Prevalence and risk factors of stroke in China: a national serial cross-sectional study from 2003 to 2018.

Stroke imposes a substantial burden worldwide. With the rapid economic and lifestyle transition in China, trends of the prevalence of stroke across different geographic regions in China remain largely unknown. Capitalizing on the data in the National Health Services Surveys (NHSS), we assessed the prevalence and risk factors of stroke in China from 2003 to 2018. In this study, data from 2003, 2008, 2013, and 2018 NHSS were collected. Stroke cases were based on participants' self-report of a previous diagnosis by clinicians. We estimated the trends of stroke prevalence for the overall population and subgroups by age, sex, and socioeconomic factors, then compared across different geographic regions. We applied multivariable logistic regression to assess associations between stroke and risk factors. The number of participants aged 15 years or older were 154,077, 146,231, 230,067, and 212,318 in 2003, 2008, 2013, and 2018, respectively, among whom, 1435, 1996, 3781, and 6069 were stroke patients. The age and sex standardized prevalence per 100,000 individuals was 879 in 2003, 1100 in 2008, 1098 in 2013, and 1613 in 2018. Prevalence per 100,000 individuals in rural areas increased from 669 in 2003 to 1898 in 2018, while urban areas had a stable trend from 1261 in 2003 to 1365 in 2018. Across geographic regions, the central region consistently had the highest prevalence, but the western region has an alarmingly increasing trend from 623/100,000 in 2003 to 1898/100,000 in 2018 (P trend<0.001), surpassing the eastern region in 2013. Advanced age, male sex, rural area, central region, hypertension, diabetes, depression, low education and income level, retirement or unemployment, excessive physical activity, and unimproved sanitation facilities were significantly associated with stroke. In conclusion, the increasing prevalence of stroke in China was primarily driven by economically underdeveloped regions. It is important to develop targeted prevention programs in underdeveloped regions. Besides traditional risk factors, more attention should be paid to nontraditional risk factors to improve the prevention of stroke.

Comparative transcriptome analysis of Liriomyza trifolii (Burgess) and Liriomyza sativae (Blanchard) (Diptera: Agromyzidae) in response to rapid cold hardening.

The ability of insets to react efficiently to fluctuation in temperature is crucial for them to survive in variable surroundings. Rapid cold hardening (RCH) is a process that increase cold tolerance in most insect species. The molecular mechanisms of RCH remain largely unknown, and whether it is associated with transcriptional changes is unclear. In this study, we compared the transcriptomes of Liriomyza trifolii and L. sativae exposed to RCH to investigate the transcript abundance due to RCH in both species. RNA-seq revealed 93,166 assembled unigenes, and 34,303 of these were annotated in the L. trifolii and L. sativae transcriptome libraries. After a 4-h treatment at 1°C (RCH) compared with control, 268 and 606 unigenes were differentially expressed in L. trifolii and L. sativae, respectively. When comparing pupae exposed to 2h cold shock directly with pupae went through 4h acclimation prior to 2h cold shock, 60 and 399 unigenes were differentially expressed in L trifolii and L sativae, respectively. Genes that were commonly expressed in both L. trifolii and L. sativae, included cytochrome P450, cuticular protein, glucose dehydrogenase, solute carrier family 22 and cationic amino acid transporter. Additionally, several pathways including galactose metabolism and peroxisome were significantly enriched during RCH. Our results show that the transcriptional response is correlated with RCH in the pupal stage of the two Liriomyza species, but more transcriptional changes were identified in L sativae than in L. trifolii.

The Novel Application of Three-Dimensional Printing Assisted Patient-Specific Instrument Osteotomy Guide in the Precise Osteotomy of Adult Talipes Equinovarus.

OBJECTIVE: This current research is aimed at assessing clinical efficacy and prognosis of three-dimensional (3D) printing assisted patient-specific instrument (PSI) osteotomy guide in precise osteotomy of adult talipes equinovarus (ATE). METHODS: We included a total of 27 patients of ATE malformation (including 12 males and 15 females) from June 2014 to June 2018 in the current research. The patients were divided into the routine group (n = 12) and 3D printing group (n = 15) based on different operative methods. The parameters, including the operative time, intraoperative blood loss, complications, time to obtain bony fusion, functional outcomes based on American Orthopedic Foot and Ankle Society (AOFAS), and International Congenital Clubfoot Study group (ICFSG) scoring systems between the two groups were observed and recorded regularly. RESULTS: The 3D printing group exhibits superiorities in shorter operative time, less intraoperative blood loss, higher rate of excellent, and good outcomes presented by ICFSG score at last follow-up (P < 0.001, P < 0.001, P = 0.019) than the routine group. However, there was no significant difference exhibited in the AOFAS score at the last follow-up and total rate of complications between the two groups (P = 0.136, P = 0.291). CONCLUSION: Operation assisted by 3D printing PSI osteotomy guide for correcting the ATE malformation is novel and feasible, which might be an effective method to polish up the precise osteotomy of ATE malformation and enhance the clinical efficacy.

Differential Response of Leafminer Flies Liriomyza trifolii (Burgess) and Liriomyza sativae (Blanchard) to Rapid Cold Hardening.

Rapid cold hardening (RCH) is a rapid and critical adaption of insects to sudden temperature changes but is often overlooked or underestimated as a component of survival. Thus, interspecific comparisons of RCH are needed to predict how phenotypes will adapt to temperature variability. RCH not only enhances cold survival but also protects against non-lethal cold injury by preserving essential functions such as locomotion, reproduction, and energy balance. This study investigated the difference in basal cold tolerance and RCH capacity of L. trifolii and L. sativae. In both species, the cold tolerance of pupae was significantly enhanced after short-term exposure to moderately cold temperatures. The effect of RCH last for 4 h in L. sativae but only 2 h in L. trifolii. Interestingly, L. trifolii adults had a RCH response but L. sativae adults failed to acclimate. Short-term acclimation also lowered the supercooling point significantly in the pupae of both species. Based on these results, we propose a hypothesis that these differences will eventually affect their competition in the context of climate change. This study also provides the basis for future metabolomic and transcriptomic studies that may ultimately uncover the underlying mechanisms of RCH and interspecific competition between L. trifolii and L. sativae.

Stress analysis of the thoracolumbar junction in the process of backward fall: An experimental study and finite element analysis.

The aim of the present study was to evaluate the biomechanical mechanism of injuries of the thoracolumbar junction by the methods of a backward fall simulation experiment and finite element (FE) analysis (FEA). In the backward fall simulation experiment, one volunteer was selected to obtain the contact force data of the sacrococcygeal region during a fall. Utilizing the fall data, the FEA simulation of the backward fall process was given to the trunk FE model to obtain the stress status of local bone structures of the thoracolumbar junction during the fall process. In the fall simulation test, the sacrococcygeal region of the volunteer landed first; the total impact time was 1.14±0.58 sec, and the impact force was up to 4,056±263 N. The stress of thoracic (T)11 was as high as 42 MPa, that of the posterior margin and the junction of T11 was as high as 70.67 MPa, and that of the inferior articular process and the superior articular process was as high as 128 MPa. The average stress of T12 and the anterior margin of lumbar 1 was 25 MPa, and that of the endplate was as high as 21.7 MPa, which was mostly distributed in the back of the endplate and the surrounding cortex. According to the data obtained from the fall experiment as the loading condition of the FE model, the backward fall process can be simulated to improve the accuracy of FEA results. In the process of backward fall, the front edge of the vertebral body and the root of vertebral arch in the thoracolumbar junction are stress concentration areas, which have a greater risk of injury.

Transcriptional regulation of small heat shock protein genes by heat shock factor 1 (HSF1) in Liriomyza trifolii under heat stress.

Small heat shock proteins (sHSPs) function as molecular chaperones in multiple physiological processes and are active during thermal stress. sHSP expression is controlled by heat shock transcription factor (HSF); however, few studies have been conducted on HSF in agricultural pests. Liriomyza trifolii is an introduced insect pest of horticultural and vegetable crops in China. In this study, the master regulator, HSF1, was cloned and characterized from L. trifolii, and the expression levels of HSF1 and five sHSPs were studied during heat stress. HSF1 expression in L. trifolii generally decreased with rising temperatures, whereas expression of the five sHSPs showed an increasing trend that correlated with elevated temperatures. All five sHSPs and HSF1 showed an upward trend in expression with exposure to 40 ℃ without a recovery period. When a recovery period was incorporated after thermal stress, the expression patterns of HSF1 and sHSPs in L. trifolii exposed to 40 °C was significantly lower than expression with no recovery period. To elucidate potential interactions between HSF1 and sHSPs, double-stranded RNA was synthesized to knock down HSF1 in L. trifolii by RNA interference. The knockdown of HSF1 by RNAi decreased the survival rate and expression of HSP19.5, HSP20.8, and HSP21.3 during high-temperature stress. This study expands our understanding of HSF1-regulated gene expression in L. trifolii exposed to heat stress.

Temperature affects the tolerance of Liriomyza trifolii to insecticide abamectin.

The leafminer fly, Liriomyza trifolii, is an invasive pest of horticultural and vegetable crops that possesses a robust competitive ability when compared to congeneric species, especially with respect to temperature and insecticide tolerance. Abamectin, which is commonly used to control L. trifolii in the field, was selected as the target insecticide in this study. Our objective was to study the effect of abamectin and high temperature stress on L. trifolii mortality and the expression of genes encoding cytochrome P450 (CYP450s) and heat shock proteins (Hsps) by quantitative real-time reverse transcriptase PCR (qRT-PCR). When L. trifolii was exposed to abamectin followed by exposure to 40 °C (LC50 +HT40), mortality showed a significant increase, whereas exposure to 40 â„ƒ followed by abamectin (HT40+LC50) reduced mortality relative to abamectin or HT40 alone. Expression of three CYP450s in the CYP4 family was highest in the HT40+LC50 treatment, followed by the LC50+HT40 treatment. The expression levels of CYP18A1 (CYP18 family) were not significantly different among treatments, and CYP301A1 (CYP301 family) was only sensitive to temperature (HT40). The expression of five sHsps showed similar expression patterns and were highly responsive to the LC50+HT40 treatment, followed by the HT40 and HT40+LC50 treatments. Based on CYP450s and Hsps expression levels, our findings that suggest that L. trifolii exhibits adaptive cross-tolerance to high temperature and abamectin. This study provides a framework for selecting the most effective application time for abamectin with respect to controlling L. trifolii, which will ultimately reduce the overuse of pesticides.

High temperature stress induces expression of CYP450 genes and contributes to insecticide tolerance in Liriomyza trifolii.

Liriomyza trifolii is an invasive leafminer fly that inflicts damage on many horticultural and vegetable crops. In this study, the effects of elevated temperatures on L. trifolii tolerance to insecticides abamectin (AB), monosultap (MO) and a mixture of abamectin and monosultap (AM) were firstly investigated, then five CYP450 genes (LtCYPs) were cloned, and expression patterns and NADPH cytochrome C reductase (NCR) activity in L. trifolii were compared in response to high temperature stress and insecticide exposure. Results showed elevated temperatures induced expression of LtCYP450s, the expression level of LtCYP4g1, LtCYP4g15 and LtCYP301A1 after exposed to different high temperature were significantly up-regulated compared with the control (25 °C), while there was no significant difference in LtCYP4E21 and LtCYP18A1. Under the joint high temperature and insecticide stress, the expression of LtCYP4g15, LtCYP18A1 and LtCYP301A1 was significantly higher under elevated temperatures than that of only under AB exposure. For MO and AM exposure, only 40 °C could induce the expression of LtCYP4g15, LtCYP18A1 and LtCYP301A1. In general, the LtCYPs expression pattern was correlated with increased NCR activity and decreased mortality in response to insecticide exposure under elevated temperatures. These all demonstrated that insecticide tolerance in L. trifolii could be mediated by high temperature. This study improves our understanding of L. trifolii physiology and offers a theoretical context for improved control that ultimately reduces the abuse of insecticides and decreases exposure to non-target organisms.

RNA Interference of Genes Encoding the Vacuolar-ATPase in Liriomyza trifolii.

The leafminer fly, Liriomyza trifolii, is an invasive pest of vegetable and horticultural crops in China. In this study, a microinjection method based on dsRNA was developed for RNA interference (RNAi) in L. trifolii using genes encoding vacuolar-ATPase (V-ATPase). Expression analysis indicated that V-ATPase B and V-ATPase D were more highly expressed in L. trifolii adults than in larvae or pupae. Microinjection experiments with dsV-ATPase B and dsV-ATPase D were conducted to evaluate the efficacy of RNAi in L. trifolii adults. Expression analysis indicated that microinjection with 100 ng dsV-ATPase B or dsV-ATPase led to a significant reduction in V-ATPase transcripts as compared to that of the dsGFP control (dsRNA specific to green fluorescent protein). Furthermore, lower dsRNA concentrations were also effective in reducing the expression of target genes when delivered by microinjection. Mortality was significantly higher in dsV-ATPase B- and dsV-ATPase D-treated insects than in controls injected with dsGFP. The successful deployment of RNAi in L. trifolii will facilitate functional analyses of vital genes in this economically-important pest and may ultimately result in new control strategies.

Comparative transcriptome analysis of three invasive leafminer flies provides insights into interspecific competition.

Liriomyza spp. (Diptera: Agromyzidae) represent a group of economically-significant highly polyphagous pests of plants grown in field and greenhouse conditions. Liriomyza spp. share similar biological and morphological characteristics, and complex interspecific interactions have been documented among these species in various geographical regions. Where the displacement of one of these species by the other has been studied, no unique mechanisms have been identified as causing it. The impact of competitive factors (such as, insecticide tolerance, thermotolerance, and adaptability to cropping systems) may be unique to specific geographic regions of Liriomyza spp., but more research is needed to confirm these hypotheses. In this study, RNA-seq was used to determine the transcriptomes of three closely-related leafminers, e.g. L. sativae, L. trifolii, and L. huidobrensis. Over 20 Gb of clean reads were generated and assembled into unique transcriptomes, and 38,747 unigenes were annotated in different databases. In pairwise comparisons, L. trifolii and L. sativae had more up-regulated genes than L. huidobrensis. With respect to common differentially-expressed genes (Co-DEGs), the three leafminers exhibited distinct groups of highly-expressed gene clusters. When genes related to competitive factors were compared, expression patterns in L. trifolii and L. sativae were more closely related to each other than to L. huidobrensis. The data suggest that DEGs involved in competitive factors may play a key role in competition and displacement of leafminers. The divergent genes identified in this study will be valuable in revealing possible mechanisms of invasion, displacement and interspecific competition in Liriomyza spp.

Transcriptome analysis of Liriomyza trifolii (Diptera: Agromyzidae) in response to temperature stress.

The leafminer Liriomyza trifolii is an important insect pest of ornamental and vegetable crops worldwide. Temperature is a critical environmental factor that impacts both the distribution and interspecific competition of Liriomyza spp. In this study, we compared the transcriptomes of L. trifolii exposed to ambient (25 °C), hot (43 °C), and cold (-7 °C) temperatures. RNA-seq revealed 100,041 assembled unigenes, and 50,546 of these were annotated in L. trifolii transcriptome libraries. A total of 207 and 2904 differentially expressed genes (DEGs) were identified in response to hot and cold stress, respectively. Functional classification indicated that "cellular process", "single organism processes" and "metabolic processes" pathways were significantly enriched, along with "binding activity" and "catalytic activity". With respect to clusters of orthologous genes (COG) classification, DEGs were assigned to "post-translational modification, protein turnover, chaperones", "carbohydrate transport and metabolism" and "lipid transport and metabolism" categories. Subsequent annotation and enrichment analyses indicated that genes encoding heat shock proteins (HSPs) and cuticular proteins were significantly up-regulated during heat and cold stress, respectively. This study expands our knowledge of gene expression in L. trifolii during temperature stress and provides a basis for further studies aimed at understanding the mechanism of thermotolerance in this important invasive leafminer fly.

Molecular Cloning and Characterization of Small Heat Shock Protein Genes in the Invasive Leaf Miner Fly, Liriomyza trifolii.

Small heat shock proteins (sHSPs) comprise numerous proteins with diverse structure and function. As molecular chaperones, they play essential roles in various biological processes, especially under thermal stresses. In this study, we identified three sHSP-encoding genes, LtHSP19.5, LtHSP20.8 and LtHSP21.7b from Liriomyza trifolii, an important insect pest of ornamental and vegetable crops worldwide. Putative proteins encoded by these genes all contain a conserved α-crystallin domain that is typical of the sHSP family. Their expression patterns during temperature stresses and at different insect development stages were studied by reverse-transcription quantitative PCR (RT-qPCR). In addition, the expression patterns were compared with those of LtHSP21.3 and LtHSP21.7, two previously published sHSPs. When pupae were exposed to temperatures ranging from -20 to 45 °C for 1 h, all LtsHSPs were strongly induced by either heat or cold stresses, but the magnitude was lower under the low temperature range than high temperatures. Developmentally regulated differential expression was also detected, with pupae and prepupae featuring the highest expression of sHSPs. Results suggest that LtsHSPs play a role in the development of the invasive leaf miner fly and may facilitate insect adaptation to climate change.

Cloning of a new HSP70 gene from western flowerthrips, Frankliniella occidentalis, and expression patterns during thermal stress.

Frankliniella occidentalis (Pergande) is an invasive pest that endangers a wide variety of horticultural and agronomic crops. HSP70 is the most important member of the heat shock protein (HSP) family and plays an important role in insect thermal tolerance. In this study, a new gene encoding HSP70 from F. occidentalis, Fohsp706, was selected from the F. occidentalis transcriptome exposed to thermal stress (40 °C) and cloned by RT-PCR and RACE. Further characterization indicated that Fohsp706 localizes to the cytoplasm and does not contain introns. Quantitative real-time reverse transcriptase PCR indicated that Fohsp706 expression was significantly up-regulated by thermal stress; furthermore, there were significant differences in Fohsp706 expression in adults and second instar nymphs after heat stress. Our results indicated that Fohsp706 contributes to thermotolerance in F. occidentalis and provides another example of how this pest adapts to unfavorable environmental conditions.

Three-Dimensional Printing-Assisted Cervical Anterior Bilateral Pedicle Screw Fixation of Artificial Vertebral Body for Cervical Tuberculosis.

BACKGROUND: Cervical tuberculosis accounts for only 4.2%-12% of the total incidence of spinal tuberculosis cases. Although antituberculosis drugs have been the mainstay treatment of cervical tuberculosis, they have been ineffective against the symptoms of existing spinal deformities and spinal cord compression, which often require surgical intervention. The conventional surgical methods have been anterior debridement and titanium mesh, cage bone graft fusion and internal fixation. However, all have certain deficiencies regarding the stability of fixation. CASE DESCRIPTION: We have presented the case of a 41-year-old Chinese man who had been experiencing neck pain and stiffness for 1 month. The symptoms had been accompanied by low-grade fever and repeated night sweats. The purified protein derivative test result was positive and the antituberculosis test result was negative. Imaging examination showed destruction of the C5 and C6 vertebral bodies and C5 andC6 intervertebral discs, with an intensive abscess at the C5-C6 vertebral level. After 3-dimensional printing-assisted anterior debridement and artificial vertebral body replacement, his preoperative symptoms of neck pain and stiffness had been alleviated. Also, his symptoms of numbness in both upper limbs had disappeared completely. At the last follow-up examination, he had recovered well and the tuberculosis focus had been completely cured. CONCLUSION: To the best of our knowledge, we have reported the first clinical application of 3-dimensional printing-assisted cervical anterior bilateral pedicle screw fixation of an artificial vertebral body. We accomplished ultrashort segment fixation, with excellent clinical outcomes obtained, which were maintained at the recent 2-year follow-up examination.

Satellite-based entanglement distribution over 1200 kilometers.

Long-distance entanglement distribution is essential for both foundational tests of quantum physics and scalable quantum networks. Owing to channel loss, however, the previously achieved distance was limited to ~100 kilometers. Here we demonstrate satellite-based distribution of entangled photon pairs to two locations separated by 1203 kilometers on Earth, through two satellite-to-ground downlinks with a summed length varying from 1600 to 2400 kilometers. We observed a survival of two-photon entanglement and a violation of Bell inequality by 2.37 ± 0.09 under strict Einstein locality conditions. The obtained effective link efficiency is orders of magnitude higher than that of the direct bidirectional transmission of the two photons through telecommunication fibers.

A genetic study of the regeneration capacity of embryonic callus from the maize immature embryo culture.

We carried out a study of regeneration capacities of embryonic callus from maize immature embryo culture with 144 different inbred lines of natural groups from different countries, and found that the regeneration capacity was affected by three factors: environment, genotype and the interaction between the environment and genotype. We found that green embryonic callus rate (GCR), embryonic callus differentiating rate (CDR) and the plantlet number of embryonic callus regeneration (CPN) have significant positive correlations with each other, and they all have significant negative correlations with embryonic callus browning rate (CBR). Moreover, embryonic callus cloning index for the first subculture (CCI1) and embryonic callus cloning index for the second subculture (CCI2) have a significant positive correlation with each other, and CCI2 is positively correlated with green GCR, and is negatively correlated with CBR. Embryonic callus rooting rate (CRR) is positively correlated with GCR, CDR and CPN to some degree. Furthermore, we calculated Broad-Sense Heritability of each trait, and uncovered that the heritability index of CCI1, CCI2 and CRR was lower, and the heritability index of others was higher. In addition, by using the Ward method for two-way cluster analysis, we found eleven inbred lines with high regenerating abilities, and the rooting situation of regenerating plantlet was excellent by rooting culture, which could be used as the elite inbred lines of the maize transgenic receptor.

Fine mapping of the qLOP2 and qPSR2-1 loci associated with chilling stress tolerance of wild rice seedlings.

KEY MESSAGE: Using leaf osmotic potential and plant survival rate as chilling-tolerant trait indices, we identified two major quantitative trait loci qLOP2 and qPSR2 - 1 (39.3-kb region) and Os02g0677300 as the cold-inducible gene for these loci. Chilling stress tolerance (CST) at the seedling stage is an important trait affecting rice production in temperate climate and high-altitude areas. To identify quantitative trait loci (QTLs) associated with CST, a mapping population consisting of 151 BC(2)F(1) plants was constructed by using chilling-tolerant Dongxiang wild rice (Oryza rufipogon Griff.) as a donor parent and chilling-sensitive indica as a recurrent parent. With leaf osmotic potential (LOP) and plant survival rate (PSR) as chilling-tolerant trait indexes, two major QTLs, qLOP2 (LOD = 3.8) and qPSR2-1 (LOD = 3.3), were detected on the long arm of chromosome 2 by composite interval mapping method in QTL Cartographer software, which explained 10.1 and 12.3% of the phenotypic variation, respectively. In R/QTL analyzed result, their major effects were also confirmed. Using molecular marker RM318 and RM106, qLOP2 and qPSR2-1 have been introgressed into chilling-sensitive varieties (93-11 and Yuefeng) by marker-assisted selection procedure (MAS), which resulted in 16 BC(5)F(3) BILs that chilling tolerance have significantly enhanced compare with wild-type parents (P < 0.01). Therefore, two large segregating populations of 11,326 BC(4)F(2) and 8,642 BC(4)F(3) were developed to fine mapping of qLOP2 and qPSR2-1. Lastly, they were dissected to a 39.3-kb candidate region between marker RM221 and RS8. Expression and sequence analysis results indicated that Os02g0677300 was a cold-inducible gene for these loci. Our study provides novel alleles for improving rice CST by MAS and contributes to the understanding of its molecular mechanisms.

Fine mapping of qRC10-2, a quantitative trait locus for cold tolerance of rice roots at seedling and mature stages.

UNLABELLED: Cold stress causes various injuries to rice seedlings in low-temperature and high-altitude areas and is therefore an important factor affecting rice production in such areas. In this study, root conductivity (RC) was used as an indicator to map quantitative trait loci (QTLs) of cold tolerance in Oryza rufipogon Griff., Dongxiang wild rice (DX), at its two-leaf stage. The correlation coefficients between RC and the plant survival rate (PSR) at the seedling and maturity stages were -0.85 and -0.9 (P = 0.01), respectively, indicating that RC is a reliable index for evaluating cold tolerance of rice. A preliminary mapping group was constructed from 151 BC2F1 plants using DX as a cold-tolerant donor and the indica variety Nanjing 11 (NJ) as a recurrent parent. A total of 113 codominant simple-sequence repeat (SSR) markers were developed, with a parental polymorphism of 17.3%. Two cold-tolerant QTLs, named qRC10-1 and qRC10-2 were detected on chromosome 10 by composite interval mapping. qRC10-1 (LOD = 3.1, RM171-RM1108) was mapped at 148.3 cM, and qRC10-2 (LOD = 6.1, RM25570-RM304) was mapped at 163.3 cM, which accounted for 9.4% and 32.1% of phenotypic variances, respectively. To fine map the major locus qRC10-2, NJ was crossed with a BC4F2 plant (L188-3), which only carried the QTL qRC10-2, to construct a large BC5F2 fine-mapping population with 13,324 progenies. Forty-five molecular markers were designed to evenly cover qRC10-2, and 10 markers showed polymorphisms between DX and NJ. As a result, qRC10-2 was delimited to a 48.5-kb region between markers qc45 and qc48. In this region, Os10g0489500 and Os10g0490100 exhibited different expression patterns between DX and NJ. Our results provide a basis for identifying the gene(s) underlying qRC10-2, and the markers developed here may be used to improve low-temperature tolerance of rice seedling and maturity stages via marker-assisted selection (MAS). KEY MESSAGE: With root electrical conductivity used as a cold-tolerance index, the quantitative trait locus qRC10-2 was fine mapped to a 48.5-kb candidate region, and Os10g0489500 and Os10g0490100 were identified as differently expressed genes for qRC10-2.

Comparison of (18)F-labeled CXCR4 antagonist peptides for PET imaging of CXCR4 expression.

PURPOSE: CXCR4 is overexpressed on tumor cells from many types of human cancers. A high level of CXCR4 expression often correlates with poor prognosis, chemotherapy resistance, and metastasis. The development of CXCR4-specific radiotracers for positron emission tomography (PET) imaging will allow in vivo evaluation of receptor expression level for diagnosis or therapeutic evaluation. PROCEDURES: Two new (18)F-labeled radiotracers based on an Ac-TC14012 peptide, [(18)F]FP-Ac-TC14012 and [(18)F]FB-Ac-TC14012, were synthesized and characterized. The affinities of the 2-fluoropropionate (FP)-conjugated or 4-fluorobenzoate (FB)-conjugated peptides to CXCR4-transfected Chinese hamster ovarian (CHO) cells were evaluated in a competitive binding assay with [(125)I]CXCL12 radioligand. The cell uptake and retention of [(18)F]FP-labeled and [(18)F]FB-labeled peptides were measured. The tumor targetability and pharmacokinetics of these two tracers were also evaluated by microPET imaging and biodistribution studies. RESULTS: The labeled peptides retained high binding affinity to CXCR4 and showed much higher uptake in CXCR4-positive CHO cells than in CXCR4-negative cells in vitro. The smaller and more hydrophilic [(18)F]FP prosthetic group resulted in higher affinity and lower nonspecific cell uptake compared to the [(18)F]FB-labeled peptide. Both radiotracers showed much higher accumulation in CXCR4-positive than CXCR4-negative tumor xenografts in mice and allowed clear visualization of CXCR4 expression by PET. Among the two, [(18)F]FP-Ac-TC14012 showed higher tumor uptake and better tumor-to-background contrast. Unlike their N-terminal 4-F-benzoate analogs, these two tracers had minimal blood retention, likely due to reduced red blood cell binding. Metabolic organs, such as the liver and kidney, also showed high uptake. When blocked with low-dose cold peptide (10 µg), the tumor uptake was significantly increased, most likely due to the increased concentration in blood circulation, as evidenced by decreased liver uptake. CONCLUSION: These results demonstrate that the [(18)F]FP-labeled Ac-TC14012 peptide with high tumor uptake, low nonspecific binding, and good tumor-to-background contrast promises [(18)F]FP-Ac-TC14012 as a PET tracer for in vivo PET imaging of CXCR4 expression.

pH-sensitive fluorescent dyes: are they really pH-sensitive in cells?

Chemically synthesized near-infrared aza-BODIPY dyes displayed off-on fluorescence at acidic pH (pKa = 6.2-6.6) through the suppression of the photoinduced electron transfer and/or internal charge transfer process. The apparent pKas of the dyes were shifted well above physiological pH in a hydrophobic microenvironment, which led to "turned-on" fluorescence in micelles and liposomes at neutral and basic pH. Bovine serum albumin also activated the fluorescence, though to a much lesser extent. When these small molecular dyes entered cells, instead of being fluorescent only in acidic organelles, the whole cytoplasm exhibited fluorescence, with a signal/background ratio as high as ∼10 in no-wash live-cell imaging. The dye 1-labeled cells remained highly fluorescent even after 3 days. Moreover, slight variations of the dye structure resulted in significantly different intracellular fluorescence behaviors, possibly because of their different cellular uptake and intracellular activation capabilities. After the separation of cellular components, the fraction of plasma membrane and endoplasmic reticulum showed the highest fluorescence, further confirming the fluorescence activation by membrane structures. The fluorescence intensity of these dyes at different intracellular pHs (6.80 and 8.00) did not differ significantly, indicating that intracellular pH did not play a critical role. Altogether, we showed here for the first time that the fluorescence of pH-sensitive aza-BODIPY dyes was switched intracellularly not by acidic pH, but by intracellular membranes (and proteins as well). The excellent membrane permeability, ultrahigh fluorescence contrast ratio, persistent fluorescent signal, and minimal biological interference of dye 1 make it an ideal choice for live-cell imaging and in vivo cell tracking. These findings also imply that the intracellular fluorescence properties of pH-sensitive dyes should be carefully examined before they are used as pH indicators.