Learning curve and initial outcomes of a novel percutaneously endoscopic-assisted total hip arthroplasty through mini bikini direct anterior approach: an observational cohort study.

OBJECTIVE: Although the direct anterior approach can reduce muscle damage and ensure accurate prosthesis placement, the steep learning curve and increased risk of complications associated with DAA necessitate careful consideration. Therefore, we describe a technique for a novel percutaneously endoscopic-assisted total hip arthroplasty through mini bikini direct anterior approach (mDAA) and report the learning curve and initial outcomes. METHODS: The first 125 THA performed by a single surgeon between September 2020 and February 2022 using the anterior approach were included, comprising the initial 41 cases of bikini DAA (bDAA) and the subsequent 84 cases of mDAA. Outcome measures included perioperative outcomes and postoperative complications. The cumulative sum analysis (CUSUM) was used to determine the learning curve of anterior approach THA for each patient's ORT. Multivariable analysis was performed to determine risk correlation. RESULTS: A total of 125 anterior approach THA completed between 2020 and 2022 were identified. Among these, 41 were performed via bDAA and 84 via mDAA. No statistically significant differences were observed between the groups in terms of age, gender distribution, BMI or follow-up time. A significant reduction in ORT was noted, from 140 min for bDAA to 130 min for mDAA. Furthermore, there was a consistent decrease in LOI, LOS, and wound-healing problems. There was no statistically significant difference between groups with respect to Harris Hip Scores and other postoperative complications. The curve inflection points of the learning curve for the bDAA and mDAA group were located in the 22nd and 68th cases, respectively. The reduction of hemoglobin indicated a predicted increase in ORT. CONCLUSIONS: In this study, ORT, LOI, LOS, and wound-healing problems decreased overall in mDAA group. After mastering the bDAA technique, approximately 27 mDAA cases are needed to acquire proficiency in this technique. Hence, mDAA is a valuable alternative for those seeking smaller incisions, resolving wound healing problems, and aiming for enhanced recovery after surgery.

The Verticillium dahliae Effector VdPHB1 Promotes Pathogenicity in Cotton and Interacts with the Immune Protein GhMC4.

Verticillium dahliae is a kind of pathogenic fungus that brings about wilt disease and great losses in cotton. The molecular mechanism of the effectors in V. dahliae regulating cotton immunity remains largely unknown. Here, we identified an effector of V. dahliae, VdPHB1, whose gene expression is highly induced by infection. The VdPHB1 protein is localized to the intercellular space of cotton plants. Knock-out of the VdPHB1 gene in V. dahliae had no effect on pathogen growth, but decreased the virulence in cotton. VdPHB1 ectopically expressed Arabidopsis plants were growth-inhibited and significantly susceptible to V. dahliae. Further, VdPHB1 interacted with the type II metacaspase GhMC4. GhMC4 gene-silenced cotton plants were more sensitive to V. dahliae with reduced expression of pathogen defense-related and programmed cell death genes. The accumulation of GhMC4 protein was concurrently repressed when VdPHB1 protein was expressed during infection. In summary, these results have revealed a novel molecular mechanism of virulence regulation that the secreted effector VdPHB1 represses the activity of cysteine protease for helping V. dahliae infection in cotton.

Circular RNA CREBBP modulates cartilage degradation by activating the Smad1/5 pathway through the TGFß2/ALK1 axis.

Osteoarthritis, characterized by articular cartilage degradation, is the leading cause of chronic disability in older adults. Studies have indicated that circular RNAs are crucial regulators of chondrocyte development and are involved in the progression of osteoarthritis. In this study, we investigated the function and mechanism of a circular RNA and its potential for osteoarthritis therapy. The expression levels of circCREBBP, screened by circular RNA sequencing during chondrogenic differentiation in adipose tissue-derived stem cells, and TGFß2 were significantly increased in the cartilage of patients with osteoarthritis and IL-1ß-induced chondrocytes. circCREBBP knockdown increased anabolism in the extracellular matrix and inhibited chondrocyte degeneration, whereas circCREBBP overexpression led to the opposite effects. Luciferase reporter assays, rescue experiments, RNA immunoprecipitation, and RNA pulldown assays confirmed that circCREBBP upregulated TGFß2 expression by sponging miR-1208, resulting in significantly enhanced phosphorylation of Smad1/5 in chondrocytes. Moreover, intra-articular injection of adeno-associated virus-sh-circCrebbp alleviated osteoarthritis in a mouse model of destabilization of the medial meniscus. Our findings reveal a critical role for circCREBBP in the progression of osteoarthritis and provide a potential target for osteoarthritis therapy.

Experimental study on the effect of the split-type air-conditioner on the transmission of smoking pollutants in a room.

Environmental tobacco smoke (ETS) has become one of the most important sources of indoor air pollution. The study aimed to obtain the variation characteristics of typical air pollutant concentrations when people smoke in a closed room and explore the effect of the air-conditioner. A closed and air-conditioned room of 21 m2 was taken as the research object. Fine particulate matter (PM2.5) and total volatile organic compound (TVOC) were measured while 10 cigarettes were burnt in smoldering or smoking mode, with the air-conditioner on or off. The contents of nicotine in condensate samples were obtained by liquid chromatography. The impact of ETS on indoor air quality lasted for hours, causing typical pollutant concentrations to far exceed the Chinese standard. The PM2.5 produced by smoking was 11 times higher than by smoldering, but the TVOC produced by smoldering was more than by smoking. After one hour of the cigarette burning off, the PM2.5 concentration would be decreased by 96.1% with the air-conditioner on, in contrast to 67.9% with the air-conditioner off. Nicotine was detected in all samples of condensate from the air-conditioner. It is concluded that smoking cigarettes cannot be replaced by smoldering to evaluate the pollution of ETS. The air-conditioner has a positive effect on reducing the concentration of air pollutants produced by cigarette burning. More than 10% of the indoor nicotine may be taken away by condensate discharge, and its possible pollution should be paid attention to.Implications: This study provides new evidence of the effect of the split-type air-conditioner on ETS. The TVOC concentrations, which were less considered previously, were measured. PM2.5 concentration in human breathing zone can be reduced more quickly with the air-conditioner on. This study shows that there is a big difference in the concentrations of typical pollutants between smoking and smoldering. And it could be a guide for the formulation of relevant research methods. This study also demonstrates that the air conditioning condensate from the smoking room may contain nicotine. Attention should be paid to the recovery and utilization of such condensate.

Liposome Loaded Ion/Temperature Dual Responsive Gellan Gum Hydrogel as Potential Nasal-To-Brain Delivery System.

Intranasal administration, which can bypass the blood-brain barrier (BBB), is widely recognized as a promising strategy for high-efficiency drug delivery to the brain. Herein, for the purpose of effectively delivering drugs to the brain via intranasal administration, glutathione (GSH)-modified gellan gum (GSH-GG) with ion/temperature dual responsive properties was synthesized and encapsulated on galanthamine hydrobromide (GH)-loaded liposomes (GH-Lipo) for effective GH delivery to the brain (GH-Lipo@GSH-GG). Our results demonstrated that GSH-GG greatly decreased the gelation temperature of GG from 44.0 °C to 22.1 °C without compromising its ion responsiveness. Moreover, GSH-GG had a good protection ability for GH-loaded liposomes without affecting its drug release. Most importantly, the finally obtained GH-Lipo@GSHGG showed acceptable targeted delivery of GH to the brain upon in vivo administration. Therefore, this formulation can be employed as a potential delivery system in nasal-to-brain delivery.

Long Non-coding RNAs LOC100126784 and POM121L9P Derived From Bone Marrow Mesenchymal Stem Cells Enhance Osteogenic Differentiation via the miR-503-5p/SORBS1 Axis.

Long non-coding RNAs (lncRNAs) play pivotal roles in mesenchymal stem cell differentiation. However, the mechanisms by which non-coding RNA (ncRNA) networks regulate osteogenic differentiation remain unclear. Therefore, our aim was to identify RNA-associated gene and transcript expression profiles during osteogenesis in bone marrow mesenchymal stem cells (BMSCs). Using transcriptome sequencing for differentially expressed ncRNAs and mRNAs between days 0 and 21 of osteogenic differentiation of BMSCs, we found that the microRNA (miRNA) miR-503-5p was significantly downregulated. However, the putative miR-503-5p target, sorbin and SH3 domain containing 1 (SORBS1), was significantly upregulated in osteogenesis. Moreover, through lncRNA-miRNA-mRNA interaction analyses and loss- and gain-of-function experiments, we discovered that the lncRNAs LOC100126784 and POM121L9P were abundant in the cytoplasm and enhanced BMSC osteogenesis by promoting SORBS1 expression. In contrast, miR-503-5p reversed this effect. Ago2 RNA-binding protein immunoprecipitation and dual-luciferase reporter assays further validated the direct binding of miR-503-5p to LOC100126784 and POM121L9P. Furthermore, SORBS1 knockdown suppressed early osteogenic differentiation in BMSCs, and co-transfection with SORBS1 small interfering RNAs counteracted the BMSCs' osteogenic capacity promoted by LOC100126784- and POM121L9P-overexpressing lentivirus plasmids. Thus, the present study demonstrated that the lncRNAs LOC100126784 and POM121L9P facilitate the osteogenic differentiation of BMSCs via the miR-503-5p/SORBS1 axis, providing potential therapeutic targets for treating osteoporosis and bone defects.

Preparation of PP-g-(AA-MAH) Fibers Using Suspension Grafting and Melt-Blown Spinning and its Adsorption for Aniline.

This paper uses polypropylene (PP) as the matrix and acrylic acid (AA) and maleic anhydride (MAH) as functional monomers to prepare PP-g-(AA-MAH) fibers by suspension grafting and melt-blown spinning technology that are easy to industrially scale-up. The fibers can be used to adsorb aniline. Results showed that the grafting ratio reached the maximum of 12.47%. The corresponding optimal conditions were grafting time of 3 h, AA: MAH = 0.75, total monomer content of 55%, benzoyl peroxide 1.4%, xylene concentration of 6 mL/g PP, and deionized water content of 8 mL/g PP. Owing to its good fluidity and thermal stability, the product of suspension grafting can be used for melt-blown spinning. Infrared spectroscopic and nuclear magnetic resonance spectroscopic analyses indicated that AA and MAH were successfully grafted onto PP fibers. After grafting, the hydrophilicity of PP-g-(AA-MAH) fiber increased. Therefore, it had higher absorptivity for aniline and the adsorption capacity could reach 42.2 mg/g at 45 min and pH = 7. Moreover, the PP-g-(AA-MAH) fibers showed good regeneration performance.

Expanded Polytetrafluoroethylene/Graphite Composites for Easy Water/Oil Separation.

Oil spills in the ocean greatly threaten local environments, marine creatures, and coastal economies. An automatic water/oil separation material system was proposed in this study, and a tubular geometry was chosen to demonstrate the water/oil separation efficiency and effectiveness. The water/oil separation tubes were made of expanded polytetrafluoroethylene (ePTFE) and graphite composites. The permeation pressures of water and oil through the tube walls were tuned by adjusting the ePTFE microstructure, which, in turn, depended on the degree of expansion and the graphite content. Fourier-transform infrared spectroscopy was performed to confirm the compositions of the ePTFE/graphite composites, and a scanning electron microscope was used to examine the microstructure and morphology of the expanded PTFE/graphite composite tubes. When a proper pressure was applied, which was higher than the oil's permeation pressure (3.0 kPa) but lower than the water's permeation pressure (57 kPa), the oil leaked out of the tube walls while the water went through the ePTFE/graphite tubes. As such, the water/oil mixture could be separated and collected in different containers or an outer tube. Due to this automatic separation, the whole process could be done continuously and conveniently, thus exhibiting great potential in the practical applications of oil spill and water separation/remediation.

Artificial small-diameter blood vessels: materials, fabrication, surface modification, mechanical properties, and bioactive functionalities.

Cardiovascular diseases, especially ones involving narrowed or blocked blood vessels with diameters smaller than 6 millimeters, are the leading cause of death globally. Vascular grafts have been used in bypass surgery to replace damaged native blood vessels for treating severe cardio- and peripheral vascular diseases. However, autologous replacement grafts are not often available due to prior harvesting or the patient's health. Furthermore, autologous harvesting causes secondary injury to the patient at the harvest site. Therefore, artificial blood vessels have been widely investigated in the last several decades. In this review, the progress and potential outlook of small-diameter blood vessels (SDBVs) engineered in vitro are highlighted and summarized, including material selection and development, fabrication techniques, surface modification, mechanical properties, and bioactive functionalities. Several kinds of natural and synthetic polymers for artificial SDBVs are presented here. Commonly used fabrication techniques, such as extrusion and expansion, electrospinning, thermally induced phase separation (TIPS), braiding, 3D printing, hydrogel tubing, gas foaming, and a combination of these methods, are analyzed and compared. Different surface modification methods, such as physical immobilization, surface adsorption, plasma treatment, and chemical immobilization, are investigated and are compared here as well. Mechanical requirements of SDBVs are also reviewed for long-term service. In vitro biological functions of artificial blood vessels, including oxygen consumption, nitric oxide (NO) production, shear stress response, leukocyte adhesion, and anticoagulation, are also discussed. Finally, we draw conclusions regarding current challenges and attempts to identify future directions for the optimal combination of materials, fabrication methods, surface modifications, and biofunctionalities. We hope that this review can assist with the design, fabrication, and application of SDBVs engineered in vitro and promote future advancements in this emerging research field.

Programmed Release of Multimodal, Cross-Linked Vascular Endothelial Growth Factor and Heparin Layers on Electrospun Polycaprolactone Vascular Grafts.

Viable tissue-engineering small-diameter vascular grafts should support rapid growth of an endothelial cell layer and exhibit long-term antithrombogenic property. In this study, multiple layers of various bioactive molecules, such as vascular endothelial growth factor (VEGF) and heparin, on an electrospun polycaprolactone scaffold have been developed through repeated electrostatic adsorption self-assembly (up to 20 layers), followed by genipin cross-linking. Programmed and sustained release of biomolecules embedded within the multilayered structure can be triggered by matrix metallopeptidase 2 enzyme in vitro. The result is an early and full release of VEGF to promote rapid endothelialization on the intended vascular grafts, followed by a gradual but sustained release of heparin for long-term anticoagulation and antithrombogenicity. This method of forming a biologically responsive, multimodal delivery of VEGF and heparin is highly suitable for all hydrophobic surfaces and provides a promising way to meet the critical requirements of engineered small-diameter vascular grafts.

GPRC5D is a target for the immunotherapy of multiple myeloma with rationally designed CAR T cells.

Early clinical results of chimeric antigen receptor (CAR) T cell therapy targeting B cell maturation antigen (BCMA) for multiple myeloma (MM) appear promising, but relapses associated with residual low-to-negative BCMA-expressing MM cells have been reported, necessitating identification of additional targets. The orphan G protein-coupled receptor, class C group 5 member D (GPRC5D), normally expressed only in the hair follicle, was previously identified as expressed by mRNA in marrow aspirates from patients with MM, but confirmation of protein expression remained elusive. Using quantitative immunofluorescence, we determined that GPRC5D protein is expressed on CD138+ MM cells from primary marrow samples with a distribution that was similar to, but independent of, BCMA. Panning a human B cell-derived phage display library identified seven GPRC5D-specific single-chain variable fragments (scFvs). Incorporation of these into multiple CAR formats yielded 42 different constructs, which were screened for antigen-specific and antigen-independent (tonic) signaling using a Nur77-based reporter system. Nur77 reporter screen results were confirmed in vivo using a marrow-tropic MM xenograft in mice. CAR T cells incorporating GPRC5D-targeted scFv clone 109 eradicated MM and enabled long-term survival, including in a BCMA antigen escape model. GPRC5D(109) is specific for GPRC5D and resulted in MM cell line and primary MM cytotoxicity, cytokine release, and in vivo activity comparable to anti-BCMA CAR T cells. Murine and cynomolgus cross-reactive CAR T cells did not cause alopecia or other signs of GPRC5D-mediated toxicity in these species. Thus, GPRC5D(109) CAR T cell therapy shows potential for the treatment of advanced MM irrespective of previous BCMA-targeted therapy.

A novel antibody-TCR (AbTCR) platform combines Fab-based antigen recognition with gamma/delta-TCR signaling to facilitate T-cell cytotoxicity with low cytokine release.

The clinical use of genetically modified T-cell therapies has led to unprecedented response rates in leukemia and lymphoma patients treated with anti-CD19 chimeric antigen receptor (CAR)-T. Despite this clinical success, FDA-approved T-cell therapies are currently limited to B-cell malignancies, and challenges remain with managing cytokine-related toxicities. We have designed a novel antibody-T-cell receptor (AbTCR) platform where we combined the Fab domain of an antibody with the γ and δ chains of the TCR as the effector domain. We demonstrate the ability of anti-CD19-AbTCR-T cells to trigger antigen-specific cytokine production, degranulation, and killing of CD19-positive cancer cells in vitro and in xenograft mouse models. By using the same anti-CD19 binding moiety on an AbTCR compared to a CAR platform, we demonstrate that AbTCR activates cytotoxic T-cell responses with a similar dose-response as CD28/CD3ζ CAR, yet does so with less cytokine release and results in T cells with a less exhausted phenotype. Moreover, in comparative studies with the clinically validated CD137 (4-1BB)-based CAR, CTL019, our anti-CD19-AbTCR shows less cytokine release and comparable tumor inhibition in a patient-derived xenograft leukemia model.

Polycaprolactone Nanofibers Containing Vascular Endothelial Growth Factor-Encapsulated Gelatin Particles Enhance Mesenchymal Stem Cell Differentiation and Angiogenesis of Endothelial Cells.

During the regeneration of tissues and organs, growth factors (GFs) play a vital role by affecting cell behavior. However, because of the low half-life time and quick degradation of GFs, their stimulations on cells are relatively short and discontinuous. In this study, a releasing scaffold platform, consisting of polycaprolactone (PCL) nanofibers and vascular endothelial growth factor (VEGF)-encapsulated gelatin particles, was developed to extend the influence of GFs on mesenchymal stem cells (MSCs) and endothelial cells (ECs). The results showed that this kind of scaffold can direct the differentiation of MSCs to ECs and maintain the stability of the tubular structure, an indicator of the angiogenesis ability of ECs, for an extended period of time. Therefore, the results suggest the potential application of PCL/VEGF-encapsulated gelatin particles (PCL/VGPs) as a growth factor (GF)-releasing scaffold platform in vascular tissue engineering.

Development and Evaluation of an Optimal Human Single-Chain Variable Fragment-Derived BCMA-Targeted CAR T Cell Vector.

B cell maturation antigen (BCMA) has recently been identified as an important multiple myeloma (MM)-specific target for chimeric antigen receptor (CAR) T cell therapy. In CAR T cell therapy targeting CD19 for lymphoma, host immune anti-murine CAR responses limited the efficacy of repeat dosing and possibly long-term persistence. This clinically relevant concern can be addressed by generating a CAR incorporating a human single-chain variable fragment (scFv). We screened a human B cell-derived scFv phage display library and identified a panel of BCMA-specific clones from which human CARs were engineered. Despite a narrow range of affinity for BCMA, dramatic differences in CAR T cell expansion were observed between unique scFvs in a repeat antigen stimulation assay. These results were confirmed by screening in a MM xenograft model, where only the top preforming CARs from the repeat antigen stimulation assay eradicated disease and prolonged survival. The results of this screening identified a highly effective CAR T cell therapy with properties, including rapid in vivo expansion (>10,000-fold, day 6), eradication of large tumor burden, and durable protection to tumor re-challenge. We generated a bicistronic construct including a second-generation CAR and a truncated-epithelial growth factor receptor marker. CAR T cell vectors stemming from this work are under clinical investigation.

Targeting Alpha-Fetoprotein (AFP)-MHC Complex with CAR T-Cell Therapy for Liver Cancer.

PURPOSE: The majority of tumor-specific antigens are intracellular and/or secreted and therefore inaccessible by conventional chimeric antigen receptor (CAR) T-cell therapy. Given that all intracellular/secreted proteins are processed into peptides and presented by class I MHC on the surface of tumor cells, we used alpha-fetoprotein (AFP), a specific liver cancer marker, as an example to determine whether peptide-MHC complexes can be targets for CAR T-cell therapy against solid tumors. EXPERIMENTAL DESIGN: We generated a fully human chimeric antigen receptor, ET1402L1-CAR (AFP-CAR), with exquisite selectivity and specificity for the AFP158-166 peptide complexed with human leukocyte antigen (HLA)-A*02:01. RESULTS: We report that T cells expressing AFP-CAR selectively degranulated, released cytokines, and lysed liver cancer cells that were HLA-A*02:01+/AFP+ while sparing cells from multiple tissue types that were negative for either expressed proteins. In vivo, intratumoral injection of AFP-CAR T cells significantly regressed both Hep G2 and AFP158-expressing SK-HEP-1 tumors in SCID-Beige mice (n = 8 for each). Moreover, intravenous administration of AFP-CAR T cells in Hep G2 tumor-bearing NSG mice lead to rapid and profound tumor growth inhibition (n = 6). Finally, in an established intraperitoneal liver cancer xenograft model, AFP-CAR T cells showed robust antitumor activity (n = 6). CONCLUSIONS: This study demonstrates that CAR T-cell immunotherapy targeting intracellular/secreted solid tumor antigens can elicit a potent antitumor response. Our approach expands the spectrum of antigens available for redirected T-cell therapy against solid malignancies and offers a promising new avenue for liver cancer immunotherapy. Clin Cancer Res; 23(2); 478-88. ©2016 AACR.

Therapeutic bispecific T-cell engager antibody targeting the intracellular oncoprotein WT1.

Intracellular tumor antigens presented on the cell surface in the context of human leukocyte antigen (HLA) molecules have been targeted by T cell-based therapies, but there has been little progress in developing small-molecule drugs or antibodies directed to these antigens. Here we describe a bispecific T-cell engager (BiTE) antibody derived from a T-cell receptor (TCR)-mimic monoclonal antibody (mAb) ESK1, which binds a peptide derived from the intracellular oncoprotein WT1 presented on HLA-A*02:01. Despite the very low density of the complexes at the cell surface, ESK1-BiTE selectively activated and induced proliferation of cytolytic human T cells that killed cells from multiple leukemias and solid tumors in vitro and in mice. We also discovered that in an autologous in vitro setting, ESK1-BiTE induced a robust secondary CD8 T-cell response specific for tumor-associated antigens other than WT1. Our study provides an approach that targets tumor-specific intracellular antigens without using cell therapy and suggests that epitope spreading could contribute to the therapeutic efficacy of this BiTE.

The lytic transcriptome of Kaposi's sarcoma-associated herpesvirus reveals extensive transcription of noncoding regions, including regions antisense to important genes.

Genomewide analyses of the mammalian transcriptome have revealed that large tracts of sequence previously annotated as noncoding are frequently transcribed and give rise to stable RNA. Although the transcription of individual genes of the Kaposi's sarcoma-associated herpesvirus (KSHV) has been well studied, little is known of the architecture of the viral transcriptome on a genomewide scale. Here we have employed a genomewide tiling array to examine the lytic transcriptome of the Kaposi's sarcoma-associated herpesvirus, KSHV. Our results reveal that during lytic growth (but not during latency), there is extensive transcription from noncoding regions, including both intergenic regions and, especially, noncoding regions antisense to known open reading frames (ORFs). Several of these transcripts have been characterized in more detail, including (i) a 10-kb RNA antisense to the major latency locus, including many of its microRNAs as well as its ORFs; (ii) a 17-kb RNA antisense to numerous ORFs at the left-hand end of the genome; and (iii) a 0.7-kb RNA antisense to the viral homolog of interleukin-6 (vIL-6). These studies indicate that the lytic herpesviral transcriptome resembles a microcosm of the host transcriptome and provides a useful system for the study of noncoding RNAs.

Making sense of antisense: seemingly noncoding RNAs antisense to the master regulator of Kaposi's sarcoma-associated herpesvirus lytic replication do not regulate that transcript but serve as mRNAs encoding small peptides.

The mammalian transcriptome is studded with putative noncoding RNAs, many of which are antisense to known open reading frames (ORFs). Roles in the regulation of their complementary mRNAs are often imputed to these antisense transcripts, but few have been experimentally examined, and such functions remain largely conjectural. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes two transcripts that lack obvious ORFs and are complementary to the gene (RTA) encoding the master regulator of the latent/lytic switch. Here, we show that, contrary to expectation, these RNAs do not regulate RTA expression. Rather, they are found on polysomes, and genetic analysis indicates that translational initiation occurs at several AUG codons in the RNA, leading to the presumptive synthesis of peptides of 17 to 48 amino acids. These findings underscore the need for circumspection in the computational assessment of coding potential and raise the possibility that the mammalian proteome may contain many previously unsuspected peptides generated from seemingly noncoding RNAs, some of which could have important biological functions. Irrespective of their function, such peptides could also contribute substantially to the repertoire of T cell epitopes generated in both uninfected and infected cells.

Overexpression of the kaposi's sarcoma-associated herpesvirus transactivator K-Rta can complement a K-bZIP deletion BACmid and yields an enhanced growth phenotype.

Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 (HHV8) ORF50 encodes a transactivator, K-Rta, which functions as the switch from latent to lytic virus replication. K-bZIP interacts with K-Rta and can repress its transactivation activity for some viral promoters. Both K-Rta and K-bZIP are required for origin-dependent DNA replication. To determine the role of K-bZIP in the context of the viral genome, we generated a recombinant HHV8 bacterial artificial chromosome (BAC) with a deletion in the K-bZIP open reading frame. This BACmid, BAC36DeltaK8, displayed an enhanced growth phenotype with respect to virus production and accumulation of virus-encoded mRNAs measured by real-time PCR when K-Rta was used to induce the virus lytic cycle. Conversely, induction of the virus lytic cycle using tetradecanoyl phorbol acetate/n-butyrate resulted in no virus production and an aberrant gene expression pattern from BAC36DeltaK8-containing cells compared to wild-type (wt) BAC. This null virus phenotype was efficiently complemented by the expression of K-bZIP in trans, restoring virus production to wt BAC levels. Immunofluorescence staining revealed that subcellular localization of K-Rta was unchanged; however, a disruption of LANA subcellular localization was observed in cells harboring BAC36DeltaK8, suggesting that K-bZIP influences LANA localization. Coimmunoprecipitation experiments confirmed that K-bZIP interacts with LANA in BCBL-1 cells and in cotransfection assays. Lastly, the chromatin immunoprecipitation assay revealed that, in an environment where K-Rta is overexpressed and in the absence of K-bZIP, K-Rta binds to CAAT enhancer binding protein alpha sites within oriLyt, suggesting that it is K-Rta that supplies an essential replication function and that K-bZIP may serve to augment or facilitate the interaction of K-Rta with oriLyt.

Human cytomegalovirus UL84 interacts with an RNA stem-loop sequence found within the RNA/DNA hybrid region of oriLyt.

Human cytomegalovirus (HCMV) lytic DNA replication is initiated at the complex cis-acting oriLyt region, which spans nearly 3 kb. DNA synthesis requires six core proteins together with UL84 and IE2. Previously, two essential regions were identified within oriLyt. Essential region I (nucleotides [nt] 92209 to 92573) can be replaced with the constitutively active simian virus 40 promoter, which in turn eliminates the requirement for IE2 in the origin-dependent transient-replication assay. Essential region II (nt 92979 to 93513) contains two elements of interest: an RNA/DNA hybrid domain and an inverted repeat sequence capable of forming a stem-loop structure. Our studies now reveal for the first time that UL84 interacts with a stem-loop RNA oligonucleotide in vitro, and although UL84 interacted with other nucleic acid substrates, a specific interaction occurred only with the RNA stem-loop. Increasing concentrations of purified UL84 produced a remarkable downward-staircase pattern, which is not due to a nuclease activity but is dependent upon the presence of secondary structures, suggesting that UL84 modifies the conformation of the RNA substrate. Cross-linking experiments show that UL84 possibly changes the conformation of the RNA substrate. The addition of purified IE2 to the in vitro binding reaction did not affect binding to the stem-loop structure. Chromatin immunoprecipitation assays performed using infected cells and purified virus show that UL84 is bound to oriLyt in a region adjacent to the RNA/DNA hybrid and the stem-loop structure. These results solidify UL84 as the potential initiator of HCMV DNA replication through a unique interaction with a conserved RNA stem-loop structure within oriLyt.