Increased Copy Number Research Articles

Ethanol Preference Leads to Alterations in Telomere Length, Mitochondria Copy Number, and Antioxidant Enzyme Activity in Zebrafish Brains.

The motivations for and effects of ethanol consumption vary considerably among individuals, and as such, a significant proportion of the population is prone to substance abuse and its negative consequences in the physical, social, and psychological spheres. In a biological context, the characterization of these phenotypes provides clues for understanding the neurological complexity associated with ethanol abuse behavior. Therefore, the objective of this research was to characterize four ethanol preference phenotypes described in zebrafish: Light, Heavy, Inflexible, and Negative Reinforcement. To do this, we evaluated the telomere length, mtDNA copy number using real-time quantitative PCR (qPCR), and the activity of these antioxidant enzymes: catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) in the brain, and the interactions between these biomarkers. Changes observed in these parameters were associated with ethanol consumption and alcohol abuse. The Heavy, Inflexible, and Negative Reinforcement phenotypes showed ethanol preference. This was particularly the case with the Inflexible phenotype, which was the group with the greatest ethanol preference. These three phenotypes showed telomere shortening as well as high SOD/CAT and/or GPx activities, while the Heavy phenotype also showed an increase in the mtDNA copy number. However, the Light phenotype, containing individuals without ethanol preference, did not demonstrate any changes in the analyzed parameters even after being exposed to the drug. Additionally, the PCA analysis showed a tendency to cluster the Light and Control groups differently from the other ethanol preference phenotypes. There was also a negative correlation between the results of the relative telomere length and SOD and CAT activity, providing further evidence of the biological relationship between these parameters. Our results showed differential molecular and biochemistry patterns in individuals with ethanol preference, suggesting that the molecular and biochemical basis of alcohol abuse behavior extends beyond its harmful physiological effects, but rather is correlated with preference phenotypes.

Extrachromosomal DNA in the cancerous transformation of Barrett’s oesophagus

Oncogene amplification on extrachromosomal DNA (ecDNA) drives the evolution of tumours and their resistance to treatment, and is associated with poor outcomes for patients with cancer1–6. At present, it is unclear whether ecDNA is a later manifestation of genomic instability, or whether it can be an early event in the transition from dysplasia to cancer. Here, to better understand the development of ecDNA, we analysed whole-genome sequencing (WGS) data from patients with oesophageal adenocarcinoma (EAC) or Barrett’s oesophagus. These data included 206 biopsies in Barrett’s oesophagus surveillance and EAC cohorts from Cambridge University. We also analysed WGS and histology data from biopsies that were collected across multiple regions at 2 time points from 80 patients in a case–control study at the Fred Hutchinson Cancer Center. In the Cambridge cohorts, the frequency of ecDNA increased between Barrett’s-oesophagus-associated early-stage (24%) and late-stage (43%) EAC, suggesting that ecDNA is formed during cancer progression. In the cohort from the Fred Hutchinson Cancer Center, 33% of patients who developed EAC had at least one oesophageal biopsy with ecDNA before or at the diagnosis of EAC. In biopsies that were collected before cancer diagnosis, higher levels of ecDNA were present in samples from patients who later developed EAC than in samples from those who did not. We found that ecDNAs contained diverse collections of oncogenes and immunomodulatory genes. Furthermore, ecDNAs showed increases in copy number and structural complexity at more advanced stages of disease. Our findings show that ecDNA can develop early in the transition from high-grade dysplasia to cancer, and that ecDNAs progressively form and evolve under positive selection.

Human cytomegalovirus infection activates NLRP3 inflammasome by releasing mtDNA into the cytosol in human THP-1 cells.

Human cytomegalovirus (HCMV) infection of monocytes results in the production of inflammatory cytokine through inflammasome. However, the mechanism of NLR family pyrin domain containing 3 (NLRP3) inflammasome activation in HCMV infection remains unclear. In this study, HCMV infection promoted the increase of mitochondrial fusion and caused mitochondrial dysfunction in THP-1 cells, including excessive reactive oxygen species production and decreased mitochondrial membrane potential (Δψm). Meanwhile, the expression of mitochondrial DNA (mtDNA)-binding protein TFAM (transcription factor A, mitochondrial) was decreased and mtDNA content in the cytoplasm was increased. Knockdown of TFAM caused an increase in mtDNA copy number in the cytoplasm and resulted in elevated NLRP3 expression, active caspase-1, and mature IL-1β. After a 3 h treatment with MCC950, an NLRP3 inhibitor, the increase of cleaved caspase-1 and mature IL-1β were suppressed. Besides, overexpression of TFAM inhibited the expression of NLRP3, cleaved caspase-1, and mature IL-1β. In addition, knockdown of NLRP3 inhibited the IL-1β process after HCMV infection. mtDNA-deficient cells showed a limited ability to produce NLRP3 and process IL-1β after HCMV infection. In conclusion, HCMV infection of THP-1 cells resulted in decreased mitochondrial TFAM protein expression and increased mtDNA release into the cytoplasm, which eventually led to the activation of NLRP3 inflammasome.

Mutation-induced infections of phage-plasmids

Phage-plasmids are extra-chromosomal elements that act both as plasmids and as phages, whose eco-evolutionary dynamics remain poorly constrained. Here, we show that segregational drift and loss-of-function mutations play key roles in the infection dynamics of a cosmopolitan phage-plasmid, allowing it to create continuous productive infections in a population of marine Roseobacter. Recurrent loss-of-function mutations in the phage repressor that controls prophage induction leads to constitutively lytic phage-plasmids that spread rapidly throughout the population. The entire phage-plasmid genome is packaged into virions, which were horizontally transferred by re-infecting lysogenized cells, leading to an increase in phage-plasmid copy number and to heterozygosity in a phage repressor locus in re-infected cells. However, the uneven distribution of phage-plasmids after cell division (i.e., segregational drift) leads to the production of offspring carrying only the constitutively lytic phage-plasmid, thus restarting the lysis-reinfection-segregation life cycle. Mathematical models and experiments show that these dynamics lead to a continuous productive infection of the bacterial population, in which lytic and lysogenic phage-plasmids coexist. Furthermore, analyses of marine bacterial genome sequences indicate that the plasmid backbone here can carry different phages and disseminates trans-continentally. Our study highlights how the interplay between phage infection and plasmid genetics provides a unique eco-evolutionary strategy for phage-plasmids.

Aneuploidy enables cross-tolerance to unrelated antifungal drugs in Candida parapsilosis.

Candida parapsilosis is an emerging major human fungal pathogen. Echinocandins are first-line antifungal drugs for the treatment of invasive Candida infections. In clinical isolates, tolerance to echinocandins in Candida species is mostly due to point mutations of FKS genes, which encode the target protein of echinocandins. However, here, we found chromosome 5 trisomy was the major mechanism of adaptation to the echinocandin drug caspofungin, and FKS mutations were rare events. Chromosome 5 trisomy conferred tolerance to echinocandin drugs caspofungin and micafungin and cross-tolerance to 5-flucytosine, another class of antifungal drugs. The inherent instability of aneuploidy caused unstable drug tolerance. Tolerance to echinocandins might be due to increased copy number and expression of CHS7, which encodes chitin synthase. Although copy number of chitinase genes CHT3 and CHT4 was also increased to the trisomic level, the expression was buffered to the disomic level. Tolerance to 5-flucytosine might be due to the decreased expression of FUR1. Therefore, the pleiotropic effect of aneuploidy on antifungal tolerance was due to the simultaneous regulation of genes on the aneuploid chromosome and genes on euploid chromosomes. In summary, aneuploidy provides a rapid and reversible mechanism of drug tolerance and cross-tolerance in C. parapsilosis.

Genomic microsatellite characteristics analysis of Dysommaanguillare (Anguilliformes, Dysommidae), based on high-throughput sequencing technology.

Microsatellite loci were screened from the genomic data of Dysommaanguillare and their composition and distribution were analysed by bioinformatics for the first time. The results showed that 4,060,742 scaffolds with a total length of 1,562 Mb were obtained by high-throughput sequencing and 1,160,104 microsatellite loci were obtained by MISA screening, which were distributed on 770,294 scaffolds. The occurrence frequency and relative abundance were 28.57% and 743/Mb, respectively. Amongst the six complete microsatellite types, dinucleotide repeats accounted for the largest proportion (592,234, 51.05%), the highest occurrence frequency (14.58%) and the largest relative abundance (379.27/Mb). A total of 1488 microsatellite repeats were detected in the genome of D.anguillare, amongst which the hexanucleotide repeat motifs were the most abundant (608), followed by pentanucleotide repeat motifs (574), tetranucleotide repeat motifs (232), trinucleotide repeat motifs (59), dinucleotide repeat motifs (11) and mononucleotide repeat motifs (4). The abundance of microsatellites of the same repeat type decreased with the increase of copy numbers. Amongst the six types of nucleotide repeats, the preponderance of repeated motifs are A (191,390, 43.77%), CA (150,240, 25.37%), AAT (13,168, 14.05%), CACG (2,649, 8.14%), TAATG (119, 19.16%) and CCCTAA (190, 19.16%, 7.65%), respectively. The data of the number, distribution and abundance of different types of microsatellites in the genome of D.anguillare were obtained in this study, which would lay a foundation for the development of high-quality microsatellite markers of D.anguillare in the future.

Abstract 3871: KRAS G12C mutant allele amplification drives resistance to sotorasib in vitro

Abstract Sotorasib is an approved KRASG12C-selective inhibitor for the treatment of KRAS p.G12C-mutant advanced and previously treated non-small cell lung cancers (NSCLC). Acquired resistance due to genomic alterations following sotorasib treatment has been observed in 28% of lung cancer patients (CodeBreaK100, ASCO 2022) and can include bypass signaling, inactivation of negative feedback loops, or compensatory mutations in KRASG12C including amplification of KRAS. Preclinical studies on the mechanisms of acquired resistance are critical for understanding these resistance patterns and can reveal new therapeutic or combination strategies in the clinic. For that reason, we developed an NCI-H358 lung cancer model of acquired resistance to KRASG12C inhibition in vitro. NCI-H358 cells were continuously cultured in the presence of elevated concentrations of sotorasib (IC90) until resistance to KRASG12C inhibitor was achieved. Expression analyses of the resistant cells by ddPCR and immunoblotting showed increases in both KRAS gene and protein levels, resulting in enhanced MAPK signaling. siRNA knockdown of KRAS G12C expression re-sensitized the resistant cells to an analog of sotorasib. Further characterization of this cell line by whole exome sequencing (WES) and fluorescence in situ hybridization (FISH) showed that resistance was not due to genetic co-alterations but amplification of the mutant allele specifically. In comparison to DMSO-treated and parental controls, the sotorasib-resistant NCI-H358 cells contained an approximately 50-fold increase in KRAS G12C mutant allele copy numbers. Preclinical data generated using a KRASG12C inhibitor-resistant lung cancer cell line is consistent with clinical observations of acquired KRAS amplification following KRASG12C inhibitor treatment as a mechanism of resistance. This model further provides an opportunity to study acquired KRAS amplification and investigate combination treatment options in vitro. Citation Format: Deanna Mohn, Anne Y. Saiki, Pragathi Achanta, Andres Plata Stapper, J. Russell Lipford, Rati Verma. KRAS G12C mutant allele amplification drives resistance to sotorasib in vitro. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3871.

Abstract 3130: Extrachromosomal DNA in the cancerous transformation of Barrett’s esophagus

Abstract Oncogene amplification on extrachromosomal DNA (ecDNA) drives rapid tumor evolution, treatment resistance, and poor outcomes for patients. Computational tools can detect ecDNA in whole-genome sequencing (WGS) data from biopsies. However, the lack of longitudinal studies tracking patients from pre-cancer to cancer have made it difficult to determine how ecDNAs contribute to the malignant transformation. Two independent surveillance studies of Barrett’s esophagus (BE) patients enabled us to study this transition, as BE is the precursor lesion of esophageal adenocarcinoma (EAC). The studies included a longitudinal case-control study and an independent, cross-sectional surveillance cohort, both with histological correlatives, providing an unprecedented opportunity to study the role of ecDNA in the transition of BE to EAC. We analyzed WGS data from 206 patients in a cross-sectional surveillance cohort from Cambridge University UK, with biopsy-validated BE, including 42 patients who never developed high-grade dysplasia (HGD) or EAC during multi-year follow-ups, 25 patients with HGD, 51 patients with early-stage EAC, and 88 patients with late-stage EAC. ecDNA frequency increased between early-stage EAC (24%) and late-stage EAC (43%), suggesting continual ecDNA formation during cancer progression. We additionally analyzed WGS and histology data collected from multi-regional sampling of BE and EAC tissue at two time points from 80 BE patients in an observational case-control study at the Fred Hutchinson Cancer Center (FHCC), where 40 of the patients developed BE-associated EAC during the study and 40 did not. Among FHCC patients, 33% who developed EAC had at least one esophageal biopsy with ecDNA prior to, or at EAC diagnosis. In biopsies collected before cancer diagnosis, ecDNA presence in the biopsy was enriched among patients who later developed EAC compared to patients who did not. When linked to histology data, ecDNA-positive biopsies associated with worsened histological states before cancer diagnosis (p=0.015), and also at cancer diagnosis (p=0.037), with the pre-cancer timepoint being on average 2.1 years prior to cancer diagnosis. The ecDNAs had diverse collections of oncogenes and immunomodulatory genes, and showed higher copy number states than other focal amplification types. Furthermore, ecDNAs showed increasing copy number and structural complexity in more advanced disease stages highlighting their positive selection and ongoing structural evolution. In both FHCC and Cambridge cohorts, we found an association of TP53 loss with ecDNA formation demonstrating the role of prior genomic instability to form ecDNA. In total, our findings illustrate that oncogenic ecDNA can develop early in the malignant transformation, and that they are tied to worsening patient disease status. Citation Format: Jens Luebeck, Alvin W. Ng, Patricia C. Galipeau, Xiaohong Li, Annalise Katz-Summercorn, Hoon Kim, Sriganesh Jammula, Yudou He, Roel Verhaak, Carlo C. Maley, Ludmil B. Alexandrov, Rebecca C. Fitzgerald, Thomas G. Paulson, Howard Y. Chang, Sihan Wu, Vineet Bafna, Paul S. Mischel. Extrachromosomal DNA in the cancerous transformation of Barrett’s esophagus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3130.

Molecular markers as predictors of response to perioperative chemotherapy in locally advanced gastric cancer

Introduction. Perioperative FLOT chemotherapy has improved prognosis in patients with locally advanced resectable gastric cancer (GC). However, in 80 % of cases, the tumor is resistant to the therapy, resulting in unnecessary toxicity and delayed surgical treatment.Aim. Evaluation of clinico-morphological patterns of microsatellite instability, HER2 gene amplification, changes in gene copy number and their relationship with the response to perioperative FLOT chemotherapy in patients with locally advanced resectable GC.Materials and methods. The retrospective study included 185 patients. All tumor samples were assessed for HER2 and microsatellite instability status. Among all cases there were 45 patients with locally advanced T2–4N1–2 M0 GC, who underwent a total or subtotal gastrectomy with D2 lymphadenectomy and perioperative chemotherapy with FLOT. Microsatellite instability detection was performed using fragment analysis, HER2 gene amplification testing – fluorescent in situ hybridization. Also 19 patients were tested for copy number changes of the FGFR1, FGFR2, KRAS, MET, EGFR, CCND1, MYC genes using Multiplex ligation-dependent probe amplification. The endpoints were progression-free survival and objective response rate.Results. Microsatellite instability was detected in 4.8 % (9/185) of GC cases. Microsatellite instability was associated with advanced age (p = 0.005), low grade of differentiation (p = 0.011), presence of tumor-infiltrating lymphocytes (p = 0.0004), and high preoperative CA 72–4 levels (p = 0.025). Prevalence of HER2 amplification was 7.5 % (14/185). It was associated with low grade of differentiation (p = 0.048) and metastasis in regional lymph nodes (p = 0.037). PFS in patients with HER2-positive (HER2 – human epidermal growth factor receptor 2) GC treated with perioperative FLOT chemotherapy (4/45) was significantly lower than in patients with HER2-negative GC: the median was 156 and 317 days, respectively (hazard ratio 0.49; 95 % confidence interval 0.16–1.47; p = 0.0006). There was no correlation between the presence of the alteration and ORR (p = 1.0). Progression-free survival in GC patients with KRAS amplification (3/19) was significantly lower comparing with patients without it: the median was 98 and 327 days, respectively (hazard ratio 0.29; 95 % confidence interval 0.07–1.19; p <0.0001). There was no association between an increase in KRAS copy number and objective response rate (p = 1.0). For microsatellite instability and other studied markers no statistically significant correlation with progression-free survival and objective response rate was found (p >0.05).Conclusion. The presence of HER2 and KRAS amplification have been shown as promising predictive markers of the treatment failure in patients treated with perioperative FLOT chemotherapy for locally advanced resectable GC.

Epithelioid and spindle rhabdomyosarcoma with TFCP2 rearrangement in abdominal wall: a distinctive entity with poor prognosis

BackgroundEpithelioid and spindle rhabdomyosarcoma (ES-RMS) with TFCP2 rearrangement is a recently discovered rare variant of rhabdomyosarcoma composed of epithelioid and spindle cells, because it shows extraordinarily adverse prognosis and is easily misdiagnosed as other epithelioid or spindle cell tumors.MethodsA rare case of ES-RMS with TFCP2 rearrangement was presented and English literatures in Pubmed online up to 01 July 2022 were gathered by two authors for a systematic review according to the inclusion and exclusion criteria.Case presentation/resultsWe report a case of ES-RMS in an early 30s-years-old female, the neoplastic cells are remarkably immunoreactive with CK(AE1/AE3), and partially with ALK protein. Unexpectedly, the tumor shows TFCP2 rearrangement with coexistence of increased copy numbers of EWSR1 and ROS1 gene and MET gene mutation. Besides, Next-generation sequencing for genetic mutational profiling revealed frequent MET exon14 mutations in chromosome 7, most of which are C > T nonsynonymous SNV, and exon42 of ROS1 in chromosome 6 showed frequent G > T mutation up to 57.54%. In addition, neither MyoD1 mutation nor gene fusions were detected. Moreover, the patient shows high tumor mutational burden (TMB) up to 14.11 counts/Mb. Finally, as many cases of ES-RMS including our case had local progression or metastasis, we find, similar to epithelioid rhabdomyosarcoma (median survival time is 10 month), ES-RMS shows a more aggressive behavior and adverse prognosis (median survival time is 17 month) than spindle cell/sclerosing rhabdomyosarcoma (median survival time is 65 month) according previous studies.ConclusionsES-RMS with TFCP2 rearrangement is a rare malignant tumor and easily confused with other epithelioid or spindle cell tumors, it may harbor additional gene alteration in addition to TFCP2 rearrangement, such as MET mutation, increased copy numbers of EWSR1 and ROS1 gene, high TMB. Most importantly, it may show very poor outcome with extensive metastasis.

Urinary extracellular vesicles carry valuable hints through mRNA for the understanding of endocrine hypertension.

Urinary extracellular vesicles (uEVs), released from cells of the urogenital tract organs, carry precious information about originating tissues. The study of molecules transported through uEVs such as proteins, lipids and nucleic acids provides a deeper understanding of the function of the kidney, an organ involved in the pathogenesis of hypertension and a target of hypertension-mediated organ damage. Molecules derived from uEVs are often proposed for the study of disease pathophysiology or as possible disease diagnostic and prognostic biomarkers. Analysis of mRNA loading within uEVs may be a unique and readily obtainable way to assess gene expression patterns of renal cells, otherwise achievable only by an invasive biopsy procedure. Interestingly, the only few studies investigating transcriptomics of hypertension-related genes through the analysis of mRNA from uEVs are inherent to mineralocorticoid hypertension. More specifically, it has been observed that perturbation in human endocrine signalling through mineralcorticoid receptors (MR) activation parallels changes of mRNA transcripts in urine supernatant. Furthermore, an increased copy number of uEVs-extracted mRNA transcripts of the 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2) gene were detected among subjects affected by apparent mineralocorticoid excess (AME), a hypertension-inducing autosomal recessive disorder due to a defective enzyme function. Moreover, by studying uEVs mRNA, it was observed that the renal sodium chloride cotransporter (NCC) gene expression is modulated under different conditions related to hypertension. Following this perspective, we illustrate here the state of the art and the possible future of uEVs transcriptomics towards a deeper knowledge of hypertension pathophysiology and ultimately more tailored investigational, diagnostic-prognostic approaches.

Effects of maternal blood selenium and zinc levels on mitochondrial DNA copy number at term and in-turn their effect on the birthweight of the baby

Introduction and Aim: The micronutrients such as selenium and zinc have the antioxidant property. They are cofactors of antioxidant enzymes, regulate the inflammatory response by counterbalancing the oxidative stress. Reduced maternal selenium and zinc levels have been shown to be associated with early pregnancy loss and low birthweight. The aim is to study the effects of maternal blood selenium and zinc levels on mitochondrial DNA copy number at term and in-turn their effect on the birth weight of the baby. Methods: An Institutional ethical clearance was obtained and the present hospital based cross sectional study was conducted in the OBG department of a medical college of North Karnataka between December 2019 to February 2022. Results: A total of 150 term pregnant women participated in the study. There was a slightly increased serum level of zinc and selenium found in the term pregnant mothers who gave birth to normal birth weight babies when compared to low-birth-weight babies, though this difference was not statistically significant. Median (IQR) values of Delta CT values of Mitochondrial DNA copy number in low-birth-weight babies were 3.07(1.7-5.74) and in normal birth weight babies was 3.71 (0.83-4.4). The difference in median values was not statistically significant (p=0.57). We observed a positive correlation between the maternal zinc, selenium, and mitochondrial DNA copy number with birth weight of the baby. Though the correlation between Delta CT means and birthweight of the baby is found to be statistically significant. Conclusion: In the present study, apparently healthy pregnant women participated. The serum selenium and zinc levels were found to be within normal limits according to lab reference values. There was an increase in Mitochondrial DNA copy number in the present study and it was positively correlated with the birthweight of the baby.

SO2 and copper tolerance exhibit an evolutionary trade-off in Saccharomyces cerevisiae.

Copper tolerance and SO2 tolerance are two well-studied phenotypic traits of Saccharomyces cerevisiae. The genetic bases of these traits are the allelic expansion at the CUP1 locus and reciprocal translocation at the SSU1 locus, respectively. Previous work identified a negative association between SO2 and copper tolerance in S. cerevisiae wine yeasts. Here we probe the relationship between SO2 and copper tolerance and show that an increase in CUP1 copy number does not always impart copper tolerance in S. cerevisiae wine yeast. Bulk-segregant QTL analysis was used to identify variance at SSU1 as a causative factor in copper sensitivity, which was verified by reciprocal hemizygosity analysis in a strain carrying 20 copies of CUP1. Transcriptional and proteomic analysis demonstrated that SSU1 over-expression did not suppress CUP1 transcription or constrain protein production and provided evidence that SSU1 over-expression induced sulfur limitation during exposure to copper. Finally, an SSU1 over-expressing strain exhibited increased sensitivity to moderately elevated copper concentrations in sulfur-limited medium, demonstrating that SSU1 over-expression burdens the sulfate assimilation pathway. Over-expression of MET 3/14/16, genes upstream of H2S production in the sulfate assimilation pathway increased the production of SO2 and H2S but did not improve copper sensitivity in an SSU1 over-expressing background. We conclude that copper and SO2 tolerance are conditional traits in S. cerevisiae and provide evidence of the metabolic basis for their mutual exclusivity. These findings suggest an evolutionary driver for the extreme amplification of CUP1 observed in some yeasts.

Increased level of RAB39B leads to neuronal dysfunction and behavioural changes in mice.

Duplications of the Xq28 region are a common cause of X-linked intellectual disability (XLID). The RAB39B gene locates in Xq28 and has been implicated in disease pathogenesis. However, whether increased dosage of RAB39B leads to cognitive impairment and synaptic dysfunction remains elusive. Herein, we overexpressed RAB39B in mouse brain by injecting AAVs into bilateral ventricles of neonatal animals. We found that at 2 months of age, neuronal overexpression of RAB39B impaired the recognition memory and the short-term working memory in mice and resulted in certain autism-like behaviours, including social novelty defect and repetitive grooming behaviour in female mice. Moreover, overexpression of RAB39B decreased dendritic arborization of primary neurons in vitro and reduced synaptic transmission in female mice. Neuronal overexpression of RAB39B also altered autophagy without affecting levels and PSD distribution of synaptic proteins. Our results demonstrate that overexpression of RAB39B compromises normal neuronal development, thereby resulting in dysfunctional synaptic transmission and certain intellectual disability and behavioural abnormalities in mice. These findings identify a molecular mechanism underlying XLID with increased copy numbers of Xq28 and provide potential strategies for disease intervention.

Characterization of centromeric DNA of Gossypium anomalum reveals sequence-independent enrichment dynamics of centromeric repeats.

Centromeres in eukaryotes are composed of highly repetitive DNAs, which evolve rapidly and are thought to achieve a favorable structure in mature centromeres. However, how the centromeric repeat evolves into an adaptive structure is largely unknown. We characterized the centromeric sequences of Gossypium anomalum through chromatin immunoprecipitation against CENH3 antibodies. We revealed that the G. anomalum centromeres contained only retrotransposon-like repeats but were depleted in long arrays of satellites. These retrotransposon-like centromeric repeats were present in the African-Asian and Australian lineage species, suggesting that they might have arisen in the common ancestor of these diploid species. Intriguingly, we observed a substantial increase and decrease in copy numbers among African-Asian and Australian lineages, respectively, for the retrotransposon-derived centromeric repeats without apparent structure or sequence variation in cotton. This result indicates that the sequence content is not a decisive aspect of the adaptive evolution of centromeric repeats or at least retrotransposon-like centromeric repeats. In addition, two active genes with potential roles in gametogenesis or flowering were identified in CENH3 nucleosome-binding regions. Our results provide new insights into the constitution of centromeric repetitive DNA and the adaptive evolution of centromeric repeats in plants.

Metabolic engineering of thermophilic Bacillus methanolicus for riboflavin overproduction from methanol.

The growing need of next generation feedstocks for biotechnology spurs an intensification of research on the utilization of methanol as carbon and energy source for biotechnological processes. In this paper, we introduced the methanol-based overproduction of riboflavin into metabolically engineered Bacillus methanolicus MGA3. First, we showed that B. methanolicus naturally produces small amounts of riboflavin. Then, we created B. methanolicus strains overexpressing either homologous or heterologous gene clusters encoding the riboflavin biosynthesis pathway, resulting in riboflavin overproduction. Our results revealed that the supplementation of growth media with sublethal levels of chloramphenicol contributes to a higher plasmid-based riboflavin production titre, presumably due to an increase in plasmid copy number and thus biosynthetic gene dosage. Based on this, we proved that riboflavin production can be increased by exchanging a low copy number plasmid with a high copy number plasmid leading to a final riboflavin titre of about 523 mg L-1 in methanol fed-batch fermentation. The findings of this study showcase the potential of B. methanolicus as a promising host for methanol-based overproduction of extracellular riboflavin and serve as basis for metabolic engineering of next generations of riboflavin overproducing strains.

Evaluation of oil displacement potential of genetically engineered strain WJPAB fermentation broth

Rhamnoolipids are the most widely studied and used biosurfactants, which can be used in microbial flooding to enhance oil recovery. Pseudomonas aeruginosa is the main species of rhamnoolipids synthesized at present. In this study, wild strain WJ was modified by directional genetic engineering, and the strong promoter PoprL was used to replace the original promoter of rhlAB gene, and the genetically engineered strain with increased copy number of Popr-Rhlab gene was constructed, which was named as the genetically engineered strain WJPAB. The optimal carbon source of WJPAB was rapeseed oil. Under the optimal fermentation conditions, the product yield of WJPAB was 57.83g/L, which was 91.17% higher than that of wild strain WJ.The metabolites of strain WJPAB are purified and analyzed by high performance liquid chromatography mass spectrometry (HPLC-MS). The products are confirmed to be rhamnose homologues, including 3 single rhamnose homologues and 5 double rhamnose homologues. The physical and chemical properties of the metabolites of strain WJPAB were studied, and it was found that the metabolites of strain WJPaB had good surface/interfacial activity, which could reduce the surface tension of water to 27.34 mN/m, and change the surface of oil-wet core from oil-wet surface to water-wet surface. The emulsification index of simulated oil, cetane and liquid paraffin is above 60%. In addition, bacterial metabolites have good stability and can maintain good surface activity even at pH 3–11, salinity 0–20g/L and temperature 120 °C, and can adapt to a wide range of reservoir environmental conditions. In the oil displacement experiment, a compound oil displacement system is established by combining fermentation broth and xanthan gum. The compound system can increase the recovery rate by 15.45%, save the separation and purification process of biosurfactant, and reduce the application cost. The results show that Pseudomonas aeruginosa WJPAB has a good application prospect in the microbial oil recovery field.

Intermittent cold exposure upregulates regulators of cardiac mitochondrial biogenesis and function in mice.

Hypothermic conditions enhance the incidence of cardiovascular diseases due to increased blood pressure. Cold-induced adaptive thermogenesis increased mitochondrial biogenesis and function in skeletal muscles and adipocytes. Here, we studied the effect of intermittent cold exposure on the regulators of cardiac mitochondrial biogenesis, function, and its regulation by SIRT-3. Intermittent cold exposed mice hearts showed normal histopathology with increased mitochondrial antioxidant and metabolic function, as evidenced by an increase in the activity and expression of MnSOD and SDH. A substantial increase in mitochondrial DNA copy number and increase in the expression of PGC-1α and its downstream targets NRF-1 and Tfam indicated the possibility of enhanced cardiac mitochondrial biogenesis and function on intermittent cold exposure. Increased mitochondrial SIRT-3 level and decreased total protein lysine acetylation indicate increased sirtuin activity in cold exposed mice hearts. Ex vivo cold mimic using norepinephrine showed a significant increase in PGC-1α, NRF-1, and Tfam levels. AGK-7, a SIRT-3 inhibitor, reversed the norepinephrine-induced upregulation of PGC-1α and NRF-1, indicating the role ofSIRT-3 on the production of PGC-1α and NRF-1. Inhibition of PKA with KT5720 in norepinephrine treated cardiac tissue slices indicates the role of PKA in regulating the production of PGC-1α and NRF-1. In conclusion, intermittent cold exposure upregulated the regulators of mitochondrial biogenesis and function through PKA and SIRT-3 mediated pathway. Our results emphasize the role of intermittent cold-induced adaptive thermogenesis in overcoming chronic cold-induced cardiac damage.

Chronic hypoxia is associated with transcriptomic reprogramming and increased genomic instability in cancer cells.

Hypoxia afflicts the microenvironment of solid tumors fueling malignancy. We investigated the impact of long hypoxia exposure on transcriptional remodeling, tumor mutational burden (TMB), and genomic instability of cancer cells that were grouped based on their inherent sensitivity or resistance to hypoxia. A hypoxia score was used as a metric to distinguish between the most hypoxia-sensitive (hypoxia high (HH)), and most resistant (hypoxia low (HL)) cancer cells. By applying whole exome sequencing and microarray analysis, we showed that the HH group was indeed more sensitive to hypoxia, having significantly higher TMB (p = 0.03) and copy number losses (p = 0.03), as well as a trend of higher transcriptional response. Globally cells adapted by decreasing expression of genes involved in metabolism, proliferation, and protein maturation, and increasing alternative splicing. They accumulated mutations, especially frameshift insertions, and harbored increased copy number alterations, indicating increased genomic instability. Cells showing highest TMB simultaneously experienced a significant downregulation of DNA replication and repair and chromosomal maintenance pathways. A sixteen-gene common response to chronic hypoxia was put forth, including genes regulating angiogenesis and proliferation. Our findings show that chronic hypoxia enables survival of tumor cells by metabolic reprogramming, modulating proliferation, and increasing genomic instability. They additionally highlight key adaptive pathways that can potentially be targeted to prevent cancer cells residing in chronically hypoxic tumor areas from thriving.

Abstract P117: Weight Loss is Associated With Increases in Mitochondrial DNA Copy Number

Background: Mitochondrial DNA copy number (mtDNA-CN) is a surrogate measure of mitochondrial function that is independently associated with increased cardiovascular disease risk. Individuals with obesity and cardiometabolic risk factors have lower mtDNA-CN levels. However, the modifiability of mtDNA-CN following a weight loss intervention has remained largely unexplored. Hypothesis: Bariatric surgery-induced weight loss is associated with longitudinal increases in circulating mtDNA-CN levels. Methods: In the BARI-Heart prospective cohort of bariatric surgery patients, we quantified mtDNA-CN from whole blood samples collected at three visits: 3 to 6 months prior to surgery, 2-4 weeks prior to surgery, and 6 months post-surgery. We used linear mixed models with random effects to assess changes in mtDNA-CN after bariatric surgery, with the pre-surgery visits while awaiting bariatric surgery serving as the control period. We used multivariable linear regression to evaluate the dose-response association between weight loss and change in mtDNA-CN. Results: We included 71 participants (mean age 45 years, 74% female, 88% White). While mtDNA-CN levels did not change significantly during the pre-surgery period (V1 to V2), mtDNA-CN significantly increased after bariatric surgery (V2 to V3) (Figure). In linear mixed models adjusting for age, sex, and smoking, mtDNA-CN was higher after bariatric surgery than immediately prior to bariatric surgery (Table: ß=0.141, p < 0.001). This increase in mtDNA-CN remained significant following further adjustment for changes in cardiometabolic risk factors (ß=0.116, p < 0.01). In linear regression, each 10% greater weight loss was associated with a 1.6% increase in mtDNA-CN. Conclusions: Weight loss via bariatric surgery is associated with significant increases in mtDNA-CN, independent of cardiometabolic risk factors. Improvements in mitochondrial function may mediate some of the cardiovascular benefits of significant weight loss.