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

Diets containing sea cucumber (Isostichopus badionotus) meals are hypocholesterolemic in young rats

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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

Merkel cell carcinoma of skin-current controversies and recommendations

The review covers the current recommendations for Merkel cell carcinoma (MCC), with detailed discussion of many controversies. The 2010 AJCC staging system is more in-line with other skin malignancies although more complicated to use. The changes in staging system over time make comparison of studies difficult. A wide excision with margins of 2.5–3 cm is generally recommended. Even for primary </= 1 cm, there is a significant risk of nodal and distant metastases and hence sentinel node biopsy should be done if possible; otherwise adjuvant radiotherapy to the primary and nodal region should be given. Difficulties of setting up trials owing to the rarity of the disease and the mean age of the patient population result in infrequent reports of adjuvant or concurrent chemotherapy in the literature. The benefit, if any, is not great from published studies so far. However, there may be a subgroup of patients with high-risk features, e.g. node-positive and excellent performance status, for whom adjuvant or concurrent chemotherapy may be considered. Since local recurrence and metastases generally occur within 2 years of the initial diagnosis, patients should be followed more frequently in the first 2 years. However delayed recurrence can still occur in a small proportion of patients and long-term follow-up by a specialist is recommended provided that the general condition of the patient allows it. In summary, physician judgment in individual cases of MCC is advisable, to balance the risk of recurrence versus the complications of treatment

CRISPR-based strategies in infectious disease diagnosis and therapy

CRISPR gene-editing technology has the potential to transform the diagnosis and treatment of infectious diseases, but most clinicians are unaware of its broad applicability. Derived from an ancient microbial defence system, these so-called "molecular scissors" enable precise gene editing with a low error rate. However, CRISPR systems can also be targeted against pathogenic DNA or RNA sequences. This potential is being combined with innovative delivery systems to develop new therapeutic approaches to infectious diseases.info:eu-repo/semantics/publishedVersio

Diurnal timing of nonmigratory movement by birds: the importance of foraging spatial scales

This is the author accepted manuscript. The final version is available from Wiley via the DOI in this recordData Availability statement: R code used in analyses can be accessed at datadryad.com. Most of the data used are publicly available at www.movebank.orgTiming of activity can reveal an organism's efforts to optimize foraging either by minimizing energy loss through passive movement or by maximizing energetic gain through foraging. Here, we assess whether signals of either of these strategies are detectable in the timing of activity of daily, local movements by birds. We compare the similarities of timing of movement activity among species using six temporal variables: start of activity relative to sunrise, end of activity relative to sunset, relative speed at midday, number of movement bouts, bout duration, and proportion of active daytime hours. We test for the influence of flight mode and foraging habitat on the timing of movement activity across avian guilds. We used 64570 days of GPS movement data collected between 2002 and 2019 for local (non‐migratory) movements of 991 birds from 49 species, representing 14 orders. Dissimilarity among daily activity patterns was best explained by flight mode. Terrestrial soaring birds began activity later and stopped activity earlier than pelagic soaring or flapping birds. Broad‐scale foraging habitat explained less of the clustering patterns because of divergent timing of active periods of pelagic surface and diving foragers. Among pelagic birds, surface foragers were active throughout the day while diving foragers matched their active hours more closely to daylight hours. Pelagic surface foragers also had the greatest daily foraging distances, which was consistent with their daytime activity patterns. This study demonstrates that flight mode and foraging habitat influence temporal patterns of daily movement activity of birds.Nature ConservancyBailey Wildlife FoundationBluestone FoundationOcean View FoundationBiodiversity Research InstituteMaine Outdoor Heritage FundDavis Conservation FoundationUS Department of EnergyDarwin InitiativePortuguese Foundation for Science and Technology (FCT)Enterprise St Helena (ESH)Hawk Mountain Sanctuar

Multi-scale genomic, transcriptomic and proteomic analysis of colorectal cancer cell lines to identify novel biomarkers

This work was partially funded by the Strategic Educational Pathways Scholarship (Malta). The scholarship is part-financed by the European Union – European Social Fund (ESF) under Operational Programme II – Cohesion Policy 2007-2013, “Empowering People for More Jobs and a Better Quality of Life”. This project was additionally funded by Medical Research Scotland.Selecting colorectal cancer (CRC) patients likely to respond to therapy remains a clinical challenge. The objectives of this study were to establish which genes were differentially expressed with respect to treatment sensitivity and relate this to copy number in a panel of 15 CRC cell lines. Copy number variations of the identified genes were assessed in a cohort of CRCs. IC50’s were measured for 5-fluorouracil, oxaliplatin, and BEZ-235, a PI3K/mTOR inhibitor. Cell lines were profiled using array comparative genomic hybridisation, Illumina gene expression analysis, reverse phase protein arrays, and targeted sequencing of KRAS hotspot mutations. Frequent gains were observed at 2p, 3q, 5p, 7p, 7q, 8q, 12p, 13q, 14q, and 17q and losses at 2q, 3p, 5q, 8p, 9p, 9q, 14q, 18q, and 20p. Frequently gained regions contained EGFR, PIK3CA, MYC, SMO, TRIB1, FZD1, and BRCA2, while frequently lost regions contained FHIT and MACROD2. TRIB1 was selected for further study. Gene enrichment analysis showed that differentially expressed genes with respect to treatment response were involved in Wnt signalling, EGF receptor signalling, apoptosis, cell cycle, and angiogenesis. Stepwise integration of copy number and gene expression data yielded 47 candidate genes that were significantly correlated. PDCD6 was differentially expressed in all three treatment responses. Tissue microarrays were constructed for a cohort of 118 CRC patients and TRIB1 and MYC amplifications were measured using fluorescence in situ hybridisation. TRIB1 and MYC were amplified in 14.5% and 7.4% of the cohort, respectively, and these amplifications were significantly correlated (p≤0.0001). TRIB1 protein expression in the patient cohort was significantly correlated with pERK, Akt, and Caspase 3 expression. In conclusion, a set of candidate predictive biomarkers for 5-fluorouracil, oxaliplatin, and BEZ235 are described that warrant further study. Amplification of the putative oncogene TRIB1 has been described for the first time in a cohort of CRC patients.Publisher PDFPeer reviewe

Target motifs affecting natural immunity by a constitutive CRISPR-Cas system in Escherichia coli

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated (cas) genes conform the CRISPR-Cas systems of various bacteria and archaea and produce degradation of invading nucleic acids containing sequences (protospacers) that are complementary to repeat intervening spacers. It has been demonstrated that the base sequence identity of a protospacer with the cognate spacer and the presence of a protospacer adjacent motif (PAM) influence CRISPR-mediated interference efficiency. By using an original transformation assay with plasmids targeted by a resident spacer here we show that natural CRISPR-mediated immunity against invading DNA occurs in wild type Escherichia coli. Unexpectedly, the strongest activity is observed with protospacer adjoining nucleotides (interference motifs) that differ from the PAM both in sequence and location. Hence, our results document for the first time native CRISPR activity in E. coli and demonstrate that positions next to the PAM in invading DNA influence their recognition and degradation by these prokaryotic immune systems.This study was supported by the Spanish Ministerio de Ciencia e Innovación (BIO2011-24417)