NEK11 as a candidate high-penetrance melanoma susceptibility gene

BackgroundA proportion of patients diagnosed with cutaneous melanoma reports a positive family history. Inherited variants in CDKN2A and several other genes have been shown to predispose to melanoma; however, the genetic basis of familial melanoma remains unknown in most cases. The objective of this study was to provide insight into the genetic basis of familial melanoma.MethodsIn order to identify novel melanoma susceptibility genes, whole exome sequencing (WES) analysis was applied in a Dutch family with melanoma. The causality of a candidate variant was characterised by performing cosegregation analysis in five affected family members using patient-derived tissues and digital droplet PCR analysis to accurately quantify mutant allele frequency. Functional in-vitro studies were performed to assess the pathogenicity of the candidate variant.ResultsApplication of WES identified a rare, nonsense variant in the NEK11 gene (c.1120C>T, p.Arg374Ter), cosegregating in all five affected members of a Dutch family. NEK11 (NIMA-related Kinase 11) is involved in the DNA damage response, enforcing the G2/M cell cycle checkpoint. In a melanoma from a variant carrier, somatic loss of the wildtype allele of this putative tumour suppressor gene was demonstrated. Functional analyses showed that the NEK11 p.Arg374Ter mutation results in strongly reduced expression of the truncated protein caused by proteasomal degradation.ConclusionThe NEK11 p.Arg374Ter variant identified in this family leads to loss-of-function through protein instability. Collectively, these findings support NEK11 as a melanoma susceptibility gene.

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Silencing of hsa_circ_0000515 Inhibits Proliferation, Migration and Invasion of Gastric Cancer Cells by Sponging miR-615-5p In Vitro

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2021.2733 ◽

2021 ◽

Vol 11 (8) ◽

pp. 1466-1476

Author(s):

Xuli Wang ◽

Aiping Wang

Keyword(s):

Gastric Cancer ◽

Underlying Mechanism ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Pcr Analysis ◽

Circular Rnas ◽

Loss Of Function ◽

Gastric Cancer Cells ◽

Novel Target

Circular RNAs (circRNAs) have been reported to participate in the molecular mechanism of human cancers. This study investigates the role of circRNA hsa_circ_0000515 in gastric cancer (GC) cells and the underlying mechanism associated with microRNA-615-5p (miR-615-5p). qRT-PCR analysis showed the upregulation of hsa_circ_0000515 and downregulation of miR-615-5p in GC cell lines. Loss-of-function experiments indicated that suppression of hsa_circ_0000515 inhibited cell proliferation, migration, and invasion. Dual-luciferase reporter assay highlighted that hsa_circ_0000515 was able to act as a ceRNA of miR-615-5p. Furthermore, hsa_circ_0000515 could interact with splicing factors and bind miR-615-5p to regulate progression of GC cells. Deficiency of miR-615-5p reverses the inhibitory roles of si-hsa_circ_0000515 on the proliferation, migration, and invasion of GC cells. The findings highlighted the promising uses of hsa_circ_0000515 as a likely novel target for gastric cancer treatment.

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The ATM/ATR Signaling Effector Chk2 Is Targeted by Epstein-Barr Virus Nuclear Antigen 3C To Release the G2/M Cell Cycle Block

Journal of Virology ◽

10.1128/jvi.00053-07 ◽

2007 ◽

Vol 81 (12) ◽

pp. 6718-6730 ◽

Cited By ~ 56

Author(s):

Tathagata Choudhuri ◽

Subhash C. Verma ◽

Ke Lan ◽

Masanao Murakami ◽

Erle S. Robertson

Keyword(s):

Cell Cycle ◽

B Lymphocytes ◽

Epstein Barr Virus ◽

Cell Cycle Checkpoint ◽

Nuclear Antigen ◽

M Cell ◽

Barr Virus ◽

Cycle Checkpoint ◽

Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) infects most of the human population and persists in B lymphocytes for the lifetime of the host. The establishment of latent infection by EBV requires the expression of a unique repertoire of genes. The product of one of these viral genes, the EBV nuclear antigen 3C (EBNA3C), is essential for the growth transformation of primary B lymphocytes in vitro and can regulate the transcription of a number of viral and cellular genes important for the immortalization process. This study demonstrates an associated function of EBNA3C which involves the disruption of the G2/M cell cycle checkpoint. We show that EBNA3C-expressing lymphoblastoid cell lines treated with the drug nocodazole, which is known to block cells at the G2/M transition, did not show a G2/M-specific checkpoint arrest. Analyses of the cell cycles of cells expressing EBNA3C demonstrated that the expression of this essential EBV nuclear antigen is capable of releasing the G2/M checkpoint arrest induced by nocodazole. This G2/M arrest in response to nocodazole was also abolished by caffeine, suggesting an involvement of the ATM/ATR signaling pathway in the regulation of this cell cycle checkpoint. Importantly, we show that the direct interaction of EBNA3C with Chk2, the ATM/ATR signaling effector, is responsible for the release of this nocodazole-induced G2/M arrest and that this interaction leads to the serine 216 phosphorylation of Cdc25c, which is sequestered in the cytoplasm by 14-3-3. Overall, our data suggest that EBNA3C can directly regulate the G2/M component of the host cell cycle machinery, allowing for the release of the checkpoint block.

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Genetic Analysis of Osteoblast Activity Identifies Zbtb40 as a Regulator of Osteoblast Activity and Bone Mass

10.1101/828525 ◽

2019 ◽

Author(s):

Madison L. Doolittle ◽

Gina M Calabrese ◽

Larry D. Mesner ◽

Dana A. Godfrey ◽

Robert D. Maynard ◽

...

Keyword(s):

Bone Formation ◽

Genetic Basis ◽

Association Studies ◽

Genome Wide Association Studies ◽

Loss Of Function ◽

Mineral Density ◽

Osteoblast Activity ◽

Genome Wide ◽

Increased Risk

ABSTRACTOsteoporosis is a genetic disease characterized by progressive reductions in bone mineral density (BMD) leading to an increased risk of fracture. Over the last decade, genome-wide association studies (GWASs) have identified over 1000 associations for BMD. However, as a phenotype BMD is challenging as bone is a multicellular tissue affected by both local and systemic physiology. Here, we focused on a single component of BMD, osteoblast-mediated bone formation in mice, and identified associations influencing osteoblast activity on mouse Chromosomes (Chrs) 1, 4, and 17. The locus on Chr. 4 was in an intergenic region between Wnt4 and Zbtb40, homologous to a locus for BMD in humans. We tested both Wnt4 and Zbtb40 for a role in osteoblast activity and BMD. Knockdown of Zbtb40, but not Wnt4, in osteoblasts drastically reduced mineralization. Additionally, loss-of-function mouse models for both genes exhibited reduced BMD. Our results highlight that investigating the genetic basis of in vitro osteoblast mineralization can be used to identify genes impacting bone formation and BMD.

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Atypical Genetic Basis of Pyrazinamide Resistance in Mono-resistant Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01916-20 ◽

2021 ◽

Author(s):

Samuel J. Modlin ◽

Tyler Marbach ◽

Jim Werngren ◽

Mikael Mansjö ◽

Sven E. Hoffner ◽

...

Keyword(s):

Genetic Background ◽

Genetic Basis ◽

Active Form ◽

Large Set ◽

R Genes ◽

Loss Of Function ◽

Resistance Development ◽

Long Read ◽

Pyrazinoic Acid

Pyrazinamide (PZA) is a widely used antitubercular chemotherapeutic. Typically, PZA resistance (PZA-R) emerges in M. tuberculosis strains with existing resistance to isoniazid and rifampicin (MDR) and is conferred by loss-of-function pncA mutations that inhibit conversion to its active form, Pyrazinoic acid (POA). PZA-R departing from this canonical scenario is poorly understood. Here, we genotype pncA and purported alternative PZA-R genes (panD, rpsA, and clpC1) with long-read sequencing of nineteen phenotypically PZA mono-resistant isolates collected in Sweden and compare their phylogenetic and genomic characteristics to a large set of MDR PZA-R (MDRPZA-R) isolates. We report the first association of ClpC1 mutations with PZA-R in clinical isolates, in the ClpC1 promoter (clpC1p-138) and N-terminal (ClpC1Val63Ala). Mutations have emerged in both these regions under POA selection in vitro and ClpC1N-terminal has been implicated further, through its POA-dependent efficacy in PanD proteolysis. ClpC1Val63Ala mutants spanned 4 Indo-oceanic sublineages. Indo-oceanic isolates invariably harbored ClpC1Val63Ala and were starkly overrepresented (OR=22.2, p <0.00001) among PZA mono-resistant isolates (11/19) compared to MDRPZA-R isolates (5/80). The genetic basis of Indo-oceanic isolates’ overrepresentation in PZA mono-resistant TB remains undetermined, but substantial circumstantial evidence suggests ClpC1Val63Ala confers low-level PZA resistance. Our findings highlight ClpC1 as potentially clinically relevant for PZA-R and reinforce the importance of genetic background in the trajectory of resistance development.

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Loss of Function of the GdpP Protein Leads to Joint β-Lactam/Glycopeptide Tolerance in Staphylococcus aureus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05148-11 ◽

2011 ◽

Vol 56 (1) ◽

pp. 579-581 ◽

Cited By ~ 52

Author(s):

J. M. Griffiths ◽

A. J. O'Neill

Keyword(s):

Signal Transduction ◽

Staphylococcus Aureus ◽

Genome Sequencing ◽

Genetic Basis ◽

Loss Of Function ◽

Gene Encoding ◽

Content Type ◽

Peptidoglycan Biosynthesis ◽

Insertional Inactivation

ABSTRACTThe genetic basis of tolerance to inhibitors of peptidoglycan biosynthesis inStaphylococcus aureuswas investigated by generating tolerant mutantsin vitroand characterizing them by comparative genome sequencing. Two independently selected tolerant mutants harbored nonsynonymous mutations ingdpP, a gene encoding a putative membrane-located signaling protein. Insertional inactivation ofgdpPalso conferred tolerance. Our findings further implicate altered signal transduction as a route to antibiotic tolerance inS. aureus.

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WEE1 kinase is a therapeutic vulnerability in CIC-DUX4 undifferentiated sarcoma.

10.1101/2021.06.21.448439 ◽

2021 ◽

Author(s):

Rovingaile Kriska Ponce ◽

Nicholas J Thomas ◽

Nam Q Bui ◽

Tadashi Kondo ◽

Ross A Okimoto

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Cell Cycle Checkpoint ◽

M Cell ◽

Undifferentiated Sarcoma ◽

Cycle Checkpoint ◽

Wee1 Kinase ◽

Apoptotic Induction

CIC-DUX4 rearrangements define an aggressive and chemotherapy-insensitive subset of undifferentiated sarcomas. The CIC-DUX4 fusion drives oncogenesis through direct transcriptional upregulation of cell cycle and DNA replication genes. Notably, CIC-DUX4-mediated CCNE1 upregulation compromises the G1/S transition, conferring a potential survival dependence on the G2/M cell cycle checkpoint. Through an integrative transcriptional and kinase activity screen using patient-derived specimens, we now show that CIC-DUX4 sarcomas depend on the G2/M checkpoint regulator, WEE1, as an adaptive survival mechanism. Specifically, CIC-DUX4 sarcomas depend on WEE1 activity to limit DNA damage and unscheduled mitotic entry. Consequently, genetic or pharmacologic WEE1 inhibition in vitro and in vivo leads to rapid DNA damage-associated apoptotic induction of patient-derived CIC-DUX4 sarcomas. Thus, we identify WEE1 as an actionable therapeutic vulnerability in CIC-DUX4 sarcomas.

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Healing effects of curcumin nanoparticles in deep tissue injury mouse model.

Current Drug Delivery ◽

10.2174/1567201818666201214125237 ◽

2020 ◽

Vol 18 ◽

Author(s):

Zirui Zhang ◽

Shangcong Han ◽

Panpan Liu ◽

Xu Yang ◽

Jing Han ◽

...

Keyword(s):

Wound Healing ◽

Mrna Expression ◽

Tissue Injury ◽

Inflammatory Cells ◽

Pcr Analysis ◽

Protective Effects ◽

Deep Tissue ◽

Deep Tissue Injury

Background: Chronic inflammation and lack of angiogenesis are the important pathological mechanisms in deep tissue injury (DTI). Curcumin is a well-known anti-inflammatory and antioxidant agent. However, curcumin is unstable under acidic and alkaline conditions, and can be rapidly metabolized and excreted in the bile, which shortens its bioactivity and efficacy. Objective: This study aimed to prepare curcumin-loaded poly (lactic-co-glycolic acid) nanoparticles (CPNPs) and to elucidate the protective effects and underlying mechanisms of wound healing in DTI models. Methods: CPNPs were evaluated for particle size, biocompatibility, in vitro drug release and their effect on in vivo wound healing. Results : The results of in vivo wound closure analysis revealed that CPNP treatments significantly improved wound contraction rates (p<0.01) at a faster rate than other three treatment groups. H&E staining revealed that CPNP treatments resulted in complete epithelialization and thick granulation tissue formation, whereas control groups resulted in a lack of compact epithelialization and persistence of inflammatory cells within the wound sites. Quantitative real-time PCR analysis showed that treatment with CPNPs suppressed IL-6 and TNF-α mRNA expression, and up-regulated TGF-β, VEGF-A and IL-10 mRNA expression. Western blot analysis showed up-regulated protein expression of TGF-β, VEGF-A and phosphorylatedSTAT3. Conclusion: Our results showed that CPNPs enhanced wound healing in DTI models, through modulation of the JAK2/STAT3 signalling pathway and subsequent upregulation of pro-healing factors.

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Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01948-20 ◽

2020 ◽

Vol 65 (1) ◽

pp. e01948-20

Author(s):

Dalin Rifat ◽

Si-Yang Li ◽

Thomas Ioerger ◽

Keshav Shah ◽

Jean-Philippe Lanoix ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Clinical Evidence ◽

Clinical Samples ◽

Loss Of Function ◽

Wild Type ◽

Content Type ◽

Solid Media ◽

High Level ◽

Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

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Cockayne syndrome B protein acts as an ATP-dependent processivity factor that helps RNA polymerase II overcome nucleosome barriers

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2013379117 ◽

2020 ◽

Vol 117 (41) ◽

pp. 25486-25493 ◽

Cited By ~ 1

Author(s):

Jun Xu ◽

Wei Wang ◽

Liang Xu ◽

Jia-Yu Chen ◽

Jenny Chong ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Neurological Diseases ◽

Transcription Elongation ◽

Cockayne Syndrome ◽

Stable Complex ◽

Loss Of Function ◽

Pol Ii ◽

Chromatin Remodelers ◽

Processivity Factor

While loss-of-function mutations in Cockayne syndrome group B protein (CSB) cause neurological diseases, this unique member of the SWI2/SNF2 family of chromatin remodelers has been broadly implicated in transcription elongation and transcription-coupled DNA damage repair, yet its mechanism remains largely elusive. Here, we use a reconstituted in vitro transcription system with purified polymerase II (Pol II) and Rad26, a yeast ortholog of CSB, to study the role of CSB in transcription elongation through nucleosome barriers. We show that CSB forms a stable complex with Pol II and acts as an ATP-dependent processivity factor that helps Pol II across a nucleosome barrier. This noncanonical mechanism is distinct from the canonical modes of chromatin remodelers that directly engage and remodel nucleosomes or transcription elongation factors that facilitate Pol II nucleosome bypass without hydrolyzing ATP. We propose a model where CSB facilitates gene expression by helping Pol II bypass chromatin obstacles while maintaining their structures.

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TRIM27 interacts with Iκbα to promote the growth of human renal cancer cells through regulating the NF-κB pathway

BMC Cancer ◽

10.1186/s12885-021-08562-5 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Chengwu Xiao ◽

Wei Zhang ◽

Meimian Hua ◽

Huan Chen ◽

Bin Yang ◽

...

Keyword(s):

Cell Lines ◽

Prognostic Indicator ◽

The Cancer Genome Atlas ◽

Mrna Levels ◽

Loss Of Function ◽

Protein Levels ◽

Kaplan Meier ◽

Cancer Genome Atlas

Abstract Background The tripartite motif (TRIM) family proteins exhibit oncogenic roles in various cancers. The roles of TRIM27, a member of the TRIM super family, in renal cell carcinoma (RCC) remained unexplored. In the current study, we aimed to investigate the clinical impact and roles of TRIM27 in the development of RCC. Methods The mRNA levels of TRIM27 and Kaplan–Meier survival of RCC were analyzed from The Cancer Genome Atlas database. Real-time PCR and Western blotting were used to measure the mRNA and protein levels of TRIM27 both in vivo and in vitro. siRNA and TRIM27 were exogenously overexpressed in RCC cell lines to manipulate TRIM27 expression. Results We discovered that TRIM27 was elevated in RCC patients, and the expression of TRIM27 was closely correlated with poor prognosis. The loss of function and gain of function results illustrated that TRIM27 promotes cell proliferation and inhibits apoptosis in RCC cell lines. Furthermore, TRIM27 expression was positively associated with NF-κB expression in patients with RCC. Blocking the activity of NF-κB attenuated the TRIM27-mediated enhancement of proliferation and inhibition of apoptosis. TRIM27 directly interacted with Iκbα, an inhibitor of NF-κB, to promote its ubiquitination, and the inhibitory effects of TRIM27 on Iκbα led to NF-κB activation. Conclusions Our results suggest that TRIM27 exhibits an oncogenic role in RCC by regulating NF-κB signaling. TRIM27 serves as a specific prognostic indicator for RCC, and strategies targeting the suppression of TRIM27 function may shed light on future therapeutic approaches.

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