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Enteroviruses are a leading cause of upper respiratory tract, gastrointestinal, and neurological infections. Management of enterovirus-related diseases has been hindered by the lack of specific antiviral treatment. The pre-clinical and clinical development of such antivirals has been challenging, calling for novel model systems and strategies to identify suitable pre-clinical candidates. Organoids represent a new and outstanding opportunity to test antiviral agents in a more physiologically relevant system. However, dedicated studies addressing the validation and direct comparison of organoids versus commonly used cell lines are lacking. Here, we described the use of human small intestinal organoids (HIOs) as a model to study antiviral treatment against human enterovirus 71 (EV-A71) infection and compared this model to EV-A71-infected RD cells. We used reference antiviral compounds such as enviroxime, rupintrivir, and 2'- C -methylcytidine (2'CMC) to assess their effects on cell viability, virus-induced cytopathic effect, and viral RNA yield in EV-A71-infected HIOs and cell line. The results indicated a difference in the activity of the tested compounds between the two models, with HIOs being more sensitive to infection and drug treatment. In conclusion, the outcome reveals the value added by using the organoid model in virus and antiviral studies.

Maintenance of immunological homeostasis between tolerance and autoimmunity is essential for the prevention of human diseases ranging from autoimmune disease to cancer. Accumulating evidence suggests that p53 can mitigate phagocytosis-induced adjuvanticity thereby promoting immunological tolerance following programmed cell death. Here we identify Inhibitor of Apoptosis Stimulating p53 Protein (iASPP), a negative regulator of p53 transcriptional activity, as a regulator of immunological tolerance. iASPP-deficiency promoted lung adenocarcinoma and pancreatic cancer tumorigenesis, while iASPP-deficient mice were less susceptible to autoimmune disease. Immune responses to iASPP-deficient tumors exhibited hallmarks of immunosuppression, including activated regulatory T cells and exhausted CD8 + T cells. Interestingly, iASPP-deficient tumor cells and tumor-infiltrating myeloid cells, CD4 + , and γδ T cells expressed elevated levels of PD-1H, a recently identified transcriptional target of p53 that promotes tolerogenic phagocytosis. Identification of an iASPP/p53 axis of immune homeostasis provides a therapeutic opportunity for both autoimmune disease and cancer., (© 2023. The Author(s).)

During the past 15 years, a plethora of innovative 3D in vitro systems has been developed. They offer the possibility of identifying crucial cellular and molecular contributors to the disease by permitting manipulation of each in isolation. However, improvements are needed particularly with respect to the predictivity and validity of those models. The major challenge now is to identify which assay and readout combination(s) best suits the current scientific question(s). A deep understanding of the different platforms along with their pros and cons is a prerequisite to make this decision. This review aims to give an overview of the most prominent systems with a focus on applications, translational relevance and adoption drivers from an industry perspective., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The main risk factor for stomach cancer, the third most common cause of cancer death worldwide, is infection with Helicobacter pylori bacterial strains that inject cytotoxin-associated gene A (CagA). As the first described bacterial oncoprotein, CagA causes gastric epithelial cell transformation by promoting an epithelial-to-mesenchymal transition (EMT)-like phenotype that disrupts junctions and enhances motility and invasiveness of the infected cells. However, the mechanism by which CagA disrupts gastric epithelial cell polarity to achieve its oncogenicity is not fully understood. Here we found that the apoptosis-stimulating protein of p53 2 (ASPP2), a host tumor suppressor and an important CagA target, contributes to the survival of cagA -positive H. pylori in the lumen of infected gastric organoids. Mechanistically, the CagA-ASPP2 interaction is a key event that promotes remodeling of the partitioning-defective (PAR) polarity complex and leads to loss of cell polarity of infected cells. Blockade of cagA -positive H. pylori ASPP2 signaling by inhibitors of the EGFR (epidermal growth factor receptor) signaling pathway-identified by a high-content imaging screen-or by a CagA-binding ASPP2 peptide, prevents the loss of cell polarity and decreases the survival of H. pylori in infected organoids. These findings suggest that maintaining the host cell-polarity barrier would reduce the detrimental consequences of infection by pathogenic bacteria, such as H. pylori , that exploit the epithelial mucosal surface to colonize the host environment., Competing Interests: The authors declare no competing interest.

Recent work has disclosed the critical role played by enamel peptides in sex classification of old skeletal remains. In particular, protein AMELY (amelogenin isoform Y) is present in the enamel dental tissue of male individuals only, while AMELX (isoform X) can be found in both sexes. AMELY can be easily detected by LC-MS/MS in the ion extracted chromatograms of the SM (ox) IRPPY peptide (monoisotopic [M + 2 H] +2 mass = 440.2233 m/z). In this paper, we exploited the dimorphic features of the amelogenin protein to determine the sex of the so-called 'Lovers of Modena', two Late Antique individuals whose skeletons were intentionally buried hand-in-hand. Upon discovery, mass media had immediately assumed they were a male-female couple, even if bad preservation of the bones did not allow an effective sex classification. We were able to extract proteins from the dental enamel of both individuals (~1600 years old) and to confidently classify them as males. Results were compared to 14 modern and archaeological control samples, confirming the reliability of the ion chromatogram method for sex determination. Although we currently have no information on the actual relationship between the 'Lovers of Modena' (affective? Kin-based?), the discovery of two adult males intentionally buried hand-in-hand may have profound implications for our understanding of funerary practices in Late Antique Italy.

Inflammatory bowel disease (IBD) are heterogenous disorders of the gastrointestinal tract caused by a spectrum of genetic and environmental factors. In mice, overlapping regions of chromosome 3 have been associated with susceptibility to IBD-like pathology, including a locus called Hiccs. However, the specific gene that controls disease susceptibility remains unknown. Here we identify a Hiccs locus gene, Alpk1 (encoding alpha kinase 1), as a potent regulator of intestinal inflammation. In response to infection with the commensal pathobiont Helicobacter hepaticus (Hh), Alpk1-deficient mice display exacerbated interleukin (IL)-12/IL-23 dependent colitis characterized by an enhanced Th1/interferon(IFN)-γ response. Alpk1 controls intestinal immunity via the hematopoietic system and is highly expressed by mononuclear phagocytes. In response to Hh, Alpk1 -/- macrophages produce abnormally high amounts of IL-12, but not IL-23. This study demonstrates that Alpk1 promotes intestinal homoeostasis by regulating the balance of type 1/type 17 immunity following microbial challenge.

Objectives: Exact symmetry and perfect balance between opposite jaw halves, as well as between antagonistic teeth, is not frequently observed in natural masticatory systems. Research results show that asymmetry in our body, skull, and jaws is often related to genetic, epigenetic, environmental and individual ontogenetic factors. Our study aims to provide evidence for a significant link between masticatory asymmetry and occlusal contact between antagonist teeth by testing the hypothesis that tooth inclination is one of the mechanisms driving distribution of wear in masticatory phases in addition to dietary and cultural habits., Materials and Methods: The present work investigates the relationship between dental macrowear patterns and tooth inclinations on a sample of complete maxillary and mandibular 3D models of dental arches from 19 young and adult Yuendumu Aboriginal individuals. The analysis was carried out on first molars (M1) from all quadrants. Occlusal Fingerprint Analysis was used for the quantification of macrowear patterns, and 2D cross-sectional geometric analysis was carried out to investigate asymmetry in dental arches., Results: The asymmetry is highly variable on both arches, and it is associated with differences in the inclination of upper M1 crowns. Each molar has variable inclination (buccal/lingual) which influence tooth to tooth contact, producing greater or lesser variation in wear pattern. Interindividual variability of morphological variation of the occlusal relationship has to be considered in macrowear analysis., Discussion: Our results suggest that overall asymmetry in the masticatory apparatus in modern humans affects occlusal contact areas between antagonist teeth influencing macrowear and chewing efficiency during ontogeny., (© 2018 The Authors. American Journal of Physical Anthropology Published by Wiley Periodicals, Inc.)

The 4th century BC marks the main entrance of Celtic populations in northern Italy. Their arrival has been suggested based on the presence of Celtic customs in Etruscan mortuary contexts, yet up to now few bioarchaeological data have been examined to support or reject the arrival of these newcomers. Here we use strontium isotopes, non-metric dental traits and funerary patterns to unravel the biocultural structure of the necropolis of Monterenzio Vecchio (Bologna, Italy). Subsamples of our total sample of 38 individuals were analyzed based on different criteria characterizing the following analyses: 1) strontium isotope analysis to investigate migratory patterns and provenance; 2) non-metric dental traits to establish biological relationships between Monterenzio Vecchio, 13 Italian Iron age necropolises and three continental and non-continental Celtic necropolises; 3) grave goods which were statistically explored to detect possible patterns of cultural variability. The strontium isotopes results indicate the presence of local and non-local individuals, with some revealing patterns of mobility. The dental morphology reveals an affinity between Monterenzio Vecchio and Iron Age Italian samples. However, when the Monterenzio Vecchio sample is separated by isotopic results into locals and non-locals, the latter share affinity with the sample of non-continental Celts from Yorkshire (UK). Moreover, systematic analyses demonstrate that ethnic background does not retain measurable impact on the distribution of funerary elements. Our results confirm the migration of Celtic populations in Monterenzio as archaeologically hypothesized on the basis of the grave goods, followed by a high degree of cultural admixture between exogenous and endogenous traits. This contribution shows that combining different methods offers a more comprehensive perspective for the exploration of biocultural processes in past and present populations.

Transition from pluripotency to differentiation is a pivotal yet poorly understood developmental step. Here, we show that the tumour suppressor RASSF1A is a key player driving the early specification of cell fate. RASSF1A acts as a natural barrier to stem cell self-renewal and iPS cell generation, by switching YAP from an integral component in the β-catenin-TCF pluripotency network to a key factor that promotes differentiation. We demonstrate that epigenetic regulation of the Rassf1A promoter maintains stemness by allowing a quaternary association of YAP-TEAD and β-catenin-TCF3 complexes on the Oct4 distal enhancer. However, during differentiation, promoter demethylation allows GATA1-mediated RASSF1A expression which prevents YAP from contributing to the TEAD/β-catenin-TCF3 complex. Simultaneously, we find that RASSF1A promotes a YAP-p73 transcriptional programme that enables differentiation. Together, our findings demonstrate that RASSF1A mediates transcription factor selection of YAP in stem cells, thereby acting as a functional "switch" between pluripotency and initiation of differentiation.

Enamel thickness figures prominently in studies of human evolution, particularly for taxonomy, phylogeny, and paleodietary reconstruction. Attention has focused on molar teeth, through the use of advanced imaging technologies and novel protocols. Despite the important results achieved thus far, further work is needed to investigate all tooth classes. We apply a recent approach developed for anterior teeth to investigate the 3D enamel thickness of Neandertal and modern human (MH) canines. In terms of crown size, the values obtained for both upper and lower unworn/slightly worn canines are significantly greater in Neandertals than in Upper Paleolithic and recent MH. The 3D relative enamel thickness (RET) is significantly lower in Neandertals than in MH. Moreover, differences in 3D RET values between the two groups appear to decrease in worn canines beginning from wear stage 3, suggesting that both the pattern and the stage of wear may have important effects on the 3D RET value. Nevertheless, the 3D average enamel thickness (AET) does not differ between the two groups. In both groups, 3D AET and 3D RET indices are greater in upper canines than in lower canines, and overall the enamel is thicker on the occlusal half of the labial aspect of the crown, particularly in MH. By contrast, the few early modern humans investigated show the highest volumes of enamel while for all other components of 3D enamel, thickness this group holds an intermediate position between Neandertals and recent MH. Overall, our study supports the general findings that Neandertals have relatively thinner enamel than MH (as also observed in molars), indicating that unworn/slightly worn canines can be successfully used to discriminate between the two groups. Further studies, however, are needed to understand whether these differences are functionally related or are the result of pleiotropic or genetic drift effects., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objectives: This study investigates the effect of allometry on the shape of lower dm2 (dm2) and lower M1 (M1) crown outlines and examines whether the trajectory and magnitude of allometric scaling are shared between Neandertals and Homo sapiens., Methods: Our sample included 164 specimens: 57 recent H. sapiens, 44 Upper Paleolithic H. sapiens, 17 early H. sapiens, and 46 Neandertals. Of these, 59 represent dm2/M1 pairs from the same individuals. Occlusal photographs were used to obtain crown shapes of dm2s and M1s. Principal components analysis (PCA) of the matrix of shape coordinates was used to explore the pattern of morphological variation across the dm2 and M1 samples. Allometry was investigated by means of the Pearson product-moment correlation coefficient. Two-block partial least squares (2B-PLS) analysis was used to explore patterns of covariation between dm2 and M1 crown outlines of matched individual pairs., Results: The PCA confirmed significant differences between Neandertal and H. sapiens dm2 and M1 shapes. Allometry accounted for a small but statistically significant proportion of the total morphological variance. The magnitude of the allometric contribution to crown shape was stronger among Neandertals than among H. sapiens. However, we could not reject the null hypothesis that the two species share the same allometric trajectory. The 2B-PLS analysis of the pooled sample of paired individuals revealed a significant correlation in crown shape between dm2 and M1. While Procrustes distances differed significantly between dm2 and M1 in Neandertals, it did not among H. sapiens groups., Conclusions: Our results confirm several of the results obtained by a similar study of upper dm2/M1 (dm(2)/M(1)), but there are differences as well. Neandertal dm2/M1 shapes are less derived than those of the dm(2)/M(1). Such differences may support previous studies, which have suggested that different developmental and/or epigenetic factors affect the upper and lower dentitions., (© 2015 Wiley Periodicals, Inc.)

The Hippo pathway, by tightly controlling the phosphorylation state and activity of the transcription cofactors YAP and TAZ is essential during development and tissue homeostasis whereas its deregulation may lead to cancer. Recent studies have linked the apicobasal polarity machinery in epithelial cells to components of the Hippo pathway and YAP and TAZ themselves. However the molecular mechanism by which the junctional pool of YAP proteins is released and activated in epithelial cells remains unknown. Here we report that the tumour suppressor ASPP2 forms an apical-lateral polarity complex at the level of tight junctions in polarised epithelial cells, acting as a scaffold for protein phosphatase 1 (PP1) and junctional YAP via dedicated binding domains. ASPP2 thereby directly induces the dephosphorylation and activation of junctional YAP. Collectively, this study unearths a novel mechanistic paradigm revealing the critical role of the apical-lateral polarity complex in activating this localised pool of YAP in vitro, in epithelial cells, and in vivo, in the murine colonic epithelium. We propose that this mechanism may commonly control YAP functions in epithelial tissues.

The Cytotoxin associated gene A (CagA) protein of Helicobacter pylori is associated with increased virulence and risk of cancer. Recent proteomic studies have demonstrated an association of CagA with the human tumor suppressor Apoptosis-stimulating Protein of p53-2 (ASPP2). We present here a genetic, biochemical, and structural analysis of CagA with ASPP2. Domain delineation of the 120-kDa CagA protein revealed a stable N-terminal subdomain that was used in a yeast two-hybrid screen that identified the proline-rich domain of ASPP2 as a host cellular target. Biochemical experiments confirm this interaction. The cocrystal structure to 2.0-Å resolution of this N-terminal subdomain of CagA with a 7-kDa proline-rich sequence of ASPP2 reveals that this domain of CagA forms a highly specialized three-helix bundle, with large insertions in the loops connecting the helices. These insertions come together to form a deep binding cleft for a highly conserved 20-aa peptide of ASPP2. ASPP2 forms an extended helix in this groove of CagA, burying more than 1,000 Å(2) of surface area. This interaction is disrupted in vitro and in vivo by structure-based, loss-of-contact point mutations of key residues in either CagA or ASPP2. Disruption of CagA and ASPP2 binding alters the function of ASPP2 and leads to the decreased survival of H. pylori-infected cells., Competing Interests: The authors declare no conflict of interest.

This review discusses the multiple roles of the CagA protein encoded by the cag pathogenicity island of Helicobacter pylori and highlights the CagA degradation activities on p53. By subverting the p53 tumor suppressor pathway CagA induces a strong antiapoptotic effect. Helicobacter pylori infection has been always associated with an increased risk of gastric cancer. The pro-oncogenic functions of CagA also target the tumor suppressor ASPP2. In the absence of tumor suppressor genes, cells survive and proliferate at times and in places where their survival and proliferation are inappropriate.

Antigen-specific memory B cells generate anamnestic responses and high affinity antibodies upon re-exposure to pathogens. Attempts to isolate rare antigen-specific memory B cells for in-depth functional analysis at the single-cell level have been hindered by the lack of tools with adequate sensitivity. We applied two independent methods of protein labeling to sensitive and specific ex vivo identification of antigen-specific memory B cells by flow cytometry: stringently controlled amine labeling, and sortagging, a novel method whereby a single nucleophilic fluorochrome molecule is added onto an LPETG motif carried by the target protein. We show that sortagged NadA, a major antigen in the meningococcal serogroup B vaccine, identifies NadA-specific memory B cells with high sensitivity and specificity, comparable to NadA amine-labeled under stringent reaction parameters in a mouse model of vaccination. We distinguish NadA-specific switched MBC induced by vaccination from the background signal contributed by splenic transitional and marginal zone B cells. In conclusion, we demonstrate that protein structural data coupled with sortag technology allows the development of engineered antigens that are as sensitive and specific as conventional chemically labeled antigens in detecting rare MBC, and minimize the possibility of disrupting conformational B cell epitopes.

Despite the general increase in digital techniques for dental morphometric analyses, only a few methods are available to study worn teeth. Moreover, permanent dentitions are studied much more frequently than deciduous teeth. In this study, we address both issues by providing a taxonomic classification of Neanderthal and modern human (MH) lower second deciduous molars (dm(2) s) through the analysis of crown and cervical outlines. Crown and cervical outlines were obtained from a three-dimensional (3D) digital sample of uniformly oriented dm(2) s. Both outlines were centered on the centroid of their area and represented by 16 pseudolandmarks obtained by equiangularly spaced radial vectors out of the centroid. We removed size information from the oriented and centered outlines with a uniform scaling of the pseudolandmark configurations to unit Centroid Size. Group shape variation was evaluated separately for the dm(2) crown and cervical outlines through a shape-space principal component (PC) analysis. Finally, quadratic discriminant analysis of a subset of PCs was used to classify the specimens. Our results demonstrate that both outlines successfully separate the two groups. Neanderthals showed a buccodistal expansion and convex lingual outline shape, whilst MHs have buccodistal reduction and straight lingual outline shape. Therefore, we confirmed that the cervical outline represents an effective parameter for distinguishing between the two taxa when dealing with worn or damaged dm(2) s., (Copyright © 2012 Wiley Periodicals, Inc.)

Protein array technology is an emerging tool that enables high-throughput screening of protein-protein or protein-lipid interactions and identification of immunodominant antigens during the course of a bacterial or viral infection. In this work, we developed an Influenza virus protein array using the sortase-mediated transpeptidation reaction known as "Sortagging". LPETG-tagged Influenza virus proteins from bacterial and eukaryotic cellular extracts were immobilized at their carboxyl-termini onto a preactivated amine-glass slide coated with a Gly3 linker. Immobilized proteins were revealed by specific antibodies, and the newly generated Sortag-protein chip can be used as a device for antigen and/or antibody screening. The specificity of the Sortase A (SrtA) reaction avoids purification steps in array building and allows immobilization of proteins in an oriented fashion. Previously, this versatile technology has been successfully employed for protein labeling and protein conjugation. Here, the tool is implemented to covalently link proteins of a viral genome onto a solid support. The system could readily be scaled up to proteins of larger genomes in order to develop protein arrays for high-throughput screening.

Type I strains of Helicobacter pylori (Hp) possess a pathogenicity island, cag, that encodes the effector protein cytotoxin-associated gene A (CagA) and a type four secretion system. After translocation into the host cell, CagA affects cell shape, increases cell motility, abrogates junctional activity, and promotes an epithelial to mesenchymal transition-like phenotype. Transgenic expression of CagA enhances gastrointestinal and intestinal carcinomas as well as myeloid and B-cell lymphomas in mice, but the mechanism of the induced cancer formation is not fully understood. Here, we show that CagA subverts the tumor suppressor function of apoptosis-stimulating protein of p53 (ASPP2). Delivery of CagA inside the host results in its association with ASPP2. After this interaction, ASPP2 recruits its natural target p53 and inhibits its apoptotic function. CagA leads to enhanced degradation of p53 and thereby, down-regulates its activity in an ASPP2-dependent manner. Finally, Hp-infected cells treated with the p53-activating drug Doxorubicin are more resistant to apoptosis than uninfected cells, an effect that requires ASPP2. The interaction between CagA and ASPP2 and the consequent degradation of p53 are examples of a bacterial protein that subverts the p53 tumor suppressor pathway in a manner similar to DNA tumor viruses. This finding may contribute to the understanding of the increased risk of gastric cancer in patients infected with Hp CagA+ strains.

Toll-like receptor (TLR) signaling plays a critical role in innate and adaptive immune responses and must be tightly controlled. TLR4 uses LPS binding protein, MD-2, and CD14 as accessories to respond to LPS. We therefore investigated the presence of an analagous soluble cofactor that might assist in the recruitment of CpG oligonucleotides (CpG-ODNs) to TLR9. We report the identification of granulin as an essential secreted cofactor that potentiates TLR9-driven responses to CpG-ODNs. Granulin, an unusual cysteine-rich protein, bound to CpG-ODNs and interacted with TLR9. Macrophages from granulin-deficient mice showed not only impaired delivery of CpG-ODNs to endolysosomal compartments, but also decreased interaction of TLR9 with CpG-ODNs. As a consequence, granulin-deficient macrophages showed reduced responses to stimulation with CpG-ODNs, a trait corrected by provision of exogenous granulin. Thus, we propose that granulin contributes to innate immunity as a critical soluble cofactor for TLR9 signaling., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The ubiquitin (Ub)-related modifier Urm1 functions as a sulfur carrier in tRNA thiolation by means of a mechanism that requires the formation of a thiocarboxylate at the C-terminal glycine residue of Urm1. However, whether Urm1 plays an additional role as a Ub-like protein modifier remains unclear. Here, we show that Urm1 is conjugated to lysine residues of target proteins and that oxidative stress enhances protein urmylation in both Saccharomyces cerevisiae and mammalian cells. Similar to ubiquitylation, urmylation involves a thioester intermediate and results in the formation of a covalent peptide bond between Urm1 and its substrates. In contrast to modification by canonical Ub-like modifiers, however, conjugation of Urm1 involves a C-terminal thiocarboxylate of the modifier. We have confirmed that the peroxiredoxin Ahp1 is such a substrate in S. cerevisiae and found that Urm1 targets a specific lysine residue of Ahp1 in vivo. In addition, we have identified several unique substrates in mammalian cells and show that Urm1 targets at least two pathways on oxidant treatment. First, Urm1 is appended to lysine residues of three components that function in its own pathway (i.e., MOCS3, ATPBD3, and CTU2). Second, Urm1 is conjugated to the nucleocytoplasmic shuttling factor cellular apoptosis susceptibility protein. Thus, Urm1 has a conserved dual role by integrating the functions of prokaryotic sulfur carriers with those of eukaryotic protein modifiers of the Ub family.

CagA is a bacterial effector protein of Helicobacter pylori that is translocated via a type IV secretion system into gastric epithelial cells. We previously described that H. pylori require CagA to disrupt the organization and assembly of apical junctions in polarized epithelial cells. In this study, we provide evidence that CagA expression is not only sufficient to disrupt the apical junctions but also perturbs epithelial differentiation. CagA-expressing cells lose apicobasal polarity and cell-cell adhesion, extend migratory pseudopodia, and degrade basement membranes, acquiring an invasive phenotype. Expression of the CagA C-terminal domain, which contains the tyrosine phosphorylated EPIYA motifs, induces pseudopodial activity but is not sufficient to induce cell migration. Conversely, the N terminus targets CagA to the cell-cell junctions. Neither domain is sufficient to disrupt cell adhesion or cell polarity, but coexpressed in trans, the N terminus determines the localization of both polypeptides. We show that CagA induces a morphogenetic program in polarized Madin-Darby canine kidney cells resembling an epithelial-to-mesenchymal transition. We propose that altered cell-cell and cell matrix interactions may serve as an early event in H. pylori-induced carcinogenesis.