Hypoxia-induced Response in Mitochondria DNA Biogenesis in Retinal Organoids Derived from Pluripotent Stem Cells from Leber Hereditary Optic Neuropathy

This study evaluated the impact of hypoxia on mitochondrial DNA (mtDNA) biogenesis in retinal organoids of patients with Leber hereditary optic neuropathy (LHON) with an mtDNA G11778A mutation. The induced pluripotent stem cells derived from the peripheral blood mononuclear cells of LHON patients and a healthy individual were differentiated into three-dimensional retinal organoids with confirmed morphology and retinal ganglion cell (RGC) markers. The retinal organoids were cultured in alternating hypoxic and normoxic states. The ratio of the mtDNA copy number in an organoid to the mtDNA copy number in the white blood cells was calculated. Under both conditions, the control and LHON organoids had similar morphology and RGC marker patterns. Under the normoxic condition, the LHON organoids had a smaller but similar mtDNA copy number ratio as the controls. Hypoxic exposure significantly reduced the mtDNA copy number ratio in both groups. However, the magnitude of reduction was significantly smaller in LHON organoids than in the controls. One LHON patient had a similar and relative mtDNA copy number in the white blood cells to the controls, but the other patient had a much higher number. Therefore, the hypoxic response of retinal mtDNA biogenesis was reduced in LHON patients.

Polyploidy-associated paramutation in Arabidopsis is determined by small RNAs, temperature, and allele structure

ABSTRACTParamutation is a form of non-Mendelian inheritance in which the expression of a paramutable allele changes when it encounters a paramutagenic allele. This change in expression of the paramutable alleles is stably inherited even after segregation of both alleles. While the discovery of paramutation and studies of its underlying mechanism were made with alleles that change plant pigmentation, paramutation-like phenomena are known to modulate the expression of other traits and in other eukaryotes, and many cases have probably gone undetected. It is likely that epigenetic mechanisms are responsible for the phenomenon, as paramutation forms epialleles, genes with identical sequences but different expression states. This could account for the intergenerational inheritance of the paramutated allele, providing profound evidence that triggered epigenetic changes can be maintained over generations. Here, we use a case of paramutation that affects a transgenic selection reporter gene in tetraploid Arabidopsis thaliana. Our data suggest that different types of small RNA are associated with paramutable and paramutagenic epialleles. In addition, deletion of a repeat within the epiallele changes its paramutability. Further, the temperature during the growth of the epiallelic hybrids determines the degree and timing of the allelic interaction. The data further make it plausible why paramutation in this system becomes evident only in the segregating F2 population of tetraploid plants containing both epialleles. In summary, the results support a model for polyploidy-associated paramutation, with similarities as well as distinctions from other cases of paramutation.AUTHOR SUMMARYA curiosity in the history of genetics is the fact that experiments with peas, Mendel’s most studied plants, resulted in one of the first observations of non-Mendelian genetics published already in 1915. Crossings with plants showing a “rogue” leaf phenotype made normal leaf shape disappear in all progeny, without recovery. This phenomenon was later demonstrated for more traits in other plants and termed paramutation. Paramutation is due to the epigenetic switch of aa active gene to a silenced version which is then maintained in the inactive state in later generations. This demonstrates that acquired epigenetic changes can become permanent. Despite its early observation and numerous studies in mainly maize and tomato, it is barely understood how paramutation is established and which parameters influence the process. We investigated a case of paramutation in Arabidopsis thaliana, crossing plants with genetically identical but epigenetically different alleles that result in resistance or sensitivity to hygromycin in the growth medium. Unexpectedly, paramutation did not become manifest in the hybrids but only in their progeny and only in plants with a doubled chromosome set. These features make this paramutation distinct from other cases and our studies revealed the involvement of several parameters: an important role for sRNAs to initiate silencing, the sequence of the allele itself, the environmental conditions during growth of the hybrids, the developmental stage, and the copy number ratio between the epialleles.

Incidence of Mite-Vectored Viruses of Wheat in the Texas High Plains and Interactions With Their Host and Vector

Mite-vectored virus diseases of wheat are common throughout the Great Plains and cause significant economic losses to growers each year. These diseases are caused by Wheat streak mosaic virus (WSMV), Triticum mosaic virus (TriMV), and Wheat mosaic virus (WMoV), all of which are transmitted by the wheat curl mite (WCM), Aceria tosichella Keifer. New wheat cultivars with tolerance or resistance to WSMV have been released recently, but their widespread cultivation and potential impact on mite-transmitted virus incidence in the Texas Panhandle was unknown. A total of 648 symptomatic wheat samples were collected from 26 counties, predominately in the Texas Panhandle, and tested by enzyme-linked immunosorbent assay (ELISA) for WSMV, TriMV, and WMoV. Samples that tested negative by ELISA were subsequently tested by real-time quantitative PCR (qPCR) for each virus. Approximately 93% of the samples tested by ELISA were positive for WSMV, 43% were positive for TriMV, and 7% were positive for WMoV. Eleven samples tested positive only for TriMV, but none were positive only for WMoV. When samples that tested negative for the different viruses by ELISA were retested by real-time qPCR, detection of each virus was significantly increased. When results of the ELISA test and qPCR were combined, 100% of the 648 samples tested positive for WSMV, approximately 94% were positive for TriMV, and 23% were positive for WMoV. This demonstrated that the incidence of TriMV in the Texas High Plains is much greater than previously reported. The fact that real-time qPCR revealed over a 2-fold increase in the incidence of TriMV and a 3-fold increase in WMoV demonstrated that the ELISA test, which is commonly used by diagnostic laboratories in the Great Plains, should not be used for studies requiring a high degree of sensitivity and accuracy in virus detection. After initial virus infection status was determined, samples that tested positive for WSMV and TriMV were further observed for WCM infestation. A total of 292 samples were inspected and a total of 101 mites were collected from 40 tillers. Individual mites and the tillers from which they were recovered were tested by real-time qPCR to determine how copy numbers of WSMV and TriMV in mites and host tissue compared, and whether the WSMV/TriMV copy number ratio in individual mites was similar to that of the host tissue from which they were collected. In all mites and tillers tested, the WSMV copy number was always higher than that of TriMV and copy numbers of both viruses were always higher in plant tissue than in mites. Although there was a significant correlation between the WSMV/TriMV copy number ratio in plant tissue and in associated mites, the correlation coefficient was very low (r = 0.31, P = 0.0248). In the majority of comparisons, the WSMV/TriMV ratio was higher in individual mites than in the tiller from which they were recovered. The reason for this increase is unknown but indicates that mites may preferentially acquire WSMV from tillers coinfected with WSMV and TriMV, a finding that could have significant implications for virus transmission and disease epidemiology.

CKS1B/CDKN2C Copy Number Ratio : A New Predictor of Survival in Transplant- and Non Transplant-Eligible Multiple Myeloma Patients

BACKGROUND Current molecular risk stratification of multiple myeloma (MM), based on the presence of t(4 ;14) and 17p deletion, cannot fully explain treatment outcome heterogeneity, as other features also predict prognosis. About 30% of genetic events map to chromosome 1 : most upregulated genes to 1q and most downregulated ones to 1p. CKS1B gains on 1q21 and CDKN2C loss on 1p32, both favoring cell cycle progression, portended impaired outcome in many but not all studies. Based on their recurrence and considering their functional convergence, we hypothesized CKS1B/CDKN2C copy number ratio to be a risk factor fitter than each aberration alone. METHODS This single-center retrospective study, enrolled 104 newly diagnosed adult patients aged ≥18 years, 48 transplant-eligible and 56 not. All patients were routinely tested for CKS1B and CDKN2C and treated according to consensus guidelines. Data were collected from 2012 to May 31, 2018. For each subject, we calculated a FISH-based ratio by CKS1B on CDKN2C copy number : it was equal to 1 with no change in copy number and >1 in case of CKS1B gains, CDKN2C loss or both. In patients with CDKN2C biallelic loss, the ratio was not equal to 0, but to CKS1B copy number, as functional consequence should prevail over arithmetic result. We, then, analyzed separately the impact of CKS1B gains, CDKN2C loss and CKS1B/CDKN2C ratio on PFS and OS. RESULTS In the transplant subgroup, the median follow-up was 22.9 (1.4-71.9) months. By FISH analysis, 15 patients had variable gains of CKS1B, 6 monoallelic and 1 biallelic CDKN2C loss ; CKS1B/CNKN2C copy number ratio was ≥1.5 in 17, ≥2 in 9 and ≥3 in 2. ROC curves evaluating the relevance of the ratio were statistically significant for PFS (p=0.008) and OS (p=0.049). Median PFS from diagnosis, overall and for patients with CKS1B gains, CDKN2C loss and ratio ≥1.5 was 21.6 (1.4-68.4), 24.8 (vs 41.5 with no CKS1B gains), 28.7 (vs 37.7 with no 1p deletion) and 25 (vs 42 with normal ratio) months, respectively. By Kaplan-Meier log-rank analyses, PFS was significantly impaired by CKS1B gains (p=0.014) and ratio ≥1.5 (p=0.007), but not by CDKN2C loss (p=0.339). Median OS from diagnosis, overall and for patients with CKS1B gains, CDKN2C loss and ratio ≥1.5 was 22.9 (1.4-71.9), 24.2 (vs 64.2 with no CKS1B gains), 44.9 (vs 54.8 with no 1p deletion) and 32 (vs 64 with normal ratio) months, respectively. By Kaplan-Meier log-rank analyses, OS was also significantly impaired by CKS1B gains (p=0.001) and ratio ≥1.5 (p=0.004), but not CDKN2C loss (p=0.339). In multivariate analyses, parameters negatively influencing PFS were age >65 years (p=0.005), CKS1B gains (p=0.001), ratio ≥1.5 (p=0.006) and ISS3 status (p=0.019) ; factors unfavorably affecting OS were age >65 years (p=0.044), ratio ≥1.5 (p=0.049) and non secretory status (p=0.018), but not CKS1B gains (p=0.174). In the non transplant subgroup, the median follow-up was 26.4 (0.2-87.3) months. By FISH analysis 30 patients had variable gains of CKS1B and 10 hemizygous loss of CDKN2C ; CKS1B/CDKN2C ratio was ≥1.5 in 34, ≥2 in 21 and ≥3 in 7. ROC curves evaluating the relevance of the ratio showed only a trend toward statistical significance for PFS (p=0.065), but not for OS (p=0.137). Median PFS from diagnosis, overall and for patients with CKS1B gains, CDKN2C loss, ratio ≥2 and ≥3 was 17.5 (0.2-79.9), 20.9 (vs 26.7 with no CKS1B gains), 15.9 (vs 24.9 with no CDKN2C loss), 16.6 (vs 26.9 for ratio <2) and 11.1 (vs 25.6 for ratio <3) months, respectively. In Kaplan-Meier log-rank analyses, PFS was significantly impaired by ratio ≥3 (p=0.000), but not by CKS1B gains (p=0.204) or CDKN2C loss (p=0.273). Median OS from diagnosis, overall and for patients with CKS1B gains, CDKN2C loss, ratio ≥2 and ≥3 was 26.4 (0.2-87.3), 34 (vs 40 with no CKS1B gains), 24 (vs 43.6 with no 1p loss), 31.2 (vs 44.6 with ratio <2) and 13 (vs 45.2 with ratio <3) months, respectively. By Kaplan-Meier log-rank analyses, OS was also negatively affected by ratio ≥3 (p<0.0001), but not by CKS1B gains (p=0.476) or CDKN2C loss (p=0.207). In multivariate analyses, factors negatively impacting PFS were platelet count <150 G/L (p=0.020) and ratio ≥3 (p=0.031) ; parameters unfavorably affecting OS were bone marrow plasma cells ≥60% (p=0.018) and ratio ≥3 (p=0.026). CONCLUSIONS Even with few patients, these results provide proof of the concept of CKS1B/CDKN2C ratio as an outcome predictor. Of note, the ratio indicated worse post-transplant PFS and OS in patients over 65 years. Disclosures No relevant conflicts of interest to declare.

Copy number profiling of oncogenes in ductal carcinoma in situ of the male breast

Characterizing male breast cancer (BC) and unraveling male breast carcinogenesis is challenging because of the rarity of this disease. We investigated copy number status of 22 BC-related genes in 18 cases of pure ductal carcinoma in situ (DCIS) and in 49 cases of invasive carcinoma (IC) with adjacent DCIS (DCIS-AIC) in males using multiplex ligation-dependent probe amplification (MLPA). Results were compared to female BC and correlated with survival. Overall, copy number ratio and aberration frequency including all 22 genes showed no significant difference between the 3 groups. Individual unpaired analysis revealed a significantly higher MTDH copy number ratio in IC compared to DCIS-AIC and pure DCIS (P = 0.009 and P = 0.038, respectively). ADAM9 showed a significantly lower copy number aberration frequency in male BC, compared to female BC (P = 0.020). In DCIS-AIC, MTDH, CPD, CDC6 and TOP2A showed a lower frequency of copy number increase in males compared to females (P < 0.001 for all 4 genes). In IC, CPD gain and CCNE1 gain were independent predictors of poor overall survival. In conclusion, male DCIS and IC showed a similar copy number profile for 21 out of 22 interrogated BC-related genes, illustrating their clonal relation and the genetically advanced state of male DCIS. MTDH showed a higher copy number ratio in IC compared to adjacent and pure DCIS and may therefore play a role in male breast carcinogenesis. Differences were detected between male and female DCIS for 4 genes pointing to differences in breast carcinogenesis between the sexes.

Genetic Modulation of the Overexpression of Tailoring Genes eryK and eryG Leading to the Improvement of Erythromycin A Purity and Production in Saccharopolyspora erythraea Fermentation

ABSTRACT Erythromycin A (Er-A) is the most potent and clinically important member in the Er family produced by Saccharopolyspora erythraea. Er-B and Er-C, which are biologically much less active and cause greater side effects than Er-A, serve as the intermediates for Er-A biosynthesis and impurities in fermentation processes of many industrial strains. In this study, systematical modulation of the amounts of tailoring enzymes EryK (a P450 hydroxylase) and EryG (an S-adenosylmethionine-dependent O-methyltransferase) was carried out by genetic engineering in S. erythraea, including alterations of gene copy number ratio and organization and integrating the locus on the chromosome by homologous recombination. Introduction of additional eryK and eryG genes into S. erythraea showed significant impacts on their transcription levels and enhanced the biotransformation process from Er-D to Er-A with gene dose effects. At the eryK/eryG copy number ratio of 3:2 as well as their resultant transcript ratio of around 2.5:1 to 3.0:1, Er-B and Er-C were nearly completely eliminated and accordingly converted to Er-A, and the Er titer was improved by around 25% in the recombinant strain ZL1004 (genotype PermK*-K-K-G + PermE*-K + PermA*-G) and ZL1007 (genotype PermK*-K-G-K + PermE*-K + PermA*-G). This study may contribute to the continuous efforts toward further evaluation of the Er-producing system, with the aims of improving Er-A purity and production at the fermentation stage and lowering the production costs and environmental concerns in industry.