as pharate adults, and again as newly-eclosed adults. Adults were maintained on the 15% sugar diet for 8–10 days prior to analysis.Virgin female flies (Adult flies were dissected in room temperature Schneider’s medium (ThermoFisher 21720024) with microdissection scissors to separate the abdomen from the thorax. The intestine was removed and the abdominal cavity was cut open to allow maximal contact of fat body with the TMRE solution (ThermoFisher T669). Dissected abdomens were placed in freshly-prepared solution of 20 nM TMRE in Schneider’s medium and incubated in the dark for 5–7 min. Samples were then washed briefly twice in Schneider’s medium, mounted on glass slides in fresh Schneider’s medium, and immediately imaged on an Olympus FV1000 confocal microscope with a 60x water objective.DHE staining was performed as previously described . BrieflyP values were calculated using a two-tailed Student’s t-test, multiple comparison P values were calculated using one-way ANOVA with Šídák multiple test correction (except th quartiles (box), and the median (midline).GraphPad PRISM 6 software was used to plot data and perform statistical analysis. Pairwise comparison  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.Drosophila HNF4 promotes glucose-stimulated insulin secretion and increased mitochondrial function in adults" for consideration by eLife. Your article has been favorably evaluated by K VijayRaghavan (Senior editor) and three reviewers, one of whom is a member of our Board of Reviewing Editors.Thank you for submitting your work entitled "The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission.SummaryDrosophila glucose homeostasis. In humans, mutations in HNF4A cause an early onset form of diabetes. The authors make the insightful observation that mutations in HNF4 result in early adult death just after eclosion. This phenotype is rescued if the adults are fed on 3% sugar diet instead of the regular diet, which contains about 15% sugars in it. Thus, reduced carbohydrate intake increases viability of HNF4 mutants suggesting that it might function in the regulation of sugar homeostasis. Further analysis suggests indeed that HNF4 mutants have increased levels of free glucose and trehalose in adults and have impaired glucose tolerance and reduced levels of ATP . The authors then use MS to determine the levels of different intermediates in glycolysis and mitochondrial metabolism and find that many of the intermediates of glycolysis and the associated pathways that function in the biosynthesis of other metabolites show a significant increase when HNF4 mutants are fed on 15% sugar diet but not when they are reared on 3% sugar diet. Consistent with these observations the authors show that loss of HNF4 causes mitochondrial fragmentation. Based on these observations that authors conclude that HNF4 functions in the metabolic reprogramming from the larval to the adult mode during development consistent with the lethality of HNF4 mutants.In this manuscript the authors study the role of HNF in Interestingly, mutant flies survive longer on a low sugar diet. Using global analyses such as metabolomics, RNA-seq and ChIP-seq the authors find that glycolysis and mitochondrial function are altered in mutant flies. Surprisingly, the RNA-seq and ChIP-seq data point to the possibility that HNF acts in the mitochondria to increase mtDNA transcription. The authors then use tissue specific knock-downs to show that glucose homeostasis is most perturbed when HNF is knocked down in the IPCs and fat body. Indeed, knock-down of HNF in the IPCs reduces DILP2 in circulation. The authors then explore the possibility that HNF triggers a developmentally regulated metabolic switch towards GSIS and OXPHOS at the onset of adulthood.The authors use a range of technologies to establish links between development, metabolic regulation and glucose homeostasis. A number of novel findings are reported. The most notable  is the finding that HNF might regulate mtDNA transcription. If so, this would alter our understanding of how mitochondrial transcription is regulated, and offer insights into how mtDNA transcription might be altered in diseases, such as diabetes. Unfortunately, the experiments that this novel claim is based on are not conclusive.The following is a list of major concerns that the reviewers have expressed and would like addressed.Essential revisions:1) A major, most exciting, finding of the paper is that dHNF4 might directly regulate mtDNA transcription. If so, this will change much about how we think mtDNA transcription is regulated. While the authors provide some evidence for this (for example ChIP-seq data), further experiments should be conducted to solidify this finding. In particular, it would be nice if the authors provide further evidence that dHNF4 is indeed targeted to mitochondria and present within the mitochondrial matrix where mtDNA resides. This could be achieved by subcellular fractionation of tissues or cells to show that dHNF4 first associates with the mitochondrial fraction and then further sub-fractionation to determine where within mitochondria it is. Along the same lines, are there any sequences in dHNF4 specifically required to target it to mitochondria? Does it possess a canonical N-terminal mitochondrial targeting sequence? If so, what are the phenotypic consequences of preventing mitochondrial targeting?The key evidence that dHNF binds to mtDNA is the ChIP-seq data. It is unclear how to interpret this mtDNA Chiq-seq data for a couple of reasons:A) The presumably high copy number of mtDNA in the sample (perhaps 1000X in excess of nuclear).B) The low signal to noise ratio in the nuclear binding sites. Presumably, most ChIP-seq experiments will detect mtDNA as a background signal. This will usually be discarded, because of the excess signal at specific nuclear DNA binding sites. In this study, in which the signal to noise ratio in nuclear genes is quite low, any background signal caused by the mtDNA might seem more important. A nice control would have been to perform the ChIP-seq in the mutant flies. The mtDNA signal should be much reduced in the mutants (assuming the mtDNA copy number is the same in mutant flies).2) The second piece of evidence supportive of HNF4 being present in the mitochondria is an IF experiment, which uses the same antibody as the ChIP-seq study. Given the novelty of the finding, it would have been useful to see independent validation of mitochondrial localization using an alternative antibody or a genetic construct. In summary, whilst it is plausible that HNF is directly binding to mtDNA and regulating mtDNA transcription, further evidence, using a method that doesn't require the antibody used in this study, would make the claim more compelling. Given the interest in such a finding, further independent evidence of direct mtDNA binding would be necessary.3) As a related issue, the argument that loss of dHNF4 specifically impairs mitochondrial metabolism needs further clarification. Loss of dHNF4 could also or instead, cause a reduction in mitochondrial number and mass. To what degree can the consequences of dHNF4 depletion be attributed to a reduction of mitochondrial number or mass as opposed to specific TCA cycle and OXPHOS impairment? Does loss of dHNF4 reduce mitochondrial number, mass or mtDNA copy number?HNF4 tissue and does scavenging of ROS suppress the phenotype?Similarly, the mitochondrial organization is significantly impaired upon loss of HNF4 functions. Do the authors also see an increase in the expression of enzymes such as Drp1 in the RNA-seq? And does the knockdown of DRP1 function result in the rescue of the phenotype? Do ROS levels increase in mutant Related to this issue, the authors should determine if the integrity of the cells that normally express it is compromised in any way such as an incorrect specification/physiology in the absence of HNF4, and please provide a more complete justification for why they think that the cells show a "mitochondrial fragmentation" phenotype.For the RNA-seq data, a systematic enrichment analysis (GO term/KEGG pathway) needs to be carried out on all differentially expressed genes in order to support claims regarding specificity of pathways regulated by dHNF4. In the current version of the manuscript, the authors seem to have cherry-picked genes  One potential caveat with the RNA-seq and ChIP-seq experiments is that they have been carried out in whole flies. dHNF4 is expressed in multiple tissues, and both its functions and targets may be tissue-specific. This is something that will not be captured by whole-fly approaches, and may in fact prevent identification of some targets. For example, the contribution of the IPCs to total fly chromatin or RNA is likely to be negligible.While it is understandable that whole adult flies are used for technical reasons, presumably, each of the tissues in question will respond differently to the loss of HNF function, with different metabolic consequences, so it is unclear how to relate these global analyses of a heterogeneous tissue extract to pathology in a particular tissue (e.g. the IPCs). For example, the metabolomics data shows that HNF deletion increases glucose-derived metabolites, but the RNA-seq data suggests that glycolytic genes are down-regulated. These data are not incorrect, since the experiments and analysis were done with care and competence, but such analyses are easier to interpret if using a homogeneous extract. The ambiguities introduced by a mixture of tissues with mixed metabolic profiles make it difficult to appreciate the importance of the genomic analyses.The Reviewing Editor does not feel that this issue can be fully solved in a reasonable period of time. So perhaps some initial experiments that address aspects of this issue, and attending to this issue in the Discussion, as a caveat will suffice.5) All the mutants characterized in this study involve constitutive dHNF4 loss or downregulation throughout development – either from all tissues or a subset thereof. Consequently, the adult phenotypes described in this manuscript could be adaptive and result from the metabolic alterations caused by dHNF4 loss during larval life . The abundance of genes involved in stress and immunity in the RNA-seq dataset is consistent with this idea. The authors should knockdown dHNF4 specifically in the adult  and test whether some of the key phenotypes  are still apparent. Related to this, the specificity of the Ilp2-Gal4 used in this study  has not been investigated/described in larvae. Why did the authors not use the Ilp2-3-Gal4 generated in the Partridge lab, which is confined to late larva/adult IPCS? This would avoid targeting non-IPC dHNF4 expression throughout development.Also, a related important but minor issue is that often a single RNAi construct is used, the authors are well aware of the possible pitfalls, and they need to be avoided. Additionally, all UAS and Gal4 lines need to be fully defined.6) The authors argue that dHNF4 is required in the fat body and insulin-producing cells of adults to maintain glucose homeostasis by supporting a developmental switch toward OXPHOS and GSIS at the transition to adulthood. Since all experiments in this paper were performed by removing dHNF4 early in development far before the onset of adulthood, it remains a possibility that the phenotypes observed are a consequence of an earlier requirement for dHNF4 during development. To show that dHNF4 is really required in adulthood we would like the authors to remove dHNF4 at the onset of adulthood in the whole organism, fat body and IPCs to assess whether the flies become diabetic and whether survival is affected. As the muscle is also a major contributor to glucose clearance the requirement of dHNF4 in muscles should be assessed as well.Cox5a knock-down does not increase glucose, but CIA30 knock-down does. It is more likely that each complex has differential control of flux in specific tissues or flux through the ETC has differential sensitivity to knock-down of specific complexes, rather than suggesting that there is a greater requirement for CI or CV than CIV per se.7) Based on RNAi to knock-down of specific OXPHOS complexes in the IPC and fat body the authors conclude that there are differential requirements for different ETC complexes in glucose homeostasis. This requires functional validation e.g. on the rate of oxygen consumption. The authors demonstrate that 8) The authors' previous study was consistent with dHNF4 as a nutrient sensor, whereas this manuscript suggests it functions as a developmental switch that converts a cell to a fate that supports responsiveness to glucose. Are dHNF4 expression, activity and/or subcellular localization nutritionally regulated in adult flies? The very low number of ChIP-seq could be explained low dHNF4 activity if a 3% sugar diet was used. RNA-seq experiments used 15% sugar. The diets used for every experiment also need to be clearly stated, and the data analyzed accordingly. Essential revisions:1) A major, most exciting, finding of the paper is that dHNF4 might directly regulate mtDNA transcription. If so, this will change much about how we think mtDNA transcription is regulated. While the authors provide some evidence for this (for example ChIP-seq data), further experiments should be conducted to solidify this finding. In particular, it would be nice if the authors provide further evidence that dHNF4 is indeed targeted to mitochondria and present within the mitochondrial matrix where mtDNA resides. This could be achieved by subcellular fractionation of tissues or cells to show that dHNF4 first associates with the mitochondrial fraction and then further sub-fractionation to determine where within mitochondria it is. Along the same lines, are there any sequences in dHNF4 specifically required to target it to mitochondria? Does it possess a canonical N-terminal mitochondrial targeting sequence? If so, what are the phenotypic consequences of preventing mitochondrial targeting?dHNF4 mutants.  predicted by both MITOPROT and TargetP protein localization programs. Importantly, one of these is dHNF4-B, one of the five protein isoforms encoded by the dHNF4 locus  but not in isolated nuclei. We have conducted these studies with a variety of different antibodies including affinity-purified guinea pig anti-dHNF4 (unpublished from our lab), affinity-purified rat anti-dHNF4 , chicken anti-GFP (Abcam 13970), and mouse anti-FLAG monoclonal (Sigma F3165). We also attempted pre-absorbing the antibodies using purified mitochondria, but this was not sufficient to fully eliminate interference from non-specific bands. Our need for additional approaches to detect dHNF4 protein has been a major motivating factor for developing the new reagents described above that tag all dHNF4 protein isoforms as well as the dHNF4-B isoform. We now include in our paper a discussion of the importance of conducting mitochondrial fractionation studies in the future .We fully agree with the reviewers that mitochondrial fractionation is important to both confirm our model and determine the location of dHNF4 within mitochondria. Indeed, we were doing these experiments while the paper was out for review and have continued our efforts over the past few months. So far, we have conducted six attempts using control animals compared to dHNF4 mutant phenotypes. We now include this information in a new section of our Discussion that is focused on mitochondria, along with our conclusion that, although our data strongly support the model that dHNF4 directly regulates mitochondrial gene expression, they do not prove it. We now present this as a working model. We have revised all parts of the text that previously referred to “direct regulation of mitochondrial gene expression” to be clear that we cannot make this conclusion based on the evidence presented. We also now state that substantial future studies will be needed to provide conclusive evidence in support of our model. We invite the reviewers to read this modified section of the Discussion .The focus of the reviewers on requesting additional evidence for the mitochondrial functions of dHNF4 is our fault, due to a vague discussion of this topic throughout the paper. At no point in our paper did we enumerate the evidence for and against a direct role for dHNF4 in mitochondria, as well as the key experiments that are needed to conclusively prove this model. These include a full functional analysis of the dHNF4-B isoform and its contributions to dHNF4 in adults. We agree that many experiments can and should be done – particularly regarding its roles in mitochondria – and it is our intention to conduct these experiments over the coming years. We have rewritten major portions of the manuscript to be sure that these points are made clear to the reader. We have also added 7 new figures to the revised manuscript, significantly expanding the scope of our study.Finally, we want to point out that, although the reviewers are focused on the mitochondrial functions for dHNF4, this is only one of three major findings in our paper. Importantly, this study provides the first accurate animal model for MODY1 and thus the first insights into the molecular mechanisms that underlie this disorder. In addition, we show that the induction of dHNF4 in early adults provides a regulatory mechanism to explain the classic “U-shaped curve” of mitochondrial oxygen consumption in holometabolous insects – consisting of high respiration rates in larvae, low levels during metamorphosis, and a dramatic increase at the onset of adulthood. Our data supports the model that dHNF4 contributes to establishing the metabolic state of the adult fly by supporting both GSIS and mitochondrial activity. This coupling of developmental progression with metabolic state is a critical regulatory connection that is poorly understood in all animals. In addition, our paper defines a developmental shift in systemic glucose homeostasis and provides new evidence to explain why adult IPCs secrete DILP2 in response to glucose, unlike those of larvae. Finally, it is important to note that this is the first paper to report functional studies of The key evidence that dHNF binds to mtDNA is the ChIP-seq data. It is unclear how to interpret this mtDNA Chiq-seq data for a couple of reasons:A) The presumably high copy number of mtDNA in the sample (perhaps 1000X in excess of nuclear).B) The low signal to noise ratio in the nuclear binding sites. Presumably, most ChIP-seq experiments will detect mtDNA as a background signal. This will usually be discarded, because of the excess signal at specific nuclear DNA binding sites. In this study, in which the signal to noise ratio in nuclear genes is quite low, any background signal caused by the mtDNA might seem more important. A nice control would have been to perform the ChIP-seq in the mutant flies. The mtDNA signal should be much reduced in the mutants (assuming the mtDNA copy number is the same in mutant flies). The reviewers are correct that the abundance of mtDNA can complicate ChIP-seq peak calling for the mitochondrial genome. Indeed, we were aware of this and thus our analysis of the ChIP-seq data was performed using the MACS2 platform, which addresses this potential artifact by accounting for local differences in read depth across the genome (including the increased abundance of mtDNA relative to nuclear DNA). This program performs peak calling by calculating dynamic Poisson distribution parameters  and thus adjusts for biases due to local differences in abundance compared to the rest of the genome. In effect, this results in peak calling relative to protein binding levels within adjacent flanking sequences of DNA. As a result, we detect specific dHNF4 enrichment that is restricted to a portion of the mtDNA control region and, importantly, this enrichment is not observed in the input control. Although the triple-stranded structure of the D-loop in the control region of mammalian mtDNA has been proposed to contribute to possible false-positive ChIP-seq peaks in mammalian datasets , the D-loop structure is not present in Drosophila mtDNA . Furthermore, the peak for dHNF4 on mtDNA represents the 5th strongest enrichment peak in our ChIP-seq dataset out of 68 total. This suggests that its significance is not overemphasized due to low signal, as the high-confidence direct targets for dHNF4 identified from this analysis show robust enrichment for proximal dHNF4 binding. In addition, only select mtDNA genes are underexpressed in dHNF4 mutants while mtDNA copy number is unaffected, suggesting a specific regulatory defect in mitochondrial gene expression. Finally, in two other ChIP-seq studies performed in our lab for other nuclear receptors in adult flies using the same protocol, we have not observed such localized enrichment to this predicted mtDNA promoter region. As a result, we feel that specific binding of dHNF4 to mtDNA is a reasonable conclusion from our studies. We have added the information cited above to our Discussion in an effort to address this important concern for readers .dHNF4 mutant flies as the reviewers request, this is an expensive experiment to conduct as a control for the specificity of dHNF4 binding to mtDNA. Indeed, we know of no comparable control studies in the literature. In addition, the low viability of dHNF4 mutants makes it difficult to get the large quantities of fresh material we need for this experiment (gram quantities of flies). We have attempted to design PCR primers to perform focused ChIP on mtDNA, followed by qPCR, but the sequences within and proximal to the region of dHNF4 binding lack suitable annealing sites due to AT-rich content.Although we agree that it would be ideal to perform a ChIP-seq study in 2) The second piece of evidence supportive of HNF4 being present in the mitochondria is an IF experiment, which uses the same antibody as the ChIP-seq study. Given the novelty of the finding, it would have been useful to see independent validation of mitochondrial localization using an alternative antibody or a genetic construct. In summary, whilst it is plausible that HNF is directly binding to mtDNA and regulating mtDNA transcription, further evidence, using a method that doesn't require the antibody used in this study, would make the claim more compelling. Given the interest in such a finding, further independent evidence of direct mtDNA binding would be necessary. We fully agree with the reviewers that additional evidence is needed to support our detection of dHNF4 in mitochondria by IF. Accordingly, we now include antibody stains of adult fat body from both controls and dHNF4 mutants, conducted in parallel and imaged under identical conditions. This data, included as In addition, we agree that our IF studies would benefit from using a second independent antibody to detect dHNF4. Unfortunately, however, no such reagent exists. In our original work, we raised antibodies against a 317 amino acid region shared by all dHNF4 protein isoforms, using both rats and guinea pigs. Both rodent antibodies were affinity-purified, and both detect similar patterns of cytoplasmic dHNF4 protein. The rat antibody, however, has a significantly higher signal to noise ratio as determined by stains of mutant tissue. We use this antibody for all our published work. In an effort to address the reviewers concerns, we obtained the GFP+FLAG-tagged dHNF4 BAC transgenic line from Bloomington (stock 38649). However, efforts to detect either GFP or FLAG tags using several antibodies  resulted in significant background signal in the cytoplasm from cells lacking this transgene. As stated above, we are currently developing multiple tagged lines with the express purpose of addressing this important issue raised by the reviewers as part of our future studies.3) As a related issue, the argument that loss of dHNF4 specifically impairs mitochondrial metabolism needs further clarification. Loss of dHNF4 could also or instead, cause a reduction in mitochondrial number and mass. To what degree can the consequences of dHNF4 depletion be attributed to a reduction of mitochondrial number or mass as opposed to specific TCA cycle and OXPHOS impairment? Does loss of dHNF4 reduce mitochondrial number, mass or mtDNA copy number? This is an important question. We agree that this is a critical experiment, which is now included as dHNF4 mutants. This new data is presented as dHNF4 mutants display defects in glycolysis and mitochondrial metabolism”. We also show reduced levels of Complex II by western blot, consistent with the reduced expression of the critical Complex II assembly factor gene dSdhaf4 in dHNF4 mutants , in the fat body results in mitochondrial fragmentation and hyperglycemia, similar to loss of dHNF4. This data is now included in dHNF4 mutant cells adjacent to wild-type cells – indicative of reduced mitochondrial membrane potential. This result is consistent with the reduced ATP levels that we detect in dHNF4 mutants and provides a mechanism that can explain the mitochondrial fragmentation, as we now include in the Discussion (in the aforementioned subsection). We have also assayed for changes in ROS as requested by the reviewers. This is shown in dHNF4 mutant phenotypes by feeding the animals N-acetyl-cysteine, which provides effective antioxidant activity in Drosophila. One possible explanation for this is that the ETC complexes that produce ROS are expressed at reduced levels in dHNF4 mutants, while the ROS-scavenging enzymes, such as catalase and SOD, remain unaffected in these animals. We now comment on this in the paper .ed genes  for Drp1Related to this issue, the authors should determine if the integrity of the cells that normally express it is compromised in any way such as an incorrect specification/physiology in the absence of HNF4, and please provide a more complete justification for why they think that the cells show a "mitochondrial fragmentation" phenotype. We see no changes in the size or morphology of any cells or tissues we have examined in dHNF4 mutants, with a particular focus on the IPCs . The mitochondrial fragmentation that we see in dHNF4 mutants or upon dHNF4 RNAi, depicted in CG5599, which encodes a protein with homology to α-ketoglutarate dehydrogenase, or disruption of the ETC by RNAi against CIA30, results in mitochondrial fragmentation that resembles that seen in dHNF4 mutant tissue .t tissue . In addiFor the RNA-seq data, a systematic enrichment analysis (GO term/KEGG pathway) needs to be carried out on all differentially expressed genes in order to support claims regarding specificity of pathways regulated by dHNF4. In the current version of the manuscript, the authors seem to have cherry-picked genes . The next most significant categories primarily represent genes involved in metabolism, with “electron carrier activity” as number 4. For the up-regulated genes, all of the top categories are involved in antibacterial and innate immune responses. We now comment on this analysis in the subsections “dHNF4 regulates nuclear and mitochondrial gene expression“, first paragraph, and “dHNF4 acts through multiple pathways to regulate glucose homeostasis”, fourth paragraph.Many of the above questions are arising, and are important to address, because the central issue has not been convincingly addressed: How does HNF4 regulate mitochondrial gene expression? As outlined in our response to comment #1, we fully agree that significant further studies are needed to definitively demonstrate that dHNF4 directly regulates mitochondrial gene expression and function. We wish to emphasize, however, that our current paper is not intended to focus on this topic. Rather, it represents the first phenotypic study of dHNF4 mutant adults and a functional analysis of the roles for dHNF4 in maintaining carbohydrate homeostasis. In addition to providing compelling evidence for a direct mitochondrial function for dHNF4, our paper provides the first animal model for MODY1, 20 years after the discovery of this genetic association in humans. Our paper also establishes an important role for dHNF4 in contributing to a developmental switch in metabolic state and provides a molecular basis to understand the U-shaped curve of mitochondrial activity. We hope that the reviewers will agree that this work, taken together, is worthy of publication in eLife.4) One potential caveat with the RNA-seq and ChIP-seq experiments is that they have been carried out in whole flies. dHNF4 is expressed in multiple tissues, and both its functions and targets may be tissue-specific. This is something that will not be captured by whole-fly approaches, and may in fact prevent identification of some targets. For example, the contribution of the IPCs to total fly chromatin or RNA is likely to be negligible.While it is understandable that whole adult flies are used for technical reasons, presumably, each of the tissues in question will respond differently to the loss of HNF function, with different metabolic consequences, so it is unclear how to relate these global analyses of a heterogeneous tissue extract to pathology in a particular tissue (e.g. the IPCs). For example, the metabolomics data shows that HNF deletion increases glucose-derived metabolites, but the RNA-seq data suggests that glycolytic genes are down-regulated. These data are not incorrect, since the experiments and analysis were done with care and competence, but such analyses are easier to interpret if using a homogeneous extract. The ambiguities introduced by a mixture of tissues with mixed metabolic profiles make it difficult to appreciate the importance of the genomic analyses. The Reviewing Editor does not feel that this issue can be fully solved in a reasonable period of time. So perhaps some initial experiments that address aspects of this issue, and attending to this issue in the Discussion, as a caveat will suffice. The reviewers make the important point that different tissues can respond in different ways to a loss-of-function mutation. Tissue-specific RNA-seq, ChIP-seq, and GC/MS metabolomic analysis would address this issue, but they require significant technical effort and financial resources. Rather, we have chosen to address this topic through tissue-specific functional studies of select target genes for dHNF4, as depicted in 5) All the mutants characterized in this study involve constitutive dHNF4 loss or downregulation throughout development – either from all tissues or a subset thereof. Consequently, the adult phenotypes described in this manuscript could be adaptive and result from the metabolic alterations caused by dHNF4 loss during larval life . The abundance of genes involved in stress and immunity in the RNA-seq dataset is consistent with this idea. The authors should knockdown dHNF4 specifically in the adult  and test whether some of the key phenotypes  are still apparent.tubGal80ts>dHNF4-RNAi stock and tested this extensively in adults. Unfortunately, however, we could not obtain a sufficient reduction in dHNF4 expression to result in reproducible phenotypes. As an alternative approach, we have used CRISPR/Cas9 to engineer two FRT sites into introns in the endogenous dHNF4 locus in an effort to generate a conditional allele that lacks all dHNF4 functions. We believe that this is a better way to conduct this experiment, allowing us to selectively eliminate dHNF4 function in a temporally and spatially regulated manner. We have successfully used this approach to conduct adult-specific functional studies of another nuclear receptor, dERR, in our lab. We thus feel confident that this approach will allow us to perform a comprehensive adult-specific functional analysis in the future. To address this concern of the reviewers, we now discuss this topic in the Discussion (fifth paragraph of the subsection “dHNF4 acts through multiple pathways to regulate glucose homeostasis”), pointing out that developmental functions for dHNF4 could contribute to the phenotypes we observe.We agree that it is important to selectively remove dHNF4 function from adults and then conduct a complete phenotypic analysis to determine how the resulting phenotypes compare to those of the germline mutant used in our paper. Accordingly, we established the recommended Related to this, the specificity of the Ilp2-Gal4 used in this study  has not been investigated/described in larvae. Why did the authors not use the Ilp2-3-Gal4 generated in the Partridge lab, which is confined to late larva/adult IPCS? This would avoid targeting non-IPC dHNF4 expression throughout development. We agree with the reviewers that restricting RNAi against dHNF4 to the adult IPCs would be ideal to avoid non-specific effects from such constructs being expressed during larval development. The dilp2-GAL4 line we use is recombined into a complex genetic background developed by Seung Kim’s lab to allow for picomolar detection of circulating DILP2 peptide (DILP2HF) upon targeted RNAi in these cells. For details, see our Materials and methods and the paper from the Kim lab . We wanted to ensure that all of our functional studies could be related to the critical phenotype of glucose-stimulated DILP secretion, given that this is a hallmark for MODY1. We thus used this same dilp2-GAL4 driver for all functional studies in our paper. Importantly, this line has been validated to drive expression specifically in adult IPCs . In addition, this line includes a UAS-Dcr transgene in the background, which we have found is critical for efficient knockdown in IPCs. Finally, it is important to note that dHNF4 is not detectable in the IPCs during larval stages   , and we The reviewers ask about a Ilp2-3-GAL4 line generated by the Partridge lab. To our knowledge, however, this line does not exist. Perhaps the reviewers are referring to the Ilp2-GAL4 line generated by the Hafen lab (Ilp215-3-GAL4), which has been misattributed to the Partridge lab in at least one previous publication. The Ilp215-3-GAL4 driver, however, has been reported to initiate expression during the late L3 and thus would not provide the desired adult-specific expression . In addition, there is no data to support this temporal induction since, to our knowledge, a systematic time course study of Ilp215-3-GAL4 expression has not been published.Also, a related important but minor issue is that often a single RNAi construct is used, the authors are well aware of the possible pitfalls, and they need to be avoided. Additionally, all UAS and Gal4 lines need to be fully defined. We thank the reviewers for this suggestion and fully acknowledge the limitations of RNAi, especially when only a single RNAi line is available. We have used multiple RNAi lines whenever possible, in particular for our most critical results, focusing on hyperglycemia and the defects in GSIS. We use two separate lines for dHNF4 RNAi and examine the effects on glucose levels and DILP2 secretion  and within the relevant figure legends.ecretion . We alsox-C RNAi . These d6) The authors argue that dHNF4 is required in the fat body and insulin-producing cells of adults to maintain glucose homeostasis by supporting a developmental switch toward OXPHOS and GSIS at the transition to adulthood. Since all experiments in this paper were performed by removing dHNF4 early in development far before the onset of adulthood, it remains a possibility that the phenotypes observed are a consequence of an earlier requirement for dHNF4 during development. To show that dHNF4 is really required in adulthood we would like the authors to remove dHNF4 at the onset of adulthood in the whole organism, fat body and IPCs to assess whether the flies become diabetic and whether survival is affected. As the muscle is also a major contributor to glucose clearance the requirement of dHNF4 in muscles should be assessed as well. Please see the first section of #5 above for our response to this request to perform adult-specific loss-of-function studies to complement our studies using germline dHNF4 null mutants.7) Based on RNAi to knock-down of specific OXPHOS complexes in the IPC and fat body the authors conclude that there are differential requirements for different ETC complexes in glucose homeostasis. This requires functional validation e.g. on the rate of oxygen consumption. The authors demonstrate that Cox5a knock-down does not increase glucose, but CIA30 knock-down does. It is more likely that each complex has differential control of flux in specific tissues or flux through the ETC has differential sensitivity to knock-down of specific complexes, rather than suggesting that there is a greater requirement for CI or CV than CIV per se. We thank the reviewers for raising this point. We have removed this speculation from the text since we cannot be sure of the effect of each RNAi knockdown on ETC activity, as the reviewers point out.8) The authors' previous study was consistent with dHNF4 as a nutrient sensor, whereas this manuscript suggests it functions as a developmental switch that converts a cell to a fate that supports responsiveness to glucose. Are dHNF4 expression, activity and/or subcellular localization nutritionally regulated in adult flies? The very low number of ChIP-seq could be explained low dHNF4 activity if a 3% sugar diet was used. RNA-seq experiments used 15% sugar. The diets used for every experiment also need to be clearly stated, and the data analyzed accordingly.Drosophila. Needless to say, we wondered the same thing and have conducted a number of nutritional studies, with a particular focus on changes in the subcellular localization of dHNF4. We could not, however, detect any reproducible differences. The levels of dHNF4 mRNA are also unaffected by fasting and starvation, as demonstrated by both larval and adult microarray studies done in our lab, and several studies done by other labs. With regard to the diet used for the ChIP-seq study, we used a normal sugar diet since this experiment was conducted using wild-type  Drosophila adults. We now clearly state the diets for each experiment in the legends and within the Materials and methods section.The reviewers raise the interesting and important possibility that dHNF4 expression, activity and/or subcellular localization might be regulated by nutrition in


To determine if the presence of certain polymorphisms in the DNA repair gene XPC and the apoptosis inductor gene p53 is associated with pre-senile cataract development.We have performed a retrospective study over three groups of patients. The group with pre-senile cataract formed by 72 patients younger than 55 with cataract surgery. The group with senile cataract formed by 101 patients older than 55 with cataract surgery. The group without cataract was formed by 42 subjects older than 55 without lens opacities. We analyzed the presence of SNP rs2228000 from XPC and rs1042522 from p53; and the relationship between risk factors such as smoking, alcohol intake, hypertension or diabetes.The comparison of the genotype distribution in XPC, within the different groups, did not show any statistically significant association in any of our analysis . The comparison of the genotype distribution in p53 within the different groups did not show any statistically significant association ; except for the comparison between the pre-senile cataract group and the group with senile cataract where the genotype Pro/Pro (C/C) in the recessive inheritance model showed a higher risk for developing pre-senile cataract . This association decreased when we performed the analysis adjusting by the studied risk factors (p = 0.056).Allelic variants in the gene XPC are not associated with an increased risk for developing pre-senile cataract. The presence of the genotype Pro/Pro in p53 might be associated with a major risk for developing pre-senile cataract. Cataract consists in a loss of the lens transparency and it is considered to be the leading cause of treatable blindness worldwide . It is uSeveral theories have been proposed for cataract development, such as excess ultraviolet radiation (UV-r) , 9 or ioIn our genome there are certain genes responsible for DNA repair to external aggressions. They encode the proteins that recognize genome cell damage and repair it. These proteins postpone cell replication until lesions have been repaired and otherwise they induce cell apoptosis. When these mechanisms fail, damage can be transmitted to progeny cells and begin processes such as carcinogenesis or cataract formation , 13. XPCXPC is a gene located in the short arm of the chromosome 3 (3p25). It encodes the protein XPC which function is to recognize DNA damage in the nucleotide excision repair (NER) , 15. XPCp53 is located in the short arm of chromosome 17 (17p13) and it encodes a nuclear transcription factor. It encodes the protein p53, which acts on genes that regulate cell replication cycle. In situations of DNA damage, p53 stops the cell cycle until the DNA is repaired or until cell death is provoked. In that way it prevents mutated cells proliferation . In unstThere are evidences that both proteins (P53 and XPC) are implicated with NER. In fact the global genomic repair (GGR) is a p53-mediated process and p53 regulated the basal and inducible levels of XPC and p48, both proteins are essential for GGR .The aim of this study is to assess whether gene polymorphisms rs2228000 in XPC and rs1042522 in p53, may be involved in the development of cataracts in early ages in a group of population from Salamanca. This is the first study conducted to connect these genes polymorphisms with pre-senile cataract formation.Our research has been approved by the Ethics Committee of the Clinic Hospital of Salamanca and adhered to the principles of the Declaration of Helsinki.Written informed consent was obtained from all our patients.Based on a preliminary study performed by our group, we calculated the size sample needed to detect differences between groups, with an α risk of 5% and β risk of 10% . There were 38 subjects in each group. In order to obtain a sufficient sample of patients, to allow us a thorough study of the natural history of disease, 72 subjects were included in the group with pre-senile cataract, 101 patients in the group of senile cataracts and 42 in the group of subjects without cataract.To form the group with pre-senile cataract, we selected, sequentially, a sample of 72 patients who had undergone cataract surgery at the Clinic Hospital of Salamanca from January 2007 to December 2012. This group included all adult patients younger than 55 years old who had undergone cataract surgery. We excluded those patients who had cataracts secondary to trauma, uveitis, previous eye surgery, steroids intake and patients with systemic diseases associated wit cataracts such as Steinert's syndrome, galactosemia, Lowe syndrome, homocystinuria, Alport syndrome, Marfan syndrome, Weil Marchesani syndrome, Peters anomaly, connective tissue diseases such as Ehlers-Danlos syndrome, Down syndrome and Cockayne syndrome.To form the senile cataract, we selected 101 patients older than 55 years old who underwent cataract surgery. We also excluded those patients whose cataracts are secondary to the above mentioned diseases; and to form the group with no cataracts, we included 42 patients older than 55 who had not yet developed cataracts. In this group we excluded all those patients who presented any type of valuable lens opacity by LOCS III system , or thosWe analyzed variables such as the presence of risk factors (RF) which may be associated with cataracts development like the presence of diabetes mellitus, arterial hypertension, smoking at least 10 cigarettes a day for 5 or more years, alcohol intake of at least one unit alcohol at least two times a week. We also analyzed the genotypic distribution of the polymorphisms rs2228000 in XPC and rs1042522 in p53.We performed a complete anamnesis to all the subjects. This anamnesis included past medical history, age and gender, and potential risk factors such as the presence of diabetes, arterial hypertension, smoking or alcohol intake. We also performed a complete ophthalmic examination, consisting on refractive error calculation, visual acuity measurement (VA) with Snellen chart, intraocular pressure measurement (IOP) by Goldman applanation tonometry, slit lamp examination to assess the degree of lens opacity using the LOCS III system , biometrInformed consent was obtained from all the participants included in the study. After that, we took a sample of peripheral blood, in order to isolate the cellular DNA following the standard protocols. The allelic discrimination of the single nucleotide polymorphism (SNP) rs2228000 in XPC was performed by PCR using Taqman probes; ID test C_16018061_10. The study was performed in a thermocycler  where we introduced a 96 cell-container. We mixed one microliter of DNA sample from each patient; 5 ml of the commercial compound PCR (Taqman ™), which provides the necessary enzyme for amplification (Taq polymerase); 0.25 ml of the commercial compound which contains the first primer oligonucleotide "forward", the first primer oligonucleotide "reverse" and the probes labeled with VIC and FAM dyes; and 4.25 ml of distilled water. After an initial denaturation of the mixture at 95°C for three minutes followed by 35 cycles of denaturation at 94°C for 10 seconds, an annealing procedure at 58°C for 20 seconds and an elongation at 72°C for 40 seconds the amplification was done. In all PCR reactions a final elongation of 7 minutes at 72°C was performed.We used restriction endonucleases to analyze p53. These enzymes recognize specific DNA sequences and cleave it at this point. The study of the restriction fragments length polymorphism (RFLP) is a technique used to discriminate different genes alleles, analyzing the size of the fragments generated after the DNA digestion with restriction enzymes. Digestions were performed by incubating 12μL of the PCR product plus 1 mL of the selected restriction endonuclease, BstUI , and 7 mL of water, used as the digestion buffer-specific endonuclease or as universal buffer. It is placed at the specific temperature for 5 to 7 hours. The fragments obtained after the digestion were separated by electrophoresis in an agarose gel at 1% or 3% stained with Syber-safe ®. In all gels we included the size marker. In order to monitor the DNA migration at the agarose gel two dyes Xylene cyanol and bromophenol blue were included in the buffer. We accomplish a digital photograph under ultraviolet light using the Kodak Science ID program . This is a one-center, retrospective, case-control study approved by the Ethics Committee of the Clinic Hospital of Salamanca and adhered to the principles of the Declaration of Helsinki.The distribution of the percentages was calculated for each qualitative variable. For the variable age we calculated indicators of central tendency (mean or median) and dispersion (Standard deviation or percentiles). Differences in risk factors frequencies between the different groups (Case-Control) were investigated using established hypothesis comparing proportions. Genetic association analysis was performed after verifying whether polymorphisms fulfilled Hardy-Weinberg equilibrium. The polymorphisms association magnitude with the disease was quantified by calculating odds ratios (OR) of each genotype with regard to the reference group (control group).In a previous study, the comparison of environmental and acquired risk factors  between the different groups showed that smoking and alcohol intake are related to the presence of pre-senile cataracts with a significant association (p<0.01). Likewise, alcohol intake also seems to influence the formation of senile cataract type (p = 0.027). Hypertension was more frequent in the groups formed with patients older than 55 years old. Hence we performed a regression model adjusting for these environmental and acquired risk factors, logistic regression models adjusting for risk factors with statistical significance in the two-dimensional analysis, and depending on the model of inheritance: dominant, recessive and codominant alleles. The accuracy of the association was expressed through a 95% of confidence interval (CI). The results were considered statistically significant when the p-value was less than or equal to 0.05. The statistical analysis was performed by an independent person using the Free Software package SNPAsocc R.For this study we have established three groups of subjects: the group with pre-senile cataract (adults younger than 55 years old diagnosed and treated of cataract); the group with senile cataract (adults older than 55 who had undergone cataract surgery), and the group with no cataract (adults older than 55 who had no lens opacification). The average age of the group with pre-senile cataracts was 49.01± 7.26 years (range 26–55 years); in the group with senile cataract was 73.79 ± 6.53 years (range 60–91 years) and in the group with no cataracts was 67.10 ± 4.64 years (range 57–77 years).The SNP rs2228000; c.1385 G>A in the gene XPC produces a change of an adenine (A) for a guanine (G). The comparison of the distribution of genotypes and alleles of this SNP between the group with pre-senile cataract and the group with senile cataract showed no significant differences (p>0.05). We also performed the analysis using the regression model adjusted to the studied risk factors ; and we obtained no statistically significant differences in any model of inheritance (p> 0.05) .The comparison of the genotype distribution for the SNP rs2228000 in XPC between the group with pre-senile cataract and the group with no cataracts showed no statistically significant differences (p>0.05). When performing the alleles’ grouping analysis no differences were observed. We also performed the analysis using the regression model adjusted to risk factors; and found no statistically significant differences (p>0.05) .The comparison of the genotype distribution for the SNP rs2228000 in XPC between the group with senile cataract and the group with no cataract showed no statistically significant differences; neither in the alleles’ grouping analysis. When performing the analysis adjusted to environmental and acquired risk factors no differences were observed (p>0.05) .The SNP rs1042522 (c.215 C>G) in the gene p53 produces a change of a citosine (C) by a guanine (G) what produces the change of a proline (Pro) by an arginine (Arg) in the 72th codon of the protein. The comparison of the genotypes and alleles distribution of this polymorphism between the group with pre-senile cataract and the group with senile cataract showed a higher frequency of the genotype CC in the group with pre-senile cataract in the recessive inheritance model . However, when we performed the regression analysis adjusted to the studied risk factors the statistical signification decreased  .The comparison of the genotypes and alleles distribution of the SNP rs1042522 in p53 between the pre-senile cataract group and the group with no cataract showed no statistical significant association, neither when we performed the analysis adjusted to the studied risk factors  .The comparison of this SNP genotypes distribution between the group with senile cataract and the group with no cataract showed no statistical significant association in any of the analysis performed  .The gene XPC is located in the short arm of chromosome 3 (3p25). It encodes a protein involved in the DNA nucleotide excision repair (NER) by recognizing DNA damage , 31. ThiWe study the SNP rs2228000 (c.1385 G> A), which produces a change of an adenine (A) by a guanine (G). SNP rs2228000 is located in the interaction domain of XPC with RAD23B, although its functional significance remains unclear. It is possible that this variant, may alter NER capacity, thereby modulating cancer and aging susceptibility. Nevertheless we did not find any statistically significant differences in this polymorphism genotypes distribution in any of our analysis. Since there are no studies linking XPC with cataract formation, our results need to be confirmed.Cellular senescence is an antiproliferative program that leads to permanent growth arrest. Senescent cells are irreversibly arrested in G1/G0 phase of the cell cycle and lose the ability to respond to growth factors. They maintain metabolic activity for long periods of time and become resistant to apoptosis . This grMost mutations in p53 occur in the central DNA-binding domain (DBD) due to p53’s major function as transcription factor that controls the expression of a multiple genes that regulate apoptosis, senescence, cell-cycle arrest and DNA repair. Functional mutations that alter the transcriptional capacity of the p53 master gene have been identified as super-transactivating, change-in-spectrum or overall downward modulation of transactivation. For example, change-in-spectrum mutants may be capable of regulating genes containing a strong response element, such as p21, but unable to regulate those with a weak response element, such as Bax. This is consistent with the observation of mutant p53s that still induce cell cycle arrest, yet lose the ability to activate apoptosis , 37. SomBecause of the relationship of p53 with senescence and its association with the gene XPC we decided to add the polymorphism in the 72th codon in p53 gene to our study. Besides some authors have studied the association between oxidative damage and an increase of apoptosis in the lens cells , 41. ThiSeveral studies have related this polymorphism with ophthalmological diseases such as retinal vitreous proliferation following retinal detachment  or with The comparison of the genotypes and alleles distribution for the SNP between the pre-senile cataract group and the senile cataract group showed significant association in the recessive model, where a higher percentage of patients with pre-senile cataract have homozygous genotype Pro/Pro. It represents an increased risk for developing pre-senile cataract of 4,083 . However, when we performed the analysis adjusted to the risk factors, the statistical significance decreased  . The decIn summary, SNP rs2228000 in XPC is not associated with pre-senile cataract development in our study. The presence of the genotype Pro/Pro in rs1042522 of p53 might be associated with a major risk for developing pre-senile cataract. Further investigations over these two genes would be useful to reject or accept their association with pre-senile cataract development.


Chronically shunted patients with functioning shunts are often characterized as having slit or smaller than normal ventricles and chronic headaches. In this study, we used Diffusion Tensor Imaging (DTI) to quantitatively analyze the directionality of water diffusion in the internal capsule (IC) and the corpus callosum (CC) in these patients which have been shown to be affected in untreated hydrocephalus patients [1].Shunt-dependent patients who developed hydrocephalus as infants were selected. Preliminary results from 17 patients who suffer from chronic headaches  are shown in comparison to age and gender matched controls. DTI data was acquired using a single-shot EPI sequence with TE/TR=69/10000ms, voxel size= 2mm x 2mm x 2mm, max b-factor=800, num. of directions=32. White matter ROIs were manually drawn on high-resolution T1-weighted images with the aid of fractional anisotropy (FA) maps to locate the internal capsule (IC) , genu and splenium of the corpus callosum (CC) and were verified by a neuroradiologist.A statistically significant decrease  in FA was found in the patient group compared to the controls in the right posterior , left posterior  and left anterior IC  as well as the splenium of the CC . No correlations were found between ventricular volumes with the DTI parameters.This study is the first to use DTI to study chronically shunted patients and shows evidence of white matter degradation based on decreases in FA in patients compared to controls. Previous studies have shown increased FA values in the IC and a decrease in the CC of patients and hypothesized that it was due to an increase of mechanical compression whilst others have hypothesized that a decrease in FA indicated secondary irreversible, degenerative changes [1-3]. These studies were performed on younger patients pre-treatment who usually have enlarged ventricles and we hypothesize that this may be the source of the difference coupled with the chronic shunt dependence within our patient group.


This study describes the transmission of border disease virus (BDV) from a persistently infected calf to seronegative heifers in early pregnancy, resulting in persistently infected fetuses. On day 50 of pregnancy (= day 0 of the infection phase), six heifers were co-housed in a free stall with a bull calf persistently infected with BDV (pi BVD) for 60 days. The heifers underwent daily clinical examination, and blood samples were collected regularly for detection of pestiviral RNA and anti-pestivirus antibodies. After day 60 (= day 110 of pregnancy), the heifers were slaughtered, and the fetuses and placentae underwent post-mortem and immunohistochemical examination and RT-PCR for viral RNA detection.Three heifers had mild viraemia from day 8 to day 14, and by day 40 all heifers had pestivirus antibodies identified as anti-BDV antibodies in the serum neutralisation test. The placenta of the three viraemic heifers had histological evidence of inflammation, and fetal organs from these heifers were positive for pestivirus antigen by immunohistochemical examination and for BD viral RNA by RT-PCR and sequencing. Thus, co-housing of heifers in early pregnancy with a pi-BDV calf led to seroconversion in all heifers and persistent fetal infection in three.Considering that pi-BDV cattle can infect other cattle and lead to persistent infection of the fetus in pregnant cows, BDV should not be ignored in the context of the mandatory BVDV eradication and monitoring program. This strongly suggests that BDV should be taken into account in BVD eradication and control programs. It has long been known that Border disease virus (BDV) is transmissible from sheep to cattle under experimental as well as natural conditions -9, but wThe Braunvieh × Limousine pi-BDV bull calf originated from a BVDV-free herd of 24 cows, which were co-housed with 20 sheep in the same barn. With the exception of this calf, ear punch biopsy samples ,11 of alSix open heifers of different breeds were acquired at the age of 382 to 748 days (means ± sd = 506 ± 126 days). Ear punch biopsy samples obtained from all the heifers had tested negative for pestivirus. The heifers were tested twice as seronegative by antibody ELISA as described below.The entire study period was divided into an acclimation and an infection phase. During the acclimation phase, the heifers were kept in quarantine without the pi-BDV calf. After estrus synchronisation, they were artificially inseminated using sperm from a BVDV-negative bull. Pregnancy was diagnosed ultrasonographically 30 days after insemination. Four of the heifers returned to estrus but conceived after the second insemination. The acclimation phase lasted until day 50 of pregnancy in each heifer.The infection phase was 60 days and lasted from day 50 (= day 0 of infection phase) to day 110 of pregnancy. During this phase the heifers were housed together with the pi-BDV calf in a separate free stall barn. Merging of the heifers with the calf was staggered because artificial insemination occurred on different days.The heifers underwent thorough clinical examination before and at the end of the acclimation phase. In addition, the demeanour, behaviour, appetite and fecal consistency were monitored daily during acclimation. During the infection phase, the demeanour, appetite, circulatory, respiratory and digestive systems, mucous membranes and skin were assessed daily and the rectal temperature was recorded twice daily; the daily mean of the latter was calculated for each heifer. Ultrasonographic pregnancy examinations were carried out every ten days and the heifers were observed daily for signs of abortion.EDTA blood for pestivirus antigen testing was collected on day 0 and then every other day until day 20 of the infection phase. Whole blood for pestivirus antibody testing was collected on day 0 and then every ten days until day 60 of the infection phase.RNA in the blood samples was isolated using a Bio-Robot-Universal-System  and the QIAamp Virus BioRobot MDx Kit (Qiagen). RNA was detected according to the instructions of the Cador BVDV RT-PCR Kit (Qiagen) running as proposed for 45 cycles using a thermocycler ABI 7300 . The primers and probes of this kit have a very high sensitivity for the detection of BVDV and BDV .Following evaluation of the raw data, the amount of the viral RNA in the sample was expressed in Ct values; values of ≤30 were rated positive and values of >30 weakly positive. Blood samples were stored at 4°C, and weakly positive samples were re-tested after repeated RNA isolation.An antibody ELISA developed at the Institute for Veterinary Virology, Vetsuisse Faculty, University of Bern , was useThe serum neutralisation test (SNT) was used to identify the pestivirus species against which the antibody was directed . The BDVThe uterus, placenta and ovaries of the heifers and fetal organs  were examined macroscopically and histologically. Tissues were fixed in formalin, embedded in paraffin, sectioned and stained with H&E and examined using light microscopy.rns). The pestivirus-specific antibody C16  is directed against the nonstructural protein p125/80 (NS2-3/NS3), and the pestivirus-specific antibody 15C5  against the highly conserved glycoprotein gp48 (Erns).Samples of the thyroid gland, tongue, aural skin and other fetal organs, and of placentomes were collected for immunohistochemistry. Pieces of thyroid gland, tongue and skin were snap frozen in liquid nitrogen, cryosectioned and processed with the antibodies Ca3/34-C42 and C16 ,19,20. SSkin, thymus and small intestines from the fetuses and placentae were used for the detection of pestiviral RNA. Skin and thymus were disintegrated and homogenised mechanically using the Tissue Lyser (Qiagen) and small intestine and placenta enzymatically using the QIAamp Cador Pathogen Mini Kit according to manufacturer’s instructions. RNA isolation and sequencing of the 5’ terminal region (5’-UTR) of the pestiviral genome was done as described . The samt-test, a general linear model and ANOVA were carried out to analyse differences in OD values as a function of day of the infection phase and presence of a pi fetus. The underlying Stata model for the t-test was < by varx, sort: ttest vary, by (varx2)>, whereby varx = day variable, vary = OD value, varx2 = independent variable, for the general linear model it was < xtmixed vary varx2##varx || varx:> and for the ANOVA it was < vary varx3 c.varx2, repeated (varx) bse (varx4)>, whereby varx3 = heifer and varx4 = time point. A P-value ≤0.05 was considered significant.Data were recorded in Office Excel 2007 (Microsoft Inc.). Descriptive statistics were used to describe continuous data  and normality was tested using the Wilk-Shapiro test. Means ± standard deviations were calculated for normally distributed data and medians, minimum and maximum values for data with non-normal distribution. The program STATA 12  was used to analyse OD values (antibody titres). A The study was approved by an ethical committee of the canton of Zurich, Switzerland.The pi-BDV calf was clinically healthy and afebrile but grew very little during the study period. Radiographic studies revealed a mild increase in radiopacity of the diaphyses, which extended to the metaphyses, and mild osteopetrosis , animal Three heifers had weakly positive Ct values ranging from 37.8 to 42.5, indicative of pestiviral RNA and thus viraemia on day 10 (heifer 3), day 8 (heifer 4) and days 8 to 14  with the generalised linear model revealed a significant effect of day of infection phase and status of fetal infection (infected or not infected) on relative OD. The 0 Figure . Based oPestiviral RNA was isolated from three heifers 3, 4, 5) on several occasions between days 8 and 14. Re-examination of the samples confirmed infection in two heifers  but not in the third, which had only been weakly positive once on day 10. These findings support pestiviral viraemia, albeit at a low level. The time frame of the viraemia was similar to that observed in calves (8 to 21 days) ,24, shee on severTwo heifers seroconverted as early as day 20, and the remaining heifers seroconverted by day 40. This variability may have been the result of differences in susceptibility to BDV, viral dose and time of exposure to the virus, but was similar to the variability in pregnant heifers that seroconverted 23 to 38 days after first contact with sheep persistently infected with BDV . HeifersSerum neutralisation testing indicated that all heifers had high titres of specific antibodies against BDV at the end of the infection phase. Four heifers also had low antibody titres against BVDV, which was thought to be due to slight cross-neutralisation within the pestivirus genus. Nonetheless, titres against BDV were 16 to 64 times higher than the titres against BVDV, indicating that the heifers were infected with BDV as a BDV titre at least four times higher than a BVDV titre is considered to be specific .All uteri, placentae and fetuses were macroscopically normal and the fetal organs were also histologically normal. There was histological evidence of inflammation in the placentomes of the three heifers carrying an infected fetus, similar to lesions seen in BDV-infected pregnant sheep  and cowsImmunohistochemical examination of tongue, skin, thyroid gland and placenta is an established and sensitive method for the detection of persistent fetal pestivirus infection . Three oReverse transcriptase PCR allowed confirmation of BDV infection of the fetuses from heifers 3, 4 and 5. The RNA sequence isolated from heifers 3 and 5 was identical to the sequence isolated from the pi-BDV calf. The base-pair substitution of thymine for adenine at position 94 might be based on a RNA-virus specific characteristic whereby viruses produced within an infected cell are not always identical but may vary because of mutation and recombination within the viral genome . SimilarConsidering that pi-BDV cattle can infect other cattle and lead to persistent infection of the fetus in pregnant cows, BDV should not be ignored in the context of the mandatory BVDV eradication and monitoring programs such as the one instituted in Switzeland. In Switzerland and other countries, sheep and cattle are commonly kept in the same barn or co-pastured on alpine summer pastures, and it is therefore likely that BDV is transmitted from sheep to cattle and subsequently from the infected cattle to other cattle. For this reason, testing for BDV should be included in BVD eradication and control programs. It is doubtful whether pestiviruses in cattle can be eradicated without differentiating BVDV and BDV infection and the inclusion of other susceptible species such as sheep in the Swiss control program. It is therefore possible that pestivirus in cattle can not be eradicated without differentiating BVDV and BDV infection and the inclusion of other susceptible species such as sheep in the Swiss control program.


Chronic obstructive pulmonary disease (COPD) patients who have had an episode of acute hypercapnic respiratory failure (AHRF) have a large 1-year risk of death or readmission. Acute non-invasive ventilation (NIV) has been shown to be an effective treatment of AHRF; and long-term NIV (LTNIV) has been shown to be an effective treatment of chronic respiratory failure in stable hypercapnic COPD. We investigated the effects of LTNIV in a group of patients with severe, unstable COPD: frequent admissions and multiple previous episodes of AHRF treated with NIV.We conducted a retrospective analysis of 20 COPD patients treated with LTNIV after two or more episodes of AHRF during 1 year.p<0.0001). The median number of admissions decreased from 5.19 to 1.88 (p=0.0092). Four patients (20%) died in 1 year. LTNIV tended to reduce arterial CO2. No changes were found in lung function.The mean number of AHRF episodes decreased from 2.44 in the year prior to LTNIV initiation to 0.44 in the year following (LTNIV seems effective in reducing recurrent AHRF and readmissions in a highly select group of patients with severe, unstable COPD and frequent AHRF. In exacerbations of severe chronic obstructive pulmonary disease (COPD), patients are at risk of developing acute hypercapnic respiratory failure (AHRF). Patients with AHRF due to COPD necessarily have to be admitted for treatment. In accordance with guidelines from the Global Initiative for Chronic Obstructive Lung Disease (GOLD), non-invasive positive pressure ventilation (NIV) is recommended as a part of the first line treatment for AHRF due to COPD, along with oxygen supply and medical treatment –3.Patients having survived an AHRF have a poor prognosis as they are highly susceptible to readmissions (79.9%), life-threatening events (63.3%), and death 49.1%) (9.1% (4).Long-term NIV (LTNIV) has been shown to reduce mortality in stable COPD patients (with no exacerbations for at least 4 weeks prior to enrollment) with chronic hypercapnic respiratory failure (CHRF) . LTNIV aIt is hypothesized that LTNIV may prevent exacerbations and recurrent AHRF in selected COPD patients with frequent episodes of AHRF. Two small randomized controlled trials have shown a significantly longer time to worsening in patients treated with LTNIV and a lower risk of readmissions , 8. A noLarge randomized controlled trials (RCTs) showed improvements of arterial gases but no effects on survival or admissions in COPD patients treated with LTNIV after an AHRF , 11.The purpose of this study was to investigate the effect of LTNIV initiated in COPD patients with two or more admissions due to AHRF within 1 year.This study was a retrospective analysis of patient journals, arterial gas samples, and lung function parameters. The study population consisted of COPD patients enrolled for LTNIV in the Department of Pulmonary Medicine at Gentofte Hospital from 2009 to 2014. LTNIV was offered to COPD patients who had had two or more episodes of AHRF treated with acute NIV within 1 year.Pa,CO2>6 kPa and an arterial pH≤7.35 despite 1–2 h of treatment with nebulized bronchodilators, intravenous methylprednisolone, and oxygen supply according to the GOLD recommendations , obesity hypoventilation syndrome (OHS), or cancer were excluded from this analysis.We reviewed each patient's journal from 1 year before LTNIV initiation to 1 year after.intention to treat.We noted baseline data for all the patients initiated with LTNIV, denoted as per protocol (PP).Patients who survived 1 year of LTNIV were denoted For PP patients, we compared the following variables before LTNIV initiation to 1 year after: the number of episodes of AHRF treated with acute NIV per year; the number of admissions to a pulmonary ward; the number of days admitted due to COPD; lung function; use of long-term oxygen treatment (LTOT); and arterial gas values.It was assumed that the clinical and physiological status of the patients would not improve spontaneously without intervention. Thus, the patients were their own controls, and any reduction in the numbers of AHRF episodes, admissions, or admission days could be attributed to the LTNIV treatment.t-test and chi-square test.By visual analysis, we found that data followed an acceptable normal distribution. Accordingly, differences were analyzed using parametric tests: the Student's We used SAS 9.4  for the statistical analyses.No ethics committee approval was needed because the study was retrospective and no data contained personally identifiable information.As of October 2015, 41 COPD patients had been treated with LTNIV for at least 1 year at the Department of Pulmonary Medicine of Gentofte Hospital. Of this number, 21 patients were not eligible for this analysis: 8 patients had OSA, 3 had OHS, and 1 had a combination of both. Five patients had not had two episodes of AHRF prior to LTNIV initiation. Two patients had initiated the LTNIV treatment a long time before being affiliated with our department. Thus, 20 patients met the inclusion criteria.The included patients’ baseline characteristics are listed in Ten patients used the average volume assured pressure support (AVAPS) mode on the ventilator and ten patients used regular pressure controlled ventilation.2O and a mean expiratory positive airway pressure of 5.4 cmH2O.The patients were treated with a mean IPAPmax (maximum inspiratory positive airway pressure) of 17.7 cmHThe variable ‘IPAPmin’ is the mean of the minimum IPAP settings in AVAPS mode.Four of the 20 patients (20%) died during the 1-year follow-up.Thus, we conducted the PP analysis on the remaining 16 patients. The results are listed in p<0.0001) (see The patients’ mean number of AHRF episodes per year decreased from 2.44 to 0.44 (001) see .p=0.0092).Likewise, the mean number of admissions to a pulmonary ward decreased from 5.19 to 1.88 (p=0.0015).The mean number of admission days decreased from 42.6 to 11.9  is known to help mobilizing mucus . As an aper se would remain unchanged, the number of severe exacerbations causing AHRF and admissions would still decrease as in the present study.Should LTNIV not prevent exacerbations, it could be hypothesized that COPD patients who are treated with oral antibiotics, prednisolone, bronchodilators, and NIV at home simply do not deteriorate to a degree where their respiratory muscles fatigue and respiratory failure occurs. Although the number of exacerbations An analogy would be a bicycle rider on a very steep hill: At the steepest part, instead of becoming tired and crashing the bike, the rider is pushed by a scooter riding by, and thus he manages to ride over the hill.crashing – that is, developing fatigue, respiratory failure, and requiring hospital admission.NIV could be the push that allows the patient to get through the exacerbation without Whatever the mechanism of LTNIV in patients with severe, unstable COPD, the seemingly clear benefits should be investigated thoroughly in an RCT: the non-controlled design of this study is inferior to that of an RCT; the patients included with repeated episodes of AHRF were highly selected and not likely to be representative of all patients having survived an episode of AHRF due to COPD. Furthermore, the patients included had access to an increased level of care compared to the patients treated at our department – for example, the 24-h nurse-led phone hotline; this might have increased treatment compliance and thus improved the outcomes.We acknowledge that the results presented are not necessarily entirely comparable to the results of other studies; however, we believe it is important to discuss the results of the study in the context of similar studies.Optimally, an RCT of LTNIV for COPD patients with multiple previous episodes of AHRF is needed. Its focus should be mortality, admissions, exacerbations, quality of life, compliance, and a cost–benefit analysis.However, we presume this RCT would be difficult to conduct, as the needed patients would be difficult to find and include.Until investigated in an RCT, the results of the study suggest that LTNIV should be considered in patients with COPD and recurrent episodes of AHRF.The results of this study suggest that LTNIV should be considered for patients with COPD and repeated episodes of AHRF, as the treatment seemingly reduces the number of recurrent episodes of AHRF and admissions.Further studies are desirable, though RCTs could be difficult to conduct.