DSResSol: A Sequence-Based Solubility Predictor Created with Dilated Squeeze Excitation Residual Networks

Protein solubility is an important thermodynamic parameter that is critical for the characterization of a protein’s function, and a key determinant for the production yield of a protein in both the research setting and within industrial (e.g., pharmaceutical) applications. Experimental approaches to predict protein solubility are costly, time-consuming, and frequently offer only low success rates. To reduce cost and expedite the development of therapeutic and industrially relevant proteins, a highly accurate computational tool for predicting protein solubility from protein sequence is sought. While a number of in silico prediction tools exist, they suffer from relatively low prediction accuracy, bias toward the soluble proteins, and limited applicability for various classes of proteins. In this study, we developed a novel deep learning sequence-based solubility predictor, DSResSol, that takes advantage of the integration of squeeze excitation residual networks with dilated convolutional neural networks and outperforms all existing protein solubility prediction models. This model captures the frequently occurring amino acid k-mers and their local and global interactions and highlights the importance of identifying long-range interaction information between amino acid k-mers to achieve improved accuracy, using only protein sequence as input. DSResSol outperforms all available sequence-based solubility predictors by at least 5% in terms of accuracy when evaluated by two different independent test sets. Compared to existing predictors, DSResSol not only reduces prediction bias for insoluble proteins but also predicts soluble proteins within the test sets with an accuracy that is at least 13% higher than existing models. We derive the key amino acids, dipeptides, and tripeptides contributing to protein solubility, identifying glutamic acid and serine as critical amino acids for protein solubility prediction. Overall, DSResSol can be used for the fast, reliable, and inexpensive prediction of a protein’s solubility to guide experimental design.

State-Targeting Stabilization of Adenosine A2A Receptor by Fusing a Custom-Made De Novo Designed α-Helical Protein

G-protein coupled receptors (GPCRs) are known for their low stability and large conformational changes upon transitions between multiple states. A widely used method for stabilizing these receptors is to make chimeric receptors by fusing soluble proteins (i.e., fusion partner proteins) into the intracellular loop 3 (ICL3) connecting the transmembrane helices 5 and 6 (TM5 and TM6). However, this fusion approach requires experimental trial and error to identify appropriate soluble proteins, residue positions, and linker lengths for making the fusion. Moreover, this approach has not provided state-targeting stabilization of GPCRs. Here, to rationally stabilize a class A GPCR, adenosine A2A receptor (A2AR) in a target state, we carried out the custom-made de novo design of α-helical fusion partner proteins, which can fix the conformation of TM5 and TM6 to that in an inactive state of A2AR through straight helical connections without any kinks or intervening loops. The chimeric A2AR fused with one of the designs (FiX1) exhibited increased thermal stability. Moreover, compared with the wild type, the binding affinity of the chimera against the agonist NECA was significantly decreased, whereas that against the inverse agonist ZM241385 was similar, indicating that the inactive state was selectively stabilized. Our strategy contributes to the rational state-targeting stabilization of GPCRs.

Formation of Shelf-Stable Pickering High Internal Phase Emulsion Stabilized by Sipunculus nudus Water-Soluble Proteins (WSPs)

To form a stable emulsion system, the water-soluble proteins (WSPs) of Sipunculus nudus were prepared as the sole effective stabilizer for the high internal phase emulsion (HIPEs), of which the influence of the WSPs concentration and environmental stability was investigated. The HIPEs were fabricated using a simple one-pot homogenization process (10,000 rpm/min, 3 min) that involved blending the WSPs (0.1, 1, 2, 3, 4, and 5 wt%) with soybean oil (60, 65, 70, 75, 80, 85, and 90%). The microstructure and properties of stable HIPEs were characterized by particle size, ζ-potential, visual observations, optical microscopy, and dynamic rheology property measurements. As the concentration of WSPs increases, the mean particle diameter of HIPEs decreases, on the contrary, the apparent viscosity and storage modulus gradually increase. At a given emulsifier concentration (3 wt%), the stable and gel-like HIPEs were formed at the oil internal phase (ϕ) values of 70–75%, all the pH range in values from 3 to 9, and the ionic strength from 100 to 500 mM. Furthermore, the HIPEs that were stabilized formed a gel-like state that was relatively stable to heat and storage (30 days). And there was a new phenomenon that the destabilized HIPE of the freeze-thaw treatments could still return to a gel-like state again after homogenizing. The study results suggest that the WSPs of S. nudus as a natural emulsifier could be widely used in the food industry.

Multiomics study of a heterotardigrade, Echinisicus testudo, suggests the possibility of convergent evolution of abundant heat-soluble proteins in Tardigrada

Background Many limno-terrestrial tardigrades can enter an ametabolic state, known as anhydrobiosis, upon desiccation, in which the animals can withstand extreme environments. Through genomics studies, molecular components of anhydrobiosis are beginning to be elucidated, such as the expansion of oxidative stress response genes, loss of stress signaling pathways, and gain of tardigrade-specific heat-soluble protein families designated CAHS and SAHS. However, to date, studies have predominantly investigated the class Eutardigrada, and molecular mechanisms in the remaining class, Heterotardigrada, still remains elusive. To address this gap in the research, we report a multiomics study of the heterotardigrade Echiniscus testudo, one of the most desiccation-tolerant species which is not yet culturable in laboratory conditions. Results In order to elucidate the molecular basis of anhydrobiosis in E. testudo, we employed a multi-omics strategy encompassing genome sequencing, differential transcriptomics, and proteomics. Using ultra-low input library sequencing protocol from a single specimen, we sequenced and assembled the 153.7 Mbp genome annotated using RNA-Seq data. None of the previously identified tardigrade-specific abundant heat-soluble genes was conserved, while the loss and expansion of existing pathways were partly shared. Furthermore, we identified two families novel abundant heat-soluble proteins, which we named E. testudo Abundant Heat Soluble (EtAHS), that are predicted to contain large stretches of disordered regions. Likewise the AHS families in eutardigrada, EtAHS shows structural changes from random coil to alphahelix as the water content was decreased in vitro. These characteristics of EtAHS proteins are analogous to those of CAHS in eutardigrades, while there is no conservation at the sequence level. Conclusions Our results suggest that Heterotardigrada have partly shared but distinct anhydrobiosis machinery compared with Eutardigrada, possibly due to convergent evolution within Tardigrada. (276/350).

DOCKGROUND Membrane Protein-Protein Set

Membrane proteins play essential role in cellular mechanisms. Despite that and the major progress in experimental structure determination, they are still significantly underrepresented in Protein Data Bank. Thus, computational approaches to protein structure determination, which are important in general, are especially valuable in the case of membrane proteins and protein-protein assemblies. Due to a number of reasons, not the least of which is much greater availability of structural data, the main focus of structure prediction techniques has been on soluble proteins. Structure prediction of protein-protein complexes is a well-developed field of study. However, because of the differences in physicochemical environment in the membranes and the spatial constraints of the membranes, the generic protein-protein docking approaches are not optimal for the membrane proteins. Thus, specialized computational methods for docking of the membrane proteins must be developed. Development and benchmarking of such methods requires high-quality datasets of membrane protein-protein complexes. In this study we present a new dataset of 456 non-redundant alpha helical binary complexes. The set is significantly larger and more representative than previously developed ones. In the future, this set will become the basis for the development of docking and scoring benchmarks, similar to the ones developed for soluble proteins in the DOCKGROUND resource http://dockground.compbio.ku.edu.

343 Multiomic biomarker signatures identify subsets of patients who may benefit from either nivolumab or sotigalimab in combination with chemotherapy in metastatic pancreatic cancer

BackgroundGemcitabine/nab-Paclitaxel (GnP) is a standard of care regimen for first-line metastatic pancreatic ductal adenocarcinoma (PDAC) and has a 1-year overall survival (OS) rate of approximately 35%. There is an urgent need for novel therapeutics and precision medicine approaches in PDAC. PRINCE, a randomized phase 2 trial, reported an increased 1-year OS relative to historical data, for patients treated with nivolumab (nivo)/GnP (57.3%, p = 0.007, n=34) and sotigalimab (sotiga) (APX005M; CD40 agonist)/GnP (48.1%, p = 0.062, n= 36).MethodsTo investigate immune modulatory and pharmacodynamic (PD) effects of nivo or sotiga in combination with GnP we used several orthogonal minimally invasive, blood-based biomarker technologies. Immune population profiles were evaluated by CyTOF and features of T cell phenotype and function by multicolor flow cytometry. Soluble proteins were evaluated with predefined panels using the Olink platform (Immuno-oncology (IO) and Immune Response) along with an unbiased mass spectrometry proteomic approach (Biognosys) that identified circulating soluble proteins of significance.ResultsRelative to baseline, patients who received nivo/GnP had numerically increased frequencies of proliferating, activated CD8+ and CD4+ effector memory T cells in circulation across multiple timepoints. These patients also had significantly increased levels of soluble proteins associated with type II interferon responses and immune cell migration and T cell activation, as well as significantly decreased levels of immunomodulatory proteins.Patients who received sotiga/GnP had increased expression of the co-stimulatory molecule CD86 on conventional dendritic cells. These patients also had significantly increased concentrations of soluble proteins associated with mature antigen presenting cells, and the activation of helper CD4+ T cells, B cells, and monocytes. Significant increases in soluble proteins associated with type-1 cell-mediated effector immunity and decreases in immunosuppressive factors were observed in both arms. Significant proteins were defined as p ≤ 0.05, log2 expression fold change ≥ 0.5 (Olink) and Sparse PLS discriminant analysis was used with zero as a threshold (Biognosys).ConclusionsThis study is a first to use multiomic minimally invasive biomarker approaches in PDAC to demonstrate PD effects and immune modulation with immunotherapy/chemotherapy combinations. Orthogonal assays demonstrate that nivo/GnP and sotiga/GnP elicit unique immune responses and the observed effects are consistent with their distinct mechanisms of action. These data suggest that multiomic biomarker signatures may identify subsets of patients who may benefit from an immunotherapy/chemo approach in PDAC. Moreover, results from these analyses will support early phase clinical study development decisions.AcknowledgementsWe extend our gratitude to the patients, their families, the clinical investigators, and their site staff members who are making this trial possible. We would also like to thank Sultan Nawabi at Parker Institute for Cancer Immunotherapy (PICI) for operations leadership of the trial. We thank Bristol Myers Squibb (BMS) and Apexigen for collaboration and study drugs. The study was funded by Cancer Research Institute, BMS and PICI.Trial RegistrationNCT03214250Ethics ApprovalThis study was approved by University of Pennsylvania Institutional Review Board; Federalwide assurance #00004028.

Phytochemical Analyses, Antioxidant and Anticancer Activities of Ethanolic Leaf Extracts of Moringa oleifera Lam. Varieties

Moringa oleifera Lam. (Moringaceae) is revered as s ‘miracle tree’ due to its remarkable nutritional, medicinal and industrial uses. In our study, a comparative analysis of the nutritional parameters (antioxidant activity, sugar content—TSS, total soluble proteins—TSP and mineral contents), phytochemicals (HPLC analysis of four anticancer compounds), and cytotoxicity of M. oleifera leaf extracts (MLEs) of five selected varieties (conventional, PKM-1, PKM-2, ODC, and Jaffna), was performed. Jaffna variety possessed the highest antioxidant activity (FRAP) followed by other four varieties. The trend observed was: Jaffna (9.47 µg/mL, 18.48 µg/mL, 29.39 µg/mL, and 35.37 µg/mL) > PKM-1 (4.82 µg/mL, 7.63 µg/mL, 22.33 µg/mL, and 27.71 µg/mL) > PKM-2 (2.10 µg/mL, 7.04 µg/mL, 13.18 µg/mL, and 21.78 µg/mL) > ODC (0.17 µg/mL, 2.10 µg/mL, 4.41 µg/mL and 13.94 µg/mL) > Conventional (0.05 µg/mL, 1.08 µg/mL, 2.86 µg/mL, and 5.40 µg/mL), total soluble proteins (TSP) [0.69 ± 0.01 and 0.94 ± 0.01 µg/mL (young and mature stage, respectively)], sugar content (TSS) [0.39 ± 0.01 and 0.51 ± 0.01 µg/mL (young and mature stage, respectively)], chlorophyll content [1.07 ± 0.01 (plantlet stage), 1.36 ± 0.003 (vegetative-stage), 0.82 ± 0.004 (reproductive stage) mg/g], followed by the other four varieties. The trend observed for cytotoxic activities of ethanolic MLEs on HepG2 cell line, based on the IC50 values, was conventional (1.22 mg/mL) > ODC (0.90 mg/mL) > PKM-2 (0.65 mg/mL) > PKM-1 (0.35 mg/mL) > Jaffna (0.15 mg/mL). The results of HPLC quantification of anticancer compounds [beta-sitosterol (0.244%), quercetin (0.216%), kaempferol (0.013%), and moringin (0.063%)] was also in consonance with that of MTT assay. In summary, the trend observed in all the parameters tested was Jaffna > PKM-1 > PKM-2 > ODC > conventional. Thus, Jaffna variety has a better potential to combat malnutrition and cancer and must be recommended for commercial plantations.

Diagnostic and Prognostic Utility of the Extracellular Vesicles Subpopulations Present in Pleural Effusion

Extracellular vesicles (EVs), comprising exosomes, microvesicles, and apoptotic bodies, are released by all cells into the extracellular matrix and body fluids, where they play important roles in intercellular communication and matrix remodeling in various pathological conditions. Malignant pleural mesothelioma (MPM) is a primary tumor of mesothelial origin, predominantly related to asbestos exposure. The detection of MPM at an early stage and distinguishing it from benign conditions and metastatic adenocarcinomas (AD) is sometimes challenging. Pleural effusion is often the first available biological material and an ideal source for characterizing diagnostic and prognostic factors. Specific proteins have previously been identified as diagnostic markers in effusion, but it is not currently known whether these are associated with vesicles or released in soluble form. Here, we study and characterize tumor heterogeneity and extracellular vesicle diversity in pleural effusion as diagnostic or prognostic markers for MPM. We analyzed extracellular vesicles and soluble proteins from 27 pleural effusions, which were collected and processed at the department of pathology and cytology at Karolinska University Hospital, representing three different patient groups, MPM (n = 9), benign (n = 6), and AD (n = 12). The vesicles were fractionated into apoptotic bodies, microvesicles, and exosomes by differential centrifugation and characterized by nanoparticle tracking analysis and Western blotting. Multiplex bead-based flow cytometry analysis showed that exosomal markers were expressed differently on EVs present in different fractions. Further characterization of exosomes by a multiplex immunoassay (Luminex) showed that all soluble proteins studied were also present in exosomes, though the ratio of protein concentration present in supernatant versus exosomes varied. The proportion of Angiopoietin-1 present in exosomes was generally higher in benign compared to malignant samples. The corresponding ratios of Mesothelin, Galectin-1, Osteopontin, and VEGF were higher in MPM effusions compared to those in the benign group. These findings demonstrate that relevant diagnostic markers can be recovered from exosomes.

Organic onion biofortification using microalgae and humic acid

Biofortification can be understood as a strategy to raise nutrients levels in vegetables edible structures, which can positively interfere in human diet. Biofertilizers are an interesting option in alternative production systems, as they can increase food nutritional quality and contribute to plant development, while aiding environment sustainability as natural products. The use of microalgae and humic acid as biofertilizers points to improvements in nutrients and biomolecules content in plants, however, their combined application is still unexplored. In that scenario, it was carried a study with onions about the influence of applications via root immersion in microalgae Scenedesmus subspicatus (Sc) and humic acid (HA) solutions, analyzing possible alterations of macro and micronutrients, total sugars, reducing sugar, free total amino acids, , total soluble solids, soluble proteins and antioxidant capacity in the bulbs. Treatments consisted of one minute seedlings roots immersion from two onion cultivars in solutions containing microalgae and humic acid, and then transplanted to organic system field. There were used three concentrations: control, 0.3 g L-1 Sc + 0.3 g L-1 HA (3SH) and 0.6 g L-1 Sc + 0.6 g L-1 HA (6SH). Results show that the treatments with microalgae with humic acid association were able to increase the content of N, carbohydrates and soluble proteins, also elevating antioxidant activity in onion bulbs.