An alternative simple method for mass production of chimeric embryos by coculturing denuded embryos and embryonic stem cells in Eppendorf vials

The generation of germline competent chimeric mice via embryonic stem (ES) cells is a crucial step in developing gene-manipulated mouse models. To date, techniques for generating chimeric mice include direct microinjection of ES cells into the cavity of 3.5-d post-coitum (dpc) blastocysts and aggregating or coculturing 2.5 dpc zona pellucida-free (denuded) embryos with ES cells. We present here a procedure that is simple and reproducible for mass producing (10-150 embryos/vial/time) chimeric embryos by coculturing denuded 8-cell embryos and morula in 0.8 mL KSOM-AA medium containing 5 x 10(5)mL-1 purified green fluorescence protein-expressing ES cells (either fresh or thawed) in an 1.7 mL Eppendorf vial for 3h. The resulting chimeras had substantial levels of chimerism and high germline transmission rates. Therefore, the method developed in this study can provide a simple and mass reproducible alternative method (to germline transmitter chimeric mice), without technological and instrumental difficulties, for generating chimeric embryos.     

CD36 is a novel and potential anti-fibrogenic target in albumin-induced renal proximal tubule fibrosis

Albumin is not only a risk factor for diabetic nephropathy (DN), but also a therapeutic target. Hence, scientists have long sought ways to elucidate the interactions between albumin and diabetic renal tubule fibrosis. CD36, a surface receptor for thrombospondin-1, has been reported to interact with latent transforming growth factor-beta1 (TGF-beta1) and activate its fibrogenic bioactivity. This study elucidates the interactions between CD36 and renal tubule fibrosis. LLC-PK1 cells were applied to represent renal proximal tubule cells. The expression of CD36 was evaluated by flow cytometry. Fibronectin was assayed by Western blot and enzyme-linked immunosorbent assay (ELISA). Bioactive TGF-beta1 was assayed by ELISA. We demonstrated that albumin was shown significantly to inhibit cell growth without affecting hypertrophy status since protein content and cell size remained unaffected under albumin treatment. Moreover, albumin dose-dependently (0, 1, or 10 mg/ml) enhanced the secretion of bioactive TGF-beta1 and fibronectin with the upregulation of CD36. Intriguingly, CD36 siRNA, a potent silencer for CD36 effectively suppressed the albumin-induced increase in CD36, TGF-beta1, and even fibronectin level. Accordingly, albumin is a pro-fibrogenic factor for proximal tubule cells since albumin per se markedly upregulated the expression of TGF-beta1 and fibronectin. Most importantly, CD36 may mediate albumin-induced cellular fibrosis since CD36 siRNA appeared to have anti-fibrosis effects. This work suggests that CD36 is a novel and potential therapeutic target for diabetic renal tubule fibrosis.     

Tuning the Photocycle Kinetics of Bacteriorhodopsin in Lipid Nanodiscs

Monodisperse lipid nanodiscs are particularly suitable for characterizing membrane protein in near-native environment. To study the lipid-composition dependence of photocycle kinetics of bacteriorhodopsin (bR), transient absorption spectroscopy was utilized to monitor the evolution of the photocycle intermediates of bR reconstituted in nanodiscs composed of different ratios of the zwitterionic lipid (DMPC, dimyristoyl phosphatidylcholine; DOPC, dioleoyl phosphatidylcholine) to the negatively charged lipid (DOPG, dioleoyl phosphatidylglycerol; DMPG, dimyristoyl phosphatidylglycerol). The characterization of ion-exchange chromatography showed that the negative surface charge of nanodiscs increased as the content of DOPG or DMPG was increased. The steady-state absorption contours of the light-adapted monomeric bR in nanodiscs composed of different lipid ratios exhibited highly similar absorption features of the retinal moiety at 560 nm, referring to the conservation of the tertiary structure of bR in nanodiscs of different lipid compositions. In addition, transient absorption contours showed that the photocycle kinetics of bR was significantly retarded and the transient populations of intermediates N and O were decreased as the content of DMPG or DOPG was reduced. This observation could be attributed to the negatively charged lipid heads of DMPG and DOPG, exhibiting similar proton relay capability as the native phosphatidylglycerol (PG) analog lipids in the purple membrane. In this work, we not only demonstrated the usefulness of nanodiscs as a membrane-mimicking system, but also showed that the surrounding lipids play a crucial role in altering the biological functions, e.g., the ion translocation kinetics of the transmembrane proteins.     

Peripapillary Retinal Nerve Fiber Layer Changes in Preclinical Diabetic Retinopathy: A Meta-Analysis

BackgroundDiabetic retinopathy is a microvascular neurodegenerative disorder in diabetic patients. Peripapillary retinal nerve fiber layer changes have been described in patients with preclinical diabetic retinopathy, but study results have been inconsistent.ObjectiveTo assess changes in peripapillary retinal nerve fiber layer thickness in diabetic patients with preclinical diabetic retinopathy.MethodsA literature search was conducted through PubMed, EMBASE, Web of Science and Cochrane Library. Case-control studies on RNFL thickness in preclinical diabetic retinopathy patients and healthy controls were retrieved. A meta-analysis of weighted mean difference and a sensitivity analysis were performed using RevMan 5.2 software.ResultsThirteen case-control studies containing 668 diabetic patients and 556 healthy controls were selected. Peripapillary RNFL thickness was significantly reduced in patients with preclinical diabetic retinopathy compared to healthy controls in studies applying Optical Coherence Tomography (-2.88μm, 95%CI: -4.44 to -1.32, P = 0.0003) and in studies applying Scanning Laser Polarimeter (-4.21μm, 95%CI: -6.45 to -1.97, P = 0.0002). Reduction of RNFL thickness was significant in the superior quadrant (-3.79μm, 95%CI: -7.08 to -0.50, P = 0.02), the inferior quadrant (-2.99μm, 95%CI: -5.44 to -0.54, P = 0.02) and the nasal quadrant (-2.88μm, 95%CI: -4.93 to -0.82, P = 0.006), but was not significant in the temporal quadrant (-1.22μm, 95%CI: -3.21 to 0.76, P = 0.23), in diabetic patients.ConclusionPeripapillary RNFL thickness was significantly decreased in preclinical diabetic retinopathy patients compared to healthy control. Neurodegenerative changes due to preclinical diabetic retinopathy need more attention.     

Clustered Regularly Interspaced Short Palindromic Repeats Are emm Type-Specific in Highly Prevalent Group A Streptococci

Clustered regularly interspaced short palindromic repeats (CRISPR) are the bacterial adaptive immune system against foreign nucleic acids. Given the variable nature of CRISPR, it could be a good marker for molecular epidemiology. Group A streptococcus is one of the major human pathogens. It has two CRISPR loci, including CRISPR01 and CRISPR02. The aim of this study was to analyze the distribution of CRISPR-associated gene cassettes (cas) and CRISPR arrays in highly prevalent emm types. The cas cassette and CRISPR array in two CRISPR loci were analyzed in a total of 332 strains, including emm1, emm3, emm4, emm12, and emm28 strains. The CRISPR type was defined by the spacer content of each CRISPR array. All strains had at least one cas cassette or CRISPR array. More than 90% of the spacers were found in one emm type, specifically. Comparing the consistency between emm and CRISPR types by Simpson’s index of diversity and the adjusted Wallace coefficient, CRISPR01 type was concordant to emm type, and CRISPR02 showed unidirectional congruence to emm type, suggesting that at least for the majority of isolates causing infection in high income countries, the emm type can be inferred from CRISPR analysis, which can further discriminate isolates sharing the same emm type.