Heterogeneity of cancer stem cell-related marker expression is associated with three-dimensional structures in malignant pleural effusion produced by lung adenocarcinoma.

INTRODUCTION: Cancer stem cells have been described in lung adenocarcinoma-associated malignant pleural effusion. They show clinically important features, including the ability to initiate new tumours and resistance to treatments. However, their correlation with the three-dimensional tumour structures in the effusion is not well understood. METHODS: Cell blocks produced from lung adenocarcinoma patients' pleural effusion were examined for cancer stem cell-related markers Nanog and CD133 using immunocytochemistry. The three-dimensional cancer cell structures and CD133 expression patterns were visualized with tissue-clearing technology. The expression patterns were correlated with tumour cell structures, genetic variants and clinical outcomes. RESULTS: Thirty-nine patients were analysed. Moderate-to-strong Nanog expression was detected in 27 cases (69%), while CD133 was expressed by more than 1% of cancer cells in 11 cases (28%). Nanog expression was more homogenous within individual specimens, while CD133 expression was detected in single tumour cells or cells within small clusters instead of larger structures in 8 of the 11 positive cases (73%). Although no statistically significant correlation between the markers and tumour genetic variants or patient survival was observed, we recorded seven cases with follow-up specimens after cancer treatment, and four (57%) showed a change in stem cell-related marker expression corresponding to treatment response. CONCLUSIONS: Lung adenocarcinoma cells in the pleural effusion show variable expression of cancer stem cell-related markers, some showing a correlation with the size of cell clusters. Their expression level is potentially correlated with cancer treatment effects.

SARS-CoV-2 proteins structural studies using synchrotron radiation.

In the process of the development of structural biology, both the size and the complexity of the determined macromolecular structures have grown significantly. As a result, the range of application areas for the results of structural studies of biological macromolecules has expanded. Significant progress in the development of structural biology methods has been largely achieved through the use of synchrotron radiation. Modern sources of synchrotron radiation allow to conduct high-performance structural studies with high temporal and spatial resolution. Thus, modern techniques make it possible to obtain not only static structures, but also to study dynamic processes, which play a key role in understanding biological mechanisms. One of the key directions in the development of structural research is the drug design based on the structures of biomolecules. Synchrotron radiation offers insights into the three-dimensional time-resolved structure of individual viral proteins and their complexes at atomic resolution. The rapid and accurate determination of protein structures is crucial for understanding viral pathogenicity and designing targeted therapeutics. Through the application of experimental techniques, including X-ray crystallography and small-angle X-ray scattering (SAXS), it is possible to elucidate the structural details of SARS-CoV-2 virion containing 4 structural, 16 nonstructural proteins (nsp), and several accessory proteins. The most studied potential targets for vaccines and drugs are the structural spike (S) protein, which is responsible for entering the host cell, as well as nonstructural proteins essential for replication and transcription, such as main protease (Mpro), papain-like protease (PLpro), and RNA-dependent RNA polymerase (RdRp). This article provides a brief overview of structural analysis techniques, with focus on synchrotron radiation-based methods applied to the analysis of SARS-CoV-2 proteins.

Structural and antigenic characterization of the avian adeno-associated virus capsid.

IMPORTANCE: AAVs are extensively studied as promising therapeutic gene delivery vectors. In order to circumvent pre-existing antibodies targeting primate-based AAV capsids, the AAAV capsid was evaluated as an alternative to primate-based therapeutic vectors. Despite the high sequence diversity, the AAAV capsid was found to bind to a common glycan receptor, terminal galactose, which is also utilized by other AAVs already being utilized in gene therapy trials. However, contrary to the initial hypothesis, AAAV was recognized by approximately 30% of human sera tested. Structural and sequence comparisons point to conserved epitopes in the fivefold region of the capsid as the reason determinant for the observed cross-reactivity.

Serodominant SARS-CoV-2 Nucleocapsid Peptides Map to Unstructured Protein Regions.

The SARS-CoV-2 nucleocapsid (N) protein is highly immunogenic, and anti-N antibodies are commonly used as markers for prior infection. While several studies have examined or predicted the antigenic regions of N, these have lacked consensus and structural context. Using COVID-19 patient sera to probe an overlapping peptide array, we identified six public and four private epitope regions across N, some of which are unique to this study. We further report the first deposited X-ray structure of the stable dimerization domain at 2.05 Å as similar to all other reported structures. Structural mapping revealed that most epitopes are derived from surface-exposed loops on the stable domains or from the unstructured linker regions. An antibody response to an epitope in the stable RNA binding domain was found more frequently in sera from patients requiring intensive care. Since emerging amino acid variations in N map to immunogenic peptides, N protein variation could impact detection of seroconversion for variants of concern. IMPORTANCE As SARS-CoV-2 continues to evolve, a structural and genetic understanding of key viral epitopes will be essential to the development of next-generation diagnostics and vaccines. This study uses structural biology and epitope mapping to define the antigenic regions of the viral nucleocapsid protein in sera from a cohort of COVID-19 patients with diverse clinical outcomes. These results are interpreted in the context of prior structural and epitope mapping studies as well as in the context of emergent viral variants. This report serves as a resource for synthesizing the current state of the field toward improving strategies for future diagnostic and therapeutic design.

Characterisation of Elevenin-Vc1 from the Venom of Conus victoriae: A Structural Analogue of α-Conotoxins.

Elevenins are peptides found in a range of organisms, including arthropods, annelids, nematodes, and molluscs. They consist of 17 to 19 amino acid residues with a single conserved disulfide bond. The subject of this study, elevenin-Vc1, was first identified in the venom of the cone snail Conus victoriae (Gen. Comp. Endocrinol. 2017, 244, 11-18). Although numerous elevenin sequences have been reported, their physiological function is unclear, and no structural information is available. Upon intracranial injection in mice, elevenin-Vc1 induced hyperactivity at doses of 5 or 10 nmol. The structure of elevenin-Vc1, determined using nuclear magnetic resonance spectroscopy, consists of a short helix and a bend region stabilised by the single disulfide bond. The elevenin-Vc1 structural fold is similar to that of α-conotoxins such as α-RgIA and α-ImI, which are also found in the venoms of cone snails and are antagonists at specific subtypes of nicotinic acetylcholine receptors (nAChRs). In an attempt to mimic the functional motif, Asp-Pro-Arg, of α-RgIA and α-ImI, we synthesised an analogue, designated elevenin-Vc1-DPR. However, neither elevenin-Vc1 nor the analogue was active at six different human nAChR subtypes (α1ß1εδ, α3ß2, α3ß4, α4ß2, α7, and α9α10) at 1 µM concentrations.

Covalent polyphenol modification of a reactive cysteine in the major apple allergen Mal d 1.

Naturally occurring polyphenols can modify the molecular properties of food allergens. For the major apple allergen Mal d 1 it has been postulated that chemical reactions with polyphenols cause permanent changes in the tertiary structure, causing a loss of conformational IgE epitopes and reducing allergenicity. In our study, we investigated the effect that reactions with oxidized polyphenols have on the structure of Mal d 1 by mass spectrometry and NMR spectroscopy. We showed that a surface-exposed cysteine residue in this allergen spontaneously reacts with oxidized polyphenols under formation of a defined covalent adduct. Chemical modification of Mal d 1 did not destabilize or perturb the three-dimensional fold, nor did it interfere with ligand binding to its internal pocket. A structural model of the chemically modified apple allergen is presented, which reveals that the bound polyphenol partially covers a conformational IgE epitope on the protein surface.

Bioinformatic analysis of SIRT7 sequence and structure.

Sirtuins are highly conserved proteins that perform very important functions in different cellular processes. Notably, SIRT7 is the least studied human sirtuin, but it is known to be involved in a wide variety of processes in both health and disease. In this way, SIRT7 activity-regulating molecules could be beneficial for the treatment of relevant diseases such as cardiovascular and bone diseases, where SIRT7 levels are reduced, or obesity and cancer, where they are increased. In this work, using bioinformatic methods, the sequence and structure of SIRT7 orthologs in a wide variety of organisms were analyzed. Thus, the catalytic domain was found to be quite conserved (83.23% identity) and key residues such as D118, Y119, R120, D170, H187, N189, C198, C225, C228, V273, G298, F239 and V237 were identified. Furthermore, a phylogenetic tree was constructed where SIRT7 orthologs from mammals, birds, reptiles, amphibians, fish, insects, and arachnids were found to cluster in different groups. Finally, predicted three-dimensional structures showed a classic structure of the central catalytic region of most sirtuins, while the flanking N- and C-terminal regions were unique to each phylogenetic group. All this helps to understand a little more how SIRT7 works and gives clues for the future design and development of small molecules that benefit human and animal health.Communicated by Ramaswamy H. Sarma.

Functional significance of the rare rs35667974 IFIH1 gene polymorphism, associated with multiple autoimmune diseases, using a structural biological approach.

Autoimmune diseases, which affect approximately 5% of human population, are a range of diseases in which the immune response to self-antigens results in damage or dysfunction of tissues. Recent genome wide association studies (GWAS) have successfully identified novel autoimmune disease-associated loci, with many of them shared by multiple disease-associated pathways but much of the genetics and pathophysiological mechanisms remain still obscure. Considering that most of the potential causal variants are still unknown, many studies showed that the missense variant rs35667974 at interferon-induced with helicase C domain 1 (IFIH1) gene is protective for type 1 diabetes (T1D), psoriasis (PS) and psoriatic arthritis (PsA). Recently, this variant was found to be also associated with ankylosing spondylitis (AS), Crohn's disease (CD) and ulcerative colitis (UC). The IFIH1 gene encodes a cytoplasmic RNA helicase otherwise known as melanoma differentiation-associated 5 (MDA5) that recognizes viral RNA and is involved in innate immunity through recognition of viral RNA. In the present study we sought to investigate the association of the rare rs35667974 variant of IFIH1 gene, which resides in exon 14 and changes a conserved isoleucine at position #923 to valine, in the development of various autoimmune diseases and give a reason for the selectivity affecting different autoimmune diseases. Evolutionary studies and three-dimensional (3 D) homology modelling were employed on the MDA5 protein product, through its association with dsRNA, recognition factor controlling cytokine and chemokine signalling, to investigate the protective role of the MDA5 variant for certain autoimmune diseases.

Enantiomeric characteristics of non-synthetic cannabidiol by two-dimensional nuclear magnetic resonance

SUMMARY Introduction: Cannabidiol (CBD) has become a promising bioactive for the next decades after the recent recognition of the medical potential of Cannabis derivatives by United Nations member countries, as it has no psychotropic potential as your isomer A9-tetrahydrocannabinol (Δ9-THC). The differentiation of these isomers has been studied for decades. Recent studies demonstrate that even with more subtle chemical characteristics, such as those of the CBD enantiomers, there are considerable bioactive differences. However, there are still not many studies on their chemical structures. Aim: This work aims to present experimental data obtained by Nuclear Magnetic Resonance (NMR) to better elucidate the three-dimensional structure of this enantiomeric bioactive. Materials and methods: For this, a sample of non-synthetic high purity CBD was subjected to different one-dimensional (1D-NMR) and two-dimensional (2D-NMR) analyses related to the hydrogen (1H) and carbon (13C) nuclei. Results and discussion: The 1D-NMR techniques used are sufficient to distinguish the CBD and Δ 9-THC isomers, but not to identify the enantiomeric characteristics of the non-synthetic CBD. Conclusions: It is concluded that the two-dimensional homonuclear (1H,1H) and heteronuclear (1H,13C) techniques analyzed are suitable to help distinguish CBD enantiomers.

Introducción: El cannabidiol (CBD) se ha convertido en un bioactivo prometedor para las próximas décadas tras el reciente reconocimiento del potencial medicinal de los derivados del Cannabis por parte de los países miembros de las Naciones Unidas, ya que no tiene potencial psicotrópico como su isómero Δ9-tetrahidrocannabinol (Δ 9-THC). La diferenciación de estos isómeros se ha estudiado durante décadas. Estudios recientes demuestran que incluso con características químicas más sutiles, como las de los enan-tiómeros del CBD, existen diferencias bioactivas considerables. Sin embargo, no existen muchos estudios sobre sus estructuras químicas. Objetivo: Este trabajo tiene como objetivo presentar datos experimentales obtenidos por Resonancia magnética nuclear (RMN) para dilucidar mejor la estructura tridimensional de este bioactivo enantiomérico. Materiales y métodos: Para ello, una muestra de CBD no sintético de alta pureza se sometió a diferentes análisis unidimensionales (RMN-1D) y bidimensionales (RMN-2D) relacionados con los núcleos del hidrógeno (1H) y carbono (13C). Resultados y discusión: Las técnicas de RMN-1D utilizadas son suficientes para distinguir los isómeros de CBD y Δ 9-THC, pero no para identificar las características enantioméricas del CBD no sintético. Conclusiones: Se concluye que las técnicas bidimensionales homonucleares (1H,1H) y heteronucleares (1H,13C) analizadas son adecuadas para ayudar a distinguir los enantiómeros del CBD.

Introdução: O canabidiol (CBD) se tornou um bioativo promissor para as próximas décadas após o recente reconhecimento do potencial medicinal dos derivados da Cannabis pelos países membros das Nações Unidas, uma vez que não tem potencial psicotrópico como seu isômero Δ 9-tetrahidrocanabinol (A9-THC). A diferenciação desses isômeros é estudada há décadas. Estudos recentes demonstram que mesmo com características químicas mais sutis, como as dos enantiômeros do CBD, há consideráveis diferenças bioativas. Todavia, ainda não há muitos estudos sobre suas estruturas químicas. Objetivo: Este trabalho tem como objetivo apresentar dados experimentais obtidos por Ressonância magnética nuclear (RMN) para melhor elucidar a estrutura tridimensional deste bioativo enantiomérico. Materiais e métodos: Para isso, uma amostra de CBD não sintético de alta pureza foi submetida a diferentes análises unidimensionais (RMN-1D) e bidimensionais (RMN-2D) relacionadas aos núcleos de hidrogênio (1H) e carbono (13C). Resultados e discussão: As técnicas de RMN-1D usadas são suficientes para distinguir os isômeros CBD e Δ 9-THC, mas não para identificar as características enantioméricas do CBD não sintético. Conclusões: Conclui-se que as técnicas bidimensionais homonucleares (1H,1H) e heteronucleares (1H,13C) analisadas são adequadas para auxiliar na distinção dos enantiômeros do CBD.

Constructing A/B-Side Heterogeneous Asynchronous Structure with Ag2Se Layers and Bushy-like PPy toward High-Performance Flexible Photo-Thermoelectric Generators.

The enthusiasm for environmental energy harvesting has triggered a boom in research on photo-thermoelectric generators (PTEGs), and the relevant applications are mainly focused on self-energy supply sensors owing to the limitations of their output performances. For this purpose, high-output hierarchical heterogeneous PTEGs were constructed by assembling separately optimized thermoelectric (TE) and photothermal (PT) layers. The pressure and temperature conditions of Ag2Se films during the pressing process were first explored, and the sample with the optimal performance and least defects was selected as the TE layer. At the same time, different morphologies of polypyrrole (PPy) PT layers were electrochemically synthesized. It is found that the three-dimensional structure of Bushy-PPy could effectively improve the light absorption and thus enhance the PT conversion performance. The final assembled PTEG can produce an output voltage of -9.03 mV and an output power of 3.53 µW under the irradiation of a near-infrared light source of 300 mW cm-2 without a cooling source, and it can also achieve considerable output power under visible light irradiation of different intensities. Combining its high retentions of electrical conductivity (99%) and output performance (97%) after 1000 bending-tension cycles, it is proven to be a promising next-generation wearable flexible energy harvesting device.

High-Risk Mucosal Human Papillomavirus 16 (HPV16) E6 Protein and Cutaneous HPV5 and HPV8 E6 Proteins Employ Distinct Strategies To Interfere with Interferon Regulatory Factor 3-Mediated Beta Interferon Expression.

Persistent infection with some mucosal α-genus human papillomaviruses (HPVs; the most prevalent one being HPV16) can induce cervical carcinoma, anogenital cancers, and a subset of head and neck squamous cell carcinoma (HNSCC). Cutaneous ß-genus HPVs (such as HPV5 and HPV8) associate with skin lesions that can progress into squamous cell carcinoma with sun exposure in Epidermodysplasia verruciformis patients and immunosuppressed patients. Here, we analyzed mechanisms used by E6 proteins from the α- and ß-genus to inhibit the interferon-ß (IFNB1) response. HPV16 E6 mediates this effect by a strong direct interaction with interferon regulatory factor 3 (IRF3). The binding site of E6 was localized within a flexible linker between the DNA-binding domain and the IRF-activation domain of IRF3 containing an LxxLL motif. The crystallographic structure of the complex between HPV16 E6 and the LxxLL motif of IRF3 was solved and compared with the structure of HPV16 E6 interacting with the LxxLL motif of the ubiquitin ligase E6AP. In contrast, cutaneous HPV5 and HPV8 E6 proteins bind to the IRF3-binding domain (IBiD) of the CREB-binding protein (CBP), a key transcriptional coactivator in IRF3-mediated IFN-ß expression. IMPORTANCE Persistent HPV infections can be associated with the development of several cancers. The ability to persist depends on the ability of the virus to escape the host immune system. The type I interferon (IFN) system is the first-line antiviral defense strategy. HPVs carry early proteins that can block the activation of IFN-I. Among mucosal α-genus HPV types, the HPV16 E6 protein has a remarkable property to strongly interact with the transcription factor IRF3. Instead, cutaneous HPV5 and HPV8 E6 proteins bind to the IRF3 cofactor CBP. These results highlight the versatility of E6 proteins to interact with different cellular targets. The interaction between the HPV16 E6 protein and IRF3 might contribute to the higher prevalence of HPV16 than that of other high-risk mucosal HPV types in HPV-associated cancers.

A Genomic Island of Vibrio cholerae Encodes a Three-Component Cytotoxin with Monomer and Protomer Forms Structurally Similar to Alpha-Pore-Forming Toxins.

Alpha-pore-forming toxins (α-PFTs) are secreted by many species of bacteria, including Escherichia coli, Aeromonas hydrophila, and Bacillus thuringiensis, as part of their arsenal of virulence factors, and are often cytotoxic. In particular, for α-PFTs, the membrane-spanning channel they form is composed of hydrophobic α-helices. These toxins oligomerize at the surface of target cells and transition from a soluble to a protomer state in which they expose their hydrophobic regions and insert into the membrane to form a pore. The pores may be composed of homooligomers of one component or heterooligomers with two or three components, resulting in bi- or tripartite toxins. The multicomponent α-PFTs are often expressed from a single operon. Recently, motility-associated killing factor A (MakA), an α-PFT, was discovered in Vibrio cholerae. We report that makA is found on the V. cholerae GI-10 genomic island within an operon containing genes for two other potential α-PFTs, MakB and MakE. We determined the X-ray crystal structures for MakA, MakB, and MakE and demonstrated that all three are structurally related to the α-PFT family in the soluble state, and we modeled their protomer state based on the α-PFT AhlB from A. hydrophila. We found that MakA alone is cytotoxic at micromolar concentrations. However, combining MakA with MakB and MakE is cytotoxic at nanomolar concentrations, with specificity for J774 macrophage cells. Our data suggest that MakA, -B, and -E are α-PFTs that potentially act as a tripartite pore-forming toxin with specificity for phagocytic cells. IMPORTANCE The bacterium Vibrio cholerae causes gastrointestinal, wound, and skin infections. The motility-associated killing factor A (MakA) was recently shown to be cytotoxic against colon, prostate, and other cancer cells. However, at the outset of this study, the capacity of MakA to damage cells in combination with other Mak proteins encoded in the same operon had not been elucidated. We determined the structures of three Mak proteins and established that they are structurally related to the α-PFTs. Compared to MakA alone, the combination of all three toxins was more potent specifically in mouse macrophages. This study highlights the idea that the Mak toxins are selectively cytotoxic and thus may function as a tripartite toxin with cell type specificity.

Three-Dimensional MoS2/Reduced Graphene Oxide Nanosheets/Graphene Quantum Dots Hybrids for High-Performance Room-Temperature NO2 Gas Sensors.

This study presents three-dimensional (3D) MoS2/reduced graphene oxide (rGO)/graphene quantum dots (GQDs) hybrids with improved gas sensing performance for NO2 sensors. GQDs were introduced to prevent the agglomeration of nanosheets during mixing of rGO and MoS2. The resultant MoS2/rGO/GQDs hybrids exhibit a well-defined 3D nanostructure, with a firm connection among components. The prepared MoS2/rGO/GQDs-based sensor exhibits a response of 23.2% toward 50 ppm NO2 at room temperature. Furthermore, when exposed to NO2 gas with a concentration as low as 5 ppm, the prepared sensor retains a response of 15.2%. Compared with the MoS2/rGO nanocomposites, the addition of GQDs improves the sensitivity to 21.1% and 23.2% when the sensor is exposed to 30 and 50 ppm NO2 gas, respectively. Additionally, the MoS2/rGO/GQDs-based sensor exhibits outstanding repeatability and gas selectivity. When exposed to certain typical interference gases, the MoS2/rGO/GQDs-based sensor has over 10 times higher sensitivity toward NO2 than the other gases. This study indicates that MoS2/rGO/GQDs hybrids are potential candidates for the development of NO2 sensors with excellent gas sensitivity.

Discovery of novel peptide neurotoxins from sea anemone species.

As primitive metazoa, sea anemones are rich in various bioactive peptide neurotoxins. These peptides have been applied to neuroscience research tools or directly developed as marine drugs. To date, more than 1100 species of sea anemones have been reported, but only 5% of the species have been used to isolate and identify sea anemone peptide neurotoxins. There is an urgent need for more systematic discovery and study of peptide neurotoxins in sea anemones. In this review, we have gathered the currently available methods from crude venom purification and gene cloning to venom multiomics, employing these techniques for discovering novel sea anemone peptide neurotoxins. In addition, the three-dimensional structures and targets of sea anemone peptide neurotoxins are summarized. Therefore, the purpose of this review is to provide a reference for the discovery, development, and utilization of sea anemone peptide neurotoxins.

Fungal effector SIB1 of Colletotrichum orbiculare has unique structural features and can suppress plant immunity in Nicotiana benthamiana.

Fungal plant pathogens secrete virulence-related proteins, called effectors, to establish host infection; however, the details are not fully understood yet. Functional screening of effector candidates using Agrobacterium-mediated transient expression assay in Nicotiana benthamiana identified two virulence-related effectors, named SIB1 and SIB2 (Suppression of Immunity in N. benthamiana), of an anthracnose fungus Colletotrichum orbiculare, which infects both cucurbits and N. benthamiana. The Agrobacterium-mediated transient expression of SIB1 or SIB2 increased the susceptibility of N. benthamiana to C. orbiculare, which suggested these effectors can suppress immune responses in N. benthamiana. The presence of SIB1 and SIB2 homologs was found to be limited to the genus Colletotrichum. SIB1 suppressed both (i) the generation of reactive oxygen species triggered by two different pathogen-associated molecular patterns, chitin and flg22, and (ii) the cell death response triggered by the Phytophthora infestans INF1 elicitin in N. benthamiana. We determined the NMR-based structure of SIB1 to obtain its structural insights. The three-dimensional structure of SIB1 comprises five ß-strands, each containing three disulfide bonds. The overall conformation was found to be a cylindrical shape, such as the well-known antiparallel ß-barrel structure. However, the ß-strands were found to display a unique topology, one pair of these ß-strands formed a parallel ß-sheet. These results suggest that the effector SIB1 present in Colletotrichum fungi has unique structural features and can suppress pathogen-associated molecular pattern-triggered immunity in N. benthamiana.

Prediction of Anticancer Peptides with High Efficacy and Low Toxicity by Hybrid Model Based on 3D Structure of Peptides.

Recently, anticancer peptides (ACPs) have emerged as unique and promising therapeutic agents for cancer treatment compared with antibody and small molecule drugs. In addition to experimental methods of ACPs discovery, it is also necessary to develop accurate machine learning models for ACP prediction. In this study, features were extracted from the three-dimensional (3D) structure of peptides to develop the model, compared to most of the previous computational models, which are based on sequence information. In order to develop ACPs with more potency, more selectivity and less toxicity, the model for predicting ACPs, hemolytic peptides and toxic peptides were established by peptides 3D structure separately. Multiple datasets were collected according to whether the peptide sequence was chemically modified. After feature extraction and screening, diverse algorithms were used to build the model. Twelve models with excellent performance (Acc > 90%) in the ACPs mixed datasets were used to form a hybrid model to predict the candidate ACPs, and then the optimal model of hemolytic peptides (Acc = 73.68%) and toxic peptides (Acc = 85.5%) was used for safety prediction. Novel ACPs were found by using those models, and five peptides were randomly selected to determine their anticancer activity and toxic side effects in vitro experiments.

The dual role of SrbA from Paracoccidioides lutzii: a hypoxic regulator.

The fungus Paracoccidioides lutzii is one of the species of the Paracoccidioides genus, responsible for a neglected human mycosis, endemic in Latin America, the paracoccidioidomycosis (PCM). In order to survive in the host, the fungus overcomes a hostile environment under low levels of oxygen (hypoxia) during the infectious process. The hypoxia adaptation mechanisms are variable among human pathogenic fungi and worthy to be investigated in Paracoccidoides spp. Previous proteomic results identified that P. lutzii responds to hypoxia and it has a functional homolog of the SrbA transcription factor, a well-described hypoxic regulator. However, the direct regulation of genes by SrbA and the biological processes it governs while performing protein interactions have not been revealed yet. The goal of this study was to demonstrate the potential of SrbA targets genes in P. lutzii. In addition, to show the SrbA three-dimensional aspects as well as a protein interaction map and important regions of interaction with predicted targets. The results show that SrbA-regulated genes were involved with several biological categories, such as metabolism, energy, basal processes for cell maintenance, fungal morphogenesis, defense, virulence, and signal transduction. Moreover, in order to investigate the SrbA's role as a protein, we performed a 3D simulation and also a protein-protein network linked to this hypoxic regulator. These in silico analyses revealed relevant aspects regarding the biology of this pathogen facing hypoxia and highlight the potential of SrbA as an antifungal target in the future.

Morphological comparison between three-dimensional structure of immortalized human lens epithelial cells and Soemmering's ring.

The incidence rate of post-cataract surgery posterior capsule opacification (PCO) and lens turbidity is about 20% in 5 years. Soemmering's ring, which is a type of PCO also called a regenerated lens with similar tissue structure to that of a human lens, is an important proxy for elucidating the mechanism of lens regeneration and maintenance of transparency. The authors created new human immortalized crystalline lens epithelial cells (iHLEC-NY1s) with excellent differentiation potential, and as a result of culturing the cells by static and rotation-floating methods, succeeded in producing a three-dimensional cell structure model (3D-iHLEC-NY1s) which is similar to Soemmering's ring in tissue structure and expression characteristics of αA-crystalline, ßB2-crystalline, vimentin proteins. 3D-iHLEC-NY1s is expected to be a proxy in vitro experimental model of Soemmering's ring to enable evaluation of drug effects on suppression of cell aggregate formation and transparency. By further improving the culture conditions, we aim to control the cell sequence and elucidate the mechanism underlying the maintenance of lens transparency.

3D Hydrangea Macrophylla-like Nickel-Vanadium Metal-Organic Frameworks Formed by Self-Assembly of Ultrathin 2D Nanosheets for Overall Water Splitting.

The development of highly efficient and low-cost bifunctional noble metal-free electrocatalysts for both the oxygen evolution reaction (OER) and hydrogen evolution reaction (HER) is an effective strategy for improving efficiency. Herein, novel three-dimensional (3D) bimetallic metal-organic frameworks containing Ni and V with adjustable stoichiometry were synthesized on nickel foam successfully. Notably, Ni2V-MOFs@NF only require rather low overpotentials of 244 and 89 mV for the OER and HER, respectively, and expedites overall water splitting with 1.55 V at 10 mA cm-2 with robust durability during the 80 h test. The high efficiency of the novel obtained electrocatalysts should be attributed to the particular morphological design of the two-dimensional (2D) ultrathin nanosheets self-assembling into a 3D nanoflower and the electronic structure regulation resulting from the synergetic interaction between nickel and vanadium. Subsequent theoretical calculations reveal the following conclusions: (I) the exceptional electronic conductivity of Ni2V-MOFs shows enhanced optimization as a result of electronic structure reconstruction, (II) the energy barrier reduction of the rate-limiting step is responsible for the enhanced dynamics of Ni2V-MOFs for the OER, and (III) the facilitation of the adsorption of H+ and H2O plays a key role in progressing the HER catalytic activity of Ni2V-MOFs.

Identification of a novel alkaline serine protease from gazami crab (Portunus trituberculatus) hepatopancreas and its hydrolysis of myofibrillar protein.

Serine proteases are thought to play a key role in the muscle softening of gazami crab (Portunus trituberculatus) during storage. A serine protease, Pt-sp2, was purified from the hepatopancreas of gazami crab using ammonium sulfate precipitation, anion-exchange and gel filtration chromatography, and was analyzed by mass spectrometry, transcriptome and bioinformatics. It revealed that Pt-sp2 was trypsin-like, with no 100% identical proteins in the NCBI database. The molecular weight of Pt-sp2 was approximately 37.2 kDa. Its optimum pH and temperature were 9.0 and 50 °C, respectively, using t-Butyloxy­carbonyl-Phe-Ser-Arg-4-methyl-coumaryl-7-amide as a substrate. Pt-sp2 was activated in the presence of Ca2+. Both soybean trypsin inhibitor and Nα-Tosyl-l-lysine chloromethyl ketone hydrochloride completely suppressed Pt-sp2 activity, while it was only partially inhibited by phenylmethylsulfonyl fluoride and EDTA. However, PMSF, Pepstatin A and cystatin inhibitor E-64 showed no inhibition on Pt-sp2 protease activity. The Km value of Pt-sp2 was 0.82 µM, and Pt-sp2 effectively hydrolyzed myofibrillar protein at 37 °C.