Double Deletion Research Articles

In vivo Firre and Dxz4 deletion elucidates roles for autosomal gene regulation.

Recent evidence has determined that the conserved X chromosome mega-structures controlled by the Firre and Dxz4 loci are not required for X chromosome inactivation (XCI) in cell lines. Here, we examined the in vivo contribution of these loci by generating mice carrying a single or double deletion of Firre and Dxz4. We found that these mutants are viable, fertile and show no defect in random or imprinted XCI. However, the lack of these elements results in many dysregulated genes on autosomes in an organ-specific manner. By comparing the dysregulated genes between the single and double deletion, we identified superloop, megadomain, and Firre locus-dependent gene sets. The largest transcriptional effect was observed in all strains lacking the Firre locus, indicating that this locus is the main driver for these autosomal expression signatures. Collectively, these findings suggest that these X-linked loci are involved in autosomal gene regulation rather than XCI biology.

Relationship of Host Genetic Factors with Severe Malaria in Nigerian Children

Host genetic factors that influence the outcome of Plasmodium falciparum malaria infection are not fully understood. These include alpha-thalassemia, sickle cell disease, glucose-6-phosphate dehydrogenase (G6PD) deficiency and cytochrome b5 reductase 3 (CYB5R3) T117S and ferroportin Q248H polymorphisms. G6PD, an X-linked gene, encodes the sole enzyme in red blood cells that produces NADPH for protection from reactive oxygen species. Hemi- or homozygosity for G6PD A+ (G6PD c. 376G) is an African specific polymorphism with reduced activity (PMID:26633385) but no apparent phenotype; hemi- or homozygosity for G6PD A- (G6PD c 202A/376G) is associated with both decreased activity and increased risk for oxidant-induced hemolysis (PMID:27040960). A Malaria Genomic Epidemiology Network (MalariaGEN) study with almost 30,000 participants reported that G6PD A- increases the risk for severe malarial anemia (PMID:25261933). CYB5R3 in red blood cells transfers electrons from NADH to cytochrome b5, which in turn converts methemoglobin to hemoglobin. The African-specific CYB5R3 T117S variant, is not associated with methemoglobinemia (PMID:9048929) but may afford some protection from malarial anemia in Zambian children. A prevalent African-specific Q248H mutation of the iron exporter, ferroportin, may protect from high malarial parasitemia (PMID: 29599243). We genotyped 212 Nigerian children (age 4 months - 16 years) for these genetic factors: 117 were asymptomatic children who were studied in the community and 95 were children who were studied at the time of admission to hospital with severe malaria (severe anemia and/or cerebral malaria). We isolated DNA from dried blood spots and crude DNA lysate was used for Taqman genotyping to screen for G6PD A+, G6PD A-, FPN Q248H, sickle cell disease and CYB5R3 T117S and multiplex PCR was used to identify the 3.7 kb and 4.2 kb α-globin gene deletions. The clinical characteristics of the two groups of children are shown in Table 1. Children with severe malaria were significantly younger than the children from the community. The gene frequency of CYB5R3 T117S was 0.303, of hemoglobin S was 0.094, and of FPN Q248H was 0.062. The prevalences of hemizygous and homozygous G6PD A+ and A- were 13.9% and 16.0%, respectively. In addition, the prevalences of single and double alpha globin gene deletions were 34.7 % and 11.7%, respectively. Table 2 compares the prevalence of these genotypes in asymptomatic children versus those admitted with severe malaria. Alpha-thalassemia tended to be more prevalent in the asymptomatic children while ferroportin Q248H and G6PD A- were more prevalent in the children with severe malaria. Some genes seemed to have heterogenous associations: sickle trait and homozygous CYB5R3 T117S tended to be more prevalent in the asymptomatic children while hemoglobin SS and heterozygous T117S tended to be more prevalent in the children with severe malaria. In a logistic regression model that adjusted for age, alpha thalassemia (odds ratio 0.6, P=0.038), hemoglobin S trait (odds ratio 0.5, P=0.203), and CYB5R3 T117S homozygous state (odds ratio 0.5, P=0.211) independently decreased the risk of severe malaria, while ferroportin Q248H (odds ratio 5.1, P=0.008) and G6PD deficiency (1.6, P=0.049) independently increased the risk of severe malaria. In conclusion, our results suggest that common genetic factors affect the risk of severe malaria in children. Research to illuminate the mechanisms of these effects may enhance our understanding of the pathogenesis of severe malaria and point to novel preventive and therapeutic measures. Disclosures Gordeuk: Global Blood Therapeutics: Consultancy, Honoraria, Research Funding; Emmaus: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Modus Therapeutics: Consultancy, Honoraria; Pfizer: Research Funding; Inctye: Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Ironwood: Research Funding; Imara: Research Funding.

Capping proteins regulate fungal development, DON-toxisome formation and virulence in Fusarium graminearum.

SummaryDeoxynivalenol (DON) is an important trichothecene mycotoxin produced by the cereal pathogen Fusarium graminearum. DON is synthesized in organized endoplasmic reticulum structures called toxisomes. However, the mechanism for toxisome formation and the components of toxisomes are not yet fully understood. In a previous study, we found that myosin I (FgMyo1)‐actin cytoskeleton participated in toxisome formation. In the current study, we identified two new components of toxisomes, the actin capping proteins (CAPs) FgCapA and FgCapB. These two CAPs form a heterodimer in F. graminearum, and physically interact with FgMyo1 and Tri1. The deletion mutants ΔFgcapA and ΔFgcapB and the double deletion mutant ΔΔFgcapA/B dramatically reduced hyphal growth, asexual and sexual reproduction and endocytosis. More importantly, the deletion mutants markedly disrupted toxisome formation and DON production, and attenuated virulence in planta. Collectively, these results suggest that the actin CAPs are associated with toxisome formation and contribute to the virulence and development of F. graminearum.

Systematic review of the status of pfhrp2 and pfhrp3 gene deletion, approaches and methods used for its estimation and reporting in Plasmodium falciparum populations in Africa: review of published studies 2010\u20132019

BackgroundMalaria rapid diagnostic tests based on histidine-rich protein-2 have played a vital role in improving malaria case management and surveillance particularly in Africa, where Plasmodium falciparum is predominant. However, their usefulness has been threatened by the emergence of gene deletion on P. falciparum histidine rich protein 2 (pfhrp2) and P. falciparum histidine rich protein 3 (pfhrp3). Use of standard and recommended methods is key for accurate investigation, confirmation and reporting of pfhrp2 and pfhrp3 gene deletion.MethodsA systematic review was conducted to assess the status, methods and approaches that have been used for investigation, confirmation and reporting of pfhrp2 and pfhrp3 gene deletion in Africa. An online search was done using PubMed and MEDLINE Google Scholar for all articles published in English on pfhrp2/3 gene deletion in Africa. Relevant articles that met the inclusion criteria were summarized and assessed based on the protocol recommended by the World Health Organization for confirmation and reporting of pfhrp2/3 gene deletion.ResultsThe search identified a total of 18 articles out of which 14 (77.7%) fulfilled the criteria for inclusion and were retained for review. The articles were distributed across 12 countries where the pfhrp2 and pfhrp3 gene deletion studies were conducted and reported. The level of pfhrp2/3 gene deletion across selected studies in Africa ranged from the highest 62% to the lowest 0.4%. There was wide variation in methods and approaches including study designs, size and sampling and whether both pfhrp2 and pfhrp3 double deletions or pfhrp2 single deletion were investigated, with a wide variation in laboratory methods.ConclusionBased on the review, there is evidence of the presence of pfhrp2/3 gene-deleted P. falciparum parasites in Africa. The approaches and methods used for investigation, confirmation and reporting of pfhrp2/3 deleted parasites have varied between studies and across countries. Countries that are considering plans to investigate, confirm and report pfhrp2/3 deletion should use recommended standard and harmonized methods to prevent unnecessary recommendations for costly switch of RDTs in Africa.

Intron 1–Mediated Regulation of EGFR Expression in EGFR-Dependent Malignancies Is Mediated by AP-1 and BET Proteins

The epidermal growth factor receptor (EGFR) is overexpressed in numerous solid tumors and is the subject of extensive therapeutic efforts. Much of the research on EGFR is focused on protein dynamics and downstream signaling; however, few studies have explored its transcriptional regulation. Here, we identified two enhancers (CE1 and CE2) present within the first intron of the EGFR gene in models of glioblastoma (GBM) and head and neck squamous cell carcinoma (HNSCC). CE1 and CE2 contain open chromatin and H3K27Ac histone marks, enhance transcription in reporter assays, and interact with the EGFR promoter. Enhancer genetic deletion by CRISPR/Cas9 significantly reduces EGFR transcript levels, with double deletion exercising an additive effect. Targeted repression of CE1 and CE2 by dCas9-KRAB demonstrates repression of transcription similar to that of genomic deletion. We identify AP-1 transcription factor family members in concert with BET bromodomain proteins as modulators of CE1 and CE2 activity in HNSCC and GBM through de novo motif identification and validate their presence. Genetic inhibition of AP-1 or pharmacologic disruption of BET/AP-1 binding results in downregulated EGFR protein and transcript levels, confirming a role for these factors in CE1 and CE2. Our results identify and characterize these novel enhancers, shedding light on the role that epigenetic mechanisms play in regulating EGFR transcription in EGFR-dependent cancers. IMPLICATIONS: We identify critical constituent enhancers present in the first intron of the EGFR gene, and provide a rationale for therapeutic targeting of EGFR intron 1 enhancers through perturbation of AP-1 and BET in EGFR-positive malignancies.

Development and evaluation of a conventional RT-PCR for differentiating emerging influenza B/Victoria lineage viruses with hemagglutinin amino acid deletion from B/Yamagata lineage viruses.

Recent influenza B/Victoria lineage viruses contain amino acid deletions at positions 162 to 164 of the haemagglutinin (HA) protein. These amino acid deletions have affected the detection of B/Victoria lineage viruses by the lineage-specific conventional reverse-transcription polymerase chain reaction (RT-PCR) that was recommended by World Health Organization (WHO). We aimed to develop and evaluate a novel lineage-specific RT-PCR for rapid differentiation of the contemporary B/Victoria lineage from B/Yamagata lineage viruses. Primers of our in-house RT-PCR were designed to avoid amino acid positions 162 to 164 and to target conserved regions of the HA gene that are specific for B/Victoria and B/Yamagata lineage viruses. Our in-house RT-PCR and WHO RT-PCR were evaluated using influenza B positive clinical specimens or virus culture isolates. Influenza B virus lineage was confirmed by Sanger sequencing. A total of 105 clinical specimens or virus culture isolates were retrieved, including 83 with B/Victoria lineage and 22 with B/Yamagata lineage viruses. Our in-house RT-PCR correctly identified B/Victoria lineage viruses in all 83 samples, including 82 samples with double or triple amino acid deletion in the HA protein. Conversely, the WHO lineage-specific conventional RT-PCR failed to detect any of the 82 samples with HA amino acid deletions. For the 22 samples with B/Yamagata lineage viruses, both RT-PCR assays have correctly identified B/Yamagata lineage in all samples. Our novel lineage-specific RT-PCR has successfully detected all contemporary B/Victoria lineage viruses with amino acid deletions in HA. This protocol is especially useful for laboratories without the equipment for real-time PCR.

Genetic interaction between Ptc2 and protein phosphatase 4 (PP4) in the regulation of DNA damage response and virulence in Candida albicans.

In the pathogenic fungus Candida albicans, phosphoregulation of the checkpoint kinase Rad53 plays a crucial role in the filamentous growth response to genotoxic stresses. The protein phosphatase 4 (PP4) complex, containing Pph3 and either Psy2 or Psy4, is proved to play a critical role in Rad53 dephosphorylation. In previous studies, we characterized CaPtc2 (the ortholog of both Ptc2 and Ptc3 in Saccharomyces cerevisiae) as a potential DNA-damage-related protein phosphatase. In this study, we checked the genetic interaction of PTC2 with the PP4 complex in the DNA damage response pathway. The results suggest that Ptc2 shows a negative genetic interaction with Pph3, but positive genetic interaction with either Psy2 or Psy4 in response to genotoxic stress. Deletion of PTC2 alone resulted in no significant change in cell virulence, but double deletion of PTC2 PPH3 significantly decreased virulence, while double deletions of either PTC2 PSY2 or PTC2 PSY4 caused virulence levels similar to that shown by PSY2 or PSY4 single-gene deletion cells. Taken together, we propose that Ptc2 in C. albicans plays a compensatory role for Pph3 but is dependent on Psy2 and Psy4 in regulation of DNA damage and cell virulence.

602. Mechanism of LiaY-Mediated Daptomycin Resistance in Enterococcus faecalis

BackgroundDaptomycin (DAP) is a lipopeptide antibiotic that targets the cell membrane (CM) at the division septum. DAP resistance (DAP-R) in E. faecalis (Efs) has been linked to mutations in genes encoding the LiaFSR stress response system and lipid biosynthetic enzymes, including cardiolipin synthase (Cls). The signature phenotype of DAP-R is redistribution of CM anionic phospholipid (APL) microdomains. Using a genetic approach, we have identified a transmembrane protein (LiaY) as a major mediator of cell membrane APL redistribution associated with DAP-R. Here, we explore the mechanism of LiaY-mediated changes in the CM under the hypothesis that CM remodeling occurs through interactions with Cls.Methods Efs encodes two cls genes (cls1 and cls2). Deletion mutants of both cls genes were generated using the Crispr/cas9 system in the daptomycin-sensitive strain Efs OG117 and Efs OG117∆liaX (a DAP-R derivative of OG117). DAP minimum inhibitory concentration (MIC) was determined using E-test on Mueller–Hinton II agar. Visualization of APL microdomains was performed by staining mid-logarithmic phase cells with 1 µM of 10-N-nonyl-acridine orange (NAO) and fluorescence microscopy. Bacterial two-hybrid system was used to study interactions between LiaY with Cls1 or Cls2.ResultsSingle or double deletion of cls1 or cls2 in Efs OG117 did not affect DAP MIC, and no changes in CM architecture were seen by NAO staining. In contrast,deletion of cls1 (alone or in conjunction with a deletion of cls2) in a DAP-R derivative of OG117 OG117∆liaX, resulted in a marked decrease in DAP MIC, and NAO staining of Efs OG117∆liaX∆cls1∆cls2 shows a restoration of septal APL microdomain localization.In the same DAP-R background, deletion of cls2 alone did not have any effect on DAP MIC or APL microdomain distribution. Additionally, bacterial two-hybrid assays showed a positive interaction of LiaY with Cls1 but not with Cls2.ConclusionWe have identified the biochemical basis for DAP-R associated CM remodeling. In a proposed model, the LiaR-mediated activation of the LiaY triggers specific interactions with Cls1 displacing the protein away from the septum, resulting in local generation of APL microdomains that prevents DAP-mediated damage to the CM.Disclosures All authors: No reported disclosures.

Physiological investigations of the influences of byproduct pathways on 3-hydroxypropionic acid production in Klebsiella pneumoniae.

Klebsiella pneumoniae can naturally synthesize 3-hydroxypropionic acid (3-HP), 1,3-propanediol (1,3-PD), and 2,3-butanediol (2,3-BD) from glycerol. However, biosynthesis of these industrially important chemicals is constrained by troublesome byproducts. To clarify the influences of byproducts on 3-HP production, in this study, a total of eight byproduct-producing enzyme genes including pmd, poxB, frdB, fumC, dhaT, ilvH, adhP, and pflB were individually deleted from theK. pneumoniae genome. The resultant eight mutants presented different levels of metabolites. In 24-h shake-flask cultivation, the adhP- and pflB-deletion mutants produced 0.41 and 0.44 g/L 3-HP, respectively. Notably, the adhP and pflB double deletion mutant K. pneumoniaeΔadhPΔpflB produced 1.58 g/L 3-HP in 24-h shake-flask cultivation. When K. pneumoniaeΔadhPΔpflB was harnessed as a host strain to overexpress PuuC, a native aldehyde dehydrogenase (ALDH) catalyzing 3-hydroxypropionaldehyde (3-HPA) to 3-HP, the resulting recombinant strain K. pneumoniaeΔadhPΔpflB(pTAC-puuC) (pTAC-puuC is PuuC expression vector) generated 66.91 g/L 3-HP with a cumulative yield of 70.84% on glycerol in 60-h bioreactor cultivation. Additionally, this strain showed 2.3-, 5.1-, and 0.67-fold decrease in the concentrations of 1,3-PD, 2,3-BD, and acetic acid compared with the reference strain K. pneumoniae(pTAC-puuC). These results indicated that the byproducts exerted differential impacts on the production of 3-HP, 1,3-PD, and 2,3-BD. Although combinatorial elimination of byproduct pathways could reprogram glycerol flux, the enzyme 1,3-propanediol oxidoreductase (DhaT) that catalyzes 3-HPA to 1,3-PD and the enzymes ALDHs, especially, PuuC are most pivotal for 3-HP production. This study provides a deep understanding of how byproducts affect the production of 3-HP, 1,3-PD, and 2,3-BD in K. pneumoniae.

The Role of TTP Phosphorylation in the Regulation of Inflammatory Cytokine Production by MK2/3.

Tristetraprolin (TTP) is an RNA-binding protein and an essential factor of posttranscriptional repression of cytokine biosynthesis in macrophages. Its activity is temporally inhibited by LPS-induced p38MAPK/MAPKAPK2/3-mediated phosphorylation, leading to a rapid increase in cytokine expression. We compared TTP expression and cytokine production in mouse bone marrow-derived macrophages of different genotypes: wild type, MAPKAP kinase 2 (MK2) deletion (MK2 knockout [KO]), MK2/3 double deletion (MK2/3 double KO [DKO]), TTP-S52A-S178A (TTPaa) knock-in, as well as combined MK2 KO/TTPaa and MK2/3 DKO/TTPaa. The comparisons reveal that MK2/3 are the only LPS-induced kinases for S52 and S178 of TTP and the role of MK2 and MK3 in the regulation of TNF biosynthesis is not restricted to phosphorylation of TTP at S52/S178 but includes independent processes, which could involve other TTP phosphorylations (such as S316) or other substrates of MK2/3 or p38MAPK Furthermore, we found differences in the dependence of various cytokines on the cooperation between MK2/3 deletion and TTP mutation ex vivo. In the cecal ligation and puncture model of systemic inflammation, a dramatic decrease of cytokine production in MK2/3 DKO, TTPaa, and DKO/TTPaa mice compared with wild-type animals is observed, thus confirming the role of the MK2/3/TTP signaling axis in cytokine production also in vivo. These findings improve our understanding of this signaling axis and could be of future relevance in the treatment of inflammation.

The mitochondrial intermembrane space-facing proteins Mcp2 and Tgl2 are involved in yeast lipid metabolism.

Mitochondria are unique organelles harboring two distinct membranes, the mitochondrial inner and outer membrane (MIM and MOM, respectively). Mitochondria comprise only a subset of metabolic pathways for the synthesis of membrane lipids; therefore most lipid species and their precursors have to be imported from other cellular compartments. One such import process is mediated by the ER mitochondria encounter structure (ERMES) complex. Both mitochondrial membranes surround the hydrophilic intermembrane space (IMS). Therefore, additional systems are required that shuttle lipids between the MIM and MOM. Recently, we identified the IMS protein Mcp2 as a high-copy suppressor for cells that lack a functional ERMES complex. To understand better how mitochondria facilitate transport and biogenesis of lipids, we searched for genetic interactions of this suppressor. We found that MCP2 has a negative genetic interaction with the gene TGL2 encoding a neutral lipid hydrolase. We show that this lipase is located in the intermembrane space of the mitochondrion and is imported via the Mia40 disulfide relay system. Furthermore, we show a positive genetic interaction of double deletion of MCP2 and PSD1, the gene encoding the enzyme that synthesizes the major amount of cellular phosphatidylethanolamine. Finally, we demonstrate that the nucleotide-binding motifs of the predicted atypical kinase Mcp2 are required for its proper function. Taken together, our data suggest that Mcp2 is involved in mitochondrial lipid metabolism and an increase of this involvement by overexpression suppresses loss of ERMES.

A stealth adhesion factor contributes to Vibrio vulnificus pathogenicity: Flp pili play roles in host invasion, survival in the blood stream and resistance to complement activation.

The tad operons encode the machinery required for adhesive Flp (fimbrial low-molecular-weight protein) pili biogenesis. Vibrio vulnificus, an opportunistic pathogen, harbors three distinct tad loci. Among them, only tad1 locus was highly upregulated in in vivo growing bacteria compared to in vitro culture condition. To understand the pathogenic roles of the three tad loci during infection, we constructed single, double and triple tad loci deletion mutants. Interestingly, only the Δtad123 triple mutant cells exhibited significantly decreased lethality in mice. Ultrastructural observations revealed short, thin filamentous projections disappeared on the Δtad123 mutant cells. Since the pilin was paradoxically non-immunogenic, a V5 tag was fused to Flp to visualize the pilin protein by using immunogold EM and immunofluorescence microscopy. The Δtad123 mutant cells showed attenuated host cell adhesion, decreased biofilm formation, delayed RtxA1 exotoxin secretion and subsequently impaired translocation across the intestinal epithelium compared to wild type, which could be partially complemented with each wild type operon. The Δtad123 mutant was susceptible to complement-mediated bacteriolysis, predominantly via the alternative pathway, suggesting stealth hiding role of the Tad pili. Complement depletion by treating with anti-C5 antibody rescued the viable count of Δtad123 in infected mouse bloodstream to the level comparable to wild type strain. Taken together, all three tad loci cooperate to confer successful invasion of V. vulnificus into deeper tissue and evasion from host defense mechanisms, ultimately resulting in septicemia.

Peroxisome Maintenance Depends on De Novo Peroxisome Formation in Yeast Mutants Defective in Peroxisome Fission and Inheritance.

There is an ongoing debate on how peroxisomes form: by growth and fission of pre-existing peroxisomes or de novo from another membrane. It has been proposed that, in wild type yeast cells, peroxisome fission and careful segregation of the organelles over mother cells and buds is essential for organelle maintenance. Using live cell imaging we observed that cells of the yeast Hansenula polymorpha, lacking the peroxisome fission protein Pex11, still show peroxisome fission and inheritance. Also, in cells of mutants without the peroxisome inheritance protein Inp2 peroxisome segregation can still occur. In contrast, peroxisome fission and inheritance were not observed in cells of a pex11 inp2 double deletion strain. In buds of cells of this double mutant, new organelles likely appear de novo. Growth of pex11 inp2 cells on methanol, a growth substrate that requires functional peroxisomes, is retarded relative to the wild type control. Based on these observations we conclude that in H. polymorpha de novo peroxisome formation is a rescue mechanism, which is less efficient than organelle fission and inheritance to maintain functional peroxisomes.

Myosin 1a Regulates Osteoblast Differentiation Independent of Intestinal Calcium Transport.

Myosin 1A (Myo1a) is a mechanoenzyme previously thought to be located exclusively in the intestinal epithelium. It is the principle calmodulin-binding protein of the brush border. Based on earlier studies in chickens, we hypothesized that Myo1a facilitates calcium transport across the brush border membrane of the intestinal epithelium, perhaps in association with the calcium channel Trpv6. Working with C2Bbe1 cells, a human intestinal epithelial cell line, we observed that overexpression of Myo1a increased, whereas the antisense construct blocked calcium transport. To further test this hypothesis, we examined mice in which either or both Myo1a and Trpv6 had been deleted. Although the Trpv6-null mice had decreased intestinal calcium transport, the Myo1a-null mouse did not, disproving our original hypothesis, at least in mice. Expecting that a reduction in intestinal calcium transport would result in decreased bone, we examined the skeletons of these mice. To our surprise, we found no decrease in bone in the Trpv6-null mouse, but a substantial decrease in the Myo1a-null mouse. Double deletions were comparable to the Myo1a null. Moreover, Myo1a but not Trpv6 was expressed in osteoblasts. In vitro, the bone marrow stromal cells from the Myo1a-null mice showed normal numbers of colony-forming units but marked decrements in the formation of alkaline phosphatase–positive colonies and mineralized nodules. We conclude that Myo1a regulates osteoblast differentiation independent of its role, if any, in intestinal calcium transport, whereas Trpv6 functions primarily to promote intestinal calcium transport with little influence in osteoblast function.

Reverse genetic analysis of yeast YPR099C/MRPL51 reveals a critical role of both overlapping ORFs in respiratory growth and MRPL51 in mitochondrial DNA maintenance.

The Saccharomyces cerevisiae genome contains 6572 ORFs, of which 680 ORFs are classified as dubious ORFs. A dubious ORF is a small, noncoding, nonconserved ORF that overlaps with another ORF of the complementary strand. Our study characterizes a dubious/nondubious ORF pair, YPR099C/MRPL51, and shows the transcript and protein level expression of YPR099C. Its subcellular localization was observed in the mitochondria. The overlapping ORF, MRPL51, encodes a mitochondrial ribosomal protein of large subunit. Deletion of any ORF from YPR099C/MRPL51 pair induces common phenotypes, i.e. loss of mtDNA, lack of mitochondrial fusion and lack of respiratory growth, due to the double deletion (ypr099cΔ/Δmrpl51Δ/Δ) caused by sequence overlap. Hence, we created the single deletions of each ORF of the YPR099C/MRPL51 pair by an alternative approach to distinguish their phenotypes and identify the specific functions. Both the ORFs were found essential for the functional mitochondria and respiratory growth, but MRPL51 showed its specific requirement in mtDNA stability. The mechanism of mtDNA maintenance by Mrpl51 is probably Mhr1 dependent that physically interacts with Mrpl51 and also regulates mtDNA repair. Overall, our study provides strong evidence for the protein level expression of a dubious ORF YPR099C and the bifunctional role of Mrpl51 in mtDNA maintenance.

7. IMPACT OF CHROMOSOMAL MOSAICISM IN IVF OUTCOMES: EXPERIENCE FROM TWO HUNDRED MOSAIC EMBRYOS TRANSFERRED PROSPECTIVELY

Introduction Chromosomal mosaic embryos are characterized by the presence of chromosomally different cell lines within the same embryo. In a recent published study, we have demonstrated that mosaic embryos hold the potential to implant and result in the birth of healthy babies. Therefore, the transfer of these embryos is now offered as an option for women who undergo in vitro fertilization (IVF) resulting in only mosaic embryos but no euploid embryos for transfer. However, there is still few data concerning the impact of mosaicism on viability, and the classification of such embryos in relation with their reproductive potential is unclear. Here we investigated if chromosomal constitution of mosaic embryos influences the clinical outcome of in vitro fertilization treatments Material and methods The transfer of mosaic embryos was offered to 200 women for whom IVF had resulted in no euploid embryos between May 2016-May 2018. Trophectoderm (TE) biopsy was performed on Day-5 of development or Day6/7 for slow growing embryos. The clinical outcome obtained after transfer of mosaic embryos with different chromosomal constitution was compared with each other and with that obtained from a control group of 500 euploid blastocysts. Comprehensive chromosome testing PGT-A was performed using high resolution next generation sequencing (NGS) methodology. TE biopsies were classified as mosaic if they had 20%-80% abnormal cells. For statistical analysis mosaic embryos were divided in four groups based on chromosomal constitution: mosaic monosomy (single and double monosomies; MM), mosaic trisomy (single and double trisomies, MT), mosaic complex aneuploidy (more than two different aneuploidies; MC) and mosaic segmental aneuploidy (single and double deletion or insertion >5Mb, MS). Results MM showed significant higher birth rate compared to MT (46% vs 24%,p=0.02), MC (46% vs 23%,p=0.03) and MS (46% vs 22%,p=0.02). No significant difference was observed in clinical outcome between the groups MT, MC and MS. A comparison of the clinical outcomes with control euploid group showed no significant difference between euploid control and MM, while a significant low implantation rate (55.4% vs 37% MT, vs 31% MC, vs 23% MS,p Conclusion The study demonstrated that embryos with MT, MS and MC aneuploidies have lower chances of resulting with birth of healthy babies compared to embryos with MM. More interestingly, no difference has been find in clinical results between MM and euploid control group. These findings should be considered for genetic counseling.

Reflection of neuroblastoma intratumor heterogeneity in the new OHC-NB1 disease model.

Accurate modeling of intratumor heterogeneity presents a bottleneck against drug testing. Flexibility in a preclinical platform is also desirable to support assessment of different endpoints. We established the model system, OHC-NB1, from a bone marrow metastasis from a patient diagnosed with MYCN-amplified neuroblastoma and performed whole-exome sequencing on the source metastasis and the different models and passages during model development (monolayer cell line, 3D spheroid culture and subcutaneous xenograft tumors propagated in mice). OHC-NB1 harbors a MYCN amplification in double minutes, 1p deletion, 17q gain and diploid karyotype, which persisted in all models. A total of 80-540 single-nucleotide variants (SNVs) was detected in each sample, and comparisons between the source metastasis and models identified 34 of 80 somatic SNVs to be propagated in the models. Clonal reconstruction using the combined copy number and SNV data revealed marked clonal heterogeneity in the originating metastasis, with four clones being reflected in the model systems. The set of OHC-NB1 models represents 43% of somatic SNVs and 23% of the cellularity in the originating metastasis with varying clonal compositions, indicating that heterogeneity is partially preserved in our model system.

HprKXcc is a serine kinase that regulates virulence in the Gram‐negative phytopathogen Xanthomonas campestris

SummaryThe HprK serine kinase is a component of the phosphoenolpyruvate phosphotransferase system (PTS) of bacteria that generally regulates catabolite repression through phosphorylation/dephosphorylation of the PTS protein PtsH at a conserved serine residue. However, many bacteria do not encode a complete PTS or even have an HprK homologue. Xanthomonas campestris pv. campestris (Xcc) is a pathogen that cause black rot disease in crucifer plants and one of the few Gram‐negative bacteria that encodes a homologue of HprK protein (herein HprKXcc). To gain insight into the role of HprKXcc and other PTS‐related components in Xcc we individually mutated and phenotypically assessed the resulting strains. Deletion of hprK Xcc demonstrated its requirement for virulence and other diverse cellular processes associated including extracellular enzyme activity, extracellular‐polysaccharide production and cell motility. Global transcriptome analyses revealed the HprKXcc had a broad regulatory role in Xcc. Additionally, through overexpression, double gene deletion and transcriptome analysis we demonstrated that hprK Xcc shares an epistatic relationship with ptsH. Furthermore, we demonstrate that HprKXcc is a functional serine kinase, which has the ability to phosphorylate PtsH. Taken together, the data illustrates the previously unappreciated global regulatory role of HprKXcc and previously uncharacterized PTS components that control virulence in this pathogen.

Outer membrane proteins YbjX and PagP co-regulate motility in Escherichia coli via the bacterial chemotaxis pathway

Mutation of the PhoP/Q two-component system decreases the expression of ybjX and pagP encoding outer membrane proteins, and mutation of ybjX or pagP attenuates avian pathogenic Escherichia coli (APEC) pathogenicity. However, whether ybjX/pagP mutation (double-deletion mutant) has a synergistic effect on pathogenicity remains unknown. Herein, electrophoresis mobility shift assay (EMSA) experiments showed that the PhoP/Q system regulated ybjX and pagP transcription indirectly. The APECΔybjX/pagP mutant strain, constructed using the Red recombination method, exhibited reduced invasion of chicken embryo fibroblast (DF-1) cells, but had no effect on virulence in a chicken model. Using RNA sequencing to identify differential mRNAs in APECΔybjXΔpagP and native strains, we revealed up-regulation of genes involved in the bacterial chemotaxis pathway. The ybjX/pagP mutant strain displayed significantly increased motility, suggesting that double deletion of ybjX and pagP enhances motility via the bacterial chemotaxis pathway.

Lorf9 deletion significantly eliminated lymphoid organ atrophy induced by meq-deleted very virulent Marek’s disease virus

Marek’s disease virus (MDV) is a highly contagious alphaherpesvirus that causes rapid onset of T cell lymphomas in chickens. MDV continues to break through vaccinal immunity due to the emergence of highly virulent field strains. Earlier studies revealed that deletion of the meq gene from MDV results in attenuated vaccines that protect against disease when chickens are infected with highly virulent strains. However, meq-deleted viruses still retain the ability to induce lymphoid organ atrophy, which raises safety concerns. In an earlier study, we found that deletion of lorf9 counteracts this lymphoid organ atrophy. Here, we describe the generation of a double deletion mutant virus lacking virus-encoded meq and lorf9. In vitro studies revealed that during replication, the mutant virus had kinetic characteristics similar to the parental virus; however, in vivo the replication capability was significantly reduced. Results of animal studies revealed no obvious MDV-specific symptoms and lesions. Importantly, the double deletion mutant virus lost the capacity to induce lymphoid organ atrophy, which has been the main obstacle during development of a good vaccine candidate.