Editing VvDXS1 for the creation of muscat flavour in Vitis vinifera cv. Scarlet Royal.

Muscat flavour represents a group of unique aromatic attributes in some grape varieties. Biochemically, grape berries with muscat flavour produce high levels of monoterpenes. Monoterpene biosynthesis is mainly through the DOXP/MEP pathway, and VvDXS1 encodes the first enzyme in this plastidial pathway of terpene biosynthesis in grapevine. A single-point mutation resulting in the substitution of a lysine with an asparagine at position 284 in the VvDXS1 protein has previously been identified as the major cause for producing muscat flavour in grapes. In this study, the same substitution in the VvDXS1 protein was successfully created through prime editing in the table grape Vitis vinifera cv. 'Scarlet Royal'. The targeted point mutation was detected in most of the transgenic vines, with varying editing efficiencies. No unintended mutations were detected in the edited alleles, either by PCR Sanger sequencing or by amplicon sequencing. More than a dozen edited vines were identified with an editing efficiency of more than 50%, indicating that these vines were likely derived from single cells in which one allele was edited. These vines had much higher levels of monoterpenes in their leaves than the control, similar to what was found in leaf samples between field-grown muscat and non-muscat grapes.

The genome of Shanputao (Vitis amurensis) provides a new insight into cold tolerance of grapevine.

Vitis amurensis (Shanputao) is the most cold tolerant Vitis species and so is of great interest to grape breeders and producers in areas with low winter temperatures. Here, we report its high-quality, chromosome-level genome assembly based on a combination of sequence data from Illumina and PacBio platforms, BioNano optical mapping and high-throughput chromosome conformation Capture (Hi-C) mapping. The 604.56-Mb genome contains 32 885 protein-coding genes. Shanputao was found to share a common ancestor with PN40024 (V. vinifera) approximately 2.17-2.91 million years ago, and gene expansion observed in Shanputao might contribute to the enhancement of cold tolerance. Transcriptome analysis revealed 17 genes involved in cold signal transduction, suggesting that there was a different response mechanism to chilling temperature and freezing conditions. Furthermore, a genome-wide association study uncovered a phosphoglycerate kinase gene that may contribute to the freezing resistance of buds in the winter. The Shanputao genome sequence not only represents a valuable resource for grape breeders, but also is important for clarifying the molecular mechanisms involved in cold tolerance.

RNA-Seq reveals new DELLA targets and regulation in transgenic GA-insensitive grapevines.

BACKGROUND: Gibberellins (GAs) and their regulator DELLA are involved in many aspects of plant growth and development and most of our current knowledge in the DELLA-facilitated GA signaling was obtained from the studies of annual species. To understand GA-DELLA signaling in perennial species, we created ten GA-insensitive transgenic grapevines carrying a DELLA mutant allele (Vvgai1) in the background of Vitis vinifera 'Thompson Seedless' and conducted comprehensive analysis of their RNA expression profiles in the shoot, leaf and root tissues. RESULTS: The transgenic lines showed varying degrees of dwarf stature and other typical DELLA mutant phenotypes tightly correlated with the levels of Vvgai1 expression. A large number of differentially expressed genes (DEGs) were identified in the shoot, leaf and root tissues of the transgenic lines and these DEGs were involved in diverse biological processes; many of the DEGs showed strong tissue specificity and about 30% them carried a DELLA motif. We further discovered unexpected expression patterns of several key flowering induction genes VvCO, VvCOL1 and VvTFL1. CONCLUSIONS: Our results not only confirmed many previous DELLA study findings in annual species, but also revealed new DELLA targets and responses in grapevine, including the roles of homeodomain transcription factors as potential co-regulators with DELLA in controlling the development of grapevine which uniquely possess both vegetative and reproductive meristems at the same time. The contrasting responses of some key flowering induction pathway genes provides new insights into the divergence of GA-DELLA regulations between annual and perennial species in GA-DELLA signaling.

Messenger RNA exchange between scions and rootstocks in grafted grapevines.

BACKGROUND: Grafting has been widely practiced for centuries in the propagation and production of many vegetable and fruit species. However, the underlying molecular and genetic mechanisms for how the graft partners interact with each other to produce a successful graft remain largely unknown. We hypothesized that genome-wide mRNA exchanges, which were recently documented in grafted model plant species, are a general phenomenon widely present in grafted plants, including those in vegetable and fruit species, and have specific genotype- and environment-dependent characteristics modulating plant performance. METHODS: Using diagnostic SNPs derived from high throughput genome sequencing, we identified and characterized the patterns of genome-wide mRNA exchanges across graft junctions in grafted grapevines grown in the in vitro and field conditions. RESULTS: We identified more than 3000 genes transporting mRNAs across graft junctions. These genes were involved in diverse biological processes and those involved in basic cellular, biosynthetic, catabolic, and metabolic activities, as well as responses to stress and signal transduction, were highly enriched. Field-grown mature grafts had much fewer genes transmitting mRNAs than the in vitro young grafts (987 vs. 2679). These mobile mRNAs could move directionally or bi-directionally between scions and rootstocks. The mRNA transmission rates of these genes were generally low, with 65% or more having transmission rates lower than 0.01. Furthermore, genotypes, graft combinations and growth environments had impact on the directions of mRNA movement as well as the numbers and species of mRNAs being exchanged. Moreover, we found evidence for the presences of both passive and selective mechanisms underlying long distance mRNA trafficking in grafted grapevines. CONCLUSIONS: We extended the studies of mRNA exchanges in model species to grapevines and demonstrated that genomic-scale mRNA exchange across graft junctions occurred in grapevines in a passive or genotype and environment-dependent manner.

Multiple loss-of-function 5-O-glucosyltransferase alleles revealed in Vitis vinifera, but not in other Vitis species.

KEY MESSAGE: Wild and loss-of-function alleles of the 5 - O - glucosyltransferase gene responsible for synthesis of diglucoside anthocyanins in Vitis were characterized. The information aids marker development for tracking this gene in grape breeding. Anthocyanins in red grapes are present in two glycosylation states: monoglucoside (3-O-glucoside) and diglucoside (3, 5-di-O-glucoside). While monoglucoside anthocyanins are present in all pigmented grapes, diglucoside anthocyanins are rarely found in the cultivated grape species Vitis vinifera. Biochemically 3-O-glucoside anthocyanins can be converted into 3,5-di-O-glucoside anthocyanins by a 5-O-glucosyltransferase. In this study, we surveyed allelic variation of the 5-O-glucosyltransferase gene (5GT) in 70 V. vinifera ssp. vinifera cultivars, 52 V. vinifera ssp. sylvestris accessions, 23 Vitis hybrid grapes, and 22 accessions of seven other Vitis species. Eighteen 5GT alleles with apparent loss-of-function mutations, including seven premature stop codon mutations and six frameshift indel mutations, were discovered in V. vinifera, but not in the other Vitis species. A total of 36 5GT alleles without apparent loss-of-function mutations (W-type) were identified. These W-type alleles were predominantly present in wild Vitis species, although a few of them were also found in some V. vinifera accessions. We further evaluated some of these 5GT alleles in producing diglucoside anthocyanins by analyzing the content of diglucoside anthocyanins in a set of representative V. vinifera cultivars. Through haplotype network analysis we revealed that V. vinifera ssp. vinifera and its wild progenitor V. vinifera ssp. sylvestris shared many loss-of-function 5GT alleles and extensive divergence of the 5GT alleles was evident within V. vinifera. This work advances our understanding of the genetic diversity of 5GT and provides a molecular basis for future marker-assisted selection for improving this important wine quality trait.

Global overview of anterior cruciate ligament reconstruction in children and adolescents over the past 20 years: a bibliometric analysis.

OBJECTIVES: The objectives of this paper is to conduct a bibliometric analysis to examine the research status and development trend of anterior cruciate ligament injury and reconstruction in children and adolescents over the past 20 years. DESIGN: Descriptive Research. METHODS: This study obtained information regarding studies on Anterior Cruciate Ligament Reconstruction in Children and Adolescents from the Web of Science Core Collection database. Visual and bibliometric analysis were conducted using VOSviewer, Origin 2022, Pajek64 5.18and Excel 2019. These analytic tools facilitated the analysis of various aspects, including countries/regions, institutions, authors, journals and keywords related to the research. RESULTS: From 2003 to 2023, a total of 1328 articles were retrieved in WOS, and 637 articles were selected by two authors. The most productive institutions are Childrens Hosp Philadelphia, Kocher, ms. Their articles have the highest number of publications and citations. The American journal of sports medicine is the most frequently cited journal for articles on anterior cruciate ligament reconstruction in children and adolescents. The most common keywords used in these articles were "anterior cruciate ligament reconstruction", "injury, children, adolescent", and "skeletally immature patients". CONCLUSIONS: This study provides valuable insights into the research focus of anterior cruciate ligament reconstruction in children and adolescents. In recent years, there has been significant attention paid to areas of "the return to sport, re-repture rate and functional recovery after anterior cruciate ligament reconstruction" in this specific population. These aspects have emerged as key directions for future research in this field.

Clinical application of a connection device consisting of a bag valve mask and nebulizer in first aid: Two case reports.

In clinical practice, several emergencies may threaten the life of patients, and these emergencies can be unpredictable and challenging. During the coronavirus disease 2019 pandemic, in January 2023, a patient developed respiratory distress caused by coronavirus, but was unable to access respiratory support due to shortages of medical resources, intensive care unit beds and ventilators. The medical staff quickly created a portable high-flow atomized oxygen therapy apparatus consisting of a simple breathing bag connected to a nebulizer to provide breathing support. In addition, the Ambulatory Surgery Center, The First Affiliated Hospital of Anhui Medical University (Hefei, China) witnessed a case of severe laryngeal spasm after tracheal extubation during the recovery period from general anesthesia. Due to the lack of an anesthesia machine nebulizer, the aforementioned device was used to provide oxygen under pressure and initiate treatment to quickly relieve the symptoms of laryngeal obstruction. The present case report describes how the medical staff quickly applied emergency airway management skills and knowledge to create a portable high-flow atomized oxygen therapy apparatus in a resource-poor setting to save the lives of two patients.

Characterization of grape Gibberellin Insensitive1 mutant alleles in transgenic Arabidopsis.

We generated 12 different mutations in the grape Gibberellin Insensitive1 (VvGAI1) sequences, transformed them into Arabidopsis under the control of 35S, Arabidopsis GAI or grape GAI1 promoter, and evaluated the impact of these mutant alleles on plant growth and development. These VvGAI1 sequence variants included some mimics of the known GAI-like mutant alleles discovered in grape, wheat, barley, corn, Brassica, and Arabidopsis. In general, plant height and related traits such as length of internodes and inflorescences were significantly reduced for most of the mutant alleles studied, regardless of which promoter was used. Interestingly, the numbers of rosette leaves and lateral branches were generally reduced when a 35S promoter was used to express the mutant alleles, but increased when an Arabidopsis or grape GAI promoter was used. Furthermore, the 35S plants often displayed curly and small leaves. In contrast, the leaves of the plants carrying mutant alleles controlled by a GAI promoter were of variable size, dark green and rarely curly. In addition, when certain VvGAI1 mutant alleles were under the control of the grape GAI1 promoter, the number of pods on inflorescences was significantly increased, but some of the pods produced few seeds due to partial sterility. On the basis of the systematic evaluation of various VvGAI1 mutant alleles in Arabidopsis, we concluded that the VvGAI1 mutant alleles mimicking the GAI or GAI-like mutant variants discovered in wheat, barley and Brassica could potentially be useful for the improvement of grapevine plant architecture.

Removal of a 10-kb Gret1 transposon from VvMybA1 of Vitis vinifera cv. Chardonnay.

Many white grape cultivars have a nonfunctional VvMybA1 gene due to the presence of a 10-kb Gret1 transposon in its promoter. In this study, we successfully demonstrated removal of the 10-kb Gret1 transposon and functional restoration of a VvMybA1 allele in Vitis vinifera cv. Chardonnay through transgenic expression of Cas9 and two gRNAs simultaneously targeting two junction sequences between Gret1 LTRs and VvMybA1. We generated 67 and 24 Cas9-positive vines via Agrobacterium-mediated and biolistic bombardment transformation, respectively. While the editing efficiencies were as high as 17% for the 5' target site and 65% for the 3' target site, simultaneous editing of both 5' and 3' target sites resulting in the removal of Gret1 transposon from the VvMybA1 promoter was 0.5% or less in most transgenic calli, suggesting that these calli had very limited numbers of cells with the Gret1 removed. Nevertheless, two bombardment-transformed vines, which shared the same unique editing features and were likely derived from a singly edited event, were found to have the Gret1 successfully edited out from one of their two VvMybA1 alleles. The edited allele was functionally restored based on the detection of its expression and a positive coloring assay result in leaves. Precise removal of more than a 10-kb DNA fragment from a gene locus in grape broadens the possibilities of using gene editing technologies to modify various trait genes in grapes and other plants.

Calcium elevation-dependent and attenuated resting calcium-dependent abscisic acid induction of stomatal closure and abscisic acid-induced enhancement of calcium sensitivities of S-type anion and inward-rectifying K channels in Arabidopsis guard cells.

Stomatal closure in response to abscisic acid depends on mechanisms that are mediated by intracellular [Ca2+] ([Ca2+]i), and also on mechanisms that are independent of [Ca2+]i in guard cells. In this study, we addressed three important questions with respect to these two predicted pathways in Arabidopsis thaliana. (i) How large is the relative abscisic acid (ABA)-induced stomatal closure response in the [Ca2+]i-elevation-independent pathway? (ii) How do ABA-insensitive mutants affect the [Ca2+]i-elevation-independent pathway? (iii) Does ABA enhance (prime) the Ca2+ sensitivity of anion and inward-rectifying K+ channel regulation? We monitored stomatal responses to ABA while experimentally inhibiting [Ca2+]i elevations and clamping [Ca2+]i to resting levels. The absence of [Ca2+]i elevations was confirmed by ratiometric [Ca2+]i imaging experiments. ABA-induced stomatal closure in the absence of [Ca2+]i elevations above the physiological resting [Ca2+]i showed only approximately 30% of the normal stomatal closure response, and was greatly slowed compared to the response in the presence of [Ca2+]i elevations. The ABA-insensitive mutants ost1-2, abi2-1 and gca2 showed partial stomatal closure responses that correlate with [Ca2+]i-dependent ABA signaling. Interestingly, patch-clamp experiments showed that exposure of guard cells to ABA greatly enhances the ability of cytosolic Ca2+ to activate S-type anion channels and down-regulate inward-rectifying K+ channels, providing strong evidence for a Ca2+ sensitivity priming hypothesis. The present study demonstrates and quantifies an attenuated and slowed ABA response when [Ca2+]i elevations are directly inhibited in guard cells. A minimal model is discussed, in which ABA enhances (primes) the [Ca2+]i sensitivity of stomatal closure mechanisms.

[Association between myocardial calpain activation and apoptosis in lipopolysaccharide-induced septic mouse model].

OBJECTIVE: in septic mice, myocardial calpain was activated and induced caspase-3 activation, the association between calpain activation and apoptosis was explored in this experiment. METHODS: in in vivo model, adult C57 mice were injected with lipopolysaccharide (LPS, 4 mg/kg, i.p.) to induce sepsis. Myocardial calpain and caspase-3 activities, protein levels of calpain-1, calpain-2, calpastatin, Bcl-2 and Bid were detected by Western blot analysis and myocardial apoptosis was detected by TUNEL, myocardiac function was evaluated by Langendorff system. In in vitro model, adult rat cardiomyocytes were incubated with LPS (1 microg/ml) or co-incubated with calpain inhibitor-III (10 micromol/L), calpain activity, caspase-3 activity, protein levels of Bcl-2 and Bid, and cardiomyocyte apoptosis were detected. RESULTS: in septic mice, myocardial calpain and caspase-3 activity were increased up to 2.7- and 1.8-folds, respectively. Both calpain inhibitor-III and PD150606 significantly attenuated the increase of caspase-3 activity. Myocardial protein levels of calpain-1, calpain-2, calpastatin, Bcl-2 and Bid were similar between control and septic mice, and no cleavage of both Bcl-2 and Bid was found in septic mice. Calpain inhibitor-III significantly improved myocardial function in septic mice. In in vitro model, calpain and caspase-3 activities were increased after 4 h LPS treatment, co-treatment with calpain inhibitor-III prevented caspase-3 activity increase, protein Bcl-2 and Bid were similar between normal cardiomyocytes and LPS-treated cardiomyocytes. Cardiomyocyte apoptosis was similar in in vivo and in vitro septic models. CONCLUSION: myocardial calpain activity is increased in LPS induced septic mice, subsequent caspase-3 activation may contribute to myocardial dysfunction in septic mice without aggravating myocardial apoptosis and Bcl-2 and Bid are not involved on calpain induced caspase-3 activation in our model.

Thermal-responsive genetic and epigenetic regulation of DAM cluster controlling dormancy and chilling requirement in peach floral buds.

The Dormancy-associated MADS-box (DAM) gene cluster in peach serves as a key regulatory hub on which the seasonal temperatures act and orchestrate dormancy onset and exit, chilling response and floral bud developmental pace. Yet, how different temperature regimes interact with and regulate the six linked DAM genes remains unclear. Here, we demonstrate that chilling downregulates DAM1 and DAM3-6 in dormant floral buds with distinct patterns and identify DAM4 as the most abundantly expressed one. We reveal multiple epigenetic events, with tri-methyl histone H3 lysine 27 (H3K27me3) induced by chilling specifically in DAM1 and DAM5, a 21-nt sRNA in DAM3 and a ncRNA induced in DAM4. Such induction is inversely correlated with downregulation of their cognate DAMs. We also show that the six DAMs were hypermethylated, associating with the production of 24-nt sRNAs. Hence, the chilling-responsive dynamic of the different epigenetic elements and their interactions likely define distinct expression abundance and downregulation pattern of each DAM. We further show that the expression of the five DAMs remains steadily unchanged or continuously downregulated at the ensuing warm temperature after chilling, and this state of regulation correlates with robust increase of sRNA expression, H3K27me3 and CHH methylation, which is particularly pronounced in DAM4. Such robust increase of repressive epigenetic marks may irreversibly reinforce the chilling-imposed repression of DAMs to ensure flower-developmental programming free from any residual DAM inhibition. Taken together, we reveal novel information about genetic and epigenetic regulation of the DAM cluster in peach, which will be of fundamental significance in understanding of the regulatory mechanisms underlying chilling requirement and dormancy release, and of practical application for improvement of plasticity of flower time and bud break in fruit trees to adapt changing climates.

A key 'foxy' aroma gene is regulated by homology-induced promoter indels in the iconic juice grape 'Concord'.

'Concord', the most well-known juice grape with a parentage of the North American grape species Vitis labrusca L., possesses a special 'foxy' aroma predominantly resulted from the accumulation of methyl anthranilate (MA) in berries. This aroma, however, is often perceived as an undesirable attribute by wine consumers and rarely noticeable in the common table and wine grape species V. vinifera. Here we discovered homology-induced promoter indels as a major genetic mechanism for species-specific regulation of a key 'foxy' aroma gene, anthraniloyl-CoA:methanol acyltransferase (AMAT), that is responsible for MA biosynthesis. We found the absence of a 426-bp and/or a 42-bp sequence in AMAT promoters highly associated with high levels of AMAT expression and MA accumulation in 'Concord' and other V. labrusca-derived grapes. These promoter variants, all with direct and inverted repeats, were further confirmed in more than 1,300 Vitis germplasm. Moreover, functional impact of these indels was validated in transgenic Arabidopsis. Superimposed on the promoter regulation, large structural changes including exonic insertion of a retrotransposon were present at the AMAT locus in some V. vinifera grapes. Elucidation of the AMAT genetic regulation advances our understanding of the 'foxy' aroma trait and makes it genetically trackable and amenable in grapevine breeding.

CDPKs CPK6 and CPK3 function in ABA regulation of guard cell S-type anion- and Ca(2+)-permeable channels and stomatal closure.

Abscisic acid (ABA) signal transduction has been proposed to utilize cytosolic Ca(2+) in guard cell ion channel regulation. However, genetic mutants in Ca(2+) sensors that impair guard cell or plant ion channel signaling responses have not been identified, and whether Ca(2+)-independent ABA signaling mechanisms suffice for a full response remains unclear. Calcium-dependent protein kinases (CDPKs) have been proposed to contribute to central signal transduction responses in plants. However, no Arabidopsis CDPK gene disruption mutant phenotype has been reported to date, likely due to overlapping redundancies in CDPKs. Two Arabidopsis guard cell-expressed CDPK genes, CPK3 and CPK6, showed gene disruption phenotypes. ABA and Ca(2+) activation of slow-type anion channels and, interestingly, ABA activation of plasma membrane Ca(2+)-permeable channels were impaired in independent alleles of single and double cpk3cpk6 mutant guard cells. Furthermore, ABA- and Ca(2+)-induced stomatal closing were partially impaired in these cpk3cpk6 mutant alleles. However, rapid-type anion channel current activity was not affected, consistent with the partial stomatal closing response in double mutants via a proposed branched signaling network. Imposed Ca(2+) oscillation experiments revealed that Ca(2+)-reactive stomatal closure was reduced in CDPK double mutant plants. However, long-lasting Ca(2+)-programmed stomatal closure was not impaired, providing genetic evidence for a functional separation of these two modes of Ca(2+)-induced stomatal closing. Our findings show important functions of the CPK6 and CPK3 CDPKs in guard cell ion channel regulation and provide genetic evidence for calcium sensors that transduce stomatal ABA signaling.

[Association between expression of chromogranin A and myocardial fibrosis in patients with dilated cardiomyopathy].

OBJECTIVE: To observe the possible correlation between expression of chromogranin A (CGA) and myocardial fibrosis and investigate the potential role of CGA in the development of myocardial fibrosis in patients with dilated cardiomyopathy (DCM). METHODS: Surgical myocardial specimen from 10 DCM patients underwent successful orthotopic cardiac transplantation, and 3 normal myocardial specimen from brain-dead organ donors were obtained. CGA-mRNA, COLI-mRNA, COLIII-mRNA and ADAMTS-1-mRNA were analyzed by real-time PCR. The location and expression of CGA were assessed by immunohistochemistry(INH)with anti-CGA antibody. The collagen specific picrosirius red staining was applied on transversal myocardial slides and the collagen volume fraction (CVF) was calculated. The correlation between CGA and CVF was analyzed. RESULTS: Cytoplasmic expression of CGA assessed by INH showed large amount of strong positive granules densely arranged in the epicardial and endocardial myocardiocytes in DCM specimen while there was only few sparse granules in the normal myocardium (P < 0.05). CVF was significantly higher in DCM myocardial specimen than that in normal specimen (P < 0.001). CGA-mRNA was significantly correlated with COLI-mRNA (r = 0.729), COLIII-mRNA (r = 0.95) and ADAMTS-1-mRNA (r = 0.665, all P < 0.05). Moreover, collagen deposition location was almost identical with the strong positive expression location of CGA. CONCLUSION: We demonstrated for the first time that the deposition of CGA was related with the myocardial fibrosis in DCM heart, therefore, CGA might play an important role by influencing myocardial remodeling and fibrosis in DCM patients.

Endothelial­to­mesenchymal transition in human idiopathic dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is characterized by left ventricular dilation and cardiac fibrosis. Emerging evidence indicated that endothelial­to­mesenchymal transition (Endo­MT) is a crucial event during organ fibrosis. This study was performed to clarify whether Endo­MT contributed to the progression of cardiac fibrosis in DCM. Cardiac samples from patients with DCM and control were obtained. The presence of endothelial markers, cluster of differentiation (CD)31 and vascular endothelial (VE)­cadherin, and mesenchymal markers, α smooth muscle actin (SMA) and fibroblast­specific protein 1 (FSP1) was performed using immunohistochemistry. Co­localization of endothelial markers and mesenchymal markers were identified using confocal immunofluorescence staining. Serum procollagen type I carboxy­terminal propeptide (PICP) and procollagen type III amino­terminal propeptide (PIIINP) were measured by ELISA. Protein levels of Wnt, ß­catenin and Snail were determined using western blot analysis. Immunohistochemistry and double­immunofluorescence staining demonstrated that the expression of CD31 and VE­cadherin were significantly decreased in DCM samples, whereas the FSP­1, and αSMA were significantly increased. CD31 and VE­cadherin labeling indexes were respectively negatively correlated with left ventricular end­diastolic diameter (LVEDD) (CD31 r=­0.82, P<0.01; VE­cadherin r=-0.73, P<0.01), while FSP­1 and αSMA were positively associated with LVEDD (αSMA r=0.65, P<0.01, FSP1 r=0.53, P<0.01) and left ventricular ejection fraction (αSMA r=­0.18, P<0.05; FSP1 r=­0.21, P<0.05). Furthermore, PICP and PIIINP levels were positively associated with the co­expression labeling indexes (CD31/SMA co­labeling index and PICP r=0.727, P<0.01; CD31/SMA co­labeling index and PIIINP r=0.741, P<0.01; VE­Cadherin/FSP­1 co­labeling index and PICP r=0.716, P<0.01; VE­cadherin/FSP­1 co­labeling index and PIIINP r=0.648, P<0.05). Western blot analysis indicated that proteins levels of Wnt signaling and snail were significantly increased in DCM samples. These results suggested that Endo­MT is potentially implicated in the pathogenesis of myocardial fibrosis and remodeling during the development of DCM, indicating a potential therapeutic target for DCM treatment.

[Effect of astragaloside on myocardial fibrosis in chronic myocarditis].

OBJECTIVE: To investigate the effect of astragaloside (Astr), one of the active components of the Chinese medical herb Astragulus membranaceus, on cardiac fibrosis in chronic myocarditis and its relevant mechanisms. METHODS: Eighty mice were randomized into 3 groups, the control group (n=20), the model group (n=30) and the Astr group (n=30). Mice in the model group and the Astr group were monthly intraperitoneally inoculated with CVB3, but to the control group equal amount of culture fluid was given instead. Mice in the control and the model group were fed with drinking water while those in the Astr group with drinking water containing Astr-sodium carboxymethycellulose at a concentration of 300 mg/L. All the survived mice were sacrificed 3 months later. Heart tissue of mice was stained by picrosirius red for calculating collagen volume fraction (CVF) with an automatic image analysis system. Expressions of transforming growth factor beta1 (TGF-beta1), platelet derived growth factor (PDGF), matrix metalloproteinase 1 (MMP-1), tissue inhibitor of metalloproteinase 1 (TIMP-1), MMP-13 and MMP-14 in heart tissue were detected by Western blot analysis. RESULTS: As compared with the model group, in the Astr group, the mortality and CVF were significantly lower (53.3% vs. 23.3%, chi2 = 4.23, P < 0.05), and (17.4 +/- 1.2% vs. 8.6 +/- 0.9%, chi2 = 5.38, P < 0.05), respectively. As compared with the control group, Western blot analysis showed that expression of TGF-beta1 was decreased, MMP-1 and TIMP-1 were down-regulated, while expressions of MMP-13 and MMP-14 were up-regulated after Astr treatment. CONCLUSION: Astr could lower the mortality and alleviate the myocardial fibrosis of mice with chronic myocarditis. Its antifibrotic effect might be realized by way of inhibiting TGF-beta1 expression and up-regulating the expressions of MMP-13 and MMP-14 in the heart tissues.

[Association between myocardial ADAMTS-1 expression and myocardial fibrosis in a murine model of viral myocarditis].

OBJECTIVE: To investigate the association between myocardial ADAMTS-1 expression and myocardial fibrosis in coxsackievirus B(3) (CVB(3))-induced acute and chronic murine myocarditis model. METHODS: Balb/c mice were infected with CVB(3) (single injection or monthly injection for 3 months) to establish acute or chronic myocarditis model. Normal controls received equal-volume Eagles minimal essential medium (EMEM) without CVB(3). Hearts were examined at 7 days or 3 months post CVB(3) infection. Heart slides were stained with collagen specific picrosirius red staining and the collagen volume fraction (CVF) was calculated with image analysis software. The expressions of ADAMTS-1 were determined by RT-PCR and immunohistochemistry. RESULTS: Compared with controls, the CVF levels and myocardial expressions of ADAMTS-1 were significant increased in two myocarditis groups, especially in mice with chronic myocarditis (P < 0.01). The increased expression of ADAMTS-1 was located in endochylema as visualized by immunohistochemistry. Myocardial ADAMTS-1 mRNA was positively correlated with CVF in both myocarditis groups (r(7 days) = 0.65, P < 0.05; r(3 months) = 0.73, P < 0.01). CONCLUSIONS: ADAMTS-1 increased in proportion with collagen accumulation in acute and chronic myocarditis, which might play an important role in the development of myocardial fibrosis by modulating the collagen metabolism.

A transcriptome analysis of two grapevine populations segregating for tendril phyllotaxy.

The shoot structure of cultivated grapevine Vitis vinifera L. typically exhibits a three-node modular repetitive pattern, two sequential leaf-opposed tendrils followed by a tendril-free node. In this study, we investigated the molecular basis of this pattern by characterizing differentially expressed genes in 10 bulk samples of young tendril tissue from two grapevine populations showing segregation of mutant or wild-type shoot/tendril phyllotaxy. One population was the selfed progeny and the other one, an outcrossed progeny of a Vitis hybrid, 'Roger's Red'. We analyzed 13 375 expressed genes and carried out in-depth analyses of 324 of them, which were differentially expressed with a minimum of 1.5-fold changes between the mutant and wild-type bulk samples in both selfed and cross populations. A significant portion of these genes were direct cis-binding targets of 14 transcription factor families that were themselves differentially expressed. Network-based dependency analysis further revealed that most of the significantly rewired connections among the 10 most connected hub genes involved at least one transcription factor. TCP3 and MYB12, which were known important for plant-form development, were among these transcription factors. More importantly, TCP3 and MYB12 were found in this study to be involved in regulating the lignin gene PRX52, which is important to plant-form development. A further support evidence for the roles of TCP3-MYB12-PRX52 in contributing to tendril phyllotaxy was the findings of two other lignin-related genes uniquely expressed in the mutant phyllotaxy background.

Enhanced myocardial cathepsin B expression in patients with dilated cardiomyopathy.

OBJECTIVE: Cathepsin B is a prominent lysosomal protease and is involved in apoptosis as well as degradation of myofibrillar proteins in myocardial infarction. The aim of this study was to investigate myocardial cathepsin B expression in failing and non-failing human hearts. METHODS: Tissue samples were taken from transplanted left ventricles from 20 patients with dilated cardiomyopathy and 5 non-failing donor hearts that could not be transplanted for technical reasons. Myocardial cathepsin B expression was determined by immunohistochemistry, the reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Apoptosis was assessed by TUNEL staining. RESULTS: Positive cathepsin B staining was found in failing and non-failing hearts. The expression of cathepsin B at mRNA and protein levels was significantly higher in failing hearts compared with non-failing hearts. Correlation analysis revealed that cathepsin B at mRNA and protein levels negatively correlated with EF (r=0.66, p=0.002 and r=0.492, p=0.028, respectively) in patients with heart failure. The apoptotic index was 0.015+/-0.006 in failing hearts and 0.002+/-0.001 in non-failing hearts (p<0.01). CONCLUSION: Increased myocardial expression of cathepsin B was found in patients with heart failure suggesting that cathepsin B might play a role in the genesis and development of heart failure.