3D Protein Research Articles

Structural informatic study of determined and AlphaFold2 predicted molecular structures of 13 human solute carrier transporters and their water-soluble QTY variants

Solute carrier transporters are integral membrane proteins, and are important for diverse cellular nutrient transports, metabolism, energy demand, and other vital biological activities. They have recently been implicated in pancreatic cancer and other cancer metastasis, angiogenesis, programmed cell death and proliferation, cell metabolism and chemo-sensitivity. Here we report the study of 13 human solute carrier membrane transporters using the highly accurate AlphaFold2 predictions of 3D protein structures. In the native structures, there are hydrophobic amino acids leucine (L), isoleucine (I), valine (V) and phenylalanine (F) in the transmembrane alpha-helices. These hydrophobic amino acids L, I, V, F are systematically replaced by hydrophilic amino acids glutamine (Q), threonine (T) and tyrosine (Y), thus the QTY code. Therefore, these QTY variant transporters become water-soluble without requiring detergents. We present the superposed structures of these native solute carrier transporters and their water-soluble QTY variants. The superposed structures show remarkable similarity with RMSD ~ 1 Å–< 3 Å despite > 46% protein sequence substitutions in transmembrane alpha-helices. We also show the differences of surface hydrophobicity between the native solute carrier transporters and their QTY variants. Our study may further stimulate designs of water-soluble transmembrane proteins and other aggregated proteins for drug discovery and biotechnological applications.

Natural Materials for 3D Printing and Their Applications.

In recent years, 3D printing has gradually become a well-known new topic and a research hotspot. At the same time, the advent of 3D printing is inseparable from the preparation of bio-ink. Natural materials have the advantages of low toxicity or even non-toxicity, there being abundant raw materials, easy processing and modification, excellent mechanical properties, good biocompatibility, and high cell activity, making them very suitable for the preparation of bio-ink. With the help of 3D printing technology, the prepared materials and scaffolds can be widely used in tissue engineering and other fields. Firstly, we introduce the natural materials and their properties for 3D printing and summarize the physical and chemical properties of these natural materials and their applications in tissue engineering after modification. Secondly, we discuss the modification methods used for 3D printing materials, including physical, chemical, and protein self-assembly methods. We also discuss the method of 3D printing. Then, we summarize the application of natural materials for 3D printing in tissue engineering, skin tissue, cartilage tissue, bone tissue, and vascular tissue. Finally, we also express some views on the research and application of these natural materials.

Characteristics of Allan-Herndon-Dudley Syndrome in Chinese children: Identification of two novel pathogenic variants of the SLC16A2 gene.

The aim of this study was to identify causative variants associated with Allan-Herndon-Dudley syndrome (AHDS) in two unrelated Chinese families, and to determine their potential pathogenicity. We also summarized the core clinical symptoms of AHDS by reviewing the related literature. Genomic DNA was isolated from the peripheral blood of AHDS patients and their family members. Whole exome sequencing (WES) was performed on the proband from each family to identify the candidate variants. Subsequently, Sanger sequencing was used to verify the identified candidate variants and to assess co-segregation among the available family members. In silico prediction combined with 3D protein modeling was conducted to predict the functional effects of the variants on the encoded protein. Two novel hemizygous variants of SLC16A2, c.1111_1112insGTCTTGT (Gly375fs*6) and c.942delA (Val315fs*28), were detected in two patients. We compared the clinical symptoms of the patients with all patients with AHDS reported in China and those reported in the literature. While both our patients presented symptoms mostly consistent with AHDS, Patient 1 had no abnormal brain structure and thyroid function, and yet showed other symptoms including lactic aciduria, conjunctival hyperemia, vomiting, laryngeal stridor, low immunoglobulin and iron levels. This study expands the mutation spectrum of AHDS and has clinical value for variant-based prenatal and postnatal screening for this condition. Doctors often have difficulty identifying AHDS by using clinical symptoms. WES can help to identify specific disorder when diagnosis cannot be made based on symptoms alone.

Bioinformatics Predicted Linear Epitopes of the Major Coat Protein of the Beet Yellows Virus for Detection of the Virus in the Cell Extract of the Infected Plant.

Beet yellows virus, which belongs to the genus Closterovirus, family Closteroviridae and has a significant negative economic impact, has proven to be challenging to detect and diagnose. To obtain antibodies against BYV, we propose an easier bioinformatics approach than the isolation and purification of the wild virus as an antigen. We used the SWISS-MODEL Workspace (Biozentrum Basel) protein 3D prediction program to discover epitopes of major coat protein p22 lying on the surface of the BYV capsid. Sequences coding these epitopes were cloned into plasmid pQE-40 (Qiagen) in frame with mouse dihydrofolate reductase gene. Fused epitopes were expressed in Escherichia coli and isolated by the Ni-NTA affinity chromatography. Murine antibodies were raised against each epitope and in a combination of both and characterized by dot-ELISA and indirect ELISA. We successively used these antibodies for diagnosis of virus disease in systemically infected Tetragonia tetragonioides. We believe the approach described above can be used for diagnostics of difficult-to-obtain and hazardous-to-health viral infections.

Detail Analysis for Energy Transfer and Pigment Assembling in C−Phycocyanin through Time−Resolved Spectroscopy and AlphaFold2

The time-resolved fluorescence spectroscopy of the C−phycocyanin (C−PC) complex from the cyanobacterium Spirulina platensis (S. platensis) hexamer with the sub-picosecond resolution was detected, and the energy transfer pathways and related transfer rates were identified through the multiexponential analysis based on Monte-Carlo method. With the purpose to construct the relationship between the functions and the structure in vivo, the three−dimension (3D) protein structure was predicted via AlphaFold2, and the arrangement of chromophores treated as the energy transfer nodes were obtained. The experiment results have been matched well with the structure prediction. This work suggests a new way to investigate structure prediction in vivo and the corresponding functions.

OSCA Genes in Liriodendron chinense: Characterization, Evolution and Response to Abiotic Stress

The hyperosmolality-gated calcium-permeable channels (OSCA) play an imperative role in the plant response to environmental stresses. Moreover, their characteristics in the ornamental woody plant Liriodendron chinense, widely dispersed in the southern region of China, is yet to be elucidated. In the present study, 399 OSCA proteins were identified from 31 plant genomes, and comparative phylogenetic analysis revealed that LchiOSCAs gene family is closely related to the Magnolia Cinnamomum kanehirae OSCAs. In L. chinense, 11 LchiOSCA genes were identified and distributed across eight chromosomes. Additionally, phylogenetic analysis of LchiOSCAs exhibited a classification into four subfamilies based on the tree arrangement, similarity in the gene structures, and conserved motif numbers and order. Gene duplication investigations were biased towards the tandem duplication events, accounting for 36% (4/11) of the LchiOSCA gene family. The interspecies collinearity analysis revealed a closer relationship between the L. chinense OSCAs and the P. trichocarpa OSCAs. Analysis in promoter regions of the LchiOSCAs showed the presence of multiple phytohormones and stress responsive elements. Specifically, the ABA-responsive elements had the greatest representation. 3D protein structures of the modeled L. chinense OSCAs exhibited a high homology with the template structures, providing a better understanding of LchiOSCAs’ functionality at the proteome level. The expression pattern analysis of LchiOSCAs based on the transcriptome data and qRT-PCR in L. chinense leaves showed differential responses to drought, cold, and heat stress at varying degrees. Specifically, LchiOSCA2 and LchiOSCA4 were highly expressed under the three abiotic stresses. This research will provide valuable resources and further the understanding of plant OSCA genes in L. chinense for agronomic breeding and bio-engineering purposes.

An Integrated Machine Learning Model To Spot Peptide Binding Pockets in 3D Protein Screening.

The prediction of peptide-protein binding sites is of utmost importance to tackle the onset of severe neurodegenerative diseases and cancer. In this work, we detail a novel machine learning model based on Linear Discriminant Analysis (LDA) demonstrating to be highly predictive in detecting the putative protein binding regions of small peptides. Starting from 439 high-quality pockets derived from peptide-protein crystallographic complexes, three sets of well-established peptide-binding regions were first selected through a Partitioning Around Medoids (PAM) clustering algorithm based on morphological and energetic 3D GRID-MIF molecular descriptors. Next, the best combination between all the putative interacting peptide pockets and related GRID-MIF scores was automatically explored by using the LDA-based protocol implemented in BioGPS. This approach proved successful to recognize the actual interacting peptide regions (that is, AUC = 0.86 and partial ROC enrichment at 5% of 0.48) from all the other pockets of the protein. Validated on two external collections sets, including 445 and 347 crystallographic peptide-protein complexes, our LDA-based model could be effective to further run peptide-protein virtual screening campaigns.

New Labeling Methods for Deep Learning Real-Valued Inter-Residue Distance Prediction.

Much of the recent success in protein structure prediction has been a result of accurate protein contact prediction-a binary classification problem. Dozens of methods, built from various types of machine learning and deep learning algorithms, have been published over the last two decades for predicting contacts. Recently, many groups, including Google DeepMind, have demonstrated that reformulating the problem as a multi-class classification problem is a more promising direction to pursue. As an alternative approach, we recently proposed real-valued distance predictions, formulating the problem as a regression problem. The nuances of protein 3D structures make this formulation appropriate, allowing predictions to reflect inter-residue distances in nature. Despite these promises, the accurate prediction of real-valued distances remains relatively unexplored; possibly due to classification being better suited to machine and deep learning algorithms. Can regression methods be designed to predict real-valued distances as precise as binary contacts? To investigate this, we propose multiple novel methods of input label engineering, which is different from feature engineering, with the goal of optimizing the distribution of distances to cater to the loss function of the deep-learning model. Since an important utility of predicted contacts or distances is to build three-dimensional models, we also tested if predicted distances can reconstruct more accurate models than contacts. Our results demonstrate, for the first time, that deep learning methods for real-valued protein distance prediction can deliver distances as precise as binary classification methods. When using an optimal distance transformation function on the standard PSICOV dataset consisting of 150 representative proteins, the precision of 'top-all' long-range contacts improves from 60.9% to 61.4% when predicting real-valued distances instead of contacts. When building three-dimensional models we observed an average TM-score increase from 0.61 to 0.72, highlighting the advantage of predicting real-valued distances.

In-silico study of potential antiviral drug compounds against EGFR kinase domain to target non-small cell lung cancer (NSCLC)

Purpose of Research: Worldwide, lung cancer is the biggest cause of cancer-related deaths. Cancer of non-small lung cells (NSCLC) is the most prevalent kind of lung cancer. Targeting NSCLC, we investigate the anticancer effect of antiviral drug compounds against the EGFR kinase domain. Scope of The Experiments: The 3D protein structure of the EGFR kinase domain (1XKK) was derived from the RCSB PDB library. First, an ADME study was conducted, followed by Lipinski's rule of five-based toxicity analysis of the compounds. After screening for ADME and toxicity, the remaining drugs were docked to the EGFR kinase domain (PDB ID: 1XKK). For docking, the Autodock Vina application was deployed. Using the application Discovery Studio 2019, the docking discovery was investigated. Results: The binding affinity of the standard drug compounds Afatinib Dimaleate, and Gemcitabine to the active site of the EGFR kinase domain was -8.9, and -8.4, respectively. In contrast, the binding affinity of our lead drug compound (Diphyllin) to the active region of the EGFR kinase domain was -10 kcal/mol, which is superior to the both selected standard drug compounds. In addition, the found chemical generates a greater number of hydrogen bonds than our chosen benchmark compounds, indicating that it is more stable. An examination of root means square fluctuation was done to appreciate the dynamic motions of the ligand-protein complex. Findings and Conclusions: Due to its capacity to suppress the activity of the target protein EGFR kinase domain, which plays a vital role in the progression of NSCLC, Diphyllin shows great potential as an anti-NSCLC medication. To validate further our promising findings based on preliminary and in-silico analysis, in-vitro and in-vivo investigations are necessary.

Characterization of the conserved regions of E1A protein from human adenovirus for reinforcement of cytotoxic T lymphocytes responses to the all genogroups causes ocular manifestation through an in silico approach.

Adenovirus species B, C, D, and E are the most common causes of ocular manifestations caused by adenoviruses. FDA-approved treatment agents for adenovirus infections are not available. Cell-mediated immunity is the major protective mechanism versus human adenoviruses (HAdVs) infection and T cells specific for peptide epitopes from nonstructural proteins can prevent adenoviral dissemination. E1A CR2 region of HAdVs Epitopes predicted for reinforcing cytotoxic T lymphocytes (CTLs) in the EKC patients. Among human adenoviruses E1 protein, four distinct E1A regions had a significantly higher level of homology than the rest of E1A protein. E1A protein inhibits IFN signal transduction. Epitope-based vaccines are designed to have flexible and simple methods to synthesize a vaccine, using an adjuvant to trigger fast immune responses. CTL epitopes were applied to create a multiepitope vaccine. Conserve region1 (CR1) and CR3 have less antigenicity compared to CR2. Additionally, CR3 in HAdV-D8 contains three toxic areas. CR4 similar to the two regions CR1 and CR3 do not show acceptable antigenic properties. Bioinformatics' tools were used to predict, refine and validate the 3D structure of the construct. Effective binding was predicted by protein-protein docking of the epitope vaccine with MHC-I molecules and revealed the safety and efficacy of the predicted vaccine construct. In silico analysis show that rising levels of cytotoxic CD8 + T cells, TH1 cells, macrophages, and neutrophils are linked to IFN-dominant TH1-type responses, which are detected in putative immune individuals. Combined with 3D protein modeling, this study predicted the epitopes of E1A CR2 protein in HAdVs.

Molecular Regulation and Evolution of Redox Homeostasis in Photosynthetic Machinery.

The recent advances in plant biology have significantly improved our understanding of reactive oxygen species (ROS) as signaling molecules in the redox regulation of complex cellular processes. In plants, free radicals and non-radicals are prevalent intra- and inter-cellular ROS, catalyzing complex metabolic processes such as photosynthesis. Photosynthesis homeostasis is maintained by thiol-based systems and antioxidative enzymes, which belong to some of the evolutionarily conserved protein families. The molecular and biological functions of redox regulation in photosynthesis are usually to balance the electron transport chain, photosystem II, photosystem I, mesophyll and bundle sheath signaling, and photo-protection regulating plant growth and productivity. Here, we review the recent progress of ROS signaling in photosynthesis. We present a comprehensive comparative bioinformatic analysis of redox regulation in evolutionary distinct photosynthetic cells. Gene expression, phylogenies, sequence alignments, and 3D protein structures in representative algal and plant species revealed conserved key features including functional domains catalyzing oxidation and reduction reactions. We then discuss the antioxidant-related ROS signaling and important pathways for achieving homeostasis of photosynthesis. Finally, we highlight the importance of plant responses to stress cues and genetic manipulation of disturbed redox status for balanced and enhanced photosynthetic efficiency and plant productivity.

Directed Compartmentalization of Mg2+ and Carbonic Anhydrase by Designed Three-Dimensional Protein Crystalline Frameworks for Efficient Conversion of CO2 into Nanoparticles

CO2 mineralization by colloidal nanoclusters of MgO or CaO represents one effective way to reduce its levels in the atmosphere. However, a high temperature (>200 °C) is required for this process. Herein, we found that the Mg2+-mediated Thermotoga maritima ferritin (TmFtn) nanocage has the ability to convert CO2 (3.128 mmol per gram of protein) into MgCO3 nanoparticles in an aqueous solution with Mg2+ as starting materials at room temperature, but the rate of such bioconversion is relatively slow. To improve the CO2 conversion efficiency, we constructed a two-compartment system based on three-dimensional (3D) protein crystalline frameworks by using dimeric TmFtn as building blocks, which consists of two types of compartments, namely, small ferritin cavity and large interglobular space. By using the system, directed compartmentalization of Mg2+ and carbonic anhydrase (CA) according to their difference in size can be realized using a one-pot method. Consequently, the CO2 conversion value of the CA-encapsulated protein crystalline frameworks reaches as high as around 8.485 mmol per gram of protein, while the conversion rate increases by 2 to 3 times. Notably, its reversible disassembly and reassembly property endows the frameworks with satisfied recyclability, allowing them to be suitable for practical applications.

Tick-borne encephalitis virus capsid protein induces translational shutoff as revealed by its structural–biological analysis

Tick-borne encephalitis virus (TBEV) is the most medically relevant tick-transmitted Flavivirus in Eurasia, targeting the host central nervous system and frequently causing severe encephalitis. The primary function of its capsid protein (TBEVC) is to recruit the viral RNA and form a nucleocapsid. Additional functionality of Flavivirus capsid proteins has been documented, but further investigation is needed for TBEVC. Here, we show the first capsid protein 3D structure of a member of the tick-borne flaviviruses group. The structure of monomeric Δ16-TBEVC was determined using high-resolution multidimensional NMR spectroscopy. Based on natural invitro TBEVC homodimerization, the dimeric interfaces were identified by hydrogen deuterium exchange mass spectrometry (MS). Although the assembly of flaviviruses occurs in endoplasmic reticulum-derived vesicles, we observed that TBEVC protein also accumulated in the nuclei and nucleoli of infected cells. In addition, the predicted bipartite nuclear localization sequence in the TBEVC C-terminal part was confirmed experimentally, and we described the interface between TBEVC bipartite nuclear localization sequence and import adapter protein importin-alpha using X-ray crystallography. Furthermore, our coimmunoprecipitation coupled with MS identification revealed 214 interaction partners of TBEVC, including viral envelope and nonstructural NS5 proteins and a wide variety of host proteins involved mainly in rRNA processing and translation initiation. Metabolic labeling experiments further confirmed that TBEVC and other flaviviral capsid proteins are able to induce translational shutoff and decrease of 18S rRNA. These findings may substantially help to design a targeted therapy against TBEV.

Analysis and comparison of electron radiation damage assessments in Cryo-EM by single particle analysis and micro-crystal electron diffraction.

Electron radiation damage to macromolecules is an inevitable resolution limit factor in all major structural determination applications using cryo-electron microscopy (cryo-EM). Single particle analysis (SPA) and micro-crystal electron diffraction (MicroED) have been employed to assess radiation damage with a variety of protein complexes. Although radiation induced sidechain density loss and resolution decay were observed by both methods, the minimum dose of electron irradiation reducing high-resolution limit reported by SPA is more than ten folds higher than measured by MicroED using the conventional dose concept, and there is a gap between the attained resolutions assessed by these two methods. We compared and analyzed these two approaches side-by-side in detail from several aspects to identify some crucial determinants and to explain this discrepancy. Probability of a high energy electron being inelastically scattered by a macromolecule is proportional to number of layers of the molecules in its transmission path. As a result, the same electron dose could induce much more site-specific damage to macromolecules in 3D protein crystal than single particle samples. Major differences in data collection and processing scheme are the key factors to different levels of sensitivity to radiation damage at high resolution between the two methods. High resolution electron diffraction in MicroED dataset is very sensitive to global damage to 3D protein crystals with low dose accumulation, and its intensity attenuation rates at atomic resolution shell could be applied for estimating ratio of damaged and total selected single particles for SPA. More in-depth systematically radiation damage assessments using SPA and MicroED will benefit all applications of cryo-EM, especially cellular structure analysis by tomography.

Phylogenetic analysis and characterization of arsenic (As) transforming bacterial marker proteins following isolation of As-tolerant indigenous bacteria.

Marker proteins play a significant role in bacterial arsenic (As) transformation. Phylogenetic analysis and three-dimensional (3D) characteristics of As transforming bacterial marker proteins guide the evolutionary origin and As transforming potential of the species. Indeed, As-tolerant bacteria also show a significant level of As transformation. Hence, characterization of As transforming bacterial marker proteins, isolation of As transforming bacteria, and proper integration of the findings may guide to elucidate how bacteria transform As. Therefore, phylogenetic analysis and 3D characterization of As transforming bacterial marker protein following isolation of potential indigenous As-tolerant indigenous bacteria were done to explore the mechanism of bacterial As transformation. Phylogenetic analysis of ten As transforming marker proteins (arsA, arsB, arsC, arsD, arsR, aioA, arrA, aioB,acr1, andacr3) in 20 potential bacterial genomes (except19 for the acr3) were studied. Some bacterial genomes featured up to five marker proteins, and therefore, 3D characteristics of the marker proteins were analyzed in those genomes having three-to-five marker proteins. In phylogeny, species in close clades represent their phylogenetic resemblances and may have similar functions. P. aeruginosa, E. coli, and K. pneumonia were found to be more effective due to having the highest number (five) of marker proteins. In 3D protein modeling, most of the marker proteins were found to be active. Among 19 indigenous bacterial isolates, multiple isolates showed tolerance up to 50mM As(III) and 250mM As(V), which may potentially transform a significant quantities of As. Hence, integration of the results of phylogenetic analysis, 3D protein characteristics, and As tolerance in the bacterial isolates could guide to explore the mechanism of how bacteria transform As at cellular and molecular levels.

A Novel Missense WFS1 Variant: Expanding the Mutational Spectrum Associated with Nonsyndromic Low-Frequency Sensorineural Hearing Loss.

Background Nonsyndromic low-frequency sensorineural hearing loss (LFSNHL) is an uncommon form of hearing loss (HL) that typically affects frequencies at 2000 Hz and below. Heterozygous variants in the WFS1 gene at the DFNA6/14/38 locus are considered a common cause of LFSNHL. To date, 34 different pathogenic genetic variants have been reported to cause LFSNHL with seven of these variants identified in the Chinese population. However, limited reports are available on the association between WFS1 gene and LFSNHL. Here, we report a five-generation Chinese family with an autosomal dominant inheritance pattern of postlingual and progressive LFSNHL. Methods Routine clinical and audiological examinations were performed on 16 affected and 7 healthy members in this family. The targeted next-generation sequencing of 127 known deafness genes was performed to identify variants in affected individuals. Sanger sequencing were further employed to confirm the pathogenic variant identified. Results A novel heterozygous pathogenic genetic variant c.2530G > T (p.Ala844Ser) was identified in the WFS1 gene in all patients of this family. The mutated Ala residue is evolutionarily conserved and cosegregated with HL. The variant was predicted to be deleterious by MutationTaster, PolyPhen-2, LRT, and Fathmm software. Conservation analysis and 3D protein structure model indicated that the variant caused a structural change in the protein. Conclusions Our present study identifies a novel heterozygous WFS1 variant associated with LFSNHL in a Chinese family.

Molecular description of melatonin receptor 1A gene in Iraqi buffalo

The water buffalo has a seasonal reproductive pattern with reduced sexual activity during the longer photoperiod. The goal of this study was to identify the single nucleotide polymorphism (SNP) of melatonin receptor 1A (MTNR1A) gene in Iraqi buffalo cows and 3D structure of its protein and phylogeny with other sequences around the world. The 824 bp fragment of exon II of the MTNR1 A gene was amplified from 190 buffalo cows (4-5 years old) genomic DNA belonging to local breeders in Al-Chibayish Marshes, Southern Iraq. Amplified PCR products underwent custom sequencing at the two ends (5′ and 3′ ends). Five separate polymorphism sites, the 1st included 52 animals with 19 mutations (12 missense), the 2nd included 39 animals with 18 mutations (11 missense), the 3rd included 35 animals with 18 mutations (12 missense), the 4th included 32 animals with 18 mutations (12 missense) and the 5th included 32 animals with 14 mutations (8 missense). These polymorphic sites with accession numbers LC565046, LC565047, LC565709, LC565710 and LC565711 respectively were registered in gene bank. The phylogenetic tree reveals that in some of the Iraqi buffalo, the sequences of MTNR1A gene has identical to the Italian buffalo (GU817415), and the Brazilian buffalo (JN689386). Data revealed a marked difference in the fifth polymorphism sites' 3D protein structure because of the mutations. In conclusion, as a result of mutations, the gene MTNR1A in Iraqi buffalo has polymorphisms; these polymorphisms may be linked to gene function, Therefore, further studies are needed to connect the polymorphisms of this gene with the productive and reproductive traits.

Homozygous Missense Variant in the N-Terminal Region of ANK3 Gene Is Associated with Developmental Delay, Seizures, Speech Abnormality, and Aggressive Behavior

Introduction: Intellectual disability (ID) is a lifelong disability that affects an individual’s learning capacity and adaptive behavior. Such individuals depend on their families for day-to-day survival and pose a significant challenge to the healthcare system, especially in developing countries. ID is a heterogeneous condition, and genetic studies are essential to unravel the underlying cellular pathway for brain development and functioning. Methods: Here we studied a female index patient, born to a consanguineous Pakistani couple, showing clinical symptoms of ID, ataxia, hypotonia, developmental delay, seizures, speech abnormality, and aggressive behavior. Whole exome sequencing (WES) coupled with Sanger sequencing was performed for molecular diagnosis. Further, 3D protein modeling was performed to see the effect of variant on protein structure. Results: WES identified a novel homozygous missense variant (c.178T>C; p.Tyr60His) in the ANK3 gene. In silico analysis and 3-dimensional (3D) protein modeling supports the deleterious impact of this variant on the encoding protein, which compromises the protein’s overall structure and function. Conclusion: Our finding supports the clinical and genetic diversity of the ANK3 gene as a plausible candidate gene for ID syndrome. Intelligence is a complex polygenic human trait, and understanding molecular and biological pathways involved in learning and memory can solve the complex puzzle of how cognition develops. Intellectual disability (ID) is defined as a deficit in an individual’s learning and adaptive behavior at an early age of onset [American Psychiatric Association, 2013]. It is one of the major medical, and cognitive disorders with a prevalence of 1–3% in the population worldwide [Leonard and Wen, 2002]. ID often exists with other disabling mental conditions such as autism, attention deficit hyperactivity disorder, epilepsy, schizophrenia, bipolar disorder, or depression. Almost half of the cases appear to have a genetic explanation that ranges from cytogenetically visible abnormalities to monogenic defects [Flint, 2001; Ropers, 2010; Tucker-Drob et al., 2013]. Intellectual disability is a genetically heterogeneous condition, and more than 700 genes have been identified to cause ID alone or as a part of the syndrome. Research in X-linked ID has identified more than 100 disease-causing genes on the X chromosome that play a role in cognition; however, research into autosomal causes of ID is still ongoing [Vissers et al., 2016].

In English

For various adsorbents, especially nanoporous, there is an applicability problem of the Brunauer-Emmett-Teller (BET) method using nitrogen as a probe adsorbate. Therefore, the nitrogen adsorption (a(p/p0)) isotherms in several pressure ranges of the BET method at p/p0 = 0.05–0.3, 0.06–0.22, and narrower are analyzed for a large set (about 200 samples) of essentially different adsorbents such as fumed oxides (individual, binary and ternary, initial and modified), porous silicas, activated carbons and porous polymers. Graphitized carbon black ENVI–Carb composed of nonporous nanoparticles aggregated into microparticles is used as a standard adsorbent characterized by the standard area occupied by nitrogen molecule σm(N2) = 0.162 nm2. For initial nanooxides composed of nonporous nanoparticles, the standard value of σm = 0.162 nm2 results in the overestimation of the SBET values by ca. 10 % because of non-parallel-to-surface orientation of slightly polarized N2 molecules interacting with polar surface functionalities (e.g., various hydroxyls). For nanooxides modified by low- and high-molecular (linear, 2D and 3D polymers and proteins) compounds, the overestimation of SBET at σm = 0.162 nm2 could reach 30 %, as well as for some activated carbons. For adsorbents possessing nanopores (at half-width x or radius R &lt; 1 nm) and narrow mesopores (1 nm &lt; R &lt; 3 nm), an overlap of monolayer and multilayer sorption (giving apparent underestimation of SBET at σm = 0.162 nm2) and non-parallel-to-surface orientation of the N2 molecules (causing σm lower than 0.162 nm2) could lead to various location of the normalized nitrogen adsorption isotherms (in the BET range) with respect to that for ENVI–Carb. It could be characterized by positive or negative values of the BET constant cBET. Two main criteria showing the inapplicability or applicability of the BET method (with nitrogen as a probe) related to the cBET values and a course of reduced adsorption a´(1- p/p0) vs. p/p0 in the BET range could not be in agreement for adsorbents, which are not pure nanoporous, but they are in agreement for pure nanoporous or meso/macroporous adsorbents

Oceanimonas sp. BPMS22-derived protein protease inhibitor induces anti-leishmanial immune responses through macrophage M2 to M1 repolarization

The present study aimed to validate the potential of a novel serine protein protease inhibitor (PPI), purified from marine Oceanimonas sp. BPMS22, induced M2 to M1 repolarization of the macrophages to treat visceral leishmaniasis (VL). Peptide mass fingerprint of the purified trypsin digested PPI peptide was obtained using matrix-assisted laser desorption ionization-time of flight combined with tandem mass spectrometry (MALDI-TOF MS/MS) and the sequence was used to construct a 3D protein model by homology modelling. The IC50 of PPI were 25.28 ± 1.675 μg/mL and 0.415 ± 0.015 μg/mL against promastigotes and intracellular amastigotes, respectively, indicating the host-directed therapy using PPI. The PPI enhanced the effector molecule i.e., nitric oxide (NO), and dampened the arginase activity in a dose-dependent manner. In vitro studies revealed that the BPMS22-derived PPI significantly (p < 0.05) decreased the mRNA expressions of M2 markers (FIZZ-1, YM-1, CD206, Arg-1) and increased the mRNA expressions of M1 markers (iNOS, IL-1β, IL-12) in rIL-4 + rIL-10 induced M2 macrophages. Interestingly, the BPMS22-derived PPI also significantly (p < 0.05) decreased the FIZZ-1, YM-1, CD206, and Arg-1; significantly (p < 0.05) increased iNOS, IL-12, and IFN-γ mRNA expression in L. donovani -infected murine macrophages, alongside the decreased parasite load in it. Hence, PPI has the potential to repolarize the cytokines (rIL-4 + rIL-10) pre-stimulated and L. donovani-infected M2 macrophages to M1 phenotype in vitro. A decrease in parasite burden after treatment with PPI indicated the acceleration of the parasite killing by enhancing the macrophage effector functions. Further, in vivo PPI treatment reduced hepatic and splenic Leishman donovan units (LDU) up to 93.34 % and 87.63 %, respectively. This was followed by a surge in pro-inflammatory cytokines and dampening anti-inflammatory cytokines (p < 0.01), which exhibited anti-VL immunity. These observations might open new perspectives on PPI in macrophage repolarization to treat VL.