Cenh3: An Emerging Player in Haploid Induction Technology.

True-breeding lines are required for the development and production of crop varieties. In a classical breeding approach these lines are obtained through inbreeding, and often 7-9 generations of inbreeding is performed to achieve the desired level of homozygosity, over a period of several years. In contrast, the chromosomes of haploids can be doubled to produce true-breeding lines in a single generation. Over the last century, scientists have developed a variety of techniques to induce haploids and doubled haploids, though these techniques apply only to particular crop varieties. Ravi and Chan (2010) discovered that haploids could be obtained in Arabidopsis through the manipulation of the centromere-specific histone 3 variant, CENH3. Their approach, which involved extensive modifications to a transgenic CENH3, held promise of being translated to crop species, and has been successfully employed in maize (see Kelliher et al., 2016). Refinements of this technology have since been developed which indicate that non-transgenic modifications to CENH3 will also induce haploids. The complementation of a cenh3 null by CENH3 from closely related plant species can result in plants that are fertile but haploid-inducing on crossing by CENH3 wt plants- suggesting that introgression of alien CENH3 may produce non-transgenic haploid inducers. Similarly, a remarkably wide variety of point mutations in CENH3, inducible by chemical agents, have recently been shown to result in haploid induction on crossing by wild-type CENH3 plants. These CENH3-variant plants grow normally, are fully fertile on self-pollination, and may be present in existing mutagenized collections

High atomic weight, high-energy radiation (HZE) induces transcriptional responses shared with conventional stresses in addition to a core "DSB" response specific to clastogenic treatments.

Plants exhibit a robust transcriptional response to gamma radiation which includes the induction of transcripts required for homologous recombination and the suppression of transcripts that promote cell cycle progression. Various DNA damaging agents induce different spectra of DNA damage as well as "collateral" damage to other cellular components and therefore are not expected to provoke identical responses by the cell. Here we study the effects of two different types of ionizing radiation (IR) treatment, HZE (1 GeV Fe(26+) high mass, high charge, and high energy relativistic particles) and gamma photons, on the transcriptome of Arabidopsis thaliana seedlings. Both types of IR induce small clusters of radicals that can result in the formation of double strand breaks (DSBs), but HZE also produces linear arrays of extremely clustered damage. We performed these experiments across a range of time points (1.5-24 h after irradiation) in both wild-type plants and in mutants defective in the DSB-sensing protein kinase ATM. The two types of IR exhibit a shared double strand break-repair-related damage response, although they differ slightly in the timing, degree, and ATM-dependence of the response. The ATM-dependent, DNA metabolism-related transcripts of the "DSB response" were also induced by other DNA damaging agents, but were not induced by conventional stresses. Both Gamma and HZE irradiation induced, at 24 h post-irradiation, ATM-dependent transcripts associated with a variety of conventional stresses; these were overrepresented for pathogen response, rather than DNA metabolism. In contrast, only HZE-irradiated plants, at 1.5 h after irradiation, exhibited an additional and very extensive transcriptional response, shared with plants experiencing "extended night." This response was not apparent in gamma-irradiated plants

Genomic stability in response to high versus low linear energy transfer radiation in Arabidopsis thaliana.

Low linear energy transfer (LET) gamma rays and high LET HZE (high atomic weight, high energy) particles act as powerful mutagens in both plants and animals. DNA damage generated by HZE particles is more densely clustered than that generated by gamma rays. To understand the genetic requirements for resistance to high versus low LET radiation, a series of Arabidopsis thaliana mutants were exposed to either 1GeV Fe nuclei or gamma radiation. A comparison of effects on the germination and subsequent growth of seedlings led us to conclude that the relative biological effectiveness (RBE) of the two types of radiation (HZE versus gamma) are roughly 3:1. Similarly, in wild-type lines, loss of somatic heterozygosity was induced at an RBE of about a 2:1 (HZE versus gamma). Checkpoint and repair defects, as expected, enhanced sensitivity to both agents. The "replication fork" checkpoint, governed by ATR, played a slightly more important role in resistance to HZE-induced mutagenesis than in resistance to gamma induced mutagenesis

High-Resolution Analysis of the Efficiency, Heritability, and Editing Outcomes of CRISPR/Cas9-Induced Modifications of NCED4 in Lettuce (Lactuca sativa).

CRISPR/Cas9 is a transformative tool for making targeted genetic alterations. In plants, high mutation efficiencies have been reported in primary transformants. However, many of the mutations analyzed were somatic and therefore not heritable. To provide more insights into the efficiency of creating stable homozygous mutants using CRISPR/Cas9, we targeted LsNCED4 (9-cis-EPOXYCAROTENOID DIOXYGENASE4), a gene conditioning thermoinhibition of seed germination in lettuce. Three constructs, each capable of expressing Cas9 and a single gRNA targeting different sites in LsNCED4, were stably transformed into lettuce (Lactuca sativa) cvs. Salinas and Cobham Green. Analysis of 47 primary transformants (T1) and 368 T2 plants by deep amplicon sequencing revealed that 57% of T1 plants contained events at the target site: 28% of plants had germline mutations in one allele indicative of an early editing event (mono-allelic), 8% of plants had germline mutations in both alleles indicative of two early editing events (bi-allelic), and the remaining 21% of plants had multiple low frequency mutations indicative of late events (chimeric plants). Editing efficiency was similar in both genotypes, while the different gRNAs varied in efficiency. Amplicon sequencing of 20 T1 and more than 100 T2 plants for each of the three gRNAs showed that repair outcomes were not random, but reproducible and characteristic for each gRNA. Knockouts of NCED4 resulted in large increases in the maximum temperature for seed germination, with seeds of both cultivars capable of germinating >70% at 37°. Knockouts of NCED4 provide a whole-plant selectable phenotype that has minimal pleiotropic consequences. Targeting NCED4 in a co-editing strategy could therefore be used to enrich for germline-edited events simply by germinating seeds at high temperature

Point Mutations in Centromeric Histone Induce Post-zygotic Incompatibility and Uniparental Inheritance.

The centromeric histone 3 variant (CENH3, aka CENP-A) is essential for the segregation of sister chromatids during mitosis and meiosis. To better define CENH3 functional constraints, we complemented a null allele in Arabidopsis with a variety of mutant alleles, each inducing a single amino acid change in conserved residues of the histone fold domain. Many of these transgenic missense lines displayed wild-type growth and fertility on self-pollination, but exhibited frequent post-zygotic death and uniparental inheritance when crossed with wild-type plants. The failure of centromeres marked by these missense mutation in the histone fold domain of CENH3 reproduces the genome elimination syndromes described with chimeric CENH3 and CENH3 from diverged species. Additionally, evidence that a single point mutation is sufficient to generate a haploid inducer provide a simple one-step method for the identification of non-transgenic haploid inducers in existing mutagenized collections of crop species. As proof of the extreme simplicity of this approach to create haploid-inducing lines, we performed an in silico search for previously identified point mutations in CENH3 and identified an Arabidopsis line carrying the A86V substitution within the histone fold domain. This A87V non-transgenic line, while fully fertile on self-pollination, produced postzygotic death and uniparental haploids when crossed to wild type

Telomere dynamics and fusion of critically shortened telomeres in plants lacking DNA ligase IV

In the absence of the telomerase, telomeres undergo progressive shortening and are ultimately recruited into end-to-end chromosome fusions via the non-homologous end joining (NHEJ) double-strand break repair pathway. Previously, we showed that fusion of critically shortened telomeres in Arabidopsis proceeds with approximately the same efficiency in the presence or absence of KU70, a key component of NHEJ. Here we report that DNA ligase IV (LIG4) is also not essential for telomere joining. We observed only a modest decrease (3-fold) in the frequency of chromosome fusions in triple tert ku70 lig4 mutants versus tert ku70 or tert. Sequence analysis revealed that, relative to tert ku70, chromosome fusion junctions in tert ku70 lig4 mutants contained less microhomology and less telomeric DNA. These findings argue that the KU-LIG4 independent end-joining pathway is less efficient and mechanistically distinct from KU-independent NHEJ. Strikingly, in all the genetic backgrounds we tested, chromosome fusions are initiated when the shortest telomere in the population reaches ∼1 kb, implying that this size represents a critical threshold that heralds a detrimental structural transition. These data reveal the transitory nature of telomere stability, and the robust and flexible nature of DNA repair mechanisms elicited by telomere dysfunction

Caregiver strain among relatives of out-of-hospital cardiac arrest survivors; the DANCAS relative survey

Background: Knowledge about caregiver strain among relatives of out-of-hospital cardiac arrest (OHCA) survivors is limited. Thus, the objectives were to i) describe differences in self-reported mental well-being, mental health, and caregiver strain at different time points (1–5 years) post-OHCA and ii) investigate characteristics associated with caregiver strain. Methods: A national cross-sectional survey (DANCAS) from October 2020 to March 2021 with OHCA survivors and their closest relatives. The relative survey included the WHO-5 Well-being Index (WHO-5), the Hospital Anxiety and Depression Scale (HADS) and the Modified Caregiver Strain Index (M−CSI). Differences in scores between time groups were explored using descriptive statistics. Associations between characteristics and caregiver strain were investigated with multivariable logistic regression models, presented as odds ratios (OR) with 95% confidence intervals (CI), adjusted for gender, age, education status, relative affiliation, and time after OHCA. Results: Of 561 relatives, 24% (n = 137) experienced caregiver strain, with no significant differences in the relatives' mental well-being, mental health, or caregiver strain with time since OHCA. In the adjusted analyses, older age (OR 0.98 95% CI 0.96;0.99) and several self-reported outcomes, including reduced mental well-being (WHO-5 OR 7.27 95% CI 4.86;11.52), symptoms of anxiety (HADS-A OR 6.01 95% CI 3.89;9.29) and depression (HADS-D OR 15.03 95% CI 7.33;30.80) were significantly associated with worse caregiver strain. Conclusion: Nearly one-quarter of relatives of OHCA survivors experience caregiver strain, with this proportion remaining unchanged with time. Several outcomes were associated with caregiver strain, emphasising the need to identify relatives at greater risk of burden following OHCA.</p

Agreement on fixation of pediatric supracondylar humerus fractures

Background Pediatric supracondylar humerus fractures (pSCHFs) may be challenging injuries to treat because of the potential residual deformity. There is debate regarding the technical aspects of adequate closed reduction and crossed Kirschner wire (K-wire) fixation. Purpose Do surgeons have an agreement on the aspects of the fixation of pSCHFs? Methods Radiographs of 20 patients from a cohort of 154 patients with pSCHFs treated with closed reduction and crossed K-wire fixation were selected. Forty-four surgeons viewed the postoperative radiographs and diagnosed the presence or absence of technical flaws and made a recommendation for or against reoperation. An expert panel of three orthopedic and trauma surgeons provided a reference standard for technical factors. Furthermore, final outcome 2 years after trauma was assessed. Results There was limited agreement on potential technical flaws (ICC 0.15-0.28), radiographic measures of alignment (ICC for anterior humeral line and Baumann angle of 0.37 and 0.23 respectively), the quality of postoperative reduction, position of the elbow in cast, and recommendation for repeat surgery (ICCs between 0.23 and 0.40). Sensitivity and specificity for these questions ranged from 0.59 to 0.90. There was no correlation between the voted quality of postoperative reduction and loss of reduction or final function. Conclusions Surgeons have limited agreement on the quality of postoperative results in pSCHFs and the indication for reoperation. Reviewing postoperative radiographs may present a good learning opportunity and could help improve skills, but it is not a validated method for quality control and has to be seen in light of clinical outcome

The effect of paternal factors on perinatal and paediatric outcomes : a systematic review and meta-analysis

BACKGROUND: Maternal factors, including increasing childbearing age and various life-style factors, are associated with poorer short- and long-term outcomes for children, whereas knowledge of paternal parameters is limited. Recently, increasing paternal age has been associated with adverse obstetric outcomes, birth defects, autism spectrum disorders and schizophrenia in children. OBJECTIVE AND RATIONALE: The aim of this systematic review is to describe the influence of paternal factors on adverse short- and long-term child outcomes. SEARCH METHODS: PubMed, Embase and Cochrane databases up to January 2017 were searched. Paternal factors examined included paternal age and life-style factors such as body mass index (BMI), adiposity and cigarette smoking. The outcome variables assessed were short-term outcomes such as preterm birth, low birth weight, small for gestational age (SGA), stillbirth, birth defects and chromosomal anomalies. Long-term outcome variables included mortality, cancers, psychiatric diseases/disorders and metabolic diseases. The systematic review follows PRISMA guidelines. Relevant meta-analyses were performed. OUTCOMES: The search included 14 371 articles out of which 238 met the inclusion criteria, and 81 were included in quantitative synthesis (meta-analyses). Paternal age and paternal life-style factors have an association with adverse outcome in offspring. This is particularly evident for psychiatric disorders such as autism, autism spectrum disorders and schizophrenia, but an association is also found with stillbirth, any birth defects, orofacial clefts and trisomy 21. Paternal height, but not BMI, is associated with birth weight in offspring while paternal BMI is associated with BMI, weight and/or body fat in childhood. Paternal smoking is found to be associated with an increase in SGA, birth defects such as congenital heart defects, and orofacial clefts, cancers, brain tumours and acute lymphoblastic leukaemia. These associations are significant although moderate in size, with most pooled estimates between 1.05 and 1.5, and none exceeding 2.0. WIDER IMPLICATIONS: Although the increased risks of adverse outcome in offspring associated with paternal factors and identified in this report represent serious health effects, the magnitude of these effects seems modest.Peer reviewe

The immune microenvironment and relation to outcome in patients with advanced breast cancer treated with docetaxel with or without gemcitabine

Preclinical studies suggest that some effects of conventional chemotherapy, and in particular, gemcitabine, are mediated through enhanced antitumor immune responses. The objective of this study was to use material from a randomized clinical trial to evaluate whether patients with preexisting immune infiltrates responded better to treatment with gemcitabine + docetaxel (GD) compared to docetaxel alone. Formalin fixed, paraffin-embedded breast cancer tissues from SBG0102 phase 3 trial patients randomly assigned to treatment with GD or docetaxel were used. Immunohistochemical staining for CD8, FOXP3, LAG3, PD-1, PD-L1 and CD163 was performed. Tumor infiltrating lymphocytes (TILs) and tumor associated macrophages were evaluated. Prespecified statistical analyses were performed in a formal prospective-retrospective design. Time to progression was primary endpoint and overall survival secondary endpoint. Correlations between biomarker status and endpoints were evaluated using the Kaplan-Meier method and Cox proportional hazards models. Biomarker data was obtained for 237 patients. There was no difference in treatment effect according to biomarker status for the whole cohort. In planned subgroup analysis by PAM50 subtype, in non-luminal (basal-like and HER2E) breast cancers FOXP3 was a significant predictor of treatment effect with GD compared to docetaxel, with a HR of 0.22 (0.09-0.52) for tumors with low FOXP3 compared to HR 0.92 (0.47-1.80) for high FOXP3 TILs (Pinteraction = 0.01). Immune biomarkers were not predictive of added benefit of gemcitabine in a cohort of mixed breast cancer subtypes. However, in non-luminal breast cancers, patients with low FOXP3+ TILs may have significant benefit from added gemcitabine