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Zhu Wei Lim,1 Wei Liang Chen2– 4 1Department of Obstetrics and Gynecology, Changhua Christian Hospital, Changhua, Taiwan, Republic of China; 2Division of Family Medicine, Department of Family and Community Medicine, Tri-Service General Hospital; and School of Medicine, National Defense Medical Center, Taipei, Taiwan, Republic of China; 3Division of Geriatric Medicine, Department of Family and Community Medicine, Tri-Service General Hospital; and School of Medicine, National Defense Medical Center, Taipei, Taiwan, Republic of China; 4Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan, Republic of ChinaCorrespondence: Wei Liang ChenDivision of Geriatric Medicine, Department of Family Medicine, Tri-Service General Hospital, National Defense Medical Center, Number 325, Section 2, Chang-Gong Road, Nei-Hu District, Taipei, 114, Taiwan, Republic of ChinaTel +886-2-87923311 ext. 16567Fax +886-2-87927057Email weiliang0508@gmail.comIntroduction: An important clinical feature of metabolic syndrome is abdominal obesity. Microalbuminuria is important in predicting the risk of cardiovascular and renal complications in abdominal obesity patients. However, the association between microalbuminuria polymorphism and abdominal obesity has not been conducted. The objective of this study is to analyze the genetic polymorphism of microalbuminuria in participants with metabolically unhealthy obesity (MUO).Methods: Among 1325 MUO participants, we identified genomic loci underlying those with microalbuminuria, compared to those without microalbuminuria. Single nucleotide polymorphisms (SNPs) were selected with P < 1 × 10− 5 from the Manhattan plot. Multivariable linear regression and analysis of variance were used to analyze the association between different SNP genotypes and microalbuminuria.Results: The analysis showed homozygous participants for the risk allele A of rs10105606 and Affx-31885823 had 1.978-fold risk and 1.921-fold increased risk of microalbuminuria, respectively. Heterozygous distribution of rs117180252, rs10105606, and Affx-31885823 also increased the risk of microalbuminuria compared to the wild type. Further analysis showed Lipoprotein lipase (LPL), RN7SL87P, and RPL30P9 were the candidate genes associated with lipid metabolism and abdominal obesity.Conclusion: In conclusion, LPL, RN7SL87P, and RPL30P9 minor allele carriers with abdominal obesity are more susceptible to microalbuminuria, explaining the inter-individual differences of microalbuminuria in MUO patients.Keywords: metabolic syndrome, abdominal obesity, microalbuminuria, metabolically unhealthy obesity, polymorphism, lipoprotein lipase

Zhu Wei Lim,1 Wei Liang Chen2 1Department of Obstetrics and Gynecology, Changhua Christian Hospital, Changhua, Taiwan; 2Division of Geriatric Medicine, Department of Family and Community Medicine, Tri-Service General Hospital and School of Medicine, National Defense Medical Center, Taipei, TaiwanCorrespondence: Wei Liang ChenDivision of Geriatric Medicine, Department of Family and Community Medicine, Tri-Service General Hospital and School of Medicine, National Defense Medical Center, Taipei, TaiwanTel +886-2-87923311 ext. 16567Fax +886-2-87927057Email weiliang0508@gmail.comBackground: Human epididymis protein 4 (HE4) has been frequently used to study in many malignant tumors, while serum nutritional markers are used to determine a person’s health status. However, the link between serum micronutrient concentrations and HE4 has not yet been clarified.Methods: A total of 2464 eligible female participants and serum concentrations of nutritional biomarkers were chosen from the National Health and Nutrition Examination Surveys (NHANES) 2001– 2002. For statistical analysis, we used the χ2 test, multivariable linear regression, and analysis of variance. Adjusted models were used, and the concentrations of serum nutritional biomarkers were divided into quartiles.Results: The mean age of the participants was 48.07 years. Among twelve micronutrients, five were negatively associated with HE4 in models 1, 2 and 3. Only α-carotene, trans-β-carotene, cis-β-carotene, trans-lycopene and retinol were associated with HE4, with beta coefficients of − 0.102, − 0.027, − 0.506, − 0.131 and − 0.054, respectively. After performing quartile-based analysis, statistical significance was only found for serum α-carotene, trans-lycopene, and retinol in the three models. In model 3, the beta coefficients [95% confidence intervals (CIs)] of the fourth quartiles compared to the first quartiles for α-carotene, trans-lycopene, and retinol were − 3.390 (− 5.053, − 1.727), − 4.036 (− 5.722, − 2.351) and − 4.146 (− 5.899, − 2.393), respectively. Serum concentrations of these three nutritional biomarkers were inversely related to serum HE4 concentration (p trend < 0.001).Conclusion: HE4 is a useful and novel biomarker that can be used with many diseases, especially ovarian cancer. Three of our selected micronutrients were inversely associated with HE4 concentration. Supplement of micronutrients may reduce the levels of HE4 and the subsequent of ovarian cancer’s risk. Therefore, a formula that correlates HE4 with nutritional biomarkers needs to be established before use in clinical applications.Keywords: human epididymis protein 4, vitamin A, vitamin E, carotenoids

Objective: Ichthyosis are complex skin diseases, characterized by hyperkeratosis with various degrees of thickening, desquamation, and erythema. The prenatal diagnosis of ichthyosis is challenged due to the clinical and genetic heterogeneity and the late-onset of fetal features on ultrasound scan. Here, we reported two fetuses with Harlequin ichthyosis (HI), a severe subtype of autosomal recessive congenital ichthyosis (ARCI), who were diagnosed prenatally by images and genetic investigations. Preimplantation genetic testing (PGT) was also performed in one case and the family to prevent the transmission of this condition., Materials and Methods: Fetal images were analyzed by detailed fetal ultrasound. Genetic defects in affected fetuses were detected using whole exome sequencing (WES) and confirmed by Sanger sequencing. PGT for monogenic disease (PGT-M) was performed using short tandem repeat (STR) markers and amplification refractory mutation system quantitative polymerase chain reaction (ARMS-qPCR)., Results: WES identified pathogenic mutations in ABCA12 gene in both fetuses. Genome Analysis ToolKit (GATK 4 version 4.2.1.0) was merged using a setting of the trio model for exome variation analysis. Confirmatory Sanger sequencing of ABCA12 gene was also applied to both parents. Disease-cause haplotyping by STR markers and ARMS-qPCR for PGT-M strategy were conducted in family 1. Linkage analysis in conjunction with STR was used in the first case, whereas unequal coverage of WES was deciphered with the inspiration of the second case a few years later. Both cases were diagnosed by high coverage WES and led to successful subsequent pregnancies by preimplantation genetic diagnosis and genetic amniocentesis., Conclusion: Preimplantation genetic testing using STR linkage analysis can rescue cases with autosomal recessive inheritance when only one mutation is found in the putative gene, while reanalysis of high-coverage WES by optimized updated bioinformatics should always be considered in addition to whole genome sequencing., Competing Interests: Declaration of competing interest The authors declared no conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

Peptide coacervates self-assembling via liquid-liquid phase separation are appealing intracellular delivery vehicles of macromolecular therapeutics (proteins, DNA, mRNA) owing to their non-cytotoxicity, high encapsulation capacity, and efficient cellular uptake. However, the mechanisms by which these viscoelastic droplets cross the cellular membranes remain unknown. Here, using multimodal imaging, data analytics, and biochemical inhibition assays, we identify the key steps by which droplets enter the cell. We find that the uptake follows a non-canonical pathway and instead integrates essential features of macropinocytosis and phagocytosis, namely active remodeling of the actin cytoskeleton and appearance of filopodia-like protrusions. Experiments using giant unilamellar vesicles show that the coacervates attach to the bounding membrane in a charge- and cholesterol-dependent manner but do not breach the lipid bilayer barrier. Cell uptake in the presence of small molecule inhibitors - interfering with actin and tubulin polymerization - confirm the active role of cytoskeleton remodeling, most prominently evident in electron microscopy imaging. These findings suggest a peculiar internalization mechanism for viscoelastic, glassy coacervate droplets combining features of non-specific uptake of fluids by macropinocytosis and particulate uptake of phagocytosis. The broad implications of this study will enable to enhance the efficacy and utility of coacervate-based strategies for intracellular delivery of macromolecular therapeutics., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Deoxyribonucleic acid (DNA) is able to form non-canonical four-stranded helical structures with diverse folding patterns known as G-quadruplexes (G4s). G4 topologies are classified based on their relative strand orientation following the 5' to 3' phosphate backbone polarity. Broadly, G4 topologies are either parallel (4+0), antiparallel (2+2), or hybrid (3+1). G4s play crucial roles in biological processes such as DNA repair, DNA replication, transcription and have thus emerged as biological targets in drug design. While computational models have been developed to predict G4 formation, there is currently no existing model capable of predicting G4 folding topology based on its nucleic acid sequence. Therefore, we introduce G4ShapePredictor (G4SP), an application featuring a collection of multi-classification machine learning models that are trained on a custom G4 dataset combining entries from existing literature and in-house circular dichroism experiments. G4ShapePredictor is designed to accurately predict G4 folding topologies in potassium ( K + ) buffer based on its primary sequence and is able to incorporate a threshold optimization strategy allowing users to maximise precision. Furthermore, we have identified three topological sequence motifs that suggest specific G4 folding topologies of (4+0), (2+2) or (3+1) when utilising the decision-making mechanisms of G4ShapePredictor., (© 2024. The Author(s).)

Background: Myopia affects 1.4 billion individuals worldwide. Notably, there is increasing evidence that choroidal thickness plays an important role in myopia and risk of developing myopia-related conditions. With the advancements in artificial intelligence (AI), choroidal thickness segmentation can now be automated, offering inherent advantages such as better repeatability, reduced grader variability, and less reliance for manpower. Hence, we aimed to evaluate the agreement between AI-automated and manual segmented measurements of subfoveal choroidal thickness (SFCT) using two swept-source optical coherence tomography (OCT) systems., Methods: Subjects aged ≥ 16 years, with myopia of ≥ 0.50 diopters in both eyes, were recruited from the Prospective Myopia Cohort Study in Singapore (PROMYSE). OCT scans were acquired using Triton DRI-OCT and PLEX Elite 9000. OCT images were segmented both automatically with an established SA-Net architecture and manually using a standard technique with adjudication by two independent graders. SFCT was subsequently determined based on the segmentation. The Bland-Altman plot and intraclass correlation coefficient (ICC) were used to evaluate the agreement., Results: A total of 229 subjects (456 eyes) with mean [± standard deviation (SD)] age of 34.1 (10.4) years were included. The overall SFCT (mean ± SD) based on manual segmentation was 216.9 ± 82.7 µm with Triton DRI-OCT and 239.3 ± 84.3 µm with PLEX Elite 9000. ICC values demonstrated excellent agreement between AI-automated and manual segmented SFCT measurements (PLEX Elite 9000: ICC = 0.937, 95% CI: 0.922 to 0.949, P < 0.001; Triton DRI-OCT: ICC = 0.887, 95% CI: 0.608 to 0.950, P < 0.001). For PLEX Elite 9000, manual segmented measurements were generally thicker when compared to AI-automated segmented measurements, with a fixed bias of 6.3 µm (95% CI: 3.8 to 8.9, P < 0.001) and proportional bias of 0.120 (P < 0.001). On the other hand, manual segmented measurements were comparatively thinner than AI-automated segmented measurements for Triton DRI-OCT, with a fixed bias of - 26.7 µm (95% CI: - 29.7 to - 23.7, P < 0.001) and proportional bias of - 0.090 (P < 0.001)., Conclusion: We observed an excellent agreement in choroidal segmentation measurements when comparing manual with AI-automated techniques, using images from two SS-OCT systems. Given its edge over manual segmentation, automated segmentation may potentially emerge as the primary method of choroidal thickness measurement in the future., (© 2024. The Author(s).)

Distinctive aspects of a culture are often reflected in the meaning and usage of words in the language spoken by bearers of that culture. Keywords such as душа (soul) in Russian, hati (heart) in Indonesian and Malay, and gezellig (convivial/cosy/fun) in Dutch are held to be especially culturally revealing, and scholars have identified a number of such keywords using careful linguistic analyses (Peeters, 2020b; Wierzbicka, 1990). Because keywords are expected to have different statistical properties than related words in other languages, we argue that a quantitative comparison of word usage across languages can help to identify cultural keywords. To support this claim, we describe a computational method that compares word frequencies across languages, and apply it to both linguistic corpora and word association data. The method identifies culturally specific words that range from "obvious" examples, such as Amsterdam in Dutch, to non-obvious yet independently proposed examples, such as hati (heart) in Indonesian. We show in addition that linguistic corpora and word association data provide converging evidence about culturally specific words. Our results therefore show how computational analyses and behavioral experiments can supplement the methods previously used by linguists to identify culturally salient words across languages., (© 2024 The Authors. Cognitive Science published by Wiley Periodicals LLC on behalf of Cognitive Science Society (CSS).)

Objectives: To evaluate the predictability of the Invisalign appliance (Align Technology, Santa Clara, Calif) in leveling the maxillary curve of Spee (COS)., Materials and Methods: A retrospective sample of adult subjects treated with the Invisalign appliance between 2013 and 2019 were selected. Patients were treated nonextraction in the maxillary arch and had either Angle Class I or II malocclusions with a minimum of 14 aligners with no bite ramps. Initial, predicted, and actual outcomes were analyzed with Geomagic Control X software (version 2017.0.3; 3D Systems, Cary, NC)., Results: A sample of 53 cases satisfied inclusion/exclusion criteria. Paired t-tests demonstrated a significant difference between mean predicted and actual maxillary COS leveling with a shortfall of 0.11 mm (SD = 0.37; P = .033). Planned intrusion tended to be more accurate posteriorly with an overexpression of 117% for the first molars. Planned extrusion was the least accurate, with the mid-arch demonstrating expressions of -14% to -48%. These teeth intruded despite a prescribed extrusive movement., Conclusions: The Invisalign appliance did not accurately predict maxillary COS leveling. Planned intrusive movements were overcorrected, and planned extrusive movements were either undercorrected or resulted in intrusion. This effect was most apparent for the upper first molar, which expressed 117% and -48% of planned intrusion and extrusion, respectively., (© 2023 by The EH Angle Education and Research Foundation, Inc.)

Introduction: Our study aimed to examine the relationship between cardiovascular diseases (CVD) with peripapillary retinal fiber layer (RNFL) and macular ganglion cell-inner plexiform layer (GCIPL) thickness profiles in a large multi-ethnic Asian population study., Methods: 6,024 Asian subjects were analyzed in this study. All participants underwent standardized examinations, including spectral domain OCT imaging (Cirrus HD-OCT; Carl Zeiss Meditec). In total, 9,188 eyes were included for peripapillary RNFL analysis (2,417 Malays; 3,240 Indians; 3,531 Chinese), and 9,270 eyes (2,449 Malays, 3,271 Indians, 3,550 Chinese) for GCIPL analysis. History of CVD was defined as a self-reported clinical history of stroke, myocardial infarction, or angina. Multivariable linear regression models with generalized estimating equations were performed, adjusting for age, gender, ethnicity, diabetes, hypertension, hyperlipidaemia, chronic kidney disease, body mass index, current smoking status, and intraocular pressure., Results: We observed a significant association between CVD history and thinner average RNFL (β = -1.63; 95% CI, -2.70 to -0.56; p  = 0.003). This association was consistent for superior (β = -1.79, 95% CI, -3.48 to -0.10; p  = 0.038) and inferior RNFL quadrant (β = -2.14, 95% CI, -3.96 to -0.32; p  = 0.021). Of the CVD types, myocardial infarction particularly showed significant association with average (β = -1.75, 95% CI, -3.08 to -0.42; p  = 0.010), superior (β = -2.22, 95% CI, -4.36 to -0.09; p  = 0.041) and inferior (β = -2.42, 95% CI, -4.64 to -0.20; p  = 0.033) RNFL thinning. Among ethnic groups, the association between CVD and average RNFL was particularly prominent in Indian eyes (β = -1.92, 95% CI, -3.52 to -0.33; p  = 0.018). CVD was not significantly associated with average GCIPL thickness, albeit a consistent negative direction of association was observed (β = -0.22, 95% CI, -1.15 to 0.71; p  = 0.641)., Discussion: In this large multi-ethnic Asian population study, we observed significant association between CVD history and RNFL thinning. This finding further validates the impact of impaired systemic circulation on RNFL thickness., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Majithia, Quek, Chee, Lim, Nusinovici, Soh, Thakur, Rim, Sabanayagam, Cheng and Tham.)

In light of growing interest in using emerging large language models (LLMs) for self-diagnosis, we systematically assessed the performance of ChatGPT-3.5, ChatGPT-4.0, and Google Bard in delivering proficient responses to 37 common inquiries regarding ocular symptoms. Responses were masked, randomly shuffled, and then graded by three consultant-level ophthalmologists for accuracy (poor, borderline, good) and comprehensiveness. Additionally, we evaluated the self-awareness capabilities (ability to self-check and self-correct) of the LLM-Chatbots. 89.2% of ChatGPT-4.0 responses were 'good'-rated, outperforming ChatGPT-3.5 (59.5%) and Google Bard (40.5%) significantly (all p < 0.001). All three LLM-Chatbots showed optimal mean comprehensiveness scores as well (ranging from 4.6 to 4.7 out of 5). However, they exhibited subpar to moderate self-awareness capabilities. Our study underscores the potential of ChatGPT-4.0 in delivering accurate and comprehensive responses to ocular symptom inquiries. Future rigorous validation of their performance is crucial to ensure their reliability and appropriateness for actual clinical use., Competing Interests: All authors declare no competing interests., (© 2023 The Authors.)

Purpose: To examine the 6-year incidence of visual impairment (VI) and identify risk factors associated with VI in a multiethnic Asian population., Design: Prospective, population-based, cohort study., Participants: Adults aged ≥ 40 years were recruited from the Singapore Epidemiology of Eye Diseases cohort study at baseline. Eligible subjects were re-examined after 6 years. Subjects included in the final analysis had a mean age of 56.1 ± 8.9 years, and 2801 (50.5%) were female., Methods: All participants underwent standardized examination and interviewer-administered questionnaire at baseline. Incidences were standardized to the Singapore Population Census 2010. A Poisson binomial regression model was used to evaluate the associations between baseline factors and incident presenting VI., Main Outcome Measures: Incident presenting VI was assessed at the 6-year follow-up visit. Visual impairment (presenting visual acuity < 20/40), low vision (presenting visual acuity < 20/40 but ≥ 20/200), and blindness (presenting visual acuity < 20/200) were defined based on United States definition., Results: A total of 5551 subjects (2188 Chinese, 1837 Indians, and 1526 Malays) were evaluated, of whom 514 developed incident presenting VI over 6 years. Malays had a higher incidence of low vision and blindness (13.0%; 0.6%) than Indians (7.0%; 0.1%) and Chinese (7.7%; 0.2%). Among Malay individuals with VI at baseline, 52.8% remained visually impaired after 6 years, which was considerably higher than Chinese (32.4%) and Indians (37.2%). Older age (per decade; relative risk [RR] = 1.59), a history of cardiovascular disease (RR = 1.38), current smoking (RR = 1.31), smaller housing type (1- to 2-room public flat; RR = 2.01), and no formal education (RR = 1.63) at baseline were associated with a higher risk of incident VI (all P  ≤ 0.027). Older age (> 60 years) contributed the highest population attributable risk to incident VI (27.1%), followed by lower monthly income (Singapore dollar < $2000; 26.4%) and smaller housing type (24.7%). Overall, undercorrected refractive error (49.1%) and cataract (82.6%) were leading causes for low vision and blindness, respectively. This was consistently observed across the 3 ethnicities., Conclusions: In this multiethnic Asian population, Malays had a higher VI incidence compared to Indians and Chinese. Leading causes of VI are mostly treatable, suggesting that more efforts are needed to further mitigate preventable visual loss., Financial Disclosures: The authors have no proprietary or commercial interest in any materials discussed in this article., (© 2023 by the American Academy of Ophthalmology.)

Background: Large language models (LLMs) are garnering wide interest due to their human-like and contextually relevant responses. However, LLMs' accuracy across specific medical domains has yet been thoroughly evaluated. Myopia is a frequent topic which patients and parents commonly seek information online. Our study evaluated the performance of three LLMs namely ChatGPT-3.5, ChatGPT-4.0, and Google Bard, in delivering accurate responses to common myopia-related queries., Methods: We curated thirty-one commonly asked myopia care-related questions, which were categorised into six domains-pathogenesis, risk factors, clinical presentation, diagnosis, treatment and prevention, and prognosis. Each question was posed to the LLMs, and their responses were independently graded by three consultant-level paediatric ophthalmologists on a three-point accuracy scale (poor, borderline, good). A majority consensus approach was used to determine the final rating for each response. 'Good' rated responses were further evaluated for comprehensiveness on a five-point scale. Conversely, 'poor' rated responses were further prompted for self-correction and then re-evaluated for accuracy., Findings: ChatGPT-4.0 demonstrated superior accuracy, with 80.6% of responses rated as 'good', compared to 61.3% in ChatGPT-3.5 and 54.8% in Google Bard (Pearson's chi-squared test, all p ≤ 0.009). All three LLM-Chatbots showed high mean comprehensiveness scores (Google Bard: 4.35; ChatGPT-4.0: 4.23; ChatGPT-3.5: 4.11, out of a maximum score of 5). All LLM-Chatbots also demonstrated substantial self-correction capabilities: 66.7% (2 in 3) of ChatGPT-4.0's, 40% (2 in 5) of ChatGPT-3.5's, and 60% (3 in 5) of Google Bard's responses improved after self-correction. The LLM-Chatbots performed consistently across domains, except for 'treatment and prevention'. However, ChatGPT-4.0 still performed superiorly in this domain, receiving 70% 'good' ratings, compared to 40% in ChatGPT-3.5 and 45% in Google Bard (Pearson's chi-squared test, all p ≤ 0.001)., Interpretation: Our findings underscore the potential of LLMs, particularly ChatGPT-4.0, for delivering accurate and comprehensive responses to myopia-related queries. Continuous strategies and evaluations to improve LLMs' accuracy remain crucial., Funding: Dr Yih-Chung Tham was supported by the National Medical Research Council of Singapore (NMRC/MOH/HCSAINV21nov-0001)., Competing Interests: Declaration of interests All authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Competing Interests: None

Placenta accreta spectrum (PAS) described the anchoring placental villi attached or penetrating into/through the myometrium. PAS is clinically important because of the unpredictable bleeding amount when manually removing the defective decidualization at the endometrial-myometrial interface. Therefore, a multidisciplinary strategy for cesarean delivery with PAS is crucial. Postoperative embolization after cesarean hysterectomy in a hybrid suite was studied by many scientists. In this study, we demonstrated two cases of intraoperative embolization without hysterectomy in a hybrid operating room for cesarean delivery with placenta accreta. Our results show that intraoperative uterine artery embolization with a hybrid suite is a time-preserving and safe method for cesarean delivery with PAS owing to avoiding the risk of morbidity and mortality during patient transfer.

Objectives: To determine the risk of having OSA in a cohort of female subjects who are infertile and the odds of being infertile in women with OSA., Patients and Methods: A nationwide, case-control study of female patients 20 years or older diagnosed with female infertility living in Taiwan, from January 1, 2000, through December 31, 2013 (N = 4,078). We identified women who were infertile and created a 2:1 matched control group with women who were not infertile. We used multivariable logistic regression analysis to further estimate the effects of OSA on female infertility., Results: In this 14- year retrospective study, we included 4,078 patients having an initial diagnosis of female infertility. Of those women with infertility, 1.38% had a history of OSA compared with 0.63% of fertile controls (p = 0.002). The mean ages in the study groups were 32.19 ± 6.20 years, whereas the mean ages in the control groups were 32.24 ± 6.37years. Women with OSA had 2.101- times the risk of female infertility compared to women without OSA (p<0.001)., Conclusion: Our study showed that OSA is more commonly seen in infertile women and increases the odds that a woman will be infertile. More studies need to be done on the whether or not diagnosing and treating OSA can decrease the rate of infertility., Competing Interests: The authors have declared that no competing interests exist.

Abstract: Non-apnea sleep disorder (NASD) increases the risk of motor vehicle accidents. However, systemic review of NASD and its risk for all causes of injury is lacking. The aim of the present study was to provide a detailed demographic data on NASD and all causes of injury in a 14-year follow up.Our study utilized outpatient and inpatient data from the Longitudinal Health Insurance Database between 2000 and 2013 in Taiwan. We enrolled 989,753 individuals aged ≥20 years who were diagnosed with NASD as outpatients ≥3 times or inpatients ≥1 time. We matched the study cohort with a comparison cohort by age, index date and comorbidities at a ratio of 1:4. We used Cox proportional hazards regression to analyze the association of NASD and the cause of injury.In this 14-year follow up study, patients with NASD had 12.96% increased risk of injury compared to that of the control cohort. Fall was the first place of the cause of injury with 670.26 per 105 PYs. In the stratified age group, patients aged ≧65 years had the highest risk of injury (adjusted HR= 1.381; P < .001). Kaplan-Meier analysis showed that the incidence of injury between the with- and without-NASD cohorts started from the first year and persisted until the end of the follow-up.Our study demonstrates that NASD patients were associated with higher risk of all causes of injuries, with falling being the most prevalent diagnosis. The general public should be more aware of this neglected issue of NASD., Competing Interests: The authors have no conflicts of interests to disclose., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

Posttranscriptional gene regulation by Adenylate Uridylate (AU) rich element RNA binding protein, Elavl1 has been implicated in embryonic development as well as progenitor cell differentiation. Elavl1 binds to hundreds of cellular messenger RNAs predominantly through interactions with AU-rich elements (AREs) found in the untranslated regions (UTRs) and functions by regulating their stability. Biological functions of Elavl1 during osteogenic differentiation of bone marrow derived mesenchymal stem cells is not well-understood. Here we report that specific knockdown of nuclear localized Elavl1 by RNA interference in multipotent BMSCs led to increased osteogenic differentiation. Differential gene expression analysis following unbiased total RNA sequencing upon Elavl1 depletion during osteogenic differentiation of BMSCs showed increased levels of multiple mRNAs that are involved in extracellular matrix organization. We further show that many of these mRNAs contain Elavl1 binding consensus motifs that are preserved in their 3' UTRs. RNA stability analyses indicated that depletion of Elavl1 prolongs the steady state RNA levels of several of these mRNAs. Together, our data points to Elavl1 mediated negative regulation of multiple genes involved in ECM organization that play a functional role in MSC osteogenic differentiation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kota, Lim and Kota.)

We evaluated the 6-year incidence and risk factors of pterygium in a multi-ethnic Asian population. Participants who attended the baseline visit of the Singapore Epidemiology of Eye Diseases Study (year 2004-2011) and returned six years later, were included in this study. Pterygium was diagnosed based on anterior segment photographs. Incident pterygium was defined as presence of pterygium at 6-year follow-up in either eye, among individuals without pterygium at baseline. Multivariable logistic regression models were used to determine factors associated with incident pterygium, adjusting for baseline age, gender, ethnicity, body mass index, occupation type, educational level, income status, smoking, alcohol consumption, presence of hypertension, diabetes and hyperlipidemia. The overall age-adjusted 6-year incidence of pterygium was 1.2% (95% confidence interval [CI] 1.0-1.6%); with Chinese (1.9%; 95% CI 1.4%-2.5%) having the highest incidence rate followed by Malays (1.4%; 95% CI 0.9%-2.1%) and Indians (0.3%; 95% CI 0.3-0.7%). In multivariable analysis, Chinese (compared with Indians; odds ratio [OR] = 4.21; 95% CI 2.12-9.35) and Malays (OR 3.22; 95% CI 1.52-7.45), male (OR 2.13; 95% CI 1.26-3.63), outdoor occupation (OR 2.33; 95% CI 1.16-4.38), and smoking (OR 0.41; 95% CI 0.16-0.87) were significantly associated with incident pterygium. Findings from this multi-ethnic Asian population provide useful information in identifying at-risk individuals for pterygium.

Human skin is a self-healing mechanosensory system that detects various mechanical contact forces efficiently through three-dimensional innervations. Here, we propose a biomimetic artificially innervated foam by embedding three-dimensional electrodes within a new low-modulus self-healing foam material. The foam material is synthesized from a one-step self-foaming process. By tuning the concentration of conductive metal particles in the foam at near-percolation, we demonstrate that it can operate as a piezo-impedance sensor in both piezoresistive and piezocapacitive sensing modes without the need for an encapsulation layer. The sensor is sensitive to an object's contact force directions as well as to human proximity. Moreover, the foam material self-heals autonomously with immediate function restoration despite mechanical damage. It further recovers from mechanical bifurcations with gentle heating (70 °C). We anticipate that this material will be useful as damage robust human-machine interfaces.

Liver cancer is an aggressive malignancy associated with high levels of mortality and morbidity. Doxorubicin (Dox) is often used to slow down liver cancer progression; however its efficacy is limited, and its severe side effects prevent its routine use at therapeutic concentrations. We present a biomimetic peptide that coacervates into micro-droplets, within which both Dox and magnetic nanoparticles (MNPs) can be sequestered. These Dox-loaded Magnetic Coacervates (DMCs) can be used for thermo-chemotherapy, with the controlled release of Dox triggered by an external Alternating Magnetic Field (AMF). The DMCs are internalized by the cells via an energy-independent mechanism which is not based on endocytosis. Application of AMF generates a temperature of 45 °C within the DMCs, triggering their disassembly and the simultaneous release of Dox, thereby resulting in dual hyperthermia and chemotherapy for more efficient cancer therapy. In vitro studies conducted under AMF reveal that DMCs are cytocompatible and effective in inducing HepG2 liver cancer cell death. Thermo-chemotherapy treatment against HepG2 cells is also shown to be more effective compared to either hyperthermia or chemotherapy treatments alone. Thus, our novel peptide DMCs can open avenues in theranostic strategies against liver cancer through programmable, wireless, and remote control of Dox release. STATEMENT OF SIGNIFICANCE: Simultaneous administration of chemical and thermal therapy (thermo-chemotherapy) is more effective in inducing liver cancer cell death and improving survival rate. Thus, there is a keen interest in developing suitable carriers for thermo-chemotherapy. Coacervate micro-droplets display significant advantages, including high loading capacity, fast self-assembly in aqueous environments, and liquid-like behavior. However, they have not yet been explored as carriers for thermo-chemotherapy. Here, we demonstrate that peptide coacervate micro-droplets can co-encapsulate Dox and magnetic nanoparticles and cross the cell membrane. Applying an alternating magnetic field to cells containing drug-loaded coacervates triggers the release of Dox as well as the localized heating by magnetic hyperthermia, resulting in efficient liver cancer cell death by dual thermo-chemotherapy., Competing Interests: Declaration of Competing Interest ZWL and AM have submitted a US Provisional patent describing DgHBP-2 peptide used in this study and biomedical applications thereof., (Copyright © 2020 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Aims: A robust inflammatory response to tissue injury is a necessary part of the repair process but the deposition of scar tissue is a direct downstream consequence of this response in many tissues including the heart. Adult zebrafish not only possess the capacity to regenerate lost cardiomyocytes but also to remodel and resolve an extracellular scar within tissues such as the heart, but this scar resolution process remains poorly understood. This study aims to characterize the scarring and inflammatory responses to cardiac damage in adult zebrafish in full and investigate the role of different inflammatory subsets specifically in scarring and scar removal., Methods and Results: Using stable transgenic lines, whole organ imaging and genetic and pharmacological interventions, we demonstrate that multiple inflammatory cell lineages respond to cardiac injury in adult zebrafish. In particular, macrophage subsets (tnfα+ and tnfα-) play prominent roles with manipulation of different phenotypes suggesting that pro-inflammatory (tnfα+) macrophages promote scar deposition following cardiac injury whereas tnfα- macrophages facilitate scar removal during regeneration. Detailed analysis of these specific macrophage subsets reveals crucial roles for Csf1ra in promoting pro-inflammatory macrophage-mediated scar deposition. Additionally, the multifunctional cytokine Osteopontin (Opn) (spp1) is important for initial scar deposition but also for resolution of the inflammatory response and in late-stage ventricular collagen remodelling., Conclusions: This study demonstrates the importance of a correctly balanced inflammatory response to facilitate scar deposition during repair but also to allow subsequent scar resolution, and full cardiac regeneration, to occur. We have identified Opn as having both pro-fibrotic but also potentially pro-regenerative roles in the adult zebrafish heart, driving Collagen deposition but also controlling inflammatory cell resolution., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Importance: With the rapidly aging population, the burden of visual impairment (VI) and cognitive decline is expected to increase. Previous cross-sectional studies suggest an association between these 2 health outcomes. However, few longitudinal reports have examined this association, and to our knowledge, no studies have been performed in Asian populations. Further investigation on this association may help to better identify individuals at risk of cognitive decline., Objective: To examine the longitudinal association between VI and decline in cognitive function in a multiethnic Asian population., Design, Setting, and Participants: In this longitudinal, population-based, prospective cohort study, Chinese, Indian, and Malay adults 60 years or older at baseline were recruited from the Singapore Epidemiology of Eye Diseases (SEED) study. At baseline, participants from the SEED study were recruited under 3 studies: the Singapore Malay Eye Study (SiMES; 2004-2006), the Singapore Indian Eye Study (SINDI; 2007-2009), and the Singapore Chinese Eye Study (SCES; 2009-2011). Eligible participants were reexamined after 6 years (2011-2013 for SiMES, 2013-2015 for SINDI, and 2015-2017 for SCES). Data analysis was performed from November 1 to 24, 2019., Exposures: Visual impariment was defined as presenting visual acuity worse than 20/40 based on the better-seeing eye., Main Outcomes and Measures: Cognitive function was assessed using a locally validated Abbreviated Mental Test (AMT). The association between baseline VI and change in AMT score was determined using the multivariable linear regression model adjusting for baseline age; sex; race/ethnicity; presence of diabetes, hyperlipidemia, hypertension, and chronic kidney disease; history of cardiovascular disease; smoking status; alcohol intake; body mass index; educational status; and AMT score., Results: A total of 2478 individuals (1256 [50.7%] male; 1073 Chinese, 768 Indian, and 637 Malay adults) with mean (SD) age of 67.6 (5.6) years were evaluated, of whom 489 (19.7%) had reduction in AMT scores over 6 years. Baseline VI was associated with a decrease in AMT score over 6 years (β = -0.27; 95% CI, -0.37 to -0.17; P < .001). When change in vision over 6 years was evaluated, unchanged or deteriorated VI was associated with a decrease in AMT score over 6 years (β = -0.29; 95% CI, -0.40 to -0.18; P < .001). Among individuals with baseline VI and a substantial decrease in AMT score of 3 units or more over 6 years, the leading causes of VI were undercorrected refractive error (14 [45.2%]) and cataract (11 [35.5%])., Conclusions and Relevance: In this study, poor vision was independently associated with a decline in cognitive function. Causes of visual loss in these cases were mostly preventable, further suggesting that preserving good vision may be an important interventional strategy for mitigating cognitive decline.

Deep learning (DL) has been shown to be effective in developing diabetic retinopathy (DR) algorithms, possibly tackling financial and manpower challenges hindering implementation of DR screening. However, our systematic review of the literature reveals few studies studied the impact of different factors on these DL algorithms, that are important for clinical deployment in real-world settings. Using 455,491 retinal images, we evaluated two technical and three image-related factors in detection of referable DR. For technical factors, the performances of four DL models (VGGNet, ResNet, DenseNet, Ensemble) and two computational frameworks (Caffe, TensorFlow) were evaluated while for image-related factors, we evaluated image compression levels (reducing image size, 350, 300, 250, 200, 150 KB), number of fields (7-field, 2-field, 1-field) and media clarity (pseudophakic vs phakic). In detection of referable DR, four DL models showed comparable diagnostic performance (AUC 0.936-0.944). To develop the VGGNet model, two computational frameworks had similar AUC (0.936). The DL performance dropped when image size decreased below 250 KB (AUC 0.936, 0.900, p  < 0.001). The DL performance performed better when there were increased number of fields (dataset 1: 2-field vs 1-field-AUC 0.936 vs 0.908, p  < 0.001; dataset 2: 7-field vs 2-field vs 1-field, AUC 0.949 vs 0.911 vs 0.895). DL performed better in the pseudophakic than phakic eyes (AUC 0.918 vs 0.833, p  < 0.001). Various image-related factors play more significant roles than technical factors in determining the diagnostic performance, suggesting the importance of having robust training and testing datasets for DL training and deployment in the real-world settings., Competing Interests: Competing interestsD.S.W.T., G.L., M.L.L., W.H., and T.Y.W. are co-inventors of a patent on the deep learning system in this paper; potential conflicts of interests are managed according to institutional policies of the Singapore Health System (SingHealth) and the National University of Singapore. M.Y., Z.L., Q.N., C.C., M.Y., V.B., Y.X., X.L., H.H., J.H., T.T., C.S., A.G., and G.T. have no conflicts of interests to disclose., (© The Author(s) 2020.)

Hearing loss becomes increasingly common with age and affects quality of life. Recently, scientists have published articles about the relationship between metabolic disease and hearing loss. Metabolic disease was previously found to be highly related to an increase in alkaline phosphatase. Thus, there may be an indirect relationship between alkaline phosphatase (ALP) and hearing loss. In this paper, we will demonstrate the relationship between ALP and hearing loss. We included 3877 National Health and Nutrition Examination Survey (NHANES) participants, who represent the noninstitutionalized civilian population in the United States from age 20 to age 69, and examined the association between ALP and frequency distributions of pure-tone air-condition (PTAC) thresholds. After adjusting for pertinent variables, the subjects who belonged to the higher quartiles of ALP tended to have worse hearing thresholds (pure tone average at high and low frequencies) than the first quartile of ALP (p < 0.001). The results showed a positive correlation between ALP and hearing loss, in both males and females (p < 0.001) and in subjects whose body mass indices (BMI) were less than 30 (p < 0.001). In conclusion, ALP may play a role in detecting hearing loss.

The rising popularity of artificial intelligence (AI) in ophthalmology is fuelled by the ever-increasing clinical "big data" that can be used for algorithm development. Cataract is one of the leading causes of visual impairment worldwide. However, compared with other major age-related eye diseases, such as diabetic retinopathy, age-related macular degeneration, and glaucoma, AI development in the domain of cataract is still relatively underexplored. In this regard, several previous studies explored algorithms for automated cataract assessment using either slit lamp of color fundus photographs. However, several other study groups proposed or derived new AI-based calculation for pre-cataract surgery intraocular lens power. Along with advancements in digitization of clinical data, data curation for future cataract-related AI developmental work is bound to undergo significant improvements in the foreseeable future. Even though most of these previous studies reported early promising performances, limitations such as lack of robust, high-quality training data, and lack of external validations remain. In the next phase of work, apart from algorithm's performance, it will also be pertinent to evaluate deployment angles, feasibility, efficiency, and cost-effectiveness of these new cataract-related AI systems.

Statins inhibiting 3-hydroxy-3-methylglutaryl-CoA reductase are the standard treatment for hypercholesterolemia in atherosclerotic cardiovascular disease (ASCVD), mediated by inflammatory reactions within vessel walls. Several studies highlighted the pleiotropic effects of statins beyond their lipid-lowering properties. However, few studies investigated the effects of statins on T cell activation. This study evaluated the immunomodulatory capacities of three common statins, pitavastatin, atorvastatin, and rosuvastatin, in activated human T cells. The enzyme-linked immunosorbent assay (ELISA) and quantitative real time polymerase chain reaction (qRT-PCR) results demonstrated stronger inhibitory effects of pitavastatin on the cytokine production of T cells activated by phorbol 12-myristate 13-acetate (PMA) plus ionomycin, including interleukin (IL)-2, interferon (IFN)-γ, IL-6, and tumor necrosis factor α (TNF-α). Molecular investigations revealed that pitavastatin reduced both activating protein-1 (AP-1) DNA binding and transcriptional activities. Further exploration showed the selectively inhibitory effect of pitavastatin on the signaling pathways of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK), but not c-Jun N-terminal kinase (JNK). Our findings suggested that pitavastatin might provide additional benefits for treating hypercholesterolemia and ASCVD through its potent immunomodulatory effects on the suppression of ERK/p38/AP-1 signaling in human T cells.

Background: Radical measures are required to identify and reduce blindness due to diabetes to achieve the Sustainable Development Goals by 2030. Therefore, we evaluated the accuracy of an artificial intelligence (AI) model using deep learning in a population-based diabetic retinopathy screening programme in Zambia, a lower-middle-income country., Methods: We adopted an ensemble AI model consisting of a combination of two convolutional neural networks (an adapted VGGNet architecture and a residual neural network architecture) for classifying retinal colour fundus images. We trained our model on 76 370 retinal fundus images from 13 099 patients with diabetes who had participated in the Singapore Integrated Diabetic Retinopathy Program, between 2010 and 2013, which has been published previously. In this clinical validation study, we included all patients with a diagnosis of diabetes that attended a mobile screening unit in five urban centres in the Copperbelt province of Zambia from Feb 1 to June 31, 2012. In our model, referable diabetic retinopathy was defined as moderate non-proliferative diabetic retinopathy or worse, diabetic macular oedema, and ungradable images. Vision-threatening diabetic retinopathy comprised severe non-proliferative and proliferative diabetic retinopathy. We calculated the area under the curve (AUC), sensitivity, and specificity for referable diabetic retinopathy, and sensitivities of vision-threatening diabetic retinopathy and diabetic macular oedema compared with the grading by retinal specialists. We did a multivariate analysis for systemic risk factors and referable diabetic retinopathy between AI and human graders., Findings: A total of 4504 retinal fundus images from 3093 eyes of 1574 Zambians with diabetes were prospectively recruited. Referable diabetic retinopathy was found in 697 (22·5%) eyes, vision-threatening diabetic retinopathy in 171 (5·5%) eyes, and diabetic macular oedema in 249 (8·1%) eyes. The AUC of the AI system for referable diabetic retinopathy was 0·973 (95% CI 0·969-0·978), with corresponding sensitivity of 92·25% (90·10-94·12) and specificity of 89·04% (87·85-90·28). Vision-threatening diabetic retinopathy sensitivity was 99·42% (99·15-99·68) and diabetic macular oedema sensitivity was 97·19% (96·61-97·77). The AI model and human graders showed similar outcomes in referable diabetic retinopathy prevalence detection and systemic risk factors associations. Both the AI model and human graders identified longer duration of diabetes, higher level of glycated haemoglobin, and increased systolic blood pressure as risk factors associated with referable diabetic retinopathy., Interpretation: An AI system shows clinically acceptable performance in detecting referable diabetic retinopathy, vision-threatening diabetic retinopathy, and diabetic macular oedema in population-based diabetic retinopathy screening. This shows the potential application and adoption of such AI technology in an under-resourced African population to reduce the incidence of preventable blindness, even when the model is trained in a different population., Funding: National Medical Research Council Health Service Research Grant, Large Collaborative Grant, Ministry of Health, Singapore; the SingHealth Foundation; and the Tanoto Foundation., (Copyright © 2019 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

In any community, the key to understanding the burden of a specific condition is to conduct an epidemiological study. The deep learning system (DLS) recently showed promising diagnostic performance for diabetic retinopathy (DR). This study aims to use DLS as the grading tool, instead of human assessors, to determine the prevalence and the systemic cardiovascular risk factors for DR on fundus photographs, in patients with diabetes. This is a multi-ethnic (5 races), multi-site (8 datasets from Singapore, USA, Hong Kong, China and Australia), cross-sectional study involving 18,912 patients ( n  = 93,293 images). We compared these results and the time taken for DR assessment by DLS versus 17 human assessors - 10 retinal specialists/ophthalmologists and 7 professional graders). The estimation of DR prevalence between DLS and human assessors is comparable for any DR, referable DR and vision-threatening DR (VTDR) (Human assessors: 15.9, 6.5% and 4.1%; DLS: 16.1%, 6.4%, 3.7%). Both assessment methods identified similar risk factors (with comparable AUCs), including younger age, longer diabetes duration, increased HbA1c and systolic blood pressure, for any DR, referable DR and VTDR ( p  > 0.05). The total time taken for DLS to evaluate DR from 93,293 fundus photographs was ~1 month compared to 2 years for human assessors. In conclusion, the prevalence and systemic risk factors for DR in multi-ethnic population could be determined accurately using a DLS, in significantly less time than human assessors. This study highlights the potential use of AI for future epidemiology or clinical trials for DR grading in the global communities., Competing Interests: Competing interestsD.T., G.L., M.L.L., W.H., and T.Y.W. are co-inventors of a patent on the deep learning system in this paper; potential conflicts of interests are managed according to institutional policies of the Singapore Health System (SingHealth) and the National University of Singapore (NUS). The remaining authors declare no competing interests.

Seahorses have a specialized morphology that includes a toothless tubular mouth, a body covered with bony plates, a male brood pouch, and the absence of caudal and pelvic fins. Here we report the sequencing and de novo assembly of the genome of the tiger tail seahorse, Hippocampus comes. Comparative genomic analysis identifies higher protein and nucleotide evolutionary rates in H. comes compared with other teleost fish genomes. We identified an astacin metalloprotease gene family that has undergone expansion and is highly expressed in the male brood pouch. We also find that the H. comes genome lacks enamel matrix protein-coding proline/glutamine-rich secretory calcium-binding phosphoprotein genes, which might have led to the loss of mineralized teeth. tbx4, a regulator of hindlimb development, is also not found in H. comes genome. Knockout of tbx4 in zebrafish showed a 'pelvic fin-loss' phenotype similar to that of seahorses.

Cichlid fishes are famous for large, diverse and replicated adaptive radiations in the Great Lakes of East Africa. To understand the molecular mechanisms underlying cichlid phenotypic diversity, we sequenced the genomes and transcriptomes of five lineages of African cichlids: the Nile tilapia (Oreochromis niloticus), an ancestral lineage with low diversity; and four members of the East African lineage: Neolamprologus brichardi/pulcher (older radiation, Lake Tanganyika), Metriaclima zebra (recent radiation, Lake Malawi), Pundamilia nyererei (very recent radiation, Lake Victoria), and Astatotilapia burtoni (riverine species around Lake Tanganyika). We found an excess of gene duplications in the East African lineage compared to tilapia and other teleosts, an abundance of non-coding element divergence, accelerated coding sequence evolution, expression divergence associated with transposable element insertions, and regulation by novel microRNAs. In addition, we analysed sequence data from sixty individuals representing six closely related species from Lake Victoria, and show genome-wide diversifying selection on coding and regulatory variants, some of which were recruited from ancient polymorphisms. We conclude that a number of molecular mechanisms shaped East African cichlid genomes, and that amassing of standing variation during periods of relaxed purifying selection may have been important in facilitating subsequent evolutionary diversification.

The Runx family genes encode transcription factors that play key roles in hematopoiesis, skeletogenesis and neurogenesis and are often implicated in diseases. We describe here the cloning and characterization of Runx1, Runx2, Runx3 and Runxb genes in the elephant shark (Callorhinchus milii), a member of Chondrichthyes, the oldest living group of jawed vertebrates. Through the use of alternative promoters and/or alternative splicing, each of the elephant shark Runx genes expresses multiple isoforms similar to their orthologs in human and other bony vertebrates. The expression profiles of elephant shark Runx genes are similar to those of mammalian Runx genes. The syntenic blocks of genes at the elephant shark Runx gene loci are highly conserved in human, but represented by shorter conserved blocks in zebrafish indicating a higher degree of rearrangements in this teleost fish. Analysis of promoter regions revealed conservation of binding sites for transcription factors, including two tandem binding sites for Runx that are totally conserved in the distal promoter regions of elephant shark Runx1-3. Several conserved noncoding elements (CNEs), which are putative cis-regulatory elements, and miRNA binding sites were identified in the elephant shark and human Runx gene loci. Some of these CNEs and miRNA binding sites are absent in teleost fishes such as zebrafish and fugu. In summary, our analysis reveals that the genomic organization and expression profiles of Runx genes were already complex in the common ancestor of jawed vertebrates.

The emergence of jawed vertebrates (gnathostomes) from jawless vertebrates was accompanied by major morphological and physiological innovations, such as hinged jaws, paired fins and immunoglobulin-based adaptive immunity. Gnathostomes subsequently diverged into two groups, the cartilaginous fishes and the bony vertebrates. Here we report the whole-genome analysis of a cartilaginous fish, the elephant shark (Callorhinchus milii). We find that the C. milii genome is the slowest evolving of all known vertebrates, including the 'living fossil' coelacanth, and features extensive synteny conservation with tetrapod genomes, making it a good model for comparative analyses of gnathostome genomes. Our functional studies suggest that the lack of genes encoding secreted calcium-binding phosphoproteins in cartilaginous fishes explains the absence of bone in their endoskeleton. Furthermore, the adaptive immune system of cartilaginous fishes is unusual: it lacks the canonical CD4 co-receptor and most transcription factors, cytokines and cytokine receptors related to the CD4 lineage, despite the presence of polymorphic major histocompatibility complex class II molecules. It thus presents a new model for understanding the origin of adaptive immunity.