AI-guided pipeline for protein-protein interaction drug discovery identifies a SARS-CoV-2 inhibitor.

Protein-protein interactions (PPIs) offer great opportunities to expand the druggable proteome and therapeutically tackle various diseases, but remain challenging targets for drug discovery. Here, we provide a comprehensive pipeline that combines experimental and computational tools to identify and validate PPI targets and perform early-stage drug discovery. We have developed a machine learning approach that prioritizes interactions by analyzing quantitative data from binary PPI assays or AlphaFold-Multimer predictions. Using the quantitative assay LuTHy together with our machine learning algorithm, we identified high-confidence interactions among SARS-CoV-2 proteins for which we predicted three-dimensional structures using AlphaFold-Multimer. We employed VirtualFlow to target the contact interface of the NSP10-NSP16 SARS-CoV-2 methyltransferase complex by ultra-large virtual drug screening. Thereby, we identified a compound that binds to NSP10 and inhibits its interaction with NSP16, while also disrupting the methyltransferase activity of the complex, and SARS-CoV-2 replication. Overall, this pipeline will help to prioritize PPI targets to accelerate the discovery of early-stage drug candidates targeting protein complexes and pathways.

Polyglutamine disease proteins: Commonalities and differences in interaction profiles and pathological effects.

Currently, nine polyglutamine (polyQ) expansion diseases are known. They include spinocerebellar ataxias (SCA1, 2, 3, 6, 7, 17), spinal and bulbar muscular atrophy (SBMA), dentatorubral-pallidoluysian atrophy (DRPLA), and Huntington's disease (HD). At the root of these neurodegenerative diseases are trinucleotide repeat mutations in coding regions of different genes, which lead to the production of proteins with elongated polyQ tracts. While the causative proteins differ in structure and molecular mass, the expanded polyQ domains drive pathogenesis in all these diseases. PolyQ tracts mediate the association of proteins leading to the formation of protein complexes involved in gene expression regulation, RNA processing, membrane trafficking, and signal transduction. In this review, we discuss commonalities and differences among the nine polyQ proteins focusing on their structure and function as well as the pathological features of the respective diseases. We present insights from AlphaFold-predicted structural models and discuss the biological roles of polyQ-containing proteins. Lastly, we explore reported protein-protein interaction networks to highlight shared protein interactions and their potential relevance in disease development.

Characterization of a spring pendulum phase-space trajectories.

We study the geometrical properties of phase-space trajectories (or orbits) of a spring pendulum as functions of the energy. Poincaré maps are used to describe the properties of the system. The points in the Poincaré maps of regular orbits (non-chaotic) cluster around separated segments or in chains of islands. Looking at how segments are formed, we conclude that the orbits are closely related to torus knots. Examining the toroidal and poloidal turns of the orbits, we introduce the definition of a rational parameter Ω, which is closely related to the concept of frequency used in the analysis of dynamical systems. Algorithms were developed to calculate Ω, and we found that this parameter naturally describes the orbits in terms of Farey sequences; also, calculations show that orbits with the same Ω have similar dynamics. Orbits corresponding to chains of islands are identified with cable knots that can be characterized using two parameters analogous to Ω. In some cases, non-trivial cable knots were found. With the analysis presented in this study, it is shown that Ω follows predictable distributions in the (z,Ω) space.

Essential Oil and Plant Extracts as Preservatives and Natural Antioxidants Applied to Meat and Meat Products: A Review.

The meat and meat product industry has evolved according to the needs of the market. Consumers are increasingly seeking quality in food. Thus, the concern regarding the excessive use of additives such as preservatives and antioxidants has driven research towards natural, healthy and safe substitutes. Essential oils and plant extracts have been shown to be a good option for resolving this problem. They are completely natural with biological activity, which mainly includes prevention of oxidation and the proliferation of microorganisms, thus arousing the interest of the industry and consumers. This review will present studies published in the last five years regarding the potential of essential oils and plant extracts to act as preservatives and antioxidants in meat and meat products. The forms of application, innovations in the area, alternatives to the incorporation of essential oils and extracts in meat products, effects caused in food, and limitations of applications will be detailed and discussed.

Severe ischemic gastritis caused by chronic mesenteric ischemia.

Ischemic gastritis is a rare illness caused by localized or systemic vascular insufficiency. This condition is rarely seen in medical practice due to the vast arterial collateral blood supply to the stomach through the celiac trunk and superior mesenteric artery and also because other etiologies are much more frequent. The classic presentation of chronic ischemia is comprises the triad of postprandial pain, weight loss, and abdominal bruit. Intervention is indicated in symptomatic patients and endovascular treatment is an alternative to surgery in patients with high comorbidity that offers good results. We report a case of a 71-year-old female patient with severe ischemic gastritis with ulcers and bleeding caused by chronic mesenteric ischemia with occlusion of the celiac trunk and inferior mesenteric artery and critical stenosis of the superior mesenteric artery. The diagnosis was confirmed by imaging, and the patient underwent endovascular treatment. This is a rare condition that is difficult to diagnose and treat and a multidisciplinary team is needed for proper management.

Protein degradation-associated mechanisms that are affected in Alzheimer´s disease.

Alzheimer's disease (AD) is the most common type of dementia associated with age-related neurodegeneration. Alteration of several molecular mechanisms has been correlated with the progression of AD. In recent years, dysregulation of proteostasis-associated pathways has emerged as a potential risk factor for neurodegenerative diseases. This review investigated the ubiquitin-proteasome system, lysosome-associated degradation, endoplasmic-reticulum-associated degradation, and the formation of advanced glycation end products. These pathways involved in proteostasis have been reported to be altered in AD, suggesting that their study may be critical for identifying new biomarkers and target molecules for AD.

Mitochondrial fusion is required for regulation of mitochondrial DNA replication.

Mitochondrial dynamics is an essential physiological process controlling mitochondrial content mixing and mobility to ensure proper function and localization of mitochondria at intracellular sites of high-energy demand. Intriguingly, for yet unknown reasons, severe impairment of mitochondrial fusion drastically affects mtDNA copy number. To decipher the link between mitochondrial dynamics and mtDNA maintenance, we studied mouse embryonic fibroblasts (MEFs) and mouse cardiomyocytes with disruption of mitochondrial fusion. Super-resolution microscopy revealed that loss of outer mitochondrial membrane (OMM) fusion, but not inner mitochondrial membrane (IMM) fusion, leads to nucleoid clustering. Remarkably, fluorescence in situ hybridization (FISH), bromouridine labeling in MEFs and assessment of mitochondrial transcription in tissue homogenates revealed that abolished OMM fusion does not affect transcription. Furthermore, the profound mtDNA depletion in mouse hearts lacking OMM fusion is not caused by defective integrity or increased mutagenesis of mtDNA, but instead we show that mitochondrial fusion is necessary to maintain the stoichiometry of the protein components of the mtDNA replisome. OMM fusion is necessary for proliferating MEFs to recover from mtDNA depletion and for the marked increase of mtDNA copy number during postnatal heart development. Our findings thus link OMM fusion to replication and distribution of mtDNA.

TEFM regulates both transcription elongation and RNA processing in mitochondria.

Regulation of replication and expression of mitochondrial DNA (mtDNA) is essential for cellular energy conversion via oxidative phosphorylation. The mitochondrial transcription elongation factor (TEFM) has been proposed to regulate the switch between transcription termination for replication primer formation and processive, near genome-length transcription for mtDNA gene expression. Here, we report that Tefm is essential for mouse embryogenesis and that levels of promoter-distal mitochondrial transcripts are drastically reduced in conditional Tefm-knockout hearts. In contrast, the promoter-proximal transcripts are much increased in Tefm knockout mice, but they mostly terminate before the region where the switch from transcription to replication occurs, and consequently, de novo mtDNA replication is profoundly reduced. Unexpectedly, deep sequencing of RNA from Tefm knockouts revealed accumulation of unprocessed transcripts in addition to defective transcription elongation. Furthermore, a proximity-labeling (BioID) assay showed that TEFM interacts with multiple RNA processing factors. Our data demonstrate that TEFM acts as a general transcription elongation factor, necessary for both gene transcription and replication primer formation, and loss of TEFM affects RNA processing in mammalian mitochondria.

On Posterior Properties of the Two Parameter Gamma Family of Distributions.

The gamma distribution has been extensively used in many areas of applications. In this paper, considering a Bayesian analysis we provide necessary and sufficient conditions to check whether or not improper priors lead to proper posterior distributions. Further, we also discuss sufficient conditions to verify if the obtained posterior moments are finite. An interesting aspect of our findings are that one can check if the posterior is proper or improper and also if its posterior moments are finite by looking directly in the behavior of the proposed improper prior. To illustrate our proposed methodology these results are applied in different objective priors.

Relationship between beta-adrenergic agonists, calpain system activity and beef texture: A systematic review.

The objective was to conduct a systematic review to evaluate the effects of dietary supplementation with beta-adrenergic agonists on calpains and calpastatin activity in bovine muscle and changes in meat tenderness. A survey was conducted in June 2019 on Science Direct, Web of Science, Scopus, PubMed and Capes Periodicals, using four keyword combinations: agonist and calpain and cattle; agonist and calpain and bovine; agonist and calpain and heifers; agonist and calpain and steers. Thirteen studies were selected, 54% concluded that supplementation with beta-adrenergic agonists increases calpastatin activity, 23% observed increase in their gene expression and 23% reported no effect on activity or expression of this enzyme. Nine studies evaluated the influence of beta-adrenergic agonists supplementation on meat texture and all found an increase in shear force values. There is strong evidence that beta-adrenergic agonists may increase calpastatin activity in the muscle, causing damage to meat tenderness.

Unexpected relevant role of gene mosaicism in patients with primary immunodeficiency diseases.

BACKGROUND: Postzygotic de novo mutations lead to the phenomenon of gene mosaicism. The 3 main types are called somatic, gonadal, and gonosomal mosaicism, which differ in terms of the body distribution of postzygotic mutations. Mosaicism has been reported occasionally in patients with primary immunodeficiency diseases (PIDs) since the early 1990s, but its real involvement has not been systematically addressed. OBJECTIVE: We sought to investigate the incidence of gene mosaicism in patients with PIDs. METHODS: The amplicon-based deep sequencing method was used in the 3 parts of the study that establish (1) the allele frequency of germline variants (n = 100), (2) the incidence of parental gonosomal mosaicism in families with PIDs with de novo mutations (n = 92), and (3) the incidence of mosaicism in families with PIDs with moderate-to-high suspicion of gene mosaicism (n = 36). Additional investigations evaluated body distribution of postzygotic mutations, their stability over time, and their characteristics. RESULTS: The range of allele frequency (44.1% to 55.6%) was established for germline variants. Those with minor allele frequencies of less than 44.1% were assumed to be postzygotic. Mosaicism was detected in 30 (23.4%) of 128 families with PIDs, with a variable minor allele frequency (0.8% to 40.5%). Parental gonosomal mosaicism was detected in 6 (6.5%) of 92 families with de novo mutations, and a high incidence of mosaicism (63.9%) was detected among families with moderate-to-high suspicion of gene mosaicism. In most analyzed cases mosaicism was found to be both uniformly distributed and stable over time. CONCLUSION: This study represents the largest performed to date to investigate mosaicism in patients with PIDs, revealing that it affects approximately 25% of enrolled families. Our results might have serious consequences regarding treatment and genetic counseling and reinforce the use of next-generation sequencing-based methods in the routine analyses of PIDs.

Sensory profile and technological characterization of boneless dry-cured ham with lactulose added as a prebiotic.

OBJECTIVE: This study investigates the technological and sensory profile of boneless dry-cured ham added with different contents of lactulose as a prebiotic ingredient. METHODS: In addition to the control samples (without the addition of lactulose), three treatments were formulated to contain 2, 4 or 6% lactulose. Technological (lactulose content, CIE color and texture profile analysis) and sensory (acceptance and check-all-that-applies tests) analyses were performed on the final product. RESULTS: The lactulose content in the finished product (1.86±0.23%, 3.16±0.18% and 2.51±1.35%) was lower than the lactulose originally added (2, 4 and 6% respectively). The addition of 4% and 6% lactulose made (P < 0.05) the products darker (lower L*) and redder (lower h) with higher hardness and chewiness values, when compared to control samples. The additions of 2% and 4% lactulose reduce the appearance acceptability of the products, but overall the treatments were well accepted. CONCLUSION: The use of up to 4% lactulose as a prebiotic in the production of boneless dry-cured hams provides an alternative to improving its nutritional value with little alteration in the technological characteristics and still meeting the sensory characteristics desired by consumers.

Protein ISGylation and free ISG15 levels are increased by interferon gamma in breast cancer cells.

The induction of ISG15 by interferon (IFN)-α/ß and subsequent protein ISGylation has been demonstrated in several cell types. However, regulation of free ISG15 levels and ISGylation by other IFNs and its implications in some carcinomas have not yet been completely evaluated. Here, we demonstrated that free ISG15 and ISGylation levels are enhanced by IFN-γ treatment in the estrogen receptor-α-positive and -negative breast cancer cells, MCF-7 and MDA-MB-231, respectively. Specifically, IFN-γ increases free ISG15 levels in the cytoplasm and ISGylation in the nucleus and cytoplasm, but in a manner distinct between MCF-7 and MDA-MB-231 cells. Therefore, free ISG15 and ISGylation may play central roles in mammary tumors by differentially modulating certain tumorigenic characteristics of estrogen receptor-α-positive and -negative breast cancer cells.

SLIRP Regulates the Rate of Mitochondrial Protein Synthesis and Protects LRPPRC from Degradation.

We have studied the in vivo role of SLIRP in regulation of mitochondrial DNA (mtDNA) gene expression and show here that it stabilizes its interacting partner protein LRPPRC by protecting it from degradation. Although SLIRP is completely dependent on LRPPRC for its stability, reduced levels of LRPPRC persist in the absence of SLIRP in vivo. Surprisingly, Slirp knockout mice are apparently healthy and only display a minor weight loss, despite a 50-70% reduction in the steady-state levels of mtDNA-encoded mRNAs. In contrast to LRPPRC, SLIRP is dispensable for polyadenylation of mtDNA-encoded mRNAs. Instead, deep RNA sequencing (RNAseq) of mitochondrial ribosomal fractions and additional molecular analyses show that SLIRP is required for proper association of mRNAs to the mitochondrial ribosome and efficient translation. Our findings thus establish distinct functions for SLIRP and LRPPRC within the LRPPRC-SLIRP complex, with a novel role for SLIRP in mitochondrial translation. Very surprisingly, our results also demonstrate that mammalian mitochondria have a great excess of transcripts under basal physiological conditions in vivo.

Restructured low-fat cooked ham containing liquid whey fortified with lactulose.

BACKGROUND: Current health concerns have driven consumers to request products with nutritional and physiological advantages, which can be achieved by using prebiotic ingredients. Lactulose is a prebiotic with excellent functional properties and can be easily incorporated into meat products through the addition of liquid whey. This study investigated the technological and sensorial quality of restructured cooked ham elaborated without liquid whey added (control) and with liquid whey containing different contents (0, 30, 60 and 100 g kg-1 ) of lactulose. RESULTS: Liquid whey did not change any technological or sensorial characteristics of the product, but the general acceptability decreased due to addition of lactulose. Samples with higher lactulose concentrations had lower moisture content, pH and refreezing loss and increased carbohydrate content. Control and whey added samples had higher lightness and lower intense color than samples with lactulose. Liquid whey additions with higher lactulose content increased hardness and chewiness of the samples. CONCLUSION: Restructured cooked hams formulated with liquid whey and 30 g kg-1 of lactulose had minimal effects on the technological properties and sensory characteristics and, due to the possible benefits conferred by the prebiotic, is a potential alternative to provide meat products with prebiotic activity. © 2017 Society of Chemical Industry.

Effect of mechanically deboned poultry meat content on technological properties and sensory characteristics of lamb and mutton sausages.

OBJECTIVE: This study aimed to develop a value-added product concerning technological and sensory characteristics changes of the use of mechanically deboned poultry meat (MDPM) as meat replacer in lamb and mutton emulsion-type sausages (mortadella). METHODS: Sausages were produced with lamb and mutton and with different contents of MDPM. Six treatments, using lamb or mutton and 0%, 30%, and 60% of MDPM in relation to the meat batter, were produced and analyzed for pH, proximal composition, calcium and residual nitrite content, water activity, 2-thiobarbituric acid reactive substances (TBARS), instrumental color and texture profile. The sensory profile of the mortadella's was also evaluated by acceptance test and check-all-that-applies (CATA) analysis. RESULTS: The MDPM addition increased (p<0.05) fat, residual nitrite and calcium content in the all sausage formulations, but mutton sausage had (p<0.05) higher fat and lower moisture content than lamb sausage. The pH, water activity, TBARS index and color was not affected by MDPM additions, while the mutton sausages were significantly redder (higher a*, C*, and lower h°) and darker (lower L*) than lamb sausages. Adding up to 60% of MDPM reduced (p<0.05) sausages hardness and chewiness. Overall, the meat replacement by MDPM increased the sausages acceptance, but the mutton sausage with 30% of MDPM replacer were the most preferred. Consumers related that pink color, glossy appearance, poultry meat-like taste, soft texture, juicy and greasy mouth feel to all sausages contain MDPM according to CATA analysis. CONCLUSION: Mutton from culled ewes can be utilized for mortadella production with 30% replacement of lean mutton and fat by MDPM.

Bioenergetic roles of mitochondrial fusion.

Mitochondria are bioenergetic hotspots, producing the bulk of ATP by the oxidative phosphorylation process. Mitochondria are also structurally dynamic and undergo coordinated fusion and fission to maintain their function. Recent studies of the mitochondrial fusion machinery have provided new evidence in detailing their role in mitochondrial metabolism. Remarkably, mitofusin 2, in addition to its role in fusion, is important for maintaining coenzyme Q levels and may be an integral player in the mevalonate synthesis pathway. Here, we review the bioenergetic roles of mitochondrial dynamics and emphasize the importance of the in vitro growth conditions when evaluating mitochondrial respiration. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016,' edited by Prof. Paolo Bernardi.

Real-Time PCR Reveals Rapid Dissemination of Leptospira interrogans after Intraperitoneal and Conjunctival Inoculation of Hamsters.

The pathogen Leptospira interrogans is a highly motile spirochete that causes acute and fulminant infections in humans and other accidental hosts. Hematogenous dissemination is important for infection by the pathogen but remains poorly understood because few animal model studies have used sensitive tools to quantify the bacteria. We evaluated the kinetics of leptospiral infection in Golden Syrian hamsters by a sensitive quantitative real-time PCR (TaqMan) with lipl32 as the target gene. The dissemination and bacterial burden were measured after intraperitoneal infection with a high dose (10(8)) or low dose (2.5 × 10(2)) of leptospires. We also examined the conjunctival challenge route to mimic the natural history of infection. Quantification of leptospires in perfused animals revealed that pathogens were detected in all organs of intraperitoneally infected hamsters, including the eye and brain, within 1 h after inoculation of 10(8) virulent L. interrogans bacteria. Peaks of 10(5) to 10(8) leptospires per gram or per milliliter were achieved in blood and all tissues between day 4 and day 8 after intraperitoneal inoculation of high- and low-dose challenges, respectively, coinciding with macroscopic and histological changes. The conjunctival route resulted in a delay in the time to peak organ burden in comparison to intraperitoneal infection, indicating that although infection could be established, penetration efficiency was low across this epithelial barrier. Surprisingly, infection with a large inoculum of high-passage-number attenuated L. interrogans strains resulted in dissemination to all organs in the first 4 days postinfection, albeit with a lower burden, followed by clearance from the blood and organs 7 days postinfection and survival of all animals. These results demonstrate that leptospiral dissemination and tissue invasion occur. In contrast, development of a critical level of tissue burden and pathology are dependent on the virulence of the infecting strain.

Macrophages and endothelial cells orchestrate tumor-associated angiogenesis in oral cancer via hedgehog pathway activation.

The present study aimed to evaluate the role of Hedgehog (Hh) molecule expression in association with the clinical aspects of oral squamous cell carcinoma (OSCC), as well as angiogenesis and CD163+ macrophages. Twenty-eight cases of OSCC, nine cases of tumor-free resection margins (TM), and four cases of non-neoplastic oral mucosa (NNM) were submitted to immunohistochemistry to detect proteins Sonic Hedgehog (SHH), Indian Hedgehog (IHH), GLI1, CD163, and CD105. Protein colocalization with respect to SHH/CD163, IHH/CD163, GLI1/CD163, and GLI1/CD105 was assessed by immunohistochemical double staining. In tumor parenchyma, SHH and IHH were present in the cytoplasm of neoplastic cells, while GLI1 was observed in cytoplasm and nucleus. Endothelial cells were found to express SHH, IHH, and GLI1 within CD105+ vessels, and a positive correlation between infiltrating macrophage density (IMD) and microvascular density (MVD) was observed in cases of OSCC and TM. When compared to TM and NNM, the OSCC cases demonstrated higher immunoreactivity for SHH (p = 0.01), IHH (p = 0.39), GLI1 (p = 0.03), IMD (p = 0.0002), and MVD (p = 0.0002). Our results suggest the participation of the Hh pathway in OSCC by way of autocrine and paracrine signaling, in addition to the participation of both SHH and IHH ligands. Endothelial cells were also found to exhibit positivity with respect to Hh pathway components and we surmise that these molecules may play a role in tumor angiogenesis. CD163+ macrophages were also observed to express IHH, a ligand of this pathway, in addition to being associated with tumor neovascularization.

The sonic hedgehog signaling pathway contributes to the development of salivary gland neoplasms regardless of perineural infiltration.

The pleomorphic adenoma (PA), mucoepidermoid carcinoma (MEC), and adenoid cystic carcinoma (ACC) are common tumors arising from salivary glands whose histopathology is heterogeneous. The sonic hedgehog signaling pathway (Hh) and signal transducer and activator of transcription 3 (STAT3) play important roles in cell proliferation, favoring tumor growth. The aim of this investigation was to study components of the Hh pathway, as well as STAT3 in salivary gland neoplasms in an attempt to add information about the biological characteristics of these neoplasms. We used 9 cases of PA, 17 cases of ACC, and 20 cases of MEC. Using immunohistochemistry, SHH, GLI1, SUFU, HHIP, and STAT3 were investigated. For comparative purposes, MCM3 (cellular proliferation marker) was also included. In PA, there was high expression of cytoplasmic SHH and SUFU and low expression of STAT3 and MCM3. In the ACC, there was high expression of GLI1, HHIP, and STAT3 and low expression of SHH, SUFU, and MCM3. In the MEC, we observed high expression of SHH, GLI1, SUFU, and HHIP and low expression of STAT3 and MCM3. There was a statistically significant difference between SHH (p = 0.0064), STAT3 (p = 0.0003), and MCM3 (p = 0.0257) when all tumors were compared and a higher expression in parenchyma for all tumors when stroma and parenchyma were compared (p < 0.05). These findings suggests a possible role of Hh pathway in the development and maintenance of the cytoarchitectural pattern of PA, ACC, and MEC, as well as the participation of STAT3 in the development of ACC, irrespective perineural infiltration.