The salt-tolerance gene rstB can be used as a selectable marker in plant genetic transformation

The salt-tolerance gene rstB under the control of the cauliflower mosaic virus 35S promoter was used as a selectable marker gene in the Agrobacterium tumefaciens-mediated transformation of tobacco (Nicotiana tabacum cv. Xanthi). The selective agent for plant regeneration was tolerance to 170 mM sodium chloride. The highest selection efficiency was 83.3%. No obvious differences in selection efficiencies were observed when those obtained using the standard selectable marker gene hpt and a selection regime of 10 mg l⁻¹ hygromycin. Transgenic events were confirmed by PCR, Southern blot, RT-PCR and green fluorescent protein studies. The rstB transgenic plants showed improved salt tolerance and a normal phenotype. Based on these results, we suggest that the rstB gene may be used as a promising selectable marker and an alternative to the antibiotic- or herbicide-resistance genes in plant transformation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The detergent-insoluble microdomains, rafts, can be used as an effective immunogen

Detergent-insoluble microdomains, or rafts, act as a platform to transduce signals from the extracellular space into the cytoplasm. In the process of developing monoclonal antibodies against raft molecules for the purpose of studying the molecular mechanism of raft-mediated signaling, we observed the uniqueness and certain advantages of immunization with rafts. Simple subcutaneous injection of mice with a phosphate-buffered saline (PBS) suspension of rafts without mixing with Freund's adjuvant made it possible to increase the titer of antiserum reacting with raft components. Interestingly, injection of rafts prepared from certain specific cell lines induced monoglycolipid-specific antibodies. Furthermore, antibodies were produced by raft-immunization of even syngeneic mice. Our findings suggest that this phenomenon does not represent a breakdown of immunological self-tolerance, but typical immune reactions accompanying the class switch from IgM antibodies to IgG antibodies.     

One year old fillies can be successfully used as embryo donors

One year old fillies are able to conceive but, usually, not to give birth to a living foal. Although embryo transfer allows the production of foals from mature mares with repeated pregnancy losses, no reports are available on the use of one year old fillies as embryo donors. To evaluate this possibility, eleven 12-16 months old Haflinger mares were inseminated with fresh semen and subjected to embryo recovery. Some of the recovered embryos were non-surgically transferred into synchronized mature recipients. Pregnancies were terminated using PGF2alpha at day 25. Fillies' embryo recovery rate and their recipients' pregnancy rate at day 25 were compared with those achieved in two years old and mature mares of the same breed, subjected to the same management. Embryo recovery rate was 21/44 (47.7%), 12/16 (75%) and 22/26 (84.6%) (P>0.01) for one year old, two years old and mature mares, respectively. Five/7 (71.4%) one year old donors' embryos resulted in a pregnancy after transfer and 4/7 (57.1%) developed until day 25. Significant differences in pregnancy rates after transfer between donors' age groups were not observed; no short term side effects resulted from the use of fillies as embryo donors. This study showed that one year old mares employed as embryo donors produce embryos both morphologically normal and able to develop in recipient mares at least up to day 25 of pregnancy.     

Pseudogene DUXAP10 can be used as a diagnostic and prognostic biomarker in human cancers

The pseudogene DUXAP10 is overexpressed in numerous types of human cancers. However, the diagnostic and prognostic value of DUXAP10 in cancers has yet to be characterized. PubMed, EMBASE, Web of Science, the Cancer Genome Atlas (TCGA), and Gene Expression Omnibus databases were comprehensively searched in this study. A total of 50 studies comprising 11,292 patients were collected in this integrated analysis. DUXAP10 was confirmed to be significantly overexpressed in various human cancers (p < .05). Summary receiver operating characteristic (SROC) curve analysis was implemented, which indicated that DUXAP10 was a potential diagnostic biomarker for human cancers (area under the curve [AUC] of SROC curve = 0.81 [0.77–0.84]; pooled sensitivity = 0.69 [0.62–0.75]; pooled specificity = 0.81 [0.73–0.87]). In addition, hazard ratios (HRs) with 95% confidence intervals (CIs) were obtained to evaluate the association of DUXAP10 expression with overall survival (OS) time of cancer patients. Outcomes of meta-analysis suggested that upregulation of DUXAP10 was closely associated with poor OS (pooled HR = 1.11 [1.03–1.18]). Our study revealed that the pseudogene DUXAP10 was upregulated in multiple types of cancers and could be a potential biomarker with good diagnostic and prognostic value for human cancers.     

Cholesterol removal by some lactic acid bacteria that can be used as probiotic

In the present study, the relationship between exopolysaccharide production and cholesterol removal rates of five strains of Lactobacillus delbrueckii subsp. bulgaricus isolated from home‐made yoghurt was studied. Test strains were selected according to their exopolysaccharide production capacity. Influence of different bile concentrations on cholesterol removal was investigated. It was confirmed that B3, ATCC 11842 and G11 strains which produce high amounts of exopolysaccharide (211, 200 and 159 mg/l, respectively) were able to remove more cholesterol from the medium compared to those that produce low amounts of exopolysaccharide (B2, A13). The highest cholesterol removal (31%) was observed by strain L. delbrueckii subsp. bulgaricus B3, producing a high amount of exopolysaccharide, in 3 mg/ml bile concentration. Cholesterol removal by resting and dead cells was investigated and it was found to be 4%–14% and 3%–10%, respectively. Cholesterol removal by immobilized and free cells of the B3 strain was studied and it was determined that immobilized cells are more effective. Influence of cholesterol on exopolysaccharide production has also been tested and it was found that cholesterol increased the production of EPS. The results indicated that: (i) there is a correlation between cholesterol removal and EPS production; and (ii) L. delbrueckii subsp. bulgaricus B3 is regarded as a suitable candidate probiotic and adjunct culture.     

Bacterial beta-lactamase fragmentation complementation strategy can be used as a method for identifying interacting protein pairs

We investigated the applicability of the TEM-1 beta- lactamase fragment complementation (BFC) system to develop a strategy for the screening of protein-protein interactions in bacteria. A BFC system containing a human Fas-associated death domain (hFADD) and human Fas death domain (hFasDD) was generated. The hFADD-hFasDD interaction was verified by cell survivability in ampicillin-containing medium and the colorimetric change of nitrocefin. It was also confirmed by His pull-down assay using cell lysates obtained in selection steps. A coiled-coil helix coiled-coil domain-containing protein 5 (CHCH5) was identified as an interacting protein of human uracil DNA glycosylase (hUNG) from the bacterial BFC cDNA library strategy. The interaction between hUNG and CHCH5 was further confirmed with immunoprecipitation using a mammalian expression system. CHCH5 enhanced the DNA glycosylase activity of hUNG to remove uracil from DNA duplexes containing a U/G mismatch pair. These results suggest that the bacterial BFC cDNA library strategy can be effectively used to identify interacting protein pairs.     

Chinchilla laniger can be used as an experimental model for Taenia solium taeniasis

Chinchilla laniger has been reported as an experimental definitive host for Taenia solium; however no information about its suitability and yield of gravid tapeworm proglottids containing viable and infective eggs has been published. In total 55 outbred female chinchillas were infected with 4 cysticerci each; hosts were immunodeppressed with 6 or 8 mg of methyl-prednisolone acetate every 14 days starting the day of infection and their discomfort was followed. Kinetics of coproantigen ELISA or expelled proglottids was used to define the infection status. Efficiency of tapeworm establishment was 21% and of parasite gravidity was 8%; chinchillas showed some degree of suffering along the infection. Viability of eggs obtained from gravid proglottids was tested comparing methods previously published, our results showed 62% viability with propidium iodide, 54% with trypan blue, 34% with neutral red, 30% by oncosphere activation and 7% with bromide 3-(4,5-dimetil-tiazol-2-il)-2,5-difenil-tetrazolio (MTT) reduction; no statistical differences were obtained between most techniques, except activation. Four piglets were infected with 50,000 eggs each, necropsy was performed 3 months later and, after counting the number of cysticerci recovered, the percentage of infection was similar to data obtained with T. solium eggs recovered from humans. Our results demonstrate that the experimental model of T. solium taeniasis in C. laniger is a good alternative for providing eggs and adult tapeworms to be used in different types of experiments; optimization of the model probably depends on the use of inbred hosts and on the reduction of infected animals' suffering.     

A rapid assay for pectinesterase activity which can be used as a prescreen for pectinesterase inhibitors.

A rapid assay for inhibition of pectinesterase has been developed to facilitate large scale screening for possible inhibitors. The assay gives quantitative results which can be confirmed by other assay techniques. The assay uses an agar plate in which pectin and an indicator have been incorporated. The hydrolysis of pectin to pectinic acid by pectinesterase causes a lowering of the pH of the medium and produces a color change. The area of the color change produced by the hydrolysis of pectin is directly proportional to the activity of the pectinesterase.     

Solid tissues can be manipulated ex vivo and used as vehicles for gene therapy

BACKGROUND: Organ fragments can be cultured for weeks in vitro if they are prepared of microscopic thickness and if the basic organ structure is preserved. Such organ fragments, which we termed micro-organs (MOs), express in culture endogenous tissue-specific gene products. We have exploited this methodology to engineer MOs ex vivo by gene transfer. METHODS: MOs prepared from spleen, lung, colon and skin were infected using: herpes simplex type-1, adeno virus, vaccinia virus and murine leukemia virus (MuLV), carrying the reporter gene beta-galactosidase. RESULTS: All four viral vectors infected MOs in culture, with adeno infection giving significantly higher values. After optimization, high levels of expression (> 15% positive cells), comparable to those obtained with the adeno construct, were also obtained using the MuLV construct both in vitro and after implantation into syngeneic hosts. After implantation, the engineered tissue was found to remain localized, become vascularized, and to express the transduced gene for several months. CONCLUSIONS: The system can be used to study interactions between viruses and tissues both ex vivo and in vivo. Furthermore, the approach proposes a novel platform for ex vivo gene therapy. Such engineered structures could be used as autologous biological pumps for continuous secretion in vivo of gene products of clinical importance.     

SINE retroposons can be used in vivo as nucleation centers for de novo methylation

SINEs (short interspersed elements) are an abundant class of transposable elements found in a wide variety of eukaryotes. Using the genomic sequencing technique, we observed that plant S1 SINE retroposons mainly integrate in hypomethylated DNA regions and are targeted by methylases. Methylation can then spread from the SINE into flanking genomic sequences, creating distal epigenetic modifications. This methylation spreading is vectorially directed upstream or downstream of the S1 element, suggesting that it could be facilitated when a potentially good methylatable sequence is single stranded during DNA replication, particularly when located on the lagging strand. Replication of a short methylated DNA region could thus lead to the de novo methylation of upstream or downstream adjacent sequences.     

Elevated paraquat resistance can be used as a bioassay for longevity in a genetically based long-lived strain of Drosophila

A long-lived (L) strain of Drosophila melanogaster, derived from a normal-lived (R) strain by artificial selection, has a significantly different adult longevity. Previous work has shown that (1) the two strains age in the same manner, (2) the major genes responsible for much of the L strain's extended longevity are located on the 3rd chromosome, and (3) the extended longevity phenotype is significantly modulated by the larval environment. In this report, we investigate the resistance of the L and R strains to the lethal effects of dietary paraquat. We show that, within the limitations of our described chromosomal and environmental manipulations, the extended longevity phenotype always accompanies the phenotype of elevated paraquat resistance. In addition, reversed selection applied to the L strain results in the simultaneous decrease of both life span and paraquat resistance. Thus, the presence or absence of the latter phenotype may be used as a bioassay for the presence or absence of the extended longevity phenotype, without any necessary implication of causality. Use of this bioassay should greatly speed up the genetic analysis of this system by allowing us to identify long-lived animals at a young age. Finally, we show that the age-related loss of elevated paraquat resistance in both strains precedes all the other age-related functional decrements which we have previously noted in this system.     

Hypothesis: synthetic aging antigen can be used to manipulate cellular lifespan

Physiologic removal of old and damaged erythrocytes, platelets, and other terminally differentiated cells is initiated by the appearance of an aging antigen that marks them for death by initiating the binding of IgG autoantibody and subsequent removal by phagocytes. We have developed a synthetic aging antigen peptide that blocks binding of IgG to senescent cells in vitro. We hypothesize that the synthetic antigen can be used to prevent cell destruction in diseases such as autoimmune hemolytic anemias and idiopathic thrombocytopenia purpura, and that the antigen itself can be used to manipulate cellular lifespan in vivo.     

Electron microscopy can be used to measure DNA supertwisting

Incorporation of starch or casein into protoplast-regeneration medium facilitated shotgun cloning of -amylase and neutral protease genes from an unidentified Bacillus sp. in Bacillus subtilis by polyethylene glycol-induced protoplast transformation. This modification and the use of the plasmid vector pPL603b enabled us to simultaneously select for promoter-bearing recombinant plasmids that expressed amylase or protease activity. The inserts were found to be 4 and 4.6 kb, respectively. Although protease activity directed by the cloned gene was only 2- to 4-fold higher than for the donor strain, that of -amylase was 28-fold higher.