IL-10-Dependent Crosstalk between Murine Marginal Zone B Cells, Macrophages, and CD8α+ Dendritic Cells Promotes Listeria monocytogenes Infection.

Type 1 CD8α+ conventional dendritic cells (cDC1s) are required for CD8+ T cell priming but, paradoxically, promote splenic Listeria monocytogenes infection. Using mice with impaired cDC2 function, we ruled out a role for cDC2s in this process and instead discovered an interleukin-10 (IL-10)-dependent cellular crosstalk in the marginal zone (MZ) that promoted bacterial infection. Mice lacking the guanine nucleotide exchange factor DOCK8 or CD19 lost IL-10-producing MZ B cells and were resistant to Listeria. IL-10 increased intracellular Listeria in cDC1s indirectly by reducing inducible nitric oxide synthase expression early after infection and increasing intracellular Listeria in MZ metallophilic macrophages (MMMs). These MMMs trans-infected cDC1s, which, in turn, transported Listeria into the white pulp to prime CD8+ T cells. However, this also facilitated bacterial expansion. Therefore, IL-10-mediated crosstalk between B cells, macrophages, and cDC1s in the MZ promotes both Listeria infection and CD8+ T cell activation.

Regulatory T-cell dysfunction and cutaneous exposure to Staphylococcus aureus underlie eczema in DOCK8 deficiency.

BACKGROUND: Dedicator of cytokinesis 8 (DOCK8)-deficient patients have severe eczema, elevated IgE, and eosinophilia, features of atopic dermatitis (AD). OBJECTIVE: We sought to understand the mechanisms of eczema in DOCK8 deficiency. METHODS: Skin biopsy samples were characterized by histology, immunofluorescence microscopy, and gene expression. Skin barrier function was measured by transepidermal water loss. Allergic skin inflammation was elicited in mice by epicutaneous sensitization with ovalbumin (OVA) or cutaneous application of Staphylococcus aureus. RESULTS: Skin lesions of DOCK8-deficient patients exhibited type 2 inflammation, and the patients' skin was colonized by Saureus, as in AD. Unlike in AD, DOCK8-deficient patients had a reduced FOXP3:CD4 ratio in their skin lesions, and their skin barrier function was intrinsically intact. Dock8-/- mice exhibited reduced numbers of cutaneous T regulatory (Treg) cells and a normal skin barrier. Dock8-/- and mice with an inducible Dock8 deletion in Treg cells exhibited increased allergic skin inflammation after epicutaneous sensitization with OVA. DOCK8 was shown to be important for Treg cell stability at sites of allergic inflammation and for the generation, survival, and suppressive activity of inducible Treg cells. Adoptive transfer of wild-type, but not DOCK8-deficient, OVA-specific, inducible Treg cells suppressed allergic inflammation in OVA-sensitized skin of Dock8-/- mice. These mice developed severe allergic skin inflammation and elevated serum IgE levels after topical exposure to Saureus. Both were attenuated after adoptive transfer of WT but not DOCK8-deficient Treg cells. CONCLUSION: Treg cell dysfunction increases susceptibility to allergic skin inflammation in DOCK8 deficiency and synergizes with cutaneous exposure to Saureus to drive eczema in DOCK8 deficiency.

Hematopoietic cell transplantation for DOCK8 deficiency: Results from a prospective clinical trial.

BACKGROUND: DOCK8 deficiency is a primary immunodeficiency in which allogeneic hematopoietic cell transplantation (HCT) represents the only known cure. We tested the ability of a busulfan-based regimen to achieve reliable engraftment and high levels of donor chimerism with acceptable toxicity in a prospective clinical trial in DOCK8 deficiency. OBJECTIVES: To both evaluate the ability of HCT to reverse the clinical phenotype and to correct the immunologic abnormalities by 1 year post HCT. METHODS: We conducted a prospective HCT trial for recipients with DOCK8 deficiency. Subjects were recruited from October 5, 2010, to December 30, 2022. Donor sources included fully matched related and unrelated donors and haploidentical donors. The reduced toxicity, myeloablative conditioning regimen contained no serotherapy. Graft-versus-host disease (GVHD) prophylaxis included either a calcineurin inhibitor with methotrexate or post-HCT cyclophosphamide (PT/Cy) followed by tacrolimus and mycophenolate mofetil. The trial was later amended to study PT/Cy in all patients. (Pilot Study of Reduced-Intensity Hematopoietic Stem Cell Transplant of DOCK8 [NCT01176006].) RESULTS: Thirty-six subjects, both children and adults (median age 16.4 years), underwent HCT for DOCK8 deficiency. Most patients, 33 of 36 (92%), achieved full (≥98%) donor chimerism in whole blood as early as day +30. With a median potential follow-up of 7.4 years, 29 (80.6%) were alive with no evidence of new DOCK8 deficiency-related complications. PT/Cy was effective in reducing the risk of acute GVHD in patients who had received matched unrelated donor and haploidentical transplants, but it was associated with transient delays in immune-reconstitution and hemorrhagic cystitis. CONCLUSIONS: A busulfan-based HCT regimen using PT/Cy for GVHD prophylaxis and a broad range of donor types and hematopoietic cell sources were well tolerated, leading to the reversal of the clinical immunophenotype.

DOCK2 Deficiency Causes Defects in Antiviral T-Cell Responses and Impaired Control of Herpes Simplex Virus Infection.

The expanding number of rare immunodeficiency syndromes offers an opportunity to understand key genes that support immune defense against infectious diseases. However, analysis of these in patients is complicated by their treatments and comorbid infections, requiring the use of mouse models for detailed investigations. We developed a mouse model of DOCK2 immunodeficiency and herein demonstrate that these mice have delayed clearance of herpes simplex virus type 1 (HSV-1) infections. We also uncovered a critical, cell-intrinsic role of DOCK2 in the priming of antiviral CD8+ T cells and in particular their initial expansion, despite apparently normal early activation of these cells. When this defect was overcome by priming in vitro, DOCK2-deficient CD8+ T cells were surprisingly protective against HSV-1 disease, albeit not as effectively as wild-type cells. These results shed light on a cellular deficiency that is likely to impact antiviral immunity in DOCK2-deficient patients.

Reconstitution of norovirus-specific T cell responses following hematopoietic stem cell transplantation in patients with inborn errors of immunity and chronic norovirus infection.

BACKGROUND: Chronic norovirus infection (CNI) causes significant morbidity in immunocompromised patients. No effective prevention or treatment currently exists. METHODS: Two patients with inborn errors of immunity, X- linked severe combined immunodeficiency (X-SCID) and DOCK8 deficiency, were followed longitudinally for clinical course, immune reconstitution, norovirus-specific T cell (NST) response, B cell reconstitution, and norovirus-specific antibody production. Samples were obtained in the peri-hematopoietic stem cell transplant setting (HSCT) before and after CNI clearance. The norovirus strain causing CNI was followed longitudinally for norovirus stool viral loads and sequencing. RESULTS: The noroviruses were identified as GII.4 Sydney[P4 New Orleans] in one patient and GII.17[P17] in the other. An exacerbation of diarrhea post-HSCT in the patient with X-SCID was consistent with norovirus infection but not with graft-vs-host-disease on pathologic samples. Both patients recovered polyfunctional NSTs in the CD4 and CD8 T cell compartments which recognized multiple norovirus structural and non-structural viral antigens. T cell responses were minimal during active CNI but detectable after resolution. Mapping of norovirus-specific T cell responses between the patient with DOCK8 and his matched sibling donor were nearly identical. B cell reconstitution or new endogenous antibody production for IgA or IgG were not observed. CONCLUSION: This report is the first to demonstrate reconstitution of norovirus-specific T cell immunity after HSCT closely temporally aligned with clearance of CNI suggesting that cellular immunity is sufficient for norovirus clearance.

The two major splice variants of scavenger receptor BI differ by their interactions with lipoproteins and cellular localization in endothelial cells.

The scavenger receptor SR-BI facilitates the transport of both HDL and LDL through endothelial cells. Its two splice variants, SR-BIvar1 and SR-BIvar2, differ in their carboxyterminal domains. Only the one of SR-BIvar1 contains the putative binding sites for the adapter proteins PDZK1 and DOCK4, which limit the cell surface abundance and internalization of the receptor. To investigate the cellular localization of the SR-BI variants and their interaction with lipoproteins in endothelial cells, EA.hy926 cells were stably transfected with vectors encoding untagged, GFP- or mCherry-tagged constructs of the two SR-BI variants. Additionally, the cells were transfected with shRNAs against PDZK1 or DOCK4. Microscopy investigation showed that SR-BIvar1 was predominantly localized on the cell surface together with clathrin whereas SR-BIvar2 was absent from the cell surface but retrieved in endosomes and lysosomes. Accordingly, only SR-BIvar1 increased lipoprotein binding to endothelial while HDL and LDL uptake were enhanced by both variants. Silencing of PDZK1 or DOCK3 only reduced HDL association in SR-BIvar2 overexpressing cells while LDL association was reduced both in wild type and SR-BIvar2 overexpressing cells. In conclusion, either SR-BI variant facilitates the uptake of HDL and LDL into endothelial cells, however by different mechanisms and trafficking routes. This dual role may explain why the loss of DOCK4 or PDZK1 differently affects the uptake of HDL and LDL in different endothelial cells.

Diverse gut pathogens exploit the host engulfment pathway via a conserved mechanism.

Macrophages clear infections by engulfing and digesting pathogens within phagolysosomes. Pathogens escape this fate by engaging in a molecular arms race; they use WxxxE motif-containing "effector" proteins to subvert the host cells they invade and seek refuge within protective vacuoles. Here, we define the host component of the molecular arms race as an evolutionarily conserved polar "hot spot" on the PH domain of ELMO1 (Engulfment and Cell Motility protein 1), which is targeted by diverse WxxxE effectors. Using homology modeling and site-directed mutagenesis, we show that a lysine triad within the "patch" directly binds all WxxxE effectors tested: SifA (Salmonella), IpgB1 and IpgB2 (Shigella), and Map (enteropathogenic Escherichia coli). Using an integrated SifA-host protein-protein interaction network, in silico network perturbation, and functional studies, we show that the major consequences of preventing SifA-ELMO1 interaction are reduced Rac1 activity and microbial invasion. That multiple effectors of diverse structure, function, and sequence bind the same hot spot on ELMO1 suggests that the WxxxE effector(s)-ELMO1 interface is a convergence point of intrusion detection and/or host vulnerability. We conclude that the interface may represent the fault line in coevolved molecular adaptations between pathogens and the host, and its disruption may serve as a therapeutic strategy.

Autism Spectrum Disorder- and/or Intellectual Disability-Associated Semaphorin-5A Exploits the Mechanism by Which Dock5 Signalosome Molecules Control Cell Shape.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that includes autism, Asperger's syndrome, and pervasive developmental disorder. Individuals with ASD may exhibit difficulties in social interactions, communication challenges, repetitive behaviors, and restricted interests. While genetic mutations in individuals with ASD can either activate or inactivate the activities of the gene product, impacting neuronal morphogenesis and causing symptoms, the underlying mechanism remains to be fully established. Herein, for the first time, we report that genetically conserved Rac1 guanine-nucleotide exchange factor (GEF) Dock5 signalosome molecules control process elongation in the N1E-115 cell line, a model line capable of achieving neuronal morphological changes. The increased elongation phenotypes observed in ASD and intellectual disability (ID)-associated Semaphorin-5A (Sema5A) Arg676-to-Cys [p.R676C] were also mediated by Dock5 signalosome molecules. Indeed, knockdown of Dock5 using clustered regularly interspaced short palindromic repeat (CRISPR)/CasRx-based guide(g)RNA specifically recovered the mutated Sema5A-induced increase in process elongation in cells. Knockdown of Elmo2, an adaptor molecule of Dock5, also exhibited similar recovery. Comparable results were obtained when transfecting the interaction region of Dock5 with Elmo2. The activation of c-Jun N-terminal kinase (JNK), one of the primary signal transduction molecules underlying process elongation, was ameliorated by either their knockdown or transfection. These results suggest that the Dock5 signalosome comprises abnormal signaling involved in the process elongation induced by ASD- and ID-associated Sema5A. These molecules could be added to the list of potential therapeutic target molecules for abnormal neuronal morphogenesis in ASD at the molecular and cellular levels.

DOCK8 deficiency due to a deep intronic variant in two kindreds with hyper-IgE syndrome.

Dedicator of cytokinesis 8 (DOCK8) deficiency underlies the majority of cases of patients with autosomal recessive form of the hyper-immunoglobulin E syndrome (HIES). Most DOCK8 mutations involve deletions and splice junction mutations that abrogate protein expression. However, a few patients whose presentation is reminiscent of DOCK8 deficiency have no identifiable mutations. Using Whole Exome Sequencing (WES), we identified a deep intronic homozygous DOCK8 variant located in intron 36 (c.4626 + 76 A > G) in two unrelated patients with features of HIES that resulted in an in-frame 75 base pair intronic sequence insertion in DOCK8 cDNA, resulting in a premature stop codon (p.S1542ins6Ter). This variant resulted in variable decrease in DOCK8 expression that was associated with impaired T cell receptor-triggered actin polymerization, decreased IL-6-induced STAT3 phosphorylation, reduced expression of the Th17 cell markers CCR6 and IL-17, and higher frequencies of GATA3+ T cells indicative of Th2 skewing. Our approach extends the reach of WES in identifying disease-related intronic variants. It highlights the role of non-coding mutations in immunodeficiency disorders, including DOCK8 deficiency, and emphasizes the need to explore these mutations in unexplained inborn errors of immunity.

Tandem C2 domains mediate dynamic organelle targeting of a DOCK family guanine nucleotide exchange factor.

Multicellular organisms use dedicator of cytokinesis (DOCK) family guanine nucleotide exchange factors (GEFs) to activate Rac/Rho-of-plants small GTPases and coordinate cell shape change. In developing tissues, DOCK signals integrate cell-cell interactions with cytoskeleton remodeling, and the GEFs cluster reversibly at specific organelle surfaces to orchestrate cytoskeletal reorganization. The domain organizations among DOCK orthologs are diverse, and the mechanisms of localization control are poorly understood. Here, we use combinations of transgene complementation and live-cell imaging assays to uncover an evolutionarily conserved and essential localization determinant in the DOCK-GEF named SPIKE1. The SPIKE1-DHR3 domain is sufficient for organelle association in vivo, and displays a complicated lipid-binding selectivity for both phospholipid head groups and fatty acid chain saturation. SPIKE1-DHR3 is predicted to adopt a C2-domain structure and functions as part of a tandem C2 array that enables reversible clustering at the cell apex. This work provides mechanistic insight into how DOCK GEFs sense compositional and biophysical membrane properties at the interface of two organelle systems.

A novel hemizygous CD40L mutation of X-linked hyper IgM syndromes and compound heterozygous DOCK8 mutations of hyper IgE syndromes in two Chinese families.

X-linked hyper-immunoglobulin M (X-HIGM) syndrome and autosomal recessive hyper-immunoglobulin E syndrome (HIES) are rare inborn errors of immunity characterized by recurrent infections due to immune system impairment. In this study, we identified a novel hemizygous CD40 ligand (CD40L) mutation and compound heterozygous dedicator of cytokinesis-8 (DOCK8) mutations in two Han Chinese families with X-HIGM and HIES, respectively. We aimed to investigate the association between their genotypes and phenotypes. Genomic DNA was extracted from peripheral blood samples obtained from the families. Whole exome sequencing and Sanger sequencing were performed to identify and verify pathogenic variants in the two families. Clinical analyses of the probands were also performed. A novel hemizygous mutation of CD40L in exon 2 (c.257delA) was identified in the first proband, resulting in the substitution of glycine with glutamic acid at codon 86 of the protein. This leads to premature termination of translation at downstream codon 9 (p.E86Gfs*9). Sanger sequencing confirmed that the variant was inherited from the mother. The second proband carried two novel compound heterozygous mutations in DOCK8: one at exon 14 (c.1546C > G) inherited from the father, and the other at intron 41 (c.5355 + 6C > T; splicing) inherited from the mother. This study enhances our understanding of the pathogenetic mutation spectrum of CD40L and DOCK8 genes, facilitating the prenatal diagnosis of X-HIGM and HIES and enabling timely treatment of patients.

Fragment and torsion biasing algorithms for construction of small organic molecules in proteins using DOCK.

The computational construction of small organic molecules (de novo design), directly in a protein binding site, is an effective means for generating novel ligands tailored to fit the pocket environment. In this work, we present two new methods, which aim to improve de novo design outcomes using (1) biasing algorithms to prioritize selection and/or acceptance of fragments and torsions during growth, and (2) parallel-based clustering and pruning algorithms to remove duplicate molecules as candidate fragment are added. Large-scale testing encompassing thousands of simulations were employed to interrogate the methods in terms of multiple metrics which include numbers of duplicate molecules generated, pairwise-similarity, focused library reconstruction rates, fragment and torsion frequencies, fragment and torsion rank scores, interaction energy and drug-likeness scores, and 3D pose comparisons. The biasing algorithms, particularly those that include fragment and torsion components simultaneously, led to molecules that more closely mimicked the distributions of fragments and torsions found in drug-like libraries. The new parallel-based clustering and pruning algorithms, compared with the existing serial approach, also led to larger ensembles comprised of topologically unique molecules with much greater efficiency by removing redundant growth paths.

DOCK1 regulates the malignant biological behavior of endometrial cancer through c-Raf/ERK pathway.

BACKGROUND: The effect of DOCK1 gene on the biological behavior of endometrial carcinoma cells and its related pathway has not been reported. METHODS: The immunohistochemical method and western blot were utilized to analyze DOCK1 protein expression in endometrial tissues and cells, respectively. CCK-8, BrdU, transwell and flow cytometry were performed to analyze the effect of DOCK1 expression changes on the viability, proliferation, invasion, migration and apoptosis of endometrial cancer cells, respectively. The effects of DOCK1 gene on Bcl-2, MMP9, Ezrin, E-cadherin and c-RAF/ERK1/2 signaling pathway were evaluated by western blot. The xenograft models were constructed to analyze the effect of DOCK1 in vivo. RESULTS: DOCK1 expression was increased in endometrial cancer tissues and cells compared with those in normal adjacent tissues and cells. DOCK1 knockout could inhibit the malignant biological behavior of endometrial cancer cells, while DOCK1 overexpression played the opposite effect. The expression of E-cadherin was upregulated and those of MMP9, Ezrin, Bcl-2, p-c-RAF (S338) and p-ERK1/2 (T202/Y204) were downregulated after DOCK1 knockout, while DOCK1 overexpression played the opposite effect. Additionally, Raf inhibitor LY3009120 reversed the function of DOCK1 on malignant biological behavior. In vivo experiment results showed that the growth and weight of transplanted tumors in nude mice were inhibited after DOCK1 knockout. The changes of E-cadherin, MMP9, Ezrin and Bcl-2 expressions in the transplanted tumors were consistent with those in vitro. CONCLUSION: DOCK1 could enhance the malignant biological behavior of endometrial cancer cells, which might be through c-RAF/ERK1/2 signaling pathways in vitro and in vivo.

DOCK3 regulates normal skeletal muscle regeneration and glucose metabolism.

DOCK (dedicator of cytokinesis) is an 11-member family of typical guanine nucleotide exchange factors (GEFs) expressed in the brain, spinal cord, and skeletal muscle. Several DOCK proteins have been implicated in maintaining several myogenic processes such as fusion. We previously identified DOCK3 as being strongly upregulated in Duchenne muscular dystrophy (DMD), specifically in the skeletal muscles of DMD patients and dystrophic mice. Dock3 ubiquitous KO mice on the dystrophin-deficient background exacerbated skeletal muscle and cardiac phenotypes. We generated Dock3 conditional skeletal muscle knockout mice (Dock3 mKO) to characterize the role of DOCK3 protein exclusively in the adult muscle lineage. Dock3 mKO mice presented with significant hyperglycemia and increased fat mass, indicating a metabolic role in the maintenance of skeletal muscle health. Dock3 mKO mice had impaired muscle architecture, reduced locomotor activity, impaired myofiber regeneration, and metabolic dysfunction. We identified a novel DOCK3 interaction with SORBS1 through the C-terminal domain of DOCK3 that may account for its metabolic dysregulation. Together, these findings demonstrate an essential role for DOCK3 in skeletal muscle independent of DOCK3 function in neuronal lineages.

Clinical, immunological, and genetic description of a Mexican cohort of patients with DOCK8 deficiency.

PURPOSE: We aimed to describe the clinical, immunological, and genetic features of patients with DOCK8 deficiency (DOCK8-Def) in a tertiary care center for children. METHODS: Retrospective chart review of patients' clinical, immunological, and genetic characteristics with DOCK8-Def. Genetic analysis was performed with targeted- or whole-exome sequencing; we also assessed DOCK8 protein expression and a lymphoproliferation assay and analyzed survival by the Kaplan-Meier method. RESULTS: We described 11 patients from 8 unrelated kindreds. The median age at symptoms' onset was 10 months (range 1-54 months). The median follow-up time was 53.4 months (4.8-118.8). All patients presented eczema and recurrent sinopulmonary and cutaneous infections. Besides those symptoms, the most frequent manifestations were bronchiectases (8/11), food allergies (6/11), and severe infections (6/11). Infrequent characteristics were detection of CMV in bronchial lavage, C. parvum-driven sclerosing cholangitis, Takayasu vasculitis, neurological syndromes, pulmonary tuberculosis, and lymphomatoid granulomatosis. CONCLUSION: DOCK8-Def has a broad spectrum of manifestations, including allergy, autoimmunity, inflammation, infection, and cancer. The hallmark of this inborn error of immunity is IEI-associated eczema with eosinophilia and increased IgE. Here, we report six new mutations causing human DOCK8 deficiency and symptoms previously unrecognized to occur in DOCK8-Def. Therefore, an early diagnosis of DOCK8-Def is essential to facilitate an adequate treatment such as HSCT.

High DOCK1 expression identifies a distinct prognostic subgroup of pediatric acute myeloid leukemia: Results of the Japanese Pediatric Leukemia/Lymphoma Study Group AML-05 trial.

BACKGROUND: The molecular pathogenesis of acute myeloid leukemia (AML) was dramatically clarified over the latest two decades. Several important molecular markers were discovered in patients with AML that have helped to improve the risk stratification. However, developing new treatment strategies for relapsed/refractory acute myeloid leukemia (AML) is crucial due to its poor prognosis. PROCEDURE: To overcome this difficulty, we performed an assay for transposase-accessible chromatin with sequencing (ATAC-seq) in 10 AML patients with various gene alterations. ATAC-seq is based on direct in vitro sequencing adaptor transposition into native chromatin, and is a rapid and sensitive method for integrative epigenomic analysis. ATAC-seq analysis revealed increased accessibility of the DOCK1 gene in patients with AML harboring poor prognostic factors. Following the ATAC-seq results, quantitative reverse transcription polymerase chain reaction was used to measure DOCK1 gene expression levels in 369 pediatric patients with de novo AML. RESULTS: High DOCK1 expression was detected in 132 (37%) patients. The overall survival (OS) and event-free survival (EFS) among patients with high DOCK1 expression were significantly worse than those patients with low DOCK1 expression (3-year EFS: 34% vs. 60%, p < .001 and 3-year OS: 60% vs. 80%, p < .001). To investigate the significance of high DOCK1 gene expression, we transduced DOCK1 into MOLM14 cells, and revealed that cytarabine in combination with DOCK1 inhibitor reduced the viability of these leukemic cells. CONCLUSIONS: Our results indicate that a DOCK1 inhibitor might reinforce the effects of cytarabine and other anti-cancer agents in patients with AML with high DOCK1 expression.

Structure-based pose prediction: Non-cognate docking extended to macrocyclic ligands.

So-called "cross-docking" is the prediction of the bound configuration of small-molecule ligands that differ from the cognate ligand of a protein co-crystal structure. This is a much more challenging problem than re-docking the cognate ligand, particularly when the new ligand is structurally dissimilar from prior known ones. We have updated the previously introduced PINC ("PINC Is Not Cognate") benchmark which introduced the idea of temporal segregation to measure cross-docking performance. The temporal set encompasses 846 future ligands for ten targets based on information from the earliest 25% of X-ray co-crystal structures known for each target. Here, we extend the benchmark to include thirteen targets where the bound poses of 128 macrocyclic ligands are to be predicted based on knowledge from structures of bound non-macrocyclic ligands. Performance was roughly equivalent for both the temporally-split non-macrocyclic ligand set and the macrocycle prediction set. Using standard and fully automatic protocols for the Surflex-Dock and ForceGen methods, across the combined 974 non-macrocyclic and macrocyclic ligands, the top-scoring pose family was correct 68% of the time, with the top-two pose families achieving a 79% success rate. Correct poses among all those predicted were identified 92% of the time. These success rates far exceeded those observed for the alternative methods AutoDock Vina and Gnina on both sets.

Phytoconstituents of Artemisia Annua as potential inhibitors of SARS CoV2 main protease: an in silico study.

BACKGROUND: In November 2019, the world faced a pandemic called SARS-CoV-2, which became a major threat to humans and continues to be. To overcome this, many plants were explored to find a cure. METHODS: Therefore, this research was planned to screen out the active constituents from Artemisia annua that can work against the viral main protease Mpro as this non-structural protein is responsible for the cleavage of replicating enzymes of the virus. Twenty-five biocompounds belonging to different classes namely alpha-pinene, beta-pinene, carvone, myrtenol, quinic acid, caffeic acid, quercetin, rutin, apigenin, chrysoplenetin, arteannunin b, artemisinin, scopoletin, scoparone, artemisinic acid, deoxyartemisnin, artemetin, casticin, sitogluside, beta-sitosterol, dihydroartemisinin, scopolin, artemether, artemotil, artesunate were selected. Virtual screening of these ligands was carried out against drug target Mpro by CB dock. RESULTS: Quercetin, rutin, casticin, chrysoplenetin, apigenin, artemetin, artesunate, sopolin and sito-gluside were found as hit compounds. Further, ADMET screening was conducted which represented Chrysoplenetin as a lead compound. Azithromycin was used as a standard drug. The interactions were studied by PyMol and visualized in LigPlot. Furthermore, the RMSD graph shows fluctuations at various points at the start of simulation in Top1 (Azithromycin) complex system due to structural changes in the helix-coil-helix and beta-turn-beta changes at specific points resulting in increased RMSD with a time frame of 50 ns. But this change remains stable after the extension of simulation time intervals till 100 ns. On other side, the Top2 complex system remains highly stable throughout the time scale. No such structural dynamics were observed bu the ligand attached to the active site residues binds strongly. CONCLUSION: This study facilitates researchers to develop and discover more effective and specific therapeutic agents against SARS-CoV-2 and other viral infections. Finally, chrysoplenetin was identified as a more potent drug candidate to act against the viral main protease, which in the future can be helpful.

Enhanced binding interaction and antibacterial inhibition for nanometal oxide particles activated with Aloe Vulgarize through one-pot ultrasonication techniques.

The interaction of green zinc oxide nanoparticles (ZnO NPs) with bacterial strains are still scarcely reported. This work was conducted to study the green-one-pot-synthesized ZnO NPs from the Aloe Vulgarize (AV) leaf peel extract assisted with different sonication techniques followed by the physicochemical, biological activities and molecular docking studies. The NPs structure was analyzed using FTIR, UV-vis and EDX. The morphology, particle size and crystallinity of ZnO NPs were identified using FESEM and XRD. It was found that the formed flower-like structure with sharp edge and fine size of particulates in ZnO NPs/AV could enhance the bacterial inhibition. The minimum inhibitory concentration (MIC) for all the tested bacterial strains is at 3.125 µg/ml and the bacterial growth curve are dependent on the ZnO NPs dosage. The results of disc diffusion revealed that the ZnO NPs/AV possess better antibacterial effect with bigger ZOI due to the presence of AV active ingredient. The molecular docking between active ingredients of AV in the NPs with the protein of IFCM and 1MWU revealed that low binding energy (Ebind = -6.56 kcal/mol and -8.99 kcal/mol, respectively) attributes to the excessive hydrogen bond from AV that highly influenced their interaction with the amino acid of the selected proteins. Finally, the cytotoxicity test on the biosynthesized ZnO NPs with concentration below 20 µg/ml are found nontoxic on the HDF cell. Overall, ZnO NPs/20 % AV (probe sonication) is considered as the best synthesis option due to its efficient one-pot method, short sonication time but own the best antibacterial effect.

Genetic change investigation in DOCK1 gene in an Iranian family with sign and symptoms of temporomandibular joint disorder (TMD).

OBJECTIVES: Temporomandibular joint disorder (TMD) is a complex condition with pain and dysfunction in the temporomandibular joint and related muscles. Scientific evidence indicates both genetic and environmental factors play a crucial role in TMD. In this study, we aimed to discover the genetic changes in individuals from 4 generations of an Iranian family with signs and symptoms of TMD and malocclusion Class III. MATERIALS AND METHODS: Whole Exome Sequencing (WES) was performed in 4 patients (IV-8, IV-9, V-4, and V-6) with TMD according to (DC/TMD), along with skeletal Class III malocclusion. Then, PCR sequencing was performed on 23 family members to confirm the WES. RESULTS: In the present study, WES results analysis detected 6 heterozygous non-synonymous Single Nucleotide Variants (SNVs) in 5 genes, including CRLF3, DNAH17, DOCK1, SEPT9, and VWDE. A heterozygous variant, c.2012T > A (p.F671Y), in Exon 20 of the DOCK1 (NM_001290223.2) gene was identified. Then, this variant was investigated in 19 other members of the same family. PCR-Sequencing results showed that 7/19 had heterozygous TA genotype, all of whom were accompanied by malocclusion and TMD symptoms and 12/19 individuals had homozygous TT genotype, 9 of whom had no temporomandibular joint problems or malocclusion. The remaining 3 showed mild TMD clinical symptoms. The 5 other non-synonymous SNVs of CRLF3, DNAH17, SEPT9, and VWDE were not considered plausible candidates for TMD. CONCLUSIONS: The present study identified a heterozygous nonsynonymous c.2012T > A (p.F671Y) variant of the DOCK1 gene is significantly associated with skeletal class III malocclusion, TMD, and its severity in affected individuals in the Iranian pedigree. CLINICAL RELEVANCE: The role of genetic factors in the development of TMD has been described. The present study identified a nonsynonymous variant of the DOCK1 gene as a candidate for TMD and skeletal class III malocclusion in affected individuals in the Iranian pedigree.