Selective cell targeting with light-absorbing microparticles and nanoparticles

We describe a new method for selective cell targeting based on the use of light-absorbing microparticles and nanoparticles that are heated by short laser pulses to create highly localized cell damage. The method is closely related to chromophore-assisted laser inactivation and photodynamic therapy, but is driven solely by light absorption, without the need for photochemical intermediates (particularly singlet oxygen). The mechanism of light-particle interaction was investigated by nanosecond time-resolved microscopy and by thermal modeling. The extent of light-induced damage was investigated by cell lethality, by cell membrane permeability, and by protein inactivation. Strong particle size dependence was found for these interactions. A technique based on light to target endogenous particles is already being exploited to treat pigmented cells in dermatology and ophthalmology. With exogenous particles, phamacokinetics and biodistribution studies are needed before the method can be evaluated against photodynamic therapy for cancer treatment. However, particles are unique, unlike photosensitizers, in that they can remain stable and inert in cells for extended periods. Thus they may be particularly useful for prelabeling cells in engineered tissue before implantation. Subsequent irradiation with laser pulses will allow control of the implanted cells (inactivation or modulation) in a noninvasive manner.     

An optical platform for cell tracking in adult zebrafish

Adult zebrafish are being increasingly used as a model in cancer and stem cell research. Here we describe an integrated optical system that combines a laser scanning confocal microscope (LSCM) and an in vivo flow cytometer (IVFC) for simultaneous visualization and cell quantification. The system is set up specifically for non-invasive tracking of both stationary and circulating cells in adult zebrafish (casper) that have been engineered to be optically transparent. Confocal imaging in this instrument serves the dual purpose of visualizing fish tissue microstructure and an imaging-based guide to locate a suitable vessel for quantitative analysis of circulating cells by IVFC. We demonstrate initial testing of this novel instrument by imaging the transparent adult zebrafish casper vasculature and tracking circulating cells in CD41-GFP/Gata1-DsRed transgenic fish whose thrombocytes/erythrocytes express the green and red fluorescent proteins. In vivo measurements allow cells to be tracked under physiological conditions in the same fish over time, without drawing blood samples or sacrificing animals. We also discuss the potential applications of this instrument in biomedical research.     

氨氮和亚硝酸盐对红螯螯虾幼虾和亚成虾的急性毒力

【目的】近年来,红螯螯虾养殖面积越来越广泛,明确不同规格的红螯螯虾对氨氮和亚硝酸盐的耐受力,有利于提高其养成率,促进其养殖业的健康发展。【方法】在水温24~25℃、p H 7.9~8.0的条件下,研究了氨氮和亚硝酸盐对红螯螯虾幼虾和亚成虾的急性毒性,分析半致死浓度(LC50)和安全浓度(SC)。【结果】总氨氮对红螯螯虾幼虾的24、48、72和96 h LC50分别为188.0、136.15、104.67和88.00 mg·L~(-1),SC为8.80 mg·L~(-1);总氨氮对亚成虾的24、48、72和96 h LC50分别为344.01、270.46、205.15和167.68 mg·L~(-1),SC为16.77 mg·L~(-1);非离子氨对幼虾的24、48、72和96 h LC50分别为10.16、7.35、5.65和4.75 mg·L~(-1),SC为0.48 mg·L~(-1);非离子氨对亚成虾的24、48、72和96 h LC50分别为18.58、14.60、11.08和9.05 mg·L~(-1),SC为0.91 mg·L~(-1);亚硝酸盐对幼虾的24、48、72和96 h LC50分别为46.76、33.88、27.97和22.81 mg·L~(-1),SC为2.28 mg·L~(-1);亚硝酸盐对亚成虾的24、48、72和96 h LC50分别为77.56、59.33、45.41和37.48 mg·L~(-1),SC为3.75 mg·L~(-1)。【结论】红螯螯虾对氨氮的耐受力高于亚硝酸盐,亚成虾对氨氮和亚硝酸盐的耐受力高于幼虾。     

H3K36me2 methyltransferase NSD2 orchestrates epigenetic reprogramming during spermatogenesis

Spermatogenesis is precisely controlled by sophisticated gene expression programs and is driven by epigenetic reprogramming, including histone modification alterations and histone-to-protamine transition. Nuclear receptor binding SET domain protein 2 (Nsd2) is the predominant histone methyltransferase catalyzing H3K36me2 and its role in male germ cell development remains elusive. Here, we report that NSD2 protein is abundant in spermatogenic cells. Conditional loss of Nsd2 in postnatal germ cells impaired fertility owing to apoptosis of spermatocytes and aberrant spermiogenesis. Nsd2 deficiency results in dysregulation of thousands of genes and remarkable reduction of both H3K36me2 and H3K36me3 in spermatogenic cells, with H3K36me2 occupancy correlating positively with expression of germline genes. Nsd2 deficiency leads to H4K16ac elevation in spermatogenic cells, probably through interaction between NSD2 and PSMA8, which regulates acetylated histone degradation. We further reveal that Nsd2 deficiency impairs EP300-induced H4K5/8ac, recognized by BRDT to mediate the eviction of histones. Accordingly, histones are largely retained in Nsd2-deficient spermatozoa. In addition, Nsd2 deficiency enhances expression of protamine genes, leading to increased protamine proteins in Nsd2-deficient spermatozoa. Our findings thus reveal a previously unappreciated role of the Nsd2-dependent chromatin remodeling during spermatogenesis and provide clues to the molecular mechanisms in epigenetic abnormalities impacting male reproductive health.     

光钳操纵生物活体的动态监测技术的研究

采用摄像、录像和视屏监控系统及显色偏振装置与光钳系统耦合,从空间分辨、色分辨和时间分辨多方面改善系统品质,实现了光钳捕获与操纵生物活体的动态监测、实时记录、资料保存和屏幕再现的功能,并能测量光钳操纵细胞的位移量和由此计算操纵速度,提高了光钳的自我调整和光钳操纵细胞的精细度。本研究为激光光钳技术在细胞工程等方面的应用研究提供了行之有效的技术手段。     

Imaging molecular expression on vascular endothelial cells by in vivo immunofluorescence microscopy

Molecular expression on the vascular endothelium is critical in regulating the interaction of circulating cells with the blood vessel wall. Leukocytes as well as circulating cancer cells gain entry into tissue by interacting with adhesion molecules on the endothelial cells (EC). Molecular targets on the EC are increasingly being explored for tissue-specific delivery of therapeutic and imaging agents. Here we use in vivo immunofluorescence microscopy to visualize the endothelial molecular expression in the vasculature of live animals. High-resolution images are obtained by optical sectioning through the intact skin using in vivo confocal and multiphoton microscopy after in situ labeling of EC surface markers with fluorescent antibodies. Other vascular beds such as the bone marrow and ocular blood vessels can be imaged with little or no tissue manipulation. Live imaging is particularly useful for following the dynamic expression of inducible molecules such as E-selectin during an inflammatory response.     

Circulation times of prostate cancer and hepatocellular carcinoma cells by in vivo flow cytometry

In metastasis, the cancer cells that travel through the body are capable of establishing new tumors in locations remote from the site of the original disease. To metastasize, a cancer cell must break away from its tumor and invade either the circulatory or lymphatic system, which will carry it to a new location, and establish itself in the new site. Once in the blood stream, the cancer cells now have access to every portion of the body. Here, we have used the "in vivo flow cytometer" to study if there is any relationship between metastatic potential and depletion kinetics of circulating tumor cells. The in vivo flow cytometer has the capability to detect and quantify continuously the number and flow characteristics of fluorescently labelled cells in vivo. We have improved the counting algorithm and measured the depletion kinetics of cancer cells with different metastatic potential. Interestingly, more invasive PC-3 prostate cancer cells are depleted faster from the circulation than LNCaP cells. In addition, we have measured the depletion kinetics of two related human hepatocellular carcinoma (liver cancer) cell lines, high-metastatic HCCLM3 cells, and low-metastatic HepG2 cells. More than 60% HCCLM3 cells are depleted within the first hour. Interestingly, the low-metastatic HepG2 cells possess noticeably slower depletion kinetics. In comparison, <40% HepG2 cells are depleted within the first hour. The differences in depletion kinetics might provide insights into early metastasis processes.     

Photostimulation by femtosecond laser triggers restorable fragmentation in single mitochondrion

Mitochondrial research is important to the study of ageing, apoptosis, and metabolic diseases. Over the years, mitochondria have been studied with stimulation by chemical agents in a global manner for basic and applied research. This approach lacks of precision and accuracy in terms of spatial and temporal resolution. Here we demonstrate a direct and well‐defined photostimulation targeting on single mitochondrial tubular structure using a tightly‐focused femtosecond (fs) laser that could precisely activate mitochondria at single tubule level to show restorable fragmentation and subsequent recovery after tens of seconds. In these two processes, a series of mitochondrial reactive oxygen species (mROS) flashes was observed and found critical to the mitochondrial fragmentation. Meanwhile, transient openings of mitochondrial permeability transition pores (mPTP) were seen with oscillations of mitochondrial membrane potential. These activities were crucial for the recovery through scavenging the mROS. Without the feedback mechanisms, the fragmented mitochondria could not return back to their original tubular structure. These interesting observations show that photostimulation by fs laser is an active, precise, clean and well‐defined approach to dissect the role of mitochondria in normal physiology and different kinds of diseases.