Regulatory overlap and functional redundancy among Bacillus subtilis extracytoplasmic function sigma factors

Bacillus subtilis encodes seven extracytoplasmic function (ECF) sigma factors that regulate partially overlapping regulons related to cell envelope homeostasis and antibiotic resistance. Here, we investigated their physiological role by constructing a mutant set of single, double, triple, and quadruple ECF sigma factor deletions in the undomesticated B. subtilis strain NCIB3610. This mutant set was subsequently screened for defects in motility, multicellular differentiation, and sensitivity to more than 200 chemicals by using Phenotype MicroArrays. A quadruple mutant strain, harboring deletions of the sigV, sigY, sigZ, and ylaC gene, behaved indistinguishably from the wild-type strain, indicative of either regulatory redundancy or very specific functions of these four ECF sigma factors. In contrast, a triple mutant, inactivated for the sigM, sigW, and sigX genes (but none of the corresponding double mutants), showed a biphasic growth behavior and a complete loss of multicellular differentiation, as judged by both colony formation and the inability to form a pellicle. This triple mutant also displayed a greatly increased sensitivity to detergents and several cell wall antibiotics including beta-lactams, polymyxin B, and d-cycloserine. In several cases, these antibiotic-sensitive phenotypes are significantly enhanced in the triple mutant strain relative to strains lacking only one or two sigma factors.     

In vitro mutagenesis of Bacillus subtilis by using a modified Tn7 transposon with an outward-facing inducible promoter

A Tn7 donor plasmid, pTn7SX, was constructed for use with the model gram-positive bacterium Bacillus subtilis. This new mini-Tn7, mTn7SX, contains a spectinomycin resistance cassette and an outward-facing, xylose-inducible promoter, thereby allowing for the regulated expression of genes downstream of the transposon. We demonstrate that mTn7SX inserts are obtained at a high frequency and occur randomly throughout the B. subtilis genome. The utility of this system was demonstrated by the selection of mutants with increased resistance to the antibiotic fosfomycin or duramycin.     

Position-dependent effects on stability in tricyclo-DNA modified oligonucleotide duplexes

A series of oligodeoxyribonucleotides and oligoribonucleotides containing single and multiple tricyclo(tc)-nucleosides in various arrangements were prepared and the thermal and thermodynamic transition profiles of duplexes with complementary DNA and RNA evaluated. Tc-residues aligned in a non-continuous fashion in an RNA strand significantly decrease affinity to complementary RNA and DNA, mostly as a consequence of a loss of pairing enthalpy ΔH. Arranging the tc-residues in a continuous fashion rescues T_m and leads to higher DNA and RNA affinity. Substitution of oligodeoxyribonucleotides in the same way causes much less differences in T_m when paired to complementary DNA and leads to substantial increases in T_m when paired to complementary RNA. CD-spectroscopic investigations in combination with molecular dynamics simulations of duplexes with single modifications show that tc-residues in the RNA backbone distinctly influence the conformation of the neighboring nucleotides forcing them into higher energy conformations, while tc-residues in the DNA backbone seem to have negligible influence on the nearest neighbor conformations. These results rationalize the observed affinity differences and are of relevance for the design of tc-DNA containing oligonucleotides for applications in antisense or RNAi therapy.     

Mutations in NOTCH1 Cause Adams-Oliver Syndrome

Notch signaling determines and reinforces cell fate in bilaterally symmetric multicellular eukaryotes. Despite the involvement of Notch in many key developmental systems, human mutations in Notch signaling components have mainly been described in disorders with vascular and bone effects. Here, we report five heterozygous NOTCH1 variants in unrelated individuals with Adams-Oliver syndrome (AOS), a rare disease with major features of aplasia cutis of the scalp and terminal transverse limb defects. Using whole-genome sequencing in a cohort of 11 families lacking mutations in the four genes with known roles in AOS pathology (ARHGAP31, RBPJ, DOCK6, and EOGT), we found a heterozygous de novo 85 kb deletion spanning the NOTCH1 5′ region and three coding variants (c.1285T>C [p.Cys429Arg], c.4487G>A [p.Cys1496Tyr], and c.5965G>A [p.Asp1989Asn]), two of which are de novo, in four unrelated probands. In a fifth family, we identified a heterozygous canonical splice-site variant (c.743−1 G>T) in an affected father and daughter. These variants were not present in 5,077 in-house control genomes or in public databases. In keeping with the prominent developmental role described for Notch1 in mouse vasculature, we observed cardiac and multiple vascular defects in four of the five families. We propose that the limb and scalp defects might also be due to a vasculopathy in NOTCH1-related AOS. Our results suggest that mutations in NOTCH1 are the most common cause of AOS and add to a growing list of human diseases that have a vascular and/or bony component and are caused by alterations in the Notch signaling pathway.     

Causes and consequences of floral damage in Aconitum lycoctonum at high and low elevations in Switzerland

The outcomes of interactions among plants and the insects that use their flowers are likely to vary among the physical environments and the communities in which they grow. In this study we quantified floral damage of Aconitum lycoctonum in high (>2000 m) and low (c. 500 m) elevation populations in Switzerland. At high elevation, floral damage was frequent and was caused by nectar-robbing short-tongued bumblebees. Nectar robbers make a hole in the flower when they collect nectar. A nectar robber exclusion experiment showed that nectar robbery by short-tongued bumblebees had no effect on the female reproductive success of plants; robbing bees rarely damaged the nectaries, and damage to the petals probably does not decrease flower longevity. In addition, nectar robbers tended to collect pollen during about 10% of their visits. Thus, these bees may act as low-efficiency pollinators and may, at times, be mutualistic associates. At low elevation, the holes in the flowers were caused by beetles (Meligethes viridescens) and not by short-tongued bumblebees. The beetles eat pollen and might also consume nectar. Since the beetles gain access to pollen and nectar by entering the flower through its opening, and later chew holes while foraging on floral tissue, the beetles are pollen eaters rather than nectar robbers. Overall, our results show that not all floral damage is caused by nectar robbers and that there can be strong altitudinal variation in the causes and consequences of floral damage.     

Pollinator abundance and behavior on Aconitum lycoctonum (Ranunculaceae): an analysis of the quantity and quality components of pollination

The yellow-flowered monkshood Aconitum lycoctonum has variable degrees of inbreeding among populations, despite being primarily bee-pollinated. Here we examined the pollination ecology of A. lycoctonum . We investigated pollinator community and frequency in four populations at two altitudes over two years. We found that flowers were more often visited at low elevation than at high elevation. However, because flowers lived longer at high elevation than at low elevation, and plants at high elevation had more flowers than at low elevation, the overall chance of a plant being visited may be greater at high elevation. Breeding system experiments showed that at least some populations of A. lycoctonum were self-compatible and also were not completely protandrous. Thus selfing, especially by geitonogamous pollen, will be common if pollinators often visit several flowers within a plant, as we found to be true for the main pollinator (bumblebees) of A. lycoctonum . Although the stereotypical behavior of bees is to move upwards, 17% of the within-plant movements of the main pollinator ( Bombus gerstaeckeri ) were downwards, i.e. from a male-phased to a female-phased flower because the flowers open from the bottom to the top of the plant. Other pollinators of A. lycoctonum moved less often within plants. We conclude that in addition to differences in self-compatibility, differences in pollinator abundance and behavior could have led to variation in the realized mating system in different A. lycoctonum populations.     

HLA class I binding promiscuity of the CD8 T-cell epitopes of human papillomavirus type 16 E6 protein

One of the critical steps in the progression to cervical cancer appears to be the establishment of persistent human papillomavirus (HPV) infection. We have demonstrated that the lack of cytotoxic T-lymphocyte response to HPV type 16 (HPV 16) E6 protein was associated with persistence and that the potential presence of dominant CD8 T-cell epitopes was most frequently found (n = 4 of 23) in the E6 16-40 region by examining the pattern of CD8 T-cell epitopes within the E6 protein in women who had cleared their HPV 16 infections. The goal of this study was to define the minimal/optimal amino acid sequences and the HLA restricting molecules of these dominant CD8 T-cell epitopes as well as those of subdominant ones if present. Three dominant epitopes, E6 29-38 (TIHDIILECV; restricted by the HLA-A0201 molecule), E6 29-37 (TIHDIILEC; restricted by B48), and E6 31-38 (HDIILECV; restricted by B4002), and one subdominant epitope, E6 52-61 (FAFRDLCIVY; restricted by B35) were characterized. Taken together with a previously described dominant epitope, E6 52-61 (FAFRDLCIVY; restricted by B57), the CD8 T-cell epitopes demonstrated striking HLA class I binding promiscuity. All of these epitopes were endogenously processed, but the presence of only two of the five epitopes could have been predicted based on the known binding motifs. The HLA class I promiscuity which has been described for human immunodeficiency virus may be more common than previously recognized.     

Heterozygous Loss-of-Function Mutations in DLL4 Cause Adams-Oliver Syndrome

Adams-Oliver syndrome (AOS) is a rare developmental disorder characterized by the presence of aplasia cutis congenita (ACC) of the scalp vertex and terminal limb-reduction defects. Cardiovascular anomalies are also frequently observed. Mutations in five genes have been identified as a cause for AOS prior to this report. Mutations in EOGT and DOCK6 cause autosomal-recessive AOS, whereas mutations in ARHGAP31, RBPJ, and NOTCH1 lead to autosomal-dominant AOS. Because RBPJ, NOTCH1, and EOGT are involved in NOTCH signaling, we hypothesized that mutations in other genes involved in this pathway might also be implicated in AOS pathogenesis. Using a candidate-gene-based approach, we prioritized DLL4, a critical NOTCH ligand, due to its essential role in vascular development in the context of cardiovascular features in AOS-affected individuals. Targeted resequencing of the DLL4 gene with a custom enrichment panel in 89 independent families resulted in the identification of seven mutations. A defect in DLL4 was also detected in two families via whole-exome or genome sequencing. In total, nine heterozygous mutations in DLL4 were identified, including two nonsense and seven missense variants, the latter encompassing four mutations that replace or create cysteine residues, which are most likely critical for maintaining structural integrity of the protein. Affected individuals with DLL4 mutations present with variable clinical expression with no emerging genotype-phenotype correlations. Our findings demonstrate that DLL4 mutations are an additional cause of autosomal-dominant AOS or isolated ACC and provide further evidence for a key role of NOTCH signaling in the etiology of this disorder.     

Molecular and morphological analyses of EuropeanAconitum species (Ranunculaceae)

The systematics of the yellow-floweredAconitum lycoctonum species complex (Ranunculaceae) has long been considered difficult because of high morphological variability and hypothesized hybridization. To elucidate the systematic value of these taxa, and to study their phylogeny in a broader context, we studied all species of the subgenusLycoctonum from Europe and the Caucasus Mountains and the European species of the subgen.Aconitum. We sequenced the ITS region and the chloroplast intergenic spacerpsbA-trnH and conducted parsimony analyses. We also measured morphological characters from 19A. lycoctonum populations. The two subgenera in Europe are clearly separated but hybridization between subgenera might have taken place in the past. Sequence variation withinA. lycoctonum and among all taxa of subgenusLycoctonum was very low and probably the result of recent speciation, leading to one morphologically highly variable species in which color morphs could be seen as subspecies. There was high morphological variability within and among populations and the morphological characters have no value as systematic characters.     

Interlaboratory reproducibility of female genital tract cytokine measurements by Luminex: Implications for microbicide safety studies

The interlaboratory reproducibility of cytokine measurements from cervicovaginal samples by Luminex has not been reported. Using cervicovaginal lavage specimens collected on three study days from 12 women participating in a Phase I microbicide study, we measured a panel of eight cytokines in three independent laboratories. Four (IFN-γ, IL-10, IL-17, and TNF) were below the limit of detection in the majority (85%) of samples in either two or all three laboratories, an observation that may guide analyte selection for future studies. Good interlaboratory agreement (intraclass correlation coefficient, r > 0.7) in absolute levels was observed for IL-1β, IL-6, and IL-8, while poor agreement was seen for IFN-α2 (r = 0.47). When considering within-subject change from baseline (pre-product, at study-day 0) to either post-product visit (study-days 7 and 14), IL-1β and IL-6 exhibited good interlaboratory agreement (r > 0.7), while IFN-α2 and IL-8 did not. Future studies addressing the clinical utility of specific biomarkers of inflammation for microbicide trials should consider reproducibility in the context of defining biologically meaningful thresholds of change for candidate biomarkers, ensuring that such change can be reliably distinguished from background variability.