Decreased Replication Origin Activity in Temporal Transition Regions

In the mammalian genome, early- and late-replicating domains are often separated by temporal transition regions (TTRs) with novel properties and unknown functions. We identified a TTR in the mouse immunoglobulin heavy chain (Igh) locus, which contains replication origins that are silent in embryonic stem cells but activated during B cell development. To investigate which factors contribute to origin activation during B cell development, we systematically modified the genetic and epigenetic status of the endogenous Igh TTR and used a single-molecule approach to analyze DNA replication. Introduction of a transcription unit into the Igh TTR, activation of gene transcription, and enhancement of local histone modifications characteristic of active chromatin did not lead to origin activation. Moreover, very few replication initiation events were observed when two ectopic replication origin sequences were inserted into the TTR. These findings indicate that the Igh TTR represents a repressive compartment that inhibits replication initiation, thus maintaining the boundaries between early and late replication domains

Regulation of DNA Replication within the Immunoglobulin Heavy-Chain Locus During B Cell Commitment

The temporal order of replication of mammalian chromosomes appears to be linked to their functional organization, but the process that establishes and modifies this order during cell differentiation remains largely unknown. Here, we studied how the replication of the Igh locus initiates, progresses, and terminates in bone marrow pro-B cells undergoing B cell commitment. We show that many aspects of DNA replication can be quantitatively explained by a mechanism involving the stochastic firing of origins (across the S phase and the Igh locus) and extensive variations in their firing rate (along the locus). The firing rate of origins shows a high degree of coordination across Igh domains that span tens to hundreds of kilobases, a phenomenon not observed in simple eukaryotes. Differences in domain sizes and firing rates determine the temporal order of replication. During B cell commitment, the expression of the B-cell-specific factor Pax5 sharply alters the temporal order of replication by modifying the rate of origin firing within various Igh domains (particularly those containing Pax5 binding sites). We propose that, within the Igh CH-3′RR domain, Pax5 is responsible for both establishing and maintaining high rates of origin firing, mostly by controlling events downstream of the assembly of pre-replication complexes

Decreased replication origin activity in temporal transition regions

Experimental attempts to activate replication origins within the temporal transition region in the IgH locus in mouse embryonic stem cells were not successful, and thus, why and how they become activated in B cells remains unclear

Single Molecule Analysis of Replicated DNA Reveals the Usage of Multiple KSHV Genome Regions for Latent Replication

Kaposi's sarcoma associated herpesvirus (KSHV), an etiologic agent of Kaposi's sarcoma, Body Cavity Based Lymphoma and Multicentric Castleman's Disease, establishes lifelong latency in infected cells. The KSHV genome tethers to the host chromosome with the help of a latency associated nuclear antigen (LANA). Additionally, LANA supports replication of the latent origins within the terminal repeats by recruiting cellular factors. Our previous studies identified and characterized another latent origin, which supported the replication of plasmids ex-vivo without LANA expression in trans. Therefore identification of an additional origin site prompted us to analyze the entire KSHV genome for replication initiation sites using single molecule analysis of replicated DNA (SMARD). Our results showed that replication of DNA can initiate throughout the KSHV genome and the usage of these regions is not conserved in two different KSHV strains investigated. SMARD also showed that the utilization of multiple replication initiation sites occurs across large regions of the genome rather than a specified sequence. The replication origin of the terminal repeats showed only a slight preference for their usage indicating that LANA dependent origin at the terminal repeats (TR) plays only a limited role in genome duplication. Furthermore, we performed chromatin immunoprecipitation for ORC2 and MCM3, which are part of the pre-replication initiation complex to determine the genomic sites where these proteins accumulate, to provide further characterization of potential replication initiation sites on the KSHV genome. The ChIP data confirmed accumulation of these pre-RC proteins at multiple genomic sites in a cell cycle dependent manner. Our data also show that both the frequency and the sites of replication initiation vary within the two KSHV genomes studied here, suggesting that initiation of replication is likely to be affected by the genomic context rather than the DNA sequences

A modal logic for role-based access control within the HOL theorem prover

The access-control logic of Lampson, Abadi, and their colleagues [LABW92, ABLP93] makes it possible to assure the correctness of access-control decisions by accounting correctly for identity, credentials, authority, delegation, and privileges. It can be used to describe a variety of access-control policies and to reason about their access-control decisions. However, it lacks an ability to reason about role-based access control (RBAC) [FSG+01, FBK99, FKC03, SCFY96], which is a popular technique for reducing the administrative complexity of associating users and privileges. This dissertation introduces extensions to the access-control logic that can be used to assure the correctness of RBAC access-control decisions. By implementing and extending the access-control logic in a computer-assisted reasoning tool such as the Higher Order Logic (HOL) theorem prover [GM93], the access-control logic and its extensions are proved to be sound. The result is a tool for design and verification engineers to reason about access-control policies including RBAC. In this dissertation, we explain how to use the logic to describe RBAC components, such as user assignments, permission assignments, role inheritance, role activations, and users’ requests. We also describe in detail the steps of implementing the access-control logic and its extensions in the HOL theorem prover. Administrative RBAC systems are also explored to see how the HOL theorem prover can be used to formally verify their properties and policies

A Modal Logic for Role-Based Access Control within the Hol Theorem Prover

The access-control logic of Lampson, Abadi, and their colleagues [LABW92, ABLP93] makes it possible to assure the correctness of access-control decisions by accounting correctly for identity, credentials, authority, delegation, and privileges. It can be used to describe a variety of access-control policies and to reason about their access-control decisions. However, it lacks an ability to reason about role-based access control (RBAC) [FSG + 01, FBK99, FKC03, SCFY96], which is a popular technique for reducing the administrative complexity of associating users and privileges. This dissertation introduces extensions to the access-control logic that can be used to assure the correctness of RBAC access-control decisions. By implementing and extending the access-control logic in a computer-assisted reasoning tool such as the Higher Order Logic (HOL) theorem prover [GM93], the access-control logic and its extensions are proved to be sound. The result is a tool for design and verification engineers to reason about access-control policies including RBAC. In this dissertation, we explain how to use the logic to describe RBAC components, such as user assignments, permission assignments, role inheritance, role activations, and users\u27 requests. We also describe in detail the steps of implementing the access-control logic and its extensions in the HOL theorem prover. Administrative RBAC systems are also explored to see how the HOL theorem prover can be used to formally verify their properties and policies

Abstaining from symptomatic implants of modified tension band wiring by nonabsorbable suture fixation for transverse patella fractures

Abstract Background Modified tension band fixation has become commonly used for transverse patella fractures. The conventional stainless steel wire provides sufficient stability but may be associated with complications. Objective The study aimed to evaluate the effectiveness of a new modified tension band fixation technique for transverse patella fractures using a nonabsorbable suture. Material and methods We present the result of a prospective series using a nonabsorbable suture (FiberWire) for transverse patella fractures. The mean follow-up period totaled 12 months. A total of 16 patients were evaluated by radiographic and clinical review. The postoperative clinical evaluation employed Lysholm and Böstman scores. Result All clinical results on follow-up were good to excellent. Minimal intra-articular joint stepping and further fracture displacement were recorded. No patient needed re-operation, and functional outcomes of the knee were satisfactory. No significant differences were found between the injured and contralateral knee range of motion. No symptomatic implants and skin complications were noted, and all fractures were completed heal within 15 weeks. Conclusion FiberWire provided sufficient stability and reduced postoperative complications. The results proved appropriate, and the technique has merit, as it obviates the need for re-operation

Radial head replacement with the 3D-printed patient-specific titanium prosthesis: Preliminary results of a multi-centric prospective study

Purpose To report preliminary clinical results and safety of 3D-printed patient-specific titanium radial head (RH) prosthesis in treatment of the irreparable RH fractures. Material and methods This multi-centric prospective study included 10 patients (6 men and four women, mean age 41 years (range, 25–64 years)). Three cases were classified as Mason type III and 7 cases as type IV. Patients were assessed preoperatively, intraoperatively, and at 1, 6, 12, 24, 36, and 48 weeks postoperatively. Range of motion (ROM), visual analog scale (VAS) score, Disabilities of the Arm, Shoulder and Hand (DASH) score, Mayo Elbow Performance Score (MEPS), radiology imaging, and laboratory blood and urine testing were evaluated. Results The prostheses were implanted utilizing cemented stems in 5 patients and cementless stems in 5 patients. Intraoperatively, well congruency of a prosthesis with capitellum and radial notch of ulna was observed in all cases. All patients had improvement of ROM, VAS score, DASH score, and MEPS during the postoperative follow-ups. At the final follow-up, mean elbow extension was 6.5° (range, 0°–30°), flexion 145° (range, 125°–150°), supination 79° (range, 70°–80°), and pronation 73.5° (range, 45°–80°). Mean VAS score was 0.3 (range, 0–3), DASH score was 12.35 (range, 1.7–23.3), and MEPS was 99.5 (range, 95–100). Postoperative radiographs demonstrated heterotopic ossification in 2 cases, periprosthetic radiolucency in 2 cases, and proximal radial neck resorption in 2 cases. No one had the evidence of capitellar erosion, implant failure, malpositioning, overstuffing, or symptomatic stem loosening. There was no significant alteration of laboratory results or adverse events related to the 3D-printed prosthesis implantation. Conclusion The preliminary results demonstrated that implantation of the 3D-printed patient-specific titanium RH prosthesis is safe and may be a potential treatment option for irreparable RH fracture

The Patient-Specific Implant Created with 3D Printing Technology in Treatment of the Irreparable Radial Head in Chronic Persistent Elbow Instability

Successful treatment of the chronic persistent elbow instability is a challenge for orthopedic surgeons. In this form, it is important to recognize and restore the osseous stabilizer in order to obtain the concentric reduction. In the present report, we describe a case of such injury with irreparable radial head treated with patient-specific radial head prosthesis which was created with 3D printing technology. To our knowledge, this is the first report in clinical use of this kind of prosthesis for the radial head fracture. At a 24-month follow-up visit, the patient was satisfied with the functional outcomes. The Mayo Elbow Performance Index (MEPI) increased from 20 points at the preoperative day to 85 points, and the patient-based Disabilities of the Arm, Shoulder, and Hand (DASH) was reduced from 88.33 points to 28.33 points. Due to the favorable result, replacement of the radial head with the patient-specific implant could be a useful treatment for the irreparable radial head in chronic persistent elbow instability