A Coalescing Filter for Liquid-Liquid Separation and Multistage Extraction in Continuous-Flow Chemistry.

Presented here is the design and performance of a coalescing liquid-liquid filter, based on low-cost and readily available meltblown nonwoven substrates for separation of immiscible phases. The performance of the coalescer was determined across three broad classes of fluid mixtures: (i) immiscible organic/aqueous systems, (ii) a surfactant laden organic/aqueous system with modification of the type of emulsion and interfacial surface tension through the addition of sodium chloride, and (iii) a water-acetone/toluene system. The first two classes demonstrated good performance of the equipment in effecting separation, including the separation of a complex emulsion system for which a membrane separator, operating through transport of a preferentially wetting fluid through the membrane, failed entirely. The third system was used to demonstrate the performance of the separator within a multistage liquid-liquid counterflow extraction system. The performance, robust nature, and scalability of coalescing filters should mean that this approach is routinely considered for liquid-liquid separations and extractions within the fine chemical and pharmaceutical industry.

Comparable low revision rates of stemmed and stemless total anatomic shoulder arthroplasties after exclusion of metal backed glenoid components: a collaboration between the Australian and Danish national shoulder arthroplasty registries.

BACKGROUND: The stemmed anatomical total shoulder arthroplasty is the gold standard in the treatment of glenohumeral osteoarthritis. However, the use of stemless total shoulder arthroplasties has increased in recent years. The number of revision procedures are relatively low and therefore it has been recommended that national joint replacement registries should collaborate when comparing revision rates. Therefore, we aimed to compare the revision rates of stemmed and stemless TSA used for the diagnosis of glenohumeral osteoarthritis using data from both the Australian Orthopaedic Association National Joint Replacement Registry (AOANJRR) and the Danish Shoulder Arthroplasty Registry (DSR). METHODS: We included all patients who were registered in the AOANJRR and the DSR from January 1 2012 to December 2021 with an anatomical total shoulder arthroplasty used for osteoarthritis. Revision for any reason was used as the primary outcome. We used the Kaplan-Meier method to illustrate the cumulative revision rates and a multivariate cox regression model to calculate the hazard ratios. All analyses were performed separately for data from AOANJRR and DSR, and the results were then reported using a qualitative approach. RESULTS: 13066 arthroplasties from AOANJRR and 2882 arthroplasties from DSR were included. The hazard ratio for revision of stemmed TSA with stemless TSA as reference, adjusted for age and gender, was 1.67 (95% CI 1.34-2.09, p<0.001) in AOANJRR and 0.57 (95% CI 0.36-0.89, p=0.014) in DSR. When including glenoid type and fixation, surface bearing (only in AOANJRR) and hospital volume in the cox regression model the hazard ratio for revision of stemmed TSA compared to stemless TSA was 1.22 (95% CI 0.85-1.75, p=0.286) in AOANJRR and 1.50 (95% CI 0.91-2.45, p=0.109) in DSR. The adjusted hazard ratio for revision of total shoulder arthroplasties with metal backed glenoid components compared to all-polyethylene glenoid components was 2.54 (95% CI 1.70-3.79, p < 0.001) in AOANJRR and 4.1 (95% CI 1.92-8.58, p<0.001) in DSR. CONCLUSION: Based on data from two national shoulder arthroplasty registries, we found no significant difference in risk of revision between stemmed and stemless total shoulder arthroplasties after adjusting for the type of glenoid component. We advocate that metal-backed glenoid components should be used with caution and not on a routine basis.

Digital pathology for reporting histopathology samples, including cancer screening samples - definitive evidence from a multisite study.

AIMS: To conduct a definitive multicentre comparison of digital pathology (DP) with light microscopy (LM) for reporting histopathology slides including breast and bowel cancer screening samples. METHODS: A total of 2024 cases (608 breast, 607 GI, 609 skin, 200 renal) were studied, including 207 breast and 250 bowel cancer screening samples. Cases were examined by four pathologists (16 study pathologists across the four speciality groups), using both LM and DP, with the order randomly assigned and 6 weeks between viewings. Reports were compared for clinical management concordance (CMC), meaning identical diagnoses plus differences which do not affect patient management. Percentage CMCs were computed using logistic regression models with crossed random-effects terms for case and pathologist. The obtained percentage CMCs were referenced to 98.3% calculated from previous studies. RESULTS: For all cases LM versus DP comparisons showed the CMC rates were 99.95% [95% confidence interval (CI) = 99.90-99.97] and 98.96 (95% CI = 98.42-99.32) for cancer screening samples. In speciality groups CMC for LM versus DP showed: breast 99.40% (99.06-99.62) overall and 96.27% (94.63-97.43) for cancer screening samples; [gastrointestinal (GI) = 99.96% (99.89-99.99)] overall and 99.93% (99.68-99.98) for bowel cancer screening samples; skin 99.99% (99.92-100.0); renal 99.99% (99.57-100.0). Analysis of clinically significant differences revealed discrepancies in areas where interobserver variability is known to be high, in reads performed with both modalities and without apparent trends to either. CONCLUSIONS: Comparing LM and DP CMC, overall rates exceed the reference 98.3%, providing compelling evidence that pathologists provide equivalent results for both routine and cancer screening samples irrespective of the modality used.

A comparison of revision rates for stemmed and stemless primary anatomic shoulder arthroplasty with all-polyethylene glenoid components: analysis from the Australian Orthopaedic Association National Joint Replacement Registry.

BACKGROUND: We compared the rate of all-cause revision of 2 classes of primary anatomic shoulder arthroplasty, stemmed (stTSA) and stemless (slTSA), undertaken with cemented all-polyethylene glenoid components. METHODS: A large national arthroplasty registry identified 2 cohort groups for comparison, stTSA and l undertaken for all diagnoses between January 1, 2011, and December 31, 2021. A subanalysis from January 1, 2017, allowed capturing of additional patient demographics including American Society of Anesthesiologists score, body mass index, and glenoid morphology. The cumulative percent revision (CPR) was determined using Kaplan-Meier estimates of survivorship and hazard ratios (HR) from Cox proportional hazard models adjusted for age and gender. RESULTS: Of the 7995 stTSA procedures, the CPR at 9 years was 5.6% (95% confidence interval [CI]: 5.0, 6.4), and for 3156 slTSA procedures, the CPR was 4.4% (95% CI: 3.6, 5.5). There was no significant difference in the rate of revision between the study groups (HR = 0.76 [95% CI: 0.51, 1.14], P = .189, adjusted for age, gender, humeral head size, humeral fixation, bearing surface, glenoid design, and mean surgeon volume [MSV]). There was an increased rate of revision for stTSA and slTSA undertaken with humeral head sizes <44 mm (stTSA <44 mm vs. 44-50 mm, HR = 1.56 [CI: 1.18, 2.08], P = .001; slTSA <44 mm vs. 44-50 mm, HR = 2.08 [CI: 1.32, 3.33], P = .001). MSV as a continuous predictor was not a revision risk to stTSA vs. slTSA, but categorically, a low MSV (<10 stTSA + slTSA cases per annum) was associated with a higher revision rate for stTSA (10-20 cases/yr vs. <10 cases/yr, HR = 0.72 [CI: 0.55, 0.95], P = .019) but was not in slTSA. Revision rates were increased for stTSA with non-crosslinked polyethylene (XLPE) glenoids vs. XPLE after 2 years (HR = 2.20 [CI: 1.57, 3.08], P < .001) but did not significantly differ for slTSA. Metal/XPLE (humeral/glenoid) bearing surface of stTSA rate of revision was not different from each combination of slTSA bearing surface. Instability/dislocation was a revision risk for slTSA vs. stTSA (HR = 1.93 [CI: 1.28, 2.91], P = .001), but from 2017, neither of American Society of Anesthesiologists score, body mass index, and glenoid morphology changed the rate of revision. CONCLUSIONS: Revision rates of stTSA and slTSA did not significantly differ and were associated with humeral head size but not patient characteristics. Surgeon inexperience of anatomic shoulder arthroplasty and non-XLPE glenoids were risk factors for stTSA revision but not slTSA. The metal/XLPE stTSA rate of revision was not found to differ significantly from slTSA regardless of polyethylene or humeral head bearing type. Revision for instability/dislocation was more common for slTSA.

Comparing the Pathology, Clinical, and Demographic Characteristics of Younger and Older-Onset Multiple Sclerosis.

OBJECTIVE: Older people with multiple sclerosis (MS) have a less active radiological and clinical presentation, but many still attain significant levels of disability; but what drives worsening disability in this group? METHODS: We used data from the UK MS Register to characterize demographics and clinical features of late-onset multiple sclerosis (LOMS; symptom onset at ≥50 years), compared with adult-onset MS (AOMS; onset 18-49 years). We performed a pathology study of a separate MS cohort with a later onset (n = 18, mean age of onset 54 years) versus AOMS (n = 23, mean age of onset 29 years). RESULTS: In the Register cohort, there were 1,608 (9.4%) with LOMS. When compared with AOMS, there was a lower proportion of women, a higher proportion of primary progressive MS, a higher level of disability at diagnosis (median MS impact scale 36.7 vs. 28.3, p < 0.001), and a higher proportion of gait-related initial symptoms. People with LOMS were less likely to receive a high efficacy disease-modifying treatment and attained substantial disability sooner. Controlling for age of death and sex, neuron density in the thalamus and pons decreased with onset-age, whereas actively demyelinating lesions and compartmentalized inflammation was greatest in AOMS. Only neuron density, and not demyelination or the extent of compartmentalized inflammation, correlated with disability outcomes in older-onset MS patients. INTERPRETATION: The more progressive nature of older-onset MS is associated with significant neurodegeneration, but infrequent inflammatory demyelination. These findings have implications for the assessment and treatment of MS in older people. ANN NEUROL 2024;95:471-486.

Determining Phase Separation Dynamics with an Automated Image Processing Algorithm.

The problems of extracting products efficiently from reaction workups are often overlooked. Issues such as emulsions and rag layer formation can cause long separation times and slow production, thus resulting in manufacturing inefficiencies. To better understand science within this area and to support process development, an image processing methodology has been developed that can automatically track the interface between liquid-liquid phases and provide a quantitative measure of the separation rate of two immiscible liquids. The algorithm is automated and has been successfully applied to 29 cases. Its robustness has been demonstrated with a variety of different liquid mixtures that exhibit a wide range of separation behavior-making such an algorithm suited to high-throughput experimentation. The information gathered from applying the algorithm shows how issues resulting from poor separations can be detected early in process development.

Phosphonate and α-fluorophosphonate analogs of d-glucose 6-phosphate as active-site probes of 1l-myo-inositol 1-phosphate synthase.

Phosphate ester analogs in which the bridging oxygen is replaced with a methylene or fluoromethylene group are well known non-hydrolyzable mimics of use as inhibitors and substrate analogs for reactions involving phosphate esters. Properties of the replaced oxygen are often best mimicked by a mono-fluoromethylene group, but such groups are challenging to synthesize and can exist as two stereoisomers. Here, we describe the protocol for our method of synthesizing the α-fluoromethylene analogs of d-glucose 6-phosphate (G6P), as well as the methylene and difluoromethylene analogs, and their application in the study of 1l-myo-inositol-1-phosphate synthase (mIPS). mIPS catalyzes the synthesis of 1l-myo-inositol 1-phosphate (mI1P) from G6P, in an NAD-dependent aldol cyclization. Its key role in myo-inositol metabolism makes it a putative target for the treatment of several health disorders. The design of these inhibitors allowed for the possibility of substrate-like behavior, reversible inhibition, or mechanism-based inactivation. In this chapter, the synthesis of these compounds, expression and purification of recombinant hexahistidine-tagged mIPS, the mIPS kinetic assay and methods for determining the behavior of the phosphate analogs in the presence of mIPS, and a docking approach to rationalizing the observed behavior are described.

Technical note on the exploration of COVID-19 in autopsy material.

Interrogation of immune response in autopsy material from patients with SARS-CoV-2 is potentially significant. We aim to describe a validated protocol for the exploration of the molecular physiopathology of SARS-CoV-2 pulmonary disease using multiplex immunofluorescence (mIF).The application of validated assays for the detection of SARS-CoV-2 in tissues, originally developed in our laboratory in the context of oncology, was used to map the topography and complexity of the adaptive immune response at protein and mRNA levels.SARS-CoV-2 is detectable in situ by protein or mRNA, with a sensitivity that could be in part related to disease stage. In formalin-fixed, paraffin-embedded pneumonia material, multiplex immunofluorescent panels are robust, reliable and quantifiable and can detect topographic variations in inflammation related to pathological processes.Clinical autopsies have relevance in understanding diseases of unknown/complex pathophysiology. In particular, autopsy materials are suitable for the detection of SARS-CoV-2 and for the topographic description of the complex tissue-based immune response using mIF.

A Type 1 Aldolase, NahE, Catalyzes a Stereoselective Nitro-Michael Reaction: Synthesis of ß-Aryl-γ-nitrobutyric Acids.

Michael addition reactions are highly useful in organic synthesis and are commonly accomplished using organocatalysts. However, the corresponding biocatalytic Michael additions are rare, typically lack synthetically useful substrate scope, and suffer from low stereoselectivity. Herein we report a biocatalytic nitro-Michael addition, catalyzed by NahE, that proceeds with low catalyst loading at room temperature in moderate to excellent enantioselectivity and high yields. A series of ß-nitrostyrenes reacted with pyruvate in the presence of NahE to give, after oxidative decarboxylation, ß-aryl-γ-nitrobutyric acids in up to 99 % yield without need for chromatography, providing a simple preparative-scale route to chiral GABA analogues. This reaction represents the first example of an aldolase displaying promiscuous Michaelase activity and opens the use of nitroalkenes in place of aldehydes as substrates for aldolases.

Using Systemised Nomenclature of Medicine (SNOMED) codes to select digital pathology whole slide images for long-term archiving.

The archiving of whole slide images represents a hurdle to digital pathology implementation largely because of the amount of data generated. The retention of glass slides is currently recommended for a minimum of 10 years, but it is for individual departments to determine how digital images are archived and for how long. In a retrospective study, we examined the combination of Systemised Nomenclature of Medicine (SNOMED) codes allocated to cases reported between July 2011 and December 2015 and recalled more than 12 months after diagnosis in comparison to non-recalled cases.Our results show that 0.2% of cases are recalled after 12 months, and SNOMED code combinations can be used to identify which cases are likely to be recalled and which are not. This approach could reduce the number of cases archived by 62% and still ensure all cases likely to be recalled remain in the archive.

The African swine fever modelling challenge: Model comparison and lessons learnt.

Robust epidemiological knowledge and predictive modelling tools are needed to address challenging objectives, such as: understanding epidemic drivers; forecasting epidemics; and prioritising control measures. Often, multiple modelling approaches can be used during an epidemic to support effective decision making in a timely manner. Modelling challenges contribute to understanding the pros and cons of different approaches and to fostering technical dialogue between modellers. In this paper, we present the results of the first modelling challenge in animal health - the ASF Challenge - which focused on a synthetic epidemic of African swine fever (ASF) on an island. The modelling approaches proposed by five independent international teams were compared. We assessed their ability to predict temporal and spatial epidemic expansion at the interface between domestic pigs and wild boar, and to prioritise a limited number of alternative interventions. We also compared their qualitative and quantitative spatio-temporal predictions over the first two one-month projection phases of the challenge. Top-performing models in predicting the ASF epidemic differed according to the challenge phase, host species, and in predicting spatial or temporal dynamics. Ensemble models built using all team-predictions outperformed any individual model in at least one phase. The ASF Challenge demonstrated that accounting for the interface between livestock and wildlife is key to increasing our effectiveness in controlling emerging animal diseases, and contributed to improving the readiness of the scientific community to face future ASF epidemics. Finally, we discuss the lessons learnt from model comparison to guide decision making.

Phosphoenolpyruvate Mutase-Catalyzed C-P Bond Formation: Mechanistic Ambiguities and Opportunities.

Phosphonates are produced across all domains of life and used widely in medicine and agriculture. Biosynthesis almost universally originates from the enzyme phosphoenolpyruvate mutase (Ppm), EC 5.4.2.9, which catalyzes O-P bond cleavage in phosphoenolpyruvate (PEP) and forms a high energy C-P bond in phosphonopyruvate (PnPy). Mechanistic scrutiny of this unusual intramolecular O-to-C phosphoryl transfer began with the discovery of Ppm in 1988 and concluded in 2008 with computational evidence supporting a concerted phosphoryl transfer via a dissociative metaphosphate-like transition state. This mechanism deviates from the standard 'in-line attack' paradigm for enzymatic phosphoryl transfer that typically involves a phosphoryl-enzyme intermediate, but definitive evidence is sparse. Here we review the experimental evidence leading to our current mechanistic understanding and highlight the roles of previously underappreciated conserved active site residues. We then identify remaining opportunities to evaluate overlooked residues and unexamined substrates/inhibitors.

A combination of probabilistic and mechanistic approaches for predicting the spread of African swine fever on Merry Island.

Over the last decade African swine fever virus, one of the most virulent pathogens known to affect pigs, has devastated pork industries and wild pig populations throughout the world. Despite a growing literature on specific aspects of African swine fever transmission dynamics, it remains unclear which methods and approaches are most effective for controlling the disease during a crisis. As a consequence, an international modelling challenge was organized in which teams analyzed and responded to a stream of data from an in silico outbreak in the fictive country of Merry Island. In response to this outbreak, we developed a modelling approach that aimed to predict the evolution of the epidemic and evaluate the impact of potential control measures. Two independent models were developed: a stochastic mechanistic space-time compartmental model for characterizing the dissemination of the virus among wild boar; and a deterministic probabilistic risk model for quantifying infection probabilities in domestic pig herds. The combined results of these two models provided valuable information for anticipating the main risks of dissemination and maintenance of the virus (speed and direction of African swine fever spread among wild boar populations, pig herds at greatest risk of infection, the size of the epidemic in the short and long terms), for evaluating the impact of different control measures and for providing specific recommendations concerning control interventions.

Lessons from a breast cell annotation competition series for school pupils.

Due to COVID-19 outbreaks, most school pupils have had to be home-schooled for long periods of time. Two editions of a web-based competition "Beat the Pathologists" for school age participants in the UK ran to fill up pupils' spare time after home-schooling and evaluate their ability on contributing to AI annotation. The two editions asked the participants to annotate different types of cells on Ki67 stained breast cancer images. The Main competition was at four levels with different level of complexity. We obtained annotations of four kinds of cells entered by school pupils and ground truth from expert pathologists. In this paper, we analyse school pupils' performance on differentiating different kinds of cells and compare their performance with two neural networks (AlexNet and VGG16). It was observed that children tend to get very good performance in tumour cell annotation with the best F1 measure 0.81 which is a metrics taking both false positives and false negatives into account. Low accuracy was achieved with F1 score 0.75 on positive non-tumour cells and 0.59 on negative non-tumour cells. Superior performance on non-tumour cell detection was achieved by neural networks. VGG16 with training from scratch achieved an F1 score over 0.70 in all cell categories and 0.92 in tumour cell detection. We conclude that non-experts like school pupils have the potential to contribute to large-scale labelling for AI algorithm development if sufficient training activities are organised. We hope that competitions like this can promote public interest in pathology and encourage participation by more non-experts for annotation.

Phosphonate and α-Fluorophosphonate Analogues of d-Glucose 6-Phosphate as Active-Site Probes of 1l-myo-Inositol 1-Phosphate Synthase.

The biosynthesis of myo-inositol (mI) is central to the function of many organisms across all kingdoms of life. The first and rate-limiting step in this pathway is catalyzed by 1l-myo-inositol 1-phosphate synthase (mIPS), which converts d-glucose 6-phosphate (G6P) into 1l-myo-inositol 1-phosphate (mI1P). Extensive studies have shown that this reaction occurs through a stepwise NAD+-dependent redox aldol cyclization mechanism producing enantiomerically pure mI1P. Although the stereochemical nature of the mechanism has been elucidated, there is a lack of understanding of the importance of amino acid residues in the active site. Crystal structures of mIPS in the ternary complex with substrate analogues and NAD(H) show different ligand orientations. We therefore proposed to use isosteric and isoelectronic analogues of G6P to probe the active site. Here, we report the synthesis of the methylenephosphonate, difluoromethylenephosphonate, and (R)- and (S)-monofluoromethylenephosphonate analogues of G6P and their evaluation as inhibitors of mIPS activity. While the CH2 and CF2 analogues were produced with slight modification of a previously described route, the CHF analogues were synthesized through a new, shorter pathway. Kinetic behavior shows that all compounds are reversible competitive inhibitors with respect to G6P, with Ki values in the order CF2 (0.18 mM) < (S)-CHF (0.24 mM) < (R)-CHF (0.59 mM) < CH2 (1.2 mM). Docking studies of these phosphonates using published crystal structures show that substitution of the oxygen atom of the substrate changes the conformation of the resulting inhibitors, altering the position of carbon-6 and carbon-5, and this change is more pronounced with fluorine substitution.

Love is in the hair: arginine methylation of human hair proteins as novel cardiovascular biomarkers.

Cardiovascular disease is the major cause of death worldwide. Extensive cardiovascular biomarkers are available using blood tests but very few, if any, investigations have described non-invasive tests for cardiovascular biomarkers based on readily available hair samples. Here we show, first, that human hair proteins are post-translationally modified by arginine methylation (ArgMe). Using western blot, proteomic data mining and mass spectrometry, we identify several ArgMe events in hair proteins and we show that keratin-83 is extensively modified by ArgMe in the human hair. Second, using a preliminary cohort (n = 18) of heterogenous healthy donors, we show that the levels of protein ArgMe in hair correlate with serum concentrations of a well-established cardiovascular biomarker, asymmetric dimethylarginine (ADMA). Compared to blood collection, hair sampling is cheaper, simpler, requires minimal training and carries less health and safety and ethical risks. For these reasons, developing the potential of hair protein ArgMe as clinically useful cardiovascular biomarkers through further research could be useful in future prevention and diagnosis of cardiovascular disease.

Inhibition of Arginine Methylation Impairs Platelet Function.

Protein arginine methyltransferases (PRMTs) catalyze the transfer of methyl groups to arginine residues in proteins. PRMT inhibitors are novel, promising drugs against cancer that are currently in clinical trials, which include oral administration of the drugs. However, off-target activities of systemically available PRMT inhibitors have not yet been investigated. In this work, we study the relevance of arginine methylation in platelets and investigate the effect of PRMT inhibitors on platelet function and on the expression of relevant platelet receptors. We show that (1) key platelet proteins are modified by arginine methylation; (2) incubation of human platelets with PRMT inhibitors for 4 h results in impaired capacity of platelets to aggregate in response to thrombin and collagen, with IC50 values in the µM range; and (3) treatment with PRMT inhibitors leads to decreased membrane expression and reduced activation of the critical platelet integrin αIIbß3. Our contribution opens new avenues for research on arginine methylation in platelets, including the repurposing of arginine methylation inhibitors as novel antiplatelet drugs. We also recommend that current and future clinical trials with PRMT inhibitors consider any adverse effects associated with platelet inhibition of these emerging anticancer drugs.

Predicting Solvent-Dependent Nucleophilicity Parameter with a Causal Structure Property Relationship.

Solvent-dependent reactivity is a key aspect of synthetic science, which controls reaction selectivity. The contemporary focus on new, sustainable solvents highlights a need for reactivity predictions in different solvents. Herein, we report the excellent machine learning prediction of the nucleophilicity parameter N in the four most-common solvents for nucleophiles in the Mayr's reactivity parameter database (R2 = 0.93 and 81.6% of predictions within ±2.0 of the experimental values with Extra Trees algorithm). A Causal Structure Property Relationship (CSPR) approach was utilized, with focus on the physicochemical relationships between the descriptors and the predicted parameters, and on rational improvements of the prediction models. The nucleophiles were represented with a series of electronic and steric descriptors and the solvents were represented with principal component analysis (PCA) descriptors based on the ACS Solvent Tool. The models indicated that steric factors do not contribute significantly, because of bias in the experimental database. The most important descriptors are solvent-dependent HOMO energy and Hirshfeld charge of the nucleophilic atom. Replacing DFT descriptors with Parameterization Method 6 (PM6) descriptors for the nucleophiles led to an 8.7-fold decrease in computational time, and an ∼10% decrease in the percentage of predictions within ±2.0 and ±1.0 of the experimental values.

Correction: Estimation of the dispersal distances of an aphid-borne virus in a patchy landscape.

[This corrects the article DOI: 10.1371/journal.pcbi.1006085.].