Levels of γ-H2AX were measured and the biodistribution of CE/A83 and NP/A83 administered intravenously was determined in subcutaneous HCT116 CRC xenografts. The radio-sensitization effect of A83B4C63 was measured following fractionated tumor irradiation using an image-guided Small Animal Radiation Research Platform (SARRP), with 24 h pre-administration of CE/A83 and NP/A83 to Luc+/HCT116 bearing mice. Therapeutic effects were analyzed by monitoring tumor growth and functional imaging using Positron Emission Tomography (PET) and [18F]-fluoro-3’-deoxy-3’-L:-fluorothymidine ([18F]FLT) as a radiotracer for cell proliferation. The results showed an increased persistence of DNA damage in cells treated with a combination of CE/A83 or NP/A83 and IR compared to those only exposed to IR. Significantly higher tumor growth delay in mice treated with a combination of IR and NP/A83 than those treated with IR plus CE/A83 was observed. [18F]FLT PET displayed significant functional changes for tumor proliferation for the drug-loaded NP. This observation was attributed to the higher A83B4C63 levels in the tumors for NP/A83-treated mice compared to those treated with CE/A83. Overall, the results demonstrated a potential for A83B4C63-loaded NP as a novel radio-sensitizer for the treatment of CRC.Inhibition of the DNA repair enzyme polynucleotide kinase/phosphatase (PNKP) increases the sensitivity of cancer cells to DNA damage by ionizing radiation (IR). We have developed a novel inhibitor of PNKP, i.e., A83B4C63, as a potential radio-sensitizer for the treatment of solid tumors. Systemic delivery of A83B4C63, however, may sensitize both cancer and normal cells to DNA damaging therapeutics. Preferential delivery of A83B4C63 to solid tumors by nanoparticles (NP) was proposed to reduce potential side effects of this PNKP inhibitor to normal tissue, particularly when combined with DNA damaging therapies. Here, we investigated the radio-sensitizing activity of A83B4C63 encapsulated in NPs (NP/A83) based on methoxy poly(ethylene oxide)- Colorectal cancer (CRC) is the second most common cause of cancer death globally  and its Inherent or acquired cellular resistance mechanisms in CRC cells can undermine the effectiveness of IR, eventually leading to cancer recurrence in CRC patients –13. IR gHuman polynucleotide kinase-phosphatase (PNKP) is identified as a key enzyme involved in DNA repair following damage by IR or topoisomerase I inhibitors (e.g. irinotecan) in many types of cancer including CRC –26. PNKP50 and KD values in the low micro and nanomolar range, respectively (b-PBCL). Passive targeting of solid tumors by NPs is attributed to the presence of leaky vasculature as well as impaired drainage of the lymphatic system at the tumor site (b-PBCL) containing methoxy-PEO (mPEO) or acetal-PEO (acPEO), and radiolabeled with 64Cu resulted in a 3-fold increased measurable accumulation into subcutaneous HCT116 tumors (perhaps due to the EPR effect) versus muscle tissue as determined with PET  (average molecular weight of 5000 g/mol), Cremophor EL: Ethanol (CE), and all research grade organic solvents were purchased from Sigma . α-Benzyl carboxylate-ε-caprolactone monomer was synthesized by Alberta Research Chemicals Inc. . Stannous octoate was purchased from MP Biomedicals Inc. .via reverse-phase HPLC as previously described , i.e., the number of repeating units in a polymer chain, for the PBCL block was synthesized by ring-opening polymerization of α-benzyl carboxylate-ε-caprolactone using mPEO (MW: 5000 g/mol) as an initiator and stannous octoate as catalyst according to the method described previously  using deuterated chloroform (CDCl3) as solvent and tetramethylsilane as an internal reference standard.The mPEO-eviously , 51 (Fib-PBCL NPs (NP/A83) were prepared as previously described  and centrifuged at 11600 × g for 20 min at 4°C in order to concentrate as required. CE/A83 formulation was prepared by previously described method . Finally, the A83B4C63 loading and loading efficiency were calculated according to the following equations:A83B4C63-encapsulated mPEO-escribed . In bried method . In brieThe morphology of self-assembled structures under study was investigated by TEM using a Morgagni TEM  with Gatan digital camera . In brief, 20 μL of micellar solution with a polymer concentration of 0.25 mg/mL or Cremophor EL at a concentration of 0.2 mg/mL was placed on a copper-coated grid. The grid was held horizontally for 1-2 min to allow the colloidal particles to settle down. The excess fluid was removed by filter paper. The copper-coated grids holding the aqueous samples were then negatively stained by 2% phosphotungstic acid. After 2 min, the excess fluid was removed by filter paper and the grid was loaded into the TEM for image analysis.+/HCT116 cells were generated as previously described (2 in a humidified incubator in a 1:1 mixture of Dulbecco’s modified Eagle medium and F12 (DMEM/F12) supplemented with 10% FBS, 50 U/mL penicillin, 50 mg/mL streptomycin, 2 mmol/L L-glutamine, 0.1 mmol/L nonessential amino acids, and 1 mmol/L sodium pyruvate. All culture supplements were purchased from Invitrogen .Wild type HCT116 cells were obtained from the American Type Culture Collection and luciferase positive Lucescribed . Cells w5 wild type HCT116 cells were seeded onto each glass coverslip in a 35-mm Petri dish and incubated overnight to attach. The cells were then pretreated with the nano-formulations for 24 h prior to 3 Gy γ-irradiation. Irradiation was carried out at room temperature at a dose rate of 0.66 Gy/min. After irradiation, the cells were incubated for two time points up to 6 h. After the incubation, the cells were fixed with 4% paraformaldehyde in PBS for 20 min, then permeabilized, and blocked with 1% BSA in 1 x PBS/0.1% Tween 20 for 20 min. After 3 washes with 1 x PBST, anti-phospho-histone H2A.X (Ser139) antibody  at 1:4000 dilution was applied to the cells and incubated for 1 h at room temperature. The cells were washed three times with 1 x PBST and then incubated with Alexa Fluor 488 goat anti-mouse secondary antibody  at a 1:200 dilution in 0.1% BSA/1 x PBST for 1 h in the dark. After washing the cells, the coverslips were mounted on the slides with DAPI-containing mounting media . The cells were harvested at either 1 or 4 h after exposure to IR. Each experiment was performed in triplicate.Western blot was used to assess the level of cleaved caspase 3/7 and PARP induced by A83B4C63 as free drug (CE/A83) and NP (NP/A83) formulation in HCT116 cells with or without radiation. Initially, 1.5 million cells were plated. Then, cells were treated with CE/A83 and NP/A83, or vehicles alone, at an A83B4C63 concentration of 10 µM, or equivalent drug free CE and NP levels. After 24 h incubation with A83 formulations or vehicle controls, cells were exposed to a fixed dose of radiation (4 Gy) using a Protein extracts for western blot analysis were prepared using commercial RIPA lysis buffer  supplemented with a cocktail of protease inhibitors . Protein concentrations were measured using the BCA assay kit  according to the manufacturer’s protocol. Equal concentrations of protein were separated by SDS-PAGE and transferred to nitrocellulose membranes. After blocking with 5% skimmed milk in TBST , the blots were incubated with the respective primary antibodies  and secondary antibody (HRP-linked anti-rabbit IgG cat# 7074S) purchased from Cell Signaling Technology . The protein bands were detected using an enhanced chemiluminescence (ECL) based system  and quantified by densitometric analysis using ImageJ software.+/HCT116 xenograft tumor mouse models were generated by subcutaneous injection of 0.5 × 106 cells in a 100 µL mixture of culture media and matrigel matrix  (1:1 v/v) in the right flank or left shoulder of 4 - 6 week-old female NIH-III nude mice. The CRC cell implanted mice were routinely monitored for tumor growth and signs of sickness. Animals reaching early endpoints as set in our animal protocol were euthanized. All animals were euthanized at day-22 following the tumor inoculation.NIH-III nude mice were purchased from Charles River Laboratories . All animal studies were conducted in accordance with the guidelines of the Canadian Council on Animal Care and with approval from the local Animal Care Committee of the Cross Cancer Institute . The HCT116 and Luc+/HCT116 xenografts developed as described above. When the tumor volume reached 80 to 150 mm3, mice were randomly assigned into test groups receiving empty NP without IR (n = 6), or empty NP (n = 6), CE/A83 (n = 6), and NP/A83 (n = 7) formulations of A83B4C63 with a fractionated radiation dose of 3 x 5 Gy q.a.d. The treatments  were started on day 0. On day -2 (2 days before starting the treatments), tumor sizes were measured with a digital slide caliper and by bioluminescence using an in vivo imaging system (IVIS®). All drugs were given via intravenous (IV) injection via tail vein and administered on days 0, 2, and 4. The IV A83B4C63 dose was 25 mg/kg, which was injected three times one day apart. Mice received three fractionated radiation doses of 5 Gy every alternative day. The excipient dose, i.e., empty NP in control groups was selected equivalent to their amounts in the NP/A83 test group. The length (L) and width (W) of the tumor were measured two times per week and the tumor volume (TV) was calculated using the formula TV = (L × W2)/2. The measurements continued until day 22 (since the day of tumor inoculation) when all mice were euthanized.This study was performed on Luc® software . The radiation target volume was defined as the tumor volume contoured from the cone beam CT scan and the isocenter defined in the center of the tumor volume and radiation doses were calculated. After therapy planning, the radiation therapy to the target tumor area was delivered using a 0.15 mm copper filter with 220 kVp X-rays and 13 mA using and two opposing dorsal beams at 45 to 60 degrees and minus 45 to 60 degrees and a 10 mm x 10 mm square-shaped collimator at a dose rate of 0.042 Gy/sec and an exposure time of 60 s per beam. The collimator size was big enough to completely cover tumor tissue for the applied irradiation.The fractionated radiation therapy using a daily dose of 5 Gy was started on day 1 and given 3x including days 3 and 5. Radiation therapy was administered using the image-guided small-animal radiation research platform  Mice were placed ventrally onto the bed of the SARRP and immobilized with continuous isoflurane with anesthesia. A cone beam computed tomography (CT) scan was acquired first for each mouse and used for radiation therapy planning per mouse using integrated Muriplan/Murislice+/HCT116 and treated as described above were also imaged for the expression of luciferase to follow their tumor growth. For the optical imaging, mice were subcutaneously injected with the XenoLight D-Luciferin - K+ salt bioluminescent substrate  at a dose of 10 µL/g of body weight before the luciferase detection. Mice were anesthetized and placed in the dark chamber of a IVIS® LUMINA XMRS optical imaging systems  for whole-body animal imaging and the emitted photons were quantified and analyzed using Living Image® Software . Imaging of live animals was performed twice a week.The animals inoculated with Luc+/HCT116 tumor-bearing female NIH-III nude mice from the radiation therapy study (as described above) were analyzed on days 10-12 after last treatment for tumor proliferation using Positron Emission Tomography (PET). Mice were anesthetized by isoflurane (100% O2). A needle catheter was placed into the tail vein of these mice and 3 - 6 MBq of [18F]FLT in 100 to 150 µL saline were injected. [18F]FLT was synthesized at the cyclotron research facility of the Cross Cancer Institute according to the previously described procedure . Acquisition data were collected in three-dimensional list mode for 10 min, reaching ~60 min post injection. Static PET images were reconstructed using a maximum a posteriori (MAP) algorithm. Image files were further processed using the ROVER v2.0.51 software . Masks defining three-dimensional regions of interest (ROI) over tumor tissue were defined and ROI’s were analyzed with 50% threshold of radioactivity uptake. Mean standardized uptake values [SUVmean = (measured radioactivity in the ROI/mL tumor tissue)/] were calculated for each ROI.Lucrocedure  using 5-+/HCT116. When the tumor volume reached 1200 to 1500 mm3, mice were randomly assigned and grouped into three test groups (n = 3). The test groups received CE/A83 or its NP form three times, one day apart at an IV dose of 25 mg/kg. The control mice received empty NPs. 4, 24, and 48 h after the last injection, all mice were euthanized, and blood, excised tumors and other organs including brain, heart, lung, liver, kidney, and spleen were collected to define drug levels using an LC/MS/MS method of quantification as previously described . The lowest limit of quantification was set at 1 ng/mL. The mass spectrometer was operated in positive mode with capillary voltage at 3.2 kV, source temperature at 120°C, desolvation temperature at 275°C, and desolvation gas flow at 800 L/h. Instrumental control and data analysis were performed using MassLynx software .An Agilent 1100 HPLC system coupled to a Waters Quattro Micro triple quadrupole mass spectrometer  and attached to an Agilent Poroshell 120 SB- C18 2.7-micron LC column with dimensions of 2.1 mm x 50 mm was used. The column was heated to 35°C. The mobile phase consisted of water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). A gradient elution was programmed to commence with 20% B for post-injection followed by a gradual increase in 3 min of B to 95%. The composition was maintained for 3 min when it was gradually decreased back to 20% of B in 0.1 min. The flow rate was 0.3 mL/min and 2 µL of sample was injected. Standard curves were linear over the range of 1 - 1000 ng/mL  fitted with an Agilent Technology: Poroshell 120 SB-C18 2.1x50 mm, 2.7-micron column. The mobile phase consisted of 50:50 v/v ratios of water with 0.1% formic acid and acetonitrile with 0.1% formic acid.The terminal elimination rate constant was estimated from the log-linear portion of the plasma concentration - time curves. Because of the destructive sampling procedure used for the collection of blood and tissues from different animals at each time point, the area under the plasma/tissue curve (AUC) was estimated using the trapezoidal rule from the average plasma concentrations at different time points and the variance of AUC was estimated using Bailer’s method based on the standard error of the mean (SEM). The ratio of tissue concentration at each time point to that of plasma (Kp) was also calculated and reported.post-hoc test, where appropriate. If a significant difference was found among the groups, median ranks between pairs of groups were compared using the Mann-Whitney U test. A value of p ≤ 0.05 was considered as statistically significant in all experiments.GraphPad Prism 9 software  was used for statistical analysis. Significance of differences between groups was assessed using one-way and two-way ANOVA followed by Tukey’s For biodistribution experiment, the AUC of plasma or tissue versus time curves were obtained using the approach outlined by Bailer . Pairwis1H NMR spectra and peak assignments of mPEO-b-PBCL  were prepared at a 1:3 w/w A83B4C63:mPEO-b-PBCL ratio. The NP/A83 were ~60 nm in diameter on average and showed a low polydispersity index (PDI), i.e., < 0.25, indicating the uniformity of the nanocarrier population in terms of diameter (p ≤ 0.0001) than that of NP/A83. However, no significant difference was measured for the PDI values obtained from CE/A83 and NP/A83. The diameter of the empty carriers from both formulation types i.e, NP, CE, were significantly lower (*p ≤ 0.05) than their drug-encapsulated counterparts. As shown in The O-b-PBCL , 59–61. Upright microscopic evaluations were performed to assess the DNA damage following treatment of cells with a combination of PNKP inhibitor and a fixed dose of IR (3 Gy). We also analyzed the induction of cleaved PARP, cleaved caspase-7, and cleaved caspase-3 expressions following treatment by A83B4C63 with and without radiation. Both CE/A83 and NP/A83 formulations slightly induced the level of cleaved PARP, cleaved caspase-7 and cleaved caspase-3, but the level of induction was low, suggesting that apoptosis does not play a major role in the cellular response to radiation with or without the repair inhibitor  before the treatment schedule as shown in To explore the radio-sensitizing anticancer activity of intravenously administered CE/A83 and NP/A83 at a dose of 25 mg/kg three times a week in mice bearing LucAs shown in 3 (n = 6), in the mice treated with empty NP, empty NP plus IR, and CE/A83 plus IR, respectively, whereas the tumor volumes remained as low as 196.56 ± 221.01 mm3 (n = 7) in mice treated with IV NP/A83 plus IR. The overall results clearly showed the in vivo radio-sensitizing activity of A83B4C63 in its NP formulation in wild type Luc+/HCT116 CRC xenografts in mice, which was in contrast to no statistically significant activity for the CE formulation of this PNKP inhibitor compared to control groups receiving empty NPs with or without IR (p > 0.05). ®, see below). The measured mean body weight variation of the mice receiving systemic treatments were within a 20% margin  in comparison to that of other pretreatment groups, including the empty NP, empty NP plus IR, and CE/A83 plus IR cohorts. Therefore, the radiance for bioluminescence of Luc+/HCT116 xenografts in the respective treatment groups of mice showed a similar pattern in tumor growth to that observed by slide caliper measurements. When comparison was made between the day -2 (2 days prior to starting treatments) and day 12 (termination day), significant increases in luciferase-tagged cancer cells (bioluminescence) were found for all treatment groups except NP/A83-treated mice, which did not show any difference in the bioluminescence of xenografts from day -2 to day 12. The data validated the anti-tumoral activity of systemic NP/A83 administration in the HCT116 CRC xenograft model as a novel radio-sensitizing nanomedicine.To further evaluate the radio-sensitizing anticancer activity of A83B4C63, tumor growth in mice was also detected by 18F]FLT to determine the tumor proliferation in vivo in highly multiplying cancer cells in the HCT116 xenografts to determine the radio-sensitizing activity of A83B4C63 formulations in these tumors. In line with the results of caliper and bioluminescence measurements, administration of NP/A83 at 25 mg/kg dose plus IR led to a significant reduction of [18F]FLT uptake in the HCT116 xenografts. This contrasted with CE/A83 plus IR that did not show any significant reduction of [18F]FLT uptake when compared to the control receiving empty NPs plus IR. Following tumor uptake levels of [18F]FLT were determined as mean standardized uptake values (SUVmean) ± SEM: 1.35 ± 0.12, 1.18 ± 0.18, 1.03 ± 0.17  and 0.62 ± 0.09 (n = 7), for empty NP, empty NP plus IR, CE/A83 plus IR, and NP/A83 plus IR, respectively. When compared for significant differences as shown in mean value than that of empty NP without IR at α level of 0.001. The difference in SUVmean value for mice that received NP/A83 plus IR was significantly lower than for the mice that received empty NPs or CE/A83 plus IR (*p < 0.05). However, no significant differences were observed between the NP plus IR and CE/A83 plus IR treatment groups.p ≤ 0.0001) significantly above the detection limits for up to 48 h. The concentration of A83B4C63 obtained by NP/A83 formulation was significantly higher at 24 h (*p ≤ 0.05) and 48 h (****p ≤ 0.0001) when compared with that of CE/A83. This resulted in a significantly higher plasma AUC level for the mice that received NP/A83 (34246.64 ± 3710.36) treatment to those that received CE/A83 (21078.86 ± 1534.31) .) showed significantly higher AUC values for NP/A83 than for CE/A83 in tumor and liver, while the AUC of NP/A83 was lower in kidney compared to CE/A83 . However, no significant differences were observed between these treatment groups in lung, heart, and spleen .Biodistribution data Table 1A83B4C63 concentrations in excised tumors from the mice were also measured and the results are shown in p ≤ 0.0001) (p ≤ 0.01) compared to that of NP/A83-treated mice. In contrast, the obtained A83B4C63 concentration was significantly higher in the liver samples (p ≤ 0.05) post dose, only. However, no significant difference was observed in liver to plasma ratio between CE/A83 and NP/A83 treatment groups.Notably, CE/A83 formulation resulted in significantly higher accumulation of A83B4C63 in the kidney at 4 h post dose time point compared to that of NP/A83  or HCT116/PTEN+/+ cell viability  of IR was used to avoid potential side-effects on normal tissues surrounding the irradiated site .in vivo radiation studies and edited the manuscript. IP, SM, NS, FS, and ZB have provided assistance in the biodistribution studies. XY has provided assistance in fluorescent microscopy measurements. GM, FJ, DM, and AG provided feedback on in vivo studies and edited the manuscript. MP has synthesized A83B4C63. DH has supervised the synthesis of A83B4C63 and edited the manuscript. MWe and AL have supervised the study and edited the manuscript. All authors contributed to the article and approved the submitted version.SS has been responsible for the design and completion of all studies in this manuscript. He has prepared the first draft of the manuscript. MWu has assisted in the conduction of This work was supported by grants funded by the Canadian Institutes of Health Research (MOP 15385) to MWe and (159757 and 178028) to AL, the Alberta Cancer Foundation Transformative Program Project (26603) to DH, FJ, AL, and MWe. Funding from Nanomedicine Innovation Network (NMIN) grant (2019-T1-06) to DH, MWe, and AL is also acknowledged.The authors declare the following competing financial interest(s): Material in this manuscript has been included in recent US patent applications. AL is Vice-President of Meros Polymers which has the license to mPEO-b-PBCL polymer used in this manuscript.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This prospective COVID-19 Health Action Response for Marines (CHARM) study enabled assessment of T cell responses against S, N and M proteins in symptomatic and asymptomatic SARS-CoV-2 infected participants. At enrollment all participants were negative by qPCR; follow-up occurred biweekly and bimonthly for the next 6 weeks. Study participants who tested positive by qPCR SARS-CoV-2 test were enrolled in an immune response sub-study. FluoroSpot interferon-gamma (IFN-γ) and IL2 responses following qPCR-confirmed infection at enrollment (day 0), day 7 and 14 and more than 28 days later were measured using pools of 17mer peptides covering S, N, and M proteins, or CD4+CD8 peptide pools containing predicted epitopes from multiple SARS-CoV-2 antigens. Among 124 asymptomatic and 105 symptomatic participants, SARS-CoV-2 infection generated IFN-γ responses to the S, N and M proteins that persisted longer in asymptomatic cases. IFN-γ responses were significantly (p = 0.001) more frequent to the N pool (51.4%) than the M pool (18.9%) among asymptomatic but not symptomatic subjects. Asymptomatic IFN-γ responders to the CD4+CD8 pool responded more frequently to the S pool (55.6%) and N pool (57.1%), than the M pool (7.1%), but not symptomatic participants. The frequencies of IFN-γ responses to the S and N+M pools peaked 7 days after the positive qPCR test among asymptomatic  and symptomatic  participants and dropped by >28 days. Magnitudes of post-infection IFN-γ and IL2 responses to the N+M pool were significantly correlated with IFN-γ and IL2 responses to the N and M pools. These data further support the central role of Th Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection [COVID-19 vaccines that induce neutralizing antibodies particularly to the spike (S) protein have been approved for emergency use in the USA, and one, the Pfizer-BioNTech COVID-19 vaccine (Comirnaty) has received U.S. Food and Drug Administration (FDA) approval. However, the efficacy of these vaccines against evolving strains with mutations in the S-protein remains to be fully elucidated . While ein silico methods have been used to predict epitopes, often using machine learning [Since T cells play a vital role in protective immunity against SARS-CoV-2 , robust learning . These mlearning , 10, or learning  using arlearning –14. Howelearning . Internalearning , 17. Deflearning –20.This study was designed to comprehensively measure IFN-γ cell responses in the COVID-19 Health Action Response for Marines (CHARM) study, a prospective, longitudinal cohort study of healthy, young adults . This coThe CHARM study was conducted at the Marine Corps Recruitment Depot, Parris Island, South Carolina and has been previously described . ParticiAs previously reported , particiThe complete design of this study is shown in PBMCs for measuring cell-mediated immunity (FluoroSpot assay) were collected at the time of enrollment (T0) and then after the participant’s first positive qPCR test , isolated from heparin tubes within 24 hours, and stored in liquid nitrogen until used. Cryopreserved PBMCs were thawed, washed, counted, and used in the FluoroSpot assay to measure cells secreting either interferon-gamma (IFN-γ), Interleukin-2 (IL2), as previously reported –25.All peptides were obtained from BEI Resources . The full-length spike glycoprotein (S), full length nucleocapsid (N) protein, and the membrane (M) protein were each covered by a series of 17-mer or 13-mer (aa) peptides overlapping by 10 amino acids that were combined into antigen-specific peptide pools  Tables. ex vivo ELISpot was modified [5 PBMCs suspended in 100 μL complete medium were incubated in the FluoroSpot plates with antigen-specific peptide pool at final concentration of 2 ug/mL suspended in 100 μL complete medium. CTL-CEF-Class I Peptide Pool Plus  consisting of 32 peptides corresponding to defined HLA class I-restricted T cell epitopes from cytomegalovirus, Epstein-Barr virus and influenza virus was used as an internal control for each participant. PHA, a mitogen, was used as a positive control for cell viability. Negative control unstimulated PBMCs received medium only. Cultures were incubated for 40–42 h at 37°C in 5% CO2. Each PBMC sample was assayed in duplicate and the number of single-staining antigen-specific IFNγ- and IL2-secreting cells and double-staining IFNγ- and IL2-secreting cells were recognized as spot-forming cells (sfcs) and enumerated using an automated FluoroSpot reader . After subtraction of the mean number of sfcs in negative control wells (no antigen), the mean of the net sfcs of the test sample was expressed as sfcs/106 PBMCs. A positive response to each individual antigen-specific peptide pool was defined as positive when there was a statistically significant difference (p = <0.05) between the average of the sfc in test and negative control wells (Student’s two tailed t-test), plus at least a doubling of sfc in test compared to control wells, plus a difference of at least 10 sfc between text and control wells [Antigen-specific circulating PBMCs were evaluated using pre-coated FluoroSpot plates and kits purchased from Mabtech  and used according to the manufacturer’s instructions. The previously described modified ; brieflyol wells .6 PBMC at T7, T14 or TLT after the initial SARS-CoV-2 qPCR positive result. All statistical analyses were interpreted using a two-tailed alpha = 0.05 and were made using SAS v9.4 or JMP v 12 . In some cases, asymptomatic and symptomatic participants who all had responses to the N+M or CD4+CD8 peptide pools were compared for responses to individual S, N or M peptide pools. In the Comparisons of the proportion of participants demonstrating a response were made using a Pearson chi-square while comparisons on the proportion of participants demonstrating responses across pools were made using a McNemar’s Test. Comparisons across study groups were made using a Wilcoxon Rank Sum Tests and comparisons before and after infection were made use paired Wilcoxon Rank Sum Tests. Spearman Rank Correlations were used to assess the correlation between maximum responses post-infection. For each participant, the single highest magnitude of response among the 4 time points was selected for that participant that were defined as greatest number of sfc/10All relevant data of the FluoroSpot assays can be found in The individual frequencies of IFN-γ and IL2 responses in asymptomatic and symptomatic participants to the S, N+M, and CD4+CD8 pools at T0, T7, T14 and TLT are shown in We next determined the frequency of positive responses at T0, T7, T14 and TLT to the S, N+M and CD4+CD8 peptide pools.Among all participants, the frequency of positive IFN-γ responses to the S pool (30.1%) or the N+M pool (37.1%), was higher among asymptomatic  than symptomatic  participants; however, only IL2 responses to S pool were significantly higher (p = 0.04)  in the aWe next determined whether the frequencies of IFN-γ and IL2 responders to the N+M pool were associated with the individual N or M pools in asymptomatic and symptomatic participants . Among aAmong CD4+CD8 pool responders, the frequency of positive IFN-γ in matched asymptomatic and symptomatic post-infection to S, N, and M pools were compared. The responses to the S pool (63.3%) were higher than the N (47.8%) or M (17.4%) pools. However, in asymptomatic participants IFN-γ responses to S and N pools were similar  and higher than to the M pool (7.1%) , whereasWe next compared the magnitudes of the maximum post-infection responses to each peptide pool in asymptomatic and symptomatic participants. As with determining frequencies of responses, we used the largest magnitude of responses at T0, T7, T14 or TLT.The magnitude of IFN-γ and IL2 responses to the S pool were significantly higher in asymptomatic than symptomatic participants ; however, only IL2 responses to the N+M pool were significantly higher in asymptomatic participants (p = 0.04) . Yet, thSignificant correlation (using Spearman Rank Correlations) was observed between maximum post-infection IFN-γ and IL2 responses to the N+M pool and the N pool and the M pool . MaximumThere as a significant correlation (using Spearman Rank Correlations) between the maximum post-infection IFN-γ responses to the CD4+CD8 pool and post-infection IFN-γ responses to the S, N and N+M pools  . These cThis study enabled comparisons of asymptomatic and symptomatic immune responses immediately after a positive SARS-CoV-2 qPCR assay. We used recovered cells from cryopreserved samples that may not reflect total peripheral T cell counts. Both groups developed frequent and robust IFN-γ responses, although IL2 responses were lower. Others have previously suggested that a significant virus-specific T cell response was not associated with disease severity , 28–30, Early induction of IFN-γ SARS-CoV-2-specific T cell responses to the S protein has been previously associated with mild disease and accelerated viral clearance , 30 and While there are no clearly-defined immune correlates of protection against COVID-19 , there iIn the CHARM study, more than 95% complete viral genomes were recovered from 18 quarantined participants and six independent monophyletic transmission clusters were identified that were transmitted to roommate pairs but not to other platoons. Since enrollment occurred in May to September 2020, likely SARS-CoV-2 strains were prior to the emergence of the Delta and Omicron variants, and were likely the S or GH clades .in vitro binding assays [A major concern of current antibody-based vaccines are mutations in the S protein that affect sequences recognized by vaccine-induced neutralizing antibodies –59. Howeg assays . The geng assays .Finally, we observed T cell IFN-γ, but not IL2, responses to the S and N+M pool in some uninfected participants, suggesting cross-reactive T cells derived from prior exposure to the common cold human coronaviruses that is in agreement with previous studies that have reported SARS-CoV-2 cross-reactive CD4+ T cells in unexposed people , 63, andTaken together, our results support previous findings on the potential importance of the N-protein for vaccine development . ImmunizThere are several limitations to this study. Firstly, the study population is young, healthy adults with few comorbidities and may not be reflective of the general population limiting the generalizability of the findings. Secondly, all illnesses were mild, limiting the ability to assess a range of clinical outcomes. Thirdly, the timing of FluoroSpot responses may not have been optimal and further post-infection analyses are warranted. Fourthly, we used cryopreserved cells, and it is possible the recovery of viable cells after thawing may have varied and affected assay readouts. Finally, although we tested immune responses to the S, N and M proteins, it is likely that responses to other proteins may also have a significant role in disease modulation, and responses may partially reflect the relevant sizes of each protein and the numbers of peptides in each pool that were higher for the S pool. We only measured IFN-γ and IL2 producing cells and it is also possible that different cytokines would help to better understand disease severity , and pheS1 Fig*** p = <0.001; * p = <0.05. The numbers of samples at each time point were based on the numbers of available samples among the 124 asymptomatic participants. Immune responses (sfc/m) and percent positive participants are shown before the first positive qPCR test (T0), 7 days (T7), 14 days (T14) and long-term (TLT) after that first positive PCR test (infection). Immune responses to S, N+M and CD4+CD8 pools were significantly (p = <0.001) higher at T7, and were not significantly different between T7, T14 and TLT after infection; the exception was the significant rise in IL2 responses to S pool between 7d and 14d after infection. There were no positive IL2 responses to the CD4+CD8 pool.(TIFF)Click here for additional data file.S2 Fig***p = <0.001; **p = <0.01. The numbers of samples at each time point were based on the numbers of available samples among the 105 symptomatic participants. Immune responses (sfc/m) and percent positive participants are shown before the first positive PCR test (T0), and 7 days (T7), 14 days (T14) and long-term (TLT) after that first positive PCR test (infection). Immune responses to S, N+M and CD4+CD8 pools were significantly (p = <0.001) higher at T7, and IFN-γ responses to S protein significantly dropped by TLT. IL2 responses to the CD4+CD8 pool were absent, except in one participant at 14d after infection.(TIFF)Click here for additional data file.S1 TableThis 181-peptide array spans the spike (S) glycoprotein of the USA-WA1/2020 strain of SARS-CoV-2 (GenPept. QH060594).(DOCX)Click here for additional data file.S2 TableThis 59-peptide array spans the nucleocapsid (N) protein of the USA-WA1/2020 strain of SARS-CoV-2 (GenPept. QH060601).(DOCX)Click here for additional data file.S3 TableThis 31-peptide array spans the membrane (M) glycoprotein of the USA-WA1/2020 strain of SARS-CoV-2 (GenPept. QH060597).(DOCX)Click here for additional data file.S4 TableThis 241-peptide array corresponds to 221 predicted HLA class II CD4+ T cell epitopes covering all proteins in the viral genome except the spike (S) glycoprotein.(DOCX)Click here for additional data file.S5 TableThis 628-peptide array spans the USA-WA1/2020 strain of SARS-CoV-2 Click here for additional data file.S6 TableCD4+ and CD8+ epitopes within each SARS-CoV-2 protein were predicted (reference (DOCX)Click here for additional data file.S1 File(XLSX)Click here for additional data file.S2 File(XLSX)Click here for additional data file.S3 File(XLSX)Click here for additional data file.S4 File(XLSX)Click here for additional data file.S5 File(XLSX)Click here for additional data file.S6 File(XLSX)Click here for additional data file.


We have recently reported that a recombinant HIV-1NL4.3 containing Met-to-Ile change at codon 50 of integrase (IN) (IN:M50I) exhibits suppression of the virus release below 0.5% of WT HIV, and the released viral particles are replication-incompetent due to defects in Gag/GagPol processing by inhibition of the initiation of autoprocessing of GagPol polyproteins in the virions and leads to replication-incompetent viruses. The coexisting Ser-to-Asn change at codon 17 of IN or Asn-to-Ser mutation at codon 79 of RNaseH (RH) compensated the defective IN:M50I phenotype, suggesting that both IN and RH regulate an HIV infectability. In the current study, to elucidate a distribution of the three mutations during anti-retroviral therapy among patients, we performed a population analysis using 529 plasma virus RNA sequences obtained through the MiSeq. The result demonstrated that 14 plasma HIVs contained IN:M50I without the compensatory mutations. Comparing the sequences of the 14 viruses with that of the defective virus illustrated that only Val-to-Ile change at codon 151 of IN (IN:V151I) existed in the recombinant virus. This IN:V151I is known as a polymorphic mutation and was derived from HIVNL4.3 backbone. A back-mutation at 151 from Ile-to-Val in the defective virus recovered HIV replication capability, and Western Blotting assay displayed that the back-mutation restored Gag/GagPol processing in viral particles. These results demonstrate that a combination of IN:M50I and IN:V151I mutations, but not IN:M50I alone, produces a defective virus. Subse) manner , and the) manner ,9.Naturally occurring polymorphisms (NOP)s and genetic diversity in the HIV genome often regulate drug susceptibility, virus evolution, and viral infection/replication ,11,12,13Approval for these studies, including all sample materials and protocols, was granted by the National Institute of Allergy and Infectious Diseases (NIAID) Institutional Review Board, and participants have informed a written consent prior to blood being drawn. All experimental procedures in these studies were approved by the National Cancer Institute at Frederick and Frederick National Laboratory for Cancer Research .gag or gag-pol regions. The reaction was performed in three steps: 1st step: 60 °C for 1 hr, 94 °C for 2 min; 2nd step: 60 cycles at 94 °C for 1 min, 61 °C for 30 s and 72 °C for 4 min 45 s; and 3rd step: 1 cycle of 72 °C for 10 min and then hold at 4 °C. The E+ primer set resulted in a 4482 bp product (HXB2: 737-5219 nt). The A4.5 kb primer set resulted in a ~4537 bp product (HXB2: 682–5219 nt). Quality of the PCR products was analyzed by agarose gel and the appropriate size of products was purified, and then PCR products were quantified using Qubit ds DNA specific assay kit . For the next generation sequence (NGS), Illumina MiSeq instrument  along with Nextera XT DNA Library Preparation Kit (cat# FC-131-1024) (Illumina), MiSeq Reagent Nano Kit v.2, 500 Cycles (cat# MS-103-1003) (Illumina), and Nextera XT Index Kit (cat# FC-131-1001) (Illumina) were used. Briefly, 1 ng of the purified amplicons were used for the Illumina MiSeq enzymatic based Tagmentation reaction that fragments and adds (tags) partial adapter sequences at the ends of amplicons. Nextra XT PCR was performed to add the index and adapter sequences required for cluster formation on the flow cells during the sequencing run. The NGS library products were cleaned up, normalized, and then treated with NaOH followed by the equimolar sample pooling in a single tube. The final products were loaded to the MiSeq cartridge and MiSeq sequencing run was performed following a standard protocol from the vendor.All HIV RNA sequences were derived plasma samples from 209 HIV-infected patients in NIAID clinical trials. The plasma samples were obtained from pre, on, or post anti-viral therapy (ART) with or without integrase inhibitors. Total analyzed time points per patient were donor dependents (the blood drawing points were from 1 to 14 times) . HIV virhttps://www.broadinstitute.org/viral-genomics/v-fat, accessed on 28 September 2021) with HXB2 reference backbone to produce an initial sample-based consensus sequence. NovoAlign  was used to align the paired reads to this initial consensus sequence. More than 200× depth coverages for all regions of interest were required for further analysis. The criteria removed any false codons in the sequences. The above steps were executed through wrappers in viral-ngs . A pileup file was then produced from the initial consensus alignment using SAMtools mpileup (v0.1.19) [https://www.bioinformatics.org/sms/iupac.html, accessed on 28 September 2021). We did not focus on minor variants for this study as our goal was to define the combination IN:M50I with a compensatory mutation.Sequence results from the MiSeq were assembled as previously described . In briev0.1.19)  and a vav0.1.19)  from theOn each sequencing run, we included one negative and one wild-type positive control. Both controls followed the same bioinformatic process for QC, assembly and final consensus calling. Either a negative control leading to an HIV consensus sequence or a positive control producing an unexpected consensus sequence led to a failure of the sequencing run.v/v) fetal bovine serum , and 50 μg/mL gentamicin (Thermo Fisher) as previously described [Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors’ apheresis packs using a lymphocyte separation medium  , CD4(+) escribed ,25.E. coli (Thermo Fisher). The plasmid purification was carried out using the EndoFree Plasmid Maxi Kit (Qiagen).Plasmids encoding HIV variants were constructed by inducing the mutations on pNL4.3  the pla as previw/v) sucrose (MiliporeSigma) in 10 mM HEPES-150 mM NaCl buffer (pH 7.4) as previously described [Recombinant HIV-1 variants were prepared by transfection of the pNL4.3 mutants into HEK293T cells using TransIT-293  and Opti-MEM I medium (Thermo Fisher) following a method previously reported . Cultureescribed ,27. Viraescribed ,27.v/v) FBS, and 50 μg/mL gentamicin (RP10). The PHA-stimulated CD4(+) T cells (10 × 106 cells) were infected with 10 ng of p24 of each HIV variant at 10 × 106 cells/mL in RP10 for two hours at 37 °C. The infected cells were washed with RP10, and then cultured at 1 × 106 cells/mL in the medium in the presence of 20 units/mL of recombinant IL-2 (MiliporeSigma) for 14 days at 37 °C in T25 flasks [Replication capability of each variant were determined using primary CD4(+) T cells as previously described . CD4(+) 5 flasks ,28. Half5 flasks .To confirm autoprocessing of Gag and GagPol polyproteins in HIV virions, Western Blotting (WB) was performed as previously described . Due to A full-length structure of IN was predicted using the currently available crystal structures of IN data from the protein database, as previously described . To predt-tests using Prism 8 software . p values < 0.05 were considered statistically significant. To determine significant differences among a correlation between viral load and mutations, one way ANOVA test was conducted using Partek software .Intergroup comparisons were performed using two-tailed unpaired Our previous study using a series of recombinant HIV-1NL4.3 mutants demonstrated that virus containing IN:M50I mutation, mNL(IN:M50I), was a defective virus that suppressed the initial cleavage of GagPol polyproteins and viral release (note: the defective recombinant virus was depicted HIV(IN:M50I) in the previous work ; howeverWe next dissected the 90 amplicons containing IN:M50I, to define the population of combination; the combinations of RH:N79S/IN:M50I and IN:S17N/IN:M50I were detected in 40 (44%) and 13 (14%) amplicons, respectively, and RH:N79S/IN:S17N/IN:M50I was detected in 26% (23) amplicons D. SurpriWe next endeavored to identify the unknown compensatory mutation in the 14 viruses containing M50I mutation alone. To identify the compensatory mutation(s) using the HIVHXB2 sequence as a reference sequence, we compared the 14 sequences from alignments A,B. The n = 4) (n = 4) and thus around 0.3% of that in HIV(WT) (p < 0.01), indicating that the combination of mutation suppressed virus release. Since we were not able to produce a high TCID50 titer of virus stock of HIV(IN:M50I/V151I), we infected 10 × 106 cells of primary CD4(+) T cells with 10 ng of p24 amounts of virus for viral replication assay as described in the Materials and Methods. To delineate whether our hypothesis, that a combination of IN:M50I and IN:V151I causes a defective virus, was correct or not, we compared virus replication capability using a series of mutants. In the current study, we constructed the mutants utilizing the clones that we have previously used . As mentn = 4) . In strip < 0.001, n = 5), while HIV(IN:M50I) and HIV(IN:V151I) replicated 89 ± 6.5%  and 90 ± 3.8%  of HIV(WT) at day 7, respectively. These data indicated that the defective recombinant virus in the previous report of HIVmNL(IN:M50I) was caused by the combination of IN:M50I and IN:V151I mutations.As shown in To confirm the combination effect of the mutations, we carried out WB analysis using 1 μg of total viral proteins of each virus with anti-p55(Gag)+p24(CA)+p17(MA) antibody to confirm that the single mutation had no impact on the GagPol processing. Mature cleaved p17 and p24 fragments were detected in HIV(WT), HIV(M50I), and HIV(IN:V151), but not in HIV(IN:M50I/V151I), and accumulation of uncleaved GagPol polyprotein was detected in the mutant B as we sTo elucidate the population diversity of HIV carrying the IN:M50I/IN:V151I mutant in the plasma virus, we compared HIV sequences focusing on the mutations using NIAID and the Los Alamos National Laboratory (LANL) HIV database. The results demonstrated that the combination was not detected in the total 529 sequences samples from 209 patients in NIAID samples ; howeverAs described above, the virus replication assay indicated that an HIV containing the combination of M50I/V151I without RH:N79S or IN:S17N was a replication incompetent virus. Thus, we further analyzed the viruses containing the combination focusing on the presence of the compensatory mutation. In subtype B, nearly half of the sequences contained either RH:N79S or IN:S17N mutation, and surprisingly, 14 out of the 27 sequences did not contain the compensatory mutations A, and inWe recently reported that the presence of IN:M50I in HIV-1 suppresses virus release and inhibits HIV maturation by interrupting the initiation of autoprocessing; subsequently, the virus is impaired in replication, and other mutations, RH:N79S or IN:S17N, restored the viral replication . In the IN:M50I and IN:V151I mutations are naturally occurring SNPs and are reported as mutations associated with resistance to integrase inhibitors ,34,35, IIN:V151I was an endogenous mutation in the HIVNL4.3 strain. Therefore, before the drug treatment, if HIV RNA sequence data indicated that IN:V151I is present in the HIV without IN:S17N or RH:S79S, IN:M50I selection during the treatment may be delayed because the combination is a suicide mutation for HIV, and the treatment effect may last longer. However, further study needs to define the molecular mechanism of the defect, since population analysis of the LANL HIV database indicated that 14 of 27 sequences in subtype B and 3 sequences of subtype C contained IN:M50I/V151I mutation without encoding RH:N79S or IN:S17N. In our previous work, HIV containing IN:M50/V151I mutants without RH:N79S or IN:S17N was defective, and the virus release and replication were significantly suppressed ; it is rcis-interaction [GagPol polyproteins are cleaved by mature PR in viral particles ,6,7. Theeraction  , the suA complete IN structure model is not available; thus, we created a predicted model by in silico modeling system. The codon 151 does not locate on IN surface , and a p


Plasmodium falciparum, which contains two plant homeodomain (PHD) proteins (PfPHD1 and PfPHD2) and a plant apetela2 (AP2)-domain transcription factor (PfAP2-LT). To dissect the functions of the PfGCN5 complex, we generated parasite lines with either the bromodomain in PfGCN5 or the PHD domain in PfPHD1 deleted. The two deletion mutants closely phenocopied each other, exhibiting significantly reduced merozoite invasion of erythrocytes and elevated sexual conversion. These domain deletions caused dramatic decreases not only in histone H3K9 acetylation but also in H3K4 trimethylation, indicating synergistic crosstalk between the two euchromatin marks. Domain deletion in either PfGCN5 or PfPHD1 profoundly disturbed the global transcription pattern, causing altered expression of more than 60% of the genes. At the schizont stage, these domain deletions were linked to specific down-regulation of merozoite genes involved in erythrocyte invasion, many of which contain the AP2-LT binding motif and are also regulated by AP2-I and BDP1, suggesting targeted recruitment of the PfGCN5 complex to the invasion genes by these specific factors. Conversely, at the ring stage, PfGCN5 or PfPHD1 domain deletions disrupted the mutually exclusive expression pattern of the entire var gene family, which encodes the virulent factor PfEMP1. Correlation analysis between the chromatin state and alteration of gene expression demonstrated that up- and down-regulated genes in these mutants are highly correlated with the silent and active chromatin states in the wild-type parasite, respectively. Collectively, the PfGCN5 complex represents a novel HAT complex with a unique subunit composition including an AP2 transcription factor, which signifies a new paradigm for targeting the co-activator complex to regulate general and parasite-specific cellular processes in this low-branching parasitic protist.The histone acetyltransferase GCN5-associated SAGA complex is evolutionarily conserved from yeast to human and functions as a general transcription co-activator in global gene regulation. In this study, we identified a divergent GCN5 complex in  Plasmodium falciparum. To better understand the epigenetic mechanisms in this parasite, we characterized the histone acetyltransferase GCN5-mediated transcription regulation during intraerythrocytic development of the parasite. Using tandem affinity purification and proteomic characterization, we identified that the PfGCN5-associated complex contains nine core components, including two PHD domain proteins (PfPHD1 and PfPHD2) and an AP2-domain transcription factor, which is divergent from the canonical GCN5 complexes evolutionarily conserved from yeast to human. To understand the functions of the PfGCN5 complex, we performed domain deletions in two subunits of this complex, PfGCN5 and PfPHD1. We found that the two deletion mutants displayed very similar growth phenotypes, including significantly reduced merozoite invasion rates and elevated sexual conversion. These two mutants were associated with dramatic decreases in histone H3K9 acetylation and H3K4 trimethylation, which led to global changes in chromatin states and gene expression. Consistent with the phenotypes, genes significantly affected by the PfGCN5 and PfPHD1 gene disruption include those participating in parasite-specific pathways such as invasion, virulence, and sexual development. In conclusion, this study presents a new model of the PfGCN5 complex for targeting the co-activator complex to regulate general and parasite-specific cellular processes in this low-branching parasitic protist.Epigenetic regulation of gene expression plays essential roles in orchestrating the general and parasite-specific cellular pathways in the malaria parasite  Toxoplasma gondii, appear to have diverged significantly from the SAGA complexes. In Toxoplasma gondii, TgGCN5b was found to interact with ADA2 and the plant apetela2 (AP2)-domain-containing transcription factors (TFs), while the parasite lacks most of the conserved SAGA components  was amplified from P. falciparum genomic DNA using primers F1 × R1. For the deletion of the PfGCN5 BrD and the PfPHD1 PHD domain, the PfGCN5 fragment (nt 3286–4044) and the PfPHD1 fragment (nt 3286–4044) were amplified using primers F2 × R2 and F3 × R3, respectively. All amplified fragments were first cloned into a modified pBluescript SK plasmid to fuse with the PTP or GFP and pDT 3’ UTR as described earlier . To measure the gametocyte development, gametocyte induction was conducted by using an established method  is the TPM at the t =  and schizont stage  were harvested and crosslinked with paraformaldehyde  at 37°C for 15 min with agitation and then immediately neutralized by adding glycine  on ice for 5 min with agitation. The fixed iRBCs were lysed with saponin  on ice for 5–10 min. Parasites were treated with a lysis buffer  and then gently homogenized using a douncer to free nuclei. Pelleted nuclei were suspended in a shearing buffer    as described [S9B Table). Fold enrichment relative to constitutively expressed reference gene seryl-tRNA synthetase  was calculated by using the 2−ΔΔCt method [-target − IP Ct-stRNA)−(control IP Ct-target–control IP Ct-stRNA)]−[ Schematic diagram of PTP tagging at C-terminal of PfGCN5. P1 and P2 are primers used for verification of integration by PCR. (B) Cartoon shows the TAP procedure for purification of the PfGCN5 complex. A, B, and C are subunits of the GCN5 complex. TEV: tobacco etch virus protease. (C) Integration-specific PCR verification of two positive clones (C1 and C3) from transfected parasites. (D) Western blot detecting PfGCN5::PTP expression in the recombinant parasite clone C3 at different developmental stages . The blot was probed with antibodies against protein C. Molecular markers in kDa are shown on the left. The expression of aldolase was used as a loading control. The PfGCN5::PTP protein bands are indicated by asterisks. (E) Live cell imaging shows the localization of PfGCN5::GFP during intraerythrocytic development . DAPI was used to stain nucleus. BF, bright field. Scale bar = 5 μm.(PDF)Click here for additional data file.S2 Fig(A) Sequence of four PHD domains in PfPHD1, C and H amino acid residues in the PHD domain are highlighted underneath the sequence. PHD-SF: PHD superfamily; ePHD: elongated PHD domain, PHD_TAF3: TAF3 type PHD domain. (B) Sequences of four PHD domains in PfPHD2. (C) Alignment of PfPHD1 PHD_TAF3 domain with other known authentic PHD domains which bind H3K4me3/2. The alignment shows the conserved Zinc-binding residues in light gray for Zinc 1 and dark gray for Zinc 2, and the two core β-strands in green. The residues involved in H3K4me3 recognition are labeled I through V (forming the aromatic cages) and the aromatic residues in the recognition cage are shadowed in green. MLL1: mixed-lineage leukemia-1; JARID1A: jumonji, AT-rich interactive domain 1A; PYGO: pygopus homolog 1; BPTF: bromodomain PHD finger transcription factor; PHF2: PHD finger protein 2; Yng1: yeast homolog of mammalian ING1; ING4: inhibitor of growth protein 4; TAF3: transcription initiation factor TFIID subunit 3.(PDF)Click here for additional data file.S3 Fig(A) The nuclear localization of PfPHD1 and PfPHD2 were interrogated by IFA using anti-PfPHD1 and PfPHD2 antibodies. Nuclei were counter-stained by DAPI. Scale bar = 5 μm. BF, bright field. (B) Immunoprecipitation (IP) of proteins from lysates of the PfGCN5::PTP parasite line using agarose conjugated with either anti-PfPHD1 or PfPHD2 antibodies. IPed proteins were separated by SDS-PAGE and probed with anti-Protein C antibodies recognizing the full-length GCN5::PTP. Pre-immune sera were used as controls. (C) IP of proteins from lysates of the 3D7 wild-type parasites using agarose conjugated with anti-PfPHD1 (left panel) or PfPHD2 (right panel) antibodies. IPed proteins were separated by SDS-PAGE and probed with anti-PfPHD2 or anti-PfPHD1 antibodies.(PDF)Click here for additional data file.S4 Fig(A) Schematic diagram of Myc tagging at C-terminal of PfPHD1. A, AvrII; S, StuI. (B) Southern blot of 3D7 and three transgenic clones (1–3). Genomic DNA was digested with AvrII and StuI and hybridized with labeled DNA shown as “Probe” in A. (C) Western blot analysis of nuclear (Nu) and cytoplasmic (Cyto) protein extracts with antibodies against the Myc tag, aldolase (for cytoplasmic compartment) and histone H3 (for nuclear compartment).(PDF)Click here for additional data file.S5 Fig(A) The diagram shows GFP tagging of the PF3D7_1019700 at its C-terminus by single-crossover homologous recombination. Purple blocks show the fragment used for homologous recombination. (B) Integration-specific PCR using primers P1 and P2. WT, Wildtype 3D7; C1 and C2 are two transgenic clones. (C) Live cell imaging shows the localization of PF3D7_1019700::GFP in the nuclei by fluorescence microscopy. R: ring; ET: early trophozoite; LT: late trophozoite; ES: early schizont; LS: late schizont. (D) Proteins identified from parasite nuclear extracts by IP and LC-MS/MS. Single-step IPs with anti-GFP beads were done using the PF3D7_1019700::GFP (R1 and R2 indicate two replicates). The wild-type 3D7 was used as the IP control. The proteomic data were analyzed by SAINT using a threshold of probability >94% and 1% FDR. Gene ID and annotations are shown on the right.(PDF)Click here for additional data file.S6 Fig(A) Schematic showing BrD deletion by single crossover homologous recombination. (B) Southern blot analysis of three positive clones from transfected parasites. Genomic DNA was digested with AvrII and SpeI, and hybridized to the probe marked in A. (C) Schematic showing the deletion of PHD-TAF3 domain. (D) Southern blot of two positive clones from transfected parasites. Genomic DNA was digested with SpeI and XhoI, and hybridized to the probe marked in C. In both cases, GFP was tagged at the ends of truncated genes. (E) Western blot shows the size changes of the truncated GCN5 protein bands and the reduced expression levels after BrD deletion. (F). Images of Giemsa-stained films of parasite cultures synchronized at the ring stage to show the extended IDC of the two domain deletion parasite lines. (G) Live cell imaging shows GFP signals in parasites with truncated GCN5 in the GCN5-ΔBrd::GFP parasite line. Compared to H) Localization of truncated PfPHD1 in PHD1-ΔPHD::GFP parasite line. Scale bar = 5 μm. R, ET, LT and S denote ring, early trophozoite, late trophozoite and schizont stages, respectively. BF, bright field.(PDF)Click here for additional data file.S7 Fig(A) IFA images show high-degree co-localization of PfPHD1, H3K9ac and euchromatin (DAPI) in the nucleus in parasite line with c-Myc-tagged PfPHD1. Numbers indicate the levels of signal correlation between two markers. Scale bar = 5 μm. (B) IFA shows decreased co-localization of PfPHD1, H3K9ac and euchromatin (denoted by DAPI staining) after PHD1 PHD domain deletion. Scale bar = 5 μm. R, ring; T, trophozoite; S, schizont. (C) The intensities of H3K9ac in the 1N or 2N+ single nucleus of wild-type (WT), PfGCN5-ΔBrD::GFP, and PfPHD1-ΔPHD::GFP trophozoite. The copy number (1N or 2N+) in a single nucleus of trophozoite was defined by the intensity of DAPI signal compared to the 1N nucleus of the ring. Bars indicate means and standard deviations (n = 12). The statistical difference was evaluated by Mann–Whitney U test.(PDF)Click here for additional data file.S8 Fig(A–D) Volcano plots show the genes with altered transcription at the early trophozoite (A) and late trophozoite (B) stages in PfGCN5-ΔBrD, and at the early trophozoite (C) and late trophozoite (D) stages in PfPHD1-ΔPHD. (E) Pearson correlation in fold change between PfGCN5-ΔBrD and PfPHD1-ΔPHD in different developmental stages.(PDF)Click here for additional data file.S9 FigA, C) and down-regulated  genes in PfGCN5-ΔBrD  and PfPHD1-ΔPHD  parasites compared to the wildtype 3D7.Gene ontology enrichment analysis of up- ((PDF)Click here for additional data file.S10 Fig(A-D) Heatmaps display the alteration of gene transcription showing normalized z-scores to illustrate the gene expression cascade observed in 3D7 and those in mutant parasite lines. (A) Genes associated with protein translation and gene transcription. (B) Genes involved in the invasion of RBCs. (C) AP2-family genes. (D) Genes specifically expression in gametocytes and ookinetes. (E) The number of up- and down-regulated genes upon PfPHD1 PHD domain deletion are shown as the red and blue bars at four developmental stages. These altered genes were further classified as the putative target genes of AP2-LT and the remaining genes. The putative target genes of AP2-LT were significantly enriched in those down-regulated genes in the late stages of PfPHD1-ΔPHD::GFP parasites. R, ring; ET, early trophozoite; LT, late trophozoite; S, schizont. ***, P <0.001 (Fisher’s exact test).(PDF)Click here for additional data file.S11 FigMSP1 (A), VAR2CSA (B), and the sexual-stage gene Pfg27/25 (C). The enrichment was determined by chromatin-immunoprecipitation (ChIP) followed by qPCR using primer pairs marked as 1, 2, 3, or 4 located in the promoters of the respective genes. The fold change indicates the enrichment relative to constitutively expressed reference gene seryl-tRNA synthetase (PF3D7_0717700). (A) Upon domain deletion, PfGCN5-ΔBrD and PfPHD1-ΔPHD were depleted in the promoters of MSP1 at the schizont  but enriched at the ring stage (left panels). The right panels show the ratio of enrichments between domain deletions (PfGCN5-ΔBrD::GFP and PfPHD1-ΔPHD::GFP) and their wild-type parasites (PfGCN5::GFP and PfPHD1::myc).  At the ring stage, PfGCN5-ΔBrD and PfPHD1-ΔPHD were enriched at the promoters of var2csa (B) and Pfg27/25 (C). .Genes selected for evaluation include (PDF)Click here for additional data file.S12 Figvar2csa (A) and rifin (PF3D7_1000200) (B). Enrichment was determined at the ring stage by ChIP followed by qPCR using primer pairs marked as 1, 2, and 3 located in the promoters of the respective genes. The fold change indicates the enrichment relative to constitutively expressed reference gene seryl-tRNA synthetase (PF3D7_0717700). (A) H3K9me3 was depleted in the promoters of var2csa upon PfGCN5 BrD and PfPHD1 PHD deletion (upper panel) compared to the wild-type parasites (PfGCN5::GFP and PfPHD1::myc), whereas H3K9ac was enriched in both deletion mutants (lower panel). (B) Depletion of H3K9me3 and enrichment of H3K9ac were observed at certain positions of the rifin promoter upon BrD and PHD deletion, respectively.Selected HP1-controlled genes for evaluation include (PDF)Click here for additional data file.S1 TableA) Proteomic data from GCN5-PTP TAPs. (B) SAINT analysis of GCN5-PTP TAPs. (C) Proteomic data from PHD1-Myc IPs. (D) SAINT analysis of PHD1-Myc IPs. (E) Proteomic data from PF3D7_1019700-GFP IPs. (F) SAINT analysis of PF3D7_1019700-GFP IPs. (G) Proteomic data from PfGCN5-GFP IPs at ring stage. (H) SAINT analysis of PfGCN5-GFP IPs.((XLSX)Click here for additional data file.S2 TableA) Proteomic data from GCN5-GFP IPs. (B) SAINT analysis of GCN5-GFP IPs. (C) Proteomic data from GCN5-ΔBrD-GFP IPs. (D) SAINT analysis of GCN5-ΔBrD-GFP IPs IPs. (E) Proteomic data from PHD1-ΔPHD-GFP IPs. (F) SAINT analysis of PHD1-ΔPHD-GFP IPs.((XLSX)Click here for additional data file.S3 TableA), early trophozoite (B), late trophozoite (C) and schizont (D) stages.Deseq2 and TPM analysis of three replicates of RNAseq data at ring ((XLSX)Click here for additional data file.S4 TableA), early trophozoite (B), late trophozoite (C) and schizont (D) stages.Deseq2 and TPM analysis of three replicates of RNAseq data at ring ((XLSX)Click here for additional data file.S5 TableA) GO enrichment analyses of altered genes upon BrD domain deletion in GCN5. (B) GO enrichment analyses of altered genes upon PHD domain deletion in PHD1. (C) GSEA of altered genes upon BrD domain deletion in GCN5. (D) Summary of GSEA on GCN5-ΔBrD. (E) GSEA of altered genes upon PHD domain deletion in PHD1. (F) Summary of GSEA on PHD1-ΔPHD.((XLSX)Click here for additional data file.S6 Table(A) Up-regulation of var gene expression at early asexual stage upon domain deletions. (B) Down-regulation of invasion-related genes upon domain deletions. (C) Down-regulation of translation-related genes upon domain deletions. (D) Downregulation of transcription-related genes upon domain deletions. (E) Alteration of AP2 gene expression upon domain deletions.(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.S9 Table(A) for tagging, domain deletion, integration checking, and probe. (B) for ChIP-qPCR and FISH.(XLSX)Click here for additional data file.


By genotyping 1198 individuals with the Qiagen Investigator® HDplex Kit, we expand the Swiss autosomal STR dataset to 32 loci, providing additional resources for complex kinship cases. We present the first high-quality allele frequency dataset for loci D2S1360, D5S2500, D7S1517, and D10S2325 that will be accessible through the ENFSI reference database STRidER. For loci D3S1744, D4S2366, D6S474, D8S1132, and D21S2055, we provide a first European STRidER dataset.The online version contains supplementary material available at 10.1007/s00414-021-02624-w. The Qiagen Investigator® HDplex Kit provides valuable additional information to assess complex kinship scenarios, by supplementing the set of forensic standard short tandem repeat (STR) loci . We genoST values for the six regional sub-populations defined in [DNA extractions were prepared as described . Multiplfined in  revealedAll genotype data for the three loci D12S391, D18S51, and SE33 are concordant with the data previously generated with Promega PowerPlex® Fusion 6C [For marker D21S2055, heterozygote balance was usually below 60% if one allele consisted of less than 30 repeats and the other one of more than 30 repeats, limiting the utility of this marker for trace analysis and particularly DNA mixtures. However, there is no clear-cut allele length boundary for this phenomenon. We also observed pronounced peak imbalance between a couple of other pairs of alleles, e.g., 17.1/29 or 31/33. A similar observation has already been made by Tillmar et al. [Expanding the STR dataset increases the number of syntenic loci. Since ignoring linkage between loci might have an impact on certain kinship scenarios , we listThe present dataset has passed the quality control for STRidER  and obtaESM 1(XLSX 14.7 kb)ESM 2(TXT 7 kb)ESM 3(DOCX 12 kb)ESM 4(DOCX 13 kb)ESM 5(DOCX 21 kb)ESM 6(DOCX 948 kb)


Epigenetic mechanisms are central to understanding the molecular basis underlying tumorigenesis. Aberrations in epigenetic modifiers alter epigenomic landscapes and play a critical role in tumorigenesis. Notably, the histone lysine methyltransferase KMT2D  is among the most frequently mutated genes in many different types of cancer. Recent studies have demonstrated how KMT2D loss induces abnormal epigenomic reprograming and rewires molecular pathways during tumorigenesis. These findings also have clinical and therapeutic implications for cancer treatment. In this review, we summarize recent advances in understanding the role of KMT2D in regulating tumorigenesis and discuss therapeutic opportunities for the treatment of KMT2D-deficient tumors. Covalent modifications of histones sophisticatedly regulate chromatin architecture –4. Of thIn particular, H3K4 methylation is critical for epigenetic and spatiotemporal activation of gene expression –10. It cKMT2D (human chromosome 12 and mouse chromosome 15) is different from KMT2B  in both size and chromosomal location, although, confusingly, both of KMT2D and KMT2B have been called MLL2 .KMT2D gene is mutated and deleted in many different types of cancer, including medulloblastoma, melanoma, lymphoma, leukemia, and lung, prostate, renal, bladder, ovarian, pancreatic, esophageal, and gastric cancers [KMT2D has two frequently occurring genomic alterations: truncation and missense mutations  [Dnmt3a whose protein, DNMT3A, downregulates expression of several oncogenic Ras activators to suppress medulloblastoma [Bcl6 and Sirt1, whose proteins repress expression of several oncogenic Notch pathway genes to suppress medulloblastoma [Consistent with its loss-of-function mutations, KMT2D can act as a tumor suppressor . We havel (GEMM) . Mechaniblastoma . In addiblastoma .Kmt2d significantly promoted K-RasG12D–driven lung tumorigenesis by upregulating tumor-promoting programs [G12D–driven lung tumors [et al. [Kmt2d loss promoted melanoma in a GEMM and that KMT2D downregulated expression of glycolytic genes by increasing expression of IGFBP5, a tumor suppressor and negative regulator of IGF1R signaling. In a study of pancreatic cancer, Koutsioumpa et al. [We also demonstrated that lung-specific loss of programs . Interesg tumors . Similar [et al.  showed ta et al.  showed tKmt2d or KMT2D knockdown accelerated the oncogene (Bcl2)-driven lymphomagenesis of B cells in mice [Kmt2d deletion in CD19+ early B cells induced spontaneous lymphoma in 58% of mice [Studies have shown that genetic ablation of  in mice , 35. In  of mice , 35. A s of mice .et al. [in vivo. Another leukemia study showed that KMT2D occupied and activated pro-tumorigenic HOXA9 target genes and that KMT2D was required for leukemogenesis mediated by co-expression of HOXA9 and MEIS1in vivo [In contrast to its tumor-suppressive function, KMT2D appears to activate tumor-promoting pathways in certain contexts or tissue-specific manners . Santos et al.  have shoin vivo .et al. [KMT2D knockout inhibited cell proliferation and migration and reduced epithelial mesenchymal transition [In addition, Lv et al.  have shoet al. , 41. In ansition .A study using estrogen receptor (ER)-positive breast cancer cell lines showed that KMT2D knockdown reduced cell viability and increased the sensitivity of xenograft tumors to a PI3Kα inhibitor. In addition, KMT2D played a critical role in recruiting ER to ER target genes and activating such genes . In a bret al. [et al. [et al. [et al. [In patient-derived xenograft (PDX) melanoma models, KMT2D was required for the growth and invasion of N-RAS-mutated melanoma and positively regulated genes for tumor growth and cell migration . This fiet al. , which s [et al.  reported [et al. , althoug [et al.  showed t [et al. . In thesAn important question is how KMT2D can have tumor-suppressive or pro-tumorigenic function for a specific tissue, depending on whether the model system is a GEMM, a human cancer cell line, or a PDX model? GEMM models can harbor tumors that result from a few defined genetic alterations, whereas human cancer cell lines and PDX models often harbor multiple mutations. Such mutations may render a cancer cell line or PDX tumor dependent on KMT2D for survival. In such cases, KMT2D deficiency can attenuate the proliferation of affected cell lines or the growth of PDX tumors, even if KMT2D is a tumor suppressor. This point can be supported by the notion that simultaneous inactivation of two tumor suppressor genes, such as BRCA1 and Poly(ADP-ribose) polymerase, leads to synthetic lethality (cell death) . AnotherAnother critical question is how KMT2D can act as a tumor suppressor or a pro-tumorigenic factor in a tissue-dependent manner. One possibility is that tissue-specific transcription factors direct the KMT2D complex to a unique set of tumor-promoting genes or tumor suppressor genes in a tissue-dependent manner. For example, KMT2D associates with the tumorigenic factor HOXA9 during leukemogenesis . Relevanin vitro [KMT2D was initially identified as ALR in a multi-protein complex containing activating signal cointegrator 2 (ASC2) and other proteins . Later, in vitro , 56. In in vitro . TherefoKMT2D loss on H3K4 methylation were examined using mammalian cells with MLL3-null states  because KMT2D and MLL3 (homologues of the Drosophila H3K4 methyltransferase Trr) are closely related to each other. Our study also showed that KMT2D loss in K-RasG12D–driven lung tumors reduced global H3K4me1 levels while not having an obvious effect on global H3K4me3 levels [Interestingly, it has been shown that KMT2D acts as a H3K4 mono-methyltransferase or a H3K4 mono- and dimethyltransferase in mammalian cells , 58, 59.3 levels .in vitro activity of KMT2D. We have shown that loss of the Kmt2d gene in the mouse brain reduced H3K4me1 and H3K4me3 levels in many genes during medulloblastoma genesis. We have also reported that KMT2D activates gene expression by upregulating H3K4me3 at gene promoters in breast cancer cells and in the neuron-committed human embryonal carcinoma cell line NT2/D1 [Kmt2d loss in mouse B cells during lymphomagenesis decreases global H3K4me1, H3K4me2, and H3K4me3 levels [In addition to KMT2D-catalyzed mono- and dimethylation of H3K4, KMT2D-mediated H3K4 trimethylation has been reported in other cell lines and animal models, consistent with the e NT2/D1 , 56. Con3 levels .These studies indicate that KMT2D monomethylates H3K4 and can trimethylate H3K4 in a cell-type-specific manner. Although it is not clear how KMT2D–catalyzed H3K4 trimethylation is regulated in a cell-type-specific manner, one possibility is that the distinct composition or expression patterns of the core subunits WDR5, ASH2L, RBBP5, and DPY30 in different cell types play a role in KMT2D-mediated H3K4 trimethylation. Future studies may determine the detailed mechanism underlying KMT2D-mediated H3K4 trimethylation.Enhancers are a critical gene-activating signature that spatiotemporally enhances gene expression by interacting with and activating the promoters. Monomethyl H3K4 (H3K4me1) and acetyl H3K27 (H3K27ac) decorate the enhancers, which are co-occupied by transcriptional activators and co-activators  , 61. H3KMFGE8 and RPL39L [MYC and cFOS) [It has been reported that KMT2D activates pro-tumorigenic enhancers in different types of cancer . For insd RPL39L . In ER-pnd cFOS) .Dnmt3a and the medulloblastoma suppressor gene Bcl6 and upregulate these genes to suppress medulloblastoma genesis [IGFBP5 for melanoma suppression [Socs3, Asxl1 and Arid1A) for lymphoma suppression [In contrast to oncogenic super-enhancers, some super-enhancers are associated with tumor suppressor genes . We have genesis . We have genesis . Moreovepression  and the pression . Therefopression , embryonpression , and adipression .H3K4me3 is generally located in gene-regulatory regions spanning the promoters and the transcription start sites , 71. Thikmt2d loss in the brain downregulates broad H3K4me3 peaks to decrease expression of tumor suppressor genes  in non-small cell lung cancer were clonal [KMT2D mutations. For other types of cancer, the clonality of KMT2D mutations remains to be investigated.If gene mutations occur as early events and are present in all cancer cells during tumorigenesis, such mutations are clonal . The clonality of mutations is important because subclonal (branched) alterations may exist in only a subset of cancer cells and thus limit treatment efficacy if tumors bearing such mutations are treated . If mutae clonal . Moreovee clonal . These sKmt2d loss increases glycolysis in lung tumorigenesis, [Consistent with our finding that genesis, , we showgenesis, . Similargenesis, . Howevergenesis, , 33.Interestingly, pharmacological inhibition of the KDM5 family of H3K4 demethylases diminished tumorigenic growth of KMT2D-mutant lymphoma cells, as certain KDM5 family members can antagonize the function and methylation activity of KMT2D . Other sAs mentioned above, KMT2D has been described as either a tumorigenic factor or a tumor suppressor on the basis of results from the use of human cancer cell lines and their xenograft tumor models. Such results have also provided mechanistic insights into how KMT2D regulates tumorigenicity. However, because experiments using human cancer cell lines do not use native tumor microenvironments and human cancer cell lines often harbor multiple uncharacterized mutations, future studies using genetically defined mouse models  are desirable. This approach may allow researchers to assess whether results from cancer cell line experiments would be reproduced using GEMM experiments.G12D-driven tumorigenesis [Histone modifiers often posttranslationally modify non-histone substrates relevant to tumorigenesis. For instance, the histone lysine methyltransferase SMYD3 methylates the kinase MAP3K2 to upregulate MAP kinase signaling and accelerate K-Rasigenesis . It is pThe use of standard chemotherapy medicines, small molecular inhibitors, and immune checkpoint inhibitors has provided survival benefits for cancer patients. To improve cancer treatment, drug-combination therapies have frequently been used. Thus, it would be useful to study drug-combination approaches as therapeutic strategies of the treatment of KMT2D-deficient tumors. Recently, Tazemetostat, which inhibits the histone methyltransferase EZH2, was approved as the first epigenetic drug of solid tumors (epithelioid sarcomas and follicular lymphoma). Other epigenetic inhibitors are in clinical trials for the treatment of different cancers . Thus, t


Pivotal factors that contribute to tumorigenesis were subjected to analysis by molecular modeling. In particular, the FAM72A-UNG2 protein–protein interaction was modeled to predict a potential solution for the treatment of cancer. We screened chemical libraries to identify withaferin B as a lead molecule capable of interfering with the FAM72A-UNG2 interaction, thus opening new therapeutic avenues for cancer.Family with sequence similarity 72 A (FAM72A) is a pivotal mitosis-promoting factor that is highly expressed in various types of cancer. FAM72A interacts with the uracil-DNA glycosylase UNG2 to prevent mutagenesis by eliminating uracil from DNA molecules through cleaving the N-glycosylic bond and initiating the base excision repair pathway, thus maintaining genome integrity. In the present study, we determined a specific FAM72A-UNG2 heterodimer protein interaction using molecular docking and dynamics. In addition, through in silico screening, we identified withaferin B as a molecule that can specifically prevent the FAM72A-UNG2 interaction by blocking its cell signaling pathways. Our results provide an excellent basis for possible therapeutic approaches in the clinical treatment of cancer. Genomic uracil bases may occur from cytosine deamination or the misincorporation of dUMP residues during DNA replication . The uraHuman UNG exists in two different isoforms, mitochondrial UNG1 and nuclear UNG2, that are both encoded from the same single 13.5-kb nuclear UNG gene as a result of two separate promoters and alternative splicing ,13,14. WFamily with sequence similarity 72 A (FAM72A) is a novel gene expressed in the brain hippocampus area in proliferating neural stem cells, particularly during the G2/M-phase of the cell cycle ,21. MostTo understand the possible disruption of the FAM72A-UNG2 interaction, the current investigation conducted an in silico prediction of FAM72A-UNG2 heterodimer–protein interaction and the identification of potential chemicals that interfere with the FAM72A-UNG2 heterodimer protein activity for the potential treatment of cancer.The crystallographic three-dimensional (3D) protein structure of the FAM72A protein (from Protein Data Bank (PDB) data ) and UNGhttps://alphafold.ebi.ac.uk/), accessed on 3 November 2021) [FAM72A 3D protein structure was used from previously designed PDB data . Unfortuer 2021) ,41.The N-terminal regulatory region of UNG2 is described as an intrinsically disordered region by several groups ,17,42. IDocking interactions for the FAM72A protein with the modeled UNG2 peptide (AA 1–45) as a heterodimer were performed by HPEPDOCK (default parameters were applied) . The FAMThe molecular mechanics/generalized Born surface area (MM/GBSA) free energy decomposition per AA residue in protein–protein interactions was predicted on the FAM72A protein and UNG2 AA 1–45) peptide heterodimer  peptide .The protein carbon distribution (CARd) analysis was performed to validate the specific FAM72A-UNG2 interaction sites using our recently described algorithm ,47.A site-specific mutagenic approach was enabled to check the hot spot residues in the FAM72A protein and UNG2 (AA 1–45) peptide heterodimer interaction. AA modifications in FAM72A  and the effect on the conformational stability of the FAM72A protein and UNG2 (AA 1–45) peptide heterodimer complex were investigated by the BIOVIA Discovery Studio software  as described previously ,49.+ and Cl− ions were added to the simulation box. The structure was relaxed through a process called energy minimization (EM). Subsequently, energy-minimized structures were used for the system equilibration performed under constant NVT and NPT  ensembles. The production run was carried out using the NPT ensemble for 50 ns with a time step of 2 fs at a constant temperature of 300 K and 1 bar pressure. Simulation trajectories were visualized using Visual Molecular Dynamics (VMD) 1.9.4a42. Analysis of features, including root mean square deviation (RMSD), root mean square fluctuation (RMSF), and radius of gyration (Rg), were performed using GROMACS (2019) tools [The starting coordinates of FAM72A and UNG2 were taken from the modeled structures, as described in 9) tools ,52. Sinc9) tools . The RMSThe MTiOpenScreen , along wFurther molecular docking studies have been undertaken in order to gain further insights into the possible FAM72A-UNG2 binding interference by molecules newly identified by protein–ligand binding site prediction and to understand their mechanisms of interaction. Identified molecules obtained by the virtual screening were docked onto the FAM72A protein and UNG2 (AA 1–45) peptide heterodimer and/or FAM72A monomer using Schrödinger to depict binding mode and calculate binding energy ,61. FAM7The UNG2 protein can be functionally divided into two domains: an N-terminal regulatory region (AA 1–92) and a C-terminal catalytic region (AA 93–313). The disordered N-terminal region has been identified as interacting with several proteins, including proliferating cell nuclear antigen (PCNA) and replication protein A (RPA) (both found at DNA replication forks), as well as with FAM72A ,18,26,27Intrinsically disordered proteins (IDPs) execute various functions in all kinds of cellular processes ,65,66. TOur molecular docking study evaluated the molecular forces responsible for specific biomolecular FAM72A-UNG2 interactions. The FAM72A monomer was exported as a PDB file (FAM72A 3D protein structure was used from previously designed PDB data ), whereaAn MM/GBSA prediction is imposed on the free binding energy calculation in the FAM72A protein and UNG2 (AA 1–45) peptide heterodimer. The MM/GBSA analysis offered a breakthrough regarding catalytic AAs in the FAM72A protein and UNG2 (AA 1–45) peptide heterodimer. AA residue–residue contacts in the FAM72A-UNG2 heterodimer were calculated in terms of free binding energy, considering van der Waals forces, electrostatic energy, solvent accessible surface areas, and polar and non-polar energies. Binding affinity values were plotted as a scatter plot indicating key AA residues in FAM72A , while AA F104 was identified as pivotal AA. In proportion, the UNG2 (AA 1–45) peptide has been verified with only a few AAs accountable for binding contribution  .Site-directed specific mutations in the FWMF motif (AA 101–104) of FAM72A were used  to evaluate the rigidity and flexibility of the interface in the FAM72A protein and UNG2 (AA 1–45) peptide heterodimer binding .We modeled the FAM72A and UNG2 (AA 1–45) peptide and the interaction of FAM72A with the UNG2 (AA 1–45) peptide. The FWMF motif (AA 101–104) appears to be key for the FAM72A protein structure and its binding to UNG2 . A mutatSince phenylalanine F104 appeared to be the key AA within the FWMF motif (AA 101–104) at the interface of the FAM72A-UNG2 interaction, we further investigated the effect of FAM72A mutations at wt AA F104 phenylalanine  within the FWMF motif (AA 101–104) on the dynamic nature of FAM72A-UNG2 binding. Dynamic conformation changes in FAM72A-UNG2 (AA 1–45) binding were simulated by GROMACS and plotted by Grace . We assessed the effect of F104 mutations in FAM72A on the dynamic confirmation changes, stability, and rigidity of core and buried regions. Trajectories recorded up to 50 ns were plotted as RMSD, RMSF, and Rg, respectively. Knowing that AAs Trp/W125 and F104 in FAM72A are important for the FAM72A-UNG2 interaction, we changed wt FAM72A-W125 (FAM72A-wt) to FAM72A-W125A or W125R, and wt FAM72A-F104 to F104A, F104R, F104N, F104G, and F104S, respectively, and performed CARd analysis .wt-UNG2 (AA 1–45) heterodimer confirmed the FAM72Awt-UNG2 (AA 1–45) interaction and the effect of the various FAM72A mutations on it, as carbon governs cohesiveness at the active site in proteins. Accordingly, ICODs of the FAM72Awt-UNG2 (AA 1–45) interaction validate a strong overlap of FAM72A-UNG2 binding. Particularly, mutations in FAM72A turn the ICODs of FAM72A at AAs 100–104 and 125–135 into non-ICODs, encouraging a less favorable and weaker FAM72Amut-UNG2 induced-fit-binding than that of FAM72Awt-UNG2. Based on CARd analysis, F104 tends to switch to F104R > F104S > F104N > F104G > F104A. The likelihood of the F104 (uuu) mutation to S104: ucu appears to be highest, as only one nucleotide would be changed compared with the others .Internal carbon optimized domains (ICODs) of the FAM72AWe performed a virtual high-throughput screening to detect a potential lead interference with the FAM72A-UNG2 interaction ,72,73. BWithania somnifera (Indian ginseng)) and contain an oxapentacyclo moiety. Besides, in their central moieties, withaferin B contains octadecan-5-yl, whereas withaferin A contains octadec-4-en-3-one. Of note, withaferin A has been reported to be a potential anti-cancer molecule that can inhibit cell proliferation, cell migration, and cell invasion [Interestingly, withaferin B has structural similarities with withaferin A. Both compounds are withanolide analogues (derived from invasion ,84,85,86invasion ,29; howeAccumulating evidence indicates the involvement of FAM72A in tumorigenesis ,29,87,88


The avian influenza virus (AIV) subtype H9N2 circulating in Indonesia has raised increasing concern about its impact on poultry and its public health risks. In this study, the H9N2 virus from chicken poultry farms in Java was isolated and characterized molecularly.Thirty-three pooled samples of chicken brain, cloacal swab, trachea, and oviduct were taken from multiple chickens infected with AIV in five regions of Java, Indonesia. The samples were isolated from specific pathogenic-free embryonated eggs that were 9 days old. Reverse transcription polymerase chain reaction and sequencing were used to identify H9N2 viruses.hemagglutinin genes revealed a 96.9%–98.8% similarity to the H9N2 AIV isolated from Vietnam in 2014 (A/muscovy duck/Vietnam/LBM719/2014). According to the phylogenetic analysis, all recent H9N2 viruses were members of the lineage Y280 and clade h9.4.2.5. Nine of the H9N2 isolates studied showed PSKSSR↓GLF motifs at the cleavage site, while four had PSKSSR↓GLF. Notably, all contemporary viruses have leucine (L) at position 216 in the receptor-binding region, indicating that the virus can interact with a human-like receptor.This study was successful in detecting and characterizing 13 H9N2 isolates. The sequencing analysis of This study described the features of recent H9N2 viruses spreading in Java’s poultry industry. Additionally, H9N2 infection prevention and management must be implemented to avoid the occurrence of virus mutations in the Indonesian poultry industry. Since the early 1980s, Indonesia’s poultry sector has risen dramatically. The poultry industry in Indonesia is projected to be worth more than 34 billion US dollars. According to CASARED, there are approximately 67.9 million birds on 99,000 farms in Indonesia that use a commercially oriented production method .Mycoplasma gallisepticum and Escherichia coli or other infections from other viruses such as infectious bronchitis (IB) and newcastle disease (ND) can increase the mortality of chickens infected with H9N2 [In the last two decades, avian influenza virus (AIV) infection has become a common problem for the global poultry industry ,4, incluith H9N2 . CurrentThere is a dearth of evidence on the characteristics of circulating AIVs subtype H9. The bulk of these virus sequences are classified as G1 (clade h9.4.1) or Y280 (h9.4.2 of clade). The h9.4.1 lineage includes viruses from India, Iran, Israel, and Pakistan, whereas the h9.4.2 lineage had all strains found in China. Prior to 2007, H9N2 was classified as clade h9.4.2.1–h9.4.2.4. After 2010, the clade H9.4.2.5 represented by Guangxi/55/2005 became well established and began spreading rapidly throughout the country. Furthermore, all h9.4.2.5 viruses discovered between 2013 and 2015 possessed human-like receptor specificity, implying the possibility of cross-species transmission [hemagglutinins and neuraminidases in indirect enzyme-linked immunosorbent assay and dot blot studies, the H9N2 subtype was believed to exist in Java, Indonesia, duck population [Based on the duck sera’s susceptibility to recombinant pulation . This isThe study was carried out in April 2017–March 2018. The materials and methods used in this study are describe below.The current study follows the requirements established by the Indonesian Law on Animal Health Research  with a history of widespread depression, respiratory and neurological disorders, and decreased egg production. The poultry flocks comprised birds ranging in age from 25 to 79 weeks. Three moribunds or dead hens were randomly selected from each group and brought to the laboratory. The chicken brain, cloacal swab, trachea, and oviduct were taken and pooled for virus isolation. The samples collected are listed in A total of 33 commercial poultry chickens were sampled in 5 provinces of Java, Indonesia  embryonated eggs via the allantoic route. The eggs were incubated at 37°C for 48–72 h and daily embryo death was monitored. Eggs that died before 24 h were discarded because the virus considered was non-specific. Harvested allantoic fluid was used in the hemagglutination (HA) experiment. Three serial passages were conducted prior to the selection of negative samples [Propagation of the sample was carried out according to conventional laboratory protocols. The swab or tissue was suspended in sterile phosphate buffer saline with penicillin (200 μg/ml) and streptomycin (100 μg/ml) in a 2:10–20 ratio and centrifuged at 1,000×  samples .®, Taunton) was used to test the RNA for H9N2 viruses by RT-PCR utilizing H9 and N2-specific primers  mini kit . MyTaqTM OneStep RT-PCR kit .Following good RT-PCR results for the H9 gene, the HA gene was amplified and sequenced using the MyTaq One-Step RT-PCR kit (Bioline) with the HA9-F and HA9-R primers . ElectroThis study investigated several critical areas, including receptor-binding sites (RBS) on the left edge of the binding pocket, the right edge of the binding pocket, and the cleavage site. Additionally, certain amino acid residues  were evaluated for their association with H9N2 virus antigenicity. The amino acid sequences of the HA genes from the 13 most recent H9N2 viruses have been uploaded to http://www.cbs.dtu.dk/services/NetNGlyc/. to analyze probable N-glycosylation sites.The result revealed that 18 samples were HA-positive after three passaged in embryonated chicken eggs with mean log 2, 4, and 8 HA units , while tThe RT-PCR experiment detected the presence of a complete HA gene with an amplicon size of 1,683 bp in the allantoic fluid . The HA The characteristics of LPAIV seen from the sequence of HA segments reveal a monobasic amino acid motif at the cleavage site (PSRSSR↓GLF) in 9 out of 13 study isolates, while 4 other isolates, A/chicken/Banten-01/17 (Accession No. MG957200), A/chicken/Banten-02/17 (Accession No. MG957201), A/chicken/Banten-04/17 (Accession No. MG957202), and A/chicken/EastJava-05/17 (Accession No. MG957210), showed a non-synonymous substitution of the amino acid (R317K) at this cleavage site to make PSKSSR↓GLF. All isolates in the present study showed H173N and Q217M mutations at receptor-binding sites while comparing the reference strains in GenBank. The E180A substitution was found in only five of our isolates, while the remaining seven isolates contain the E180T substitution . The antigenic epitope of HA of H9N2 is mapped in two parts: site I and site II . The antThe potential glycosylation site of the HA gene analysis from 13 study isolates revealed 6 sites with N-X-T / S motif (where X is any amino acid except proline) in the HA1 gene section with positions in 11–13 (NST), 123–125 (NVS), 200–202 (NRT), 280–282 (NTT), 287–289 (NVS), and 295–297 (NCS) .Detection of H5N1 AIV from H9N2 positive cases indicated continuous co-circulation of the two subtypes in commercial chicken flocks. These study results agreed with previous studies  and provThe H9N2 virus has spread widely in poultry throughout the Asian region, providing the fact that the virus is potentially rapidly evolving with virulence rising ,26,27. IThe motif of the cleavage site of the recent isolate study was different from those of clades representative isolates. All H9N2 AIV fit the characteristic of LPAI. The proteases (such as trypsin), which are only secreted by the respiratory organs and intestines, will recognize a single arginine (R) residue .The h9.4.2 was categorized as h9.4.2.1–h9.4.2.6 . The recWhile vaccination is the optimum technique for preventing AIV, earlier research indicates that inadequate protective immunization may result in an antigenic drift of the H9N2 virus . To asseThe receptor-binding specificity was associated with amino acids in the RBS of HA proteins. In general, AIVs specifically recognize sialylα2,3-galactose receptors, while the human virus recognizes the sialyl-α2,6-galactose receptor . HemagluLeucine (L) at position 216 was found mainly in the HA protein of H2 and H3 viruses isolated from humans. In a previous study, Leucine (L) at 216 caused a van der Waals bond with the C-6 atom to form a sialyl-α2,6-galactose linkage. HA having leucine (L) at position 216 tends to bind to sialyl-α2,6-galactose, while Q in that position tends to bind to sialyl-α2,3-galactose. The substitution of Q to L in position 216 allows the H9N2 virus to replicate more efficiently in human cell culture . The subThe N183D mutation with Q216L at antigenic site II is involved in inhibiting viral interactions with epitope-specific antibodies and can result in mutant virus escape; both of these residues are identical to those in the most closely related vaccine. The 127 amino acid residue is another critical location in the HA H9N2 gene . Recent In general, N-linked glycosylation has a substantial effect on the virus’ biological features. It may be impacted by the N-X-S/T-X amino acid structure . AdditioIn five regions of Java, the H9N2 virus was isolated from chickens. The H9N2 virus that recently circulated in Java is classified as clade h9.4.2.5 and lineage Y280. Additionally, all recent viruses have a genetic link with viruses from China and Vietnam.AIV: Avian influenza viruses, HA: Hemagglutination, HAU; Hemagglutination unit, IBV: Infectious bronchitis virus, LPAIV: Low pathogenic avian influenza virus, ND: Newcastle disease, PBS: Phosphate buffer saline, RBS: Receptor-binding site, RNA: Ribonucleic acid, RT-PCR: Reverse transcription polymerase chain reaction, SPF: Specific pathogenic-free.