Diverse endophytic bacteria isolated from a leguminous tree Conzattia multiflora grown in Mexico

Conzattia multiflora is a leguminous tree present only in Mexico and Guatemala. There is no record about its symbiotic or pathogenic microbes. In this study, we found that numerous bacteria with 10⁴–10⁶ individuals per gram of fresh epidermis were distributed in the tissue of this plant. All the bacteria isolated from the Conzattia epidermis were Gram-negative, facultative anaerobic rods and formed yellow or colorless colonies. They were identified as endophytes by inoculation tests. Some of the bacteria could significantly promote the growth of Conzattia seedlings. Nine different groups were defined by PCR-based RFLP, which were classified as Pantoea, Erwinia, Salmonella, Enterobacter, Citrobacter and Klebsiella by the phylogenetic analysis of 16S rRNA genes. The existence of plant-borne lineages of Salmonella indicates that the unexplored plants may harbor some unknown microbes.     

A systematic, ligation-based approach to study RNA modifications

Over 100 different chemical types of modifications have been identified in thousands of sites in tRNAs, rRNAs, mRNAs, small nuclear RNAs, and other RNAs. Some modifications are highly conserved, while others are more specialized. They include methylation of bases and the ribose backbone, rotation, and reduction of uridine, base deamination, elaborate addition of ring structures, carbohydrate moieties, and more. We have developed a systematic approach to detect and quantify the extent of known RNA modifications. The method is based on the enzymatic ligation of oligonucleotides using the modified or unmodified RNA as the template. The efficiency of ligation is very sensitive to the presence and the type of modifications. First, two oligo pairs for each type of modification are identified. One pair greatly prefers ligation using the unmodified RNA template over the modified RNA template or vice versa. The other pair has equal reactivity with unmodified and modified RNA. Second, separate ligations with each of the two oligo pairs and the total RNA mixture are performed to detect the presence or absence of modifications. Multiple modification sites can be examined in the same ligation reaction. The feasibility of this method is demonstrated for three 2'O-methyl modification sites in yeast rRNA.     

Design and synthesis of novel potent and selective integrin alphanubeta3 antagonists--novel synthetic routes to isoquinolinone, benzoxazinone, and quinazolinone acetates

An unexpected ring contraction of benzazepinone based alpha(nu)beta(3) antagonists led to the design of quinolinone-type derivatives. Novel and efficient synthetic routes to isoquinolinone, benzoxazinone, and quinazolinone acetates were established. Nanomolar alpha(nu)beta(3) antagonists based on these new scaffolds were prepared. Moreover, benzoxazinones 15a and 15b exhibited high microsomal stability and good permeability.     

A Novel In Vivo Vascular Imaging Approach for Hierarchical Quantification of Vasculature Using Contrast Enhanced Micro-Computed Tomography

The vasculature of body tissues is continuously subject to remodeling processes originating at the micro-vascular level. The formation of new blood vessels (angiogenesis) is essential for a number of physiological and pathophysiological processes such as tissue regeneration, tumor development and the integration of artificial tissues. There are currently no time-lapsed in vivo imaging techniques providing information on the vascular network at the capillary level in a non-destructive, three-dimensional and high-resolution fashion. This paper presents a novel imaging framework based on contrast enhanced micro-computed tomography (micro-CT) for hierarchical in vivo quantification of blood vessels in mice, ranging from largest to smallest structures. The framework combines for the first time a standard morphometric approach with densitometric analysis. Validation tests showed that the method is precise and robust. Furthermore, the framework is sensitive in detecting different perfusion levels after the implementation of a murine ischemia-reperfusion model. Correlation with both histological data and micro-CT analysis of vascular corrosion casts confirmed accuracy of the method. The newly developed time-lapsed imaging approach shows high potential for in vivo monitoring of a number of different physiological and pathological conditions in angiogenesis and vascular development.     

Bringing European river quality into line: an exercise to intercalibrate macro-invertebrate classification methods

The Water Framework Directive (WFD) requires intercalibration to be performed to ensure that ecological status, as defined by the boundary values of national biological assessment systems, is consistent with the definitions outlined in the WFD and comparable between Member States (MS). This article describes an intercalibration of 17 national river macro-invertebrate assessment methods from the Central and Baltic regions of Europe. We explore the hypothesis that intercalibration should be successful if ratios of the observed biota to that expected in reference condition are used to compare assessments of different national assessment systems. National boundaries expressed as ecological quality ratios (EQRs) were converted to values of a common multi-metric for the purpose of comparison. Twelve MS for the High/Good boundary and nine MS for the Good/Moderate boundary (and four MS who subsequently harmonised their boundaries) were within ±0.05 EQR units of the intercalibration boundaries and were deemed to be of comparable ecological standard. The use of a reference-based approach was deemed to be successful given that all the critical pre-requisites for intercalibration were satisfied. The boundaries derived from this intercalibration represent the first common interpretation of the ecological status of rivers based on macro-invertebrate assessment methods across Europe.     

Detection of 5'- and 3'-UTR-derived small RNAs and cis-encoded antisense RNAs in Escherichia coli

Evidence is accumulating that small, noncoding RNAs are important regulatory molecules. Computational and experimental searches have led to the identification of ~60 small RNA genes in Escherichia coli. However, most of these studies focused on the intergenic regions and assumed that small RNAs were >50 nt. Thus, the previous screens missed small RNAs encoded on the antisense strand of protein-coding genes and small RNAs of <50 nt. To identify additional small RNAs, we carried out a cloning-based screen focused on RNAs of 30–65 nt. In this screen, we identified RNA species corresponding to fragments of rRNAs, tRNAs and known small RNAs. Several of the small RNAs also corresponded to 5′- and 3′-untranslated regions (UTRs) and internal fragments of mRNAs. Four of the 3′-UTR-derived RNAs were highly abundant and two showed expression patterns that differed from the corresponding mRNAs, suggesting independent functions for the 3′-UTR-derived small RNAs. We also detected three previously unidentified RNAs encoded in intergenic regions and RNAs from the long direct repeat and hok/sok elements. In addition, we identified a few small RNAs that are expressed opposite protein-coding genes and could base pair with 5′ or 3′ ends of the mRNAs with perfect complementarity.     

The fate of effector T cells in vivo is determined during activation and differs for CD4+ and CD8+ cells

Effector T cells generated in the mesenteric lymph nodes (mLN) are known to accumulate in mLN and the tissue drained by them after circulating in the blood. Their accumulation is due less to preferential entry into mLN but more to preferential proliferation within mLN. The factors regulating the proliferation of effector T cells in vivo are unclear, and it is unknown whether they are different for CD4(+) and CD8(+) effector T cells. Rat T cells from mLN or peripheral lymph nodes (pLN) were stimulated polyclonally via the TCR and CD28 and injected i.v. into congenic recipients. Using three-color flow cytometry and immunohistochemistry, they were identified in mLN, pLN, and blood over time, and proliferation was determined by measuring bromodeoxyuridine incorporation. Only effector mLN T cells showed a significantly increased proliferation rate after entry into mLN compared with that in pLN (2.4 +/- 1.8% vs 0.8 +/- 0.4%). Proliferation among the injected cells was higher when they had contact with dendritic cells within mLN (9.0 +/- 4.3%) than when they did not (4.1 +/- 2.1%). Furthermore, effector mLN T cells which were observed 56 days after injection maintained the capacity for preferential proliferation within mLN. Interestingly, CD4(+) effector mLN T cells proliferated at a higher rate (4.8 +/- 0.7%), remaining in mLN, whereas CD8(+) effector mLN T cells proliferated at a lower rate (3.3 +/- 1.0%) and were able to leave the mLN into the blood. Elucidating the factors regulating the proliferation of effector T cells in vivo will help to modify their distribution for therapeutic purposes.     

Investigation of the immunostimulatory properties of oxihumate

A unique process has been developed to convert bituminous coal by controlled wet oxidation followed by base treatment to a water-soluble humate called oxihumate. The effects of oxihumate on the proliferative response of lymphocytes has been studied in vitro and ex vivo. Oxihumate increased the proliferative response of phytohaemagglutinin-stimulated human lymphocytes, from a concentration of 20 microg/ml and upwards. This response was even more striking in the case of lymphocytes from HIV-infected patients and was not limited to the in vitro setting since similar effects were observed ex vivo following administration of a non-toxic dosage of 4 g oxihumate per day to HIV-positive individuals for two weeks. Mechanistic studies revealed that stimulation of the proliferative response of lymphocytes by oxihumate is associated with an increased production of IL-2, as well as expression of the IL-2 receptor in the setting of decreased production of IL-10. Oxihumate therefore holds promise for the treatment of immunocompromized patients.