The HSV-1 (VC2) live-attenuated vaccine strain was engineered with specific deletions in the amino termini of glycoprotein K (gK) and membrane protein UL20, rendering the virus unable to enter neurons and establish latency. VC2 replicates efficiently in epithelial cell culture but produces lower viral titers and smaller viral plaques than its parental HSV-1 (F) wild-type virus. VC2 is an effective live-attenuated vaccine against HSV-1 and HSV-2 infections in mice and guinea pigs and an anti-tumor immunotherapeutic and oncolytic virus against melanoma and breast cancer in mouse models. Previously, we reported that the gK/UL20 complex interacts with the UL37 tegument protein, and this interaction is essential for virion intracellular envelopment and egress. To investigate the potential role of the UL37 deamidase functions, the recombinant virus FC819S and VC2C819S were constructed with a C819S substitution to inactivate the UL37 predicted deamidase active site on an HSV-1(F) and HSV-1(VC2) genetic background, respectively. FC819S replicated to similar levels with HSV-1(F) and produced similar size viral plaques. In contrast, VC2C819S replication was enhanced, and viral plaques increased in size, approaching those of the wild-type HSV-1(F) virus. FC819S infection of cell cultures caused enhanced GM-CSF secretion in comparison to HSV-1(F) across several cell lines, including HEp2 cells and cancer cell lines, DU145 (prostate) and Panc 04.03 (pancreas), and primary mouse peritoneal cells. VC2 infection of these cell lines caused GM-CSF secretion at similar levels to FC819S infection. However, the VC2C819S virus did not exhibit any further enhancement of GM-CSF secretion compared to the VC2 virus. These results suggest that the UL37 deamidation functions in conjunction with the gK/UL20 complex to facilitate virus replication and GM-CSF secretion.  Herpes simplex virus type 1 (HSV-1) is an ancient human pathogen of the Alphaherpesvirinae subfamily that evolved alongside the first humans . HoweverHSV-1 has a wide range of tissue tropism and can enter cells via fusion of the viral envelope with cellular plasma membranes or endocytosis ,4. FusioThe HSV-1(VC2) strain has specific deletions in the amino termini of glycoprotein K (gK) and the membrane protein UL20. These deletions alter gB-mediated fusion resulting in VC2 being unable to enter cells, including neuronal axons, via fusion of the viral envelope with plasma membranes. However, VC2 can enter epithelial and fibroblast cells in cell culture via endocytosis and replicate efficiently ,10. IntrHSV-1 structure includes many viral proteins embedded in the viral envelope, several capsid proteins surrounding the genome, and a layer of tegument proteins between the envelope and capsid. These tegument proteins serve many essential roles in the virus life cycle, such as facilitating virion transport, initiating viral gene transcription, virus assembly, and egress. While many of their essential functions are well described, these proteins often include multiple distinct functional domains with separate roles. The HSV-1 UL37 tegument protein is 1123aa long and is highly conserved among all members of the neurotropic Alphaherpesvirinae subfamily. UL37 is a deamidase involved in immunomodulation ,21,22,23The UL37 protein is required for cytoplasmic virion envelopment and can be transported to the cis-phase of the Golgi apparatus as a complex with the UL36 tegument protein in the absence of capsid formation . The UL3HSV-1 has developed a highly complex and multi-faceted approach to evading, suppressing, and modulating the immune response. A wide range of pathogen recognition receptors (PRRs) recognizes HSV-1, including TLR2, TLR3, TLR4, TLR9, RIG-I, MDA5, and cGAS (reviewed in ). AdditiOur laboratory has shown that VC2 has exceptional promise as an oncolytic and immunotherapeutic for the treatment of melanoma and other cancers; specifically, we have reported that VC2 is efficacious in the treatment of melanoma in a mouse immunocompetent and syngeneic model system and can generate strong anti-tumor immune responses ,28. ThisOne of the major cytokines in the recruitment of antigen-presenting cells, specifically dendritic cells, is granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF expression by Talimogene laherparepvec  stimulates anti-tumor immune responses. GM-CSF is 127aa in length with many glycosylation sites and is secreted by many cell types, including lymphocytes, fibroblasts, and endothelial cells  , but not VC2, results in enhanced GM-CSF secretion. These data indicate that this UL37 deamidation-dependent GM-CSF secretion is functionally associated with the UL37/gK/UL20 interactome.Vero (African green monkey kidney cells), HEp-2 , Panc 04.03 (human pancreatic cancer), and DU 145 (human prostatic cancer) were obtained from the American Type-Culture Collection (ATCC) . L929-/-cGAS  and L929-/-cGAS reconstituted with human cGAS  were a gift from Pinghui Feng; L929 cells are mouse subcutaneous fibroblasts. Veros, HEp-2, L929KO, and L929R, were maintained in Dulbecco’s Modified Eagle medium  supplemented with 10% fetal bovine serum  and 100 ug/mL Primocin . DU 145 cells were maintained on Minimum Essential medium supplemented with 10% fetal bovine serum  and 100 ug/mL Primocin . Panc 04.03 were maintained on RPMI-1640 complete medium  supplemented with 20 U/mL recombinant human insulin, 15% fetal bovine serum , and 100 ug/mL Primocin .Peritoneal cells were harvested from female Balb/CJ mice (8–10) weeks old. Mice were purchased from Jackson Laboratories  and housed at the Louisiana State University School of Veterinary Medicine laboratory animal facility.The wild-type virus used in this study was HSV-1 (F). HSV-1 (VC2) was previously generated in our lab on an HSV-1 (F) background to include gK Δ31–68 and UL20 Δ4–22 . PreviouFC819S and VC2C819S were developed using two-step Red recombination mutagenesis and synthetic oligonucleotides in Escherichia coli. The forward primer was 5′-GGGGCCCTGG CCCCCCGAGGCCATGGGGGACGCGGTGAGTCAGTACAGCAGCATGTATCACGAC GCCAAGCGCGCGCTGGTCGCGTCCCTAGGATGACGACGATAAGTAGGG-3′, and the reverse was 5′-GTGCGCCGTGGTTTCGGTGATGACGGAACGCAGGCTCGCG AGGGACGCGACCAGCGCGCGCTTGGCGTCGTGATACATGCTGCTGTACTGACTCACCGCGTCCCCCATGGCCTCGGGGGGCAACCAATTAACCAATTCTGATTAG-3′. These oligonucleotides were implemented on the bacterial artificial chromosome (BAC) plasmid pYEbac102-VC2 carrying HSV-1 (VC2) previously developed in our lab. The sequence was confirmed using viral genome sequencing.Growth kinetics were performed on confluent 12 well plates of Hep2, L929R, and L929KO in triplicate. Viruses were infected at an MOI of 1.0 and then adsorbed by rocking for 1 h at 4 °C. Plates were then rocked at RT for 1 h. Then plates were moved to incubate at 37 °C for 24 h post-infection (hpi). For whole-cell titers, plates were frozen at −80 °C and thawed three times, and cell lysates were collected at that time point. For secreted titers, cell media was collected and frozen at −80 °C. Fresh media was added to the plates. Then, plates were frozen and thawed three times to collect cell lysates. All samples were titrated on Vero cells and stained with crystal violet. The average and standard error of the mean for each sample was calculated for each virus and time point.The levels of secreted cytokines were determined by ELISA. Confluent monolayers were infected at an MOI of 5, and conditioned media was collected at 12 hpi. These samples were analyzed for secreted GM-CSF using the GMCSF human ELISA kit and following the manufacturer’s guidelines .z-axis) was generated to contain the protein. The protein was contained in the box, and the x and y axes were determined by providing a distance of 30 Å from the protein. Dipalmitoylphosphatidylcholine (DPPC) lipid models were used to insert the protein into a bilipid membrane. The bilipid membrane was built with the following using the MARTINI 2.1 forcefield: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), cardiolipin (CL) and glucosyl-lipopolysaccharide (UDP1). The coarse-grained molecular dynamics (CGMD) simulation was completed at 60 ns. At the end of the simulation, a snapshot was taken to convert the coarse-grained resolution to atomistic resolution using CG2AT-align.To compare differences in the structure of WT UL37 and UL37 C819S, we generated tertiary structural models using AlphaFold2 (Google Colaboratory). UL37 WT and UL37 C819S were aligned using PyMol to determine the root mean standard deviation value (RMSD). To determine structural changes and differences in membrane interaction of the gK/UL20 complex, we predicted protein–protein complex formation using AlphaFold2- multimer (Google Colaboratory). The amino acid sequences of human alphaherpesvirus-1 glycoprotein K (gK) (Accession Number AFH41179.1) and UL20 (Accession Number QFQ61390.1) were retrieved from GenBank and used as input, alongside the gKΔ31–68/UL20Δ4–22 amino acid sequences. The predicted models were inserted into a bilipid membrane using MemProtMD. Briefly, MEMEMBED was used to orient the protein relative to a lipid membrane. A box of 80 Å in size  software .Experiments were in experimental triplicate unless indicated otherwise. Replication kinetics were performed in technical triplicate and averaged. ELISA data were performed in technical duplicate and averaged. Replication kinetics were log-transformed. Statistical analyses were performed using a two-way analysis of variance (ANOVA) or a Student’s The HSV-1 (VC2) strain has been derived from HSV-1(F) by engineering the gK Δ31–68 and UL20 Δ4–22 amino acid deletions . To abroAs previously reported, VC2 produces smaller non-syncytial plaques than F A 9]. Th. Th9]. TNext, we investigated the impact of gKΔ31–68, UL20Δ4–22, and UL37C819S mutations on viral replication. Viruses were grown in HEp-2 cells at an MOI of 1 for 0, 6, 12, 24, and 48 h post-infection. HSV-1(F) and FC819S viruses exhibited similar growth kinetics, while VC2 titers were approximately ten-fold lower at 24 hpi but approached wild-type titers at 48 hpi B. The C8The UL37 C819 deamidates the pathogen recognition receptor cGAS. Using L929 cells without CGAS (L929KO) and those reconstituted with human cGAS (L929R), we tested the ability of different viruses to replicate in the presence and absence of cGAS. VC2 and VC2C819S replicated to a similar extent and less efficiently than F and FC819S in L929R cells, as seen in Vero and other cell lines. F, FC819S, and VC2 replicated to similar levels in L929KO and L929R cells. However, VC2C819S replicated significantly more efficiently than both VC2 and F viruses A,B. ThesVC2 produces a substantial adjuvant effect . TherefoViruses with deletions of the entire gK, UL20, or UL37 genes all exhibit similar phenotypes, with unenveloped capsids accumulating in the cytoplasm. All three proteins are involved in viral egress. We investigated the impact of the partial deletions of gKΔ31–68, UL20Δ4–22, and UL37C819S mutations on virus egress using TEM. At 18 h post-infection, cells infected with FC819S and VC2C819S appeared to have many virions attached to the extracellular plasma membrane in comparison to their respective parental strains HSV-(F) and VC2 .HSV-1 UL37 tertiary structure was modeled using AlphaFold2 as previously described . The C81Because gK and UL20 are known to complex together, we used AlphaFold2-multimer to model the tertiary structure of the gK/UL20 complex for both wild-type gK/UL20 and ΔgK31–68/ΔUL204–22. The gK/UL20 complex is embedded in the viral envelope. Therefore, these protein complexes were modeled in association with bilipid membranes using MemProtMD C,D. The Previous work in our laboratory has focused on identifying distinct functional domains and binding sites of gK and its binding partner, UL20. While best characterized for their roles in egress, they function in physical association with the UL37 tegument protein to facilitate cytoplasmic virion envelopment; they are also important modulators of viral entry and fusion through their interactions with the sole fusion glycoprotein gB ,39. DeleWe have shown previously that the gK/UL20 heterodimer interacts with the UL37 protein, and both function in cytoplasmic virion envelopment and egress . SpecifiThe VC2 virus produces a smaller plaque and replicates less efficiently than its parental wild-type HSV-1(F) . The FC8GM-CSF plays a vital role in monocyte, macrophage, and dendritic cell maturation and is critical for antigen presentation. For this reason, the oncolytic HSV-1 virus Talimogene laherparepvec  was engineered to express GM-CSF to enhance anti-tumor immune responses . We haveGM-CSF is not highly conserved across species, with humans and mice only sharing 56% homology. This species-specificity necessitates the creation of human or mouse species-specific versions of GM-CSF-inserted viruses. However, because FC819S, VC2, and VC2C819S can stimulate mouse GM-CSF as effectively as human GM-CSF, the viruses can be used across various models without the need for specialized gene tailoring. These qualities lend greater versatility to these models and their broader applications.We have previously predicted the structure of gK and UL20 using domain-specific modeling. In this manuscript, we utilized newer predictive algorithms to derive the predicted structure of the gK and UL20 heterodimer formation. Notably, the prediction of the gK/UL20 heterodimer having the gK and UL20 amino terminal deletions engineered in the VC2 virus in juxtaposition to their association with cellular membranes reveals that the gK/UL20 heterodimer is shifted inwards into the cellular membranes, potentially altering the availability of cytoplasmic domains of gK/UL20 to bind to other viral and cellular proteins. Notably, the horizontal alpha helix of gK that is shifted from the intracellular space into the plasma membrane is the most highly conserved region of gK, suggesting that it forms a critical function . AdditioThe observed enhanced secretion of GM-CSF by VC2 and FC819S viruses suggests that the UL37/gK/UL20 complex functions not only in virion morphogenesis and egress but also in the release of GM-CSF into extracellular spaces. Interestingly, VC2 does not replicate efficiently in cGAS-null cell lines, and the UL37C819S mutation restores replication. This result suggests that the UL37/gK/UL20 protein complex has additional functions in immunomodulation, partially by suppressing GM-CSF secretion from infected cells.


In addition, IL-6 and IL-10 mRNA expression also did not vary significantly in koala PBMCs based on KoRV subtype differences. We also investigated the Toll-like receptors (TLRs) response, including TLR2–10, and TLR13 mRNA in koala PBMCs infected with multiple KoRV subtypes. Significant differential expression of TLR5, 7, 9, 10, and 13 mRNA was observed in the PBMCs from koalas infected with different KoRV subtypes. Therefore, based on the findings of this study, it is assumed that co-infection of multiple KoRV subtypes might modify the host innate immune response, including IFN-γ and TLRs responses. However, to have a more clear understanding regarding the effect of multiple KoRV subtypes on host cytokines and TLR response and pathogenesis, further large-scale studies including the koalas negative for KoRV and koalas infected with other KoRV subtypes  are required.Koala retrovirus (KoRV) exists in both endogenous and exogenous forms and has appeared as a major threat to koala health and conservation. Currently, there are twelve identified KoRV subtypes: an endogenous subtype (KoRV-A) and eleven exogenous subtypes . However, information about subtype-related immune responses in koalas against multiple KoRV infections is limited. In this study, we investigated KoRV-subtype -related immunophenotypic changes, including CD4, CD8b, IFN-γ, IL-6, and IL-10 mRNA expression, in peripheral blood mononuclear cells (PBMCs) obtained from captive koalas (n = 37) infected with multiple KoRV subtypes (KoRV-A to F) reared in seven Japanese zoos. Based on KoRV subtype infection profiles, no significant difference in CD4 and CD8b mRNA expression was observed in the study populations. Based on the different KoRV subtype infections, we found that the IFN-γ mRNA expression in koala PMBCs differs insignificantly ( Phascolarctos cinereus) with leukemia, lymphoma, and immunodeficiency-like diseases [Koala retrovirus (KoRV) is a recently discovered gammaretrovirus found in koalas  have been reported ,12. KoRVCD4 and CD8 are important coreceptor molecules expressed on T helper cells and cytotoxic T lymphocytes, respectively, and are used to enhance responses mediated via the TCR ,21,22. TIn this study, we targeted captive koalas (n = 37) dwelling in seven different Japanese zoos, including Kanazawa Zoo, Tama Zoological Park, Kobe Oji Zoo, Awaji Farm England Hill Zoo, Hirakawa Zoological Park, Saitama Children’s Zoo, and Nagoya Higashiyama Zoo, and these represent all seven zoos in Japan which rear koalas. In all seven zoos, the koalas were maintained in an air conditioning system (23~25 °C). In addition, as much as possible, each habitat is designed to simulate the koalas’ natural habitat, and koalas were provided with an ad libitum supply of eucalyptus leaves. These koalas were available for blood sampling one time during the study period (June to October 2021) for the characterization of the innate immune response against multiple KoRV subtypes infections. From the captive koalas, whole blood samples were obtained by venipuncture using EDTA/heparin between June and October 2021. The koalas used in this study have previously been investigated for multiple KoRV subtype infections, including KoRV-A to -F . Figure Hematological examination was performed following standard protocols to determine white blood cell (WBC) count. Clinical signs of each koala used in this study were observed by trained zoo veterinarians to understand the health status of koalas. Genomic DNA (gDNA) was extracted from whole blood 300 µL) using a Wizard Genomic DNA Purification Kit (Promega), according to the manufacturer’s instructions. The purity and concentration of the extracted DNA were measured using a NanoDrop ND-1000 spectrophotometer . Extracted gDNA was used as a template for KoRV provirus determination by PCR analysis, and also for KoRV subtyping. KoRV provirus was confirmed by PCR analysis, using primers targeting KoRV pol gene, pol F (5′-CCTTGGACCACCAAGAGACTTTTGA-3′) and pol R (5′-TCAAATCTTGGACTGGCCGA-3′), as described previously 00 µL usi. The infPBMCs were isolated from whole blood samples obtained from the koalas, as reported previously  but withTo investigate the mRNA expression patterns of CD4, CD8b, cytokines , and TLRs (TLR2–10 and TLR13) in koala PBMCs, a one-step qRT-PCR was performed, as described previously ,30. The p < 0.05.A one-way ANOVA was performed using GraphPad Prism  to compare gene expression among different groups based on the KoRV subtype infection profiles, including KoRV-A, KoRV-A+B, KoRV-A+B+D, KoRV-A+B+F, KoRV-A+D+F, KoRV-A+B+C+D, KoRV-A+B+D+F, and KoRV-A+B+C+D+F. A Mann–Whitney U test was performed using GraphPad Prism (version 9) software to compare gene expression difference between the groups. Statistical significance was set at In this study, 37 captive koalas, including 13 male koalas between the ages of 11 months and 12 years (median age: 4 years 6 months) and 24 female koalas between 10 months and 24 years (median age: 5 years 7 months) across seven zoos in Japan, were used. Using KoRV pol gene-specific primers, PCR analysis of the gDNA extracted from whole blood showed that all the koalas were positive for the KoRV provirus. The infection status of the individual koalas for the KoRV subtypes, including KoRV-A, KoRV-B, KoRV-C, KoRV-D, KoRV-E, and KoRV-F, was determined in a previous study and confirmed by genotyping PCR and sequencing . Data onTo investigate the CD4 and CD8b mRNA expressions in koala PBMCs infected with multiple subtypes, we measured the expression levels of these molecules by qRT-PCR. The expression levels of CD4 and CD8b mRNA were normalized against koala beta actin mRNA levels. In one-way ANOVA analysis among the groups, CD4 and CD8b showed no significant difference based on infection subtype profiles A,B.p = 0.0534)  A. IL-6 a 0.0534) B,C.To investigate immune response based on KoRV subtype differences, we measured the expression patterns of TLR (TLR2–10 and TLR13) mRNAs in koala PBMCs by qRT-PCR. It was found that TLR2–10 and TLR13 were expressed at the mRNA level in koala PBMCs A–J. One-We also performed analysis between the groups such as KoRV-A+B vs. KoRV-A+B+D, KoRV-A+B vs. KoRV-A+B+F, KoRV-A+B+D vs. KoRV-A+B+C+D, KoRV-A+B+D vs. KoRV-A+B+D+F, and KoRV-A+B+D+F vs. KoRV-A+B+C+D+F using the Mann–Whitney U test to determine the effect of individual serotypes on CD4, CD8b, IFN-γ, IL-6, IL-10, and TLRs (TLR2–10 and TLR13) mRNA expressions in koala PBMCs. When comparing the KoRV-A+B and KoRV-A+B+D-infected groups, significant difference in the mRNA expression of IFN-γ, TLR3, TLR4, TLR7, and TLR10 was observed . HoweverWhen comparing the KoRV-A+B and KoRV-A+B+F-infected groups, only TLR6 mRNA expression showed a significant difference, and no other molecules showed any significant difference .When comparing the KoRV-A+B+D and KoRV-A+B+C+D-infected groups, IFN-γ, IL-6, TLR5, TLR7, and TLR10 mRNA expressions showed significant differences, and no other molecules showed any significant difference .When comparing the KoRV-A+B+D and KoRV-A+B+D+F-infected groups, TLR3, TLR5, and TLR10 mRNA expressions showed significant differences, and no other molecules showed any significant difference .When comparing the KoRV-A+B+D+F and KoRV-A+B+C+D+F-infected groups, no molecules showed any significant difference in gene expression patterns .Until recently, 12 KoRV subtypes are identified ,12, whicThis study will also extend the understanding of our recently published data , where wIn this study, the difference in CD4 and CD8b expression among different groups of multiple KoRV subtypes was not found to be significant in these koala populations. However, it was found that the presence of KoRV-C might influence a decrease in CD4 mRNA expression A. The saSignificant differences in the expression of TLR5, TLR7, TLR9, TLR10, and TLR13 mRNAs were observed, suggesting the effect of different KoRV subtypes on TLR expression. We also cannot rule out the possibility of the presence of genetically susceptible koalas, which would influence the host–virus interaction modulating immune response. However, the findings of the present study indicate that there are different expressions of TLRs, CD4, CD8b, and cytokines in these captive koala populations, which might be associated with different KoRV subtype infections. However, further large-scale studies including the koalas negative for KoRV and koalas infected with other KoRV subtypes  are required to gain a deeper understanding regarding the effect of KoRV subtypes on host immune response as well as koala health and conservation.


Cereibacter sphaeroides with two site-directed mutations Ile-L177–His and M197 Phe–His is of double interest. The substitution I(L177)H results in strong binding of a bacteriochlorophyll molecule with L-subunit. The second mutation F(M197)H introduces a new H-bond between the C2-acetyl carbonyl group of the bacteriochlorophyll PB and His-M197, which is known to enhance the stability of the complex. Due to this H-bond, π -electron system of P finds itself connected to an extensive H-bonding network on the periplasmic surface of the complex. The crystal structure of the double mutant reaction center obtained with 2.6 Å resolution allows clarifying consequences of the Ile L177 – His substitution. The value of the P/P+ midpoint potential in the double mutant RC was found to be ~20 mV less than the sum of potentials measured in the two RCs with single mutations I(L177)H and F(M197)H. The protein environment of the BChls PA and BB were found to be similar to that in the RC with single substitution I(L177)H, whereas an altered pattern of the H-bonding networks was found in the vicinity of bacteriochlorophyll PB. The data obtained are consistent with our previous assumption on a correlation between the bulk of the H-bonding network connected with the π-electron system of the primary electron donor P and the value of its oxidation potential.The photosynthetic reaction center of the purple bacterium  Cereibacter (C.) sphaeroides shows that this complex consists of three subunits and ten cofactors  P/P+ which is related to the energy level of P* after light excitation. In the wild-type RCs of purple bacteria, cofactors interact with integral L- and M-subunits through relatively weak non-covalent contacts. C. sphaeroides RC and some amino acid residues from their environment. Histidine residues, L173 and M202, coordinate central Mg2+ atoms of BChls PA and PB, respectively, and His-M182 is the ligand of BChl BB. In native C. sphaeroides RC, the C2-acetyl carbonyl group of the BChl PA forms an H-bond with His-L168. The C2-acetyl carbonyl group of BChl PB is free of H-bonds with its environment since the residue closest to this group and symmetry related to His-L168 is Phe-M197 [Photosynthetic reaction centers are integral membrane complexes responsible for the extremely efficient conversion of light energy into the chemical energy of separated charges. The crystal structure of the reaction center (RC) of the purple nonsulfur bacterium ofactors A 1]. Th. ThCereiPhe-M197  H mutant RC structure, shown in previous work 7H mutant. It is kpressure ,9. Basedhl dimer . HoweverC. sphaeroides RC to increase the stability of its structure and thereby increase the probability of obtaining the crystal structure of this complex with higher resolution. This work introduces the new double mutant RC and describes its spectral properties and oxidation potential of the primary electron donor P. X-ray crystal structure of the RC I(L177)H + F(M197)H, obtained with a resolution of 2.6 Å, clarifies some details of unusually strong pigment-protein interaction resulting from the Ile→His substitution at L177 position and shows that a new water molecule appears from β–side of BChl BB, which is responsible for hexa-coordination of the central Mg2+ atom of this cofactor. In addition, the crystal structure provides data in support of the previously stated assumption about the correlation between the bulk of the H-bonding network in the vicinity of the BChl dimer and the value of the oxidation potential of the primary electron donor.We have combined I(L177)H and F(M197)H mutations in puf-operon by simple cloning using available unique restriction sites [puf-M gene, similar to what was previously described [puf-operon with the mutations was cloned into the broad-host-range vector pRK415, containing DNA fragment that included the pufLMX genes. The resulting plasmid was transferred into C. sphaeroides strain DD13 [pufLMX genes.The I(L177)H and F(M197)H mutations previously introduced ,12 were on sites . Poly-hiescribed . The modain DD13  through The growth of recombinant bacterial strains under dark semi-aerobic conditions has been described elsewhere . The celY band amplitude at 48 °C as previously described [+ midpoint potential was determined by chemical titrations using potassium ferricyanide and sodium ascorbate as previously described [Thermal stability of the RCs was studied in Tris-Triton buffer with 250 mM NaCl by monitoring thermo-dependent changes of the Qescribed . Absorptescribed .121, were grown using the hanging drop vapor diffusion method as previously described in detail [C. sphaeroides (PDB ID: 3V3Z) [Trigonal crystals of the reaction center with the double mutation IL177)H + FM197)H, space group P3n detail . X-ray dn detail . Molecul7H + F(M1D: 3V3Z)  as the sD: 3V3Z) . Manual 7H, spaceD: 3V3Z) . The cooY P band at 865 nm, the QY B band at 804 nm, and the QY H band at 759 nm [Y P band near 847 nm, a diminished amplitude of the QY B band, as well as decreased absorption in the QX region of BChl absorption near 600 nm. The absorption spectrum of RC I(L177)H + F(M197)H showed similarity to that of RC I(L177)H, except that the blue shift of the QY P band was less pronounced and the maximum of this band was observed at 854 nm (The mutant RC with the double substitution I(L177)H + F(M197)H was stably assembled in photosynthetic membranes and the yield of the RC after purification was similar to that of the wild-type RC from the same amount of the cell mass. The absorption spectrum of the wild-type RC measured at room temperature shows the Qt 759 nm . In the t 759 nm . As desct 759 nm , the root 854 nm .Y B band amplitude at 48 °C in Tris-Triton buffer. It was shown that after 60 min of incubation in the spectrum of the wild-type RC, the amplitude of the QY B band at 804 nm decreased by 50%, whereas the spectra of the RCs I(L177)H, F(M197)H, and I(L177)H+F(M197)H demonstrated 55%, 40%, and 45% decreases, respectively H, F(M197)H, and I(L177)H+F(M197)H was determined by recording thermo-dependent changes of the Qectively . These d+ midpoint potential (Em P/P+) of the double mutant RC was determined by redox titrations as 555 ± 10 mV H results in a decrease of the Em P/P+ value by 50 mV [m P/P+ value to be ~575 mV. The crystal structure of RC I(L177)H + F(M197)H resolved in this study allows us to suggest a possible reason for the discrepancy between expected and experimentally observed mid-point potential P/P+ (discussed below).The value of the P/P ± 10 mV . Previouy 125 mV  due to tby 50 mV . AssuminB and PA is similar to that in the previously described structure of RC I(L177)H [B, which suggests the incorporation of a new water molecule H in the proximity of BChl Protation , which iB H + F(M197)H structure in the vicinity of PB  shows thC. sphaeroides, an H-bonding network also presents on the periplasmic side of BChl PA [A, the imidazole group of His-L168, side groups of Asn-L166, Asp-M184, and Tyr-L169 and a water molecule assigned as water-D. In the crystal structure of RC I(L177)H + F(M197)H, the positions of the side groups of the aforementioned residues did not change significantly, compared with those in the wild-type and F(M197)H RC structures (A and BB is similar to that in the RC with single substitution I(L177)H.It has been shown that in the wild-type RC of  BChl PA . The netructures . Althoug+ of the double mutant RC I(L177)H + F(M197)H as well as presented the crystal structure of the complex with 2.6 Å resolution. It is shown that the spectral properties of the double mutant RC are similar to those of RC I(L177)H, except that the blue shift of the QY P band is less pronounced, namely 11 nm compared with 18 nm for RC I(L177)H. It was noticed that while a single mutation F(M197)H did not lead to evident changes in the spectral properties of RC, the addition of this mutation to RC I(L177)H led to a significant red shift of the QY P band. This spectral shift of the QY P band is likely a result of a much more substantial out-of-plane rotation of the C2-acetyl carbonyl group of BChl PB (29.7º) compared with that observed in the structure of RC F(M197)H (20.7º) under the same conditions. As discussed previously in detail [Y transition in the mutant RC F(M197)H appears to be affected both by the rotation of the acetyl group of PB, which causes a red shift of the P QY band, and by a small increase in the distance between the two BChls of the dimer that leads to a blue shift of the band. In RC F(M197)H, these opposing effects compensate for each other, and as a result, the absorption spectrum remained practically the same as in the wild-type RC [B and suggest that it causes an additional 7 nm red-shift of the P QY band compared to the absorption spectrum of RC I(L177)H.We have characterized spectral properties and mid-point potential P/Pn detail , the ene-type RC . The resC. sphaeroides RC was performed based on its crystal structure obtained with 2.9 Å resolution (PDB ID: 3V3Z) [B and is suggested to be the 6th –ligand of the Mg2+atom of this BChl. This water appears to donate H-bond to the imidazole group of His-L177, which is also in contact with the methyl group of the pyrrole ring-1 of BChl PA H and F(M197)H mutations to increase the stability of the mutant RC to improve the resolution of its crystal structure. The data obtained demonstrate that RC I(L177)H + F(M197)H is slightly more thermostable than the wild-type RC but not as stable as RC F(M197)H. Nevertheless, after the introduction of an additional H-bond of P with its protein environment, we succeeded in improving the resolution of the crystal structure up to 2.6 Å. For the first time, an analysis of the structural consequences of the I(L177)H mutation in the  BChl PA . The dis present .C. sphaeroides RC was reported [m P/P+ and the patterns, sizes, and, apparently, the total strength of H-bonds in the clusters. The crystal structure of RC I(L177)H + F(M197)H provides data in support of this assumption.The mutant RC I(L177)H + F(M197)H presented in this work turned out to be of interest as an object that has a modified H-bonding network near the BChl dimer P. Recently, the presence of H-bonding clusters on the periplasmic surface of the reported . ComparaB in the double mutant RC structure (B through imidazole His-M197 became not as extended as in the F(M197)H RC. We assume that this can explain why the value of Em P/P+ in double mutant RC is ~20 mV lower than the sum of changes in the mid-point potentials caused by each of the two mutations. One should also not exclude the possibility that a more significant out-of-plane rotation of the acetyl group of PB reduces its conjugation into the π-electron system of the macrocycle, affecting both the total strength of H-bonds in the network and the value of Em P/P+. The loss of one H-bond in the network may also serve as an explanation for the fact that the thermal stability of RC I(L177)H + F(M197)H turned out to be less than that of RC F(M197)H [A with His L177, which is expected to shift P towards BChl BB. This assumption is consistent with more significant out-of-plane rotation of the C2-acetyl carbonyl group of PB, compared with that of RC F(M197)H.The main pattern of the H-bonding network in the protein environment of BChl Ptructure  was fountructure B. If theF(M197)H . A possiC. spheroids RC is an area of short-term binding of soluble cytochrome c2, which donates electrons for oxidized P [2. In the recent work of Allen et al., it was proposed that the wide-spread network on the periplasmic side of RC participate in a hydrogen-bonded linear pathway that transfers protons between the environment of P+ and the membrane surface [B H. In addition, the crystal structure of RC I(L177)H + F(M197)H illustrates that protein surroundings can affect the oxidation potential of the primary electron donor by altering the parameters of the H-bonding networks connected with the π-electron system of P.


Rhizoctonia solani Khün has a wide host range and threatens crop production. Various glycosyltransferases secreted by phytopathogenic fungi play an essential role in pathogenicity. Previously, we identified a glycosyltransferase RsIA_GT (AG11A_09161) as a secreted protein-encoding gene of R. solani AG-1 IA, whose expression levels increased during infection in rice. In this study, we further characterized the virulence function of RsIA_GT. It is conserved not only in Basidiomycota, including multiple anastomosis groups of R. solani, but also in other primary fungal taxonomic categories. RsIA_GT possesses a signal peptide (SP) for protein secretion, and its functionality was proven using yeast and Nicotiana benthamiana. The SP-truncated form of RsIA_GT (RsIA_GT(ΔS)) expressed in Escherichia coli-induced lesion-like phenotype in rice leaves when applied to punched leaves. However, Agrobacterium-mediated transient expressions of both the full-length RsIA_GT and RsIA_GT(ΔS) did not induce cell death in N. benthamiana leaves. Instead, only RsIA_GT(ΔS) suppressed the cell death induced by two reference cell death factors BAX and INF1 in N.benthamiana. RsIA_GT(ΔS)R154A D168A D170A, a mutant RsIA_GT(ΔS) for the glycosyltransferase catalytic domain, still suppressed the BAX- or INF1-induced cell death, suggesting that the cell death suppression activity of RsIA_GT(ΔS) would be independent from its enzymatic activity. RsIA_GT(ΔS) also suppressed the H2O2 production and callose deposition and showed an effect on the induction of defense genes associated with the expression of BAX and INF1. The transient expression of RsIA_GT(ΔS) in N. benthamiana enhanced the lesion area caused by R. solani AG-1 IA. The secreted glycosyltransferase, RsIA_GT, of R. solani AG-1 IA is likely to have a dual role in virulence inside and outside of host cells.Anastomosis group AG-1 IA of  Rhizoctonia solani Khün, the basidiomycete soil-borne fungal pathogen, infects many plants belonging to over 180 genera from 32 families . To see the distribution and conserved degree of the homologs of this protein in fungi, BLASTP searches against the non-redundant protein database ‘nr’ were performed with E-value of 1.0 × 10−10 and the minimum query coverage of 75%. We found homologous proteins in the fungal categories of Basidiomycota [386 proteins in 293 species], Ascomycota [881 proteins in 642 species], Mucoromycota [78 proteins in 63 species], Zoopagomycota [11 proteins in 10 species], and Chytridiomycota [14 proteins in 8 species]. In contrast, no homologous proteins were found in the earliest-diverging categories Microsporidia and Cryptomycota. These results implicate that fungi acquired and evolved this glycosyltransferase during the process of adaptation and co-evolution between pathogen fungi and hosts. To check the molecular evolutionary relationship of the glycosyltransferase in basidiomycetes, we analyzed selected 16 glycosyltransferase-homologous proteins in 13 fungal species in a phylogenetic tree. The maximum likelihood tree formed two major clusters for (1) proteins in Ceratobasidiaceae and Tulasnellaceae, and (2) proteins in other Agaricomycetes , one protein in R. solani AG-3 , and one protein in R. solani AG-8  (RsIA_GT encodes a 346-aa protein that contains a glycosyltransferase domain (57–241 aa). It belongs to ‘glycosyltransferase family 2’ in CAZymes classification  to a final OD600 of 0.5. All suspensions were incubated for 2–3 h in the dark prior to infiltration. Then, N. benthamiana leaves were infiltrated with the MMA incubation buffer containing pro::GFP-FLAG35S, pro::RsIA_GT(35SΔS), and pro::RsIA_GT-FLAG,35S respectively. The infiltration sites were challenged one day after with the MMA incubation buffer containing A. tumefaciens carrying pro::INF1-FLAG35S or pro::BAX-FLAG35S. A. tumefaciens strain GV3101 carrying the vector pro::GFP-FLAG35S was used as a negative control. Cell death status was observed and compared over the next few days.Strains of BLASTP searches of amino acid sequence of RsIA_GT against the GenBank  non-reduN. benthamiana, respectively, expressed RsIA_GT-FLAG, RsIA_GT(ΔS)-FLAG, INF1-FLAG, BAX-FLAG, and GFP-FLAG were harvested at two days post infiltration and powdered in liquid nitrogen using mortars and pestles. Resultant powders were re-suspended by 1.0 mL of extraction buffer  and centrifuged at 4 °C for 15 min. The supernatant containing the transiently expressed proteins was, respectively, collected into new centrifuge tubes. The extracted proteins were separated by electrophoresis in 12.5%, 10%, or 15% sodium dodecyl sulfate–polyacrylamide gels. Then, the separated proteins were transferred to nitrocellulose membranes at 25 volts for 10 min. The nitrocellulose membranes were washed three times by TBS-T buffer  and then blocked with TBS-T buffer containing 5% milk for 1.5 h at room temperature. The membranes were washed three times by TBS-T buffer and then incubated at 4 °C overnight with TBS-T buffer containing 5% milk and an anti-FLAG antibody . The membranes were washed three times with TBS-T buffer and then incubated with the mouse-anti-rabbit second antibody for 1 h at room temperature. Then, these membranes were visualized with BeyoECL Star kit  and photographed on X-ray films.Proteins were extracted, according to Moffett et al.  describeR. solani AG-1 IA, inhibits plant defense responses and promotes the pathogen infection. RsIA_GT(ΔS)R154A D168A D170A, the mutant of glycosyltransferase catalytic domain, still inhibited cell death, H2O2 production, and callose deposition induced by INF1 or BAX in N.benthamiana. Although further studies on the GT activity of the mutant RsIA_GT(ΔS)R154A D168A D170A are needed, this suggests that RsIA_GT(ΔS) might contribute to virulence independently of its enzymatic activity. Future studies on receptors or interacting proteins of RsIA_GT could be helpful to elucidate the pathogenic mechanism of R. solani AG-1 IA and provide a valuable target for disease resistance breeding.Our study provided evidence that RsIA_GT(ΔS), a secreted glycosyltransferase from


CACNA1H mutation represents a minor etiology in primary aldosteronism, it plays a significant role in causing uPAs in sporadic cases.Somatic mutations for excess aldosterone production have been frequently identified as important roles in the pathogenesis of unilateral primary hyperaldosteronism (uPA). Although CACNA1H mutation in patients with uPA and investigate the pathophysiological, immunohistological, and clinical characteristics of the variant.To identify novel somatic in vitro studies, demonstrating aldosterone production in variant-transfected human adrenal cell lines.We applied a customized and targeted gene panel next-generation sequencing approach to detect mutations from the uPA cohort in Taiwan Primary Aldosteronism Investigation study group. Information from pre-diagnostic to postoperative data was collected, including past history, medications, blood pressure readings, biochemical data, and image studies. The functional role of the variant was confirmed by CACNA1H mutation c.5809G>A  in a uPA case. The CACNA1H gene encodes the pore-forming alpha-1H subunit of the voltage-dependent T-type calcium channel Cav3.2. This somatic CACNA1H p.V1937M variant showed excellent clinical and biochemical outcomes after ipsilateral adrenalectomy. The functional effect of somatic CACNA1H p.V1937M variant results in increased CYP11B2 expression and aldosterone biosynthesis in HAC15 cells. A distinct heterogeneous foamy pattern of CYP11B2 and CYP17A1 expression was identified in immunohistological staining, supporting the pathological evidence of aldosterone synthesis.We identified a novel somatic CACNA1H p.V1937M might be a pathogenic driver in aldosterone overproduction. This study provides new insight into the molecular mechanism and disease outcomes of uPA.The somatic mutation of  KCNJ5 , CACNA1D , ATP1A1 (encoding α1 subunit of the Na+/K+ ATPase), and ATP2B3  genes, have been identified in at least 50% of patients with excess aldosterone production  is one of the common causes of primary aldosteronism (PA), while PA accounts for 5-10% of patients referred for evaluation of hypertension . In patiynthesis .CACNA1H p.Met1549Val, encoding the pore-forming α1H subunit of the voltage-dependent T-type calcium channel Cav3.2, was found in children with early-onset PA via autosomal dominant transmission, as a cause of familial hyperaldosteronism (FH) type IV , because the large gene size of CACNA1H escalates the challenges of sequencing through conventional Sanger’s approach, which could only target selected exons or hotspot regions of the gene  mutation in a patient with sporadic uPA. We further explored the pathophysiologic function of the classical uPA harboring CACNA1H variant on expressing aldosterone synthase.By using CYP11B2 immunohistochemistry (IHC)-guided targeted next-generation sequencing (NGS), aldosterone-driving somatic mutations were identified in over 90% of PAs . The detThis study was approved by the Institutional Review Board of National Taiwan University Hospital  and was conducted according to the principles of the Declaration of Helsinki. Written informed consent for clinical data collection, genomic analysis, and research use was obtained from the patient.Patients with primary aldosteronism were detected and diagnosed by following the updated consensus of the Taiwan Society of Aldosteronism  and werevia adrenal vein sampling (AVS) or dexamethasone suppression NP-59 SPECT/CT targeting adrenal tumor found on CT scan  at screening were regarded as possible cases of PA , testing CT scan . SubsequPatients were followed in the ambulatory office every month during the first three months after adrenalectomy and then every 3 months for at least one year. Their outcomes were measured by clinical and biochemical parameters according to the Primary Aldosteronism Surgery Outcome (PASO) criteria . Complet® Boost IHC Detection Reagent  was used . The sections were developed with the Liquid DAB+ Substrate Chromogen System  and counterstained with hematoxylin. Images were captured by using Olympus BX51 microscope with Olympus DP72 camera and processed using cell Sens Standard 1.14 software .The formalin-fixed paraffin-embedded (FFPE) sections were blocked with 10% goat serum  for 1 hour at room temperature, then incubated overnight at 4°C with mouse monoclonal antibody for CYP11B2 and 17α-hydroxylase (CYP17A1) (generous gifts from Professor Celso Gomez-Sanchez) , and ratAdrenal tumors were freshly preserved at −80°C. Genomic DNA was extracted from the adrenal tumor and peripheral whole blood. Genomic DNA from the adrenal tumor was extracted by using QIAamp DNA Mini Kit ; genomic DNA from whole blood was extracted by using Blood DNA Isolation Kit  according to the instructions from manufacturers.CACNA1H gene was investigated by a customized and targeted gene panel next-generation sequencing approach, the targeted panel of aldosterone-driving genes including KCNJ5, ATP1A1, ATP2B3, CACNA1D, CACNA1H, CLCN2, and CTNNB1. The library construction contained amplicons targeting the full coding regions of these genes. PCR-based amplified products using Fluidigm Access-Array technology are followed by barcoding and next-generation resequencing on an Illumina MiSeq platform as previously reported . PCR cycling conditions were as follows: initial denaturation at 95°C for 5 minutes followed by 20 cycles of denaturation at 95 °C for 30 seconds, annealing at 52 °C for 30 seconds, and extension at 72 °C for 1 minute. This is followed by final extension at 72 °C for 7 minutes and the amplified product was stored at 4 °C. Direct sequencing of PCR products was performed using the BigDye Terminator version 3.1 cycle sequencing kit  with a 3730 DNA Analyzer . Sequences were analyzed using DNAStar Lasergene SeqMan Pro 7.1.0 softwareThe somatic in silico) programs of Combined Annotation Dependent Depletion   protein with the mutated sequence was performed by a Protter tool  , PolymorD, v1.2) , SortingD, v1.2) , and theD, v1.2)  were appter tool HAC15 human adrenocortical cells  were culCACNA1H as well as CACNA1H p.V1937M mutation were constructed into the pIRES-EGFP-puro vector (Addgene plasmid #45567) using PCR-assisted, site-directed mutagenesis. We used PCR-based direct sequencing to confirm the mutation was successfully cloned into the vector. To evaluate the effect of mutant CACNA1H on the expression of CYP11B2 and aldosterone secretion, the HAC15 cells were transfected with pIRES-EGFP-wild-type CACNA1H or pIRES-EGFP CACNA1H p.V1937M using an Amaxa Nucleofector I  according to the manufacturer’s instructions. The pIRES-EGFP empty vector was used as a control. After transfection, HAC15 cells were seeded at a density of 1 × 106 cells/well into a 6-well plate. The cells were exposed to 10 nM angiotensin II  48 hours after transfection. The culture supernatant was collected 72 hours after transfection for measuring the concentrations of aldosterone, and cells were harvested for Western blot analysis.The plasmids expressing the wild-type Whole-cell lysates were extracted by RIPA buffer  containing a protease inhibitor . A 30 μg total protein was separated using 10% SDS-PAGE gels and electrophoretically transferred onto PVDF membranes. The membranes were then blocked for 1 hour at room temperature by using the BlockPRO™ blocking buffer  and then incubated overnight at 4°C with mouse monoclonal antibody for CYP11B2 (a kind gift from Professor Celso Gomez-Sanchez) at 1:1,000 dilution, and anti-GAPDH antibody  at 1:10,000 dilution in blocking buffer. After washing in Tris-buffered saline containing 0.1% Tween-20 (TBST) buffer, the membranes were incubated with HRP-conjugated anti-mouse secondary antibody  at 1:3,000 dilution or HRP-conjugated anti-rabbit secondary antibody  at 1:10,000 dilution for 1.5 hours at room temperature. Levels of proteins were detected by using the chemiluminescent detection reagents  which were used to detect the protein levels and visualized using a UVP Biospectrum 810 imaging system . The intensity of each protein band was quantified by UVP software and normalized to GAPDH levels.CACNA1H or p.V1937M mutation plasmid for 72 hours, the culture supernatant was collected to measure the aldosterone concentrations by using ALDO-RIACT RIA kit . Aldosterone levels were normalized against protein concentrations of cell lysates.After HAC15 cells transfection with wild-type post-hoc least significant difference (LSD) tests. Statistical analyses were performed with commercial statistical software SPSS . A value of p < 0.05 was considered to indicate statistical significance.Experimental data were presented as mean ± standard error of mean (SEM) and the differences among groups with different transfected genes were analyzed by one-way ANOVA with CACNA1H c.5809G>A   single photon-emission computed tomography (SPECT) and adrenal venous sampling both showed a hyperfunctioning left unilateral PA  were applied to aid in the impact of every presumed polymorphism, revealing damaging and functional consequences of this missense variant. Besides, sequence analysis of the patient’s germline DNA has confirmed this variant is truly somatic. Therefore, this novel somatic variant of CACNA1H p.V1937M could be considered a possible driver in patients with uPA.The NGS-derived variant of idelines . First, CACNA1H leads to a clear gain-of-function effect of calcium signaling on aldosterone production and cell proliferation  type II   –46. Germoperties .CACNA1H p.V1937M somatic mutation revealed a distinct pattern of heterogeneous immunoreactivity of CYP11B2 and CYP17A1 expression with foamy cells. The heterogeneity of CYP11B2 and CYP17A1 expression was observed in the somatic mutation of CACNA1H I1430T as well  can be found below: National Center for Biotechnology Information (NCBI) BioProject database under accession number SCV002062061.1.The studies involving human participants were reviewed and approved by the institutional review board of National Taiwan University Hospital. The patients/participants provided their written informed consent to participate in this study.Conceptualization, C-ST, K-YP, and V-CW; methodology, K-YP; software, K-YP; validation, C-ST, K-YP, and V-CW; formal analysis, K-YP; investigation, W-CL, and V-CW; resources, S-MW, Y-CT, K-HH, W-CL, and V-CW; writing—original draft preparation, C-ST and K-YP; writing—review and editing, V-CW and JC; visualization, C-ST and K-YP; supervision, V-CW and JC; project administration, V-CW; funding acquisition, V.-C.W. All authors have read and agreed to the published version of the manuscript.This research was funded by the Ministry of Science and Technology, Taiwan, R.O.C. , National Health Research Institutes [PH-102-SP-09)], National Taiwan University Hospital , Grant MOHW110-TDU-B-212-124005 and Mrs. Hsiu-Chin Lee Kidney Research Fund, National Taiwan University New Faculty Grant (110L7460 and 111L7432), and Most 110-2314-B-002-239.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


CACNA1C encoding Cav1.2 cause syndromic or non-syndromic type-8 long QT syndrome (LQTS) (sLQT8 or nsLQT8). The cytoplasmic domain (D)Ⅰ-Ⅱ linker in Cav1.2 plays a pivotal role in calcium channel inactivation, and mutations in this site have been associated with sLQT8 (such as Timothy syndrome) but not nsLQT8.Gain-of-function mutations in CACNA1C mutation, located in the DⅠ-Ⅱ linker, associated with nsLQTS, we sought to reveal its biophysical defects.Since we identified a novel 2+ or Ba2+ currents (ICa or IBa) were recorded using whole-cell patch-clamp techniques.Target panel sequencing was employed in 24 genotype-negative nsLQTS probands (after Sanger sequencing) and three family members. Wild-type (WT) or R511Q Cav1.2 was transiently expressed in tsA201 cells, then whole-cell CaCACNA1C mutations, a previously reported R858H mutation and a novel R511Q mutation located in the DⅠ-Ⅱ linker. Four members of one nsLQTS family harbored the CACNA1C R511Q mutation. The current density and steady-state activation were comparable to those of WT-ICa. However, persistent currents in R511Q-ICa were significantly larger than those of WT-ICa . The steady-state inactivation of R511Q-ICa was weak in comparison to that of WT-ICa at higher prepulse potentials, resulting in increased window currents in R511Q-ICa. Slow component of inactivation of R511Q-ICa was significantly delayed compared to that of WT-ICa . Inactivation of R511Q-IBa was still slower than that of WT-IBa, indicating that voltage-dependent inactivation (VDI) of R511Q-ICa was predominantly delayed.We identified two Ca cause nsLQT8. Our data provide novel insights into the structure-function relationships of Cav1.2 and the pathophysiological roles of the DⅠ-Ⅱ linker in phenotypic manifestations.Delayed VDI, increased persistent currents, and increased window currents of R511Q-I KCNQ1 for LQT1, KCNH2 for LQT2 and SCN5A for LQT3—account for approximately 90% of genetically affected LQTS patients, while those in other causal genes have rarely been identified } or y = 1/{1+exp[(Vm-V1/2)/K]}, respectively, where y is the relative current, Vm is the membrane potential, V1/2 is the voltage at which half of the channels are available to open, and K is the slope factor. The time course of inactivation was fitted with a single or double exponential function of the following form: I(t)/Imax = A0+A1[1-exp(-t/τ)] or I(t)/Imax = A0+Af[1-exp(-t/τf)]+As[1-exp(-t/τs)], where A and τ refer to the amplitudes and time constants, respectively, and f and s refer to the fast and slow components, respectively. I refers to the current, and t refers to the time. The time course of recovery from inactivation was fitted with a single exponential function of the following form: I(t)/Imax = A0+A1exp(-t/τ), as described previously . , 8. Ca icurrents  17]. Mu. MuCa in density  18]. In. InCa in or COTS , which mKorkosh et al. recently built structural models of Cav1.2 and proposed mechanisms underlying the VDI: The cytoplasmic N-terminal part of VSDII (DIIS0) in the DⅠ-Ⅱ linker is bound with the α1-interaction domain (AID). Following voltage-dependent channel activation, the cytoplasmic face of DIIS0 would perturb and shift DIIS0-bound AID toward the pore axis. The AID-linked DIS6 would bend at the flexible G402 and G406, facilitating the activation-gate closure and thus the VDI . The R51CACNA1C R511Q mutation)-derived hiPSC-CMs and mutant allele-specific knockout using a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (CRISPR-Cas9) system may reveal the pathophysiological roles of the CACNA1C R511Q in QT prolongation and arrhythmogenesis.TS patient- and COTS patient-derived human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been established , 38. An CACNA1C R511Q mutation, but not all carried the KCNE1 D85N variant which was reported to be a disease-causing or a drug-induced LQTS variant [CACNA1C R511Q mutation might be a main contributor to QT prolongation in this family, and the KCNE1 D85N variant may have contributed less to QT prolongation in comparison to the CACNA1C R511Q mutation. Although the father (Ⅱ-3)   harbors CACNA1C mutations: An R858H mutation in one proband and a novel R511Q mutation in one family. This supported the notion that nsLQT8 is more prevalent than previously expected. Despite the fact that CACNA1C mutations in the DⅠ-Ⅱ linker have been associated with sLQTS , the R511Q mutation in this site is associated with nsLQT8. The biophysical defects of the R511Q mutation were a delay of slow component of ICa inactivation through predominant impairment of the VDI, increased persistent currents, and increased window currents, without a change of the current density. The degree of functional impairment associated with the R511Q was more modest in comparison to that seen with TS, aTS and COTS mutations. These findings provide novel insights into the structure-function relationships of Cav1.2 and the pathophysiological roles of the DⅠ-Ⅱ linker in phenotypic manifestations.Target panel sequencing in 24 genotype-negative nsLQTS patients after Sanger screening identified two


Neoepitopes formed by the conversion of glutamine (Q) residues to glutamic acid (E) by deamidation are central to the immune pathogenesis of coeliac disease and have been implicated in autoimmune responses in T1D. Here, we asked if the immunogenicity of full-length C-peptide, which comprises four glutamine residues, was enhanced by deamidation, which we mimicked by substituting glutamic acid for glutamine residue. First, we used a panel of 18 well characterized CD4+ T-cell lines specific for epitopes derived from human C-peptide. In all cases, when the substitution fell within the cognate epitope the response was diminished, or in a few cases unchanged. In contrast, when the substitution fell outside the epitope recognized by the TCR responses were unchanged or slightly augmented. Second, we compared CD4+ T-cell proliferation responses, against deamidated and unmodified C-peptide, in the peripheral blood of people with or without T1D using the CFSE-based proliferation assay. While, as reported previously, responses were detected to unmodified C-peptide, no deamidated C-peptide was consistently more stimulatory than native C-peptide. Overall responses were weaker to deamidated C-peptide compared to unmodified C-peptide. Hence, we conclude that deamidated C-peptide does not play a role in beta-cell autoimmunity in people with T1D.Type 1 diabetes (T1D) is a T-cell mediated autoimmune disease in which the insulin-producing beta cells are destroyed. While it is clear that full-length C-peptide, derived from proinsulin, is a major antigen in human T1D it is not clear how and why C-peptide becomes a target of the autoimmune CD4 While c+ T cells in the immune pathogenesis of T1D, including the following. Genome-wide association studies (GWAS) have revealed that the risk of developing T1D is strongly associated with the HLA class II specifically the haplotypes: HLA-DR3-DQ2 (HLA-DRB1*03:01-DQA*05:01-DQB*02:01) and HLA-DR4-DQ8  [trans) [+ T cells ‘help’ is required for antibody isotype switching. Isotype switched autoantibodies, specific for beta-cell antigens, arise before the clinical onset of T1D, suggesting that CD4+ T cells play a direct role [+ T cells, restricted by T1D-associated HLA allomorphs, infiltrate the pancreatic islets of people with T1D, putting CD4+ T cells at the ‘scene of the crime’ [Several lines of evidence point at a central role for CD4ect role . Finallye crime’ .+ T cells that infiltrated the residual pancreatic islets of deceased organ donors who suffered from T1D revealed that many islet-infiltrating CD4+ T cells were specific for epitopes derived from the C-peptide of proinsulin [+ T-cells recognized C-peptide epitopes presented by HLA-DQ8, or HLA-DQ8trans [+ T cells infiltrate human islets in T1D has subsequently been confirmed by others [+ T-cell responses against C-peptide we asked if CD4+ T-cell responses against full-length C-peptide (PI33-63) could be detected in the peripheral blood of people with, or without, T1D. We found that C-peptide specific responses could be detected in PBMC samples from over 60% of people with T1D [trans and DQ2trans [+ T-cell responses against full-length C-peptide [+ T-cell responses play a direct role in the autoimmune pathogenesis of human T1D.Insulin, and its precursor proinsulin, are important targets of autoimmune responses in T1D . C-peptioinsulin . Many ofDQ8trans . The obsy others . To furtwith T1D . C-peptiDQ2trans . More re-peptide . Hence t+ T cells in T1D [+ T-cell responses associated with coeliac disease targeted gliadin epitopes which are rendered immunogenic by deamidation of glutamine residues [A central question in autoimmunity is: why does the immune system target otherwise healthy tissues? One compelling hypothesis is that the autoimmune response targets, to some extent, self-antigens that have been modified. These modified self-epitopes, known collectively as neoepitopes, could be the targets of autoimmune response in autoimmune diseases ,15. Thiss in T1D ,15,17. Ns in T1D ,18. One s in T1D ,20. Coels in T1D . Like T1residues ,22,23.+ T cells in people with T1D [33-45 (EAEDLEVGEVELG) bound more strongly to HLA-DQ8 and HLA-DQ8trans than the native sequence (EAEDLQVGQVELG) [30-43, TRREAEDLEVGEVE) peptide, than native PI30-43 peptide (TRREAEDLQVGQVE) in samples from subjects with recent onset T1D [+ T-cell responses to deamidated C-peptide initiated anti-beta cell autoimmunity which then spread to unmodified C-peptide. More recently, exposure of human islets, in vitro, to inflammatory cytokines promoted the formation of deamidated C-peptide, suggesting a mechanistic link between T-cell infiltration/inflammatory cytokines and the deamidation of C-peptide and autoimmune T-cell responses [Deamidation of beta-cell antigens, and proinsulin specifically, have been suggested to create neoepitopes that are recognized by T1D-associated CD4with T1D ,24. Betawith T1D . It has VGQVELG) . They alnset T1D . This leesponses . Furtheresponses .+ T-cell responses against full-length C-peptide and the role of neoepitopes formed by deamidation in the immune pathogenesis of T1D, we asked the following question. Compared to unmodified C-peptide, does deamidated C-peptide stimulated stronger, or more frequent, CD4+ T-cell responses in PBMC of people with T1D? We took two approaches to answering this question. First, we analyzed the impact of glutamine deamidation on the responses of C-peptide specific T-cell lines that express TCRs derived from human islet-infiltrating CD4+ T cells. Second, we compared CD4+ T-cell responses against unmodified full-length C-peptide (PI33-63) to responses against variants of C-peptide comprising glutamine (Q) to glutamic acid (E) substitutions in PBMC from individuals with recent onset T1D. Neither approach provided any evidence that deamidated C-peptide was more immunogenic than unmodified C-peptide. Hence, we conclude that deamidated C-peptide, in contrast to unmodified C-peptide, is not an important target of autoimmune CD4+ T-cell responses in people who develop T1D.Given the strong evidence for CD422.1Ethical approval was given by St Vincent's Hospital (HREC-A 135/08), Royal Melbourne Hospital (2009.026) and Monash Health (12185B) ethics committees. All participants provided written informed consent. T1D was diagnosed according to American Diabetes Association criteria . DetailsSupplementary Tables 1 and 22.2Epstein Barr Virus (EBV) transformed B cell lines were used as antigen presenting cells (APC). 2.3BamHI/SalI digestion and replaced by human TRAC or TRBC with either a PmeI (TRAC) or SbfI (TRBC) site at the 5′ end of the TCR constant regions. All TCR genes were synthesised by IDT as geneblocks and cloned into the modified pRRLSIN vectors by In-fusion cloning according to the manufacturer's (Takara) protocol. Plasmids were extracted from growing E. coli and the inserts were verified by Sanger sequencing. For transfection of HEK293T cells, TCR gene-containing plasmids were purified using Macherey-Nagel kits according to the manufacturer's recommendations. HEK293T cells were transfected with the appropriate pRRLSIN construct and the packaging plasmids: pMDLg/pRRE, pRSV-Rev, and pMD2.g using Lipofectamine 2000 (Invitrogen) as per manufacturer's instructions. After 7–8 h incubation at 37 °C, 5% CO2, the medium was changed. After a further 1–2.5 days incubation, supernatant was collected and filtered through a 0.45 μm low protein binding filter. Lentivirus-containing supernatant was either used fresh, or frozen at -80oC until required. Plasmids used for making lentiviruses are listed in 2.46/ml with 5 μg/ml polybrene. Cells were centrifuged at 1200 rpm (300×g) for 60min at room temperature, then diluted 1:1 in fresh medium and incubated overnight at 37 °C, 5% CO2. Cells were grown for a week; during this time, they were washed six times. Transduced cells were purified either by flow cytometry or magnetic bead enrichment, as described below.Jurkat cells were resuspended in viral supernatant at 1–2 x 102.5+/TCRαβ+ cells.Flow cytometry was performed on a Becton Dickinson LSR Fortessa. The following anti-human mAbs were used for FACS staining: CD3-PE  anti CD4-PE  anti-TCRαβ-AF647 . Cells were stained with the appropriate mAb in PBS 0.1% FBS for 20 min on ice, then washed twice. Dead cells were excluded by propidium iodide staining. Compensation settings were determined using single color controls. TCR transduced Jurkat cells were purified by sorting viable (propidium iodide negative) CD32.66 cells were labelled with REALease CD3-biotin followed by REALease anti-biotin MicroBeads. Labelled cells were applied to a MS MACS column on a magnetic stand. After washing through the unlabeled cells, the column was removed from the magnet and the CD3-expressing cells were eluted. Then the microbeads and the REALease Complex were removed. After washing in PBS, the purified cells were cultured for 6–7 days before purity was checked by staining for CD3 and TCRαβ (as above) and FACS analysis. In some experiments, the number of viable cells were counted by Trypan Blue exclusion using a hemocytometer at different times after purification.A REALease CD3 Microbead Kit (Miltenyi) was used to purify TCR-transduced Jurkat cells. Briefly, 3–5x102.74/well with an equal number of EBV-transformed B cells  and optical density (OD) measured using a FLUOstar Omega microplate reader (BMG LabTech). The amount of IL-2 secreted was determined using a linear regression of all OD values (minus the background) of the standard curve. The maximum response was defined as the amount of IL-2 secreted by the PMA/Ionomycin stimulated Jurkat T cells. The 50% effective concentration (EC50) was calculated and the fold change of the amount of IL-2 secreted in response to the deamidated peptides relative to the amount secreted in response to native C-peptide was determined.ELISAs were performed using the ELISA MAX™ Deluxe set Human IL-2 (BioLegend) as per the manufacturer's instructions. Briefly, 96-well ELISA plates (Greiner Bio-One) were coated with 100μL/well of purified anti-human IL-2 antibody diluted 1:200. The following day, the plate was washed three times in 0.05% Tween/PBS, then blocked with 200μL/well of 1%BSA/PBS at room temperature for 1 h. The Jurkat supernatant (100 μL) was transferred to the ELISA plates. An IL-2 standard was prepared by two-fold serial dilutions of 1,000 pg/mL recombinant IL-2. were prepared and added, in triplicates, to the mAb coated ELISA plate. After adding detection reagents, the plate was washed and 100μL/well of 3,3′,5,5′-Tetramethylbenzidine (TMB) solution was added. The reaction was stopped by the addition of 2 M H2.9PI33-63), or PI33-63 Q38E (referred to as Q6E), PI33-63 Q41E (referred to as Q9E), PI33-63 Q54E, (referred to as Q22E) PI33-63 Q63E (referred to as Q31E), or PI33-63 with all glutamines substituted for glutamic acid, . See + T cells that have proliferated in the presence of antigen to the number of CD4+ T cells that have proliferated in the absence of antigen [Blood was obtained by venepuncture. Peripheral blood mononuclear cells (PBMC) were isolated over Ficoll-paque  and washed twice in phosphate buffered saline (PBS) as described . The CFS antigen ,33.2.1010 transformed before using the Wilcoxon matched-pairs signed rank test. Statistical significance was defined as p < 0.05.Prism 9.4.1 was used to analyze the data and perform statistical analysis. Comparisons between group data were made using a two tailed Mann-Whitney test. Data from CFSE-based proliferation assays was log33.1−/− Jurkat cells that expressed human CD4 and transduced them with lentiviruses to express the TCRαβ genes of the TCRs derived from a panel of human CD4+ T-cell clones specific for epitopes derived from human proinsulin C-peptide  C-peptide). Six of these clones were isolated from CD4+ T cells that infiltrated the pancreatic islets of an organ donor who suffered from T1D [CRISPR/Cas9 was used to knock out the endogenous TRAC, TRBC and CD4 genes in the human T-cell line Jurkat. This gave us a recipient cell line to which CD4 or CD8αβ was introduced by lentiviral transduction. Here we used TCRfrom T1D , and 12 from T1D ,12 and t3.2+ T-cell clone ACD4_2. Jurkat cells produce abundant IL-2 when TCR signaling is activated, but no IL-2 is produced without TCR activation. Jurkat cells also produce IL-8 [33-63), but weakly to an 18mer fragment of C-peptide (PI38-54) [To validate our assay, we used a Jurkat line that expressed the TCR from the human islet-infiltrating CD4uce IL-8 , but we uce IL-8 . It can PI38-54) . This al3.350). The EC50 for each peptide in two independent experiments is shown in 41-50). The first residue of this epitope is Q9, which when substituted for E dramatically diminishes T-cell recognition.To determine if C-peptide specific TCRs were more sensitive to Q to E variants of C-peptide we tested graded concentrations of wild type C-peptide and five deamidated variants. Four variants had a single naturally occurring glutamine substituted for glutamic acid  and one peptide had all four glutamines substituted for glutamic acid  A. The re50 dropped 10–100 fold. Because the epitopes were known for these clones, we separated the clones which had a substitution within the cognate epitope from those where the substitution fell outside the epitope. In all cases, when the Q to E substitution fell within the epitope, the fold change in EC50 relative to WT C-peptide decreased (50 between clones that recognized epitopes containing a substitution (labelled ‘overlapping’) to the remaining clones whose epitope did not contain a substitution . Our findings do not support the notion that deamidated C-peptide primes CD4+ T-cell responses in people with T1D [+ T-cell clones specific for C-peptide. These CD4+ T cells were isolated from human islet-infiltrating CD4+ T cells of a cadaveric organ donor [+ T cells were all selected based on their capacity to recognize WT C-peptide.Here we show that full length C-peptide comprising glutamic acid, in place of glutamine, does not increase the immunogenicity of full-length C-peptide  in PBMC using ELISpot for IFNγ and IL-10 [+ T-cell proliferation in responses to full-length C-peptide using the CFSE-based proliferation assay [Why do our results differ from earlier work which reached the conclusion that deamidated C-peptide is a triggering antigen in human T1D? There are several technical differences in our approach compared to van Lummel et al. . For exand IL-10 , whereason assay . The IFNon assay  were rarThe strength of our approach is that it allowed us to measure the relative potency of WT full-length C-peptide and compare it directly to peptides with individual Q to E substitutions. In all cases the epitope recognized by each clone had been mapped ,12. This+ T-cell responses in the PBMC of people with and without T1D. Using a polyclonal population and the highly sensitive CFSE-based proliferation assay [An important caveat from our T-cell clone analysis is that the clones were selected based on their capacity to respond to WT C-peptide. It may not be surprising then that these clones are not potently stimulated by C-peptide variants. To address this possibility, we investigated the capacity of C-peptide, incorporating different Q to E substitutions to stimulate CD4on assay  allowed on assay , and com+ T cells that responded to deamidated C-peptide. It remains unknow if the responses to these peptides are due to the Q to E substitution, or if the unmodified region of the peptide is being recognized by the responding CD4+ T cells. Nonetheless, it is clear at a polyclonal level that C-peptide variants that contain Q to E substitutions are not more immunogenic than unmodified C-peptide. Second, our conclusions only apply to C-peptide. We have not examined other parts of insulin, nor other beta-cell antigens, hence we cannot comment on the putative immunogenicity of other beta-cell proteins upon deamidation.Our study has some limitations. First, we have not attempted to clone and analyze the specificity of CD45In conclusion, functional analysis of C-peptide specific TCRs and analysis of peripheral blood from people with recent onset T1D showed that substitution of glutamine with glutamic acid does not increase the immunogenicity of C-peptide. We conclude that deamidation of C-peptide does not play a direct role in the autoimmune beta-cell destruction in type 1 diabetes.Abby Foster: Investigation, writing-Review and editing, formal analysis; Pushpak Bhattacharjee: Investigation, Project administration; Miha Pakusch: Investigation, Project administration; Eleonora Tresoldi: Investigation, Project administration; Fergus Cameron: Resources; Project administration; Stuart Mannering: Conceptualization, Writing-original draft, Visualization, Supervision, formal analysis, funding acquisition.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


The aim of this study was to investigate the biodistribution of [18F]F-AraG in healthy volunteers and assess the preliminary safety and radiation dosimetry.  [18F]F-AraG (dose range: 244.2–329.3 MBq) prior to four consecutive PET/MR whole-body scans. Blood samples were collected at regular intervals and vital signs monitored before and after tracer administration. Regions of interest were delineated for multiple organs, and the area under the time-activity curves was calculated for each organ and used to derive time-integrated activity coefficient (TIAC). TIACs were input for absorbed dose and effective dose calculations using OLINDA.  Six healthy subjects  between the ages of 24 and 60 participated in the study. Each subject received a bolus venous injection of [18F]F-AraG were noted by the study participants. The biodistribution was generally reflective of the expression and activity profiles of the enzymes involved in [18F]F-AraG's cellular accumulation, mitochondrial kinase dGK, and SAMHD1. The highest uptake was observed in the kidneys and liver, while the brain, lung, bone marrow, and muscle showed low tracer uptake. The estimated effective dose for [18F]F-AraG was 0.0162 mSv/MBq .  PET/MR examination was well tolerated, and no adverse effects to the administration of [18F]F-AraG in healthy volunteers was consistent with its association with mitochondrial metabolism. PET/MR [18F]F-AraG imaging was well tolerated, with a radiation dosimetry profile similar to other commonly used [18F]-labeled tracers. [18F]F-AraG's connection with mitochondrial biogenesis and favorable biodistribution characteristics make it an attractive tracer with a variety of potential applications. Biodistribution of [ Nelarabine, AraG's prodrug with improved solubility, is FDA-approved for treatment of patients with T cell acute lymphoblastic leukemia (ALL) and T cell lymphoblastic lymphoma [18F]F-AraG as an imaging agent for activated T cells [18F]F-AraG, without toxicity, allows in vivo probing of T cell activity. Activated T cells play a key role in a range of processes that include both host-beneficial antitumor and antiviral immunity, and host-damaging immune response in graft vs. host disease (GvHD) and multiple sclerosis. Non-invasive tracking of activated T cells may allow assessment of proper or aberrant immune function and enable timely treatment interventions or modifications.Intracellular nucleotide pools are intricately controlled as they critically affect cells' genomic stability, growth, proliferation, and survival . The inclymphoma . AraG's  T cells . Adminis18F]F-AraG, enters T cells via nucleoside transporters and is trapped intracellularly through phosphorylation primarily by deoxyguanosine kinase (dGK) [18F]F-AraG's ability to visualize activated T cells lies in its association with mitochondrial biogenesis mediated through the action of mitochondrial dGK. Mitochondrial metabolism and biogenesis are tightly coupled to T cell function F-se (dGK) –9. dGK ise (dGK) . Geneticse (dGK) . The keyfunction , 13. In function , 15. Immfunction . T cell function , 18.FF-se  . SAMHD1, T cells , a rare 18F]F-AraG has been evaluated in preclinical models of rheumatoid arthritis, GvHD, multiple sclerosis, and cancer F-AraG was prepared by UCSF Radiochemistry facility according to the approved IND Chemistry Manufacturing and Control (CMC) procedures. A published procedure was modified for the use on Neptis PET synthesizer [18F]fluoride (n.c.a.) in [18O]water was passed through a QMA-carbonate cartridge, eluted with 0.55 μL of a phase transfer catalyst solution containing Kryptofix222 (6 mg) and K2CO3 (1.25 mg) in 1 : 1 acetonitrile/water. The K222/K[18F]F complex was heated and dried under nitrogen at 95°C for 3 min, followed by 3 × 100 μL additions of acetonitrile and azeotropic evaporation under nitrogen flow and vacuum. After cooling to 60°C, a 1.2 mL solution containing [18F]F-AraG precursor (7-10 mg) in acetonitrile/2-methyl-2-butanol (1 : 5) is added to the reactor, and the solution was heated to 115°C for 30 min. After cooling down to 60°C, 1.2 mL of 0.5 M sodium methoxide was added to the reaction mixture and the solution heated at 100°C for 10 min. Reaction mixture was cooled to 60°C, 1.7 mL of 1 N HCl was added, and the solution was heated at 100°C for 10 min. The reaction mixture was cooled to 50°C and diluted with a solution of 1 mL of 1 N NaHCO3 and 2.5 mL water for injection. The solution was injected and purified on an HPLC Luna C18(2) semipreparative reversed-phase column . Desired peak was collected between 14 and 16 minutes and filtered on-line through a 0.22 μm Millex GV sterile filter. The radiochemical yield was approximately 4% (decay corrected to End of Synthesis) with a synthesis time of 90 minutes. Approximately 0.6 mL of final product was removed aseptically for quality control tests.F-AraG image acquisition and safety monitoring. Each enrolled subject received a bolus venous injection of [18F]F-AraG (dose range: 244.2–329.3 MBq), followed by four consecutive whole-body scans, covering the vertex to the midthigh on positron emission tomography/magnetic resonance imaging . The duration of scan at each bed position was at minimum 180 seconds. Dixon MRI was acquired for MR-based attenuation correction implemented in PET reconstruction. During the PET imaging session blood samples were collected at 1, 3, 5, and 10 minutes after [18F]F-AraG injections and then at every 30 minutes interval up to 3 hours after [18F]F-AraG injections for blood time activity analysis.Following voluntary informed consent, each enrolled subject was prepared for F-AraG images acquired at 4 consecutive time points starting upon injection of [18F]F-AraG were used to quantify 18F radioactivity present in various organs of each healthy subject. Activity was computed in the heart, liver, intestine, kidneys, lungs, bladder, brain, and spleen of each subject at all four time points, and the remainder of the body was computed by subtracting measured organ activities from the whole-body activity. The area under the time-activity curves were calculated for each organ and used to derive time-integrated activity coefficient . TIACs were then input for absorbed dose and effective dose calculations using OLINDA.Reconstructed and attenuation corrected whole-body [18F]F-AraG's uptake and retention [18F]F-AraG's cellular accumulation, we determined the level of SAMHD1, activation markers PD-1 and CD69 and tracer uptake in CD8+ cells cocultured with flu peptide pulsed dendritic cells. Compared to naïve cells, antigen stimulated CD8+ cells had lower expression of SAMHD1, higher expression of PD-1 and CD69, and increased uptake of [18F]F-AraG , the mean weight 63.3 kg (range: 46.4–92.1 kg), and the mean administered dose 307.1 MBq  . ParticiThe whole-body scans were obtained at four time points post tracer injection, each scan lasting 30 minutes. The start time for scans 2 to 4 differed slightly between participants and is shown in 18F]F-AraG at different time points after tracer administration in a representative female and male participant are shown in Maximum intensity projection (MIP) and whole-body distribution of F-AraG, a radiolabeled AraG analog, was developed as an agent for imaging activated T cells and is currently being investigated as a biomarker of response to immunotherapies. Here, we report the results of a pilot study that evaluated biodistribution, dosimetry, and safety of [18F]F-AraG in six healthy participants. Administration of [18F]F-AraG was found to be well tolerated and safe. No adverse effects to the study participants were noted.[18F]F-AraG was reflective of the expression , the recent preclinical findings  as well he liver  into thehe liver . SAMHD1 he liver . SAMHD1 detected . As dGK e organs .RM) are a special subset of long-lived effector memory T cells that do not circulate in the blood and allow rapid response upon antigen re-encounter F-AraG uptake, such as the salivary glands [18F]F-AraG uptake in the liver and other TRM-rich organs could provide information useful for assessing systemic response to T cell-modulating therapies.In addition to the mitochondrial biogenesis in the tissue regulated by the functional interplay between dGK and SAMHD1, the signal observed in various organs may also reflect the presence and functional status of the T cells transiting or residing there. Tissue-resident T cells , similar to estimates for other nucleoside tracers such as FLT (0.0305 mSv/MBq) F-AraG in healthy human subjects showed its suitability for clinical imaging. [18F]F-AraG was well tolerated, with a radiation dosimetry profile similar to other commonly used [18F]-labeled tracers. Its biodistribution was reflective of [18F]F-AraG's proposed mechanism of intracellular accumulation. [18F]F-AraG's association with mitochondrial biogenesis and favorable biodistribution characteristics make it an attractive tracer with a variety of potential applications.Evaluation of [