Eurydema gebleri and Rubiconia intermedia, and present the first comparative analysis of the 11 pentatomoid mitogenomes that have been sequenced to date.Insect mitochondrial genomes (mitogenomes) are the most extensively used genetic marker for evolutionary and population genetics studies of insects. The Pentatomoidea superfamily is economically important and the largest superfamily within Pentatomomorpha with over 7,000 species. To better understand the diversity and evolution of pentatomoid species, we sequenced and annotated the mitogenomes of Eurydema gebleri  and a nearly complete mitogenome of Rubiconia intermedia . Our results show that gene content, gene arrangement, base composition, codon usage, and mitochondrial transcription termination factor sequences are highly conserved in pentatomoid species, especially for species in the same family. Evolutionary rate analyses of protein-coding genes reveal that the highest and lowest rates are found in atp8 and cox1 and distinctive evolutionary patterns are significantly correlated with the G + C content of genes. We inferred the secondary structures for two rRNA genes for eleven pentatomoid species, and identify some conserved motifs of RNA structures in Pentatomidea. All tRNA genes in pentatomoid mitogenomes have a canonical cloverleaf secondary structure, except for two tRNAs (trnS1 and trnV) which appear to lack the dihydrouridine arm. Regions that are A + T-rich have several distinct characteristics (e.g. size variation and abundant tandem repeats), and have potential as species or population level molecular markers. Phylogenetic analyses based on mitogenomic data strongly support the monophyly of Pentatomoidea, and the estimated phylogenetic relationships are: (Urostylididae + (Plataspidae + (Pentatomidae + (Cydnidae + (Dinidoridae + Tessaratomidae))))).We obtained the complete mitogenome of This comparative mitogenomic analysis sheds light on the architecture and evolution of mitogenomes in the superfamily Pentatomoidea. Mitogenomes can be effectively used to resolve phylogenetic relationships of pentatomomorphan insects at various taxonomic levels. Sequencing more mitogenomes at various taxonomic levels, particularly from closely related species, will improve the annotation accuracy of mitochondrial genes, as well as greatly enhance our understanding of mitogenomic evolution and phylogenetic relationships in pentatomoids.The online version of this article (doi:10.1186/s12864-015-1679-x) contains supplementary material, which is available to authorized users. Insect mitochondrial genomes (mitogenomes) are circular double-stranded molecules 15–18 kb in size that usually code for 37 genes: 13 protein-coding genes (PCGs), two ribosomal RNA genes (rRNAs), and 22 transfer RNA genes (tRNAs) –6.et al. [et al. [rrnL and rrnS) to examine the position of Dinidoridae within Pentatomoidea, and identified Dinidoridae as monophyletic and sister to the Tessaratomidae. Analysis of the partial cox2 sequence, however, did not support a close affinity between Dinidoridae and Tessaratomidae [Pentatomoidea (Hemiptera: Heteroptera: Pentatomomorpha) consists of about 7,000 known species in 15 families, of which Pentatomidae is the largest family containing over 4,700 species in ca. 900 genera  used moret al.  performe [et al.  used twoatomidae . Currentatomidae , 13–15. atomidae . Much ofTo date, only 24 complete or nearly complete mitogenomes have been sequenced for Pentatomomorpha , of which nine are from Pentatomoidea. The number of sequenced pentatomoid mitogenomes is still quite limited compared to the species-richness of Pentatomoidea and restricts our understanding of evolution in pentatomoid species at the genomic level. In addition, the accurate delimitation of mitochondrial genes may be difficult and error prone when annotated mitogenomes are not available from closely related species –18. TherEurydema gebleri (Strachiini) and Rubiconia intermedia (Carpocorini) from the family Pentatomidae. These two species are important alfalfa pests in China. We compared the newly sequenced mitogenomes with nine previously published sequences to check and refine the original annotation of gene boundaries. We provided a detailed comparative analysis of eleven pentatomoid mitogenomes, including nucleotide composition, codon usage, RNA secondary structure, evolutionary pattern among 13 PCGs, and structural elements within the A + T-rich regions. Finally, we evaluated the phylogenetic utility of mitogenomic sequence data at the taxonomic levels of Pentatomomorpha, Pentatomoidea and Pentatomidae.In the present study, we sequenced and annotated the mitogenomes of E. gebleri  and the nearly complete mitogenome of R. intermedia  were obtained . The overlap sequences in the three gene junctions are highly conserved among all sequenced pentatomoid mitogenomes  and an A + T-rich region. Gene arrangement is highly conserved within Pentatomoidea and is identical to the gene order of the putative ancestral insect mitogenome [M. cribraria to 76.9 % in N. viridula , TTA-Leu (10.2), ATT-Ile (9.5), and ATA-Met (7.4), are the most frequently used codons. Comparative analyses among other sequenced pentatomoid species also reveal a similar pattern, with values ranging from 25.6 % in M. cribraria to 34.9 % in N. viridula. Differences in the four most frequently used codons in pentatomoid species seem to be directly linked to the A + T content of all PCGs (R2 = 0.96), as has been reported in other insects and chelicerates [Nucleotide composition bias is also reflected in the codon usage pattern. Relative synonymous codon frequencies (RSCU) reveal that four- and two-fold degenerate codon usage is biased to use more As and Ts than Gs and Cs in the third codon positions    , CBI (codon bias index), the G + C content of all codons, and the G + C content of the 3rd codon positions Fig. . We founnad2, cox1, atp6, nad3, nad5, nad4L, nad6, cob and nad1) for nine previously sequenced pentatomoid species , ATCA (nad2), GTG (nad1 and nad6), and TTG  are also proposed as initiation codons. While most PCGs end with the termination codon TAN, truncated termination codons TA or T are also common , but ω) were calculated for each PCG, respectively  had the lowest evolutionary rates, suggesting that they are potentially useful barcoding markers. The ω values for all PCGs were far lower than one (<0.48), indicating that these genes are evolving under purifying selection. Therefore, all mitochondrial PCGs could be used to investigate phylogenetic relationships within Pentatomoidea. Furthermore, a negative correlation has been found between ω and the G + C content of each PCG (R2 = 0.89), indicating that variation in G + C content probably causes the different evolutionary patterns among genes.In order to investigate the evolutionary patterns among the mitochondrial PCGs in pentatomoid species, the values of Ka, Ks, and Ka/Ks , trnA (M. cribraria), trnR (C. bifaria and Urochelas quadrinotata), trnF and trnH , and trnS1   appear to lack the dihydrouridine (DHU) arm  has been considered a typical feature of metazoan mitogenomes [trnS1 in eleven pentatomoid mitogenomes possesses an unusual anticodon stem  with a bulged nucleotide  DHU stem, suggesting that this tRNA may have lost the DHU arm, as has been observed in other pentatomoid species  that are located at a conserved position between trnL1 (CUN) and trnV, and between trnV and the A + T-rich region, respectively  and rrnL  .As in other insect mitogenomes, pentatomoid mitogenomes have two genes encoding the large and small rRNA subunits  and 44 helices . The seces Fig. , whereasrrnL, domains IV and V are more conserved within Pentatomoidea than domains I, II, and VI. Six helices  of rrnL are highly conserved with 0–2 nucleotide substitutions. Some helices  are highly variable in terms of their sequence and secondary structure, as has been frequently observed in other insects [In  insects , 32.rrnS is structurally more conserved within Pentatomoidea, especially in the helices H921-960, H1047 and H1399. The helix H47 is highly variable between different insects; in fact no consistent structure has been found for this region [E. gebleri consists of a long stem and a short terminal loop, as is also found in Stenopirates sp. [C. fallax [rrnS of E. gebleri, as found in Stenopirates sp. [C. salicicola [E. gebleri, H673 forms a long stem and a very small loop. Comparison of this helix among pentatomoid species shows that its nucleotide sequences are highly variable, and thus this structure might be taxon-specific.Compared to the 5′-end, the 3′-end of s region . The estates sp.  and C. f. fallax . The oth. fallax , 36, 37.. fallax , 36–38, ates sp.  and C. slicicola . In mosttrnS2 (UCN) and nad1 in all pentatomoid species, except for M. cribraria and E. gebleri  that is present in all of the eight species  in the eight pentatomoid mitogenomes is located at a conserved position as in most insects Fig.  and 2. A region 44–632 bp rrnS) did not improve resolution in our analyses  and two inference methods (BI and ML) Fig.  and 11. All analyses consistently recover the five superfamilies  with high support Fig.  and 11. Although some conflicting results are observed from different datasets and inference methods, our results largely agree with the traditional morphological classification and recent molecular studies, suggesting that mitogenome sequences are promising genetic markers for resolving phylogenetic relationships at various taxonomic levels of Pentatomomorpha. Further researches with denser taxon sampling and molecular markers from across the nuclear genome, as well as appropriate phylogenetic inference methods are needed to provide more precise estimates for phylogenetic relationships within Pentatomomorpha and Heteroptera.Eurydema gebleri and the nearly complete mitogenome of Rubiconia intermedia. We present the first comparative analysis of eleven pentatomoid mitogenomes and our results show that gene content, gene arrangement, base composition, codon usage, RNA structure, and mitochondrial transcription termination factor sequences are highly conserved in pentatomoids, especially among closely-related species. Although most tRNAs have a canonical cloverleaf secondary structure, abnormal tRNAs are also present in pentatomoid mitogenomes. Phylogenetic relationships within Pentatomomorpha based on mitogenomic data are consistent with the traditional morphological classification, suggesting that mitogenome sequences are useful for resolving phylogenetic relationships at various taxonomic levels. Sequencing more mitogenomes representing various taxonomic levels, particularly from closely related species, will not only improve the accuracy of annotations for mitochondrial genes, but will also greatly improve our understanding of mitogenomic evolution and phylogenetic relationships in pentatomoids.In this study we sequenced and annotated the complete mitogenome of Eurydema gebleri and Rubiconia intermedia were collected from an alfalfa field in Xiaguanying Town  in July, 2013 and from Shishe Town  in August, 2013. Samples and voucher specimens are deposited in the State Key Laboratory of Grassland Agro-Ecosystems, College of Pastoral Agricultural Science and Technology, Lanzhou University, Lanzhou, China. All specimens were initially preserved in 100 % ethanol in the field, and transferred to −20 °C until used for DNA extraction. For each species, total genomic DNA was extracted from the thorax muscle of a single specimen using a DNeasy Tissue Kit (Qiagen) according to the manufacturer’s protocols.Adult specimens of E. gebleri and R. intermedia were amplified in overlapping fragments by a set of universal insect mitochondrial primers [The mitogenome sequences of  primers  and specSequence files were proof read and assembled into contigs with BioEdit 7.0.9.0 . PCGs wehttp://tandem.bu.edu/trf/trf.html) [http://mfold.rna.albany.edu/) [Nucleotide composition and codon usage were analyzed with MEGA 6.06 . The numrf.html)  and the ny.edu/) .rrnL and rrnS) were predicted following the models proposed for Drosophila melanogaster (Diptera: Drosophilidae) [Apis mellifera (Hymenoptera: Apidae) [Manduca sexta (Lepidoptera: Sphingidae) [The secondary structure of the large and small subunits of rRNAs (hilidae) , Apis me Apidae) , Manducaingidae) , and othingidae) , 24, 32)ingidae) . Regionsingidae) .Adelphocoris fasciaticollis and Apolygus lucorum, were used as outgroups. Details of the species used in this study are listed in Table Twenty-six Pentatomomorpha species with complete or nearly complete mitogenomes were used in phylogenetic analyses, representing five superfamilies and seventeen families. Two species of Cimicomorpha, http://mafft.cbrc.jp/alignment/server/) using the Q-INS-i method [http://molevol.cmima.csic.es/castresana/Gblocks_server.html), allowing gap positions within the final blocks. Alignments of individual genes were then concatenated as a combined matrix. To determine if sequence saturation exists in our alignments, transitions and transversions were plotted against genetic distance estimated with a GTR model of nucleotide evolution, as implemented in DAMBE 5.3.74 [rrnS  and (4) P123, two rRNAs, and 22 tRNAs (P123RT).The complete sequences of 13 PCGs (excluding stop codons), two rRNAs and 22 tRNAs were used for phylogenetic analyses. Each PCG was aligned individually with codon-based multiple alignments using MAFFT as implemented in the TranslatorX online server . Gaps ani method , which hi method , and theE 5.3.74 . SaturatWe used PartitionFinder 1.1.1  to deter7 generations. Each set was sampled every 100 generations. Stationarity was considered to be reached when the ESS (estimated sample size) value was above 100 and the PSRF  approached 1.0 as suggested by the authors of MrBayes 3.2.2 [Phylogenetic analyses were conducted with ML and BI methods available on the CIPRES Science Gateway 3.3 . ML anales 3.2.2 . The firhttp://purl.org/phylo/treebase/phylows/study/TB2:S17642).Phylogenetic data  have been deposited in the TreeBASE (


P < 0.05). When mitotic cell counts were assessed in the renal tissues, it was found that mitotic cell count was significantly higher in the D group while it was lower in the D + CAPE, D + IL, and D + IL + CAPE groups when compared to the control group (P < 0.05). Also, immune reactivity showed increased apoptosis in D group and low profile of apoptosis in the D + CAPE group when compared to the Control group. The apoptosis level was significantly lower in D + IL + CAPE compared to D group (P < 0.05) in the kidneys. As a result, we concluded that Dichlorvos can be used either alone or in combination with CAPE and IL as supportive therapy or as facilitator for the therapeutic effect of the routine treatment in the patients presenting with pesticide poisoning.The protective effects of Caffeic Acid Phenethyl Ester (CAPE) and intralipid (IL) on nephrotoxicity caused by acute Dichlorvos (D) toxicity were investigated in this study. Forty-eight Wistar Albino rats were divided into 7 groups as follows: Control, D, CAPE, intralipid, D + CAPE, D + IL, and D + CAPE + IL. When compared to D group, the oxidative stress index (OSI) values were significantly lower in Control, CAPE, and D + IL + CAPE groups. When compared to D + IL + CAPE group, the TOS and OSI values were significantly higher in D group ( Organophosphorus pesticides (OPs) have been widely and effectively used for applications in agricultural settings, public health, commerce, and individual households worldwide in order to increase efficiency of agricultural production and maintain hygienic conditions , 2. DichSeveral antidotes have been evaluated for the routine treatment of OP poisoning and the currently recommended drugs are atropine and pralidoxime chloride . AtropinRecently, oral IL emulsion was introduced as a novel method in the treatment of intoxication from several lipophilic agents. Since it was shown to be effective in bupivacaine toxicity, IL may be a promising approach for other lipophilic drug intoxications, including herbicides and pesticides. Moreover, it has been suggested that IL binds lipophilic agents and confines liposoluble toxic elements. It is also reported that it is administered by a bolus dose of 1.5 mL/kg and an infusion dose of 0.25 mL/kg/min in liposoluble drugs . However, IL is not currently used in the treatment protocols because of insufficient evidence in intoxications .Caffeic acid phenethyl ester (CAPE) is a compound that is structurally similar to the flavonoid found in bee propolis. It is an active component of propolis extract, which is one of the reactive oxygen species (ROS) that occurs as a result of oxidative stress in toxic failure and ischemia-reperfusion injury and has tissue protective effect . CAPE reThis study concluded that CAPE and IL are capable to exert protective effects against nephrotoxicity caused by acute D toxicity in rats. Therefore, this study shed light on the literature in terms of the prophylactic use of these two agents in the disorders induced by oxidative damage and distant organs damage.The study was approved (ethic number: 2012-065) by Necmettin Erbakan University, Experimental Medical Research Center's Experimental Animals Ethics Committee, Konya, Turkey. The forty-eight mature Wistar Albino rats weighing 200–250 g were randomly divided into seven groups. The animals were kept under laboratory conditions of 12-hour light-dark cycle at a room temperature (21°C ± 2°C).n = 6) as Control (C), Dichlorvos (n = 7), intralipid (n = 7), CAPE (n = 7), Dichlorvos + intralipid (n = 7), Dichlorvos + CAPE (n = 7), and Dichlorvos + intralipid + CAPE (n = 7) groups. Doses and durations of medication were adjusted according to the literature so the rats were given Dichlorvos  [μmol/kg via intraperitoneal route) [The forty-eight rats were randomly divided into seven groups (l route) , 12, intl route) , 13, andl route) , 11. IL 2O2 equivalent/milligram of protein. The units of TOS and TAS were micromoles of H2O2 equivalent/gram protein and millimole of H2O2 equivalents/gram protein, respectively. The Oxidative stress index (OSI) value was calculated as follows: OSI = TOS/TAS [The TAS of supernatant fractions was evaluated by using a novel automated and colorimetric measurement method developed by Erel. Hydroxyl radicals, the most potent biological radicals, are produced in this method. In the assay, the ferrous ion solution present in reagent 1 is mixed with hydrogen peroxide, which is present in reagent 2. The radicals produced subsequently, such as brown-colored dianisidinyl radical cations produced by the hydroxyl radicals, are also potent radicals. Using this method, the antioxidative effect of the sample is measured against the potent-free radical reactions initiated by the produced hydroxyl radicals. The assay has excellent precision values (lower than 3%). The TAS results are expressed as nmol Trolox equivalent/mg protein. The TOS of supernatant fractions was also evaluated by using a novel automated and colorimetric measurement method developed by Erel. Oxidants present in the sample oxidize the ferrous ion-o-dianisidine complex to ferric ion. The oxidation reaction is amplified by glycerol molecules, which are abundantly present in the reaction medium. The ferric ion produces a colored complex with xylenol orange in an acidic medium. The color intensity, which can be measured spectrophotometrically, is related to the total amount of oxidant molecules present in the sample. The assay is calibrated with hydrogen peroxide, and the results are expressed in terms of nanomoles of H TOS/TAS , 15. Ell TOS/TAS .μm thick sections were obtained from paraffin blocks by using a microtome . Collected sections were stained with hematoxylin-eosin (H&E) and examined under light microscope (×100) and photos were captured. Histopathology of the tissue samples was rated semiquantitatively according to normal tissue composition. In the pathological examinations of the sample parameters of brush border loss, extravasation, tubular cast structures, nucleus loss in the tubule epithelial cells, tubular dilatation and interstitial accumulation of lymphocytes, and tubular necrosis were rated from 0 to 5 points; thus, normal kidneys and other applied kidneys were compared. This scoring  was carried out according to similar scoring of the other studies in kidney [For histopathologic examinations, routine histologic paraffin block preparation method was used after fixation of kidney samples that are kept in 10% formalin. 5 n kidney .Immunohistochemical examination was performed on a Leica Bond-Max automated IHC/ISH platform . Four-micrometer paraffin sections were dewaxed in a Bond Dewax solution and rehydrated in alcohol and Bond Wash solution (Leica Microsystems). Antigen retrieval was performed using a high pH (ER2) retrieval solution for 15 minutes followed by endogenous peroxidase blocking for 5 minutes on the machine. Anti-mouse monoclonal antibody Bcl-2 , anti-mouse monoclonal antibody Bax , and anti-mouse caspase-3 (CPP32) monoclonal antibody  were applied at 1 : 50 dilution for 60 minutes at room temperature. Detection was performed using the Bond Polymer Refine Red Detection system (Leica Microsystems) with a 15-minute postprimary step followed by 25-minute incubation with alkaline phosphatase-linked polymers. Sections were then counterstained with hematoxylin on the machine, dehydrated in alcohols, and mounted with mounting medium . Prepared tissues were observed by histopathologists blinded to the experimental study groups. The numbers of apoptotic cells were counted in ten randomly selected microscope fields under a ×400 magnification in a blind fashion. The average number of stained neurons for each set of ten fields was calculated and expressed as the number of the positive cells/high-power field.U test. P < 0.05 was considered to be statistically significant.SPSS 11.5 software was used for statistical analysis. Data were expressed as mean ± SD. Kruskal-Wallis test was applied to determine the abnormal distribution followed by post hoc Tukey's and Mann-Whitney P < 0.05). the TAS values were significantly higher in Control, CAPE, and D + IL  +  CAPE groups when compared to the D group (P < 0.05). Also the TAS values were significantly higher in the CAPE and IL groups when compared to the D + IL + CAPE group (P < 0.05) (The TAS level was significantly lower in the D group than those in the other groups ( < 0.05) .P < 0.05), while no significant difference was observed among other groups (P > 0.05) (P < 0.05)  . Also, t < 0.05) .P < 0.05) while no significant difference was observed among other groups (P < 0.05)  .When the mitotic counts were assessed in the renal tissues, it was found that mitotic count was higher in the D group Figures  and signUsing caspase-3, Bcl-2, and Bax, immune reactivity showed increased apoptosis in the kidneys from the D group Figures  and low When organophosphate is absorbed through the skin or by means of digestion, mucosal membranes, conjunctiva, or respiration, organophosphate (OP) intoxication leads to quite a serious clinical picture, even leading to sudden onset of respiratory failure which requires admission to intensive care unit , 19. OP μmol/kg via intraperitoneal route) is effective in reversing this injury [μmol/kg via intraperitoneal route) in the present study.The oral acute LD50 dose of D, which is an OP compound, is 80 mg/kg in rats . Previous injury . TherefoSimilarly, Tuzcu et al. claim that IL diminishes the pancreatic injury caused by malathion, an OP, and that IL exerts these effects through decreasing malathion absorption by acting as a chelate with malathion in the stomach . SimilarToxic cell death is known to appear by only two main mechanisms: necrosis and apoptosis, which can be divided by their characteristic morphological and biochemical situations. These aspects of apoptosis are observed in various tissues from animal models, and while there is widespread necrosis occurring within the area of injury, apoptosis also plays an important role in toxic cell death in various tissues .Hou et al. have demonstrated that rat exposure to D caused renal injury, including renal tubular, glomerular filtration, and oxidative stress. Also, these toxic effects were also regulated by high-dose quercetin. In that study, histopathological examination revealed that D induced extensive cell vacuolar denaturation; however, milder histopathological alterations were observed in the kidney tissues of rats by combined D + quercetin (50 mg/kg bw) . Fiore eThe results of above referred studies were in agreement with our results. In present study, the D group revealed cytoplasmic hypereosinophilic changes, loss of intracellular boundaries, nuclear pyknosis, diffusely increased mitotic activity in histopathologic examinations, and diffusely increased apoptosis in immunohistochemical analysis. However, in the rats given D + CAPE, D + IL, and D  +  IL  +  CAPE the mitotic density was decreased and regenerative changes were observed. Similarly, in the immunohistochemical analysis, apoptosis was increased in the D group while it was lower in D + CAPE, D + IL, and D  +  IL  +  CAPE groups compared to the D group. These results show that CAPE and IL may exclusively ameliorate the renal injury caused by D in the present study.When biochemical results were evaluated in some studies, it was reported that OPs cause increase of oxidative stress, ROS, and depletion of antioxidant enzymes . Celik aOur results were in agreement with abovementioned studies. The present study showed that the administration of D causes increase in the TOS levels and decrease in the TAS levels compared to the Control, IL, CAPE, D + CAPE, and D  +  IL  +  CAPE groups. Also, the TAS levels in D + CAPE, D + IL, and D + IL + CAPE groups were found higher than in the D group. In the present study, histopathological results were consistent with biochemical results.Based on our results, it can be concluded that CAPE and IL are similarly capable of preventing the renal injuries caused by D by means of their antioxidant effects. Therefore, we suggest that it can be used either alone or in combination with CAPE and IL or it can be used as supportive therapy or as facilitator for the therapeutic effect of the routine treatment in the patients presenting with pesticide poisoning. However, further studies are needed about protective effects of CAPE and IL on OP intoxications.


In this study we evaluated the morphological changes and the expression pattern of Nrf2 and correlated them with the Cd concentrations in different ages of developing rats in heart, lung, kidney, liver, and brain. The Cd content in different organs of rats treated with the metal was increased in all ages assayed. Comparatively, lower Cd brain levels were found in rats intoxicated at the age of 12 days, then pups treated at 5, 10, or 15 days old, at the same metal dose. No evident changes, as a consequence of cadmium exposure, were evident in the morphological analysis in any of the ages assayed. However, Nrf2-ARE immunoreactivity was observed in 15-day-old rats exposed to Cd. Our results support that fully developed blood-brain barrier is an important protector against Cd entrance to brain and that Nrf2 increased expression is a part of protective mechanism against cadmium-induced toxicity.In developing animals, Cadmium (Cd) induces toxicity to many organs including brain. Reactive oxygen species (ROS) are often implicated in Cd-inducedtoxicity and it has been clearly demonstrated that oxidative stress interferes with the expression of genes as well as transcriptional factors such as Nrf2-dependent Antioxidant Response Element (Nrf2-ARE). Cd-generated oxidative stress and elevated Nrf2 activity have been reported  Cadmium, a heavy metal, is present in increasingly hazardous concentrations in soils, sediments, air, and water , 2. A wiκB), and NF-E2-related factor (Nrf2). ROS may function as secondary messengers that deregulate gene expression and induce cell transformation when cells are exposed to Cd [Oxidative stress is generally defined as an imbalance that favors the production of Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS) over antioxidant defenses , 11, 12.ed to Cd , 21–23. ed to Cd . The nuced to Cd , 26. In ed to Cd , 28.One important factor linked to brain Cd toxicity is age; experimental evidence has shown that in adult rats only small amounts of Cd reach brain, whereas in the newborn and developing animals Cd readily reaches brain –31. This ad libitum. Rats were individually housed in polycarbonate cages. Animals were handled according to the National Institutes of Health (USA) Guidelines for the Care and Use of Laboratory Animals, and this protocol was approved by the Bioethics Committee of the National Institute of Neurology and Neurosurgery of Mexico.Experiments were performed using male and female Wistar rats; NIH bred in-house strain newborn or 1, 5, 10, 15 and 21 days old. Animals were maintained in standard conditions  during all the experimental period. The newborn rats were allowed to remain with their dams throughout the experiment. All animals were fed on a standard chow diet (Purine Chow) and given waterNewborns were randomly assigned to different experimental groups and were injected i.p. with cadmium chloride (1 mg Cd/kg) dissolved in saline (0.9% NaCl w : v); the control group received saline solution (0.9% NaCl w : v); similar doses have been used previously by us , 16, 32.On the other hand, to find out the participation of age into Cd tissue distribution, rats aged 1, 5, 10, 15, and 21 days were injected at the appropriate times with cadmium chloride (1 mg Cd/kg) and then sacrificed 24 h after the metal injection to determine tissue cadmium content. From every experimental group, four animals were randomly selected for histological examination, and six animals were used for cadmium content analysis.3. Calibration curves were constructed using an aqueous Cd reference standard . As biological-matrix external standard, we used nitric acid-digested bovine liver (NIST 1577b) standard reference material, and this certified standard was analyzed in every analytical session; values obtained from that analysis were 95–105% from those reported by manufacturer. Cd content was expressed as μg of Cd/g wet tissue. All glassware was cleaned by soaking in 3% nitric acid and rinsing several times in deionized water. The limit of quantification for Cd analysis was 0.01 μg/g wet tissue.Cadmium content in tissues was analyzed by graphite furnace atomic absorption spectrophotometry (GFAAS) , according to the technique described by Christian, 1969  and usinμm slices and hematoxylin-eosin stained [For histological examination, animals were anesthetized with pentobarbital (40 mg/Kg) and then perfused with cold saline solution followed by 10% formaldehyde at 4°C and the heart, lung, kidney, liver, and brain were rapidly removed. Tissue samples were processed separately in a Histokinette 200 apparatus (Reichert Jung). Sections were paraffin-embedded, cut into 5  stained , and exaOn the other hand, sections were incubated with monoclonal antibody against Nrf2 1 : 100 (Nrf2 Santa Cruz Laboratory). Sections were then incubated with a Biotin-conjugated secondary antibody and Streptavidin-Enzyme Conjugate . The immune reaction resulted in the oxidation of the 3,3′-diaminobenzidine by peroxidase  into an insoluble brown precipitate. The reaction sites were visualized as a brown staining. Counterstaining with hematoxylin was performed after immunostaining.p < 0.05 was considered to be statistically significant.Statistical analyses were performed by using SPSS V.19.0 statistical software package. Data are presented as mean ± standard error of the mean. Normal distribution and homogeneity of data were confirmed before one-way ANOVA, followed by Tukey's test. A level of μg/g, followed by elimination phase decreasing to 4 μg/g 72 hours after metal injection. Kidney also showed a peak at two hours but the concentration found was one-tenth from liver. Heart and lung showed comparatively no appreciable content of the metal.The time-course of Cd content in lung, heart, liver, and kidney of newborn rats at different times after single dose Cd administration is observed in Cd distribution in the different brain regions assayed is shown in μg/g wet tissue), 10- (1.55 ± 0.31 μg/g), 15- (0.95 ± 0.3 μg/g), and 21-day-old (1.04 ± 0.07 μg/g) rats. In contrast, the highest Cd concentration observed in this study was in the liver at all ages analyzed.The Cd content in different organs in function of age is shown in p < 0.05) lower than those other ages assayed. No differences were observed among brain regions.In Figures The morphological findings from the current study revealed slight changes in the kidney of 21-day-old Cd-exposed rats. In the control group normal structure of glomeruli, the proximal tubular epithelium, tubules, and basement membrane showed normal appearance ; in contWe performed immunohistochemistry to identify the Nrf2 in several organs from Cd-exposed rats. Immunohistochemical examination revealed negative immunostaining for Nrf2 in heart, lung, and kidney sections of control groups from 1- and 5-day-old rats, whereas slight immunoreactivity was observed in liver of 1- and 5-day-old rats both exposed to Cd . In contWe found scarce immunoreactivity for Nrf2 in brain regions on both 1- and 5-day-old rat groups. Increased immunoreactivity was observed in oligodendrocytes from Ce brain region in 10-day-old rats . HoweverIn this study, we investigated the distribution of Cd in several organs of the developing rat. Our results showed that the administration of Cd to the developing newborn rats was able to increase the concentrations of this metal in some organs during early postnatal development. High content of Cd, from 2 h after metal injection, suggested a fast distribution to liver and kidney. In brain we also observed Cd in the different brain areas, from 2 h and maintained along 72 h after metal injection. In cerebellum, which also was the brain area with the highest Cd content from brain areas assayed, it showed a peak 48 h after Cd treatment. The presence of Cd in the newborn rats in short periods suggests a fast distribution to organs, including brain.The ability of Cd to accumulate is dependent on its binding to ligands present inside the cell; Cd is widely distributed in the body with the major body burden located in the liver and kidney. Cd content builds up with age and the rate of accumulation is significantly higher during fast growing phases of animals ; similarOxidative stress is considered the major event underlying Cd toxicity, being able to produce tissue damage. However, in our histopathological examination, no apparent changes were observed after Cd treatment in the morphology of heart, lung, kidney, liver, or brain. Only minor renal damage was observed in 21-day-old rats Figures  and 4. TCd-induced ROS production is associated with increased oxidative stress and in turn with transcription factors such as Nrf2 . Nrf2-ARIn summary, this study shows that the exposure to Cd in developing rats induce the accumulation of this metal in several tissues including the central nervous system. However, the Cd entrance to brain is limited by a developed blood-brain barrier. Immunohistochemical staining demonstrated that high level expression of Nrf2 was present in several cellular types including glial cells and that Nrf2 immunoreactivity was increased in rats exposed to Cd. Our results support that Nrf2 increased expression is an intracellular response against Cd toxicity.


Although aged garlic extract (AGE) shares some active components with fresh garlic and in spite of its palatability and milder side effects, the anti-diabetic and related anti-oxidant properties of AGE have not been investigated extensively, and the reported findings are inconsistent.This study investigated the anti-diabetic effects of 3 incremental doses of AGE in streptozotocin (STZ)-induced diabetic rats (fasting blood sugar > 20 mM).Diabetic rats were divided into a control diabetic group (CD) and AGE-treated diabetic group (AGE-D). The AGE-D was divided into 3 groups and accordingly treated with AGE i.p. at 100, 300 and 600 mg/kg daily for 8 weeks. A control normal group (CN) was also included for reference.Compared to the CN group, the CD group showed significant loss of body weight (over 50 %); and decreased serum insulin concentration (10 fold) and total anti-oxidant level and catalase activity (45–70 %) in serum, kidney and liver. Conversely, the CD rats had an elevated blood glucose (nearly 4 fold), serum cholesterol (nearly 2 fold) and triglycerides (>2 fold), erythrocyte glycated hemoglobin  and kidney and liver lipid peroxidation (MDA levels). Treatment with AGE positively reversed the diabetic changes in the targeted parameters to levels significantly lower than those measured in the CD group and the degrees of attenuation were almost dose dependent especially with the two higher doses.AGE exhibits a dose-dependent ameliorative action on indicators of diabetes in STZ-induced diabetic rats. Allium sativum L.), a widely used condiment and spice, is well known for its health-promoting properties as reported in numerous human and animal studies , increase in intracellular cAMP and reduction in the interaction of fibrinogen with the CPIIb/IIIa receptor [In vitro and in vivo studies on AGE have demonstrated potent anti-thrombotic  and antireceptor , 20. In receptor . This acreceptor –21. Furtreceptor  and lowereceptor . AGE canreceptor .Diabetes mellitus (DM), an increasingly prevalent medical condition, is characterized by hyperglycemia, dyslipidemia and OS. Clinical and experimental studies have shown that diabetic hyperglycemia is a major source of OS, and that increased generation of free radicals plays a significant role in the pathogenesis and complications of DM –28. UratDuring the last 20 years, many studies have investigated the anti-diabetic potential of fresh garlic. Our group and others have shown that raw garlic extract is active in reducing hyperglycemia and alleviating many diabetic indicators, such as dyslipidemia and OS in both animal models and humans , 31–35. Based on its acceptable palatability and milder side effects, content of potent bioactive components and conflicting reported effects in DM, the present study was carried out to assess the anti-diabetic potential of AGE by conducting an 8-week daily intraperitoneal (IP) three incremental dose–response study in STZ-induced diabetic rats. The doses investigated were selected based on a pilot study. In addition to examining the effect of AGE on the general indicators of diabetes, such as serum insulin and glucose, erythrocyte GHb, food and water intake, urine output in addition to body weight changes, this study also looked at the indicators of OS and dyslipidemia to further delineate the scope of the anti-diabetic activity of this garlic preparation.AGE (Kyolic AGE) was purchased as a liquid suspension  containing 240 mg AGE/ml. All analysis kits were purchased from the different sources as indicated in their respective usage in this section.ad libitum were used in the study. The rats were kept under standard ambient conditions .Healthy 3 month old male Sprague–Dawley rats (150–180 g BW) maintained on standard rodent diet and tap water Diabetes was induced in 60 rats by a single IP injection of STZ, 60 mg/kg in 0.5 ml of 0.01 M sodium citrate buffer, pH 4.5. After 5 days, blood glucose was quantitated after overnight fasting in a drop of tail-blood under mild diethyl ether sedation using One-Touch Ultra-Easy Glucometer .n = 12–16): Control diabetic (CD) group injected IP daily with normal saline (NS); low-dose AGE-diabetic (AGE100-D) group injected IP daily with 100 mg AGE /kg; medium dose AGE-diabetic (AGE300-D) group injected IP daily with 300 mg AGE/kg; high dose AGE-diabetic (AGE600-D) group injected IP daily with 600 mg AGE/kg. A group of control normal rats (CN) that received NS was included as a reference.Diabetic rats (blood sugar level > 20 mM) were divided into four groups . The blood was collected by cardiac puncture into two separate tubes for serum and red blood cell (RBC) preparation. For serum, 3 ml of blood was collected in a tube and allowed to clot for 30 min at room temperature. The tubes were centrifuged at 1000 × g for 15 min and the serum was separated, and stored as 0.5 ml aliquots in Eppendorf tubes at −80 °C for later analysis. For RBCs, 1 ml of whole blood was collected in a separate glass tube containing 0.2 ml of 3.8 % sodium citrate as an anticoagulant and mixed well. The tubes were centrifuged at 1000 × g for 10 min and the plasma and buffy coat were carefully removed from the cells. RBCs was mixed well and analyzed for GHb on the same day.Immediately after sacrifice, the liver and kidneys of each rat were collected separately. The capsule of each kidney was removed and the tissues were washed with buffer, blotted with filter paper and stored at −40 °C for later analysis. One gram of kidney or liver tissue was diced and mixed with 3 ml Tris–HCl . The mixture was homogenized, allowed to stand on ice for a few minutes and then centrifuged for 15 min at 8000 × g at 4 °C. The supernatant was stored in small aliquots at −40 °C for later analysis.et al. [et al. [Glucose levels in whole blood were determined after overnight fasting at 2, 4, 8 weeks as described above. Total water-soluble antioxidants were determined by the method of Rice-Evans and coworkers as modified by Drobiova et al.  based onet al. . Serum s [et al. . Total s [et al. .P < 0.05 being significant.Data are expressed as mean +/− SEM. Statistical analysis was carried out using SPSS V. 21 (IBM). Readings within each group were compared using the independent sample test, whereas the readings of the different groups were compared using One-Way ANOVA: Post hoc multiple comparisons test. In Post Hoc, equal variances were assumed by Fisher’s Least Significant Difference, (LSD) with During the 8 weeks of the study, the CN rats more than doubled in weight Fig.  while thThe CD rats greatly increased water (235 ± 3 ml/24 h) and food (46 ± 2 g/24 h) intake during the experiment compared to CN rats   compared to CN rats . Total serum antioxidants and catalase activity were significantly increased by treatment of diabetic rats with 300  and 600 mg/kg of AGE . Serum SOD activity was only significantly increased in diabetic rats treated with 600 mg AGE/kg. The lowest AGE dose (100 mg/kg) had no effect on serum antioxidant parameters.Figure In parallel with serum anti-oxidants and catalase, total kidney and liver anti-oxidants and catalase activity were significantly lower in CD rats Figs.  and 7. TDuring the last two decades, considerable research has been devoted to studying the effects of natural herbs and spices as alternative forms of treatment for chronic diseases such as DM. Extensive research has established that fresh garlic extract and some garlic constituents have anti-diabetic and anti-oxidant properties. However, AGE, an over the counter available stable garlic preparation with known composition, in addition to acceptable ingestion and side effects attributes, has received little attention as an anti-diabetic agent. The goal of the present study was to assess the anti-diabetic and related anti-oxidant potential of AGE, which is either lacking in certain aspects or controversially reported in with respect to other aspects in the literature. Toward that objective and to define the most active dose or range of doses, a three incremental-dose–response study of AGE was investigated in STZ-induced diabetic rats.Prolonged hyperglycemia in both human conditions and animal models has been shown to result in the physical symptoms of diabetes including weight loss, polyphagia, polydipsia and polyuria , 43. As et al. [et al. [In the present study, STZ-injected rats developed a rapid hyperglycemia and hypoinsulinemia as shown previously by our group and others –46. Treaet al.  who did  [et al.  showed t [et al. , 48.et al. [Dyslipidemia, a major symptom and risk factor for the development of cardiovascular diseases in DM , 50 is cet al. , treatmeet al. . These oin vitro and in vivo studies have indicated that AGE can reduce both the formation of superoxide and lipid peroxidation, and thus alleviate OS [et al. [Oxidative stress has been implicated as a key contributor to many diabetic complications, including nephropathy . Thus, aviate OS . In spitviate OS , 44. Tre [et al.  also repet al. [In an attempt to specify the active components of AGE, Saravanan et al.  investiget al.  suggesteIn conclusion, the observed attenuating effects of AGE on the diabetic indicators in STZ-induced diabetic rats suggest that this ready-to-use, fixed concentration, stable preparation has the potential to be used as a palatable anti-diabetic agent. Further biochemical and pharmacological investigations into the insulin-glucose pathway are necessary to further elucidate the anti-diabetic and connected anti-oxidant mechanism of action of AGE to support its use as a possible supplementary treatment in DM.


Purpose. To elucidate the characteristics and risk factors for positive biopsy outcomes in Chinese patients with prostate specific antigen (PSA) 4–10 ng/mL and develop a risk-stratification score model. Methods. The data of 345 patients who underwent transrectal ultrasound-guided prostate biopsy between 2011 and 2013 was retrospectively analyzed. Digital rectal examination (DRE), prostate volume (PV), magnetic resonance imaging (MRI), and smoking status were also collected. Positive biopsy outcomes were defined as prostate cancer (PCa) and high grade PCa . Results. The median PSA was 7.15 (IQR 5.91–8.45) ng/mL. Overall 138 patients (40.0%) were shown to have PCa, including 100 patients (29.0%) with HGPCa. Smaller PV, elder age, MRI results, and positive DRE were proved to be predictive factors for positive biopsy outcomes in both univariate and multivariate analysis. We developed a “PAMD” score which combined the four factors to categorize patients into three risk groups, and the model performed good predictive sensitivity and specificity. Conclusion. The prevalence of prostate cancer in Chinese patients with PSA 4–10 ng/mL was 40%, including 29% patients with high grade disease. DRE, age, MRI, and PV were predictive factors for positive biopsy outcomes, and the PAMD score model could be utilized for risk-stratification and decision-making. Systematic transrectal ultrasound- (TRUS-) guided prostate biopsy is currently the standard practice for the diagnosis of prostate cancer (PCa) . AccordiAs the most commonly used screening tool, the elevated value of PSA often indicated the need for prostate biopsy. Traditionally 4 ng/mL was often regarded as the cut-off to consider biopsy , but conBesides, there are few prostate cancer screening protocols in China, and the epidemiologic characteristics in this unique population are unknown –14. It iThus based on this large cohort of Chinese patients, we sought to elucidate the characteristics and risk factors for positive biopsy outcomes and develop a risk-stratification score model for clinical use.Following institutional review board approval and written informed consent from patients, we initially collected information of 1348 patients who underwent TRUS-guided prostate biopsy between January 2011 and November 2013. All patients were referred for a prostate evaluation because of elevated PSA and/or suspicious DRE or TRUS. We exclude patients who received previous prostate biopsy or neoadjuvant hormonal therapy before biopsy and patients with incomplete data or extreme values . Three hundred and forty-five patients with prebiopsy PSA between 4 ng/mL and 10 ng/mL were finally enrolled for evaluation. Eligible patients were unselected and were accrued consecutively.PSA levels including the percentage of free PSA (%fPSA) were measured before DRE and TRUS. DRE result was defined as unsuspicious versus suspicious. Prostate volume (PV) was determined by TRUS and was calculated using the formula (width × length × height × 0.52). By two experienced doctors, TRUS-guided systematic biopsies of ≥10 cores  plus targeted biopsies at any suspicious area for malignancy were performed in all patients. All biopsy specimens were evaluated by a dedicated genitourinary pathologist to determine the presence of PCa and the Gleason score in positive cases. High grade PCa (HGPCa) was defined as Gleason score ≥7. Because of the extremely low prevalence in Chinese population, family history was not analyzed. Positive smoking history was defined as over 10 years' duration of smoking with over 20 cigarettes per day.MRI studies were performed at 1.5T or 3T; all MRI studies involved the sequences and acquisition parameters of axial T1-weighted imaging, axial and coronal T2-weighted fast spin-echo imaging, axial diffusion weighted imaging, and axial dynamic contrast enhanced imaging. Two experienced radiologists retrospectively and independently interpreted the MR images. Any disagreement was resolved by the adjudicating senior radiologist. The MRI diagnosis was evaluated by using a three-point scale in consideration of clinical use and the PI-RADS criteria : Grade 0U test was used for continuous variables. Multivariate logistic regression was used to calculate the predictive factors. Multivariate logistic regression coefficients were used to generate a risk-stratification score. Receiver operating characteristic (ROC) curves were generated to illustrate the predictive accuracy. All statistical tests were performed with SPSS 20.0 . All reported p values were two-sided with statistical significance considered at p < 0.05.Pearson's test and Chi-square test were used to test the distribution of categorical variables, and the Mann-Whitney The median age of this cohort of patients was 67  years. The median PSA was 7.15 (IQR 5.91–8.45) ng/mL, and the median %fPSA was 0.15 (IQR 0.10–0.19) . The medOverall 138 patients (40.0%) were shown to have PCa, and particularly 100 patients (29.0%) were diagnosed with HGPCa.In univariate analysis, elder age, lower %fPSA, suspicious DRE, MRI grades, and smaller PV were related to the presence of PCa and HGPCa . It is nPV, age, MRI grades, and DRE remained independent predictor in multivariate analysis in predicting both PCa and HGPCa, while %fPSA exhibited no statistical correlation when controlling for other factors .According to the multivariate relative risk, we proposed a risk factor-based stratification model: a “PAMD” score. Each score was defined as the approximate value of their coefficient. Finally each risk factor was scored as follows: PV > 50 mL = 0 points, PV≦50 mL = 2 points; age ≤ 68 = 0 points, age > 68 = 2 points; negative MRI = 0 points, suspicious MRI = 1 point, and positive MRI = 2 points; negative DRE = 0 points, positive DRE = 1 point. PAMD score was defined as the sum of the scores, which represented the combination of the four independent risk factors .As is shown in p < 0.001 (All 345 patients could be divided into three risk groups according to the PAMD score: low (0-1 point), intermediate (2-3 points), and high (over 4 points). There is significant difference in biopsy outcomes between the three risk groups with  < 0.001 .The commonly regarded cut-off value of PSA to consider biopsy is 4 ng/mL , but to For patients with PSA 4–10 ng/mL, several articles have focused on risk factors for biopsy outcomes –9, and mThe prevalence of PCa in this cohort of Chinese patients was 40%, including 29% high grade disease, which indicates clinicians should pay attention to the risk for PCa in this PSA range. The sensitivity and specificity at different cut-offs of PAMD score could help decision-making. It is notable that patients with PAMD score over 2 points (intermediate or high risk) would have at least 24.8% possibility to have PCa and 12.4% possibility to have HGPCa. Clinicians should fully consider the option for biopsy even if for patients with only 1 risk factor . For low-risk patients, the decision to undergo biopsy could be based on clinical judgment of treating physician in consideration of clinical characteristics and thorough discussion with patient about possible options and expectations.The %fPSA was recommended in many previous papers which exhibit good predictive accuracy , 20, 21.In current guidelines there is no recommendation for the use of MRI for early detection , 24, 25.The limitations of this study include the retrospective design and data collection, which did not allow us to evaluate some potentially useful variables such as PSA-velocity, PCA3, treatment outcomes, and survival information; and our study cohort might be subject to selection and recall bias. The relatively small study sample size might have affected the analysis and further external validation is required. Future prospective multicenter studies and maybe some screening trials would be required to fully elucidate the prevalence of PCa in Chinese population.The prevalence of prostate cancer in Chinese patients with PSA 4 ng/mL–10 ng/mL was 40%, including 29% patients with high grade disease. DRE, age, MRI, and PV were predictive factors for positive biopsy outcomes, and the PAMD score model could be utilized for risk-stratification and decision-making.


P = 0.06 to 0.41). However, our review included two cases of patients who experienced symptoms probably related to QTc prolongation after IM ziprasidone. Thus, careful screening and close monitoring, including baseline ECG, should be considered in patients receiving IM ziprasidone for the first time.Intramuscular (IM) ziprasidone has been associated with QTc interval prolongations in patients with preexisting risk factors. A 23-year-old male Chinese schizophrenia patient experienced an increase of QTc interval of 83 milliseconds (ms) after receiving 20 mg IM ziprasidone . This was rated as a probable adverse drug reaction (ADR) by the Liverpool ADR causality assessment tool. A systematic review including all types of trials reporting the effect of IM ziprasidone on the QTc interval prolongation identified 19 trials with a total of 1428 patients. Mean QTc change from baseline to end of each study was −3.7 to 12.8 ms after IM ziprasidone. Four randomized trials (3 of 4 published in Chinese) were used to calculate a meta-analysis of QTc interval prolongation which showed no significant differences between IM ziprasidone and IM haloperidol groups (risk ratio 0.49 to 4.31, 95% confidence interval 0.09 to 19.68,  The second-generation antipsychotic ziprasidone is available for intramuscular (IM) use in the management of acute agitation associated with schizophrenia . IM ziprIn China, IM ziprasidone is the first and only atypical antipsychotic agent in clinical use for agitation associated with schizophrenia. Although previous studies have demonstrated that IM ziprasidone is well-tolerated for treatment of agitation in schizophrenia in Western populations , cliniciMr. A, a 23-year-old Chinese male, was brought in by his family to the emergency department (ED) with acute psychotic symptoms including disorganized speech, delusion, and agitation. There was no history of substance abuse. A provisional diagnosis of schizophrenia was made. He had never taken antipsychotics and had no preexisting cardiovascular conditions and no known medical illness. Physical examination was unremarkable. His weight was 68 kg and his body mass index (BMI) was 22.2. The basic metabolic panel values, including serum potassium and glucose levels, were all within normal limits. Serum magnesium was not measured but he had no clinical condition associated with hypomagnesemia. Although psychotic symptoms were prominent at admission, his vital signs remained relatively stable at arrival: blood pressure (BP): 100/70 mmHg; pulse: 76 beats per minute (bpm); respiration rate: 18 respirations/minute; and temperature 36.0°C. A baseline electrocardiogram (ECG) was obtained which showed normal sinus rhythm and a QT/QTc of 384/418 milliseconds (ms) at a pulse of 78 bpm.During the ED stay, an initial IM ziprasidone dose of 10 mg was given and then IM doses of 10 mg with oral oxazepam of 30 mg were prescribed every 6 hours as needed for agitation. The total IM ziprasidone dose was 20 mg. An ECG obtained 24 hours later showed a QT/QTc of 450/501 ms with a pulse rate of 94 bpm. The increased QTc from baseline was 83 ms, a significant prolongation, but no symptoms occurred. Ziprasidone was then switched to oral olanzapine 5 mg/day, with oxazepam 30 mg/day. His QT/QTc returned to 366/416 ms at a pulse of 93 bpm 48 hours after the last ziprasidone injection. The QT/QTc returned to 382/402 ms 72 hours after the last dose of ziprasidone. This QTc of 402 ms is 16 ms lower than at baseline and 99 ms lower than during ziprasidone treatment. The patient was later transferred to inpatient services for further treatment.All authors agreed that this was a probable ADR, according to the Liverpool ADR causality assessment tool . Therefohttp://www.crd.york.ac.uk/prospero/). The registration number was CRD42014007542 at the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA), which provides an evidence-based minimum set of items for reporting in systematic reviews and meta-analyses.A systematic review on this topic was conducted. Our protocol of reviewing IM ziprasidone for acute psychosis has been published online . We excluded meta-analyses and systematic reviews. We searched the PubMed, Embase, and Cochrane Library databases. We also searched the Chinese databases (CBM and CNKI databases) using the same key words. The search included all studies published between January 2000 and December 2012, regardless of language. The keywords used for the searches included schizophrenia, intramuscular ziprasidone, QTc, and agitation. The keywords were used in combination with the Boolean operators AND, OR, and NOT. We used the “related article” function to supplement the research. We also manually searched bibliographies of RCTs, meta-analyses, case reports, and systematic reviews for studies that were missed in the initial electronic search.Review authors X-BL and WZ considered all included studies initially, without seeing comparison data, to judge clinical, methodological, and statistical heterogeneity, which provided the basis for deciding whether each study would be included for meta-analysis or other data synthesis. Data from all included studies was extracted and organized into standard, simple forms. In addition, to ensure reliability, X-BL independently extracted data from a random sample of these studies, comprising 30% of the total. Data presented only in graphs and figures was extracted whenever possible but was included only if two authors independently had the same result. We also attempted to contact authors through an open-ended request in order to obtain missing information or for clarification whenever deemed necessary.P < 0.05.The meta-analysis was performed according to the recommendations of the Cochrane Collaboration, using the Review Manager Version 5.1.7.0 software. Two authors (X-BL and WZ) independently extracted data for analysis. We presented the summary statistic of dichotomous outcomes as a risk ratio for QTc interval prolongation ≥ 450 ms, 450 ms ≤ QTc interval < 480 ms, and QTc interval ≥ 480 ms. The Mantel-Haenszel method was used to combine the summary statistic. We used I2 methods to assess statistical heterogeneity. We used a fixed effect model if no heterogeneity existed or I2 < 50% . All staThe search yielded 132 articles, 71 records remained after duplicates were removed, 46 full-text publications were assessed for eligibility, and finally 19 articles were included in qualitative synthesis . The decIn summary, in the IM ziprasidone group, only three subjects (0.2%) had a QTc ≥ 500 ms; 3 subjects (0.2%) reached a QTc ≥ 480 ms; 10 (0.7%) had a QTc ≥ 450 ms. On the other hand, within the haloperidol group, one subject had a QTc ≥ 500 ms, 3 reached a QTc ≥ 480 ms, and 3 had a QTc ≥ 450 ms. Seven patients (0.4%) of the IM ZPD group had QTc changes that exceeded 60 ms relative to the time-matched baseline values. By combining all the studies included in Overall, IM ziprasidone had a mild effect on the QTc interval prolongation. First, ziprasidone was associated with modest concentration-related QTc increases . Second,We paid particular attention to cardiac ADRs that may be linked to IM ziprasidone. In the prospective trials and RCTs, no patients reported symptoms related to QTc prolongation. Two cases from other studies did have symptoms associated with prolonged QTc interval. One patient lost consciousness within 45 min of IM ziprasidone injection of 20 mg; she was pulseless and hypotensive and her physical examination was remarkable for absent pulses and signs of poor perfusion , commentP = 0.06 to 0.41)  .This patient, a young, healthy male with no preexisting cardiac conditions, received only the recommended dose of IM ziprasidone for acute agitation (20 mg IM). The only concurrent use of medication was oxazepam, which has no known effects on the QT interval . HoweverWhile QT/QTc prolongation has been reported in patients receiving IM ziprasidone, the extent of prolongation in this patient is perhaps among the greatest in China, especially considering that this occurred in a young, healthy patient with no known preexisting conditions. Some unknown factors, including genetic defects at the heart potassium ion channel, may have played a role , 29. ZipOur subsequent systematic review found that, among 1428 patients, only three subjects (0.2%) had a QTc ≥ 500 ms. In our calculations, the mean change in QTc from baseline to end of study was −3.7 to 12.8 ms after the ziprasidone injection. This is comparable to other less comprehensive reviews such as the one completed by Camm et al. . Our sysFurthermore, meta-analysis of QTc interval prolongation of four RCTs, including three only published in Chinese, showed no significant differences in the IM ziprasidone group compared with the haloperidol group. However, similarity to haloperidol does not guarantee safety, as the US IM haloperidol prescribing information has a warning about torsades de pointes .Changes from baseline QTc interval were clinically modest with both drugs, which is comparable to an important RCT which showed that both ziprasidone and haloperidol were generally well tolerated . In geneAlthough several patients experienced significant QTc interval prolongation in the current review, no cardiac ADRs were reported in the prospective trials and RCTs. Nevertheless, our review found two cases , 11; botMost reported cases of prolonged QT interval associated with ziprasidone use have occurred in patients with preexisting cardiovascular conditions, hypokalemia, hypomagnesemia, concurrent use of other QT prolonging medications, higher doses of ziprasidone (>40 mg IM), rapid titration of the dosage, or repeated dosing , 12. ForMost cases of drug-induced torsades de pointes occur in the context of substantial prolongation of the QTc interval, typically to values >500 ms, but QTc alone is a relatively poor predictor of arrhythmic risk in any individual patient. Some drugs that substantially prolong the QTc interval produce very low rates of torsades de pointes while others have much smaller QTc effects but are considerably more proarrhythmic .Although all authors agreed that this was a probable ziprasidone-induced ADR, there is no absolute proof that it was. One could argue that the QTc prolongation was an artifact, but this conclusion does not appear likely since the patient had 3 ECGs using the same equipment and no other patient at that time showed similar QTc prolongations.Significant heterogeneity of the results in QTc ≥ 480 ms was seen in this meta-analysis, suggesting the effect of relevant moderator and mediator variables. This may be because meta- analyses combine results from trials that differ in their methodology, sample size and year, patient and treatment selection, and outcome variables. This meta-analysis only included 288 patients from four RCTs because these were the only ones identified in our search. The sample size was relatively small for meta-analysis, which prevented further data exploration. However, we included well-defined RCTs comparing ziprasidone with haloperidol. Although there were about 12 published RCTs, only 4 trials were included in this meta-analysis after a more strict quality assessment.As the first and only IM atypical antipsychotic medication available in China, IM ziprasidone offers some advantages over haloperidol , 9, whic


The study documents the persistence of inflammation and plasma amino acid abnormalities in post-EHA rehabilitation phase. EAAs enhance hip function retrieval and improve plasma amino acid abnormalities.It is not known whether postsurgery systemic inflammation and plasma amino acid abnormalities are still present during rehabilitation of individuals after elective hip arthroplasty (EHA). Sixty subjects  were randomized to receive 14-day oral EAAs (8 g/day) or a placebo . At admission to and discharge from the rehabilitation center, serum C-reactive protein (CRP) and venous plasma amino acid concentrations were determined. Post-EHA hip function was evaluated by Harris hip score (HHS) test. Ten matched healthy subjects served as controls. At baseline, all patients had high CRP levels, considerable reduction in several amino acids, and severely reduced hip function (HHS 40.78 ± 2.70 scores). After treatment, inflammation decreased both in the EAA group and in the placebo group. Only EAA patients significantly improved their levels of glycine, alanine, tyrosine, and total amino acids. In addition, they enhanced the rate of hip function recovery (HHS) (from baseline 41.8 ± 1.15 to 76.37 ± 6.6 Elective hip arthroplasty (EHA) is a surgical technique used for patients with damaged hip joints after degenerative osteoarthritis or an injury. One and half million EHA operations are carried out all over the world each year . In ItalEHA and post-EHA rehab allow patients to regain physical ability and improve their quality of life. However, a number of factors may hinder both the duration and outcome of rehabilitation, including preoperative functional state, postoperative anemia, and any infection or metabolic alterations. Among the latter, the possible persistence of inflammation and alterations of plasma amino acids –4 can leHere, we have formulated two hypotheses. Firstly, rehab subjects after EHA could have alterations of circulating amino acids from persistent inflammation. To test this hypothesis, we measured plasma amino acid levels and inflammation markers. Secondly, this study investigated whether the administration of essential amino acids (EAAs) could enhance the recovery of hip-joint dysfunction of the operated hip and correct/limit plasma amino acid alterations. The rationale for this choice was based on the fact that these substrates are well-known to enhance body and muscle protein synthesis  , even duThe ultimate purpose of this study was to stimulate further research on the topic and to provide added recommendations for rehabilitation management.n = 5), hyperthyroidism (n = 1), chronic renal disease (n = 2), and all conditions which could alter plasma amino acid levels. As a result, a total of 60 patients were studied.Sixty-eight subjects at 17 ± 1.12 days after elective hip arthroplasty (EHA) were recruited at admission to the rehabilitation center. After operation and before the admission to our rehabilitation department, patients stayed an average of 12 days in orthopedic department to achieve clinical stabilization and to start training for erected posture and gait. The patients were selected on the basis of the type of surgical access they had had  in order to establish a more uniform recovery time. The causes for EHA included aseptic necrosis of the femoral head (5%), rheumatoid arthritis (1.6%), congenital hip dysplasia (1.6%), and grade 3 and 4 osteoarthritis (91.8%) diagnosed through preoperatory X-ray. Eight subjects were excluded due to diabetes on insulin and/or oral hypoglycemic drugs  Anthropometric Measurements. They are body weight  and height (m). Body mass index (BMI) was calculated as kg/m2. (b) Biohumoral Variables(1)Routine variables, including serum total protein (g/dL) and protein electrophoresis.(2)C-reactive protein (CRP) as a marker of systemic inflammation .(3)μL of the derivatized mixture and measuring absorbance simultaneously at 338 and 262 nm. Plasma concentrations were expressed as μmol/L. Baseline plasma amino acids were compared to those of 10 healthy subjects matched for sex distribution , age (69 ± 7.5 years), and body mass index (27.5 ± 3.5 kg/m2).Plasma amino acids: the concentration of free amino acids in the plasma was measured using an AminoQuant II amino acid analyzer, based on the HP 1090 HPLC system, with fully automated precolumn derivatization. Both orthopthalaldehyde (OPA) and 9-fluorenyl-methyl-chloroformate (FMOC) reaction chemistries were used, according to the manufacturer's protocol. Measurements were made by injecting 1 (c) Clinical-Functional Status. This was measured using the Harris hip score (HHS) [re (HHS) . This isre (HHS) .(d) Nutritional Intake. A 3-day alimentary diet was kept by the rehab nurses, trained ad hoc. The nurses recorded the type and weight of cooked or uncooked food selected by patients from the hospital catering menu on a diet sheet for 3 days before and after the patients' meals [s' meals .Nutritional analysis on the amount of food actually ingested was used to calculate actual ingested calories and macronutrients . NutritiPatients were then assigned to the treatment or placebo according to a randomized allocation procedure established before patient admission to our rehabilitation department. A randomization list was generated using SAS statistical software, A and B being the identifiers of the blinded treatment. The list was made available to both the physiatrist (E. B.) and the hospital pharmacist. The physiatrist allocated patients sequentially to treatment A or B according to the randomization list. The author of the article (A. R.) who interpreted all the results and the physiatrist (S. C) who evaluated the clinical-functional state (HHS) were blinded to the patient allocation. The treatment consisted of 14-d EAA supplementation. Eight g/d (2 packages) of EAAs  or a placebo  was supplied. One package (4 g) of EAAs contained the essential leucine (1.250 g), lysine (0.65 g), isoleucine (0.625 g), valine (0.625 g), threonine (0.35 g), phenylalanine (0.1 g), methionine (0.05 g), and tryptophan (0.02 g) as well as cysteine (0.15 g), tyrosine (0.03 g), and histidine (0.15 g). The 14-day treatment was dictated by the rehabilitation center policy, which permitted patients to stay for a maximum of 20 days.With regard to the placebo, we decided not to use isonitrogenous nonessential amino acids as, under physiological conditions, these amino acids are inferior to essential amino acids in boosting protein synthesis or limiting protein breakdown. The use of maltodextrin as a placebo would have been a more appropriate choice since this carbohydrate, like essential amino acids , 13, 14,(e) The Rehabilitation Protocol. This protocol aimed to restore complete functional recovery of the altered body statics and the resumption of a normal walking pattern. The rehabilitation program included 2 daily therapy sessions lasting 45 minutes each (morning and afternoon), with patients assisted by the same physiotherapist. The entire rehabilitation cycle encompassed 24 sessions. The rehabilitation program consisted of the following:Passive mobilization of hip movements in triple flexion .Stretching the adductor and flexor muscles of the operated limb.Hip extension using isotonic contraction of the gluteus.Isotonic contraction of the quadriceps muscles against a resistance of 1 kg.Assisted gait training with the use of walking sticks and training for the stairs.Maintenance of cardiorespiratory capacity.Within 2 days before patient discharge, all procedures except for nutritional intake were repeated. The local ethics scientific committee approved the protocol  after the subjects gave their informed consent. t-test was used to compare differences between admission and discharge in patient's plasma amino acids. Baseline variable differences between the randomized patient subgroups were tested, when appropriate, by unpaired Student's t-test, chi-square, and Mann-Whitney test.Descriptive statistics were carried out for all recorded variables, reporting mean and standard deviations for quantitative variables and distribution frequencies for qualitative variables. Chi-squared test was used for categorical variables. Wilcoxon signed rank test was used to compare the difference in HHS of the entire patient population between admission to and discharge from rehabilitation institute. Paired Student'sp < 0.05.Repeated measures analysis of variance tested the over time changes in plasma amino acids of the randomized subgroups. Kruskal-Wallis test was used to compare the over time gains of HHS in randomized EAA and placebo subgroups. Statistical significance was set at n = 21; 12 in EAA group) were on antibiotic therapy for infection (mainly of urinary tract). The used antibiotics were amoxicillin  or levofloxacin . Seventy percent  had received blood transfusions  during their acute stage.All patients had systemic inflammation as indicated by elevated serum CRP, which was more than 25 times higher than the normal maximal value. The erythrosedimentation rate (ESR) and the positive reactants of inflammatory response  were above normal, whereas negative proteins  were normal. All patients had mild anemia .Functionally, the patients had severe reduction of hip function as indicated by their HHS test (40.78 ± 2.70 scores). Within the test, the domain relative to pain showed that patients suffered with moderate pain (19.5 ± 1.7 scores).p < 0.05) and citrulline (p < 0.05) were significantly improved in patients.Plasma amino acid levels can be seen in (a) Differences in Baseline Characteristics between EAA and Placebo Groups. After randomization . Compared to the placebo group, the EAA subjects had lower serum transferrin (p = 0.04) and circulating total proteins (p = 0.04). Other biohumoral variables including CRP and plasma amino acids were similar. Hip dysfunction was similar between the two patient groups . Within the test, all domains including that which is relative to pain were similar between placebo and EAA subjects.The EAA group was different from the placebo one in the time from index event ((b) After 14-d Treatment. All patients randomized to receive EAAs or placebo completed the study. versus −9.2 ± 6.5 mg/dL in EAA group, interaction p = 0.6; ESR −11.03 ± 5.4 mm in placebo versus −13.02 ± 6.4 mm in EAA group, interaction p = 0.7). Blood Hb and serum transferrin increased by +0.84 ± 0.3 g/dL in placebo versus +0.87 ± 0.19 g/dL in EAA group, interaction p = 0.8 for Hb; +1.34 ± 2.5 mg/dL in placebo versus +11.45 ± 3.1 mg/dL in EAA group, interaction p = 0.005 for transferrin. All other biohumoral variables did not show significant changes in both intra- and intergroups.During rehab, the inflammation rate decreased similarly for both groups (CRP −7.8 ± 4 mg/dL in placebop = 0.001), alanine (interaction p = 0.02), tyrosine (interaction p = 0.05), total AAs (interaction p = 0.02), and total EAAs (interaction p = 0.05). Arginine in the EAA group decreased towards the control value. The over time changes of other amino acids were similar between the two groups.As can be seen from p < 0.001) but the EAA patients improved more (from baseline 41.8 ± 1.15 to 76.37 ± 6.6 scores versus 39.78 ± 4.89 to 70.0 ± 7.10 scores) (p < 0.006); (p = 0.01). The over time changes in the other HHS items resulted similar between placebo and EAA groups ; . In the A groups .This study shows that, in the rehabilitation phase of EHA, patients may have abnormal plasma levels of several amino acids that could be associated with systemic inflammation and may persist until the end of rehabilitation. While supplementation with EAAs may optimize post-EHA rehabilitation by enhancing the functional retrieval of hip-joint function, EAAs cannot correct amino acid abnormalities.Inflammation is clearly a sequela of surgical trauma , althougThe reduced availability of these amino acids may, therefore, increase the risk of impaired and/or delayed wound repair and low quality of scar formation. The low glutamine level in patients combines with glycine and lysine in negatively influencing wound repair. Indeed, glutamine is an important fuel for fibroblast activities . ReducedThe coexistence of amino acid abnormalities with the patients' normal protein intake indicates that the intake of protein (1 g/kg body weight) is not enough to correct alterations of amino acids. This raises the question of whether, in post-EHA, the observed altered amino acids could behave as semiessential amino acids.In synthesis, rehabilitative patients after EHA may have an abnormal plasma amino acid profile, potentially increasing the risk for poor quality of wound repair.After randomization, the two patient groups had different amounts of circulating transferrin and total proteins. Given the similarities for the other measured variables, these differences could be due to both different preoperative protein status and different postoperative metabolic response rates. This is suggested by both the time from index event and the trend to stay longer in acute setting observed in EAA patients.The study shows that a supplementation with EAAs improved the levels of the circulating amino acids and enhanced hip function retrieval. The treatment with EAAs probably gave the patients a more balanced protein turnover. This is suggested by both the maintenance of branched chain amino acid  concentrations and the improved concentration of glycine, threonine, and alanine, with normalization of arginine. This was not the case for controls.However, the EAA treatment failed to normalize plasma glutamine. This would suggest that body requirements for this amino acid were higher than that provided both exogenously by diet and endogenously by essential BCAA metabolism . de novo synthesis of an enormous amount of peptides and proteins. To support this, a very recent study has reported that the infusion of amino acid mixtures  has anabolic effect during acute inflammation [Several mechanisms may explain the enhanced retrieval rate of hip-joint dysfunction associated with EAA supplementation. One mechanism is related to the effects of EAAs on wound repair. The phases of wound healing  require high EAA and semiessential glutamine and arginine  availabiammation .Another mechanism underlying the beneficial effects of EAA on hip retrieval may be the potentiation of the increase of muscle mass and strength of the operated limb, damaged by factors including surgical stress, reduced or absent muscle contraction, being bed-ridden, unloading, and inflammation. In support of this, the 12-week administration of 7.5 g EAAs (very similar to the amount used here) to older women was shown to increase muscle mass (as measured by Dexa), while they failed to improve clinical outcomes such as knee extension and handgrip . This isA further mechanism primed by EAAs to improve hip retrieval may be the attenuation of muscle weakness and soreness , which aA preferential/prevalent use of supplemented EAAs in wound areas and regional muscles could also help explain the coexistence of plasma amino acid alterations and EAA's failure to correct these alterations as well as better retrieval of hip-joint dysfunction.The study has several limitations that need to be addressed in future investigations.These results need to be confirmed in a larger sample size, which also takes into account the changes of these variables in relation to gender and patient body composition. This requires a well-planned powered study. Understanding the patients' preoperative physical dysfunction rate would have strengthened the discussion. Pre- and postoperative assessment of muscle mass and strength could help us predict the gain in physical recovery better.Notwithstanding these limitations, we believe this study can provide some useful suggestions for clinical practice.The persistence of unmet metabolic alterations during the rehabilitation phase of EHA may predispose patients to a persistence of impairment and functional limitations at short- and long-term follow-up . Thus, EIn conclusion, it could be useful to provide patients with a supplementation of EAAs, both during postexercise recovery to stimulate muscle protein synthesis  and duriThis investigation would prompt us to look at two different research areas concerning changes in the dose and/or composition of the formula we used. For example, EAAs need to be tested at a dose higher than 8 g/d and/or for longer, to document whether plasma amino acid alterations may be corrected and functional recovery further enhanced. In addition, it could be clinically important to seek whether increasing the formula used here, that is, the amount of lysine and phenylalanine and adding the nonessential glycine, serine, asparagine, and glutamine, could further improve retrieval of postsurgery hip function. This could be particularly important for those patients who return home for full-hip function recovery and for subjects who, for any reason, do not have access to a post-EHA rehabilitation program.This study shows that recovery of hip dysfunction after elective surgery of EHA may be enhanced by short-term supplemented essential amino acids which, however, fail to correct plasma amino acid alterations.


The objective of our study was to evaluate the prevalence of abnormal maternal echocardiographic findings in triplet pregnancies presenting with dyspnoea.Between 2003 and 2013, patients’ records of 96 triplet pregnancies at our department were analysed including maternal and fetal outcome, echocardiographic parameters and N-terminal pro-B-type natriuretic peptide (NT-proBNP) levels. After exclusion of triplet pregnancies with fetal demise before 23 + 0 weeks, selective feticide or missing outcome data, the study population consisted of 60 triplet pregnancies. All women with dyspnoea underwent echocardiography and measurement of NT-proBNP.p < 0.001 by Mann-Whitney-U Test).Dyspnoea towards the end of pregnancy was observed in 13.3 % (8/60) of all women with triplet pregnancies, and all of these women underwent echocardiography. The prevalence of abnormal echocardiographic findings in women with dyspnoea was 37.5 % (3/8) with peripartum cardiomyopathy in one woman. Median serum NT-proBNP was significantly higher in women with abnormal echocardiographic findings compared with those without (1779 ng/ml, range 1045–6076 ng/ml vs 172 ng/ml, range 50–311 ng/ml; We conclude that triplet pregnancies presenting with dyspnoea show a high prevalence of abnormal echocardiographic findings. Since dyspnoea is a common sign in triplet pregnancies and is associated with a high rate of cardiac involvement, echocardiography and evaluation of maternal NT-proBNP could be considered to improve early diagnosis and perinatal management. The prevalence of multiple pregnancies has increased over the past three decades due to increases in ovulation induction, in vitro fertilisation and childbearing at older ages , 2. TripWomen with triplet pregnancies have significantly higher frequencies of hypertension, pre-eclampsia and diabetes . FurtherSymptoms of reduced cardiac function or heart failure may mimic normal physiological findings of late pregnancy, including persistent tiredness, oedema, orthopnoea and dyspnoea on exertion .One cause of heart failure that affects women late in pregnancy or in the early puerperium is peripartum cardiomyopathy with a reported maternal mortality rate of 9 % . It is dN-terminal pro-B-type natriuretic peptide (NT-proBNP) is secreted from the cardiac ventricles in response to ventricular volume expansion and pressure overload –15. NowaTo our knowledge, there are no data about the prevalence of peripartum cardiomyopathy or abnormal cardiac function in women with triplet pregnancies. Thus, this study aimed to evaluate the prevalence of abnormal echocardiographic findings as well as NT-proBNP levels in a large cohort of women with triplet pregnancies.This retrospective cohort study aims to evaluate the prevalence of abnormal maternal echocardiographic findings in triplet pregnancies treated at our tertiary referral centre as part of routine antenatal care. Ethical approval was obtained from the Ethics Committee of the Medical University of Vienna, reference number 1633/2012.During a study period of 10 years, over 26.000 pregnancies were referred to our hospital. Of those, 96 women (0.38 %) presented with a triplet pregnancy. Medical records of all women with triplet pregnancies who attended our department between December 2003 and December 2013 were reviewed. Demographic characteristics and data on pregnancy outcome were collected from the hospital maternity records.Of all triplet pregnancies, 14.6 % (14/96) were excluded because of fetal demise before 23 + 0 weeks, and 20.8 % (20/96) were excluded because of selective feticide (elective determination). Furthermore, 2.1 % (2/96) were excluded because of missing outcome data. Finally, the study population consisted of 60 women with triplet pregnancies.Women with triplet pregnancies routinely underwent ultrasound screening at 11 + 0 to 13 + 6 weeks since first trimester screening has been established. Chorionicity and amnionicity were routinely determined at first trimester screening by ultrasound examination of the dividing membranes characteristics and by examination of the placenta after birth. At 20–22 weeks, pregnant women routinely underwent anatomy scan. From 16 weeks of gestation onward, triplet pregnancies were routinely seen in 2-weekly intervals at our institution’s outpatient clinic. All women were seen by operators specialised in the field of multiple pregnancies. If a woman presented with dyspnoea , clinical assessment using 2-dimensional transthoracic and Doppler echocardiography was performed.All patients’ medical records were reviewed for maternal parameters such as maternal age, body mass index (BMI), pre-existing cardiac disease, smoking behaviour, parity, method of conception, pregnancy complications , use of tocolytics, mode of delivery and neonatal outcome parameters such as live birth, gestational age at delivery, birth weight, apgar 5 min after birth, arterial cord blood pH and admission to the neonatal intensive care unit.Furthermore, all patients’ medical records were reviewed for reports of dyspnoea, maternal echocardiographic ultrasound examinations and maternal serum NT-proBNP levels.The main outcome parameter of this study was the prevalence of abnormal maternal echocardiographic findings in women with triplet pregnancies.Furthermore, we evaluated the prevalence of peripartum cardiomyopathy. We used the definition of the European Society of Cardiology Study Group on Peripartum Cardiomyopathy for defining peripartum cardiomyopathy: ‘Peripartum cardiomyopathy is an idiopathic cardiomyopathy presenting with heart failure secondary to left ventricular systolic dysfunction towards the end of pregnancy or in the months following delivery, where no other cause of heart failure is found. It is a diagnosis of exclusion. The left ventricle may not be dilated but the ejection fraction is nearly always reduced below 45 %’ .2 body surface area [Echocardiographic results were analysed according to the guidelines of the National Heart, Lung and Blood Institute and Office of Rare Diseases  Workshop on Peripartum Cardiomyopathy including an ejection fraction of 45 % or less, fractional shortening of less than 30 %, or both and end-diastolic dimension of greater than 2.7 cm/mace area .Statistical analyses were performed with SPSS software . Parametric continuous variables were summarised as means (± standard deviation), non-parametric continuous variables as medians (minimum and maximum) and categorical data as percentages. Categorical variables were analysed using Fisher’s exact test, continuous variables were compared using unpaired T-test, Kruskal-Wallis or Mann-Whitney U-test. For infant outcomes, the correlation within sets of triplets was accounted for by considering robust estimates of variance based on the method of generalised estimating equations (GEE) .Sixty triplet pregnancies were analysed, including 63.3 % (38/60) trichorionic and 36.7 % (22/60) dichorionic triplet pregnancies. Patients’ characteristics are shown in Table The prevalence of abnormal echocardiographic findings in women with dyspnoea was 37,5 % (3/8), with peripartum cardiomyopathy in one woman. The whole study population showed a high rate of diabetes  and hypertension . One of the three women with abnormal echocardiographic findings had diabetes and one had pre-eclampsia.Dyspnoea was observed in 13.3 % (8/60) of all women with triplet pregnancies, and all of these women underwent clinical assessment using 2-dimensional transthoracic and Doppler echocardiography. Mean gestational age at examination was 27.8 (± 4.6) weeks of gestation. Three women had abnormal echocardiographic findings . All other women with dyspnoea had normal echocardiographic results, and in one woman the previously described mitral insufficiency was confirmed without worsening.Women with abnormal echocardiographic findings are described in detail:Patient no. 1 was a 30-year-old woman with a dichorionic triamniotic triplet pregnancy and mild pre-eclampsia from 26 weeks of gestation onwards. As antihypertensive therapy she received methyldopa. At 30.3 weeks of gestation, she presented with dyspnoea and oedema. Echocardiography showed normal left ventricular ejection fraction, impaired cardiac relaxation and pericardial effusion, and NT-proBNP was 1045 ng/ml. Maternal infections were excluded. The next day, dyspnoea and oedema worsened, additionally the woman reported palpitations, and therefore caesarean section was performed. Clinical improvement began right after delivery, and after 6 days the mother was free of symptoms and discharged from hospital.Patient no. 2 was a 28-year-old woman with a trichorionic triamniotic triplet pregnancy who presented with dyspnoea at 33.6 weeks of gestation. Echocardiography showed abnormal left ventricular diastolic function and impaired cardiac relaxation, and NT-proBNP was 1779 ng/ml. The next day the woman reported worsened dyspnoea, and caesarean section was performed. She recovered soon after delivery, and NT-proBNP decreased to 372 ng/ml. Five days after delivery the mother was free of symptoms and discharged from hospital.Patient no. 3 was a 37-year-old woman with a trichorionic triamniotic triplet pregnancy with insulin-dependent gestational diabetes. She presented with dyspnoea at 31.9 weeks of gestation. Echocardiography showed a left ventricular ejection fraction of 50 %. One day later, caesarean section was performed due to more pronounced dyspnoea resulting in marked limitation of physical activity in accordance with New York Heart Association functional class III . After delivery, the patient was transferred to the Department of Cardiology, and echocardiography showed a reduced left ventricular ejection fraction of 36 % and pericardial effusion, and the diagnosis of peripartum cardiomyopathy was made. NT-proBNP was 6076 ng/ml. Therapy with ramipril and bisoprolol was started, and the symptoms got better. Twenty-four days after delivery, echocardiography showed a left ventricular ejection fraction of 50 %, abnormal septal movements and mitral insufficiency. The patient felt much better and could be discharged. In the following months she had several check-ups with mild or no symptoms of heart failure reported.p < 0.001 by Mann-Whitney-U Test).NT-proBNP levels were measured in all women with dyspnoea  at the time of maternal echocardiography. Median NT-proBNP levels were significantly higher in women with abnormal echocardiographic findings compared with those without . In one of these (1/3), the diagnosis of peripartum cardiomyopathy was made. Abnormal echocardiographic findings were associated with diabetes and pre-eclampsia. All women with abnormal echocardiographic findings had markedly elevated NT-proBNP levels.This is one of the largest studies evaluating the prevalence of abnormal echocardiographic findings in triplet pregnancies. All pregnant women with dyspnoea underwent echocardiography. As typical for a retrospective study design, our report shows potential limitations. Since we did not perform echocardiography in all triplet pregnancies, we could not exclude that even women with shortness of breath which seems appropriate for pregnancy might have reduced cardiac function. Nevertheless, in those who presented with more pronounced symptoms, the percentage of abnormal echocardiographic findings was very high. Furthermore NT-proBNP levels were not measured longitudinally and not in all women with triplet pregnancies; therefore we cannot present normal values for NT-proBNP in triplet pregnancies.The high rate of dyspnoea (13.3 %) in triplet pregnancies could be explained by haemodynamic factors—even in singleton pregnancies there is a significant increase in blood volume and cardiac output resulting in transient left ventricular remodelling and hypertrophy –23. ThisHypertension and pre-eclampsia have been described as risk factors for peripartum cardiomyopathy , 11. We In our study, abnormal echocardiographic findings were observed after 30 weeks of gestation. Since triplet pregnancies were delivered at a median gestational age of 32–34 weeks, reduced cardiac function occurred in the last month of pregnancy in our study population, which is in accordance with the time of diagnosis of peripartum cardiomyopathy in singletons .We showed a prevalence of peripartum cardiomyopathy of 1.7 % in triplet pregnancies, which is higher than previously reported prevalence of 0.1–0.3 % in different populations including mainly singleton pregnancies , 25. It It has been described that NT-proBNP, which is a quantitative serum marker of cardiac disease reflecting systolic and diastolic ventricular dysfunction, is elevated in patients with heart failure and in women with peripartum cardiomyopathy , 30. ForAlthough only one woman with abnormal echocardiographic findings fulfilled the diagnostic criteria of peripartum cardiomyopathy, one could assume that also the other women with abnormal echocardiographic findings may have similar pathophysiological processes since some shared risk factors for peripartum cardiomyopathy, and all women with abnormal echocardiographic findings had significantly elevated levels of NT-proBNP. However, this remains speculative.Since reduced cardiac function can mimic normal physiological findings of late pregnancy , and these symptoms are even more pronounced in triplet pregnancies, diagnosis of reduced cardiac function could be challenging. Nevertheless, 37.5 % (3/8) of all women who presented with remarkable dyspnoea had abnormal echocardiographic findings, and 62.5 % (5/8) had normal echocardiographic results.Triplet pregnancies presenting with dyspnoea show a high prevalence of abnormal echocardiographic findings. Since dyspnoea is a common sign in triplet pregnancies and is associated with a high rate of cardiac involvement, echocardiography and evaluation of maternal NT-proBNP could be considered to improve early diagnosis and perinatal management.This research received no specific grant from any funding agency, commercial or not-for-profit sectors.