Functional study of the soybean stamen-preferentially expressed gene GmFLA22a in regulating male fertility.

China has a high dependence on soybean imports, yield increase at a faster rate is an urgent problem that need to be solved at present. The application of heterosis is one of the effective ways to significantly increase crop yield. In recent years, the development of an intelligent male sterility system based on recessive nuclear sterile genes has provided a potential solution for rapidly harnessing the heterosis in soybean. However, research on male sterility genes in soybean has been lagged behind. Based on transcriptome data of soybean floral organs in our research group, a soybean stamen-preferentially expressed gene GmFLA22a was identified. It encodes a fasciclin-like arabinogalactan protein with the FAS1 domain, and subcellular localization studies revealed that it may play roles in the endoplasmic reticulum. Take advantage of the gene editing technology, the Gmfla22a mutant was generated in this study. However, there was a significant reduction in the seed-setting rate in the mutant plants at the reproductive growth stage. The pollen viability and germination rate of Gmfla22a mutant plants showed no apparent abnormalities. Histological staining demonstrated that the release of pollen grains in the mutant plants was delayed and incomplete, which may due to the locule wall thickening in the anther development. This could be the reason of the reduced seed-setting rate in Gmfla22a mutants. In summary, our study has preliminarily revealed that GmFLA22a may be involved in regulating soybean male fertility. It provides crucial genetic materials for further uncovering its molecular function and gene resources and theoretical basis for the utilization of heterosis in soybean.

Progress on genic male sterility gene in soybean.

Male sterility refers to the phenomenon that stamens cannot grow normally and produce viable pollen grains in plants. Hybrid seed production by taking advantage of the trait of male sterility is an effective and quick strategy to increase crop yield. Up to date, the yield of rice (Oryza sativa L.), maize (Zea mays L.), wheat (Triticum aestivum L.) and other crops has been greatly increased based on hybrid vigor utilization. Soybean (Glycine max (L.) Merr.) is a self-pollination species, artificial emasculation is not only time-consuming, but also labor-intensive and economically impracticable. So far, large scale hybrid breeding has not been performed in soybean due to the shortage of male sterile lines suitable for hybrid production. Therefore, it is urgent to identify a stable male sterile system for the rapid utilization of heterosis in soybean. In this review, we summarize the progress on the discovery of soybean genic male sterility (GMS) mutants and GMS genes. Combining with the investigation of GMS genes in Arabidopsis, rice and maize, we provide important insights into the identification and potential utilization of GMS genes in soybean in the perspective of reverse genetics.

Functional characterization of CEP250 variant identified in nonsyndromic retinitis pigmentosa.

Retinitis pigmentosa (RP) is the most common manifestation of inherited retinal diseases with high degree of genetic, allelic, and phenotypic heterogeneity. CEP250 encodes the C-Nap1 protein and has been associated with various retinal phenotypes. Here, we report the identification of a mutation (c.562C>T, p.R188*) in the CEP250 in a consanguineous family with nonsyndromic RP. To gain insights into the molecular pathomechanism underlying CEP250 defects and the functional relevance of CEP250 variants in humans, we conducted a functional characterization of CEP250 variant using a novel Cep250 knockin mouse line. Remarkably, the disruption of Cep250 resulted in severe impairment of retinal function and significant retinal morphological alterations. The homozygous knockin mice showed significantly reduced retinal thickness and ERG responses. This study not only broadens the spectrum of phenotypes associated with CEP250 mutations, but also, for the first time, elucidates the function of CEP250 in photoreceptors using a newly established animal model.

Retroperitoneal laparoendoscopic single-site ureterolithotomy and ureteroureterostomy for retrocaval ureter with ureteral calculus: first case report.

A 53-year-old man presented with a 6-month history of intermittent right flank pain. Radiological imaging confirmed the diagnosis of retrocaval ureter (RCU) and ureteral calculus. Retroperitoneal laparoendoscopic single-site surgery (LESS) ureterolithotomy and ureteroureterostomy was successfully performed. The operative time was 185 min and the blood loss was approximately 20 ml. The patient's postoperative course was uneventful. Postoperative analgesia was not needed. The patient was discharged on the third postoperative day. The drain and double-J stent were respectively removed at 1 and 8 weeks postoperatively. At the 3-month follow-up, nuclear scan showed no evidence of obstruction of the right kidney and the patient also remained symptom free. It may be concluded that retroperitoneal LESS repair for RCU is a feasible and safe procedure, which can be considered as a option for the management of RCU even if it is complicated by the presence of a ureteral calculus.

Targeting neuronal and glial cell types with synthetic promoter AAVs in mice, non-human primates and humans.

Targeting genes to specific neuronal or glial cell types is valuable for both understanding and repairing brain circuits. Adeno-associated viruses (AAVs) are frequently used for gene delivery, but targeting expression to specific cell types is an unsolved problem. We created a library of 230 AAVs, each with a different synthetic promoter designed using four independent strategies. We show that a number of these AAVs specifically target expression to neuronal and glial cell types in the mouse and non-human primate retina in vivo and in the human retina in vitro. We demonstrate applications for recording and stimulation, as well as the intersectional and combinatorial labeling of cell types. These resources and approaches allow economic, fast and efficient cell-type targeting in a variety of species, both for fundamental science and for gene therapy.

Retinal miRNAs variations in a large cohort of inherited retinal disease.

BACKGROUND: Although great efforts have been paid on identification of genetic predisposition in the inherited retinal disease (IRD), genetic causes of a large proportion of patients remain a mystery. This dilemma makes us attempt to speculate that genetic components other than coding genes might be an additional pool predisposing IRD. In this study, we aim to perform a mutational screening in a large cohort of IRD patients with a particular focus on retina-specific or abundant microRNAs (miRs). MATERIAL AND METHODS: A total of 324 unrelated patients with IRD were recruited. Targeted next-generation sequencing (tNGS) was performed to survey genetic mutations in 32 known miRs highly expressed in the retina, followed by validation with Sanger sequencing, co-segregation analysis in each family, and computational assessments. RESULTS: Novel genotype-phenotype associations have been uncovered. In total, six different variants in the miRs were identified, including four rare ones, miR-216a (n.56C>A), miR-216b (n.43_44insG), miR-7-2 (n.107C>T), and miR-7-3 (n.95G>A). The other two variants, miR-182 (n.106G>A) and miR-216a (n.105T>A), were considered as polymorphic. CONCLUSIONS: We for the first time screened candidate retinal miRs in patients with IRD. Although there is no convincing evidence that these variants are responsible for the IRD, the results enhance the current knowledge of the associations between IRD and miRNAs variants.

Mutations in LRP5,FZD4, TSPAN12, NDP, ZNF408, or KIF11 Genes Account for 38.7% of Chinese Patients With Familial Exudative Vitreoretinopathy.

Purpose: Familial exudative vitreoretinopathy (FEVR) is a severe hereditary retinal disorder characterized by defects in retinal vascular development. To date, six genes have been reported to be responsible for this disease, including LRP5, FZD4, TSPAN12, NDP, ZNF408, and KIF11. The purpose of our study was to investigate the genetic defects in Chinese patients with FEVR through mutational analyses of 31 pedigrees. Methods: Clinical data and peripheral blood were collected from 31 pedigrees with FEVR. All coding sequences and intron/exon junctions were amplified and sequenced comprehensively, followed by cosegregation testing to verify suspected variants in the family members. Finally, we assessed clinical relevance of the identified mutations, according to the standards and guidelines from the American College of Medical Genetics and Genomics. Results: Twelve index cases (12/31, 38.7%) were confirmed to harbor mutations in the known genes, including one previously reported mutation and 11 novel mutations. Among the detected mutations, LRP5 accounted for the largest proportion with a mean mutation rate of 16.1% (5/31, 16.1%), followed by NDP (3/31, 9.7%), FZD4 (2/31, 6.5%), TSPAN12 (1/31, 3.2%), and KIF11 (1/31, 3.2%). All the novel changes were predicted to be pathogenic by a series of bioinformatics analyses. Conclusions: We comprehensively screened six known disease-causing genes in 31 pedigrees with FEVR and achieved a clear picture of the mutation spectrum in Chinese patients with FEVR, which highlights the importance and utility of clinical genetic diagnosis.

Laparoendoscopic single-site retroperitoneoscopic adrenalectomy for pheochromocytoma: case selection, surgical technique, and short-term outcome.

PURPOSE: To present our experience with case selection and operative skills of laparoendoscopic single-site (LESS) retroperitoneoscopic adrenalectomy for pheochromocytoma and evaluate its feasibility. PATIENTS AND METHODS: Between June 2011 and December 2012, we performed LESS retroperitoneoscopic adrenalectomy for 16 patients with pheochromocytoma. In all patients, the diameter of the pheochromocytoma was less than 4.0 cm. During the operation, a single-port access was inserted through a 2.5-3.0 cm transverse incision below the tip of the 12th rib. Internally, the operative procedure duplicates the conventional retroperitoneoscopic adrenalectomy for pheochromocytoma. RESULTS: No conversions to open surgery or standard laparoscopy with additional trocars were necessary. The mean operative duration was 68.1 minutes (range 41-125 min). The mean blood loss was negligible (<50 mL), and no patient needed blood transfusion. Intraoperative hypertension (SBP>180 mmHg) occurred in 12.5% (2/16) of the patients. No patient had sustained hypertension, and none experienced intraoperative hypotension (systolic blood pressure <80 mm Hg). The only postoperative complication was one case of pneumonia successfully treated with antibiotics. The average postoperative hospital stay was 3.1 days (range 2-5 days). All patients left the hospital with a good cosmetic appearance. CONCLUSIONS: In properly selected patients, LESS retroperitoneoscopic adrenalectomy is a feasible and safe procedure for pheochromocytoma.

Retroperitoneoscopic upper pole nephroureterectomy in duplex kidney: focus on the role of dilated upper pole ureter.

PURPOSE: To describe our technique and experience with retroperitoneoscopic upper pole nephroureterectomy in duplex kidney, focusing on the role of dilated upper ureter. MATERIALS AND METHODS: From November 2004 to August 2011, retroperitoneoscopic upper pole nephroureterectomy was performed in 31 patients with a duplex kidney by a single, experienced laparoscopic surgeon. We developed our own surgical technique to suit this technically challenging procedure. Follow-up studies were performed using renal ultrasonography, intravenous urography (IVU) and/or dimercaptosuccinic acid (DMSA) renal scan in all patients at 3 months postoperatively and annually thereafter. RESULTS: All procedures were completed laparoscopically without conversion to open surgery and blood transfusion. The mean operative time was 106 (90-157) min. The estimated blood loss was < 50 mL in all cases. The mean postoperative hospital stay was 4.2 (3-7) days. Perioperative complications were limited to 1 case of peritoneal tear during a procedure and 1 case of transient postoperative fever. No major intraoperative and postoperative complication occurred. With the mean follow-up period of 41 months (range 3 to 80), no case was observed to have functional loss of the remaining lower moiety on postoperative IVU or DMSA renal scan. CONCLUSION: Retroperitoneoscopic upper pole nephroureterectomy using our technique is safe and effective.

Laparoendoscopic single-site ureteroureterostomy with intraoperative retrograde ureteroscopy-assisted technique for benign proximal and middle ureteral strictures: a single-center experience.

OBJECTIVE: To report a minimally invasive and reproducible technique that greatly facilitates the identification of the stricture during laparoendoscopic single-site ureteroureterostomy (LESS-UU) for benign proximal and middle ureteral strictures, using the intraoperative retrograde ureteroscopy-assisted technique. PATIENTS AND METHODS: Between April 2011 and January 2013, 13 patients with a benign proximal or middle ureteral stricture underwent LESS-UU at our institution. A combination of diuretic renal scans, antegrade/retrograde ureteropyelography and/or computed tomography, and stent placement or exchange was preoperatively performed to assess all patients. The intraoperative retrograde ureteroscopy-assisted technique was used to identify the exact position of the stricture and place the stenting during LESS-UU. RESULTS: Intraoperative retrograde ureteroscopy was successfully performed in all cases. The mean operative time was 156 minutes (range, 125-190 minutes), and the estimated blood loss was 80 mL (range, 20-160 mL). The mean hospital stay was 5 days (range, 4-7 days). One patient required conversion to open surgery because of the severe adhesions surrounding the stricture that resulted in failure to progress. Urine leakage occurred in 1 patient postoperatively and was successfully treated by conservative management. Postoperative fever occurred in another patient, who was treated with a dose of oral antibiotics. No major intraoperative or postoperative complication occurred. Clinical and radiographic success was achieved in 100% (13/13) of patients during a mean follow-up of 13.1 months (range, 9-27 months). CONCLUSIONS: LESS-UU is feasible and safe for repairing benign proximal and middle ureteral stricture. The intraoperative retrograde ureteroscopy-assisted technique during LESS-UU is useful for localizing the stricture.

Transperitoneal mini-laparoscopic pyeloplasty and concomitant ureteroscopy-assisted pyelolithotomy for ureteropelvic junction obstruction complicated by renal caliceal stones.

OBJECTIVE: To present our experience of combining transperitoneal mini-laparoscopic pyeloplasty (mini-LP) and concomitant ureteroscopy-assisted pyelolithotomy (U-P) for ureteropelvic junction obstruction (UPJO) complicated by renal caliceal stones in the same session. METHODS: Between May 2007 and December 2011, mini-LP and concomitant U-P was performed in nine patients with UPJO and ipsilateral renal caliceal stones. Stone location and burden were preoperatively assessed. After pyelotomy with appropriate length (about 4 mm), a 16-Fr catheter sheath replaced the uppermost or lowermost laparoscopic trocar and was introduced directly into the renal pelvis under the guidance of a guide wire and laparoscopic vision. A 7.5F rigid ureteroscopy passed through the catheter sheath into the plevis. Intracorporeal lithotripsy and/or pressure irrigation via a pump was used for caliceal stone removal. Subsequently, laparoscopic pyeloplasty was performed in a standard fashion. Postoperative imaging was assessed. RESULTS: The calculi sizes ranged from 2 to 11 mm (mean, 7.1 mm) and an average of 3 stones per patient was removed (range, 1 to 6 stones). Complete stone clearance confirmed by postoperative imaging was achieved in all patients. Mean operative time was 210 minutes, and estimated blood loss was 20 mL. Mean hospital stay was 5 days (4-7). Stent was removed after 4-8 weeks. No intraoperative or postoperative complications were noted during a mean follow-up of 18.5 months (range, 6 to 24 months). CONCLUSIONS: Mini-LP and concomitant U-P are simple and effective alternatives for the simultaneous management of UPJO complicated by coexisting ipsilateral renal caliceal stones.