ATP13A3 is a major component of the enigmatic mammalian polyamine transport system

Polyamines, such as putrescine, spermidine, and spermine, are physiologically important polycations, but the transporters responsible for their uptake in mammalian cells remain poorly characterized. Here, we reveal a new component of the mammalian polyamine transport system using CHO-MG cells, a widely used model to study alternative polyamine uptake routes and characterize polyamine transport inhibitors for therapy. CHO-MG cells present polyamine uptake deficiency and resistance to a toxic polyamine biosynthesis inhibitor methylglyoxal bis-(guanylhydrazone) (MGBG), but the molecular defects responsible for these cellular characteristics remain unknown. By genome sequencing of CHO-MG cells, we identified mutations in an unexplored gene, ATP13A3, and found disturbed mRNA and protein expression. ATP13A3 encodes for an orphan P5B-ATPase (ATP13A3), a P-type transport ATPase that represents a candidate polyamine transporter. Interestingly, ATP13A3 complemented the putrescine transport deficiency and MGBG resistance of CHO-MG cells, whereas its knockdown in WT cells induced a CHO-MG phenotype demonstrated as a decrease in putrescine uptake and MGBG sensitivity. Taken together, our findings identify ATP13A3, which has been previously genetically linked with pulmonary arterial hypertension, as a major component of the mammalian polyamine transport system that confers sensitivity to MGBG

Evolving trends in the management of acute appendicitis during COVID-19 waves. The ACIE appy II study

Background: In 2020, ACIE Appy study showed that COVID-19 pandemic heavily affected the management of patients with acute appendicitis (AA) worldwide, with an increased rate of non-operative management (NOM) strategies and a trend toward open surgery due to concern of virus transmission by laparoscopy and controversial recommendations on this issue. The aim of this study was to survey again the same group of surgeons to assess if any difference in management attitudes of AA had occurred in the later stages of the outbreak. Methods: From August 15 to September 30, 2021, an online questionnaire was sent to all 709 participants of the ACIE Appy study. The questionnaire included questions on personal protective equipment (PPE), local policies and screening for SARS-CoV-2 infection, NOM, surgical approach and disease presentations in 2021. The results were compared with the results from the previous study. Results: A total of 476 answers were collected (response rate 67.1%). Screening policies were significatively improved with most patients screened regardless of symptoms (89.5% vs. 37.4%) with PCR and antigenic test as the preferred test (74.1% vs. 26.3%). More patients tested positive before surgery and commercial systems were the preferred ones to filter smoke plumes during laparoscopy. Laparoscopic appendicectomy was the first option in the treatment of AA, with a declined use of NOM. Conclusion: Management of AA has improved in the last waves of pandemic. Increased evidence regarding SARS-COV-2 infection along with a timely healthcare systems response has been translated into tailored attitudes and a better care for patients with AA worldwide

Prediction of compressive strength of cementitious grouts for semi-flexible pavement application using machine learning approach

This study involves the preparation of cement grout samples by varying proportions of superplasticizer (SP) and water-cement (w/c) ratio (0.25–0.45) which were tested for computing flow value and 1-day, 7-day, and 28-day compressive strength. With 75 data points a neural network model was developed to predict the 28-day compressive strength based on w/c ratio, superplasticizer percentage, flow value, and compressive strengths of 1-day and 7-day as input variables. Due to the better performance of Artificial Neural Networks (ANN) over other applied models, this study predicts a confident pattern of relationship between w/c ratio (indirect), superplasticizer (direct up to 3%), and the corresponding strength characteristics of the study samples. The prediction power of the developed model has been improved from R2 = 0.959 to R2 = 0.984 by optimizing the number of neurons, activation function, and optimizer. The best performance was observed in the case of Model45-R_A. The same model was used for assessing the effect of the percentage of superplasticizer and water cement ratio on 28 days compressive strength where the compressive strength of 73.941 MPa was recorded in the case of 3% superplasticizer and w/c of 0.25

Novel Green Fluorescent Polyamines to Analyze ATP13A2 and ATP13A3 Activity in the Mammalian Polyamine Transport System

Cells acquire polyamines putrescine (PUT), spermidine (SPD) and spermine (SPM) via the complementary actions of polyamine uptake and synthesis pathways. The endosomal P5B-type ATPases ATP13A2 and ATP13A3 emerge as major determinants of mammalian polyamine uptake. Our biochemical evidence shows that fluorescently labeled polyamines are genuine substrates of ATP13A2. They can be used to measure polyamine uptake in ATP13A2- and ATP13A3-dependent cell models resembling radiolabeled polyamine uptake. We further report that ATP13A3 enables faster and stronger cellular polyamine uptake than does ATP13A2. We also compared the uptake of new green fluorescent PUT, SPD and SPM analogs using different coupling strategies (amide, triazole or isothiocyanate) and fluorophores (symmetrical BODIPY, BODIPY-FL and FITC). ATP13A2 promotes the uptake of various SPD and SPM analogs, whereas ATP13A3 mainly stimulates the uptake of PUT and SPD conjugates. However, the polyamine linker and coupling position on the fluorophore impacts the transport capacity, whereas replacing the fluorophore affects polyamine selectivity. The highest uptake in ATP13A2 or ATP13A3 cells is observed with BODIPY-FL-amide conjugated to SPD, whereas BODIPY-PUT analogs are specifically taken up via ATP13A3. We found that P5B-type ATPase isoforms transport fluorescently labeled polyamine analogs with a distinct structure–activity relationship (SAR), suggesting that isoform-specific polyamine probes can be designed

ATP13A3 variants promote pulmonary arterial hypertension by disrupting polyamine transport.

AIMS: Potential loss-of-function variants of ATP13A3, the gene encoding a P5B-type transport ATPase of undefined function, were recently identified in pulmonary arterial hypertension (PAH) patients. ATP13A3 is implicated in polyamine transport but its function has not been fully elucidated. Here, we sought to determine the biological function of ATP13A3 in vascular endothelial cells and how PAH-associated variants may contribute to disease pathogenesis. METHODS AND RESULTS: We studied the impact of ATP13A3 deficiency and overexpression in endothelial cell (EC) models (human pulmonary ECs, blood outgrowth ECs (BOECs) and HMEC-1 cells), including a PAH patient-derived BOEC line harbouring an ATP13A3 variant (LK726X). We also generated mice harbouring an Atp13a3 variant analogous to a human disease-associated variant to establish whether these mice develop PAH.ATP13A3 localised to the recycling endosomes of human ECs. Knockdown of ATP13A3 in ECs generally reduced the basal polyamine content and altered the expression of enzymes involved in polyamine metabolism. Conversely, overexpression of wild-type ATP13A3 increased polyamine uptake. Functionally, loss of ATP13A3 was associated with reduced EC proliferation, increased apoptosis in serum starvation and increased monolayer permeability to thrombin. Assessment of five PAH-associated missense ATP13A3 variants (L675V, M850I, V855M, R858H, L956P) confirmed loss-of-function phenotypes represented by impaired polyamine transport and dysregulated EC function. Furthermore, mice carrying a heterozygous germ-line Atp13a3 frameshift variant representing a human variant spontaneously developed a PAH phenotype, with increased pulmonary pressures, right ventricular remodelling and muscularisation of pulmonary vessels. CONCLUSION: We identify ATP13A3 as a polyamine transporter controlling polyamine homeostasis in ECs, deficiency of which leads to EC dysfunction and predisposes to PAH. This suggests a need for targeted therapies to alleviate the imbalances in polyamine homeostasis and EC dysfunction in PAH.Great-Britain-China Educational Trust; Henry Lester Trust, Leche Trus

Evolving trends in the management of acute appendicitis during COVID-19 waves. The ACIE appy II study (vol 46, pg 2021, 2022)

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Global attitudes in the management of acute appendicitis during COVID-19 pandemic: ACIE Appy Study

Background: Surgical strategies are being adapted to face the COVID-19 pandemic. Recommendations on the management of acute appendicitis have been based on expert opinion, but very little evidence is available. This study addressed that dearth with a snapshot of worldwide approaches to appendicitis. Methods: The Association of Italian Surgeons in Europe designed an online survey to assess the current attitude of surgeons globally regarding the management of patients with acute appendicitis during the pandemic. Questions were divided into baseline information, hospital organization and screening, personal protective equipment, management and surgical approach, and patient presentation before versus during the pandemic. Results: Of 744 answers, 709 (from 66 countries) were complete and were included in the analysis. Most hospitals were treating both patients with and those without COVID. There was variation in screening indications and modality used, with chest X-ray plus molecular testing (PCR) being the commonest (19\ub78 per cent). Conservative management of complicated and uncomplicated appendicitis was used by 6\ub76 and 2\ub74 per cent respectively before, but 23\ub77 and 5\ub73 per cent, during the pandemic (both P < 0\ub7001). One-third changed their approach from laparoscopic to open surgery owing to the popular (but evidence-lacking) advice from expert groups during the initial phase of the pandemic. No agreement on how to filter surgical smoke plume during laparoscopy was identified. There was an overall reduction in the number of patients admitted with appendicitis and one-third felt that patients who did present had more severe appendicitis than they usually observe. Conclusion: Conservative management of mild appendicitis has been possible during the pandemic. The fact that some surgeons switched to open appendicectomy may reflect the poor guidelines that emanated in the early phase of SARS-CoV-2