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Introduction: The Maximum Entropy models (MaxEnt) is commonly employed for early detection of disease transmission, particularly effective in assessing the risk zones and intensity of tick-borne disease transmission based on climatic factors.Methods: Utilizing the diversity data of common ticks in Qinghai, along with surveillance statistics tick-borne pathogens of ticks and epidemiological information, we have charted and predicted the prevalence of tick and tick-borne pathogens across Qinghai province.Results and Discussion: The findings indicate that the pivotal environmental factors influencing the distribution of ticks and tick-borne pathogens include land use and land cover (Lucc), elevation (Elev), annual precipitation (Bio12) and annual mean temperature (Bio1). Notably, for Dermacentor nuttalli, the significant environmental factors accounted for 35.2% for Lucc, 24.7% for Elev and 18.6% for Bio12. In the case of Dermacentor silvaru, the pivotal factors contributed 33.2% for Lucc, 23.7% for Elev and 22.6% for Bio1. For Haemaphysalis qinghaiensis, the key environmental variables were 45% for Elev, 30.9% for Lucc and 18.4% for Bio12. Regarding the pathogens, the environmental factors influencing Borrelia burgdorferi contributed 61.5% for Lucc, 13.3% for Elev and 11.9% for Bio1. For Piroplasmida, the contribution was 62.1% for Lucc, 16.7% for Bio1 and 9.9% for Bio12. Rickettsia was influenced by factors accounting for 34.2% for Lucc, 29.7% for Elev and 17.3% for Bio12, while Anaplasma variables contributed 38.2% for Bio1, 26.6% for Lucc and 18.9% for Bio12. The finding indicated that the three predominantly ticks species (D. nuttalli, D. silvaru, and H. qinghaiensis) and the primary tick-borne pathogens (B. burgdorferi, Piroplasmida, Rickettsia, and Anaplasma) are predominantly concentrated in the source regions of the Yellow River, the Yangtze River, the Lancang River, and the eastern part of the Haixi Autonomous Prefecture. Projected under the ssp245 scenario, there is a notable expansion trend in the risk areas for ticks and tick-borne pathogens These findings are consistent with previous epidemiological studies on major ticks and tick-borne diseases in Qinghai conducted by our laboratory, this suggests the feasibility of using the MaxEnt model to predict the distribution of tick-borne disease transmission and compensates for the paucity of research on the Maxent model in the Qinghai Province.

BackgroundLong term hypertension seriously promotes target organ damage in the brain and heart, and has increasingly become serious public health problem worldwide. The anti-hypertensive effects of capsaicin has been reported, however, the role and mechanism of capsaicin within the brain on salt-induced hypertension have yet to be elucidated. This study aimed to verify the hypothesis that capsaicin attenuates salt-induced hypertension via the AMPK/Akt/Nrf2 pathway in hypothalamic paraventricular nucleus (PVN).MethodsDahl salt-sensitive (Dahl S) rats were used as animal model for the present study. Rats were randomly divided into four groups based on their dietary regimen (0.3% normal salt diet and 8% high salt diet) and treatment methods (infusion of vehicle or capsaicin in the PVN). Capsaicin was chronically administered in the PVN throughout the animal experiment phase of the study that lasted 6 weeks.ResultsOur results demonstrated that PVN pretreatment with capsaicin can slow down raise of the blood pressure elevation and heart rate (HR) of Dahl S hypertensive rats given high salt diet. Interestingly, the cardiac hypertrophy was significantly improved. Furthermore, PVN pretreatment with capsaicin induced decrease in the expression of mRNA expression of NADPH oxidase-2 (NOX2), inducible nitric oxide synthase (iNOS), NOX4, p-IKKβ and proinflammatory cytokines and increase in number of positive cell level for Nrf2 and HO-1 in the PVN of Dahl S hypertensive rats. Additionally, the protein expressions of phosphatidylinositol 3-kinase (p-PI3K) and phosphorylated protein kinase-B (p-AKT) were decreased, phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK) were increased after the PVN pretreatment with capsaicin.ConclusionCapsaicin pretreatment attenuates salt-sensitive hypertension by alleviating AMPK/Akt/iNOS pathway in the PVN.

IntroductionWhole-genome resequencing technology covers almost all nucleotide variations in the genome, which makes it possible to carry out conservation genomics research on endangered species at the whole-genome level.MethodsIn this study, based on the whole-genome resequencing data of 101 critically endangered Artocarpus nanchuanensis individuals, we evaluated the genetic diversity and population structure, inferred the demographic history and genetic load, predicted the potential distributions in the past, present and future, and classified conservation units to propose targeted suggestions for the conservation of this critically endangered species.ResultsWhole-genome resequencing for A. nanchuanensis generated approximately 2 Tb of data. Based on abundant mutation sites (25,312,571 single nucleotide polymorphisms sites), we revealed that the average genetic diversity (nucleotide diversity, π) of different populations of A. nanchuanensis was relatively low compared with other trees that have been studied. And we also revealed that the NHZ and QJT populations harboured unique genetic backgrounds and were significantly separated from the other five populations. In addition, positive genetic selective signals, significantly enriched in biological processes related to terpene synthesis, were identified in the NHZ population. The analysis of demographic history of A. nanchuanensis revealed the existence of three genetic bottleneck events. Moreover, abundant genetic loads (48.56% protein-coding genes) were identified in Artocarpus nanchuanensis, especially in genes related to early development and immune function of plants. The predication analysis of suitable habitat areas indicated that the past suitable habitat areas shifted from the north to the south due to global temperature decline. However, in the future, the actual distribution area of A. nanchuanensis will still maintain high suitability.DiscussionBased on total analyses, we divided the populations of A. nanchuanensis into four conservation units and proposed a number of practical management suggestions for each conservation unit. Overall, our study provides meaningful guidance for the protection of A. nanchuanensis and important insight into conservation genomics research.

Background and purpose: Testosterone is an essential sex hormone in maintaining masculine characteristics, which is prescribed for male hypogonadism as testosterone replacement treatment (TRT). Herein, we investigated long-standing controversies about the association between TRT and major adverse cardiovascular events (MACEs), based on real world adverse event (AE) reports, registered in the Food and Drug Administration Adverse Event Reporting System (FAERS).Methods: Publicly available FAERS data from 1 January 2004 to 31 December 2022 were retrieved from the Food and Drug Administration (FDA) website. The data mining protocol including the reporting odds ratio (ROR) and the Bayesian confidence propagation neural network (BCPNN) was applied to analyze overreporting caused by risk factors and MACEs, including TRT, morbidities, and ages. The ROR and the BCPNN were also applied to investigate the annually developing trend of pharmacovigilance (PV) signals in the real world, retrospectively.Results: A total of 3,057 cases referring to MACEs, with a median age of 57 years old (yo), were identified from 28,921 cases of testosterone users. MACEs related to PV signals have emerged since 2014, including cardiac death, non-fatal myocardial infarction, and non-fatal stroke. Myocardial infarction (MI) (ROR: 9.46; IC025: 3.08), acute myocardial infarction (AMI) (ROR: 16.20; IC025: 3.72), ischemic cardiomyopathy (ROR: 11.63; IC025: 2.20), and cardiomyopathy (ROR: 5.98; IC025: 1.96) were the most significant signals generated, and weaker signals included cardiac failure acute (ROR: 4.01; IC025: 0.71), cardiac arrest (ROR: 1.88; IC025: 0.56), and ventricular fibrillation (VF) (ROR: 2.38; IC025: 0.38). The time-to-onset (TTO) of MACEs was calculated with a median of 246 days for AMI.Conclusion: For myocardial infarction and cardiomyopathy, TRT statistically tended to increase the risk of MACEs, while for cardiac arrhythmia, cardiac failure, and stroke, TRT demonstrated beneficial effects among the population with morbidities, such as testosterone deficiency (TD), diabetes mellitus (DM), and hypertension. MACEs were rare but led to serious outcomes including significant increase in death and disability. Since 2018, and before 2014, reports referring to TRT associated with MACEs were relatively scarce, which indicated that there might be a considerable number of cases that went unrecorded, due to neglection. Health workers and testosterone users might pay more attention to testosterone-induced MACEs.

Protozoan parasites are a well-known threat to human health, particularly for people working at or visiting zoos, and potentially cause zoonotic diseases in humans. Captive wildlife may be potential reservoirs for human infection with protozoan parasites. Therefore, focusing on zoonotic protozoan infections in zoo animals is critical. However, there is no report on this topic in the Qinghai-Tibetan Plateau region. In this study, a total of 167 and 103 fecal samples were collected from 12 animal species from Qinghai-Tibet Plateau Wildlife Park in winter and summer, respectively, to detection the prevalence of infections and subtype distribution with Entamoeba sp., Cryptosporidium sp., Giardia duodenalis, Enteromicrosporidia bieneusi sp., Blastocystis sp. by PCR assay. The results showed that a total of 21 fecal samples collected in winter, including from 2 white-lipped deer, 8 Sika deer, 6 blue sheep, 2 wolves and 3 bears, were positive for Entamoeba, with a 12.6% (21/167) positive rate. However, 4.9% (5/103) of animals in summer were positive for Entamoeba, including 1 snow leopard, 1 tiger, 1 Tibetan argali and 2 mouflon. Moreover, 1 white-lipped deer and 1 bear were found to be positive for Blastocystis sp., one zoonotic STs (ST10) was identified and found in white-lipped deer. We found no effect on season on Blastocystis sp. and Entamoeba sp. colonization. To the best of our knowledge, this study is the first description of Blastocystis sp. and Entamoeba sp. infecting zoo animals in the plateau area. The findings provide the latest data on Entamoeba sp. and Blastocystis sp. in zoo animals in China.

Sleep inertia (SI) is a time period during the transition from sleep to wakefulness wherein individuals perceive low vigilance with cognitive impairments; SI is generally identified by longer reaction times (RTs) in attention tasks immediately after awakening followed by a gradual RT reduction along with waking time. The sluggish recovery of vigilance in SI involves a dynamic process of brain functions, as evidenced in recent functional magnetic resonance imaging (fMRI) studies in within-network and between-network connectivity. However, these fMRI findings were generally based on the presumption of unchanged neurovascular coupling (NVC) before and after sleep, which remains an uncertain factor to be investigated. Therefore, we recruited 12 young participants to perform a psychomotor vigilance task (PVT) and a breath-hold task of cerebrovascular reactivity (CVR) before sleep and thrice after awakening (A1, A2, and A3, with 20 min intervals in between) using simultaneous electroencephalography (EEG)-fMRI recordings. If the NVC were to hold in SI, we hypothesized that time-varying consistencies could be found between the fMRI response and EEG beta power, but not in neuron-irrelevant CVR. Results showed that the reduced accuracy and increased RT in the PVT upon awakening was consistent with the temporal patterns of the PVT-induced fMRI responses (thalamus, insula, and primary motor cortex) and the EEG beta power (Pz and CP1). The neuron-irrelevant CVR did not show the same time-varying pattern among the brain regions associated with PVT. Our findings imply that the temporal dynamics of fMRI indices upon awakening are dominated by neural activities. This is the first study to explore the temporal consistencies of neurovascular components on awakening, and the discovery provides a neurophysiological basis for further neuroimaging studies regarding SI.

Dermacentor nuttalli has been a focus of study because tick-borne pathogens have been widely identified in this tick from northern and southwestern China. The aim of this study was to characterize the life cycle of D. nuttalli under laboratory conditions and to detect spotted fever group (SFG) Rickettsia in the midgut and salivary glands of both field-collected and first laboratory generation adults. D. nuttalli ticks were collected in the field on the Qinghai-Tibetan Plateau from March to April 2021 and their life cycle was studied under laboratory conditions. Tick identify was molecularly confirmed, and SFG Rickettsia were detected in the midgut and salivary glands of males and females by PCR targeting different rickettsial genes. The results showed that the life cycle of D. nuttalli under laboratory conditions was completed in an average of 86.1 days. High positivity of Rickettsia spp. was detected in the midgut and salivary glands of both males (92.0%) and females (93.0%) of field-collected D. nuttalli ticks. However, a relatively lower positivity (4.0–6.0%) was detected in first laboratory generation adults. Furthermore, sequencing analysis showed that the Rickettsia sequences obtained in this study shared 98.6 to 100% nucleotide identity with Rickettsia slovaca and Rickettsia raoultii isolated from Dermacentor spp. in China. Phylogenetic analysis of Rickettsia spp. based on the gltA, ompA, ompB and sca4 genes revealed that the Rickettsia sequences obtained could be classified as belonging to R. slovaca and R. raoultii clades. This study described for the first time the life cycle of D. nuttalli from the Qinghai-Tibetan Plateau under laboratory conditions. Two species of SFG Rickettsia were detected in the midgut and salivary glands of males and females in both field-collected and first laboratory-generation adults of D. nuttalli. Our study provides new insights into pathogen detection in ticks in the Qinghai-Tibet Plateau, and the relationships among hosts, ticks, and pathogens.

Background and purpose: Several clinical trials have indicated that the use of canagliflozin increases the risk of lower extremity amputation. Although the US Food and Drug Administration (FDA) has withdrawn its black box warning about amputation risk for canagliflozin, the risk still exists. We sought to estimate the association between hypoglycemic medications, especially sodium-glucose co-transporter-2 inhibitors (SGLT2is), and adverse events (AEs) before the irreversible outcome of amputation as a promising early warning, based on the FDA Adverse Event Reporting System (FAERS) data.Methods: Publicly available FAERS data were analyzed using a reporting odds ratio (ROR) method and validated by a Bayesian confidence propagation neural network (BCPNN) method. The developing trend of the ROR was investigated by a series of calculations based on the accumulation of data in the FAERS database quarter by quarter.Results: Ketoacidosis, infection, peripheral ischemia, renal impairment, and inflammation including osteomyelitis might be more likely to occur among users of SGLT2is, especially canagliflozin. Osteomyelitis and cellulitis are AEs unique to canagliflozin. Among 2,888 osteomyelitis-related reports referring to hypoglycemic medications, 2,333 cases were associated with SGLT2is, with canagliflozin accounting for 2,283 of these cases and generating an ROR value of 360.89 and a lower limit of information component (IC025) of 7.79. No BCPNN-positive signal could be generated for drugs other than insulin and canagliflozin. Reports suggesting that insulin could generate BCPNN-positive signals span from 2004 to 2021, whereas reports with BCPNN-positive signals emerged only since the second quarter (Q2) of 2017, 4 years since the approval of SGLT2is in Q2 of 2013, for canagliflozin and drug groups containing canagliflozin.Conclusion: This data-mining investigation revealed a strong association between canagliflozin treatment and developing osteomyelitis that might be a crucial forewarning to lower extremity amputation. Further studies with updated data are needed to better characterize the risk of osteomyelitis associated with SGLT2is.

Purpose: 1) to explore the test-retest reliability of a new device for measuring ankle inversion proprioception during walking, i.e., the Ankle Inversion Discrimination Apparatus—Walking (AIDAW) in individuals with or without Chronic Ankle instability (CAI); 2) to assess its discriminant validity in differentiating individuals with or without CAI; 3) to investigate its convergent validity by examining its association with Cumberland Ankle Instability Tool (CAIT) and the Y Balance Test (YBT).Methods: For test-retest reliability, 15 participants with CAI and 15 non-CAI healthy controls were recruited. Participants completed the AIDAW test twice with a 7-day interval. The area under the receiver operating curve (AUC) was obtained as the AIDAW score. The intraclass correlation coefficient (ICC) and MDC90 were calculated. For the validity study, another 20 individuals with CAI and 20 non-CAI healthy controls were involved. The AIDAW scores were analyzed by an independent samples t-test, and the optimal cutoff value of AIDAW scores to best distinguish individuals with CAI was calculated by Youden’s index. Spearman or Pearson correlation analysis was used to analyze the correlation between AIDAW proprioceptive scores and the CAIT and final YBT scores.Results: For test-retest reliability, the ICC values for the CAI, non-CAI, and the whole group were 0.755, 0.757, and 0.761 respectively. The MDC90 of the CAI and non-CAI group was 0.04 and 0.05. Regarding discriminant validity, the AIDAW proprioceptive discrimination scores in the CAI group were significantly lower than those in the non-CAI group (p = 0.003); and the cutoff score for distinguishing CAI from the non-CAI participants was 0.759. For convergent validity, the AIDAW scores were significantly correlated with the functional balance YBT final scores (p = 0.001) and the CAIT scores (p = 0.009).Conclusion: The AIDAW is a reliable and valid device for evaluating ankle inversion proprioception during walking in individuals with and without CAI. AIDAW can be used as a clinical assessment tool to discriminate CAI from non-CAI individuals and to monitor effects of rehabilitation. The AIDAW proprioceptive discrimination scores were significantly and positively correlated with YBT and CAIT scores.

Toxoplasmosis is a zoonotic disease caused by the obligate intracellular protozoan parasite T. gondii which is widely prevalent in humans and animals worldwide. The diagnosis of toxoplasmosis and distinguishing acute or chronic T. gondii infections have utmost importance for humans and animals. The TgSAG1, TgGRA7, and TgBAG1 proteins were used in the present study to develop the serological rSAG1-ELISA, rGRA7-ELISA and rBAG1-ELISA methods for the testing of T. gondii specific IgG and IgM antibodies and differentiating acute or chronic toxoplasmosis in 3733 animals, including Tibetan sheep, yaks, pigs, cows, cattle, horses, chickens, camels and donkeys from the Qinghai-Tibetan Plateau. The ELISA tests showed that the overall positivity of IgG antibody was 21.1% (786/3733), 15.3% (570/3733) and 18.2% (680/3733) for rSAG1-, rGRA7- and rBAG1-ELISA, respectively, and the positivity of IgM antibody was 11.8% (439/3733), 13.0% (486/3733) and 11.8% (442/3733) for rSAG1-, rGRA7- and rBAG1-ELISA, respectively. A total of 241 animals (6.5%) positive for all rSAG1-, rGRA7- and rBAG1-IgG were found in this study, and the 141 animals (3.8%) tested were anti-T. gondii IgM positive in all three ELISAs. Moreover, the 338, 284 and 377 animals were IgG positive in rSAG1 + rGRA7-, rBAG1 + rGRA7- and rSAG1 + rBAG1- ELISAs respectively, and the 346, 178 and 166 animals in rSAG1 + rGRA7-, rBAG1 + rGRA7- and rSAG1 + rBAG1-ELISAs were IgM positive respectively. The results confirmed that the application of SAG1, GRA7, and BAG1 recombinant antigens could successfully be used in the detection of specific IgG and IgM antibodies for distinguishing between acute or chronic T. gondii infections. It is inferred that the forms in which current animal species in the plateau area were infected with T. gondii, and the period of infection or the clinical manifestations of the current infections may be different. The present study provides substantial clinical evidence for the differential diagnosis of toxoplasmosis, and the classification of acute and chronic T. gondii infections.

Background and purpose: Serious adverse events following immunization (AEFI) associated with the COVID-19 vaccines, including BNT162b2 (Pfizer-BioNTech), Ad26.COV2.S (Janssen), and mRNA-1273 (Moderna), have not yet been fully investigated. This study was designed to evaluate the serious AEFI associated with these three vaccines.Methods: A disproportionality study was performed to analyze data acquired from the Vaccine Adverse Event-Reporting System (VAERS) between 1 January 2010 and 30 April 2021. The reporting odds ratio (ROR) method was used to identify the association between the COVID-19 vaccines BNT162b2, Ad26.COV2.S, and mRNA-1273 and each adverse event reported. Moreover, the ratio of the ROR value to the 95% CI span was applied to improve the credibility of the ROR. The median values of time from vaccination to onset (TTO) for the three vaccines were analyzed.Results: Compared with BNT162b2 and mRNA-1273, Ad26.COV2.S vaccination was associated with a lower death frequency (p < 0.05). Ad26.COV2.S vaccination was associated with a lower birth defect and emergency room visit frequency than BNT162b2 (p < 0.05). There were 6,605, 830, and 2,292 vaccine recipients who suffered from COVID-19-related symptoms after vaccination with BNT162b2, Ad26.COV2.S, and mRNA-1273, respectively, including people who were infected by COVID-19, demonstrated a positive SARS-CoV-2 test, and were asymptomatic. Serious AEFI, including thromboembolism, hemorrhage, thrombocytopenia, cardiac arrhythmia, hypertension, and hepatotoxicity, were associated with all three vaccines. Cardiac failure and acute renal impairment events were associated with BNT162b2 and mRNA-1273, while seizure events were associated with BNT162b2 and Ad26.COV2.S. The median values of TTO associated with the three vaccinations were similar.Conclusion: These findings may be useful for health workers and the general public prior to inoculation, especially for patients with underlying diseases; however, the risk/benefit profile of these vaccines remains unchanged. The exact mechanism of SARS-CoV-2 vaccine-induced AEFI remains unknown, and further studies are required to explore these phenomena.

Though the karst regions in south and southwest China are plant diversity hotspots, our understanding of the phylogeography and evolutionary history of the plants there remains limited. The genus Heteroplexis (Asteraceae) is one of the typical representative plants isolated by karst habitat islands, and is also an endangered and endemic plant to China. In this study, species-level phylogeographic analysis of the genus Heteroplexis was conducted using restriction site-associated DNA sequencing (RADseq). The genetic structure showed a clear phylogeographic structure consistent with the current species boundaries in the H. microcephala, H. incana, H. vernonioides, H. sericophylla, and H. impressinervia. The significant global (R = 0.37, P < 0.01) and regional (R = 0.650.95, P < 0.05) isolation by distance (IBD) signals among species indicate strong geographic isolation in the karst mountains, which may result in chronically restricted gene flow and increased genetic drift and differentiation. Furthermore, the phylogeographic structure of Heteroplexis suggested a southward migration since the last glacial period. Demographic analysis revealed the karst mountains as a refuge for Heteroplexis species. Finally, both Treemix and ABBA-BABA statistic detected significant historical gene flow between species. Significant historical gene flow and long-term stability of effective population size (Ne) together explain the high genome-wide genetic diversity among species (π = 0.05370.0838). However, the recent collapse of Ne, widespread inbreeding within populations, and restricted contemporary gene flow suggest that Heteroplexis species are probably facing a high risk of genetic diversity loss. Our results help to understand the evolutionary history of karst plants and guide conservation.

The dysfunction of islet β-cells is one of the causes of diabetes, and lncRNA Gm10451 is also a participant in the occurrence and the development of various diseases. This study was carried out to reveal the correlation within β-cells and Gm10451. Our study was started with the cellular cultivation of MIN6 cells in vitro, where this islet β-cell line was randomly divided into the groups of control, hyperglycemia, Gm10451 siRNA tansfection, and Gm10451 tansfection. Of all these treatments, cells in the groups of Gm10451 siRNA tansfection and Gm10451 tansfection were given with lentiviral transfection under hyperglycemia condition. Further explorations were established using PCR assay and MTT method to evaluate Gm10451 expression and estimate cellular proliferation. It ended up with the enzyme-linked immunosorbent assay (ELISA) to assess Caspase 3 activity, superoxide dismutase (SOD) activity, and reactive oxygen species (ROS) content and the secretion of IL-10 and IL-1. It was found that Gm10451 expression in MIN6 cells under hyperglycemia cultivation was notably higher than the control group; likewise, a transfection with the lentivirus of Gm10451 also resulted in the upregulation of Gm10451 expression, succeeded with inhibiting cellular proliferation, enhancing Caspase 3 activity, and decreasing SOD activity. In the lentivirus transfection groups, transfection of Gm10451 elevated the ROS content and promoted IL-1 expression, and it also decreased both IL-10 expression and insulin secretion, leading to a consequence of statistically significant difference in contrast to the high-glucose group; on the contrary, transfection of Gm10451 siRNA in a high-glucose environment downregulated the expression of Gm10451 and inversed those change before, whose results were statistically significant when compared with the high-glucose group. Hyperglycemia promotes the expression of Gm10451. Targeting inhibition toward Gm10451 alleviates cellular apoptosis and the oxidative stress of islet cells, promoting proliferation and insulin secretion of islet cells.

Neosporosis is a worldwide infectious disease caused by intracellular parasite Neospora caninum that is a major pathogen of abortion in cattle and neurological disorders in other hosts. However, limited data are available on animals exposed to N. caninum in the Qinghai-Tibetan Plateau Area (QTPA), and little is known about whether animals in the plateau area play an important role in the epidemiology of N. caninum. Therefore, indirect ELISAs based on a combination of NcSAG1 and NcGRA7 antigens were developed to examine both N. caninum-specific IgG and IgM antibodies in Tibetan sheep, yak, cow, pig, cattle, horse, chicken, camel, and donkey from the QTPA in this study. The results showed that all current species present- IgG and IgM-positive animals, and that the overall seroprevalence of N. caninum were 18.6 (703/3,782) and 48.1% (1,820/3,782) for the IgG and IgM antibodies, respectively. Further analysis found significant differences from different altitudes in IgG in Tibetan sheep and IgM in the yak. Hence, the present serological results indicate that the tested animal populations in the QTPA are suffering from N. caninum infections or have become carriers of N. caninum antibodies. To the best of our knowledge, this is the first report on current N. caninum-infected animals in the QTPA, the first epidemiology of neosporosis in cow and camel in China, and the first record of N. caninum IgM antibodies in all the surveyed animals in China. This study provides the latest valuable data on the epidemiology of neosporosis in China and in plateau areas of the world.

In patients with congenital blindness (CB), the lack of any visual experience may affect brain development resulting in functional, structural, or even psychological changes. Few studies to date have addressed or focused on the synchronicity of regional brain activity in patients with CB. Our study aimed to investigate regional brain activity in patients with CB in a resting state and try to explain the possible causes and effects of any anomalies. Twenty-three CB patients and 23 healthy control (HC) volunteers agreed to undergo resting state functional magnetic resonance imaging (fMRI) scans. After the fMRI data were preprocessed, regional homogeneity (ReHo) analysis was conducted to assess the differences in brain activity synchronicity between the two groups. Receiver operating characteristic (ROC) curve analysis was used to explore whether the brain areas with statistically significant ReHo differences have diagnostic and identification values for CB. All CB patients were also required to complete the Hospital Anxiety and Depression Scale (HADS) to evaluate their anxiety and depression levels. The results showed that in CB patients mean ReHo values were significantly lower than in HCs in the right orbital part of the middle frontal gyrus (MFGorb), bilateral middle occipital gyrus (MOG), and the right dorsolateral superior frontal gyrus (SFGdl), but significantly higher in the left paracentral lobule (PCL), right insula and bilateral thalamus. The ReHo value of MFGorb showed a negative linear correlation with both the anxiety score and the depression score of the HADS. ROC curve analysis revealed that the mean ReHo values which differed significantly between the groups have excellent diagnostic accuracy for CB (especially in the left PCL and right SFGdl regions). Patients with CB show abnormalities of ReHo values in several specific brain regions, suggesting potential regional structural changes, functional reorganization, or even psychological effects in these patients. FMRI ReHo analysis may find use as an objective method to confirm CB for medical or legal purposes.

Introduction Pharmacotherapy is one of the main treatments for patients with young-onset Parkinson’s disease (YOPD). Although numerous studies on the treatment of YOPD have been published, the real-world prescription patterns of these populations remain unclear in China.Methods A national comprehensive evaluation was performed to reveal the pharmacological treatment patterns in Chinese patients with Parkinson’s disease from 1 January 2014 to 31 December 2019, with patients aged 21–50 years classified as having YOPD for the subgroup analysis. Information on patients and drugs was extracted to analyse the demographic characteristics, prescription patterns, and levodopa equivalent daily dose (LED) during disease progression.Results A total of 1,134 patients with YOPD were included, and the majority were aged 41–50 years. Prescription of L-DOPA/benserazide and pramipexole accounted for more than 30 and 20%, respectively, in each year from 2014 to 2019. There was no difference in prescription patterns in terms of age, sex and geographical areas. Half of the patients with YOPD were on monotherapy, but the proportion decreased from 2016. Correspondingly, the proportion of patients receiving polytherapy increased, especially those who were prescribed more than two anti-Parkinson’s disease drugs. During the disease course, LED showed high variability, which increased over time.Conclusion L-DOPA/benserazide and pramipexole were the most frequently prescribed anti-PD drugs for patients with YOPD in China. There was a slight trend in the transition from monotherapy to polytherapy. LED increased with disease duration. Thus, we provided an overview of the prescription patterns for patients with YOPD in China.

The Qinghai–Tibet Plateau Area (QTPA) has a complex natural ecosystem, causing a greatly increased risk of spreading various tick-borne diseases including rickettsial infections, which are regarded as one of the oldest known vector-borne zoonoses. However, the information of one of its pathogen, spotted fever group Rickettsia (SFG Rickettsia), is limited in tick vectors and animals in this area. Therefore, this study focused on the investigation of SFG Rickettsia in tick vectors, yaks (Bos grunniens), and Tibetan sheep (Ovis aries) in the QTPA. A total of 1,000 samples were collected from nine sampling sites, including 425 of yaks, 309 of Tibetan sheep, 266 of ticks. By morphological examination, PCR, and sequencing, we confirmed the species of all collected ticks. All tick samples, all yak and Tibetan sheep blood samples were detected based on SFG Rickettsia ompA and sca4 gene. The results showed that all tick samples were identified to be Haemaphysalis qinghaiensis, and the positive rates of SFG Rickettsia were 5.9% (25/425), 0.3% (1/309), and 54.1% (144/266) in yaks, Tibetan sheep, and ticks, respectively. All positive samples were sequenced, and BLASTn analysis of the ompA gene sequences of SFG Rickettsia showed that all positive samples from animals and ticks had 99.04–100% identity with yak and horse isolates from Qinghai Province, China. BLASTn analysis of the sca4 gene sequences of SFG Rickettsia showed that all positive samples had 97.60–98.72% identity with tick isolates from Ukraine. In addition, the phylogenetic analysis showed that all the SFG Rickettsia ompA and sca4 sequences obtained from this study belong to the same clade as Rickettsia raoultii isolated from livestock and ticks from China and other countries. Molecularly, this study detected and characterized SFG Rickettsia both in the tick vectors and animals, suggesting that the relationship between SFG Rickettsia, tick species and animal hosts should be explored to understand their interrelationships, which provide a theoretical basis for preventing control of this pathogen.

The effects of condiment (salt) and processing technic (homogenization) on digestion and interfacial properties of micro/nano-sized colloidal particles (MNCPs) in bigeye tuna head soup (BTHS) using simulated gastrointestinal digestion model in vitro were investigated. For MNCPs in BTHS, the triglycerides were wrapped with proteins in the form of a ring. After salting, the average particle size of the MNCPs in salted BTHS (SBTHS) decreased compared with BTHS. However, the partial demulsification phenomenon existed, and part of the protein was encapsulated in some MNCPs. After further homogenization, the average particle size of the MNCPs in homogenous SBTHS (HSBTHS) was further decreased based on SBTHS and the MNCP was rearranged, which changed the original membrane structure. After gastrointestinal digestion, adding salt decreased the release of total fatty acids compared with unsalted. But homogenization processing increased the release of total fatty acids in HSBTHS and there was no significant difference (p ≥ 0.05) between HSBTHS and BTHS. Thus, the decrease in the release of some fatty acids due to adding salt was compensated by homogenization. Therefore, the changes in composition and microstructure of MNCPs induced by salt and homogenization might contribute to the digestion difference of MNCPs.

The incidence rate and mortality of hypertension increase every year. Hypothalamic paraventricular nucleus (PVN) plays a critical role on the pathophysiology of hypertension. It has been demonstrated that the imbalance of neurotransmitters including norepinephrine (NE), glutamate (Glu) and γ-aminobutyric acid (GABA) are closely related to sympathetic overactivity and pathogenesis of hypertension. N-methyl-D-aspartate receptor (NMDAR), consisting of GluN1 and GluN2 subunits, is considered to be a glutamate-gated ion channel, which binds to Glu, and activates neuronal activity. Studies have found that the synthesis of respiratory chain enzyme complex was affected and mitochondrial function was impaired in spontaneously hypertensive rats (SHR), further indicating that mitochondria is associated with hypertension. Nuclear respiratory factor 1 (Nrf1) is a transcription factor that modulates mitochondrial respiratory chain and is related to GluN1, GluN2A, and GluN2B promoters. However, the brain mechanisms underlying PVN Nrf1 modulating sympathoexcitation and blood pressure during the development of hypertension remains unclear. In this study, an adeno-associated virus (AAV) vector carrying the shRNA targeting rat Nrf1 gene (shNrf1) was injected into bilateral PVN of male rats underwent two kidneys and one clip to explore the role of Nrf1 in mediating the development of hypertension and sympathoexcitation. Administration of shNrf1 knocked down the expression of Nrf1 and reduced the expression of excitatory neurotransmitters, increased the expression of inhibitory neurotransmitters, and reduced the production of reactive oxygen species (ROS), and attenuated sympathoexcitation and hypertension. The results indicate that knocking down Nrf1 suppresses sympathoexcitation in hypertension by reducing PVN transcription of NMDAR subunits (GluN1, GluN2A, and GluN2B), rebalancing PVN excitatory and inhibitory neurotransmitters, inhibiting PVN neuronal activity and oxidative stress, and attenuating sympathetic activity.

BackgroundAll agents engaging sphongosine-1-phospate receptors (S1PRs) will have some cardiovascular effect. This study aimed to elucidate the risk of cardiovascular adverse events (AEs) in patients with multiple sclerosis (MS) treated with S1PR modulators (S1PRMs).MethodsWe systematically searched the PubMed, EMBASE, and Cochrane Library databases for randomised controlled trials (RCTs) published through January 5, 2021. Relative risks (RRs) and 95% confidence intervals (CIs) were calculated using the random-effects model. Sensitivity analyses and meta-regression were performed.ResultsSeventeen RCTs (12 for fingolimod; 3 for ozanimod; 2 for siponimod) involving 13,295 patients were included. Compared with the control treatment, S1PRMs significantly increased the risk of cardiovascular AEs (RR, 2.21; 95% CI, 1.58–3.10; I2, 75.6%). Notably, the high-risk cardiovascular AEs associated with S1PRMs were primarily bradyarrhythmia (RR, 2.92; 95% CI, 1.91–4.46; I2, 30.8%) and hypertension (RR, 2.00; 95% CI, 1.49–2.67; I2, 56.5%). Subgroup analysis results were consistent with the primary outcomes except that ozanimod was associated with a higher risk of hypertension only (RR, 1.76; 95% CI, 1.10–2.82; I2, 0.0%), while siponimod was associated with a higher risk of bradyarrhythmia only (RR, 2.75; 95% CI, 1.75–4.31; I2, 0.0%). No significant inter-subgroup differences were observed (Pinteraction > 0.05).ConclusionsS1PRM use increased the risk of cardiovascular AEs by 1.21 times in patients with MS, and increased risks for bradyarrhythmia and hypertension were at 2.92- and 2.00-fold, respectively. These findings can help clinicians assess the risk of cardiovascular AEs in patients treated with S1PRMs.Systematic Review RegistrationThe PROSPERO ID is CRD42020183215.

Aims: Long-term salt diet induces the oxidative stress in the paraventricular nucleus (PVN) and increases the blood pressure. Extracellular superoxide dismutase (Ec-SOD) is a unique antioxidant enzyme that exists in extracellular space and plays an essential role in scavenging excessive reactive oxygen species (ROS). However, the underlying mechanism of Ec-SOD in the PVN remains unclear.Methods: Sprague–Dawley rats (150–200 g) were fed either a high salt diet (8% NaCl, HS) or normal salt diet (0.9% NaCl, NS) for 6 weeks. Each group of rats was administered with bilateral PVN microinjection of AAV-Ec-SOD (Ec-SOD overexpression) or AAV-Ctrl for the next 6 weeks.Results: High salt intake not only increased mean arterial blood pressure (MAP) and the plasma noradrenaline (NE) but also elevated the NAD(P)H oxidase activity, the NAD(P)H oxidase components (NOX2 and NOX4) expression, and ROS production in the PVN. Meanwhile, the NOD-like receptor protein 3 (NLRP3)–dependent inflammatory proteins (ASC, pro-cas-1, IL-β, CXCR, CCL2) expression and the tyrosine hydroxylase (TH) expression in the PVN with high salt diet were higher, but the GSH level, Ec-SOD activity, GAD67 expression, and GABA level were lower than the NS group. Bilateral PVN microinjection of AAV-Ec-SOD decreased MAP and the plasma NE, reduced NAD(P)H oxidase activity, the NOX2 and NOX4 expression, and ROS production, attenuated NLRP3-dependent inflammatory expression and TH, but increased GSH level, Ec-SOD activity, GAD67 expression, and GABA level in the PVN compared with the high salt group.Conclusion: Excessive salt intake not only activates oxidative stress but also induces the NLRP3-depensent inflammation and breaks the balance between inhibitory and excitability neurotransmitters in the PVN. Ec-SOD, as an essential anti-oxidative enzyme, eliminates the ROS in the PVN and decreases the blood pressure, probably through inhibiting the NLRP3-dependent inflammation and improving the excitatory neurotransmitter release in the PVN in the salt-induced hypertension.

Cycads represent one of the most ancestral living seed plants as well as one of the most threatened plant groups in the world. South China is a major center and potential origin of Cycas, the most rapidly diversified lineage of cycads. However, genomic-wide diversity of Cycas remains poorly understood due to the challenge of generating genomic markers associated with their inherent large genomes. Here, we perform a comprehensive conservation genomic study based on restriction-site associated DNA sequencing (RADseq) data in six representative species of Cycas in South China. Consistently low genetic diversity and strong genetic differentiation were detected across species. Both phylogenetic inference and genetic structure analysis via several methods revealed generally congruent groups among the six Cycas species. The analysis with ADMIXTURE showed low mixing of genetic composition among species, while individuals of C. dolichophylla exhibited substantial genetic admixture with C. bifida, C. changjiangensis, and C. balansae. Furthermore, the results from Treemix, f4-statistic, and ABBA-BABA test were generally consistent and revealed the complex patterns of interspecific gene flow. Relatively strong signals of hybridization were detected between C. dolichophylla and C. szechuanensis, and the ancestor of C. taiwaniana and C. changjiangensis. Distinct patterns of demographic history were inferred for these species by Stairway Plot, and our results suggested that both climate fluctuation and frequent geological activities during the late Pleistocene exerted deep impacts on the population dynamics of these species in South China. Finally, we explore the practical implications of our findings for the development of conservation strategies in Cycas. The present study demonstrates the efficiency of RADseq for conservation genomic studies on non-model species with large and complex genomes. Given the great significance of cycads as a radical transition in the evolution of plant biodiversity, our study provides important insights into the mechanisms of diversification in such recently radiated living fossil taxa.

Excessive sympathetic activation plays crucial roles in the pathogenesis of hypertension. Chemical stimulation of renal afferents increases the sympathetic activity and blood pressure in normal rats. This study investigated the excitatory renal reflex (ERR) in the development of hypertension in the spontaneously hypertensive rat (SHR). Experiments were performed in the Wistar-Kyoto rat (WKY) and SHR aged at 4, 12, and 24 weeks under anesthesia. Renal infusion of capsaicin was used to stimulate renal afferents, and thus, to induce ERR. The ERR was evaluated by the changes in the contralateral renal sympathetic nerve activity and mean arterial pressure. At the age of 4 weeks, the early stage with a slight or moderate hypertension, the ERR was more enhanced in SHR compared with WKY. The pressor response was greater than the sympathetic activation response in the SHR. At the age of 12 weeks, the development stage with severe hypertension, there was no significant difference in the ERR between the WKY and SHR. At the age of 24 weeks, the later stage of hypertension with long-term several hypertensions, the ERR was more attenuated in the SHR compared with the WKY. On the other hand, the pressor response to sympathetic activation due to the ERR was smaller at the age of 12 and 24 weeks than those at the age of 4 weeks. These results indicate that ERR is enhanced in the early stage of hypertension, and attenuated in the later stage of hypertension in the SHR. Abnormal ERR is involved in the sympathetic activation and the development of hypertension.

Oxidative stress plays an important role in the pathogenesis of hypertension. Oligomeric proantho cyanidins (OPC) is the main polyphenol presents in grape seed and is known for its potent antioxidant and anti-inflammatory properties. In the present study, we hypothesize that OPC can attenuate oxidative stress in the paraventricular nucleus of hypothalamus (PVN), ameliorate neurotransmitter imbalance, decrease the blood pressure and sympathetic activity in renovascular hypertensive rats. After induction of renovascular hypertension by the two-kidney one-clip (2K-1C) method, male Sprague-Dawley rats received chronic bilateral PVN infusion of OPC (20 μg/h) or vehicle via osmotic minipump for 4 weeks. We found that hypertension induced by 2K-1C was associated with the production of reactive oxygen species (ROS) in the PVN. Infusion of OPC in the PVN significantly reduced the systolic blood pressure and norepinephrine in plasma of 2K-1C rats. In addition, PVN infusion of OPC decreased the level of ROS and the expression of stress-related nicotinamide adenine dinucleotide phosphate (NADPH) oxidases subunit NOX4, increased the levels of nuclear factor E2-related factor 2 (Nrf2) and antioxidant enzyme, balanced the content of cytokines, increased expression of glutamic acid decarboxylase and decreased the expression of tyrosine hydroxylase in the PVN of 2K-1C rats. Our findings provided strong evidence that PVN infusion of OPC inhibited the progression of renovascular hypertension through its potent anti-oxidative and anti-inflammatory function in the PVN.

Background and Aims: There is a controversy regarding whether fingolimod is associated with an increased risk of infection in patients with multiple sclerosis (MS). We performed a systematic review and meta-analysis of data from randomized controlled trials (RCTs) to determine the risk of infection in these patients.Methods: We systematically searched PubMed, EMBASE, the Cochrane Library, and clinicaltrials.gov from inception to April 8, 2020, to identify RCTs that reported the occurrence of infection in patients with MS treated with fingolimod. Relative risks (RRs) and 95% confidence intervals (95% CIs) were calculated using the random-effects model.Results: Twelve RCTs including 8,448 patients were eligible. Compared with the control (placebo and other active treatments), fingolimod significantly increased the risk of infection (RR, 1.16; 95% CI, 1.07–1.27; I2, 81%), regardless of whether the infection was a general infection (RR, 1.14; 95% CI, 1.05–1.25; I2, 78%), or a serious infection (RR, 1.49; 95% CI, 1.06–2.10; I2, 0%). Analyses of subgroups found that fingolimod significantly increased the risk of lower respiratory infection (RR, 1.48; 95% CI, 1.19–1.85; I2, 0%) and herpes virus infection (RR, 1.34; 95% CI, 1.01–1.78; I2, 9%). There appears to be no dose-dependent increase in the risk of infection associated with fingolimod (0.5 mg: RR, 1.15; 95% CI, 1.07–1.25; I2, 91%; 1.25 mg: RR, 1.11; 95% CI, 0.97–1.28; I2, 81%; Pinteraction = 0.66).Conclusions: Compared with a placebo and other active treatments, fingolimod was associated with a 16% increase in the risk of infection, especially lower respiratory infection and herpes virus infection. The risk of infection associated with fingolimod might not be dose related.

Multipotent mesenchymal stem/stromal cells (MSCs) exhibit great potential for cell-based therapy. Proper epigenomic signatures in MSCs are important for the maintenance and the subsequent differentiation potential. The DNA methyltransferase 3-like (DNMT3L) that was mainly expressed in the embryonic stem (ES) cells and the developing germ cells plays an important role in shaping the epigenetic landscape. Here, we report the reduced colony forming ability and impaired in vitro osteogenesis in Dnmt3l-knockout-mice-derived MSCs (Dnmt3l KO MSCs). By comparing the transcriptome between undifferentiated Dnmt3l KO MSCs and the MSCs from the wild-type littermates, some of the differentially regulated genes (DEGs) were found to be associated with bone-morphology-related phenotypes. On the third day of osteogenic induction, differentiating Dnmt3l KO MSCs were enriched for genes associated with nucleosome structure, peptide binding and extracellular matrix modulation. Differentially expressed transposable elements in many subfamilies reflected the change of corresponding regional epigenomic signatures. Interestingly, DNMT3L protein is not expressed in cultured MSCs. Therefore, the observed defects in Dnmt3l KO MSCs are unlikely a direct effect from missing DNMT3L in this cell type; instead, we hypothesized them as an outcome of the pre-deposited epigenetic signatures from the DNMT3L-expressing progenitors. We observed that 24 out of the 107 upregulated DEGs in Dnmt3l KO MSCs were hypermethylated in their gene bodies of DNMT3L knock-down ES cells. Among these 24 genes, some were associated with skeletal development or homeostasis. However, we did not observe reduced bone development, or reduced bone density through aging in vivo. The stronger phenotype in vitro suggested the involvement of potential spreading and amplification of the pre-deposited epigenetic defects over passages, and the contribution of oxidative stress during in vitro culture. We demonstrated that transient deficiency of epigenetic co-factor in ES cells or progenitor cells caused compromised property in differentiating cells much later. In order to facilitate safer practice in cell-based therapy, we suggest more in-depth examination shall be implemented for cells before transplantation, even on the epigenetic level, to avoid long-term risk afterward.

Exercise training (ExT) is beneficial for cardiovascular health, yet the central mechanism by which aerobic ExT attenuates the hypertensive responses remains unclear. Activation of pro-inflammatory cytokines (PICs) in the hypothalamic paraventricular nucleus (PVN) is important for the sympathoexcitation and hypertensive response. We thus hypothesized that aerobic ExT can decrease the blood pressure of hypertensive rats by reducing the levels of PICs through TLR4/MyD88/NF-κB signaling within the PVN. To examine this hypothesis, two-kidney-one-clip (2K1C) renovascular hypertensive rats were assigned to two groups: sedentary or exercise training and examined for 8 weeks. At the same time, bilateral PVN infusion of vehicle or TAK242, a TLR4 inhibitor, was performed on both groups. As a result, the systolic blood pressure (SBP), renal sympathetic nerve activity (RSNA) and plasma levels of norepinephrine (NE), epinephrine (EPI) were found significantly increased in 2K1C hypertensive rats. These rats also had higher levels of Fra-like activity, NF-κB p65 activity, TLR4, MyD88, IL-1β and TNF-α in the PVN than SHAM rats. Eight weeks of ExT attenuated the RSNA and SBP, repressed the NF-κB p65 activity, and reduced the increase of plasma levels of NE, EPI, and the expression of Fra-like, TLR4, MyD88, IL-1β and TNF-α in the PVN of 2K1C rats. These findings are highly similar to the results in 2K1C rats with bilateral PVN infusions of TLR4 inhibitor (TAK242). This suggests that 8 weeks of aerobic ExT may decrease blood pressure in hypertensive rats by reducing the PICs activation through TLR4/MyD88/NF-κB signaling within the PVN, and thus delays the progression of 2K1C renovascular hypertension.

Hydrological conditions determine the distribution of plant species in wetlands, where conditions such as water depth and hydrological fluctuations are expected to affect the interspecific interactions among emergent wetland species. To test such effects, we conducted a greenhouse experiment with three treatment categories, interspecific interaction (mixed culture or monoculture), water depth (10 or 30 cm depth), and hydrological fluctuation (static or fluctuating water level), and two common emergent wetland plant species, Scirpus planiculumis Fr. (Cyperaceae) and Phragmites australis var. baiyangdiansis (Gramineae). An increase in the water depth significantly restrained the growth of both S. planiculumis and P. australis, while hydrological fluctuations did not obviously alter the growth of either species. In addition, both water depth and hydrological fluctuations significantly affected the interspecific interaction between these two wetland species. P. australis benefited from interspecific interaction under increasing water depth and hydrological fluctuations, and the RII values were clearly positive for plants grown at a water depth that fluctuated around 30 cm. The results may have some implications for understanding how S. planiculumis and P. australis, as well as wetland communities, respond to the natural variation or human modification of hydrological conditions.

Introduction Pharmacotherapy is one of the main treatments for patients with young-onset Parkinson's disease (YOPD). Although numerous studies on the treatment of YOPD have been published, the real-world prescription patterns of these populations remain unclear in China. Methods A national comprehensive evaluation was performed to reveal the pharmacological treatment patterns in Chinese patients with Parkinson's disease from 1 January 2014 to 31 December 2019, with patients aged 21–50 years classified as having YOPD for the subgroup analysis. Information on patients and drugs was extracted to analyse the demographic characteristics, prescription patterns, and levodopa equivalent daily dose (LED) during disease progression. Results A total of 1,134 patients with YOPD were included, and the majority were aged 41–50 years. Prescription of L-DOPA/benserazide and pramipexole accounted for more than 30 and 20%, respectively, in each year from 2014 to 2019. There was no difference in prescription patterns in terms of age, sex and geographical areas. Half of the patients with YOPD were on monotherapy, but the proportion decreased from 2016. Correspondingly, the proportion of patients receiving polytherapy increased, especially those who were prescribed more than two anti-Parkinson's disease drugs. During the disease course, LED showed high variability, which increased over time. Conclusion L-DOPA/benserazide and pramipexole were the most frequently prescribed anti-PD drugs for patients with YOPD in China. There was a slight trend in the transition from monotherapy to polytherapy. LED increased with disease duration. Thus, we provided an overview of the prescription patterns for patients with YOPD in China. [ABSTRACT FROM AUTHOR]

Quercus is considered economically and ecologically one of the most important genera in the Northern Hemisphere. Oaks are taxonomically perplexing because of shared interspecific morphological traits and intraspecific morphological variation, which are mainly attributed to hybridization. Universal plastid markers cannot provide a sufficient number of variable sites to explore the phylogeny of this genus, and chloroplast genomescale data have proven to be useful in resolving intractable phylogenetic relationships. In this study, the complete chloroplast genomes of four Quercus species were sequenced, and one published chloroplast genome of Quercus baronii was retrieved for comparative analyses. The five chloroplast genomes ranged from 161,072 bp (Q. baronii) to 161,237 bp (Q. dolicholepis) in length, and their gene organization and order, and GC content, were similar to those of other Fagaceae species. We analyzed nucleotide substitutions, indels, and repeats in the chloroplast genomes, and found 19 relatively highly variable regions that will potentially provide plastid markers for further taxonomic and phylogenetic studies within Quercus. We observed that four genes (ndhA, ndhK, petA, and ycf1) were subject to positive selection. The phylogenetic relationships of the Quercus species inferred from the chloroplast genomes obtained moderate-to-high support, indicating that chloroplast genome data may be useful in resolving relationships in this genus. [ABSTRACT FROM AUTHOR]

Flow cytometry (FCM) is a commonly used method for estimating genome size in many organisms. The use of FCM in plants is influenced by endogenous fluorescence inhibitors and may cause an inaccurate estimation of genome size; thus, falsifying the relationship between genome size and phenotypic traits/ecological performance. Quantitative optimization of FCM methodology minimizes such errors, yet there are few studies detailing this methodology. We selected the genus Primulina, one of the most representative and diverse genera of the Old World Gesneriaceae, to evaluate the methodology effect on determining genome size. Our results showed that buffer choice significantly affected genome size estimation in six out of the eight species examined and altered the 2C-value (DNA content) by as much as 21.4%. The staining duration and propidium iodide (PI) concentration slightly affected the 2C-value. Our experiments showed better histogram quality when the samples were stained for 40 min at a PI concentration of 100μg ml-1. The quality of the estimates was not improved by 1-day incubation in the dark at 4°C or by centrifugation. Thus, our study determined an optimum protocol for genome size measurement in Primulina: LB01 buffer supplemented with 100μ ml-1 PI and stained for 40 min. This protocol also demonstrated a high universality in other Gesneriaceae genera. We report the genome size of nine Gesneriaceae species for the first time. The results showed substantial genome size variation both within and among the species, with the 2C-value ranging between 1.62 and 2.71 pg. Our study highlights the necessity of optimizing the FCM methodology prior to obtaining reliable genome size estimates in a given taxon. [ABSTRACT FROM AUTHOR]