Changes in the function of the nucleus and chromatin of human cells under the action of hormonal factors in vitro

The influence of hormones--adrenaline, noradrenaline, hydrocortisone on the electronegativity of cell nuclei (ENN) was shown by the method of intracellular microelectrophoresis. Changes in heterochromatin state were estimated on the basis of heterochromatin granules (chromocenters) quantity. Adrenaline and noradrenaline induced the decrease in ENN, the maximum effect was revealed with hormones concentration observed in human blood during physical training. The hormonal effect increased with gradual increase of the exposition time from 5 to 120 min. Hydrocortisone also decreased ENN. Adrenaline, and in some cases hydrocortisone, induced the significant increase of heterochromatin granule quantity. The correlation of this process with ENN decrease was shown only in the case of adrenaline. Obtained data are discussed in connection with regulation mechanisms of cell nucleus functional activity.     

Multicellular dosimetric chain for molecular radiotherapy exemplified with dose simulations on 3D cell spheroids

PurposeAbsorbed radiation dose-response relationships are not clear in molecular radiotherapy (MRT). Here, we propose a voxel-based dose calculation system for multicellular dosimetry in MRT. We applied confocal microscope images of a spherical cell aggregate i.e. a spheroid, to examine the computation of dose distribution within a tissue from the distribution of radiopharmaceuticals.MethodsA confocal microscope Z-stack of a human hepatocellular carcinoma HepG2 spheroid was segmented using a support-vector machine algorithm and a watershed function. Heterogeneity in activity uptake was simulated by selecting a varying amount of the cell nuclei to contain ¹¹¹In, ¹²⁵I, or ¹⁷⁷Lu. Absorbed dose simulations were carried out using vxlPen, a software application based on the Monte Carlo code PENELOPE.ResultsWe developed a schema for radiopharmaceutical dosimetry. The schema utilizes a partially supervised segmentation method for cell-level image data together with a novel main program for voxel-based radiation dose simulations. We observed that for ¹⁷⁷Lu, radiation cross-fire enabled full dose coverage even if the radiopharmaceutical had accumulated to only 60% of the spheroid cells. This effect was not found with ¹¹¹In and ¹²⁵I. Using these Auger/internal conversion electron emitters seemed to guarantee that only the cells with a high enough activity uptake will accumulate a lethal amount of dose, while neighboring cells are spared.ConclusionsWe computed absorbed radiation dose distributions in a 3D-cultured cell spheroid with a novel multicellular dosimetric chain. Combined with pharmacological studies in different tissue models, our cell-level dosimetric calculation method can clarify dose-response relationships for radiopharmaceuticals used in MRT.     

FK506 confers chemosensitivity to anticancer drugs in glioblastoma multiforme cells by decreasing the expression of the multiple resistance-associated protein-1

Malignant rhabdoid tumor (MRT) is a rare and highly aggressive neoplasm of young children. MRT is characterized by inactivation of integrase interactor 1 (INI1). Cyclin-dependent kinase 4 (CDK4), which acts downstream of INI1, is required for the proliferation of MRT cells. Here we investigated the effects of PD 0332991 (PD), a potent inhibitor of CDK4, against five human MRT cell lines (MP-MRT-AN, KP-MRT-RY, G401, KP-MRT-NS, KP-MRT-YM). In all of the cell lines except KP-MRT-YM, PD inhibited cell proliferation >50%, (IC₅₀ values 0.01 to 0.6 μM) by WST-8 assay, and induced G1-phase cell cycle arrest, as shown by flow cytometry and BrdU incorporation assay. The sensitivity of the MRT cell lines to PD was inversely correlated with p16 expression (r = 0.951). KP-MRT-YM cells overexpress p16 and were resistant to the growth inhibitory effect of PD. Small interfering RNA against p16 significantly increased the sensitivity of KP-MRT-YM cells to PD (p < 0.05). These results suggest that p16 expression in MRT could be used to predict its sensitivity to PD. PD may be an attractive agent for patients with MRT whose tumors express low levels of p16.     

Optimizing dose enhancement with Ta2O5 nanoparticles for synchrotron microbeam activated radiation therapy

Microbeam Radiation Therapy (MRT) exploits tumour selectivity and normal tissue sparing with spatially fractionated kilovoltage X-ray microbeams through the dose volume effect. Experimental measurements with Ta₂O₅ nanoparticles (NPs) in 9L gliosarcoma treated with MRT at the Australian Synchrotron, increased the treatment efficiency. Ta₂O₅ NPs were observed to form shells around cell nuclei which may be the reason for their efficiency in MRT. In this article, our experimental observation of NP shell formation is the basis of a Geant4 radiation transport study to characterise dose enhancement by Ta₂O₅ NPs in MRT. Our study showed that NP shells enhance the physical dose depending microbeam energy and their location relative to a single microbeam. For monochromatic microbeam energies below ∼70 keV, NP shells show highly localised dose enhancement due to the short range of associated secondary electrons. Low microbeam energies indicate better targeted treatment by allowing higher microbeam doses to be administered to tumours and better exploit the spatial fractionation related selectivity observed with MRT. For microbeam energies above ∼100 keV, NP shells extend the physical dose enhancement due to longer-range secondary electrons. Again, with NPs selectively internalised, the local effectiveness of MRT is expected to increase in the tumour. Dose enhancement produced by the shell aggregate varied more significantly in the cell population, depending on its location, when compared to a homogeneous NP distribution. These combined simulation and experimental data provide first evidence for optimising MRT through the incorporation of newly observed Ta₂O₅ NP distributions within 9L cancer cells.     

Cortisol Is Not Associated with Telomere Shortening or Chromosomal Instability in Human Lymphocytes Cultured under Low and High Folate Conditions

Chronic psychological stress and nutritional deficiencies are factors that impact negatively on human health and disease risk. Chronic stress has been associated with accelerated leukocyte telomere shortening in numerous cohorts, however, a mechanistic link has proven elusive. This study tested the hypotheses that chronic exposure to the stress hormone, cortisol, causes telomere shortening and chromosome instability (CIN) in vitro, and that these effects would be further exacerbated by folate (vitamin B9) deficiency. Primary human lymphocytes were maintained in vitro for 12 days in medium containing either 25 nM folic acid (FA(low)) or 100 nM FA (FA(high)), together with either 0, 400, 1000 or 3500 nM cortisol. The interactive effects of cortisol and FA were examined by comparing telomere length (TL), biomarkers of DNA damage, and cytostasis. At day 12 TL was 5-17% longer in lymphocytes cultured in FA(low) conditions (mean ± SD;10.2% ± 1.6), compared with those in FA(high) medium (9.1% ± 1, p = 0.02). Refuting the hypothesis, TL was consistently greater in the presence of cortisol. The effect of FA deficiency on the frequency of DNA damage was significant for nucleoplasmic bridges, circular nuclei, micronuclei and nuclear buds, (p < 0.0001 – 0.001). The effect of cortisol, however, was negligible, only reaching statistical significance for the frequency of fused nuclei (p = 0.04). Cortisol was significantly associated with reduced cell division and growth and had an apparent protective effect on cell viability in the FA(low) conditions. Conclusions: Both chronic cortisol exposure, and folate deficiency, resulted in telomere elongation, however, the effect of cortisol was marginal relative to that of folate. Cortisol was not associated with increased chromosomal instability, but caused a significant reduction in cell division and growth. Together these results indicate that cortisol is not directly genotoxic and that the telomere shortening associated with increased psychological stress in vivo may not be explained by a direct effect of cortisol.     

Mathematical morphologic segmentation dedicated to quantitative immunohistochemistry

OBJECTIVE: To develop automatic segmentation sequences for fully automated quantitative immunohistochemistry of cancer cell nuclei by image analysis. STUDY DESIGN: The study focused on the automated delineation of cancer cell lobules and nuclei, taking breast carcinoma as an example. A hierarchic segmentation was developed, employing mainly the chaining of mathematical morphology operators. The proposed sequence was tested on 22 images of various situations, collected from 18 different cases of breast carcinoma. A quality control procedure was applied, comparing the automated method with manual outlining of cancer cell foci and with manual pricking of cancer cell nuclei. RESULTS: Good concordance was found between automated and manual segmentation procedures (90% for cancer cell clumps, 97% for cancer cell nuclei on average), but the rate of false positive nuclei (small regions labeled as nuclei by the segmentation procedure) could be relatively high (11% on average, with a maximum of 35%) and can result in underestimation of the immunostaining ratio. CONCLUSION: This study examined a preliminary approach to automated immunoquantification, limited to automated segmentation without any color characterization. The automated hierarchic segmentation presented here leads to good discrimination of cancer cell nuclei at the chosen magnification.     

水土流失区生态修复后植被健康的遥感判别

基于遥感技术和WorldView-2卫星影像,提出一个新型植被健康指数(VHI),以快速、大面积地监测与评价水土流失区的植被健康状况.该指数由归一化山地植被指数、氮素反射指数和黄光波段反射率3个因子构成,通过主成分变换将3个因子集成为VHI,以避免主观加权求和集成法所产生的偏差.将VHI应用于福建省长汀县河田盆地一带,对水土流失区的植被健康状况进行监测.结果表明: VHI可以很好地揭示植被的健康状况,其判别总精度可达91%.河田盆地健康等级为好、中、差的植被面积占植被总面积的比例分别为10.1%、49.2%和40.7%,说明研究区的植被健康总体状况仍不理想,主要因为水土流失区土壤贫瘠、新种植的植被生长不良.     

Differential Effects of selenium on the proliferation of human pulmonary adenocarcinoma cells and human embryonic lung diploid cells in vitro

The effect of different concentrations of Na₂SeO₃ on human pulmonary adenocarcinoma cells and human embryonic lung diploid cells in vitro was investigated. For human pulmonary adenocarcinoma cells, mitotic index and cell count decreased with increasing selenium concentrations. At 1 μg/mL sodium selenite, mitotic activity and growth of human lung cancer cells were partially inhibited, and the progression of human lung cancer cell cycle was partially arrested. When human embryonic lung diploid cells were treated with 1 μg/mL sodium selenite for five continuous days, cell counts of the treated group were closely parallel to those of the control group. After treating human embryonic lung diploid cells with 1–5μg/mL sodium selenite for 1–3 d, the mitotic index (MI), labeled index (LI), and average silver grain (SG) number per 20 labeled nuclei were the same as those of the control. In mixed cultures of human embryonic lung diploid cells and human pulmonary adenocarcinoma cells, treated with 3 and 5 μg/mL sodium selenite for 24 h, the lung diploid cells showed a normal fusiform morphology, whereas the lung cancer cells showed heavily vacuolated cytoplasms and distorted nuclei.     

Hepatoprotective and free radical scavenging effects of Nelumbo nucifera.

Ethanol extracts from Nelumbo nucifera (ENN) seeds were studied for possible antioxidative and hepatoprotective effects. Antioxidative effects were measured spectrophotometrically by reduction of 2,2'-Diphenyl-1-picrylhydrazyl (DPPH) radicals. Hepatoprotective effects were tested using carbon tetrachloride (CCl4) and aflatoxin B1 (AFB1)-induced hepatocyte toxicity models. ENN showed potent free radical scavenging effects with a median inhibition concentration of 6.49 microg/ml. Treatment of hepatocytes with ENN inhibited both the production of serum enzymes and cytotoxicity by CCl4. The genotoxic and cytotoxic effects of AFB1 were also inhibited by ENN in dose-dependent manners. These hepatoprotective effects of ENN against CCl4 and AFB1 might result from its potent antioxidative properties.     

基于多元回归树的公益林群落结构解析

为揭示不同生境类型下公益林群落物种多样性及优势种种间关系特征, 以临安市公益林125个固定监测样地为研究对象, 利用多元回归树(MRT)进行群落分类, 以物种多样性指数和种间联结系数为指标, 对不同群落类型的结构特征进行探索。结果表明: 临安市公益林125个典型样地可划分为5个类型; 类型I、II、III与类型V的多样性指数之间均无显著差异, 但类型III的多样性指数均最高, 类型IV的物种多样性指数均显著低于其它类型, 海拔是影响群落多样性水平的主要因子。种间联结初步分析显示, 研究区群落中稳定性最高的层片是乔木层, 且毛竹入侵极可能是影响5个类型植物群落稳定性的关键因素。研究结果可为临安市公益林分类经营管理提供理论依据, 同时为区域公益林群落数量结构研究奠定基础。     

Phorbol esters and mitogenesis: comparison of the proliferative response of parental and Na+K+Cl- -cotransport-defective BALB/c 3T3 cells to 12-0-tetradecanoylphorbol-13-acetate

The ability of the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) to stimulate the growth of quiescent BALB/c 3T3 cell lines lacking Na+K+Cl- cotransport activity was tested. We have previously isolated and characterized two mutant cell lines defective in this important ion transport system by mutagenesis and selection in medium containing low K+. To test our hypothesis that loss of this transport activity might abrogate the proliferative response to TPA, two kinds of mitogenesis assays were performed. First, the effect of 0.16 microM TPA on the saturation density of parental vs. mutant cell lines was determined. TPA caused a small but reproducible 30-35% increase in the saturation density of mutant cells compared to the 100-120% increase seen in parental cell lines. Second, the effect of TPA on the incorporation of 3H-thymidine into cell nuclei (labeling index) was measured. While some variability from experiment to experiment in the extent and time course of the response of mutant cells was noted, TPA either had no effect or only a small effect on the labeling index when compared to the response of parental cells. When a range of concentrations of TPA (0.016-1.6 microM) was tested, neither cell line exhibited a large response to any concentration. These results suggest that loss of Na+K+Cl- cotransport activity decreases the response of these cells to the mitogenic action of TPA.     

天津市典型湖库湿地生态完整性评价

对湖库湿地进行生态健康评价,可为湖库湿地生态系统修复和管理提供决策依据。本文以天津市为例,选取20个典型的湖库湿地采样点,基于2018年8—9月期间调查获取的物理、化学和生物群落指标(浮游动物、浮游植物、底栖动物、鱼类、水生大型植物、河岸带植物)数据,构建包含物理完整性、化学完整性和生物完整性在内的生态完整性指数(IEI)对采样点生态健康状况进行评价。根据栖息地环境质量(QHEI)、水质状况和人类活动干扰3方面选取参照点,采用标准化方法筛选候选指标,应用层次分析法计算各指标权重,最终得出天津市典型湖库湿地生态完整性评价结果。结果表明: 1)所有样点中,“健康”点位占5.0%,“较好”点位占20.0%,“一般”点位占35.0%,“较差”点位占30.0%,“差”点位占10.0%。天津市典型湖库湿地生态健康状况整体处于一般水平,呈现出西部优于东部的趋势,空间差异显著;2)基于栖息地评分、水质状况与人类活动干扰相结合选取参照点是可行的,依据水质指标可降低选择参照点时的主观性;3)适用性验证结果表明,IEI可较好表征各点位的健康状况,区分效率明显,适用于评价研究区湖库湿地生态健康状况。     

一株葡萄藻的分离、鉴定及产烃性能评价

研究从我国海南博鳌海边的淡水池塘水样分离获得一株绿藻ENN41。显微形态观察表明,ENN41的形态特征属于葡萄藻。进一步克隆ENN41的核糖体小亚基18S rRNA片段,利用分子生物学软件进行比对分析,结果表明ENN41的18S rRNA基因序列与布朗葡萄藻（Botryococcus braunii）同源性最高,说明ENN41为一株布朗葡萄藻（B.braunii）。ENN41在柱式反应器中培养16d,单位体积产率为0.483 g/（Ld）,粗烃占干重的含量为56.6%;主要脂肪酸为油酸（C18:1）、十八碳四烯酸（C18:4）和棕榈酸（C16:0）,三者之和占总脂肪酸的72.6%;利用尼罗红染色,清晰可见大量的油脂分布在细胞内和胞外基质中。ENN41在板式反应器中培养16d,单位体积产率为0.234 g/（Ld）,粗烃占干重的含量为20.0%。上述研究表明,ENN41是具有较高的生长速度和粗烃积累能力的布朗葡萄藻（B.braunii）藻株,具有产业化应用潜力。     

基于近海健康评价现有体系的我国普适海洋健康评价“双核”新框架的构建

海洋生态环境保持健康状态是人类可持续开发利用海洋资源环境的基础,而近海海洋生态环境的健康状况综合评价可为海洋生态环境良性发展以及社会经济决策提供科学依据。首先对国际上3种代表性的海洋环境健康综合评价方法的应用特点进行了对比分析,并归纳了我国海洋生态环境健康评价的状况,在此基础上,以指标体系法重构了一个双核结构的综合评价框架,其中内核评价以海洋生态系统的自身状态为主,外核评价则主要反映海洋之于人类经济社会的贡献,以期能够科学、准确、全面的评价我国近海海洋环境健康状况。     

Differential reaction of condensed and diffuse chromatin to polyamines. I. Reaction of interphase nuclei chromatin to putrescine]

Condensation of the interphase nuclei chromatin under putrescine treatment was studied in cultured human fibroblasts 46, XX: 47, XXX: 49, XXXXY, and aneuploid cells of the Chinese hamster. The effect was tested separately for diffuse and condensed chromatin. Putrescine treatment did not affect the percentage of cell nuclei with X-chromatin bodies in the human cell strains while significantly increasing the percentage of nuclei with coarse chromatin network and chromocenters. In cultured Chinese hamster cells, putrescene did not change the percentage of nuclei with identified chromocenters and no significant condensation of diffuse chromatin was observed either.     

Rapid Assessment of Seed Removal and Frugivore Activity as a Tool for Monitoring the Health Status of Tropical Forests

Ecological processes in tropical forests are being affected at unprecedented rates by human activities. Yet, the continuity of ecological functions like seed dispersal is crucial for forest regeneration. It thus becomes increasingly urgent to be able to rapidly assess the health status of these processes in order to take appropriate management measures. We tested a method to rapidly evaluate seed removal rates on two animal‐dispersed tree species, Virola kwatae and V. michelii (Myristicaceae), at three sites in French Guiana with increasing levels of anthropogenic disturbance. We counted fallen fruits, fruit valves, and seeds of each focal fruiting tree in a single 1 m² quadrat, and calculated two indices: the proportion of seeds removed and the proportion of fruits opened by mammals. They both provide an indirect and rapid assessment of frugivore activity. Our results showed a significant decrease in the proportion of removed seeds (16%) and fruits opened (19%) at the most impacted site in comparison with the other two sites (79% for seeds, 60% and 35% for fruits). This testifies to an increased impoverishment of the primate and toucan communities at the disturbed sites. This standardized protocol provides fast information about the health status of the community of seed dispersers and predators and of their seed removal services. It is time‐ and cost‐effective and is not species‐specific, allowing comparisons among sites or over time. We suggest using it with the pantropical Myristicaceae and any other capsule‐producing family to rapidly assess the health status of seed removal processes across the tropics.     

基于耦合模型和遥感技术的江苏中部海岸带生态系统健康评价

以生态系统健康理论为基础,从资源环境、景观生态、人类活动3个方面,按活力、组织力和恢复力3个子系统选取了10个代表性指标,构建了遥感技术支持下的海岸带陆域生态系统健康评价指标体系,并耦合TOPSIS模型和VOR生态系统健康度量模型对江苏中部海岸新洋港至川东港岸段进行了应用评价。研究结果表明:江苏中部海岸生态系统健康状态处于健康和良好的区域占27.62%,一般占60.94%,较差和差占11.44%,整体生态健康状况中等偏好。从地物类型和空间分布来看,斗龙港至四卯酉河岸段以滩涂植被、农田和围海养殖区为主,植被和水体对气候调节有积极作用,整体生态健康状况良好;四卯酉河至王港岸段由于大丰港建设,港区陆域植被覆盖率低、热岛效应强,建筑将原本连通的自然景观隔断,导致斑块数量增多,斑块面积减小,加剧了景观破碎化,对原有海岸带生态系统产生一定的破坏,生态健康状况相对较差;此外,新洋港至斗龙港岸段以及川东港岸段以自然保护区湿地为主,植被覆盖度高,人为干扰程度小,生态健康状况也较好。由于该耦合模型评价方法直接基于遥感监测数据,且无需赋予指标权重,因此研究结果相对更加客观,更能反映海岸带生态系统的实际健康状况。     

Digital morphonuclear analyses of sensitive versus resistant neoplastic cells to vinca-alkaloid, alkylating, and intercalating drugs

We tested 12 resistant cell lines in vitro in order to evaluate common morphonuclear characteristics induced by various cytotoxic drugs on cell lines of different origins. We used the MXT mouse mammary cancer and the neoplastic J82 and T24 human bladder cell lines, whose variants are either sensitive or resistant to a vinca alkaloid derivative (Navelbine, NVB), to an investigational alkylating agent (PE1001), and to Adriamycin (ADR). We tested cell population variants resistant to NVB + PE1001 + ADR. The level of chemoresistance was evaluated by a colorimetric assay assessing the 50% concentration-induced inhibition of cellular growth (IC50) brought about by each drug on the growth of each cell variant under study. We show that resistant neoplastic cell nuclei present common morphonuclear characteristics, independent of cell origin (neoplastic mouse mammary versus human bladder cells) and the drug used (vinca alkaloid, alkylating, and intercalating derivatives). Our results further indicate that the phenotype of resistant versus sensitive cells corresponds to cell nuclei populations with smaller nuclei and less nuclear DNA content and, as a consequence, a chromatin texture showing large pale areas with some hyperchromatic clumps.     

基于压力-状态-响应和物元可拓模型的城市河流健康评价

城市河流作为人类活动和自然过程相互作用强烈的区域,承受着人类资源开发利用和污染物排放的压力,但近些年来随着对河流健康的重视,针对城市河流实施了大量的生态治理措施。综合考虑人类正负面影响对河流健康状态的影响,进行河流健康状态的评价,对于城市河流的修复效果检验和管理保护具有重要意义。以南京市鼓楼区典型城市河流西北护城河为研究区,根据河流周边土地利用类型、物理形态差异及城市管网分布将其划分为6个河段,考虑人类修复措施对河流健康水平的影响,构建了基于压力-状态-响应(Pressure-State-Response,PSR)模型的城市河流健康评价指标体系,采用物元可拓模型确定了河段健康等级,并根据各等级综合关联度之间的距离判断河流健康水平的发展趋势。结果表明:压力、状态以及响应准则层的权重分别为0.458、0.311、0.231,压力指标为影响城市河流健康水平的主要因素;西北护城河6个河段的健康水平表现出较强的空间异质性,河段A-F的健康等级分别为中等、健康、亚健康、中等、亚病态和病态,流经公园绿地的河段健康水平明显高于流经居民住宅区的河段,河段B、C、D的健康水平有进一步提升的趋势,而河段A、E、F的健康状态则有进一步恶化的趋势,需要进行重点关注。     

A rapid and simple staining method, using Toluidine Blue, for analysing mitotic figures in tissue sections

Summary Mitotic index is a clinically important parameter in cancer pathology. We developed a staining method using Toluidine Blue to detect efficiently and rapidly mitotic figures in sections of formalin-fixed paraffin-embedded human and rat tisues. Sections were stained at acid pH with a 0.01% Toluidine Blue solution after removal of RNA with hydrochloric acid or ribonuclease. The optimal pH of the TB staining solution was found to be 4.5 for rat tissues and 3.5 for human tissues. This procedure stained mitotic figures much more intensely than other (extra)cellular structures. A quantitative estimate of the total number of nuclei in the field where mitotic figures were counted, was obtained in an adjacent section hydrolysed in 5 N hydrochloric acid and stained by the Feulgen reaction with a Schiff-type reagent containing 0.01% Toluidine Blue. This method specifically stained interphase and mitotic nuclei and the field cellularity could be quantified by image cytometry. When these procedures were performed on two consecutive serial sections, a mitotic index could be determined accurately by relating the count of mitotic figures to the number of tumour cells.