Repurposing endogenous type II CRISPR-Cas9 system for genome editing in Streptococcus thermophilus.

Streptococcus thermophilus has been extensively used in industrial milk fermentation. However, lack of efficient genetic manipulation approaches greatly hampered the industrial application of this species. Here, we repurposed the endogenous CRISPR1 and CRISPR3 systems, both belong to type II-A CRISPR-Cas9, by delivering a self-targeting CRISPR array with DNA repair template into S. thermophilus LMD-9. We achieved 785-bp deletion in lacZ gene by repurposing CRISPR1 and CRISPR3 systems with efficiencies of 35% and 59%, respectively, when 1-kb DNA repair template was provided. While providing with 1.5-kb repair template, the editing efficiency for deletion in lacZ gene reached 90% using CRISPR3 systems. Diverse editing outcomes encompassing a stop code insertion and single nucleotide variation within lacZ, as well as a 234-bp DNA fragment insertion upstream of ster_0903, were generated with high efficiencies of 75%-100% using the CRISPR3 system. Harnessing the customized endogenous CRISPR3 system to target six genes of eps gene cluster, we obtained six single-gene knockout mutants with efficiencies of 29%-80%, and proved that the epsA, epsE, and epsG were the key genes affecting exopolysaccharides biosynthesis in S. thermophilus LMD-9. Altogether, repurposing the native type II-A CRISPR-Cas9 can be served as a toolkit for precise genome engineering in S. thermophilus for biotechnological applications.

Study on association of the central cornea histology change with age in adults by in vivo confocal microscopy / 眼科研究

Background Corneal confocal microscopy can denamically offer the morphological image of corneal cells at cellular level.To find out the dynamic change of corneal thickness and morphology of corneal cells with aging is very important for the design of corneal refractive surgery.ObjectivePresent study is to find out the important relevance of dynamic change of corneal thickness and morphology of corneal cells with aging and the design of corneal refractive surgery and investigate the influence of age on central corneal tissue and characterize precisely the anatomy of cornea by in vivo confocal microscopy.Methods122 eyes of 122 normal subjects were enrolled in this study with the age from 18 through 80 years old.The subjects were diagnosed as emmetropia in the Center of Tianjin Medical University from August 2003 to December 2007.All eyes were examined in vivo by confocal microscopy.The cell morphology,cell density and corneal thickness were measured by confocal microscopy.The relationship of central corneal tissue change and age was evaluated.ResultsThe density of corneal superficial basement epithelial cells showed a significantly negative correlation with age (r=-0.355,P=0.017).The keratocyte density in the anterior and posterior stroma indicated a significantly negative correlation with age (r_1=-0.462,P=0.001;r_2=-0.403,P=0.016).The thickness of corneal epithelium cells had a significantly negative correlation with age (P=0.02).The mean value of total corneal thickness and stromal thickness in high myopic eyes was (523.2±26.20)μm and (468.4±20.72)μm respectively,presenting a significantly negative correlation with age (r_1=-0.552,P=0.014;r_2=-0.545,P=0.035).A significantly negative correlation was also found in endothelial cell areas and density with age (r_1=0.417,P=0.004;r_2=-0.598,P=0.002).The percentage of polymorphological corneal endothelial cells was considerably increased(r=0.417,P=0.004)but that of pleomorphism cells was significantly decreased in >60-year-old population compared with younger subjects (r=-0.598,P=0.002).The morphology of corneal basement cell,anterior stroma cytocyte and endothelial cell were normal in younger subjects,and enlargement of the cells in size,decreases of number and density of the cells were found in >60-year-old subject.ConclusionThe anatomy and histology of the central cornea in young subjects exist discrepancy from older ones.The corneal hisological changes with aging include thinner thickness of epithelium,thinner thickness of cornea,decreased density of keratocyte and endothelial cells and increased size of endothelial cells.Corneal thickness is asymmetric and seems to undergo age-related anatomic changes.

The Thinking of Promoting the Medical Students' Humanities Quality Training in 21st Century / 中华医学教育探索杂志

Medical science is the science which closely combines scientific knowledge with humanistic spirit. Becausemodern science and technology have been more and more applied in medical research and practice, the medical sciencehas been developed rapidly. But medical science calls us to concern more about medical humanistic spirit, so how wecombine medical scientific knowledge with humanistic spirit is a very important topic in our medical university education.[

Long-time follow-up study of the changes of morphology of central corneal after LASIK using an in vivo confocal microscopy / 解放军医学杂志

Objective To morphologically observe the changes of cornea tissue a long period after myopic LASIK with confocal microscopy. Methods 34 patients (68 eyes) from April 1999 to July 2004, who had undertook LASIK in our Hospital, were involved in present study. The confocal microscopy was employed to examine the changes of cornea tissue. The average spherical equivalence was -6.81?2.88D (ranged from -3.0 to -16D). The average age of the patients was 24.17?5.93 (ranged from 18 to 42). The average follow-up time was 39?3 months (ranged from 6 to 73 months). Results The Micro-folds at the Bowman's layer were found in 68 eyes (100%), as well as variously reflectivity particles located at the interface in all eyes examined after operation. The density of high reflective particles did not decrease significantly with time. In the middle stroma, the mean depth of acellular area was 44.01?10.67?m in all 68 eyes (100%). The appearance of morphology of subbasel corneal nerve fiber bundles after operation recovered. Conclusions Morphologic changes were found in central cornea. Acellular area, micro-folds as well as variously reflectivity particles located at the interface in all eyes existed in the central cornea for long time.