Incidence of biopsy-proven giant cell arteritis (GCA) in South Australia 2014-2020.

Objective: To determine the incidence of biopsy proven giant cell arteritis (GCA) in South Australia. Methods: Patients with biopsy-proven GCA were identified from pathology reports of temporal artery biopsies at state-based pathology laboratories, from 1 January 2014 to 31 December 2020. Incidence rates for biopsy-proven GCA were calculated using Australian Bureau of Statistics data for South Australian population sizes by age, sex, and calendar year. Seasonality was analyzed by cosinor analysis. Results: There were 181 cases of biopsy-proven GCA. The median age at diagnosis of GCA was 76 years (IQR 70, 81), 64% were female. The estimated population incidence for people over 50 was 5.4 (95% CI 4.7, 6.1) per 100,000-person years. The female: male incidence ratio was 1.6 (95% CI 1.2, 2.2). There was no ordinal trend in GCA incidence rates by calendar year (p = 0.29). The incidence was, on average, highest in winter, but not significantly (p = 0.35). A cosinor analysis indicated no seasonal effect (p = 0.52). Conclusion: The incidence of biopsy-proven GCA remains low in Australia. A higher incidence was noted compared to an earlier study. However, differences in ascertainment and methods of GCA diagnosis may have accounted for the change.

Dynamic conformational switching underlies TFIIH function in transcription and DNA repair and impacts genetic diseases.

Transcription factor IIH (TFIIH) is a protein assembly essential for transcription initiation and nucleotide excision repair (NER). Yet, understanding of the conformational switching underpinning these diverse TFIIH functions remains fragmentary. TFIIH mechanisms critically depend on two translocase subunits, XPB and XPD. To unravel their functions and regulation, we build cryo-EM based TFIIH models in transcription- and NER-competent states. Using simulations and graph-theoretical analysis methods, we reveal TFIIH's global motions, define TFIIH partitioning into dynamic communities and show how TFIIH reshapes itself and self-regulates depending on functional context. Our study uncovers an internal regulatory mechanism that switches XPB and XPD activities making them mutually exclusive between NER and transcription initiation. By sequentially coordinating the XPB and XPD DNA-unwinding activities, the switch ensures precise DNA incision in NER. Mapping TFIIH disease mutations onto network models reveals clustering into distinct mechanistic classes, affecting translocase functions, protein interactions and interface dynamics.

Diagnosis of giant cell arteritis by temporal artery biopsy is associated with biopsy length.

Aims: Temporal artery biopsy (TAB) is a widely used method for establishing a diagnosis of Giant Cell Arteritis (GCA). The optimal TAB length for accurate histological GCA diagnosis has been suggested as 15 mm post-fixation (15-20 mm pre-fixation). The aim of this study was to determine the relationship between a histological GCA diagnosis and optimal TAB length in the South Australian (SA) population. Materials and methods: Pre-fixation TAB length (mm) was reported in 825/859 of all samples submitted to SA Pathology between 2014 and 2020 from people aged 50 and over. When more than one biopsy was taken, the longest length was recorded. Analyses of both TAB length and TAB positive proportions were performed by multivariable linear and logistic regression analysis, including covariates sex, age, and calendar year. Results: The median age of participants was 72 (IQR 65, 79) years, 549 (66%) were female. The TAB positive proportion was 172/825 (21%) with a median biopsy length of 14 mm (IQR 9, 18). Biopsy length (mm) was shorter in females (p = 0.001), increased with age (p = 0.006), and a small positive linear trend with calendar year was observed (p = 0.015). The TAB positive proportion was related to older age (slope/decade: 6, 95% CI 3.6, 8.3, p < 0.001) and to TAB length (slope/mm 0.6, 95% CI 0.2, 0.9, p = 0.002), but not sex or calendar year. Comparison of models with TAB length cut-points at 5, 10, 15, 20 mm in terms of diagnostic yield, receiver operating characteristics and Akaike Information Criteria confirmed ≥ 15 mm as an appropriate, recommended TAB length. However, only 383 (46%) of the biopsies in our study met this criteria. The diagnostic yield at this cut-point was estimated as 25% which equates to an expected additional 30 histologically diagnosed GCA patients. Conclusion: This study confirms that TAB biopsy length is a determinant of a histological diagnosis of temporal arteritis, and confirms that a TAB length ≥ 15 mm is optimal. Approximately half the biopsies in this study were shorter than this optimal length, which has likely led to under-diagnosis of biopsy-proven GCA in SA. Further work is needed to ensure appropriate TAB biopsy length.

Single-cell multiomics reveals the complexity of TGFß signalling to chromatin in iPSC-derived kidney organoids.

TGFß1 plays a regulatory role in the determination of renal cell fate and the progression of renal fibrosis. Here we show an association between SMAD3 and the histone methyltransferase, EZH2, during cell differentiation; ChIP-seq revealed that SMAD3 and EZH2 co-occupy the genome in iPSCs and in iPSC-derived nephron progenitors. Through integration of single cell gene expression and epigenome profiling, we identified de novo ACTA2+ve/POSTN+ve myofibroblasts in kidney organoids treated with TGFß1, characterised by increased SMAD3-dependent cis chromatin accessibility and gene expression associated with fibroblast activation. We have identified fibrosis-associated regulons characterised by enrichment of SMAD3, AP1, the ETS family of transcription factors, and NUAK1, CREB3L1, and RARG, corresponding to enriched motifs at accessible loci identified by scATACseq. Treatment with the EZH2 specific inhibitor GSK343, blocked SMAD3-dependent cis co-accessibility and inhibited myofibroblast activation. This mechanism, through which TGFß signals directly to chromatin, represents a critical determinant of fibrotic, differentiated states.

Mechanism of Rad26-assisted rescue of stalled RNA polymerase II in transcription-coupled repair.

Transcription-coupled repair is essential for the removal of DNA lesions from the transcribed genome. The pathway is initiated by CSB protein binding to stalled RNA polymerase II. Mutations impairing CSB function cause severe genetic disease. Yet, the ATP-dependent mechanism by which CSB powers RNA polymerase to bypass certain lesions while triggering excision of others is incompletely understood. Here we build structural models of RNA polymerase II bound to the yeast CSB ortholog Rad26 in nucleotide-free and bound states. This enables simulations and graph-theoretical analyses to define partitioning of this complex into dynamic communities and delineate how its structural elements function together to remodel DNA. We identify an allosteric pathway coupling motions of the Rad26 ATPase modules to changes in RNA polymerase and DNA to unveil a structural mechanism for CSB-assisted progression past less bulky lesions. Our models allow functional interpretation of the effects of Cockayne syndrome disease mutations.

Polymerization and editing modes of a high-fidelity DNA polymerase are linked by a well-defined path.

Proofreading by replicative DNA polymerases is a fundamental mechanism ensuring DNA replication fidelity. In proofreading, mis-incorporated nucleotides are excised through the 3'-5' exonuclease activity of the DNA polymerase holoenzyme. The exonuclease site is distal from the polymerization site, imposing stringent structural and kinetic requirements for efficient primer strand transfer. Yet, the molecular mechanism of this transfer is not known. Here we employ molecular simulations using recent cryo-EM structures and biochemical analyses to delineate an optimal free energy path connecting the polymerization and exonuclease states of E. coli replicative DNA polymerase Pol III. We identify structures for all intermediates, in which the transitioning primer strand is stabilized by conserved Pol III residues along the fingers, thumb and exonuclease domains. We demonstrate switching kinetics on a tens of milliseconds timescale and unveil a complete pol-to-exo switching mechanism, validated by targeted mutational experiments.

Structural mechanism of helicase loading onto replication origin DNA by ORC-Cdc6.

DNA replication origins serve as sites of replicative helicase loading. In all eukaryotes, the six-subunit origin recognition complex (Orc1-6; ORC) recognizes the replication origin. During late M-phase of the cell-cycle, Cdc6 binds to ORC and the ORC-Cdc6 complex loads in a multistep reaction and, with the help of Cdt1, the core Mcm2-7 helicase onto DNA. A key intermediate is the ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) complex in which DNA has been already inserted into the central channel of Mcm2-7. Until now, it has been unclear how the origin DNA is guided by ORC-Cdc6 and inserted into the Mcm2-7 hexamer. Here, we truncated the C-terminal winged-helix-domain (WHD) of Mcm6 to slow down the loading reaction, thereby capturing two loading intermediates prior to DNA insertion in budding yeast. In "semi-attached OCCM," the Mcm3 and Mcm7 WHDs latch onto ORC-Cdc6 while the main body of the Mcm2-7 hexamer is not connected. In "pre-insertion OCCM," the main body of Mcm2-7 docks onto ORC-Cdc6, and the origin DNA is bent and positioned adjacent to the open DNA entry gate, poised for insertion, at the Mcm2-Mcm5 interface. We used molecular simulations to reveal the dynamic transition from preloading conformers to the loaded conformers in which the loading of Mcm2-7 on DNA is complete and the DNA entry gate is fully closed. Our work provides multiple molecular insights into a key event of eukaryotic DNA replication.

Simulation-Based Methods for Model Building and Refinement in Cryoelectron Microscopy.

Advances in cryoelectron microscopy (cryo-EM) have revolutionized the structural investigation of large macromolecular assemblies. In this review, we first provide a broad overview of modeling methods used for flexible fitting of molecular models into cryo-EM density maps. We give special attention to approaches rooted in molecular simulations-atomistic molecular dynamics and Monte Carlo. Concise descriptions of the methods are given along with discussion of their advantages, limitations, and most popular alternatives. We also describe recent extensions of the widely used molecular dynamics flexible fitting (MDFF) method and discuss how different model-building techniques could be incorporated into new hybrid modeling schemes and simulation workflows. Finally, we provide two illustrative examples of model-building and refinement strategies employing MDFF, cascade MDFF, and RosettaCM. These examples come from recent cryo-EM studies that elucidated transcription preinitiation complexes and shed light on the functional roles of these assemblies in gene expression and gene regulation.

Transcription preinitiation complex structure and dynamics provide insight into genetic diseases.

Transcription preinitiation complexes (PICs) are vital assemblies whose function underlies the expression of protein-encoding genes. Cryo-EM advances have begun to uncover their structural organization. Nevertheless, functional analyses are hindered by incompletely modeled regions. Here we integrate all available cryo-EM data to build a practically complete human PIC structural model. This enables simulations that reveal the assembly's global motions, define PIC partitioning into dynamic communities and delineate how structural modules function together to remodel DNA. We identify key TFIIE-p62 interactions that link core-PIC to TFIIH. p62 rigging interlaces p34, p44 and XPD while capping the DNA-binding and ATP-binding sites of XPD. PIC kinks and locks substrate DNA, creating negative supercoiling within the Pol II cleft to facilitate promoter opening. Mapping disease mutations associated with xeroderma pigmentosum, trichothiodystrophy and Cockayne syndrome onto defined communities reveals clustering into three mechanistic classes that affect TFIIH helicase functions, protein interactions and interface dynamics.

Small molecule binders recognize DNA microstructural variations via an induced fit mechanism.

Regulation of gene-expression by specific targeting of protein-nucleic acid interactions has been a long-standing goal in medicinal chemistry. Transcription factors are considered "undruggable" because they lack binding sites well suited for binding small-molecules. In order to overcome this obstacle, we are interested in designing small molecules that bind to the corresponding promoter sequences and either prevent or modulate transcription factor association via an allosteric mechanism. To achieve this, we must design small molecules that are both sequence-specific and able to target G/C base pair sites. A thorough understanding of the relationship between binding affinity and the structural aspects of the small molecule-DNA complex would greatly aid in rational design of such compounds. Here we present a comprehensive analysis of sequence-specific DNA association of a synthetic minor groove binder using long timescale molecular dynamics. We show how binding selectivity arises from a combination of structural factors. Our results provide a framework for the rational design and optimization of synthetic small molecules in order to improve site-specific targeting of DNA for therapeutic uses in the design of selective DNA binders targeting transcription regulation.

Uncovering universal rules governing the selectivity of the archetypal DNA glycosylase TDG.

Thymine DNA glycosylase (TDG) is a pivotal enzyme with dual roles in both genome maintenance and epigenetic regulation. TDG is involved in cytosine demethylation at CpG sites in DNA. Here we have used molecular modeling to delineate the lesion search and DNA base interrogation mechanisms of TDG. First, we examined the capacity of TDG to interrogate not only DNA substrates with 5-carboxyl cytosine modifications but also G:T mismatches and nonmismatched (A:T) base pairs using classical and accelerated molecular dynamics. To determine the kinetics, we constructed Markov state models. Base interrogation was found to be highly stochastic and proceeded through insertion of an arginine-containing loop into the DNA minor groove to transiently disrupt Watson-Crick pairing. Next, we employed chain-of-replicas path-sampling methodologies to compute minimum free energy paths for TDG base extrusion. We identified the key intermediates imparting selectivity and determined effective free energy profiles for the lesion search and base extrusion into the TDG active site. Our results show that DNA sculpting, dynamic glycosylase interactions, and stabilizing contacts collectively provide a powerful mechanism for the detection and discrimination of modified bases and epigenetic marks in DNA.

EU ambition to build the world's leading bioeconomy-Uncertain times demand innovative and sustainable solutions.

This article outlines the current context and the development of the European Bioeconomy Strategy. It analyses the current situation, challenges and needs for EU action and concludes with the next steps that the European Commission will undertake to review and update the Bioeconomy Strategy. Bioeconomy offers great opportunities to realising a competitive, circular and sustainable economy with a sound industrial base that is less dependent on fossil carbon. A sustainable bioeconomy also contributes to climate change mitigation, with oceans, forests and soils being major carbon sinks and fostering negative CO2 emissions. The EU has invested significantly in research and innovation in this field and the European Commission is committed to lead on European bioeconomy strategy.

Mortality in patients with biopsy-proven giant cell arteritis: a south australian population-based study.

OBJECTIVE: To compare mortality rates and cause of death in patients with biopsy-proven giant cell arteritis (GCA) with those in the general population. METHODS: Patients with biopsy-proven GCA were identified from pathology reports of temporal artery biopsies in South Australia, from January 1, 1992, to December 31, 2006. All patients with biopsy-proven GCA were linked to the South Australian Births, Death and Marriage Registry to identify deaths until December 31, 2006. Standardized mortality ratios and relative survival (ratio of observed survival in GCA group to expected survival of general South Australian population, matched by age, sex, and calendar time) were calculated. The cause of death recorded on the death certificate was also documented. RESULTS: There were 225 cases of biopsy-proven GCA (163 women and 62 men). The mean age at diagnosis of GCA was 78.2 years. The mean followup period was 66.2 months (SD 47.1 mo). During the followup period, there were 71 deaths in the GCA group (50 women, 21 men). The standardized mortality ratio was 0.99 (95% CI 0.77-1.25). The relative survival for different followup periods demonstrates that patients with GCA experienced similar mortality to the general population (age-matched and sex-matched). Death from cardiovascular causes (45%) was the most common, followed by infection (17%) and cancer (17%). Infection was a significantly more common cause of death in the first year (chi-squared, p = 0.0002). CONCLUSION: Our population-based cohort study did not demonstrate any increased mortality risk for patients diagnosed with biopsy-proven GCA. The risk of death from infection early in the disease may be increased.

Lacrimal gland involvement in lymphomatoid granulomatosis and review of the literature.

Objective. To describe the clinicoradiological and histopathological findings in a case of lacrimal gland enlargement secondary to lymphomatoid granulomatosis (LG) and to review the literature. Design. Case report and systematic literature review. Methods. A 75-year-old woman presented with right ptosis. Computerised tomography showed lacrimal gland enlargement, and biopsy done was inconclusive. She subsequently developed pulmonary symptoms and underwent transbronchial biopsy that was diagnosed as LG. Pub Med and OVID databases were searched using the term "orbit/eye involvement in lymphomatoid granulomatosis". Articles that predated the databases were gathered from current references. Results. The patient underwent lacrimal gland biopsy which revealed necrotic and inflamed tissue with no further categorisation but transbronchial biopsy helped in establishing the diagnosis of LG. On initiation of prednisolone and cyclophosphamide, her orbital lesion resolved but the patient died following massive pulmonary hemorrhage within a month of diagnosis. Conclusion. Ophthalmic involvement in LG is very rare. Varied presentations are due to central nervous system involvement, vasculitis, or infiltration of ocular or orbital structures. LG is an angiocentric and angiodestructive granulomatous disorder and can involve any tissue, thus accounting for the variable presentations reported in literature.

Comparative biomarker expression and RNA integrity in biospecimens derived from radical retropubic and robot-assisted laparoscopic prostatectomies.

BACKGROUND: Knowledge of preanalytic conditions that biospecimens are subjected to is critically important because novel surgical procedures, tissue sampling, handling, and storage might affect biomarker expression or invalidate tissue samples as analytes for some technologies. METHODS: We investigated differences in RNA quality, gene expression by quantitative real-time PCR, and immunoreactive protein expression of selected prostate cancer biomarkers between tissues from retropubic radical prostatectomy (RRP) and robot-assisted laparoscopic prostatectomy (RALP). Sections of tissue microarray of 23 RALP and 22 RRP samples were stained with antibodies to androgen receptor (AR) and prostate-specific antigen (PSA) as intersite controls, and 14 other candidate biomarkers of research interest to three laboratories within the Australian Prostate Cancer BioResource tissue banking network. Quantitative real-time PCR was done for AR, PSA (KLK3), KLK2, KLK4, and HIF1A on RNA extracted from five RALP and five RRP frozen tissue cores. RESULTS: No histologic differences were observed between RALP and RRP tissue. Biomarker staining grouped these samples into those with increased (PSA, CK8/18, CKHMW, KLK4), decreased (KLK2, KLK14), or no change in expression (AR, ghrelin, Ki67, PCNA, VEGF-C, PAR2, YB1, p63, versican, and chondroitin 0-sulfate) in RALP compared with RRP tissue. No difference in RNA quality or gene expression was detected between RALP and RRP tissue. CONCLUSIONS: Changes in biomarker expression between RALP and RRP tissue exist at the immunoreactive protein level, but the etiology is unclear. IMPACT: Future studies should account for changes in biomarker expression when using RALP tissues, and mixed cohorts of RALP and RRP tissue should be avoided.

Are alternative investments the right approach for your organization?

The decision by a healthcare provider to implement and monitor an alternative investment program requires careful consideration and planning. There are several things an organization should do when making this decision: Begin slowly. Consider access and liquidity. Don't force the investment to fill a target allocation. View an alternative investment as an opportunity investment."

Risk of cancer in patients with biopsy-proven giant cell arteritis.

OBJECTIVE: Results of previous studies investigating the association between GCA and malignancy are conflicting. We performed a study of the risk of cancer in patients with biopsy-proven GCA. METHODS: Patients with biopsy-proven GCA were identified from pathology reports of temporal artery biopsies from the major pathology laboratories in South Australia (SA). All subjects with biopsy-proven GCA were linked to the SA Cancer Registry to identify cases of cancer until 31 December 2006. Standardized incidence ratios (SIRs) for cancer were determined using the age- and gender-specific rates for SA. RESULTS: There were 226 cases of biopsy-proven GCA (163 females and 63 males). Thirty-one cases were diagnosed with cancer, following the diagnosis of biopsy-proven GCA. There was no increased risk of cancer among those with biopsy-proven GCA, following the diagnosis of GCA compared with the general population (SIR 1.2; 95% CI 0.8, 1.6). CONCLUSION: This cohort study did not demonstrate any increased risk for malignancy in subjects with biopsy-proven GCA.

Diffuse bilateral orbital inflammation in Churg- Strauss syndrome.

A 33-year-old man was diagnosed with asthma and within 5 weeks developed bilateral periocular swelling. Examination revealed bilateral axial proptosis with conjunctival nodules. His blood tests revealed a positive p-antineutrophil cytoplasmic autoantibody with significant eosinophilia. MRI of the orbit showed enlarged extraocular muscles, lacrimal glands, and infiltrative changes in the orbital fat. Biopsy demonstrated granulomatous inflammation with eosinophil predominance. A diagnosis of diffuse bilateral inflammation in Churg-Strauss syndrome was made and the patient responded dramatically to prednisolone with resolution of systemic and orbital findings. The second case was a 72-year-old woman with a prolonged prodromal phase of asthma, paranasal sinus disease, and bilateral orbital involvement by a process consistent with reactive lymphoid hyperplasia on initial biopsy. Three years later she developed rapidly worsening orbital disease, marked peripheral eosinophilia, and orbital biopsy showed evidence of granulomatous inflammation with marked eosinophil infiltration and vasculitic changes, and a weakly positive antineutrophil cytoplasmic autoantibody. Hence, diffuse bilateral orbital inflammation occurring in the setting of asthma and peripheral eosinophilia should raise the possibility of Churg-Strauss syndrome and warrants biopsy as early institution of therapy can reduce both systemic and ophthalmic complications.

Primary orbital liposarcoma.

PURPOSE: To describe 6 new cases of primary orbital liposarcoma and provide a review of the relevant literature. DESIGN: Noncomparative consecutive case series and literature review. PARTICIPANTS: Six patients with primary orbital liposarcoma. METHODS: Review of patient charts, imaging, and histopathology; literature review. MAIN OUTCOME MEASURES: Patient demographics; clinical presentations; results of radiologic imaging; histopathology; surgical techniques used and their complications; other treatment modalities; outcomes and recurrences. RESULTS: Six cases of primary orbital liposarcoma were identified, 5 of which were primary presentations and 1 of which was a recurrence. In 4 cases, exenteration was deferred, resulting in recurrence of disease in all 4. All cases were exenterated, and 2 cases had local recurrence despite exenteration. Two cases were associated with the Li-Fraumeni syndrome and other malignancies. Literature review identified 34 other cases of primary orbital liposarcoma, which, partly because of its rarity, is frequently initially misdiagnosed. The most common subtype is myxoid (56.8%); other types are pleomorphic (10.8%) and well differentiated (29.7%). Well-differentiated tumors have the best prognosis. Non-exenterating surgery was associated with recurrence, although recurrence post-exenteration also occurred. Although radiotherapy has an established role in the treatment of nonorbital liposarcoma, the role of both radiotherapy and chemotherapy in the management of primary orbital liposarcoma is still unclear. CONCLUSIONS: Orbital liposarcoma remains a diagnostic and surgical challenge. Exenteration remains the treatment of choice, but clinicians must also be aware that liposarcoma may herald the diagnosis of the Li-Fraumeni familial cancer syndrome.