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1.
J Am Coll Health ; : 1-8, 2023 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-36701421

RESUMO

Objective: Research indicates that coping styles mediate self-control and health outcomes. Emotion- and problem-focused coping strategies (eg, getting advice or planning) are used to address stressors. In contrast, avoidance-focused strategies (eg, substance use) are used to escape distress and are associated with greater alcohol problems. The purpose of this study was to examine associations between college students' levels of self-control, coping styles, and alcohol use and problems. Participants and Methods: 183 undergraduates completed questionnaires regarding self-control, coping styles, and alcohol consumption and problems. We hypothesized that self-control would be associated with alcohol problems through avoidance-focused coping, but not emotion- or problem-focused coping. Results: Our results were consistent with our hypothesis with and without controlling for alcohol consumption. Undergraduates lower in self-control who engage in avoidance-focused coping may experience greater alcohol problems. Conclusions: University programs dedicated to addressing substance use among undergraduates may develop workshops that promote problem- or emotion-focused coping strategies as alternatives to avoidance-focused strategies.

2.
Cancer Discov ; 13(1): 14-16, 2023 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-36620883

RESUMO

SUMMARY: p53 mutant proteins are widely expressed in human cancer. In this issue, Guiley and Shokat describe the development of compounds that rescue the function of the Y220C mutant p53 protein by forming covalent complexes with the target protein. See related article by Guiley and Shokat, p. 56 (3).


Assuntos
Neoplasias , Proteína Supressora de Tumor p53 , Humanos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética
3.
Sci Rep ; 12(1): 22564, 2022 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-36581692

RESUMO

The RON receptor tyrosine kinase is an exceptionally interesting target in oncology and immunology. It is not only overexpressed in a wide variety of tumors but also has been shown to be expressed on myeloid cells associated with tumor infiltration, where it serves to dampen tumour immune responses and reduce the efficacy of anti-CTLA4 therapy. Potent and selective inhibitory antibodies to RON might therefore both inhibit tumor cell growth and stimulate immune rejection of tumors. We derived cloned and sequenced a new panel of exceptionally avid anti-RON antibodies with picomolar binding affinities that inhibit MSP-induced RON signaling and show remarkable potency in antibody dependent cellular cytotoxicity. Antibody specificity was validated by cloning the antibody genes and creating recombinant antibodies and by the use of RON knock out cell lines. When radiolabeled with 89-Zirconium, the new antibodies 3F8 and 10G1 allow effective immuno-positron emission tomography (immunoPET) imaging of RON-expressing tumors and recognize universally exposed RON epitopes at the cell surface. The 10G1 was further developed into a novel bispecific T cell engager with a 15 pM EC50 in cytotoxic T cell killing assays.


Assuntos
Anticorpos Monoclonais , Transdução de Sinais , Linhagem Celular Tumoral , Proliferação de Células
4.
Sci Justice ; 62(5): 582-593, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36336451

RESUMO

The need to forensically search soil for small artefacts at a burial site or traces of evidence in a deposition site is a common task shared by investigators and forensic archaeologists. In forensic casework, the importance of finding small pieces of evidence, such as personal effects or ballistic fragments, cannot be overstated as it can assist in the positive identification of the deceased, give an insight into the manner and cause of death, and identify any perpetrators. The soil search methods known as wet and dry sieving, are cumbersome, time-consuming and have limited success for some soil types. This often leads to the decision not to search, resulting in missed opportunities to identify potential evidence. The primary aim of this study was to investigate if a dual energy X-ray baggage scanner could be used to search for items of potential forensic interest in soil. A trial was conducted using a Smiths Detection ScanTrailer 100100 V-2is mobile X-ray inspection system to establish if it could be used to detect organic, inorganic, and metallic items located within soil. The soil type and natural variables such as water and organic content were adjusted to simulate different environments. The baggage scanner was found to provide a quick and easy way to detect items contained within various soil types, particularly in a sand rich matrix. It is estimated that using this method to search 1 m3 of soil, when broken down into samples that are < 13 cm in depth, would take around one hour to complete, compared with 100 to 150 person-hours by manual sieving. This is believed to be the first use of dual energy X-ray technology for this purpose and shows the potential for further research and use of this method in forensic archaeology.


Assuntos
Arqueologia , Solo , Humanos , Raios X , Sepultamento , Medicina Legal/métodos
5.
Nat Commun ; 13(1): 4854, 2022 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-35982046

RESUMO

An attractive approach to target intracellular macromolecular interfaces and to model putative drug interactions is to design small high-affinity proteins. Variable domains of the immunoglobulin heavy chain (VH domains) are ideal miniproteins, but their development has been restricted by poor intracellular stability and expression. Here we show that an autonomous and disufhide-free VH domain is suitable for intracellular studies and use it to construct a high-diversity phage display library. Using this library and affinity maturation techniques we identify VH domains with picomolar affinity against eIF4E, a protein commonly hyper-activated in cancer. We demonstrate that these molecules interact with eIF4E at the eIF4G binding site via a distinct structural pose. Intracellular overexpression of these miniproteins reduce cellular proliferation and expression of malignancy-related proteins in cancer cell lines. The linkage of high-diversity in vitro libraries with an intracellularly expressible miniprotein scaffold will facilitate the discovery of VH domains suitable for intracellular applications.


Assuntos
Fator de Iniciação 4E em Eucariotos , Fator de Iniciação 4F em Eucariotos , Técnicas de Visualização da Superfície Celular , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4F em Eucariotos/metabolismo , Biblioteca Gênica , Cadeias Pesadas de Imunoglobulinas/genética
6.
Front Pharmacol ; 13: 883083, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35571133

RESUMO

The process of sprouting angiogenesis can be measured in vitro using endothelial cells in sprouting assays such as the fibrin bead assay and the spheroid-based assay. While the technical aspects of these sprouting assays have been well-optimized, the analysis aspects have been limited to manual methods, which can be time-consuming and difficult to reproduce. Here, we developed an automated analysis tool called AQuTAS to quantify sprouting parameters from the spheroid-based sprouting assay. We trained and validated the algorithm on two subsets of data, and tested its sensitivity by measuring changes in sprouting parameters over a range of concentrations of pro- and antiangiogenic compounds. Our results demonstrate that the algorithm detects known differences in sprouting parameters in endothelial spheroids treated with pro- and antiangiogenic compounds. Moreover, it is sensitive to biological changes that are ≥40%. Among the five quantified parameters, cumulative sprout length is likely the most discriminative parameter for measuring differences in sprouting behavior because it had the highest effect size (>1.5 Cohen's d). In summary, we have generated an automated tool that quantifies sprouting parameters from the spheroid-based assay in a reproducible and sensitive manner.

7.
Open Vet J ; 12(1): 105-113, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35342724

RESUMO

Background: In humans, multiple researchers have not only determined that there is a relationship between urinary incontinence (UI) and lower back pain (LBP), but that by treating the LBP, clinicians are able to improve or resolve the UI. Up until now, no equivalent canine research has investigated whether treatment of LBP can improve the clinical signs of acquired, non-neurologic UI in dogs. Aim: To determine if a relationship exists between LBP and urethral sphincter mechanism incompetence (USMI) in dogs. Methods: Review of medical records of all patients that presented to Points East West Veterinary Services with a history of naturally occurring acquired UI from May 2013 to December 2019. Thirty-nine patients treated for LBP using combined acupuncture and manual therapy, and 33/39 patients that also received concurrent photobiomodulation (PBM) therapy, qualified for this study. Results: Treated patients showed a statistically significant reduction in both the frequency (p < 0.01) and volume (p < 0.01) of UI episodes. Treatment responses ranged from no improvement, to complete resolution of the USMI clinical signs. Conclusion: The reduction of USMI clinical signs following LBP treatment suggests a relationship between these two conditions. Combined acupuncture, manual therapy, with or without PBM was shown to be an effective treatment for USMI. By corollary, USMI incontinence should be considered a potential pain symptom.


Assuntos
Doenças do Cão , Dor Lombar , Incontinência Urinária , Animais , Doenças do Cão/diagnóstico , Cães , Feminino , Humanos , Dor Lombar/terapia , Dor Lombar/veterinária , Masculino , Resultado do Tratamento , Uretra , Incontinência Urinária/diagnóstico , Incontinência Urinária/terapia , Incontinência Urinária/veterinária
8.
Structure ; 30(5): 733-742.e7, 2022 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-35290795

RESUMO

Disordered proteins pose a major challenge to structural biology. A prominent example is the tumor suppressor p53, whose low expression levels and poor conformational stability hamper the development of cancer therapeutics. All these characteristics make it a prime example of "life on the edge of solubility." Here, we investigate whether these features can be modulated by fusing the protein to a highly soluble spider silk domain (NT∗). The chimeric protein displays highly efficient translation and is fully active in human cancer cells. Biophysical characterization reveals a compact conformation, with the disordered transactivation domain of p53 wrapped around the NT∗ domain. We conclude that interactions with NT∗ help to unblock translation of the proline-rich disordered region of p53. Expression of partially disordered cancer targets is similarly enhanced by NT∗. In summary, we demonstrate that inducing co-translational folding via a molecular "spindle and thread" mechanism unblocks protein translation in vitro.


Assuntos
Neoplasias , Proteína Supressora de Tumor p53 , Humanos , Ligação Proteica , Domínios Proteicos , Proteína Supressora de Tumor p53/metabolismo
9.
Proc Natl Acad Sci U S A ; 119(10): e2113233119, 2022 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-35235448

RESUMO

SignificanceOur work focuses on the critical longstanding question of the nontranscriptional role of p53 in tumor suppression. We demonstrate here that poly(ADP-ribose) polymerase (PARP)-dependent modification of p53 enables rapid recruitment of p53 to damage sites, where it in turn directs early repair pathway selection. Specifically, p53-mediated recruitment of 53BP1 at early time points promotes nonhomologous end joining over the more error-prone microhomology end-joining. Similarly, p53 directs nucleotide excision repair by mediating DDB1 recruitment. This property of p53 also correlates with tumor suppression in vivo. Our study provides mechanistic insight into how certain transcriptionally deficient p53 mutants may retain tumor-suppressive functions through regulating the DNA damage response.


Assuntos
Dano ao DNA , Reparo do DNA por Junção de Extremidades , Proteína Supressora de Tumor p53/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação a DNA , Humanos , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Domínios Proteicos , Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética
11.
Cell Chem Biol ; 29(4): 572-585.e8, 2022 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-34265272

RESUMO

The optimal use of many cancer drugs is hampered by a lack of detailed understanding of their mechanism of action (MoA). Here, we apply a high-resolution implementation of the proteome-wide cellular thermal shift assay (CETSA) to follow protein interaction changes induced by the antimetabolite 5-fluorouracil (5-FU) and related nucleosides. We confirm anticipated effects on the known main target, thymidylate synthase (TYMS), and enzymes in pyrimidine metabolism and DNA damage pathways. However, most interaction changes we see are for proteins previously not associated with the MoA of 5-FU, including wide-ranging effects on RNA-modification and -processing pathways. Attenuated responses of specific proteins in a resistant cell model identify key components of the 5-FU MoA, where intriguingly the abrogation of TYMS inhibition is not required for cell proliferation.


Assuntos
Antineoplásicos , Neoplasias , Antineoplásicos/farmacologia , Fluoruracila/farmacologia , Proteoma , Proteômica , RNA
12.
Dermatology ; 238(3): 527-533, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34610598

RESUMO

BACKGROUND: The incidence of cutaneous malignant melanoma continues to increase worldwide and in Canada. It is unclear whether the increase in incidence and clinical characteristic trends of cutaneous malignant melanoma are similar in the province of Newfoundland and Labrador. OBJECTIVE: The objective of this study is to examine the incidence and trends of cutaneous malignant melanoma in Eastern Newfoundland and Labrador. METHODS: Patients aged 18 years or older diagnosed with cutaneous malignant melanoma were identified from the Eastern Health Authority's Cancer Registry. The diagnosis was confirmed by a pathologist via histological subtype. Patients were excluded if the diagnosis was unspecified, a nonmelanoma skin cancer or if there was a recurrence in the same lesion location. In total 298 patients diagnosed with cutaneous malignant melanoma from 2007 to 2015 were included in the analysis. RESULTS: The total incidence increased from 4.1 to 15.6 cases/100,000 person-years, which represents a 283.0% increase from 2007 to 2015. The largest increases in incidence were seen in males and patients aged from 60 to 79 years. The most common lesion anatomical locations were the trunk in males and the lower extremity in females. The majority of cases had a Breslow thickness below 1.0 mm. CONCLUSION: The incidence of cutaneous malignant melanoma in Eastern Newfoundland and Labrador is increasing at a faster rate than in any other region in Canada. Health care providers should work to be aware of the clinical trends and risk factors associated with this disease to facilitate early detection and prevent morbidity.


Assuntos
Melanoma , Neoplasias Cutâneas , Canadá/epidemiologia , Feminino , Humanos , Incidência , Masculino , Melanoma/epidemiologia , Terra Nova e Labrador/epidemiologia , Neoplasias Cutâneas/epidemiologia
13.
Blood Adv ; 6(2): 704-715, 2022 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-34731882

RESUMO

Protein S is a cofactor in the tissue factor pathway inhibitor (TFPI) anticoagulant pathway. It enhances TFPIα-mediated inhibition of factor (F)Xa activity and generation. The enhancement is dependent on a TFPIα-protein S interaction involving TFPIα Kunitz 3 and protein S laminin G-type (LG)-1. C4b binding protein (C4BP), which binds to protein S LG1, almost completely abolishes its TFPI cofactor function. However, neither the amino acids involved in TFPIα enhancement nor the mechanisms underlying the reduced TFPI cofactor function of C4BP-bound protein S are known. To screen for functionally important regions within protein S LG1, we generated 7 variants with inserted N-linked glycosylation attachment sites. Protein S D253T and Q427N/K429T displayed severely reduced TFPI cofactor function while showing normal activated protein C (APC) cofactor function and C4BP binding. Based on these results, we designed 4 protein S variants in which 4 to 6 surface-exposed charged residues were substituted for alanine. One variant, protein S K255A/E257A/D287A/R410A/K423A/E424A, exhibited either abolished or severely reduced TFPI cofactor function in plasma and FXa inhibition assays, both in the presence or absence of FV-short, but retained normal APC cofactor function and high-affinity C4BP binding. The C4BP ß-chain was expressed to determine the mechanisms behind the reduced TFPI cofactor function of C4BP-bound protein S. Like C4BP-bound protein S, C4BP ß-chain-bound protein S had severely reduced TFPI cofactor function. These results show that protein S Lys255, Glu257, Asp287, Arg410, Lys423, and Glu424 are critical for protein S-mediated enhancement of TFPIα and that binding of the C4BP ß-chain blocks this function.


Assuntos
Laminina , Proteína S , Proteína de Ligação ao Complemento C4b , Fator V/metabolismo , Lipoproteínas , Proteína S/química , Proteína S/metabolismo , Trombina/metabolismo
14.
Cancer Res ; 82(1): 36-45, 2022 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-34750099

RESUMO

Inactivating p53 mutations are the most abundant genetic alterations found in cancer. Here we show that CRISPR/Cas9-induced double-stranded DNA breaks enrich for cells deficient in p53 and in genes of a core CRISPR-p53 tumor suppressor interactome. Such enrichment could predispose to cancer development and thus pose a challenge for clinical CRISPR use. Transient p53 inhibition could suppress the enrichment of cells with these mutations. The level of DNA damage response induced by an sgRNA influenced the enrichment of p53-deficient cells and could be a relevant parameter in sgRNA design to limit cellular enrichment. Furthermore, a dataset of >800 human cancer cell lines identified additional factors influencing the enrichment of p53-mutated cells, including strong baseline CDKN1A expression as a predictor for an active CRISPR-p53 axis. Taken together, these data provide details about p53 biology in the context of CRISPR-induced DNA damage and identify strategies to enable safer CRISPR use. SIGNIFICANCE: CRISPR-mediated DNA damage enriches for cells with escape mutations in a core CRISPR-p53 interactome, which can be suppressed by transient inhibition of p53.


Assuntos
Sistemas CRISPR-Cas/genética , Dano ao DNA/genética , Proteína Supressora de Tumor p53/genética , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Mutação , Transfecção
16.
Biomolecules ; 11(11)2021 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-34827693

RESUMO

p53 is involved in DNA damage response and is an exciting target for radiosensitization in cancer. Targeted radionuclide therapy against somatostatin receptors with 177Lu-DOTATATE is currently being explored as a treatment for neuroblastoma. The aim of this study was to investigate the novel p53-stabilizing peptide VIP116 in neuroblastoma, both as monotherapy and together with 177Lu-DOTATATE. Five neuroblastoma cell lines, including two patient-derived xenograft (PDX) lines, were characterized in monolayer cultures. Four out of five were positive for 177Lu-DOTATATE uptake. IC50 values after VIP116 treatments correlated with p53 status, ranging between 2.8-238.2 µM. IMR-32 and PDX lines LU-NB-1 and LU-NB-2 were then cultured as multicellular tumor spheroids and treated with 177Lu-DOTATATE and/or VIP116. Spheroid growth was inhibited in all spheroid models for all treatment modalities. The most pronounced effects were observed for combination treatments, mediating synergistic effects in the IMR-32 model. VIP116 and combination treatment increased p53 levels with subsequent induction of p21, Bax and cleaved caspase 3. Combination treatment resulted in a 14-fold and 1.6-fold induction of MDM2 in LU-NB-2 and IMR-32 spheroids, respectively. This, together with differential MYCN signaling, may explain the varying degree of synergy. In conclusion, VIP116 inhibited neuroblastoma cell growth, potentiated 177Lu-DOTATATE treatment and could, therefore, be a feasible treatment option for neuroblastoma.


Assuntos
Proteína Supressora de Tumor p53 , Humanos , Neuroblastoma , Tomografia por Emissão de Pósitrons , Cintilografia , Receptores de Somatostatina
17.
Biomolecules ; 11(7)2021 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-34356680

RESUMO

Proteins of the major histocompatibility complex (MHC) class I, or human leukocyte antigen (HLA) in humans interact with endogenous peptides and present them to T cell receptors (TCR), which in turn tune the immune system to recognize and discriminate between self and foreign (non-self) peptides. Of especial importance are peptides derived from tumor-associated antigens. T cells recognizing these peptides are found in cancer patients, but not in cancer-free individuals. What stimulates this recognition, which is vital for the success of checkpoint based therapy? A peptide derived from the protein p53 (residues 161-169 or p161) was reported to show this behavior. T cells recognizing this unmodified peptide could be further stimulated in vitro to create effective cancer killing CTLs (cytotoxic T lymphocytes). We hypothesize that the underlying difference may arise from post-translational glycosylation of p161 in normal individuals, likely masking it against recognition by TCR. Defects in glycosylation in cancer cells may allow the presentation of the native peptide. We investigate the structural consequences of such peptide glycosylation by investigating the associated structural dynamics.


Assuntos
Antígeno HLA-A24/química , Antígeno HLA-A24/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Acetilglucosamina/metabolismo , Glicosilação , Proteínas do Vírus da Imunodeficiência Humana/química , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Simulação de Dinâmica Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Receptores de Antígenos de Linfócitos T/química , Proteína Supressora de Tumor p53/química
19.
Cancers (Basel) ; 13(12)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34203762

RESUMO

Extracellular vesicles (EVs) shed by cancer cells play a major role in mediating the transfer of molecular information by reprogramming the tumor microenvironment (TME). TP53 (encoding the p53 protein) is the most mutated gene across many cancer types. Mutations in TP53 not only result in the loss of its tumor-suppressive properties but also results in the acquisition of novel gain-of-functions (GOF) that promote the growth of cancer cells. Here, we demonstrate that GOF mutant p53 proteins can be transferred via EVs to neighboring cancer cells and to macrophages, thus modulating them to release tumor supportive cytokines. Our data from pancreatic, lung, and colon carcinoma cell lines demonstrate that the mutant p53 protein can be selectively sorted into EVs. More specifically, mutant p53 proteins in EVs can be taken up by neighboring cells and mutant p53 expression is found in non-tumor cells in both human cancers and in non-human tissues in human xenografts. Our findings shed light on the intricate methods in which specific GOF p53 mutants can promote oncogenic mechanisms by reprogramming and then recruiting non-cancerous elements for tumor progression.

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