Spatiotemporal proteomic profiling of the pro-inflammatory response to lipopolysaccharide in the THP-1 human leukaemia cell line.

Protein localisation and translocation between intracellular compartments underlie almost all physiological processes. The hyperLOPIT proteomics platform combines mass spectrometry with state-of-the-art machine learning to map the subcellular location of thousands of proteins simultaneously. We combine global proteome analysis with hyperLOPIT in a fully Bayesian framework to elucidate spatiotemporal proteomic changes during a lipopolysaccharide (LPS)-induced inflammatory response. We report a highly dynamic proteome in terms of both protein abundance and subcellular localisation, with alterations in the interferon response, endo-lysosomal system, plasma membrane reorganisation and cell migration. Proteins not previously associated with an LPS response were found to relocalise upon stimulation, the functional consequences of which are still unclear. By quantifying proteome-wide uncertainty through Bayesian modelling, a necessary role for protein relocalisation and the importance of taking a holistic overview of the LPS-driven immune response has been revealed. The data are showcased as an interactive application freely available for the scientific community.

OPTN recruitment to a Golgi-proximal compartment regulates immune signalling and cytokine secretion.

Optineurin (OPTN) is a multifunctional protein involved in autophagy and secretion, as well as nuclear factor κB (NF-κB) and IRF3 signalling, and OPTN mutations are associated with several human diseases. Here, we show that, in response to viral RNA, OPTN translocates to foci in the perinuclear region, where it negatively regulates NF-κB and IRF3 signalling pathways and downstream pro-inflammatory cytokine secretion. These OPTN foci consist of a tight cluster of small membrane vesicles, which are positive for ATG9A. Disease mutations in OPTN linked to primary open-angle glaucoma (POAG) cause aberrant foci formation in the absence of stimuli, which correlates with the ability of OPTN to inhibit signalling. By using proximity labelling proteomics, we identify the linear ubiquitin assembly complex (LUBAC), CYLD and TBK1 as part of the OPTN interactome and show that these proteins are recruited to this OPTN-positive perinuclear compartment. Our work uncovers a crucial role for OPTN in dampening NF-κB and IRF3 signalling through the sequestration of LUBAC and other positive regulators in this viral RNA-induced compartment, leading to altered pro-inflammatory cytokine secretion.

Role of HIF-1α and CASPASE-3 in cystogenesis of odontogenic cysts and tumors.

OBJECTIVES: Odontogenic cysts and tumors are the most relevant lesions that affect the gnathic bones. These lesions have in common the formation of cystic areas and this common feature may suggest involvement of similar mechanisms. The hypoxia inducible factor 1 alpha (HIF-1α), a responsive protein to hypoxia and caspase-3, an irreversible apoptosis marker, may contribute to cyst formation. Thus, this study aimed to investigate the immunoexpression of these proteins in odontogenic cysts and tumors. MATERIAL AND METHODS: Twenty cases of ameloblastoma, keratocystic odontogenic tumor (KOT) (n = 20), radicular cyst (RC) (n = 18), dentigerous cyst (DC) (n = 11), calcifying cystic odontogenic tumor (n = 8), and dental follicle (DF) (n = 10) were used to investigate HIF-1α and caspase-3 expression in sequential serial cuts by immunohistochemistry. RESULTS: HIF-1α was overexpressed in RC, DC, and ameloblastoma when compared with DF. The basal and sometimes the lower suprabasal layer showed no or very low expression in DC, KOT, and ameloblastoma, the last also showing strong expression in solid epithelial areas and initial cystic formation regions. Caspase-3 was found to be overexpressed in all lesions, with the highest expression in odontogenic cysts compared to tumors. HIF-1α and caspase-3 were localized in similar areas of the same lesions, especially in the epithelium surrounding cystic formations. CONCLUSIONS: This study showed distinct immunoexpression of HIF-1α and caspase-3 in odontogenic cyst and tumors, with higher expression observed in odontogenic cysts. CLINICAL RELEVANCE: These findings suggest a possible correlation between hypoxia, apoptosis, and cystogenesis, leading to understand the mechanisms responsible to cystic formation in odontogenic lesions.

The Human Salivary Microbiome Is Shaped by Shared Environment Rather than Genetics: Evidence from a Large Family of Closely Related Individuals.

The human microbiome is affected by multiple factors, including the environment and host genetics. In this study, we analyzed the salivary microbiomes of an extended family of Ashkenazi Jewish individuals living in several cities and investigated associations with both shared household and host genetic similarities. We found that environmental effects dominated over genetic effects. While there was weak evidence of geographical structuring at the level of cities, we observed a large and significant effect of shared household on microbiome composition, supporting the role of the immediate shared environment in dictating the presence or absence of taxa. This effect was also seen when including adults who had grown up in the same household but moved out prior to the time of sampling, suggesting that the establishment of the salivary microbiome earlier in life may affect its long-term composition. We found weak associations between host genetic relatedness and microbiome dissimilarity when using family pedigrees as proxies for genetic similarity. However, this association disappeared when using more-accurate measures of kinship based on genome-wide genetic markers, indicating that the environment rather than host genetics is the dominant factor affecting the composition of the salivary microbiome in closely related individuals. Our results support the concept that there is a consistent core microbiome conserved across global scales but that small-scale effects due to a shared living environment significantly affect microbial community composition.IMPORTANCE Previous research shows that the salivary microbiomes of relatives are more similar than those of nonrelatives, but it remains difficult to distinguish the effects of relatedness and shared household environment. Furthermore, pedigree measures may not accurately measure host genetic similarity. In this study, we include genetic relatedness based on genome-wide single nucleotide polymorphisms (SNPs) (rather than pedigree measures) and shared environment in the same analysis. We quantify the relative importance of these factors by studying the salivary microbiomes in members of a large extended Ashkenazi Jewish family living in different locations. We find that host genetics plays no significant role and that the dominant factor is the shared environment at the household level. We also find that this effect appears to persist in individuals who have moved out of the parental household, suggesting that aspects of salivary microbiome composition established during upbringing can persist over a time scale of years.

EGFR signaling downstream of EGF regulates migration, invasion, and MMP secretion of immortalized cells derived from human ameloblastoma.

Ameloblastoma is an odontogenic tumor characterized by local invasiveness and frequent recurrence. The surrounding stroma, composed of different cell types and extracellular matrix (ECM), may influence ameloblastoma invasive behavior. Furthermore, tumor and stromal cells secrete matrix metalloproteases (MMPs), which, in turn, can modulate the matrix and promote the release of ECM-bound growth factors. Among these growth factors, epidermal growth factor (EGF) and its receptor, EGFR, have already been shown to stimulate MMP synthesis, suggesting that an interdependent mechanism, involving MMP activity and growth factors release, may contribute to tumor invasiveness. The aim of this study was to evaluate the effects of the EGF/EGFR signaling pathway on migration, invasion, and MMP activity, in a primary cell line derived from human ameloblastoma. We established and characterized a primary cell line (AME-1) from a human ameloblastoma sample. This cell line was transduced with human papillomavirus type 16 (HPV16) E6/E7 oncogenes, generating the AME-HPV continuous cell line. EGF, MMP2, and MMP9 expression in ameloblastoma biopsies and in the AME-HPV cell line was analyzed by immunohistochemistry and immunofluorescence, respectively. Migratory activity of EGF-treated AME-HPV cells was investigated using monolayer wound assays and Transwell chambers. EGF-induced invasion was assessed in Boyden chambers coated with Matrigel. Conditioned medium from EGF-treated cells was subjected to zymography. EGFR expression in AME-HPV cells was silenced by small interfering RNA (siRNA), to verify the relationship between this receptor and MMP secretion. Ameloblastoma samples and AME-HPV cells expressed EGF, EGFR, MMP2, and MMP9. AME-HPV cells treated with EGF showed increased rates of migration and invasion, as well as enhanced MMP2 and MMP9 activity. EGFR knockdown decreased MMP2 and MMP9 levels in AME-HPV cells. EGFR signaling downstream of EGF probably regulates migration, invasion, and MMP secretion of ameloblastoma-derived cells.

Life-threatening expansive sublingual hematoma: a stab wound with lingual artery injury.

Vascular injuries are a constant risk in facial trauma, although bone and soft tissues of the face have provided some protection to the larger blood vessels. However, penetrating injuries usually do not have this type of protection and can damage significant vascular arteries. This article presents a case of a stab wound, which led to airway obstruction arising to a large sublingual hematoma due to lingual artery injury. A healthy 44-year-old man was stabbed in the submandibular region and admitted with an airway obstruction. He was subjected to an emergency tracheotomy and evolved with progressive sublingual edema. Computed tomography (CT) angiography showed a left lingual artery injury with the formation of an expansive hematoma. The CT angiography findings helped to identify the cause of the hematoma and guided the surgery to drain the hematoma after ligation of the lingual artery. The treatment was safely performed as planned and evolved uneventfully. The patient recovered fast and well and presented normal functions 6 months after the treatment. This surgical technique is an effective method for treating such injuries because it can be safely performed when guided by CT angiography. The authors argue that the demand for vascular lesions should be routine in patients who have facial trauma.