E-cadherin variants associated with oral facial clefts trigger aberrant cell motility in a REG1A-dependent manner.

BACKGROUND: Germline mutations of E-cadherin contribute to hereditary diffuse gastric cancer (HDGC) and congenital malformations, such as oral facial clefts (OFC). However, the molecular mechanisms through which E-cadherin loss-of-function triggers distinct clinical outcomes remain unknown. We postulate that E-cadherin-mediated disorders result from abnormal interactions with the extracellular matrix and consequent aberrant intracellular signalling, affecting the coordination of cell migration. METHODS: Herein, we developed in vivo and in vitro models of E-cadherin mutants associated with either OFC or HDGC. Using a Drosophila approach, we addressed the impact of the different variants in cell morphology and migration ability. By combining gap closure migration assays and time-lapse microscopy, we further investigated the migration pattern of cells expressing OFC or HDGC variants. The adhesion profile of the variants was evaluated using high-throughput ECM arrays, whereas RNA sequencing technology was explored for identification of genes involved in aberrant cell motility. RESULTS: We have demonstrated that cells expressing OFC variants exhibit an excessive motility performance and irregular leading edges, which prevent the coordinated movement of the epithelial monolayer. Importantly, we found that OFC variants promote cell adhesion to a wider variety of extracellular matrices than HDGC variants, suggesting higher plasticity in response to different microenvironments. We unveiled a distinct transcriptomic profile in the OFC setting and pinpointed REG1A as a putative regulator of this outcome. Consistent with this, specific RNAi-mediated inhibition of REG1A shifted the migration pattern of OFC expressing cells, leading to slower wound closure with coordinated leading edges. CONCLUSIONS: We provide evidence that E-cadherin variants associated with OFC activate aberrant signalling pathways that support dynamic rearrangements of cells towards improved adaptability to the microenvironment. This proficiency results in abnormal tissue shaping and movement, possibly underlying the development of orofacial malformations.

Integrin ß1 orchestrates the abnormal cell-matrix attachment and invasive behaviour of E-cadherin dysfunctional cells.

BACKGROUND: Tumour progression relies on the ability of cancer cells to penetrate and invade neighbouring tissues. E-cadherin loss is associated with increased cell invasion in gastric carcinoma, and germline mutations of the E-cadherin gene are causative of hereditary diffuse gastric cancer. Although E-cadherin dysfunction impacts cell-cell adhesion, cell dissemination also requires an imbalance of adhesion to the extracellular matrix (ECM). METHODS: To identify ECM components and receptors relevant for adhesion of E-cadherin dysfunctional cells, we implemented a novel ECM microarray platform coupled with molecular interaction networks. The functional role of putative candidates was determined by combining micropattern traction microscopy, protein modulation and in vivo approaches, as well as transcriptomic data of 262 gastric carcinoma samples, retrieved from the cancer genome atlas (TCGA). RESULTS: Here, we show that E-cadherin mutations induce an abnormal interplay of cells with specific components of the ECM, which encompasses increased traction forces and Integrin ß1 activation. Integrin ß1 synergizes with E-cadherin dysfunction, promoting cell scattering and invasion. The significance of the E-cadherin-Integrin ß1 crosstalk was validated in Drosophila models and found to be consistent with evidence from human gastric carcinomas, where increased tumour grade and poor survival are associated with low E-cadherin and high Integrin ß1 levels. CONCLUSIONS: Integrin ß1 is a key mediator of invasion in carcinomas with E-cadherin impairment and should be regarded as a biomarker of poor prognosis in gastric cancer.

Hereditary diffuse gastric cancer: updated clinical practice guidelines.

Hereditary diffuse gastric cancer (HDGC) is an autosomal dominant cancer syndrome that is characterised by a high prevalence of diffuse gastric cancer and lobular breast cancer. It is largely caused by inactivating germline mutations in the tumour suppressor gene CDH1, although pathogenic variants in CTNNA1 occur in a minority of families with HDGC. In this Policy Review, we present updated clinical practice guidelines for HDGC from the International Gastric Cancer Linkage Consortium (IGCLC), which recognise the emerging evidence of variability in gastric cancer risk between families with HDGC, the growing capability of endoscopic and histological surveillance in HDGC, and increased experience of managing long-term sequelae of total gastrectomy in young patients. To redress the balance between the accessibility, cost, and acceptance of genetic testing and the increased identification of pathogenic variant carriers, the HDGC genetic testing criteria have been relaxed, mainly through less restrictive age limits. Prophylactic total gastrectomy remains the recommended option for gastric cancer risk management in pathogenic CDH1 variant carriers. However, there is increasing confidence from the IGCLC that endoscopic surveillance in expert centres can be safely offered to patients who wish to postpone surgery, or to those whose risk of developing gastric cancer is not well defined.

E-cadherin deregulation in breast cancer.

E-cadherin protein (CDH1 gene) integrity is fundamental to the process of epithelial polarization and differentiation. Deregulation of the E-cadherin function plays a crucial role in breast cancer metastases, with worse prognosis and shorter overall survival. In this narrative review, we describe the inactivating mechanisms underlying CDH1 gene activity and its possible translation to clinical practice as a prognostic biomarker and as a potential targeted therapy.

Clinical spectrum and pleiotropic nature of CDH1 germline mutations.

CDH1 encodes E-cadherin, a key protein in adherens junctions. Given that E-cadherin is involved in major cellular processes such as embryogenesis and maintenance of tissue architecture, it is no surprise that deleterious effects arise from its loss of function. E-cadherin is recognised as a tumour suppressor gene, and it is well established that CDH1 genetic alterations cause diffuse gastric cancer and lobular breast cancer-the foremost manifestations of the hereditary diffuse gastric cancer syndrome. However, in the last decade, evidence has emerged demonstrating that CDH1 mutations can be associated with lobular breast cancer and/or several congenital abnormalities, without any personal or family history of diffuse gastric cancer. To date, no genotype-phenotype correlations have been observed. Remarkably, there are reports of mutations affecting the same nucleotide but inducing distinct clinical outcomes. In this review, we bring together a comprehensive analysis of CDH1-associated disorders and germline alterations found in each trait, providing important insights into the biological mechanisms underlying E-cadherin's pleiotropic effects. Ultimately, this knowledge will impact genetic counselling and will be relevant to the assessment of risk of cancer development or congenital malformations in CDH1 mutation carriers.

S100P is a molecular determinant of E-cadherin function in gastric cancer.

BACKGROUND: E-cadherin has been awarded a key role in the aetiology of both sporadic and hereditary forms of gastric cancer. In this study, we aimed to identify molecular interactors that influence the expression and function of E-cadherin associated to cancer. METHODS: A data mining approach was used to predict stomach-specific candidate genes, uncovering S100P as a key candidate. The role of S100P was evaluated through in vitro functional assays and its expression was studied in a gastric cancer tissue microarray (TMA). RESULTS: S100P was found to contribute to a cancer pathway dependent on the context of E-cadherin function. In particular, we demonstrated that S100P acts as an E-cadherin positive regulator in a wild-type E-cadherin context, and its inhibition results in decreased E-cadherin expression and function. In contrast, S100P is likely to be a pro-survival factor in gastric cancer cells with loss of functional E-cadherin, contributing to an oncogenic molecular program. Moreover, expression analysis in a gastric cancer TMA revealed that S100P expression impacts negatively among patients bearing Ecad- tumours, despite not being significantly associated with overall survival on its own. CONCLUSIONS: We propose that S100P has a dual role in gastric cancer, acting as an oncogenic factor in the context of E-cadherin loss and as a tumour suppressor in a functional E-cadherin setting. The discovery of antagonist effects of S100P in different E-cadherin contexts will aid in the stratification of gastric cancer patients who may benefit from S100P-targeted therapies.

Blue intensity matters for cell cycle profiling in fluorescence DAPI-stained images.

In the past decades, there has been an amazing progress in the understanding of the molecular mechanisms of the cell cycle. This has been possible largely due to a better conceptualization of the cycle itself, but also as a consequence of technological advances. Herein, we propose a new fluorescence image-based framework targeted at the identification and segmentation of stained nuclei with the purpose to determine DNA content in distinct cell cycle stages. The method is based on discriminative features, such as total intensity and area, retrieved from in situ stained nuclei by fluorescence microscopy, allowing the determination of the cell cycle phase of both single and sub-population of cells. The analysis framework was built on a modified k-means clustering strategy and refined with a Gaussian mixture model classifier, which enabled the definition of highly accurate classification clusters corresponding to G1, S and G2 phases. Using the information retrieved from area and fluorescence total intensity, the modified k-means (k=3) cluster imaging framework classified 64.7% of the imaged nuclei, as being at G1 phase, 12.0% at G2 phase and 23.2% at S phase. Performance of the imaging framework was ascertained with normal murine mammary gland cells constitutively expressing the Fucci2 technology, exhibiting an overall sensitivity of 94.0%. Further, the results indicate that the imaging framework has a robust capacity to both identify a given DAPI-stained nucleus to its correct cell cycle phase, as well as to determine, with very high probability, true negatives. Importantly, this novel imaging approach is a non-disruptive method that allows an integrative and simultaneous quantitative analysis of molecular and morphological parameters, thus awarding the possibility of cell cycle profiling in cytological and histological samples.

E-cadherin-defective gastric cancer cells depend on Laminin to survive and invade.

Epithelial-cadherin (Ecad) deregulation affects cell-cell adhesion and results in increased invasiveness of distinct human carcinomas. In gastric cancer, loss of Ecad expression is a common event and is associated with disease aggressiveness and poor prognosis. However, the molecular mechanisms underlying the invasive process associated to Ecad dysfunction are far from understood. We hypothesized that deregulation of cell-matrix interactions could play an important role during this process. Thus, we focussed on LM-332, which is a major matrix component, and in Ecad/LM-332 crosstalk in the process of Ecad-dependent invasion. To verify whether matrix deregulation was triggered by Ecad loss, we used the Drosophila model. To dissect the key molecules involved and unveil their functional significance, we used gastric cancer cell lines. The relevance of this relationship was then confirmed in human primary tumours. In vivo, Ecad knockdown induced apoptosis; nonetheless, at the invasive front, cells ectopically expressed Laminin A and ßPS integrin. In vitro, we demonstrated that, in two different gastric cancer cell models, Ecad-defective cells overexpressed Laminin γ2 (LM-γ2), ß1 and ß4 integrin, when compared with Ecad-competent ones. We showed that LM-γ2 silencing impaired invasion and enhanced cell death, most likely via pSrc and pAkt reduction, and JNK activation. In human gastric carcinomas, we found a concomitant decrease in Ecad and increase in LM-γ2. This is the first evidence that ectopic Laminin expression depends on Ecad loss and allows Ecad-dysfunctional cells to survive and invade. This opens new avenues for using LM-γ2 signalling regulators as molecular targets to impair gastric cancer progression.

Multivariate optimization of headspace-GC for the determination of monoaromatic compounds (benzene, toluene, ethylbenzene, and xylenes) in waters and wastewaters.

The objective of this study was to optimize, by employing a central composite rotatable design, and validate an analytical method to detect and quantify monoaromatic compounds (benzene, toluene, ethylbenzene, and xylenes) in waters and wastewaters by using headspace extraction followed by GC coupled with photoionization detection. The extraction parameters optimized were: salinity, sample volume, incubation time, and extraction temperature. The results revealed that the sample volume was the most significant parameter in the extraction process, whereas the salinity effect was negligible, which extends the applicability of the analytical method to waters with different salinities. Finally, the studied method was very selective and, at the optimal extraction conditions (15 mL sample volume, 15 min incubation time, and temperature of 70°C), presented excellent repeatability (<4%), linearity (R > 0.999 for each compound), and sensitivity, since very low LODs (0.13-0.48 µg/L) and LOQs (0.43-1.61 µg/L) were achieved.

Protective effects of amphetamine and methylphenidate against dopaminergic neurotoxicants in SH-SY5Y cells.

Full treatment of the second most common neurodegenerative disorder, Parkinson's disease (PD), is still considered an unmet need. As the psychostimulants, amphetamine (AMPH) and methylphenidate (MPH), were shown to be neuroprotective against stroke and other neuronal injury diseases, this study aimed to evaluate their neuroprotective potential against two dopaminergic neurotoxicants, 6-hydroxydopamine (6-OHDA) and paraquat (PQ), in differentiated human dopaminergic SH-SY5Y cells. Neither cytotoxicity nor mitochondrial membrane potential changes were seen following a 24-hour exposure to either therapeutic concentration of AMPH or MPH (0.001-10 µM). On the other hand, a 24-hour exposure to 6-OHDA (31.25-500 µM) or PQ (100-5000 µM) induced concentration-dependent mitochondrial dysfunction, assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, and lysosomal damage, evaluated by the neutral red uptake assay. The lethal concentrations 25 and 50 retrieved from the concentration-toxicity curves in the MTT assay were 99.9 µM and 133.6 µM for 6-OHDA, or 422 µM and 585.8 µM for PQ. Both toxicants caused mitochondrial membrane potential depolarization, but only 6-OHDA increased reactive oxygen species (ROS). Most importantly, PQ-induced toxicity was partially prevented by 1 µM of AMPH or MPH. Nonetheless, neither AMPH nor MPH could prevent 6-OHDA toxicity in this experimental model. According to these findings, AMPH and MPH may provide some neuroprotection against PQ-induced neurotoxicity, but further investigation is required to determine the exact mechanism underlying this protection.

Amphetamine and methylphenidate potential on the recovery from stroke and traumatic brain injury: a review.

The prevalence of stroke and traumatic brain injury is increasing worldwide. However, current treatments do not fully cure or stop their progression, acting mostly on symptoms. Amphetamine and methylphenidate are stimulants already approved for attention deficit hyperactivity disorder and narcolepsy treatment, with neuroprotective potential and benefits when used in appropriate doses. This review aimed to summarize pre-clinical and clinical trials testing either amphetamine or methylphenidate for the treatment of stroke and traumatic brain injury. We used PubMed as a database and included the following keywords ((methylphenidate) OR (Ritalin) OR (Concerta) OR (Biphentin) OR (amphetamine) OR (Adderall)) AND ((stroke) OR (brain injury) OR (neuroplasticity)). Overall, studies provided inconsistent results regarding cognitive and motor function. Neurite outgrowth, synaptic proteins, dendritic complexity, and synaptic plasticity increases were reported in pre-clinical studies along with function improvement. Clinical trials have demonstrated that, depending on the brain region, there is an increase in motor activity, attention, and memory due to the stimulation of the functionally depressed catecholamine system and the activation of neuronal remodeling proteins. Nevertheless, more clinical trials and pre-clinical studies are needed to understand the drugs' full potential for their use in these brain diseases namely, to ascertain the treatment time window, ideal dosage, long-term effects, and mechanisms, while avoiding their addictive potential.

Biological Response Following the Systemic Injection of PEG-PAMAM-Rhodamine Conjugates in Zebrafish.

Numerous therapeutic and diagnostic approaches used within a clinical setting depend on the administration of compounds via systemic delivery. Biomaterials at the nanometer scale, as dendrimers, act as delivery systems by improving cargo bioavailability, circulation time, and the targeting of specific tissues. Although evaluating the efficacy of pharmacological agents based on nanobiomaterials is crucial, conducting toxicological assessments of biomaterials is essential for advancing clinical translation. Here, a zebrafish larvae model was explored to assess the biocompatibility of poly(amido amine) (PAMAM), one of the most exploited dendrimers for drug delivery. We report the impact of a systemic injection of polyethylene glycol (PEG)-modified G4 PAMAM conjugated with rhodamine (Rho) as a mimetic drug (PEG-PAMAM-Rho) on survival, animal development, inflammation, and neurotoxicity. A concentration- and time-dependent effect was observed on mortality, developmental morphology, and innate immune system activation (macrophages). Significant effects in toxicological indicators were reported in the highest tested concentration (50 mg/mL PEG-PAMAM-Rho) as early as 48 h post-injection. Additionally, a lower concentration of PEG-PAMAM-Rho (5 mg/mL) was found to be safe and subsequently tested for neurotoxicity through behavioral assays. In accordance, no significative signs of toxicity were detected. In conclusion, the dose response of the animal was assessed, and the safe dosage for future use in theragnostics was defined. Additionally, new methodologies were established that can be adapted to further studies in toxicology using other nanosystems for systemic delivery.

CDH1 Genotype Exploration in Women With Hereditary Lobular Breast Cancer Phenotype.

Importance: Pathogenic or likely pathogenic (P/LP) germline CDH1 variants are associated with risk for diffuse gastric cancer and lobular breast cancer (LBC) in the so-called hereditary diffuse gastric cancer (HDGC) syndrome. However, in some circumstances, LBC can be the first manifestation of this syndrome in the absence of diffuse gastric cancer manifestation. Objectives: To evaluate the frequency of germline CDH1 variants in women with the hereditary LBC (HLBC) phenotype, somatic CDH1 gene inactivation in germline CDH1 variant carriers' tumor samples, and the association of genetic profiles with clinical-pathological data and survival. Design, Setting, and Participants: This single-center, longitudinal, prospective cohort study was conducted from January 1, 1997, to December 31, 2021, with follow-up until January 31, 2023. Women with LBC seen at the European Institute of Oncology were included. Testing for germline CDH1, BRCA1, and BRCA2 genes was performed. Somatic profiling was assessed for germline CDH1 carriers. Main Outcomes and Measures: Accurate estimates of prevalence of germline CDH1 variants among patients with HLBC and the association of somatic sequence alteration with HLBC syndrome. The Kaplan-Meier method and a multivariable Cox proportional hazards regression model were applied for overall and disease-free survival analysis. Results: Of 5429 cases of primary LBC, familial LBC phenotype accounted for 1867 (34.4%). A total of 394 women with LBC were tested, among whom 15 germline CDH1 variants in 15 unrelated families were identified. Among these variants, 6 (40.0%) were P/LP, with an overall frequency of 1.5% (6 of 394). Of the 6 probands with P/LP CDH1 LBC, 5 (83.3%) had a positive family history of BC and only 1 (16.7%) had sporadic juvenile early-onset LBC. No germline BRCA1 and BRCA2 variants were identified in CDH1 carriers. An inactivating CDH1 mechanism (second hit) was identified in 4 of 6 explored matched tumor samples (66.7%) in P/LP germline carriers. The P/LP CDH1 LBC variant carriers had a significantly lower age at diagnosis compared with the group carrying CDH1 variants of unknown significance or likely benign (42.5 [IQR, 38.3-43.0] vs 51.0 [IQR, 45.0-53.0] years; P = .03). Conclusions and Relevance: In this cohort study, P/LP germline CDH1 variants were identified in individuals not fulfilling the classic clinical criteria for HDGC screening, suggesting that identification of these variants may provide a novel method to test women with LBC with early age at diagnosis and/or positive family history of BC.

Effect of metabolic syndrome risk factors and MMP-2 genetic variations on circulating MMP-2 levels in childhood obesity.

Matrix metalloproteinase-2 is involved in the development of the adipose tissue, and associated with cardiovascular diseases. Metabolic risk factors (MRFs) and functional polymorphisms in the MMP-2 gene may affect its expression and activity. We investigated whether traditional MRFs and two MMP-2 gene polymorphisms (C(-1306)T; rs243865, and C(-735)T; rs2285053) affect circulating MMP-2 levels in children and adolescents, and whether MMP-2 polymorphisms and/or haplotype are associated with susceptibility to childhood obesity. We studied 114 healthy controls, 43 obese, and 83 obese with ≥ 3 MRFs children and adolescents. Genotypes were determined by Taqman allele discrimination assay and real-time PCR. Plasma MMP-2 was measured using zymography. We found positive correlations between MMP-2 concentrations and mean blood pressure in all children and adolescents group (r = 0.132; P < 0.05) and in obese children and adolescents (r = 0.247; P < 0.01). We found that the CC genotype for the C(-1306)T polymorphism was more common in subjects with higher MMP-2 concentrations in controls (P = 0.003) and in the obese group (P = 0.013). The CT genotype (OR = 0.40; P < 0.01) and the T allele (OR = 0.48; P < 0.01) for the C(-735)T polymorphism were less common in obese children and adolescents than in controls. The haplotypes distribution did not show significant differences between control and obese (P > 0.05). Ours findings show that blood pressure is associated with circulating MMP-2 concentrations, and that the CC genotype for the C(-1306)T polymorphism was more common subjects (controls and obese) with higher MMP-2 concentrations, whereas the CT genotype and the T allele for the C(-735)T polymorphism are less common in obesity.

The ECM and tissue architecture are major determinants of early invasion mediated by E-cadherin dysfunction.

Germline mutations of E-cadherin cause Hereditary Diffuse Gastric Cancer (HDGC), a highly invasive cancer syndrome characterised by the occurrence of diffuse-type gastric carcinoma and lobular breast cancer. In this disease, E-cadherin-defective cells are detected invading the adjacent stroma since very early stages. Although E-cadherin loss is well established as a triggering event, other determinants of the invasive process persist largely unknown. Herein, we develop an experimental strategy that comprises in vitro extrusion assays using E-cadherin mutants associated to HDGC, as well as mathematical models epitomising epithelial dynamics and its interaction with the extracellular matrix (ECM). In vitro, we verify that E-cadherin dysfunctional cells detach from the epithelial monolayer and extrude basally into the ECM. Through phase-field modelling we demonstrate that, aside from loss of cell-cell adhesion, increased ECM attachment further raises basal extrusion efficiency. Importantly, by combining phase-field and vertex model simulations, we show that the cylindrical structure of gastric glands strongly promotes the cell's invasive ability. Moreover, we validate our findings using a dissipative particle dynamics simulation of epithelial extrusion. Overall, we provide the first evidence that cancer cell invasion is the outcome of defective cell-cell linkages, abnormal interplay with the ECM, and a favourable 3D tissue structure.

Genetic control of IL-1 beta production and inflammatory response by the mouse Irm1 locus.

Genome-wide linkage analysis using single nucleotide polymorphism arrays was carried out in pedigrees of mice differing in the extent of acute inflammatory response (AIRmax or AIRmin). The AIR phenotype was determined by quantifying the number of infiltrating cells in the 24-h exudate induced by Biogel P-100 s.c. injection and by ex vivo IL-1beta production by leukocytes stimulated with LPS and ATP. We mapped the major inflammatory response modulator 1 locus on chromosome 7, at the 1-logarithm of odds (LOD) confidence interval from 116.75 to 139.75 Mb, linked to the number of infiltrating cells (LOD = 3.61) through the production of IL-1beta (LOD = 9.35). Of several interesting candidate genes mapping to the inflammatory response modulator 1 locus, 28 of these were differentially expressed in the bone marrow of AIRmax and AIRmin mice. These findings represent a step toward the identification of the genes underlying this complex phenotype.

Proteomic Identification of a Gastric Tumor ECM Signature Associated With Cancer Progression.

The extracellular matrix (ECM) plays an undisputable role in tissue homeostasis and its deregulation leads to altered mechanical and biochemical cues that impact cancer development and progression. Herein, we undertook a novel approach to address the role of gastric ECM in tumorigenesis, which remained largely unexplored. By combining decellularization techniques with a high-throughput quantitative proteomics approach, we have performed an extensive characterization of human gastric mucosa, uncovering its composition and distribution among tumor, normal adjacent and normal distant mucosa. Our results revealed a common ECM signature composed of 142 proteins and indicated that gastric carcinogenesis encompasses ECM remodeling through alterations in the abundance of 24 components, mainly basement membrane proteins. Indeed, we could only identify one de novo tumor-specific protein, the collagen alpha-1(X) chain (COL10A1). Functional analysis of the data demonstrated that gastric ECM remodeling favors tumor progression by activating ECM receptors and cellular processes involved in angiogenesis and cell-extrinsic metabolic regulation. By analyzing mRNA expression in an independent GC cohort available at the TGCA, we validated the expression profile of 12 differentially expressed ECM proteins. Importantly, the expression of COL1A2, LOX and LTBP2 significantly correlated with high tumor stage, with LOX and LTBP2 further impacting patient overall survival. These findings contribute for a better understanding of GC biology and highlight the role of core ECM components in gastric carcinogenesis and their clinical relevance as biomarkers of disease prognosis.

Anti-SARS-CoV-2 equine F (Ab')2 immunoglobulin as a possible therapy for COVID-19.

The new outbreak of coronavirus disease 2019 (COVID-19) has infected and caused the death of millions of people worldwide. Intensive efforts are underway around the world to establish effective treatments. Immunoglobulin from immunized animals or plasma from convalescent patients might constitute a specific treatment to guarantee the neutralization of the virus in the early stages of infection, especially in patients with risk factors and a high probability of progressing to severe disease. Worldwide, a few clinical trials using anti-SARS-CoV-2 immunoglobulins from horses immunized with the entire spike protein or fragments of it in the treatment of patients with COVID-19 are underway. Here, we describe the development of an anti-SARS-CoV-2 equine F(ab')2 immunoglobulin using a newly developed SARS-CoV-2 viral antigen that was purified and inactivated by radiation. Cell-based and preclinical assays showed that the F(ab')2 immunoglobulin successfully neutralizes the virus, is safe in animal models, and reduces the severity of the disease in a hamster model of SARS-CoV-2 infection and disease.

Geographical Distribution of E-cadherin Germline Mutations in the Context of Diffuse Gastric Cancer: A Systematic Review.

Hereditary diffuse gastric cancer (HDGC) is a complex and multifactorial inherited cancer predisposition syndrome caused by CDH1 germline mutations. Nevertheless, current CDH1 genetic screening recommendations disregard an unbalanced worldwide distribution of CDH1 variants, impacting testing efficacy and patient management. In this systematic review, we collected and analyzed all studies describing CDH1 variants in gastric cancer patients originating from both high- and low-prevalence countries. Selected studies were categorized as family study, series study, and unknown study, according to the implementation of HDGC clinical criteria for genetic testing. Our results indicate that CDH1 mutations are more frequently identified in gastric cancer low-incidence countries, and in the family study group that encompasses cases fulfilling criteria. Considering the type of CDH1 alterations, we verified that the relative frequency of mutation types varies within study groups and geographical areas. In the series study, the missense variant frequency is higher in high-incidence areas of gastric cancer, when compared with non-missense mutations. However, application of variant scoring for putative relevance led to a strong reduction of CDH1 variants conferring increased risk of gastric cancer. Herein, we demonstrate that criteria for CDH1 genetic screening are critical for identification of individuals carrying mutations with clinical significance. Further, we propose that future guidelines for testing should consider GC incidence across geographical regions for improved surveillance programs and early diagnosis of disease.

Germline CDH1 G212E Missense Variant: Combining Clinical, In Vitro and In Vivo Strategies to Unravel Disease Burden.

E-cadherin, encoded by CDH1, is an essential molecule for epithelial homeostasis, whose loss or aberrant expression results in disturbed cell-cell adhesion, increased cell invasion and metastasis. Carriers of CDH1 germline mutations have a high risk of developing diffuse gastric cancer and lobular breast cancer, associated with the cancer syndrome Hereditary Diffuse Gastric Cancer (HDGC). The ubiquitous availability of cancer panels has led to the identification of an increasing amount of "incidental" CDH1 genetic variants that pose a serious clinical challenge. This has sparked intensive research aiming at an accurate classification of the variants and consequent validation of their clinical relevance. The present study addressed the significance of a novel CDH1 variant, G212E, identified in an unusually large pedigree displaying strong aggregation of diffuse gastric cancer. We undertook a comprehensive pipeline encompassing family data, in silico predictions, in vitro assays and in vivo strategies, which validated the deleterious phenotype induced by this genetic alteration. In particular, we demonstrated that the G212E variant affects the stability and localization, as well as the adhesive and anti-invasive functions of E-cadherin, triggering epithelial disruption and disorganization. Our findings illustrate the clinical implication of a complementary approach for effective variant categorization and patient management.