Findings from 18F-FBPA and 18F-FDG PET/CT were compared with pathological diagnoses in patients with malignant tumors or benign lesions.The goal of the study was to evaluate the diagnostic ability of 18F-FDG and 18F-FBPA PET/CT. Tumor uptake of FDG or FBPA was quantified using the maximum standardized uptake value (SUVmax). The final diagnosis was confirmed by cytopathology or histopathological findings of the specimen after biopsy or surgery. A ROC curve was constructed from the SUVmax values of each PET image, and the area under the curve (AUC) and cutoff values were calculated.A total of 82 patients  with various types of malignant tumors or benign lesions, such as inflammation and granulomas, were examined by 18F-FDG PET/CT did not differ significantly for malignant tumors and benign lesions (10.9 ± 6.3 vs. 9.1 ± 2.7 P = 0.62), whereas SUVmax for 18F-FBPA PET/CT was significantly higher for malignant tumors . The best SUVmax cutoffs for distinguishing malignant tumors from benign lesions were 11.16 for 18F-FDG PET/CT  and 3.24 for 18F-FBPA PET/CT . ROC analysis showed significantly different AUC values for 18F-FDG and 18F-FBPA PET/CT .The SUVmax for 18F-FBPA PET/CT showed superior diagnostic ability over 18F-FDG PET/CT in differential diagnosis of malignant tumors and benign lesions. The results of this study suggest that 18F-FBPA PET/CT diagnosis may reduce false-positive 18F-FDG PET/CT diagnoses. Patients were recruited for the study from March 2020 through March 2022 with agreement from oncologists and after determination of eligibility.18F-FDG PET/CT findings suspicious for primary and/or metastatic lesions and/or benign lesions such as inflammation or granuloma with abnormal 18F-FDG uptake; (ii) suspected or newly diagnosed or previously treated malignancies and/or benign lesions; (iii) age ≥ 20 years old; (iv) agreement to undergo biopsy; (v) provision of informed consent according to the guidelines of the Clinical Research Ethics Committee. The exclusion criteria were: (i) pregnant women or those who wish to become pregnant; and (ii) treatment started before PET/CT was performed.The inclusion criteria were: (i) 18F-FBPA PET/CT and 18F-FDG PET/CT studies from March 2020 through March 2022. These patients included five with recurrence after treatment who underwent repeated PET examinations. The final definitive diagnosis was confirmed by cytopathological or histopathological findings of the specimen after biopsy or resection.A total of 82 patients  with various types of malignant tumors and benign lesions were examined in 87 18F-FDG was produced using the standard method in our laboratory. 18F-FBPA was synthesized as previously described  using a YMC-Pack ODS-A column  eluted with 0.1% acetic acid at a flow rate of 10 mL/min. The radiochemical purity was > 95% for 18F-FDG and 18F-FBPA, and the final product was sterile and pyrogen-free.18F-FDG and 18F-FBPA PET/CT scans was 5 days . The 18F-FDG PET/CT scan was performed first. Patients fasted for at least 4 h before 18F-FDG PET/CT scans to ensure a normal glucose level in the peripheral blood. 18F-FBPA PET/CT was also performed in patients who had fasted for at least 4 h to align the conditions with 18F-FDG PET/CT. The dose of intravenously injected 18F-FDG or 18F-FBPA was calculated based on body weight (3.7–5.0 MBq/kg for 18F-FDG and 18F-FBPA)  and reconstructed using the ordered subset expectation maximization algorithm (two iterations and 18 subsets) using CT data for attenuation correction. The reconstructed images were then co-registered and displayed. PET images were analyzed qualitatively  and semi-quantitatively using a volumetric volume of interest (VOI) placed over the target lesion and tailored to the extent of each lesion. The maximum tumor standardized uptake value (SUVmax) for each VOI was automatically generated by the tomography software. Standard vital signs  were checked between 18F-FDG and 18F-FBPA on PET images and quantification of the SUV were performed using AW Volume Share software . VOIs were delineated on axial 3-D images. The SUV was defined as regional radioactivity divided by injected radioactivity normalized to body weight. 18F-FDG and 18F-FBPA uptake were evaluated using SUVmax at 1 h after injection , negative predictive value (NPV) and accuracy of 18F-FDG and 18F-FBPA PET/CT were calculated and compared to evaluate the diagnostic efficacy.Statistical analyses were performed using R software (ver. 4.0.3) and Excel. Results for P values < 0.05 were considered significant in all analyses.Receiver-operating characteristic (ROC) curves were generated from the SUVmax of the target lesion for each PET method, and the area under the curve (AUC) and the best SUVmax cutoff for differentiating malignant tumors and benign lesions were obtained to give optimal sensitivity and specificity. The AUCs of the two markers for malignant tumors and benign lesions were compared statistically to evaluate the diagnostic performance of each PET method. Two-tailed 18F-FDG and 18F-FBPA PET/CT well. There were no signs of any drug-related pharmacologic effects or physiological responses. All observed vital signs  remained within normal limits during and after 18F-FBPA PET/CT. None of the patients reported any abnormal symptoms.All patients tolerated n = 22) and recurrent/metastatic tumors (n = 55) did not differ significantly for 18F-FDG  or 18F-FBPA   on 18F-FBPA PET/CT occurred for external auditory canal cancer  was found in well-differentiated lung cancer, mantle cell lymphoma, olfactory neuroblastoma, ovarian cancer, chronic lymphocytic leukemia, pancreatic cancer, gallbladder cancer and benign lesions.Patient numbers were insufficient to compare the SUVs of primary tumors vs. recurrent or metastatic tumors for individual cancers. The details are summarized in Table 18F-FDG PET/CT did not differ significantly for malignant tumors and benign lesions . In contrast, SUVmax on 18F-FBPA PET/CT was significantly higher for malignant tumors compared to benign lesions  . The respective SUVmax cutoffs were 11.16 and 3.24. Representative cases are shown in Figs. In a lesion-based analysis, the sensitivity, specificity, PPV, NPV and accuracy of the diagnosis were 98.7, 22.2, 87.4, 66.7 and 86.4% for 18F-FDG is a substrate for GLUT1 and is taken up by normal tissues such as the brain, resulting in high background values. Therefore, 18F-FDG PET has limited utility in diagnosis of malignant brain tumors , indicating that differentiation is difficult, as also found in previous reports , 18F-FAMT (methyltyrosine) and 18F-FBPA. 11C-MET is taken up by multiple amino acid transporters in normal cells since it is a natural amino acid used for protein synthesis in the cell, which leads to high background values in the liver, pancreas, and salivary gland tissue on PET , but others showed low 18F-FBPA accumulation  and could not be visualized . Conversely, a small number of cases of granulomatous inflammatory disease in the brainstem and cervical lymph node areas gave false-positive results. In such cases, it is important to evaluate the results in conjunction with other examinations and the clinical course. The expression level of LATI was not examined in this study, but is likely to vary among types of tumor, and this may also have caused differences in accumulation of 18F-FBPA. Well-differentiated lung cancers with frosted appearances on CT and 18F-FDG PET have low accumulation and cannot be visualized. This suggests that tumor size, cell density, and the limited spatial resolution of PET can also affect the diagnostic performance.BPA used in boron neutron capture therapy (BNCT) is transported by LAT1, LAT2 and another transporter of neutral amino acids, amino acid transporter B0 (ATB0). BPA is not specific for LAT1, but is mainly transported into cancer cells via LAT1  is a type II membrane-bound glycoprotein belonging to the dipeptidylpeptidase 4 family and is highly expressed in many epithelial cancer-associated fibroblasts. It is characterized by a strong desmoplastic response, and the association between FAP overexpression and a poor cancer prognosis has led to development of FAP-specific inhibitors (FAPIs) (Kratochwil et al. 18F-FBPA PET/CT was superior to 18F-FDG PET/CT for differential diagnosis of malignant tumors and benign lesions. There are some limitations due to low 18F-FBPA accumulation in certain tumors, but the additional information obtained from 18F-FBPA PET/CT can reduce false positives from 18F-FDG PET/CT in tumor diagnosis.The results of this study showed that


Hepatitis-hydropericardium syndrome (HHS) induced by fowl adenovirus serotype 4 (FAdV-4) has caused huge economic losses to poultry industries. The key genes responsible for different virulence of FAdV-4 strains are not fully elucidated. Previous studies indicated that hexon of pathogenic FAdV-4 has a conserved arginine (R) at position 188, and a conserved isoleucine (I) is present at this position in reported nonpathogenic FAdV-4. Recently, it was reported that R188 of hexon is the determinant site for pathogenicity of the emerging Chinese FAdV-4 strain. However, the role of hexon amino acid 188 (aa188) has not been examined in the nonpathogenic FAdV-4 strain. In this study, three recombinant FAdV-4 viruses, H/H/R188I, O/O/I188R, and H/O/I188R, were constructed by mutating hexon aa188 of FAdV-4 pathogenic strain CH/HNJZ/2015 (H) and nonpathogenic strain ON1 (O), and pathogenicity was assessed in specific-pathogen-free (SPF) chickens. Consistent with previous findings, H/O/I188R exhibited pathogenicity similar to that of CH/HNJZ/2015, yet H/H/R188I induced no mortality. Unexpectedly, all chickens infected with O/O/I188R survived. Postmortem examination of O/O/I188R-infected chickens showed typical lesions of inclusion body hepatitis rather than HHS. Expression of proinflammatory cytokines in CH/HNJZ/2015- and H/O/I188R-infected chickens was significantly higher than that in H/H/R188I-, ON1-, and O/O/I188R-infected chickens. Analysis of predicted hexon protein structures indicated that aa188 mutation leads to conformational changes in the L1 loop of HNJZ-hexon but not in ON1-hexon. In summary, the present study demonstrated that the role of hexon aa188 in the virulence of FAdV-4 varies between different strains. Induction of HHS requires factors aside from hexon aa188 in the emerging Chinese FAdV-4 strain.IMPORTANCE HHS induced by FAdV-4 has caused huge economic losses to the poultry industry. The key determinants for the different virulence of FAdV-4 have not been fully elucidated. Here, we investigated the role of hexon aa188 in FAdV-4 strains with different virulence and showed that the role of hexon aa188 varies in FAdV-4 strains with different genetic contents. The hexon R188 may be the key amino acid for causing inclusion body hepatitis by the pathogenic FAdV-4 strain, and induction of HHS by FAdV-4 may need other viral cofactors. Moreover, the hexon R188I mutation greatly affected the expression of proinflammatory cytokines induced by the pathogenic strain CH/HNJZ/2015, but no significant difference was observed between the nonpathogenic strain ON1 and ON1 with hexon I188R mutation. We found that hexon aa188 mutation induced conformational changes to hexon protein in CH/HNJZ/2015 but not in ON1, which might be the underlying reason for the changing virulence. Aviadenovirus and family Adenoviridae are categorized into five species designated FAdV-A to E and 12 serotypes, including FAdV-1 to 7, 8a, 8b, and 9 to 11. Hepatitis-hydropericardium syndrome (HHS) induced by FAdV-4 of FAdV-C and inclusion body hepatitis (IBH) induced by FAdV-8 of FAdV-E and FAdV-11 of FAdV-D are ubiquitous worldwide, causing significant economic losses to the world poultry industry (1–Fowl adenoviruses (FAdV) of the genus ndustry 14. Up to ndustry 1–7. IBH iFAdV-4 is a nonenveloped, double-stranded DNA virus with an approximately 43- to 45-kb genome encoding 11 structural proteins and 32 nonstructural proteins . IdentifIn the present study, the role of hexon aa188 was examined in both the pathogenic and nonpathogenic strains of FAdV-4. Hexon aa188 of the pathogenic strain and that of the nonpathogenic strain were substituted with one another by the FAdV-4 reverse genetic platform established previously . The vir6, 105.3, 105.7, 104.9, and 105.7 median tissue culture infective dose (TCID50)/100 μL, respectively. The viral titers and in vitro replication capacity of the 3rd passage of the recombinant viruses were similar to those of the parental viruses on LMH cells , including severe depression, fluffy feathers, and diarrhea with green-yellowish excrement. Chickens in the H/H/R188I group showed milder depression and slightly reduced feed consumption at 48 hpi but recovered completely at 4 dpi, indicating that the hexon R188I mutation greatly reduced the virulence of HNJZ. Interestingly, chickens in the O/O/I188R group became depressed at 48 hpi with reduced feed intake and gradually recovered to normal at 6 dpi. Chickens in the ON1 and control groups showed no symptoms throughout the experiment. The mortality of chickens infected with HNJZ or H/O/I188R reached 100% at 70 hpi or 90% at 72 hpi, respectively. All chickens in the ON1, H/H/R188I, O/O/I188R, and control groups survived the experiment . HoweverThe viral copy numbers in the heart, liver, spleen, kidney, bursa of Fabricius, and proventriculus of the dead chickens and chickens euthanized at 7 dpi were determined. Only background levels of viral DNA were detected in the H/H/R188I, O/O/I188R, and control groups at 7 dpi. The viral loads detected in chickens in the H/H/R188I group were significantly lower than those in the HNJZ group, indicating that the hexon R188I mutation significantly suppressed the viral replication capacity of HNJZ in all tested viral targeting organs. However, the amount of viral DNA detected in the organs of chickens in the O/O/I188R group was at the same level as that in the ON1 group, suggesting that the hexon I188R mutation had almost no effect on the viral replication capacity of ON1 in the viral targeting organs . The effAt necropsy, typical characteristics for HHS were observed in chickens infected with HNJZ and H/O/I188R, including accumulation of the clear, amber-colored liquid in the pericardial sac and swollen livers showing multifocal areas of necrosis . HistopaTo investigate the effect of hexon aa188 mutation on the expression of proinflammatory cytokines in chickens, total RNA was extracted from the liver tissues of chickens infected with HNJZ, ON1, H/H/R188I, O/O/I188R, and H/O/I188R for quantitative RT-PCR analysis. The results indicated that the expression levels of interleukin 1 beta (IL-1β), IL-8, and IL-18 were extremely significantly higher in the livers of HNJZ- and H/O/I188R-infected chickens than in those of the ON1-, H/H/R188I-, and O/O/I188R-infected chickens. Only the expression of IL-1β in the livers of O/O/I188R-infected chickens showed significant upregulation compared to that in those of the ON1-infected chickens, and the other two cytokines showed no significant changes .To elucidate the reason that the role of hexon aa188 mutation varied in FAdV-4 with different virulence, we predicted the 3D protein structure of hexon with or without aa188 mutations by Phyre2 software and visualized it in PyMol . The hypKU991797) and HNJZ (GenBank accession no. KU558760) is 100%. This prompted us to determine the role of hexon aa188 in FAdV-4 strains with different virulence and explore whether induction of HHS requires factors aside from the hexon aa188 in the pathogenic FAdV-4 strain.HHS caused by FAdV-4 has been spread worldwide after the first report in 1987 in Pakistan –23. To e–in vivo.To elucidate the role of hexon aa188 in FAdV-4 strains with different virulence, recombinant FAdV-4 viruses H/H/R188I, O/O/I188R, and H/O/I188R were constructed by using the FAdV-4 reverse genetic technique established previously . The hexPathogenic FAdV-4 infection induces significant upregulations of proinflammatory cytokines in the primary viral targeting organs such as the liver, spleen, and bursa of Fabricius –27. The –Hexon is one of the major structural proteins constituting the capsid of adenoviruses and playing an important role in the genome organization of adenoviruses . Most ad35–In summary, the present study showed that the role of hexon aa188 mutation varies in FAdV-4 strains with different virulence, and the induction of HHS by FAdV-4 might need a certain hexon conformation and participation of other viral cofactors.KU558760), was isolated from chickens suffering from HHS in Henan province, China as described previously (GU188428) was kindly provided by Éva Nagy from the Department of Pathobiology, University of Guelph, Guelph, Canada  cells  cultured in DME/F12 medium  supplemented with 10% fetal bone serum  at 37°C with 5% CO2.The pathogenic FAdV-4 strain CH/HNJZ/2015, denoted as HNJZ  in E. coli GBred-gyrA462 as described previously  overnight at 37°C and purified by ethanol precipitation before transfection to LMH cells. LMH cells were seeded in T25 flasks a day before transfection. The transfection mixture was carried out with 5 μg of the linearized infectious clones and Lipofectamine 3000 according to the manufacturer’s instruction . The cells were harvested when obvious cytopathic effects occurred. The harvested viruses were centrifuged at 10,000 rpm for 1 min, and the supernatant was aliquoted and stored at −80°C. The viral DNA was extracted using TIANamp genomic DNA extraction kit  for sequencing identification. The successfully rescued recombinant viruses were named H/H/R188I, O/O/I188R, and H/O/I188R, respectively.The constructed infectious clones p15A-cm-H/H/R188I, p15A-cm-O/O/I188R, and p15A-cm-H/O/I188R were linearized with in vitro replication capacity of the 3rd passage of recombinant viruses were determined on LMH cells. LMH cell monolayers (105 cells/well) were seeded in 96-well plates and inoculated with the parental viruses HNJZ and ON1 and the recombinant viruses H/H/R188I, O/O/I188R, and H/O/I188R, and each was serially diluted by 10-fold from 10−1 to 10−10 with 8 replicates. The cytopathic effects of LMH cells were observed under a light microscope at 96 hpi. The median tissue culture infective dose (TCID50) of the recombinant viruses was determined by the Reed-Muench method. The in vitro replication capacity of recombinant viruses was determined by the growth curve measurement. LMH cell monolayers (2 × 106 cells/well) in 6-well plates were inoculated with HNJZ, ON1, H/H/R188I, O/O/I188R, or H/O/I188R at a multiplicity of infection (MOI) of 0.001. The viruses were harvested at 12, 24, 36, 48, 60, 72, 84, and 96 hpi for TCID50 measurement as described above.The viral titer and 5 TCID50 of the corresponding virus and observed for 7 days. Tissue samples of the heart, liver, spleen, kidney, bursa of Fabricius, and proventriculus were collected from the dead chickens during the experiment and chickens euthanized at 7 dpi for viral load analysis, histopathological examination, and study of cytokine expression.The pathogenicity of the rescued recombinant viruses was assessed in comparison with the parental viruses HNJZ and ON1 on 21-day-old SPF chickens . The animal study was conducted under the approval of the Animal Care and Use Committee of Henan Agricultural University (HNND2022030814). Sixty chickens were randomly divided into five infection groups, including H/H/R188I, O/O/I188R, H/O/I188R, HNJZ, and ON1, and one control group, with 10 chickens in each group. Chickens were separately housed in negative pressure isolators with sufficient supplies of food, water, and light. Chickens in the infection groups were inoculated intramuscularly with 0.2 mL of 2 × 10To determine the viral loads of viral targeting organs, total DNA was extracted from 100 mg of tissue samples of the heart, liver, spleen, kidney, bursa of Fabricius, and proventriculus with TIANamp genomic DNA extraction kit . A quantitative real-time PCR standard curve method for viral load measurement was established previously using the ORF14 gene of FAdV-4 as an indicator for the presence of HNJZ, ON1, and corresponding derivatives . Each reTo examine the microscopic lesions of viral targeting organs, the heart, liver, spleen, kidney, bursa of Fabricius, and proventriculus samples were placed in 10% formalin for fixation and embedded in paraffin. Sections of paraffin-embedded tissue samples were stained with hematoxylin and eosin following standard procedures. The stained samples were examined under light microscopy for the presence of lesions.2O). The reaction conditions were set as 1 cycle at 95°C for 30 s and then 40 cycles at 95°C for 10 s and 60°C for 30 s. GAPDH  was used as the internal reference gene for the relative mRNA expression level analysis by the 2−ΔΔCt method.To examine the expression levels of proinflammatory cytokines IL-1β, IL-8, and IL-18, the total RNA from 100 mg of liver tissue of chickens in each group was extracted using RNAsimple total RNA kit . Reverse transcription was performed with 2 μg RNA in a 20 μL reaction system using FastKing RT kit  according to the manufacturer’s instruction. The primers for detecting IL-1β, IL-8, and IL-18 were reported in previous studies , 33. Thehttp://www.pymol.org/) for structural comparison.The 3-dimensional structures of hexon of parental viruses HNJZ and ON1 and recombinant viruses H/H/R188I, O/O/I188R, and H/O/I188R were predicted with Phyre2 software and visualized in PyMol (t test. The normality of data was confirmed by the D’Agostino-Pearson normality test. Parametric and unpaired t test was utilized to compare the viral loads and cytokine expressions between different groups. A P value smaller than 0.05 was considered to be significantly different.The statistical analysis was performed in GraphPad Prism 7 using the Student’s


Medulloblastoma (MB) is the most common malignant brain tumor in children. It is classified into core molecular subgroups (wingless activated (WNT), sonic hedgehog activated (SHH), Group 3 (G3), and Group 4 (G4)). In this study, we analyzed the tumor-infiltrating immune cells and cytokine profiles of 70 MB patients in Taiwan using transcriptome data. In parallel, immune cell composition in tumors from the SickKids cohort dataset was also analyzed to confirm the findings. The clinical cohort data showed the WNT and G4 MB patients had lower recurrence rates and better 5-year relapse-free survival (RFP) compared with the SHH and G3 MB patients, among the four subgroups of MB. We found tumor-infiltrating B cells (TIL-Bs) enriched in the G4 subgroups in the Taiwanese MB patients and the SickKids cohort dataset. In the G4 subgroups, the patients with a high level of TIL-Bs had better 5-year overall survival. Mast cells presented in G4 MB tumors were positively correlated with TIL-Bs. Higher levels of CXCL13, IL-36γ, and CCL27 were found compared to other subgroups or normal brains. These three cytokines, B cells and mast cells contributed to the unique immune microenvironment in G4 MB tumors. Therefore, B-cell enrichment is a G4-subgroup-specific immune signature and the presence of B cells may be an indicator of a better prognosis in G4 MB patients. MB with myogenic and/or melanotic differentiation is very rare ,2. Medules 3–17) ,7,8. Thees 3–17) ,10.G4 MB makes up the largest population of MB patients and commonly occurs in children and adolescents (ages 3–19). The overall survival of G4 MB in children is better than G3 MB . In our The composition of the tumor microenvironment (TME) has been proven to be highly associated with prognosis and affects the incidence of refractory and recurrent disease in MB patients. Due to the small sample size and lack of molecular subgroup information, information on immune and stromal cells in the MB TME is still insufficient. Molecular subgrouping results indicate a correlation between M1 or M2 types of tumor-associated macrophage (TAM) recruitment and survival. CD163-positive M2 macrophages have been found in SHH MB, but high M1 macrophage recruitment has worse overall survival (OS) and progression-free survival (PFS) in SHH MB . Transcr+ T cells, cytotoxic CD8+ T cells, and γδ-T cells have been associated with clinical benefit and favorable prognosis in the tumor microenvironment [+ T cells [+ and CD8+ T cells to secrete interferon-γ, resulting in immediate cytotoxicity to tumor cells [+ B cells) infiltrated with CD20+ B cells and CD4+ and CD8+ T cells in tumors improved survival of the patients by 65% at 10 years in ovarian cancer [Recently, tumor-infiltrating lymphocytes (TIL), especially CD4ironment ,16,17. H T cells , and theor cells ,19; meanor cells . Furthern cancer . TIL-Bs n cancer ,23. Thus+ IL-10+ or CD19+ CD24hiCD38hi) increased the immune regulatory protein of PD-L1 and CTLA4 to promote the regulatory T (Treg) cells, myeloid-derived suppressor cells (MDSC), and tumor-associated macrophages (TAMs) [+ T cells into Treg cells via TGF-β secretion [On the other hand, some of the literature also shows that B cells are harmful to cancer prognosis. Adoptive transfer with B cells or anti-serum from K14-HPV16 mice into K-14-HPV16/Rag1−/− mice promoted epithelial carcinogenesis . B cell s (TAMs) . Furtherecretion ,31. Therecretion .Although the G4 MB had high tumor metastasis at first diagnosis, the mortality rate of G4 MB was lower than the SHH group or G3 subgroups. Information about the composition of the tumor microenvironment and tumor-infiltrating immune cells is still unclear. In this study, we studied 70 patients with MB in Taiwan to analyze the tumor microenvironment and tumor-infiltrating immune cells through gene expression data. We also evaluated the SickKids cohort dataset to confirm our findings. We characterized the immune signature of MB patients and investigated the role of TIL-Bs in the G4 tumor. The unique immune signature of B cell enrichment in G4 tumors and the association with mast cells were also explored.n = 34, 48.6%), desmoplastic/nodular MB , MB with extensive nodularity , large cell/anaplastic (LCA) MB , and MB with melanotic myogenic differentiation . Tumor recurrence occurred in 27 (38.6%) cases. In a median follow-up period of 5.1 years , the 5-year overall survival (OS) and relapse-free survival (RFS) of the whole cohort series were 74.1% and 63.0%, respectively. Among the four subgroups of MB, the WNT and G4 MB patients had lower recurrence rates and better 5-year relapse-free survival (RFS) compared with the SHH and G3 MB patients , but high levels of Tregs were also seen (+ T subsets (CD8+ Tcm and CD8+ Tm), Th1, and NKT cells compared to WNT and G4 tumors (p = 0.018) in MB VI/VIII tumors had higher infiltration than other subgroups , membrane-bound receptors and ligands , and granule products (histamine and proteases). These interactions can promote B cell proliferation, survival, and class-switch . However T cells ,46. In o T cells .+ and some TIL-CD8+ T cells are major CXCL13 producers in tumors. CXCL13 potentially promotes TLS formation and is correlated with B cell infiltration and germinal center maturation at the tumor site [+, NK, and γδ-T cells [+ B cells within TLS in tumors, indicating that local IL-36γ supports TLS formation within the TME [CXCL13, IL36γ, and CCL27 expression in the G4 tumor was higher than in the normal brain and other MB subgroups . CXCL13,mor site . In addimor site . CXCL13-mor site . IL-36γ mor site . Several-T cells . Moreove the TME . Higher  the TME . CCL27  production via the activation of STAT3 [IL-22 and IL-12B expression in G4 tumors was higher than in the SHH tumors . IL-22, of STAT3 . It has of STAT3 . IL-12B of STAT3 . IL-12B of STAT3 ,62,63,64of STAT3 . Therefo+ T cells and neutrophils compared to other subgroup tumors [+ T cells and neutrophils were similarly found in the G4 subgroup compared to the SHH and G3 sub-groups  and Taipei Medical University Hospital (TMUH) between 1989–2019. This cohort comprised 64 primary tumors, 5 first recurrence, and 1 metastasis. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The samples were fully encoded and used under a protocol approved by the Institutional Review Board of Human Subjects Research Ethics Committee of the Taipei Medical University Hospital and Chang Gung Memorial Hospital, Taiwan .Clinical data and tumor images were retrospectively retrieved and reviewed from medical records and reports. Histological variants were classified according to the WHO 2007 classification . The datRNA-Seq was performed in this cohort of 70 MB cases as described in a previous study . Brieflyp value of less than 0.05 was considered as statistically significant.Immune cell deconvolution was estimated with RNA-Seq profiling by xCell . The scoIllumina Infinium MethylationEPIC array was performed for 66 MB cases in this cohort series as described in a previous study . Raw datn = 216) and probes (n = 3211) from a previous study [The similarity network fusion (SNF) method was performed in the cohort series with a combination of gene expression and DNA methylation data. Subtype clustering was performed based on the top 1% of the most differentially expressed common genes  which were previously reported was applied in this cohort [2 transition to the classifier with adjustment for frozen derivatives and patient gender. The most differential 10,000 probes were implemented to calculate RF score between 0 and 1 with a threshold of ≥0.9 for non-WNT/non-SHH subtype prediction.To predict subtypes of non-WNT/non-SHH MBs, a web-based random forest (RF) classifier of MB G3/4 subgroups , CD20 , CD27 , and CD138  and secondary antibodies, and visualized with diaminobenzydine .p-value of less than 0.05 was considered as statistically significant.Overall survival (OS) analysis was calculated by the date of first tumor surgery (diagnosis date), last follow-up, and death. Survival analysis based on the scores of various cell types was performed using the Kaplan–Meier method by using survminer package in R environment. The differences in survival were assessed using the log-rank test. The association between categorized variables was determined by Kruskal–Wallis test. A In this study, we characterized TIL-B and the unique immune microenvironment in G4 MB tumors. It was ascertained that the enrichment of B and mast cells contributes to the G4 subgroup-specific tumor signature. High expression levels of B cells in tumors increased 5-year overall survival in G4 MB patients. Therefore, the presence of B cells may be an indicator of a better prognosis in G4 MB patients.


Due to structural similarities of flortaucipir with some monoamine oxidase A (MAO-A) inhibitors, this study aimed to evaluate flortaucipir binding to MAO-A and MAO-B and any potential impact on PET interpretation.[18F]Flortaucipir autoradiography was performed on frozen human brain tissue slices, and PET imaging was conducted in rats. Dissociation constants were determined by saturation binding, association and dissociation rates were measured by kinetic binding experiments, and IC50 values were determined by competition binding.[18F]flortaucipir binding was observed on tau NFT-rich Alzheimer’s disease tissue and not control tissue. In vivo PET experiments in rats revealed no evidence of [18F]flortaucipir binding to MAO-A; pre-treatment with MAO inhibitor pargyline did not impact uptake or wash-out of [18F]flortaucipir. [18F]Flortaucipir bound with low nanomolar affinity to human MAO-A in a microsomal preparation in vitro but with a fast dissociation rate relative to MAO-A ligand fluoroethyl-harmol, consistent with no observed in vivo binding in rats of [18F]flortaucipir to MAO-A. Direct binding of flortaucipir to human MAO-B was not detected in a microsomal preparation. A high concentration of flortaucipir (IC50 of 1.3 μM) was found to block binding of the MAO-B ligand safinamide to MAO-B on microsomes suggesting that, at micromolar concentrations, flortaucipir weakly binds to MAO-B in vitro.Under stringent wash conditions, specific [18F]flortaucipir.These data suggest neither MAO-A nor MAO-B binding will contribute significantly to the PET signal in cortical target areas relevant to the interpretation of [The online version contains supplementary material available at 10.1007/s00259-022-05822-9. Off-target binding has been noted in areas of the brain where aggregated tau is unexpected, and there have been conflicting reports in the literature about possible MAO-A and MAO-B binding relationships with [18F]flortaucipir [18F]THK5351 has been reported to be greatly reduced following treatment with the MAO-B inhibitor selegiline (deprenyl) [. The present study aimed to investigate and characterize any binding relationship between [18F]flortaucipir and MAO-A or MAO-B in vivo using PET imaging and by in vitro competition and saturation binding experiments.FFlortauctaucipir . Furthereprenyl) . The pre18F]Flortaucipir was produced as described previously [18F]Fluoroethyl-harmol was produced following the procedure as described by Schieferstein et al. [[eviously . [18F]Fln et al. .N = 3, PHF tau-rich as determined by AT8 immunohistochemistry) and age-matched non-AD control donors . Brains slices were obtained from the National Disease Research Interchange .Autoradiography was performed on frozen human brain slices from AD donors (18F]flortaucipir (0.74 MBq [20 μCi]) in 2.5% dimethyl sulfoxide (DMSO)/2.5% ethanol (EtOH)/phosphate-buffered saline (PBS), pH 7.4, was pipetted onto each slide. Adjacent sections were incubated with [18F]flortaucipir and non-radioactive flortaucipir (1 μM). After 60-min incubation at room temperature, the tissue was washed under mild (4 × 2 min in PBS) or stringent wash conditions . After drying under ambient conditions, the sections were placed in a cassette, exposed to a phosphor imaging plate overnight, and then scanned with a GE Typhoon FLA7000 Bio-Imaging System.In brief, 500 μL of [18F]flortaucipir binding to normal human brain was used to determine the IC50 values of flortaucipir, MAO-A inhibitors clorgyline (Sigma-Aldrich) and fluoroethyl-harmol  with varying concentrations of competing compound  in 2.5% DMSO/2.5% EtOH/PBS, pH 7.4. After 60-min incubation at room temperature, PBS washes (4 × 2 min) were used to remove any unbound ligand. After drying under ambient conditions, the sections were placed in a cassette and exposed to a phosphor imaging plate overnight. The plate was then scanned with a GE Typhoon FLA7000 Bio-Imaging System. Signal intensity (counts/pixel) on each section was measured using Multi Gauge V3.0 Imaging software. Total binding was evaluated by nonlinear regression using GraphPad Prism v8 to determine IC50 values.Autoradiography competition binding assay of fluoroethyl-harmol or [18F]flortaucipir via tail vein injection . For blocking studies, rats were pre-treated with pargyline  30 min prior to scans; control rats were pre-treated with 0.9% saline injection . A short high-resolution CT scan was first conducted for anatomical registration, followed by a 120-min dynamic PET scan. PET images were generated for each minute of the acquisition time. Uptake of the tracers was determined by visually drawing regions of interest based on the fused PET/CT images, and corresponding activity values were determined using Inveon Research Workplace software. All values are represented as % injected dose per gram (ID/g). A total of 6 rats were studied  for [18F]fluoroethyl-harmol and 8 rats for [18F]flortaucipir .All procedures in this study followed the guidelines of the Institutional Animal Care and Use Committee of the University of the Sciences, Philadelphia, PA. A Siemens Inveon Multimodality Scanner was used for micro-PET/computed tomography (CT) imaging. Male wild-type Sprague-Dawley rats (128 ± 58 g) were anesthetized with 3% isoflurane with oxygen. Rats were administered flortaucipir were assayed and analyzed for radioactivity as previously noted.The association . It is noted that only approximately 50% of the [18F]flortaucipir bound to normal brain sections under mild wash conditions was displaced, while the remainder was non-specific and non-saturable since it could not be displaced by high levels of MAO-A ligand or flortaucipir itself.To further understand the off-target binding observed under mild wash conditions, inhibition of fluoroethyl-harmol, to that of [18F]flortaucipir in both control and 50 mg/kg pargyline (an irreversible MAO-A/B inhibitor) pre-treated rats  wash conditions demonstrated binding of [18F]flortaucipir that could be blocked by excess non-radioactive flortaucipir, the MAO-A inhibitor clorgyline and the MAO-A/B inhibitor pargyline but not by the MAO-B inhibitor deprenyl. These results are consistent with the binding pattern observed on human brain tissue using mild washing conditions in Fig. PET studies were conducted in rats to compare retention of a selective and reversible MAO-A tracer, flortaucipir to isolated human PHF was determined to be 0.57 nM compared to 2.0 nM to recombinant MAO-A. The on/off rates (kon/koff) for [18F]flortaucipir binding to PHF tau and MAO-A  were observed for each ligand-protein pair (1.8E07–3.9E07 M−1min−1), the dissociation rate (koff) for [18F]flortaucipir:MAO-A was found to be approximately 8–9 times faster than that of [18F]fluoroethyl-harmol:MAO-A and [18F]flortaucipir:PHF.The KO-A Fig.  were meaing Fig. . While s18F]THK5351 have shown that its affinity toward MAO-B confounds its quantification of tau by PET flortaucipir and [18F]THK5351 to MAO-B microsomes was conducted . Similar Kd and Bmax were determined when 10 μM deprenyl  or 1 μM THK5351  were used to define NSB. In contrast, no specific binding of [18F]flortaucipir to MAO-B was detected using the same assay conditions.Studies using the putative tau tracer flortaucipir with MAO-A or MAO-B and evaluate if this would impact the interpretation of [18F]flortaucipir PET. We observed binding of [18F]flortaucipir to MAO-A (2.0 nM Kd) in a microsomal preparation in vitro, however with a fast dissociation rate relative to that of [18F]fluoroethyl-harmol:MAO-A and [18F]flortaucipir:PHF, consistent with the lack of binding in our in vivo experiment. Direct binding of flortaucipir to MAO-B was not detected in vitro, but a high concentration of flortaucipir (IC50 of 1.3 μM) was found to block safinamide binding to MAO-B in microsomes suggesting that, at micromolar concentrations, flortaucipir weakly binds to MAO-B in vitro.This study aimed to characterize any binding relationship of [18F]flortaucipir PET images in AD patients showing a pattern of distribution consistent with the pathological observations of Braak [18F]flortaucipir is seen also in striatum and choroid plexus in both AD patients and those with no evidence of disease [18F]flortaucipir, [18F]MK-6240, and [18F]THK5351, have also been observed in structures rich in neuromelanin and melanin-containing cells [18F]flortaucipir PET have not been definitively identified; multiple labs have been unable to detect the same pattern of off-target binding observed in [18F]flortaucipir PET by autoradiography studies on post-mortem tissue flortaucipir also interacts with MAO-B; thus far, conflicting evidence has failed to clarify any possible interaction [18F]THK5351, a radiotracer that has been investigated as a possible tau imaging agent, is known to bind to both NFT tau and MAO-B [18F]THK5351 in humans flortaucipir PET studies in non-human primates to elucidate potential binding to MAOs [in vivo binding and retention of flortaucipir to MAOs or specific displacement of flortaucipir binding by known MAO inhibitors in any brain region. Drake et al. [11C]deprenyl-d2 [18F]FSL25.1188 [11C]harmine [18F]flortaucipir PET studies probing potential off-target binding to MAOs have not convincingly demonstrated an association in vivo, prompting the current study.Concerns about off-target binding associations to MAO-B or MAO-A have prompted Fflortauc to MAOs . We do nrenyl-d2  and harmine , which d]harmine –52, acti]harmine , whereas]harmine , a lack 18F]flortaucipir binding to MAO-B could not be evaluated due to low levels of MAO-B expression. In autoradiography studies of normal human brain tissue using mild aqueous wash conditions, there was minimal displacement of [18F]flortaucipir by MAO-B ligands (IC50 > 10μM). In saturation binding experiments, whereas [18F]THK5351 bound to a preparation of human MAO-B in microsomes with a Kd of 37 nM, no significant binding of [18F]flortaucipir to MAO-B observed; there was no difference in binding of [18F]flortaucipir to MAO-B microsomes than to control microsomes devoid of MAO-B or to MAO-B microsomes in the presence of 10 μM of the MAO-B inhibitor deprenyl. In another competition binding assay, flortaucipir only weakly inhibited the binding of safinamide to MAO-B with an IC50 of 1.3 µM. Under the same assay conditions, MAO-A inhibitors clorgyline and fluoroethyl-harmol inhibited with IC50 results of 3.3 µM and > 10 µM, respectively, whereas the MAO-B inhibitor deprenyl inhibited with an IC50 of 66 nM. Taken altogether, these data indicate that [18F]flortaucipir does not bind significantly to MAO-B, and any weak interaction would not be expected to impact the interpretation of [18F]flortaucipir PET images in humans.In PET imaging studies using normal rodents, [d for [18F]flortaucipir binding to recombinant human MAO-A, we found no evidence of significant binding to MAO-B. Vermeiren et al. used excess non-radioactive flortaucipir to define non-specific binding [18F]flortaucipir non-specific binding below that of the control microsomes and produced an artifactual specific binding curve in buffer only (without any MAO or PHF). Under similar conditions as Vermeiren et al. , a disease characterized by TDP-43 deposition [18F]flortaucipir may be binding to TDP-43 or MAO-B associated with astrogliosis in these cases. However, Schaeverbeke et al. [18F] flortaucipir bound to TDP-43 in autopsy tissue from svPPA patients and no effect of blocking with the MAO-B inhibitor deprenyl on [18F]flortaucipir binding by autoradiography [18F]flortaucipir PET scan for an svPPA patient was unaffected by pre-treatment with deprenyl, an MAO-B inhibitor. These data from Pascual and Schaeverbeke are consistent with our preclinical data and clinical studies from Hansen [18F]flortaucipir does not bind to MAO-B.Additionally, studies have demonstrated flortaucipir and MAO-A would be of much less concern with respect to imaging with [18F]flortaucipir; unlike MAO-B, MAO-A is largely expressed in neurons, not astrocytes [18F]flortaucipir and the MAO-A ligands harmine and fluoroethyl-harmol, we also sought to characterize the binding of [18F]flortaucipir to MAO-A. In autoradiography experiments, displacement of [18F]flortaucipir binding by MAO-A ligands could be observed only using mild aqueous washing conditions and even then, only weakly (IC50 0.1–1 μM). However, [18F]flortaucipir bound to MAO-A accounted for ~50% of binding observed, while the remaining was non-specific and non-saturable since it could not be displaced by high levels of non-radioactive flortaucipir. In vivo PET experiments in rats revealed no evidence of [18F]flortaucipir binding to MAO-A. In vitro studies revealed low nanomolar affinity of [18F]flortaucipir for MAO-A in microsomes, but kinetic binding studies indicated that the off rate of [18F]flortaucipir from MAO-A is much faster than [18F]flortaucipir from PHF tau or [18F]fluoroethyl-harmol from MAO-A. This fast dissociation rate from MAO-A may explain why binding of flortaucipir to MAO-A was not observed in PET imaging studies in animals, and consequently, it would not be expected that [18F]flortaucipir would produce images of MAO-A in humans.A possible interaction between flortaucipir localization or binding in tau-rich areas of AD tissue but not in normal control tissue under stringent wash conditions. In direct binding studies, flortaucipir had low nanomolar affinity toward MAO-A but with an off rate 9 times faster than for tau and PET imaging showed no significant retention consistent with binding to MAO-A in a rat model, in contrast to MAO-A ligand [18F]fluoroethyl-harmol. Although we could not detect any direct binding of [18F]flortaucipir to MAO-B or significant displacement from tissue by MAO-B ligands, a high concentration of flortaucipir blocked safinamide binding to MAO-B microsomes indicating that, at micromolar concentrations, flortaucipir may bind weakly to MAO-B in vitro. Overall, the in vitro and in vivo data presented here suggest that neither MAO-A nor MAO-B binding contribute significantly to [18F]flortaucipir PET images. The present study, together with human PET blocking studies [In summary, autoradiography experiments showed [ studies , 55, cleSupplementary file1 (PDF 189 kb)Below is the link to the electronic supplementary material.


The c-actin variants inhibited DNase I, indicating maintenance of their native state. Electron microscopy showed the formation of normal appearing actin filaments though they showed mutant specific differences in length and straightness correlating with their polymerization rates. TRITC-phalloidin staining showed that p.A295S and p.R312H exhibited reduced and the p.E361G mutant increased lengths of their formed filaments. Decoration of c-actins with cardiac tropomyosin (cTm) and troponin (cTn) conveyed Ca2+-sensitivity of the myosin-S1 ATPase stimulation, which was higher for the HCM p.A295S mutant and lower for the DCM p.R312H and p.E361G mutants than for wt c-actin. The lower Ca2+-sensitivity of myosin-S1 stimulation by both DCM actin mutants was corrected by the addition of levosimendan. Ca2+-dependency of the movement of pyrene-labeled cTm along polymerized c-actin variants decorated with cTn corresponded to the relations observed for the myosin-S1 ATPase stimulation though shifted to lower Ca2+-concentrations. The N-terminal C0C2 domain of cardiac myosin-binding protein-C increased the Ca2+-sensitivity of the pyrene-cTM movement of bovine, recombinant wt, p.A295S, and p.E361G c-actins, but not of the p.R312H mutant, suggesting decreased affinity to cTm.Human wild type (wt) cardiac α-actin and its mutants p.A295S or p.R312H and p.E361G correlated with hypertrophic or dilated cardiomyopathy, respectively, were expressed by using the  Hypertrophic cardiomyopathy (HCM) is frequently characterized by a thickening of the left ventricular wall due to cardiomyocyte hypertrophy accompanied by their disarray. Additional fibrosis leads to increased stiffness of the cardiac muscle and reduced ventricular filling during diastole ,2.In conThe hereditary cardiomyopathies (CM) are caused in the vast majority by mutations in genes encoding sarcomeric ,2 or cytACTC gene coding for cardiac α-actin (c-α-actin). Mutations of the c-α-actin gene have an incidence of 4–6%, an infrequent finding in patients with familial HCM, and the incidence may be even lower in patients with familial DCM [ACTA1) [Mutations leading to HCM or DCM have also been identified within the same gene, for instance, in the lial DCM ,13,14. T [ACTA1) . The fir [ACTA1) . The fir [ACTA1) . Subsequ [ACTA1) . Present [ACTA1) . For exa [ACTA1) ,13,14,18The development of a certain cardiomyopathy phenotype does not proceed by a single pathway. It is complicated by the fact that missense mutations in different sarcomeric proteins can lead to the same disease phenotype. Conversely, point mutations at different locations in the same protein can induce diverse outcomes ,19. TherSf21/baculovirus system and their purification in the native state, allowing for their biochemical and cell biological characterization [Previously, we described the expression of two mutants of c-actin causing HCM (p.Y166C and p.M305L) by the rization . In the rization . The p.Rrization ,21 with rization . Here, we compared wild-type (wt) c-α-actin with the p.A295S, p.R312H, and p.E361G mutants in a number of different assays. We also included in this study the p.R312K mutant, which had been suspected to cause DCM . So far, however, no report exists indicating that this mutant causes a form of CM. Instead, it appears to represent a polymorphic, not disease related variant, probably because the exchange does not lead to a significant charge alteration. Nevertheless, the data obtained with this variant are given in  because it highlights the fact that the side chain of a missense residue has a considerable effect on its properties.sf21-cells and purification, the c-α-actin variants were in the native state and able to polymerize albeit with different kinetics and formed normally appearing filaments though of varying lengths. In polymerized form, they stimulated the myosin-subfragment-1 ATPase activity to different extents. After decoration with cardiac tropomyosin (cTm) and troponin complex (cTn), they showed slightly altered Ca2+-sensitivities of the stimulation of the myosin-S1 ATPase activity and the azimuthal shift of cTm on their filamentous forms. These alterations might, however, have a larger impact when interacting with other regulatory proteins during cardiomyocyte development or within the intact sarcomere.Our data indicate that after expression in insect Sf21 insect cells by using the baculovirus system and purified after affinity binding to His-tagged gelsolin G4-6 as shown for wt and the p:E361G mutant (Expression of the c-α-actins (wt plus the mutants) was achieved in G mutant C and werG mutant D. The imSf21-cells. Indeed, after expression in Sf21-cells, the purified cardiac actins possessed β-actin immune-reactivity  were determined separately see . The datFilament formation was further verified by electron microscopy after negative staining (EM) before and after decoration with cardiac tropomyosin (cTm) and troponin complex (cTn) at a molar ratio of 6:1:1 D–H. The After decoration with cTm/cTn, all mutants appeared to form more regular and longer filaments, as particularly evident for the p.E361G mutant H. Bindinbaculovirus/Sf9 system and subsequently purified. The R312K c-actin variant possesses a conservative exchange and was suspected to cause DCM . Since the p.R312K c-actin variant has not been characterized to date, we investigated some of its biochemical properties. The data shown in We also analyzed some properties of the p.R312K variant, which was also expressed by the Since filament lengths cannot be reliably determined by EM, we analyzed the length distribution of the c-actin filaments by using fluorescence microscopy after labelling with TRITC-phalloidin . The time dependent changes in the filament length distribution shown in 2+-concentration (at pCa2+ = 4.3) before and after decoration with cTm/cTn (at a molar ratio of 6:1:1), in order to compare the standard kinetic parameters. The data (M (apparent binding affinity of myosin-S1 to the particular F-c-actin) and Vmax values . A compilation of these parameters for the cardiac actins is given in M values varied considerably. In the absence of cTm/cTn, the Vmax values were rather similar for all c-actins, but indicated a decrease in the apparent affinity (KM) of the mutant c-actins to myosin-S1 after decoration with cTm/cTn, except for the p.R312H mutant  ATPase activity by the F-c-actin variants at high CaThe data  showed tThe data A′,A″, whH mutant . After d2+-ion concentration released from the sarcoplasmic reticulum. Therefore, we determined the Ca2+-concentration dependence of the myosin-S1 ATPase stimulation after decoration of the filamentous c-actins with cTm/cTn (at 7:1:1 ratio). The c-actins were decorated by native cTm or pyrene-labelled cTm (see below) without any difference in their ability to convey Ca2+-dependent stimulation of the myosin-S1 ATPase. The absolute ATPase rates (shown in 2+-dependence (2+-dependences of the stimulation of the myosin-S1 ATPase by wt and the three c-α-actin mutants (2+-concentration) and the two DCM mutants (p.R312H and p.E361G) a similar or slightly lower Ca2+-sensitivity in comparison to wt c-α-actin  analysis indicated that the measurements were significant for each special condition but not of high significance when comparing different mutants with each other or under different conditions . All measurements were performed in triplicate. Skeletal muscle myosin-S1 ATPase activity was determined by the enzyme-linked assay. ATPase stimulation by (A) recombinant wt c-actin, (B) bovine c-actin, and (C) comparison of (A) and (B). ATPase stimulation by (D) p.A295S, (E) p.R312H, and (F) p.E361G. (G) Comparison of the mutant forms of c-actin. Final F-c-actin concentrations in the assay were 1.5 μM and of rabbit skeletal muscle myosin-S1 0.5 μM. ATPase activity (ordinate) is given as μM ATP hydrolyzed/sec/μM myosin-S1 in  and normalized in (C and G) setting the minimal ATPase activity  as zero and the maximal at 1 . Abscissa gives pCa-values . Next, we tested whether the cTn-binding Ca mutants . In the 2+-ion to the cardiac troponin subunit TnC induces a conformational alteration of the cTn complex, leading to reduced binding of TnI to actin and an azimuthal movement of Tm along the actin filament and the exposure of binding sites for myosin heads [2+-ion increase and binding to TnC, tropomyosin moves to the closed (C-)state position that allows binding of myosin heads with low affinity; and (iii) finally induced by further myosin head binding the open (M-)state position, allowing their binding with high affinity [Binding of one Cain heads . Functioaffinity ,33,34,35affinity . 2+-sensitivities of the myosin-S1 ATPase stimulation are paralleled by a similar behavior of the cTm movement . For this aim, polymerized c-actin variants were decorated with pyrene-labeled cTm and cTn at a 6:1:1 molar ratio. The Ca2+-dependent changes in pyrene-cTm fluorescence were followed by using a microtiter assay read by a microplate-reader .In cardiac muscle, most of the tropomyosin is a homodimer composed of two α-chains. Labeling of cTm with pyrenyl-maleinimide occurs at Cys190 of both chains and produces a fluorescent eximer  that has2+-sensitivity for the pyrene-cTm movement than that determined by the myosin-S1 ATPase stimulation (for an explanation see Discussion). 2+-sensitivity of pyrene-cTm movement bound to recombinant wt c-actin in the absence and presence of myosin-S1 than for the myosin-S1 ATPase stimulation. −8 M versus 10−7 M CaCl2 for half maximal ATPase stimulation  when bound to bovine but not when attached to recombinant wt c-actin , suggesting that cTm bound to wt c-actin had attained the M-state irrespective of the presence of myosin-S1 , suggesting that this mutation allowed, only at higher Ca2+-concentration, a sudden transition from B- to C-state, probably due to a weakened cTm binding in the C-state. This effect is probably due to a charge alteration of the so-called A-triad—a cluster of three positive residues on subdomain 3 of actin forming the binding site of Tm during the C-state [In addition, this assay allowed for the determination of the Ca C-state ,20. In t C-state   Similar comparison for bovine c-actin and wt c-actin decorated with pyrene-cTm and cTn in the absence and the presence of N-cMyBP-C.  Identical experiments to (A) and (B) using the recombinant c-actins.  Identical experiment to (C) and (D) using the recombinant c-actins in the additional presence of myosin-S1. The ordinates give the fluorescence intensities (F) normalized to Fmax = 1, Fmin = 0. The abscissa gives the pCa . The c-actins  were decorated with pyrene-labeled cTm and cTn at a molar ratio of 6:1:1  and the Ca Methods . (A) Com2+-concentration was determined  in the absence or additional presence of myosin-S1 alone and/or plus N-cMyBP-C. Hill coefficients, pCa50-values, and the R2-coefficient were calculated by Sigma Plot Systat software . n gives the number of experiments. Variations are given as SEM.The c-actin variants were decorated with pyrene-labelled cTm at a ratio of 6:1 and the dependence of the increase in pyrene-fluorescence on Ca2+-concentration [Within the intact sarcomere, a number of associated proteins modulate the activity of the contractile machinery such as the myosin-binding protein-C, which extends from the thick to the thin filaments within the so-called C-region of the A-band (overlap region) . The carntration ,41,42. Sntration ,44. Bind2+-dependency of muscle activation, most probably due to the ability of the C1-domain to also bind to cTm and to shift its position toward the C-state, thereby increasing the probability of myosin-heads to bind to actin at low Ca2+-concentrations [It is generally accepted that MyBP-C broadens the Catrations ,45. Of ntrations .2+-sensitivity of pyrene-cTm movement bound to the c-actin variants. The measurements were performed under four different conditions: in the absence and presence of N-cMyBP-C and of cardiac myosin-S1. The F-c-actins were decorated with pyrene-cTm, cTn, N-cMyBP-C, and myosin-S1 at a six-fold lower molar concentration to actin subunits. A broadening of the Ca2+-sensitivity of the pyrene-cTm movement will be reflected by a decrease in the Hill coefficient of the sigmoidal fluorescence increase. The data obtained are compiled in 50 and the Hill coefficient was observed in the presence of myosin-S1. For recombinant wt F-c-actin, we detected, only in the presence of myosin-S1, a decrease in the pCa50, but no change in the Hill coefficient. In contrast, the p.A295S and p.E361G mutants showed a lowering of the Hill coefficient in the absence and presence of myosin-S1, the p.R312H mutant only in its absence. N-cMyBP-C, however, showed no significant effects on the pCa50-values of the mutants.Because our data indicated alterations of the interaction of the mutant c-actins with cTm, we analyzed whether N-cMyPB-C altered the CaSf21/baculovirus system, isolated in native state, and analyzed. It was hoped that the analysis of actin mutants, causative for either of these two CM forms, might reveal distinctly different properties that could be specifically attributed to a particular CM disease phenotype. Hypertrophic and dilated cardiomyopathies are the most frequent genetic diseases of the heart and caused to a high percentage by point mutations of genes encoding sarcomeric proteins. Here, we analyzed the biochemical and physiological properties of three mutations of cardiac α-actin correlated to cardiomyopathies. For this study, wild type cardiac α-actin, the p.A295S mutant causing HCM, and the p.R312H and p.E361G mutants correlated to DCM were expressed using the After isolation, the cardiac α-actins were pure as judged by SDS-PAGE, but appeared to contain about 10% cytoplasmic ß-actin as revealed by western blotting with an anti-ß-actin specific antibody. Nevertheless, we are confident that the properties and activities of the purified α-actins are attributable to the cardiac actins, since the observed deviations from the wild type behavior can only be due to alterations of the properties of the cardiac isoforms.Point mutations of proteins can lead to loss or alterations of the native state, restricting their functionality. The purified c-α-actins appeared to be in native state as verified by their ability to inhibit DNase I activity though with different efficiencies. These data may suggest slight structural alterations, since none of the point mutations were localized in subdomain 2, the binding region of DNase I . Bovine,Drosophila cardiac tube or flight muscles [The length distribution was analyzed by fluorescence microscopy after labeling with TRITC-phalloidin because such measurements cannot reliably be performed by EM due to its much higher magnification. The data showed decreased mean lengths for the p.R312H mutant, particularly for F-p.A295S, and considerably higher percentage of long filaments for F-p.E361G. The data concerning the length of filaments of the p.A295S mutant are in contrast to an earlier report suggesting normal or even longer filament length after expression in  muscles . This re muscles .max) of p.A295S before and after decoration with cTm/cTn than the wt and the DCM c-α-actin variants. Surprisingly, the p.R312H mutant showed the lowest apparent KM-value, suggesting a considerably stronger affinity of myosin-S1 than the other c-actins. Furthermore, decoration of the p.R312H mutant with cTm/cTn led to a further decrease in the apparent KM-value, probably causing the inhibitory effect of cTm/cTn on its myosin-S1 ATPase stimulatory activity  and thereby to increase the Ca2+-sensitivity of native thin filaments at low Ca2+-concentrations [We also determined the Cailaments . MyBP-C ilaments ,41. Cardilaments ,41. In o50 of bovine and wt c-α-actin to lower Ca2+-concentrations (increase the Ca2+-sensitivity), whereas its effect on the mutant c-α-actins varied from none to a slight increase in Ca2+-sensitivity of p.R312H and p.E361G in the absence of myosin-S1 , anti-α-actin (clone AC15), and rabbit anti-actin pan (clone C11) antibodies were obtained from Sigma-Aldrich . Mouse anti-cardiac α-actin monoclonal antibody was purchased from Progen Biotechnik GmbH . Pyrene-maleinimide was obtained from Sigma Aldrich . All other reagents were of analytical grade.®-WT-α-cardiac actin and the mutants p.A295S, p.R312K, and p.E361G were donated from Dr. Cora-Ann Schoenenberger . The p.R312H mutant was generated by site-directed mutagenesis from the p.R312K variant. The c-α-actin-containing plasmids served as templates for cloning the c-α-actin variants into the p3xHA-C1 plasmid. The p3xHA-C1 plasmid was a kind gift from Dr. T. Engel , who deleted cDNA of EGFP from the pEGFP-C1 plasmid  and instead cloned into this plasmid the cDNA of a three times repeated hemagglutinin-tag (HA). The primers used for amplifying the actin cDNAs were as follows: 5′-GTTATGTGTGACGACGAGGAGACC-3′ and 5′-ATTGCCCTTTTAGAAGCATTTGCG-3′. PCR inserts were cloned into p3xHA-C1 using XbaI and XhoI sites.The pcDNA3.1/NT-GFP-TOPOBamHI and HindIII enzymes obtained from Fermentas . The pCOLD II plasmid provides a His-Tag sequence for affinity chromatography, which was fused to the N-terminus of G4-6 and subsequently used to affinity purify the c-α-actins [The deletion construct of human gelsolin G4-6 was kindly supplied by Dr. A.G. Weeds  and subcloned from shuttle vector pKN172 into the cold-shock expression plasmid pCOLD II  using the restriction sites for α-actins ,46.baculovirus/Sf21-system [Rabbit skeletal muscle and bovine c-α-actin were prepared from dried acetone powder and human cardiac muscle wt α-actin and its mutants were expressed in the 1-system ,52 as de1-system ,45 and t1-system ,53,54, t1-system , and the1-system  were per2, 0.5 mM DTT, 0.2 mM NADH and an ATP regeneration system consisting of 0.05 mg/mL pyruvate kinase, 0.5 mM PEP, and 0.02 mg/mL lactate dehydrogenase (LDH). The reaction was started by the addition of myosin-S1 to a final concentration of 0.5 μM. NADH oxidation was measured by the decrease in absorption at 340 nm (1 μM = 6220 M−1 cm−1) [Stimulation of the ATPase activity of α-cardiac or skeletal muscle myosin-S1 activated by human wt cardiac α-actin or the cardiomyopathy inducing mutants was performed at 25 °C using a modified version of the NADH-coupled assay according to  in a buf−1 cm−1) ,57 using2, and 0.1 mM CaCl2. To determine the endonuclease activity of DNase I, a 10 μL pre-incubation reaction containing 3.2 μM DNase I from bovine pancreas (Sigma-Aldrich DN25) and zero to 6.4 μM of G-actin was prepared in G-buffer and incubated at room temperature for 20 min. Aliquots of the samples were added to 0.8 mL of 50 μg/mL DNA-solution and the absorbance was immediately monitored at 260 nm for 10 min using the Beckman DU 640 spectrophotometer. The DNase I activity was determined from the initial linear rates of OD 260 nm increase and expressed as Kunitz units .The DNase I inhibition assay was performed as described . The DNaPolyacrylamide gel electrophoresis in the presence of SDS (SDS-PAGE) was performed as given . Native 4P2O7, 2 mM Na3VO4, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate) and vortexed for 30 s and frozen until use. After thawing, the extracts were vortexed again and centrifuged at 20,817× g at 4 °C for 5 min. The protein concentration was estimated according to Bradford [Cells were lysed 10 mM Tris–HCl, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM NaBradford . A totalBradford . Subsequ2, 7 mM β-mercaptoethanol, 1 mM ATP) and centrifugation at 100,000× g for 30 min. In these tests, we used pyrene-labelled skeletal muscle actin that was added to the c-actins at a ratio of 20:1 (0.25 to 5 c-actin). Since pyrene-labeled skeletal-actin on its own at 0.25 μM did not show significant polymerization , we therefore assumed that the increase in fluorescence observed after mixing it with globular c-α-actin in G-buffer was solely due to the polymerization of the c-α-actins. Polymerization was induced by the addition of 2 mM MgCl2 and 0.05 M KCl and the subsequent increase in pyrene-fluorescence was monitored with excitation and emission wavelengths of 365 nm and 385 nm, respectively, using a Shimadzu RF5001PC spectrofluorometer.Actin polymerization rates were determined by the increase in fluorescence caused by the incorporation of pyrene-labeled actin into actin filaments ,27. Pyre2 and 0.05 M KCl overnight. The actin concentrations varied from 0.1 to 10 μM. The steady-state fluorescence of polymerized actin was plotted versus monomeric actin concentration and the critical concentration was calculated from the intersections with the abscissa.The critical concentration of c-α-actin polymerization was determined after varying the concentrations of the c-α-actins supplemented with 5% pyrene-actin and polymerized in the presence of 2 mM MgClFor negative staining F-actin samples were diluted to 0.1 mg/mL and adsorbed to freshly glow-discharged carbon-coated copper grids (200 mesh) for 45 s. Negative staining with 1.0% uranyl acetate was performed as described earlier ,62. The 2+-dependence of pyrene-labeled cTm movement on polymerized c-actin variants was determined by the increase in the eximer pyrene-fluorescence at excitation and emission wavelengths of 340 nm and 480 nm, respectively, using an Infinite 200 microplate reader  [2+-concentrations in the presence of 1 mM ATP were generated in black 96-well plates [max = 1 and Fmin = 0. Nine experiments were performed for each c-α-actin variant and condition. The data were fitted using a normalized Hill equation . The Cazerland) . Thin fil plates . Fluores2, and 0.1 mM CaCl2 (Buffer A) The F-actins were stained in the concentration of 1 μM in buffer A containing 2 μM TRITC-labeled phalloidin . Immediately and ca. 60 min after staining, the TRITC-phalloidin stained actins ware diluted to 20 to 100 nM in buffer A and 3 μL was dropped on a glass-slide, mixed with 3 μL of a DAKO fluorescence mounting medium  then covered with a coverslip.Freshly purified bovine cardiac actin, recombinant wild-type, and mutant cardiac actins were polymerized in the concentration of 5 μM in 10 mM Hepes buffer pH 7.4 containing 50 mM KCl, 2 mM MgClFluorescence measurements were performed using a Zeiss Axio Imager Z2m microscope equipped with a Zeiss LD LCI Plan-Apochromat 63×/1.2 multi-immersion objective and Zeiss Axiocam 503 color camera using glycerol as an immersion medium. Rhodamine fluorescence was excited using the LED 555 of the solid-state light source Colibri 7 and the quadruple bandpass filter set 90 HE, both from Zeiss. Images were recorded as gray-scale pictures with the microscope-associated ZEN software. The image size was 1936 × 1460 pixels, and the pixel size was 0.116 µm/pixel.http://fiji.sc/Ridge_Detection, accessed on 01.11.2021). This plugin is based on the detection algorithm described by Steger [The recorded images were analyzed to obtain the length and number of the filaments using ImageJ and the available plugin Ridge Detection . In addition, the significance of the Ca2+-dependency data were analyzed by the Student’s t-test and the R-square (R2) analysis using Sigma Plot software .DNA sequences were analyzed in DNAstar Lasergene software . Densitometric analysis of bands was performed with the help of the Ultra Quant 6.0 software . Graphs were plotted in Excel 2007 (Microsoft) or in Origin 8.5 (OriginLab). In both the myosin-S1 ATPase activity and Cap.A295S mutant possesses, at low Ca2+-concentrations, a higher Ca2+-sensitivity of cTm movement and myosin-S1 ATPase stimulation. It has been proposed that this effect was due to a charge disturbance of the A-triad, since residue 295 is located on a helix opposite the A-triad containing loop [2+-sensitivity, supporting the notion that a higher contractility paves the path to HCM [2+-sensitivity of the p.A295S mutant may be causative for HCM development. Summarizing the special properties of each c-actin mutant at the protein level, we can conclude the following. (1) The ing loop ,32,38, wing loop . Its dish to HCM . Our resp.R312H mutant, which was characterized by a lower Ca2+-sensitivity and a steep Hill coefficient (of 3.9). The p.R312H residue is located in subdomain 3 of actin  An opposite behavior may account for DCM induction by the of actin  and alsocapacity . Further2+-sensitivity at low Ca2+-concentrations than recombinant wt c-actin for the DCM p.E361G mutant, in line with a reduced contractility typical for a DCM causing mutant. Previous reports have revealed a particular property of the E361G mutant, namely its insensitivity toward the Ca2+-ion regulatory effect of cTnI phosphorylation [2+-sensitivity but increases the rate of relaxation of cardiomyocytes [2+-concentration and binds to three consecutive actin subunits parallel to cTm [2+-sensitivity of pyrene-cTm movement (pCa50 = 7.6) in the absence of myosin-S1 ((3) Our data demonstrate a slightly lower Carylation . Beta-admyocytes ,55. Sincmyocytes ) forms al to cTm . The binyosin-S1 . In addi2+-sensitizer levosimendan might correct the decreased Ca2+-sensitivity of the DCM causing c-actin mutants. Our data indeed suggest that levosimendan increased the Ca2+-sensitivity of the myosin-S1 ATPase stimulation of the p.R312H and p.E361G mutants. Therefore, this result might open new approaches for the treatment of DCM caused by these or similarly acting c-actin mutants.Finally, we want to emphasize that we tested the possibility that the cTn-binding Ca2+-sensitivity and filament length and stability of the cardiac actin mutants causing either HCM or DCM. In summary, our data demonstrate that each c-actin mutation induces slight biochemical albeit specific alterations of its properties. Within the sarcomere, the functional unit of striated muscle is composed of a large assembly of interacting proteins and metabolites. Therefore, small mutation-specific alterations might influence cardiac development and lead to the progression of cardiomyopathies. Our data indeed demonstrate disparate properties concerning the Ca


Introduction: The perpetual appearance of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-COV-2), and its new variants devastated the public health and social fabric around the world. Understanding the genomic patterns and connecting them to phenotypic attributes is of great interest to devise a treatment strategy to control this pandemic.Materials and Methods: In this regard, computational methods to understand the evolution, dynamics and mutational spectrum of SARS-CoV-2 and its new variants are significantly important. Thus, herein, we used computational methods to screen the genomes of SARS-CoV-2 isolated from Pakistan and connect them to the phenotypic attributes of spike protein; we used stability-function correlation methods, protein-protein docking, and molecular dynamics simulation.Results: Using the Global initiative on sharing all influenza data (GISAID) a total of 21 unique mutations were identified, among which five were reported as stabilizing while 16 were destabilizing revealed through mCSM, DynaMut 2.0, and I-Mutant servers. Protein-protein docking with Angiotensin-converting enzyme 2 (ACE2) and monoclonal antibody (4A8) revealed that mutation G446V in the receptor-binding domain; R102S and G181V in the N-terminal domain (NTD) significantly affected the binding and thus increased the infectivity. The interaction pattern also revealed significant variations in the hydrogen bonding, salt bridges and non-bonded contact networks. The structural-dynamic features of these mutations revealed the global dynamic trend and the finding energy calculation further established that the G446V mutation increases the binding affinity towards ACE2 while R102S and G181V help in evading the host immune response. The other mutations reported supplement these processes indirectly. The binding free energy results revealed that wild type-RBD has a TBE of −60.55 kcal/mol while G446V-RBD reported a TBE of −73.49 kcal/mol. On the other hand, wild type-NTD reported −67.77 kcal/mol of TBE, R102S-NTD reported −51.25 kcal/mol of TBE while G181V-NTD reported a TBE of −63.68 kcal/mol.Conclusions: In conclusion, the current findings revealed basis for higher infectivity and immune evasion associated with the aforementioned mutations and structure-based drug discovery against such variants. Coronavirus disease (COVID-19), caused by a new beta coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was declared a pandemic by the World Health Organization (WHO) on 11 March 2020 . After e–3 per year for SARS-CoV-2 (Genome sequencing insights have shown the nucleotide substitution rate as ∼1 × 10RS-CoV-2 . VariatiRS-CoV-2 . Single RS-CoV-2 . As a reRS-CoV-2 .a), beta (ß), gamma (γ), and delta (∆) variants. Among these, alpha (B1.1.7) emerged in the United Kingdom in September 2020 and increased transmissibility and virulence was reported due to mutations in the spike protein  of the spike protein  by the WHO . These V protein . Mutatio protein . These v protein . They ar protein .Mutational alterations in amino acids are anticipated to influence the structure and function of the related proteins. Therefore, it is imperative to discover the mutational landscape while creating novel antiviral therapies, and hence it is important to determine the mutations that have been observed in the spike protein polyprotein and subsequent influence in the protein structure and interaction with the host body. Therefore, the current work seeks to identify the mutations found in the spike protein to forecast the structural changes of SARS-COV-2 spike protein owing to the mutations and determine the signature pattern. Mutational analysis of the spike protein in SARS-CoV-2 isolates from Pakistan was performed using Global Initiative on Sharing All Influenza Data (GISAID), and mutations were identified. The impact of these substitutions on the structure and function of the spike protein was then investigated using various structural modeling tools. The current study provides the basis for therapeutics development to control the SARS-CoV-2 pandemic in Pakistan and worldwide.https://www.ncbi.nlm.nih.gov/) (https://www.gisaid.org/epiflu-applications/covsurver-mutations-app/) was used for single nucleotide substitution in the spike protein  , and the protein . The ser protein .https://www.uniprot.org/) (6XRA (The spike protein sequence with a full length of (1273aa) was collected in the FASTA format from UniProt (ot.org/) . The 3D /) (6XRA . The mis/) (6XRA . The fin/) (6XRA .https://www.uniprot.org/uniprot/P0DTC2#family_and_domains). Mutations identified through comparative analysis were mapped to their respective domains.All the retrieved variants were classified based on the domain’s information, retrieved from UniProt’s Family and Domain option (http://biosig.unimelb.edu.au/mcsm/stability) was used to accurately predict the impact of mutations on the protein stability and interaction by using graph-based signatures. For each mutation, relative solvent accessibility (RSA) and DDG values were computed (http://biosig.unimelb.edu.au/dynamut2/submit_prediction) was used for assessing the impact of the mutation on protein stability and dynamics by using the normal mode analysis (NMA) method. Predicted Gibbs free energy (ΔΔG) values of mutants less than zero (0) were classified as destabilizing, while those greater than 0 were classified as stabilizing (The mCSM server (computed . Furtherbilizing .http://folding.biofold.org/i-mutant/i-mutant2.0.html)   was usedStructures of spike protein subunits of SARS-COV-2 were collected from RCSB with host receptor, the wild-type structure of RBD–ACE2 (PDB ID: 6M0J), and NTD–monoclonal antibody (4A8) (PDB ID: 7C2L) in a PDB file. The wild-type RBD and NTD structures were used as templates for mutant modeling using Chimera software. After each mutant modeling, each structure was prepared using an AMBER force field; hydrogen and charges were fixed. Finally, the prepared structures were minimized.https://life.bsc.es/pid/pydockweb) algorithm was used for molecular docking (http://www.ebi.ac.uk/thornton-srv/databases/pdbsum/Generate.html).To check the binding efficiency of wild-type and mutant RBD with the human ACE2 receptor, the PyDock ( docking . PyDock  docking . The samD) is an important parameter to estimate the strength of biological macromolecules association. Thus, herein, to determine KD, we used the PROtein binDIng enerGY prediction (PRODIGY) server (Determination of the dissociation constant (K) server .+ ions. Gentle minimization using 6,000 and 3,000 steps of steepest descent and conjugate gradient algorithms was achieved, and heating at 300K, followed by the equilibration, was completed. Lastly, a production run at a time scale of 50 ns was executed. Simulation trajectories were analyzed using the CPPTRAJ and PTRAJ modules of AMBER  to identify single amino acid changes throughout the whole protein sequence (1273aa). There were 21 different types of single amino acid substitutions (single mutant) identified in the protein. The mutations include H49Y, L54F, N74K, D80Y, R102S, G142A, G181V, A222V, G261R, G446V, D614G, D614A, V622F, Q67H, S813N, C840V, A846V, A890T, S943T, A1078S, and T117I. Of these, five mutations stabilized the structure and 16 mutations destabilized it , including, H49Y, L54F, N74K, D80Y, G142A, G181V, A222V, and G261R. Among four of them, mainly H49Y “predicted ΔΔG value” (.945), D80Y (.091), N74K (.096), and A222V (.096) were responsible for stabilizing the S protein structure and the rest of the mutants. L54F (−1.298), R102S (−1.345), G142A (−.908), G181V (−.488), and G261R (−.309) were found to be destabilizing. For instance, the H49Y mutation was also previously reported to stabilize the structure and is responsible for enhanced transmission as also reported in the B.1.618 variant . Similardy titer . The A22dy titer . G261R tdy titer . The S1 dy titer  , A890T (−.309), A1078S (−1.33), S943T (−.306), C840V (−.418), and A846V (−.127) were analyzed to be destabilizing, while T1117I (.082) stabilizes the S protein structure . The S81Central helices contain D614G (−.665), D614A (−.445), V622F (−.821), and Q677H (−.179), which were analyzed to destabilize the S protein . The D61Additionally, the DynaMut2 server was also used for mutation verification and their related effects on the spike protein structure and dynamics. Out of 21 mutations, seven were found to be responsible for stabilizing, and the remaining mutants (14) were found to destabilize the protein .In the S1 domain, NTD H49Y (.43), N74K (.03), D80Y (.16), and A222V (.17) were analyzed to be stabilizing, while NTD R102S (−1.02), G142A (−.28), G181V (−1.07), G261R (−.51), L54F (−.69), and RBD G446V (−.75) were found to destabilize the S protein .The S2 domain contains S813N (−.48), A1078S (−.7), S943T (−.49), C840V (−.59), and A846V (−.73), which were found to be destabilizing, while A890T (.16) and T1117I (.43) were found to stabilize the S protein .The central helices contain D614G (−.22), D614A (−.4), and V622F (−.3), which were analyzed as destabilizing, while Q677H (.02) was found to stabilize the S protein .The I-Mutant server shows that these all identified mutations (21) in the spike glycoprotein decreased structural stability . In thisThe 3D structures of spike protein RBD–ACE2 and NTD–mAbs were collected from RCSB using PDB ID: 6M0J and PDB ID: 7C2L. Furthermore, these experimentally reported structures of S1 regions (NTD and RBD) were used for mutant modeling using Chimera software. The NTD domain mutants include H49Y, L54F, N74K, D80Y, R102S, G142A, G181V, A222V, and G261R, while G446V was modeled in the wild-type RBD domain.https://life.bsc.es/pid/pydockweb), we docked the spike (S1) RBD with the human ACE2 receptor to check the effect of 10 identified single amino acid substitutions in the S1 subunit of SARS-COV-2 of Pakistani isolates on the binding efficiency of RBD and NTD domains with human ACE2. The mutants were generated using Chimera software. By using the PyDock server, ACE2 was docked with a wild-type spike (RBD); and then, PDBsum was used (http://www.ebi.ac.uk/thornton-srv/databases/pdbsum/Generate.html) to explore interaction analysis of complex. The PDBsum interaction analysis revealed that two salt bridges, 10 hydrogen bonds, and 145 non-bonded contacts were formed. The complex formed 10 double hydrogen bonds between Asn487–Gln24, Lys417–Asp30, Gly446–Gln42, Gln493–Glu35, Tyr449–Asp38, Tyr449–Gln42, Asn487–Tyr83, Gly502–Lys353, Gly496–Lys353, Tyr505–Arg393, and Thr500–Tyr41 residues. The two salt bridges formed between Lys417–Asp30 residues . The interaction analysis of the PyDock server revealed that the mutant (G446V) RBD–ACE2 complex forms 12 hydrogen bonds and 135 non-bonded contacts. The hydrogen bonds include Lys417–Glu30, Val446–Gln42, Ala475–Ser19, Asn487–Tyr83, Tyr489–Tyr83, Gly496–Glu38, Gly496–Lys353, Thr500–Tyr41, Ser494–Asp157, Tyr505–Tyr158, Lys417–Asn250, and Glu406–Ser254 residues, while a salt bridge was formed between Lys417 and Glu30 . Similar findings were also reported previously by different studies; thus, our results are consistent and accurate  NTD–ACE2 complex was as follows: electrostatics (−32.118), desolvation (8.856), and VdW (68.920). The interaction analysis of mutant G181V–mAbs revealed that both structures formed one salt bridge, three hydrogen bonds, and 305 non-bonded contacts. The hydrogen bonds formed between Lys150–Gly26, Thr250–Asp55, and Lys147–Val102 residues. The salt bridge forms between Lys150 and Glu31 residues . The interaction analysis of H49Y NTD–mAbs by PDBsum revealed that the interaction formed one salt bridge, four hydrogen bonds, and 266 non-bonded contacts. The hydrogen bonds formed between Asn149–Tyr27, Ser151–Glu31, and Lys187–Asp55 and Tyr145–Gly104 residues. The single salt bridge forms between Lys187 and Asp55 residues. The PyDock interaction analysis of mutant (L54F) with mAbs by PDBsum analysis revealed that both structures form one salt bridge, four hydrogen bonds, and 268 non-bonded contacts. The interaction analysis of this complex exhibits a binding pattern similar to the wild-type complex. The PDBsum analysis of mutant D80Y–mAbs revealed that both structures form one salt bridge, four hydrogen bonds, and 266 non-bonded contacts. In this complex the salt-bridges and hydrogen bonds determined the same pattern as the wild type complex, and the hydrogen binding pattern is similar to the wild complex.The PyDock server was used to dock wild-type NTD with mAbs. The PyDock docking score for the wild-type NTD–mAbs complex was as follows: electrostatic score (−36.092), desolvation (−14.139), and VdW (95.823). The interaction analysis revealed that the interaction forms one salt bridge, four hydrogen bonds, and 265 non-bonded contacts. The hydrogen bonds formed by the wild NTD–mAbs complex include Asn149–Tyr27, Ser151–Glu31, and Lys187–Asp55 and Tyr145–Gly104 residues. The single salt bridge forms between Lys187 and Asp55 in the wild NTD–mAbs complex . On theontacts . Moreovesidues . The inThe interaction analysis of mutant G142A–mAbs revealed that both structures form the same number of hydrogen bonds and salt bridges with hydrogen binding patterns similar to the wild-type NTD–mAbs complex. The interaction analysis of the mutant A222V–ACE2 receptor revealed that both structures form one salt bridge, four hydrogen bonds, and 264 non-bonded contacts. In this, the complex exhibits a binding pattern similar to that of the wild-type NTD–mAbs complex. The interaction analysis of mutant G261R–mAbs receptor revealed that the complex formed one salt bridge, four hydrogen bonds, and 268 non-bonded contacts. This complex shows a hydrogen binding pattern similar to that of the wild-type NTD–mAbs complex. In the PyDock docking score for mutant (N74K) NTD–mAbs, the complex shows electrostatics energy (−50.511), desolvation (−6.857), and vdW (121.522). The PDBsum analysis of mutant N74K–mAbs revealed that both structures formed three salt bridges, seven hydrogen bonds, and 274 non-bonded contacts. Among single hydrogen bonds, Ser71–Tyr27, Lys74–Tyr27, His69–Glu31, Asn185–Asp55, Tyr145–Thr74, Tyr145–Ser75, and His146–Ser75 residues were involved. The key residues Lys74–Glu1, His69–Glu31, and Lys147–Asp77 formed salt bridges. Consequently, the other mutations help the virus escape the neutralizing antibodies, while R102S, G181V, and G446V directly affect the binding. Thus, these three mutations, along with the wild type, were subjected to further analysis using a molecular dynamics simulation tool. The docking scores, including vdW, electrostatic, desolvation, and the docking scores of the wild type and mutant (RBDs and NTDs), are given in To further see the structural stability and compactness of the deleterious mutations, we performed MD simulation. We calculated the stability as root mean square deviation (RMSD) and compactness as the radius of gyration (Rg) as a function of time. Comparison of the wild-type and G446V-RBD complexes revealed more comparable RMSD. During the first 30 ns, both the complexes remained more stable; however, the G446V-RBD complex converged during the last 20 ns. The average RMSD for both the complexes was 2.0 and 2.1 Å, respectively. For instance, the unstable behavior of the G446V justifies the radical function of the variants, as reported previously. The RMSD graphs of the wild-type RBD–ACE2 and G446V-RBD–ACE2 complexes are given in Moreover, to see the structural compactness differences, a dynamic environment Rg for each complex was computed. Variations in the wild-type RBD–ACE2 and G446V-RBD–ACE2 complexes were observed during the first stage of simulation particularly between 0 and 30 ns. In this time period, the wild type remained more compact than the G446V complex. Then, after 30 ns, the G446V complex also achieved the compact state and remained comparable with the wild type. The average Rg(s) for both the complexes was reported to be 31.3 Å and 31.6 Å, respectively. Increase or decrease in the compactness is due to the binding and unbinding events that happened during the simulation, respectively. The Rg(s) of all the complexes are given in Understanding the residual flexibility helps in assessing the biological mechanism of different molecules and plays an important role in enzyme engineering, drug designing, interface study, and peptide inhibitor discovery. Thus, herein, we also calculated residual flexibility as root mean square fluctuation (RMSF) using the simulation trajectories. The wild-type RBD–ACE2 and G446V-RBD–ACE2 demonstrated a similar pattern of flexibility, except for minor variations in different regions. As can be seen, the regions 190–200 and 450–500 displayed higher fluctuation in the wild type. This shows that the G446V mutations have induced stable binding and thus stabilized the fluctuations. The RMSF of the wild-type RBD–ACE2 and G446V-RBD–ACE2 complexes is given in via increment in the number of hydrogen bonds. To check the bonding differences in the wild-type NTD–mAb, R102S-NTD–mAb, and G181V-NTD–mAb complexes, we also calculated hydrogen bonds in each complex. In each complex, the number of hydrogen bonds was recorded as wild-type NTD–mAb (256), R102S-NTD–mAb (249), and G181V-NTD–mAb (247). Consequently, this shows that SARS-CoV-2 strains with these mutations reduce the binding of monoclonal antibodies and thus escape the immune response to increase the infectivity. The hydrogen bonds calculated in each complex are given in Hydrogen bonding is important to maintain the binding complex and has been previously reported by various studies to understanding the binding pattern of the wild type and other variants . The hydEstimation of the binding free energy defines the correction conformation and rescoring of the binding molecules. It is a widely used method to understand the binding of two or more molecules. To redefine the binding affinity predicted by docking, herein, we used the MM/GBSA approach using 2,500 frames. The binding free energy calculations revealed that G446V-RBD binds to the ACE2 receptor more strongly than the wild-type RBD. The total binding energy for the wild-type RBD was reported to be −60.55 kcal/mol, while it was −73.49 kcal/mol for the G446V-RBD complex. This shows that SARS-CoV-2 variants with the G446V mutation increase the binding affinity to enhance the infectivity. Moreover, it can be seen that both the vdW and electrostatic energies are increased in the mutant complex, thus revealing that these findings are consistent with the previous findings reported for other variants . The binin silico virtual screening approaches. In conclusion, the current findings revealed the basis for higher infectivity and immune evasion associated with the mutations mentioned previously and structure-based drug discovery against such variants.The perpetual appearance of SARS-CoV-2 and its new variants devastated the public health and social fabric around the world. Herein, we connected spike protein’s genomic patterns and phenotypic attributes to understand the evolution, dynamics, and mutational spectrum of SARS-CoV-2 and its new variants circulating in Pakistan. Using the GISAID, a total of 21 unique mutations were identified, and docking with ACE2 and monoclonal antibody (4A8) revealed that mutation G446V in RBD, R102S, and G181V in NTD significantly affect the binding and thus increase the infectivity. The structural-dynamic features of these mutations revealed the global dynamic trend, and the resulting energy calculation further established that the G446V mutation increases the binding affinity toward ACE2, while R102S and G181V help in evading the host immune response. The already available antibodies can be engineered to design potent synthetic antibodies. Moreover, the specified mutant residues in the interface of RBD and ACE2 can be targeted by using