Monitoring signal transduction in cancer: cDNA microarray for semiquantitative analysis.

This study targeted the development of a novel microarray tool to allow rapid determination of the expression levels of 58 different tyrosine kinase (tk) genes in small tumor samples. The goals were to define a reference probe for multi-sample comparison and to investigate the variability and reproducibility of the image acquisition and RT-PCR procedures. The small number of tk genes on our arrays enabled us to define a reference probe by artificially mixing all genes on the arrays. Such a probe provided contrast reference for comparative hybridization of control and sample DNA and enabled cross-comparison of more than two samples against one another. Comparison of signals generated from multiple scanning eliminated the concern of photo bleaching and scanner intrinsic noise. Tests performed with breast, thyroid, and prostate cancer samples yielded distinctive patterns and suggest the feasibility of our approach. Repeated experiments indicated reproducibility of such arrays. Up- or downregulated genes identified by this rapid screening are now being investigated with techniques such as in situ hybridization.     

Seasonal abundance and vertical distribution of mesopelagic calycophoran siphonophores in Monterey Bay, CA

The seasonal abundance and vertical distribution patterns ofa group of small calycophoran siphonophores (principally Chuniphyesmultidentata and Lensia conoidea) were investigated using aremotely operated vehicle (ROV) deployed in Monterey Bay, California.Abundance was assessed along 295 horizontal transects coveringa depth range of 100–1000 m over a three and a half yearperiod. The vertical distribution of the study animals changedseasonally, coupled to the onset and cessation of upwellingin the bay. While numerical abundance peaked after upwelling,there was no significant difference between seasons. The siphonophoreswere more broadly distributed over the depth range sampled duringthe upwelling or Shallow Mixed Layer (SML) period, than duringthe non-upwelling or Deep Mixed Layer (DML) period. There wereno significant differences in abundance or distribution patternsbetween years except in 1993, when there were significantlymore siphonophores observed during the SML period than duringthe DML period. This may reflect effects resulting from the1992–1993 El Niño event. The abundance of thesesiphonophores was negatively correlated with that of Nanomiabijuga, a physonect siphonophore of similar size and feedingbehavior found in the bay. The siphonophores studied here appearfrom preliminary data to migrate vertically, possibly with twoseparately migrating groups.     

Glycosylation of the phosphate binding protein, PstS, in Streptomyces coelicolor by a pathway that resembles protein O-mannosylation in eukaryotes

Previously mutations in a putative protein O -mannosyltransferase (SCO3154, Pmt) and a polyprenol phosphate mannose synthase (SCO1423, Ppm1) were found to cause resistance to phage, φC31, in the antibiotic producing bacteria Streptomyces coelicolor A3(2). It was proposed that these two enzymes were part of a protein O-glycosylation pathway that was necessary for synthesis of the phage receptor. Here we provide the evidence that Pmt and Ppm1 are indeed both required for protein O-glycosylation. The phosphate binding protein PstS was found to be glycosylated with a trihexose in the S. coelicolor parent strain, J1929, but not in the pmt ⁻ derivative, DT1025. Ppm1 was necessary for the transfer of mannose to endogenous polyprenol phosphate in membrane preparations of S. coelicolor . A mutation in ppm1 that conferred an E218V substitution in Ppm1 abolished mannose transfer and glycosylation of PstS. Mass spectrometry analysis of extracted lipids showed the presence of a glycosylated polyprenol phosphate (PP) containing nine repeated isoprenyl units (C₄₅-PP). S. coelicolor membranes were also able to catalyse the transfer of mannose to peptides derived from PstS, indicating that these could be targets for Pmt in vivo .     

Induction of high-frequency somatic embryos in cassava for cryopreservation

Methods for inducing high-frequency somatic embryos in cassava on cotyledons and 33 clonal accessions by the addition of supplementary copper sulphate to the induction medium were investigated. The addition of copper sulphate enhanced primary embryo induction and significantly increased secondary embryo production. All accessions from Latin America (CIAT) were embryogenically competent on medium supplemented with 8 mg l^-1 2,4-dichlorophenoxyacetic acid (2,4-D) plus 1 µM copper sulphate as were 15 of the 18 accessions from Africa. The percentage of calli producing somatic embryos ranged from 7.5% in M. Bra 12 to 100% in M. Col. 1505, while the number of embryos produced per callus ranged from 0.3 in M. Bra 383 to 13.5 in TEK. The frequency of embryo production was dependent on the concentration of copper sulphate. The number of primary embryos produced per callus was also comparatively higher in the medium supplemented with copper sulphate than in the controls. The optimal concentration of copper sulphate for number of embryos produced in most accessions was 5 µM, and at this concentration the number of embryos produced was double that of the controls. Copper sulphate also reduced the maturation time of somatic embryos to 25 days from embryo initiation. High levels of 2,4-D were detrimental to embryo production. Similarly, fragmented embryos incubated in the dark produced more embryos tan those incubated under light conditions. On the basis of these results, the use of cassava somatic embryo micropropagules for germplasm conservation and synthetic seed development seems to be a strong possibility.     

Profiling SARS-CoV-2 HLA-I peptidome reveals T cell epitopes from out-of-frame ORFs

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A mini-Tn5-derived transposon with reportable and selectable markers enables rapid generation and screening of insertional mutants in Gram-negative bacteria

We re-engineered a classic tool for mutagenesis and gene expression studies in Gram-negative bacteria. Our modified Tn5-based transposon contains multiple features that allow rapid selection for mutants, direct quantification of gene expression and straightforward cloning of the inactivated gene. The promoter-less gfp-km cassette provides selection and reporter assay depending on the activity of the promoter upstream of the transposon insertion site. The cat gene facilitates positive antibiotic selection for mutants, while the narrow R6Kγ replication origin forces transposition in recipient strains lacking the pir gene and enables cloning of the transposon flanked with the disrupted gene from the chromosome. The suicide vector pCKD100, a plasmid that could be delivered into recipient cells through biparental mating or electroporation, harbours the modified transposon. We used the transposon to mutagenize Pectobacterium versatile KD100, Pseudumonas coronafaciens PC27R and Escherichia coli 35150N. The fluorescence intensities of mutants expressing high GFP could be quantified and detected qualitatively. Transformation efficiency from conjugation ranged from 1600 to 1900 CFU per ml. We sequenced the upstream flanking regions, identified the putative truncated genes and demonstrated the restoration of the GFP phenotype through marker exchange. The mini-Tn5 transposon was also utilized to construct mutant a library of P. versatile for forward genetic screens.     

Control of the growth of Alicyclobacillus acidoterrestris in industrialized orange juice using rosemary essential oil and nisin

The use of rosemary essential oil (RO) and its combination with nisin (RO+N) in preventing the multiplication of Alicyclobacillus acidoterrestris in orange juice was evaluated. The minimum inhibitory and bactericidal concentrations (MIC and MBC) for RO were both 125 μg ml⁻¹ while RO+N displayed a synergistic effect. The use of RO and RO+N at concentrations of 1, 4 and 8× MIC in orange juice for 96 h was evaluated in terms of their sporicidal effectiveness. With regard to the action against A. acidoterrestris spores, RO at 8× MIC was sporostatic, whereas RO+N at 1× MIC was sporicidal. Morphological changes in the structure of the micro-organism after treatment were also observed by microscopy. Furthermore, flow cytometric analysis showed that most cells were damaged or killed after treatment. In general, the antioxidant activity after addition of RO+N decreased with time. The results demonstrate that using the combination of RO and nisin can prevent the A. acidoterrestris growth in orange juice.