Achieving a textbook outcome in patients undergoing gastric resections in a low incidence, high-volume Australian Upper GI unit.

BACKGROUND: Textbook outcome (TBO) has been proposed as a composite measure of quality in esophagogastric surgery, and achieving a TBO has been associated with improved overall survival (OS). The Dutch Upper Gastrointestinal Cancer Audit group determined their TBO rate for gastrectomy to be 32.1%, using 10 parameters. Our study aimed to assess the TBO rate in patients who had a gastrectomy for cancer in an Australian Upper GI unit, allowing for comparisons with international specialist centers. METHODS: Retrospective analysis of a prospectively maintained database of patients who had a gastrectomy for cancer performed by the surgeons in a single Australian center between 2013 and 2018. Postoperative complications were analyzed using Clavien-Dindo (CD) ≥2 and CD ≥3 definitions. Baseline factors and their association with TBO were analyzed using multivariable logistical regression. The association between TBO and survival rates was determined by Cox proportional hazards regression analysis. RESULTS: In 136 patients, 84 (62%) achieved a TBO when complications were graded as CD ≥2. Greatest negative impact on TBO was the complication rate, lymph node yield, and length of stay. Patients more likely to achieve a TBO were younger, with an increased body mass index and absence of underlying respiratory disease. A nonsignificant trend toward improved OS was seen when TBO was achieved. CONCLUSION: Our TBO rate compares favorably with published data from high-volume centers. Assessment of a unit's TBO may provide a stronger evaluation of quality when assessing where complex surgery should be performed within Australia.

Investigating Protein Binding with the Isothermal Ligand-induced Resolubilization Assay.

Target engagement assays typically detect and quantify the direct physical interaction of a protein of interest and its ligand through stability changes upon ligand binding. Commonly used target engagement methods detect ligand-induced stability by subjecting samples to thermal or proteolytic stress. Here we describe a new variation to these approaches called Isothermal Ligand-induced Resolubilization Assay (ILIRA), which utilizes lyotropic solubility stress to measure ligand binding through changes in target protein solubility. We identified distinct buffer systems and salt concentrations that compromised protein solubility for four diverse proteins: dihydrofolate reductase (DHFR), nucleoside diphosphate-linked moiety X motif 5 (NUDT5), poly [ADP-ribose] polymerase 1 (PARP1), and protein arginine N-methyltransferase 1 (PRMT1). Ligand-induced solubility rescue was demonstrated for these proteins, suggesting that ILIRA can be used as an additional target engagement technique. Differences in ligand-induced protein solubility were assessed by Coomassie blue staining for SDS-PAGE and dot blot, as well as by NanoOrange, Thioflavin T, and Proteostat fluorescence, thus offering flexibility for readout and assay throughput.

Investigating the anti-cancer potential of pyrimethamine analogues through a modern chemical biology lens.

Pharmacological inhibition of dihydrofolate reductase (DHFR) is an established approach for treating a variety of human diseases, including foreign infections and cancer. However, treatment with classic DHFR inhibitors, such as methotrexate (MTX), are associated with negative side-effects and resistance mechanisms that have prompted the search for alternatives. The DHFR inhibitor pyrimethamine (Pyr) has compelling anti-cancer activity in in vivo models, but lacks potency compared to MTX, thereby requiring higher concentrations to induce therapeutic responses. The purpose of this work was to investigate structural analogues of Pyr to improve its in vitro and cellular activity. A series of 36 Pyr analogues were synthesized and tested in a sequence of in vitro and cell-based assays to monitor their DHFR inhibitory activity, cellular target engagement, and impact on breast cancer cell viability. Ten top compounds were identified, two of which stood out as potential lead candidates, 32 and 34. These functionalized Pyr analogues potently engaged DHFR in cells, at concentrations as low as 1 nM and represent promising DHFR inhibitors that could be further explored as potential anti-cancer agents.

Protein arginine N-methyltransferase 2 plays a noncatalytic role in the histone methylation activity of PRMT1.

Protein arginine N-methyltransferases are a family of epigenetic enzymes responsible for monomethylation or dimethylation of arginine residues on histones. Dysregulation of protein arginine N-methyltransferase activity can lead to aberrant gene expression and cancer. Recent studies have shown that PRMT2 expression and histone H3 methylation at arginine 8 are correlated with disease severity in glioblastoma multiforme, hepatocellular carcinoma, and renal cell carcinoma. In this study, we explore a noncatalytic mechanistic role for PRMT2 in histone methylation by investigating interactions between PRMT2, histone peptides and proteins, and other PRMTs using analytical and enzymatic approaches. We quantify interactions between PRMT2, peptide ligands, and PRMT1 in a cofactor- and domain-dependent manner using differential scanning fluorimetry. We found that PRMT2 modulates the substrate specificity of PRMT1. Using calf thymus histones as substrates, we saw that a 10-fold excess of PRMT2 promotes PRMT1 methylation of both histone H4 and histone H2A. We found equimolar or a 10-fold excess of PRMT2 to PRMT1 can improve the catalytic efficiency of PRMT1 towards individual histone substrates H2A, H3, and H4. We further evaluated the effects of PRMT2 towards PRMT1 on unmodified histone octamers and mononucleosomes and found marginal PRMT1 activity improvements in histone octamers but significantly greater methylation of mononucleosomes in the presence of 10-fold excess of PRMT2. This work reveals the ability of PRMT2 to serve a noncatalytic role through its SH3 domain in driving site-specific histone methylation marks.

Textbook outcomes for oesophagectomy: A valid composite measure assessment tool for surgical performance in a specialist unit.

INTRODUCTION: In 2017 the Dutch Upper Gastrointestinal Cancer Audit Group proposed a ten-item composite measure for a 'textbook outcome' (TBO) following oesophago-gastric resection. Studies have shown associations between TBO and improved conditional and overall survival. The aim of this study was to evaluate the use of TBO to assess the outcomes from a single specialist unit in a country, with low incidence of disease, allowing comparisons with international specialist centres. MATERIALS AND METHODS: Retrospective analysis of prospectively collected oesophageal cancer surgery data at a single centre, in Australia, between 2013 and 2018. Multivariable logistical regression assessed association between baseline factors and TBO. Post-operative complications were analysed in two separate groups as Clavien-Dindo ≥2 (CD ≥ 2) and Clavien-Dindo ≥3 (CD ≥ 3). Cox-proportional hazards regression analysis determined the association between TBO and survival. RESULTS: 246 patients were analysed, with 50.8% (n = 125) achieving a TBO when complications were defined as CD ≥ 2 and 58.9% (n = 145) when using CD ≥ 3. Patients aged ≥75, and those with a pre-operative respiratory co-morbidity were less likely to achieve a TBO. Overall survival was not influenced by TBO when complications were defined as CD ≥ 2, however it was higher when a TBO was achieved, and complications were defined as CD ≥ 3 (HR 0.54, 95% CI, 0.35 to 0.84, P = 0.007). CONCLUSION: TBO is a multi-parameter metric that allowed benchmarking of the quality of oesophageal cancer surgery in our unit, providing favourable outcomes compared with other published data. There was an association between TBO and improved overall survival when the definition of severe complications was CD ≥ 3.

Primary and subsequent secondary aorto-enteric fistulae in the setting of chronic Q fever.

We report the case of an 80-year-old male with stage three kidney disease, who survived a primary aorto-enteric fistula (AEF) in the setting of chronic Q fever after presenting with melena and syncope. His initial surgical treatment included endovascular aortic repair. Type 2 endoleak was present post-operatively. Six months later, he was diagnosed with a secondary AEF after syncope and large volume hematemesis. He was definitively treated with an open explant of his stent, repair of the duodenum and bilateral axillofemoral bypass. Two years later, he remains active and independent on life-long antibiotics.

Laparoscopic fundoplication after lung transplantation does not appear to alter lung function trajectory.

BACKGROUND: The primary aim of this study was to determine if allograft function in lung transplant (LTx) recipients improves or stabilizes after laparoscopic fundoplication (LF). The secondary aim was to examine the differences in forced expiratory volume in 1 second (FEV1) before and after LF for various subgroups to identify patients who obtained a superior respiratory outcome after LF, and potential predictive factors for this outcome. METHODS: Retrospective analysis of consecutive LTx recipients undergoing LF at a single centre in Brisbane, Australia between 2004 and 2018. 149/431 proceeded to LF after clinical review and pH study. Regular pre- and post-fundoplication pulmonary function tests were collected from participants. Data were analyzed with linear mixed models, random intercept models, the Reliable Change Index (RCI), and graphical and visual analysis of the trajectory of FEV1. RESULTS: There was 100% follow-up. After Bonferroni adjustment for multiple comparison was performed, none of the models demonstrated statistical significance. The Reliable Change Index showed one patient had a significant improvement in lung function across that time period, while nine had a significant reduction. The rate of change before and after LF was similar for the 132/149 patients for whom the first and last pre- and post-LF FEV1 values were available. A subset of patients had a considerable reduction in their FEV1 in the peri-operative period (i.e., a large difference between the first measurement post-LF and the final measurement pre-LF). CONCLUSION: In the largest published cohort to date, LF performed in a high-volume center did not appear to alter the reduction in allograft function seen with time.

Methylarginine efflux in nutrient-deprived yeast mitigates disruption of nitric oxide synthesis.

Protein arginine N-methyltransferases (PRMTs) have emerged as important actors in the eukaryotic stress response with implications in human disease, aging, and cell signaling. Intracellular free methylarginines contribute to cellular stress through their interaction with nitric oxide synthase (NOS). The arginine-dependent production of nitric oxide (NO), which is strongly inhibited by methylarginines, serves as a protective small molecule against oxidative stress in eukaryotic cells. NO signaling is highly conserved between higher and lower eukaryotes, although a canonical NOS homologue has yet to be identified in yeast. Since stress signaling pathways are well conserved among eukaryotes, yeast is an ideal model organism to study the implications of PRMTs and methylarginines during stress. We sought to explore the roles and fates of methylarginines in Saccharomyces cerevisiae. We starved methyltransferase-, autophagy-, and permease-related yeast knockouts by incubating them in water and monitored methylarginine production. We found that under starvation, methylarginines are expelled from yeast cells. We found that autophagy-deficient cells have an impaired ability to efflux methylarginines, which suggests that methylarginine-containing proteins are degraded via autophagy. For the first time, we determine that yeast take up methylarginines less readily than arginine, and we show that methylarginines impact yeast NO production. This study reveals that yeast circumvent a potential methylarginine toxicity by expelling them after autophagic degradation of arginine-modified proteins.

Efficacy of laparoscopic fundoplication in patients with chronic cough and gastro-oesophageal reflux.

BACKGROUND: The outcome of anti-reflux surgery in patients with suspected gastro-oesophageal reflux-induced cough is frequently uncertain. The aims of this study were to assess the efficacy of laparoscopic fundoplication for controlling cough in patients with chronic cough without asthma, who have pathologic gastro-oesophageal reflux, and to identify predictors of response. METHODS: From a prospective database of 1598 patients who have undergone laparoscopic fundoplication, 66 (4%) with proven gastro-oesophageal reflux disease (GORD) and chronic cough without asthma were studied. All patients underwent gastroscopy and 24-h pH monitoring before operation. Heartburn and regurgitation were assessed using a modified DeMeester score. Severity of cough before and after surgery was self-assessed by the patient using a visual analog scale at a minimum of 12 months post-operatively (median 43 mo; range: 14-104 mo). Patients were considered to have responded to fundoplication if they had no cough or the cough had improved by 50% or more after operation. RESULTS: Cough and heartburn/regurgitation were relieved in 61% (40/66) and 90% (44/49) of the patients, respectively. The presence of typical GORD symptoms or oesophagitis, and pH study variables did not predict the response of the cough to fundoplication. CONCLUSION: Refinement in the aetiological diagnosis of chronic cough due to GORD is necessary for improved outcome. Patients diagnosed with GORD-related chronic cough need to be counseled regarding their expectations from anti-reflux surgery.

Laparoscopic fundoplication versus laparoscopic Roux-en-Y gastric bypass for gastro-oesophageal reflux disease in obese patients: protocol for a randomized clinical trial.

BACKGROUND: Laparoscopic fundoplication (LF) is the standard surgical procedure for the treatment of gastro-oesophageal reflux disease (GORD). Laparoscopic Roux-en-Y gastric bypass (LRYGB) is commonly performed to achieve weight loss in obese patients, but it also has anti-reflux properties. Hence, in the obese population suffering from GORD, LRYGB could be an alternative to LF. The aim of this trial will be to compare LF and LRYGB in an obese population presenting with GORD and being considered for surgery. METHODS: This will be an investigator-initiated randomized clinical trial. The research population will be obese patients (BMI 30-34.9 with waist circumference more than 88 cm (women) or more than 102 cm (men), or BMI 35-40 with any waist circumference) referred to a public hospital for consideration of anti-reflux surgery. The primary aim of the study will be to determine the efficacy of LF compared with LRYGB on subjective and objective control of GORD. Secondary aims include determining early and late surgical morbidity and the side-effect profile of LF compared with LRYGB and to quantify any non-reflux benefits of LRYGB (including overall quality of life) compared with LF. CONCLUSION: This trial will determine whether LRYGB is effective and acceptable as an alternative to LF for the surgical treatment of GORD in obese patients Registration number: Australian New Zealand Clinical Trials Registry (ANZCTR): ACTRN12622000636752p (https://www.anzctr.org.au/).

Protein arginine N-methyltransferase activity determination with filter binding and phosphor screening (FBAPS) assay.

We developed a cost-effective assay to measure protein arginine N-methyltransferase (PRMT) activity in a medium-throughput manner by combining P81 filter binding and phosphor screening (FBAPS). Recombinantly-expressed PRMT1 and coactivator-associated arginine methyltransferase 1 (CARM1) were used to develop the FBAPS assay using GST fusions of glycine- and arginine-rich (GAR) protein and polyA binding protein 1 (PABP1(437-488)) as substrates, respectively, and radiolabelled S-adenosyl-L-[methyl-14C]-methionine as cofactor. Methylation reactions were spotted onto P81 filter paper in a dot blot apparatus and radioactive signals were measured both by phosphor imaging and liquid scintillation counting. Kinetic parameters (KM, kcat) for enzymes and substrates were determined, and IC50 values were obtained for well-characterized inhibitors. FBAPS yielded kinetic parameters with no statistically significant difference to what was obtained using liquid scintillation counting. The IC50 values obtained by the FBAPS assay for PRMT1 and CARM1 were comparable to values reported in literature. The FBAPS assay is a modification to the P81 filter binding assay with a dot blot apparatus that allows for processing of samples in a multi-well format, moderately increasing throughput. Signal detection by phosphor imaging offers an affordable and quantitative method that can be used to screen several inhibitors simultaneously against PRMT enzymes with high accuracy.

Esophageal perforations - a population-based nationwide study in Sweden with survival analysis.

BACKGROUND: Esophageal perforation is a rare and life-threatening condition with several treatment options. The aim was to assess the incidence, type of treatment and mortality of esophageal perforations in Sweden and to identify risk factors for 90-day mortality. METHOD: All patients admitted with an esophageal perforation from 2007 to 2017 were identified from the National Patient Register. Mortality was assessed by linkage with the Cause of Death Registry. We analyze the incidence and the impact of age, sex, comorbidities on mortality. RESULTS: 879 patients with esophageal perforation were identified, giving an incidence rate of 1.09 per 100,000 person-years. The median age at diagnosis was 68.8 years and 60% were men. The mortality was 26% at 90 days. Independent risk factors for death within 90 days were age (odds ratio (OR): 6.20; 95% (confidence interval) CI: 2.16-17.79 at 60-74 years and OR: 11.58; 95% CI: 4.04-33.15 at 75 years or older), peripheral vascular disease (OR: 2.92; 95% CI: 1.44-5.92) and underlying malignant disease (OR: 5.91; 95% CI: 3.86-9.03). In patients younger than 45 years, survival was lower among women than among men (at 5 years 73 and 93%, respectively). The cause of death among young women was often drug-related or suicide. CONCLUSIONS: 90-day mortality was 26%, old age, vascular disease and underlying malignant disease were risk factors.

A rare case of a giant retroperitoneal lipoma with multiple limb and trunk lipomata without familial multiple lipomatosis.

Retroperitoneal lipoma is exceedingly rare, and due to the difficulty in distinguishing between retroperitoneal lipoma and well-differentiated liposarcoma (WDLS), recommendation is en-bloc resection. A 58-year-old male was investigated for scrotal swelling, ultrasound and computed tomography showed a well-defined lipomatous mass occupying much of the left side of the lower abdomen. At laparotomy, a large left-sided retroperitoneal mass was found. Histology reported a 160 mm × 150 mm × 90 mm fatty tumour weighing 1540 g. MDM2 gene amplification was not present on fluorescence in situ hybridization. No significant somatic signatures were identified on whole exome sequencing. Retroperitoneal fatty tumours represent a diagnostic dilemma. Sampling via core biopsy has been recorded at 85% accuracy for WDLS. Positive amplification of the MDM2 gene supports a diagnosis of WDLS; however, a negative biopsy does not exclude the diagnosis due to varied amplification among different cells in the same tumour.

Protein arginine methyltransferase 8 modulates mitochondrial bioenergetics and neuroinflammation after hypoxic stress.

Protein arginine methyltransferases (PRMTs) are a family of enzymes involved in gene regulation and protein/histone modifications. PRMT8 is primarily expressed in the central nervous system, specifically within the cellular membrane and synaptic vesicles. Recently, PRMT8 has been described to play key roles in neuronal signaling such as a regulator of dendritic arborization, synaptic function and maturation, and neuronal differentiation and plasticity. Here, we examined the role of PRMT8 in response to hypoxia-induced stress in brain metabolism. Our results from liquid chromatography mass spectrometry, mitochondrial oxygen consumption rate, and protein analyses indicate that PRMT8(-/-) knockout mice presented with altered membrane phospholipid composition, decreased mitochondrial stress capacity, and increased neuroinflammatory markers, such as tumor necrosis factor alpha and ionized calcium binding adaptor molecule 1 (Iba1, a specific marker for microglia/macrophage activation) after hypoxic stress. Furthermore, adenovirus-based overexpression of PRMT8 reversed the changes in membrane phospholipid composition, mitochondrial stress capacity, and neuroinflammatory markers. Together, our findings establish PRMT8 as an important regulatory component of membrane phospholipid composition, short-term memory function, mitochondrial function, and neuroinflammation in response to hypoxic stress.

Heterotopic ossification within the gallbladder - First reported Australian case.

INTRODUCTION: We report the rare and unusual case of heterotopic ossification within the gallbladder secondary to chronic calculi debris. PRESENTATION OF CASE: A 35-year-old female underwent routine laparoscopic cholecystectomy for recurrent intermittent right upper quadrant pain which had persisted for three months and was worse post prandial with associated nausea. Abdominal ultrasound prior to surgery was reported by a consultant radiologist as demonstrating a thin-walled gallbladder and cholelithiasis, without features of cholecystitis. At four-week review, she had recovered well with no concerns. The histopathology report revealed fibromuscular hyperplasia and patchy chronic inflammation. Rokitansky-Aschoff sinuses were present and cholesterosis was noted. Additionally, there was a focus of eroded mucosa showing adherent microlithiasis with an incidental focus of heterotopic ossification within the mucosa, there was no evidence of dysplasia or malignancy. DISCUSSION: Gallbladder heterotopic ossification is exceedingly rare, with few cases reported. To our knowledge this is the first reported case in Australia. CONCLUSION: In conclusion, we report the rare and unusual finding of heterotopic ossification of the gallbladder, and suspect that inflammation secondary to calculous debris initiated the ossification. Current technical limitations preclude diagnosis prior to surgery. Appropriate follow-up is unclear, but we feel a single report associated the finding with adenocarcinoma was sufficient to warrant follow-up.

Intra-abdominal desmoplastic small round cell tumour in a 56-year-old female: Case report of a very rare presentation of an unusual tumour.

INTRODUCTION: This case report discusses the rare diagnosis of intra-abdominal desmoplastic small round cell tumour (DSRCT) in a 56-year-old female. PRESENTATION OF CASE: An incidental intra-abdominal lesion was found during investigation of joint pain. Ultrasound-guided biopsy suggested desmoid tumour, after undergoing laparotomy and en-bloc excision of the tumour due to concerning radiological progression, the final histology was desmoplastic small round cell tumour. At six-week follow-up imaging, no recurrence or metastatic disease was noted. She declined chemotherapy and specialist follow-up, electing to have routine follow up with her General Practitioner only. DISCUSSION: Intra-abdominal DSRCT is rare and mainly seen in young males. To our knowledge, this is the only reported case of DSRCT in a female over the age of 50. CONCLUSION: There should be timely discussion between different surgical units to provide efficient care. Any disparity between radiological and histological appearance should prompt further review and investigation in order to ensure misdiagnosis is avoided and appropriate treatment is provided. Despite cytoreductive surgery, survival is dismal due to the aggressive nature of the tumour, and its low numbers limiting adequate study into post diagnosis care.

Characterization of impact pile driving signals during installation of offshore wind turbine foundations.

Impact pile driving creates intense, impulsive sound that radiates into the surrounding environment. Piles driven vertically into the seabed generate an azimuthally symmetric underwater sound field whereas piles driven on an angle will generate an azimuthally dependent sound field. Measurements were made during pile driving of raked piles to secure jacket foundation structures to the seabed in waters off the northeastern coast of the U.S. at ranges between 500 m and 15 km. These measurements were analyzed to investigate variations in rise time, decay time, pulse duration, kurtosis, and sound received levels as a function of range and azimuth. Variations in the radiated sound field along opposing azimuths resulted in differences in measured sound exposure levels of up to 10 dB and greater due to the pile rake as the sound propagated in range. The raked pile configuration was modeled using an equivalent axisymmetric FEM model to describe the azimuthally dependent measured sound fields. Comparable sound level differences in the model results confirmed that the azimuthal discrepancy observed in the measured data was due to the inclination of the pile being driven relative to the receiver.