Measurement of S-phase duration of adult stem cells in the flatworm Macrostomum lignano by double replication labelling and quantitative colocalization analysis

Platyhelminthes are highly attractive models for addressing fundamental aspects of stem cell biology in vivo. These organisms possess a unique stem cell system comprised of neoblasts that are the only proliferating cells during adulthood. We have investigated T-s (S-phase duration) of neoblasts during homoeostasis and regeneration in the flatworm, Macrostomum lignano. A double immunohistochemical technique was used, performing sequential pulses with the thymidine analogues CldU (chlorodeoxyuridine) and IdU (iododeoxyuridine), separated by variable chase times in the presence of colchicine. Owing to the localized nature of the fluorescent signals (cell nuclei) and variable levels of autofluorescence, standard intensity-based colocalization analyses could not be applied to accurately determine the colocalization. Therefore, an object-based colocalization approach was devised to score the relative number of double-positive cells. Using this approach, T-s (S-phase duration) in the main population of neoblasts was similar to 13 h. During early regeneration, no significant change in T-s was observed

Timed inhibition of CDC7 increases CRISPR-Cas9 mediated templated repair.

Repair of double strand DNA breaks (DSBs) can result in gene disruption or gene modification via homology directed repair (HDR) from donor DNA. Altering cellular responses to DSBs may rebalance editing outcomes towards HDR and away from other repair outcomes. Here, we utilize a pooled CRISPR screen to define host cell involvement in HDR between a Cas9 DSB and a plasmid double stranded donor DNA (dsDonor). We find that the Fanconi Anemia (FA) pathway is required for dsDonor HDR and that other genes act to repress HDR. Small molecule inhibition of one of these repressors, CDC7, by XL413 and other inhibitors increases the efficiency of HDR by up to 3.5 fold in many contexts, including primary T cells. XL413 stimulates HDR during a reversible slowing of S-phase that is unexplored for Cas9-induced HDR. We anticipate that XL413 and other such rationally developed inhibitors will be useful tools for gene modification

Identification of the Er1 resistence gene and RNase S-alleles in Malus prunifolia var. ringo rootstock

Woolly apple aphid (WAA; Eriosoma lanigerum Hausm.) is a major insect pest that has significant economic impact on apple growers worldwide. Modern breeding technologies rely on several molecular tools to help breeders select genetic determinants for traits of interest. Consequently, there is a need for specific markers linked to the genes of interest. Apple scions and rootstocks have an additional barrier to the introduction of pest resistance genes due to the presence of self-incompatibility S-RNase alleles. The genetic characterization and early identification of these alleles can amplify the contribution of a breeding program to the selection of resistant genitors that are as compatible as possible. In this study, we identified the Er1 gene involved in the resistance to WAA in Malus prunifolia var. ringo, also known as ‘Maruba Kaido’ rootstock, and we analyzed the inheritance pattern of the WAA resistance Er1 gene in a segregant population derived from Malus pumila ‘M.9’ and ‘Maruba Kaido’ rootstocks. The self-incompatibility of S-RNase alleles S6S26 of ‘Maruba Kaido’ were also identified along with their inheritance pattern. We also confirmed the identification of the S1S3 alleles in the ‘M.9’ rootstock. To the best of our knowledge, this is the first study to characterize WAA resistance and RNase S-alleles in ‘Maruba Kaido’. Furthermore, we discuss the potential use of the genetic markers for these genes and their potential impact on apple breeding programs

Comparison of immature and mature bone marrow-derived dendritic cells by atomic force microscopy

A comparative study of immature and mature bone marrow-derived dendritic cells (BMDCs) was first performed through an atomic force microscope (AFM) to clarify differences of their nanostructure and adhesion force. AFM images revealed that the immature BMDCs treated by granulocyte macrophage-colony stimulating factor plus IL-4 mainly appeared round with smooth surface, whereas the mature BMDCs induced by lipopolysaccharide displayed an irregular shape with numerous pseudopodia or lamellapodia and ruffles on the cell membrane besides becoming larger, flatter, and longer. AFM quantitative analysis further showed that the surface roughness of the mature BMDCs greatly increased and that the adhesion force of them was fourfold more than that of the immature BMDCs. The nano-features of the mature BMDCs were supported by a high level of IL-12 produced from the mature BMDCs and high expression of MHC-II on the surface of them. These findings provide a new insight into the nanostructure of the immature and mature BMDCs

Immature and Maturation-Resistant Human Dendritic Cells Generated from Bone Marrow Require Two Stimulations to Induce T Cell Anergy In Vitro

Immature dendritic cells (DC) represent potential clinical tools for tolerogenic cellular immunotherapy in both transplantation and autoimmunity. A major drawback in vivo is their potential to mature during infections or inflammation, which would convert their tolerogenicity into immunogenicity. The generation of immature DC from human bone marrow (BM) by low doses of GM-CSF (lowGM) in the absence of IL-4 under GMP conditions create DC resistant to maturation, detected by surface marker expression and primary stimulation by allogeneic T cells. This resistence could not be observed for BM-derived DC generated with high doses of GM-CSF plus IL-4 (highGM/4), although both DC types induced primary allogeneic T cell anergy in vitro. The estabishment of the anergic state requires two subsequent stimulations by immature DC. Anergy induction was more profound with lowGM-DC due to their maturation resistance. Together, we show the generation of immature, maturation-resistant lowGM-DC for potential clinical use in transplant rejection and propose a two-step-model of T cell anergy induction by immature DC

Causes of genome instability: the effect of low dose chemical exposures in modern society.

Genome instability is a prerequisite for the development of cancer. It occurs when genome maintenance systems fail to safeguard the genome's integrity, whether as a consequence of inherited defects or induced via exposure to environmental agents (chemicals, biological agents and radiation). Thus, genome instability can be defined as an enhanced tendency for the genome to acquire mutations; ranging from changes to the nucleotide sequence to chromosomal gain, rearrangements or loss. This review raises the hypothesis that in addition to known human carcinogens, exposure to low dose of other chemicals present in our modern society could contribute to carcinogenesis by indirectly affecting genome stability. The selected chemicals with their mechanisms of action proposed to indirectly contribute to genome instability are: heavy metals (DNA repair, epigenetic modification, DNA damage signaling, telomere length), acrylamide (DNA repair, chromosome segregation), bisphenol A (epigenetic modification, DNA damage signaling, mitochondrial function, chromosome segregation), benomyl (chromosome segregation), quinones (epigenetic modification) and nano-sized particles (epigenetic pathways, mitochondrial function, chromosome segregation, telomere length). The purpose of this review is to describe the crucial aspects of genome instability, to outline the ways in which environmental chemicals can affect this cancer hallmark and to identify candidate chemicals for further study. The overall aim is to make scientists aware of the increasing need to unravel the underlying mechanisms via which chemicals at low doses can induce genome instability and thus promote carcinogenesis

Global patient outcomes after elective surgery: prospective cohort study in 27 low-, middle- and high-income countries.

BACKGROUND: As global initiatives increase patient access to surgical treatments, there remains a need to understand the adverse effects of surgery and define appropriate levels of perioperative care. METHODS: We designed a prospective international 7-day cohort study of outcomes following elective adult inpatient surgery in 27 countries. The primary outcome was in-hospital complications. Secondary outcomes were death following a complication (failure to rescue) and death in hospital. Process measures were admission to critical care immediately after surgery or to treat a complication and duration of hospital stay. A single definition of critical care was used for all countries. RESULTS: A total of 474 hospitals in 19 high-, 7 middle- and 1 low-income country were included in the primary analysis. Data included 44 814 patients with a median hospital stay of 4 (range 2-7) days. A total of 7508 patients (16.8%) developed one or more postoperative complication and 207 died (0.5%). The overall mortality among patients who developed complications was 2.8%. Mortality following complications ranged from 2.4% for pulmonary embolism to 43.9% for cardiac arrest. A total of 4360 (9.7%) patients were admitted to a critical care unit as routine immediately after surgery, of whom 2198 (50.4%) developed a complication, with 105 (2.4%) deaths. A total of 1233 patients (16.4%) were admitted to a critical care unit to treat complications, with 119 (9.7%) deaths. Despite lower baseline risk, outcomes were similar in low- and middle-income compared with high-income countries. CONCLUSIONS: Poor patient outcomes are common after inpatient surgery. Global initiatives to increase access to surgical treatments should also address the need for safe perioperative care. STUDY REGISTRATION: ISRCTN5181700