Rhodnius prolixus is a main vector of Chagas disease, which affects several million people, mostly in Latin-America. Host searching, pheromone communication, and microclimatic preferences are aspects of its behaviour that depend on multimodal sensory inputs. The molecular bases of these sensory processes are largely unknown. The expression levels of genes transcribed in antennae were compared between 5th instar larvae, and female and male adults by means of RNA-Seq. The antennae of R. prolixus showed increased expression of several chemosensory-related genes in imaginal bugs, while both sexes had similar expression patterns for most target genes. Few cases suggest involvement of target genes in sexually dimorphic functions. Most odorant and ionotropic receptor genes seemed to be expressed in all libraries. OBPs and CSPs showed very high expression levels. Other sensory-related genes such as TRPs, PPKs and mechanoreceptors had consistent levels of expression in all libraries. Our study characterises most of the sensory gene repertoire of these insects, opening an avenue for functional genetics studies. The increase in expression of chemosensory genes suggests an enhanced role in adult bugs. This knowledge allows developing new behaviour interfering strategies, increasing the options for translational research in the vector control field.The triatomine bug  Rhodnius prolixus Stål, 1859  is the second most important vector of Chagas disease, transmitting its etiological agent, i.e., Trypanosoma cruzi, to humans in Colombia and Venezuela1T. cruzi transmission.The haematophagous bug R. prolixus adult bugsNumerous neuroethological studies have improved our understanding of diverse aspects of triatomine olfactory-mediated behaviours, such as host searching121314R. prolixus genome has recently been sequencedR. prolixus genome, including 5 pseudogenesDrosophila melanogaster OSNs located in coeloconic sensillaR. prolixus genome, 33 IRs have been identified. Other sensory-related genes annotated for this species include gustatory receptors (GRs = 28); odorant binding proteins (OBPs = 27); chemosensory proteins (CSPs = 19); sensory neuron membrane proteins (SNMPs = 1); secreted esterases belonging to the detoxification/pheromone hormone processing class (10); CYP4 clade members (22); transient receptor potential channels (TRPs = 9); and pickpocket receptors (PPKs = 6)18The th instar larvae, and female and male adults of R. prolixus and characterized transcript levels for the main sensory protein families mentioned above.In triatomines, as in other hemimetabolous insects, the imaginal moult represents significant changes in physiology and behaviour. Our hypothesis is that the more complex behavioural repertoire of adult bugs should be correlated with changes in gene expression profiles at their sensory structures. To characterize these molecular changes at the peripheral level, we have sequenced the antennal transcriptomes of 5The sequencing yield was 353.6 M read-pairs, distributed as 103 M for larval, 125 M for female, and 125.6 M for male libraries. A total of 128,047 contigs, with an average length of 744 bp, were assembled using SOAPdenovo. The length of contigs ranged from 100 to 35,978 bp and N50 value was 2,518. Using Trinity, a total of 303,403 contigs with an average length of 368 bp were assembled. The length of contigs ranged from 101 to 29,400 bp and N50 value was 649.R. prolixus genome. Those transcripts enriched in the antennal transcriptomes and their Gene Ontology annotations are detailed in 2+ flux activates a number of signalling pathways including but not restricted to nucleoside diphosphate kinase activity and nucleoside diphosphate phosphorylation, which appeared only in the male library  was shared by all libraries . AdditioPiezo and narrow abdomen genes. In addition two IRs identified as pseudogenes in the genome  had coding sequences in the antennal transcriptome that allowed their modelling as intact genes.The transcript sequences allowed us to manually improve the gene models for 16 ORs, two GRs, 12 IRs, seven OBPs, three CSPs, two ammonium transporters, four TRPs, two PPKs, and R. prolixus genome by tBLASTn searches against the VectorBase database using orthologous sequences from other insects222324R. prolixus genome  and annotated as CheB1-CheB5 respectively (CheB1 (RPRC004662) was manually edited. No sequence belonging to the CheA protein family was found in the bug genome.Additional sensory genes were identified in the ectively . The seqSnmp1b (RPRC013910); Snmp1c (RPRC000399); Snmp1d (RPRC002720); and Snmp2 (RPRC002754) (Amt1 (RPRC006389). The previously identified ammonium transporter (Amt)Amt3 according to tBLASTn results. The sequences of Rh50 and both Amts were manually edited after comparing them to our transcriptome assemblies.Only one SNMP sequence (RPRC013907) had been annotated in the bug genomeC002754) . SeveralC002754) . A new aTRPA1 isoform were identified and annotated , raising the number of R. prolixus TRPs to 14. The sequence described as pyrexia in the genome paper (identified as RPRC000570) was properly annotated as TRPA5a based on its sequence similarity to other TRPA5 members. Additionally, the sequence of TRPML needed to be manually edited after comparison with the originally described gene. Four new PPK receptors were identified, bringing the total of R. prolixus PPKs to ten. Finally, new orthologous sequences of known insect mechanoreceptor genes were identified: mrityu (RPRC014507), two type B chloride channels (RPRC0065358 and RPRC013530), and three NMDA receptors . The sequences of mirtyu and both chloride channel-b genes were manually edited after comparison to our transcriptome assemblies. The nucleotide sequences of all genes analysed in this work are included in R. prolixus genome generic file format (GFF) was created and all the adjustments included  to analyse gene expression profiles. The BioProject PRJNA281760 in the Sequence Read Archive at NCBI contains all the RNA-Seq reads produced in this study. A high proportion of reads mapped to the R. prolixus genome, being 92% for larval, 94.6% for female and 94.3% for male libraries. A total of 17,190 genes and 17,353 isoforms were predicted by Cufflinks-Cuffmerge in the consensus transcriptome based on the three conditions studied.The Illumina reads were mapped to the modified version of the RproC1 vs. female (2 = 0.903) and larval vs. male (2 = 0.872) antennal transcriptomes showed an overall similar gene expression for antennae of larvae and adults. Regarding male and female antennae, both transcriptomes showed a similar global expression profile (2 = 0.918). Interestingly, several transcripts showed higher expression in the male than in the female transcriptome  were compared in pair-wise regression analyses and visualized using scatter plots . The comvs. male results  and larval vs. female results  were compared (see The expression levels of 25 genes and two reference genes  were evaared see .2 = 0.586) and male (2 = 0.584) bugs, when compared to those seen in the larval transcriptome. As observed in the overall comparison, the expression levels of this set of sensory receptor genes were similar in the antennae of male and female adult bugs (2 = 0.888).The expression levels of a set of 217 transcripts belonging to OR, IR, GR, PPK, TRP, CheB and SNMP protein families were compared in pair-wise regression analyses and visualized using scatter plots represented as Log10 FPKM +1 . This seR. prolixus genome contains 116 ORs, including Orco and five pseudogenes. Our transcriptome analyses suggest that at least 83 ORs (75%) present transcripts in R. prolixus antennae  did so exclusively in female antennae and, six  augmented only in male antennae , and not the IR co-receptor genes, which showed a maximum FPKM of 13  showed expression profiles similar to that of Ir25a  . Many ofincrease . The antincrease , a thirdincrease .Gr26, Gr27, and Gr28 was relatively high (Gr1 (orthologue of D. melanogaster fructose receptor), Gr2, Gr20 and Gr24 seemed to have increased expression in the antennae of adult bugs  showed significantly increased expression in the antennae of females when compared to those of larvae  were found to be expressed in the antennae , part of the Ir75 gene subfamily, as well as other specific receptor genes like Ir41a and Ir41b . More than 10,000 genes were expressed in antennae, meaning that at least 60% of all genome genes were expressed, similarly as seen for D. melanogaster antennaede novo transcriptome assemblies and the subsequent manual curation process have allowed validating a large number of gene models, while many additional sensory-related genes have also been identified here. An edited version of the R. prolixus genome generic file format (GFF) was created including these adjustments  is similar to that reported  in the last version of the ustments , improviRhodnius prolixus were obtained from a colony held at the Centro de Pesquisas René Rachou - FIOCRUZ. Insects were reared under controlled conditions at 26 ± 1 °C, 65 ± 10% relative humidity and a 12 h:12 h light/dark cycle provided by artificial lights . Colony insects were routinely fed ad libitum with citrated rabbit blood , using an artificial membrane feeder. This procedure does not require approval by the institutional ethics committee, as no live vertebrates are used. Experiments were performed using 5th instar larvae and female and male adult insects, all being unfed and 21 days-old when antennae were excised (adult bugs were sorted by sex at 5th instar and kept virgin until their antennae were excised). A total of sixty antennae were collected per sample and used for RNA extraction for subsequent RNA-Seq library preparation. Tissues were manually homogenized using sterilized pestles and total RNA was extracted using TRIzol® Reagent  according to the manufacturer’s instructions. Then, extracted RNA was resuspended in 22 μL of DEPC-treated water (Life Technologies), and its concentration determined at 260 nm using a Nanodrop . Extraction yield for the larval antennae sample was 4.5 μg , while it reached 2.8 μg for samples of male and female adult antennae. RNA integrity and quality were assessed by means of an agarose electrophoresis gel and a Bionalyzer using the RNA 6000 Nano Kit .Library construction and Illumina HiSeq2000 sequencing services were hired at the W. M. Keck Centre for Comparative and Functional Genomics . RNA-Seq libraries were constructed using the TruSeq Stranded RNA Sample Preparation Kit . Briefly, messenger RNA was selected from one microgram of high quality total RNA. First-strand cDNA was synthesized with a random hexamer and SuperScript II (Life Technologies). Double stranded DNA was blunt-ended, 3′-end A-tailed, and ligated to indexed adaptors. The adaptor-ligated double-stranded cDNA was amplified by PCR for 10 cycles with the Kapa HiFi polymerase . The final libraries were quantified on a Qubit (Life Technologies), and the average size, 280 nt, was determined on an Agilent bioanalyzer DNA7500 DNA chip. Individual libraries were diluted to 10 nM, and the indexed libraries were pooled in equimolar concentration. The pooled libraries were further quantitated by qPCR on an ABI 7900 (Life Technologies). The multiplexed libraries were loaded onto one lane of an 8-lane flowcell for cluster formation and sequenced on an Illumina HiSeq2000 with TruSeq SBS sequencing kits version 3. The libraries were sequenced from both ends of the molecules to a total read length of 100 nt from each end. The raw.bcl files were converted into demultiplexed fastq files with Casava 1.8.2 (Illumina).The single lane of paired-end sequencing yielded 353,628,668 total raw reads. The larval sample produced 103,016,074 reads; the female 124,995,224 reads; and the male 125,617,370 reads.www.bioinformatics.babraham.ac.uk/projects/fastqc/) was used to assess the reads bias for the sequencing lanes as a whole. Subsequently, FASTX Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/index.html) was used to trim off biased reads at the 5′ end, and then using the quality score trimmer to remove low-quality reads at the 3′ end of the read (−t 20). After trimming processes, the final number of reads was 102,457,875; 124,386,666; and 124,742,367 for larvae, female and male libraries, respectively. Using all trimmed reads obtained from the three libraries, two de novo transcriptome assemblies were elaborated using SOAPdenovo v.1.02K-mer sizes were assessed; 49-mer yielded the best assembly for the desired application, and was chosen to construct the de Bruijn graph. Trinity was used with default parameters, except minimum reported contig length = 100.The FASTQ software  were created for these new models and these were subsequently included in the RproC1.2 version of the R. prolixus genome GFF file (downloaded from http://Vectorbase.org/downloads/) which was used in the subsequent read mapping analysis. The nucleotide sequences of all genes analysed in this work and the edited GFF file are included in the The sequences of all target genes were compared to our SOAPdenovo and Trinity assemblies using BLASTn searches. Subsequently, incomplete or inconsistent sequences found in that genome version were manually corrected/extended based on our transcriptome assemblies. Suffixes were added to gene names according to their gene model characteristics: NTE – N-terminus missing in gap, CTE – C-terminus missing in gap, INT – internal exon missing in gap, FIX – genome assembly was repaired; JOI – gene model spans scaffolds; FX - gene model repaired based on R. prolixus genome assembly (version RproC1.2) by means of TopHat v. 2.0.11The filtered and trimmed Illumina reads from the three libraries were mapped independently to the R. prolixus antennae . Subsequently, a linear regression analysis was performed to calculate the slope coefficient and R2 values for each comparison. Heat maps showing gene expression  of the different protein families in the three conditions were prepared using ggplot2 (www.ggplo2.org).The Cufflinks-predicted transcripts and genes and their FPKM values from larvae, female and male libraries were used for gene expression comparison. R package was used to generate scatter plots to compare expression levels (represented as Log10 FPKM +1) of each transcript in pair-wise analyses: larval transcriptome Rhodnius prolixus insects from the same colony, reared in the same conditions and whose antennae were excised in the same developmental/physiological conditions of those used for RNA-Seq samples were utilized for qRT-PCR validation. Each treatment  was replicated 6 times using 60 antennae  per sample. Total RNA was extracted using 500 μL of TRIzol® Reagent (Life Technologies) according to the manufacturer’s instructions. Later, each RNA sample was resuspended in 30 μL of DEPC-treated water (Life Technologies), and its concentration determined using a Qubit® 2.0 Fluorometer (Life Technologies). Subsequently, all samples were treated with RQ1 RNase-Free DNase . All treated RNA (33 μL per sample) was immediately used to synthesize cDNA using the SuperScript III Reverse Transcriptase (Life Technologies) and a 1:1 mix of Random Hexamers and 10 μM Oligo(dT)20 primers in a final volume of 60 μL.In order to validate RNA-Seq data and expression profiles, the expression levels of 25 genes were determined by means of the qRT-PCR technique . Rhodniu2) for each primer were determined using the slope of a linear regression model. Information about primers, amplicons and calibration curves is presented in −ΔΔCt method. First, the gene expression levels were normalized to the geometric mean of two reference genes, α-tubulin and G6PDH, for each condition. Subsequently, expression levels of each gene were normalized to the expression levels of larvae.For qPCR reactions, 5 μL of SYBR Green PCR Master Mix (Life Technologies) were used in the reaction mixture that also contained 0.4 μL of a 10 μM primer solution and 1 μL of cDNA sample diluted 2-fold into a final volume of 10 μL. The reactions were conducted using a ViiA™ 7 Real-Time PCR System (Life Technologies) under the following conditions: one 10 min cycle at 95 °C, followed by 40 cycles of 15 s at 95 °C, 20 s at 60 °C and 30 s at 72 °C. Following the amplification step, a melting curve analysis was performed to confirm the specificity of the reaction. In all qPCR experiments, non-template controls (NTC) were included in triplicate for each primer set to verify the absence of exogenous DNA. The PCR efficiencies (E) and repeatability  and NCBI under the project accesion number PRJNA281760/SRP057515 and the SRA accession numbers for the three experiments can be accessed at SRS923612/SRX1011796/SRR2001242 ; SRS923595/SRX1011769/SRR2001240 ; and SRS923599/SRX1011778/SRR2001241 .How to cite this article: Latorre-Estivalis, J. M. et al. The molecular sensory machinery of a Chagas disease vector: expression changes through imaginal moult and sexually dimorphic features. Sci. Rep.7, 40049; doi: 10.1038/srep40049 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Additionally, VP patients were less likely to have received influenza vaccination. VP patients had a systemic inflammation response involving IL-6, TNF-α, and MCP-1 which may be due to virus-induced activation of macrophages. There are important opportunities for further investigating AECOPD mechanisms and for the development of better strategies in the management and prevention of virus-related AECOPD.Viral infection is a common trigger for acute exacerbations of chronic obstructive pulmonary disease (AECOPD). The aim of this study is to investigate the expression of cytokines in AECOPD. Patients with AECOPD requiring hospitalization were recruited. Meanwhile healthy volunteers of similar age that accepted routine check-ups and showed no clinical symptoms of inflammatory diseases were also recruited. Induced sputum and serum were collected. Induced sputum of participants was processed and tested for thirteen viruses and bacteria. Forty cytokines were assayed in serum using the Quantibody Human Inflammation Array 3 . The most common virus detected in virus positive AECOPD (VP) was influenza A (16%). No virus was found in controls. Circulating levels of IL-6, TNF- Acute exacerbation of chronic obstructive pulmonary disease (AECOPD) is characterized by changes in sputum production, dyspnoea, and cough , 2. AECOViral infection has been confirmed as a cause of AECOPD and considered a major cause of AECOPD hospitalizations . Viral dα (TNF-α) and interleukin-6 (IL-6) are higher in COPD than those of healthy controls (HC) [The host inflammatory response is an important component of AECOPD. The profile of inflammatory markers may be used to classify airway inflammation in AECOPD with heterogeneous inflammation . Many cyols (HC) –11. HoweIn this study, we aimed to determine the prevalence of several respiratory viruses in AECOPD and to detect the distribution of systemic inflammatory markers.1)/forced vital capacity (FVC) < 70%, and postbronchodilator FEV1 < 80%) [Patients diagnosed with COPD and clinical symptoms of AECOPD requiring hospitalization were recruited from June 2012 to May 2013 in Jilin Provincial People's Hospital. Patients were recruited within 4–12 h after presentation. All of the patients were diagnosed with COPD according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria (forced expiratory volume in 1 second (FEV1 < 80%) . Patient2 by inoculating in eosin methylene blue agar, blood agar, Brucella agar, and chocolate agar media. Participants whose samples were identified as having bacterial infection or bacterial-viral coinfection were excluded.Sputum was taken to a sterile container and was analyzed immediately microscopically by Gram staining. After Gram staining and direct microscopic examination, sputum was incubated for 18–36 hours at 37°C in 10% CO1 every 5 minutes. Nebulization continued if the level of FEV1 had not fallen by more than 15% for a maximum of 20 minutes. All sputum was processed within 2 h. Mucus clumps were separated from saliva using a forceps, and a minimum of 200 μL sample was transferred to a test tube; 4 times the volume of Sputolysin  working solution was added to the tube and was mixed at room temperature for 30 minutes. Subsequently an equal volume of PBS was added and fully mixed. The samples were then filtered and centrifuged at 400 ×g for 10 minutes. After removing the supernatant, the cell pellet was resuspended in Lysis Buffer . Later total nucleic acid extraction was performed in 200 μL sample suspended in Lysis Buffer.Recruited patients received nebulized 0.9% saline for 30 seconds . Spirome10 mL venous blood was collected into a heparin-lithium anticoagulant tube and the serum was stored at −80°C for later analysis of inflammatory markers. All samples were obtained within 24 h of enrolment.Measurement of virus was performed on RNA by Viral RNA Mini Kit (QIAamp®). RNA was extracted from induced sputum from AECOPD participants and controls. Thirteen viruses were tested, using RT-PCR (ABI AgPath-ID™ One-Step RT-PCR Kit) to detect influenza A and B; RT-PCR  to detect hMPV, coronavirus, adenovirus, and bocavirus; Multiplex RT-PCR  to detect parainfluenza −1, −2, −3, and −4, rhinovirus, respiratory syncytial virus, and enterovirus (Ev71). Sputum was used for bacterial culture as well. According to the results, participants were divided into virus positive AECOPD (VP) participants, bacteria positive AECOPD (BP) participants, virus-bacteria negative AECOPD (VN) participants, coinfection (CI) participants, and controls.Serum was obtained after centrifugation  of whole blood and was kept at −80°C prior to inflammatory marker profiling. The levels of cytokines were evaluated by Quantibody Human Inflammation Array 3 , according to the manufacturer's instructions.t-test. For cytokine expression assay, data were of normal distribution, ANOVA was used to evaluate the expression of different cytokines, and the levels were measured through the multiple comparison method. Data were expressed as mean values ± standard error of mean. Statistical analysis was performed using Graph pad 6.0 and p < 0.05 was considered statistically significant.Normality of data was tested by the Kolmogorov-Smirnov test. For basic characteristics of participants in There were 100 eligible AECOPD patients during June 2012 to May 2013. The recruitment process is shown in  Haemophilus influenzae (p < 0.05). No AECOPD were observed in May and July, and no VP were observed in March, April, May, and July.The most common virus was influenza A , and the most common bacterium isfluenzae . The numfluenzae  (p < 0.0p > 0.05) (p < 0.05) and sore throat  when compared with VN participants. Moreover, CI participants had the highest rate of fever (40%) and sore throat (46%).Baseline characteristics and treatment of the recruited patients are shown in  > 0.05) . VP partp < 0.05). The number of participants with exacerbation symptoms that lasted for more than 4 days was 70% in VP, which was higher than that of VN (p < 0.05). The percentages of VP participants that received treatments showed no difference when compared with that of VN participants (p > 0.05).The rate of current smokers in CI participants (40%) and VP participants (35%) was higher than VN participants (30%) . 11 , and overexpression of IL-6, TNF-α, and MCP-1 was also observed between VP and VN (p < 0.05) , and the levels of these three markers demonstrated no difference between VP and CI (p > 0.05).Forty measured markers are shown in  < 0.05) . The difStudies specifically related to viral infection and cytokines expression in AECOPD are uncommon, especially in Asia. To the best of our knowledge, this is the first study conducted in the Alpine region in Asia, in relation to the prevalence of respiratory viruses and the expression of cytokines for AECOPD.Interestingly, the viral distribution in our study is also quite different from most of other studies. Influenza A was the most common respiratory virus in our study and was found in 10% of AECOPD subjects , which iIn our study, 29% of recruited AECOPD patients were vaccinated against influenza last year, and among these, 80% had been vaccinated in VN, 46% had been vaccinated in CI, and however only 23% participants were vaccinated in VP. Cohorts with higher vaccine coverage for influenza have been found to have a lower prevalence of influenza , 19, whiWe also note the odd phenomenon that RSV was not found in viral AECOPD in our study all the year round. In a study conducted from January to June 2014 in China, despite low rate, three cases of RSV infections were found in nasopharyngeal samples of 81 participants admitted for AECOPD , which wViral symptoms include increased rhinorrhoea, sore throat, and fever/chills . Besidesα, IL-6, and MCP-1 than those without , indicating that the difference of marker expression might be caused by viral infection. IL-6 is a pleiotropic acute-phase cytokine involved in the host immune response to infection, reflecting the severity of lower airway inflammation [α is a mononuclear-phagocyte-origin cytokine that has pleiotropic effects on innate host responses to microbes [α may be associated with the activation of macrophages. Influenza virus, which was the most common virus in our study, induces the activation of macrophages via Toll-like receptor 3 (TLR3) [α, and MCP-1 [The profiling of 40 inflammatory markers was conducted to assess the association between viral infection and host cytokine responses in AECOPD. In previous studies the major objective of detection was to investigate the types of microorganisms involved in AECOPD, and less emphasis was placed on proinflammatory cytokines. We found that patients with viral infections showed higher levels of TNF- without . Patientammation , 28. Higammation , 29. TNFmicrobes  and it rmicrobes  and is omicrobes . MCP-1 imicrobes , 34. Higmicrobes , 36. Themicrobes . Higher 3 (TLR3) . Yageta 3 (TLR3)  found thnd MCP-1 . Tissue nd MCP-1 . Taken tα, and MCP-1, suggesting a prominent role for macrophage activation in AECOPD. Future work can define the clinical and treatment implications of these result, and lay the basis for further investigating the role of immune system in the pathogenesis and development of AECOPD.In summary, we have established that viral infection is common and has an altered viral profile in AECOPD that occurs in Alpine China. Viral AECOPD is associated with a systemic immune response that involves IL-6, TNF-α, and MCP-1 which may be due to the activation of macrophages that was caused by the viral infection. There are important opportunities for further investigation of AECOPD mechanisms and for the development of better strategies in the management and prevention of AECOPD that is associated with viral infection.Influenza A is the most prevalent viral pathogen in AECOPD in Northern China. Patients with viral AECOPD were less likely to have received influenza vaccination. They had a prominent systemic immune response involving IL-6, TNF-


Accelerometer measured physical activity (PA) studies particularly in non-western populations are lacking. Therefore, this study investigated stepping activity in a multi-ethnic urban Asian population.Adult participants from the Singapore Health Study 2 consented to accelerometer activity monitoring for 7-consecutive days. Mean daily step count, peak stepping intensity (i.e. cadence) over 1-min, 30-min and 60-min and time spent in each cadence band: 0 (non-movement), 1–19, 20–39, 40–59, 60–79, 80–99 and ≥ 100 steps/minute (moderate to vigorous PA) were calculated.p = 0.02) but not Malays 7140 (adjusted p = 0.052) compared to Chinese (7745 steps). The proportion of Malays, Indians, and Chinese achieving < 5000 daily steps was 26%, 23% and 14%, respectively (p < 0.01). Regardless of ethnicity, approximately half of the recorded time was spent undertaking 0-steps/minute (7.9 h).A total of 713 participants  were included. Overall, the mean daily step count was 7549. Mean daily step count was significantly lower in Indians (7083 adjusted Greater promotion of brisk walking is required in light of the low step volume and pace observed in this multi-ethnic Asian population. Ethnic differences in stepping activity were also identified which indicates a need for targeted ethnic specific health promotion interventions. Regular physical activity (PA) has repeatedly been shown to be associated with good overall health. International guidelines, such as those formulated by the World Health Organisation (WHO), recommend that adults undertake at least 150-min of moderate intensity aerobic PA/week, which can reduce the risk of developing certain cancers, type 2 diabetes, cardiovascular diseases, falling and mental health issues . A PA suDespite clearly defined guidelines and the ability to accumulate PA from a number of sources, a large proportion of global populations do not reach PA goals and inactivity has contributed to the rise in non-communicable diseases (NCDs) . An estiMeasuring steps is a popular way to determine PA, especially considering the increasingly widespread use of consumer PA trackers. However, assessment of stepping volume alone is limited as it does not factor in the intensity of the PA; the focus of PA recommendations, nor does it consider periods of sedentary behaviour. To address this gap, research has recently applied cadence measurement in free living populations i.e. the number of steps taken/minute. Incorporating a measure of cadence into accelerometer measured PA research can then be used to assess whether PA goals (i.e. 150 min/week) are met through stepping based activities . LaboratTo address identified gaps in the literature the aim of this study was to investigate stepping activity in a multi-ethnic urban Asian population using accelerometers and to examine if differences in stepping volume and intensity were apparent between different ethnic groups.n = 2686) were approached to take part in a further investigation into accelerometer measured PA, which formed the population for this study. Participants of SHS2 were offered to participate in different additional studies as part of the consent procedure (one by one), including this PA study. Those who agreed to participate in the accelerometer measured PA study were subsequently enrolled.Participants of the Singapore Health Study 2 (SHS2) (n = 29).The SHS2, is a cross-sectional representative health survey of participant’s residing in Singapore. A random sample of households were mailed regarding the survey between 2014 and 2015 and followed up through home visits with a trained interviewer. Eligible participants were permanent residents born between 1933 and 1994. Exclusion criteria for participation in the survey were: being pregnant, having a severe mental retardation or mental illness, having had a stroke or injury resulting in speech impairment or being bedridden or wheelchair bound. Information on socio-demographic characteristics and clinical history were captured during a home visit with a researcher and included: age (years), gender , marital status (married or single: separated/divorced/widowed), educational level , employment status , monthly household income (in Singapore dollars), Body Mass Index (BMI) determined from self-reported height and weight, diagnosis of hypertension, diabetes, asthma or arthritis (yes/no), tobacco use (smoker: currently smoking cigarettes), alcohol use . Only those of Chinese, Malay and Indian ethnicity, the main ethnic groups in Singapore, were included in the current analysis. Other ethnicities were excluded due to small sample size  and instructed to wear the device continuously for 7 consecutive days, positioned at the hip except when bathing or swimming. Participants were advised to continue their usual routine whilst wearing the device. Raw accelerometer data were extracted from the devices and re-integrated in 1-min epochs using ActiLife software™ (version 6) and processed using the package ‘accelerometry’ in R . Validit2 test were used to test differences between participant demographics as appropriate. Analysis of covariance and pairwise comparisons were performed to test for differences in continuous stepping parameters by ethnicity and differences in mean peak cadence by step categories. Multivariate logistic regression was used to test for associations between categorical stepping parameters and ethnic group. All analyses were adjusted for age, gender, marital status, education, diagnosis of arthritis, hypertension, diabetes or asthma, smoking status, alcohol drinking status, BMI and wear-time. A p value of < 0.05 was considered significant. Mean daily step counts were calculated for each participant by averaging the total steps/day across the days of wear, subsequently deriving the mean value. Mean peak cadences were calculated by ranking the individual cadence minutes and identifying the top 1 min for peak 1-min cadence and the mean of the top 30 and 60-min steps/minute (not necessarily consecutive minutes) for each participant. The mean peak 1-min, 30-min and 60-min cadence was then derived.All analyses were conducted in STATA 14.2. Pearson’s pairwise correlation was performed to investigate the relationship between daily step count and peak 1-min, 30-min and 60-min cadence for the whole sample and by ethnic group. Assumptions were checked prior to calculating correlations. Descriptive statistics were calculated as frequencies (%) and mean (with standard deviation). Data is presented by the total population and by ethnic group . Two-sample t-test and chiApproval was obtained from the National University of Singapore Institutional Review board. All participants provided written informed consent to take part in the study.Out of the 895 participants who agreed to take part in the accelerometer measured PA study, 742 (83%) had valid accelerometer data. As per the inclusion criteria, 29 participants from other ethnic groups were removed leaving a total of 713 participants in the analyses. Table p = 0.02). Other ethnic group comparisons did not reach statistically significant differences for daily step count. The largest step category across all ethnic groups (35–39% of participants) was 5000–7499 daily steps, which is classified as ‘low activity’ [p = 0.15, Table p < 0.05). The overall proportion of participants achieving 30 min of moderate intensity PA through steps (≥100 steps/minute) was 27%. The proportion meeting this activity level was statistically significantly lower in Indian (15%) and Malay (16%) participants versus Chinese (32%) (unadjusted chi2p < 0.001).The mean daily step count was 7549 steps and did not exceed 10,000 steps/day in any ethnic group Table . Indiansp < 0.001). Peak 1-min, 30-min and 60-min cadences are presented by stepping group categories in Fig. p < 0.001).Significant correlations were found between daily step count and peak cadences overall: 1-min (0.66), 30-min (0.79), 60-min (0.86) (steps/minute) and by ethnic groups: Chinese 0.62, 0.77, 0.84, Malay 0.68, 0.83, 0.89 and Indian participants 0.78, 0.84, 0.88, peak 1-min, 30-min and 60-min cadence respectively  . One of In terms of the relationship between step volume and intensity significant correlations between the two dimensions of step activity were found in this study. Increasing step volume could therefore also result in meaningful improvements in intensity and thus meeting PA goals. However, in this population the mean peak 1-min and peak 30-min cadence only exceeded ≥100 steps/minute (moderate intensity PA) in those categorised in the 10,000 daily steps group and only 16% of the study population were in the 10,000 daily step group. The combination of limited PA levels and increased metabolic risk in a population of Asian descent is concerning . One stuOther factors such as culture could also influence PA levels and what is perceived as PA. A previous investigation into self-reported PA in Singapore found that Malays were the most physically active with 79% meeting PA guidelines compared to 75% of Indians and 73% of Chinese , which iThis study investigated stepping patterns in a mixed ethnic population using an accelerometer. Other activities such as cycling, swimming and occupational activities which do not require stepping are not accurately captured by the accelerometer and thus some participants may be undertaking more PA than is reflected by the stepping activity reported. In addition, only a proportion of those who took part in the Singapore Health Study survey were included in this accelerometer based study. This may limit the generalisability of the study findings to the wider Asian population in Singapore and beyond as, for example, only those interested in PA may have taken part.Promotion of walking activity is an important strategy for achieving PA recommendations. This study suggests a need to improve walking volumes and intensity which by logic will also reduce large accumulations of non-movement. Future strategies should consider tailoring health promotion strategies by ethnicity to engage the population more broadly.


Between 2009 and June 2010, patients received bortezomib  together with low-dose IVIG and PPH . In July 2010, we increased the IVIG dose and treated all subsequent patients with bortezomib, high-dose IVIG (1.5 g/kg), and PPH . Graft survival at three years after treatment was 73% in group BHP as compared to 45% in group BLP and 25% in group RLP. At six months after treatment median serum creatinine was 2.1 mg/dL, 2.9 mg/dL, and 4.2 mg/dL in groups BHP, BLP, and RLP, respectively (p = 0.02). Following treatment, a significant decrease of donor-specific HLA antibody (DSA) mean fluorescence intensity from 8467 ± 6876 to 5221 ± 4711 (p = 0.01) was observed in group BHP, but not in the other groups. Our results indicate that graft survival, graft function, and DSA levels could be improved along with stepwise modifications to our treatment regimen, that is, the introduction of bortezomib and high-dose IVIG treatment.Throughout the past years we stepwise modified our immunosuppressive treatment regimen for patients with antibody-mediated rejection (ABMR). Here, we describe three consecutive groups treated with different regimens. From 2005 until 2008, we treated all patients with biopsy-proven ABMR with rituximab (500 mg), low-dose (30 g) intravenous immunoglobulins (IVIG), and plasmapheresis  (group RLP,  Patients of all groups additionally received a three-day pulse of methylprednisolone (500 mg/day i.v.). After discharge all patients were regularly monitored in our outpatient clinic.Between January 2005 and November 2008 nine consecutive patients with biopsy-proven ABMR were treated with a fixed dose of rituximab (500 mg i.v.), six sessions of PPH , and low-dose (30 g) polyvalent human IVIG (KIOVIG®) after the last PPH (group RLP). Since 2009, all of our patients with a diagnosis of ABMR have received bortezomib-based treatment. However, three patients received rituximab between 2010 and 2013 because of a diagnosis of preexisting polyneuropathy, which is a well-known side effect and consequently a contraindication for bortezomib treatment. Therefore, group RLP finally comprised 12 patients. Between January 2009 and June 2010 eleven consecutive patients received bortezomib  with and without dithiothreitol using T- and B-lymphocytes with current and historical serum. In addition, graft allocation was based on a negative virtual crossmatch by considering current and historical unacceptable antigens as defined by Luminex® based single antigen bead assays. Based on this procedure, none of the patients underwent any kind of desensitization before transplantation. Consequently, only patients with de novo DSA were included.n = 22) or daclizumab (n = 4). The remaining eight patients were transplanted before the introduction of basiliximab or daclizumab and received no induction therapy. The distribution of patients, who received no induction therapy, was not significantly different between groups .Altogether, 76% (26/34) of all patients received induction therapy with either basiliximab , proteinuria (≥1 g/d), or primary nonfunction in the early phase after transplantation. The diagnosis of ABMR was based on the presence of circulating DSA and significant allograft pathology according to the definitions of the Banff classification . C4d staSerum samples before and after treatment were screened for HLA antibodies (HLAab) by the Luminex bead-based assay LABScreen® Mixed . In addition, HLAab specificities were determined by LABScreen Single Antigen beads assay (One Lambda). As an indicator for the antibody level, the normalized MFI was used. HLAab were considered positive when exceeding an MFI value of 500. The DSA showing the highest MFI at the time of ABMR diagnosis (DSAmax) and the MFI sum of all DSA (DSAsum) were tracked to indicate the effectiveness of treatment.End of follow-up was June 30th, 2016. Renal allograft survival was defined as the interval between diagnosis of ABMR and return to maintenance dialysis treatment or end of follow-up. The estimated glomerular filtration rate (eGFR) was calculated according to the chronic kidney disease epidemiology collaboration (CKD-EPI) formula . All advp = 0.049). The applied IVIG dose for group BHP on average was four times as high as that of both other groups . Following diagnosis, all patients received triple drug maintenance immunosuppression comprising a calcineurin inhibitor or mammalian target of rapamycin inhibitor in combination with steroids and mycophenolic acid. In group RLP more patients received tacrolimus as compared to group BLP . Median observation time following diagnosis was 101, 88, and 64 months for groups RLP, BLP, and BHP, respectively. Due to the sequential nature of our treatment protocol modifications follow-up was shortest for the BHP group.Relevant patient characteristics are shown in p = 0.003). Graft survival at one and three years after treatment was 91% and 73% in group BHP, as compared to 55% and 45% in group BLP and 33% and 25% in group RLP, respectively. At the end of follow-up, graft survival was 55% (6/11) in group BHP, 18% (2/11) in group BLP, and 8% (1/12) in group RLP. At diagnosis, serum creatinine  (p = 0.79] (p = 0.02] (2 (26.9–41.4) versus 26.0 mL/min/1.73 m2 (15.5–32.0) versus 16.3 mL/min/1.73 m2 (15.4–19.0), p = 0.03]. In addition, proteinuria was significantly lower in group BHP as compared to both other groups at six months after diagnosis   and the increase of the IVIG dose from a low-dose (30 g fixed dose) to a high-dose (1.5 g/kg) IVIG regimen (group BLP → BHP) . A signi = 0.23]  and prot = 0.79]  were not = 0.79] . A signi = 0.02] . In acco= 0.049] .p = 0.07). Concerning the level of DSAmax and DSAsum before treatment as indicated by MFI, there was no significant difference between groups. Interestingly, following treatment, DSAmax and DSAsum could be decreased significantly as compared to the pretreatment status in group BHP, but not in the other two groups. The BHP treatment scheme was statistically more efficient in the decrease of DSAmax MFI levels than the RLP scheme . In all three groups, there were a few patients with a posttreatment DSAmax level below 500 MFI.The immunological characteristics with respect to HLA mismatches and HLA antibodies are summarized in p = 0.009). Reversible peripheral sensory neuropathy (grade I-II) occurred in two patients of the BHP group. One patient in each group experienced a mild allergic reaction during the IVIG infusion. These events were successfully treated with antihistamines and prednisolone. The number of hospitalizations as well as the number of hospitalized patients was not significantly different between groups. In two patients of group BHP a nonmelanoma skin cancer was diagnosed and successfully treated by local excision.The observed side effects during the first year after treatment are shown in 2) at the time of ABMR diagnosis as well as treatment with rituximab, bortezomib, and high-dose IVIG as the only statistically significant predictors for allograft survival following ABMR treatment. Impaired graft function at diagnosis and ABMR treatment by rituximab were identified as risk factors for subsequent allograft survival. On the contrary, bortezomib and high-dose IVIG revealed a beneficial effect. Subsequent multivariate analysis was performed using impaired graft function at diagnosis and the treatment options rituximab, bortezomib, and IVIG dose as combined covariates. Taken together, impaired allograft function contributed significantly as a risk factor to the regression model  and bortezomib plus high-dose IVIG treatment revealed the strongest beneficial effect on allograft survival following ABMR treatment  as compared to rituximab plus low-dose IVIG.To correct for any covariate with a potential impact on allograft outcome following ABMR treatment, we performed a Cox proportional hazard analysis with clinical, immunological, and therapeutic covariates as summarized in The existing literature on the treatment of ABMR is characterized by a marked heterogeneity concerning the definition of ABMR as well as the applied treatment protocols . NotablyTo date, IVIG preparations are widely used for treatment of ABMR . Yet, itOur results indicate that treatment with bortezomib in combination with high-dose IVIG and PPH (group BHP) is statistically superior as compared to treatment with rituximab, low-dose IVIG, and PPH (group RLP). In addition, high-dose IVIG with bortezomib and PPH seemed to be advantageous as compared to low-dose IVIG with an identical regimen comprising bortezomib and PPH. Both the change from rituximab-based treatment to bortezomib-based treatment and the increase of the IVIG dose from a low-dose to a high-dose regimen resulted in a stepwise improvement of graft survival and graft function. The observed reduction of DSA following treatment in the BHP group but to a lower extent in both other groups reflects the clinical course and is in concordance with the literature. Terasaki and colleagues demonstrated that a decrease in HLA antibody levels can be used as a surrogate marker for the efficacy of an antirejection treatment and directly correlates with superior allograft survival . TherefoTheoretically, it would have been interesting to investigate a group of patients treated with rituximab, PPH, and high-dose IVIG, in order to further elucidate the efficacy of rituximab and high-dose IVIG treatment. However, in our previous study  we obserFirst reports on the effects of IVIG in the context of renal transplantation have been published 20–30 years ago. In 1984, Steiner et al. investigated the impact of prophylactic IVIG administration on the rate of infection in a prospective randomized trial . Because2 ×2) reduced PRA levels from 77% to 44% [ Nocardia brain abscess.The effectiveness of IVIG therapy on circulating HLAab has also been investigated in various studies on pretransplant desensitization. In 1993, Glotz et al. described that pretransplant administration of high-dose IVIG (1.6 g/kg) suppresses HLAab formation . More th% to 44% . Recentl% to 44% . PatientIn group BLP, two patients received maintenance immunosuppression including everolimus following diagnosis. One of these patients returned to maintenance hemodialysis at 6 months after diagnosis; in the other patient serum creatinine at 88 months after diagnosis is stable at 2.4 mg/dL. In this patient we did not reinitiate calcineurin-inhibitor treatment because of a history of cyclosporine A-induced hemolytic uremic syndrome soon after transplantation. Notably, we were not yet aware of the fact that everolimus-based immunosuppression is associated with an increased risk for the development of DSA and ABMR in 2009 . In addiTaken together, our results demonstrate that graft survival of patients with a diagnosis of ABMR following renal transplantation improved during the past years along with the introduction of bortezomib and high-dose IVIG treatment resulting in a 5-year graft survival of about 50% following diagnosis. In conclusion, the use of high-dose IVIG in combination with a bortezomib-based treatment regimen seems to be useful, especially when compared to a historical group of patients treated with low-dose IVIG, PPH, and rituximab. The degree of DSA reduction in group BHP supports this conclusion and may be used as biomarker for the efficacy of treatment. In our view, stepwise controlled modifications of the established treatment protocols are helpful to gradually improve the prognosis following a diagnosis of ABMR. The main limitation of our study is the fact that it is a retrospective study with a limited number of patients comprising episodes of early and late ABMR and comparing cohorts treated at different time periods that differ in some important variables. Therefore, the results must be interpreted with caution, and further studies are necessary to confirm our findings. In future, IVIG preparations containing a higher proportion of Fc fragments with terminal sialic acid residues may be even more effective. Until such preparations will be available, the amount of applied IVIG seems to be crucial for the success of treatment.


Sepsis, an infection-induced systemic disease, leads to pathological, physiological, and biochemical abnormalities in the body. Organ dysfunction is caused by a dysregulated host response to infection during sepsis which is a major contributing factor to acute kidney injury (AKI) and the mortality rate for sepsis doubles due to coincidence of AKI. Sepsis-induced AKI is strongly associated with increased mortality and other adverse outcomes. More timely diagnosis would allow for earlier intervention and could improve patient outcomes. Sepsis-induced AKI is characterized by a distinct pathophysiology compared with other diseases and may also have unique patterns of plasma and urinary biomarkers. This concise review summarizes properties and perspectives of the biomarkers for their individual clinical utilization. Sepsis is a serious medical condition and is caused by a dysregulated host response to infection. Despite advances in antibiotic therapy and life support, the fatality rate of patients with sepsis has remained at least 25% and is increasing in incidence . Sepsis The recognition of sepsis dates back to 2000 years, when Hippocrates claimed that sepsis was the process by which flesh rots and wounds fester . After t⩾26.4 μmol/l (0.3 mg/dl) or a percentage increase in serum creatinine level of ⩾50% (1.5-fold from baseline) or a reduction in urine output (documented oliguria of <0.5 ml/kg/h for >6 h) [AKI is defined as “an abrupt and persistent decline in renal function.” In 2002, the Acute Dialysis Quality Initiative (ADQI) AKI RIFLE classification criteria were defined; the access criteria was as follows: creatinine rose to or more than 1.5 times the original or GFR decreased by >25%, or urine output < 0.5 ml/kg/h and continued for more than 6 hours, depending on severity and AKI duration was divided into three levels: risk, injury, failure, and 2 prognosis: loss and end-stage renal disease . In 2004or >6 h)  (Table 1μmol/L) within 48 h; or an increase in serum creatinine to 1.5 times baseline, which is known or presumed to have occurred within 7 d; or urine output < 0.5 ml/(kg·h) for 6–12 h. According to the severity, the condition is divided into stages 1, 2, and 3, similar to the AKIN staging; however, the KDIGO staging clarified the admittance criterion, and the diagnostic criteria for each stage are more explicit  group performed a retrospective analysis on clinical trials published before 2011 based on the kidney risk, injury, failure, loss, and end-stage renal disease (RIFLE) and Acute Kidney Injury Network (AKIN) criteria. Combined with the evidence in evidence-based medicine, the KDIGO published the KDIGO guidelines in March 2012 and established the diagnostic criteria for acute kidney injury (AKI) : an increxplicit .Based on the published research before March 2017 in Pubmed, Embase, and Cochrane Central Register of Controlled Trails (CENTRAL) databases, a comprehensive computer search was conducted. The keywords we used were “acute kidney injury”, “sepsis”, “biomarkers”, and the names of the biomarkers. After the results have been collated and analyzed, we classified biomarkers of acute kidney injury according to the mechanism and the injury site of AKI. More importantly, we selected the markers that have an early diagnostic value for the sepsis-induced acute kidney injury and summarized them in the hope of helping in choosing the best management in early clinical stage.The KDIGO guidelines highlight early AKI diagnosis and treatment, and the diagnostic marker remains at serum creatinine level. Because the serum creatinine test is convenient and inexpensive, it provides a practical clinical indicator. However, some limitations exist. Renal hypoperfusion due to a prerenal cause may lead to an increase of creatinine, despite noninjured renal parenchyma . When thEvidently, AKI occurs via complex mechanisms often due to multiple factors. Different mechanisms lead to injury in different parts of the kidney. Using the same marker to diagnose injury in all renal subregions caused by all diseases is problematic for establishing a clear diagnosis and accurate injury localization. Discrete study on a specific disease and its associated kidney injury will certainly increase the diagnostic accuracy. Approximately 45–70% of AKI is associated with sepsis, which is one of the most important causes of AKI , 24. Furα and IL-1β) and activating other white blood cells. Neutrophils play a major role in tissue injury. After activation, neutrophils interact with the microvascular endothelium and migrate out of blood vessels to produce superoxide. The superoxide may react with the nitric oxide (NO) produced under induction by mitochondrial NO synthase to form peroxynitrite salt [During the course of sepsis, immune disorders occur in the body, namely, the imbalance between proinflammatory and anti-inflammatory regulation mechanisms . To mainite salt , which dite salt . PAMPs aite salt . DAMPs mite salt . PAMPs aite salt . Cell cyite salt . In sepsite salt . Maintaiite salt .Some of the factors generated in the above processes represent biomarkers for predicting sepsis-associated AKI. Here, we summarize the studies of specific biomarkers for AKI secondary to sepsis:sTREM-1. sTREM-1 is the soluble form of the triggering receptor expressed on myeloid cells 1 (TREM-1). TREM-1, first reported by Bouchon et al. in 2000, is selectively expressed on the surface of neutrophils and monocytes. TREM-1 is a receptor of the immunoglobulin superfamily associated with the inflammatory response, with a relative molecular weight of 26 kDa [f 26 kDa . TREM-1 f 26 kDa . sTREM-1f 26 kDa . When AKf 26 kDa . Given tf 26 kDa . In 2015f 26 kDa . NGAL. Neutrophil gelatinase-associated lipocalin (NGAL) is a glycoprotein composed of 178 amino acid residues. NGAL is a member of the lipid carrier protein superfamily with a molecular weight of 25 kDa and is expressed on the surface of neutrophils [trophils . NGAL istrophils . NGAL istrophils . NGAL getrophils . The NGAtrophils . Thus, Ntrophils . Clinicatrophils , 49. A mtrophils . Anothertrophils . A prosptrophils . Cell Cycle Arrest Protein. Tissue inhibitor metalloproteinase-2 (TIMP-2) is a 21-kDa nonglycosylated protein comprising 194 amino acid residues, and it regulates cell growth and apoptosis [poptosis . Urine ipoptosis . IGFBP7 poptosis . In patipoptosis , 57. Thipoptosis . A multipoptosis . In 2014poptosis . KIM-1. Kidney injury molecule-1 (KIM-1) is a type 1 transmembrane glycoprotein. KIM-1 expression is upregulated in renal proximal tubular cells in ischemic and nephrotoxic AKI [oxic AKI . The extoxic AKI . A meta-oxic AKI . Howeveroxic AKI . Netrin-1. According to its initial description, netrin-1 may affect axonal migration and central nervous system development in the course of neurogenesis [ogenesis . The kidogenesis . A studyogenesis . A studyogenesis .All of the above biomarkers have been validated using large clinical sample sizes, which have efficient and reliable predictive value for sepsis-associated AKI. Impaired kidney function is well known to be undetectable until more than 50% of the renal parenchyma has been injured. The aforementioned biomarkers appeared in renal parenchyma injury, which is more conducive to early diagnosis, on account of creatinine, and its similar markers are not abnormal until renal function is impaired. Should these biomarkers enter clinical use, they would permit early detection of structural injury within the renal parenchyma, with no need to await the emergence of kidney dysfunction. This prospect is favorable for early intervention, such as the withdrawal or reduced use of nephrotoxic drugs. However, how might clinicians proceed after finding increased levels of these biomarkers? Might the increased level of a particular biomarker, which indicates the opening of a particular signaling pathway by sepsis, guide clinical targeted therapy? Might the increase of these indicators guide the implementation of hemopurification interventions? These are questions to which research must be directed in the next steps toward biomarker clinical utility.


Amaranthus dubius, Vigna unguiculata, and Cucurbita pepo are traditionally used to manage obesity in Kenya but lack scientific validation to support their use. The aim of this study was to determine the antiobesity activity of methanolic leaf extracts of these plants in progesterone-induced obese mice. The activity of the methanolic leaf extracts was orally bioscreened in progesterone-induced obese mice at 200 mg/kg/bw and 400 mg/kg/bw. Body mass index was calculated once per week for four weeks and blood samples were obtained at the end of the experiment for lipid profile analysis. Antiobesity activities of the extracts were compared with the controls. Leaf extracts of A. dubius, C. pepo, and V. unguiculata, at dose concentrations of 200 mg/kgbw and 400 mg/kgbw, showed significant effects on body mass index (p < 0.05). There was no significant difference between the three extracts on lipid parameter profiles (p > 0.05). The present study showed high food intake in the negative control group as compared with normal control, positive control, and treatment groups. These extracts contained various phytochemicals such as saponins, flavonoids, alkaloids, and steroids and therefore validate use of aforementioned plants in the suppression of obesity and their use for management of obesity is recommended. Obesity is a serious problem in the world and has been associated with increase in morbidity, mortality, and reduced life expectancy . It occuThe prevalence of obesity is increasing rapidly worldwide. Presently, 300 million people are medically obese while more than one billion adults are overweight . WHO als Camellia sinensis (L) [ Citrus aurantium L. [ Salix matsudana [ Hibiscus sabdariffa L [ Nelumbo nucifera, [Previous studies also indicate that people increase their intake of high energy snack foods when stressed, thereby leading to obesity . In addinsis (L) , Citrus ntium L. , Salix matsudana , Hibiscuariffa L , and Nelucifera,  among ot Vigna unguiculata seeds whose results indicated decreased serum LDL-cholesterol, serum total cholesterol concentration, and serum triacylglycerol level as well as glucose concentration in rats compared with rats fed on high fat control diet. These plants, therefore, showed hypolipidemic and hypoglycemic effects [ Cucurbita pepo peel extracting male diabetic mice, whose results indicated pumpkin significantly reduced LDL, triglycerides, increased HDL, and reduced glucose levels as compared with the control group [ Amaranthus dubius leaves have also shown antidiabetic effect on alloxan induced diabetic mice [ cucurbitae have been attributed to the hypoglycemic activities in diabetic rats [ A. dubius have also been attributed to its antidiabetic effect [Most studies have reported that bioactive compounds such as steroids, flavonoids, alkaloids, saponins, and tannins have promising effects in tackling obesity by several mechanisms. Literature survey also indicates that countries that retain African leafy vegetables (ALVs) are not likely to be affected by obesity, diabetes, and cardiovascular diseases. For example, obese mice were introduced to ethanolic extract of effects . Anotherol group . Amaranttic mice . This istic rats . Alkaloic effect . C. pepo, V. unguiculata, and A. dubius leaves in relation to their antiobesity effects. This study, therefore, aimed at determining whether methanolic leaf extracts of A. dubius, V. unguiculata, and C. pepo possess antiobesity effects. Progesterone-induced obese mice were used as in vivo models in this study.Although these studies provide useful information, no study had been done onThis study was carried out at the Department of Biochemistry and Biotechnology at the School of Pure and Applied Sciences of Kenyatta University from June, 2016, to September, 2016. Kenyatta University is located 19.3 km from Nairobi city off Thika Super Highway. C. pepo, V. unguiculata, and A. dubius were obtained from Marula village, Eldoret subcounty, Uasin Gishu County, Kenya. They were then packaged in khaki bags and transported to Kenyatta University, department of Biotechnology and Biochemistry. Botanical identification and authentication of the plants were done by an acknowledged taxonomist and voucher specimens deposited at the Kenyatta University Herbarium, Nairobi. Voucher specimens numbers assigned were KWO1 (C. pepo), KWO2 (V. unguiculata), and KWO3 (A. dubius). The samples were then separately washed using tap water and shade dried for two weeks and later ground into fine homogeneous powder using an electric mill. The powdered plant materials were stored at room temperature away from direct sunlight in closed and dry air tight bags ready for extraction.Fresh leaves ofExtraction was carried out in the Chemistry departmental Laboratories of Kenyatta University using a protocol described by Grayer and Harborne (1994). Briefly, five hundred grams (500 g) of each powdered plant material was soaked in 1000 ml of methanol at room temperature for 48 hours. Filtration was then done using Whatman number 1 filter paper and later concentrated under vacuum by rotary evaporation at 40°C. The concentrate was then weighed and transferred to an air tight sample bottle and stored at 4°C awaiting use in bioassays.A total of 45 female Swiss albino mice weighing an average of 23 g were used in this study. The animals were housed at the animal handling facility of the Department of Biochemistry and Biotechnology at Kenyatta University. The animals were kept in cages under standard laboratory conditions . They were then acclimatized to the environmental conditions for one week before the initiation of the experiment. Standard rodent pellets were used to feed the experimental animals and were supplied with water ad libitum. Guidelines by Organization for Cooperation and Development (OECD) and ethics committee of the Kenyatta University on research on animal models were used NACOSTI/P/17/26547/16553.Obesity was induced in laboratory animals by subcutaneous administration of progesterone (DPMA) at a dose of 10 mg/kgbw. This was done daily, 30 minutes after oral administration of the extracts for 28 days except for the negative controls, which were not administered with the extract . A. dubius extracts at dosages of 200 mg/kgbw and 400 mg/kgbw, respectively. This design was repeated in C. pepo and V. unguiculata extracts as summarized in The mice of female sex were randomly divided into nine groups of five mice each and treated as follows: Group I  no treatment and Group II (negative control) depo-medroxyprogesterone acetate (DPMA) was administered subcutaneously in the dorsal neck region and the mice received oral administration of water (0.1 ml/mice). Group III (positive control) was given depo-medroxyprogesterone acetate (DPMA) (10 mg/kgbw) subcutaneously at the dorsal neck region and received standard drug orlistat (0.1 ml/mice); Groups IV and V  were given DPMA (10 mg/kgbw) and receivedAnimal weights and lengths  were monitored weekly for 4 weeks using an electronic precision balance and a ruler. To determine body mass index of mice, Lee index was used, which was defined ased obese .Food consumption behavior of mice was studied on days 7, 14, 21, and 28. The mice were deprived of food 1 hour prior to the experiment. 30 minutes after progesterone administration, 10 g of rat chow was given to groups of mice in their plastic cages and food intake was recorded at 0.5, 1, and 1.5 h intervals . The graAt the end of the experimental period (on the 29th day), the tail of each mouse was nipped and venous blood from the tail was collected. Blood glucose test was carried out using a glucose analyzer model . The mice were then euthanized using chloroform to minimize stress and pain during sacrificing. The mice were laid on a dissecting board using pins on a bench and then sacrificed. Blood samples were then collected by cardiac puncture and transferred into plain microvacutainer tubes immediately.The blood samples were then centrifuged at 2400 rpm for 10 minutes to collect clear serum. The clear serum was then aspirated off, packed in Eppendorf tubes and stored frozen at −20°C awaiting analysis. Olympus 640 chemistry auto analyzer was used for analysis of lipid profiles . All assays were performed based on the standard operating procedures (SOPs) written and maintained at the Department of Biochemistry, Thika Levels Hospital.The crude extracts were subjected to qualitative phytochemical screening to identify presence or absence of selected bioactive compounds using standard methods . SecondaSaponins (Froth Test). One gram (1 g) of each plant extract was separately added in 2 ml of distilled water in a test tube, sodium bicarbonate solution was added drop wise, and the mixture shaken vigorously. The occurrence of frothing which persisted for at least 15–20 minutes indicated saponins presence.Alkaloids. One gram (1 g) of each plant extract was separately added to 2 ml of 1 molar aqueous concentrated hydrochloric acid. The mixture was stirred and heated in a water bath for 5 minutes and then cooled. Thereafter, the mixture was filtered with Whitman's filter paper number 1 and two drops of Dragendorff's reagent were added. A color change to orange after addition of Dragendorff's reagent indicated presence of alkaloids.Terpenoids . One gram (1 g) of each plant extract was separately added to 1 ml of ethyl acetate/petroleum ether and mixed into 2 ml of chloroform. Three milliliters (3 ml) of concentrated sulphuric acid was added alongside to form a layer. A reddish brown coloration of the interface was formed to show presence of terpenoids.Anthraquinones. Crude extracts were tested for anthraquinones by boiling 1 g of each plant extract with 10% HCl for a few minutes in a water bath. It was then filtered and allowed to cool. Thereafter, 1 ml chloroform (CHCl3) and 10% ammonia were added dropwise to the filtrate followed by heating. Rose-pink color formation indicated presence of anthraquinones.Flavonoids (Sodium Hydroxide Test). Crude extracts were tested for flavonoids by mixing 1 g of each plant extract with 2 ml of diluted sodium hydroxide. An intense golden yellow precipitate indicated presence of flavonoids.Steroids. One gram (1 g) of each plant extract was dissolved in 2 ml of chloroform. Thereafter, 3 ml of concentrated sulphuric acid was added by the sides of the test tube to form a layer. A reddish brown color at the interface indicates the presence of steroids.Phenols. The crude extracts were screened for phenols by adding 1 ml of ferric chloride solution to 1 g of each plant extract. Formation of blue to green color indicated the presence of phenols.Tannins. One milliliter (1 ml) of distilled water was added to each plant extract followed by two drops of 5% iron chloride. Blue-black coloration indicated presence of tannins.Diterpenes. One gram (1 g) of each plant extract was dissolved in water. Thereafter, 3 to 4 drops of copper acetate (Cu(CH3COO)2) solution were added. Change of color from blue to emerald green indicated presence of diterpenes.p ≤ 0.05 were considered significant. The data was presented in tables and figures.Raw data on body mass index and biochemical parameters were tabulated on MS Excel spread sheets where it was organized for statistical analysis. The data was then exported to Minitab statistical software version 17.0  for analysis. The data was subjected to descriptive statistics and the results were expressed as mean ± standard error of mean (SEM). One-way analysis of variance (ANOVA) was used to determine whether there were significant differences between the means of different groups. This was followed by Tukey's post hoc tests for pairwise separation and comparison of means. The values of  A. dubius caused changes in the body mass index (BMI) of progesterone-induced obese mice  compared to the normal control mice  was observed compared to normal mice  had high food intake in comparison with the mice in normal control group, mice treated with the plant extracts (200 mg/kgbw and 400 mg/kgbw), and orlistat treated mice positive control . V. unguiculata, C. pepo, and A. dubius showed presence of alkaloids, steroids, saponins, phenolics, flavonoids, terpenoids, and diterpenes. However, tannins and anthraquinones were absent , and entered in the brain and, upon reaching the brain, acted on receptors in the hypothalamus to curtail appetite . It is aα-melanocyte-stimulating hormone (α-MSH) pathway, leading to a reduction of the arcuate hypothalamic α-MSH immunoreactivity and leptin hormone. Therefore, the decreased leptin level probably associated with methanolic leaf extracts of African leafy vegetable supplementation may be attributed to the decrease of adipose tissue and thus the antiobesity effect [γ [In addition, this study postulates that hypothalamic neural networks downstream from the leptin receptor were affected, such as the y effect . Thus, tffect [γ . A. dubius, C. pepo, and V. unguiculata have antiobesity activity and reveal the presence of vital phytochemicals. The antiobesity activity of the studied plant may have resulted from its phytochemicals constituents. It provided evidence that these extracts decrease body mass index, food intake, and lipid levels in serum in progesterone-induced obese mice. The study, therefore, confirmed the use of African leafy vegetables in prevention and management of obesity. Consideration should be made to subject the plant to a more nonpolar solvent extraction and compare activities of both methanolic and nonpolar solvents. A bioassay guided fractionation should also be done.We conclusively demonstrate that methanolic leaf extracts of


Worldwide, pertussis remains a major health problem among children. During the recent outbreaks of pertussis, maternal antenatal immunisation was introduced in several industrial countries. This systematic review aimed to synthesize evidence for the efficacy and safety of the pertussis vaccination that was given to pregnant women to protect infants from pertussis infection.We searched literature in the Cochrane Central Register of Controlled Trials, Medline, Embase, and OpenGrey between inception of the various databases and 16 May 2016. The search terms included ‘pertussis’, ‘whooping cough’, ‘pertussis vaccine,’ ‘tetanus, diphtheria and pertussis vaccines’ and ‘pregnancy’ and ‘perinatal’.n = 48, no incidence in either group) and pertussis-related severe complications  or mortality (no study) in infants. Meanwhile, there is no evidence of increased risk of serious complications such as stillbirth , or preterm birth  related to administration of the vaccine during pregnancy.We included 15 articles in this review, which represented 12 study populations, involving a total of 203,835 mother-infant pairs from the US, the UK, Belgium, Israel, and Vietnam. Of the included studies, there were two randomised controlled trials (RCTs) and the rest were observational studies. Existing evidence suggests that vaccinations administered during 19–37 weeks of gestation are associated with significantly increased antibody levels in the blood of both mothers and their newborns at birth compared to placebo or no vaccination. However, there is a lack of robust evidence to suggest whether these increased antibodies can also reduce the incidence of pertussis  contains supplementary material, which is available to authorized users. Worldwide, pertussis remains a major health problem among children . DespiteIn seeking potential strategies for preventing pertussis in infants, vaccinating babies at birth was not considered as the best protection against pertussis. This is because newborns’ immune systems cannot create antibodies until they are 2 months old , so vaccn = 5992/12,089) who delivered in Wisconsin from November 2013 to March 2014 received a pertussis-containing vaccine (tetanus-diphtheria-acellular pertussis: Tdap) during pregnancy  but after the primary infant vaccination schedule was complete [five to 12 months of age]; or antibodies in breast milk in colostrum and up to 12 months after giving birth)Adverse vaccine-related outcomes  following injectionsObstetric or perinatal complicationsWe included randomised control trials (RCTs) in which individual pregnant women were randomly assigned to a vaccination or a no-vaccination (placebo or no vaccination) group so that differences in outcomes of babies born to women with or without the pertussis vaccination could be examined. If a RCT adopted a crossover design, we only included outcome data from the first randomisation period. We excluded cluster-randomised trials because the purpose of our review was to examine the vaccine’s efficacy and safety at the individual level. Apart from RCTs, we also included quasi-RCTs and observational studies that evaluated the vaccine’s efficacy and safety. We did not apply restrictions based on study settings, but we included only studies written in English.Using data extraction forms designed specifically for this review, MF extracted information on study design, participants, interventions or exposures, and outcomes measured. JS double-checked the accuracy of data extraction. Two authors  independently assessed the risk of bias in the included studies, using the Cochrane collaboration risk of bias tool for RCTs, cohort studies and case-control studies , 21 as aThe extracted outcome data were further checked by a statistician (ESWN) who conducted statistical analyses. We used the Mantel-Haenszel method to estimate the pooled risk ratios (RRs) with 95% confidence intervals (CIs) for all dichotomous outcomes, except for one case-control study for which we produced odds ratios (ORs), because with this type of study design, the entire at-risk population cannot be defined and, thus, we could not calculate RR. For the continuous outcome data, i.e. anti-pertussis antibodies, underlying data were skewed. We therefore calculated the ratios of the geometric means and 95%CIs where group-specific geometric means and associated CIs were reported for the included studies . Test ofWe performed random effects meta-analyses, where possible, using Review Manager (RevMan) 5.3 to produce the average effect sizes of the interventions across studies. We analysed the data from observational studies separately from the trial data. Clinical heterogeneity  and methodological heterogeneity (variability in study design and risk of bias) were assessed within each comparison. We also assessed statistical heterogeneity using I-squared and chi-squared statistics , as welln = 1347), we examined 91 full-text articles and finally, a total of 15 articles met our inclusion criteria  were drawn from the participants in Maertens et al. , antibodies in the maternal blood: 51.0 EU/mL [95% CI: 37.1–70.1] in the Tdap-vaccinated group and 9.1 EU/mL [4.6–17.8] in the placebo group; ratio of geometric means 5.6 [3.0–10.5]; p < 0.001) and in their infants at birth . Only oth (e.g. .Table 2GTable p = 0.006) lower in the Tdap group than in the tetanus toxoid group. One cohort study  vs. 6.52 EU/mL [95% CI 2.19–19.41]; p = 0.01) and IgG to pertactin . In the breast milk at 2 weeks postpartum, the geometric mean concentrations of IgA to filamentous hemagglutinin remains significantly higher in the Tdap-vaccinated women compared with women with no vaccination . At 4 weeks postpartum, there were no statistically significant differences in any of the measured antibodies in breast milk  vs. 0.19 EU/mL [95% CI 0.16–0.23]; p = 0.01]), whereas one study in Israel  of 33 in the vaccine group vs. 3 [20.0%] of 15 in the placebo group; RR 3.9 [95% CI 1.41–11.01]). Other adverse outcomes reported in the RCT  of 26,229 in the Tdap-vaccinated group vs. 5326 [5.5%] of 972,653 in the unvaccinated group; adjusted RR 1.19 [95% CI: 1.13–1.26]). However, importantly, the study did not demonstrate the association between the antenatal pertussis vaccine and increased risk of preterm birth, a clinically important consequence of chorioamnionitis. Two additional RCTs  of 85 in the Tdap-vaccinated group vs. 2 [3.0%] of 66 in the unvaccinated group; RR 0.86 [95% CI 0.14–5.21]; two cohort studies: n = 124,133; 1535 [5.8%] of 26,365 in the vaccine group vs. 7582 [7.8%] of 97,770 in the control group; RR 0.75 [95% CI: 0.75–0.79], Fig. n = 128,154; RR 1.03 [95% CI 0.95–1.13], Fig. n = 103, RR 0, meaning no stillbirth in the vaccine group; two cohort studies, n = 25,398; RR 0.82 [95% CI 0.44–1.54], Fig. Table nal RCTs , 31 and nal RCTs , in addinal RCTs , also reFollowing the GRADE criteria, the quality of the evidence for our prespecified obstetric or perinatal complications was rated as low for neonatal death and small-for-gestational-age because only one observational study contributed to the data; but moderate for hypertensive disorders, preterm birth and stillbirth because three different studies (including one RCT for preterm birth and stillbirth) showed consistent results.This review demonstrates that antenatal pertussis vaccination in middle to late pregnancy is associated with significantly higher antibody titres in maternal and infant blood than placebo or no vaccination. Studies testing maternal vaccinations at an early stage of pregnancy are small in number and hence evidence, if any for or against vaccination at an early stage of pregnancy, is scarce. Regarding safety concerns, there may be relatively minor injection site reactions, such as pain associated with the vaccine injection. However, the existing evidence has not demonstrated that having a maternal pertussis vaccination during pregnancy is associated with an increased risk of any serious maternal or perinatal complications such as hypertensive disorder, stillbirth, neonatal death or pre-term birth.The UK Department of Health , 40 and This is the first systematic review, to the authors’ knowledge, evaluating the efficacy and safety of pertussis vaccination during pregnancy. We developed a protocol  or comparators (placebo versus unvaccinated during pregnancy versus vaccination before pregnancy). Some of the included studies reported an insufficient amount of background information. The lack of data also prohibited our plan to assess the interaction effects of the types and timings of vaccination and study settings through subgroup analyses. We were also not able to use sensitivity analysis to assess either study quality or potential bias caused by missing data. Finally, in this current review, we were not able to assess publication bias with a funnel plot because a small number of studies (n ranges from 1 to 3) contributed to each outcome. This was the case in which hypothesis testing for publication bias was deemed inappropriate due to the low power to detect real asymmetry .Recently, there has been a marked increase in the incidence of pertussis in the world, and newborn babies are at greatest risk of catching this highly infectious disease. Although pertussis vaccination in pregnancy is now recommended in several countries worldwide, pertussis vaccine has not yet been universally provided to the pregnant women. Our review findings aid evidence-based decision making for clinicians and public health commissioners, as well as for the women themselves regarding pertussis vaccination during pregnancy. Given that the existing evidence has not demonstrated that having a pertussis-containing vaccine during pregnancy is associated with an increased risk of any serious maternal and perinatal complications, maternal vaccination in pregnancy should continue to be supported, especially during the outbreak period, while further research endeavours to fill knowledge gaps and strengthen evidence of the vaccination’s efficacy and safety. Further studies need to focus on establishing the vaccine’s efficacy and effectiveness in relation to the prevention of pertussis and pertussis-related complications in infants through large, sufficiently powered, placebo-controlled RCTs. RCTs are also required to allow subgroup analyses that investigate whether vaccine efficacy differs according to the timing of vaccine administration, types of pertussis vaccination, and/or different settings. In particular, there is currently little evidence from low-income countries, where the risk of pertussis is higher and cases are more likely to be fatal; thus, the implications for clinical practice based on the findings of this review may be different . There aAdditional file 1:Search strategies. Systematic electronic search stragegies for the CENTRAL, Medline, Embase and OpenGrey. (DOCX 20 kb)Additional file 2:Excluded studies after full-texts retrieved and reasons for exclusion. A list of 76 excluded full-text articles with reasons for exclusion (DOCX 26 kb)Additional file 3:Risk of bias assessment. The risk of bias assessment for all included studies using the Cochrane collaboration risk of bias tools (DOCX 16 kb)Additional file 4:Efficacy and safety of pertussis vaccination for pregnant women – A systematic review of randomised controlled trials and observational studies (Protocol). A protocol developed prior to the review. (PDF 783 kb)