Comparative Transcriptome Analysis Uncovers the Regulatory Roles of MicroRNAs Involved in Petal Color Change of Pink-Flowered Strawberry.

The pink-flowered strawberry is popular in China due to its high ornamental value. In the present study, sRNAome, transcriptome, and degradome sequencing were performed to understand the functions of microRNAs (miRNAs) and their target genes during flower development in pink-flowered strawberry. Nine small RNA libraries and a mixed degradome library from flower petals at different developmental stages were constructed and sequenced. A total of 739 known miRNAs and 964 novel miRNAs were identified via small RNA sequencing, and 639 miRNAs were identified to cleave 2,816 target genes based on the degradome data. Additionally, 317 differentially expressed miRNAs among the various stages of flower development were identified, which regulated 2,134 differentially expressed target genes. These target genes were significantly enriched in the transcriptional regulation, phenylpropanoid biosynthesis, and plant hormone signal transduction pathways. Furthermore, integrated microRNAomic and transcriptomic analyses suggested that 98 miRNAs targeted several transcription factors, including MYBs (26), bHLHs (12), NACs (14), and SPLs (19), related to anthocyanin accumulation. In addition, 27 differentially expressed miRNAs might affect anthocyanin biosynthesis by regulating 23 targets involved in the hormone signal transduction pathway. The quantitative real-time PCR (qRT-PCR) analysis confirmed the expression changes of 21 miRNA-target pairs. Furthermore, the transient expression of candidate miRNAs was performed in the pink-flowered strawberry cultivar "Fenyun" at the bud stage. Introduction of FamiR156a, FamiR396e, and FamiR858_R-2 in the "Fenyun" increased flower color intensity, while transient expression of FamiR828a decreased flower color intensity. Overall, the present study uncovers the regulatory functions of microRNAs, including anthocyanin biosynthesis, hormone signaling, and regulation factors during flower development and coloration in pink-flowered strawberry. This work expands the knowledge of miRNAs affecting coloration in strawberry and provides rich resources for future functional studies.

Indoor and Outdoor Rodent Hosts of Orientia tsutsugamushi, Shandong Province, China.

During December 2012-July 2016, we tested small indoor and outdoor mammals in Qingdao, China, for Orientia tsutsugamushi infection. We found that outdoor Apodemus agrarius mice, Cricetulus barabensis hamsters, and Niviventer confucianus rats, as well as indoor Mus musculus mice, tested positive for O. tsutsugamushi by PCR.

Leisure-Time Physical Activity and Cardiovascular Disease Risk Among Hypertensive Patients: A Longitudinal Cohort Study.

Objective: Few studies estimated the effect of leisure-time physical activity (LTPA) on cardiovascular disease (CVD) risk among hypertensive patients in a longitudinal cohort. This study aims to evaluate the association between LTPA and CVD in a longitudinal management cohort of hypertensive patients. Methods: A total of 58,167 hypertensive patients without baseline CVD from a longitudinal cohort were included in this study. LTPA and other covariates were measured at the follow-up four times annually. The primary outcome was CVD events. The association between LTPA and CVD was assessed by the marginal structure model (MSM) and Cox model with adjustment for age, gender, body mass index (BMI), smoking, drinking, diabetes, hyperlipidemia, and antihypertensive medication. The restricted cubic spline and segmented regression were used to assess the dose-response relationship between LTPA and CVD. Results: We recorded 16,332 CVD events; crude incidence of CVD were 89.68, 80.39, 62.64, and 44.04 per 1,000 person-years for baseline 0, 1-150, 151-300, and >300 min/week LTPA, respectively. Compared with inactive LTPA, the adjusted hazard ratios (HRs) estimated by Cox model and MSM-Cox model for CVD associated with 1-150,151-300, and 300 min/week LTPA were 0.85 (95% CI, 0.83-0.88), 0.67 (95% CI, 0.64-0.71), 0.47 (95% CI, 0.44-0.51), and 0.83 (95% CI, 0.76-0.91), 0.58 (95% CI, 0.52-0.63), and 0.39 (95% CI, 0.35-0.44), respectively. Per 60 min/week increase in LTPA was associated with a 13% reduction in CVD risk. LTPA breakpoint was 417 min/week for CVD. Before and after the break-point, the slopes of the piecewise-linear relationship between LTPA and CVD risk were -0.0017 and -0.0003, respectively. Conclusion: LTPA was more strongly associated with the CVD risk than that estimated by conventional analyses based on baseline LTPA; 417 min/week is a breakpoint, after which the incremental health benefits on CVD prevention obtained from the increase in LTPA are much less than before.

Galli gigeriae endothelium corneum: its intestinal barrier protective activity in vitro and chemical composition.

BACKGROUND: Galli gigeriae endothelium corneum (GGEC) has been effectively used for centuries for the treatment of functional dyspepsia (FD) in clinical practice in Asian countries. However, its potential mechanism and chemical composition remains undertermined. METHODS: In this study, the chemical profile of GGEC ethyl acetate extract (EAE) was evaluated by HPLC-Q-TOF-MS/MS. The effects of EAE on intestinal barrier function and inflammation were investigated in IEC-6 cells and RAW264.7 cells. RESULTS: The results showed that 33 compounds were tentatively identified, including 12 soy isoflavones, 7 bile acids for the first time in EAE. EAE significantly reinforced intestinal barrier function via increasing the tight junction protein levels of ZO-1 and Occludin, reducing the mRNA expression levels of interleukin (IL)-1ß and IL-6 in tumor necrosis factor alpha (TNF-α)-challenged IEC-6 cells. The scratch wound assay showed that EAE accelerated wound healing of IEC-6 cells. EAE evidently reduced the level of NO in a dose-dependent manner with an IC50 value of 18.12 µg/mL, and the mRNA expression of TNF-α, IL-1ß, IL-6, iNOS and COX-2 in LPS-treated RAW264.7 cells. CONCLUSION: This study revealed the intestinal barrier protective effects and chemical profile of GGEC, and the results indicated that GGEC strengthened the intestinal barrier by up-regulating protein expression of tight junctions and limiting inflammatory responses.

Antihypertensive medication adherence and cardiovascular disease risk: A longitudinal cohort study.

BACKGROUND AND AIMS: Few studies estimated the impact of antihypertensive adherence on cardiovascular diseases (CVD) in a longitudinal cohort with presence of time-dependent confounders. This study aims to assess the association between antihypertensive adherence and CVD using marginal structural Cox model (MSM-Cox) and to characterize blood pressure (BP) trajectories of patients with different adherence. METHODS: This longitudinal study included 16,896 hypertensive patients receiving antihypertensive medication. The median follow-up time was 3.5 years (25th to 75th, 1.75-4.75 years). BP and medication adherence were measured four times every year. We used MSM-Cox and Cox model to assess association between antihypertensive adherence and CVD events. The linear mixed-effects model was used to characterize BP trajectories of patients with different adherence, and the area under curves (AUC) was calculated as BP burden. RESULTS: We documented 4735 CVD events, crude incidence of CVD was 80.8 (95% CI, 78.1-83.4) and 112.6 (95% CI, 107.2-118.0) per 1000 person-years for baseline high-adherence and low-adherence, respectively. Compared with high adherence, the adjusted hazard ratio (HR) for association between low adherence with CVD was 1.75 (95%CI, 1.62-1.89) and 1.34 (95%CI, 1.26-1.42) based on the MSM-Cox and the Cox model, respectively. The BP burden and fluctuation range of BP trajectory in low-adherence patients were larger than those of high-adherence patients. Patients with high adherence got 28% greater reduction of BP burden than low-adherence patients. CONCLUSIONS: Antihypertensive adherence was more strongly associated with the risk of CVD than conventional regression analyses based on a single adherence measurement.

Relationship between Lipid Profiles and Glycemic Control Among Patients with Type 2 Diabetes in Qingdao, China.

Glycosylated hemoglobin (HbA1c) was the best indicator of glycemic control, which did not show the dynamic relationship between glycemic control and lipid profiles. In order to guide the health management of Type 2 diabetes (T2D), we assessed the levels of lipid profiles and fasting plasma glucose (FPG) and displayed the relationship between FPG control and lipid profiles. We conducted a cross-sectional study that included 5822 participants. Descriptive statistics were conducted according to gender and glycemic status respectively. Comparisons for the control of lipid profiles were conducted according to glycemic control. Four logistic regression models were generated to analyze the relationship between lipid profiles and glycemic control according to different confounding factors. The metabolic control percentage of FPG, triglyceride (TG), total cholesterol (TC), high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) was 27.50%, 73.10%, 28.10%, 64.20% and 44.80% respectively. In the fourth model with the most confounding factors, the odds ratios (ORs) and 95% confidence intervals (CIs) of TG, TC, LDL-C and HDL-C were 0.989 (0.935, 1.046), 0.862 (0.823, 0.903), 0.987 (0.920, 1.060) and 2.173 (1.761, 2.683). TC and HDL-C were statistically significant, and TG and LDL-C were not statistically significant with adjustment for different confounding factors. In conclusion, FPG was significantly associated with HDL and TC and was not associated with LDL and TG. Our findings suggested that TC and HDL should be focused on in the process of T2D health management.

Differentiation, chemical profiles and quality evaluation of five medicinal Stephania species (Menispermaceae) through integrated DNA barcoding, HPLC-QTOF-MS/MS and UHPLC-DAD.

Stephania species is one of the alkaloid-rich genus of the family Menispermaceae. Most plants of the genus Stephania possess medicinal value, whose main components are alkaloids. However, the non-medical species are often mistakenly used as herbs because of the difficulty in identification of the species. A systematic method which involved the combination of DNA barcoding, HPLC-QTOF-MS/MS and UHPLC was established for differentiation, chemical profiles and quality evaluation of medicinal Stephania species. Firstly, twenty batches of Stephania species samples were classified into five Stephania species by DNA barcoding. Secondly, 114 alkaloids including 22 tetrahydroprotoberberines, 13 protoberberines, 27 aporphines, 13 benzylisoquinolines, 12 hasubanans, 3 morphines and 24 other alkaloids were clearly or tentatively identified. Thirdly, thirteen representative components were simultaneously detected by UHPLC-DAD to characterize the differences of chemical compositions among five Stephania species. In conclusion, this method was comprehensive and effective for identification, chemical profiles and quality evaluation of medicinal Stephania species. It will provide a basis for holistic quality evaluation of medicinal Stephania species.

Rickettsia japonica and Novel Rickettsia Species in Ticks, China.

PCR amplification indicated the minimum infection rate of Rickettsia spp. was 0.66% in Haemaphysalis longicornis ticks collected from Shandong Province, China. Phylogenetic analysis based on the rrs, gltA, ompA, and ompB genes indicated that the ticks carried R. japonica, Candidatus Rickettsia longicornii, and a novel Rickettsia species related to R. canadensis.

Anaplasma species detected in Haemaphysalis longicornis tick from China.

Anaplasma are tick-borne obligatory intracellular bacteria, which infect humans and other animals. The Anaplasma species carried by ticks in China are not well studied. We collected 3145 questing Haemaphysalis longicornis ticks including 120 larvae, 2460 nymphs and 565 adults from vegetation in Jiaonan County, Shandong Province, China from 2013 to 2015. All ticks were examined for the presence of Anaplasma species by nested PCR amplification of the 16S rRNA gene. For further differentiation of A. capra from A. centrale, gltA and msp2 genes were sequenced for A. capra. Three Anaplasma species were detected in the nymph and/or adult ticks with the minimum infection rate of ticks as follows: 1.55% for A. bovis, 0.10% for A. phagocytophilum, and 0.03% for A. capra. These results indicated that the H. longicornis tick in Jiaonan County carried multiple Anaplasma species, which may be a challenge for public health in the studying area.

Meteorological factors affect the epidemiology of hemorrhagic fever with renal syndrome via altering the breeding and hantavirus-carrying states of rodents and mites: a 9 years' longitudinal study.

The incidence of hemorrhagic fever with renal syndrome (HFRS) in Qingdao, China was three times higher than that of the average national level. Here we characterized the epidemiology, ecological determinants and pathogen evolution of HFRS in Qingdao during 2007-2015. In this longitudinal study, a total of 1846 HFRS patients and 41 HFRS-related deaths were reported. HFRS in Qingdao peaked once a year in the fourth quarter. We built a time series generalized additive model, and found that meteorological factors in the previous quarter could accurately predict HFRS occurrence. To explore how meteorological factors influenced the epidemic of HFRS, we analyzed the relationship between meteorological factors and hantavirus-carrying states of the hosts (including rodents and shrews). Comprehensive analysis showed humidity was correlated to high host densities in the third quarter and high hantavirus-carrying rates of animal hosts in the third to fourth quarters, which might contribute to HFRS peak in the fourth quarter. We further compared the L segments of hantaviruses from HFRS patients, animal hosts and ectoparasites. Phylogenetic analysis showed that hantaviruses in gamasid and trombiculid mites were the same as those from the hosts. This indicated mites also contributed to the transmission of hantavirus. Furthermore, Hantaan virus from HFRS patients, hosts and mites in Qingdao formed a distinct phylogenetic cluster. A new clade of Seoul virus was also identified in the hosts. Overall, meteorological factors increase HFRS incidence possibly via facilitating hosts' reproduction and consequent mite-mediated hantavirus transmission. New hantavirus subtypes evolved in Qingdao represent new challenges of fighting against HFRS.

Detection of a Novel Rickettsia From Leptotrombidium scutellare Mites (Acari: Trombiculidae) From Shandong of China.

Leptotrombidium scutellare mites, the vector of Orientia tsutsugamushi, have rarely been reported to associate with Rickettsia species. Three hundred nineteen chiggers were collected from the ears of 32 rodents captured in Huangdao District of Qingdao City, China, in October 2015. The chigger samples were tested for Rickettsia, severe fever with thrombocytopenia syndrome virus, and hantavirus by PCR or RT-PCR amplification. All mites were classified morphologically and molecularly as L. scutellare chiggers. Rickettsial DNA sequences were amplified for four genes including 16S rRNA, ompB, gltA, and 17 kD protein genes. The minimum infection rate (MIR; number of positive pools/total specimens tested) of the Rickettsia species in the chiggers were 2.8% (9/319). Phylogenetic analysis indicated that individual genes were closely related to different Rickettsia species including R. felis (with 16S rRNA gene), R. australis (with gltA gene), an unnamed Rickettsia sp. TwKM02 (with ompB gene), and Rickettsia endosymbiont of soft tick Ornithodoros erraticus (with 17 kD protein gene). Phylogenic analysis of the concatenated sequence of 16S rRNA, gltA, ompB, and 17 kD protein genes indicated that the Rickettsia species from L. scutellare chigger was most closely related to R. australis and R. akari. These results indicated that the Rickettsia species in chiggers was unique; it was named Candidatus Rickettsia leptotrombidium. Severe fever with thrombocytopenia syndrome virus and hantavirus were not amplified from the chiggers, suggesting lack of infection of these pathogens in the chiggers. A unique Rickettsia species was detected in L. scutellare, which expanded the knowledge on the vector distribution of Rickettsia.

Seroprevalence of Severe Fever with Thrombocytopenia Syndrome Virus in Hedgehog from China.

Severe fever with thrombocytopenia syndrome, an emerging hemorrhagic fever, is caused by severe fever with thrombocytopenia syndrome virus (SFTSV), a tick-borne bunyavirus. Information regarding SFTSV animal hosts is very limited. In this study, we showed that 64% (9/14) of hedgehogs in Shandong Province, China were seropositive to SFTSV antibody, suggesting that hedgehog could be a vertebrate parasitifer for SFTSV.

Detection of Imjin Virus and Seoul Virus in Crocidurine Shrews in Shandong Province, China.

INTRODUCTION: Recently, hantaviruses have been discovered in insectivores in Europe, Asia, Africa, and North America. Imjin virus (MJNV) was first isolated from the lung tissues of Ussuri white-toothed shrew (Crocidura lasiura) from South Korea in 2009. We aim to detect the species and prevalence of insectivore- and rodent-borne hantaviruses in shrews and rodents. MATERIALS AND METHODS: Shrews and rodents were captured in Jiaonan County of Shandong Province, China, in 2014. RT-PCR was used to amplify viral RNA of Hantavirus species, including insectivore-borne Imjin virus (MJNV), rodent-borne Hantaan virus (HTNV), and Seoul virus (SEOV) from shrews and rodents. RESULTS AND DISCUSSION: We found that MJNV infected 10.7% (19/178) of Crocidura shrews, but it infected none of rodents (0/475); we also found that 2 of 178 (1.1%) Crocidura shrews were PCR positive to SEOV. This study indicated that the major animal hosts of Imjin virus are shrews, and rodent-borne SEOV can infect shrews.

Prevalence of hemorrhagic fever with renal syndrome in Qingdao City, China, 2010-2014.

Hemorrhagic fever with renal syndrome (HFRS) was considered to be transmitted by Apodemus agrarius and Rattus norvegicus, the principal animal hosts of Hantaan virus and Seoul virus, respectively. The aim of this study is to determine the correlation of HFRS incidence with capture rate and hantavirus infection rate of rodent species in Qingdao City, China. We collected HFRS patients' information and captured field and residential rodents in Qingdao City, China from 2010 to 2014. The correlations of HFRS incidence to rodent capture rate and hantavirus infection rate of rodents were analyzed statistically. The main findings of this study are that the high HFRS incidence (19.3/100,000) is correlated to the capture rate of field Mus musculus (p = 0.011, r = 0.037); but surprisingly it did not correlated to the capture rate of the principal rodent hosts Apodemus agrarius and Rattus norvegicus and the hantavirus infection rate of these rodent species in the field or residential area. These novel findings suggest that Mus musculus, a nontraditional animal host of hantavirus may play an important role in hantavirus transmission in Qingdao City.

Detection of a Novel Ehrlichia Species in Haemaphysalis longicornis Tick from China.

We collected 2460 Haemaphysalis longicornis ticks from vegetation in Jiaonan County, Shandong Province, in June of 2013 and Daishan County, Zhejiang Province, China, in May of 2015. The tick DNA was subsequently amplified with nested polymerase chain reaction using Ehrlichia common 16S rRNA gene primers and Ehrlichia ewingii species-specific groEL and gltA primers. We found 0.4% (3/780) of the ticks from Zhejiang Province contained Ehrlichia DNA that was different from all known Ehrlichia species, but most closely related to E. ewingii. We concluded that a novel Ehrlichia species exists in H. longicornis ticks in China.

Haemaphysalis longicornis Ticks as Reservoir and Vector of Severe Fever with Thrombocytopenia Syndrome Virus in China.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging hemorrhagic fever in East Asia caused by SFTS virus (SFTSV), a newly discovered phlebovirus. The Haemaphysalis longicornis tick has been suspected to be the vector of SFTSV. To determine whether SFTSV can be transmitted among ticks, from ticks to animals, and from animals to ticks, we conducted transmission studies between developmental stages of H. longicornis ticks and between ticks and mice. Using reverse transcription PCR, we also analyzed the prevalence of SFTSV infection among H. longicornis ticks collected from vegetation in Shandong Province, China. Our results showed a low prevalence of SFTSV among collected ticks (0.2%, 8/3,300 ticks), and we showed that ticks fed on SFTSV-infected mice could acquire the virus and transstadially and transovarially transmit it to other developmental stages of ticks. Furthermore, SFTSV-infected ticks could transmit the virus to mice during feeding. Our findings indicate ticks could serve as a vector and reservoir of SFTSV.

Prevalence of SFTSV among Asian house shrews and rodents, China, January-August 2013.

To evaluate the role of small mammals as hosts of severe fever with thrombocytopenia syndrome virus (SFTSV), we tested serum samples from rodents and shrews in China, collected in 2013. SFTSV antibodies and RNA were detected, suggesting that rodents and shrews might be hosts for SFTSV.

Meteorological factors are associated with hemorrhagic fever with renal syndrome in Jiaonan County, China, 2006-2011.

This study examined the effect of meteorological factors on the occurrence of hemorrhagic fever with renal syndrome (HFRS) using a generalized additive model with penalized smoothing splines in Jiaonan, China, from 2006 to 2011. The dose-response relationship was first examined, and then the association between daily meteorological variables and HFRS occurrence was investigated according to the dose-response curves. There were two linear segments in the temperature-HFRS relationship curve. When daily temperature was lower than 17 °C, a positive association was found [with excessive risk (ER) for 1 °C increase on the current day being 2.56 %, 95 % confidence interval (CI): 0.36 % to 4.80 %]. An inverse association was found when daily temperature was higher than 17 °C [ER for 1 °C increase on the current day was -12.82 % (95 % CI: -17.51 % to -7.85 %)]. Inverse associations were observed for relative humidity [ER for 1 % increase on lag day 4 was -1.21 % (95 % CI: -1.63 % to -0.79 %)] and rainfall [ER for 1 mm increase on lag day 1 was -2.20 % (95 % CI: -3.56 % to -0.82 %)]. Meteorological factors might be important predictor of HFRS epidemics in Jiaonan County.