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1.
Angew Chem Int Ed Engl ; 61(44): e202211774, 2022 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-36083191

RESUMO

Obesity is a chronic health condition characterized by the accumulation of excessive body fat which can lead to and exacerbate cardiovascular disease, type-II diabetes, high blood pressure, and cancer through systemic inflammation. Unfortunately, visualizing key mediators of the inflammatory response, such as monoacylglycerol lipase (MGL) and fatty acid amide hydrolase (FAAH), in a selective manner is a profound challenge owing to an overlapping substrate scope that involves arachidonic acid (AA). Specifically, these enzymes work in concert to generate AA, which in the context of obesity, has been implicated to control appetite and energy metabolism. In this study, we developed the first selective activity-based sensing probes to detect MGL (PA-HD-MGL) and FAAH (PA-HD-FAAH) activity via photoacoustic imaging. Activation of PA-HD-MGL and PA-HD-FAAH by their target enzymes resulted in 1.74-fold and 1.59-fold signal enhancements, respectively. Due to their exceptional selectivity profiles and deep-tissue photoacoustic imaging capabilities, these probes were employed to measure MGL and FAAH activity in a murine model of obesity. Contrary to conflicting reports suggesting levels of MGL can be attenuated or elevated, our results support the latter. Indeed, we discovered a marked increase of both targets in the gastrointestinal tract. These key findings set the stage to uncover the role of the endocannabinoid pathway in obesity-mediated inflammation.


Assuntos
Endocanabinoides , Monoacilglicerol Lipases , Animais , Camundongos , Humanos , Monoacilglicerol Lipases/química , Monoacilglicerol Lipases/metabolismo , Ácido Araquidônico , Modelos Animais de Doenças , Amidoidrolases/metabolismo , Obesidade/diagnóstico por imagem , Inflamação
2.
Health Info Libr J ; 39(4): 336-346, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-35808921

RESUMO

BACKGROUND: Responding to the COVID-19 pandemic, Health Education England (HEE) mobilised a group of expert searchers from NHS libraries in England to develop a platform for librarians to share peer reviewed search strategies and results on the Knowledge for Healthcare website. OBJECTIVES: (1) To document the origins of the COVID-19 search bank, (2) evaluate attitudes of NHS librarians in England towards the search bank and (3) identify lessons learned and consider whether the initiative might be developed further. METHODS: Structured interviews with the peer reviewers (n = 10) were conducted, and a questionnaire survey of the NHS library community using the search bank was undertaken. RESULTS: The interviews confirmed the value of collaboration. Expert searchers worked in pairs to peer review submitted search strategies. The survey (85 responses) indicated that a majority had used the search bank, and approved of the project, with some differences of opinion on functionality and future developments. DISCUSSION: Collaborative working for the search bank probably saved time for individual NHS librarians. The quality of the searches submitted was variable as were librarians' approaches to presentation and development of search strategies. Peer review benefits from a buddy approach among expert searchers and agreement about feedback provided to contributors. CONCLUSION: Search strategies are the most useful element of a search bank. Peer review can be challenging and would benefit from a formal structure, but it is professionally rewarding.


Assuntos
COVID-19 , Bibliotecários , Bibliotecas Médicas , Humanos , Medicina Estatal , Pandemias , Revisão por Pares
3.
J Am Chem Soc ; 144(31): 14351-14362, 2022 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-35905456

RESUMO

Near-infrared (NIR) fluorophores absorbing maximally in the region beyond 800 nm, i.e., deep-NIR spectral region, are actively sought for biomedical applications. Ideal dyes are bright, nontoxic, photostable, biocompatible, and easily derivatized to introduce functionalities (e.g., for bioconjugation or aqueous solubility). The rational design of such fluorophores remains a major challenge. Silicon-substituted rhodamines have been successful for bioimaging applications in the red spectral region. The longer-wavelength silicon-substituted congeners for the deep-NIR spectral region are unknown to date. We successfully prepared four silicon-substituted bis-benzannulated rhodamine dyes (ESi5a-ESi5d), with an efficient five-step cascade on a gram-scale. Because of the extensive overlapping of their HOMO-LUMO orbitals, ESi5a-ESi5d are highly absorbing (λabs ≈ 865 nm and ε > 105 cm-1 M-1). By restraining both the rotational freedom via annulation and the vibrational freedom via silicon-imparted strain, the fluorochromic scaffold of ESi5 is highly rigid, resulting in an unusually long fluorescence lifetime (τ > 700 ps in CH2Cl2) and a high fluorescence quantum yield (ϕ = 0.14 in CH2Cl2). Their half-lives toward photobleaching are 2 orders of magnitude longer than the current standard (ICG in serum). They are stable in the presence of biorelevant concentration of nucleophiles or reactive oxygen species. They are minimally toxic and readily metabolized. Upon tail vein injection of ESi5a (as an example), the vasculature of a nude mouse was imaged with a high signal-to-background ratio. ESi5 dyes have broad potentials for bioimaging in the deep-NIR spectral region.


Assuntos
Corantes Fluorescentes , Silício , Animais , Fluorescência , Camundongos , Rodaminas
4.
Microb Genom ; 8(7)2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35876490

RESUMO

The COVID-19 pandemic continues to expand globally, with case numbers rising in many areas of the world, including the Eastern Mediterranean Region. Lebanon experienced its largest wave of COVID-19 infections from January to April 2021. Limited genomic surveillance was undertaken, with just 26 SARS-CoV-2 genomes available for this period, nine of which were from travellers from Lebanon detected by other countries. Additional genome sequencing is thus needed to allow surveillance of variants in circulation. In total, 905 SARS-CoV-2 genomes were sequenced using the ARTIC protocol. The genomes were derived from SARS-CoV-2-positive samples, selected retrospectively from the sentinel COVID-19 surveillance network, to capture diversity of location, sampling time, sex, nationality and age. Although 16 PANGO lineages were circulating in Lebanon in January 2021, by February there were just four, with the Alpha variant accounting for 97 % of samples. In the following 2 months, all samples contained the Alpha variant. However, this had changed dramatically by June and July 2021, when all samples belonged to the Delta variant. This study documents a ten-fold increase in the number of SARS-CoV-2 genomes available from Lebanon. The Alpha variant, first detected in the UK, rapidly swept through Lebanon, causing the country's largest wave to date, which peaked in January 2021. The Alpha variant was introduced to Lebanon multiple times despite travel restrictions, but the source of these introductions remains uncertain. The Delta variant was detected in Gambia in travellers from Lebanon in mid-May, suggesting community transmission in Lebanon several weeks before this variant was detected in the country. Prospective sequencing in June/July 2021 showed that the Delta variant had completely replaced the Alpha variant in under 6 weeks.


Assuntos
COVID-19 , SARS-CoV-2 , COVID-19/epidemiologia , Genoma Viral/genética , Humanos , Líbano/epidemiologia , Pandemias , Filogenia , Estudos Prospectivos , Estudos Retrospectivos , SARS-CoV-2/genética
5.
FASEB J ; 36 Suppl 12022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35555578

RESUMO

Cholesterol is a key biological molecule that is a vital precursor of steroid molecule biosynthesis, including steroid hormones, bile acids and vitamin D3. These processes require 3ß-hydroxysteroid dehydrogenase (3ß-HSD) enzymes, which catalyze dehydrogenation of hydroxysteroids. This family of enzymes catalyzes a wide variety of reaction mechanisms, carrying out a combination of oxidation, isomerization and decarboxylase reactions that result in the formation of cholesterol, steroid hormones and bile acids. 3ß-hydroxy-Δ5 -C27 steroid dehydrogenase/isomerase type VII (HSD3B7) is responsible for the NAD+ dependent conversion of 7α-hydroxycholesterol (7-OHC) to 7α-hydroxy-3-oxo-4-cholestenoate (7-HOC), the second step of the bile acid synthesis pathway, which is vital for nutrient absorption and excretion of excess lipids. This oxidation reaction is one of many steps that increases the solubility of cholesterol for either storage or secretion. HSD3B7 shows substantial functional differences from other family members leaving significant gaps in our understanding of its mode of catalysis and substrate specificity. To this end, I have characterized HSD3B7 using Michaelis-Menten kinetics, thermal shift assays, and begun x-ray crystallographic studies. These studies reveal that HSD3B7 displays activity towards multiple bile acid precursors with differing hydrocarbon tail oxidation, with Km values in the low micromolar range. HSD3B7 retains its catalytic efficiency towards these substrates, suggesting that its substrate binding pocket can withstand changes in polarity upon alterations to this hydrocarbon tail. Lastly, HSD3B7 can be crystallized after modification to its membrane associated region, which will result in one of the first structures of a 3ß-HSD enzyme. Together these studies provide key insight into the mechanism of substrate specificity of HSD3B7 and appears amenable for X-ray crystal structure determination.

6.
Sci Transl Med ; 14(641): eabn6150, 2022 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-35258323

RESUMO

Breakthrough infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have been reported frequently in vaccinated individuals with waning immunity. In particular, a cluster of over 1000 infections with the SARS-CoV-2 delta variant was identified in a predominantly fully vaccinated population in Provincetown, Massachusetts in July 2021. In this study, vaccinated individuals who tested positive for SARS-CoV-2 (n = 16) demonstrated substantially higher serum antibody responses than vaccinated individuals who tested negative for SARS-CoV-2 (n = 23), including 32-fold higher binding antibody titers and 31-fold higher neutralizing antibody titers against the SARS-CoV-2 delta variant. Vaccinated individuals who tested positive also showed higher mucosal antibody responses in nasal secretions and higher spike protein-specific CD8+ T cell responses in peripheral blood than did vaccinated individuals who tested negative. These data demonstrate that fully vaccinated individuals developed robust anamnestic antibody and T cell responses after infection with the SARS-CoV-2 delta variant. Moreover, these findings suggest that population immunity will likely increase over time by a combination of widespread vaccination and breakthrough infections.


Assuntos
COVID-19 , SARS-CoV-2 , Anticorpos Antivirais , Formação de Anticorpos , Humanos
7.
Sci Transl Med ; 14(638): eabm4996, 2022 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-35191769

RESUMO

Ad26.COV2.S has demonstrated durability and clinical efficacy against symptomatic COVID-19 in humans. In this study, we report the correlates of durability of humoral and cellular immune responses in 20 rhesus macaques immunized with single-shot Ad26.COV2.S and the immunogenicity of a booster shot at 8 to 10 months after the initial immunization. Ad26.COV2.S elicited durable binding and neutralizing antibodies as well as memory B cells and long-lived bone marrow plasma cells. Innate immune responses and bone marrow plasma cell responses correlated with durable antibody responses. After Ad26.COV2.S boost immunization, binding and neutralizing antibody responses against multiple SARS-CoV-2 variants increased 31- to 69-fold and 23- to 43-fold, respectively, compared with preboost concentrations. Antigen-specific B cell and T cell responses also increased substantially after the boost immunization. Boosting with a modified Ad26.COV2.S.351 vaccine expressing the SARS-CoV-2 spike protein from the beta variant led to largely comparable responses with slightly higher beta- and omicron-specific humoral immune responses. These data demonstrate that a late boost with Ad26.COV2.S or Ad26.COV2.S.351 resulted in a marked increase in humoral and cellular immune responses that were highly cross-reactive across multiple SARS-CoV-2 variants in rhesus macaques.


Assuntos
Ad26COVS1 , COVID-19 , Imunidade Humoral , Imunização Secundária , SARS-CoV-2 , Ad26COVS1/imunologia , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19/prevenção & controle , Macaca mulatta , Glicoproteína da Espícula de Coronavírus
8.
NPJ Vaccines ; 7(1): 2, 2022 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-35013325

RESUMO

SARS-CoV-2 Spike-specific binding and neutralizing antibodies, elicited either by natural infection or vaccination, have emerged as potential correlates of protection. An important question, however, is whether vaccine-elicited antibodies in humans provide direct, functional protection from SARS-CoV-2 infection and disease. In this study, we explored directly the protective efficacy of human antibodies elicited by Ad26.COV2.S vaccination by adoptive transfer studies. IgG from plasma of Ad26.COV2.S vaccinated individuals was purified and transferred into naïve golden Syrian hamster recipients, followed by intra-nasal challenge of the hamsters with SARS-CoV-2. IgG purified from Ad26.COV2.S-vaccinated individuals provided dose-dependent protection in the recipient hamsters from weight loss following challenge. In contrast, IgG purified from placebo recipients provided no protection in this adoptive transfer model. Attenuation of weight loss correlated with binding and neutralizing antibody titers of the passively transferred IgG. This study suggests that Ad26.COV2.S-elicited antibodies in humans are mechanistically involved in protection against SARS-CoV-2.

9.
Plant Dis ; 106(2): 357-359, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34340566

RESUMO

In this Short Communication we describe the occurrence of mummy berry associated with huckleberry (Vaccinium membranaceum) caused by Monilinia spp. in Oregon. To our knowledge, this is the first report of a Monilinia spp. associated with mummy berry of huckleberry in Oregon. Sequence data from our specimens reveal the closest identity was Monilinia vaccinii-corymbosi, a pathogen of commercial blueberry (Vaccinium corymbosum). This may be a new species of Monilinia, not previously reported on huckleberry, and further investigation is needed. Of specific importance, the huckleberry holds cultural importance as a sacred First Food of the Confederated Tribes of the Umatilla Indian Reservation and other Pacific Northwest tribes. Although plant pathogen management in natural landscapes presents unique challenges, we will work with tribal authorities to determine whether cultural management techniques may mitigate yield loss due to Monilinia spp.


Assuntos
Huckleberry (Planta) , Vaccinium , Frutas , Oregon
10.
J Virol ; 96(2): e0159921, 2022 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-34705557

RESUMO

Live oral vaccines have been explored for their protective efficacy against respiratory viruses, particularly for adenovirus serotypes 4 and 7. The potential of a live oral vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), however, remains unclear. In this study, we assessed the immunogenicity of live SARS-CoV-2 delivered to the gastrointestinal tract in rhesus macaques and its protective efficacy against intranasal and intratracheal SARS-CoV-2 challenge. Postpyloric administration of SARS-CoV-2 by esophagogastroduodenoscopy resulted in limited virus replication in the gastrointestinal tract and minimal to no induction of mucosal antibody titers in rectal swabs, nasal swabs, and bronchoalveolar lavage fluid. Low levels of serum neutralizing antibodies were induced and correlated with modestly diminished viral loads in nasal swabs and bronchoalveolar lavage fluid following intranasal and intratracheal SARS-CoV-2 challenge. Overall, our data show that postpyloric inoculation of live SARS-CoV-2 is weakly immunogenic and confers partial protection against respiratory SARS-CoV-2 challenge in rhesus macaques. IMPORTANCE SARS-CoV-2 remains a global threat, despite the rapid deployment but limited coverage of multiple vaccines. Alternative vaccine strategies that have favorable manufacturing timelines, greater ease of distribution, and improved coverage may offer significant public health benefits, especially in resource-limited settings. Live oral vaccines have the potential to address some of these limitations; however, no studies have yet been conducted to assess the immunogenicity and protective efficacy of a live oral vaccine against SARS-CoV-2. Here, we report that oral administration of live SARS-CoV-2 in nonhuman primates may offer prophylactic benefits, but the formulation and route of administration will require further optimization.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Vacinas contra COVID-19/administração & dosagem , COVID-19/prevenção & controle , Administração Oral , Animais , Feminino , Macaca mulatta , Masculino
11.
J Wound Care ; 30(Sup11): S1-S25, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34756097

RESUMO

This consensus document is endorsed by The Queen's Nursing Institute (QNI) and The Queen's Nursing Institute Scotland (QNIS).


Assuntos
Enfermagem em Saúde Comunitária , Perna (Membro) , Humanos , Escócia
12.
World J Hepatol ; 13(10): 1439-1449, 2021 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-34786178

RESUMO

BACKGROUND: Management of single small hepatocellular carcinoma (HCC) is straightforward with curative outcomes achieved by locoregional therapy or resection. Liver transplantation is often considered for multiple small or single large HCC. Management of two small HCC whether presenting synchronously or sequentially is less clear. AIM: To define the outcomes of patients presenting with two small HCC. METHODS: Retrospective review of HCC databases from multiple institutions of patients with either two synchronous or sequential HCC ≤ 3 cm between January 2000 and March 2018. Primary outcomes were overall survival (OS) and transplant-free survival (TFS). RESULTS: 104 patients were identified (male n = 89). Median age was 63 years (interquartile range 58-67.75) and the most common aetiology of liver disease was hepatitis C (40.4%). 59 (56.7%) had synchronous HCC and 45 (43.3%) had sequential. 36 patients died (34.6%) and 25 were transplanted (24.0%). 1, 3 and 5-year OS was 93.0%, 66.1% and 62.3% and 5-year post-transplant survival was 95.8%. 1, 3 and 5-year TFS was 82.1%, 45.85% and 37.8%. When synchronous and sequential groups were compared, OS (1,3 and 5 year synchronous 91.3%, 63.8%, 61.1%, sequential 95.3%, 69.5%, 64.6%, P = 0.41) was similar but TFS was higher in the sequential group (1,3 and 5 year synchronous 68.5%, 37.3% and 29.7%, sequential 93.2%, 56.6%, 48.5%, P = 0.02) though this difference did not remain during multivariate analysis. CONCLUSION: TFS in patients presenting with two HCC ≤ 3 cm is poor regardless of the timing of the second tumor. All patients presenting with two small HCC should be considered for transplantation.

14.
medRxiv ; 2021 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-34704104

RESUMO

BACKGROUND: A cluster of over a thousand infections with the SARS-CoV-2 delta variant was identified in a predominantly fully vaccinated population in Provincetown, Massachusetts in July 2021. Immune responses in breakthrough infections with the SARS-CoV-2 delta variant remain to be defined. METHODS: Humoral and cellular immune responses were assessed in 35 vaccinated individuals who were tested for SARS-CoV-2 in the Massachusetts Department of Public Health outbreak investigation. RESULTS: Vaccinated individuals who tested positive for SARS-CoV-2 demonstrated substantially higher antibody responses than vaccinated individuals who tested negative for SARS-CoV-2, including 28-fold higher binding antibody titers and 34-fold higher neutralizing antibody titers against the SARS-CoV-2 delta variant. Vaccinated individuals who tested positive also showed 4.4-fold higher Spike-specific CD8+ T cell responses against the SARS-CoV-2 delta variant than vaccinated individuals who tested negative. CONCLUSIONS: Fully vaccinated individuals developed robust anamnestic antibody and T cell responses following infection with the SARS-CoV-2 delta variant. These data suggest important immunologic benefits of vaccination in the context of breakthrough infections.

15.
Sci Transl Med ; 13(618): eabj2641, 2021 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-34546094

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that result in increased transmissibility and partial evasion of neutralizing antibodies have recently emerged. Whether natural immunity induced by the original SARS-CoV-2 WA1/2020 strain protects against rechallenge with these SARS-CoV-2 variants remains a critical unresolved question. In this study, we show that natural immunity induced by the WA1/2020 strain leads to partial but incomplete protection against the SARS-CoV-2 variants B.1.1.7 (alpha) and B.1.351 (beta) in rhesus macaques. We challenged rhesus macaques with B.1.1.7 and B.1.351 and showed that infection with these variants resulted in high viral replication in the upper and lower respiratory tract. We then infected rhesus macaques with the WA1/2020 strain and rechallenged them on day 35 with the WA1/2020, B.1.1.7, or B.1.351 variants. Natural immunity to WA1/2020 led to robust protection against rechallenge with WA1/2020 but only partial protection against rechallenge with B.1.351. An intermediate degree of protection was observed in rhesus macaques against rechallenge with B.1.1.7. These data demonstrate partial but incomplete protective efficacy of natural immunity induced by WA1/2020 against SARS-CoV-2 variants of concern. Our findings have important implications for both vaccination and public health strategies in the context of emerging SARS-CoV-2 variants of concern.


Assuntos
COVID-19 , SARS-CoV-2 , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , Humanos , Macaca mulatta , Reinfecção
16.
J Vis Exp ; (174)2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34398156

RESUMO

Tumor-associated macrophages (TAM) can switch their expression and cytokine profile according to external stimuli. This remarkable plasticity enables TAM to adapt to ongoing changes within the tumor microenvironment. Macrophages can have either primarily pro-inflammatory (M1-like) or anti-inflammatory (M2-like) attributes and can continually switch between these two main states. M2-like macrophages within the tumor environment are associated with cancer progression and poor prognosis in several types of cancer. Many different methods for inducing differentiation and polarization of THP-1 cells are used to investigate cellular and intercellular mechanisms and the effects of TAM within the microenvironment of tumors. Currently, there is no established model for M2-like macrophage polarization using the THP-1 cell line, and the results of expression and cytokine profiles of macrophages due to certain in vitro stimuli vary between studies. This protocol serves as detailed guidance to differentiate THP-1 monocyte-like cells into M0 macrophages and to further polarize cells into an M2-like phenotype within 14 days. We demonstrate the morphological changes of THP-1 monocyte-like cells, differentiated macrophages, and polarized M2-like macrophages using light microscopy. This model is the basis for cell line models investigating the anti-inflammatory effects of TAM and their interactions with other cell populations of the tumor microenvironment.


Assuntos
Leucemia , Monócitos , Diferenciação Celular , Linhagem Celular Tumoral , Humanos , Macrófagos , Fenótipo , Células THP-1 , Microambiente Tumoral
17.
Nature ; 596(7872): 423-427, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34161961

RESUMO

The emergence of SARS-CoV-2 variants that partially evade neutralizing antibodies poses a threat to the efficacy of current COVID-19 vaccines1,2. The Ad26.COV2.S vaccine expresses a stabilized spike protein from the WA1/2020 strain of SARS-CoV-2, and has recently demonstrated protective efficacy against symptomatic COVID-19 in humans in several geographical regions-including in South Africa, where 95% of sequenced viruses in cases of COVID-19 were the B.1.351 variant3. Here we show that Ad26.COV2.S elicits humoral and cellular immune responses that cross-react with the B.1.351 variant and protects against B.1.351 challenge in rhesus macaques. Ad26.COV2.S induced lower binding and neutralizing antibodies against B.1.351 as compared to WA1/2020, but elicited comparable CD8 and CD4 T cell responses against the WA1/2020, B.1.351, B.1.1.7, P.1 and CAL.20C variants. B.1.351 infection of control rhesus macaques resulted in higher levels of virus replication in bronchoalveolar lavage and nasal swabs than did WA1/2020 infection. Ad26.COV2.S provided robust protection against both WA1/2020 and B.1.351, although we observed higher levels of virus in vaccinated macaques after B.1.351 challenge. These data demonstrate that Ad26.COV2.S provided robust protection against B.1.351 challenge in rhesus macaques. Our findings have important implications for vaccine control of SARS-CoV-2 variants of concern.


Assuntos
Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , COVID-19/virologia , Imunidade Celular , Imunidade Humoral , Macaca mulatta/imunologia , SARS-CoV-2/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Líquido da Lavagem Broncoalveolar/virologia , COVID-19/imunologia , COVID-19/patologia , Feminino , Macaca mulatta/virologia , Masculino , Nariz/virologia , SARS-CoV-2/crescimento & desenvolvimento , SARS-CoV-2/patogenicidade , Linfócitos T/imunologia , Replicação Viral
18.
J Med Chem ; 64(12): 8263-8271, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34110812

RESUMO

Protein N-terminal acetyltransferase D (NatD, NAA40) that specifically acetylates the alpha-N-terminus of histone H4 and H2A has been implicated in various diseases, but no inhibitor has been reported for this important enzyme. Based on the acetyl transfer mechanism of NatD, we designed and prepared a series of highly potent NatD bisubstrate inhibitors by covalently linking coenzyme A to different peptide substrates via an acetyl or propionyl spacer. The most potent bisubstrate inhibitor displayed an apparent Ki value of 1.0 nM. Biochemical studies indicated that bisubstrate inhibitors are competitive to the peptide substrate and noncompetitive to the cofactor, suggesting that NatD undergoes an ordered Bi-Bi mechanism. We also demonstrated that these inhibitors are highly specific toward NatD, displaying about 1000-fold selectivity over other closely related acetyltransferases. High-resolution crystal structures of NatD bound to two of these inhibitors revealed the molecular basis for their selectivity and inhibition mechanism, providing a rational path for future inhibitor development.


Assuntos
Coenzima A/farmacologia , Inibidores Enzimáticos/farmacologia , Acetiltransferase N-Terminal D/antagonistas & inibidores , Peptídeos/farmacologia , Sequência de Aminoácidos , Linhagem Celular Tumoral , Coenzima A/síntese química , Coenzima A/metabolismo , Cristalografia por Raios X , Desenho de Fármacos , Ensaios Enzimáticos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Humanos , Cinética , Estrutura Molecular , Acetiltransferase N-Terminal D/química , Acetiltransferase N-Terminal D/metabolismo , Peptídeos/síntese química , Peptídeos/metabolismo , Ligação Proteica , Relação Estrutura-Atividade
19.
Angew Chem Int Ed Engl ; 60(34): 18860-18866, 2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34089556

RESUMO

Most photoacoustic (PA) imaging agents are based on the repurposing of existing fluorescent dye platforms that exhibit non-optimal properties for PA applications. Herein, we introduce PA-HD, a new dye scaffold optimized for PA probe development that features a 4.8-fold increase in sensitivity and a red-shift of the λabs from 690 nm to 745 nm to enable ratiometric imaging. Computational modeling was used to elucidate the origin of these enhanced properties. To demonstrate the generalizability of our remodeling efforts, we developed three probes for ß-galactosidase activity (PA-HD-Gal), nitroreductase activity (PA-HD-NTR), and H2 O2 (PA-HD-H2 O2 ). We generated two cancer models to evaluate PA-HD-Gal and PA-HD-NTR. We employed a murine model of Alzheimer's disease to test PA-HD-H2 O2 . There, we observed a PA signal increase at 735 nm of 1.79±0.20-fold relative to background, indicating the presence of oxidative stress. These results were confirmed via ratiometric calibration, which was not possible using the parent HD platform.


Assuntos
Doença de Alzheimer/diagnóstico por imagem , Carbocianinas/química , Corantes Fluorescentes/química , Imagem Óptica , Técnicas Fotoacústicas , Doença de Alzheimer/metabolismo , Animais , Encéfalo/diagnóstico por imagem , Modelos Animais de Doenças , Peróxido de Hidrogênio/química , Camundongos , Estrutura Molecular , Estresse Oxidativo
20.
Cell Death Discov ; 7(1): 61, 2021 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-33771981

RESUMO

Colon adenocarcinoma is a common cause of cancer-related deaths worldwide. Epithelial-mesenchymal transition is a major regulator of cancer metastasis, and increased understanding of this process is essential to improve patient outcomes. Long non-coding RNA (lncRNA) are important regulators of carcinogenesis. To identify lncRNAs associated with colon carcinogenesis, we performed an exploratory differential gene expression analysis comparing paired colon adenocarcinoma and normal colon epithelium using an RNA-sequencing data set. This analysis identified lncRNA ZFAS1 as significantly increased in colon cancer compared to normal colon epithelium. This finding was validated in an institutional cohort using laser capture microdissection. ZFAS1 was also found to be principally located in the cellular cytoplasm. ZFAS1 knockdown was associated with decreased cellular proliferation, migration, and invasion in two colon cancer cell lines (HT29 and SW480). MicroRNA-200b and microRNA-200c (miR-200b and miR-200c) are experimentally validated targets of ZFAS1, and this interaction was confirmed using reciprocal gene knockdown. ZFAS1 knockdown regulated ZEB1 gene expression and downstream targets E-cadherin and vimentin. Knockdown of miR-200b or miR-200c reversed the effect of ZFAS1 knockdown in the ZEB1/E-cadherin, vimentin signaling cascade, and the effects of cellular migration and invasion, but not cellular proliferation. ZFAS1 knockdown was also associated with decreased tumor growth in an in vivo mouse model. These results demonstrate the critical importance of ZFAS1 as a regulator of the miR-200/ZEB1/E-cadherin, vimentin signaling cascade.

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