Hypothenemus hampei, is the most economically important insect pest of coffee worldwide. We present an analysis of the draft genome of the coffee berry borer, the third genome for a Coleopteran species. The genome size is ca. 163 Mb with 19,222 predicted protein-coding genes. Analysis was focused on genes involved in primary digestion as well as gene families involved in detoxification of plant defense molecules and insecticides, such as carboxylesterases, cytochrome P450, gluthathione S-transferases, ATP-binding cassette transporters, and a gene that confers resistance to the insecticide dieldrin. A broad range of enzymes capable of degrading complex polysaccharides were identified. We also evaluated the pathogen defense system and found homologs to antimicrobial genes reported in the Drosophila genome. Ten cases of horizontal gene transfer were identified with evidence for expression, integration into the H. hampei genome, and phylogenetic evidence that the sequences are more closely related to bacterial rather than eukaryotic genes. The draft genome analysis broadly expands our knowledge on the biology of a devastating tropical insect pest and suggests new pest management strategies.The coffee berry borer,  Hypothenemus  consists of 181 described species, of which only H. hampei (Ferrari), commonly known as the coffee berry borer, is considered an important agricultural pestWorldwide, the genus Tribolium castaneumDendroctonus ponderosaeWe have sequenced the genome of female coffee berry borers in an attempt to gain a better understanding of the basic biology of the insect. Only two other Coleopteran genomes have been published to date: the red flour beetle, D. ponderosae genome size of 204 Mbet al.et al.After quality filtering, a total of 62 Gbp of sequence data (36 Gbp from a 350 bp insert library and 26 Gbp from a 550 bp insert library) was used for genome assembly. With an estimated genome size of 200 Mb used for the assembly software , a method to identify highly conserved eukaryotic proteins using the NCBI KOGs databaseH. hampei genome using the Infernal 1.1 software packagehttp://rfam.xfam.org)Functional ncRNA sequences were predicted on the H. hampei proteins (https://genome.nyumc.org/CBB.htm) were functionally characterized in the KEGG PATHWAY Database (http://www.genome.jp/kegg/pathway.html) by the BlastKOALA sequence similarity tool  and by alignment to the PANTHER database of protein HMM models (http://pantherdb.org/panther/) with the PANTHER scoring tool (http://pantherdb.org/tools/hmmScoreForm.jsp). The KEGG BlastKOALA tool mapped 27% of the H. hampei predicted proteins  to KEGG ortholog groups, while PANTHER assigned 68% of the proteins to PANTHER families. In The set of 19,222 predicted et al., submitted), we focused on genes potentially involved in caffeine detoxification as well as the detoxification of other plant allelochemicals and insecticides, such as the carboxylesterase gene family (CE), the cytochrome P450 gene family (CYP), the gluthathione S-transferase gene family (GST), the ATP-binding cassette transporter gene superfamily (ABC), and a gene (Rdl) that confers resistance to the insecticide dieldrin. In addition, we identified genes involved in pathogen defense, including homologs to antimicrobial genes reported in the Drosophila genome.The coffee berry borer spends most of its life cycle inside the coffee berry, where it consumes the seed. We therefore examined digestive enzyme composition, focusing on two digestive enzymes (amylases and mannanases), essential for catabolism of two representative coffee seed carbohydrates. Mannanases are discussed under horizontal gene transfer (below). Seeds are typically well defended against insect predators, so we also examined different detoxifying systems. While recent research indicates that coffee berry borer relies on bacteria in the midgut to detoxify caffeine (Ceja-Navarro T. castaneum amylase gene (XP_969234) as query, we found exactly one orthologous match to assembled transcript C129694, with a BLAST e-value of 0.0 and 72% conserved amino acid matches. The T. castaneum amylase also matches by tblastn to exactly one genomic location on scaffold 1599, where the gene is predicted by GeneMark as evmscaffold1599.1. The gene is highly expressed, with an FPKM in our RNA-seq data of 52997. This transcript also shows homologous matches to α-amylase proteins from other Coleoptera, including the spruce bark beetle (Ips typographus) amylase B with a tBLASTn e-value of 6e−142 and 70% (predicted) amino acid identity; boll weevil (Anthonomus grandis) α-amylase (AAN77139) at e = 0.0 (55% amino acid identity); rice weevil (Sitophilus oryzae) at e = 0.0 (69% amino acid identity); corn rootworm (Diabrotica virgifera virgifera) α-amylase (AAF20998) at e = 0.0 (56% amino acid identity); azuki bean weevil  (BAD83655) at e = 0.0 (58% amino acid identity); Mexican bean weevil (Zabrotes subfasciatus) (AAF73435) at e = 0.0 (57% amino acid identity); and mustard beetle (Phaedon cochleariae) (CAA76926) at e = 0.0 (54% amino acid identity).The α-amylases are a family of simple carbohydrate (starch)-metabolizing enzymes common in animals, microorganisms, and plantsH. hampei genome.As expected, several additional digestive enzyme genes were present, however some unexpected ones were found that are likely important in the insect’s ability to use the seeds as a food source. Seeds typically have high levels of protease and amylase inhibitors, as well as lectins, designed to interfere with insect digestion and absorption. A wide variety of digestive proteinases were found, and included serine proteinases , thiol proteinases , and metalloproteinases. While we cannot easily determine which of these are involved in digestion and which may have other roles until a gut transcriptome becomes available, it is clear that the insect has a potent suite of proteinases likely to be capable of dealing with plant defensive proteins. Most beetles have mainly thiol proteinasesD. ponderosae contained polysaccharide endo-β-1,4 glucanases, endopolygalacturonases, glycoside hydrolases in family 48 , rhamnogalacturonan lyases, and pectin methylesterasesH. hampei genome, namely four glycosyl hydrolases in family 48, seven polygalacturonases, one pectin esterase, five rhamnogalacturonan lyases, and two beta glucanases. Additionally we found two mannanase and two xylanase genes that appear to be the result of horizontal gene transfer (see below). Although microbial associates of insects are reported to produce microcrystalline cellulases to the benefit of the associated insectTriboliumThe green coffee seed has high levels of hemicellulose (complex polysaccharides), pectins, and celluloses, which could serve as an important nutrient source if the complex matrix can be degradedH. hampei genome, as were those for olfactory and pheromone receptors. Determination of these sequences allows for the potential development of feeding inhibitors, as well as attractants useful for monitoring, or attraction to insecticides of the free flying females.Genes for gustatory receptors were also found in the Seeds are typically well defended by plant secondary metabolites. The coffee seed is no exception, and contains high concentrations of a variety of chlorogenic acid derivatives2325et al., submitted), we have detected expression of methylxanthine N-demethylase genes (ndm) responsible for caffeine demethylation in H. hampei. However, we observed no homologs of this gene in the draft genome, suggesting that unlike mannanase , caffeine demethylation has not been laterally transferred to the genome and that this function resides in the bacterial community. This supports observations of elimination of this function through antibiotic treatment of coffee berry borers .In another related study (Ceja-Navarro ABC transporters are membrane bound proteins that use adenosine triphosphate (ATP) binding and hydrolysis for the translocation of amino acids, lipids, sterols, sugars, peptides, xenobiotics, etc., across membranes, with the efflux of xenobiotics having been implicated in resistance to toxins (including insecticides)27H. hampei predicted proteins using the hmmsearch program from the HMMER3 software package (http://hmmer.janelia.org)http://pfam.xfam.org)H. hampei genome (D. melanogaster for each of the eight established ABC transporter subfamilies (ABCA-ABCH). The sequences were multiple aligned with CLUSTALW and a maximum likelihood tree (with 100 bootstraps) was built using MEGA6 (http://www.megasoftware.net)H. hampei ABC proteins ABC-A: six; ABC-B: 14; ABC-C: 24, ABC-D: two; ABC-E/F: four; ABC-G: 11, ABC-H: four; unidentified: 30. A total of 73 ABC genes were reported in T. castaneumH. hampei; their specific functions await further study to determine their role in transporting toxic molecules out of cells.We searched for ABC transporters in the translated set of i genome . We combApis mellifera has 24 genes that are members of this gene family while D. melanogaster has 35Aedes aegypti has 49Anopheles gambiae has 51Tetranychus urticae has 76H. hampei predicted proteins using the hmmsearch program from the HMMER3 software packageT. castaneum genome assembly (NCBI version 4.0), there are 10 esterase genes that are highly similar to the H. hampei proteins , six of which form a tandem cluster on chromosome ChLG7, indicating a probable ancestral amplification of this gene. This means that the coffee berry borer has the appropriate gene resources to develop resistance to several insecticides.Carboxylesterase genes are involved in insecticide resistance and metabolism32H. hampei predicted proteins using the hmmsearch program from the HMMER3 software packageH. hampei draft genome (H. hampei CYP genes were expressed at a wide range of different levels from FPKM 1eth most highly expressed gene) down to FPKM 10. Expression level was not correlated with similarity to the CYP consensus HMM model. Of the 54 full-length CYP genes, three were mitochondrial, seven were CYP2, 20 CYP3, 17 CYP4, and seven undetermined, in contrast to D. ponderosae, whose 85 CYPs showed a different distribution , positives = 397/480 (83%), gaps = 3/480 (1%)).Cyp301a1 is a protein involved in cuticle formation and has been detected in all insect genomesThe proteins coded by some of these CYP genes are likely responsible for detoxifying the chlorogenic acids present in the seed. Again, some of these could potentially contribute to insecticide resistance. Insect P450s involved in the detoxification of a wide range of xenobiotics have nonpolar residues in the hydrophilic cage surrounding the aromatic network responsible for substrate positioning, and nonaromatic residues in regions SRS2, SRS5 and SRS6H. hampei has N-demethylase capability of its own. Thus, using contact insecticides that contain N-methyl groups attached to aromatic molecules, compared to other structural moieties, may be preferable for longer lifespan controls.Determining the substrate capabilities of the P450s may help guide choices for insecticides for control and selecting those that are less likely to develop resistance due to an absence of capable P450s. For example, based on studies reported above, it does not appear that the Drosophila and HMM searches with PFAM GST families, 19 predicted GST genes were identified in the H. hampei genome and transcriptome, distributed throughout all six classes as follows: one Delta, four Epsilon, two Omega, three Sigma, one Theta, and one Zeta (H. hampei genes are similar to both Epsilon and Delta classes (44% amino acid identity to both Drosophila GST D and E genes) but cannot be clearly distinguished between them. Homologs of these unclassified genes (63% amino acid identity) in D. ponderosae are annotated as hypothetical proteins. At least some of these GSTs are likely involved in detoxifying the chlorogenates in the seed and could contribute to detoxification of insecticides as well. Delta class GSTs are associated with resistance to DDT and are involved in resistance to some organophosphate insecticides, while omega forms can readily act on aromatic substratesH. hampei has GSTs likely to be involved in plant defensive compound and insecticide detoxification.The GST gene family is present in all aerobic organisms and is involved in the mediation of oxidative stress responses, hormone biosynthesis, and in the detoxification of plant allelochemicals and insecticides43one Zeta . Five of9H6Cl6O3S) has been used in many countries in an attempt to control H. hampei, but due to its human and environmental toxicity, the insecticide has been banned in at least 70 countriesRdl gene (resistance to dieldrin), which encodes the γ-aminobutyric acid subtype A (GABAA) receptor, are responsible for the resistance to the cyclodiene group insecticides in many insects, including A. aegypti, D. melanogaster, Musca domestica, Periplaneta americana, and T. castaneumH. hampei Rdl gene from an endosulfan resistant strain was deposited in GenBank (AF037324)Hypothenemus hampei resistance to endosulfan has been reported in New CaledoniaRdl has been demonstrated in insects from New Caledonia47The broad-spectrum chlorinated cyclodiene insecticide endosulfan  and shows a serine at an equivalent site to the resistance mutation that has been reported in endosulfan resistant flies, beetles and other insects. The insects used to establish the laboratory colony used in the present study originated in Colombia and have been under continuous rearing in the laboratory for ca. 10 years and hundreds of generations, indicating that even though there is no longer selective pressure, the resistant form of the gene has been maintained.The H. hampei draft genome. These include lectin C, mannose and galactose binding lectins, as well as peptidoglycan receptor proteins (see below). The regulatory protein gene Spätzle was also present. A prophenoloxidase gene with 77% homology to two Tribolium prophenoloxidases (XM_008196766.1 and NM_001039433) was present in the draft genome, although it was annotated during our analysis as Hemocyanin_C , 6586. Several serpin genes, which can regulate the phenoloxidase activity in insects and shrimpsInsects have a complex pathogen defense system that consists of pathogen recognition, humoral responses , cellular responses, melanization, and intracellular defenses (RNAi)D. melanogaster genes annotated with the following Gene Ontology (GO) database terms (http://amigo.geneontology.org/amigo/landing): antimicrobial humoral response (GO:0019730), antibacterial humoral response (GO:0019731), or defense response to bacterium (GO:0042742). A total of 175 genes were identified including 18 w, akirin, andropin, attacin, cecropin, defensin, diptericin, drosomycin, listericin, lysozyme, etc. Of these 175 Drosophila antimicrobial genes, we find that 120 of them match predicted H. hampei proteins with BLAST significance scores of 5e−10 or lower. For the reciprocal search, a total of 749 predicted H. hampei proteins match the set of Drosophila anti-microbial genes with BLAST significance scores of 5e−10 or lower, showing that these gene families are conserved and have abundant members in the H. hampei genome. A search of the T. castaneum proteome  with the same set of 175 D. melanogaster antimicrobial proteins finds 1,075 matches with BLAST e-values of 5e−10 or lower.We identified additional putative antimicrobial-related genes by BLAST using the set of all Drosophila melanogaster has five attacin proteins . Four uncharacterized homologs are found in the genome of T. castaneum and three in the genome of D. ponderosae. Three significantly similar H. hampei predicted proteins . Phylogenetic analysis does not show a clear orthology relationship among the attacin proteins from these species, rather the sequences from each species cluster together.Tribolium castaneum has an annotated cecropin gene (NP_001164146), but no significant similarity to this gene was found by blastp to H. hampei predicted proteins or by tblastn to the H. hampei transcript assemblies or to the H. hampei draft genome sequence. No PFAM matches to the cecropin HMM model (PF00272) were found for H. hampei predicted proteins.T. castaneum (XP_008199794) and D. ponderosae (ERL87200). All of these proteins have significant blastp matches to H. hampei predicted protein evm.model.scaffold348.1 . No PFAM matches to the coleoptericin HMM model (PF06286) were found for H. hampei predicted proteins.Coleoptericin is a glycine rich antimicrobial protein found in many Coleopteran species. Homologs have been identified in the draft genomes of H. hampei predicted proteins with the defensin peptide sequence from the Japanese rhinoceros beetle Allomyrina dichotoma (gb|AAB36306) produced one significant match (evm.scaffold2042.7), which is highly expressed (FPKM 10520). A hmmsearch with PFAM HMM model PF01097 (Defensin_2) also matches only to evm.model.scaffold2042.7, whose predicted protein has significant blastp similarity to defensin from several insect species with 50–60% amino acid identity, including holotricin (T. castaneum), coprisin (Copris tripartitus), tenecin (Tenebrio molitor), and phormin (Protophormia terraenovae).A blastp search of Drosophila drosomycin protein (AAF47767) was used as a query, but no significant similarity was found by blastp to H. hampei predicted proteins or by tblastn to the H. hampei transcript assemblies or to the H. hampei draft genome sequence. Similarly, moricins and gloverins have been found only in Lepidoptera . BLAST searches of H. hampei predicted proteins, assembled transcripts, and draft genome sequence did not yield any significant similarities to these proteins. Proline rich antimicrobial proteins such as lebocin (NP_001037468) and drosocin (CAA79936) are found in Diptera, Hymenoptera, Hemiptera, and Lepidoptera. No homologs of these genes have been reported in Coleoptera. BLAST searches with sequences of drosocin and lebocin did not find any significant matches in the H. hampei predicted proteins, assembled transcripts, or draft genome sequence.Drosomycin is found primarily in flies. The The detection of antimicrobial peptides genes reveals that the coffee berry borer possesses a wide array of mechanisms that could be used for antimicrobial defence. These peptides have been reported to have activity against both fungi and bacteriaBacillus circulans protein was identified from a H. hampei cDNAStreptomyces bingchenggensis was isolated from a H. hampei genomic cloneH. hampei proteins from the draft genome were screened for HGT candidates by identifying proteins with more significant BLAST e-values to a set of bacterial proteins  than to a set of arthropod proteins (UniRef50 arthropod proteins). To remove genes originating from contaminating bacterial DNA in the H. hampei genomic library, only gene models with >10% of exon bases covered by aligned RNA-seq reads were considered as HGT candidates.A mannanase gene (GenBank ID ACU52527) similar to a H. hampei genome, and phylogenetic evidence that the sequences are more closely related to bacterial rather than eukaryotic genes . These predicted proteins have 89% and 86% amino acid identity respectively to the xylanase gene reported by Padilla-Hurtado et al.H. hampei genes are highly expressed  was identified in the gut microbiota of the termite Globitermes brachycerastes, but is incorrectly annotated in GenBank ID 4HU8_A as of arthropod originPhaedon cochleariae (Chrysomelidae) also appear to have been acquired from bacteriaA total of 10 HGT candidate genes were identified with evidence for expression, integration into the ic genes . Two copBacillus . This gene has ~94% DNA sequence identity to GQ375156 over a much smaller region of 562 bases and 91% amino acid identity to the HhMAN1 protein (GenBank ID ACU52526) over the coding sequence. This second mannanase gene is expressed at FPKM 3.7eTwo copies of mannanase genes of possible bacterial origin were found on two different contigs of the draft genome ; Fig. 6.Streptomyces  and no significant similarity to any arthropod genes  (Predicted gene scaffold3436.2 has strong similarity to hydrolase proteins from ue >2.0) . The genH. hampei gene (scaffold7971.1) has stronger similarity to a Proteobacteria lipase/acylhydrolase  than to its closest arthropod match, platelet activating factor acetylhydrolase   . A maximic genes . Alignedic genes  and has Rickettsia   , but is Wolbachia regulatory protein RepA   , but hasCitrobacter Ig-like protein  invasin   with moderate RNA-seq coverage (FPKM 2.3ePredicted protein C2660673.1 has strong similarity to permease from dentity) . The cloH. hampei genome confirm the close relationship the insect has had with bacterial populations. For example, Ceja-Navarro et al. (submitted) identified several bacteria isolated from the alimentary canal that are able to subsist on caffeine as the sole source of carbon and nitrogen, and demonstrated that caffeine detoxification by the coffee berry borer is meditated by its microbiota.The 10 HGT candidate genes in the H. hampei genome is the third Coleoptera genome and the second bark beetle genome to have been analyzed. The most important findings in the coffee berry borer genome include (1) evidence for 10 cases of horizontal gene transfer from diverse bacteria; (2) paralogous expansion of antimicrobial peptides; (3) presence of a broad range of enzymes capable of degrading complex polysaccharides; and (4) the presence of a gene (Rdl) that confers resistance to a cyclodiene insecticide. The genome should be a useful tool for other researchers studying Coleoptera, and in particular, to those studying economically important bark beetles. The genome has also provided further understanding as to how H. hampei is able to successfully exploit its food resource and defend against pathogens. The information provided also can help guide the development of management strategies related to use of insecticides, biological control agents, attractants and plant resistance mechanisms that could be developed by breeding or genetic engineering.The draft later®  and incubated overnight at 4°C. After the initial incubation, the RNAlater® was removed, and the insects’ cuticle was sterilized by treating each batch with four sequential washing steps consisting of 500 μl of DEPC-treated water, commercial bleach, absolute ethanol and DEPC-water. At each step, the sample was vigorously shaken for ca. 30 sec. The washing of the samples was done twice. The insects from each batch were then transferred to a Lysing Matrix E tube  containing 500 μl of lysis solution (2% CTAB in 0.5 M NaCl). The samples were bead-beaten in a FastPrep Instrument  at 4 m/s for 30 seconds. Lysozyme buffer , 2mM EDTA, 1.2% Triton X-100) containing 10 mg/ml of lysozyme was added to each tube and the mixture incubated at 37 °C for 30 min. Five μl of proteinase K  was added to the tubes, mixed by tapping and incubated at 56°C for 30 minutes. After the proteinase K lysis, 500 μl of phenol:chloroform:isoamyl alcohol (25:24:1) was added to each tube, and the samples bead-beaten in the FastPrep instrument as before. The samples were centrifuged at 10,000 × g for 1 min at 4 °C and the supernatants transferred to a MaXtract high density tube  containing 500 μl of chloroform:isoamyl alcohol (24:1). The samples were centrifuged 10,000 × g for 1 min at 4 °C, and the supernatants transferred to a microcentrifuge tube containing 1 ml of isopropanol and 1 μl of linear acrylamide . The DNA/RNA mixture was precipitated by incubating for 10 min at room temperature and centrifuged at 10,000 × g for 5 min at 4 °C, and the isopropanol removed. The obtained pellet was washed with 70% ethanol and centrifuged at 10,000 × g for 1 min at 4 °C. The ethanol was completely removed, and the pellet dissolved in DEPC-treated water. The DNA and RNA were purified and separated with the AllPrep DNA/RNA kit (Qiagen) following the manufacturers protocol that was modified to treat the samples with either DNase or RNase (Ambion) for 5 min before the washing steps. The DNA and RNA concentration was determined with the use of a Qubit fluorometer (Qiagen) and the RNA quality assessed by bioanalyzer. The extracted DNA and RNA were finally pooled to a final concentration of 10 μg for genomic and transcriptomic library preparation.Female coffee berry borers had been continuously reared on a meridic diet in the laboratoryGenomic DNA from whole coffee berry borers was sheared to 350 and 550 bp sized fragments. To remove any amplification bias at the PCR step of the library preparation, noPCR libraries were prepared from coffee berry borer DNA using Illumina TruSeq DNA PCR-Free LT Sample Preparation Kit according to manufacturer’s instructions. Briefly, 1–2 ug of genomic DNA was fragmented using Covaris shearing with prescribed settings for 350 or 550 bp insert sizes. The fragmented DNA was cleaned, end-repaired, and selected for appropriate insert sizes (350 bp and 550 bp) using different ratios of Sample Purification Beads. This was followed by 3′ end adenylation and adapter ligation. Libraries were sequenced in Illumina Hiseq2000 with 100 bp paired-end reads.Strand-specific mRNA sequencing libraries were prepared from total RNA of coffee berry borer using TruSeq Stranded mRNA Sample Prep Kit LT (Illumina) according to manufacturer’s instructions. Briefly, polyA + mRNA was purified from total RNA using oligo(dT) Dynabeads (Life Technologies) selection. First strand cDNA was synthesized using randomly primed oligos followed by second strand synthesis where dUTPs were incorporated to achieve strand-specificity. The cDNA was adapter-ligated and the libraries amplified by PCR. Libraries were sequenced in Illumina Hiseq2000 with paired-end 100 bp read chemistry.The genomic reads were cleaned with TrimmomaticAfter quality filtering, a total of 62 Gbp of sequence data (36 Gbp from the 350 bp insert library and 26 Gbp from the 550 bp insert library) was used for genome assembly. With an estimated genome size of 200 Mb, this corresponds to an average depth of coverage of 180-fold. Sequence reads were assembled using SOAPdenovo2 to form contigs and scaffolds resulting in 156 Mb in contigs and 163 Mb in scaffolds .


To examine whether elevated anxiety and/or depressive symptoms are related to all-cause mortality in people with Type 2 diabetes, not using insulin.1c, cardiovascular disease and microvascular complications.948 participants in the community-wide Nord-Trøndelag Health Survey conducted during 1995–97 completed the Hospital Anxiety and Depression Scale with subscales of anxiety (HADS-A) and depression (HADS-D). Elevated symptoms were defined as HADS-A or HADS-D ≥8. Participants with type 2 diabetes, not using insulin, were followed until November 21, 2012 or death. Cox regression analyses were used to estimate associations between baseline elevated anxiety symptoms, elevated depressive symptoms and mortality, adjusting for sociodemographic factors, HbA1c attenuated this relation . Those with elevated depression symptoms alone had an increased mortality risk . Having both elevated anxiety and depressive symptoms was not associated with increased mortality risk .At baseline, 8% (n = 77/948) reported elevated anxiety symptoms, 9% (n = 87/948) elevated depressive symptoms and 10% (n = 93/948) reported both. After a mean follow-up of 12 years , 541 participants (57%) had died. Participants with elevated anxiety symptoms only had a decreased mortality risk . Adjustment for HbAElevated depressive symptoms were associated with excess mortality risk in people with Type 2 diabetes not using insulin. No significant association with mortality was found among people with elevated anxiety symptoms. Having both elevated anxiety and depressive symptoms was not associated with mortality. The hypothesis that elevated levels of anxiety symptoms leads to behavior that counteracts the adverse health effects of Type 2 diabetes needs further investigation. People with Type 2 diabetes are more likely to experience elevated depression and/or anxiety symptoms or an anxiety disorder, than people without diabetes –3. While1c levels [n = 125) or included clinical samples only, and only one study included only patients with Type 2 diabetes [Depression was found to be associated with a higher risk of adverse diabetes outcomes and higher mortality rates . Depressc levels , developc levels  and incrc levels . Howeverc levels . Unfortudiabetes . More rediabetes –12. LongThere is evidence from longitudinal cardiovascular studies that having symptoms of both anxiety and depression predicts poor health outcomes –15. It iThe Second Nord Trøndelag Health Study (HUNT2) was conducted in one large Norwegian county during 1995–1997, inviting all residents aged ≥ 20 years. The population is ethnically homogenous, with only a small percentage (3%) of people of non-Caucasian origin . In totaOf 1124 participants with diabetes who were not treated with insulin, 948 completed the Hospital Anxiety and Depression Scale (HADS) with valid answers on the anxiety (HADS-A) and the HADS depression (HADS-D) subscale  ; the anaThe HADS was used to assess symptoms of anxiety and depression . This se1c). HbA1c measurements were reported in International Federation of Clinical Chemistry units (mmol/mol) in addition to derived NGSP units (%) (http://www.ngsp.org/convert1.asp).In the general baseline questionnaire participants were asked about age, gender and their level of education. In the analyses education was categorized as a low (<10 years) or high (≥10 years) level. Living alone was categorized as yes/no. Questions related to cardiovascular disease included having a history of angina pectoris, myocardial infarction or stroke. These three conditions were combined into a composite measure of any cardiovascular disease (yes/no). The diabetes-specific questionnaire included a question about the presence of eye problems due to diabetes. Participants reported having diabetes received tubes for collecting three consecutive first-morning urine samples, which were analyzed for albumin and creatinine levels . We defiInformation on date of death was obtained from the Norwegian Population Registry. We followed participants from the baseline examination to time of death (end point) or November 21, 2012 (end of the follow-up period), whichever came first.The Mid Norway Regional Committee for Medical and Health Research Ethics approved the study protocol (2011/1157), and the Norwegian Social Science Data Services approved merging the HUNT-2 data and the NorPD registry.p-value <0.05 was considered statistically significant. Using earlier definitions of elevated symptoms of anxiety and depression [2 test for categorical variables. Three planned comparisons were performed in case of statistically significant overall results, comparing the depressed-only, anxious-only and comorbid group with the non-anxious and non-depressed group as the reference group. The log-rank test was used for an overall comparison of survival curves for the four groups .All analyses were performed using IBM SPSS Statistics version 19.0), Somers, NY, USA. A .0, Somer1c (model 2); and (iii) after additional adjustment for the presence of any cardiovascular disease or microvascular complications (model 3).A hierarchical Cox regression analysis was then used to examine whether group status (using the group without anxiety and depressive symptoms as the reference category) was associated with increased mortality risk in an unadjusted analysis; (i) after adjustment for the demographic variables age (continuous), male sex (no or yes), level of education (high or low), and living alone (no or yes) (model 1); (ii) after adjustment for demographic variables and HbAWe compared those who completed the HADS-A and HADS-D with those who did not (fewer than 5 items), with regard to demographics and clinical variables. Furthermore, to assess the validity of the findings, we examined whether those not completing either the HADS-A or HADS-D had increased mortality risk. Cox regression analyses were used, unadjusted and with adjustments (model 3).We conducted additional sensitivity analyses with a higher cut-off point (≥ 11) on either or both of the sub-scales to indicate moderate-severe symptomatology or clinically significant disorder .1c level was 61mmol/mol (SD 19) (i.e. 7.7% (SD 1.7)). Cardiovascular disease and microvascular complications were present in 27% and 33% of participants, respectively. Seventy-three percent of the sample (n = 691/948) did not have elevated anxiety or depressive symptoms, 8% (n = 77) had elevated anxiety symptoms only, 9% (n = 87) had elevated depressive symptoms only, and 10% (n = 93) had both. When comparing these four groups with respect to demographic and clinical characteristics, significant overall differences were found for age, HbA1c level and eye problems due to diabetes (1c levels (p<0.001); those with elevated depressive symptoms only were older (p<0.001) and those with both elevated anxiety and depressive symptoms had more eye problems due to diabetes (p<0.001) compared with individuals in the reference group , mean age was 67 (SD 12) years (range 20–90 years) and 49% were men. Mean HbAdiabetes . Plannedce group .p<0.001) . Crude survival analyses for these three comparisons showed that compared to the reference group of people with neither elevated anxiety nor depressive symptoms, the mortality risk was decreased for participants with elevated anxiety symptoms only  (p = 0.03), and increased for participants with elevated depressive symptoms only  (p = 0.001) (p = 0.58). Adjustment for demographic variables gave similar results for all three comparisons  . In the fully adjusted Model 3, persons with elevated depressive symptoms only had an increased mortality risk compared to the reference group  (p = 0.02).After a mean follow-up of 12 years , 541 people had died (57%). The survival curves for the four groups differed significantly (p<0.001) . The pro= 0.001) . MortaliModel 1; . HoweverSome individuals did not complete either the HADS-A (15%) or HADS-D (10%) subscale, or neither. Those not completing either scale were older, more likely to be women, to have lower education, more cardiovascular disease and microvascular complications. Those not completing the HADS-D were more likely to live alone. Non-responders on HADS-A had an increased mortality risk compared to responders in the unadjusted analysis  (p<0.001) and in the fully adjusted Model 3  (p = 0.02). Non-responders on HADS-D had an increased mortality risk compared to responders in the unadjusted analysis  (p<0.001) and in the fully adjusted Model 3  (p<0.001).The results from the sensitivity analysis with groups defined by HADS ≥ 11 were analogous to our results for HADS ≥ 8. Compared to the reference group (n = 790), we observed an increased mortality rate among participants with elevated depression symptoms only (n = 30)  (model 3). Probably due to a lack of power, this association was not significant. The mortality rate did also not differ statistically between the reference group and participants with elevated anxiety symptoms only (n = 39)  and those with co-morbid elevated anxiety and depressive symptoms (n = 25) .1c, and comorbidities. In contrast, anxiety only  was associated with a 34% decreased mortality risk in unadjusted analysis. However, this association was attenuated to a 27% and no longer statistically significant reduced risk after adjustment for differences in demographics and baseline HbA1c. Having both elevated anxiety and depression symptoms was not associated with increased or decreased mortality risk.In this 17-year follow-up study of 948 people with Type 2 diabetes not using insulin, baseline depression only  was associated with a 39% increased mortality risk after adjustment for demographics, HbAOur study is the first to investigate the associations between increased baseline symptom levels of anxiety, depression (and their co-morbidity) and subsequent mortality in a large community-based population of people with insulin naïve Type 2 diabetes. The finding that particularly depression is associated with increased mortality rates is in line with results of a meta-analysis which showed that depression was associated with a 46% higher mortality rate in persons with diabetes . Cuijper1c in those reporting elevated anxiety symptoms. Two recent cross-sectional studies among people with Type 1 diabetes investigated the association between anxiety and HbA1c levels [1c. However, results in the study of Anderbro [1c. Similarly in our study, participants with elevated anxiety symptoms only had lower HbA1c levels compared with individuals with no elevated level of anxiety or depressive symptoms. In contrast to participants with Type 1 diabetes in the study of Anderbro [1c may be that anxious persons with diabetes are more cautious about their blood glucose level and worry about diabetes-related complications such as macro- and microvascular complications. Also, anxious people with insulin-naïve Type 2 diabetes more often fear hypoglycemia, insulin injections or blood glucose self-testing, a fear which has shown to be a barrier to insulin treatment in previous studies [1c levels increased and people subsequently started with insulin treatment which is associated with a worse prognosis or more advanced disease. Treatment adherence may be a mediator into the association between elevated anxiety symptoms and lower HbA1c levels. In this case, elevated anxiety scores might be a marker for a dedicated attitude towards minimizing health risks. It could be speculated that better treatment adherence is more often adopted as coping style in anxious people compared to other participants with less anxiety [Longitudinal studies focusing on the relation between anxiety and health outcomes are lacking in people with Type 2 diabetes. In this follow-up study, reporting elevated anxiety symptoms only was associated with a decreased mortality risk, partly explained by more optimal levels of HbAc levels , 23. Thec levels  showed nAnderbro . One exp studies , 25. Pre studies . In a lo studies . Other s studies  and it i anxiety . Treatme1c levels.Furthermore, we found that having elevated symptoms of both anxiety and depression was not associated with an increased mortality rate. Lou et al.  found dia priori aim to test depressive symptoms as a risk factor for mortality, with depression being measured in an early, relatively uncomplicated stage of diabetes. Depression may be a consequence of complications of diabetes, which again is associated with increased mortality risk, but this was not the focus of the paper. We therefore requested data from participants with type 2 diabetes who were insulin-naïve at baseline and data from participants who using insulin at baseline are thus not available to us. Hence, we do not have the possibility to re-run the analysis including those using insulin. It is a strength is that the self-reported assessments used do not include somatic symptoms of depression or anxiety thus diminishing the effect of somatic illness such as e.g. diabetes. On the other hand, assessment of depression and anxiety symptoms was by means of a self-report measure, while a psychiatric diagnostic interview is regarded as the gold standard for assessing mood and anxiety disorders. We are aware of the fact that the validity of the HADS questionnaire has been the subject of a recent debate. Coyne et al [Our study has several strengths. We have studied anxiety and depression as risk factors early in the course of the disease by including the large, relatively homogenous sample of insulin naïve people with Type 2 diabetes who were followed for a relatively long period with mortality as the endpoint. At the same time, this is also a limitation, as we could not compare our sample to people using insulin. However, it was our ne et al  stated tne et al  stated ine et al  has showne et al . Fourth,1c levels. This may be interpreted as lending weight to a hypothesis of elevated levels of anxiety symptoms leading to behavior that counteract the adverse health effects of Type 2 diabetes. This hypothesis needs further investigations in future research.In sum, in a cohort of insulin-naïve participants with Type 2 diabetes followed over a period of 17 years, an elevated level of depressive symptoms (without elevated anxiety symptoms) was associated with a 39% increased mortality risk. A similar association was not observed among people with elevated anxiety symptoms only. While decreased mortality was observed in this group, it was in part explained by lower HbA


The aim of this study was to provide evidence for further in vivo maturation of insulin-producing cells (IPCs) derived from human bone marrow-derived mesenchymal stem cells (HBM-MSCs). HBM-MSCs were obtained from three insulin-dependent type 2 diabetic volunteers. Following expansion, cells were differentiated according to a trichostatin-A/GLP protocol. One million cells were transplanted under the renal capsule of 29 diabetic nude mice. Blood glucose, serum human insulin and c-peptide levels, and glucose tolerance curves were determined. Mice were euthanized 1, 2, 4, or 12 weeks after transplantation. IPC-bearing kidneys were immunolabeled, number of IPCs was counted, and expression of relevant genes was determined. At the end of in vitro differentiation, all pancreatic endocrine genes were expressed, albeit at very low values. The percentage of IPCs among transplanted cells was small (≤3%). Diabetic animals became euglycemic 8 ± 3 days after transplantation. Thereafter, the percentage of IPCs reached a mean of ~18% at 4 weeks. Relative gene expression of insulin, glucagon, and somatostatin showed a parallel increase. The ability of the transplanted cells to induce euglycemia was due to their further maturation in the favorable in vivo microenvironment. Elucidation of the exact mechanism(s) involved requires further investigation. Human embryonic stem cells (h-ESCs) can be expanded and differentiated into all cell types, including insulin-producing cells (IPCs) [β-cells is very attractive, the most successful differentiation protocols have produced only a modest number of functional IPCs [In the year 2000, it was estimated that 150 million people were affected by DM, and this number is expected to double in 2025 [1C level . Recent s (IPCs) –6. Howevs (IPCs) . The sucs (IPCs)  However,s (IPCs) . IPCs cas (IPCs) , 11. This (IPCs) –14 and anal IPCs . In a prnal IPCs . Despitenal IPCs , 19. TheThe required approvals for all the procedures in this study were obtained from the ethical committee of the University of Mansoura. Bone marrow aspirates (BMAs) were collected in heparin from the iliac crests of three consenting donors. All donors were type II, insulin-requiring diabetic patients.6 nucleated cells. The collected cells were cultured in complete DMEM at a density of 5 × 105 cells/mL (10 mL in 25 cm2 tissue culture flasks) and incubated at 37°C in a 5% CO2 incubator. Aliquots were preserved in liquid nitrogen for subsequent expansion and examination. After 3 days, the nonadherent cells were discarded. The adherent MSCs were cultured to 80% confluence before passaging using trypsin. The cells were resuspended in complete DMEM and reseeded at a ratio of 1 : 2 and cultured for another 8 days to reach 80% confluence. This step was repeated for a second passage. At this point, the cells were spindle-shaped and displayed a fibroblast-like appearance. The samples from each donor were examined in duplicate for the in vitro and in vivo components of this study.The BMAs were diluted 1 : 1 in low-glucose Dulbecco's modified Eagle's medium  layered atop a density gradient   and centrifuged for 20 min at 600 ×g. The cells were collected from the DMEM/Ficoll interface, washed twice in phosphate-buffered saline (PBS), and resuspended in 10 mL of low-glucose complete DMEM , 100 U/mL penicillin, and 100 U/mL streptomycin (Sigma)). One milliliter of BMAs yielded ~1.5 × 106 cells/mL. Aliquots of 100 μL were incubated for 30 min in 20 μL of antibodies against CD14, CD45 (FITC) or CD73, CD34 phycoerythrin (PE) or in 5 μL of CD105 PE or CD90 (FITC) , washed with 1 mL of stain buffer , and resuspended in 500 μL of stain buffer. The labeled cells were analyzed using an argon ion laser at a wavelength of 488 nm . A total of ten thousand events were obtained and analyzed using CellQuest software . Control staining using the appropriate isotype-matched monoclonal antibodies was included.HBM-MSCs at passage 3 were trypsinized, centrifuged at 300 ×g for 8 min, and resuspended in PBS at a concentration of 1 × 10HBM-MSCs were induced to differentiate into adipocytes, chondrocytes, and osteocytes using a previously described differentiation protocol . Oil-RedDifferentiation was performed according to a protocol previously reported by Tayaramma et al. . Initial6 of cells obtained at the end of in vitro differentiation were suspended in 20 μL of culture medium and implanted beneath the renal capsule of each mouse. The surviving animals were sacrificed 1, 2, 4, or 12 weeks after transplantation. Before euthanization, blood samples were obtained from the tail vein and measured for blood glucose levels using glucometer strips , serum human insulin, serum human c-peptide, and serum mouse insulin levels by ELISA . In addition, an oral glucose tolerance test was performed: 1 g/kg glucose was administered orally via gavage. Blood samples were collected before glucose administration and after 30, 60, 90, and 120 min. Measurements of the glucose and human c-peptide levels were performed on the obtained samples.The ability of the differentiated cells to induce normoglycemia in diabetic nude mice  was examined following implantation of these cells into the renal subcapsular space. Diabetes was chemically induced using a single dose of 220 mg/kg of streptozotocin . The mice were considered diabetic once the blood glucose levels exceeded 350 mg/dL for 2 consecutive readings. Twenty-nine animals at an average age of 12 weeks were utilized. The diabetic mice were anesthetized via intraperitoneal injection of ketamine (100 mg/kg) and diazepam (5 mg/kg). A total of 1 × 10The HBM-MSC-bearing kidneys of the euthanized animals were divided into halves. One-half was immunolabeled for histological analysis and to count the insulin-positive cells. The expression of relevant endocrine genes was determined in the other half. The pancreas of these animals was also harvested and immunostained for insulin.An Alexa Fluor 488-conjugated rabbit monoclonal antihuman insulin antibody  was used for flow cytometry. The primary antibodies utilized for immunocytochemistry and immunohistochemistry included mouse monoclonal antihuman insulin, rabbit monoclonal anti-human GCG, rabbit polyclonal anti-human c-peptide , and rabbit polyclonal antihuman somatostatin (SST) . The employed secondary antibodies were Alexa Fluor 488-conjugated anti-mouse IgG (H + L) and Alexa Fluor 555-conjugated anti-rabbit IgG (H + L) .At the end of in vitro differentiation, the cells were fixed in 4% formaldehyde for 10 min at 37°C, permeabilized using chilled 90% methanol for 30 min and blocked in incubation buffer for 10 min at RT. Then, the cells were incubated in the conjugated antibody for 60 min at RT. Next, the cells were washed with incubation buffer and, after centrifugation, were resuspended in 0.5 mL PBS. The labeled cells were evaluated using a 15 mW argon ion laser at a wavelength of 488 nm . A total of ten thousand events were obtained and analyzed using CellQuest software . Mouse pancreatic islets served as a positive control.Cell preparations were cultured on chamber slides . Then, the cells were fixed in 4% paraformaldehyde, permeabilized using chilled 100% methanol for 10 min, blocked with 5% normal goat serum for 60 min at RT, and incubated overnight in the primary antibodies at 4°C. Subsequently, the cells were washed with PBS and incubated in the secondary antibodies for 2 hours at RT. Negative controls were performed by omitting treatment with the primary antibody.The harvested organs were fixed in formalin and sectioned on coated on positively charged adhesion slides . Then, the slides were deparaffinized using xylene and a decreasing ethanol gradient. The antigens were unmasked by boiling the slides in 10 millimoles sodium citrate buffer (pH 6.0) and maintaining subboiling temperature for 10 min. The sections were blocked with 5% normal goat serum and incubated overnight in the primary antibody at 4°C. Then, the slides were washed 3 times in PBS and incubated in the secondary antibody for 2 hours at RT. The nuclei were counterstained using DAPI. ImageJ software (developed by NIH) was used to determine the proportion of transplanted cells beneath the renal capsule that intracytoplasmically expressed insulin. To this end, ten fields were randomly selected for cell counting which was carried out by two independent histopathologists. The results from all fields were calculated and expressed as the mean proportion of insulin-positive cells out of the total transplanted cells. In all the above studies, confocal images were captured using a Leica TCS SP8 microscope .For immunolabeling of the native pancreas, the primary antibody used was mouse monoclonal anti-insulin (L6B10) , and the secondary antibody was the power-stain 1.0 poly-HRP DAB Kit for mouse . The sections were examined under light microscopy.μL reaction volume consisting of 12.5 μL of 2× TaqMan Master Mix , 1 μL of cDNA template, 1.5 μL of the primers, and 10 μL of nuclease-free water. The plate was inserted into a real-time thermal cycler  that was programmed according to the manufacturer's instructions. The procedure was performed in duplicate for each sample. A mathematical model introduced by Pfaffl was used for the relative quantification of target genes [Total RNA was extracted from the undifferentiated cells at the end of in vitro differentiation and from the cells transplanted beneath the renal capsule using the RNeasy plus mini kit . Three micrograms of total RNA were converted to cDNA using the RT first Strand kit . Custom gene arrays were designed and supplied as the Quantifast Probe Assay . Gene expression was examined for endocrine hormones , transcription factors , an endocrine precursor marker (nestin), a glucose transporter (Glut-2), and a pancreatic enzyme (glucokinase (GCK)). Human islets and GAPDH were included as positive and internal controls, respectively. Amplifications were performed in each well using a 25 et genes . In thisP values were corrected using Bonferroni adjustments. A P value of <0.05 was considered significant. The mean values were used as a measure of variation. The median values were utilized only if there were extreme observations.Nonparametric data were evaluated using Friedman's test. Post hoc analysis was performed using the Wilcoxon signed-rank test, and the http://dx.doi.org/10.1155/2015/575837). These cells could be differentiated to form adipocytes, chondrocytes, and osteocytes when the appropriate growth factors were added . Accordingly, evidence for their multilineage potential was confirmed.At the end of expansion, the cultured cells became spindle-shaped, fibroblast-like cells that arranged in monolayers. Flow cytometry revealed that these cells expressed high levels of CD73, CD90, and CD105 but negligible levels of CD14, CD34, and CD45 . Thereafter, the animals remained euglycemic throughout the observation period. The serum levels of human insulin and human c-peptide were measurable one week after transplantation, and these values also remained unchanged throughout the observation period. Serum levels of mouse insulin became negligible after induction of diabetes . The resImmunohistochemistry of the HBM-MSC-bearing kidneys revealed that the percentage of IPCs increased gradually, peaking at 4 weeks after transplantation (~18%) without any substantial change thereafter . ImmunolAt the end of the in vitro differentiation period, relevant endocrine genes were expressed, although at approximately 1/1000 that in human islets. After transplantation, there was a significant increase in the relative gene expression values of the transcription factor PDX1, the endocrine hormones insulin, GCG, and SST, the glucose transporter GLUT-2, and the pancreatic enzyme GCK. This increase peaked by the fourth week after transplantation. At 12 weeks after transplantation, there was a decline in the gene expression of insulin, GCG, and GCK  . It was IPCs can be generated from HBM-MSCs via directed differentiation, although the yield is meager. However, transplanting these cells results in the normalization of blood glucose levels in diabetic animals. Evidence was provided for the further maturation of these cells in vivo. The proportion of IPCs increased by 10-fold 4 weeks after transplantation. All relevant pancreatic endocrine genes were expressed, and their expression levels were significantly increased in vivo. The glucose-tolerance curves and the simultaneously measured c-peptide levels demonstrated that these cells are glucose-responsive and insulin-secreting. Our experiments indicated that only a subpopulation of MSCs are capable of differentiation into the pancreatic lineage. The identification, sorting, and expansion of this cellular subpopulation before differentiation would optimize the yield. However, several questions remain: how long will these cells will retain their function? What is the optimal site for their transplantation? What are the risks of teratogenicity? Once these questions are adequately addressed, meaningful clinical applications can be developed.Supplementary Material: In figure (1), the multilineage potentials of HBM-MSCs intoadipocytes, chondrocytes and osteocytes had been provided. The cytoflowmetric characterization of HBM-MSCs is shown in table (1). Table 2 shows the glucose tolerance data of the transplanted animals at different time points. In addition to glucose levels, c-peptide values were also determined. The percentage of insulin-positive cells at end of differentiation and following transplantation are given in table 3. Table 4, demonstrates data of the relative gene expression of relevant pancreatic endocrine genes at end of differentiation as well as following transplantation.


A representative cross-sectional study showed that chronic itch (lasting for a minimum of 6 weeks) affects 25.2 %  of hemodialysis (HD) patients. Pathophysiology and etiology of chronic itch (CI) in HD are still unclear.We investigated 860 HD patients from a representative randomly selected cluster-sample considering the regional distributions of dialysis units in GermanyThe current analyses report comorbidities, laboratory values and dialysis characteristics of HD patients in relation to CI.Diabetes was the only comorbidity that was associated with the occurrence of itch but interestingly with less CI. Except for creatinine, phosphorus, and parathormone, there were no significant associations between the occurrence and characteristics of CI and any laboratory value. Kt/V was not associated with the presence of CI. Patients dialyzed with polyarylethersulfone-membrane showed significantly more CI in all prevalence estimates and those dialyzed with polysulfone-membrane were significantly less affected by CI.Long-term follow-up studies will show if the type of dialysis membrane influences the development of CI in HD patients. It is most likely that several factors e.g. elevated parathormone, origin of end stage renal disease (ESRD), type of dialysis membrane, and a neuropathic component all contribute to the occurrence of CI in HD patients. Future research should consider a multifactorial origin of itch in HD. GEHIS  is a cross-sectional study that investigated 860 hemodialysis (HD) patients from a randomly selected cluster-sample  aiming to identify different prevalence estimates of chronic itch . AccordiThe role of comorbidities in the occurrence of itch in HD patients is unclear and has so far only been investigated for selected comorbidities –5. SinglThe study (GEHIS) was established as a prospective observational prevalence study in 2012 and was conducted between May and September 2013. Eligible patients were diagnosed with ESRD and were undergoing chronic hemodialysis (HD) treatment. The study was established according to the guidelines of good clinical practice and conducted in full accordance with the protocols of the World Medical Association’s Declaration of Helsinki. Ethical approval was obtained from the University of Heidelberg, Germany (No. S-648/2012). Patients gave their written informed consent. All results were reported in line with the “Strengthening the Reporting of Observational Studies in Epidemiology (STROBE)” recommendations [The current analyses are based on a data set obtained in every patient including the etiology of ESRD  and sociodemographic data  Routine laboratory investigations were usually performed every four weeks (except for PTH). Laboratory values closest to the date of investigation were transferred from the laboratory chart: leukocytes (G/l), haemoglobin (Hb) (g/dl), haematocrit (Hct) (%), platelets (G/l), albumin (g/dl), creatinine (mg/dl), urea (mg/dl), glucose (mg/dl), sodium (mmol/l), potassium (mmol/l), calcium (mmol/l), phosphate (mmol/l), C-reactive-protein (CRP) (mg/dl), iPTH (ng/L). PTH values older than 6 month were not considered. Different assays have been used for PTH measurement with different reference values. This was considered in our analyses. The following laboratory values were also collected if available in the chart: magnesium, alkaline phosphatase, transaminases, gamma-GT, serum albumin, IgE, eosinophils, hepatitis and HIV-status.An adapted version of the Charlson Comorbidity Index was used to assess comorbidities in patients with ESRD , 16. AddKt/V was extracted from the laboratory files selecting the last available value within the last 4 months. Calculation of Kt/V was performed in accordance to the German quality assurance regulation (single pool) using the Daugirdas approximation by each centre . The folSPSS (version 20) for Windows. Nominal and ordinal data were analyzed by computing absolute (n) and relative frequencies (%), respectively, and 95 % confidence intervals were computed. First chi2 statistics were used to identify variables that were significantly associated with CI in univariate analysis. P-values below 0.05 were considered significant. Comparisons between HD patients suffering from CI and HD patients not affected were conducted by independent t-tests/ANOVA for the continuous variables  and by chi2-test for binary distributions. Associations between laboratory findings and the occurrence of CI are reported by Pearson’s correlation coefficients. To deal with the problem of multiple testing when analyzing differences in laboratory values, a Bonferroni correction was conducted, setting the significance cut-off at α/n.For data management, a Microsoft Access 2003 database was used. Data entry was conducted twice by two independent persons. To maximize data quality, all observed random or potentially systematic inconsistencies within resulting data were solved. Statistical analyses were performed using The study sample has been described in detail elsewhere . 860 HD n = 327), congestive heart failure in 24.7 % (n = 212) and peripheral arterial occlusive disease in 21.2 % (n = 182) (Table Leading comorbidities according to Charlson Comorbidity Index adapted for patients with ESRD were diabetes mellitus in 38.0 % (7 % n = 22 and pern = 753) of the HD patients suffered from arterial hypertension, 93.7 % (n = 806) from anemia and 83.8 % (n = 721) from hyperparathyroidism. Dyslipidemia was observed in 40.8 % (n = 351), osteoporosis in 5.5 % (n = 47). These comorbidities did not show any significant association with the prevalence of CI  Table . There w % n = 72 from hypThere were no associations of any comorbidities, laboratory values, or type of dialyzer membrane used with the characteristics of itch e.g. localization and severity of CI.All laboratory values are presented in Table According to the current quality criteria on HD treatment in Germany, HD patients undergo at least 4 h HD dialysis treatment 3 times weekly and HD treatment aims at a Kt/V of >1.3. The mean dialysis efficacy (Kt/V) was 1.5  Tables  and 3. TRegarding the methods of dialyzing used, HD treatment was the leading procedure (80.0 %), followed by post-dilution hemodiafiltration (17.2 %) and pre-dilution hemodiafiltration (2.0 %). A significant association of the dialyzing method and the occurrence of CI did not occur. Also differences in dialyzers used (high-flux 88.7 % and low-flux 11.3 %) were not significantly associated with the occurrence of CI. However, the difference in sample size of these two groups may explain why we could not detect significant differences.Steam was the most commonly used method of sterilization in both blood lines 43.9 %) and dialyzers (79.5 %) (Table .9 % and n = 668) and low molecular weight heparin in 16.5 % (n = 135). 1.8 % received another medication for anticoagulation (n = 15). There was no significant association of the type of heparin and current CI  but low molecular heparin use was significantly associated with a higher prevalence of CI ever experienced in life. Multiple testing showed that CI was significantly more prevalent  in low molecular heparin use compared to those patients who received another type of anticoagulation . There was no significant association between heparin used and any other factors previously identified e.g. self-reported dry skin, eczema Anticoagulation was documented for 818 patients. Out of those, unfractionated heparin (UFH) was used for anticoagulation in 81.7 % (n = 544), polyarylethersulfone , polyethersulfone , polymethylmethacrylat  and polyacrylonitril   . It has The current analyses confirm previous research about the high frequency of comorbidities like congestive heart failure and hypertension in HD patients. Unlike others showing significant differences between patients with and without CI for congestive heart failure and peripheral vascular disease (significantly more frequent in patients with CI), we cannot confirm this. This may be explained by the large numbers of patients investigated in the DOPPS study increasing the likelihood of detecting significant differences , 5. WherPrior investigations suggest optimal dialysis efficacy in terms of higher Kt/V and better nutrition leading to a reduction of uremic itch . It is nSo far, there is no systematic study investigating the role of dialyzer membranes and the development of itch, especially chronic itch. A smaller study observed that patients dialyzed with polysulfone membranes suffered significantly more frequent from CI than those on hemophane and cuprophane membranes Heparin respectively the type of heparin used during HD treatment has not been reported to be relevant for CI in HD patients but interestingly, heparin treatment was suggested for treatment of uremic itching , 30. We As previously discussed some limitations of the study need to be considered and for the current analyses the following ones need to be mentioned . We were


TMEM67 .We report the first genetically proven case of COACH syndrome from the Indian subcontinent in a 6-year-old girl who presented with typical features of Joubert syndrome along with hepatic involvement. Mutation analysis revealed compound heterozygous missense mutation in the known gene  TMEM67 gene are responsible for the majority of COACH syndrome, with minor contributions from CC2D2A and RPGRIP1L [ TMEM67 gene.COACH syndrome  is a rare autosomal recessive multisystemic disorder first proposed by Verloes and Lambotte . MutatioRPGRIP1L . Our patA 6-year-old girl presented with global developmental delay, large head, and ataxic gait. The proband was the only child born to nonconsanguineous couple at term after LSCS with birth weight of 3.5 kg. Her motor developmental milestones and speech were grossly delayed.On examination, her weight was 18.2 kg (50th centile), height 107 cm (50th centile), and head circumference 53 cm (75th–95th centile). Facial features revealed large head, frontal bossing, and hypertelorism with squint. There was pectus excavatum, bilateral clinodactyly, and hepatomegaly (liver span: 10 cm). Neurologically, there was ataxic gait with generalized hypotonia. Eye evaluation showed squint in the right eye (30-degree exotropia) with normal fundus.Investigations showed normal metabolic parameters except elevated liver enzymes  with normal bilirubin (0.68 mg). MRI brain revealed “molar tooth appearance”  and “Bat TMEM67 with father and mother being carriers for c.1413-1G>A and c.434T>G, respectively.In view of MRI findings with deranged liver function test, COACH syndrome was suspected. Mutation analysis showed the patient to have a compound heterozygous missense mutation at position c.1413-1G>A and c.434T>G in the geneJoubert syndrome (JS) is a rare genetic disorder affecting the cerebellum characterised by the absence or underdevelopment of cerebellum and malformed brain stem. Diagnosis is based on neurologic signs of hypotonia, ataxia, developmental delay, and oculomotor apraxia along with the “molar tooth sign” (MTS) on MRI, which is the neuroradiologic hallmark of the condition. Disorders that share the MTS have been termed Joubert syndrome (JS). JS comes under the ciliopathies, which are a group of disorders with genetic mutations encoding defective proteins which result in abnormal formation or function of cilia .Cilia are classified into motile and immotile (primary). All the causative JS genes identified to date encode proteins that are localized to the base or axoneme of the cilium . CombinaMotile cilia when defective cause primary ciliary dyskinesias, which comprise a heterogenous group of disorders characterised by bronchiectasis, left right asymmetry, and infertility . Immotil TMEM67. Brancati et al. [ TMEM67 gene.COACH syndrome OMIM 21630) is considered by some to be a subtype of Joubert syndrome with congenital hepatic fibrosis. The key feature of COACH is congenital hepatic fibrosis (CHF), resulting from malformation of the embryonic ductal plate [630 is coi et al.  identifiIn our patient, we found hepatomegaly and raised liver enzymes with typical features of Joubert syndrome in MRI (molar tooth sign and batwing sign) suggesting COACH syndrome. Clinically, the liver disease can vary from raised liver enzymes to features of portal hypertension . Identif TMEM67 gene in this patient further delineates the genotype-phenotype correlation in COACH syndrome. JSRD patients with known liver involvement should be tested first for TMEM67 mutations, followed by CC2D2A and RPGRIP1L. Presymptomatic, gene based diagnosis should make it possible to deliver improved care for systemic complications and prenatal identification.Renal manifestations, reported in subjects with JSRD, were not present in our case. Thus, the presence of mutation in


Psoriatic arthritis (PsA) disease activities at baseline may determine physical function over time. There is no longitudinal data on course of physical function in PsA patients from Asia. We aim to describe variables associated with a deterioration of physical function in PsA in Chinese over a 6-year period.125 consecutive patients with PsA fulfilled the CASPAR criteria from a rheumatology outpatient center were recruited to give sociodemographic and clinical data in 2006 to 2008. Follow up interviews were conducted in 2012 to 2013 to assess physical function using Health Assessment Questionnaire (HAQ). Regression models were constructed to determine baseline variables that predict physical function on follow up.A total of 97 patients completed the follow up survey, with mean follow up time of 6.2 (±0.7) years, response rate 77.6%. PsA patients had poor physical function and health related quality of life (HRQoL) compared to normal population. There were 33% who improved in disability status and 41.2% had persistent minimal disability by HAQ categories (HAQ 0–0.49) over time. There were 14.4% of the patients who had persistent moderate disability (HAQ 0.5-1.50) and 10.3% had deterioration in disability status. There were 17.5% of patients who had deterioration in physical function as defined by an increment of HAQ score of more than 0.2 at follow up survey. Age, physical function at baseline and the number of damaged joint were significantly related HAQ at follow up.Chinese patients with PsA had had poor physical function and quality of life. One fifth of patient experienced deterioration of physical function over time. Joint damage and baseline physical function were important factors associated with poor physical function in PsA over time. Psoriatic arthritis (PsA) is an inflammatory arthritis associated with psoriasis. It affects young adults in their working ages. It has deleterious effects on patients and joint deformities and disease progression have been shown to develop over time–4. We anOne hundred and twenty five consecutive out-patients with PsA followed up in a single center were recruited to assessment using a standardized protocol from January 2006 to May 2008. This rheumatology center overlooks a population size of 628,634 and is the only secondary and tertiary rheumatology referral center in the district. All patients were adults, over 18 year-old and fulfilled the Classification of Psoriatic Arthritis (CASPAR) criteria for PsA[Physical function was assessed by the Health Assessment Questionnaire (HAQ). The ChiSociodemographic variables included age, gender, education level, duration of psoraitc arthritis. Clinical features included swollen, tender and damaged joint count in 66/68/68 diaarthrodial joints and the number of digits with dactylitis (0–20). Damaged joints were defined as joints ankylosed, subluxed, having > 20% decreased range of movement and joints that have undergone joint replacement, arthrodesis, arthroplasty or bone modification alteration. Psoriasis was assessed by using the psoriasis area-and-severity index (PASI). EntheseOn follow up interview, patients were invited to self-fill questionnaires including HAQ, SF36, pain score and patients’ perception of disease activity on 11-point numeric rating scales. Patients were asked if they have ever used disease modifying anti-rheumatic drugs (DMARDs), including methotrexate, sulfasalazine, leflunomide, cyclosporine A, hydroxychloroquine, chloroquine, azathioprine, and others; or biological DMARD, including infliximab, etanercept, adalimumab and golimumab.The study protocols were reviewed and approved by the Joint Chinese University of Hong Kong - New Territories East cluster (CUHK-NTEC) clinical research ethics committees. Prior to the entry to the study, patients were informed of the nature and purpose of the study and signed informed consents.Demographic and clinical characteristics were performed using descriptive parametric or non-parametric statistics as appropriate. The HAQ scores were divided into 3 categories: 0–0.49, 0.5–1.5, and 1.51–3, representing minimal disability, moderate and severe disability. These cut-offs were consistent with past studies on rheumatology arthritis and inflammatory arthritis–30. The Associations of functional outcomes with potential clinical variables were tested with Mann–Whitney U test for categorical variables or Spearman’s correlations for continuous variables. Variables with p value < 0.05 were taken as significant. As for multivariate analysis, the HAQ at follow up were not normally distributed and therefore transformed to Ln (1 + HAQ at follow up) for analysis. In a multivariate analysis, independent variables predicting Ln (1 + HAQ at follow up) were entered into backwards linear regression analysis. All hypotheses were 2-tailed and p value < 0.05 were considered significant.Independent variables entered into linear regression model includes age, gender, duration of PsA (years), Education (years), concurrent illness (yes/no), body mass index, sacroiliitis (yes/no), tender joint count, swollen joint count, damaged joint count, ESR, MASES, PASI, PhGA, PGA, pain score, PCS, MCS, ever used DMARDs and ever used biological DMARDs. Binary logistic regression models were built for the same variables predictive of 1). A deterioration of HAQ disability status; and 2). Deterioration of HAQ scores of more than 0.2 over time. Analyses were performed using the IBM SPSS Statistic Package, version 21.A total of 97 patients completed the follow up survey, after a mean (±SD) follow up time of 6.2 (±0.7) years. PsA affect adults in their middle age and affected individual had poor physical function and consistently poorer health related quality of life (HRQoL) compared to normal population over time. There were 56.7%, 59.8% and 35.1% who had clinically damaged joints, radiographic erosion of hand/ wrist joints and radiographic sacroiliitis at baseline respectively. At baseline, high percentages (41%) of patient have used DMARD therapy; while utilization of biological agents was low due to the non-reimbursement of medical expense. Table There were 41.7% of patients who had persistent minimal disability by HAQ categories (HAQ 0–0.49) over time, while 14.6% had persistent moderate disability (HAQ 0.5-1.50) Table . There wIndependent variables associated with physical function at follow up as measured by HAQ included years of education (p = 0.042), number of tender joint p = 0.033), number of damaged joint (p = 0.008), ESR (p = 0.031), pain score (p < 0.0001), physicians’ global assessment (p = 0.004), the PCS (p <0.0001) and MCS (p = 0.033) of SF36. In multivariate analysis, age, baseline physical function as measured by the PCS, and the number of damaged joint were related to HAQ at follow up  were also associated with poorer physical function over time; but the association became insignificant after adjustment of other factors. This is consistent of our previous cross sectional study, demonstrating the significant social and economic impact of PsA in Chinese, and damaged joint were associated with poor functional status[There have been a few studies evaluating the longitudinal course of physical function in PsA from Caucasian countries, 12. HowWe demonstrated age, educational level, disease activities , disease damage (damaged joint count) and baseline physical function were associated with HAQ over time. In general this is consistent with the Western literature. AdvanciThe strength of our study is the representative sample of PsA in the region with reasonable sample size. Our center is the only secondary and tertiary referral center for PsA in the region, while there is essentially no primary health care other setting that provide long term care for PsA. The response rate at baseline was high and there was an acceptable response rate (77.6%) at the 6-year follow up survey with high completion of dataset. We used standardized and mostly validated outcome measures according to standardized protocol and clinical assessments at baseline were limited to three clinicians experienced in PsA clinical trials. The main limitation of our study is the small sample size. It is the main reason why we were unable to demonstrate predictive variables for deterioration of HAQ using two definitions. Finally, we only have physical function data at 2 time points and thus unable to tell whether functional statuses were fluctuating or gradual declining with time.We demonstrated Chinese patients with PsA had poor physical function and quality of life. One fifth of patient experienced deterioration of physical function over time. Advancing age, baseline physical function and joint damage were important factors associated with poor physical function in PsA over time.


The microbiological procedures for cystic fibrosis (CF) samples of 17 participating Spanish centers were examined to verify their compliance with current international and national guidelines and to implement the best standards of care for microbiology practices. A 47-item questionnaire covering different CF microbiology aspects was sent to participant laboratories. Telephone interviews were performed when necessary. Data about samples processing for bacteria, mycobacteria and fungi were collected.Staphylococcus aureus (59%) and Burkholderia cepacia complex (71%), and of epidemiological typing methods (41%) was reported.Gene sequencing (71%), MALDI-TOF (59%) or both (94%) were available for most laboratories. Susceptibility testing was performed by automated microdilution systems (94%) and manual diffusion methods (59%). However, a low use of selective media for Most Spanish laboratories are in agreement with consensus guidelines for the processing of CF respiratory samples, but need to improve in the use of specific selective media and typing methods for epidemiologic studies.The online version of this article (doi:10.1186/s12866-014-0335-y) contains supplementary material, which is available to authorized users. Cystic fibrosis (CF) disease is produced by mutations in the CF transmembrane conductance regulator gene (CFTR). Altered CFTR leads to the production of viscous secretions in respiratory airways that cannot be cleared by the mucociliary system and patients get chronically colonized by different microorganisms  which cause inflammation, progressive lung destruction and, finally, death by respiratory failure .Pseudomonas aeruginosa and Staphylococcus aureus [Microbiological diagnosis of CF has evolved far beyond the isolation and identification of classic pathogens such as s aureus . Early ds aureus ,3. Contis aureus ,5.Burkholderia cepacia complex (BCC), other non-fermenting Gram negative rods (NFGNR) and nontuberculous-mycobacteria (NTM) [P. aeruginosa or S. aureus can exhibit altered phenotypic characteristics as a result of time-dependent adaptive phenotypic changes to the CF lung, including small-colony variants (SCVs) and hyper-mutable and mucoid strains variants. Correct diagnosis of these phenotypic variants is difficult and has clinical relevance as there is growing evidence that correlates them with multi-drug resistance, persistency phenomena and poorer lung function [High antibiotic pressure and the special environment of the CF lung allow the establishment of multi-drug resistant bacteria that require special techniques for their isolation and/or identification, such as ia (NTM) ,3. In adfunction -9. Moreofunction -14.Laboratories working with CF samples need special procedures and installations as well as specialized microbiologists to provide a correct patient assessment for clinicians ,10-14. ASeventeen hospital microbiology laboratories covering all the Spanish territory were requested to participate by answering to our questionnaire Table  and all http://www.seimc.org) in all cases.The number of CF samples processed weekly by each laboratory varies from 10 to 20 in the majority of cases , N-acetylcysteine (NAC) and dithiothreitol being the most common chemical agents used . Samples pretreatment for NTM culture was reported by 13 laboratories; the combination of NAC plus 2% NaOH being the preferred option , followed by the combination of 3% sodium lauryl sulphate plus 1% NaOH .Quantitative culture for bacterial pathogens was routinely carried out by only 14 laboratories (82%), using calibrated loops (n = 7) or serial dilutions plus whole plate seeding (n = 7). Fungal CF colonizers were cultured routinely in 13 centers and on the clinician’s request in 4. NTM culture of CF samples was reported in 15 (88%) laboratories. Anaerobic bacteria in CF samples were never considered as relevant in 14 centers and cultured in 3 laboratories only under clinician’s request.S. aureus and BCC were not present in all laboratories . For culturing NTM, automated systems with liquid enrichment media were the most frequently used . Sabouraud-chloramphenicol agar was universally used for culturing fungi.The use of culture media for bacterial, mycobacterial and fungal pathogens is shown in Table Identification techniques used by the clinical laboratories are shown in Table Antibiotic susceptibility testing of bacterial CF pathogens was performed by all participant laboratories. Most of them reported the use of automated microdilution systems (n = 16) and agar diffusion techniques, either disk diffusion (n = 2) or gradient strips , or both (n = 10). Automated systems were MicroScan (n = 8), VITEK2 (n = 3) or both (n = 5).Scedosporium spp. (n = 1) and pre-transplant patient status (n = 1). Susceptibility assays are performed by broth microdilution (n = 3), agar diffusion with antifungal gradient strips  or both (n = 4).Antifungal susceptibility is routinely evaluated in 2 (12%) centers and under special conditions in 8 (47%) which include clinician’s request (n = 4), clinically significant isolates (n = 2), treatment refractory cases (n = 2), isolation of Most laboratories (n = 11) report culture results in 3–5 days and the rest in 1–3 days (n = 5) or >5 days (n = 1). Information sent to clinicians of bacterial culture is summarized in Table Clonal relationships between strains for epidemiological studies were performed in 7 (41%) laboratories, the pulse field gel electrophoresis (PFGE) being the main technique used, alone (n = 3) or in combination with multi-locus sequence typing (MLST) (n = 4).P. aeruginosa, S. aureus or BCC, could be misidentified due to their phenotypic variation or to the limitations of the classical culture techniques with these organisms [P. aeruginosa NFGNR, filamentous fungi, certain yeasts and NTM, could also be missed. Standardized microbiological procedures could help to avoid this situation and to better understand its clinical and epidemiological importance. The present work is an overview of the proceedings of the main CF Spanish laboratories that might be used to improve the clinical microbiological procedures in line with current guidelines and with the recommendations of standards of care from the European Cystic Fibrosis Society [A correct processing of CF samples is critical to identify the maximum number of potential pathogens in the respiratory airways. Incorrect results in the isolation, identification and susceptibility testing of CF organisms have negative consequences in the patient’s clinical management and quality of life, and can affect the whole CF community by delaying the implementation of appropriate infection control measures to prevent patient-to-patient transmission -14,16-18 Society ,10,14.Culturing CF samples is one of the most labor-intensive procedures of the Microbiology Laboratory . It requP. aeruginosa. Moreover, it can also serve for monitoring treatment efficacy [Almost all laboratories homogenize sputum with mucolytic agents as currently recommended for the more viscous CF airways secretions, in which microorganisms grow as biofilm-like microcolonies ,17. Howeefficacy ,17.P. aeruginosa morphotype to clinicians, few of them (24%) inform about hyper-mutable traits or SCVs of P. aeruginosa or S. aureus isolates. These variants could modify treatment strategies as they are related to antibiotic resistance and persistency, so informing clinicians about their presence could be clinically and epidemiologically important [While all laboratories report the presence of mucoid mportant -9. Perfomportant ,14,19.S. aureus, P. aeruginosa, BCC and Haemophilus influenzae [P. aeruginosa and H. influenzae isolation, not all of them use specific selective media for S. aureus (59%) and BCC (71%). These rates are considerably low when compared with the corresponding figures in Germany  and USA  [P. aeruginosa. Missing pathogens like BCC or methicillin-resistant S. aureus (MRSA) is especially worrisome since it could have an impact not only at patient level but affecting also the whole CF community by patient-to-patient transmission [P. aeruginosa, and are unreliable for identifying single species of the BCC [American, European and Spanish CF guidelines strongly recommend selective media for the isolation of fluenzae ,14,20. Wctively) ,13. Lacksmission ,11,18. A the BCC ,18,21. P the BCC -25.P. aeruginosa due to unacceptable major  and very major  errors [Another positive result of the study is the use in nearly all laboratories of agar diffusion tests in addition to automated microdilution systems for antimicrobial susceptibility testing. Use of both techniques is especially relevant. Automated microdilution systems alone are not recommended for ) errors ,14. On t) errors .S. aureus, including MRSA, P. aeruginosa and BCC are pathogens with a proved spreading between CF patients but patient-to-patient transmission of S. maltophilia, A. xylosoxidans and NTM has also been suggested [uggested ,27,28. HMycobacterium avium complex and M. abscessus being the most commonly isolated [P. aeruginosa [P. aeruginosa [On the other hand, NTM prevalence has been rising during the last years and has been recently estimated as 10% of all CF patients, isolated . The onlruginosa . Most Spruginosa . Recentlruginosa .Although CF consensus guidelines also recommend fungal cultures ,10,14, nPrevotella spp. [In our study few laboratories cultured respiratory samples under anaerobic conditions. Currently, there are no specific recommendations for this practice in CF samples. However, recent microbiome studies suggested a possible role of these organisms, especially lla spp. . Since illa spp. .S. aureus and BCC and implementation of typing methods for epidemiologic studies. These actions will facilitate Spanish CF microbiology laboratories to be in line with recommended standards of care for Microbiology laboratories within the framework of European CF centers [In summary, there are few studies regarding the assessment of microbiological procedures in CF samples and this is the first one performed in our country ,13,17. T centers . Moreove, and those included in National and International Guidelines for the management and treatment of CF patients [http://www.surveymonkey.com) was used for sending the questionnaire to the participating laboratories and for analyzing the data obtained. Telephone interviews were performed when necessary to clarify specific answers to the questionnaire. This work was part of a multicenter study which was approved by Ramón y Cajal Hospital Clinical Research Ethical Committee (reference n° 341/12).A questionnaire for the participating laboratories was elaborated based on the recommendations for the microbiological diagnosis of bronchopulmonary infection in CF patients published by the Spanish Society of Clinical Microbiology and Infectious Diseases (SEIMC), by the Cystic Fibrosis Foundationpatients ,10,14,17