Is Pooled CRISPR-Screening the Dawn of a New Era for Functional Genomics.

Functional genomics aims to develop an in-depth understanding of how specific gene dysfunctions are related to diseases. A common method for investigating the genome and its complex functions is via perturbation of the interactions between the DNA, RNA and their protein respective protein derivatives. Commonly, arrayed and pooled genetic screens are utilized to achieve this and in recent years have been fundamental in achieving the current level of understanding for gene dysfunctions. However, they are limited in specific aspects which scientists have attempted to address. Clustered regularly palindromic repeats (CRISPR)-based methods for genetic screens have in recent years become more prevalent but crucially shared similar properties to previous methods and failing to provide a distinct advantage over previous methods. CROP-seq, Perturb-seq, and CRISPR-seq have combined CRISPR and single-cell RNA-sequencing (scRNA-seq) and is the newest addition to the geneticist's arsenal, providing scientists with methods to edit DNA with improved speed, accuracy, and efficiency which could usher us into a new era of study methods for functional genomics. We briefly overview the CRISPR-Cas9 systems, the evolution of genetic screening in recent years, and evaluate and discuss the significance of CROP-seq, Perturb-seq, and CRISPR-seq.

[Differentially expressed urine protein of contrast induced acute kidney injury by two-dimensional differential in-gel electrophoresis and mass spectrometry].

OBJECTIVE: To explore the differentially expressed proteins of contrast-induced nephropathy through a comparison of urinary proteome so as to further elucidate the pathogenesis and discover the disease biomarker. METHODS: The urine samples of 12 patients were collected before and after coronary angiography. Two dimensional electrophoresis was performed after the urine samples were labeled by different dyes. The differences of urine proteome were analyzed by Decyder software and the differentially expressed spots identified by mass spectrometry. RESULTS: A total of 56 differentially expressed spots were detected. Among them, 39 spots were up-regulated and 17 spots down-regulated. And mannose binding lectin and mannose binding lectin associated serine protease 2, key proteins in complement body activation, were both significantly up-regulated. CONCLUSION: Urine proteomic study methods are constructed based on 2D-DIGE and mass spectrometry. The lectin pathway of complement body may be associated with contrast-induced acute kidney injury.

[Analysis of incidence and risk factor in hospitalized patients with acute kidney injury].

OBJECTIVE: To investigate the epidemiology and the risk factors of acute kidney injury (AKI) in hospitalized patients in order to help clinicians better understand and prevent AKI. METHODS: All patients hospitalized in Renji Hospital of Shanghai Jiao Tong University, which is a three-level General Hospital in Shanghai, during January to December of 2008 were screened by Lab Administration Network. Study group was comprised of the patients with full clinical data of AKI, as defined by Acute Kidney Injury Network (AKIN). The incidence, etiology and distribution characteristics of hospitalized patients with AKI were retrospectively analyzed. Logistic regression analysis was used to investigate the risk factors in severity of AKI. RESULTS: Nine hundred and thirty-four patients suffering from AKI for 1 001 episodes were enrolled. The incidence of AKI in hospitalized patients was 2.4% (934/38 734). The ratio of male to female was 1.88:1. The mean age was (60.82 ± 16.94) years old. Higher incidence was seen with an increase in age. Three hundred and thirty-one(35.4%) patients with AKI were found in medical department, 592(63.4%) patients in surgical department and 11(1.2%) patients in department of gynecologic and obstetrics. Analysis of the causes of AKI showed that pre-AKI accounted for 52.0%, followed by renal parenchyma AKI (44.7%) and postrenal AKI (3.3%). The most common reason for AKI was acute tubular necrosis (ATN, 37.5%), followed by absolute (33.6%) and relative inadequacy of blood volume (13.4%). Multivariate logistic regression analysis showed that chronic kidney disease (CKD) [odds ratio (OR)=2.085, 95% confidence interval (95%CI): 1.536-2.830,P<0.01], renal injurious drugs (OR=1.438, 95%CI: 1.087-1.901 ,P<0.05), and failure of organs other than kidney (OR=1.327, 95%CI: 1.014-1.737,P<0.05) were independent risk factors for stage II-III AKI. CONCLUSION: AKI is one of the most common clinical syndromes in hospitalized patients. With the increase of age, the incidence increases gradually. The most common reasons for hospitalized AKI are pre-AKI and ATN. CKD, renal injurious drugs and failure of other organs are independent risk factors of medium to serious AKI.

[Icodextrin improve angiogenesis of peritoneal membrane in continuous ambulatory peritoneal dialysis patients].

OBJECTIVE: To observe the effect of icodextrin on peritoneal membrane angiogenesis in continuous ambulatory peritoneal dialysis (CAPD) patients. METHODS: This was a randomized double-blind perspective study of CAPD patients at our center between January 2006 to December 2006. The patients were randomized to receive either 7.5% icodextrin (ICO, n = 27) or glucose (GLU, n = 27) solution at night for 4 weeks. Peritoneal membrane function was defined as dialysate dwell for 4 hours to plasma ratio of creatinine (4 h D/Pcr) at baseline. Ultrafiltration volume, creatinine clearance (Ccr), VEGF and IL-6 in peritoneal effluent during the long night dwell (UF) dialysate were measured at baseline and after 4 weeks. The VEGF appearance was used to adjust the influences of dwell time and ultrafiltration volume. RESULTS: A total of 54 patients were enrolled. The baseline conditions showed no difference between the groups. After 2 and 4 weeks of therapy, both net UF and peritoneal creatinine clearance of long dwell were significantly higher in the ICO group than the GLU group. VEGF in night dwell PD solution was positively correlated with D/PCr (r = 0.68, P < 0.01)and negatively correlated to 4 hour ultrafiltration volume (r = -0.51, P < 0.01). The VEGF appearance was comparable between two groups at baseline. After a follow-up of 4 weeks, the VEGF appearance had an increasing tendency in the GLU group and a decreasing tendency in the ICO group but there was no significant difference. The ΔVEGF appearance (VEGF appearance in 4 week-VEGF appearance at baseline) was different between the GLU and ICO groups (9.5 ± 20.2 vs -13.4 ± 26.1, P < 0.01). IL-6 in night dwell dialysate had no difference between two groups. CONCLUSION: As compared with glucose-based solution, 7.5% icodextrin significantly decreases the local VEGF level in dialysate.

[Effect of ethyl pyruvate on expression of inflammatory factors and mitogen-activated protein kinase proteins in renal ischemic/reperfusion injury in BABL/c mice].

OBJECTIVE: To investigate the effects of ethyl pyruvate (EP) on expression of proinflammatory related gene and proteins of mitogen-activated protein kinase (MAPK) in renal tissues in ischemic/reperfusion (I/R) injury in mice. METHODS: Fifty male BABL/c mice were randomly divided into sham operation group (n=8), model group (n=10), and EP treatment group (n=32). EP treatment group was subdivided into EP pretreatment group (administration of 40 mg/kg EP 30 minutes before reproduction of model, n=8), and 4, 6 and 12 hours treatment groups (administration of 40 mg/kg EP 4, 6 and 12 hours after reproduction of model, respectively, n=8 in each group). Bilateral renal artery was occluded with a microvascular clamp for 30 minutes to reproduce kidney I/R injury model, and the kidney was harvested at 24 hours after I/R. The mRNA expressions of interleukins (IL-1ß, IL-6), tumor necrosis factor-α (TNF-α), intercellular adhesion molecule-1 (ICAM-1) and high-mobility group box 1 (HMGB1) were determined by real time reverse transcription-polymerase chain reaction (RT-PCR). The changes in protein levels of MAPKs [extracellular regulated protein kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), p38MAPK] were determined by Western blotting analysis. RESULTS: Real-time PCR assay showed that the mRNA expressions of IL-1ß, IL-6, TNF-α, ICAM-1, HMGB1 in renal tissue were much higher than those in sham operation group (IL-1ß: 12.05±8.08 vs. 3.18±1.13, IL-6: 10.26±6.85 vs. 0.81±0.34, TNF-α: 5.83±3.85 vs. 0.67±0.34, ICAM-1: 3.87±2.02 vs. 0.29±0.13, HMGB1: 652.82±78.50 vs. 112.31±32.50, all P<0.05); and the expression in EP treatment groups was markedly down-regulated than that in model group, especially in 12-hour treatment group (0.45±0.26, 0.66±0.13, 0.21±0.11, 0.05±0.02, 212.26±3.20, respectively, all P<0.05). Western blotting analysis revealed that the expression of the phosphorylated forms of ERK1/2, JNK, p38MAPK proteins was significantly higher than in sham operation group (p-ERK1/2: 1.13±0.38 vs. 0.48±0.34, p-JNK: 1.40±0.15 vs. 0.36±0.15, p-p38MAPK: 0.47±0.15 vs. 0.21±0.17, all P<0.05); the expression of the phosphorylated forms of ERK1/2, JNK, p38MAPK in each EP treatment group was significantly down-regulated compared with that in model group (all P<0.05). CONCLUSION: EP can effectively protect kidney from acute injury produced by I/R, which may be related to the regulation of proinflammatory genes and the MAPKs in renal tissue.

[Analysis of independent risk factor in patients with poor prognosis after liver transplantation].

OBJECTIVE: To investigate the prognosis after orthotopic liver transplantation (OLT), and to elucidate the risk factors of poor prognosis in these patients. METHODS: Adult recipients of OLT in Renji Hospital of Shanghai Jiaotong University were retrospectively analyzed. Data in pre-, intra- and post-OLT periods of these patients were collected. Acute kidney injury net (AKIN) criteria were used to analyze the post-OLT acute kidney injury (AKI). By following up all the patients for over a year, Kaplan-Meier survival analysis was used to evaluate the prognosis within 28 days and 1 year. Cox regression analysis was performed to evaluate risk factors of patient death, especially the influence of AKI on patient prognosis. RESULTS: There were 193 patients enrolled, the average age was (48.07+/-10.02) years old. The ratio of male to female was 4:1. One hundred and sixteen patients (60.1%) were found to have AKI after OLT. Twenty-eight-day mortality of post-OLT AKI patients was higher than that without AKI patients [15.5%(18/116) vs. 0, P<0.05], 1 year survival rate of post-OLT AKI patients was lower than that without AKI patients [(70.7% (82/116) vs. 90.9 (70/77), P<0.05). Kaplan-Meier survival analysis showed the survival rate of non-AKI (77 patients), AKI stage 1, 2 and 3 patients (58, 25 and 33 patients respectively) post-OLT were 90.9%, 81.0%, 84.0% and 42.4%, respectively. All the non-survivors were discovered to have AKI within 28 days post-OLT. Cox regression analysis showed pre-OLT hypertension [hazard ratio (HR)=4.398, 95% confidence interval (CI)ú 1.535-12.604, P=0.006], post-OLT AKI (HR=12.100, 95%CI: 1.565-93.540, P=0.017), infection (HR=4.709, 95%CI: 1.813-12.226, P=0.001) and acute physiology and chronic health evaluation II (APACHE II) score > or =10 (HR=3.627, 95%CI: 1.244-10.573, P=0.018) were risk factors of 1 year death. CONCLUSION: AKI is an independent risk factor of poor prognosis after liver transplantation. Prevention of AKI may improve the survival rate of OLT patients.

[Effects of Astragalus on expression of renal angiopoietin receptor Tie-2 in diabetic rats].

OBJECTIVE: To study the expression of angiopoietin receptor Tie-2 in the renal tissue of diabetic rats and the effects of Astragalus. METHODS: SD rats were randomly divided into normal control group, diabetes group and Astragalus-treated group. The expression of receptor Tie-2 in the renal tissue was assessed by using real-time quantitative polymerase chain reaction and immunohistochemical method. RESULTS: Glomerule Tie-2 protein expression was significantly elevated in the diabetes group as compared with the normal control group (P<0.01). Glomerule Tie-2 protein expression in the Astragalus-treated group was decreased as compared with the diabetes group (P<0.01). CONCLUSION: Tie-2 may play an important role in the pathogenesis of the early stage diabetic renal injury. The reno-protection effect of Astragalus may be mediated by down-regulating the expression of Tie-2 in the kidney tissue of diabetic rats.

Testosterone regulates the autophagic clearance of androgen binding protein in rat Sertoli cells.

Dysregulation of androgen-binding protein (ABP) is associated with a number of endocrine and andrology diseases. However, the ABP metabolism in Sertoli cells is largely unknown. We report that autophagy degrades ABP in rat Sertoli cells, and the autophagic clearance of ABP is regulated by testosterone, which prolongs the ABP biological half-life by inhibiting autophagy. Further studies identified that the autophagic clearance of ABP might be selectively regulated by testosterone, independent of stress (hypoxia)-induced autophagic degradation. These data demonstrate that testosterone up-regulates ABP expression at least partially by suppressing the autophagic degradation. We report a novel finding with respect to the mechanisms by which ABP is cleared, and by which the process is regulated in Sertoli cells.

Renal tissue thawed for 30 minutes is still suitable for gene expression analysis.

Some biosamples obtained from biobanks may go through thawing before processing. We aim to evaluate the effects of thawing at room temperature for different time periods on gene expression analysis. A time course study with four time points was conducted to investigate the expression profiling on 10 thawed normal mice renal tissue samples through Affymetrix GeneChip mouse gene 2.0 st array. Microarray results were validated by quantitative real time polymerase chain reactions (qPCR) on 6 candidate reference genes and 11 target genes. Additionally, we used geNorm plus and NormFinder to identify the most stably expressed reference genes over time. The results showed RNA degraded more after longer incubation at room temperature. However, microarray results showed only 240 genes (0.91%) altered significantly in response to thawing at room temperature. The signal of majority altered probe sets decreased with thawing time, and the crossing point (Cp) values of all candidate reference genes correlated positively with the thawing time (p<0.05). The combination of B2M, ACTB and PPIA was identified as the best choice for qPCR normalization. We found most target genes were stable by using this normalization method. However, serious gene quantification errors were resulted from improper reference genes. In conclusion, thirty minutes of thawing at room temperature has a limited impact on microarray and qPCR analysis, gene expression variations due to RNA degradation in early period after thawing can be largely reduced by proper normalization.

Sperm donation and its application in China: a 7-year multicenter retrospective study.

Sperm donation in China is different from that in other countries due to cultural, social and political factors. This research presents the current status of sperm donation in Mainland China and highlights some problems. Between January 2003 and December 2009, 19 471 sperm donors were screened totally and 6467 donors (33.2%) were recruited. The primary reasons for non-recruitment were either inadequate semen parameters (55.0%) or positive results for sexually transmitted diseases (7.9%). There were 327 (1.7%) qualified donors who withdrew from the program because of frustration related to failed semen parameters, participation merely for free medical tests or job transfer. A questionnaire investigating donor intention, as well as other concerns associated with sperm donation, was distributed to 516 potential donors. All potential donors indicated their primary motivation as altruism, while 90.9% mentioned monetary reward as a second motivating factor. Approximately 93.4% of donors expressed some apprehension about the risk of consanguineous mating and the protection of their identity. Over the past 7 years, 488 389 vials of donors' semen have been cryopreserved. In 36 438 artificial insemination with donor sperm (AID) cycles, the clinical pregnancy rate was 23.9% and the live birth rate was 16.6%. In 7148 in vitro fertilization cycles, the clinical pregnancy rate was 45.8% and the live birth rate was 35.2%. Human sperm banks have been strictly monitored to ensure that each sperm donor can only impregnate five women nationwide. There is still a large gap between the supply and demand for sperm donation which may be solved by updated guidelines.