A cytological comparative analysis of male meiocytes was performed for Arabidopsis wild type and the ahp2 (hop2) mutant with emphasis on ahp2's largely uncharacterized prophase I. Leptotene progression appeared normal in ahp2 meiocytes; chromosomes exhibited regular axis formation and assumed a typical polarized nuclear organization. In contrast, 4',6'-diamidino-2-phenylindole-stained ahp2 pachytene chromosome spreads demonstrated a severe reduction in stabilized pairing. However, transmission electron microscopy (TEM) analysis of sections from meiocytes revealed that ahp2 chromosome axes underwent significant amounts of close alignment (44% of total axis). This apparent paradox strongly suggests that the Ahp2 protein is involved in the stabilization of homologous chromosome close alignment. Fluorescent in situ hybridization in combination with Zyp1 immunostaining revealed that ahp2 mutants undergo homologous synapsis of the nucleolus-organizer-region-bearing short arms of chromosomes 2 and 4, despite the otherwise "nucleus-wide" lack of stabilized pairing. The duration of ahp2 zygotene was significantly prolonged and is most likely due to difficulties in chromosome alignment stabilization and subsequent synaptonemal complex formation. Ahp2 and Mnd1 proteins have previously been shown, "in vitro," to form a heterodimer. Here we show, "in situ," that the Ahp2 and Mnd1 proteins are synchronous in their appearance and disappearance from meiotic chromosomes. Both the Ahp2 and Mnd1 proteins localize along the chromosomal axis. However, localization of the Ahp2 protein was entirely foci-based whereas Mnd1 protein exhibited an immunostaining pattern with some foci along the axis and a diffuse staining for the rest of the chromosome.

Background:Low buffy coat mitochondrial DNA copy number (mtDNA-CN) is associated with incident risk of stroke and post-stroke mortality; however, its prognostic utility has not been extensively explored.Objective:To investigate whether low buffy coat mtDNA-CN is a marker and causal determinant of post-stroke outcomes using epidemiological and genetic studies.Methods:First, we performed association testing between baseline buffy coat mtDNA-CN measurements and 1-month post-stroke outcomes in 3498 acute, first stroke cases from 25 countries from the international, multicenter case-control study, “Importance of Conventional and Emerging Risk Factors of Stroke in Different Regions and Ethnic Groups of the World” (INTERSTROKE). Then, we performed two-sample Mendelian Randomization analyses to evaluate potential causative effects of low mtDNA-CN on 3-month modified Rankin Scale (mRS). Genetic variants associated with mtDNA-CN levels were derived from the UKBiobank study (N=383476), and corresponding effects on 3-month mRS were ascertained from the Genetics of Ischemic Stroke funCtional Outcome study (GISCOME; N=6021).Results:A 1-standard deviation (SD) lower mtDNA-CN at baseline was associated with stroke severity (baseline mRS; OR=1.27; 95% CI, 1.19-1.36; P=4.7x10-12). Independent of baseline stroke severity, lower mtDNA-CN was associated with increased odds of greater 1-month disability (ordinal mRS; OR=1.16; 95% CI, 1.08-1.24; P=4.4x10-5), poor functional outcome status (mRS 3-6 vs. 0-2; OR=1.21; 95% CI, 1.08-1.34; P=6.9x10-4), and mortality (OR=1.35; 95% CI, 1.14-1.59; P=3.9x10-4). Subgroup analyses demonstrated consistent effects across stroke type, sex, age, country income level, and education level. In addition, mtDNA-CN significantly improved reclassification of poor functional outcome status (Net Reclassification Index (NRI)=0.16; 95% CI, 0.08-0.23; P=3.6x10-5) and mortality (NRI=0.31; 95% CI, 0.19-0.43; P=1.7x10-7) beyond known prognosticators. Using independent datasets, Mendelian Randomization revealed that a 1 SD decrease in genetically determined mtDNA-CN was associated with increased odds of greater 3-month disability quantified by ordinal mRS (OR=2.35; 95% CI, 1.13-4.90; P=0.02) and poor functional outcome status (OR=2.68; 95% CI, 1.05-6.86; P=0.04).Conclusions:Buffy coat mtDNA-CN is a novel and robust marker of post-stroke prognosis that may also be a causal determinant of post-stroke outcomes.Classification of Evidence:This study provides class II evidence that low buffy coat mtDNA-CN (>1-standard deviation) was associated with worse baseline severity and 1-month outcomes in patients with ischemic or hemorrhagic stroke.