Development of 3D hydrogel culture systems with on-demand cell separation

Recently there has been an increased interest in the effects of paracrine signaling between groups of cells, particularly in the context of better understanding how stem cells contribute to tissue repair. Most current 3D co-culture methods lack the ability to effectively separate two cell populations after the culture period, which is important for simultaneously analyzing the reciprocal effects of each cell type on the other. Here, we detail the development of a 3D hydrogel co-culture system that allows us to culture different cell types for up to 7 days and subsequently separate and isolate the different cell populations using enzyme-sensitive glues. Separable 3D co-culture laminates were prepared by laminating PEG-based hydrogels with enzyme-degradable hydrogel adhesives. Encapsulated cell populations exhibited good segregation with well-defined interfaces. Furthermore, constructs can be separated on-demand upon addition of the appropriate enzyme, while cell viability remains high throughout the culture period, even after laminate separation. This platform offers great potential for a variety of basic cell signaling studies as the incorporation of an enzyme-sensitive adhesive interface allows the on-demand separation of individual cell populations for immediate analysis or further culture to examine persistence of co-culture effects and paracrine signaling on cell populations. See accompanying commentary by Danielle R. Bogdanowicz and Helen H. Lu DOI: 10.1002/biot.201300054     

A method for large-scale multiplication of Curculigo orchioides through bulbil formation from leaf explant in shake flask culture

An efficient method has been developed for large-scale multiplication of Curculigo orchioides (Hypoxidaceae), an endangered medicinal plant, through direct bulbil formation from leaf explants in shake flask cultures. Leaf-segments (7 x 10 mm) were cultured in B5 liquid medium containing KNO3 (200 mgNL-1), (NH4)2SO4 (50 mgNL-1), benzyl adenosine (2.2 microM), adenine (0.11 mM), indole butyric acid (1.0 microM) and polyvinyl pyrrolidone (250 mgL-1). About 95% of explants produced maximum number of bulbils (546/flask at 6 weeks growth) in the medium. Shake flask cultures yielded 2737 bulbils/L medium whereas static cultures yielded 624 bulbils/L medium. Germination of bulbils was maximum (90.62%) on agar-gelled B5 medium containing benzyl adenosine (2.2 microM) and gibberellic acid (3.5 microM). Plantlets developed in vitro were successfully transferred to soil with a high rate of survivability (90%) and were comparable to natural population in growth and vigour.     

SUMMARY OF GREEN PLANT PHYLOGENY AND CLASSIFICATION

Abstract— A cladogram of green plants involving all major extant groups of green algae, bryophytes, pteridophytes, and seed plants is presented. It is partly based on contributions by B. Mishler and S. Churchill, H. Wagner, and P. Crane. The relationships of green plants to other green organisms ( Prochloron , euglenophytes) are discussed. The characters and subclades of the cladogram are briefly discussed, with an attempt to indicate weak points. The possibility of including some major extinct groups is considered. A cladistic classification consistent with the cladogram is presented. Grades are abandoned as taxa and major clades like the division Chlorophyta (green algae excluding micro-monadophytes and charophytes sensu Mattox and Stewart), the division Streptophyta (charophytes + embryophytes), the subdivision Embryophytina (land plants or embryophytes), the superclass Tracheidatae (tracheophytes), and the class Spermatopsida (seed plants) are recognized.     

Atypical glandular cells in cervical smears: histological correlation and a suggested plan of management based on age of the patient in a low-resource setting

Objectives:  To perform an audit of all smears reported as atypical glandular cells (AGC) using the Bethesda system (TBS) 2001.
Methods:  A total of 18 376 cervical smears were screened from January 2005 to June 2007, of which 65 cases were reported as AGC. Follow-up histology was available in 31 cases (47.7%), in whom a detailed cytological/histological correlation was carried out.
Results:  AGC constituted 0.35% of all Pap smears. Follow-up histology was normal or benign in 20 cases, whereas a squamous or glandular abnormality was seen in 11 cases. Squamous abnormalities included one case each of cervical intraepithelial neoplasia (CIN)1, CIN2 and CIN3 and five cases of squamous cell carcinoma. All glandular epithelial abnormalities were endometrial in origin and included two endometrial adenocarcinomas and one uterine serous carcinoma. Neither in situ nor invasive adenocarcinoma of the endocervix was observed. Review of smears and reclassification as AGC, not otherwise specified and favour neoplasia revealed a higher proportion of abnormality in the latter group, reaffirming the utility of subtyping. The median age of women with AGC was 41 years. The outcome was analysed with respect to the median age. In women aged equal or more than 40 years, AGC reflected a high-grade squamous or glandular epithelial abnormality in 50% of cases compared with none in those less than 40 years old ( P  = 0.010).
Conclusion:  The age of the woman as well as the subtype of atypical glandular cells influences outcome and hence must be taken into consideration while formulating an acceptable management strategy in these women in a low-resource setting.     

TREATMENT OF RAT PROXIMAL AND DISTAL COLONIC CELLS WITH SODIUM ORTHOVANADATE ENHANCES THEIR ADHESION AND SURVIVAL IN PRIMARY CULTURE

We have studied the effect of sodium orthovanadate, an inhibitor of protein tyrosine phosphatases, on primary cultures of colonocytes and stromal cells. Everted proximal and distal colonic tissue of adult rats were disintegrated by a collagenase/dispase solution for 60 min at 37°C to prepare viable gland fragments and isolated cells. Cell preparations were inoculated onto plastic substratum or cytodex-3 microcarriers in a defined maintenance medium or in 1% fetal calf serum media. Incorporation of sodium orthovanadate (≥50 μm) in these media constantly enhanced the survival (cell enumeration and trypan blue exclusionP<0.05) and the adhesion (up to four-fold by crystal violet staining,P<0.01) of colonocytes (characterized by cytokeratin-18, transforming growth factor-α or alkaline phosphatase expression) and stromal cells. Removal of sodium orthovanadate from culture media restored cellular death processes. Incorporation of 10 mmn-butyric acid did not promote cell adhesion and survival except for distal cells exposed to 2 mm sodium orthovanadate. Besides studies in the regulation of anoikis in primary culture, the model will help to assay the influences of dietary and growth factors on the biology of non-cancerous colonic cells.     

Zinc and cadmium hyperaccumulation by Thlaspi caerulescens from metalliferous and nonmetalliferous sites in the Mediterranean area: implications for phytoremediation

Growth, tolerance and zinc and cadmium hyperaccumulation of Thlaspi caerulescens populations from three metal contaminated soils and three normal soils were compared under controlled conditions. Individuals of six populations were cultivated on five soils with increasing concentrations of zinc (50–25000 μg g⁻¹) and cadmium (1–170 μg g⁻¹). There was no mortality of normal soil populations in the four metal-contaminated soils, but plant growth was reduced to half that of populations from metal-contaminated soils. However, in noncontaminated soil, the growth of individuals from normal soils was greater than that of individuals from metal-contaminated soils. Individuals from normal soils concentrated three times more zinc in the aboveground biomass than those from metal-contaminated soils, but the latter accumulated twice as much cadmium. We conclude that populations of T. caerulescens from both normal and metal-contaminated soils are interesting material for phytoextraction of zinc and cadmium, but to optimize the process of phytoextraction it is necessary to combine the extraction potentials of both type of populations.     

利用AFLP遗传连锁图定位大麦苗期对叶锈病的部分抗性基因

借助大麦染色体ＡＦＬＰ标记遗传连锁图和ＭａｐＱＴＬＶ３．０作图软件,对大麦叶病的数量抗性基因进行了定位分析,明确了大麦部分抗性品种Ｖａｄａ对叶锈病的潜育期由分别位于染色体１、２、６、７上离短臂末端７９ｃＭ、１８６ｃＭ、５８ｃＭ和１１７ｃＭ处的４个数量抗性基因所控制。     

Sesquiterpene lactone from Salmalia malbarica

A new cadalane type sesquiterpenoid, 1, 6-dihydroxy-3-methyl-5-(1-methylethyl)-7-methoxy-8-carboxylic acid (8 → 1 lactone) has been isolated from a 50% aqueous methanolic extract of 6–9 months old cultivated roots of Salmalia malbarica (Bombax malbaricum).