Generation and characterization of an inducible transgenic model for studying mouse esophageal biology

ABSTRACT: BACKGROUND: To facilitate the in vivo study of esophageal (stem) cell biology in homeostasis and cancer, novel mouse models are necessary to elicit expression of candidate genes in a tissue-specific and inducible fashion. To this aim, we developed and studied a mouse model to allow labeling of esophageal cells with the histone 2B-GFP (H2B-GFP) fusion protein. RESULTS: First, we generated a transgenic mouse model expressing the reverse tetracycline transactivator rtTA2-M2 under control of the promoter (ED-L2) of the Epstein-Barr virus (EBV) gene encoding the latent membrane protein-1 (LMP-1). The newly generated ED-L2-rtTA2-M2 (ED-L2-rtTA) mice were then bred with the previously developed tetOHIST1H2BJ/GFP (tetO-H2B-GFP) model to assess inducibility and tissue-specificity. Expression of the H2B-GFP fusion protein was observed upon doxycycline induction but was restricted to the terminally differentiated cells above the basal cell layer. To achieve expression in the basal compartment of the esophagus, we subsequently employed a different transgenic model expressing the reverse transactivator rtTA2S-M2 under the control of the ubiquitous, methylation-free CpG island of the human hnRNPA2B1-CBX3 gene (hnRNPrtTA). Upon doxycycline administration to the compound hnRNP-rtTA/tetO-H2B-GFP mice, near-complete labeling of all esophageal cells was achieved. Pulse-chase experiments confirmed that complete turnover of the esophageal epithelium in the adult mouse is achieved within 7-10 days. CONCLUSIONS: We show that the esophagus-specific promoter ED-L2 is expressed only in the differentiated cells above the basal layer. Moreover, we confirmed that esophageal turn-over in the adult mouse does not exceed 7-10 days.     

The transcriptional landscape of the deep-sea bacterium Photobacterium profundum in both a toxR mutant and its parental strain

Background

Mutans streptococci are a group of gram-positive bacteria including the primary cariogenic dental pathogen Streptococcus mutans and closely related species. Two component systems (TCSs) composed of a signal sensing histidine kinase (HK) and a response regulator (RR) play key roles in pathogenicity, but have not been comparatively studied for these oral bacterial pathogens.

Results

HKs and RRs of 8 newly sequenced mutans streptococci strains, including S. sobrinus DSM20742, S. ratti DSM20564 and six S. mutans strains, were identified and compared to the TCSs of S. mutans UA159 and NN2025, two previously genome sequenced S. mutans strains. Ortholog analysis revealed 18 TCS clusters (HK-RR pairs), 2 orphan HKs and 2 orphan RRs, of which 8 TCS clusters were common to all 10 strains, 6 were absent in one or more strains, and the other 4 were exclusive to individual strains. Further classification of the predicted HKs and RRs revealed interesting aspects of their putative functions. While TCS complements were comparable within the six S. mutans strains, S. sobrinus DSM20742 lacked TCSs possibly involved in acid tolerance and fructan catabolism, and S. ratti DSM20564 possessed 3 unique TCSs but lacked the quorum-sensing related TCS (ComDE). Selected computational predictions were verified by PCR experiments.

Conclusions

Differences in the TCS repertoires of mutans streptococci strains, especially those of S. sobrinus and S. ratti in comparison to S. mutans, imply differences in their response mechanisms for survival in the dynamic oral environment. This genomic level study of TCSs should help in understanding the pathogenicity of these mutans streptococci strains.     

Preparation and biological evaluation of (64)Cu labeled Tyr(3)-octreotate using a phosphonic acid-based cross-bridged macrocyclic chelator

Somatostatin receptors (SSTr) are overexpressed in a wide range of neuroendocrine tumors, making them excellent targets for nuclear imaging and therapy, and radiolabeled somatostatin analogues have been investigated for positron emission tomography imaging and radionuclide therapy of SSTr-positive tumors, especially of the subtype-2 (SSTr2). The aim of this study was to develop a somatostatin analogue, Tyr(3)-octreotate (Y3-TATE), conjugated to a novel cross-bridged macrocyclic chelator, 11-carboxymethyl-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4-methanephosphonic acid (CB-TE1A1P). Unlike traditional cross-bridged macrocycles, such as 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CB-TE2A), CB-TE1A1P-Y3-TATE was radiolabeled with (64)Cu in high purity and high specific activity using mild conditions. Saturation binding assays revealed that (64)Cu-CB-TE1A1P-Y3-TATE had comparable binding affinity but bound to more binding sites in AR42J rat pancreatic tumor cell membranes than (64)Cu-CB-TE2A-Y3-TATE. Both radiopharmaceuticals showed comparable uptake in SSTr2 positive tissues in AR42J tumor-bearing rats. (64)Cu-CB-TE1A1P-Y3-TATE demonstrated improved blood clearance compared to (64)Cu-CB-TE2A-Y3-TATE, as the tumor/blood ratios of (64)Cu-CB-TE1A1P-Y3-TATE were shown to be significantly higher than those of (64)Cu-CB-TE2A-Y3-TATE at 4 and 24 h postinjection. (64)Cu-CB-TE1A1P-Y3-TATE, in spite of a relatively high kidney uptake, accumulated less in nontarget organs such as liver, lung, and bone. Small animal PET/CT imaging of (64)Cu-CB-TE1A1P-Y3-TATE in AR42J tumor bearing rats validated significant uptake and good contrast in the tumor. This study suggests that CB-TE1A1P is a promising bifunctional chelator for (64)Cu-labeled for Y3-TATE, owing to high binding affinity and target tissue uptake, the ability to radiolabel the agent at lower temperatures, and improved tumor/nontarget organ ratios over (64)Cu-CB-TE2A-Y3-TATE.     

Rosaceae conserved orthologous set (RosCOS) markers as a tool to assess genome synteny between Malus and Fragaria

Intertribal comparisons of genome synteny between phylogenetically distant genera in Rosaceae, such as Malus (apple) and Fragaria (strawberry), have previously been hampered by a lack of transferable markers that can be used as anchor points between genetic maps. The availability of conserved orthologous set (COS) markers recently developed for this family, coupled with the release of the Malus?×?domestica and Fragaria vesca draft genome sequences, provide new tools for comprehensive pairwise comparisons. The genetic mapping of 56 Rosaceae COS (RosCOS) markers revealed 21 regions of genomic synteny between apple and strawberry. Information concerning the location of RosCOS markers on 15 of 17 apple linkage groups (LG) and all seven LG of strawberry was used to assess the ancestral relationships between the two genera. Four differences in orientation of ancestral chromosome fragments on extant LG were identified in comparison with previous studies, as well as two potential insertions, two potential translocations, and two potential inversions. The set of orthologous markers developed for use in genetic mapping in Rosaceae, in combination with high-throughput analysis, will allow the exploration of chromosome evolution and refinement of ancestral relationships within the family, orientation, and anchoring of genome sequences as they become available and provide resources to develop markers for nonsequenced genomes within the family.     

The Silk Road, Marco Polo, a Bible and its proteome: a detective story

Around the end of XIII century (at the time of young Marco Polo's first trip to China at the court of Khubilai Khan in Khan Baliq) a pocket Bible was delivered by a Franciscan friar to the Mogul Emperor, in the framework of the evangelization program of the Far East. Four centuries later, in 1685, this Bible was rediscovered by the Jesuit Philippe Couplet in the house of a rich Chinese in Nanchin and donated to Cosimo III, Grand Duke of Tuscany. This Bible was recently "unearthed" in the Biblioteca Medicea Laurenziana in Florence, wrapped up in a precious yellow silk cloth, in a rather ruined state. After two years of restoration, the Bible will return to China in 2012 for a celebration of its >700years of life and of its remarkable return trip on the Silk Road. On account of the thinness of the parchment (barely 80μm thickness, the size of each foil being 16.5×11cm) it was widely held that the pages were produced from foetal lambskins. On tiny fragments of the margins of a foil, after several unsuccessful attempts at digesting the vellum, we were able to obtain a tryptic peptide mixture, which, upon mass spectrometry analysis, yielded the identity of 8 unique proteins, belonging to the genus Bos taurus, thus confirming the origin of the vellum from calfskins rather than from foetal lambskins. Our results prove that it is possible to obtain reliable protein extraction and IDs from ancient parchment documents.     

La micro-lithiase testiculaire: à propos de 4 cas

Testicular microlithiasis (TMI) is a rare and little known condition. We verified its clinical and pathogenetic characteristics according to our experience. We performed testicular echography on four patients. Three of them had subfertlity and presented oligoasthenospermia in the spermiogram, while one patient had prostatitis with emospermia. Three patients had a high density TMI and one had a low density TMI. None of them showed neoplastic testicular lesions in the first echography. Anamnesis and examination revealed no outstanding data, one had been treated with calcium and Vit.D when a child for rachitis and another reported acute orchyepididymitis in his past history. Oncological markers were negative. 6–18 months follow-up with examination and echography didn’t show any testicular nodules. TMI is easy to diagnose and classify thanks to echography: Isolated TMI (the commonest, of flogistic, vascular or post-traumatic origin) Low-density TMI (<5calcifications per scan) High-density TMI (>5 calcifications per scan). Aetiology, pathogenesis of TMI and its connection to testicular cancer are not yet known. Differential diagnosis trhough echography is possible with both pathological hyperechogenic lesions of testicles (i.e. Tumors, hoematomas, partial atrophy, torsion, calcified hydatid) and non pathological ones (ilum, testicular mediastinum). The structure of the calcifications is constituted by hydroxyapatite which develop on degenerated spermatogones. They measure 1–3mm in diameter and can be found on 30–75% of the seminifer tubules. TMI is typical of patients with subfertility although the connection between this condition and male sterility is not clear. Similarly, TMI can be found in testicles’ tumours although even in this case, the connection between these conditions is not clear. That is why all authors agree in suggesting an echographical check every 6–12 months. Although the above case history can’t be considered significant due to the small number of patients, it is interesting to notice how 3 out of 4 patients had subfertility. Follow-up never showed probable neoplastic lesions. Finally what we also found worthy of notice is the possible connection between past orchyepididymitis and TML and connection between therapy for rachitis and TMI.     

Hand-held echocardiography: added value in clinical cardiological assessment

In the present article we review the main published data on the application of Tissue Doppler Imaging (TDI) to stress echocardiography for the detection of myocardial ischemia. TDI has been applied to stress echocardiography in order to overcome the limitations of visual analysis for myocardial ischemia. The introduction of a new technology for clinical routine use should pass through the different phases of scientific assessment from feasibility studies to large multicenter studies, from efficacy to effectiveness studies. Nonetheless the pro-technology bias plays a major role in medicine and expensive and sophisticated techniques are accepted before their real usefulness and incremental value to the available ones is assessed. Apparently, TDI is not exempted by this approach : its applications are not substantiated by strong and sound results. Nonetheless, conventional stress echocardiography for myocardial ischemia detection is heavily criticized on the basis of its subjectivity. Stress echocardiography has a long lasting history and the evidence collected over 20 years positioned it as an established tool for the detection and prognostication of coronary artery disease. The quantitative assessment of myocardial ischemia remains a scientific challenge and a clinical goal but time has not come for these newer ultrasonographic techniques which should be restricted to research laboratories.     

Intrinsic competition between the parasitoids Eretmocerus mundus and Encarsia formosa in Bemisia tabaci

Immature stages of Eretmocerus mundus Mercet and Encarsia formosa Gahan (both Hymenoptera: Aphelinidae) compete in larvae of their host, Bemisia tabaci (Gennadius) (Homoptera: Aleyrodidae). Laboratory tests were carried out on excised tree tobacco leaves, exposing B. tabaci nymphs to one of the parasitoid species alone or to both species, one after the other, to obtain multi‐parasitization. Parasitization by E. mundus and E. formosa was allowed on specific host stages in order to obtain interactions between different immature stages of the two parasitoids (eggs, and first, second, and third instars). The outcome from each multi‐parasitization treatment was verified by analysing data on parasitoid adult emergence. Observations under a stereomicroscope and dissections of multi‐parasitized hosts were also performed in order to demonstrate any factors potentially determining the outcome of competition. Eretmocerus mundus clearly prevailed over E. formosa when multi‐parasitism occurred. A higher percentage of adults emerging from multi‐parasitized hosts belonged to this parasitoid species (68.0–88.9% depending on the treatment). The lowest percent emergence by E. mundus (68.0%) and total percent emergence of parasitoid adults (52.2%) were obtained when E. mundus first instars interacted with hosts parasitized by E. formosa third instars. Observations and dissections showed that first‐instar E. mundus induced mortality in E. formosa immatures at penetration into the hosts, although they encountered greater difficulty in exploiting hosts inside which E. formosa had reached the third stage of development. In contrast, development of E. formosa immatures was not immediately inhibited if parasitization took place on hosts inside which E. mundus larvae had already penetrated. In this case, however, E. mundus also prevailed over E. formosa (72.5% of the emerged adults). Implications for the use of these parasitoid species against B. tabaci in biological control programmes are discussed.