Tetrabenzoporphyrin and -mono-, - Cis -di- and tetrabenzotriazaporphyrin derivatives: Electrochemical and spectroscopic implications of meso CH Group replacement with nitrogen

Nonperipherally hexyl-substituted metal-free tetrabenzoporphyrin (2H-TBP, 1a) tetrabenzomonoazaporphyrin (2H-TBMAP, 2a), tetrabenzo-cis-diazaporphyrin (2H-TBDAP, 3a), tetrabenzotriazaporphyrin (2H-TBTAP, 4a), and phthalocyanine (2H-Pc, 5a), as well as their copper complexes (1b-5b), were synthesized. As the number of meso nitrogen atoms increases from zero to four, Îmax of the Q-band absorption peak becomes red-shifted by almost 100 nm, and extinction coefficients increased at least threefold. Simultaneously the blue-shifted Soret (UV) band substantially decreased in intensity. These changes were related to the relative electron-density of each macrocycle expressed as the group electronegativity sum of all meso N and CH atom groups, âχR. X-ray photoelectron spectroscopy differentiated between the three different types of macrocyclic nitrogen atoms (the Ninner, (NH)inner, and Nmeso) in the metal-free complexes. Binding energies of the Nmeso and Ninner,Cu atoms in copper chelates could not be resolved. Copper insertion lowered especially the cathodic redox potentials, while all four observed redox processes occurred at larger potentials as the number of meso nitrogens increased. Computational chemical methods using density functional theory confirmed 1b to exhibit a Cu(II) reduction prior to ring-based reductions, while for 2b, Cu(II) reduction is the first reductive step only if the nonperipheral substituents are hydrogen. When they are methyl groups, it is the second reduction process; when they are ethyl, propyl, or hexyl, it becomes the third reductive process. Spectro-electrochemical measurements showed redox processes were associated with a substantial change in intensity of at least two main absorbances (the Q and Soret bands) in the UV spectra of these compounds

Cas3 is a limiting factor for CRISPR-Cas immunity in Escherichia coli cells lacking H-NS

Background: CRISPR-Cas systems provide adaptive immunity to mobile genetic elements in prokaryotes. In many bacteria, including E. coli, a specialized ribonucleoprotein complex called Cascade enacts immunity by “an interference reaction" between CRISPR encoded RNA (crRNA) and invader DNA sequences called “protospacers”. Cascade recognizes invader DNA via short “protospacer adjacent motif” (PAM) sequences and crRNA-DNA complementarity. This triggers degradation of invader DNA by Cas3 protein and in some circumstances stimulates capture of new invader DNA protospacers for incorporation into CRISPR as “spacers” by Cas1 and Cas2 proteins, thus enhancing immunity. Co-expression of Cascade, Cas3 and crRNA is effective at giving E. coli cells resistance to phage lysis, if a transcriptional repressor of Cascade and CRISPR, H-NS, is inactivated (Δhns). We present further genetic analyses of the regulation of CRISPR-Cas mediated phage resistance in Δhns E. coli cells. Results: We observed that E. coli Type I-E CRISPR-Cas mediated resistance to phage λ was strongly temperature dependent, when repeating previously published experimental procedures. Further genetic analyses highlighted the importance of culture conditions for controlling the extent of CRISPR immunity in E. coli. These data identified that expression levels of cas3 is an important limiting factor for successful resistance to phage. Significantly, we describe the new identification that cas3 is also under transcriptional control by H-NS but that this is exerted only in stationary phase cells. Conclusions: Regulation of cas3 is responsive to phase of growth, and to growth temperature in E. coli, impacting on the efficacy of CRISPR-Cas immunity in these experimental systems

Identification of DHX9 as a cell cycle regulated nucleolar recruitment factor for CIZ1

CIP1-interacting zinc finger protein 1 (CIZ1) is a nuclear matrix associated protein that facilitates a number of nuclear functions including initiation of DNA replication, epigenetic maintenance and associates with the inactive X-chromosome. Here, to gain more insight into the protein networks that underpin this diverse functionality, molecular panning and mass spectrometry are used to identify protein interaction partners of CIZ1, and CIZ1 replication domain (CIZ1-RD). STRING analysis of CIZ1 interaction partners identified 2 functional clusters: ribosomal subunits and nucleolar proteins including the DEAD box helicases, DHX9, DDX5 and DDX17. DHX9 shares common functions with CIZ1, including interaction with XIST long-non-coding RNA, epigenetic maintenance and regulation of DNA replication. Functional characterisation of the CIZ1-DHX9 complex showed that CIZ1-DHX9 interact in vitro and dynamically colocalise within the nucleolus from early to mid S-phase. CIZ1-DHX9 nucleolar colocalisation is dependent upon RNA polymerase I activity and is abolished by depletion of DHX9. In addition, depletion of DHX9 reduced cell cycle progression from G1 to S-phase in mouse fibroblasts. The data suggest that DHX9-CIZ1 are required for efficient cell cycle progression at the G1/S transition and that nucleolar recruitment is integral to their mechanism of action

CRISPR Interference Directs Strand Specific Spacer Acquisition

Background: CRISPR/Cas is a widespread adaptive immune system in prokaryotes. This system integrates short stretches of DNA derived from invading nucleic acids into genomic CRISPR loci, which function as memory of previously encountered invaders. In Escherichia coli, transcripts of these loci are cleaved into small RNAs and utilized by the Cascade complex to bind invader DNA, which is then likely degraded by Cas3 during CRISPR interference. Results: We describe how a CRISPR-activated E. coli K12 is cured from a high copy number plasmid under non-selective conditions in a CRISPR-mediated way. Cured clones integrated at least one up to five anti-plasmid spacers in genomic CRISPR loci. New spacers are integrated directly downstream of the leader sequence. The spacers are non-randomly selected to target protospacers with an AAG protospacer adjacent motif, which is located directly upstream of the protospacer. A cooccurrence of PAM deviations and CRISPR repeat mutations was observed, indicating that one nucleotide from the PAM is incorporated as the last nucleotide of the repeat during integration of a new spacer. When multiple spacers were integrated in a single clone, all spacer targeted the same strand of the plasmid, implying that CRISPR interference caused by the first integrated spacer directs subsequent spacer acquisition events in a strand specific manner. Conclusions: The E. coli Type I-E CRISPR/Cas system provides resistance against bacteriophage infection, but also enables removal of residing plasmids. We established that there is a positive feedback loop between active spacers in a cluster – i

Beta-carotene affects gene expression in lungs of male and female Bcmo1−/− mice in opposite directions

Molecular mechanisms triggered by high dietary beta-carotene (BC) intake in lung are largely unknown. We performed microarray gene expression analysis on lung tissue of BC supplemented beta-carotene 15,15′-monooxygenase 1 knockout (Bcmo1−/−) mice, which are—like humans—able to accumulate BC. Our main observation was that the genes were regulated in an opposite direction in male and female Bcmo1−/− mice by BC. The steroid biosynthetic pathway was overrepresented in BC-supplemented male Bcmo1−/− mice. Testosterone levels were higher after BC supplementation only in Bcmo1−/− mice, which had, unlike wild-type (Bcmo1+/+) mice, large variations. We hypothesize that BC possibly affects hormone synthesis or metabolism. Since sex hormones influence lung cancer risk, these data might contribute to an explanation for the previously found increased lung cancer risk after BC supplementation (ATBC and CARET studies). Moreover, effects of BC may depend on the presence of frequent human BCMO1 polymorphisms, since these effects were not found in wild-type mice

Next Generation Mapping of Enological Traits in an F2 Interspecific Grapevine Hybrid Family

In winegrapes (Vitis spp.), fruit quality traits such as berry color, total soluble solids content (SS), malic acid content (MA), and yeast assimilable nitrogen (YAN) affect fermentation or wine quality, and are important traits in selecting new hybrid winegrape cultivars. Given the high genetic diversity and heterozygosity of Vitis species and their tendency to exhibit inbreeding depression, linkage map construction and quantitative trait locus (QTL) mapping has relied on F1 families with the use of simple sequence repeat (SSR) and other markers. This study presents the construction of a genetic map by single nucleotide polymorphisms identified through genotyping-by-sequencing (GBS) technology in an F2 mapping family of 424 progeny derived from a cross between the wild species V. riparia Michx. and the interspecific hybrid winegrape cultivar, ‘Seyval’. The resulting map has 1449 markers spanning 2424 cM in genetic length across 19 linkage groups, covering 95% of the genome with an average distance between markers of 1.67 cM. Compared to an SSR map previously developed for this F2 family, these results represent an improved map covering a greater portion of the genome with higher marker density. The accuracy of the map was validated using the well-studied trait berry color. QTL affecting YAN, MA and SS related traits were detected. A joint MA and SS QTL spans a region with candidate genes involved in the malate metabolism pathway. We present an analytical pipeline for calling intercross GBS markers and a high-density linkage map for a large F2 family of the highly heterozygous Vitis genus. This study serves as a model for further genetic investigations of the molecular basis of additional unique characters of North American hybrid wine cultivars and to enhance the breeding process by marker-assisted selection. The GBS protocols for identifying intercross markers developed in this study can be adapted for other heterozygous species

Improved Constraints on the 21 cm EoR Power Spectrum and the X-Ray Heating of the IGM with HERA Phase I Observations

We report the most sensitive upper limits to date on the 21 cm epoch of reionization power spectrum using 94 nights of observing with Phase I of the Hydrogen Epoch of Reionization Array (HERA). Using similar analysis techniques as in previously reported limits (HERA Collaboration 2022a), we find at 95% confidence that $\Delta^2(k = 0.34$ $h$ Mpc$^{-1}$) $\leq 457$ mK$^2$ at $z = 7.9$ and that $\Delta^2 (k = 0.36$ $h$ Mpc$^{-1}) \leq 3,496$ mK$^2$ at $z = 10.4$, an improvement by a factor of 2.1 and 2.6 respectively. These limits are mostly consistent with thermal noise over a wide range of $k$ after our data quality cuts, despite performing a relatively conservative analysis designed to minimize signal loss. Our results are validated with both statistical tests on the data and end-to-end pipeline simulations. We also report updated constraints on the astrophysics of reionization and the cosmic dawn. Using multiple independent modeling and inference techniques previously employed by HERA Collaboration (2022b), we find that the intergalactic medium must have been heated above the adiabatic cooling limit at least as early as $z = 10.4$, ruling out a broad set of so-called "cold reionization" scenarios. If this heating is due to high-mass X-ray binaries during the cosmic dawn, as is generally believed, our result's 99% credible interval excludes the local relationship between soft X-ray luminosity and star formation and thus requires heating driven by evolved low-metallicity stars.Comment: 57 pages, 37 figures. Updated to match the accepted ApJ version. Corresponding author: Joshua S. Dillo

The diversity-generating benefits of a prokaryotic adaptive immune system

Published onlineJOURNAL ARTICLEProkaryotic CRISPR-Cas adaptive immune systems insert spacers derived from viruses and other parasitic DNA elements into CRISPR loci to provide sequence-specific immunity. This frequently results in high within-population spacer diversity, but it is unclear if and why this is important. Here we show that, as a result of this spacer diversity, viruses can no longer evolve to overcome CRISPR-Cas by point mutation, which results in rapid virus extinction. This effect arises from synergy between spacer diversity and the high specificity of infection, which greatly increases overall population resistance. We propose that the resulting short-lived nature of CRISPR-dependent bacteria-virus coevolution has provided strong selection for the evolution of sophisticated virus-encoded anti-CRISPR mechanisms.S.v.H. has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement number 660039. E.R.W. received funding from the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme (FP7/2007-2013) under Research Executive Agency grant agreement number 327606. E.R.W., A.B. and M.B. also acknowledge the Natural Environment Research Council, the Biotechnology and Biological Sciences Research Council, the Royal Society, the Leverhulme Trust, the Wellcome Trust and the AXA research fund for funding. J.M.B.-D. was supported by the University of California San Francisco Program for Breakthrough in Biomedical Research, the Sandler Foundation, and a National Institutes of Health Director’s Early Independence Award (DP5-OD021344). H.C. was funded by the Erasmus+ programme (European Union), the Explora’Sup programme (Région Rhône-Alpes) and the Centre Régional des Œuvres Universitaires et Scolaires (CROUS; French State)

Are food-deceptive orchid species really functionally specialized for pollinators?

Food-deceptive orchid species have traditionally been considered pollination specialized to bees or butterflies. However, it is unclear to which concept of specialization this assumption is related; if to that of phenotypic specialization or of functional specialization. The main aim of this work was to verify if pollinators of five widespread food-deceptive orchid species (Anacamptis morio (L.) R.M. Bateman, Pridgeon & M.W. Chase, Anacamptis pyramidalis (L.) Rich., Himantoglossum adriaticum H. Baumann, Orchis purpurea Huds. and Orchis simia Lam.) predicted from the phenotypic point of view matched with the observed ones. We addressed the question by defining target orchids phenotypic specialization on the basis of their floral traits, and we compared the expected guilds of pollinators with the observed ones. Target orchid pollinators were collected by conducting a meta-analysis of the available literature and adding unpublished field observations, carried out in temperate dry grasslands in NE Italy. Pollinator species were subsequently grouped into guilds and differences in the guild spectra among orchid species grouped according to their phenotype were tested. In contradiction to expectations derived from the phenotypic point of view, food-deceptive orchid species were found to be highly functionally generalized for pollinators, and no differences in the pollinator guild spectra could be revealed among orchid groups. Our results may lead to reconsider food-deceptive orchid pollination ecology by revaluating the traditional equation orchid-pollination specialization