HaloClass: Salt-Tolerant Protein Classification with Protein Language Models.

Salt-tolerant proteins, also known as halophilic proteins, have unique adaptations to function in high-salinity environments. These proteins have naturally evolved in extremophilic organisms, and more recently, are being increasingly applied as enzymes in industrial processes. Due to an abundance of salt-tolerant sequences and a simultaneous lack of experimental structures, most computational methods to predict stability are sequence-based only. These approaches, however, are hindered by a lack of structural understanding of these proteins. Here, we present HaloClass, an SVM classifier that leverages ESM-2 protein language model embeddings to accurately identify salt-tolerant proteins. On a newer and larger test dataset, HaloClass outperforms existing approaches when predicting the stability of never-before-seen proteins that are distal to its training set. Finally, on a mutation study that evaluated changes in salt tolerance based on single- and multiple-point mutants, HaloClass outperforms existing approaches, suggesting applications in the guided design of salt-tolerant enzymes.

Protein stability prediction by fine-tuning a protein language model on a mega-scale dataset.

Protein stability plays a crucial role in a variety of applications, such as food processing, therapeutics, and the identification of pathogenic mutations. Engineering campaigns commonly seek to improve protein stability, and there is a strong interest in streamlining these processes to enable rapid optimization of highly stabilized proteins with fewer iterations. In this work, we explore utilizing a mega-scale dataset to develop a protein language model optimized for stability prediction. ESMtherm is trained on the folding stability of 528k natural and de novo sequences derived from 461 protein domains and can accommodate deletions, insertions, and multiple-point mutations. We show that a protein language model can be fine-tuned to predict folding stability. ESMtherm performs reasonably on small protein domains and generalizes to sequences distal from the training set. Lastly, we discuss our model's limitations compared to other state-of-the-art methods in generalizing to larger protein scaffolds. Our results highlight the need for large-scale stability measurements on a diverse dataset that mirrors the distribution of sequence lengths commonly observed in nature.

Plasma Soluble ST2 Levels Are Higher in Neurodegenerative Disorders and Associated with Poorer Cognition.

BACKGROUND: Suppressor of tumorgenicity 2 (ST2) is highly expressed in brain tissue and is a receptor for interleukin 33 (IL-33). ST2 exists in two forms, a transmembrane receptor (ST2L) and a soluble decoy receptor (sST2). IL-33 binds to ST2L, triggering downstream signaling pathways involved in amyloid plaque clearance. Conversely, sST2 binds competitively to IL-33, attenuating its neuroprotective effects. High sST2 levels have been reported in mild cognitive impairment (MCI) and Alzheimer's disease (AD), suggesting that the IL-33/ST2 signaling pathway may be implicated in neurodegenerative diseases. OBJECTIVE: To investigate plasma sST2 levels in controls and patients with MCI, AD, frontotemporal dementia (FTD), and Parkinson's disease (PD). METHODS: Plasma sST2 levels were measured using ELISA in 397 subjects (91 HC, 46 MCI, 38 AD, 28 FTD, and 194 PD). Cerebrospinal fluid (CSF) levels of sST2 were measured in 22 subjects. Relationship between sST2 and clinical outcomes were analyzed. RESULTS: Plasma sST2 levels were increased across all disease groups compared to controls, with highest levels seen in FTD followed by AD and PD. Dementia patients with higher sST2 had lower cross-sectional cognitive scores in Frontal Assessment Battery and Digit Span Backward. At baseline, PD-MCI patients had higher sST2, associated with worse attention. In the longitudinal PD cohort, higher sST2 significantly associated with decline in global cognition and visuospatial domains. Plasma sST2 levels correlated with CSF sST2 levels. CONCLUSION: Plasma sST2 is raised across neurodegenerative diseases and is associated with poorer cognition. Higher baseline sST2 is a potential biomarker of disease severity in neurodegeneration.

C9orf72 expansions are the most common cause of genetic frontotemporal dementia in a Southeast Asian cohort.

OBJECTIVE: Frontotemporal dementia (FTD) encompasses a spectrum of neurodegenerative disorders, including behavioural variant FTD (bvFTD), semantic variant primary progressive aphasia (svPPA) and non-fluent variant PPA (nfvPPA). While a strong genetic component is implicated in FTD, genetic FTD in Asia is less frequently reported. We aimed to investigate the frequency of Southeast Asian FTD patients harbouring known genetic FTD variants. METHODS: A total of 60 FTD-spectrum patients (25 familial and 35 sporadic) from Singapore and the Philippines were included. All underwent next-generation sequencing and repeat-primed PCR for C9orf72 expansion testing. Neurofilament light chain (NfL) levels were measured in a subset of patients. RESULTS: Overall, 26.6% (16/60 cases) carried pathogenic or likely pathogenic variants in a FTD-related gene, including: MAPT Gln351Arg (n = 1); GRN Cys92Ter (n = 1), Ser301Ter (n = 2), c.462 + 1G > C (n = 1); C9orf72 expansion (35-70 repeats; n = 8); TREM2 Arg47Cys (n = 1); and OPTN frameshift insertion (n = 2). Genetic mutations accounted for 48% (12/25) of patients with familial FTD, and 11.4% (4/35) of patients with sporadic FTD. C9orf72 repeat expansions were the most common genetic mutation (13.3%, 8/60), followed by GRN (6.7%, 4/60) variants. Within mutation carriers, plasma NfL was highest in a C9orf72 expansion carrier, and CSF NfL was highest in a GRN splice variant carrier. INTERPRETATION: In our cohort, genetic mutations are present in one-quarter of FTD-spectrum cases, and up to half of those with family history. Our findings highlight the importance of wider implementation of genetic testing in FTD patients from Southeast Asia.

Structural plasticity enables evolution and innovation of RuBisCO assemblies.

Oligomerization is a core structural feature that defines the form and function of many proteins. Most proteins form molecular complexes; however, there remains a dearth of diversity-driven structural studies investigating the evolutionary trajectory of these assemblies. Ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO) is one such enzyme that adopts multiple assemblies, although the origins and distribution of its different oligomeric states remain cryptic. Here, we retrace the evolution of ancestral and extant form II RuBisCOs, revealing a complex and diverse history of oligomerization. We structurally characterize a newly discovered tetrameric RuBisCO, elucidating how solvent-exposed surfaces can readily adopt new interactions to interconvert or give rise to new oligomeric states. We further use these principles to engineer and demonstrate how changes in oligomerization can be mediated by relatively few mutations. Our findings yield insight into how structural plasticity may give rise to new oligomeric states.

The effect of pregnancy on metabolic scaling and population energy demand in the viviparous fish Gambusia affinis.

Metabolism is a fundamental attribute of all organisms that influences how species affect and are affected by their natural environment. Differences between sexes in ectothermic species may substantially alter metabolic scaling patterns, particularly in viviparous or live-bearing species where females must support their basal metabolic costs and that of their embryos. Indeed, if pregnancy is associated with marked increases in metabolic demand and alters scaling patterns between sexes, this could in turn interact with natural sex ratio variation in nature to affect population-level energy demand. Here, we aimed to understand how sex and pregnancy influence metabolic scaling and how differences between sexes affect energy demand in Gambusia affinis (Western mosquitofish). Using the same method, we measured routine metabolic rate in the field on reproductively active fish and in the laboratory on virgin fish. Our data suggest that changes in energy expenditure related to pregnancy may lead to steeper scaling coefficients in females (b = 0.750) compared to males (b = 0.595). In contrast, virgin females and males had similar scaling coefficients, suggesting negligible sex differences in metabolic costs in reproductively inactive fish. Further, our data suggest that incorporating sex differences in allometric scaling may alter population-level energy demand by as much as 20-28%, with the most pronounced changes apparent in male-biased populations due to the lower scaling coefficient of males. Overall, our data suggest that differences in energy investment in reproduction between sexes driven by pregnancy may alter allometric scaling and population-level energy demand.

Perceived usefulness of open educational resources: Impact of switching to online learning for face-to-face and distance learners.

This paper reports a study on the perceived usefulness of university students on open educational resources (OER) in relation to the switch of learning mode to online learning during the COVID-19 pandemic. The participants involved two groups of students, one studying in a face-to-face mode and the other in a distance learning mode. They took part in a survey which was conducted in 2019 before the pandemic (with a total of 912 responses) and 2021 during the pandemic (with a total of 1,018 responses). The results show that both groups of students generally perceived OER to be more useful during the pandemic. The specific types of OER which were perceived as relatively more useful include open online courses and open access textbooks. Face-to-face students showed a higher level of perceived usefulness of OER for preparing tests and examinations, while distance learning students perceived OER as more useful for supplementing course materials. They both concerned about the limitations of OER, especially on accuracy and comprehensiveness. The findings suggest the importance of recognizing the diverse needs of the two groups of students and offering appropriate OER support for them.

Consumer trait responses track change in resource supply along replicated thermal gradients.

Rising temperatures may alter consumer diets through increased metabolic demand and altered resource availability. However, current theories assessing dietary shifts with warming do not account for a change in resource availability. It is unknown whether consumers will increase consumption rates or consume different resources to meet increased energy requirements and whether the dietary change will lead to associated variation in morphology and nutrient utilization. Here, we used populations of Gambusia affinis across parallel thermal gradients in New Zealand (NZ) and California (CA) to understand the influence of temperature on diets, morphology and stoichiometric phenotypes. Our results show that with increasing temperature in NZ, mosquitofish consumed more plant material, whereas in CA mosquitofish shifted towards increased consumption of invertebrate prey. In both regions, populations with plant-based diets had fuller guts, longer relative gut lengths, better-orientated mouths and reduced body elemental %C and N/P. Together, our results show multiple pathways by which consumers may alter their feeding patterns with rising temperatures, and they suggest that warming-induced changes to resource availability may be the principal determinant of which pathway is taken.

Warfarin control in Hong Kong clinical practice: a single-centre observational study.

INTRODUCTION: Time in therapeutic range (TTR) assesses the safety and effectiveness of warfarin therapy using the international normalised ratio. This study investigated the TTR in Hong Kong patients using both European and Japanese therapeutic ranges and patients' economic and clinical outcomes. Predictors of poor warfarin control and patient knowledge concerning warfarin therapy were assessed. METHODS: A 5-month observational study with retrospective and prospective components was conducted in the Prince of Wales Hospital. The study examined electronic patient records of patients who received warfarin for at least 1 year during the period from January 2010 to August 2015. Patient knowledge was assessed via phone interview using the Oral Anticoagulation Knowledge (OAK) test. RESULTS: In total, 259 patients were included; 174 completed the OAK test. The calculated mean TTR was 40.2±17.1% (European therapeutic range), compared with 49.1±16.1% (Japanese therapeutic range) [P<0.001]. Mean TTR was higher in patients with atrial fibrillation than in patients with prosthetic heart valve (P<0.001). The abilities of TTR to predict clinical and economic outcomes were comparable between European and Japanese therapeutic ranges. Patients with ideal TTR had fewer clinical complications and lower healthcare costs. Patients with younger age exhibited worse TTR, as did those with concurrent use of furosemide, famotidine, or simvastatin. Mean OAK test score was 54.1%. Only 24 (13.8%) patients achieved a satisfactory overall score of ≥75% in the test. CONCLUSION: Warfarin use in Hong Kong patients was poorly controlled, regardless of indication. Patient knowledge concerning warfarin use was suboptimal; thus, additional patient education is warranted regarding warfarin.

Phenotypic bases of NOTCH2NLC GGC expansion positive neuronal intranuclear inclusion disease in a Southeast Asian cohort.

Neuronal intranuclear inclusion disease (NIID) is a neurodegenerative disorder associated with GGC repeats of >60 to 500 copies in the 5'-untranslated region of NOTCH2NLC. The clinical and genetic characterization of NIID outside of East Asia remains unknown. We identified twelve patients who underwent genetic testing using long-read sequencing or repeat primed polymerase chain reaction. All were positive for a GGC repeat expansion; the median repeat length was 107 (range 92-138). Ten were Chinese and two of Malay ethnicity. Age at onset ranged from 50 to 69 years. Eight (66.7%) patients had dementia, while four (33.3%) patients were oligosymptomatic, without typical NIID symptoms of dementia, Parkinsonism, or muscle weakness. GGA interruptions within the GGC expansion were present in four patients; the number of GGA interruptions was highest (6.71%) in the patient with the earliest age at onset (50 years). Median plasma neurofilament light level was 47.3 pg/mL in seven patients (range 26-380 pg/mL). The highest level (380 pg/mL) was found in one patient who experienced an encephalitic episode. Overall, we describe a cohort of genetically confirmed NIID patients from Southeast Asia and provide further information that the presence of GGA interruptions within GGC repeat expansions may serve as a potential genetic modifier in NIID.

NOTCH2NLC GGC Repeat Expansions Are Associated with Sporadic Essential Tremor: Variable Disease Expressivity on Long-Term Follow-up.

We screened 662 subjects comprising 462 essential tremor (ET) subjects (285 sporadic, 125 with family history, and 52 probands from well-characterized ET pedigrees) and 200 controls and identified pathogenic NOTCH2NLC GGC repeat expansions in 4 sporadic ET patients. Two patients were followed up for >1 decade; one with 90 repeats remained an ET phenotype that did not evolve after 40 years, whereas another patient with 107 repeats developed motor symptoms and cognitive impairment after 8 to 10 years. Neuroimaging in this patient revealed severe leukoencephalopathy; diffusion-weighted imaging hyperintensity in the corticomedullary junction and skin biopsy revealed intranuclear inclusions suggestive of intranuclear inclusion body disease (NIID). No GGC repeats of >60 units were detected in familial ET cases and controls, although 4 ET patients carried 47 to 53 "intermediate" repeats. NOTCH2NLC GGC repeat expansions can be associated with sporadic ET. Carriers presenting with a pure ET phenotype may or may not convert to NIID up to 4 decades after initial tremor onset. ANN NEUROL 2020;88:614-618.

Machine Learning Clustering for Blood Pressure Variability Applied to Systolic Blood Pressure Intervention Trial (SPRINT) and the Hong Kong Community Cohort.

Visit-to-visit blood pressure variability (BPV) has been shown to be a predictor of cardiovascular disease. We aimed to classify the BPV levels using different machine learning algorithms. Visit-to-visit blood pressure readings were extracted from the SPRINT study in the United States and eHealth cohort in Hong Kong (HK cohort). Patients were clustered into low, medium, and high BPV levels with the traditional quantile clustering and 5 machine learning algorithms including K-means. Clustering methods were assessed by Stability Index. Similarities were assessed by Davies-Bouldin Index and Silhouette Index. Cox proportional hazard regression models were fitted to compare the risk of myocardial infarction, stroke, and heart failure. A total of 8133 participants had average blood pressure measurement 14.7 times in 3.28 years in SPRINT and 1094 participants who had average blood pressure measurement 165.4 times in 1.37 years in HK cohort. Quantile clustering assigned one-third participants as high BPV level, but machine learning methods only assigned 10% to 27%. Quantile clustering is the most stable method (stability index: 0.982 in the SPRINT and 0.948 in the HK cohort) with some levels of clustering similarities (Davies-Bouldin Index: 0.752 and 0.764, respectively). K-means clustering is the most stable across the machine learning algorithms (stability index: 0.975 and 0.911, respectively) with the lowest clustering similarities (Davies-Bouldin Index: 0.653 and 0.680, respectively). One out of 7 in the population was classified with high BPV level, who showed to have higher risk of stroke and heart failure. Machine learning methods can improve BPV classification for better prediction of cardiovascular diseases.

Management of large renal stones with super-mini percutaneous nephrolithotomy: an international multicentre comparative study.

OBJECTIVES: To comparatively evaluate the clinical outcomes of super-mini percutaneous nephrolithotomy (SMP) and mini-percutaneous nephrolithotomy (Miniperc) for treating urinary tract calculi of >2 cm. PATIENTS AND METHODS: An international multicentre, retrospective cohort study was conducted at 20 tertiary care hospitals across five countries (China, the Philippines, Qatar, UK, and Kuwait) between April 2016 and May 2019. SMP and Miniperc were performed in 3525 patients with renal calculi with diameters of >2 cm. The primary endpoint was the stone-free rate (SFR). The secondary outcomes included: blood loss, operating time, postoperative pain scores, auxiliary procedures, complications, tubeless rate, and hospital stay. Propensity score matching analysis was used to balance the selection bias between the two groups. RESULTS: In all, 2012 and 1513 patients underwent SMP and Miniperc, respectively. After matching, 1380 patients from each group were included for further analysis. Overall, there was no significant difference in the mean operating time or SFR between the two groups. However, the hospital stay and postoperative pain score were significantly in favour of SMP (both P < 0.001). The tubeless rate was significantly higher in the SMP group (72.6% vs 57.8%, P < 0.001). Postoperative fever was much more common in the Miniperc group (12.0% vs 8.4%, P = 0.002). When the patients were further classified into three subgroups based on stones diameters (2-3, 3-4, and >4 cm). The advantages of SMP were most obvious in the 2-3 cm stone group and diminished as the size of the stone increased, with longer operating time in the latter two subgroups. Compared with Miniperc, the SFR of SMP was comparable for 3-4 cm stones, but lower for >4 cm stones. There was no statistical difference in blood transfusions and renal embolisations between the two groups. CONCLUSIONS: Our data showed that SMP is an ideal treatment option for stones of <4 cm and is more efficacious for stones of 2-3 cm, with lesser postoperative fever, blood loss, and pain compared to Miniperc. SMP was less effective for stones of >4 cm, with a prolonged operating time.

Evaluating Protein Engineering Thermostability Prediction Tools Using an Independently Generated Dataset.

Engineering proteins to enhance thermal stability is a widely utilized approach for creating industrially relevant biocatalysts. The development of new experimental datasets and computational tools to guide these engineering efforts remains an active area of research. Thus, to complement the previously reported measures of T 50 and kinetic constants, we are reporting an expansion of our previously published dataset of mutants for ß-glucosidase to include both measures of T M and ΔΔG. For a set of 51 mutants, we found that T 50 and T M are moderately correlated, with a Pearson correlation coefficient and Spearman's rank coefficient of 0.58 and 0.47, respectively, indicating that the two methods capture different physical features. The performance of predicted stability using nine computational tools was also evaluated on the dataset of 51 mutants, none of which are found to be strong predictors of the observed changes in T 50, T M, or ΔΔG. Furthermore, the ability of the nine algorithms to predict the production of isolatable soluble protein was examined, which revealed that Rosetta ΔΔG, FoldX, DeepDDG, PoPMuSiC, and SDM were capable of predicting if a mutant could be produced and isolated as a soluble protein. These results further highlight the need for new algorithms for predicting modest, yet important, changes in thermal stability as well as a new utility for current algorithms for prescreening designs for the production of mutants that maintain fold and soluble production properties.

Case-control analysis of leucine-rich repeat kinase 2 protective variants in Alzheimer's disease.

Amyloid is the main pathological substrate of Alzheimer's disease (AD) and has been described in leucine-rich repeat kinase 2 (LRRK2) carriers with Parkinson's disease. LRRK2 has been linked with amyloid precursor protein pathways in neurodegeneration. Two common LRRK2 variants, R1398H and N551K, have been shown to be protective in multiple Parkinson's disease cohorts. We hypothesized that R1398H and N551K may be protective in AD. In a case-control study involving 1390 subjects (719 controls and 671 AD cases), R1398H was demonstrated in 16.8% of AD cases compared to 16.7% in controls (odds ratio = 1.01, 95% confidence interval = 0.76-1.34, p = 0.94), whereas N551K was demonstrated in 17.3% of AD cases compared to 17.2% of controls (odds ratio = 1.00, 95% confidence interval = 0.76-1.32, p = 0.98). Overall, these results suggest that LRRK2 R1398H or N551K variants do not appear to modulate the risk of AD.