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INTRODUCTION: In this study, we used metatranscriptomics for the first time to investigate microbial composition, functional signatures, and antimicrobial resistance gene expression in endodontic infections. METHODS: Root canal samples were collected from ten teeth, including five primary and five persistent/secondary endodontic infections. RNA from endodontic samples was extracted, and RNA sequencing was performed on a NovaSeq6000 system (Illumina). Taxonomic analysis was performed using the Kraken2 bacterial database. Then, sequences with a taxonomic classification were annotated against the Universal Protein Knowledgebase for functional annotation and the Comprehensive Antibiotic Resistance Database for AR-like gene identification. RESULTS: Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria represented the dominant phyla, whereas Fusobacteria, Spirochetes, and Synergistetes were among the nondominant phyla. The top ten species were mainly represented by obligate (or quasiobligate) anaerobes, including Gram-negative (eg, Capnocytophaga sp. oral taxon 323, Fusobacterium nucleatum, Prevotella intermedia, Prevotella oris, Tannerella forsythia, and Tannerella sp. oral taxon HOT-286) and Gram-positive species (eg, Olsenella uli and Parvimonas micra). Transcripts encoding moonlighting proteins (eg, glycolytic proteins, translational elongation factors, chaperonin, and heat shock proteins) were highly expressed, potentially affecting bacterial adhesion, biofilm formation, host defense evasion, and inflammation induction. Endodontic bacteria expressed genes conferring resistance to antibiotic classes commonly used in dentistry, with a high prevalence and expression of tetracycline and lincosamide resistance genes. Antibiotic efflux and antibiotic target alteration/protection were the main resistance mechanisms. CONCLUSIONS: Metatranscriptomics revealed the activity of potential endodontic pathogens, which expressed putative virulence factors and a wide diversity of genes potentially involved in AR.
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Cavidade Pulpar , Microbiota , Transcriptoma , Humanos , Cavidade Pulpar/microbiologia , Farmacorresistência Bacteriana/genética , Antibacterianos/farmacologia , Doenças da Polpa Dentária/microbiologiaRESUMO
[This corrects the article DOI: 10.3389/fphar.2024.1328460.].
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The inner ear is the organ responsible for hearing and balance. Inner ear dysfunction can be the result of infection, trauma, ototoxic drugs, genetic mutation or predisposition. Often, like for Ménière disease, the cause is unknown. Due to the complex access to the inner ear as a fluid-filled cavity within the temporal bone of the skull, effective diagnosis of inner ear pathologies and targeted drug delivery pose significant challenges. Samples of inner ear fluids can only be collected during surgery because the available procedures damage the tiny and fragile structures of the inner ear. Concerning drug administration, the final dose, kinetics, and targets cannot be controlled. Overcoming these limitations is crucial for successful inner ear precision medicine. Recently, notable advancements in microneedle technologies offer the potential for safe sampling of inner ear fluids and local treatment. Ultrasharp microneedles can reach the inner ear fluids with minimal damage to the organ, collect µl amounts of perilymph, and deliver therapeutic agents in loco. This review highlights the potential of ultrasharp microneedles, combined with nano vectors and gene therapy, to effectively treat inner ear diseases of different etiology on an individual basis. Though further research is necessary to translate these innovative approaches into clinical practice, these technologies may represent a true breakthrough in the clinical approach to inner ear diseases, ushering in a new era of personalized medicine.
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The interaction between the microbial communities in the human body and the onset and progression of cancer has not been investigated until recently. The vast majority of the metagenomics research in this area has concentrated on the composition of microbiomes, attempting to link the overabundance or depletion of certain microorganisms to cancer proliferation, metastatic behaviour, and its resistance to therapies. However, studies elucidating the functional implications of the microbiome activity in cancer patients are still scarce; in particular, there is an overwhelming lack of studies assessing such implications directly, through analysis of the transcriptome of the bacterial community. This review summarises the contributions of metagenomics and metatranscriptomics to the knowledge of the microbial environment associated with several cancers; most importantly, it highlights all the advantages that metatranscriptomics has over metagenomics and suggests how such an approach can be leveraged to advance the knowledge of the cancer bacterial environment.
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Microbiota , Neoplasias , Humanos , Neoplasias/genética , Metagenômica , Microbiota/genética , TranscriptomaRESUMO
Pimozide is a conventional antipsychotic drug largely used in the therapy for schizophrenia and Tourette's syndrome. Pimozide is assumed to inhibit synaptic transmission at the CNS by acting as a dopaminergic D2 receptor antagonist. Moreover, pimozide has been shown to block voltage-gated Ca2+ and K+ channels in different cells. Despite its widespread clinical use, pimozide can cause several adverse effects, including extrapyramidal symptoms and cardiac arrhythmias. Dizziness and loss of balance are among the most common side effects of pimozide. By using the patch-clamp whole-cell technique, we investigated the effect of pimozide [3 µM] on K+ channels expressed by chicken embryo vestibular type-II hair cells. We found that pimozide slightly blocks a transient outward rectifying A-type K+ current but substantially increases a delayed outward rectifying K+ current. The net result was a significant hyperpolarization of type-II hair cells at rest and a strong reduction of their response to depolarizing stimuli. Our findings are consistent with an inhibitory effect of pimozide on the afferent synaptic transmission by type-II hair cells. Moreover, they provide an additional key to understanding the beneficial/collateral pharmacological effects of pimozide. The finding that pimozide can act as a K+ channel opener provides a new perspective for the use of this drug.
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Cyst-forming Apicomplexa (CFA) of the Sarcocystidae have a ubiquitous presence as pathogens of humans and farm animals transmitted through the food chain between hosts with few notable exceptions. The defining hallmark of this family of obligate intracellular protists consists of their ability to remain for very long periods as infectious tissue cysts in chronically infected intermediate hosts. Nevertheless, each closely related species has evolved unique strategies to maintain distinct reservoirs on global scales and ensuring efficient transmission to definitive hosts as well as between intermediate hosts. Here, we present an in-depth comparative mRNA expression analysis of the tachyzoite and bradyzoite stages of Besnoitia besnoiti strain Lisbon14 isolated from an infected farm animal based on its annotated genome sequence. The B. besnoiti genome is highly syntenic with that of other CFA and also retains the capacity to encode a large majority of known and inferred factors essential for completing a sexual cycle in a yet unknown definitive host. This work introduces Besnoitia besnoiti as a new model for comparative biology of coccidian tissue cysts which can be readily obtained in high purity. This model provides a framework for addressing fundamental questions about the evolution of tissue cysts and the biology of this pharmacologically intractable infectious parasite stage.
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Besnoitia , Estágios do Ciclo de Vida , Animais , Humanos , Estágios do Ciclo de Vida/genética , Cadeia Alimentar , Expressão GênicaRESUMO
BACKGROUND: Movile Cave (SE Romania) is a chemoautotrophically-based ecosystem fed by hydrogen sulfide-rich groundwater serving as a primary energy source analogous to the deep-sea hydrothermal ecosystems. Our current understanding of Movile Cave microbiology has been confined to the sulfidic water and its proximity, as most studies focused on the water-floating microbial mat and planktonic accumulations likely acting as the primary production powerhouse of this unique subterranean ecosystem. By employing comprehensive genomic-resolved metagenomics, we questioned the spatial variation, chemoautotrophic abilities, ecological interactions and trophic roles of Movile Cave's microbiome thriving beyond the sulfidic-rich water. RESULTS: A customized bioinformatics pipeline led to the recovery of 106 high-quality metagenome-assembled genomes from 7 cave sediment metagenomes. Assemblies' taxonomy spanned 19 bacterial and three archaeal phyla with Acidobacteriota, Chloroflexota, Proteobacteria, Planctomycetota, Ca. Patescibacteria, Thermoproteota, Methylomirabilota, and Ca. Zixibacteria as prevalent phyla. Functional gene analyses predicted the presence of CO2 fixation, methanotrophy, sulfur and ammonia oxidation in the explored sediments. Species Metabolic Coupling Analysis of metagenome-scale metabolic models revealed the highest competition-cooperation interactions in the sediments collected away from the water. Simulated metabolic interactions indicated autotrophs and methanotrophs as major donors of metabolites in the sediment communities. Cross-feeding dependencies were assumed only towards 'currency' molecules and inorganic compounds (O2, PO43-, H+, Fe2+, Cu2+) in the water proximity sediment, whereas hydrogen sulfide and methanol were assumedly traded exclusively among distant gallery communities. CONCLUSIONS: These findings suggest that the primary production potential of Movile Cave expands way beyond its hydrothermal waters, enhancing our understanding of the functioning and ecological interactions within chemolithoautotrophically-based subterranean ecosystems.
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Mieloma Múltiplo , Paraproteinemias , Humanos , Paraproteinemias/genética , Medicina de PrecisãoRESUMO
Chronic kidney disease (CKD) affects approximately 10-13% of the population worldwide and halting its progression is a major clinical challenge. Metabolic acidosis is both a consequence and a possible driver of CKD progression. Alkali therapy counteracts these effects in CKD patients, but underlying mechanisms remain incompletely understood. Here we show that bicarbonate supplementation protected renal function in a murine CKD model induced by an oxalate-rich diet. Alkali therapy had no effect on the aldosterone-endothelin axis but promoted levels of the anti-aging protein klotho; moreover, it suppressed adhesion molecules required for immune cell invasion along with reducing T-helper cell and inflammatory monocyte invasion. Comparing transcriptomes from the murine crystallopathy model and from human biopsies of kidney transplant recipients (KTRs) suffering from acidosis with or without alkali therapy unveils parallel transcriptome responses mainly associated with lipid metabolism and oxidoreductase activity. Our data reveal novel pathways associated with acidosis in kidney disease and sensitive to alkali therapy and identifies potential targets through which alkali therapy may act on CKD and that may be amenable for more targeted therapies.
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Acidose , Insuficiência Renal Crônica , Acidose/complicações , Acidose/tratamento farmacológico , Álcalis/uso terapêutico , Animais , Feminino , Humanos , Inflamação , Rim/metabolismo , Masculino , CamundongosRESUMO
Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS). Its first clinical presentation (clinically isolated syndrome, CIS) is often followed by the development of relapsing-remitting MS (RRMS). The periphery-to-CNS transmission of inflammatory molecules is a major pathophysiological pathway in MS. This could include signalling via extracellular vesicle (EV) microRNAs (miRNAs). In this study, we investigated the serum EV miRNome in CIS and RRMS patients and matched controls, with the aims to identify MS stage-specific differentially expressed miRNAs and investigate their biomarker potential and pathophysiological relevance. miRNA sequencing was conducted on serum EVs from CIS-remission, RRMS-relapse, and viral inflammatory CNS disorder patients, as well as from healthy and hospitalized controls. Differential expression analysis was conducted, followed by predictive power and target-pathway analysis. A moderate number of dysregulated serum EV miRNAs were identified in CIS-remission and RRMS-relapse patients, especially relative to healthy controls. Some of these miRNAs were also differentially expressed between the two MS stages and had biomarker potential for patient-control and CIS-RRMS separations. For the mRNA targets of the RRMS-relapse-specific EV miRNAs, biological processes inherent to MS pathophysiology were identified using in silico analysis. Study findings demonstrate that specific serum EV miRNAs have MS stage-specific biomarker potential and contribute to the identification of potential targets for novel, efficacious therapies.
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Vesículas Extracelulares/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , MicroRNAs/sangue , Esclerose Múltipla Recidivante-Remitente/sangue , Adolescente , Adulto , Idoso , Biomarcadores/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Immune-inflammatory activation impacts extracellular vesicles (EVs), including their miRNA cargo. There is evidence for changes in the EV miRNome in inflammation-associated neuropsychiatric disorders. This mouse study investigated: (1) effects of systemic lipopolysaccharide (LPS) and chronic social stress (CSS) on plasma EV miRNome; and (2) physiological, transcriptional, and behavioural effects of peripheral or central delivered LPS-activated EVs in recipient mice. LPS or CSS effects on the plasma EV miRNome were assessed by using microRNA sequencing. Recipient mice received plasma EVs isolated from LPS-treated or SAL-treated donor mice or vehicle only, either intravenously or into the nucleus accumbens (NAc), on three consecutive days. Bodyweight, spleen or NAc transcriptome and reward (sucrose) motivation were assessed. LPS and CSS increased the expression of 122 and decreased expression of 20 plasma EV miRNAs, respectively. Peripheral LPS-EVs reduced bodyweight, and both LPS-EVs and SAL-EVs increased spleen expression of immune-relevant genes. NAc-infused LPS-EVs increased the expression of 10 immune-inflammatory genes. Whereas motivation increased similarly across test days in all groups, the effect of test days was more pronounced in mice that received peripheral or central LPS-EVs compared with other groups. This study provides causal evidence that increased EV levels impact physiological and behavioural processes and are of potential relevance to neuropsychiatric disorders.
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Vesículas Extracelulares/genética , Perfilação da Expressão Gênica/métodos , Lipopolissacarídeos/efeitos adversos , MicroRNAs/genética , Estresse Psicológico/genética , Animais , Vesículas Extracelulares/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , Tamanho do Órgão/efeitos dos fármacos , Projetos Piloto , Análise de Sequência de RNA , Baço/efeitos dos fármacos , Baço/metabolismo , Estresse Psicológico/psicologiaRESUMO
Signal transmission by sensory auditory and vestibular hair cells relies upon Ca2+-dependent exocytosis of glutamate. The Ca2+ current in mammalian inner ear hair cells is predominantly carried through Ca V 1.3 voltage-gated Ca2+ channels. Despite this, Ca V 1.3 deficient mice (Ca V 1.3-/- ) are deaf but do not show any obvious vestibular phenotype. Here, we compared the Ca2+ current (I Ca ) in auditory and vestibular hair cells from wild-type and Ca V 1.3-/- mice, to assess whether differences in the size of the residual I Ca could explain, at least in part, the two phenotypes. Using 5 mM extracellular Ca2+ and near-body temperature conditions, we investigated the cochlear primary sensory receptors inner hair cells (IHCs) and both type I and type II hair cells of the semicircular canals. We found that the residual I Ca in both auditory and vestibular hair cells from Ca V 1.3-/- mice was less than 20% (12-19%, depending on the hair cell type and age investigated) compared to controls, indicating a comparable expression of Ca V 1.3 Ca2+ channels in both sensory organs. We also showed that, different from IHCs, type I and type II hair cells from Ca V 1.3-/- mice were able to acquire the adult-like K+ current profile in their basolateral membrane. Intercellular K+ accumulation was still present in Ca V 1.3-/- mice during I K,L activation, suggesting that the K+-based, non-exocytotic, afferent transmission is still functional in these mice. This non-vesicular mechanism might contribute to the apparent normal vestibular functions in Ca V 1.3-/- mice.
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Necrotizing gingivitis (NG) is a necrotizing periodontal disease that differs from chronic gingivitis (CG). To date, both the microbiological causes and the involved host cytokine response of NG still remain unclear. Here, we investigated corresponding interdental plaque and serum samples from two groups of Chinese patients with CG (n = 21) or NG (n = 21). The microbiota were studied by 16S rRNA Illumina MiSeq sequencing of the microbial metagenome and by assessing quantitatively the abundance of the phylum Bacteroidetes, the genus Prevotella and the species T. forsythia, P. endodontalis, and P. gingivalis using fluorescence in situ hybridization (FISH). With respect to the associated host response, the levels of 30 inflammatory mediators were quantified by multiplex immunoassay analysis. Differential microbial abundance analysis of the two disease groups revealed at the phylum level that Proteobacteria accounted for 67% of the differentially abundant organisms, followed by organisms of Firmicutes (21%) and Actinobacteria (9%). At the species level, significant differences in abundance were seen for 75 species of which 58 species were significantly more abundant in CG patients. Notably, the FISH analysis revealed that Bacteroidetes was the most prevalent phylum in NG. The multiplex cytokine assay showed significant quantitative differences between the disease groups for eight analytes (GM-CSF, G-CSF, IFN-α, IL-4, IL-13, TNF-α, MIG, and HGF). The G-CSF was found to be the most significantly increased inflammatory protein marker in NG. The next-generation sequencing (NGS) data supported the understanding of NG as a multi-microbial infection with distinct differences to CG in regard to the microbial composition.
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BACKGROUND: Metabolic acidosis occurs frequently in patients with kidney transplant and is associated with a higher risk for and accelerated loss of graft function. To date, it is not known whether alkali therapy in these patients improves kidney function and whether acidosis and its therapy are associated with altered expression of proteins involved in renal acid-base metabolism. METHODS: We retrospectively collected kidney biopsies from 22 patients. Of these patients, nine had no acidosis, nine had metabolic acidosis [plasma bicarbonate (HCO3- <22 mmol/L) and four had acidosis and received alkali therapy. We performed transcriptome analysis and immunohistochemistry for proteins involved in renal acid-base handling. RESULTS: We found that the expression of 40 transcripts significantly changed between kidneys from non-acidotic and acidotic patients. These genes are mostly involved in proximal tubule (PT) amino acid and lipid metabolism and energy homoeostasis. Three transcripts were fully recovered by alkali therapy: the Kir4.2 potassium channel, an important regulator of PT HCO3- metabolism and transport, acyl-CoA dehydrogenase short/branched chain and serine hydroxymethyltransferase 1, genes involved in beta oxidation and methionine metabolism. Immunohistochemistry showed reduced staining for the PT NBCe1 HCO3- transporter in kidneys from acidotic patients who recovered with alkali therapy. In addition, the HCO3- exchanger pendrin was affected by acidosis and alkali therapy. CONCLUSIONS: Metabolic acidosis in kidney transplant recipients is associated with alterations in the renal transcriptome that are partly restored by alkali therapy. Acid-base transport proteins mostly from PT were also affected by acidosis and alkali therapy, suggesting that the downregulation of critical players contributes to metabolic acidosis in these patients.
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Acidose , Transplante de Rim , Equilíbrio Ácido-Base , Acidose/etiologia , Álcalis , Humanos , Transplante de Rim/efeitos adversos , Estudos RetrospectivosRESUMO
Current body fluid identification methods do not reveal any information about the time since deposition (TsD) of biological traces, even though determining the age of traces could be crucial for the investigative process. To determine the utility of microbial RNA markers for TsD estimation, we examined RNA sequencing data from five forensically relevant body fluids (blood, menstrual blood, saliva, semen, and vaginal secretion) over seven time points, ranging from fresh to 1.5 years. One set of samples was stored indoors while another was exposed to outdoor conditions. In outdoor samples, we observed a consistent compositional shift, occurring after 4 weeks: this shift was characterized by an overall increase in non-human eukaryotic RNA and an overall decrease in prokaryotic RNA. In depth analyses showed a high fraction of tree, grass and fungal signatures, which are characteristic for the environment the samples were exposed to. When examining the prokaryotic fraction in more detail, three bacterial phyla were found to exhibit the largest changes in abundance, namely Actinobacteria, Proteobacteria and Firmicutes. More detailed analyses at the order level were done using a Lasso regression analysis to find a predictive subset of bacterial taxa. We found 26 bacterial orders to be indicative of sample age. Indoor samples did not reveal such a clear compositional change at the domain level: eukaryotic and prokaryotic abundance remained relatively stable across the assessed time period. Nonetheless, a Lasso regression analysis identified 32 bacterial orders exhibiting clear changes over time, enabling the prediction of TsD. For both indoor and outdoor samples, a larger number (around 60%) of the bacterial orders identified as indicative of TsD are part of the Actinobacteria, Proteobacteria and Firmicutes. In summary, we found that the observed changes across time are not primarily due to changes associated with body fluid specific bacteria but mostly due to accumulation of bacteria from the environment. Orders of these environmental bacteria could be evaluated for TsD prediction, considering the location and environment of the crime scene. However, further studies are needed to verify these findings, determine the applicability across samples, replicates, donors, and other variables, and also to further assess the effect of different seasons and locations on the samples.
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Sangue/microbiologia , Muco do Colo Uterino/microbiologia , RNA Bacteriano/análise , Saliva/microbiologia , Sêmen/microbiologia , Análise de Sequência de RNA , Crime , Impressões Digitais de DNA , Exposição Ambiental , Feminino , Marcadores Genéticos , Humanos , Masculino , Menstruação , Repetições de Microssatélites , Fatores de TempoRESUMO
Knowledge about the age of a stain, also termed as time since deposition (TsD), would provide law-enforcing authorities with valuable information for the prosecution of criminal offenses. Yet, there is no reliable method for the inference / assessment of TsD available. The aim of this study was to gain further insight into the RNA degradation pattern of forensically relevant body fluids and to find candidate markers for TsD estimation. Blood, menstrual blood, saliva, semen and vaginal secretion samples were exposed to indoor (dark, room temperature) and outdoor (exposed to sun, wind, etc. but protected from rain) conditions for up to 1.5 years. Based on expression and degradation analyses, we were able to identify body fluid specific signatures and RNA degradation patterns. The indoor samples showed a marked drop in RNA integrity after 6 months, while the outdoor samples were difficult to interpret and therefore excluded for some of the analyses. Up to 4 weeks, indoor samples showed more stable and less degrading transcripts than outdoor samples. Stable transcripts tended to be significantly shorter than degrading ones or transcripts, which are neither degrading nor stable. We reinforced the body fluid specific and the housekeeping gene nature of previously reported markers. With an unbiased approach, we selected stable and degrading genes for each body fluid in the short term and assessed their integrity during extended storage. We identified several stable and degrading gene transcripts, which could be tested in a targeted assay to assess the TsD interval e.g. by analyzing the ratio of degrading vs stable transcripts. In conclusion, we were able to detect RNA degradation patterns in samples being aged up to 1.5 years and identified several candidate markers, which could be evaluated for TsD estimation.
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Genética Forense , Perfilação da Expressão Gênica , Estabilidade de RNA , Análise Química do Sangue , Muco do Colo Uterino/química , Crime , Feminino , Marcadores Genéticos , Humanos , Masculino , Menstruação , RNA Mensageiro/genética , Saliva/química , Sêmen/química , Fatores de TempoRESUMO
Sudden unexplained death (SUD) takes up a considerable part in overall sudden death cases, especially in adolescents and young adults. During the past decade, many channelopathy- and cardiomyopathy-associated single nucleotide variants (SNVs) have been identified in SUD studies by means of postmortem molecular autopsy, yet the number of cases that remain inconclusive is still high. Recent studies had suggested that structural variants (SVs) might play an important role in SUD, but there is no consensus on the impact of SVs on inherited cardiac diseases. In this study, we searched for potentially pathogenic SVs in 244 genes associated with cardiac diseases. Whole-exome sequencing and appropriate data analysis were performed in 45 SUD cases. Re-analysis of the exome data according to the current ACMG guidelines identified 14 pathogenic or likely pathogenic variants in 10 (22.2%) out of the 45 SUD cases, whereof 2 (4.4%) individuals had variants with likely functional effects in the channelopathy-associated genes SCN5A and TRDN and 1 (2.2%) individual in the cardiomyopathy-associated gene DTNA. In addition, 18 structural variants (SVs) were identified in 15 out of the 45 individuals. Two SVs with likely functional impairment were found in the coding regions of PDSS2 and TRPM4 in 2 SUD cases (4.4%). Both were identified as heterozygous deletions, which were confirmed by multiplex ligation-dependent probe amplification. In conclusion, our findings support that SVs could contribute to the pathology of the sudden death event in some of the cases and therefore should be investigated on a routine basis in suspected SUD cases.
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Morte Súbita/patologia , Variação Estrutural do Genoma/genética , Cardiopatias/genética , Polimorfismo de Nucleotídeo Único , Adolescente , Adulto , Alquil e Aril Transferases , Proteínas de Transporte/genética , Criança , Pré-Escolar , Estudos de Coortes , Proteínas Associadas à Distrofina/genética , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Proteínas Musculares/genética , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Neuropeptídeos/genética , Fases de Leitura Aberta , Suíça/epidemiologia , Canais de Cátion TRPM , Sequenciamento do ExomaRESUMO
Feline infectious peritonitis (FIP) is a coronavirus-induced disease of cats, in which the immune system is known to play a crucial, but complex, role in the pathogenesis. This role is still incompletely understood, with involvement of both host and viral factors. To evaluate differential gene expression and pathway involvement in feline coronavirus (FCoV) infection and FIP, we applied next-generation RNA-sequencing of the mesenteric lymph nodes from cats with naturally-acquired FIP, as well as those with systemic FCoV infection without FIP, and those with neither. Viral infection was associated with upregulation of viral defenses regardless of the disease state, but to a greater degree in FIP. FIP was associated with higher pro-inflammatory pathway enrichment, whilst non-FIP FCoV-positive cats showed lower enrichment of humoral immunity pathways, below that of uninfected cats in the case of immunoglobulin production pathways. This host response is presumed to be protective. In FIP, downregulation of T cell-related processes was observed, which did not occur in non-FIP FCoV-positive cats. These results emphasize the importance of the host's immune balance in determining the outcome of the FCoV infection.
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Pre-treatment of tumors with hyperthermia is often used to increase the efficacy of radiotherapy. One of the main proteins induced in response to hyperthermia is heat shock protein 70 (HSP70). The aim of our study was to investigate up- and down-regulated genes in response to (thermo)radiotherapy in HSP70 proficient and deficient canine osteosarcoma cell line (Abrams), and functional role of HSP70 in the mechanism of thermoradiosensitization. Cells were transfected with negative control siRNA or siRNA targeting HSP70 and treated with hyperthermia (HT), radiotherapy (RT), and thermoradiotherapy (HTRT). RNA sequencing was used to analyze gene expression. Hyperthermia and thermoradiotherapy, but not radiotherapy alone, induced differential gene expression. We identified genes differentially expressed only in HSP70 knockdown (thus HSP70-dependent) cells in response to hyperthermia and thermoradiotherapy. Interestingly, cell proliferation but not clonogenicity and apoptosis/necrosis was affected by the HSP70 knockdown in response to thermoradiotherapy. The results suggest that HSP70 regulates expression of specific genes in response to hyperthermia and thermoradiotherapy. Further investigations into the role of specific genes regulated in a HSP70-dependent manner in response to thermoradiotherapy could pave a way into new, combinatorial treatment options for (canine) osteosarcoma and other cancer types.
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Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/radioterapia , Proteínas de Choque Térmico HSP70/metabolismo , Osteossarcoma/metabolismo , Osteossarcoma/radioterapia , Animais , Proliferação de Células , Análise por Conglomerados , Modelos Animais de Doenças , Cães , Regulação Neoplásica da Expressão Gênica , Hipertermia Induzida , Metaloproteinase 1 da Matriz/metabolismo , Glicoproteínas de Membrana/metabolismo , Fótons , Análise de Componente Principal , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , RNA-Seq , Radioterapia , Proteínas de Transporte Vesicular/metabolismoRESUMO
The clinical course of prion diseases is accurately predictable despite long latency periods, suggesting that prion pathogenesis is driven by precisely timed molecular events. We constructed a searchable genome-wide atlas of mRNA abundance and splicing alterations during the course of disease in prion-inoculated mice. Prion infection induced PrP-dependent transient changes in mRNA abundance and processing already at eight weeks post inoculation, well ahead of any neuropathological and clinical signs. In contrast, microglia-enriched genes displayed an increase simultaneous with the appearance of clinical signs, whereas neuronal-enriched transcripts remained unchanged until the very terminal stage of disease. This suggests that glial pathophysiology, rather than neuronal demise, could be the final driver of disease. The administration of young plasma attenuated the occurrence of early mRNA abundance alterations and delayed signs in the terminal phase of the disease. The early onset of prion-induced molecular changes might thus point to novel biomarkers and potential interventional targets.