Simultaneous measurements of cytoplasmic viscosity and intracellular vesicle sizes for live human brain cancer cells

Intracellular vesicles, comprised of protein clusters, were individually tracked inside human brain cancer cells and characterized to simultaneously determine the average vesicle size and effective cytoplasmic viscosity. The cells were transfected with a TGF‐β superfamily gene, non‐steroidal anti‐inflammatory drug‐Activated Gene‐1 (NAG‐1) tagged with green fluorescent proteins (GFPs). Using total internal reflection fluorescent microscopy (TIRFM) the individual movements of the vesicles were categorized into either Brownian, caged, or directional type motion. In the near‐field region confined by the evanescent wave field of TIRFM, the hindrance of these vesicles was created by interactions with the glass coverslip and/or sub‐cellular structures. Measured particle motions were compared with theoretical predictions of hindered motion to estimate the unknown size and viscosity parameters using a nonlinear regression technique. For the tested human brain cancer cells, the average vesicle size and effective intracellular fluid viscosity were calculated to be 496 nm and 0.068 Pa s, respectively. This finding suggests that most of the hindrance experienced by vesicles can be due to non‐hydrodynamic interactions with microtubules and other intracellular structures. It should be also noted that this method provides a way to examine changes in vesicle size due to outside stimulus such as drug interaction, cytotoxicity, etc., unlike standard measurement techniques which require fixing the cells themselves. Biotechnol. Bioeng. 2011;108: 2504–2508. © 2011 Wiley Periodicals, Inc.     

青藏高原高寒荒漠区藏羚适宜生境识别及其保护状况评估

藏羚作为青藏高原高寒荒漠区濒危有蹄类动物的典型代表,其生境保护对于维持其种群存续具有重要意义.本文考虑食物、地形、水系等藏羚关键生境因子及道路、居民点等人为干扰因子,基于生境适宜性模型,对青藏高原高寒荒漠区藏羚的潜在及有效生境适宜性进行了模拟分析.同时,基于保护比例及单位面积对适宜生境的捕获效率,评估了研究区内阿尔金山、可可西里和羌塘国家级自然保护区及其各功能分区的保护情况.结果表明： 青藏高原高寒荒漠区潜在和有效适宜生境面积分别为2.84×10⁵和2.08×10⁵ km²,人为干扰造成的生境退化达16.1%.其中,阿尔金山、可可西里和羌塘保护区所覆盖的潜在适宜生境面积分别为2.01×10⁴、3.13×10⁴和1.26×10⁵ km²;考虑道路、居民点等人为干扰因素,其有效适宜生境面积分别为1.75×10⁴、2.81×10⁴和9.95×10⁴ km²,上述干扰因素导致的生境减损率分别为12.9%、10.2%和21.1%,表明羌塘道路、居民点等人为干扰相对较严重.尽管目前该区域3大保护区保护了藏羚2/3以上的适宜生境,体现了良好的保护效率,但仍存在一定游离于保护体系之外的保护空缺.在保护区功能区划水平上,除核心区外,缓冲区和实验区的保护比例和保护效率也不容忽视.为强化对藏羚等濒危有蹄类的保护,有必要在保护区和功能分区水平上对现有保护体系进行优化调整,减少保护空缺、优化功能分区,提高保护体系对生境保护的有效性,并预先保护物种适应气候变化的潜在庇护所.     

A new dimension in suspension fusion techniques with polyethylene glycol.

Polyethylene glycol (PEG) induces the hybridization of mammalian cells at a much higher frequency when the cells are attached to a substrate during treatment than when the cells are treated in suspension. Since many cell types, e.g., lymphocytes, cannot attach to a substrate, a new technique for the PEG-induced fusion of cells in suspension was developed. This technique, referred to as "pancake fusion," is based on the centrifugation of suspended cells onto a coverslip and the PEG treatment of the cells on the coverslip as if they were attached to a substrate. With this technique, the frequency of hybridization of human white blood cells, which are incapable of attaching to a substrate, can be greatly increased.     

Establishment of the Neural Differentiation Pattern in the Prospective Prosencephalic Ectoderm of the Chick Embryo (Gallus domesticus)

Small graft pieces (size: 0.15 × 0.2 mm to 0.1 × 0.4 mm and 0.25 × 0.25 mm) were isolated from the prospective prosencephalic ectoderm of the definitive primitive streak (st. 4) and head process (st. 5) chick blastoderms, and cultured in vivo intracoelomically. In all 1437 graft pieces isolated from 448 donor blastoderms were transplanted into the coelom of 941 host embryos; 216 hosts died before reaching 12 days. The 725 surviving hosts, which carried 1241 implants, yielded 304 analysable grafts. The very small size of the isolates affected the recovery rate. The histological analysis of the recovered grafts reveals that the frequency of neuralization was higher in the grafts isolated from the central region of the prospective prosencephalic ectoderm than in those from its peripheral region. A centrifugal extension of differentiation tendencies of prosencephalic structures was observed from st. 4 to st. 5. At st. 5 frequency of telencephalic structures was high in the grafts of the anterior region, whereas that of diencephalic structures was high in those of the more posterior region. Eye structures (retina as well as tapetum) differentiated in an area measuring ca. 0.4 × 0.1 mm extending transversely about 0.1 mm anterior to the head process at st. 5. Structural elements of the lens were not observed in any graft.     

MicroRNA-30e Functions as a Tumor Suppressor in Cervical Carcinoma Cells through Targeting GALNT7

Cervical cancer is the third most common cancer in women worldwide. However, the underlying mechanism of occurrence and development of cervical cancer is obscure. In this study, we observed that miR-30e was downregulated in clinical cervical cancer tissues and cervical cancer cells. Next, overexpression of miR-30e reduced the cervical cancer cell growth through MTT, colony formation, EdU, and Transwell assay in SiHa and Caski cells. Subsequently, UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase 7 (GALNT7) was identified as a potential miR-30e target by bioinformatics analysis. Moreover, we showed that miR-30e was able to bind to the 3′UTR of GALNT7 by luciferase reporter assay. In addition, the mRNA and protein levels of GALNT7 in cervical cancer cells were downregulated by miR-30e. And we validated that downregulation of GALNT7 repressed the proliferation of SiHa and Caski cells by MTT, colony formation, and Transwell assay. We identified that the restoration of GALNT7 expression was able to counteract the effect of miR-30e on cell proliferation of cervical cancer cells. Furthermore, we found that the expression levels of GALNT7 were frequently upregulated and negatively correlative to those of miR-30e in cervical cancer tissues. In addition, we validated that restoration of GALNT7 rescued the miR-30e–suppressed growth of cervical cancer xenografts in vivo. In conclusion, the current results suggest that miR-30e may function as tumor suppressors in cervical cancer through downregulation of GALNT7. Both miR-30e and its novel target, GALNT7, may play an important role in the process of cervical cancer.     

Use of Dimethylsulfoxide to Prevent Clumping during Feulgen Staining of Ceratocystis ulmi

The dimorphic fungus Ceratocystis ulmi is the causative agent of Dutch Elm Disease. As part of a study on the regulation of this developmental phenomenon, we attempted to stain the nuclei of cells growing vegetatively in the yeast phase by a modification of the Feulgen technique described by Gauger (1975). The cells were harvested by centrifugation, washed twice, and resuspended in 0.05 M phosphate buffer (pH 6.5). A small portion of this cell suspension was placed on a clean No. 2 glass coverslip (22 ± 22 mm) and allowed to air dry. The coverslip was flamed briefly to heat fix the cells whereupon they were fixed in glacial acetic acid: 95% ethanol (1:3 v/v) for one hour, hydrolyzed in 1 N hydrochloric acid at 60 C for 5 minutes, and stained for 30 minutes. The Feulgen stain was prepared according to Stevens (1974). Subsequently, the coverslip was rinsed briefly with distilled water and dehydrated for 30 seconds in 70% ethanol. After air drying, the coverslip was mounted on a glass microscope slide with Permount (Fisher Scientific Co.) and examined.     

The permeability of capillaries among the small granule-containing cells in rat superior cervical ganglia: an ultrastructural lanthanum tracer study.

The permeability of blood capillaries associated with small granule-containing (SGC) cells in rat superior cervical ganglia was investigated at ultrastructural level by employing ionic lanthanum as an electron dense tracer. In rat superior cervical ganglia, the majority of blood capillaries were nonfenestrated. Both fenestrated and nonfenestrated capillaries were observed in the area associated with SGC cells. Lanthanum tracer was observed in the luminal surface, the interendothelial cleft and the subendothelial perivascular spaces of both fenestrated and nonfenestrated capillaries associated with SGC cells. The external lamina of the Schwann cell which surrounded the neurons, nerve fibres and SGC cells were clearly delineated by the lanthanum tracer. Furthermore, the perineuronal space, the periaxonal space, and the pericellular space of the SGC cells were readily accessible to the lanthanum ion. The results demonstrated an absence of blood-nerve barrier, blood-ganglionic and blood-SGC cell barrier to the lanthanum ion in the parenchymal area of the SGC cells in rat superior cervical ganglia. It is proposed that lanthanum may pass through the endothelial cells via 1) the fenestrae of fenestrated capillaries, 2) the intercellular junctions of both fenestrated and nonfenestrated capillaries, i.e., a paracellular pathway; and 3) the process of endocytosis/exocytosis, i.e., a transcellular pathway, to reach the subendothelial space and be distributed in the parenchyma of SGC cells in rat superior cervical ganglia.     

NEUROTRANSMITTER UPTAKE INTO SLICES OF RAT CEREBRAL CORTEX IN VITRO: EFFECT OF SLICE SIZE

Abstract— The uptake of [¹⁴C]GABA, [¹⁴C]taurine, [³H] β -alanine and [¹⁴C]dopamine was compared in slices of rat cerebral cortex of three different sizes (0.1 × 0.1 × 2 mm, 0.2 × 0.2 × 2 mm and 0.4 × 0.4 × 2 mm prepared with a mechanical tissue chopper). [¹⁴C]Taurine and [³H] β -alanine uptake increased whereas [¹⁴C]GABA uptake decreased with increasing slice size. [¹⁴C]Dopamine uptake was optimal in 0.2 × 0.2 × 2 mm slices. Increasing slice size was shown to decrease inhibition of [³H] β -alanine and [¹⁴C]GABA uptake by l -2,4-diaminobutyric acid. Lactate dehydrogenase activity increased with increasing slice size indicating decreased tissue damage or increased cellular integrity. The possibility that varying slice size can be used to distinguish between neuronal and glial uptake is discussed. It is suggested that taurine uptake in the cerebral cortex is predominantly glial.     

Facile generation of cell microarrays using vacuum degassing and coverslip sweeping

A simple method to generate cell microarrays with high-percentage well occupancy and well-defined cell confinement is presented. This method uses a synergistic combination of vacuum degassing and coverslip sweeping. The vacuum degassing step dislodges air bubbles from the microwells, which in turn enables the cells to enter the microwells, while the physical sweeping step using a glass coverslip removes the excess cells outside the microwells. This low-cost preparation method provides a simple solution to generating cell microarrays that can be performed in basic research laboratories and point-of-care settings for routine cell-based screening assays.     

基于免疫组化和癌症基因组图谱数据分析的脾酪氨酸激酶在宫颈癌中的表达研究

摘要 目的：探讨Syk 在宫颈癌中的表达及其临床意义。方法：应用免疫组化检测Syk在宫颈癌、癌前病变(CIN)和相应的正常宫颈组织中的表达。借助R2生物信息平台挖掘Syk在TCGA数据库305例宫颈鳞癌中的mRNA表达及其与预后的关系。结果：免疫组化结果显示,Syk在宫颈癌巢分化较好的中心区表达较强,在分化较低的癌巢周边区表达较弱。Syk 染色主要定位在宫颈癌和正常宫颈组织的细胞质和细胞膜,正常宫颈组织基底细胞无 Syk 表达,8例CIN组织细胞核中可见Syk表达, 但宫颈癌组织细胞核中未见Syk表达。Syk在宫颈癌、CIN和正常宫颈组织中的阳性率分别是76%、54%、40%,三组间的表达差异具有统计学意义（P=0.001）。Syk 在深度浸润和淋巴结转移中表达较强。数据挖掘结果显示,Syk mRNA在305例不同临床分期的宫颈癌中均表达,Syk mRNA高表达组219例,Syk mRNA低表达组73例,其中13例生存数据缺失,Syk高表达组的患者预后较差。结论：Syk在宫颈癌中的表达提示Syk在宫颈癌中具有致癌蛋白的作用,Syk在某些CIN中的核表达可能与更好的预后相关。     

Scattering of exciting light by live cells in fluorescence confocal imaging: phototoxic effects and relevance for FRAP studies

As exciting light in a scanning confocal microscope encounters a cell and its subcellular components, it is refracted and scattered. A question arises as to what proportion of the exciting light is scattered by subcellular structures and whether cells in the vicinity of the imaged area, i.e., cells that are not directly illuminated by the laser beam, can be affected by either an exposure to scattered light and ensuing phototoxic reactions, or by the products of photoactivated reactions diffusing out of the directly illuminated area. We have designed a technique, which allows us to detect subtle cell photodamage and estimate the extent and range of phototoxic effects inflicted by interaction between scattered exciting light and fluorescent probes in the vicinity of the illuminated area. The technique is based on detecting an increased influx of acridine orange into photodamaged cells, which is manifested by a change of color. We demonstrate that phototoxic effects can be exerted not only on the illuminated cell, but also on fluorescently labeled neighboring cells. The damage inflicted on neighbors is due to exposure to light scattered by the imaged (i.e., directly illuminated) cell, but not phototoxic products diffusing out of the directly illuminated area. When light encounters a cell nucleus, scattering is so intense that photodamage can be inflicted even on fluorescently labeled cells located within a radius of approximately 90 microm, i.e., several cell diameters away. This range of scattering is comparable with that caused by the glass bead resting on a coverslip (up to 120 microm). The intense scattering of exciting light imposes limits on FRAP, FLIP, and other techniques employing high intensity laser beams.     

Determinations of Volumes and Minimal Surface Areas of Individual Compressed Live Tetrahymena1

ABSTRACT. A method to determine the volume of individual live Tetrahymena with improved precision has been developed. Cells are compressed between a microslide and coverslip and their volume calculated from their thickness and area determined by interference microscopy and from a photomicrograph, respectively. A formula has been derived to correct for the “edge error” which arises because the rounded edges of the cells are projected on the film as if they were rectangular. The minimal surface area of compressed cells, i.e. that necessary to surround the cells smoothly, can also be calculated. Cells may be recovered after measurements. The method may also be applied to other cells.     

Cytological study of early cervical adenocarcinoma: special reference to the depth of invasion

OBJECTIVE: Early cervical adenocarcinoma (ECA) with a tumour depth of <3 mm has a good prognosis. To clarify the cytological features of ECAs with depth <3 mm, these were compared with those of ECA with 3-5 mm and invasive adenocarcinoma (IA) invading the cervical wall with more than 5 mm in depth. METHODS: The cervical cytological features of ECAs with depth <3 mm (14 cases) were compared with those of ECA with 3-5 mm (four cases) and IA (13 cases). Cytologically, the presence or absence of tumour diathesis, number of atypical cells, crowded cell groups, groups with glandular structures, feathering, groups with palisading borders, rosettes, clusters, cell shape and size, nuclear shape and size, nucleolar shape and size, chromatin distribution, border and character of cytoplasm, and single cell pattern were evaluated. RESULTS: A tumour diathesis was seen in five of 14 ECA <3 mm in depth (36%), all four ECA with 3-5 mm (100%) and 11 of 13 IA with more than 5 mm (85%). Single cells, macronucleoli and coarsely granular chromatin pattern were less frequent in ECA of <3 mm than that in ECA with 3-5 mm and IA. The number of atypical cells and glandular structures in ECA was significantly less than that in IA. Cell crowding, feathering, palisading and rosettes were common in both ECA and IA. CONCLUSION: The characteristic cytological features of ECA with depth <3 mm, having a good prognosis, were clean background, fewer single cells and macronucleoli, and less frequent coarsely granular chromatin pattern compared with those in ECA with 3-5 mm and IA. The number of atypical cells and glandular structures in ECA was significantly less than that in IA. Familiarity with the cytological features of ECA and its mimics is essential.     

A poly(dimethylsiloxane)-based device enabling time-lapse imaging with high spatial resolution

We have developed a regulator-free device that enables long-term incubation of mammalian cells for epi-fluorescence imaging, based on a concept that the size of sample to be gassed and heated is reduced to observation scale. A poly(dimethylsiloxane) block stamped on a coverslip works as a long-lasting supplier of CO₂-rich gas to adjust bicarbonate-containing medium in a tiny chamber at physiological pH, and an oil-immersion objective warms cells across the coverslip. A time-lapse imaging experiment using HeLa cells stably expressing fluorescent cell-cycle indicators showed that the cells in the chamber proliferated with normal cell-cycle period over 2 days.     

Myxosoma microspora n. sp. (Myxosporidia: Protozoa) parasitic in the gills of Mugil cephalus L.

A new histozoic species of myxosporidian, Myxosoma microspora n.sp., infecting the gill filaments of Mugil cephalus is described. Cysts measuring 0.5–1.0 mm in diameter were found attached to the gill filaments. Spherical or slightly oval, spores 4.8–5.2 μm in diameter, were present and possessed a thin outer mucous envelope which appeared as small conical protuberances at the ends of the equatorial axis. Polar capsules were pyriform in shape, equal in size and measured 1.6–2.0 × 1.0–1.2 μm; the polar filaments were 22–28 μm in length. There was a bean-shaped sporoplasm measuring 3.5 × 1.5 μm. No iodine vacuole was detected when the parasite was stained with Lugol's iodine.     

人间充质干细胞对乳腺癌细胞生长的影响研究

目的通过构建间充质干细胞(MSC)与乳腺癌细胞间相互作用的共培养模型,探讨MSC对乳腺癌细胞生长的影响。方法用含荧光基因第三代自身失活慢病毒载体感染人类脐带分离提取的MSC和乳腺癌细胞MDA-MB-231、MCF-7,以单独培养的乳腺癌细胞MDA-MB-231和MCF-7分别设立对照,2种乳腺癌细胞分别与MSC共培养,检测乳腺癌细胞在MSC作用下增生能力的改变,流式细胞术检测共培养后细胞表面标记物表达。两组间比较采用独立样本t检验,多组间比较采用单因素方差分析,多重比较采用Dunnet-t检验。结果MSC在与乳腺癌细胞共培养过程中促进肿瘤细胞生长,第3天共培养组乳腺癌MDA-MB-231细胞数高于单独MDA-MB-231培养组[(5.50±0.71)×10^3个比(1.63±0.41)×10^3个],培养至第7天,两组间MDA-MB-231细胞数差异进一步增大[(81.25±7.40)×10^3个比(26.25±4.15)×10^3个],差异具有统计学意义(P均<0.001);共培养后MSC促进乳腺癌细胞表达干细胞特有标记物CD90,MCF-7从共培养第2天CD90表达率(1.38±0.30)﹪升高至第9天(92.45±2.04)﹪。在共培养中MSC围绕肿瘤细胞集落方式生长,在形态上变长,并发现一种新型混合细胞(hybrid融合细胞)同时表达绿色和红色荧光,且对化疗药物更敏感。结论MSC促进乳腺癌细胞的生长,伴随MSC形态学改变和hybrid融合细胞出现,乳腺癌细胞获得MSC特有CD90表达。     

Winter Ciliates in a British Columbian Fjord: Six New Species and an Analysis of Ciliate Putative Prey

ABSTRACT. This work provides the first study of North Pacific planktonic ciliates by quantitative protargol staining. Triplicate water bottle samples were collected at a depth of 2 m (above the shallow pycnocline) at six stations in Indian Arm, British Columbia, on February 15, 1990, and February 26, 1991. Thirty-six ciliate species were observed. Six new species are described from protargolstained specimens: Strombidium lynni n. sp., Strombidium taylori n. sp., Strombidium basimorphum n. sp., Slrombidiurn ventropinnum n. sp., Strobilidium undinum n. sp., and Urotricha cyrtonucleata n. sp.
Ciliate abundance varied significantly (ANOVA, α= 0.05) between sampling sites, ranging from 550 to 6,800 cells/liter in 1990 and from 1,800 to 7,900 cells/liter in 1991. Biomass also varied significantly (ANOVA, α= 0.05) ranging from 3.7 × 10⁵ to 3.3 × 10⁶ pg carbon/liter in 1990 and 3.04 × 10⁶− 6.97 × 10⁶ pg carbon/liter in 1991. Putative prey were enumerated in three size fractions (1.5–5 μm, 5–10 μm and 10–25 μm). The source of variation in ciliate abundance and biomass was not identified. Parameters of salinity, temperature, putative prey, chlorophyll a and pycnocline depth did not significantly correlate with ciliate biomass or abundance (α= 0.05).     

Adaptogenic activity of withaferin A on human cervical carcinoma cells using high-definition vibrational spectroscopic imaging

Despite invaluable advances in cervical cancer therapy, treatment regimens for recurrent or persistent cancers and low-toxicity alternative treatment options are scarce. In recent years, substances classified as adaptogens have been identified as promising drug sources for preventing and treating cancer-based diseases on their ability to attack multiple molecular targets.This paper establishes the effectiveness of inhibition of the neoplastic process by a withaferin A (WFA), an adaptogenic substance, based on an in vitro model of cervical cancer. This study explores for the first time the potential of high-definition vibrational spectroscopy methods, i.e. Fourier-transform infrared (FT-IR) and Raman spectroscopic (RS) imaging at the single-cell level to evaluate the efficacy of the adaptogenic drug. HeLa cervical cancer cells were incubated with various concentrations of WFA at different incubation times.The multimodal spectroscopic approach combined with partial least squares (PLS) regression allowed the identification of molecular changes (e.g., lipids, protein secondary structures, or nucleic acids) induced by WFA at the cellular level. The results clearly illustrate the enormous potential of WFA in inhibiting the proliferation of cervical cancer cells. WFA inhibited the growth of the studied cancer cell line in a dose-dependent manner. Such studies provide comprehensive information on the sensitivity of cells to adaptogenic drugs. This is a fundamental step towards determining the rate and nature of adaptogen-induced changes in cancer cells.     

Emigration of bilayered epidermal cell sheets from tadpole tails (Xenopus laevis)

Summary Migration of bilayered epidermal cell sheets out of explants of tadpole tails (Xenopus laevis) were investigated with time-lapse cinemicrography using reflection-contrast optics. Cell-sheet formation begins beneath the explant in a region where it is closely attached to the coverslip. A single basal cell extends a lamellipodium through the outer (surface) epidermal layer and starts moving in a direction free of attached cells. This cell remains connected to the following basal cell, which the also extends a lamellipodium onto the glass. The cell sheet develops as increasingly more adjacent basal cells start to migrate. Surface cells do not actively locomote but they remain attached to the basal cells and to adjacent surface cells. Thus, they are transported as an intact cell layer, and consequently the in situ arrangement of the tadpole epidermis is largely preserved in the cell sheet, i.e., basal cells adhere to the substratum and are covered by outer cells (surface cells) which face the culture medium. Basal cells extend lamellae beneath the rear end of the preceding cell, which is slightly fifted off the substratum. The direction of locomotion is determined by the frontal cells. Cell-sheet enlargement and locomotion cease when all the epidermal cells facing the coverslip have left the explant, and the cell sheet and epidermis covering the explant form a continuous layer.     

Coccidia (Eimeria tachyglossi n. sp., E. echidnae n. sp., and Octosporella hystrix n. sp.) in the Echidna, Tachyglossus aculeatus (Monotremata: Tachyglossidae)

Oocysts of Octosporella hystrix n. sp., Eimeria tachyglossi n. sp., and E. echidnae n. sp. are described from the feces of the echidna Tachyglossus aculeatus (Monotremata: Tachyglossidae) from Australia. Eimeria tachyglossi has subspherical oocysts, 26.4 × 23.7 μm in size, with a single oocyst wall; no micropyle; four ellipsoidal sporocysts 13.2 × 9.7, slightly pointed at one end, each containing two sporozoites. Eimeria echidnae has subspherical oocysts, 19.4 × 17.8 in size, with a single oocyst wall; no micropyle; four ellipsoidal sporocysts 9.8 × 7.8, blunt at both ends, each containing two sporozoites. Octosporella hystrix has ovoid or subspherical oocysts 32.9 × 29.7 in size with a thick outer and thin inner oocyst wall; no micropyle; eight sporocysts spherical or slightly subspherical 11.3 × 11.2 each containing two sporozoites lying in embrace, with an extensive granular sporocyst residuum about the equator of the sporocyst. Endogenous stages considered to be of E. tachyglossi at least, were recognized in the lamina propria and epithelium on villi in the small intestine of three echidnas.