The contrasting effects of fluctuating temperature on bacterial diversity and performances in temperate and subtropical soils.

The extremely high species diversity of soil bacterial community has fascinated and puzzled community ecologists. Although theory predicts that fluctuations in environments can facilitate diversity maintenance, the effects of fluctuating temperature on species diversity have rarely been investigated in species-rich microbial communities. Here, we examined whether fluctuating temperature had positive effects on species diversity relative to constant temperatures in soil bacterial communities, and investigated the effects of fluctuating temperature on bacterial performances (changes in relative abundance). We performed a temperature manipulation experiment with soils collected from temperate and subtropical zones, where the soils were subjected to constant high, low or fluctuating temperatures. We found that fluctuating temperatures showed significant positive effects on species diversity. The time-averaged effect of fluctuating temperatures (i.e., averaging out the differences between species in their environment-dependent performances) appeared to delay species loss in both the temperate and the subtropical communities. In addition, we found that the performances of temperature-responsive species at fluctuating temperatures significantly deviated from their time-weighted average performances at constant high and low temperatures, which was defined as fluctuation-dependent effects in our study. Intriguingly, fluctuation-dependent effects beyond time-averaged effect led to an opposite trend: differences in temperature-responsive species' performances decreased in the temperate communities, but increased in the subtropical communities. Our findings provide new insights into diversity maintenance in soil bacterial communities, and imply that the effects of fluctuating temperature on species diversity in soil bacterial community might vary across latitude.

Seasonal Climate Variations Promote Bacterial α-Diversity in Soil.

Ecological theory suggests that temporal environmental fluctuations can contribute greatly to diversity maintenance. Given bacteria's short generation time and rapid responses to environmental change, seasonal climate fluctuations are very likely to play an important role in maintaining the extremely high α-diversity of soil bacterial community, which has been unfortunately neglected in previous studies. Here, with in-depth analyses of two previously published soil bacterial datasets at global scale, we found that soil bacterial α-diversity was positively correlated with both seasonal variations of temperature and precipitation. Furthermore, piecewise structural equation models showed that seasonal variations of temperature or precipitation had weak but significant positive effect on soil bacterial α-diversity in each dataset. However, it is noteworthy that the importance of seasonal climate variations might be underestimated in the above analyses, due to the potential confounding factors (such as vegetation type) and the lack of sampling across seasons. As a supplement, we analyzed a previously published wheat cropland dataset with samples collected in both winter and the following summer across North China Plain. As expected, bacterial α-diversity was positively correlated with seasonal climate variations in the cropland dataset, and climate seasonality explained a larger proportion of variations in bacterial α-diversity. Collectively, these findings implied that fluctuation-dependent mechanisms of diversity maintenance presumably operate in soil bacterial communities. Based on existing evidence, we speculated that the storage effect may be the main mechanism responsible for diversity maintenance in soil bacterial community, but rigorous experimental tests are needed in the future.

Specific adaptation to strong competitors can offset the negative effects of population size reductions.

Competition plays a crucial role in determining adaptation of species, yet we know little as to how adaptation is affected by the strength of competition. On the one hand, strong competition typically results in population size reductions, which can hamper adaptation owing to a shortage of beneficial mutations; on the other hand, specificity of adaptation to competitors may offset the negative evolutionary consequences of such population size effects. Here, we investigate how competition strength affects population fitness in the bacterium Pseudomonas fluorescens Our results demonstrate that strong competition constrains adaptation of focal populations, which can be partially explained by population size reductions. However, fitness assays also reveal specific adaptation of focal populations to particular competitors varying in competitive ability. Additionally, this specific adaptation can offset the negative effects of competitor-mediated population size reductions under strong competition. Our study, therefore, highlights the importance of opposing effects of strong competition on species adaptation, which may lead to different outcomes of colonization under intense and relaxed competitive environments in the context of population dispersal.

Field experimental evidence that stochastic processes predominate in the initial assembly of bacterial communities.

To assess the relative importance of environmental selection, dispersal and stochastic processes in structuring ecological communities, we conducted a bacterial community assembly experiment using microcosms filled with sterile liquid medium under field conditions in the Inner Mongolian grasslands. Multiple replicate microcosms containing different carbon substrates were placed at nine locations across three spatial scales (10, 300 and 10 000 m distance between locations) in such a way that the environment of microcosms varies independently of the geographical distance. The operational taxonomic units within the experimental communities were assessed via the terminal restriction fragment length polymorphism techniques on the 10th and 17th days after the onset of the experiment. We found no evidence of distance decay in community similarity, and communities within a given location were more similar to each other regardless of environment than communities at other locations within the same spatial scale. Variance partitioning indicated that location explained more compositional variation in microbial communities than environment, particularly on the 17th day, despite that environment and location in combination could only explain less than half of the total variation. These results suggest that bacterial dispersal is not limited by distance in this experiment, and community assembly in microcosms is not environmentally determined but governed by stochastic processes.

[Establishment and evaluation of haloalkane dehalogenase tagged α1A-adrenergic receptor chromatography].

Receptor chromatography is an efficient analytical technique that combines the high separation ability of chromatography with the high specificity of receptors for drug recognition. In addition, this technique offers the advantages of active recognition, online separation, and convenient multidimensional target tracking. This strategy allows target active ingredients in complex systems, such as traditional Chinese medicines, to be efficiently screened and accurately identified. Furthermore, the interactions between ligands and immobilized proteins can be studied. To avoid a loss in function, receptor chromatography requires efficient, mild, and simple immobilization methods that do not damage the structure of the immobilized receptors. Improvements in the activity, stability, and ligand-recognition specificity of immobilized functional proteins can be achieved by selecting appropriate immobilization conditions. Notably, the protein immobilization method is not only closely related to the recognition ability of receptor chromatography but also determines the accuracy of the technique. Common methods for immobilizing functional proteins include physical adsorption, chemical reactions, biological affinity reactions, and click chemistry. Despite being easy to operate under mild reaction conditions, these methods have shortcomings, including poor reaction specificity and the necessity of using high-purity functional proteins to prepare chromatography columns. Maintaining the high activity of immobilized receptors and ensuring excellent identification and separation abilities are key challenges in the further development of receptor chromatography. In this work, these issues were addressed by introducing a specific bioorthogonal reaction involving haloalkane dehalogenase (Halo) and 6-chlorohexanoic acid for the immobilization of the α1A-adrenergic receptor (α1A-AR). Specifically, Halo-α1A-AR was immobilized on the surface of 6-chlorohexanoic acid-modified aminopropyl silica gel in one step. The stationary phase with immobilized Halo-α1A-AR was characterized using scanning electron microscopy. Moreover, the activity of the Halo-α1A-AR chromatographic column was evaluated using specific ligands (terazosin hydrochloride, phentolamine mesylate, tamsulosin hydrochloride, and urapidil) and nonspecific ligands (yohimbe and metoprolol) for α1A-AR. Halo-α1A-AR was successfully immobilized on the silica gel surface with good stability over 30 days, and the Halo-α1A-AR chromatographic column exhibited good ligand-recognition activity. The nonlinear chromatography results indicated that prazosin hydrochloride, terazosin hydrochloride, and urapidil interacted with immobilized Halo-α1A-AR through one type of binding site, with association constants of 3.85×105, 5.00×105, and 5.90×105L/mol, respectively. In contrast, phentolamine mesylate and tamsulosin hydrochloride interacted with immobilized Halo-α1A-AR through two types of binding site. The association constants with the high- and low-affinity binding sites were 3.12×106 and 6.01×105L/mol, respectively, for phentolamine mesylate and 9.98×105 and 0.21×105L/mol, respectively, for tamsulosin hydrochloride. Compared with the traditional carbonyldiimidazole method, the immobilization method developed in this work did not require receptor purification and thus minimized the loss of receptor activity. The affinity constants obtained with immobilized Halo-α1A-AR were consistent with the values determined for receptor-ligand binding in solution, indicating that the Halo-α1A-AR chromatography column is suitable for studying drug-protein interactions. This approach also provides a foundation for the efficient screening and accurate determination of target active ingredients in complex systems.

Revealing binding interaction between seven drugs and immobilized ß2-adrenoceptor by high-performance affinity chromatography using frontal analysis.

The development of new approaches to study the affinity between ligands and G-protein-coupled receptors proves to be of growing interest for pharmacologists, chemists, and biologists. The aim of this work was to determine the binding of seven drugs to ß2-adrenoceptors by frontal analysis using immobilized receptor stationary phase. The dissociation constants (Kd ) were determined to be (3.16 ± 0.09) × 10(-4) M for salbutamol, (4.29 ± 0.12) × 10(-4) M for terbutaline, (6.19 ± 0.16) × 10(-4) M for methoxyphenamine, (2.11 ± 0.07) × 10(-4) M for tulobuterol, (1.82 ± 0.11) × 10(-4) M for fenoterol, (9.75 ± 0.24) × 10(-6) M formoterol, and (9.84 ± 0.26) × 10(-5) M for clenbuterol. These results showed a good correlation with the data determined by radioligand binding assay. Further investigations revealed that the dissociation constant mainly attributed to the number of hydrogen bonds in the structures of ligands. This study indicates that affinity chromatography using immobilized receptor stationary phase can be used for the direct determination of drug-receptor binding interactions and has the potential to become a reliable alternative for quantitative studies of ligand-receptor interactions.

N-methyl-D-aspartic acid receptor 2A functionalized stationary phase: A reliable method for pursuing potential ligands against Alzheimer's disease from natural products.

AIMS: N-methyl-D-aspartic acid (NMDA) receptors play subunit-specific role in central neuronal development. However, insights into the pharmacological modulation of NMDA receptors were mainly lack of subunit and synaptic selectivity. The purpose of the present study was to develop a novel strategy to rapidly recognize NMDA subunit 2A (NMDA-2A) ligands from natural products and provide subunit-selective drug candidates for Alzheimer's disease (AD). METHODS: The recombinant NMDA-2A containing a tag of epidermal growth factor receptor (EGFR) was expressed in Escherichia coli cells and immobilized on ibrutinib-modified microspheres based on the specific reaction between EGFR and its inhibitor ibrutinib. A novel affinity stationary phase was synthesized to screen NMDA-2A ligands from Gardenia jasminoides Ellis. RESULTS: The immobilized receptor column exhibited excellent receptor selectivity and ligand-binding activity. Crocetin was screened by using this method. In a cellular model of AD, the protein level of NMDA-2A was significantly decreased compared with the control group, while treatment with crocetin significantly increased NMDA-2A level in a concentration-dependent manner, confirming that crocetin could bind to NMDA-2A in vitro. CONCLUSION: In the present study, we developed a reliable method for the rapid identification of NMDA-2A ligands from natural products, which may be used as a platform for new drug discovery to generate high-quality drug candidates.

Gut microbiome responds compositionally and functionally to the seasonal diet variations in wild gibbons.

Wild animals may encounter multiple challenges especially food shortage and altered diet composition in their suboptimal ranges. Yet, how the gut microbiome responds to dietary changes remains poorly understood. Prior studies on wild animal microbiomes have typically leaned upon relatively coarse dietary records and individually unresolved fecal samples. Here, we conducted a longitudinal study integrating 514 time-series individually recognized fecal samples with parallel fine-grained dietary data from two Skywalker hoolock gibbon (Hoolock tianxing) groups populating high-altitude mountainous forests in western Yunnan Province, China. 16S rRNA gene amplicon sequencing showed a remarkable seasonal fluctuation in the gibbons' gut microbial community structure both across individuals and between the social groups, especially driven by the relative abundances of Lanchnospiraceae and Oscillospiraceae associated with fluctuating consumption of leaf. Metagenomic functional profiling revealed that diverse metabolisms associated with cellulose degradation and short-chain fatty acids (SCFAs) production were enriched in the high-leaf periods possibly to compensate for energy intake. Genome-resolved metagenomics further enabled the resolving metabolic capacities associated with carbohydrate breakdown among community members which exhibited a high degree of functional redundancy. Our results highlight a taxonomically and functionally sensitive gut microbiome actively responding to the seasonally shifting diet, facilitating the survival and reproduction of the endangered gibbon species in their suboptimal habitats.

The alleviation of acute and chronic kidney injury by human Wharton's jelly-derived mesenchymal stromal cells triggered by ischemia-reperfusion injury via an endocrine mechanism.

BACKGROUND AIMS: The effects of human Wharton's jelly-derived mesenchymal stromal cells (WJ-MSC) on acute and chronic kidney injury induced by ischemia-reperfusion injury (IRI) were assessed. METHODS: WJ-MSC were injected intravenously immediately after solitary kidney ischemia for 45 min. Cells were labeled with 5-bromo-2'deoxy-uridine (BrdU) for tracing in vivo. At 48 h post-IRI, serum creatinine and blood urea nitrogen (BUN) were measured. Tubular cell proliferation and apoptosis as well as activation of the Akt signal were identified by immunostaining. Real-time polymerase chain reaction (PCR) was employed to determine gene expression of inflammation-related cytokines and hepatocyte growth factor (HGF). Levels of human HGF were assayed by enzyme-linked immunosorbant assay (ELISA). Twenty-two weeks later, renal fibrosis was assessed by Masson's tri-chrome staining, collagen content and α-smooth muscle actin (α-SMA) staining. RESULTS: There was no sign of labeled cells residing in the damaged kidney. Acute renal dysfunction elicited by IRI was considerably improved by WJ-MSC, in parallel with a stronger proliferative response and less apoptotic events. Additionally, phosphoAkt staining in injured tubular cells was substantially intensified. Cell treatment also caused a remarkable up-regulation of kidney interleukin (IL)-10, heme oxygenase (HO)-1 and HGF expression. Human HGF was detected in cell supernatants and the serum of cell-infused rats. Moreover, IRI-initiated fibrosis was abrogated by cell therapy, coincident with function amelioration. CONCLUSIONS: WJ-MSC alleviate acute kidney injury, thereby rescuing the ensuing fibrotic lesions in an endocrine manner. The Akt signal in impaired tubular cells is reinforced by WJ-MSC, facilitating cell resistance to apoptosis and cell proliferation. HGF, either delivered or induced by WJ-MSC, is an important contributor.

Preparation and characterization of immobilized 5-HT1A receptor stationary phase for high throughput screening of the receptor-binding ligands from complex systems like Curcuma wenyujin Y. H. Chen et C. Ling extract.

The incidence of depression has increased significantly during the COVID-19 pandemic. This disease is closely associated with serotonin 1A (5-HT1A) receptor and often treated by complex prescription containing Curcuma wenyujin Y. H. Chen et C. Ling. Therefore, we hypothesized that this herb contains bioactive compounds specially binding to the receptor. However, the rapid discovery of new ligands of 5-HT1A receptor is still challenging due to the lack of efficient screening methods. To address this problem, we developed and characterized a novel approach for the rapid screening of ligands by using immobilized 5-HT1A receptor as the chromatographic stationary phase. Briefly, haloalkane dehalogenase was fused at the C-terminal of 5-HT1A receptor, and the modified 5-HT1A receptor was immobilized on amino-microspheres by the reaction between haloalkane dehalogenase and 6-chlorohexanoic acid linker. Scanning electron microscope and X-ray photo-electron were used to characterize the morphology and element of the immobilized receptor. The binding of three specific ligands to 5-HT1A receptor was investigated by two different methods. Moreover, we examined the feasibility of 5-HT1A receptor colume in high throughput screening of new ligands from complex systems as exemplified by Curcuma wenyujin Y. H. Chen et C. Ling. Gweicurculactone, 2-hydroxy-1-(3,4-dihydroxybenzene)-7-(4'-hydroxybezene)-heptane and curcuminol F were identified as the ligands of 5-HT1A receptor with the binding energies of -7.06 kcal/mol, -7.77 kcal/mol and -5.26 kcal/mol, respectively. Collectively, these results indicated that the immobilized 5-HT1A receptor was capable of screening bioactive compound from complex system, providing an effective methodology for high throughput screening.

Microbial biogeography of acid mine drainage sediments at a regional scale across southern China.

Investigations of microbial biogeography in extreme environments provide unique opportunities to disentangle the roles of environment and space in microbial community assembly. Here, we reported a comprehensive microbial biogeographic survey of 90 acid mine drainage (AMD) sediment samples from 18 mining sites of various mineral types across southern China. We found that environmental selection was strong in determining the AMD habitat species pool. However, microbial alpha diversity was primarily explained by mining sites rather than environmental factors, and microbial beta diversity correlated more strongly with geographic than environmental distance at both large and small spatial scales. Particularly, the presence/absence of widespread AMD habitat generalists was only correlated with geographic distance and independent of environmental variation. These distance-decay patterns suggested that spatial processes played a more important role in determining microbial compositional variation across space; which could be explained by the reinforced impacts of dispersal limitation in less fluid, spatially structured sediment habitat with diverse pre-existing communities. In summary, our findings suggested that the deterministic assembling and spatial constraints interact to shape microbial biogeography in AMD sediments; and provided implications that spatial processes should be considered when predicting microbial dynamics in response to severe environmental change across large spatial scales.

Isopropyl 3-(3,4-dihydroxy-phen-yl)-2-hydroxy-propanoate.

The title compound, C(12)H(16)O(5), is a derivative of ß-(3,4-dihydroxy-phen-yl)-α-hydr-oxy acid. The crystal packing is stabilized by inter-molecular O-H⋯O hydrogen bonds.

Local biotic interactions drive species-specific divergence in soil bacterial communities.

It is well accepted that environmental heterogeneity and dispersal are key factors determining soil bacterial community composition, yet little is known about the role of local biotic interactions. Here we address this issue with an abundance-manipulation experiment that was conducted in a semiarid grassland. We manually increased the abundance of six randomly chosen resident bacterial species in separate, closed, communities and allowed the communities to recover in situ for 1 year. The single episode of increase in the abundance of different species drove species-specific community divergence accompanied by a decline in local diversity. Four of the six added species caused a decrease in the abundance of their closely related species, suggesting an important role of interspecific competition in driving the observed community divergence. Our results also suggested a lack of effective population regulations to force the relative abundance of manipulated species to revert to original level, which would allow persistence of the divergence among soil bacterial communities. We concluded that biotic interactions were important in determining soil bacterial community composition, which could result in substantial variation in soil bacterial community composition in abiotically homogenous environment.

Vasorelaxant effect of isopropyl 3-(3, 4-dihydroxyphenyl)-2-hydroxypropanoate, a novel metabolite from Salvia miltiorrhiza, on isolated rat mesenteric artery.

The present study was designed to investigate the relaxant effect of isopropyl 3-(3, 4-dihydroxyphenyl)-2-hydroxypropanoate (IDHP), a new metabolite from Salvia miltiorrhiza, on rat mesenteric artery. Isolated mesenteric arterial rings were mounted in organ baths and the isometric tension changes were measured continuously by a sensitive myograph system. The results showed that IDHP at concentrations greater than 0.1 nM produced a concentration-dependent relaxation of artery contracted by norepinephrine with pEC(50) of 7.41+/-0.08. Removal of the endothelium did not affect this relaxation, suggesting that IDHP exerted a direct effect on vascular smooth muscle cells. Meanwhile, the vasorelaxant effect of IDHP was unaffected by pre-treatment with ATP-sensitive K(+) channel inhibitor glibenclamide, delayed rectifier K(+) channel inhibitor 4-aminopyridine, inwardly rectifying K(+) channel inhibitor barium chloride and beta-adrenoceptor antagonist propranolol. However, the non-specific K(+) channel inhibitor tetraethylammonium (TEA, 3 mM) produced a rightward shift of 1.8 fold on the concentration-response curve of IDHP. Moreover, IDHP shifted the concentration-response curve of CaCl(2) as well as two receptor-mediated constrictors, phenylephrine and 5-hydroxytryptamine, to the right in a non-parallel manner. In the absence of extracellular Ca(2+), IDHP depressed the contractions induced by norepinephrine and CaCl(2), and the maximal inhibitions were 48.3+/-18.9% and 58.4+/-10.9%, respectively. These results suggest that IDHP exerts a vasorelaxant effect by inhibiting both Ca(2+) release from intracellular stores and Ca(2+) influx through voltage-dependent calcium channels, and receptor-operated calcium channels in vascular smooth muscle cells. In addition, activation of vascular TEA-sensitive K(+) channels may be partially involved in the relaxant effect of IDHP.

Stability of A Coevolving Host-parasite System Peaks at Intermediate Productivity.

Habitat productivity may affect the stability of consumer-resource systems, through both ecological and evolutionary mechanisms. We hypothesize that coevolving consumer-resource systems show more stable dynamics at intermediate resource availability, while very low-level resource supply cannot support sufficiently large populations of resource and consumer species to avoid stochastic extinction, and extremely resource-rich environments may promote escalatory arms-race-like coevolution that can cause strong fluctuations in species abundance and even extinction of one or both trophic levels. We tested these ideas by carrying out an experimental evolution study with a model bacterium-phage system (Pseudomonas fluorescens SBW25 and its phage SBW25Φ2). Consistent with our hypothesis, this system was most stable at intermediate resource supply (fewer extinction events and smaller magnitude of population fluctuation). In our experiment, the rate of coevolution between bacterial resistance and phage infectivity was correlated with the magnitude of population fluctuation, which may explain the different in stability between levels of resource supply. Crucially, our results are consistent with a suggestion that, among the two major modes of antagonistic coevolution, arms race is more likely than fluctuation selection dynamics to cause extinction events in consumer-resource systems. This study suggests an important role of environment-dependent coevolutionary dynamics for the stability of consumer-resource species systems, therefore highlights the importance to consider contemporaneous evolutionary dynamics when studying the stability of ecosystems, particularly those under environmental changes.

[Tumor targeting gene transfer mediated by electroporation for growth suppression of retinoblastoma in vivo].

OBJECTIVE: To explore the effects and mechanisms of electroporation on the therapy of transplanted retinoblastoma tumor (RB) by transferring the expressive plasmids of sFlk-1 and ExTek. METHODS: The effects and mechanisms of sFlk-1 and ExTek on RB is validated both in vitro and in vivo. In vitro, pCI-sFlk-1 was transfected into HEK293 (human embryonic kidney 293) cells with DMRIE-C Reagent, and its supernatant was collected 24 h later to mix with RPMI-1640 medium as 1:1, 2:1 and 3:1. RB cells were cultured in the mixed medium and the cell growth curve were analyzed 6 days later. The suspension of 1 x 10(7) RB cells were inoculated in 40 nude mice subcutaneously. The nude mice with RB were divided into 2 groups. The experimental group was treated with transfer of pCI-sFlk-1 (15 microg) and pCI-ExTek (15 microg) by electroporation once a week, while the control group was treated with physiological saline solution. After 3 weeks' therapy, the animals were observed for one more week. During this period the tumors were measured to draw the growth curve of the tumors and to calculate the anti-tumor rate. The vessel endothelium were tagged with smooth muscle actin (SMA) by immunohistochemical methods to inspect the micro vessel density (MVD) of the tumors. Moreover, the expression of vascular endothelial growth factor (VEGF) and its receptor Flk-1 of the tumors was confirmed by immunohistochemical staining. RESULTS: sFlk-1 exhibited no inhibiting effects on the growth of RB cells in vitro, while sFlk-1 combined with ExTek restrained the growth of the transplanted tumors obviously in vivo, and the anti-growth rate is 80.04%. The MVD of the experimental group is 26.69 +/- 2.95, which is evidently lower than that of the control group (44.51 +/- 6.11). The difference between these 2 groups was statistically significant (P < 0.05). As to the expression of VEGF, there was no significant difference between these 2 groups, while the expression of Flk-1 of the experimental group was much stronger than that of the control group. CONCLUSION: Transfer the expressive plasmids of sFlk-1 and ExTek by electroporation in vivo restrained the growth of the transplanted RB tumor, and the effect is due to the inhibition of angiogenesis process.

Study of the determination and pharmacokinetics of Compound Danshen Dripping Pills in human serum by column switching liquid chromatography electrospray ion trap mass spectrometry.

Compound Danshen Dripping Pill (CDDP) is an important drug widely used for the treatment of cardiovascular diseases. An active component Danshensu (DS) of CDDP was separated by reversed-phase high performance liquid chromatography using column-switching system and analyzed by electrospray mass spectrometry. With this validated assay the pharmacokinetics of CDDP was studied in 10 healthy volunteers after a single oral administration of 250 mg. After trichloroacetic acid precipitation of serum proteins, the analytes were preconcentrated and black-flushed on a reversed-phase column for separation using a switching valve. The analytes were ionized using negative electrospray ionization (ESI) mode. The precursor ion of m/z 196.6 was used to quantify DS in serum. The linear calibration curve ranged from 1.25 to 175 microg/mL. The limit of quantification (LOQ) for DS was 0.15 microg/mL. The intra-day and inter-day precision (R.S.D.) was less than 7.4 and 7.9%, respectively.

[Subretinal transplantation of genetically modified human retinal pigment epithelium cells delay photoreceptor degeneration and functional deterioration in rats].

OBJECTIVE: To investigate whether subretinal transplantation of genetically modified human retinal pigment epithelium cells can rescue photoreceptor degeneration in RCS rats. METHODS: A spontaneously derived adult human retinal pigment epithelium (RPE) cell line (CRL-2302) was infected by gfp retrovirus, then transfected by liposome mediated CNTF expression plasmid transfer. Around 1 x 10(5) genetic modified cells were injected to subretinal space of the right eye at 4 - 5 weeks old, the left eye was left without injection or injected with PBS as controls. RESULTS: The GFP expression cells under the retina were observed from 1 to 6 weeks after transplantation. Histologic and ultrastructural assessment demonstrated substantial sparing of photoreceptors and correction of RPE phagocytosis defect. Electrophysiological assessment revealed an increased sensitivity of treated eyes to white light. CONCLUSION: The results demonstrate that potential of genetically modified RPE cells for ultimate application in therapeutic transplantation strategies for human retinal degenerative diseases.

[A new orthotopic retinoblastoma model expressing green fluorescent protein].

OBJECTIVE: In order to observe the growth and metastasis of the tumor directly, a new orthotopic retinoblastoma model was established with human RB cells expressing green fluorescent protein (GFP). METHODS: pEGFP-N(1), the eukaryotic expressive plasmid of GFP, was transferred into human RB cell line HXO-RB(44) by liposome Dosper. Then, the cell clones expressing GFP steadily were selected by means of neomycin, fluorescence microscope, and flow cytometer. Two microliters of RB cells (density at 4.5 x 10(8) - 5.5 x 10(8) cells per ml) were injected into the subretinal space of 30 nude mice (60 eyes) under binocular operating microscope. The growth of transplanted RB was observed in vivo using fluorescence stereomicroscope. The nude mice were killed at different times post-operatively to investigate the metastasis process of the tumor to the optic nerve, brain, and other organs including lung, liver and kidney. RESULTS: The spread process of the tumor in the subretinal space was successfully observed under stereomicroscope. Transplanted RB developed into extra-ocular stage phase at 34 - 37 days after the operation. And metastasis to the cranium along the optic nerve was observed, with green RB cells distributing along the optic nerve sheath and long posterior ciliary artery. The histopathological characteristics of the transplanted tumor were similar to the human RB. Immunohistochemical staining showed positive expression of GFP in the tumor cells. CONCLUSION: The established orthotopic RB model expressing GFP via injection of human RB cells into the subretinal space of nude mice provides a new approach to exploring the growth and metastasis processes of RB in natural situations.

The role of late-acting self-incompatibility and early-acting inbreeding depression in governing female fertility in monkshood, Aconitum kusnezoffii.

Reduced seed yields following self-pollination have repeatedly been observed, but the underlying mechanisms remain elusive when self-pollen tubes can readily grow into ovaries, because pre-, post-zygotic late-acting self-incompatibility (LSI), or early-acting inbreeding depression (ID) can induce self-sterility. The main objective of this study was to differentiate these processes in Aconitum kusnezoffii, a plant lacking stigmatic or stylar inhibition of self-pollination. We performed a hand-pollination experiment in a natural population of A. kusnezoffii, compared seed set among five pollination treatments, and evaluated the distribution of seed size and seed set. Embryonic development suggested fertilization following self-pollination. A partial pre-zygotic LSI was suggested to account for the reduced seed set by two lines of evidence. The seed set of chase-pollination treatment significantly exceeded that of self-pollination treatment, and the proportion of unfertilized ovules was the highest following self-pollination. Meanwhile, early-acting ID, rather than post-zygotic LSI, was suggested by the findings that the size of aborted selfed seeds varied continuously and widely; and the selfed seed set both exhibited a continuous distribution and positively correlated with the crossed seed set. These results indicated that the embryos were aborted at different stages due to the expression of many deleterious alleles throughout the genome during seed maturation. No signature of post-zygotic LSI was found. Both partial pre-zygotic LSI and early-acting ID contribute to the reduction in selfed seed set in A. kusnezoffii, with pre-zygotic LSI rejecting part of the self-pollen and early-acting ID aborting part of the self-fertilized seeds.