Carbohydrate receptor-mediated gene transfer to human T leukaemic cells

The mucin-type carbohydrate Tn cryptantigen (GalNAc1-O-Ser/Thr,where GalNAc is N-acetyl-D-galactosamine) is expressed in manycarcinomas, in haemopoietic disorders including the Tn syndrome,and on human immunodeficiency virus (HIV) coat glycoproteins,but is not expressed on normal, differentiated cells becauseof the expression of a Tn-processing galactosyltransferase.Using Jurkat T leukaemic cells which express high levels ofTn antigen due to deficient Tn galactosylation, we have establishedthe Tn antigen-mediated gene transfer and demonstrate the considerableefficiency of this approach. We used poly(L-lysine) conjugatesof the monoclonal antibody 1E3 directed against the Tn antigento deliver the luciferase and ß-galactosidase reportergenes to Jurkat cells by receptor-mediated endocytosis. Additionof unconjugated 1E3 reduced transfection efficiency in a concentration-dependentmanner and incubation with free GalNAc abolished DNA transfercompletely, indicating that gene delivery is indeed mediatedby the Tn antigen. Pre-treatment of Jurkat cells with Vibriocholerae sialidase, which uncovers additional Tn antigens, resultedin an improvement of gene transfection. Both human and chickenadenovirus particles attached to the DNA/polylysine complexstrongly augmented transgene expression. When the ß-galactosidase(lacZ) gene was delivered to Jurkat cells by Tn-mediated endocytosis,up to 60% of the cells were positive in the cytochemical stainusing 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside(X-gal) as a chromogenic substrate. The efficiency of the transferrinreceptor-mediated DNA uptake into Jurkat cells was comparativelylow, although these cells were shown to express considerableamounts of transferrin receptor. We show here that a mucin-typecarbohydrate antigen mediates highly efficient DNA uptake byendocytosis into Jurkat T cells. This method represents a 50-foldimprovement of Jurkat cell transfection efficiency over otherphysical gene transfer techniques. Specific gene delivery toprimary cancer cells exhibiting Tn epitopes may especially bedesirable in immunotherapy protocols. adenovirus endocytosis gene transfer T cell Tn antigen     

Antepartum glucose tolerance test results as predictors of type 2 diabetes mellitus in women with a history of gestational diabetes mellitus: A systematic review

Background: Women with a history of gestational diabetes mellitus (GDM) are at high risk for type 2 diabetes mellitus (T2DM).Objective: We reviewed prospective studies of antepartum glucose tolerance test results as risk factors for development of T2DM among women with a history of GDM.Methods: We searched 4 electronic databases and hand-searched 13 journals for literature published through January 2007. The search strategy consisted of medical subject headings and text words for GDM, T2DM, and other relevant terms. Articles were excluded for the following reasons: (1) not written in English; (2) no human data; (3) no original data; (4) <90% of sample was diagnosed with GDM without a separate analysis for women with GDM; (5) case report or series; (6) diagnosis of GDM not based on 3-hour 100-g oral glucose tolerance test (OGTT) or 2-hour 75-g OGTT; (7) T2DM not evaluated as outcome; (8) no relative measure of association or incidence reported; or (9) design did not address antepartum OGTT as a predictor of T2DM. Two investigators independently reviewed citations, performed serial data abstraction on full articles, and assessed the quality of each article. Data were abstracted for study participants and characteristics, T2DM diagnosis, length of follow-up, regression model covariates, and measures of association and variability.Results: Of 11,400 unique citations, we identified 11 articles that evaluated antepartum glucose testing and risk of T2DM in women with a history of GDM. Five studies found that the fasting blood glucose (FBG) on the antepartum diagnostic OGTT was a significant predictor of T2DM (odds ratio [OR] range: 11.1–21.0; relative risk [RR] range: 1.37–1.5; relative hazard [RH] = 2.47). Risk of incident T2DM was predicted by the antepartum 2-hour OGTT plasma glucose in 3 studies (OR range: 1.02–1.03; RR = 1.3) and by the antepartum OGTT glucose AUC in 3 other studies (OR range: 3.64–15; RH = 2.13). Overall, study quality was limited by high losses to follow-up (>20% in 6 studies) and short duration. Few studies adjusted for adiposity, an established diabetes risk factor.Conclusion: FBG, OGTT 2-hour blood glucose, and OGTT glucose AUC appeared to be strong and consistent predictors of subsequent T2DM among women who met diagnostic criteria for GDM using the OGTT.     

Conditional deletion of Shp2 tyrosine phosphatase in thymocytes suppresses both pre-TCR and TCR signals

It is well known that T cell differentiation and maturation in the thymus is tightly controlled at multiple checkpoints. However, the molecular mechanism for the control of this developmental program is not fully understood. A number of protein tyrosine kinases, such as Zap-70, Lck, and Fyn, have been shown to promote signals required for thymocyte development, whereas a tyrosine phosphatase Src homology domain-containing tyrosine phosphatase (Shp)1 has a negative effect in pre-TCR and TCR signaling. We show in this study that Shp2, a close relative of Shp1, plays a positive role in T cell development and functions. Lck-Cre-mediated deletion of Shp2 in the thymus resulted in a significant block in thymocyte differentiation/proliferation instructed by the pre-TCR at the beta selection step, and reduced expansion of CD4(+) T cells. Furthermore, mature Shp2(-/-) T cells showed decreased TCR signaling in vitro. Mechanistically, Shp2 acts to promote TCR signaling through the ERK pathway, with impaired activation of ERK kinase observed in Shp2(-/-) T cells. Thus, our results provide physiological evidence that Shp2 is a common signal transducer for pre-TCR and TCR in promoting T cell maturation and proliferation.     

Controlling Neglected Tropical Diseases (NTDs) in Haiti: Implementation Strategies and Evidence of Their Success

Lymphatic filariasis (LF) and soil-transmitted helminths (STH) have been targeted since 2000 in Haiti, with a strong mass drug administration (MDA) program led by the Ministry of Public Health and Population and its collaborating international partners. By 2012, Haiti’s neglected tropical disease (NTD) program had reached full national scale, and with such consistently good epidemiological coverage that it is now able to stop treatment for LF throughout almost all of the country. Essential to this success have been in the detail of how MDAs were implemented. These key programmatic elements included ensuring strong community awareness through an evidence-based, multi-channel communication and education campaign facilitated by voluntary drug distributors; strengthening community trust of the drug distributors by ensuring that respected community members were recruited and received appropriate training, supervision, identification, and motivation; enforcing a “directly observed treatment” strategy; providing easy access to treatment though numerous distribution posts and a strong drug supply chain; and ensuring quality data collection that was used to guide and inform MDA strategies. The evidence that these strategies were effective lies in both the high treatment coverage obtained– 100% geographical coverage reached in 2012, with almost all districts consistently achieving well above the epidemiological coverage targets of 65% for LF and 75% for STH—and the significant reduction in burden of infection– 45 communes having reached the target threshold for stopping treatment for LF. By taking advantage of sustained international financial and technical support, especially during the past eight years, Haiti’s very successful MDA campaign resulted in steady progress toward LF elimination and development of a strong foundation for ongoing STH control. These efforts, as described, have not only helped establish the global portfolio of “best practices” for NTD control but also are poised to help solve two of the most important future NTD challenges—how to maintain control of STH infections after the community-based LF “treatment platform” ceases and how to ensure appropriate morbidity management for patients currently suffering from lymphatic filarial disease.     

Spermatogenesis in Sceloporus variabilis (Squamata,Phrynosomatidae): A non-quiescent pattern

Gaining a deeper understanding of spermatogenic cycles within squamates has aided in our knowledge of the controls of reproduction and has bettered our understanding of reproductive phenology. One of the most studied genera of squamates, Sceloporus, is widely distributed along a latitudinal and elevational gradient in temperate, tropical, low-elevation and high-elevation habitats. Due to this wide distribution and varying habitats, Sceloporus exhibit differences in their spermatogenic activity (including both cyclical and acyclical patterns) and may be one of the most useful genera for understanding the abiotic correlations with spermatogenesis. The spermatogenic activity in Sceloporus variabilis was studied histologically (in a population that inhabits a tropical region at Los Tuxtlas, Veracruz, Mexico) and found to exhibit a unique cyclical pattern with an extended period of maximum activity (from November to July) and the absence of regression and quiescence. Furthermore, these data corroborate previous works on the spermatogenic cycles of S. variabilis despite different populations utilised. These data suggest that although abiotic factors may play a role in the timing of spermatogenesis, phylogenetic signal may be equally as important. More data concerning spermatogenic cycles in phylogenetically related taxa from differing habitats will elucidate the patterns of spermatogenic diversity.     

Receptor-specific targeting with complementary peptide nucleic acids conjugated to peptide analogs and radionuclides

Summary Genomic sequencing makes it possible to identify all the genes of an organism, now includingHomo sapiens. Yet measurement of the expression of each gene of interest still presents a daunting prospect. Northern blots, RNase protection assays, as well as microarrays and related technologies permit measurement of gene expression in total RNA extracted from cultured cells or tissue samples. It would be most valuable, however, to quantitate gene expression noninvasively in living cells and tissues. Unfortunately, no reliable method has been available to measure levels of specific mRNAsin vivo. Peptide nucleic acids (PNAs) display superior ruggedness and hybridization properties as a diagnostic tool for gene expression, and could be used for this purpose. On the down side, they are negligibly internalized by normal or malignant cells in the absence of conjugated ligands. Nevertheless, we have observed that Tc-99m-peptides can delineate tumors, and PNA-peptides designed to bind to IGF-1 receptors on malignant cells are taken up specifically and concentrated in nuclei. We have postulated that antisense Tc-99m-PNA-peptides will be taken up by human cancer cells, will hybridize to complementary mRNA targets, and will permit scintigraphic imaging of oncogene mRNAs in human cancer xenografts in a mouse model. The oncogenes cyclin D1,ERBB2, c-MYC, K-RAS, and tumor suppressor p53 are being probed initially. These experiments provide a proof-of-principle for noninvasive detection of oncogene expression in living cells and tissues. This scintigraphic imaging technique should be applicable to any particular gene of interest in a cell or tissue type with characteristic receptors.     

A genetic map of tetraploid <Emphasis Type="Italic">Paspalum notatum</Emphasis> Flügge (bahiagrass) based on single-dose molecular markers

Paspalum notatum Flügge is a warm-season forage grass with mainly diploid (2n = 20) and autotetraploid (2n = 40) representatives. Diploid races reproduce sexually and require crosspollination due to a self-incompatible mating system, while autotetraploids reproduce by aposporous apomixis. The objectives of this work were to develop a genetic linkage map of Paspalum notatum Flügge at the tetraploid level, identify the linkage/s group/s associated with apomixis and carry out a general characterization of its mode of inheritance. A pseudo test-cross F₁ family of 113 individuals segregating for the mode of reproduction was obtained by crossing a synthetic completely sexual tetraploid plant (Q4188) as female parent with a natural aposporous individual (Q4117) as pollen donor. Map construction was based on single-dose markers (SDAFs) segregating from both parents. Two linkage maps (female and male) were constructed. Within each map, homologous groups were assembled by detecting repulsion-phase linked SDAFs. Putative Q4188 and Q4117 homolog groups were identified by mapping shared single dose markers (BSDF). The Q4188 map consisted of 263 markers distributed on 26 co-segregation groups over a total genetic distance of 1.590.6 cM, while the Q4117 map contained 216 loci dispersed on 39 co-segregation groups along 2.265.7 cM, giving an estimated genome coverage of 88% and 83%, respectively. Seven and 12 putative homologous chromosomes were detected within Q4188 and Q4117 maps, respectively. Afterward, ten female and male homologous chromosomes were identified by mapping BSDFs. In the Q4117 map, a single linkage group was associated with apospory. It was characterized by restriction in recombination and preferential chromosome pairing. A BPSD marker mapping within this group allowed the detection of the female homolog and the putative four male groups of the set carrying apospory.     

PCR-Denaturing Gradient Gel Electrophoresis Profiling of Inter- and Intraspecies 18S rRNA Gene Sequence Heterogeneity Is an Accurate and Sensitive Method To Assess Species Diversity of Arbuscular Mycorrhizal Fungi of the Genus Gigaspora

Despite the importance of arbuscular mycorrhizal fungi in the majority of terrestrial ecosystems, their ecology, genetics, and evolution are poorly understood, partly due to difficulties associated with detecting and identifying species. We explored the inter- and intraspecies variations of the 18S rRNA genes of the genus Gigaspora to assess the use of this marker for the discrimination of Gigaspora isolates and of Gigasporaceae populations from environmental samples. Screening of 48 Gigaspora isolates by PCR-denaturing gradient gel electrophoresis (DGGE) revealed that the V3-V4 region of the 18S rRNA gene contained insufficient variation to discriminate between different Gigaspora species. In contrast, the patterns of 18S ribosomal DNA (rDNA) heterogeneity within the V9 region of this marker could be used for reliable identification of all recognized species within this genus. PCR-DGGE patterns provided insight into some putative misidentifications and could be used to differentiate geographic isolates of G. albida, G. gigantea, and G. margarita but not G. rosea. Two major clusters were apparent based upon PCR-DGGE ribotype patterns, one containing G. albida, G. candida, G. ramisporophora, and G. rosea and the other containing G. decipiens and G. margarita. Dissection of the DGGE patterns by cloning, DGGE screening, and sequencing confirmed these groupings and revealed that some ribotypes were shared across species boundaries. Of the 48 isolates examined, only two displayed any spore-to-spore variation, and these exceptions may be indicative of coisolation of more than one species or subspecies within these cultures. Two Brazilian agricultural soils were also analyzed with a Gigasporaceae-specific nested PCR approach, revealing a dominance of G. margarita within this family.     

The first species of Hapalodectes (Mesonychia,Mammalia) from the middle Paleocene of China (Qianshan Basin,Anhui Province) sheds light on the initial radiation of hapalodectids

A lower jaw of the mesonychian Hapalodectes is reported from Nongshanian sediments (Upper Doumu Formation; middle Paleocene) of the Qianshan Basin (Anhui Province, China). The fragmentary mandible is only the third specimen of Hapalodectidae discovered in Paleocene deposits, and the first in south east China; it is moreover the oldest, the two other specimens having been found in Gashatan (late Paleocene) localities. The premolars and molars of the new fossil are morphologically similar to Hapalodectes dux (late Paleocene of Mongolia), which has been considered to be the most primitive hapalodectid, but their relative proportions recall H. paleocenus and the Eocene Hapalodectes species. As a result, the fossil described herein appears to be different from the other previously described species of Hapalodectes in being morphologically intermediate between H. dux and the other Hapalodectes species, notably the Bumbanian Hapalodectes hetangensis and H. huanghaiensis from China; it is thus identified as a new species, Hapalodectes lopatini (possibly a male individual). Its discovery is important because it sheds light on the initial radiation of hapalodectids. The presence of one primitive hapalodectid in Mongolia previously suggested the Mongolian Plateau as the centre of origination of this carnivorous family, but the discovery of H. lopatini in older sediments from south‐east China challenges this hypothesis. In the earliest Eocene, Hapalodectes dispersed from Asia to North America; this event being part of the ‘East of Eden’ dispersals. This event resulted in the geographical separation of two distinct Hapalodectes groups, in North America and south‐eastern China respectively.