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During the evolution of SARS-CoV-2 in humans, a D614G substitution in the spike glycoprotein (S) has emerged; virus containing this substitution has become the predominant circulating variant in the COVID-19 pandemic1. However, whether the increasing prevalence of this variant reflects a fitness advantage that improves replication and/or transmission in humans or is merely due to founder effects remains unknown. Here we use isogenic SARS-CoV-2 variants to demonstrate that the variant that contains S(D614G) has enhanced binding to the human cell-surface receptor angiotensin-converting enzyme 2 (ACE2), increased replication in primary human bronchial and nasal airway epithelial cultures as well as in a human ACE2 knock-in mouse model, and markedly increased replication and transmissibility in hamster and ferret models of SARS-CoV-2 infection. Our data show that the D614G substitution in S results in subtle increases in binding and replication in vitro, and provides a real competitive advantage in vivo-particularly during the transmission bottleneck. Our data therefore provide an explanation for the global predominance of the variant that contains S(D614G) among the SARS-CoV-2 viruses that are currently circulating.
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COVID-19/transmissão , COVID-19/virologia , Mutação , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/genética , Replicação Viral/genética , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Brônquios/citologia , Brônquios/virologia , COVID-19/epidemiologia , Linhagem Celular , Células Cultivadas , Cricetinae , Modelos Animais de Doenças , Células Epiteliais/virologia , Feminino , Furões/virologia , Efeito Fundador , Técnicas de Introdução de Genes , Aptidão Genética , Humanos , Masculino , Mesocricetus , Camundongos , Mucosa Nasal/citologia , Mucosa Nasal/virologia , Ligação Proteica , RNA Viral/análise , Receptores de Coronavírus/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidadeRESUMO
Engineering of catalytically active inorganic nanomaterials holds promising prospects for biomedicine. Catalytically active metal oxides show applications in enhancing wound healing but have also been employed to induce cell death in photodynamic or radiation therapy. Upon introduction into a biological system, nanomaterials are exposed to complex fluids, causing interaction and adsorption of ions and proteins. While protein corona formation on nanomaterials is acknowledged, its modulation of nanomaterial catalytic efficacy is less understood. In this study, proteomic analyses and nano-analytic methodologies quantify and characterize adsorbed proteins, correlating this protein layer with metal oxide catalytic activity in vitro and in vivo. The protein corona comprises up to 280 different proteins, constituting up to 38% by weight. Enhanced complement factors and other opsonins on nanocatalyst surfaces lead to their uptake into macrophages when applied topically, localizing >99% of the nanomaterials in tissue-resident macrophages. Initially, the formation of the protein corona significantly reduces the nanocatalysts' activity, but this activity can be partially recovered in endosomal conditions due to the proteolytic degradation of the corona. Overall, the research reveals the complex relationship between physisorbed proteins and the catalytic characteristics of specific metal oxide nanoparticles, providing design parameters for optimizing nanocatalysts in complex biological environments.
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Óxidos , Coroa de Proteína , Óxidos/química , Animais , Catálise , Coroa de Proteína/química , Coroa de Proteína/metabolismo , Agregados Proteicos , Macrófagos/metabolismo , Camundongos , Metais/química , Humanos , Células RAW 264.7RESUMO
Acetaminophen (APAP) is one of the most commonly used analgesic and anti-pyretic drugs, and APAP intoxication is one of the main reasons for liver transplantation following liver failure in the Western world. While APAP poisoning ultimately leads to liver necrosis, various programmed cell death modalities have been implicated, including ER stress-triggered apoptosis. The BCL-2 family member BOK (BCL-2-related ovarian killer) has been described to modulate the unfolded protein response and to promote chemical-induced liver injury. We therefore investigated the impact of the loss of BOK following APAP overdosing in mice. Surprisingly, we observed sex-dependent differences in the activation of the unfolded protein response (UPR) in both wildtype (WT) and Bok-/- mice, with increased activation of JNK in females compared with males. Loss of BOK led to a decrease in JNK activation and a reduced percentage of centrilobular necrosis in both sexes after APAP treatment; however, this protection was more pronounced in Bok-/- females. Nevertheless, serum ALT and AST levels of Bok-/- and WT mice were comparable, indicating that there was no major difference in the overall outcome of liver injury. We conclude that after APAP overdosing, loss of BOK affects initiating signaling steps linked to ER stress, but has a more minor impact on the outcome of liver necrosis. Furthermore, we observed sex-dependent differences that might be worthwhile to investigate.
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Acetaminofen/efeitos adversos , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Overdose de Drogas/complicações , Predisposição Genética para Doença , Proteínas Proto-Oncogênicas c-bcl-2/deficiência , Acetaminofen/administração & dosagem , Animais , Apoptose/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/patologia , Citocromo P-450 CYP2E1/genética , Citocromo P-450 CYP2E1/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Feminino , Regulação da Expressão Gênica , Genes p53 , Masculino , Camundongos , Camundongos Knockout , Índice de Gravidade de Doença , Fatores Sexuais , Resposta a Proteínas não Dobradas/efeitos dos fármacosRESUMO
Chicken proventricular necrosis virus (CPNV) is a recently described birnavirus, which has been proposed to be the cause of transmissible viral proventriculitis (TVP). The understanding of the epidemiology of both the virus and the disease is very limited. A retrospective investigation on TVP and CPNV in broiler chicken submissions from the UK from between 1994 and 2015 was performed with the aims of assessing the longitudinal temporal evolution of TVP and CPNV, and to review the histological proventricular lesions in the studied chickens. Ninety-nine of the 135 included submissions (73.3%) fulfilled the TVP-diagnostic criteria, while the remaining 36 submissions (26.7%) displayed only lymphocytic proventriculitis (LP). The first detection of CPNV by PCR dated from 2009. Results showed a rise in the number of both TVP and positive CPNV RT-PCR submissions from 2009 with a peak in 2013, suggesting that they may be an emerging or re-emerging disease and pathogen, respectively. Twenty-two out of the 99 submissions displaying TVP lesions (22%) and four out of the 36 (11%) submissions with LP gave positive CPNV RT-PCR results, further supporting the association between CPNV and TVP and confirming that CPNV is present in a low proportion of proventriculi that do not fulfil the TVP-diagnostic criteria. In addition, intranuclear inclusion bodies were observed in 22 of the submissions with TVP. The vast majority of these cases (21 of 22, 96%) gave negative CPNV RT-PCR results, raising the question of whether a virus other than CPNV is responsible for some of these TVP-affected cases.RESEARCH HIGHLIGHTSTVP and CPNV have been present in British broilers since at least 1994 and 2009, respectively.TVP and CPNV seem to be an emerging and re-emerging disease and pathogen, respectively.CPNV was detected in proventriculi with both TVP and LP-lesions.Viruses other than CPNV may be responsible for some TVP-affected cases.
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Infecções por Birnaviridae/veterinária , Birnaviridae/isolamento & purificação , Galinhas , Doenças das Aves Domésticas/virologia , Proventrículo/virologia , Gastropatias/veterinária , Animais , Birnaviridae/classificação , Birnaviridae/genética , Infecções por Birnaviridae/patologia , Infecções por Birnaviridae/virologia , Filogenia , Doenças das Aves Domésticas/patologia , Proventrículo/patologia , RNA Viral/química , RNA Viral/isolamento & purificação , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Alinhamento de Sequência/veterinária , Análise de Sequência de RNA/veterinária , Gastropatias/patologia , Gastropatias/virologiaRESUMO
Increasing evidence suggests that a new birnavirus, named chicken proventricular necrosis virus (CPNV), is the aetiological agent of transmissible viral proventriculitis (TVP). The present work aimed to explore the possible presence of both TVP and CPNV in the UK. Forty-four chickens showing TVP-compatible gross lesions were classified into three groups based on the histological lesions: (i) TVP-affected chickens: lymphocytic infiltration and glandular necrosis (n = 15); (ii) lymphocytic proventriculitis (LP)-affected chickens: lymphocytic infiltration without necrosis (n = 18); and (iii) without proventriculitis (WP): no lymphocytic infiltration or necrosis (n = 11). Nine proventriculi (seven out of 15 corresponding to TVP, and two out of 11 corresponding to LP) were positive for CPNV by reverse transcriptase polymerase chain reaction (RT-PCR). These results support the previously suggested idea of CPNV as causative agent of TVP. Moreover, these data show that CPNV can also be detected in a number of cases with LP, which do not fulfil the histological TVP criteria. Phylogenetic analysis of partial sequences of gene VP1 showed that British CPNV sequences were closer to other European CPNV sequences and might constitute a different lineage from the American CPNV. TVP cases with negative CPNV PCR results may be due to chronic stages of the disease or to the reduced PCR sensitivity on formalin-fixed paraffin-embedded tissues. However, involvement of other agents in some of the cases cannot totally be ruled out. As far as the authors are aware, this is the first peer-reviewed report of TVP as well as of CPNV in the UK, and the first exploratory CPNV phylogenetic study.
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Infecções por Birnaviridae/veterinária , Birnaviridae/isolamento & purificação , Galinhas/virologia , Doenças das Aves Domésticas/virologia , Animais , Birnaviridae/classificação , Birnaviridae/genética , Infecções por Birnaviridae/diagnóstico , Infecções por Birnaviridae/patologia , Infecções por Birnaviridae/virologia , Necrose/veterinária , Filogenia , Doenças das Aves Domésticas/patologia , Estudos Prospectivos , Proventrículo/patologia , Proventrículo/virologia , Análise de Sequência de RNA/veterinária , Reino Unido/epidemiologiaRESUMO
Acute kidney injury (AKI) is a common and serious condition with no specific treatment. An episode of AKI may affect organs distant from the kidney, further increasing the morbidity associated with AKI. The mechanism of organ cross talk after AKI is unclear. The renal and immune systems of pigs and humans are alike. Using a preclinical animal (porcine) model, we tested the hypothesis that early effects of AKI on distant organs is by immune cell infiltration, leading to inflammatory cytokine production, extravasation, and edema. In 29 pigs exposed to either sham surgery or renal ischemia-reperfusion (control, n = 12; AKI, n = 17), we assessed remote organ (liver, lung, brain) effects in the short (from 2- to 48-h reperfusion) and longer term (5 wk later) using immunofluorescence (for leukocyte infiltration, apoptosis), a cytokine array, tissue elemental analysis (e.g., electrolytes), blood hematology and chemistry (e.g., liver enzymes), and PCR (for inflammatory markers). AKI elicited significant, short-term (â¼24 h) increments in enzymes indicative of acute liver damage (e.g. , AST: ALT ratio; P = 0.02) and influenced tissue biochemistry in some remote organs (e.g., lung tissue [Ca(2+)] increased; P = 0.04). These effects largely resolved after 48 h, and no further histopathology, edema, apoptosis, or immune cell infiltration was noted in the liver, lung, or hippocampus in the short and longer term. AKI has subtle biochemical effects on remote organs in the short term, including a transient increment in markers of acute liver damage. These effects resolved by 48 h, and no further remote organ histopathology, apoptosis, edema, or immune cell infiltration was noted.
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Injúria Renal Aguda/complicações , Injúria Renal Aguda/fisiopatologia , Animais , Apoptose , Contagem de Células Sanguíneas , Citocinas/biossíntese , Modelos Animais de Doenças , Edema/etiologia , Edema/patologia , Eletrólitos/sangue , Feminino , Hipocampo/patologia , Imunidade Celular/imunologia , Fígado/patologia , Pulmão/patologia , Traumatismo por Reperfusão/complicações , Traumatismo por Reperfusão/fisiopatologia , SuínosRESUMO
BACKGROUND: Skin malformations that resembled manifestations of Ehlers-Danlos-Syndrome were described in a variety of domestic animals during the last century as cutis hyperelastica, hyperelastosis cutis, dermatosparaxis, dermal/collagen dysplasia, dermal/cutaneous asthenia or Ehlers-Danlos-like syndrome/s. In 2007, the mutation responsible for Hereditary Equine Regional Dermal Asthenia (HERDA) in Quarter Horses was discovered. Several case reports are available for similar malformations in other breeds than Quarter Horses (Draught Horses, Arabians, and Thoroughbreds) including four case reports for Warmblood horses. Since 2013, a genetic test for the Warmblood Fragile Foal Syndrome Type 1 (WFFS), interrogating the causative point mutation in the equine procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (PLOD1, or lysyl hydroxylase 1) gene, has become available. Only limited data are available on the occurrence rate and clinical characteristics of this newly detected genetic disease in horses. In humans mutations in this gene are associated with Ehlers-Danlos Syndrome Type VI (kyphoscoliotic form). CASE PRESENTATION: This is the first report describing the clinical and histopathological findings in a foal confirmed to be homozygous positive for WFFS. The Warmblood filly was born with very thin, friable skin, skin lesions on the legs and the head, and an open abdomen. These abnormalities required euthanasia just after delivery. Histologic examination revealed abnormally thin dermis, markedly reduced amounts of dermal collagen bundles, with loosely orientation and abnormally large spaces between deep dermal fibers. CONCLUSION: WFFS is a novel genetic disease in horses and should be considered in cases of abortion, stillbirth, skin lesions and malformations of the skin in neonatal foals. Genetic testing of suspicious cases will contribute to evaluate the frequency of occurrence of clinical WFFS cases and its relevance for the horse population.
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Síndrome de Ehlers-Danlos/veterinária , Doenças dos Cavalos/patologia , Pele/patologia , Animais , Animais Recém-Nascidos/genética , Síndrome de Ehlers-Danlos/genética , Síndrome de Ehlers-Danlos/patologia , Feminino , Homozigoto , Doenças dos Cavalos/genética , Cavalos/genética , LinhagemRESUMO
A dwarf bearded dragon (Pogona henrylawsoni) was presented with a white subcutaneous mandibular mass and multiple nodules in the oral mucosa, heart, liver, kidney, intestine, and visceral fat. Histologically, the tumor consisted of densely packed spindle-shaped cells with brow intracytoplasmic pigment that exhibited white-blue birefringence with polarized light. Immunohistochemical staining was negative for S-100 and weakly positive with melan A. Electron microscopic examination revealed cytoplasmic irregular and oblong empty spaces, laminated and often arranged into short stacks, compatible with reflecting platelet profiles typically seen in iridophores. However, in unstained ultrathin sections, electron-dense crystalline material was present, which filled the empty spaces described for stained sections before. Based on histology, immunohistochemistry, and biologic behavior, a malignant iridophoroma was diagnosed. To the authors' knowledge, iridophoromas in lizards have rarely been characterized by using electronic microscopy. Moreover, this is the first description of an iridophoroma in a dwarf bearded dragon.
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Cromatóforos/patologia , Lagartos , Neoplasias Cutâneas/veterinária , Animais , Feminino , Neoplasias Cutâneas/diagnóstico , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/ultraestruturaRESUMO
Etomidate, an agonist of the GABA A receptors, is available for clinical use either in combination with 35% propylene glycol or in a lipid emulsion. Its recognized ability to minimally impact the cardiovascular system made etomidate a suitable option for cardiac-compromised patients. Myoclonus and pain at the injection site are recognized side effects of etomidate in propylene glycol, affecting both human and veterinary species. There is no information available concerning potential side effect in minipigs. In the present case series, we report the side effects related to the use of etomidate in 35% propylene glycol in five Ellegaard Göttingen Minipigs that underwent general anesthesia for cardiac magnetic resonance imaging days or weeks after experimentally induced myocardial infarction. Following intravenous injection of etomidate, laryngeal edema and hyperemia were observed in one case. In another case, tachycardia, apnea, and decreased oxygen saturation, accompanied by laryngeal edema and hyperemia, were observed, which resolved spontaneously in a few minutes. In the arterial or venous samples collected shortly after the induction of general anesthesia, hemolysis was macroscopically visible and subsequently confirmed with a hematological exam in all five cases, as well as hemoglobinuria. Necropsies carried out immediately after euthanasia confirmed macroscopic laryngeal edema, marked diffuse lung alveolar and interstitial edema and hyperemia at histology in one animal, and marked acute lung congestion in another animal. These side effects were not observed when etomidate in a lipid emulsion was injected into another 24 animals. The role played by the different formulations (propylene glycol versus lipidic formulation) has not yet been fully elucidated. Based on our observations, we recommend caution in using the formulation of etomidate in 35% propylene glycol in Göttingen Minipigs.
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Molecular biomarkers are central to personalised medicine for human cancer patients. It is gaining traction as part of standard veterinary clinical practice for dogs and cats with cancer. Molecular biomarkers can be somatic or germline genomic alterations and can be ascertained from tissues or body fluids using various techniques. This review discusses how these genomic alterations can be determined and the findings used in clinical settings as diagnostic, prognostic, predictive, and screening biomarkers. We showcase the somatic and germline genomic alterations currently available to date for testing dogs and cats in a clinical setting, discussing their utility in each biomarker class. We also look at some emerging molecular biomarkers that are promising for clinical use. Finally, we discuss the hurdles that need to be overcome in going 'bench to bedside', i.e., the translation from discovery of genomic alterations to adoption by veterinary clinicians. As we understand more of the genomics underlying canine and feline tumours, molecular biomarkers will undoubtedly become a mainstay in delivering precision veterinary care to dogs and cats with cancer.
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Genetic molecular testing is starting to gain traction as part of standard clinical practice for dogs with cancer due to its multi-faceted benefits, such as potentially being able to provide diagnostic, prognostic and/or therapeutic information. However, the benefits and ultimate success of genomic analysis in the clinical setting are reliant on the robustness of the tools used to generate the results, which continually expand as new technologies are developed. To this end, we review the different materials from which tumour cells, DNA, RNA and the relevant proteins can be isolated and what methods are available for interrogating their molecular profile, including analysis of the genetic alterations (both somatic and germline), transcriptional changes and epigenetic modifications (including DNA methylation/acetylation and microRNAs). We also look to the future and the tools that are currently being developed, such as using artificial intelligence (AI) to identify genetic mutations from histomorphological criteria. In summary, we find that the molecular genetic characterisation of canine neoplasms has made a promising start. As we understand more of the genetics underlying these tumours and more targeted therapies become available, it will no doubt become a mainstay in the delivery of precision veterinary care to dogs with cancer.
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Canine urothelial carcinoma (UC) and prostate carcinoma (PC) frequently exhibit the BRAFV595E mutation, akin to the BRAFV600E mutation common in various human cancers. Since the initial discovery of the BRAF mutation in canine cancers in 2015, PCR has been the standard method for its detection in both liquid and tissue biopsies. Considering the similarity between the canine BRAFV595E and human BRAFV600E mutations, we hypothesized that immunohistochemistry (IHC) using a BRAFV600E-specific antibody could effectively identify the canine mutant BRAFV595E protein. We tested 122 canine UC (bladder n = 108, urethra n = 14), 21 PC, and benign tissue using IHC and performed digital droplet PCR (ddPCR) on all 122 UC and on 14 IHC positive PC cases. The results from ddPCR and IHC were concordant in 99% (135/136) of the tumours. Using IHC, BRAFV595E was detected in 72/122 (59%) UC and 14/21 (65%) PC. Staining of all benign bladder and prostate tissues was negative. If present, mutant BRAF staining was homogenous, with rare intratumour heterogeneity in three (4%) cases of UC. Additionally, the BRAFV595E mutation was more prevalent in tumours with urothelial morphology, and less common in glandular PC or UC with divergent differentiation. This study establishes that BRAFV600-specific IHC is a reliable and accurate method for detecting the mutant BRAFV595E protein in canine UC and PC. Moreover, the use of IHC, especially with tissue microarrays, provides a cost-efficient test for large-scale screening of canine cancers for the presence of BRAF mutations. This advancement paves the way for further research to define the prognostic and predictive role of this tumour marker in dogs and use IHC to stratify dogs for the treatment with BRAF inhibitors.
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Doenças do Cão , Imuno-Histoquímica , Mutação , Neoplasias da Próstata , Proteínas Proto-Oncogênicas B-raf , Neoplasias da Bexiga Urinária , Cães , Animais , Doenças do Cão/genética , Doenças do Cão/diagnóstico , Doenças do Cão/patologia , Proteínas Proto-Oncogênicas B-raf/genética , Masculino , Neoplasias da Próstata/veterinária , Neoplasias da Próstata/genética , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Imuno-Histoquímica/veterinária , Neoplasias da Bexiga Urinária/veterinária , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/diagnóstico , Feminino , Carcinoma/veterinária , Carcinoma/genética , Carcinoma/patologia , Carcinoma/metabolismo , Carcinoma/diagnóstico , Carcinoma de Células de Transição/veterinária , Carcinoma de Células de Transição/genética , Carcinoma de Células de Transição/metabolismo , Carcinoma de Células de Transição/diagnóstico , Carcinoma de Células de Transição/patologiaRESUMO
The tumor-draining lymph nodes (tdLN) are the initial site of metastases and are the prime site for generating robust antitumor responses. In this study, we explored the efficacy of a universal immune activator (ImmAct) targeted to the tdLN. This approach can be viewed as an attempt to turn a cold, unresponsive tdLN into a hot, responsive site. The adjuvant antitumor efficacy of our novel intranodal injection was evaluated in an aggressive metastatic mammary carcinoma murine model. The cancer cells were inoculated subcutaneously in the lower quadrant of the mouse to provoke the tdLN (inguinal lymph node). The study encompasses a range of methodologies, including in vivo and in vitro assays and high-dimensional flow cytometry analysis. Our findings demonstrated that intranodal administration of ImmAct following the dissection of the primary tumor led to improved tumor-free survival and minimized weight loss. ImmAct led to both local and systemic alterations in the cellular and humoral immunity. Additionally, after ImmAct treatment, non-responders showed a higher rate of exhausted CD8+ T cells compared to responders. Indeed, our innovative approach surpassed the gold standard surgery of sentinel lymph node excision. Overall, intranodal administration of ImmAct yielded a robust antitumor immune response, offering protection against micrometastases and relapse.
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The global incidence of human papillomavirus (HPV) associated head and neck carcinoma is on the rise, in response to this a tetravalent therapeutic vaccine named Qß-HPVag was developed. This vaccine, utilizing virus-like particles (VLPs) loaded with toll-like receptor ligands and chemically coupled to four HPV16-derived peptides, demonstrated strong anti-tumor effects in a murine head and neck cancer model. Qß-HPVag impeded tumor progression, increased infiltration of HPV-specific T cells, and significantly improved survival. The vaccine`s efficacy was associated with immune repolarization in the tumor microenvironment, characterized by expanded activated dendritic cell subsets (cDC1, cDC2, DC3). Notably, mice responding to treatment exhibited a higher percentage of migratory DC3 cells expressing CCR7. These findings suggest promising prospects for optimized VLP-based vaccines in treating HPV-associated head and neck cancer.
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Early identification of drug toxicity is essential yet challenging in drug development. At the preclinical stage, toxicity is assessed with histopathological examination of tissue sections from animal models to detect morphological lesions. To complement this analysis, toxicogenomics is increasingly employed to understand the mechanism of action of the compound and ultimately identify lesion-specific safety biomarkers for which in vitro assays can be designed. However, existing works that aim to identify morphological correlates of expression changes rely on qualitative or semi-quantitative morphological characterization and remain limited in scale or morphological diversity. Artificial intelligence (AI) offers a promising approach for quantitatively modeling this relationship at an unprecedented scale. Here, we introduce GEESE, an AI model designed to impute morphomolecular signatures in toxicology data. Our model was trained to predict 1,536 gene targets on a cohort of 8,231 hematoxylin and eosin-stained liver sections from Rattus norvegicus across 127 preclinical toxicity studies. The model, evaluated on 2,002 tissue sections from 29 held-out studies, can yield pseudo-spatially resolved gene expression maps, which we correlate with six key drug-induced liver injuries (DILI). From the resulting 25 million lesion-expression pairs, we established quantitative relations between up and downregulated genes and lesions. Validation of these signatures against toxicogenomic databases, pathway enrichment analyses, and human hepatocyte cell lines asserted their relevance. Overall, our study introduces new methods for characterizing toxicity at an unprecedented scale and granularity, paving the way for AI-driven discovery of toxicity biomarkers.
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Background: Seroma formation is a common postoperative complication. Fibrin-based glues are typically employed in an attempt to seal the cavity. Recently, the first nanoparticle (NP)-based treatment approaches have emerged. Nanoparticle dispersions can be used as tissue glues, capitalizing on a phenomenon known as 'nanobridging'. In this process, macromolecules such as proteins physically adsorb onto the NP surface, leading to macroscopic adhesion. Although significant early seroma reduction has been shown, little is known about long-term efficacy of NPs. The aim of this study was to assess the long-term effects of NPs in reducing seroma formation, and to understand their underlying mechanism. Methods: Seroma was surgically induced bilaterally in 20 Lewis rats. On postoperative day (POD) 7, seromas were aspirated on both sides. In 10 rats, one side was treated with NPs, while the contralateral side received only NP carrier solution. In the other 10 rats, one side was treated with fibrin glue, while the other was left untreated. Seroma fluid, blood and tissue samples were obtained at defined time points. Biochemical, histopathological and immunohistochemical assessments were made. Results: NP-treated sides showed no macroscopically visible seroma formation after application on POD 7, in stark contrast to the fibrin-treated sides, where 60% of the rats had seromas on POD 14, and 50% on POD 21. At the endpoint (POD 42), sides treated with nanoparticles (NPs) exhibited significant macroscopic differences compared to other groups, including the absence of a cavity, and increased fibrous adhesions. Histologically, there were more macrophage groupings and collagen type 1 (COL1) deposits in the superficial capsule on NP-treated sides. Conclusion: NPs not only significantly reduced early manifestations of seroma and demonstrated an anti-inflammatory response, but they also led to increased adhesion formation over the long term, suggesting a decreased risk of seroma recurrence. These findings highlight both the adhesive properties of NPs and their potential for clinical therapy.
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INTRODUCTION: Continuous extracorporeal perfusion (ECP), or machine perfusion, holds promise for prolonged skeletal muscle preservation in limb ischemia-reperfusion injury. This study aimed to extend the amputation-to-replantation time window from currently 6 hours to 33 hours using a 24-hour ECP approach. MATERIALS AND METHODS: Six large white pigs underwent surgical forelimb amputation under general anesthesia. After amputation, limbs were kept for 9 hours at room temperature and then perfused by 24-hour ECP with a modified histidine-tryptophan-ketoglutarate (HTK) solution. After ECP, limbs were orthotopically replanted and perfused in vivo for 12 hours. Clinical data, blood, and tissue samples were collected and analyzed. RESULTS: All 6 forelimbs could be successfully replanted and in vivo reperfused for 12 hours after 9 hours of room temperature ischemia followed by 24 hours ECP. Adequate limb perfusion was observed after replantation as shown by thermography and laser Doppler imaging. All pigs survived without severe organ failure, and no significant increase in inflammatory cytokines was found. Macroscopy and histology showed marked interstitial muscular edema of the limbs, whereas myofiber necrosis was not evident, implying the preservation of muscular integrity. CONCLUSIONS: The use of a 24-hour ECP has successfully extended limb preservation to 33 hours. The modified histidine-tryptophan-ketoglutarate perfusate demonstrated its ability for muscle protection. This innovative approach not only facilitates limb replantation after combat injuries, surmounting geographical barriers, but also broadens the prospects for well-matched limb allotransplants across countries and continents.
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Amputação Traumática , Reimplante , Animais , Reimplante/métodos , Suínos , Amputação Traumática/cirurgia , Fatores de Tempo , Perfusão/métodos , Procaína/farmacologia , Procaína/uso terapêutico , Cloreto de Potássio/farmacologia , Cloreto de Potássio/uso terapêutico , Traumatismo por Reperfusão , Membro Anterior/fisiopatologia , Glucose , ManitolRESUMO
In drug development, assessing the toxicity of candidate compounds is crucial for successfully transitioning from preclinical research to early-stage clinical trials. Drug safety is typically assessed using animal models with a manual histopathological examination of tissue sections to characterize the dose-response relationship of the compound - a time-intensive process prone to inter-observer variability and predominantly involving tedious review of cases without abnormalities. Artificial intelligence (AI) methods in pathology hold promise to accelerate this assessment and enhance reproducibility and objectivity. Here, we introduce TRACE, a model designed for toxicologic liver histopathology assessment capable of tackling a range of diagnostic tasks across multiple scales, including situations where labeled data is limited. TRACE was trained on 15 million histopathology images extracted from 46,734 digitized tissue sections from 157 preclinical studies conducted on Rattus norvegicus. We show that TRACE can perform various downstream toxicology tasks spanning histopathological response assessment, lesion severity scoring, morphological retrieval, and automatic dose-response characterization. In an independent reader study, TRACE was evaluated alongside ten board-certified veterinary pathologists and achieved higher concordance with the consensus opinion than the average of the pathologists. Our study represents a substantial leap over existing computational models in toxicology by offering the first framework for accelerating and automating toxicological pathology assessment, promoting significant progress with faster, more consistent, and reliable diagnostic processes.
RESUMO
Prostate cancer (PCa) is the second most common cancer diagnosed in men. While radical prostatectomy and radiotherapy are often successful in treating localised disease, post-treatment recurrence is common. As the androgen receptor (AR) and androgen hormones play an essential role in prostate carcinogenesis and progression, androgen deprivation therapy (ADT) is often used to deprive PCa cells of the pro-proliferative effect of androgens. ADTs act by either blocking androgen biosynthesis (e.g. abiraterone) or blocking AR function (e.g. bicalutamide, enzalutamide, apalutamide, darolutamide). ADT is often effective in initially suppressing PCa growth and progression, yet emergence of castrate-resistant PCa and progression to neuroendocrine-like PCa following ADT are major clinical challenges. For this reason, there is an urgent need to identify novel approaches to modulate androgen signalling to impede PCa progression whilst also preventing or delaying therapy resistance. The mechanistic convergence of androgen and epitranscriptomic signalling offers a potential novel approach to treat PCa. The epitranscriptome involves covalent modifications of mRNA, notably, in the context of this review, the N(6)-methyladenosine (m6A) modification. m6A is involved in the regulation of mRNA splicing, stability, and translation, and has recently been shown to play a role in PCa and androgen signalling. The m6A modification is dynamically regulated by the METTL3-containing methyltransferase complex, and the FTO and ALKBH5 RNA demethylases. Given the need for novel approaches to treat PCa, there is significant interest in new therapies that target m6A that modulate AR expression and androgen signalling. This review critically summarises the potential benefit of such epitranscriptomic therapies for PCa patients.
Assuntos
Androgênios , Epigênese Genética , Neoplasias da Próstata , Receptores Androgênicos , Transdução de Sinais , Humanos , Masculino , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Neoplasias da Próstata/terapia , Androgênios/metabolismo , Receptores Androgênicos/metabolismo , Receptores Androgênicos/genética , Regulação Neoplásica da Expressão Gênica , Antagonistas de Androgênios/uso terapêutico , Antagonistas de Androgênios/farmacologia , Transcriptoma , AnimaisRESUMO
Nonsense-mediated mRNA decay (NMD) is a eukaryotic RNA decay pathway with roles in cellular stress responses, differentiation, and viral defense. It functions in both quality control and post-transcriptional regulation of gene expression. NMD has also emerged as a modulator of cancer progression, although available evidence supports both a tumor suppressor and a pro-tumorigenic role, depending on the model. To further investigate the role of NMD in cancer, we knocked out the NMD factor SMG7 in the HT1080 human fibrosarcoma cell line, resulting in suppression of NMD function. We then compared the oncogenic properties of the parental cell line, the SMG7-knockout, and a rescue cell line in which we re-introduced both isoforms of SMG7. We also tested the effect of a drug inhibiting the NMD factor SMG1 to distinguish NMD-dependent effects from putative NMD-independent functions of SMG7. Using cell-based assays and a mouse xenograft tumor model, we showed that suppression of NMD function severely compromises the oncogenic phenotype. Molecular pathway analysis revealed that NMD suppression strongly reduces matrix metalloprotease 9 (MMP9) expression and that MMP9 re-expression partially rescues the oncogenic phenotype. Since MMP9 promotes cancer cell migration and invasion, metastasis and angiogenesis, its downregulation may contribute to the reduced tumorigenicity of NMD-suppressed cells. Collectively, our results highlight the potential value of NMD inhibition as a therapeutic approach.