Membrane cholesterol enrichment and folic acid functionalization lead to increased accumulation of erythrocyte-derived optical nano-constructs within the ovarian intraperitoneal tumor implants in mice.

Cytoreductive surgery remains as the gold standard to treat ovarian cancer, but with limited efficacy since not all tumors can be intraoperatively visualized for resection. We have engineered erythrocyte-derived nano-constructs that encapsulate the near infrared (NIR) fluorophore, indocyanine green (ICG), as optical probes for NIR fluorescence imaging of ovarian tumors. Herein, we have enriched the membrane of these nano-constructs with cholesterol, and functionalized their surface with folic acid (FA) to target the folate receptor-α. Using a mouse model, we show that the average fraction of the injected dose per tumor mass for nano-constructs with both membrane cholesterol enrichment and FA functionalization was ~ sixfold higher than non-encapsulated ICG, ~ twofold higher than nano-constructs enriched with cholesterol alone, and 33 % higher than nano-constructs with only FA functionalization at 24-h post-injection. These results suggest that erythrocyte-derived nano-constructs containing both cholesterol and FA present a platform for improved fluorescence imaging of ovarian tumors.

Proteomes of Micro- and Nanosized Carriers Engineered from Red Blood Cells.

Red blood cell (RBC)-derived systems offer a potential platform for delivery of biomedical cargos. Although the importance of specific proteins associated with the biodistribution and pharmacokinetics of these particles has been recognized, it remains to be explored whether some of the key transmembrane and cytoskeletal proteins responsible for immune-modulatory effects and mechanical integrity of the particles are retained. Herein, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and quantitative tandem mass tag mass spectrometry in conjunction with bioinformatics analysis, we have examined the proteomes of micro- and nanosized erythrocyte ghosts doped with indocyanine green and compared them with those of RBCs. We identified a total of 884 proteins in each set of RBCs, micro-, and nanosized particles, of which 8 and 45 proteins were expressed at significantly different relative abundances when comparing micro-sized particles vs RBCs and nanosized particles vs RBCs, respectively. We found greater differences in relative abundances of some mechano-modulatory proteins, such as band 3 and protein 4.2, and immunomodulatory proteins like CD44, CD47, and CD55 in nanosized particles as compared to RBCs. Our findings highlight that the methods utilized in fabricating RBC-based systems can induce substantial effects on their proteomes. Mass spectrometry data are available at ProteomeXchange with the identifier PXD038780.

The Radial Synergy Test: An Aid to Diagnose de Quervain's Tenosynovitis.

BACKGROUND: Diagnosis of de Quervain's tenosynovitis is made clinically. Finkelstein's and Eichoff's tests are commonly utilized examination maneuvers. Their specificity has been questioned due to a propensity to provoke pain in asymptomatic patients. Using the principle of synergism, the novel radial synergy test takes advantage of isometric contraction of the first dorsal compartment with resisted abduction of the small finger. METHODS: Electromyography was performed on 3 authors and the first dorsal compartment sampled during the maneuver. Sensitivity evaluation was performed via retrospective chart review for patients diagnosed with de Quervain's from 2013 to 2018. Inclusion criteria were documented radial synergy test, Eichoff's test, and ≥90% pain relief after lidocaine/corticosteroid injection. We enrolled 222 patients with 254 affected extremities. Specificity evaluation was performed via a prospective cohort of volunteers undergoing radial synergy and Eichoff's tests. Inclusion criterion was lack of preexisting wrist pain. Score > 0 on Visual Analog Scale was considered positive. We enrolled 48 volunteers with 93 tested extremities. RESULTS: Electromyography revealed positive recruitment of the first dorsal compartment. Sensitivity of the radial synergy test was inferior to Eichoff's test (97% vs 91%, relative risk [RR] = 0.93 [95% confidence interval [CI] = 0.89-0.97], P < .01). Specificity of the radial synergy test was superior to Eichoff's test (99% vs 74%, RR = 1.33 [95% CI = 1.18-1.51], P < .001). CONCLUSIONS: We describe and evaluate the radial synergy test, a novel examination maneuver to aid the diagnosis of de Quervain's. This serves as an adjunct for future diagnostic evaluations with its high specificity. LEVEL OF EVIDENCE: Level II, diagnostic study.

Erythrocyte-Derived Nanoparticles with Folate Functionalization for Near Infrared Pulsed Laser-Mediated Photo-Chemotherapy of Tumors.

Despite its common side effects and varying degrees of therapeutic success, chemotherapy remains the gold standard method for treatment of cancer. Towards developing a new therapeutic approach, we have engineered nanoparticles derived from erythrocytes that contain indocyanine green as a photo-activated agent that enables near infrared photothermal heating, and doxorubicin hydrochloride (DOX) as a chemotherapeutic drug. We hypothesize that milliseconds pulsed laser irradiation results in rapid heating and photo-triggered release of DOX, providing a dual photo-chemo therapeutic mechanism for tumor destruction. Additionally, the surface of the nanoparticles is functionalized with folate to target the folate receptor-α on tumor cells to further enhance the therapeutic efficacy. Using non-contract infrared radiometry and absorption spectroscopy, we have characterized the photothermal response and photostability of the nanoparticles to pulsed laser irradiation. Our in vitro studies show that these nanoparticles can mediate photo-chemo killing of SKOV3 ovarian cancer cells when activated by pulsed laser irradiation. We further demonstrate that this dual photo-chemo therapeutic approach is effective in reducing the volume of tumor implants in mice and elicits an apoptotic response. This treatment modality presents a promising approach in destruction of small tumor nodules.

Morphological Characteristics, Hemoglobin Content, and Membrane Mechanical Properties of Red Blood Cell Delivery Systems.

Red blood cell (RBC)-based systems are under extensive development as platforms for the delivery of various biomedical agents. While the importance of the membrane biochemical characteristics in relation to circulation kinetics of RBC delivery systems has been recognized, the membrane mechanical properties of such carriers have not been extensively studied. Using optical methods in conjunction with image analysis and mechanical modeling, we have quantified the morphological and membrane mechanical characteristics of RBC-derived microparticles containing the near-infrared cargo indocyanine green (ICG). We find that these particles have a significantly lower surface area, volume, and deformability as compared to normal RBCs. The residual hemoglobin has a spatially distorted distribution in the particles. The membrane bending modulus of the particles is about twofold higher as compared to normal RBCs and exhibits greater resistance to flow. The induced increase in the viscous characteristics of the membrane is dominant over the elastic and entropic effects of ICG. Our results suggest that changes to the membrane mechanical properties are a result of impaired membrane-cytoskeleton attachment in these particles. We provide a mechanistic explanation to suggest that the compromised membrane-cytoskeleton attachment and altered membrane compositional and structural asymmetry induce curvature changes to the membrane, resulting in mechanical remodeling of the membrane. These findings highlight the importance of membrane mechanical properties as an important criterion in the design and engineering of future generations of RBC-based delivery systems to achieve prolonged circulation.

Membrane Cholesterol Enrichment of Red Blood Cell-Derived Microparticles Results in Prolonged Circulation.

Particles fabricated from red blood cells (RBCs) can serve as vehicles for delivery of various biomedical cargos. Flipping of phosphatidylserine (PS) from the inner to the outer membrane leaflet normally occurs during the fabrication of such particles. PS externalization is a signal for phagocytic removal of the particles from circulation. Herein, we demonstrate that membrane cholesterol enrichment can mitigate the outward display of PS on microparticles engineered from RBCs. Our in-vitro results show that the phagocytic uptake of cholesterol-enriched particles by murine macrophages takes place at a lowered rate, resulting in reduced uptake as compared to RBC-derived particles without cholesterol enrichment. When administered via tail-vein injection into healthy mice, the percent of injected dose (ID) per gram of extracted blood for cholesterol-enriched particles was ∼1.5 and 1.8 times higher than the particles without cholesterol enrichment at 4 and 24 h, respectively. At 24 h, ∼43% ID/g of the particles without cholesterol enrichment was eliminated or metabolized while ∼94% ID/g of the cholesterol-enriched particles were still retained in the body. These results indicate that membrane cholesterol enrichment is an effective method to reduce PS externalization on the surface of RBC-derived particles and increase their longevity in circulation.

Phototheranostics Using Erythrocyte-Based Particles.

There has been a recent increase in the development of delivery systems based on red blood cells (RBCs) for light-mediated imaging and therapeutic applications. These constructs are able to take advantage of the immune evasion properties of the RBC, while the addition of an optical cargo allows the particles to be activated by light for a number of promising applications. Here, we review some of the common fabrication methods to engineer these constructs. We also present some of the current light-based applications with potential for clinical translation, and offer some insight into future directions in this exciting field.

DMTMM-Mediated Intramolecular Cyclization of Acidic Residues in Peptides/Proteins.

The formation of succinimide in proteins has attracted considerable attention in protein aging and biopharmaceutical research. The succinimide formation occurs spontaneously in proteins and is prone to hydrolysis to yield aspartate and isoaspartate, resulting in altered protein functions. Herein, we demonstrated that the coupling reagent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) can mediate intramolecular cyclization of aspartic acid to form succinimide efficiently in the LL37-derived short antimicrobial peptide KR12. The formation of succinimide in KR12 was confirmed by liquid chromatography tandem mass spectrometry and nuclear magnetic resonance. Moreover, the succinimide-containing KR12 displayed decreased antimicrobial activity, helicity, and serum stability in comparison with unmodified KR12. The succinimide formation usually changes the protein structure and function, and only in rare cases, it can help to maintain the protein stability. In addition to succinimide, DMTMM can also mediate intraresidue cyclization of N-terminal glutamate to form pyroglutamate. Our work thus provides a convenient and efficient method for preparation of succinimide/pyroglutamate-containing peptides, which can be used for studying their impact on peptide/protein function.

Do Mid-term Outcomes of Lateral Ankle Stabilization Procedures Differ Between Military and Civilian Populations?

BACKGROUND: Ankle instability is common and previous studies have documented greater than 85% good-to-excellent outcomes based upon both patient-reported outcome measures and subjective evaluation of ability to return to previous activity levels after lateral ankle stabilization in the civilian population. However, patient-reported outcomes and performance may differ in the military population. The military oftentimes requires servicemembers to navigate uneven terrain and ladderwells, which can stress ankles differently than in their civilian counterparts. There has been limited evidence regarding patient outcomes after lateral ankle stabilization within a military population and its elucidation is important in optimizing outcomes for our servicemembers. Furthermore, the potential benefit of fibular periosteum augmentation with lateral ankle stabilization procedures in a military population has not been described. The results of using this extra tissue to reinforce the repair are important in determining whether its routine incorporation is indicated in the military. QUESTIONS/PURPOSES: In an active-duty military population, we asked: (1) What proportion of patients who underwent lateral ankle stabilization using anatomic repair techniques with or without fibular periosteum augmentation achieved good-to-excellent outcomes based on the Foot and Ankle Disability Index (FADI) score at a minimum follow-up interval of 2 years? (2) Was the proportion of patients who achieved a good-to-excellent FADI score higher among those treated with fibular periosteum augmentation than those treated without? (3) Did the likelihood of achieving a good-to-excellent outcome after lateral ankle stabilization vary based on whether the procedure was performed by a fellowship-trained sports or foot and ankle orthopaedic surgeon versus a podiatrist? METHODS: Between 2007 and 2017, 15 surgeons (six orthopaedic surgeons and nine podiatrists) performed 502 lateral ankle stabilizations. We excluded 4% (18 of 502) of patients because they were not active-duty at the time of surgery, and we excluded 12% (56 of 502) of lateral ankle stabilizations because they were performed as part of other potentially confounding foot or ankle procedures. We considered 60% (303 of 502) as lost to follow-up because the patients could not be contacted at least 2 years after surgery, they declined to participate, or they did not fully answer the questionnaires. This left 125 patients for analysis. Of those, 79% (99 of 125) had a procedure with fibular periosteum augmentation and 21% (26 of 125) had a procedure without augmentation. During the study period, five fellowship-trained orthopaedic foot and ankle surgeons and two podiatrists always used fibular periosteum augmentation. Orthopaedic surgeons performed 75% (94 of 125) of the procedures, and the other 25% (31 of 125) were performed by podiatrists. Whether a servicemember was treated by one specialty or the other was simply based upon whom they were referred to for care. Orthopaedic surgeons tended to perform procedures with augmentation (five with versus one without) and podiatrists tended to perform procedures without augmentation (two with versus seven without). To help account for this confounding factor, we performed separate analyses for procedures performed with versus without augmentation in addition to procedures performed by orthopaedic surgeons versus podiatrists. We retrospectively contacted each patient to obtain their self-reported overall result, FADI outcome score, and postoperative military capabilities. The minimum follow-up duration was 2 years; overall mean follow-up duration was 7 years. The fibular periosteum augmentation group mean follow-up was 7 ± 4 years and without augmentation was 6 ± 3 years. The orthopaedic surgeons group mean follow-up was 7 ± 3 years and the podiatrists group was 7 ± 3 years. We obtained postoperative FADI scores via phone interview along with data regarding the patients' postoperative military capabilities, but did not have preoperative FADI scores. RESULTS: Pooling both surgical treatments, 67% (84 of 125) of the patients reported good-to-excellent results and 33% (41 of 125) reported very poor-to-fair results. We found no difference in the proportion of patients treated with fibular periosteal augmentation who achieved a good or excellent score on the FADI than was observed among the patients treated without periosteal augmentation (68% [67 of 99] versus 65% [17 of 26]; odds ratio 1 [95% CI 0 to 2]; p = 0.81). The proportion of patients who achieved a good or excellent score on the FADI did not differ depending on whether the procedure was performed by an orthopaedic surgeon or a podiatric surgeon (66% [62 of 94] versus 71% [22 of 31]; OR 1 [95% CI 1 to 2]; p = 0.66). CONCLUSION: The patient-reported outcome scores after lateral ankle stabilization in our study of military servicemembers at a minimum of 2 years and a mean of 7 years were far lower than have been reported in studies on civilians. Indeed, our findings may represent a best-case scenario because more patients were lost to follow-up than were accounted for, and in general, surgical results among missing patients are poorer than among those who return for follow-up. Variability in the addition of fibular periosteum augmentation and whether an orthopaedic surgeon or podiatrist performed the procedure did not account for these findings. With one third of patients reporting very-poor-to-fair results after these reconstructions, and many patients lost to follow-up, we recommend surgeons counsel their servicemember patients accordingly before surgery. Specifically, that there is a one third chance they will need permanent restrictions or have to leave the military postoperatively; analogously, we believe that our findings may apply to similarly active patients outside the military, and we question whether these procedures may not be serving such patients as well as previously believed. LEVEL OF EVIDENCE: Level III, therapeutic study.

MicroRNA-146a suppresses tumor malignancy via targeting vimentin in esophageal squamous cell carcinoma cells with lower fibronectin membrane assembly.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is widely prevalent in Taiwan, and high metastatic spread of ESCC leads to poor survival rate. Fibronectin (FN) assembly on the cell membrane may induce ESCC mobility. MicroRNAs (MiRNAs) are abundant in and participate in tumorigenesis in many cancers. However, the role of MiRNA in FN assembly-related ESCC mobility remains unexplored. METHODS: We divided ESCC CE81T cells into high-FN assembly (CE81FN+) and low-FN assembly (CE81FN-) groups by flow cytometry. MiRNA microarray analysis identified miR-146a expression as the most down-regulated miRNA in comparison of CE81FN+ and CE81FN- cells. RESULTS: Cell proliferation and migration were decreased when CE81FN+ cells overexpressed transgenic miR-146a compared to the parental cells, indicating an inverse correlation between low miR-146a expression and high proliferation as well as motility of FN assembly ESCC cells. Furthermore, vimentin is the target gene of miR-146a involved in ESCC tumorigenesis. MiR-146a suppressed cell proliferation, migration and invasion of CE81FN+ cells through the inhibition of vimentin expression, as confirmed by real-time PCR, Western blotting and Transwell™ assay. Analysis of one hundred and thirty-six paired ESCC patient specimens revealed that low miR-146a and high vimentin levels were frequently detected in tumor, and that the former was associated with late tumor stages (III and IV). Notably, either low miR-146a expression or high vimentin level was significantly associated with poor overall survival rate among ESCC patients. CONCLUSIONS: This is the first report to link FN assembly in the cell membrane with miR-146a, vimentin and ESCC tumorigenesis both in vitro and in ESCC patients.

No Difference in Risk of Implant Removal Between Orthogonal Mini-fragment and Single Small-fragment Plating of Midshaft Clavicle Fractures in a Military Population: A Preliminary Study.

BACKGROUND: Clavicle fractures are common, especially in military personnel. A persistent problem of open reduction and internal fixation (ORIF) of the clavicle is the high percentage of symptomatic implants needing removal. Prominent implants can lead to discomfort in military service members performing activities such as carrying rucksacks and firing high-caliber weapons, potentially resulting in a high removal of implant rate. Alternative approaches to plate fixation may mitigate this, but to our knowledge, only limited evidence is available comparing various plate-fixation approaches. QUESTIONS/PURPOSES: In a military population, we asked (1) Is there a difference in implant removal rates after midshaft clavicle fixation using orthogonal plating with 2.7-mm reconstruction plates versus a single 3.5-mm locking compression plate? (2) What complications are associated with each fixation approach? (3) Is there a difference in surgical time between the approaches? METHODS: Between January 2010 and May 2015, three surgeons performed 99 ORIF procedures of midshaft clavicle fractures, always using a single small-fragment plate, and one surgeon performed 34 procedures, always using two mini-fragment plates with an orthogonal plating construct. Of those, 89 (90%) in the small-fragment plating group were available for analysis in this retrospective study and 33 (97%) were available for analysis in the mini-fragment plating group, both groups with a minimum of 2 years of followup. There were no between-group differences in terms of gender, tobacco use, injured side, hand dominance, 100% displacement, comminution, shortening, and active-duty status. We analyzed the proportion of patients who had their plates removed for any complications recorded in their charts. Assessment bias for indications for symptomatic removal of implant was minimized as there was always another fellowship-trained trauma surgeon at our institution available for a second opinion if the operative surgeon did not agree with a patient request for implant removal. A posthoc power calculation indicated that with the numbers available, we had 80% power to detect a between-group difference in implant removal proportion of 14.5% at the p < 0.05 level. RESULTS: We found no difference between the small-fragment plating group and the mini-fragment group in the frequency of plate removal (9% [8 of 89] versus 0% [0 of 33]; odds ratio, 3.38 [95% confidence interval 0.41 to 27.68]; p = 0.11); a worst-case analysis that assumed all patients lost to follow-up underwent plate removal did not change this no-difference finding. All cases of implant removal were performed secondary to a symptomatic implant. With the numbers available, there were no differences between the two groups in nonunion, delayed union, infection, or other complications. There was a longer mean operative time in the mini-fragment group than in the small-fragment group (173.7 minutes versus 118.7 minutes; mean difference, 55 minutes [95% CI 38.71 to 71.23]; p < .001). Our overall implant removal percentage for the two groups combined was 6.6% (8 of 122). CONCLUSIONS: Our study was underpowered to show differences in implant removal but may serve as a pilot for larger randomized controlled trials or multi-institutional studies on this topic. Although there was increased operative time to insert two plates, there was no difference in overall complications. LEVEL OF EVIDENCE: Level III, therapeutic study.

New paradigm of functional regulation by DNA mimic proteins: Recent updates.

For many, "DNA mimic protein" (DMP) remains an unfamiliar term. The key feature of these proteins is their DNA-like shape and charge distribution, and they affect the activity of DNA-binding proteins by occupying their DNA-binding domains. Functionally, DMPs regulate mechanisms such as gene expression, restriction, and DNA repair as well as the nucleosome package. Although a few DMPs, such as phage uracil DNA glycosylase inhibitor (UGI) and overcome classical restriction (Ocr), were reported about 20 years ago, only a small number of DMPs have been studied to date. In 2014, we reviewed the functional and structural features of 16 DMPs that were known at the time. Now, seven new DMPs, namely anti-CRISPR suppressors AcrF2, AcrF10 and AcrIIA4, human immunodeficiency virus essential factor VPR, multi-functional inhibitor anti-restriction nuclease (Arn), translational regulator AbbA, and putative Z-DNA mimic MBD3, have been reported. In addition, further study of two previously known DMPs, DMP19 and SAUGI, increased our knowledge of their importance and function. Here, we discuss these updated results and address how several characteristics of the structure/sequence of DMPs (e.g. the DNA-like charge distribution and structural D/E-rich repeats) might someday be used to identify new DMPs using bioinformatic approach. © 2018 IUBMB Life, 71(5):539-548, 2019.

The C-terminal D/E-rich domain of MBD3 is a putative Z-DNA mimic that competes for Zα DNA-binding activity.

The Z-DNA binding domain (Zα), derived from the human RNA editing enzyme ADAR1, can induce and stabilize the Z-DNA conformation. However, the biological function of Zα/Z-DNA remains elusive. Herein, we sought to identify proteins associated with Zα to gain insight into the functional network of Zα/Z-DNA. By pull-down, biophysical and biochemical analyses, we identified a novel Zα-interacting protein, MBD3, and revealed that Zα interacted with its C-terminal acidic region, an aspartate (D)/glutamate (E)-rich domain, with high affinity. The D/E-rich domain of MBD3 may act as a DNA mimic to compete with Z-DNA for binding to Zα. Dimerization of MBD3 via intermolecular interaction of the D/E-rich domain and its N-terminal DNA binding domain, a methyl-CpG-binding domain (MBD), attenuated the high affinity interaction of Zα and the D/E-rich domain. By monitoring the conformation transition of DNA, we found that Zα could compete with the MBD domain for binding to the Z-DNA forming sequence, but not vice versa. Furthermore, co-immunoprecipitation experiments confirmed the interaction of MBD3 and ADAR1 in vivo. Our findings suggest that the interplay of Zα and MBD3 may regulate the transition of the DNA conformation between B- and Z-DNA and thereby modulate chromatin accessibility, resulting in alterations in gene expression.

Irreducible Ankle Fracture from an Interposed Chaput Fragment: A Case Report.

CASE: A 23-year-old active-duty military service member sustained an unusual irreducible fracture-dislocation of the ankle joint. Preoperative computed tomography showed an anterolateral tibial (Chaput) fragment entrapped within the distal syndesmosis, preventing adequate reduction. An open reduction of the fragment was performed in the operating room, which allowed near-anatomic reduction and internal fixation of the tibiotalar joint. At the 21-month follow-up visit, the patient remained on active duty in the Navy without limitations. CONCLUSION: To our knowledge, this rare injury pattern, an interposed Chaput fragment preventing closed reduction of the syndesmosis in a skeletally mature patient, has not previously been described in the literature. An open reduction and internal fixation of the Chaput fragment allowed near-anatomic reduction of the tibiotalar joint.

Epigenetic regulation of the Drosophila chromosome 4 by the histone H3K9 methyltransferase dSETDB1.

The polytene chromosomes of Drosophila melanogaster consist of condensed heterochromatin regions most of which are in the chromocenter, telomeres, and the fourth chromosome. Whereas suppressor of variegation 3-9 [SU(VAR)3-9], a histone methyltransferase, is mainly responsible for lysine 9 of histone H3 (H3K9) methylation of the chromocenter and consequent binding of the heterochromatin-protein HP1, the enzyme for painting of the fourth chromosome by H3K9 methylation has been elusive. We show here that dSETDB1, the Drosophila ortholog of the mammalian SETDB1, is an authentic H3K9 methyltransferase and a pleiotropic regulator of the fly's development. Drosophila mutants hypomorphic or null in dSETDB1 expression lose most of the H3K9 methylation as well as HP1-binding on the fourth chromosome. We also show that binding of painting of fourth (POF), a known fourth chromosome-specific protein, and the dSETDB1-controlled H3K9 methylation of this chromosome are interdependent. Furthermore, POF and dSETDB1 interact with each other in vivo. The deregulation of H3K9 methylation, HP1-binding, and POF-binding resulted in, on the average, a global reduction of gene expression from the fourth chromosome but not the other chromosomes. Deficiency of dSETDB1 also up-regulated the expression of HP1. These results have suggested an interactive network, as controlled in part by dSETDB1, regulating the epigenetic modification and gene expression of Drosophila chromosome 4.

The double-stranded RNA-activated kinase, PKR, can phosphorylate hepatitis D virus small delta antigen at functional serine and threonine residues.

Hepatitis D virus (HDV) encodes two proteins, the 24-kDa small delta antigen (S-HDAg) and 27-kDa large delta antigen (L-HDAg) in its single open reading frame. Both of them had been identified as nuclear phosphoproteins. Moreover, the phosphorylated form of S-HDAg was shown to be important for HDV replication. However, the kinase responsible for S-HDAg phosphorylation remains unknown. Therefore, we employed an in-gel kinase assay to search candidate kinases and indeed identified a kinase with a molecular mass of about 68 kDa. Much evidence demonstrated this kinase to be the double-stranded RNA-activated kinase, PKR. The immunoprecipitated endogenous PKR was sufficient to catalyze S-HDAg phosphorylation, and the kinase activity disappeared in the PKR-depleted cell lysate. The S-HDAg and PKR could be co-immunoprecipitated together, and both of them co-located in the nucleolus. The LC/MS/MS analysis revealed that the serine 177, serine 180, and threonine 182 of S-HDAg were phosphorylated by PKR in vitro. This result was consistent with previous phosphoamino acid analysis indicating that serine and threonine were phosphorylation targets in S-HDAg. Furthermore, serine 177 was also shown to be the predominant phosphorylation site for S-HDAg purified the from cell line. In dominant negative PKR-transfected cells, the level of phosphorylated S-HDAg was suppressed, but replication of HDV was enhanced. Other than human immunodeficiency virus type 1 trans-activating protein (Tat), S-HDAg is another viral protein phosphorylated by PKR that may regulates HDV replication and viral response to interferon therapy.