Outcomes of a Novel Spine Virtual Fracture Clinic in an Australian Tertiary Hospital.

STUDY DESIGN: This was a retrospective cohort study. OBJECTIVE: To determine the proportion of referrals diverted by the SVFC from traditional outpatient clinic management. SUMMARY OF BACKGROUND DATA: The consistent rise in demand for orthopaedic outpatient clinic services is creating marked challenges to the provision of quality care. Virtual fracture clinics for upper and lower limb fractures have reduced the burden on outpatient clinics through telephone-based management of these conditions. To date, no study describes the expansion of virtual care to the spine trauma population. METHODS: A study of spine fractures referred to the RMH Department of Orthopaedic Surgery was conducted comparing outcomes prior to (January to December 2021) and following (July 2022 to November 2023) implementation of a spine virtual fracture clinic (SVFC). The primary aim of this study was to investigate the effects of a telephone-based SVFC on outpatient clinic activity, represented by the proportion of referrals discharged without requiring in-person clinic review. Secondary aims included appointment utilisation, lost to follow-up rates, duration of care, missed or mis-diagnoses, unplanned operations and complications. RESULTS: A total of 91.9% (n=666) referrals managed by the SVFC were discharged without in-person clinic attendance. Compared to outpatient clinic management (n=150 referrals), SVFC implementation was associated with reductions in the average number of consultations per referral (1.8 versus 2.4, P<0.001), appointments not attended (5% versus 13%, P<0.001), referrals lost to follow-up (0 versus 10.7%, P<0.001) and a shorter duration of care (median 48 d versus 58 d, P<0.001). A total of 65 patients (8.1%) were redirected to in-person clinics of which three underwent surgical intervention. No diagnostic errors, complications or adverse events were identified. CONCLUSION: This study demonstrates that a SVFC is an effective and safe alternative pathway to traditional hospital-based outpatient clinics with low-risk for any adverse outcomes.

The clinical and radiographic degenerative spondylolisthesis classification and its predictive value.

INTRODUCTION: The clinical and radiographic degenerative spondylolisthesis (CARDS) classification is a new classification that has been introduced for degenerative spondylolisthesis (DS). It has four categories. Our study aimed to analyse the functional and radiographic outcome following DS surgery based on the preoperative CARDS classification. METHODS: A retrospective study of the prospectively collected Australian Spine Registry database was performed. Data on demographics, patient reported outcome measures including the Oswestry Disability Index (ODI) and EQ-5D-3 L scores, and changes in radiographic measurements were analysed. Based on the preoperative findings all x-rays were classified applying the CARDS classification. RESULTS: Between 2018 and 2021 a total of 54-patients were identified as having had surgery for DS at L4/5. The mean age was 65.3 ± 11.3years and females were predominantly affected (61%). Most cases were of CARDS type C (46%), followed by type B (29%). CARDS type A and D were observed in 18% and 6% respectively. Preoperatively, the L4/5 lordosis was 19.8 ± 6.3° and lumbar lordosis 43.9 ± 12.8°. Postoperatively the L4/5 lordosis alignment changed significantly to 23.5 ± 8.8° (p < 0.05). Preoperatively, the CARDS classification was 34.8 ± 17.4 (type A), 40.5 ± 11.0 (type B), 43.8 ± 12.9 and 50.0 ± 14.4 for type D (Pearson-coefficient 0.284, p = 0.041). Postoperatively this changed to 22.7 ± 16.1, 28.7 ± 21.2, 12.5 ± 13.1, and 6.5 ± 2.1 respectively. Similar improvements were observed for the EQ-5D-3 L. CONCLUSION: This study shows that the CARDS classification correlates with preoperative functional scores as well as helping to predict response to surgery. CARDS will likely assist in operative planning and prognostication. LEVEL OF EVIDENCE: III, therapeutic and prognostic study.

Spinal fractures in fused spines: nonoperative treatment is a reliable alternative.

INTRODUCTION: Spinal fractures in fused spines such as in ankylosing spondylitis or DISH are typically of type B or C fractures where operative treatment is recommended. The mortality rate in non-operatively treated patients is reported to be 51%. The purpose of this study was to investigate the mortality rate, complication rate and demographics of patients following non-operatively treatment in fused spine injuries. METHODS: Between 2019 and 2021, a retrospective study was conducted including all patients who presented to our trauma center with a spinal fracture of a fused spine. Radiology and patient charts were analyzed for fracture pattern, complications, neurological deficit, comorbidities, and mortality rate. RESULTS: A total of 49 patients were found at a mean age was 79.8 ± 10.9 years and primarily males were affected in 65.3%. All fractures were of type B and the thoracic spine was involved in 85.7%. The mean follow-up was 6.3 ± 8.2 months and fusion was obtained in all patients. No neurological deficit was observed in any. A total of 13 patients died at a mean age of 86.5 ± 10.0 years after 157.1 ± 158.1 days. 6 patients (10.2%) deceased within the first 6 weeks at a mean age of 91.8 ± 3.8 years. One patient each suffered from heart failure, an acute delirium, end stage colon cancer and subdural hemorrhage. CONCLUSION: This study shows that the mortality rate in the first 6 weeks following a fracture in a fused spine is 10.2% for patients above the age of 90 years. Therefore, non-operative treatment should be taken into consideration as the mortality rate in other studies may be overestimated. LEVEL OF EVIDENCE: III, retrospective study.

Subacute Posttraumatic Ascending Myelopathy: Case Report and Systematic Review of the Literature.

STUDY DESIGN: Case report and literature review. OBJECTION: Aim of this study was to summarize the current evidence base behind subacute posttraumatic ascending myelopathy (SPAM) including the epidemiology, presentation, diagnosis, prognosis, and etiology. SUMMARY OF BACKGROUND DATA: SPAM is a rare, potentially fatal disorder which is not attributable to ongoing mechanical instability, syrinx formation, or iatrogenic causes. METHODS: A systematic literature search on SPAM was performed on Medline, Ovid, Cochrane, Embase, and PubMed databases between 1969 and 2021. Cases were reviewed and the findings summarized. Further evidence was reviewed to support the hypothesis that disruption of cerebrospinal fluid (CSF) circulation is the underlying etiology of the condition. RESULTS: It is estimated to occur in 0.4%-0.7% of spinal cord injuries and may have a mortality of up to 10%. The most likely etiology disruption of CSF circulation leading to further damage to the spinal cord presumably through pressure mediated effects such as a reduction in cellular perfusion. CONCLUSION: There is effectively no treatment of this condition, however, with interest developing in monitoring of CSF pressures during spinal cord injury this may help confirm the etiology, and allow the suggestion of therapies such as drains or expansion duraplasty to reduce spinal cord pressures. LEVEL OF EVIDENCE: Level II-case report and systematic review.

DExD/H Box Helicases DDX24 and DDX49 Inhibit Reactivation of Kaposi's Sarcoma Associated Herpesvirus by Interacting with Viral mRNAs.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic gammaherpesvirus that is the causative agent of primary effusion lymphoma and Kaposi's sarcoma. In healthy carriers, KSHV remains latent, but a compromised immune system can lead to lytic viral replication that increases the probability of tumorigenesis. RIG-I-like receptors (RLRs) are members of the DExD/H box helicase family of RNA binding proteins that recognize KSHV to stimulate the immune system and prevent reactivation from latency. To determine if other DExD/H box helicases can affect KSHV lytic reactivation, we performed a knock-down screen that revealed DHX29-dependent activities appear to support viral replication but, in contrast, DDX24 and DDX49 have antiviral activity. When DDX24 or DDX49 are overexpressed in BCBL-1 cells, transcription of all lytic viral genes and genome replication were significantly reduced. RNA immunoprecipitation of tagged DDX24 and DDX49 followed by next-generation sequencing revealed that the helicases bind to mostly immediate-early and early KSHV mRNAs. Transfection of expression plasmids of candidate KSHV transcripts, identified from RNA pull-down, demonstrated that KSHV mRNAs stimulate type I interferon (alpha/beta) production and affect the expression of multiple interferon-stimulated genes. Our findings reveal that host DExD/H box helicases DDX24 and DDX49 recognize gammaherpesvirus transcripts and convey an antiviral effect in the context of lytic reactivation.

App-Based Rehabilitation in Back Pain, a Systematic Review.

Smartphones and their associated applications are used frequently by patients and clinicians alike. Despite the technology being widely accessible, their use to aid in rehabilitation is yet to be adopted. The SARS CoV-2 pandemic has presented an opportunity to expedite their integration given the difficulty patients currently have in accessing healthcare. The aim of this study was to perform a systematic literature review on the use of smartphone rehabilitation applications compared to standard physiotherapy for back pain. We conducted a search of Medline/Pubmed and google databases using the search terms [APP] AND [[Orthopaedic] OR [Neurosurgery]], following the PRISMA guidelines. All prospective studies investigating rehabilitation applications for back pain or following spine surgery were included. A total of nine studies met the inclusion criteria which investigated 7636 patients, of which 92.4% were allocated to the interventional group (n = 7055/7636) with a follow up of 4 weeks to 6 months. All except one study reported on patients experiencing back pain on average for 19.6 ± 11.6 months. The VAS-pain score was presented in all studies without significance between the interventional and control group (p = 0.399 before and p = 0.277 after intervention). Only one research group found significantly higher improvement in PROMs for the application group, whereas the remaining showed similar results compared to the control group. Using application-based rehabilitation programs provides an easily accessible alternative or substitute to traditional physiotherapy for patients with back pain. Given that smartphones are so prevalent in activities in our daily lives, this will enhance and improve rehabilitation if patients are self-dedicated and compliant.

Modified Cross-Linking, Ligation, and Sequencing of Hybrids (qCLASH) to Identify MicroRNA Targets.

This protocol was designed to identify microRNA (miRNA) targetomes from smaller-input samples by performing a simplified workflow of the Cross-Linking and Sequencing of Hybrids (CLASH) technique developed in the Tollervey group. In this ribonomics-based technique, Cross-Linking and Immunoprecipitation (CLIP) of Argonaute (Ago) is combined with an RNA ligase reaction that yields covalently bound "hybrids" between miRNAs and their target RNAs. While this iteration of CLIP identifies "high-confidence" or "unambiguous" miRNA targets, the added ligation step is highly inefficient and therefore requires large numbers of cultured cells. To make this powerful approach applicable to smaller cell numbers, we created qCLASH, incorporating a workflow that performs all enzymatic reactions on bead-bound complexes and omits gel purification of immunoprecipitated Ago complexes associated with major loss of RNA. At a sequencing depth of 100 million reads per library, which is highly feasible with rapidly decreasing sequencing costs, qCLASH, when used with three biological replicates, results in thousands of high-confidence miRNA targets. qCLASH was first developed to identify viral miRNA targetomes of endothelial cells infected with Kaposi's sarcoma-associated herpesvirus. Since then, qCLASH has been applied to Epstein-Barr virus- and MHV68-infected cells, and more recently to metastatic melanoma and breast cancer cells. Currently, protocols are under development to apply qCLASH to human solid tumor specimens. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Quick Cross-Linking and Sequencing of Hybrids (qCLASH) Support Protocol: Optimization of Ago immunoprecipitation.

Promising Anticancer Activity of [Bis(1,8-quinolato)palladium (II)] Alone and in Combination.

Due to similar coordination chemistry of palladium and platinum, a large number of palladium compounds as well have been investigated for their anticancer activity. In the present study, we describe synthesis, characterization, and anticancer activity of palladium complex [Bis(1,8-quinolato)palladium (II)], coded as NH3 against seven different cancer cell lines. NH3 is found to have higher antitumor activity than cisplatin against both parent ovarian A2780 cell line and cisplatin-resistant cell lines. Also, NH3 has the lower IC50 value in HT-29 colorectal cancer cell line. The higher antitumor activity of NH3 is due to the presence of bulky 8-Hydroxyquinoline ligand, thus reducing its reactivity. Proteomic study has identified significantly expressed proteins which have been validated through bioinformatics. NH3 has been found to be less toxic than cisplatin at 2.5 mg/kg and 5 mg/kg dosages on mice models. Binary combinations of NH3 with curcumin and epigallocatechin gallate (EGCG) have demonstrated dose and sequence-dependent synergism in ovarian and colorectal cancer models. All of the preclinical studies indicate promising therapeutic potential of NH3 [Bis(1,8-quinolato)palladium (II)] as an anticancer drug.

Kaposi's Sarcoma-associated Herpesvirus microRNA mutants modulate cancer hallmark phenotypic differences in human endothelial cells.

Kaposi's sarcoma (KS) results from the transformation of Kaposi's sarcoma-associated herpesvirus (KSHV)-infected endothelial cells. The contribution of the KSHV microRNAs (miRNAs) to the process of oncogenesis in endothelial cells has not been fully elucidated. To better understand the contributions of individual miRNAs to oncogenesis-related cellular phenotypes, we used KSHV miRNA knockout mutants, each one lacking one of the twelve miRNA genes. An additional mutant lacked all miRNAs. Since KSHV infection causes a variety of phenotypic changes in endothelial cells, we tested the mutants for their ability to effect such changes in Telomerase-Immortalized Vein Endothelial (TIVE) cells infected with each of the mutant viruses. Wild type- and mutant-infected as well as uninfected cells were evaluated for perturbations to proliferation, migration, tubule formation, and glycolysis. We found broad variation between the different viruses in these aspects. With respect to proliferation rate, ΔmiR-K12-3, ΔmiR-K12-8, and ΔmiR-K12-11 showed significant impairment. Cells infected with ΔmiR-K12-11 had reduced migration. In tubule formation, the ΔmiR-K12-5, -6, and -7 viruses were deficient. At the same time, cells infected with the ΔmiR-K12-10 virus showed dysregulated glycolysis. By combining these observations with previously published KSHV miRNA targetome lists from ribonomics data, we were able to functionally validate a number of new miRNA targets in specific pathways. As proof of concept, miR-K12-3 was shown to target Cathepsin D, a strong promoter of apoptosis. Taken together, the results demonstrate that KSHV miRNAs play different roles in inducing the phenotypic changes which are characteristic of transformed cells.Importance: Kaposi's sarcoma-associated herpesvirus (KSHV) causes Kaposi's sarcoma (KS). The contribution of KSHV microRNAs (miRNAs) to oncogenesis is not fully understood. This is particularly true for human endothelial cells, the cell type from which KS tumors are derived. Here we used a panel of KSHV miRNA knockout viruses in order to shed light on the roles of individual miRNAs in the process of transformation. Latently infected endothelial cells were studied for phenotypic changes related to cancer, including proliferation, migration, angiogenesis, glycolysis, and apoptosis. The mutant-infected cell lines displayed a wide range of phenotypes in these selected measures of oncogenesis which differed from wild type-infected cells and from each other. These results indicate that KSHV miRNAs contribute to different aspects of oncogenesis, and that each one has a unique role to play.

Voltage-Switchable HCl Transport Enabled by Lipid Headgroup-Transporter Interactions.

Synthetic anion transporters that facilitate transmembrane H+ /Cl- symport (cotransport) have anti-cancer potential due to their ability to neutralize pH gradients and inhibit autophagy in cells. However, compared to the natural product prodigiosin, synthetic anion transporters have low-to-modest H+ /Cl- symport activity and their mechanism of action remains less well understood. We report a chloride-selective tetraurea macrocycle that has a record-high H+ /Cl- symport activity similar to that of prodigiosin and most importantly demonstrates unprecedented voltage-switchable transport properties that are linked to the lack of uniport activity. By studying the anion binding affinity and transport mechanisms of four other anion transporters, we show that the lack of uniport and voltage-dependent H+ /Cl- symport originate from strong binding to phospholipid headgroups that hampers the diffusion of the free transporters through the membrane, leading to an unusual H+ /Cl- symport mechanism that involves only charged species. Our work provides important mechanistic insights into different classes of anion transporters and a new approach to achieve voltage-switchability in artificial membrane transport systems.

Contemporary Ribonomics Methods for Viral microRNA Target Analysis.

Numerous cellular processes are regulated by microRNAs (miRNAs), both cellular and viral. Elucidating the targets of miRNAs has become an active area of research. An important method in this field is cross-linking and immunoprecipitation (CLIP), where cultured cells or tissues are UV-irradiated to cross-link protein and nucleic acid, the RNA binding protein of interest is immunoprecipitated, and the RNAs pulled down with the protein are isolated, reverse-transcribed, and analyzed by sequencing. CLIP using antibody against Argonaute (Ago), which binds to both miRNA and mRNA as they interact in RISC, has allowed researchers to uncover a large number of miRNA targets. Coupled with high-throughput sequencing, CLIP has been useful for revealing miRNA targetomes for the γ-herpesviruses Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Variants on the CLIP protocol are described, with the benefits and drawbacks of each. In particular, the most recent methods involving RNA⁻RNA ligation to join the miRNA and its RNA target have aided in target identification. Lastly, data supporting biologically meaningful interactions between miRNAs and long non-coding RNAs (lncRNAs) are reviewed. In summary, ribonomics-based miRNA targetome analysis has expanded our understanding of miRNA targeting and has provided a rich resource for EBV and KSHV research with respect to pathogenesis and tumorigenesis.

Modified Cross-Linking, Ligation, and Sequencing of Hybrids (qCLASH) Identifies Kaposi's Sarcoma-Associated Herpesvirus MicroRNA Targets in Endothelial Cells.

Kaposi's sarcoma (KS) tumors are derived from endothelial cells and express Kaposi's sarcoma-associated herpesvirus (KSHV) microRNAs (miRNAs). Although miRNA targets have been identified in B cell lymphoma-derived cells and epithelial cells, little has been done to characterize the KSHV miRNA targetome in endothelial cells. A recent innovation in the identification of miRNA targetomes, cross-linking, ligation, and sequencing of hybrids (CLASH), unambiguously identifies miRNAs and their targets by ligating the two species while both species are still bound within the RNA-induced silencing complex (RISC). We developed a streamlined quick CLASH (qCLASH) protocol that requires a lower cell input than the original method and therefore has the potential to be used on patient biopsy samples. Additionally, we developed a fast-growing, KSHV-negative endothelial cell line derived from telomerase-immortalized vein endothelial long-term culture (TIVE-LTC) cells. qCLASH was performed on uninfected cells and cells infected with either wild-type KSHV or a mutant virus lacking miR-K12-11/11*. More than 1,400 cellular targets of KSHV miRNAs were identified. Many of the targets identified by qCLASH lacked a canonical seed sequence match. Additionally, most target regions in mRNAs originated from the coding DNA sequence (CDS) rather than the 3' untranslated region (UTR). This set of genes includes some that were previously identified in B cells and some new genes that warrant further study. Pathway analysis of endothelial cell targets showed enrichment in cell cycle control, apoptosis, and glycolysis pathways, among others. Characterization of these new targets and the functional consequences of their repression will be important in furthering our understanding of the role of KSHV miRNAs in oncogenesis.IMPORTANCE KS lesions consist of endothelial cells latently infected with KSHV. Cells that make up these lesions express KSHV miRNAs. Identification of the targets of KSHV miRNAs will help us understand their role in viral oncogenesis. The cross-linking and sequencing of hybrids (CLASH) protocol is a method for unambiguously identifying miRNA targetomes. We developed a streamlined version of CLASH, called quick CLASH (qCLASH). qCLASH requires a lower initial input of cells than for its parent protocol. Additionally, a new fast-growing KSHV-negative endothelial cell line, named TIVE-EX-LTC cells, was established. qCLASH was performed on TIVE-EX-LTC cells latently infected with wild-type (WT) KSHV or a mutant virus lacking miR-K12-11/11*. A number of novel targets of KSHV miRNAs were identified, including targets of miR-K12-11, the ortholog of the cellular oncogenic miRNA (oncomiR) miR-155. Many of the miRNA targets were involved in processes related to oncogenesis, such as glycolysis, apoptosis, and cell cycle control.

The influence of the ancillary ligand on the potential of cobalt(iii) complexes to act as chaperones for hydroxamic acid-based drugs.

Cobalt(iii) chaperones are a promising class of bioreductive prodrugs under investigation for the delivery of cytotoxic ligands to hypoxic solid tumours. Here we investigate a series of cobalt complexes as chaperones for hydroxamic acid ligands, comparing the properties of the cyclic cyclen (1,4,7,10-tetraazacyclododecane) ancillary ligand with the tripodal tpa (tris-(2-pyridylmethyl)amine) and tren (tris-(2-aminoethyl)amine). A small library of complexes containing several different hydroxamic acids, including the MMP inhibitor Marimistat and the fluorescent ligand C343haH2, were prepared and their pKa values, reduction potentials, and in some cases X-ray crystal structures, were determined. The antiproliferative actitivity of the series was evaluated against DLD-1 colon cancer cells and the cellular accumulation of the fluorescent C343haH2 complexes was monitored by ICPMS and confocal fluorescence microscopy, revealing that the nature of the ancillary ligand significantly influences the complexes' properties, cytotoxicity and cellular distribution.

Locking stand-alone cages versus anterior plate constructs in single-level fusion for degenerative cervical disease: a systematic review and meta-analysis.

PURPOSE: To conduct a meta-analysis to compare the clinical and radiological outcomes in single-level anterior cervical discectomy and fusion (ACDF) surgery for degenerative cervical disease performed by either single-level locking stand-alone cage (LSC) or anterior plate construct (APC). METHODS: We performed a comprehensive database search of Medline, PubMed, EMBASE and Cochrane Database of Systematic Reviews according to PRISMA guidelines and identified six articles that satisfied our inclusion criteria. We excluded all non-English language articles and articles which did not directly compare LSC and APC. Only papers which focussed on single-level ACDF were included in the study. RESULTS: There were no significant differences in blood loss, clinical outcomes (JOA, VAS, NDI scores) or radiological outcomes (cervical lordosis, segmental Cobb angle, subsidence and fusion) between the two groups. Operative time was significantly shorter in the LSC group (MD 7.2 min, 95% CI 0.3-14.1, p = 0.04). APC was associated with a statistically significant increase in dysphagia in the follow-up period (OR 6.2, 95% CI 1.0-36.6, p = 0.05). CONCLUSION: LSC and APC have similar clinical and radiological outcomes. Further blinded randomised trials are required to establish conclusive evidence in favour of LSC with regards to minimising post-operative dysphagia. We also encourage future studies to make use of formalised dysphagia outcome measures in reporting complications.

A Toolbox for Herpesvirus miRNA Research: Construction of a Complete Set of KSHV miRNA Deletion Mutants.

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes 12 viral microRNAs (miRNAs) that are expressed during latency. Research into KSHV miRNA function has suffered from a lack of genetic systems to study viral miRNA mutations in the context of the viral genome. We used the Escherichia coli Red recombination system together with a new bacmid background, BAC16, to create mutants for all known KSHV miRNAs. The specific miRNA deletions or mutations and the integrity of the bacmids have been strictly quality controlled using PCR, restriction digestion, and sequencing. In addition, stable viral producer cell lines based on iSLK cells have been created for wildtype KSHV, for 12 individual miRNA knock-out mutants (ΔmiR-K12-1 through -12), and for mutants deleted for 10 of 12 (ΔmiR-cluster) or all 12 miRNAs (ΔmiR-all). NGS, in combination with SureSelect technology, was employed to sequence the entire latent genome within all producer cell lines. qPCR assays were used to verify the expression of the remaining viral miRNAs in a subset of mutants. Induction of the lytic cycle leads to efficient production of progeny viruses that have been used to infect endothelial cells. Wt BAC16 and miR mutant iSLK producer cell lines are now available to the research community.

LANA binds to multiple active viral and cellular promoters and associates with the H3K4methyltransferase hSET1 complex.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a γ-herpesvirus associated with KS and two lymphoproliferative diseases. Recent studies characterized epigenetic modification of KSHV episomes during latency and determined that latency-associated genes are associated with H3K4me3 while most lytic genes are associated with the silencing mark H3K27me3. Since the latency-associated nuclear antigen (LANA) (i) is expressed very early after de novo infection, (ii) interacts with transcriptional regulators and chromatin remodelers, and (iii) regulates the LANA and RTA promoters, we hypothesized that LANA may contribute to the establishment of latency through epigenetic control. We performed a detailed ChIP-seq analysis in cells of lymphoid and endothelial origin and compared H3K4me3, H3K27me3, polII, and LANA occupancy. On viral episomes LANA binding was detected at numerous lytic and latent promoters, which were transactivated by LANA using reporter assays. LANA binding was highly enriched at H3K4me3 peaks and this co-occupancy was also detected on many host gene promoters. Bioinformatic analysis of enriched LANA binding sites in combination with biochemical binding studies revealed three distinct binding patterns. A small subset of LANA binding sites showed sequence homology to the characterized LBS1/2 sequence in the viral terminal repeat. A large number of sites contained a novel LANA binding motif (TCCAT)3 which was confirmed by gel shift analysis. Third, some viral and cellular promoters did not contain LANA binding sites and are likely enriched through protein/protein interaction. LANA was associated with H3K4me3 marks and in PEL cells 86% of all LANA bound promoters were transcriptionally active, leading to the hypothesis that LANA interacts with the machinery that methylates H3K4. Co-immunoprecipitation demonstrated LANA association with endogenous hSET1 complexes in both lymphoid and endothelial cells suggesting that LANA may contribute to the epigenetic profile of KSHV episomes.

One-pot peptide ligation-desulfurization at glutamate.

An efficient methodology for ligation at glutamate (Glu) is described. A γ-thiol-Glu building block was accessed in only three steps from protected glutamic acid and could be incorporated at the N-terminus of peptides. The application of these peptides in one-pot ligation-desulfurization chemistry is demonstrated with a range of peptide thioesters, and the utility of this methodology is highlighted through the synthesis of the osteoporosis peptide drug teriparatide (Forteo).

The Uncertainty of the eGFR.

The estimated glomerular filtration rate (eGFR) is a parameter derived from the serum creatinine, patient age and gender and is used to ascertain renal function. It is subject to variation because of the analytical error of the creatinine measurement and biological variation. The widespread use of the eGFR to classify renal disease has led to the identification of more patients with marginal chronic kidney disease but because of the uncertainty of the eGFR it has also led to over-diagnosis of some kidney disease. There is a well described age relation with eGFR.The uncertainty of the eGFR at the critical decision level of 60 mL/min/1.73 m(2) is calculated to be 11. Caution needs to be exercised when interpreting an eGFR between 49 and 71 mL/min/1.73 m(2).

Viral cross-class serpin inhibits vascular inflammation and T lymphocyte fratricide; a study in rodent models in vivo and human cell lines in vitro.

Poxviruses express highly active inhibitors, including serine proteinase inhibitors (serpins), designed to target host immune defense pathways. Recent work has demonstrated clinical efficacy for a secreted, myxomaviral serpin, Serp-1, which targets the thrombotic and thrombolytic proteases, suggesting that other viral serpins may have therapeutic application. Serp-2 and CrmA are intracellular cross-class poxviral serpins, with entirely distinct functions from the Serp-1 protein. Serp-2 and CrmA block the serine protease granzyme B (GzmB) and cysteine proteases, caspases 1 and 8, in apoptotic pathways, but have not been examined for extracellular anti-inflammatory activity. We examined the ability of these cross-class serpins to inhibit plaque growth after arterial damage or transplant and to reduce leukocyte apoptosis. We observed that purified Serp-2, but not CrmA, given as a systemic infusion after angioplasty, transplant, or cuff-compression injury markedly reduced plaque growth in mouse and rat models in vivo. Plaque growth was inhibited both locally at sites of surgical trauma, angioplasty or transplant, and systemically at non-injured sites in ApoE-deficient hyperlipidemic mice. With analysis in vitro of human cells in culture, Serp-2 selectively inhibited T cell caspase activity and blocked cytotoxic T cell (CTL) mediated killing of T lymphocytes (termed fratricide). Conversely, both Serp-2 and CrmA inhibited monocyte apoptosis. Serp-2 inhibitory activity was significantly compromised either in vitro with GzmB antibody or in vivo in ApoE/GzmB double knockout mice. Conclusions The viral cross-class serpin, Serp-2, that targets both apoptotic and inflammatory pathways, reduces vascular inflammation in a GzmB-dependent fashion in vivo, and inhibits human T cell apoptosis in vitro. These findings indicate that therapies targeting Granzyme B and/or T cell apoptosis may be used to inhibit T lymphocyte apoptosis and inflammation in response to arterial injury.

Cobalt complexes with tripodal ligands: implications for the design of drug chaperones.

Extensive research is currently being conducted into metal complexes that can selectively deliver cytotoxins to hypoxic regions in tumours. The development of pharmacologically suitable agents requires an understanding of appropriate ligand-metal systems for chaperoning cytotoxins. In this study, cobalt complexes with tripodal tren (tris-(2-aminoethyl)amine) and tpa (tris-(2-pyridylmethyl)amine) ligands were prepared with ancillary hydroxamic acid, ß-diketone and catechol ligands and several parameters, including: pK(a), reduction potential and cytotoxicity were investigated. Fluorescence studies demonstrated that only tpa complexes with ß-diketones showed any reduction by ascorbate in situ and similarly, cellular cytotoxicity results demonstrated that ligation to cobalt masked the cytotoxicity of the ancillary groups in all complexes except the tpa diketone derivative [Co(naac)tpa](ClO(4))(2) (naac = 1-methyl-3-(2-naphthyl)propane-1,3-dione). Additionally, it was shown that the hydroxamic acid complexes could be isolated in both the hydroxamate and hydroximate form and the pK(a) values (5.3-8.5) reveal that the reversible protonation/deprotonation of the complexes occurs at physiologically relevant pHs. These results have clear implications for the future design of prodrugs using cobalt moieties as chaperones, providing a basis for the design of cobalt complexes that are both more readily reduced and more readily taken up by cells in hypoxic and acidic environments.