Coxiella burnetii, a bacterial agent for which ruminants are the most prevalent natural reservoir. Data regarding Q fever infection in camels in Algeria are limited. Therefore, a survey to detect seroprevalence of C. burnetii antibodies was conducted among healthy camel populations in a vast area in southeastern Algeria to determine distribution of the Q fever causative organism and to identify risk factors associated with infection. Between January and March 2016, blood samples were collected from 184 camels and serum samples were subsequently analysed using a commercial Enzyme-Linked Immunosorbent Assay (ELISA) kit. At the time of blood collection, a questionnaire investigating 13 potential predisposing factors associated with C. burnetii seropositivity was completed for every dromedary camel and herd. Results were analysed by a chi-square (χ2) test and multivariate logistic regression. The seroprevalence of C. burnetii at the animal level was 71.2% (95% CI: 65.2–78.3) and 85.3% (95% CI: 72.8–97.8) at the herd level. At the animal level, differences in seroprevalence were observed because of herd size, animal age, animal sex, presence of ticks and contact with other herds. A multivariable logistic regression model identified three main risk factors associated with individual seropositivity: (1) age class > 11 years , (2) herd size > 50 head  and (3) infestation with ticks . This study of seroprevalence of C. burnetii infection in camels in Algeria revealed a high seroprevalence of Q fever in camel populations in southeastern Algeria and provided strong evidence that Q fever represents an economic, public health and veterinary concern. Appropriate measures should be taken to prevent the spread of C. burnetii and to reduce the risk of Q fever in farm animals and humans in this agro-ecologically and strategically important region of North Africa.Query (Q) fever is a globally distributed zoonotic disease caused by  Coxiella burnetii, the causative agent of Q fever, is an obligate intracellular bacterium. Recently, this bacterium was classified into the Legionellales order and the Coxiellaceae family  fever is a zoonotic disease with worldwide distribution with the exception of New Zealand. C. burnetii infection in sheep and goats, however, includes late gestation abortion, reduced reproductive efficiency because of stillbirths, delivery of weak offspring and premature delivery  in southeastern Algeria. These provinces are located at 002° 04 to 007° 35 E and 28° 32 to 34° 56 N . This reC. burnetii antibodies was calculated according to the formula (see below) given by Thrusfield  following the manufacturer’s recommendations and protocols.Detection of Results were expressed as optical density (OD) and absorbance was read at 450 nm (wavelength) with an EL-800 ELISA plate reader . Positive and negative controls (provided by the manufacturer) were used to validate each test.Samples were considered ‘positive’ if they had an OD value ≥ 40%, ‘questionable’ for values between 30% and 40%, and ‘negative’ for OD value < 30%. These percentages were calculated according to the manufacturer’s instructions. Any sample that initially was classified as ‘questionable’ was re-assayed; after the second assay, any sample that still fell in the OD value of 30% – 40% was assigned as either ‘positive’ or ‘negative’ in an alternating fashion.The sensitivity and specificity of this ELISA test  were used to convert the apparent seroprevalence to the true seroprevalence using the formula developed by Rogan and Gladen . A herd C. burnetii seropositivity was developed using both closed and open-ended questions. Questions pertaining to individual camels included age, breed , sex, history of abortion and presence of ticks. Additional data were gathered on general herd and management data such as geographical location of herd, herd size, husbandry system, contact with small ruminants (yes or no), contact with others camel herds (yes or no) and herd size were categorised into three groups: small (< 20 head), medium (20–50 head) and large (> 50 head). The questionnaire was completed by face-to-face interviews with the camel farm owner or manager.Information regarding potential risk factors was collected at the time of blood sample collection. A structured questionnaire containing 13 variables potentially associated with P £ 0.25) in a univariate analysis. The logistic regression model was developed in a stepwise forward approach using a likelihood ratio test at each step (with P < 0.05 to enter and P > 0.10 to exit). Model fit was assessed with the Hosmer and Lemeshow goodness-of-fit test. All statistical analyses were performed using the statistical software SPSS version 22.0 . In all analyses, two-tailed P values < 0.05 were considered as statistically significant.Individual- and herd-level seroprevalence of Q fever was estimated based on ELISA results. Pearson’s chi-square test or Fisher’s exact test was applied to check for significant associations between the potential risk factors and the outcome variables (status of Q fever seropositivity in camels) in a univariate analysis. Multivariate logistic regression was conducted using all variables showing moderate statistical significance  of all camels investigated and the true prevalence was calculated as 71.1% (95% CI: 65.1–78.3). The herd-level seroprevalence was estimated at 85.3% (95% CI: 72.8–97.8).Antibodies to C. burnetii seroprevalence in camels in southeastern Algeria are summarised in C. burnetii: sex (P = 0.013), age (P = 0.002), herd size (P = 0.012), presence of ticks (P = 0.019) and contact with other camel herds (P = 0.036). Individual seroprevalence was higher (P < 0.05) in females (74.1%) than in males (42.9%) and in camels > 11 years old (85.9%) than in camels < 3 years (47.1%). Seropositivity was greater in camels infested with ticks, whereas there was no difference in seroprevalence of Q fever among breeds (P > 0.05). Likewise, geographical region was not predictive of seropositivity against C. burnetii among camels. No significant differences were seen for other risk factors, including history of abortion, husbandry system, introduction of newly purchased animals and contact with small ruminants.Results for the univariate analysis of individual-level risk factors for P = 0.001) progressively with age  and seropositivity of camels reared in large herds (74.7%) was higher  than that in camels from small herds (42.7%). The odds ratio for the presence of ticks showed that infection was nearly 2.2 times higher in animals with ticks present on their bodies at the time of sampling.Results of the multivariate logistic regression analyses are shown in C. burnetii infection in camels in Algeria. This study may therefore represent an important contribution to our scientific knowledge because there is a paucity of published information regarding the status of Q fever in humans and animals in Algeria. The few published epidemiological studies conducted in ruminants were performed in sheep and goats  falls within the limits previously reported for camels, which ranged in Ethiopia from a low of 18.6%  seroprevalence than male. Our results are in agreement with previous studies in camels  have also found the same pattern  potentially associated with seropositivity were not significant in our study. We failed to observe differences as a result of breed in distribution of C. burnetii because of quarantine of newly purchased animals, use of disinfectants, treatment of newly purchased animals, movement of animals, camel parity, level of milk production and lactation stage, we could not do so because of a paucity of data.Although our original intent in this study was to investigate the potential risk of infection with C. burnetii  that share common production systems and where camels may serve as a reservoir of the zoonotic agent causing Q fever. Veterinarians and epidemiologists should work together to develop effective strategies for control of this disease.Our findings constitute the first known investigation on seroprevalence of


Most defined systems have identified microbial elements as the primary factors determining disease suppression, but the involvement of the soil abiotic environment is less defined. The significance of this work is that the soil abiotic environment plays a critical role in the establishment of the soil microbial community and key microbial agents that directly contribute to the prevention of soilborne diseases. We highlight the importance of the soil abiotic environment in disease suppression. Furthermore, we provide a framework for the characterization of disease-suppressing agents in artificially managed soil. These results will gradually close the gap in knowledge on soil environment-microbe interactions. Sphingobacteriales and fungal order Sordariales, as well as their affiliated genera Sphingobacterium, unclassified genus within Sphingobacteriaceae, Zopfiella, and unclassified genera within Lasiosphaeriaceae and Chaetomiaceae, were negatively correlated with disease incidence and positively associated with RSD-conditioned soil environment. Furthermore, we validated that both the microbial disease-suppressive agent and its adapted abiotic environment contributed to disease suppression. Our results elucidate the abiotic and biotic foundations of soilborne disease suppression under artificial management and highlight that the abiotic environment is as important as the microbial agents in disease suppression.Many agricultural soil management strategies have been shown to be effective in preventing soilborne diseases. However, their underlying mechanisms of action remain unknown. In this study, we used reductive soil disinfestation (RSD), also named anaerobic soil disinfestation (ASD) and biological soil disinfestation (BSD), as a representative method for disease management and cucumber damping-off diseased soil as a model system to identify the disease-suppressive agents in artificially managed soil. The results showed that RSD created a soil environment that was different from that of the diseased soil, where the pH level and the carbon content were greater. Heat treatment and pathogen or soil microbiota self- and cross-reinoculations resulted in the expansion of various soil microbial communities harbored by the two soil environments, as well as various disease incidences. Environmental factors were the primary determinant of the reassembled bacterial community, followed by initial microbiota, whereas initial microbiota was the key driver of the reassembled fungal community. The relative abundances of the bacterial order IMPORTANCE Most defined systems have identified microbial elements as the primary factors determining disease suppression, but the involvement of the soil abiotic environment is less defined. The significance of this work is that the soil abiotic environment plays a critical role in the establishment of the soil microbial community and key microbial agents that directly contribute to the prevention of soilborne diseases. We highlight the importance of the soil abiotic environment in disease suppression. Furthermore, we provide a framework for the characterization of disease-suppressing agents in artificially managed soil. These results will gradually close the gap in knowledge on soil environment-microbe interactions. Rhizoctonia solani and Fusarium oxysporum, respectively, often lead to economic losses in many important crops  and ferrous (Fe2+) cations, and ammonia, effectively suppresses a wide range of disease-causing soilborne pathogens  R. solani damping-off diseased soil and RSD-treated diseased soil are considered two original soils in this study. Heat treatment and pathogen or soil microbiota self- and cross-reinoculations created diverse microbial communities in the two types of soil  higher than those in the other soils, in which the R. solani abundances ranged from 4.40 × 105 to 1.88 × 106 copies g−1  soils was significantly (P < 0.01) higher than those in the ER  soils, and cucumber seedlings in the heat-treated and pathogen-reinoculated soils (EDP and ERP) were largely infected .A schematic diagram of the experimental design and detailed definitions of the abbreviations used for treated soils are shown in pies g−1 . The disinfected . The relinfected . FurtherP < 0.05) higher in the ER  soils  than those in the ED  soils  after planting . The R soil considerably (P < 0.05) reduced fungal observed species numbers, as well as their diversity and evenness, compared with those in D soil (Table S1). Furthermore, Chao and Shannon indices for bacteria and fungi in the heat-treated soils were significantly lower (P < 0.05) than those for the non-heat-treated soils .The abundances of bacteria and fungi were significantly  altered the bacterial and fungal communities, and heat treatment followed by microbiota reinoculation, especially microbiota cross-reinoculation, also changed the bacterial and fungal communites (P < 0.01) (P < 0.05) affected several physicochemical properties by increasing the soil pH and the contents of several carbon fractions and by decreasing the soil electrical conductivity (EC) and inorganic nitrogen content (Tables S2 and S3), whereas heat treatment and microbiota reinoculation rarely affected the soil physicochemical properties compared with those in the nonheat-treated soils (RSD treatment significantly ( < 0.01)  and C. T < 0.01)  and D. Ied soils . FurtherD  and ER   and MR  . Furthermore, the top 50 bacterial and fungal genera that significantly differed between ED and ER soils were also identified, and we also found several genera whose relative abundances were significantly different between ERMD and ERMR soils, such as Sphingobacterium, Pseudomonas, Zopfiella, and Uc_Sarcosomataceae (currently unclassified genera belonging to Sarcosomataceae)  bacterial and fungal genera (End ERMR)  or MD (Dnd ERMR)  soils reataceae)  and D. Il genera .D and ER soils, microbes could be classified into three typical groups, namely, microbes having a broad environmental adaptation range covering two types of soil environments that could be mostly transferred from each other, such as those having no significant differences in abundance between ED and ER soils (group I); microbes preferring or only favoring the abiotic environment of RSD-treated soils that could not be or partly be transferred from RSD-treated soils to the diseased soils, such as Zopfiella, Uc_Lasiosphaeriaceae, Sphingobacterium, and Uc_Sphingobacteriaceae, whose abundances were significantly higher in ER soils (group II); and, conversely, microbes restricted to conditions in the diseased soils that could not be or partly be transferred from the diseased soils to the RSD-treated soils, such as Uc_Xanthomonadaceae and Uc_Nocardiodaceae (group III).In addition, according to their distributions in ESphingobacteriales and the fungal order Sordariales were significantly (P < 0.001) and negatively correlated with the DI  negatively correlated with the DI (P < 0.05) positively correlated with the soil pH and total organic carbon (TOC) content  sequence (Zopfiella sp. per kg of soil in the diseased soil (CK) did not significantly (P > 0.05) prevent the development of damping-off disease during the successive cropping seasons, although the DI in the Zop10 soil was lower than that in the CK  had no effect on DI during the first growth season compared with alfalfa-amended CK soil (CK+Al); however, it resulted in a significant (P < 0.05) decrease in the DI during the second season. Furthermore, soil inoculation with 7.5 g of Zopfiella sp. per kg of soil combined with alfalfa amendment (Zop10+Al) considerably (P < 0.05) prevented cucumber damping-off disease during both growth seasons , with physicochemical properties similar to but microbiota different from those of R soil, did not prevent disease, indicating that the biotic properties of the RSD-treated soil, rather than the abiotic properties, may be directly responsible for disease suppression. Furthermore, it has long been believed that the inoculum level of soilborne pathogens is the key determinant of disease occurrence  could be transferred from disease-suppressive soils to disease-conducive soils through microbial community exchange processes similar to those reported here. We inferred that the transfer of microbial species from one soil to another depended on its range of environmental adaptation and the degree of environmental difference between the two soils. Differences in the soil pH, which is considered the primary environmental determinant of bacterial distribution , Jiangsu Province, China. Crop residues and stones were removed from the soil by passing through a 2-mm sieve. The characteristics of the soil were as follows: moisture content of 14.9%, pH of 5.26, EC of 0.89 mS cm−1, TOC content of 18.11 g kg−1, total nitrogen (TN) content of 2.44 g kg−1, nitrate (NO3−-N) content of 592 mg kg−1, and 1.36 × 107 ITS copies of R. solani g−1. Alfalfa (Medicago sativa), composed of a TOC content of 399.1 g kg−1 and a TN content of 13.45 g kg−1, was powdered and used as the organic matter of the RSD treatment. RSD treatment was performed in a box (length by width by height = 25 by 25 by 25 cm), where the diseased soil was combined with 2% (wt/wt) alfalfa, irrigated to saturation, and sealed with a plastic film. The untreated diseased soil and the RSD-treated soil were considered two original soils, defined as D and R soils, respectively. The D and R soils were incubated for 18 days at 35°C and then respectively drained, passed through a 2-mm sieve, and homogenized for the next step of the experiment.Salinized and acidified soil containing the damping-off disease-causing pathogen DMD and ERMD, respectively), 10% (wt/wt) of raw R soil , or a suspension of R. solani mycelium  to the levels present in D soil detected based on real-time quantitative PCR (qPCR). Soils involving the abiotic environment of the diseased soil  and RSD-treated soil  were aggregately defined as ED and ER, respectively. Soils contained the initial microbiota of the diseased soil  and RSD-treated soil  were aggregately defined as MD and MR, respectively.The schematic diagram of the experimental design is listed in R. solani in that replicate)/(DI in D soil/the abundance of R. solani in D soil)] × 100.All of the culture bottles were incubated for 60 days at 20°C and irrigated with 5 ml of sterile water every 10 days (approximately 15% soil water content). After incubation, approximately 10 g of soil was collected from each bottle, and those soils from 10 bottles in a replicate were mixed to form a composite biological replicate (this time point was defined as “after incubation”), and a single, pregerminated cucumber seed (cv. JinChun 5) was placed in each bottle and cultivated for 20 days, with average day and night temperatures of 30 and 18°C, respectively, before the soil was mixed thoroughly and collected for analysis (this time point was defined as “after planting”). The disease indices, including DI and RPR, were respectively calculated using the following formulae: DI = (the number of infected plants in a replicate/10) × 100; RPR = [(DI in each replicate/the abundance of 2SO4-K2Cr2O7 (−1 (EOC333), 167 mmol liter−1 (EOC167), and 33.3 mmol liter−1 (EOC33.3) of KMnO4, following the method reported by Blair et al. (333 content. The light fraction organic carbon (LFOC) content was measured using the approach described by Compton and Boone (3−-N and ammonium (NH4+-N) were extracted using 2 mol liter−1 of KCl solution (1:5 wt/vol), followed by shaking at 250 revolutions min−1 for 1 h and filtering for 30 min. The content was measured using a continuous flow analyzer .We quantified the soil physicochemical properties as surrogate measures of the abiotic environmental factors. The soil pH and EC were measured in slurries  using a S220K pH meter  and a conductivity meter , respectively. The soil TOC content was measured using wet digestion with H-K2Cr2O7 , and frar et al. . The soind Boone , and thend Boone . NO3−-N R. solani (ST-RS1/ITS4R) primer sets are listed in Microbial DNA was extracted from 0.25 g of soil using the PowerSoil DNA isolation kit , according to the manufacturer’s instructions. Real-time PCR amplification was carried out in 8-well tubes using the CFX96 real-time system ; qPCR mixtures and thermal conditions followed those reported by Huang et al. . The bacDP and ERP soils, microbial DNA from soil samples (n = 18) collected after planting was sequenced using individual barcoded primers 515F and 907R and primers ITS3F and ITS4R , following the approach described by Caporaso et al. , where pZopfiella sp. as a potential disease-suppressing agent. First, the strain was isolated from R soil using gradient dilution coating in soil extract agar medium , diseased soil inoculated with 0.75 or 7.5 g of Zopfiella sp. mycelia per kg of soil , diseased soil amended with 2% (wt/wt) alfalfa (CK+Al), and CK+Al inoculated with 0.75 or 7.5 g of Zopfiella sp. mycelia per kg of soil , were arranged in a completely randomized design. Treatment boxes (25 by 25 by 6 cm) containing 2.5 kg of soil were planted with 9 pregerminated cucumber seeds and cultivated as previously described. After 20 days, the DI was recorded, the seedlings were removed, and the soil in each box was mixed in preparation for a repeat cultivation of the cucumber seedlings.Based on the analysis of the aforementioned results, we tested the function of the r medium  and iden10 transformed prior to statistical analysis. The treatment effects were tested using one-way analysis of variance (ANOVA) and Duncan’s test (P < 0.05) using SPSS 19.0 software . The treatment effects (excluding EDP and ERP) on the soil environmental factors and microbial community were estimated by principal-coordinate analysis (PCoA) using the pco function within the R package labdsv  and ER  soils and between MD  and MR  soils were compared using likelihood ratio tests (LRT) within the edgeR package  was further used to identify the microbial taxonomic differences (from phylum to family) between ED and ER soils. In addition, heat maps of top 50 bacterial and fungal genera significantly different between ED and ER soils were generated to visualize dissimilarities in the microbial composition using the R package Pheatmap .The raw sequencing data were deposited into the NCBI Sequence Read Archive (SRA) database (accession number


Salmonella is generally considered as a human pathogen causing typhoid fever and gastrointestinal infections called salmonellosis, with S. Enteritidis and S. Typhimurium strains as the main causative agents. Salmonella enterica strains have a wide host array including humans, birds, pigs, horses, dogs, cats, reptiles, amphibians and insects. Up to 90% of reptiles are the carriers of one or more serovars of Salmonella. Extraintestinal bacterial infections associated with reptiles pose serious health threat to humans. The import of exotic species of reptiles as pet animals to Europe correlates with the emergence of Salmonella serotypes, which not found previously in European countries. The presented study is a new report about Salmonella serotypes associated with exotic reptiles in Poland. The goal of this research was to examine the zoonotic potential of Salmonella strains isolated from reptiles by comparative analysis with S. Enteritidis strains occurring in human population and causing salmonellosis.Salmonella serovars other than correlated with salmonellosis in humans . Among the isolated Salmonella strains we identified serovars that have not been reported earlier in Poland, for example belonging to subspecies diarizonae and salamae. Restriction analysis with Pulsed-field Gel Electrophoresis (PFGE), showed a great diversity among Salmonella strains isolated from reptiles. Almost all tested strains had distinct restriction patterns. While S. Enteritidis strains were quite homogeneous in term of phylogenetic relations. Most of the tested VGs were common for the two tested groups of Salmonella strains.The main findings of our work show that exotic reptiles are asymptomatic carriers of Salmonella strains isolated from reptiles share most of virulence genes with the S. Enteritidis strains and exhibit a greater phylogenetic diversity than the tested S. Enteritidis population.The obtained results show that The online version of this article (10.1186/s12917-019-2019-1) contains supplementary material, which is available to authorized users. Salmonella is a Gram-negative bacteria responsible for a wide variety of infectious diseases: typhoid fever, gastroenteritis, food poisoning and septicaemia. Multiple genes required for full virulence of Salmonella strains are encoded on Salmonella pathogenicity islands (SPI) and could be acquired by horizontal transfer from other organisms. Infections, caused by nontyphoidal Salmonella strains (NTS), are recorded worldwide (94 million cases/year) but the epidemiological data are probably underestimated as many milder cases are neither diagnosed nor reported  and also from birds with infection [n = 76]. Results obtained by Skyberg’s group revealed, that all of tested bacterial strains from both groups of birds had following VGs: spiA, pagC, msgA, invA, sipB, prgH, spaN, orgA, tolC, iroN, sitC. Krawiec et al.  needed in GLC-MS analysis.For genetic assays, the bacteria were cultured overnight at 37°C in Lysogeny Broth (LB) or nutrient agar (Biocorp). For gas-liquid chromatography mass spectrometry analysis (GLC-MS), the bacteria were cultivated in minimal medium [KSalmonella strains were identified, using biochemical tests and serotyping with specific O and H antisera, and classified according to the Kauffmann-White-Le Minor scheme [Salmonella strains isolated from reptiles was performed in the National Veterinary Research Institute  and National Salmonella Centre . A complete list of the tested strains is presented in Table r scheme . SerotypE. coli DH5-alpha standard . Biotyper ver. 3.1 (MSP 4613) database software  was used for the identification of bacterial isolates.Bacterial sample were prepared according to the manufacturer’s protocol . Shortly: two to five bacterial colonies were suspended in water and precipitated with ethanol. After drying, equal volumes of 70% formic acid and acetonitrile were added and, after centrifugation, 1 μl of supernatant was transferred to ground steel MALDI plate for analysis, with α-cyano-4-hydroxy-cynnamic acid in 50% ethanol with 2,5% TFA used as a matrix. Identification of bacterial strains using MALDI–TOF MS Biotyper was conducted with the application of ultraflExtreme . Spectra were recorded in the positive linear mode for a mass range of 2000–20.000 Da. Each spectrum was obtained by averaging 600 laser shots acquired from the automatic mode under control of FlexControl software ver. 3.4 . The spectra were externally calibrated using an Bacterial DNA was extracted using commercially available Genomic Mini Kit  according to the manufacturer’s protocol from overnight  culture.To amplify the entire ~ 1500-bp region of the 16S rRNA gene universal primers were used: 16S_Start (5’AGAGTTTGATCMTGGCTCAG3’) and 16S_Stop (5’AAGGAGGTGWTCCARCC3’). In brief, samples underwent an initial denaturation of 5 min at 98°C and 35 cycles of 10 s at 98°C (denaturation), 30 s at 60°C , 45 s at 72°C (extension), followed by 5 min at 72°C . The products were sequenced (Sanger’s method). Sequences were aligned using DNA Baser v4.36.0. The isolate was identified using the EzTaxon server [Salmonella. The genes invA, sipB, prgH, spaN, orgA, tolC, iroN, sitC, lpfC, sifA, sopB, pefA, spvB, spiA, pagC, cdtB and msgA were targeted by three multiplex-PCR reactions using the protocol given below according to literature [Strains were subjected to the testing of 17 virulence genes related to pathogenicity of terature  with autThe amplified products from all types of the PCR reactions were resolved on a 2% or 0.8% agarose gel  and visualized with Midori Green DNA  under UV light using a Gel Doc camera system  and analyzed with Quantity One software . PCR assays were repeated twice to confirm the correctness of the assignment of the investigated strains to their respective patterns.http://insilico.ehu.es/.All bacterial isolates were fingerprinted by the PFGE method, using the PulseNet protocol developed by Centers for Diseases Control and Prevention . ChromosHafnia alvei PCM 2386 with confirmed presence of NeuAc in the O-antigen [Samples for the analysis of NeuAc content were prepared according to Pawlak et al. . In brie-antigen  were usem/z 446 was isolated and fragmented. The secondary fragment of m/z 386 was used for the quantitation of NeuAc derivative in the sample [Thermo FOCUS GC with ITQ 700 ion trap detector with external ionization  was used for sample analysis by GLC-MS/MS. For MS/MS analysis primary ion e sample , 62.Additional file 1:Figure S1-S6. The electrophoregrams of amplification products of the tested virulence genes. (PDF 332 kb)


Haemoproteus compared to Plasmodium genus, but this only held true among males. Female TL was independent of the infection type. Our results indicate that parasitic infections could bring about other types of costs to females than to males with respect to TL. Additionally, we detected linear telomere loss with age, however a random regression analysis did not confirm significant heterogeneity in TL of first breeders and telomere shortening rates in further life.Parasitic infections potentially drive host’s life-histories since they can have detrimental effects on host’s fitness. Telomere dynamics is a candidate mechanism to underlie life-history trade-offs and as such may correlate with observed fitness reduction in infected animals. We examined the relationship of chronic infection with two genera of haemosporidians causing avian malaria and malaria-like disease with host’s telomere length (TL) in a longitudinal study of free-ranging blue tits. The observed overall infection prevalence was 80% and increased with age, constituting a potentially serious selective pressure in our population. We found longer telomeres in individuals infected with a parasite causing lesser blood pathologies i.e. The online version of this article (10.1007/s00114-019-1601-5) contains supplementary material, which is available to authorized users. Parasitic infections have been reported to impair survival in a number of taxa  of Haemoproteus occurs in other tissues, whereas Plasmodium multiplies also in red blood cells leading to their disintegration  and telomere length (TL) in a wild population of a small, short-lived passerine, the blue tit (Cyanistes caeruleus). To this end, we examined if the presence of blood parasites correlates with TL of host’s red blood cells. We expected to (i) detect shorter telomeres among infected individuals while compared to uninfected ones and (ii) find differences in TL among hosts infected with parasite genera with varying life cycles, as the action of each parasite is expected to have diverse direct impact on red blood cells. A longitudinal setup of our study allowed to test if (iii) red blood cell telomeres become shorter with chronological age of an individual and (iv) components of telomere dynamics: first breeders’ TL and TL erosion rate vary among individuals.Here, we aim to determine the relationship between infection with blood parasites  and louse flies (Hippoboscidae) in the case of Haemoproteus  with a maximum longevity of 12 years recorded in the wild (EURING database). The median lifespan of birds that were observed in our population as adults is 2 years, and maximum lifespan recorded in the study period is at least 6 years. Birds seem to contract infection locally . The DNA concentration and purity was measured with a NanoDrop 1000 Spectrophotometer  and the integrity of each sample was confirmed by an electrophoresis on a 1% agarose gel.T/S) of a telomere copy number (T) and a single control gene copy number  of the standard curve were 96% (range 82–109%) and 0.985 (range 0.963–0.990) for GAPDH and 86% (range 77–103%) and 0.974 (range 0.949–0.993) for telomeres respectively. Mean intra-plate SD for the Ct values  exceeded 0.5, all samples for each individual were measured again. In total, we ran 21 plates. For further details on primers, reagent concentration, reaction setup and T/S ratio calculation please refer to Sudyka et al. . We use Cawthon , 2016. Ta et al. . From tha et al. ) was R =Haemoproteus and Plasmodium) by amplifying a 478-bp fragment of the mitochondrial cyt b gene, using a nested polymerase chain reaction  were included in Plasmodium category because more detrimental effects are expected to arise from the action of this parasite  interaction terms. Additionally, to evaluate infection status dynamics (response variable: infected vs uninfected) with individual age (explanatory continuous variable), we performed an analysis accounting for individual id in random effects structure. Analyses were performed in R (v.3.3.1) , we employed random regression analysis, a type of a linear mixed model allowing to study inter-individual variation in intercepts (first breeder TL) and slopes (TL loss with advancing age). To this end, we modelled TL  as a response variable and fitted age , sex and infection type as fixed explanatory variables. Infection type was categorised into three levels: none, Data are available in the online Plasmodium  and Haemoproteus genera , whereas 2.8% of birds yielded mixed infection with the two genera . Among 102 individuals, that were recaptured in the subsequent year, probability of contracting infection was high (0.71), whereas losing infection was less likely (0.12) .Overall, haemosporidian prevalence reached 80% (please see the Electronic Supporting Information (ESI), Table ra Table B, ESI. P2) Table , S3, ESI2) Table , ESI. InP = 0.597), thus we applied the model without the covariance. Random regression analysis , and more importantly, it was also not related to the type of parasitic infection , so these interactions were not included in the final model. We found no association between infection intensity and TL  of a model including the correlation = 284.13, and of a model without the correlation = 283.99, likelihood-ratio test Haemoproteus had longer telomeres than males infected with Plasmodium. Such a finding can be readily explained, considering differential life cycles of the two genera. Haemoproteus causes less blood pathologies during its developmental cycle


Here, we tested the usefulness of small non-coding RNAs as references in quantitative RT-PCR expression analyses in hypothermia and chronic cardiac ischemia as the primary causes of death. Cq values of RNU6B, SCARNA17, SNORD25, and SNORA73A were determined from human cadaver samples of hypothermia and cardiac deaths. Average Cq values of RNU6B were higher in hypothermic and average SCARNA17 Cq values in chronic ischemic samples, but no difference in SNORD25 and SNORA73A Cq values could be seen between the groups. RNU6B expression levels were calculated using SNORD25, SNORA73A, and their combination as the reference in normalization. Expression of RNU6B, a widely used reference, was found to be significantly lower in hypothermia than in chronic cardiac ischemia. In these conditions, RNU6B is a useful marker differentiating hypothermia deaths from chronic ischemic heart disease deaths, but not a valid reference for normalization in expression studies. The expression of genes is influenced by various physiological and pathological conditions such as hypothermia and myocardial ischemia. Small non-coding RNAs  participate in the regulation of gene expression at post-transcriptional level. Small ncRNAs (length less than 400 nucleotides) are a versatile class of non-coding RNAs including, among others, microRNAs (miRNA), small nucleolar RNAs (snoRNA and scaRNA), and spliceosomal RNAs (RNU) .Molecular level changes elucidate different death processes in general and have potential impact on death investigations in the future . In routThe quality of RNA is variable in post mortem samples. RNA integrity is considered more critical than post mortem interval (PMI) for defining suitability of tissue for molecular biology applications , 8. The Coronary artery disease and signs of chronic myocardial ischemia are frequent findings in medico-legal autopsies in the western population . Our aimThe study cases were chosen on the bases of autopsy reports and the causes of death. The total number of hypothermia cases in this study was 25: 20 of them having hypothermia as the primary cause of death (group H) and 5 as a contributing cause of death (group cH). Of the 44 ischemic heart disease cases, 17 had atherosclerotic heart disease as the primary cause of death by itself (group AHD) and 27 with acute myocardial infarction as direct cause of death (group AHD + AMI).The permissions to use cadaver tissue samples and data from medico-legal death investigations were granted by the National Supervisory Authority for Welfare and Health (Dnro 2932/06.01.03.01/2013) and the National Institute for Health and Welfare (Dnro THL/479/5.05.01/2013), respectively. The Northern Ostrobothnia Hospital research ethics committee approved the study protocol (31/2007).Fresh, non-formalin-fixed tissue samples from the anterior wall of the left ventricle were collected for research purposes in medico-legal autopsies in the Department of Forensic Medicine, University of Oulu, Finland, and the National Institute for Health and Welfare, Oulu, Finland. The samples were stored at − 80 °C.A miScript II Reverse Transcription Kit  was used for the synthesis of cDNAs from previously extracted (miRNeasy kit) and quality checked (RIS) RNA.-ΔΔCq method [Four small ncRNAs were randomly selected from a miScript PCR Control Set kit (Qiagen) recommended to be used in miRNA studies. miScript SYBR Green PCR kit (Qiagen) and miScript Primer Assays (Qiagen) were used for RNU6B, SCARNA17, SNORD25, and SNORA73A amplifications (< 250 bp) with a Rotor Gene Q (Qiagen) instrument. The RNU6B Cq values were normalized to the Cq values of SNORD25, SNORA73A, and their average value. cDNA from commercial Human Heart Total RNA  was used as reference sample to calculate fold changes of relative expressions for RNU6B using the 2q method . The expp < 0.05).Statistical analyses were performed with IBM SPSS statistics, version 24 . The hypothermia death groups were compared with the ischemic heart disease death groups. The SPSS tests Shapiro Wilk and Kolmogorov-Smirnov were used to test the normality of the data. Only part of the data followed normal distribution. In addition, the groups were small. For these reasons, the pair-wise comparisons between the groups were made with the non-parametric Mann-Whitney test  or combined Fig.  in normar = − 0.881, p = 0.048; SCARNA17: r = − 0.984, p = 0.003; SNORA73A: r = − 0.969, p = 0.006 and the average value of SNORD25 and SNORA73A: r = − 0.959, p = 0.010).The average RIS values with standard errors and the statistical differences between the groups are presented in Table Small ncRNAs, particularly miRNAs, have lately been frequently evaluated as markers in forensic body fluid identification . Their tRNUs function as guides in the spliceosome formation and intron removal from mRNA . RNUs arWhen the RNU6B expression was determined in the myocardium, normalization with SNORD25, SNORA73A, or their combination gave comparable results. In this study, the myocardial RNU6B expression levels seemed to be higher in hypothermia than the level of the trauma death reference. However, a much striking elevation in the expression levels was seen in chronic cardiac ischemia, possibly due to adaptation mechanisms activating spliceosome formation and intron removal in this chronic condition.Our results show that small ncRNA RNU6B is not suitable for a reference in cadaver myocardial expression studies of hypothermia and chronic cardiac ischemia. Instead, RNU6B acts as a differentiating marker under these conditions.


Male gender and younger age at the initiation of intervention showed a non-significant trend towards greater success in weight loss. Long-term benefits were demonstrated in around one-quarter of obese adolescents after lifestyle modification treatment, with associated improvements in body composition and metabolic parameters.We undertook a feasibility study to reassess metabolic outcomes in young people with early onset obesity who attended a hospital-based lifestyle weight-loss intervention during adolescence. Comparisons of metabolic assessments, including body mass index standard deviation scores (BMI–SDSs), blood pressure (BP), oral glucose tolerance tests (OGTTs), lipid profile, and alanine transaminase (ALT), before and after treatment were made. Twenty-five subjects  with median ages  of 14.5 (12.6–15.4) years at the beginning of intervention and 18.2 (17.2–18.9) years at reassessment and who were 3.5 (2.4–6.5) years post-intervention were recruited. Twenty-eight percent had a ≥0.25 reduction in BMI–SDS from baseline (responders). Responders demonstrated significantly lower BMI–SDS, systolic BP, and glucose disposal at reassessment compared with baseline. They also showed significantly lower total fat percentage SDSs, trunk fat percentages, 120 min insulin, and ALT, as well as higher insulin sensitivity index (ISI Obesity increases the risk of many long-term health conditions, such as early onset type 2 diabetes mellitus, atherosclerotic cardiovascular disease, stroke, degenerative arthritis, obstructive sleep apnea, non-alcoholic fatty liver disease, infertility, and various cancers  . FurtherLifestyle and behavioural interventions based on modification of diet and physical activity are recommended as the first-line treatment for obesity during childhood and adolescence . SystemaAll subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki and was approved by the National Research Ethics Committee South West–Central, Bristol, UK . We aimed to recruit 25–30 young adults aged 16–25 years for metabolic reassessments with childhood-onset obesity who had previously undergone a lifestyle weight-loss intervention during adolescence in a children’s hospital clinic. All had a BMI of ≥98th centile at the beginning of treatment. Referrals were from community health professionals: health visitors, school nurses, and general practitioners. The multidisciplinary team consisted of a paediatrician, a dietician, and an exercise specialist, with access to psychological services if clinically indicated. Management included an initial general health assessment and metabolic screening for obesity-related comorbidity followed by four-monthly follow up for a minimum of one year. At each clinic visit, all subjects underwent anthropometry and received behavioural modification advice for their diet  and exercise habits . The subjects for the present study were identified from the department’s patient database and were approached in reverse chronological order from the last clinic appointment date. All female subjects underwent a pregnancy test on the assessment day. Informed consent was obtained from all participants.2)) were measured and converted to SDSs according to UK 1990 reference data  [comp) according to the following formula: 10,000 ÷ Demographic data was collected in clinic. Weight, height, and BMI (weight (kg) divided by height squared  . Metaboldon, UK) . Bioimpedon, UK) . Trunk fWe recorded dates of the first and last attended appointments as well as the total number of clinic appointments offered and attended by the patients during intervention. Compliance with intervention was defined as the number of attended appointments divided by the total number of appointments offered over the duration of intervention.U tests, Fisher’s exact tests, and Spearman’s ρ non-parametric correlation using SPSS Statistical Software version 23  at 5% significance with results reported in medians and interquartile ranges (IQRs).Results were compared by Mann–Whitney The 133 eligible subjects from the clinical database were approached, among which 65 were non-contactable, 31 did not wish to take part, 7 withdrew after having initially agreed to take part, and 5 cases were excluded as they received weight-loss interventions for under one year, resulting in a total of 25 cases  for the analysis of metabolic outcomes, representing 18.7% of the clinical cohort. No female participants were pregnant. There were 21 Caucasian, two South Asian, and two mixed Caucasian/Black participants of median (IQR) ages of 14.5 (12.6–15.4) years at the beginning of intervention and 18.2 (17.2–18.9) years at reassessment. Subjects were 3.5 (2.4–6.5) years post-intervention at the time of metabolic assessment in the study. Body mass index standard deviation scores reduction was greater than 0.25 from baseline in 28% (7/25) of the subjects (referred to as responders in the rest of this report).comp, but there were no group differences in diastolic BP, TG, and HDL or fat free mass %. There were no patients with impaired glucose tolerance or diabetes in either group at baseline or reassessment.All results are listed in p = 0.075). There were no statistical differences in pubertal status, BMI–SDS at the start of intervention, or social deprivation scores between the responders and non-responders. There was a trend towards greater BMI reduction in subjects treated at a younger age, but again this did not reach statistical significance .More males (5/10) than females (2/15) maintained a >0.25 BMI–SDS reduction, although the result was not statistically significant (p = 0.36) or compliance with the intervention (0.75 (0.54–0.9) years vs. 0.75 (0.59–1.0) years; p = 0.91).There were no differences between the responders and non-responders in terms of the length of intervention offered (1.82 (1.3–4.87) years vs. 2.44 (1.97–4.94) years; Our study suggested that slightly over one in four obese adolescents may benefit in the longer term after lifestyle modification interventions with associated improvements in body composition and metabolic parameters. Although the BMI–SDSs of all subjects in this cohort remained in the obese range at reassessment, those who achieved and maintained weight loss after intervention still benefitted from improved metabolic outcomes three to four years later. This message may motivate some obese individuals who will realistically never achieve an ideal BMI.comp values in subjects who maintained weight loss. Responders also showed a significantly lower ALT, a marker of non-alcoholic fatty liver disease. Non-alcoholic fatty liver disease is another well-known comorbidity of obesity that may lead to liver cirrhosis. Previous data have shown that multidisciplinary lifestyle interventions resulting in a reduction in BMI–SDS improve transaminitis in children and adolescents with obesity [Type 2 diabetes is the most common complication associated with obesity and is characterised by reduced insulin sensitivity and increased visceral fat. Our results showed significantly lower trunk fat % and improved insulin sensitivity, as indicated by lower stimulated insulin levels and higher ISI obesity . HoweverAs a feasibility study, the number of subjects was too small and underpowered to demonstrate statistical significance. The low participation percentage in this study may be a real-life reflection on the challenges of engaging individuals in lifestyle changes to enable fat loss and maintenance of a healthy weight. There were possible trends indicating greater success in maintenance of weight loss among male subjects and those referred for intervention at a younger age. Interestingly, these factors were associated with greater success in a different cohort from the same obesity clinic . Body coPerhaps surprisingly, the maintenance of BMI–SDS reduction was not related to the length of attendance or compliance to the intervention. In clinical practice, this means that patients resistant to weight loss are unlikely to succeed over a longer period of attendance. This may enable clinicians to justify discharging patients after a limited period of follow up regardless of the outcome for weight reduction. Further studies should evaluate the cost-effectiveness of weight-loss interventions by identifying factors that influence success and failure in order to inform screening criteria for entry into specialised clinics as well as to explore the optimal amount of weight loss and its longer-term benefits in later adulthood.In summary, we demonstrate positive effects on metabolic health in early adulthood for individuals with early onset obesity who maintained BMI–SDS changes after a lifestyle weight intervention during early adolescence. Whilst by no means ubiquitous in effect, a simple intervention that improves the anthropometry and metabolic health in the longer term of one in four obese adolescents undergoing therapy is relatively encouraging, given the pandemic of adult obesity. Further studies are needed to assess the overall cost-effectiveness of weight-loss interventions implemented for obese children.C.W. was involved in the study design, patient recruitment, conduction of clinical investigations, data collection, and statistical analysis and wrote the first draft of the manuscript. T.C. assisted in data collection and contributed to the writing of the manuscript. E.C. and J.P.H.-S. were involved in the study design and writing of the manuscript and had overall responsibility in the supervision of the study.


Patients (59.22%) presented with a high CIMT. In the adjusted regression model, age ≥60 years , ACR ≥30 mg/g , and GFR/CKD-EPI using cystatin C  were independently associated with carotid atherosclerosis. The occurrence of atherosclerotic lesions was high in the studied group. Age, albuminuria, and GFR (estimated by the formula CKD-EPI using cystatin C) influenced the prevalence of carotid atherosclerosis.Ethnicity appears to play an important role in the prevalence and severity of hypertension, renal disease, and atherosclerosis. A cross-sectional study was conducted, including 206 Afro-descendants with hypertension, living in the remaining quilombo communities. These subjects underwent a carotid intima-media thickness (CIMT) assessment. The presence of renal injury was assessed by: (1) The glomerular filtration rate (GFR) estimated by the formula CKD-EPI using creatinine and cystatin C and (2) Albuminuria (ACR ≥30 mg/g). The Poisson distribution model was set with robust variance to identify factors associated with carotid atherosclerosis. The statistical analysis was performed using the Stata 12.0 software, adopting a significance level of 5%. Most subjects were women (61.65%); the average age was 61.32 (±12.44) years. Subjects (12.62%) were identified with GFR <60 mL/min/1.73 m Cardiovascular diseases have been historically considered as diseases of the developed countries. However, these disorders are rapidly contributing to an increased morbidity in the developing regions with varied racial/ethnic groups ,2.Ethnicity appears to play an important role in the prevalence and severity of systemic arterial hypertension (SAH), atherosclerosis, and renal disease. The Black race is a strong predisposing factor to SAH, leaving the Afro-descendant population exposed to the risk of a more severe hypertension, as well as a greater risk for angina, heart attack, and sudden death .Atherosclerosis Risk in Communities Study (ARIC) cohort. They observed that the carotid intima-media thickness (CIMT) was greater in Black men with an aggressive profile, when compared to other sexes and ethnicity groups. In a cardiovascular health study, which evaluated 4926 White Caucasian subjects and 244 Black subjects of both sexes, aged 65 years or over, it was found that the CIMT was significantly greater in the Black population when compared to the White population, even after adjusting for cardiovascular risk factors [The assessment of carotid atherosclerotic disease has revealed an association with ethnicity in several studies . William factors .American Heart Association has recommended the investigation of atherosclerotic cardiovascular diseases in all ethnic groups, including short-term and long-term risk analyses to fill this knowledge gap [The differences in the occurrence of cardiovascular disease between ethnic groups cannot be completely explained by traditional risk factors, despite a higher occurrence of arterial hypertension and diabetes mellitus in the Black population . In concedge gap .This study aims to investigate the influence of renal disease markers on the prevalence of carotid atherosclerotic disease among African-American residents in the remaining quilombo communities in the northern state of Maranhão, Brazil.Prevalence of Chronic Renal Disease in the municipality of São Luís and Alcântara Quilombola communities, State of Maranhão’ study, which evaluated 1539 African-American people from the 32 remaining quilombo communities in northeastern Brazil , between August 2012 and April 2013.This is a cross-sectional analysis including the Afro-descendants with hypertension who participated in the PREVRENAL: ‘n = 439), using a two-tailed hypothesis test . Individuals with BMI >30 kg/m2 were classified as obese [Education was presented in years of study. Age was categorized into age groups. Smoking was defined as current consumption of ≥1 cigarette/d. Weight (kg) and height (m) were measured to calculate the body mass index , history of stroke (S), acute myocardial infarction (AMI)/angina, and use of medications were referred to by the study participants, based on their history of a previous medical diagnosis.The BP assessment was performed by the oscillometric method, using an Omron 705-IT device. Three measures of systolic (SBP) and diastolic (DBP) blood pressure were made, in which the average of the last two measurements was used.Patients who had a previous diagnosis, or blood pressure levels equal to or exceeding 140 mmHg (SBP) and/or 90 mmHg (DBP), in two measurements at different times were considered to have systemic arterial hypertension .Venous blood samples were taken after a 12 h fasting, and included the following biochemical dosages: creatinine, cystatin C, blood count, glucose, uric acid, lipid profile, ultra-sensitive c-reactive protein (CRP). The uric acid was considered high, when it was above 7 mg/dL for men and above 6 mg/dL for women.The dyslipidemia was categorized as (1) isolated hypercholesterolemia: LDL isolated elevation (≥160 mg/dL); (2) isolated hypertriglyceridemia: triglycerides elevation (≥150 mg/dL); (3) mixed hyperlipidemia: LDL ≥160 mg/dL and triglycerides ≥150 mg/dL; and (4) low HDL: <40 mg/dL in men and <50 mg/dL in women .2.The glomerular filtration rate (GFR) was estimated from the formula derived from the CKD-EPI study, using the value of creatinine  and cystThe value of urinary albumin was obtained from the albuminuria/creatinine ratio (ACR), of an isolated sample of the second urine of the day. Patients with laboratory urinary value ≥30 mg/g were defined as having albuminuria.To assess the CIMT, a GE Vingmed Ultrasound device, model Vivid3  was used. The ultrasound examination of the carotid was performed by a single experienced examiner, blinded to the clinical and laboratory data and risk classification of each patient. After a rest of at least 10 min in supine position with the neck in discreet hyperextension, an evaluation of the carotid system was performed bilaterally. The CIMT was measured in the distal wall  of the common carotid, 1 cm proximally to its bifurcation, according to the current recommendations. The measurement consisted of the distance between two echogenic lines represented by the lumen-intima and media-adventitia interfaces of the arterial wall. Values were considered normal when CIMT <0.9 mm and amended when CIMT ≥ 0.9 mm .p < .20 in the bivariate analysis, and only those with p < .05 remained in the model. The data were analyzed using the Stata 12.0 software.First, a descriptive analysis of the study variables was carried out. Numeric variables were presented as mean and standard deviation, and the categorical variables were presented as frequencies and percentages. The association between independent variables and carotid atherosclerosis was examined in the bivariate analysis by the chi-squared test, with a significance level of 5%. The Poisson regression with robust variance was adopted to investigate how the prevalence of carotid atherosclerosis could be influenced by various explanatory variables. Considering this, the multiple Poisson regression was carried out for the adjusted analysis. In the multiple model, the variables with All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The research protocol was approved by the Research Ethics Committee of the University Hospital of Federal University of Maranhão (consolidated opinion No. 41492/2012), in compliance with the requirements of the Resolution of the National Health Council No. 466/12 and its supplementary norms for research involving human beings.n = 127; 61.65%), with a mean age of 61.32 (±12.44) years. Ninety-four (45.63% of the sample) individuals were aged less than 60 years. Twenty-six subjects with diabetes mellitus (12.62% of the sample), 19 subjects with a history of stroke (9.22% of the sample), 46 subjects with obesity (22.33% of the sample), and 58 smoker subjects (28.15% of the sample) were identified. The measurement of systolic blood pressure (SBP) revealed SBP values of ≥140 mmHg in 158 individuals (76.70% of the sample) and diastolic blood pressure (DBP) ≥ 90 mmHg in 92 cases (44.66% of the sample). The distribution of the clinical-epidemiological characteristics, according to the CIMT, are shown in The study sample consisted of 206 Afro-descendants with hypertension, mostly women  had a reduced GFR assessed by the CKD-EPI formula using cystatin C as a marker. Albuminuria (≥30 mg/g of creatinine) occurred in 47 cases (22.8% of the sample).In 16 cases (7.77% of the sample), the patients had a GFR <60 mL/min/1.73 m2 estimated by formula CKD-EPI creatinine (87.50%) and CKD-EPI cystatin C (92.31%), those without isolated hypertriglyceridemia (59.55%), those with mixed hyperlipidemia (75.00%), and those with hyperuricemia (75.86%), as shown in CIMT was assessed in 122 patients (59.22% of the sample), and calcified plates were identified in 100 patients (48.54% of the samples). Carotid atherosclerosis was more frequent in male Afro-descendants (69.62% of the sample), those aged between 60 and 79 years (73.74% of the sample), those aged ≥80 years (92.31% of the sample), those with a history of diabetes mellitus (80.77% of the sample), those using acetylsalicylic acid (ASA) (82.35%), those with SBP ≥140 mmHg (63.29%), those with albuminuria/creatinine ratio (ACR) ≥ 30 mg/g (72.34%), those with GFR <60 mL/min/1.73 mp = .014), in individuals aged between 60 and 79 years  and ≥80 years , those with diabetes mellitus , those using ASA , those with ACR ≥30 mg/g of creatinine , those with GFR <60 mL/min/1.73 m2 by the formula using creatinine , and using the CKD-EPI cystatin C , those with mixed hyperlipidemia  and hyperuricemia .p values = .001), ACR ≥30 mg/g  and GFR <60 mL/min/1.73 m2 assessed by formula using cystatin C only .In the adjusted analysis, the following remained statistically associated: age ≥60 years , for which the independently associated factors are age, albuminuria, and reduced glomerular filtration rate estimated by the CKD-EPI formula, using cystatin C as a serum marker.Except for the study by Wagenknecht et al. , which iAge is an important non-modifiable risk factor for atherosclerosis. Some authors consider atherosclerosis a part of the aging process . The isoSimilar to this study, several authors have demonstrated the association between age and carotid atherosclerotic disease . GijsberMulti-Ethnic Study of Atherosclerosis (average of 9.8 years of follow-up), concluded that albuminuria was an independent risk factor for the development of peripheral arterial disease, after finding that patients with higher ACR quintile had a significant increased risk of the progression of ankle-brachial index below 0.90  and above 1.40 , which is a reflection of atherosclerotic disease.The influence of urinary loss of albumin on the prevalence of atherosclerotic lesions in the carotid arteries of the Afro-descendants was also demonstrated. Severe albuminuria is considered a structural renal damage marker . It is aBogalusa Heart Study [p values = .005). Moreover, in the multivariate logistic regression model, only the Black race was associated with the presence of albuminuria . This study, which included only African-American subjects, also showed a statistically significant association between CIMT ≥ 0.9 mm and ACR ≥30 mg/g, with a p values = .037.The rt Study , includiThe African-American-diabetes heart study [p < .01), but not among African-Americans (p = .33), after adjustments for age, sex, glomerular filtration rate, and body mass index. In addition, the calcium metabolism clearly differs between African-Americans and European Americans. Although African-Americans typically resent lesser intake of calcium in the diet than Caucasians, they have a higher bone density, with lower rates of osteoporosis, and skeletal resistance to the effects of the parathyroid hormone [The relationship between albuminuria and calcified atherosclerotic plaque has shown ethnic differences, with lower levels of calcified plaque found in African-American individuals in relation to Caucasians with or without diabetes mellitus . The Afrrt study , includi hormone . The intThe findings of this study indicate that the GFR estimated by cystatin C has a strong association with CIMT, unlike the glomerular filtration rate estimated using the serum creatinine. Several studies have pointed out that cystatin C shows a stronger association to creatinine in the occurrence of cardiovascular events and mortality ,38, becaVitamins to Prevent Stroke . Its main objective was to evaluate the effect of a decrease in homocysteine by supplementation with complex B vitamins in reducing the incidence of greater vascular events. However, it did not identify a correlation between cystatin C and CIMT.Nevertheless, the number of studies that attempted to correlate the cystatin C with CIMT is still limited. Their results are conflicting. While two small cross-sectional studies involving middle-aged patients with hypertension reported a significant correlation between cystatin C and CMIT , 41, larr = 0.491, p < .001). However, the serum cystatin C was not correlated with the CIMT and flow-mediated dilation of the brachial artery, corroborating the role of cystatin C as a renal function marker. Nevertheless, it did not show an association with atherosclerosis markers.Monteiro et al.  evaluatep values < .001). In our study, the use of statin did not alter the prevalence of carotid intima-media thickness.Only 5 of the 122 subjects diagnosed with CIMT ≥0.9 mm were using statins. Statins have been shown in primary and secondary cardiovascular prevention trials to reduce the incidence of cardiovascular events . It is nAlthough previous studies indicate diabetes mellitus , smokingThis study has some limitations. The first is related to its cross-sectional design, which prevents forming conclusions on the impact of the markers evaluated on the occurrence of cardiovascular events. Additionally, since a highly specific group of patients was assessed, the results cannot be automatically extrapolated to the Afro-descendant populations. Conversely, our work shows some positive aspects that deserve to be mentioned. This is the first study to diagnose carotid atherosclerotic disease in the Black population descending from the African slave community who live in the isolated places of Brazil. Furthermore, the association of glomerular filtration rate, estimated by cystatin C (not by the creatinine) and albuminuria as renal function markers in an isolated sample, with carotid thickening must be highlighted. These markers might be used in the assessment of atherosclerotic disease in patients with similar social, demographic, and clinical characteristics.In conclusion, the occurrence of carotid atherosclerotic lesions measured using CIMT was high in the studied group. Renal disease markers  and age were independently associated with these lesions. The reduced GFR influenced the prevalence of carotid atherosclerosis only when calculated by the CKD-EPI formula using cystatin C as a serum marker. Therefore, the Afro-descendants belonging to the ethnic minority groups, of advanced age, with albuminuria and a reduced GFR (estimated by the CKD-EPI formula using cystatin C) should be strictly monitored and treated to reduce the risk of cardiovascular events, considering their association with carotid atherosclerotic disease.


Because the assessment of sperm DNA fragmentation (SDF) plays a key role in male fertility, our study was designed to find the relationships between SDF and standard semen parameters. The receiver operating characteristic (ROC) curve showed that 18% SDF is a prognostic parameter for discriminating between men with normal and abnormal standard semen parameters (n = 667). Men with > 18% SDF had significantly lower quality semen, a higher prevalence of abnormal semen characteristics, and a higher odds ratio for abnormal semen parameters compared to men with ≤ 18% SDF. An ROC analysis provided predictive values for age and semen parameters to distinguish between men with SDF > 18% and men with ≤ 18% SDF. SDF was positively correlated with male age and teratozoospermia index but negatively with sperm concentration, total number of spermatozoa, sperm morphology, progressive motility, and vitality. Our study shows that 18% SDF has a predictive value for distinguishing between men with normal and abnormal semen characteristics. Men with >18% SDF have a higher risk for abnormal semen parameters, while age and obtained semen parameters have a predictive value for SDF. There is a relationship between SDF and conventional sperm characteristics, and thus, SDF can be incorporated into male fertility assessment. Approximately up to 20% of couples trying to achieve pregnancy suffer from infertility ,2,3. It Many authors have shown that knowledge about the level of DNA damage is essential and provides the possibility of an optimal approach to an infertility problem ,19,20,21The study was performed on ejaculated sperm cells obtained from a general population of men  attending the Andrology Laboratory of Department of Histology and Developmental Biology . The laboratory is a research and scientific unit and access to research was open and unlimited—any adult man could take part in the research. The groups of men with normal standard semen parameters (n = 234) and with abnormal standard semen parameters (n = 434) were created according to the World Health Organization (WHO)  criteriaThe semen samples were collected after 2–7 days of sexual abstinence by masturbation. Sperm concentration was calculated in an improved Neubauer haemocytometer . In turn, sperm motility , vitality (live sperm cells: eosin-negative or hypo-osmotic-reactive sperm cells (HOS) test) were examined with phase-contrast microscope , but sperm morphology with a bright light microscope , respectively. Papanicolaou-stained spermatozoa were used for sperm morphology and teratozoospermia index (TZI) assessment. The concentration of leukocytes in the semen samples (peroxidase-positive cells) was calculated using the Endtz test .Normozoospermia (n = 242) was considered according to the following WHO  criteriaThe SCD test was used to verify SDF. The evaluation of DNA dispersion after denaturation was carried out using a Halosperm G2 kit  following the manufacturer’s guidelines: (1) preparation of a mixture containing sperm cells (≤ 20 mln/mL) and melted agarose (1:2); (2) placement of the sperm suspension (10 µL) on the centre of a super-coated slide; and (3) denaturation, lysis, dehydration, and staining of sperm cells with eosin and thiazine.The smears were evaluated under a bright light microscope at x1000 magnification . A minimum of 300 spermatozoa per sample were counted. Sperm cells without SDF can produce the characteristic halo of dispersed DNA loops , while spermatozoa with damaged DNA fail to form a halo of dispersed DNA loops . The results are presented as the total number of spermatozoa with small or no halo, that is, degraded, divided by the total number of assessed sperm cells, and multiplied by 100% [p < 0.05. The quantitative variables are expressed as the mean ± standard deviation (SD) and median (range), while categorical data are reported as percentages. The conformity of numerical variables with the normal distribution was examined using the Shapiro–Wilk test. Therefore, the nonparametric Mann–Whitney U test was used to compare data from two independent groups. A chi-square test was performed to compare the categorical data. The interdependences of the variables were examined by calculating the rank Spearman correlation coefficient (rs). To interpret the strength dependence between the study parameters, the following levels of correlation were presumed: <0.2—lack of linear dependence, 0.2–0.4—weak dependence, >0.4–0.7—moderate dependence, >0.7–0.9—strong dependence, >0.9—very strong dependence. The predictive values of obtained parameters were verified using the receiver operating characteristic (ROC) curve and the area under curve (AUC), taking into account the standard error (SE) and 95% confidence interval (CI 95%). The following levels of AUC were: 0.9–1.0—excellent predictive value, >0.8–0.9—good predictive value, >0.7–0.8—satisfactory predictive value, >0.6–0.7—moderate predictive value, 0.5–0.6—insufficient predictive value. The odds ratios (OR) for SDF levels  to define the relative risk in predicting the abnormal standard semen parameters in study group with respect to the SDF level were calculated.The statistical analyses were performed using the software Statistica version 13.3  and MedCalc version 15.2.2 , with significance set at p < 0.05) in evaluated parameters except for semen volume  to distinguish between men with abnormal and normal standard semen parameters . Based on volume . Men witMoreover, men with > 18% SDF presented a significantly higher prevalence of abnormal standard semen parameters than men with SDF ≤ 18%  . AdditioThe second ROC analysis provided information about the predictive value of age and standard semen analysis for sperm DNA fragmentation . The cals = 0.211) and TZI (rs = 0.339), but was negatively correlated with sperm concentration (rs = −0.289), total number of spermatozoa (rs = −0.255), sperm morphology (rs = −0.457), sperm progressive motility (rs = −0.524), and eosin-negative and HOS-test reactive sperm cells   .Generally, > 30% sperm cells with DNA damage is considered to be a cut-off point for a high risk of infertility ,33,34,35It should be emphasized that our obtained findings could have clinical utility. The suggested threshold of 18% SDF was lower than the reference value given by the Halosperm G2 kit manufacturer (30% SDF). Based on our findings, it seems that 30% SDF may be unsatisfactory for discriminating men with normal fertility from those with reduced fertility potential. This suggestion is partly consistent with the studies of other authors ,38,39,40Our second ROC evaluation revealed the predictive value of age and standard semen parameters for distinguishing between men with > 18% SDF and ≤ 18% SDF. Importantly, the cut-off points were as follows: sperm concentration 13.80 mln/mL, total sperm count 27.50 mln, and sperm morphology 2%. Our findings suggest that with the above values, which are slightly lower than the reference range given by WHO , we can Some limitations of our study must be addressed. Firstly, the method we used to reveal SDF was the SCD, and we have to highlight that this method has some known disadvantages, such as the indirect assessment of sperm DNA and the susceptibility to subjective assessment of sperm DNA dispersion, where there is a risk of over-interpretation of the results ,46,47. AIt is also worth mentioning that the clinically useful threshold of SDF is difficult to estimate ,27,28,48In light of our findings, we can conclude that men with >18% SDF have a higher risk for abnormal standard semen parameters, while age and obtained standard semen parameters have a predictive value for SDF. Our statistical data indicate association between SDF and parameters of basic semen analysis; however, it is possible that men with normal standard semen parameters may have reduced fertility potential due to diminished sperm chromatin integrity. Therefore, DNA fragmentation testing and conventional semen analysis can be considered as complementary tools in the evaluation of male fertility potential.


Logistic regression analysis was used to assess the combined effects and multiplicative interactions of pre-pregnancy OWO + HDP on offspring high BMI. Relative excess risk due to interaction (RERI) or attributable proportion (AP) was used to estimate additive interactions. RERI > 0 or AP > 0 indicates a significant additive interaction. Compared with the non-OWO and normal blood pressure group, the combination of OWO + HDP was positively associated with offspring high BMI at 12 months of age [OR 3.10 ], with 51% of the effects attributed to an additive interaction [AP 0.51 ]. An interactive effect was found between the pre-pregnancy OWO + HDP and offspring high BMI in infancy. Interventions to control pre-pregnancy OWO and HDP are important to prevent obesity and associated adverse outcomes in offspring.We aimed to assess whether hypertensive disorders of pregnancy (HDP) could modify the effect of pre-pregnancy overweight or obesity (OWO) on the risk of offspring high body mass index (BMI) in infancy. A total of 3,765 mother-child pairs were recruited from two Chinese birth cohorts. BMI ≥ 85 OWO that develops in childhood increases the early risk of cardiovascular damage resulting in an increased incidence of cardiovascular morbidity and mortality in adulthood4.The prevalence of overweight or obesity (OWO) in both children and adults has been increasing substantially worldwide6, etc. Pre-pregnancy overweight status is considered as a risk factor for the high BMI status in offspring by influencing the intrauterine environment. However, results have not been consistent. For instance, McCloskey et al. reported that high maternal pre-pregnancy BMI was a risk factor for obesity in newborn offspring7. Yet others reported no association between pre-pregnancy high BMI and offspring OWO at 1 year of age8. These inconsistent findings suggest that other variables influencing the intrauterine environment might be modifying the association between maternal BMI and BMI status in offspring. A number of studies have found that hypertensive disorders of pregnancy (HDP) could also increase the risk of offspring obesity, which might be partly attributed to a change in the intrauterine environment10.To reduce the incidence of obesity and associated adverse outcomes, exposure to potential risk factors early in life that contribute to a high body mass index (BMI) status should first be considered. There is an increasing interest in understanding the role of the intrauterine environment on the development of obesity in offspring, e.g., maternal pre-pregnancy weight, gestational weight gain and blood pressure (BP) during pregnancyTherefore, in this study, we aimed to investigate the independent and combined effects of pre-pregnancy OWO and HDP on the risk of offspring high BMI status in infancy and explore the related additive and multiplicative interactions further.Data were extracted from two Chinese birth cohorts. The first birth cohort (Cohort A) was conducted in the cities of Shenyang, Wuhan, and Guangzhou from April 2009 to March 2010. The second cohort (Cohort B) was performed in Zhuhai city from May 2014 to December 2016. In Cohort A, maternal age, infant sex, birthdate, delivery mode, gestational weight gain (GWG), BP measurements during pregnancy and gestational condition  were obtained from the patient Perinatal Health Booklets; maternal height and infant weight and length were measured by trained nurses; and maternal pre-pregnancy weight, gestational age, household income, educational level, maternal secondhand smoke exposure during pregnancy, the feeding practices during the first month of life, and cities were obtained using self-reported questionnaires. In Cohort B, maternal age, infant sex, birthdate, delivery mode, BP measurements during pregnancy, and gestational conditions were obtained from the Maternal and Child Health Information System; infant weight and length were measured by trained nurses; and maternal pre-pregnancy weight and height, gestational age, household income, educational level, maternal secondhand smoke exposure during pregnancy, the feeding practices during the first month of life, and GWG were obtained from self-reported questionnaires.A total of 4,036 mother-child pairs  were originally recruited with information on maternal pre-pregnancy weight and height, BP measurements during pregnancy, infant sex, infant weight and length measured at least twice at birth, 1, 3, 6 and 12 months, and questionnaires were filled out during the study recruitment, before delivery, and 1 month after giving birth. Among all participants, 97 mother-child pairs in Cohort A and 174 mother-child pairs in Cohort B were excluded for mothers using antihypertensive medications, having diabetes mellitus, tuberculosis, thyroid or hepatobiliary diseases, or a non-singleton pregnancy, or missing data in infants’ weight and length at birth. A total of 3,765 mother-child pairs  were finally included in this analysis. No significant differences were observed in the main variables between the samples used for analysis and those originally included  × (365.25/370) + 3.5 = 10.9 kg (12-month exact days were: 12 × 30.4375 = 365.25days). Maternal height in Cohort B was measured by trained staff using a stadiometer .In Cohort A, infant weight and length at birth, 3, 6, and 12 months were measured twice by trained health staff with an electronic scale  to the nearest 0.05 kg and a length measurement instrument (WHS-I) to the nearest 0.1 cm, respectively. In Cohort B, infant weight and length at the corresponding months were measured twice by trained health staff with an electronic weighing scale (Hengxin HCS-20-YE) and a horizontal baby bed (Hengxin HX-II), respectively. For all measurements, average values were used. The weight and length at each visit were linearly adjusted by the measured intervals to the exact duration of the corresponding months based on World Health Organization criteria12. Feeding patterns at 1 month were classified as exclusive breastfeeding (WHO definition13), mixed feeding (both formula and breast milk), and formula feeding (exclusively formula without breast milk). Household income was categorized as <3000RMB ($426.6), 3001–5000RMB ($426.7–711.0) and 5001–8000RMB ($711.1–1137.6), or ≥8001RMB ($1137.7). The adjusted models were performed with the mentioned confounders and cohort variable adjusted. The missing data was compensated using a non-parametric missing value imputation (missForest based on random forest package in R3.5.2)14. The included data and the imputed datasets representing the main variables did not show significant differences  and OWO (BMI ≥ 24 kg/m2), according to the Working Group on Obesity in China20. Gestational BP status was divided into the HDP (systolic BP ≥ 140 mmHg and/or diastolic BP ≥ 90 mmHg during pregnancy) and normal BP  groups. We were unable to distinguish specific gestational hypertensive disorders, such as gestational hypertension and preeclampsia since urine protein data were not available. Therefore, the definition of HDP in this study represents a combination of gestational hypertensive disorders during pregnancy, which was similarly defined in a previous study10.BMI was calculated by dividing weight in kilograms by height in meters squared. An expert panel of the World Health Organization appealed for additional research on the health implications of infants using a BMI indicator based on length21 were assessed in this study. A total of three logistic regression models were used to evaluate the effects of pre-pregnancy BMI status and gestational BP status and their multiplicative interactions on the high BMI status of the offspring at 12 months of age, adjusting for potential confounders. The three models are Model 1, the unadjusted model; Model 2, adjusted for birth-related variables , maternal variables  and cohort variable and; Model 3, a model similar to Model 2 with further adjustments for family environment-related variables . These potential variables were included because there were either well-established associations with the offspring BMI-z values or because there was at least a 10% variation in the interest estimates if removed22. P-values <0.05 were considered statistically significant.All data analyses were conducted using SPSS software, version 16.0 . Maternal age, GWG and gestational age (continuous variables) with non-normal distributions were expressed as the median and inter-quartile range (IQR). Maternal education level, feeding patterns at 1 month, mode of delivery, and maternal secondhand smoke exposure during pregnancy (category variables) were presented as N (%). The Kruskal-Wills test was used to compare continuous variables, and the Chi-square test was used to compare categorical variables between the groups. The generalized estimating equation models were used to assess the association of pre-pregnancy BMI and BP during pregnancy with the BMI-z at birth, 3, 6, and 12 months. Both multiplicative  and additive  interactions00, OR10, OR01, and OR11 were calculated for the Non-OWO + NBP (reference group), Non-OWO + HDP, OWO + NBP and OWO + HDP groups, respectively. The calculation of the relative excess risk of interaction (RERI = OR11 − OR10 − OR01 + 1), attributable proportion (AP = RERI/OR11) and the 95% confidence interval(CI) was performed using a Microsoft Excel spreadsheet generated by Andersson et al.23. RERI > 0 or AP > 0 indicates a biologically additive interaction, which had been widely used in previous studies25.The odds ratios (OR) of OR2], more likely to live in low income household (46.7%), and have a higher proportion of infants with low birth weight (2.2%). Gestational age, maternal secondhand smoke exposure during pregnancy, maternal educational level, delivery mode, feeding pattern during the first month of life, and infant sex are also presented.Table β 0.31, 95%CI ] and 12 months of age . Maternal HDP was independently associated with offspring BMI-z values at 12 months of age [β 0.24 ] compared with the NBP group . Compared with infants from Non-OWO + NBP mothers, the BMI-z values of infants from OWO + HDP mothers were on average 0.62 units greater at 12 months of age (P = 0.002).As shown in Table As shown in Table The comparison of the baseline characteristics between Cohort A and Cohort B were performed and the results are presented in Supplementary Table Our findings indicated that maternal pre-pregnancy OWO and HDP were independent risk factors for offspring high BMI-z values in infancy. A significant combined effect of OWO and HDP on high BMI-z values of the infants was observed at 12 months of age. We also found that the OWO + HDP in mothers synergistically increased the risk of having offspring with a high BMI status at 12 months compared with Non-OWO + NBP mothers. A total of 51.0% of the increased risk was attributable to additive interactions.2 during pre-pregnancy, had a higher risk of being obese at 2 to 4 years of age26. However, Lindberg et al. reported pre-pregnancy OWO was not a risk factor for offspring OWO at 1 year old of age8. In this study, we showed that maternal pre-pregnancy OWO was associated with higher offspring BMI-z values at 12 months of age, which suggests that the effects of pre-pregnancy OWO on offspring high BMI could emerge early in life. The discrepancy between the findings of Lindberg et al. and ours might be due to the different ethnic groups studied (American Indian children vs. Chinese children), the potential factors considered , sample sizes , classification references (CDC growth charts for the United States vs. WHO Child-Growth Standards), as well as other factors.Previous studies have inconsistently reported the effects of maternal pre-pregnancy OWO on offspring high BMI status. Offspring of mothers, who had a BMI ≥ 30 kg/m2) or obese (BMI ≥ 30 kg/m2) in early middle age27. A meta-analysis demonstrated that mothers with preeclampsia during pregnancy had an increased risk of having children with higher BMI at ≥10 years of age, but not in children <10 years of age28. In the present study, we found that mothers with HDP had an independently increased risk of having offspring with higher BMI-z values as early as 12 months of age. These different findings could be explained by the diverse backgrounds of the study populations, the various ages of the measured outcomes, and the different definitions of the hypertensive disorders, etc. The findings similar to our results have also demonstrated a positive association of HDP with high BMI-z in offspring during childhood29. A birth cohort in Jiaxing, China showed that HDP was a risk factor for obesity in 4–7 year-old children10. However, the results were based on one small city in China, and some potential confounders such as gestational weight gain were not considered. Based on the data from four representative cities in China, our results showed a significant association between maternal HDP and high infant BMI-z scores, which suggests the importance of early detection of HDP.To our knowledge, most previous research has primarily focused on preeclampsia. For example, a Swedish research group showed that preeclampsia during pregnancy had a 1.54- and 1.40-fold increased risk of having offspring that became overweight  of offspring, which has been linked to OWO in offspring34. Second, maternal OWO could also affect the orexigenic factors (NPY) in offspring appetite control systems by altering the hypothalamic architecture, which leads to overeating and subsequent rapid weight gain35. Third, mothers with unhealthy eating habits and pre-pregnancy obesity could cause offspring to have increased energy intakes and BMI as well through unhealthy feeding practice37. Although the mechanisms between HDP and high BMI in offspring is not clear, several possible mechanisms have been proposed. First, altered adipose tissue gene (e.g. leptin gene) expression in the placenta of pregnant women with preeclampsia40 might influence offspring body weight43. Second, HDP was shown to be related to oxidative stress in offspring, which was associated with obesity45. In general, both OWO and HDP in mothers might impair fetal growth through a changed intrauterine nutrition environment47, which could synergistically alter adipose tissue morphology and metabolism, modify the hormones and epigenome, and regulate appetite pathways49. Thus, through these mechanisms, maternal obesity and HDP might jointly increase the risk of high BMI status in offspring.Direct intrauterine mechanisms could partly explain the potential effects of maternal pre-pregnancy OWO, HDP, and the interactions of these two disorders50. Fifth, there are 21 individuals with a BMI ≥ 85th percentile at 12 months, which limits our ability to assess the effect of maternal OWO + HDP on a higher BMI status (e.g. BMI ≥ 90th percentile) in infancy. In this study, we used a BMI ≥ 85th percentile, which has been demonstrated as a cut-off for predicting obesity in early childhood17. Finally, we only adjusted feeding patterns during the first month because feeding practices were not provided at 3 months or longer in Cohort A. Further studies are needed to consider feeding practices for a longer period.We used pooled data from the two birth cohorts in four cities located in Northern, Central and Southern China that were more representative of Chinese populations, thus increasing the external validity of our results. However, several limitations should also be mentioned. First, since urine protein concentrations were unavailable, we were unable to differentiate the different types of hypertensive disorders, such as chronic hypertension, gestational hypertension or preeclampsia, and therefore, could not determine which disorders would be better predictors of offspring obesity than a BP ≥ 140/90 mmHg. Second, we did not consider BP status at different trimesters because only a limited number of participants received BP measurements at all trimesters. The prevalence of HDP might have been overestimated; however, the primary aim of this study was not to make a clinical diagnosis of hypertension but to assess the interactive effects of maternal pre-pregnancy OWO and HDP in a general population. Third, to improve statistical power, we combined overweight and obese women during pre-pregnancy into one group. Larger populations with more detailed categories of maternal pre-pregnancy weight status and BP measurements during different trimesters are needed for future studies. Fourth, maternal pre-pregnancy weight in both cohorts was self-reported. However, the self-reported weight has been demonstrated to be reliable and valid in epidemiologic studiesIn conclusion, there was an additive interaction of maternal pre-pregnancy OWO and HDP on offspring high BMI status. Therefore, monitoring maternal weight status before pregnancy and measuring BP during pregnancy has important implications to identify high-risk infant offspring for the early prevention of a high BMI status to protect them from health risks in later life.Supplementary Table S1–4


The consumption of non-carbonated sugar-sweetened beverages (NCSSBs) has many adverse health effects. However, the sugar and energy content in NCSSBs sold in China remain unknown. We aimed to investigate the sugar and energy content of NCSSBs in China and how these contents were labelled.A cross-sectional survey was conducted in 15 supermarkets in Haidian District, Beijing from July to October 2017. The product packaging and nutrient information panels of NCSSBs were recorded to obtain type of products , serving size, nutrient contents of carbohydrate, sugar and energy. For those NCSSBs without sugar content information, we used carbohydrate content as a replacement.A total of 463 NCSSBs met the inclusion criteria and were included in our analysis. The median of sugar content and energy content was 9.6 [interquartile range (IQR): 7.1–11.3] g/100 ml and 176 (IQR: 121–201) kJ/100 ml. The median of sugar contents in juice drinks, tea-based beverages, sports drinks and energy drinks were 10.4, 8.5, 5.0 and 7.4 g/100 ml. Imported products had higher sugar and energy content than local products. There were 95.2% products of NCSSBs receiving a ‘red’(high) label for sugars per portion according to the UK criteria, and 81.6% products exceeding the daily free sugar intake recommendation from the World Health Organization (25 g). There were 82 (17.7%) products with sugar content on the nutrition labels and 60.2% of them were imported products.NCSSBs had high sugar and energy content, and few of them provided sugar content information on their nutrition labels especially in local products. Measures including developing better regulation of labelling, reducing sugar content and restricting the serving size are needed for reducing sugar intakes in China.The online version of this article (10.1186/s12889-019-7486-6) contains supplementary material, which is available to authorized users. Free sugar in sugar-sweetened beverages (SSBs) received considerable attention from public and emerged as a popular health issue. The term ‘free sugar’ refers to monosaccharides and disaccharides added to foods and beverages by the manufacturer, cook or consumer, and sugars naturally present in honey, syrups, fruit juices and fruit juice concentrates . With thThe most well-known type of SSBs are carbonated products such as soda. With the increasing studies and rapid spread of mass media, the public has improved the awareness of the high sugar content and hazard of carbonated SSBs (CSSBs) –8. As a Importantly, the sugar contents of the same NCSSBs were different across different countries . AlthougThe information of the total number of markets and their addresses in Haidian district were gathered by using the Location Based Service open platform of Amap . We thenA total of 15 supermarkets were included in this study and they were Wu Mart, Yonghui Superstores, Wal-Mart, Cuiwei, Merry Mart, Carrefour, Xingfu, Hua Lian, Century Mart, Chaoshifa, Jingkelong, Vanguard, Auchan, Shijijiajia, and Shijihualian.: Table S1 for comparison with other studies [The categories of NCSSBs included juice drinks, tea-based beverages, sports drinks, and energy drinks , whose d1 studies , 27.The data were collected from product packaging and nutrient information panels of each NCSSBs by a snapshot in time. For each NCSSBs, its company name, product name, serving size, nutrient content of carbohydrate, sugar and energy per 100 ml were extracted. The information of country of origin on the package of products and nutrition labels were used to determine if the NCSSBs were local or imported. For those NCSSBs without sugar content information on their nutrition labels, we used carbohydrate content as a replacement. NCSSBs without the information (carbohydrate/sugar/energy) we needed on their nutrition labels were not investigated in this study. When any product which had been photographed in a previous supermarket showed in subsequent supermarkets, it would not be photographed again. Some brands sold the same formulation in different serving sizes, and we only included one product of one formulation.Two different recommendations for free sugar intakes were used in our study to evaluate sugar content in different categories of NCSSBs. The Food Standards Agency in the UK released a guideline on front of pack color-coded labelling for drinks . Color cP value< 0.05 was considered statistically significant.Epidata was used for data entry and all data were double checked by the authors (Jin and Peng). Sugar and energy content in different NCSSBs were presented as median and interquartile ranges (IQR). The nonparametric Kruskal-Wallis test was used to determine differences across four NCSSBs categories and between imported and local products. Products that meet the recommendations for free sugar intakes were presented as number and proportion. McNemar–Bowker test was used for comparison of the proportion of products with ‘Red’ label between per 100 ml criterion and per serving criterion according to the UK front of pack color-coded labelling. For NCSSBs with sugar and carbohydrate contents information on their nutrition labels, Spearman’s rank correlation coefficient and related-samples Wilcoxon signed-rank test were used to explore the relationship between two contents. Sensitivity analysis was performed to compare the results between NSCCBs with and without sugar information. We tested whether the replacement of sugar content with carbohydrate content in NCSSBs induced bias or not. Statistical analyses were conducted using SAS, version 9.4, and a A total of 463 NCSSBs met the inclusion criteria and were included in our analysis. Of the 463 products, 323 were juice drinks, 96 were tea-based beverages, 32 were sports drinks and 12 were energy drinks.P < 0.05) and energy content .Table 2 = 7.810, P < 0.05). Juice drinks had the highest percentage of products (32.2%) exceeding the UK per 100 ml criterion while tea-based beverages had the highest percentage of products (83.3%) exceeding the WHO criterion. All energy drinks received ‘red’ label according to the UK per serving criterion.Table 2 = 14.540, P < 0.01). In those 82 NCSSBs with sugar content information, the Spearman’s rank correlation coefficient between carbohydrate and sugar content was 0.899 (P < 0.0001). The paired differences between sugar and carbohydrate contents was 0.76 ± 0.16 g/ 100 ml .Among 463 NCSSBs, 82 (17.7%) products had sugar content on the nutrition labels. Juice drinks had highest proportion of products with sugar labelling (19.5%) while none of the energy drinks had sugar content on the label (shown in Table : Table S3).The sensitivity analyses showed that the sugar content in NCSSBs with both sugar and carbohydrate information was insignificantly different from those without sugar information, which we used carbohydrate content as alternatives . Similar results were found in the proportion of products exceeding recommendation levels between NCSSBs with and without sugar information Additional file 2:Table S2. Sugar content information in different categories of NCSSBs. *: P < 0.05. Abbreviation: NCSSBs, non-carbonated sugar-sweetened beverages. (DOCX 14 kb)Additional file 3:Table S3. Comparison between NCSSB with and without sugar information according to different recommendations. α:The serving criterion of UK guidance of front of pack color-coded labelling for drinks was> 13.5 g/portion if serving size> 150 ml. β: The recommendation for daily free sugar intakes from the WHO was 25 g. Abbreviation: NCSSBs, non-carbonated sugar-sweetened beverages. (DOCX 14 kb)