Germline homozygosity and allelic imbalance of HLA-I are common in esophagogastric adenocarcinoma and impair the repertoire of immunogenic peptides.

BACKGROUND: The individual HLA-I genotype is associated with cancer, autoimmune diseases and infections. This study elucidates the role of germline homozygosity or allelic imbalance of HLA-I loci in esophago-gastric adenocarcinoma (EGA) and determines the resulting repertoires of potentially immunogenic peptides. METHODS: HLA genotypes and sequences of either (1) 10 relevant tumor-associated antigens (TAAs) or (2) patient-specific mutation-associated neoantigens (MANAs) were used to predict good-affinity binders using an in silico approach for MHC-binding (www.iedb.org). Imbalanced or lost expression of HLA-I-A/B/C alleles was analyzed by transcriptome sequencing. FluoroSpot assays and TCR sequencing were used to determine peptide-specific T-cell responses. RESULTS: We show that germline homozygosity of HLA-I genes is significantly enriched in EGA patients (n=80) compared with an HLA-matched reference cohort (n=7605). Whereas the overall mutational burden is similar, the repertoire of potentially immunogenic peptides derived from TAAs and MANAs was lower in homozygous patients. Promiscuity of peptides binding to different HLA-I molecules was low for most TAAs and MANAs and in silico modeling of the homozygous to a heterozygous HLA genotype revealed normalized peptide repertoires. Transcriptome sequencing showed imbalanced expression of HLA-I alleles in 75% of heterozygous patients. Out of these, 33% showed complete loss of heterozygosity, whereas 66% had altered expression of only one or two HLA-I molecules. In a FluoroSpot assay, we determined that peptide-specific T-cell responses against NY-ESO-1 are derived from multiple peptides, which often exclusively bind only one HLA-I allele. CONCLUSION: The high frequency of germline homozygosity in EGA patients suggests reduced cancer immunosurveillance leading to an increased cancer risk. Therapeutic targeting of allelic imbalance of HLA-I molecules should be considered in EGA.

Investigation of the interaction between S-isoalkyl derivatives of the thiosalicylic acid and human serum albumin.

S-isoalkyl derivatives of thiosalicylic acid (isopropyl-(L1), isobutyl-(L2) and isoamyl-(L3)) were selected in order to investigate the binding interaction with the human serum albumin (HSA) using different spectroscopic methods and molecular docking simulation. Association constants and number of binding sites were used to analyze the quenching mechanism. The experimental results showed that the fluorescence quenching of HSA by L1, L2 and L3 occurs because of static quenching and that binding processes were spontaneous, with the leading forces in bonding by hydrogen bonding, hydrophobic interactions, and electrostatic interactions. Fluorescence spectroscopy, UV-Vis spectroscopy and synchronous fluorescence spectroscopy showed that ligands (L1, L2 and L3) can bind to HSA and that the binding of ligands induced some microenvironmental and conformational changes in HSA. The calculated distance between the donor and the acceptor according to fiFörster's theory confirms the energy transfer efficiency between the acceptor and HSA. Results of site marker competitive experiments showed that the tested compounds bind to HSA in domain IIA (Site I). Molecular dynamics and docking calculations demonstrated that L3 binds to the Sudlow site I of HSA with lower values of binding energies compared to L1 and L2, indicating the formation of the most stable ligand-HSA complex. Understanding the binding mechanisms of S-isoalkyl derivatives of the thiosalicylic acid to HSA may provide valuable data for the future studies of their biological activity and application as potential antitumor drugs.Communicated by Ramaswamy H. Sarma.

Sacubitril/valsartan promotes white adipose tissue browning in rats with metabolic syndrome through activation of mTORC1.

In addition to their usual use in the treatment of cardiovascular disease, weak evidence is available for the potential of combined use of neprilysin inhibitor (sacubitril) and AT1 receptor antagonist (valsartan) to promote browning of white adipose tissue (WAT) in rats with metabolic syndrome (MetS). This study involved 32 male Wistar albino rats divided into four groups: CTRL-healthy control rats; ENT-healthy rats treated with sacubitril/valsartan; MS-rats with MetS; MS + ENT-rats with MetS treated with sacubitril/valsartan. After finishing the experimental protocol, different WAT depots were isolated for further analysis of molecular pathways. Molecular docking and molecular dynamics studies were used for in silico assessment of the binding affinity of sacubitril and valsartan towards subunits of mechanistic target of rapamycin complex 1 (mTORC1). Sacubitril/valsartan treatment markedly diminished morphological changes in adipose tissue, resulting in smaller lipid size and multilocular lipid droplet structure in WAT. We showed significantly higher protein expression of uncoupling protein-1 (UCP-1) and mTORC1 in WAT of MS + ENT rats, correlating with increased relative gene expression of browning-related markers in tissue of rats treated with sacubitril/valsartan compared with MS group of rats. In silico analysis showed that sacubitrilat and valsartan exhibited the highest binding affinity against mTOR and mLST8, forming stable complexes with these mTORC1 subunits. The observed results confirmed strong potential of combined sacubitril/valsartan treatment to increase browning markers expression in different WAT depots in MetS condition and to form permanent complexes with mTOR and mLST8 subunits over the time.

Native and endemic Iranian Nepeta spp.: powerful antimicrobial agents.

Pathogenic microorganisms are more or less successfully treated by synthetic chemical compounds, whose residues often cause serious health problems. Plant specialized metabolites with antimicrobial properties have for a long time been the focus of both medicine and pharmacology. This study was conducted to evaluate the in vitro antimicrobial activity of methanol extracts of selected endemic and native Iranian Nepeta species against some of the most important pathogenic bacteria and fungi. The results indicated that N. kotschyi leaf extract was the most efficient against the tested bacteria, with Pseudomonas aeruginosa being the most sensitive and fungal species were more susceptible to the extracts than bacterial strains. Nepeta spp. extracts showed a strong antifungal activity against micromycetes, except for quite resistant Aspergillus niger. Antibacterial MIC values (mg.mL-1) ranged from 0.01 (N. kotschyi) to 0.20 (N. crassifolia), while antifungal MIC values ranged from 0.02 (N. crassifolia, N. kotschyi, N. menthoides, and N. cataria) to 0.13 (N. crassifolia and N. menthoides). When compared to positive controls, in most cases the extracts performed much better. The recorded antimicrobial activity candidates the selected 4 endemic and native Iranian Nepeta spp. as prospective and promising antimicrobial agents to be used in both pharmacology and biotechnology.

Serum Zinc Concentration in Patients with Alopecia Areata.

Alopecia areata is an autoimmune non-scarring disease in which the exact mechanism that induces loss of immune privilege is unknown. Zinc is important for DNA stability and repair mechanisms that are essential in maintaining normal hair growth. Zinc deficiency has been investigated as an important factor in many autoimmune diseases, and may have a possible role in the aetiopathogenesis of alopecia areata. This study included 32 patients with severe forms of alopecia areata, and 32 age- and sex-matched healthy controls. When comparing serum zinc levels in these 2 groups, statistically significantly lower zinc concentrations were found in the alopecia areata group (p = 0.017). Detected zinc deficiency was statistically more prevalent in patients with alopecia areata (p = 0.011). Evaluating patients with alopecia areata, a statistically significant negative correlation between serum zinc levels and severity of the disease was found (ρ = 0.006). The results indicate that zinc serum assessment is necessary in patients with alopecia areata. Low serum zinc levels were found to correlate with severity of alopecia areata. Given that most severe forms of alopecia areata are frequently most treatment-resistant, additional randomized control trials examining zinc supplementation are necessary to investigate its potential role in the restoration of hair follicles.

A novel rapamycin cream formulation improves facial angiofibromas associated with tuberous sclerosis complex: a double-blind randomized placebo-controlled trial.

BACKGROUND: Facial angiofibromas (FAs) are a major feature of tuberous sclerosis complex (TSC). Topical rapamycin can successfully treat FAs. A new stabilized cream formulation that protects rapamycin from oxidation has been developed in 0.5% and 1% concentrations. OBJECTIVES: To assess the efficacy and safety of a novel, stabilized topical rapamycin cream formulation. METHODS: This multicentre double-blind randomized placebo-controlled dose-response phase II/III study with a parallel design included participants aged 6-65 years with FAs of mild or moderate severity according to the Investigator's Global Assessment (IGA) scale. Participants were randomized to one of three treatment arms: topical rapamycin 0.5%, topical rapamycin 1% or placebo. Treatment was applied once daily for 26 weeks. Safety and efficacy measures were assessed at days 14, 56, 98, 140 and 182. The primary endpoint was the percentage of participants achieving IGA scores of 'clear' or 'almost clear' after 26 weeks of treatment. Secondary measures included Facial Angiofibroma Severity Index (FASI) and participant- and clinician-reported percentage-based improvement. Safety measures included the incidence of treatment-emergent adverse events and blood rapamycin concentration changes over time. RESULTS: Participants (n = 107) were randomized to receive either rapamycin 1% (n = 33), rapamycin 0.5% (n = 36) or placebo (n = 38). All treated participants were included in the final analysis. The percentage of participants with a two-grade IGA improvement was greater in the rapamycin 0.5% treatment group (11%) and rapamycin 1% group (9%) than in the placebo group (5%). However, this was not statistically significant [rapamycin 0.5%: odds ratio (OR) 1.71, 95% confidence interval (CI) 0.36-8.18 (P = 0.50); rapamycin 1%: OR 1.68, 95% CI 0.33-8.40 (P = 0.53)]. There was a statistically significant difference in the proportion of participants treated with rapamycin cream that achieved at least a one-grade improvement in IGA [rapamycin 0.5%: 56% (OR 4.73, 95% CI 1.59-14.10; P = 0.005); rapamycin 1%: 61% (OR 5.14, 95% CI 1.70-15.57; P = 0.004); placebo: 24%]. Skin adverse reactions were more common in patients following rapamycin application (64%) vs. placebo (29%). CONCLUSIONS: Both rapamycin cream formulations (0.5% and 1%) were well tolerated, and either strength could lead to clinical benefit in the treatment of FA.

Autophagy Receptor p62 Regulates SARS-CoV-2-Induced Inflammation in COVID-19.

As autophagy can promote or inhibit inflammation, we examined autophagy-inflammation interplay in COVID-19. Autophagy markers in the blood of 19 control subjects and 26 COVID-19 patients at hospital admission and one week later were measured by ELISA, while cytokine levels were examined by flow cytometric bead immunoassay. The antiviral IFN-α and proinflammatory TNF, IL-6, IL-8, IL-17, IL-33, and IFN-γ were elevated in COVID-19 patients at both time points, while IL-10 and IL-1ß were increased at admission and one week later, respectively. Autophagy markers LC3 and ATG5 were unaltered in COVID-19. In contrast, the concentration of autophagic cargo receptor p62 was significantly lower and positively correlated with TNF, IL-10, IL-17, and IL-33 at hospital admission, returning to normal levels after one week. The expression of SARS-CoV-2 proteins NSP5 or ORF3a in THP-1 monocytes caused an autophagy-independent decrease or autophagy-inhibition-dependent increase, respectively, of intracellular/secreted p62, as confirmed by immunoblot/ELISA. This was associated with an NSP5-mediated decrease in TNF/IL-10 mRNA and an ORF3a-mediated increase in TNF/IL-1ß/IL-6/IL-10/IL-33 mRNA levels. A genetic knockdown of p62 mimicked the immunosuppressive effect of NSP5, and a p62 increase in autophagy-deficient cells mirrored the immunostimulatory action of ORF3a. In conclusion, the proinflammatory autophagy receptor p62 is reduced inacute COVID-19, and the balance between autophagy-independent decrease and autophagy blockade-dependent increase of p62 levels could affect SARS-CoV-induced inflammation.

Synthesis and Investigation of Anti-Inflammatory Activity of New Thiourea Derivatives of Naproxen.

The aim of the study was a synthesis and investigation of the dose-dependent anti-inflammatory effect of new thiourea derivatives of naproxen with selected aromatic amines and esters of aromatic amino acids. The results of the in vivo study indicate that derivatives of m-anisidine (4) and N-methyl tryptophan methyl ester (7) showed the most potent anti-inflammatory activity four hours after injection of carrageenan, with the percentage of inhibition of 54.01% and 54.12%, respectively. In vitro assays of COX-2 inhibition demonstrated that none of the tested compounds achieved 50% inhibition at concentrations lower than 100 µM. On the other hand, the aromatic amine derivatives (1-5) accomplished significant inhibition of 5-LOX, and the lowest IC50 value was observed for compound 4 (0.30 µM). High anti-edematous activity of compound 4 in the rat paw edema model, together with potent inhibition of 5-LOX, highlight this compound as a promising anti-inflammatory agent.

Rapid biomechanical imaging at low irradiation level via dual line-scanning Brillouin microscopy.

Brillouin microscopy is a technique for mechanical characterization of biological material without contact at high three-dimensional resolution. Here, we introduce dual line-scanning Brillouin microscopy (dLSBM), which improves acquisition speed and reduces irradiation dose by more than one order of magnitude with selective illumination and single-shot analysis of hundreds of points along the incident beam axis. Using tumor spheroids, we demonstrate the ability to capture the sample response to rapid mechanical perturbations as well as the spatially resolved evolution of the mechanical properties in growing spheroids.

Manuka Honey/2-Hydroxyethyl Methacrylate/Gelatin Hybrid Hydrogel Scaffolds for Potential Tissue Regeneration.

Scaffolding biomaterials are gaining great importance due to their beneficial properties for medical purposes. Targeted biomaterial engineering strategies through the synergy of different material types can be applied to design hybrid scaffolding biomaterials with advantageous properties for biomedical applications. In our research, a novel combination of the bioactive agent Manuka honey (MHo) with 2-hydroxyethyl methacrylate/gelatin (HG) hydrogel scaffolds was created as an efficient bioactive platform for biomedical applications. The effects of Manuka honey content on structural characteristics, porosity, swelling performance, in vitro degradation, and in vitro biocompatibility (fibroblast and keratinocyte cell lines) of hybrid hydrogel scaffolds were studied using Fourier transform infrared spectroscopy, the gravimetric method, and in vitro MTT biocompatibility assays. The engineered hybrid hydrogel scaffolds show advantageous properties, including porosity in the range of 71.25% to 90.09%, specific pH- and temperature-dependent swelling performance, and convenient absorption capacity. In vitro degradation studies showed scaffold degradability ranging from 6.27% to 27.18% for four weeks. In vitro biocompatibility assays on healthy human fibroblast (MRC5 cells) and keratinocyte (HaCaT cells) cell lines by MTT tests showed that cell viability depends on the Manuka honey content loaded in the HG hydrogel scaffolds. A sample containing the highest Manuka honey content (30%) exhibited the best biocompatible properties. The obtained results reveal that the synergy of the bioactive agent, Manuka honey, with 2-hydroxyethyl methacrylate/gelatin as hybrid hydrogel scaffolds has potential for biomedical purposes. By tuning the Manuka honey content in HG hydrogel scaffolds advantageous properties of hybrid scaffolds can be achieved for biomedical applications.

Mastocytosis in children: a single-center long-term follow-up study.

BACKGROUND: Mastocytosis is a heterogeneous group of rare disorders characterized by the accumulation of clonal mast cells in organs such as the skin and bone marrow. The diagnosis of cutaneous mastocytosis (CM) is based on clinical findings, positive Darier's sign, and histopathology, if necessary. METHODS: Medical records of 86 children with CM diagnosed during a 35-year long period were reviewed. Most patients (93%) developed CM during the first year of life (median age 3 months). Clinical features at presentation and during the follow-up period were analyzed. Baseline serum tryptase level was measured in 28 patients. RESULTS: A total of 85% of patients had maculopapular cutaneous mastocytosis/urticaria pigmentosa (MPCM/UP), 9% had mastocytoma, and 6% had diffuse cutaneous mastocytosis (DCM). Boy to girl ratio was 1.1:1. Fifty-four of 86 patients (63%) were followed from 2 to 37 years (median 13 years). Complete resolution was registered in 14% of mastocytoma cases, 14% of MCPM/UP, and in 25% of DCM patients. After the age of 18, skin lesion persisted in 14% mastocytoma, 7% MCPM/UP, and 25% children with DCM. Atopic dermatitis was diagnosed in 9.6% of patients with MPCM/UP. Three of 28 patients had elevated serum tryptase. Prognosis in all patients was good, and there were no signs of progression to systemic mastocytosis (SM). CONCLUSION: To the best of our knowledge, our results represent the longest single-center follow-up study of childhood-onset CM. We found no complications of massive mast cell degranulation or progression to SM.

Synthesis, Characterization, and Investigation of Anti-Inflammatory and Cytotoxic Activities of Novel Thiourea Derivatives of Naproxen.

The objective of this study was to synthesize seven novel thiourea derivatives of naproxen (8-14), examine the anti-inflammatory activity of the newly synthesized compounds, investigate the cytotoxic potential of both sets of synthesized compounds (1-7 and 8-14), and select the most promising anti-inflammatory and antitumor drug candidates. The results of the in vivo anti-inflammatory study clearly showed that compounds 8 and 9 were capable of decreasing paw edema, as evident from a high percentage of inhibition (44.83% and 49.29%, respectively). In addition, the results of in vitro enzyme inhibition assays demonstrated that neither of the newly synthesized compounds reached 50% inhibition of 5-LOX at concentrations lower than 100 µM. In terms of antitumor potential, derivatives 3 and 8 exhibited strong cytotoxic effects on the HeLa cell line, suggesting the involvement of the extrinsic pathway of apoptosis. According to the overall results obtained for both sets of synthesized molecules, derivatives 4 and 8 can be underlined as molecules with the strongest anti-inflammatory activity, while derivatives 3 and 8 are the most promising cytotoxic agents.

Scale invariance in early embryonic development.

The body plan of the fruit fly is determined by the expression of just a handful of genes. We show that the spatial patterns of expression for several of these genes scale precisely with the size of the embryo. Concretely, discrete positional markers such as the peaks in striped patterns have absolute positions along the anterior-posterior axis that are proportional to embryo length, with better than 1% accuracy. Further, the information (in bits) that graded patterns of expression provide about position can be decomposed into information about fractional or scaled position and information about absolute position or embryo length; all of the available information is about scaled position, again with ~1% accuracy. These observations suggest that the underlying genetic network exhibits scale invariance in a deeper mathematical sense. Taking this mathematical statement seriously requires that the network dynamics have a zero mode, which connects to many other observations on this system.

Multimodal microscale mechanical mapping of cancer cells in complex microenvironments.

The mechanical phenotype of the cell is critical for survival following deformations due to confinement and fluid flow. One idea is that cancer cells are plastic and adopt different mechanical phenotypes under different geometries that aid in their survival. Thus, an attractive goal is to disrupt cancer cells' ability to adopt multiple mechanical states. To begin to address this question, we aimed to quantify the diversity of these mechanical states using in vitro biomimetics to mimic in vivo two-dimensional (2D) and 3D extracellular matrix environments. Here, we used two modalities Brillouin microscopy (∼GHz) and broadband frequency (7-15 kHz) optical tweezer microrheology to measure microscale cell mechanics. We measured the response of intracellular mechanics of cancer cells cultured in 2D and 3D environments where we modified substrate stiffness, dimensionality (2D versus 3D), and presence of fibrillar topography. We determined that there was good agreement between two modalities despite the difference in timescale of the two measurements. These findings on cell mechanical phenotype in different environments confirm a correlation between modalities that employ different mechanisms at different temporal scales (Hz-kHz versus GHz). We also determined that observed heterogeneity in cell shape is more closely linked to the cells' mechanical state. Moreover, individual cells in multicellular spheroids exhibit a lower degree of mechanical heterogeneity when compared with single cells cultured in monodisperse 3D cultures. The observed decreased heterogeneity among cells in spheroids suggested that there is mechanical cooperativity between cells that make up a single spheroid.

Directing Multicellular Organization by Varying the Aspect Ratio of Soft Hydrogel Microwells.

Multicellular organization with precise spatial definition is essential to various biological processes, including morphogenesis, development, and healing in vascular and other tissues. Gradients and patterns of chemoattractants are well-described guides of multicellular organization, but the influences of 3D geometry of soft hydrogels are less well defined. Here, the discovery of a new mode of endothelial cell self-organization guided by combinatorial effects of stiffness and geometry, independent of protein or chemical patterning, is described. Endothelial cells in 2 kPa microwells are found to be ≈30 times more likely to migrate to the edge to organize in ring-like patterns than in stiff 35 kPa microwells. This organization is independent of curvature and significantly more pronounced in 2 kPa microwells with aspect ratio (perimeter/depth) < 25. Physical factors of cells and substrates that drive this behavior are systematically investigated and a mathematical model that explains the organization by balancing the dynamic interaction between tangential cytoskeletal tension, cell-cell, and cell-substrate adhesion is presented. These findings demonstrate the importance of combinatorial effects of geometry and stiffness in complex cellular organization that can be leveraged to facilitate the engineering of bionics and integrated model organoid systems with customized nutrient vascular networks.

Extracellular vesicles and PD-L1 suppress macrophages, inducing therapy resistance in TP53-deficient B-cell malignancies.

Genetic alterations in the DNA damage response (DDR) pathway are a frequent mechanism of resistance to chemoimmunotherapy (CIT) in B-cell malignancies. We have previously shown that the synergy of CIT relies on secretory crosstalk elicited by chemotherapy between the tumor cells and macrophages. Here, we show that loss of multiple different members of the DDR pathway inhibits macrophage phagocytic capacity in vitro and in vivo. Particularly, loss of TP53 led to decreased phagocytic capacity ex vivo across multiple B-cell malignancies. We demonstrate via in vivo cyclophosphamide treatment using the Eµ-TCL1 mouse model that loss of macrophage phagocytic capacity in Tp53-deleted leukemia is driven by a significant downregulation of a phagocytic transcriptomic signature using small conditional RNA sequencing. By analyzing the tumor B-cell proteome, we identified a TP53-specific upregulation of proteins associated with extracellular vesicles (EVs). We abrogated EV biogenesis in tumor B-cells via clustered regularly interspaced short palindromic repeats (CRISPR)-knockout (KO) of RAB27A and confirmed that the EVs from TP53-deleted lymphoma cells were responsible for the reduced phagocytic capacity and the in vivo CIT resistance. Furthermore, we observed that TP53 loss led to an upregulation of both PD-L1 cell surface expression and secretion of EVs by lymphoma cells. Disruption of EV bound PD-L1 by anti-PD-L1 antibodies or PD-L1 CRISPR-KO improved macrophage phagocytic capacity and in vivo therapy response. Thus, we demonstrate enhanced EV release and increased PD-L1 expression in TP53-deficient B-cell lymphomas as novel mechanisms of macrophage function alteration in CIT resistance. This study indicates the use of checkpoint inhibition in the combination treatment of B-cell malignancies with TP53 loss.

Comparison of Different Machine Learning Models in Prediction of Postirradiation Recurrence in Prostate Carcinoma Patients.

After primary treatment of localized prostate carcinoma (PC), up to a third of patients have disease recurrence. Different predictive models have already been used either for initial stratification of PC patients or to predict disease recurrence. Recently, artificial intelligence has been introduced in the diagnosis and management of PC with a potential to revolutionize this field. The aim of this study was to analyze machine learning (ML) classifiers in order to predict disease progression in the moment of prostate-specific antigen (PSA) elevation during follow-up. The study cohort consisted of 109 PC patients treated with external beam radiotherapy alone or in combination with androgen deprivation therapy. We developed and evaluated the performance of two ML algorithms based on artificial neural networks (ANN) and naïve Bayes (NB). Of all patients, 72.5% was randomly selected for a training set while the remaining patients were used for testing of the models. The presence/absence of disease progression was defined as the output variable. The input variables for models were conducted from the univariate analysis preformed among two groups of patients in the training set. They included two pretreatment variables (UICC stage and Gleason's score risk group) and five posttreatment variables (nadir PSA, time to nadir PSA, PSA doubling time, PSA velocity, and PSA in the moment of disease reevaluation). The area under the receiver operating characteristic curve, sensitivity, specificity, positive predictive value, negative predictive value, and predictive accuracy was calculated to test the models' performance. The results showed that specificity was similar for both models, while NB achieved better sensitivity then ANN (100.0% versus 94.4%). The ANN showed an accuracy of 93.3%, and the matching for NB model was 96.7%. In this study, ML classifiers have shown potential for application in routine clinical practice during follow-up when disease progression was suspected.