Deep Learning Based Cystoscopy Image Enhancement.

Background: Endoscopy image enhancement technology provides doctors with clearer and more detailed images for observation and diagnosis, allowing doctors to assess lesions more accurately. Unlike most other endoscopy images, cystoscopy images face more complex and diverse image degradation because of their underwater imaging characteristics. Among the various causes of image degradation, the blood haze resulting from bladder mucosal bleeding make the background blurry and unclear, severely affecting diagnostic efficiency, even leading to misjudgment. Materials and Methods: We propose a deep learning-based approach to mitigate the impact of blood haze on cystoscopy images. The approach consists of two parts as follows: a blood haze removal network and a contrast enhancement algorithm. First, we adopt Feature Fusion Attention Network (FFA-Net) and transfer learning in the field of deep learning to remove blood haze from cystoscopy images and introduce perceptual loss to constrain the network for better visual results. Second, we enhance the image contrast by remapping the gray scale of the blood haze-free image and performing weighted fusion of the processed image and the original image. Results: In the blood haze removal stage, the algorithm proposed in this article achieves an average peak signal-to-noise ratio of 29.44 decibels, which is 15% higher than state-of-the-art traditional methods. The average structural similarity and perceptual image patch similarity reach 0.9269 and 0.1146, respectively, both superior to state-of-the-art traditional methods. Besides, our method is the best in keeping color balance after removing the blood haze. In the image enhancement stage, our algorithm enhances the contrast of vessels and tissues while preserving the original colors, expanding the dynamic range of the image. Conclusion: The deep learning-based cystoscopy image enhancement method is significantly better than other traditional methods in both qualitative and quantitative evaluation. The application of artificial intelligence will provide clearer, higher contrast cystoscopy images for medical diagnosis.

Comparison of AirSeal versus conventional insufflation system for retroperitoneal robot-assisted laparoscopic partial nephrectomy: a randomized controlled trial.

PURPOSE: AirSeal is a valve-less trocar insufflation system which is widely used in robotic urologic surgeries. More evidence is needed concerning the application and cost of AirSeal in retroperitoneal robot-assisted laparoscopic partial nephrectomy. METHODS: We conducted a randomized controlled trial enrolling 62 patients who underwent retroperitoneal robot-assisted laparoscopic partial nephrectomy from February 2022 to February 2023 in the Peking Union Medical College Hospital. Patients were randomly assigned into AirSeal insufflation (AIS) group and conventional insufflation (CIS) group. The primary outcome was the rate of subcutaneous emphysema (SCE). RESULTS: The SCE rate in the AIS group (12.9%) was significantly lower than that in the CIS group (35.5%) (P = 0.038). Lower maximum end-tidal carbon dioxide (CO2) (41 vs 45 mmHg, P = 0.011), PaCO2 at the end of the operation (40 vs 45 mmHg, P < 0.001), maximum tidal volume (512 vs 570 ml, P = 0.003), frequency of lens cleaning (3 vs 5, P < 0.001), pain score at 8 h (3 vs 4, P = 0.025), 12 h (2 vs 3, P = 0.029) postoperatively and at time of discharge (1 vs 2, P = 0.002) were observed in the AIS group, despite a higher hospitalization cost (68,197 vs 64658RMB, P < 0.001). Logistic regression analysis identified insufflation approach was the only influencing factor for the occurrence of SCE events. CONCLUSION: AirSeal insufflation system exhibited similar efficacy and improved safety for retroperitoneal robot-assisted laparoscopic partial nephrectomy than conventional insufflation system, despite an affordable increase of hospitalization costs.

Prevalence of Urolithiasis in a China Hospital: A Cross-Sectional Study.

BACKGROUND: This study aimed to investigate the prevalence of urolithiasis and its association with systemic diseases among inpatients in a top hospital of China. METHODS: This cross-sectional study included all inpatients in Peking Union Medical College Hospital (PUMCH) from 1 January 2017 to 31 December 2017. Patients were divided in two groups, the urolithiasis group and non-urolithiasis group. Subgroup analysis according to payment type (General or VIP ward), hospitalization department (surgical or not) and age was performed with patients in the urolithiasis group. Additionally, Univariable and multivariable regression analyses were performed to determine factors associated with urolithiasis prevalence. RESULTS: 69518 hospitalized cases were included in this study. The age was 53.40 ± 15.05 and 48.00 ± 18.12 years, and the male-to-female ratio was 1.7:1 and 0.55:1 in the urolithiasis and non-urolithiasis groups, respectively (p < 0.01). Among all patients, urolithiasis had a prevalence of 1.78%. The rate depending on payment type (5.73% versus 9.05%, p < 0.01) and hospitalization department (56.37% versus 70.91%, p < 0.01) were significantly lower in urolithiasis group compared to the non-urolithiasis. Urolithiasis rate also showed age differences. Female was independent protective factor of urolithiasis, while age, hospitalization in the non-surgical department and payment type of general ward were risk factors of urolithiasis (all p < 0.01). CONCLUSIONS: Gender, age, hospitalization in the non-surgical department and the socioeconomic status, specifically, the payment type of general ward, are independently associated with urolithiasis.

Prevalence of Urolithiasis in a China Hospital: A Cross-Sectional Study

Background: This study aimed to investigate the prevalence of urolithiasis and its association with systemic diseases among inpatients in a top hospital of China. Methods: This cross-sectional study included all inpatients in Peking Union Medical College Hospital (PUMCH) from 1 January 2017 to 31 December 2017. Patients were divided in two groups, the urolithiasis group and non-urolithiasis group. Subgroup analysis according to payment type (General or VIP ward), hospitalization department (surgical or not) and age was performed with patients in the urolithiasis group. Additionally, Univariable and multivariable regression analyses were performed to determine factors associated with urolithiasis prevalence. Results: 69518 hospitalized cases were included in this study. The age was 53.40 ± 15.05 and 48.00 ± 18.12 years, and the male-to-female ratio was 1.7:1 and 0.55:1 in the urolithiasis and non-urolithiasis groups, respectively (p < 0.01). Among all patients, urolithiasis had a prevalence of 1.78%. The rate depending on payment type (5.73% versus 9.05%, p < 0.01) and hospitalization department (56.37% versus 70.91%, p < 0.01) were significantly lower in urolithiasis group compared to the non-urolithiasis. Urolithiasis rate also showed age differences. Female was independent protective factor of urolithiasis, while age, hospitalization in the non-surgical department and payment type of general ward were risk factors of urolithiasis (all p < 0.01). Conclusions: Gender, age, hospitalization in the non-surgical department and the socioeconomic status, specifically, the payment type of general ward, are independently associated with urolithiasis (AU)

Subcutaneous Adipose Tissue Accumulation Is an Independent Risk Factor of Urinary Stone in Young People.

Background: Urinary stones usually start at a young age and tend to recur. Therefore, preventing stone occurrence and recurrence in young people is crucial. We aimed to investigate the association between subcutaneous adipose tissue, visceral adipose tissue, and stone episodes in young people. Methods: We retrospectively studied patients aged below 40 years with kidney or ureteral stones. Data on demographic and metabolic characteristics, urolithiasis history, subcutaneous fat area (SFA), and visceral fat area (VFA) were collected. We evaluated the association between SFA or VFA and the occurrence or recurrence of stone episodes using binary logistic regression and Poisson regression analyses. Results: In total, 120 patients were included. Abdominal obesity, overweight or obesity, dyslipidemia, metabolic syndrome, SFA, and VFA increased with the number of stone episodes (all p < 0.05). The increase in SFA was independently associated with episode occurrence (p = 0.015). Patients with an SFA > 97 cm2 had a higher risk of episode occurrence. SFA and VFA accumulation were independently associated with episode recurrence (all p < 0.05), and SFA had a stronger association than VFA did. Conclusions: In young people, SFA accumulation is an independent and early risk factor for the occurrence and recurrence of stone episodes. Subcutaneous fat could be a convenient and effective indicator to assess the risk of stone episodes before the development of metabolic disorders.

Surface Oxygen Vacancies Confined by Ferroelectric Polarization for Tunable CO Oxidation Kinetics.

Surface oxygen vacancies have been widely discussed to be crucial for tailoring the activity of various chemical reactions from CO, NO, to water oxidation by using oxide-supported catalysts. However, the real role and potential function of surface oxygen vacancies in the reaction remains unclear because of their very short lifetime. Here, it is reported that surface oxygen vacancies can be well confined electrostatically for a polarization screening near the perimeter interface between Pt {111} nanocrystals and the negative polar surface (001) of ferroelectric PbTiO3. Strikingly, such a catalyst demonstrates a tunable catalytic CO oxidation kinetics from 200 °C to near room temperature by increasing the O2 gas pressure, accompanied by the conversion curve from a hysteresis-free loop to one with hysteresis. The combination of reaction kinetics, electronic energy loss spectroscopy (EELS) analysis, and density functional theory (DFT) calculations, indicates that the oxygen vacancies stabilized by the negative polar surface are the active sites for O2 adsorption as a rate-determining step, and then dissociated O moves to the surface of the Pt nanocrystals for oxidizing adsorbed CO. The results open a new pathway for tunable catalytic activity of CO oxidation.

The Small Size and Superficial Location Suggest That Laparoscopic Partial Nephrectomy Is the First Choice for the Treatment of Juxtaglomerular Cell Tumors.

Background: Juxtaglomerular cell tumor (JGCT) is a very rare disease, and surgical resection is the only possible way to cure this tumor. Open nephrectomy and partial nephrectomy have been reported to manage JGCTs with excellent results in the previous reviews. Laparoscopic surgery has been popularized in recent years, while critical issues associated with laparoscopic surgical management have been seldom reported. We summarized the JGCTs in our center to discover the optimal surgical management and its anatomic foundation. Methods: In this retrospective study, we enrolled a total of 14 JGCT patients. All patients received surgeries and were followed up for up to 11 years. We mainly summarized the size and location of tumors, imaging features, and surgical strategies. A descriptive statistical analysis was performed. Results: The JGCTs in this study had a median size of 1.35 cm and all located superficially, mainly in the cortical or subcortical area of the kidney. All 14 patients had hypertension, ten had hypokalemia, and seven had elevated plasma renin activity. Pathologically, JGCT cells were polygonal or spindle shape, with positive CD34 and vimentin immunostaining. All patients received partial nephrectomy; nine were laparoscopic, and five were open. Laparoscopic partial nephrectomy (LPN) was performed in seven out of eight patients over the last nine years. Postoperative blood pressure, serum potassium, and plasma renin activity were normal in all patients. No recurrence occurred within a median follow-up of 60 months. Conclusion: The small size and superficial location are the characteristic anatomic features of JGCT; they suggest that LPN is the preferred surgical strategy. Laparoscopic ultrasound is helpful for the intraoperative detection of small JGCTs. Longer follow-up is required to examine the biological behavior of JGCTs and the effect of LPN.

Long non-coding RNA MEG3 suppresses the development of bladder urothelial carcinoma by regulating miR-96 and TPM1.

We aimed at investigating effects of long non-coding RNA maternally expressed 3 (MEG3) on the proliferation, cell cycle and apoptosis of bladder urothelial carcinoma cells and regulatory relationships among lncRNA MEG3, miR-96 and α-tropomyosin 1 (TPM1). Human clinical data from The Cancer Genome Atlas (TCGA) which contains bladder urothelial carcinoma tissues and adjacent tissues were used for analysis. The expression profiles of MEG3, miR-96, TPM1, cell cycle-related genes and apoptosis-related genes were examined by real-time quantitative polymerase chain reaction (RT-qPCR) and western blot. Regulating relationship among MEG3, miR-96 and TPM1 was confirmed by dual luciferase reporter assay. MTT assay and flow cytometry were performed to observe cell proliferation, cell cycle and apoptosis. The effects of lncRNA MEG3 on bladder urothelial carcinoma were confirmed both in vivo and in vitro. The mRNA expression and protein expression of MEG3, TPM1 were down-regulated in carcinoma tissues, whereas miR-96 expression was up-regulated. MEG3 overexpression resulted in miR-96 downregulation along with TPM1 upregulation, which inhibited cell proliferation and cell cycle but promoted cell apoptosis of bladder urothelial carcinoma cells in vitro, and at the same time inhibited tumor growth in vivo. In this process, expressions of apoptosis-related protein BCL2 associated X (Bax), cleaved-caspase 3 was up-regulated, whereas apoptosis regulator protein (Bcl-2) expression was suppressed when MEG3 was overexpressed, and cell cycle-related protein Cyclin D1 was down-regulated. LncRNA MEG3 low-expression promotes the proliferation and inhibits apoptosis of bladder urothelial carcinoma cells by regulating miR-96 along with TPM1.

LncRNA TP73-AS1 Promotes Cell Proliferation and Inhibits Cell Apoptosis in Clear Cell Renal Cell Carcinoma Through Repressing KISS1 Expression and Inactivation of PI3K/Akt/mTOR Signaling Pathway.

BACKGROUND/AIMS: Emerging evidence suggests that long non-coding RNAs (lncRNAs) play a vital regulatory role in the pathogenesis and progression of renal cell carcinoma (RCC). We aim to determine lncRNA profiles in clear cell RCC (ccRCC) and investigate key lncRNAs involved in ccRCC tumorigenesis and progression. METHODS: RNA sequencing technique and qPCR were used to determine the candidate lncRNAs in ccRCC tissues. The correlations between lncRNA P73 antisense RNA 1T (TP73-AS1) levels and survival outcomes were analyzed to elucidate its clinical significance. The underlying mechanisms of TP73-AS1 in ccRCC were analyzed through in vitro functional assays. RESULTS: We found TP73-AS1 was upregulated in 40 ccRCC tissues compared with adjacent normal renal tissues and increased TP73-AS1 was correlated to aggressive clinicopathologic features and unfavorable prognosis. Knockdown of TP73-AS1 suppressed cell proliferation, invasion and induced cell apoptosis. We also identified KISS-1 metastasis-suppressor (KISS1) was significantly upregulated in TP73-AS1 knockdown cells. Further, we revealed that TP73-AS1 suppressed KISS1 expression through the interaction with Enhancer of zeste homolog 2 (EZH2) and the specific binding to KISS1 gene promoter region. Knockdown of KISS1 partly reversed TP73-AS1 knockdown-induced inhibition of cell proliferation and promotion of apoptosis. We further determined that TP73-AS1 knockdown activated PI3K/Akt/mTOR signaling pathway, while overexpression of TP73-AS1 induced inhibition of PI3K/Akt/mTOR pathway and these effects could be partly abolished by overexpression of KISS1. CONCLUSION: In conclusion, we identified that TP73-AS1 as an oncogenic lncRNA in the development of ccRCC and a potential target for human renal carcinoma treatment.

Preservation of type H vessels and osteoblasts by enhanced preosteoclast platelet-derived growth factor type BB attenuates glucocorticoid-induced osteoporosis in growing mice.

Survival of chronic diseases in childhood is often achieved utilizing glucocorticoids, but comes with significant side effects, including glucocorticoid-induced osteoporosis (GIO). Knowledge of the mechanism of GIO is limited to the adult skeleton. We explored the effect of genetic loss and inhibition of cathepsin K (Ctsk) as a potential treatment target in a young GIO mouse model as genetic loss of cathepsin K results in a mild form of osteopetrosis secondary to impaired osteoclast bone resorption with maintenance of bone formation. We first characterized the temporal osteoclast and osteoblast progenitor populations in Ctsk-/- and wild type (WT) mice in the primary and secondary spongiosa, as sites representative of trabecular bone modeling and remodeling, respectively. In the primary spongiosa, Ctsk-/- mice had decreased numbers of osteoclasts at young ages (2 and 4 weeks) and increased osteoblast lineage cells at later age (8 weeks) relative to WT littermates. In the secondary spongiosa, Ctsk-/- mice had greater numbers of osteoclasts and osteoblast lineage cells relative to WT littermates. We next developed a young GIO mouse model with prednisolone 10 mg/m2/day injected intraperitoneally daily from 2 through 6 weeks of age. Overall, WT-prednisolone mice had lower bone volume per tissue volume, whereas Ctsk-/--prednisolone mice maintained a similar bone volume relative to Ctsk-/--vehicle controls. WT-prednisolone mice exhibited a decreased number of osteoclasts, tartrate-resistant acid phosphatase and platelet-derived growth factor type BB (PDGF-BB) co-positive cells, type H endothelial cells, and osteoblasts relative to WT-vehicle mice in both the primary and secondary spongiosa. Interestingly, Ctsk-/--prednisolone mice demonstrated a paradoxical response with increased numbers of all parameters in primary spongiosa and no change in secondary spongiosa. Finally, treatment with a cathepsin K inhibitor prevented WT-prednisolone decline in osteoclasts, osteoblasts, type H vessels, and bone volume. These data demonstrate that cells in the primary and secondary spongiosa respond differently to glucocorticoids and genetic manipulation. Inhibition of osteoclast resorption that preserves osteoclast coupling factors, such as through inhibition of cathepsin K, may be a potential preventive treatment strategy against GIO in the growing skeleton.

SP1-induced up-regulation of lncRNA SNHG14 as a ceRNA promotes migration and invasion of clear cell renal cell carcinoma by regulating N-WASP.

Clear cell renal cell carcinoma (ccRCC) is one of the leading causes of genitourinary cancer-related death, largely due to the metastasis of ccRCC. Previous profiling study showed that lncRNAs are critical regulators in lots of cancers. However, the roles of specific lncRNAs in ccRCC migration and invasion are still unknown. In this study, we utilized the high-throughput genome sequencing to identify the potential differentially expressed lncRNAs in ccRCC and further determined the underlying regulatory mechanism. We found that lncRNA SNHG14 was significantly up-regulated in ccRCC cell lines in contrast to normal renal epithelial cells. By performing bioinformatics analysis and luciferase reporter assays, we revealed that the transcription factor SP1 can bind to the promoter region of SNHG14, resulting in the overexpression of SNHG14 in ccRCC. Functionally, enhanced expression of lncRNA SNHG14 promoted cell migration and invasion through promoting N-WASP protein level. More importantly, RT-qPCR and in situ RNA FISH analysis showed that SNHG14 was predominantly abundant in the cytoplasm of ccRCC cells. The subsequent RNA immunoprecipitation assay, and gain or loss-function assays showed that SNHG14 functioned as ceRNA to regulate N-WASP expression and cell motility ability via a miR-203-dependent manner. Our results imply that SNHG14 is a critical lncRNA that promotes ccRCC migration and invasion via sponging miR-203 and elevating N-WASP. Therefore, SNHG14 could serve as a promising therapeutic target for ccRCC.

CrWSKP1, an SKP1-like Gene, Is Involved in the Self-Incompatibility Reaction of "Wuzishatangju" (Citrus reticulata Blanco).

Plant S-phase kinase-associated protein 1 (SKP1) genes play crucial roles in plant development and differentiation. However, the role of SKP1 in citrus is unclear. Herein, we described a novel SKP1-like gene, designated as CrWSKP1, from "Wuzishatangju" (Citrus reticulata Blanco). The cDNA sequence of CrWSKP1 is 779 base pairs (bp) and contains an open reading frame (ORF) of 477 bp. The genomic sequence of the CrWSKP1 gene is 1296 bp with two exons and one intron. CrWSKP1 has high identity with SKP1-like genes from other plant species within two conserved regions. Approximately 85% of pollen tubes of self-pollinated CrWSKP1 transgenic tobaccos became twisted at four days after self-pollination. Pollen tube numbers of self-pollinated CrWSKP1 transformants entering into ovules were significantly fewer than that of the control. Seed number of self-pollinated CrWSKP1 transformants was significantly reduced. These results suggested that the CrWSKP1 is involved in the self-incompatibility (SI) reaction of "Wuzishatangju".

Plasma-Derived Fibronectin Stimulates Chondrogenic Differentiation of Human Subchondral Cortico-Spongious Progenitor Cells in Late-Stage Osteoarthritis.

Migration and chondrogenesis of human subchondral cortico-spongious progenitor cells (SPCs) are the key steps in the repair of microfracture-induced articular cartilage defects. The aim of this study was to evaluate the effect of human plasma-derived fibronectin (Fn) on the chondrogenic differentiation of SPCs, which was isolated from subchondrol cortico-spongious bone of late-stage osteoarthritis (OA) patients. SPCs were isolated and cultured for three passages. Stem cell surface antigens of SPCs were analyzed by flow cytometry. The osteogenic, chondrogenic and adipogenic differentiation potential were detected by histological staining. The chondrogenesis potential of SPCs with or without stimulation of either Fn or BMP-2 were studied by immunochemical staining and gene expression analysis. Cells isolated from subchondral bone presented to be positive for CD44, CD73, CD90, and CD166, and showed high capacity of osteogenic, adipogenic and chondrogenic differentiation, which suggested this cell population to be MSC-like cells. Stimulating with Fn increased the expression of SOX-9, aggrecan, collagen II while decreased the formation of collagen I by immunochemical staining. Gene expression analysis showed similar results. These results suggest that plasma-derived Fn can increase the chondrogenic differentiation of SPCs isolated from late-stage OA and improve cartilage repair after microfracture.

CD146 as a new marker for an increased chondroprogenitor cell sub-population in the later stages of osteoarthritis.

Cartilage-derived mesenchymal stem cells (MSCs) have been isolated with different methods. In this study lateral and medial femoral condyles were respectively collected from patients with late-stage osteoarthritis during the total knee arthroplasty. After digestion of the cartilage tissues with type II collagenase and analysis by fluorescence-activated cell sorting (FACS) with CD146, a chondroprogenitor cell sub-population were isolated and purified. The expression of other MSC-associated markers in the CD146+ chondroprogenitors was analyzed by flow cytometry. Multi-lineage differentiation capacity of CD146+ chondroprogenitors was compared with that of unsorted chondrocytes and adipose-derived MSCs (ADMSCs). Higher percentage of CD146+ chondroprogenitors isolated from the medial femoral condyles was observed than that from the lateral. CD146+ chondroprogenitors expressed high levels of MSC-specific surface antigens, and showed higher chondrogenesis capacity than ADMSCs and unsorted chondrocytes in a 3D cell pellet culture model. Thus CD146 might be a new cell surface marker for cartilage progenitor cell population in the late-stage osteoarthritis.

Mixed phenotype acute leukemia.

OBJECTIVE: To highlight the current understanding of mixed phenotype acute leukemia (MPAL). DATA SOURCES: We collected the relevant articles in PubMed (from 1985 to present), using the terms "mixed phenotype acute leukemia", "hybrid acute leukemia", "biphenotypic acute leukemia", and "mixed lineage leukemia". We also collected the relevant studies in WanFang Data base (from 2000 to present), using the terms "mixed phenotype acute leukemia" and "hybrid acute leukemia". STUDY SELECTION: We included all relevant studies concerning mixed phenotype acute leukemia in English and Chinese version, with no limitation of research design. The duplicated articles are excluded. RESULTS: MPAL is a rare subgroup of acute leukemia which expresses the myeloid and lymphoid markers simultaneously. The clinical manifestations of MPAL are similar to other acute leukemias. The World Health Organization classification and the European Group for Immunological classification of Leukaemias 1998 criteria are most widely used. MPAL does not have a standard therapy regimen. Its treatment depends mostly on the patient's unique immunophenotypic and cytogenetic features, and also the experience of individual physician. The lack of effective treatment contributes to an undesirable prognosis. CONCLUSION: Our understanding about MPAL is still limited. The diagnostic criteria have not been unified. The treatment of MPAL remains to be investigated. The prognostic factor is largely unclear yet. A better diagnostic criteria and targeted therapeutics will improve the therapy effect and a subsequently better prognosis.

Identifying differentially expressed genes in pollen from self-incompatible "Wuzishatangju" and self-compatible "Shatangju" mandarins.

Self-incompatibility (SI) is one of the important factors that can result in seedless fruit in Citrus. However, the molecular mechanism of SI in Citrus is not yet clear. In this study, two suppression subtractive hybridization (SSH) libraries (forward, F and reverse, R) were constructed to isolate differentially expressed genes in pollen from "Wuzishatangju" (SI) and "Shatangju" (self-compatibility, SC) mandarins. Four hundred and sixty-eight differentially expressed cDNA clones from 2077 positive clones were sequenced and identified. Differentially expressed ESTs are possibly involved in the SI reaction of "Wuzishatangju" by regulating pollen development, kinase activity, ubiquitin pathway, pollen-pistil interaction, and calcium ion binding. Twenty five SI candidate genes were obtained, six of which displayed specific expression patterns in various organs and stages after self- and cross-pollination. The expression level of the F-box gene (H304) and S1 (F78) in the pollen of "Wuzishatangju" was 5-fold higher than that in "Shatangju" pollen. The F-box gene, S1, UBE2, UBE3, RNaseHII, and PCP were obviously up-regulated in pistils at 3 d after self-pollination of "Wuzishatangju", approximately 3-, 2-, 10-, 5-, 5-, and 2-fold higher, respectively than that at the same stage after cross-pollination of "Wuzishatangju" × "Shatangju" pistils. The potential involvement of these genes in the pollen SI reaction of "Wuzishatangju" is discussed.

Identification of differentially expressed genes in pistils from self-incompatible Citrus reticulata by suppression subtractive hybridization.

Self-incompatibility (SI) is one important factor that can result in Citrus seedlessness. However, the molecular mechanism of SI in Citrus is not clear yet. To isolate the pistil's SI-related genes, a suppression subtractive hybridization library was constructed using mature pistils of 'Wuzishatangju' mandarin (SI) as the tester and mature pistils of 'Shatangju' mandarin (self-compatibility, SC) as the driver. 229 differentially expressed cDNA clones from 967 positive clones were sequenced and identified. Differentially expressed ESTs are possibly involved in the SI reaction of 'Wuzishatangju' through a regulating signaling pathway, serine/threonine phosphatase activity, receptor kinase, embryonic development, gibberellin stimulus, or transcription. 11 out of 36 SI candidate genes displayed different expression patterns in various tissues and stages after self- and cross-pollination of 'Wuzishatangju'. The expression of CaBP (WY65), a senescence-protease (WY372), an unknown gene (WY283), and a WRKY (WY17) were up-regulated in the styles of 'Wuzishatangju' while higher expression of WY190 was observed in styles of 'Shatangju'. Highest expression levels of WY65, WY372, an annexin (WY598), the zinc-finger protein (WY376), a C2-protein (WY291), and an unknown gene (WY318) were detected in styles at 3 days after self-pollination of 'Wuzishatangju' while lowest levels were observed in styles at 3 days after cross-pollination of 'Wuzishatangju' × 'Shatangju'. The potential involvement of these genes in the SI reaction is discussed.

Molecular characterization and expression analysis of ubiquitin-activating enzyme E1 gene in Citrus reticulata.

Ubiquitin-activating enzyme E1 (UBE1) catalyzes the first step in the ubiquitination reaction, which targets a protein for degradation via a proteasome pathway. UBE1 plays an important role in metabolic processes. In this study, full-length cDNA and DNA sequences of UBE1 gene, designated CrUBE1, were obtained from 'Wuzishatangju' (self-incompatible, SI) and 'Shatangju' (self-compatible, SC) mandarins. 5 amino acids and 8 bases were different in cDNA and DNA sequences of CrUBE1 between 'Wuzishatangju' and 'Shatangju', respectively. Southern blot analysis showed that there existed only one copy of the CrUBE1 gene in genome of 'Wuzishatangju' and 'Shatangju'. The temporal and spatial expression characteristics of the CrUBE1 gene were investigated using semi-quantitative RT-PCR (SqPCR) and quantitative real-time PCR (qPCR). The expression level of the CrUBE1 gene in anthers of 'Shatangju' was approximately 10-fold higher than in anthers of 'Wuzishatangju'. The highest expression level of CrUBE1 was detected in pistils at 7days after self-pollination of 'Wuzishatangju', which was approximately 5-fold higher than at 0 h. To obtain CrUBE1 protein, the full-length cDNA of CrUBE1 genes from 'Wuzishatangju' and 'Shatangju' were successfully expressed in Pichia pastoris. Pollen germination frequency of 'Wuzishatangju' was significantly inhibited with increasing of CrUBE1 protein concentrations from 'Wuzishatangju'.

Cloning and expression analysis of S-RNase homologous gene in Citrus reticulata Blanco cv. Wuzishatangju.

S-RNase-based self-incompatibility is the most widespread form of genetically controlled mate selection in plants and that S-RNase controls pollination specificity in the pistils. 'Wuzishatangju' (Citrus reticulata Blanco), a nature bud mutant from a self-compatible (SC) cultivar 'Shatangju', displays gametophytic self-incompatibility (GSI). In this study, full-length sequences of cDNA and DNA of the S-RNase homologous gene were obtained from 'Wuzishatangju' and 'Shatangju'. There was no difference in ORF sequences of the S-RNase cDNA between 'Wuzishatangju' and 'Shatangju'. However, 13, 9 and 6 consecutive bases were missing in 'Wuzishatangju' cDNA 5' UTR, 3' UTR and genomic DNA, respectively. Tissue-specific expression of the S-RNase gene was detected using semi-quantitative RT-PCR and quantitative real-time PCR. The expression level of the S-RNase gene in styles of 'Wuzishatangju' was approximately 10- and 5-fold higher than that in leaves and pollen, respectively. When 'Wuzishatangju' was self-pollinated, the expression of S-RNase in pistils peaked at 3 days, which was approximately 10-fold higher than that at 4h and 7 days, while in cross-pollination of 'Wuzishatangju' x 'Shatangju' the expression was very weak at 3 days. Results from a Southern blot showed that two copies of the S-RNase gene existed in genomic DNA of both 'Wuzishatangju' and 'Shatangju'.