Genome editing in rice using CRISPR/Cas12i3.

The CRISPR/Cas type V-I is a family of programmable nuclease systems that prefers a T-rich protospacer adjacent motif (PAM) and is guided by a short crRNA. In this study, the genome-editing application of Cas12i3, a type V-I family endonuclease, was characterized in rice. We developed a CRIPSR/Cas12i3-based Multiplex direct repeats (DR)-spacer Array Genome Editing (iMAGE) system that was efficient in editing various genes in rice. Interestingly, iMAGE produced chromosomal structural variations with a higher frequency than CRISPR/Cas9. In addition, we developed base editors using deactivated Cas12i3 and generated herbicide-resistant rice plants using the base editors. These CRIPSR/Cas12i3-based genome editing systems will facilitate precision molecular breeding in plants.

Cortical blindness after percutaneous vertebroplasty: a case report and comprehensive review of the literature.

OBJECTIVE: Many complications but cortical blindness after percutaneous vertebroplasty has been rarely reported. Here, we describe a case who developed cortical blindness after percutaneous vertebroplasty. We also reviewed the literature to find the possible causes of this complication and its treatment. METHODS: Case report and literature review. RESULTS: A 71-year-old woman experienced cortical blindness after percutaneous vertebroplast. She developed dizziness, nausea, sweating, blood pressure changes, and vision loss during the procedure. MRI confirmed bilateral cerebral infarctions. The patient recovered with conservative treatment. CONCLUSIONS: Percutaneous vertebroplasty, though helpful, carries a rare risk of cortical blindness. Surgeon awareness is crucial for informing patients of this potential complication.

Genome-wide identification and analysis of TMT-based proteomes in longissimus dorsi tissue from Kazakh cattle and Xinjiang brown cattle.

With the gradual completion of the human genome project, proteomes have gained extremely important value in the fields of human disease and biological process research. In our previous research, we performed transcriptomic analyses of longissimus dorsi tissue from Kazakh cattle and Xinjiang brown cattle and conducted in-depth studies on the muscles of both species through epigenetics. However, it is unclear whether differentially expressed proteins in Kazakh cattle and Xinjiang brown cattle regulate muscle production and development. In this study, a proteomic analysis was performed on Xinjiang brown cattle and Kazakh cattle by using TMT markers, HPLC classification, LC/MS and bioinformatics analysis. A total of 13,078 peptides were identified, including 11,258 unique peptides. We identified a total of 1874 proteins, among which 1565 were quantifiable. Compared to Kazakh cattle, Xinjiang brown cattle exhibited 75 upregulated proteins and 44 downregulated proteins. These differentially expressed proteins were enriched for the functions of adrenergic signaling in cardiomyocytes, fatty acid degradation and glutathione metabolism. In our research, we found differentially expressed proteins in longissimus dorsi tissue between Kazakh cattle and Xinjiang brown cattle. We predict that these proteins regulate muscle production and development through select enriched signaling pathways. This study provides novel insights into the roles of proteomes in cattle genetics and breeding.

Effectiveness of Rex shunt for improving the abnormal portal hemodynamics and portal venous pathology in EHPVO animal model.

PURPOSE: To investigate the feasibility of the re-patent EHPVO (r-EHPVO) as an animal model of Rex shunt and the effectiveness of Rex shunt in improving abnormal portal hemodynamics and portal venous pathology of EHPVO. METHODS: A total of 18 New Zealand white rabbits were randomly divided into three groups: normal control (NC) group, extrahepatic portal venous obstruction (EHPVO) group, and r-EHPVO group. The main portal vein was dissected only in the NC group. The main portal vein was narrowed by a cannula in the EHPVO group. The cannula narrowing the main portal vein was removed to restore the portal blood flow into the liver on day 14 in the r-EHPVO group. The portal pressure, splenic size, blood flow velocity, and diameter of the portal vein were measured on days 14 and 28. The shear stress (SS) and circumferential stress (CS) of the portal vein were calculated. The proximal end of the main portal vein was collected on day 28 for further pathological analysis, and the thickness and area of the intima and media were measured by Image J software. The portal pressure, splenic size, SS, CS, intima and media thickness, the ratio of intimal to medial area (I/M), and the ratio of intimal area to the sum of intimal and medial area (I/I + M) were compared among the three groups. The correlation between SS and intimal thickness and between CS and medial thickness were analyzed. RESULTS: On day 28, the portal pressure of the EHPVO group was significantly higher than that of the NC and r-EHPVO groups, but no significant difference was detected in the portal pressure between r-EHPVO and NC groups. The length and thickness of the spleen in the EHPVO and r-EHPVO groups were significantly higher than those in the NC group (P < 0.01) but were significantly lower in the r-EHPVO group than those in the EHPVO group (P < 0.05). The SS was significantly lower in the EHPVO group than in NC and r-EHPVO groups (P < 0.05) but was significantly higher in the NC group than in the r-EHPVO group (P = 0.003). The CS was significantly higher in the EHPVO and r-EHPVO groups than that in the NC group (P < 0.05) but was significantly lower in the r-EHPVO group than that in the EHPVO group (P < 0.001). The intimal thickness, I/M, and I/I + M of the EHPVO group were significantly higher than those of the NC and r-EHPVO groups (P < 0.05), but no significant difference was observed between the NC and r-EHPVO groups (P > 0.05). The SS is negatively related to intimal thickness (r = - 0.799, P < 0.001). CONCLUSION: The r-EHPVO model is feasible as an animal model of the Rex shunt. The Rex shunt could be beneficial to improving the abnormal portal hemodynamic and portal venous intimal hyperplasia by restoring the portal blood flow into the liver.

Serum amyloid A-to-albumin ratio as a potential biomarker to predict the activity, severity, and poor prognosis of systemic lupus erythematosus.

OBJECTIVES: To evaluate the predictive value of serum amyloid A-to-albumin ratio (SAR) for active systemic lupus erythematosus (SLE), severe active SLE, and poor prognosis of SLE. METHODS: One hundred and eighty-six patients with SLE undergoing treatment in our hospital were selected. The demographic characteristics, clinical data, and disease prognosis of all patients were collected and analyzed. RESULTS: There were significant differences in SLEDAI, total glyceride (TG), serum amyloid A (SAA), SAR, urinary microalbumin-to-creatinine ratio (ACR), erythrocyte sedimentation rate (ESR), albumin (ALB), complement 3 (C3), anti-dsDNA, anti-Sm positive rate, and anti-dsDNA positive rate between active SLE and stable SLE patients. TG, SAR, C3, ACR, and positive anti-dsDNA were independent influencing factors of active SLE, and the odds ratio (OR) values were 2.342, 10.921, 0.832, 1.451, and 2.476, respectively. The area under curves (AUCs) of SAA, ALB, and SAR for predicting active SLE and severe active SLE were 0.743, 0.724, 0.787, 0.711, 0.686, and 0.733, respectively. The AUC of SAR for predicting the poor prognosis of active SLE was 0.719. High SAR, high ACR, low C3, and positive anti-dsDNA were high risk factors for poor prognosis. Kaplan-Meier (K-M) survival analysis showed that patients with high SAR, high ACR, low C3, and positive anti-dsDNA had shorter continuous remission time than that with low SAR, low ACR, high C3, and negative anti-dsDNA. CONCLUSION: SAR had high predictive value for active SLE, severe active SLE, and poor prognosis of SLE. High SAR may be a potential marker for predicting the activity and prognosis of Chinese patients with SLE.

Intragenic heterochromatin-mediated alternative polyadenylation modulates miRNA and pollen development in rice.

Despite a much higher proportion of intragenic heterochromatin-containing genes in crop genomes, the importance of intragenic heterochromatin in crop development remains unclear. Intragenic heterochromatin can be recognised by a protein complex, ASI1-AIPP1-EDM2 (AAE) complex, to regulate alternative polyadenylation. Here, we investigated the impact of rice ASI1 on global poly(A) site usage through poly(A) sequencing and ASI1-dependent regulation on rice development. We found that OsASI1 is essential for rice pollen development and flowering. OsASI1 dysfunction has an important impact on global poly(A) site usage, which is closely related to heterochromatin marks. Intriguingly, OsASI1 interacts with the intronic heterochromatin of OsXRNL, a nuclear XRN family exonuclease gene involved in the processing of an miRNA precursor, to promote the processing of full-length OsXRNL and regulate miRNA abundance. We found that OsASI1-mediated regulation of pollen development partially depends on OsXRNL. Finally, we characterised the rice AAE complex and its involvement in alternative polyadenylation and pollen development. Our findings help to elucidate an epigenetic mechanism governing miRNA abundance and rice development, and provide a valuable resource for studying the epigenetic mechanisms of many important processes in crops.

Age-related trabecular bone loss is associated with a decline in serum Galectin-1 level.

BACKGROUND: Senile osteoporosis with age-related bone loss is diagnosed depending on radiographic changes of bone and bone mineral density (BMD) measurement. However, radiographic alterations are usually signs of medium-late stage osteoporosis. Therefore, biomarkers have been proposed as indicators of bone loss. In the current study, Galectin-1 (Gal-1) showed age-related decline in mice serum. The role of Gal-1 in osteoporosis has not been investigated so far. Hence, the current study illustrated the relationship of serum Gal-1 level with bone loss. METHODS: We employed 6- and 18-month-old mice to establish an animal model of age-related trabecular bone loss, whose bone density and microstructure were investigated by micro-CT. ELISA was used to measure the levels of Gal-1 in serum. The correlation analysis was performed to illustrate the relationship between serum Gal-1 levels and trabecular bone loss. In addition, immunohistochemistry was used to investigate the abundance of Gal-1 in bone marrow of mice. ELISA and western blot were performed to measure the secretion ability and protein expression of Gal-1 in bone marrow stromal cells (BMSC), hematopoietic stem cells (HSC) and myeloid progenitor (MP) respectively. Flow cytometry was used to measure BMSC number in bone marrow. Finally, male volunteers with age-related BMD decrease were recruited and the relationship between serum Gal-1 and BMD was analyzed. RESULTS: Gal-1 showed age-related decline in mice serum. Serum Gal-1 was positively associated with BV/TV of femur, tibia and L1 vertebrae in mice. BMSC secreted more Gal-1 compared with HSC and MP. BMSC number in bone marrow was significantly lower in aged mice compared with young mice. Significant attenuation of Gal-1 protein expression was observed in BMSC and HSC from aged mice compared with young mice. Further, we found a decline in serum Gal-1 levels in men with age-related BMD decrease. There was positive correlation between BMD and serum Gal-1 levels in these men. CONCLUSIONS: Age-related trabecular bone loss is associated with a decline in serum Gal-1 level in mice and men. Our study suggested Gal-1 had great potential to be a biomarker for discovering BMSC senescence, diagnosing early osteoporosis and monitoring trabecular bone loss.

Novel Wx alleles generated by base editing for improvement of rice grain quality.

Amylose content (AC), which is regulated by the Waxy (Wx) gene, is a major indicator of eating and cooking quality (ECQ) in rice (Oryza sativa). Thus far, only a limited number of mutations in the N-terminal domain of Wx were found to have a major impact on the AC of rice grains and no mutations with such effects were reported for other regions of the Wx protein. Here, nucleotide substitutions in the middle region of Wx were generated by adenine and cytosine base editors. The nucleotide substitutions led to changes in 15 amino acid residues of Wx, and a series of novel Wx alleles with ACs of 0.3%-29.43% (wild type with AC of 19.87%) were obtained. Importantly, the waxyabe2 allele showed a "soft rice" AC, improved ECQ, favorable appearance, and no undesirable agronomic traits. The transgenes were removed from the waxyabe2 progeny, generating a promising breeding material for improving rice grain quality.

Adoptive Transfer of NKG2D CAR mRNA-Engineered Natural Killer Cells in Colorectal Cancer Patients.

By fusing the extracellular domain of the natural killer (NK) cell receptor NKG2D to DAP12, we constructed a chimeric antigen receptor (CAR) to improve NK cell tumor responses. An RNA electroporation approach that provides transient expression of the CAR was adopted as a risk mitigation strategy. Expression of the NKG2D RNA CAR significantly augmented the cytolytic activity of NK cells against several solid tumor cell lines in vitro and provided a clear therapeutic benefit to mice with established solid tumors. Three patients with metastatic colorectal cancer were then treated with local infusion of the CAR-NK cells. Reduction of ascites generation and a marked decrease in number of tumor cells in ascites samples were observed in the first two patients treated with intraperitoneal infusion of low doses of the CAR-NK cells. The third patient with metastatic tumor sites in the liver was treated with ultrasound-guided percutaneous injection, followed by intraperitoneal infusion of the CAR-NK cells. Rapid tumor regression in the liver region was observed with Doppler ultrasound imaging and complete metabolic response in the treated liver lesions was confirmed by positron emission tomography (PET)- computed tomographic (CT) scanning. Our results highlight a promising therapeutic potential of using RNA CAR-modified NK cells to treat metastatic colorectal cancer.

Short tandem target mimic rice lines uncover functions of miRNAs in regulating important agronomic traits.

Improvements in plant agricultural productivity are urgently needed to reduce the dependency on limited natural resources and produce enough food for a growing world population. Human intervention over thousands of years has improved the yield of important crops; however, it is increasingly difficult to find new targets for genetic improvement. MicroRNAs (miRNAs) are promising targets for crop improvement, but their inactivation is technically challenging and has hampered functional analyses. We have produced a large collection of transgenic short tandem target mimic (STTM) lines silencing 35 miRNA families in rice as a resource for functional studies and crop improvement. Visual assessment of field-grown miRNA-silenced lines uncovered alterations in many valuable agronomic traits, including plant height, tiller number, and grain number, that remained stable for up to five generations. We show that manipulation of miR398 can increase panicle length, grain number, and grain size in rice. In addition, we discovered additional agronomic functions for several known miRNAs, including miR172 and miR156. Our collection of STTM lines thus represents a valuable resource for functional analysis of rice miRNAs, as well as for agronomic improvement that can be readily transferred to other important food crops.

Laparoscopic ligation of portosystemic shunt for the treatment of congenital intrahepatic portosystemic shunt in one newborn infant.

BACKGROUND: Congenital portosystemic shunt (CPSS) is a rare congenital portal malformation. 35.5% of CPSS is intrahepatic. Certain intrahepatic CPSS cases can be spontaneously closed within 2 years after birth. However, the intrahepatic CPSS with clinical symptoms or large shunt requires immediate treatment. In the present study, we used a laparoscopic ligation of portosystemic shunt to treat one newborn case. MATERIALS AND METHODS: An abnormal shunt between the portal and the hepatic vein was initially detected by a prenatal ultrasound in one newborn infant. The postnatal ultrasound and CT indicated a connection between the left portal and hepatic vein and a venous cyst at the anterior edge of the left liver. Owing to the hyperammonemia and the large shunt from the portal into the hepatic vein, the infant was treated with a laparoscopic ligation of the shunt. During the operation, the left portal vein and its branches were dissected and the anterior branch of the left portal vein was clamped using a Hem-o-lok. The venous cyst was sutured and ligated using a prolene suture. RESULTS: The surgery was completed successfully. The duration of the operation time was 60 min. The postoperative hospital stay was 3 days. The preoperative portal angiography indicated an intrahepatic shunt between the left portal and the left hepatic vein and a venous cyst. Postoperative portal angiography indicated the disappearance of the abnormal shunt and the venous cyst. The portal pressure was increased from the preoperative 6 cmH2O to the postoperative 12 cmH2O. The duration of the follow-up was 22 months. Following surgery, the level of serum ammonia returned to normal. The postoperative CT demonstrated that the abnormal shunt had disappeared. No symptoms of portal hypertension were noted following surgery, such as splenomegaly, hypothrombocytopenia, and upper gastrointestinal bleeding. CONCLUSION: The laparoscopic ligation of portosystemic shunt is suitable for the treatment of the intrahepatic CPSS in the newborn patient.

Laparoscopic ligation of splenic vessels for the treatment of hereditary spherocytosis in children.

BACKGROUND: Total splenectomy is the most effective surgical treatment for hereditary spherocytosis (HS). Nevertheless, post-splenectomy sepsis and hypoimmunity can pose a great risk to children. Some alternative treatments have been proposed to avoid the post-splenectomy complications. In this study, we propose such a procedure (laparoscopic ligation of splenic vessels, L-LSV) for the treatment of HS in children and investigate its effectiveness and feasibility. MATERIALS AND METHODS: A total of 17 children with HS who underwent the L-LSV at our hospital between May 2015 and Apr 2018 were enrolled in the current study. All patients were followed-up for 3-38 months (mean 19.8 months). The volume of spleen was preoperatively and postoperatively measured using the AW VolumeShare5. The size of functional spleen and the condition of splenic infarction were evaluated using ultrasound and computed tomography (CT) after surgery. The routine blood, biochemistry and coagulation tests were carried out after surgery. RESULTS: The L-LSV was successfully performed in all patients. The average operative time was 115 min (range 60-180 min). No patients underwent blood transfusion during surgery, and the length of hospital stay varied 5-9 days after surgery (mean 7 days). Postoperatively, the red blood cells, platelet and hemoglobin were significantly increased (P < 0.05). The postoperative volume of functional spleen was significantly smaller than preoperative volume of spleen (307.393 ± 177.634 cm3 vs. 581.242 ± 270.260 cm3, P = 0.000). The recent volume of functional spleen was significantly bigger than the postoperative 1 month volume of functional spleen in ten children who were followed-up for more than 1 year (P = 0.004). The index of splenic infarction (the proportion of the postoperative 1-month volume of splenic infarction in the preoperative volume of spleen) was 0.31-0.99 (mean 0.53). There were no patients undergoing the blood transfusion after surgery. CONCLUSIONS: The L-LSV is an effective treatment for HS in children; however, future studies should re-evaluate the long-term prognosis.

Knockdown of Rice MicroRNA166 Confers Drought Resistance by Causing Leaf Rolling and Altering Stem Xylem Development.

MicroRNAs are 19- to 22-nucleotide small noncoding RNAs that have been implicated in abiotic stress responses. In this study, we found that knockdown of microRNA166, using the Short Tandem Target Mimic (STTM) system, resulted in morphological changes that confer drought resistance in rice (Oryza sativa). From a large-scale screen for miRNA knockdown lines in rice, we identified miR166 knockdown lines (STTM166); these plants exhibit a rolled-leaf phenotype, which is normally displayed by rice plants under drought stress. The leaves of STTM166 rice plants had smaller bulliform cells and abnormal sclerenchymatous cells, likely causing the rolled-leaf phenotype. The STTM166 plants had reduced stomatal conductance and showed decreased transpiration rates. The STTM166 lines also exhibited altered stem xylem and decreased hydraulic conductivity, likely due to the reduced diameter of the xylem vessels. Molecular analyses identified rice HOMEODOMAIN CONTAINING PROTEIN4 (OsHB4), a member of HD-Zip III gene family, as a major target of miR166; moreover, rice plants overexpressing a miR166-resistant form of OsHB4 resembled the STTM166 plants, including leaf rolling and higher drought resistance. The genes downstream of miR166-OsHB4 consisted of polysaccharide synthesis-related genes that may contribute to cell wall formation and vascular development. Our results suggest that drought resistance in rice can be increased by manipulating miRNAs, which leads to developmental changes, such as leaf rolling and reduced diameter of the xylem, that mimic plants' natural responses to water-deficit stress.

Effects of Ghrelin on the Apoptosis of Rheumatoid Arthritis Fibroblast-Like Synoviocyte MH7A Cells.

Ghrelin is a circulating peptide hormone, which involved in promoting feeding and regulating energy metabolism in human and rodents. Abnormal synovial hyperplasia is the most important pathologic hallmark of rheumatoid arthritis (RA), which is characterised by tumor-like expansion. Existing studies indicated that there may exist some relation between the decreased ghrelin and the abnormally proliferating synovial cells in RA. Therefore, the aim of this study is to explore the apoptotic effects of ghrelin on MH7A synovial cells in vitro. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate the effects of ghrelin on the viability of MH7A cells. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling (TUNEL) and flow cytometry were used to test the apoptotic effects of ghrelin. At last, Western blot and real-time PCR were performed to explore the expression of caspases-8, -9, and -3 after the treatment of ghrelin. MTT experiments showed that ghrelin could inhibit viability of MH7A cells. The results of flow cytometry and TUNEL showed that ghrelin could induce apoptosis of MH7A synovial cells. Western blot showed that expression of cleaved-caspases-8, -9, and -3 were increased in ghrelin stimulation group compared with the control group, while expression of pro-caspases-8, -9, and -3 had no significant difference. In mRNA levels, ghrelin can decrease pro-caspases-8, -9, and -3 mRNA expression, which confirmed the results of protein levels. Then these apoptotic effects were significantly reversed by [D-Lys3] GHRP-6 (ghrelin receptor antagonist). This study found that ghrelin can induce apoptosis of MH7A cells through caspase signaling pathways.

Rex Shunt as a Treatment of Type II Abernethy Malformation with Severe Dysplasia of Portal Vein.

BACKGROUND: Ligation of abnormal portosystemic shunt has been used to treat type II Abernethy malformation, but it may not be suitable for all patients. In this study, Rex shunt was carried out to manage type II Abernethy malformation with portal venous dysplasia. The outcomes are evaluated retrospectively. METHODS: Between June 2014 and January 2015, 2 boys (age: 4.8 and 12.8 years, respectively) with type II Abernethy malformation underwent Rex shunt with ligation of inferior mesenteric vein (IMV). The portal venous pressures were measured intraoperatively to decide the extent of inferior mesenteric venous ligation. An ileal vein (6 mm in diameter) and a venae sigmoideae (7 mm in diameter) were interposed between the left portal vein and superior mesenteric vein, respectively. To minimize postoperative portal hypertension, the IMV was partially ligated. Five months later, the IMV was completely ligated in a second operation because of persistent bloody stool and anemia (hemoglobin < 100 g/L). RESULTS: The duration of the operations was 180 and 240 min. The extrahepatic portal pressure increased after bypass, but the portal pressure was less than 24 cm H2O in both patients. The fecal blood loss and frequency of hemafecia decreased after surgery. The bypass vein was patent, and the portal blood flow was shown increased on postoperative ultrasound and computed tomography. There was no hypersplenism and esophageal gastric varices. CONCLUSIONS: The surgical management of Abernethy type II malformation should be individualized. Rex shunt with ligation of portosystemic shunt is feasible and effective in patients with severe dysplasia of portal vein.

Surgical treatment for rebleeding caused by bypass failure after Rex shunt: re-Rex shunt or Warren shunt?

PURPOSE: Although Rex shunt is an effective treatment for extrahepatic portal venous obstruction (EHPVO), 4-20% children develop rebleeding postoperatively. This study was used to evaluate the surgical treatment of rebleeding after Rex shunt in our center. METHOD: From June 2008 to Jan 2017, 12 of 122 children with EHPVO underwent a second operation due to graft stenosis and occlusion after Rex shunt in our center. The abdominal ultrasound and computed tomography (CT) showed the occlusion of bypass vein in nine children, stenosis of bypass vein in two children, and the patency of bypass vein in one child with dysplasia of intrahepatic portal vein. A re-Rex shunt was performed in eight children, of which one child required conversion to Warren shunt due to postoperative rebleeding. Five children underwent Warren shunt due to a narrowed left portal vein shown by intraoperative portal angiography and surgical exploration. All patients were followed up in this study. RESULTS: The postoperative incidences of re-bleeding and esophageal varices in children with Warren shunt were significantly lower than those in children undergoing re-Rex shunt (P = 0.027 and 0.015). After a second operation, the rate of bypass vein patency in children with re-Rex shunt was lower than that in children with Warren shunt (50 vs. 100%). The postoperative reduced size of spleen in children undergoing Warren shunt was significantly higher than that of children undergoing re-Rex shunt (P < 0.05). CONCLUSIONS: Comparing to re-Rex shunt, Warren shunt was a better treatment for rebleeding caused by bypass failure after Rex shunt.

Generation of new glutinous rice by CRISPR/Cas9-targeted mutagenesis of the Waxy gene in elite rice varieties.

In rice, amylose content (AC) is controlled by a single dominant Waxy gene. We used Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated 9 (Cas9) to introduce a loss-of-function mutation into the Waxy gene in two widely cultivated elite japonica varieties. Our results show that mutations in the Waxy gene reduce AC and convert the rice into glutinous ones without affecting other desirable agronomic traits, offering an effective and easy strategy to improve glutinosity in elite varieties. Importantly, we successfully removed the transgenes from the progeny. Our study provides an example of generating improved crops with potential for commercialization, by editing a gene of interest directly in elite crop varieties.

Elevated Expression of Zinc Finger Protein 703 Promotes Cell Proliferation and Metastasis through PI3K/AKT/GSK-3ß Signalling in Oral Squamous Cell Carcinoma.

BACKGROUND/AIMS: Zinc finger protein 703 (ZNF703), initially identified as a novel oncogene in human breast cancer, is a member of the NET/NlZ family of zinc finger transcription factors. It is recognized that the overexpression of ZNF703 is associated with various types of human cancers, but the role and molecular mechanism of ZNF703 in oral squamous cell carcinoma (OSCC) are unknown. METHODS: ZNF703 expression levels were examined in OSCC tissues and non-cancerous tissues by qRT-PCR and immunohistochemistry (IHC). The molecular mechanisms of ZNF703 and its effects on cell growth and metastasis were explored in vitro and in vivo using the CCK8 assay, colony formation assay, cell cycle analysis, migration and invasion assays, wound-healing assay, western blotting and xenograft experiments in nude mice. RESULTS: In this study, ZNF703 was found to be upregulated in OSCC tissues compared to that in normal tissues at both mRNA and protein levels, and its expression level was closely correlated with the overall survival of patients with OSCC. Silencing of the ZNF703 gene in OSCC cells significantly inhibited cell growth and metastasis in vitro and in vivo. Conversely, the overexpression of ZNF703 in OSCC cells promoted cancer growth and metastasis in vitro. Mechanistically, ZNF703 activated the PI3K/AKT/GSK-3ß signalling pathway and its downstream effectors, thus regulating the cell cycle and epithelial-mesenchymal transition (EMT). Furthermore, the promotive effects of ZNF703 on cellular proliferation and metastasis could be rescued by LY294002 (a PI3K-specific inhibitor) and MK2206 (an Akt-specific inhibitor). CONCLUSION: The results show that ZNF703 promotes cell growth and metastasis through PI3K/Akt/GSK-3ß signalling in OSCC and that it may be a promising target in the treatment of patients with OSCC.

TALEN-mediated targeted mutagenesis produces a large variety of heritable mutations in rice.

CRISPR/Cas9 and TALEN are currently the two systems of choice for genome editing. We have studied the efficiency of the TALEN system in rice as well as the nature and inheritability of TALEN-induced mutations and found important features of this technology. The N287C230 TALEN backbone resulted in low mutation rates (0-6.6%), but truncations in its C-terminal domain dramatically increased efficiency to 25%. In most transgenic T0 plants, TALEN produced a single prevalent mutation accompanied by a variety of low-frequency mutations. For each independent T0 plant, the prevalent mutation was present in most tissues within a single tiller as well as in all tillers examined, suggesting that TALEN-induced mutations occurred very early in the development of the shoot apical meristem. Multigenerational analysis showed that TALEN-induced mutations were stably transmitted to the T1 and T2 populations in a normal Mendelian fashion. In our study, the vast majority of TALEN-induced mutations (~81%) affected multiple bases and ~70% of them were deletions. Our results contrast with published reports for the CRISPR/Cas9 system in rice, in which the predominant mutations affected single bases and deletions accounted for only 3.3% of the overall mutations.