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A high leaf vein density is both an essential feature of C4 photosynthesis and a foundation trait to C4 evolution, ensuring the optimal proportion and proximity of mesophyll and bundle sheath cells for permitting the rapid exchange of photosynthates. Two rice mutant populations, a deletion mutant library with a cv. IR64 background (12,470 lines) and a T-DNA insertion mutant library with a cv. Tainung 67 background (10,830 lines), were screened for increases in vein density. A high throughput method with handheld microscopes was developed and its accuracy was supported by more rigorous microscopy analysis. Eight lines with significantly increased leaf vein densities were identified to be used as genetic stock for the global C4 Rice Consortium. The candidate population was shown to include both shared and independent mutations and so more than one gene controlled the high vein density phenotype. The high vein density trait was found to be linked to a narrow leaf width trait but the linkage was incomplete. The more genetically robust narrow leaf width trait was proposed to be used as a reliable phenotypic marker for finding high vein density variants in rice in future screens.

Objective: To assess the efficacy and clinical outcome of PGT-M undertaken on Day-3, Day-4 and Day-4 "delayed" embryos that were unsuitable for biopsy on Day-3., Design and Setting: Cohort-historical study of all consecutive patients admitted to the IVF-PGT-M program in a large tertiary center., Main Outcome Measure(s): The pregnancy rates and the percentages of complete, incomplete diagnosis, PCR failure, abnormal embryos in PGT of Day-3 cleavage-stage, Day-4 and Day-4 "delayed" embryos., Patients and Methods: We reviewed the medical files of all consecutive patients admitted to our IVF for a fresh IVF-PGT-M cycle. Patients were divided into 3 groups according to the day of blastomere biopsy: Day 3 cleavage-stage, Day-4 morula and Day-4 "delayed" embryos. The laboratory data, genetic diagnostic and clinical results were collected and compared between the different study groups., Results: Nine hundred and six patients underwent PGT-M cycles in our PGT program: 747, 127 and 32 in the Day-3, Day- 4 and Day-4 "delayed" groups, respectively. Ongoing pregnancy rates per transfer and per patient (15.8% and 9.4%, respectively) were non-significantly lower in the Day-4 "delayed", compared to Day-3 (21.4% and 17.5%, respectively) and Day-4 (24.3% and 19.7%, respectively). When comparing ALL morulas (Day-4 and Day-4 "delayed") to ALL cleavage-stage embryos (Day-3, Day-4 and Day-4 "delayed"), a significantly higher ongoing pregnancy rate was demonstrated following the transfer of embryos derived from morula biopsy, as compared to biopsy at the cleavage-stage (33.3% vs 20.5%, p<0.03, respectively)., Conclusion: Day-4 embryo biopsy is feasible and yields comparable and even higher ongoing pregnancy rate if undertaken at the morula stage. Further studies evaluating the cumulative live-birth rate per started cycles in Day-3 vs Day-4 embryo biopsy for PGT-M are warranted., Competing Interests: The authors have declared that no competing interests exist.

Aims: To identify clinically meaningful clusters of patients with similar glycated hemoglobin (HbA1c) trajectories among patients with type 2 diabetes., Methods: A retrospective cohort study using unsupervised machine learning clustering methodologies to determine clusters of patients with similar longitudinal HbA1c trajectories. Stability of these clusters was assessed and supervised random forest analysis verified the clusters' reproducibility. Clinical relevance of the clusters was assessed through multivariable analysis, comparing differences in risk for a composite outcome (macrovascular and microvascular outcomes, hypoglycemic events, and all-cause mortality) at HbA1c thresholds for each cluster., Results: Among 60,423 patients, three clusters of HbA1c trajectories were generated: stable (n = 45,679), descending (n = 6,084), and ascending (n = 8,660) trends, which were reproduced with 99.8% accuracy using a random forest model. In the clinical relevance assessment, HbA1c levels demonstrated a J-shape association with the risk for outcomes. HbA1c level thresholds for minimizing outcomes' risk differed by cluster: 6.0-6.4% for the stable cluster, <8.0% for the descending cluster, and <9.0 for the ascending cluster., Conclusions: By applying unsupervised machine learning to longitudinal HbA1c trajectories, we have identified clusters of patients who have distinct risk for diabetes-related complications. These clusters can be the basis for developing individualized models to personalize glycemic targets., Competing Interests: None of the authors are employed by or consult with Eli Lilly and Company, nor do they have patents, products in development or marketed, or any other commercial interests that would be considered a conflict of interest to the current study and its findings. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

The Three Delays Model has proven a useful framework for examining barriers to seeking obstetric care and preventing maternal and child mortality. This article demonstrates the applicability of the Three Delays Model to the case of civil registration in rural Indonesia and examines ways that efforts to strengthen civil registration services can draw on lessons from maternal and child health programming. Twenty focus group discussions were conducted using a participatory ranking exercise in four Indonesian districts. Focus groups were stratified into four groups: (1) government officials involved in civil registration, (2) civil society organization members that assist communities in civil registration, and (3) female and (4) male community members. Transcripts were analyzed using constant comparative method and thematic analysis, revealing barriers that communities commonly faced in accessing civil registration services. In examining the categories and themes related to these barriers, the research team found a significant overlap with the factors and phases of the Three Delays Model. Participants were delayed from seeking registration services by a range of sociocultural factors and by the perceived inaccessibility and poor quality of services. Once they decided to seek care, long distances to services and poor transportation options delayed their access to registration offices. Finally, a series of bottlenecks in service provision created extended delays once applicants reached registration offices. Ownership of civil registration documents in Indonesia remains exceptionally low, with just over half of children and youth possessing a birth certificate. To strengthen civil registration and health systems more generally, it is important to understand the factors that enable and constrain civil registration, how these factors relate to one another, and how they change over a child's life., Competing Interests: The authors have declared that no competing interests exist.

Aim: To assess the role of mRNA accumulation in granulosa cells as the cause of low ovarian response among FMR1 premutation carriers undergoing pre-implantation genetic diagnosis (PGD)., Design: Case control study in an academic IVF unit. Twenty-one consecutive FMR1 premutation carriers and 15 control women were included. After oocyte retrieval the granulosa cells mRNA levels of FMR1 was measured using RT-PCR., Results: In FMR1 premutation carriers, there was a significant non-linear association between the number of CGG repeats and the number of retrieved oocytes (p<0.0001) and a trend to granulosa cells FMR1 mRNA levels (p = 0.07). The lowest number of retrieved oocytes and the highest level of mRNA were seen in women with mid-size CGG repeats (80-120). A significant negative linear correlation was observed between the granulosa cells FMR1 mRNA levels and the number of retrieved oocytes (R2 linear = 0.231, P = 0.02)., Conclusion: We suggest that there is a no-linear association between the number of CGG repeats and ovarian function, resulting from an increased granulosa cells FMR1 mRNA accumulation in FMR1 carriers in the mid-range (80-120 repeats).

Coding region alterations of ZIC2 are the second most common type of mutation in holoprosencephaly (HPE). Here we use several complementary bioinformatic approaches to identify ultraconserved cis-regulatory sequences potentially driving the expression of human ZIC2. We demonstrate that an 804 bp element in the 3' untranslated region (3'UTR) is highly conserved across the evolutionary history of vertebrates from fish to humans. Furthermore, we show that while genetic variation of this element is unexpectedly common among holoprosencephaly subjects (6/528 or >1%), it is not present in control individuals. Two of six proband-unique variants are de novo, supporting their pathogenic involvement in HPE outcomes. These findings support a general recommendation that the identification and analysis of key ultraconserved elements should be incorporated into the genetic risk assessment of holoprosencephaly cases.