Metformin Preserves Mitochondrial Integrity at Old Age in Male Rats

Metformin is being deployed in clinical trials to ameliorate aging in older humans who do not have diabetes. In C. elegans, metformin treatment at old ages exacerbated mitochondrial dysfunction, led to respiratory failure, and shortened lifespan. Metformin is a commonly used, well-tolerated treatment for diabetes in older adults. Mitochondrial effects of metformin treatment in aged mammals has not been sufficiently investigated. We hypothesized that metformin treatment would not be toxic to older mammals. To define a therapeutic dose in aged hybrid rats, we evaluated two doses of metformin (0.1%, 0.75% of the diet) at 30-months of age. Body mass decreased at the 0.75% dose. Neither dose affected mortality between 30- and 34-months of age. We assessed mitochondrial quality, quantity, and function in aged rats treated with metformin at the 0.75% dose by measuring mitochondrial DNA copy number, deletion mutation frequency, and respirometry in skeletal muscle and heart. In skeletal muscle, we observed no effect of metformin on quadriceps mass, mtDNA copy number or deletion frequency. In the heart, metformin treated rats had higher mtDNA copy number, lower cardiac mass and no effect on deletion frequency. Metformin treatment resulted in lower mitochondrial complex I activity in both heart and quadriceps. Metformin did not compromise mitochondrial integrity, was well tolerated, and may have cardiac benefits to rats at old ages.

Muscle Mitochondrial DNA Copy Number, Deletion Mutation Frequency, and Physical Performance in Older Adults

Mitochondrial DNA (mtDNA) quantity and quality influence hallmarks of aging – mitochondrial dysfunction and genomic instability. The interactions between mtDNA quantity and quality and physical performance have not been extensively examined in humans. The aim of this study was to test the interactions between skeletal muscle mtDNA copy number, mtDNA deletion mutation frequency, and physical performance measures in older adults. Total DNA was isolated from muscle biopsies and used for quantitation of mtDNA copy number and mutation frequency by digital PCR. The biopsies were obtained from a cross-sectional cohort of 53 adults aged 50 to 86 years. Before the biopsy, physical performance measures were collected. MtDNA deletions increased exponentially with advancing age. On average, mtDNA deletion frequency increased 18-fold between 50 and 80, with a trend toward lower deletion frequency in females. MtDNA deletion frequency predicted declines in VO2 max, where 4.7% of the variation in VO2 max was explained by mtDNA deletion frequency. MtDNA copy number was negatively correlated with age and mtDNA deletion frequency, but positively correlated with lean mass. There was a trend to lower mtDNA deletion frequency in females, consistent with increased longevity in females. Larger studies may better delineate sex effects. These data are consistent with a role for mitochondrial function and genome integrity in the maintenance of physical performance with age. Analyses of mtDNA quality and quantity in longitudinal studies could extend our understanding of the importance of mitochondria in human aging and longevity.

Cas9-induced large deletions and small indels are controlled in a convergent fashion

Repair of Cas9-induced double-stranded breaks results primarily in formation of small indels, but can also cause potentially harmful large deletions. While mechanisms leading to the creation of small indels are relatively well understood, very little is known about the origins of large deletions. Using a novel library of clonal mouse embryonic stem cells bona fide deficient for 32 DNA repair genes, we have shown that large deletion frequency increases in cells impaired for non-homologous end joining and decreases in cells deficient for the central resection gene Nbn and the microhomology-mediated end joining gene Polq. Across deficient clones, increase in large deletion frequency was closely correlated with the increase in the extent of microhomology and the size of small indels, implying a continuity of repair processes across different genomic scales. Furthermore, by targeting diverse genomic sites, we identified examples of repair processes that were highly locus-specific, discovering a novel role for exonuclease Trex1. Finally, we present evidence that indel sizes increase with the overall efficiency of Cas9 mutagenesis. These findings may have impact on both basic research and clinical use of CRISPR-Cas9, in particular in conjunction with repair pathway modulation.

Improving FnCas12a genome editing by exonuclease fusion

Among current reported Cas12a orthologs, Francisella novicida Cas12a (FnCas12a) is less restricted by protospacer adjacent motif (PAM), which will help target previously inaccessible genomic sites. However, the activity of FnCas12a nuclease is relatively low or undetectable in human cells, limiting its application as desirable genome engineering tools. Here, we describe TEXT (Tethering EXonuclease T5 with FnCas12a), a fusion strategy that significantly increased the knockout efficiency of FnCas12a in human cells, at multiple genomic loci in three different cell lines. TEXT shows higher insertions and deletions (indels) efficiency than FnCas12a using different spacer lengths from 18nt to 23nt, in which 18nt results in highest fold increase, with up to 11 folds higher efficiency than FnCas12a. Deep sequencing shows that TEXT substantially increased the deletion frequency and deletion size at the targeted locus. TEXT enhances the activity of FnCas12a nuclease and expand its application in human cell genome engineering.

RESPONSE TO NEOADYAVANT CHEMOTHERAPY WITH PLATINUM-BASED DRUGS IN BREAST CANCER PATIENTS WITH BRCA1 DELETION IN TUMOR

Currently, the presence of the germinal mutation BRCA1 5382insC in breast cancer patients is one of the determining factors for prescribing platinum-based drugs. However, this type of mutation is found in no more than 10 % of patients, thus limiting the feasibility of administering platinum-based drugs. Various somatic changes in the BRCA1 gene in breast tumors, in particular the deletions of this gene, can play an important role in the tumor sensitivity to platinum drugs.Case description. We present the case of a 42-year-old woman diagnosed with breast cancer. The deletion of the BRCA1 gene was detected in the tumor. The patient had a complete response to preoperative chemotherapy according to the CP regimen.Conclusion. The frequency of the germline mutation of the BRCA1 gene does not exceed 10 %, and the deletion frequency of this gene can vary from 30 to 45 %, thus greatly increasing the feasibility of using platinum-based drugs in mutationnegative patients to achieve complete pathologic response and high survival rates.

CRISPR/Cas9-Mediated Deletion of Large Genomic Fragments in Soybean

At present, the application of CRISPR/Cas9 in soybean (Glycine max (L.) Merr.) has been mainly focused on knocking out target genes, and most site-directed mutagenesis has occurred at single cleavage sites and resulted in short deletions and/or insertions. However, the use of multiple guide RNAs for complex genome editing, especially the deletion of large DNA fragments in soybean, has not been systematically explored. In this study, we employed CRISPR/Cas9 technology to specifically induce targeted deletions of DNA fragments in GmFT2a (Glyma16g26660) and GmFT5a (Glyma16g04830) in soybean using a dual-sgRNA/Cas9 design. We achieved a deletion frequency of 15.6% for target fragments ranging from 599 to 1618 bp in GmFT2a. We also achieved deletion frequencies of 12.1% for target fragments exceeding 4.5 kb in GmFT2a and 15.8% for target fragments ranging from 1069 to 1161 bp in GmFT5a. In addition, we demonstrated that these CRISPR/Cas9-induced large fragment deletions can be inherited. The T2 ‘transgene-free’ homozygous ft2a mutants with a 1618 bp deletion exhibited the late-flowering phenotype. In this study, we developed an efficient system for deleting large fragments in soybean using CRISPR/Cas9; this system could benefit future research on gene function and improve agriculture via chromosome engineering or customized genetic breeding in soybean.

Genome Editing in Model Strain Myxococcus xanthus DK1622 by a Site-Specific Cre/loxP Recombination System

Myxococcus xanthus DK1622 is a rich source of novel secondary metabolites, and it is often used as an expression host of exogenous biosynthetic gene clusters. However, the frequency of obtaining large genome-deletion variants by using traditional strategies is low, and progenies generated by homologous recombination contain irregular deletions. The present study aims to develop an efficient genome-engineering system for this bacterium based on the Cre/loxP system. We first verified the functionality of the native cre system that was integrated into the chromosome with an inducible promoter PcuoA. Then we assayed the deletion frequency of 8-bp-spacer-sequence mutants in loxP by Cre recombinase which was expressed by suicide vector pBJ113 or self-replicative vector pZJY41. It was found that higher guanine content in a spacer sequence had higher deletion frequency, and the self-replicative vector was more suitable for the Cre/loxP system, probably due to the leaky expression of inducible promoter PcuoA. We also inspected the effects of different antibiotics and the native or synthetic cre gene. Polymerase chain reaction (PCR) and sequencing of new genome joints confirmed that the Cre/loxP system was able to delete a 466 kb fragment in M. xanthus. This Cre/loxP-mediated recombination could serve as an alternative genetic manipulation method.

Association of P16-RBSP3 inactivation with phosphorylated RB1 overexpression in basal–parabasal layers of normal cervix unchanged during CACX development

To understand the molecular mechanism of RB1 phosphorylation in basal–parabasal layers of normal cervix and during cervical cancer (CACX) development, we analyzed the alterations (expression/methylation/deletion/mutation) of RB1/phosphorylated RB1 (p-RB1) (ser807/811 and ser567) and two RB1 phosphorylation inhibitors, P16 and RBSP3, in disease-free normal cervical epithelium (n = 9), adjacent normal cervical epithelium of tumors (n = 70), cervical intraepithelial neoplasia (CIN; n = 28), CACX (n = 102) samples and two CACX cell lines. Immunohistochemical analysis revealed high/medium expression of RB1/p-RB1 (ser807/811 and ser567) and low expression of P16 and RBSP3 in proliferating basal–parabasal layers of majority of normal cervical epitheliums, irrespective of HPV16 infection. Interestingly, 35–52% samples showed high/medium expression of P16 in basal–parabasal layers of normal and had significant association with deleterious non-synonimous SNPs of P16. Methylation of P16 and RBSP3 in basal–parabasal layers of normal cervix (32 and 62%, respectively) showed concordance with their respective expressions in basal–parabasal layers. The methylation frequency of P16 and RBSP3 in basal–parabasal layers of normal did not change significantly in CIN and CACX. The deletion frequency of P16 and RB1 increased significantly with CACX progression. While, deletion of RBSP3 was high in CIN and comparable during CACX progression. P16 showed scattered and infrequent mutation in CACX. The alteration of P16 and RBSP3 was synergistic and showed association with overexpression of p-RB1 in tumors and associated with poor prognosis of patients. Thus, our data suggest that overexpression of p-RB1 in basal–parabasal layers of normal cervical epithelium was due to methylation/low functional-linked non-synonimous SNPs of P16 and RBSP3. This pattern was maintained during cervical carcinogenesis by additional deletion/mutation.