Binary decisions of artificial intelligence to classify third molar development around the legal age thresholds of 14, 16 and 18 years.

Third molar development is used for dental age estimation when all the other teeth are fully mature. In most medicolegal facilities, dental age estimation is an operator-dependent procedure. During the examination of unaccompanied and undocumented minors, this procedure may lead to binary decisions around age thresholds of legal interest, namely the ages of 14, 16 and 18 years. This study aimed to test the performance of artificial intelligence to classify individuals below and above the legal age thresholds of 14, 16 and 18 years using third molar development. The sample consisted of 11,640 panoramic radiographs (9680 used for training and 1960 used for validation) of males (n = 5400) and females (n = 6240) between 6 and 22.9 years. Computer-based image annotation was performed with V7 software (V7labs, London, UK). The region of interest was the mandibular left third molar (T38) outlined with a semi-automated contour. DenseNet121 was the Convolutional Neural Network (CNN) of choice and was used with Transfer Learning. After Receiver-operating characteristic curves, the area under the curve (AUC) was 0.87 and 0.86 to classify males and females below and above the age of 14, respectively. For the age threshold of 16, the AUC values were 0.88 (males) and 0.83 (females), while for the age of 18, AUC were 0.94 (males) and 0.83 (females). Specificity rates were always between 0.80 and 0.92. Artificial intelligence was able to classify male and females below and above the legal age thresholds of 14, 16 and 18 years with high accuracy.

The axis of complement C1 and nucleolus in antinuclear autoimmunity.

Antinuclear autoantibodies (ANA) are heterogeneous self-reactive antibodies that target the chromatin network, the speckled, the nucleoli, and other nuclear regions. The immunological aberration for ANA production remains partially understood, but ANA are known to be pathogenic, especially, in systemic lupus erythematosus (SLE). Most SLE patients exhibit a highly polygenic disease involving multiple organs, but in rare complement C1q, C1r, or C1s deficiencies, the disease can become largely monogenic. Increasing evidence point to intrinsic autoimmunogenicity of the nuclei. Necrotic cells release fragmented chromatins as nucleosomes and the alarmin HMGB1 is associated with the nucleosomes to activate TLRs and confer anti-chromatin autoimmunogenecity. In speckled regions, the major ANA targets Sm/RNP and SSA/Ro contain snRNAs that confer autoimmunogenecity to Sm/RNP and SSA/Ro antigens. Recently, three GAR/RGG-containing alarmins have been identified in the nucleolus that helps explain its high autoimmunogenicity. Interestingly, C1q binds to the nucleoli exposed by necrotic cells to cause protease C1r and C1s activation. C1s cleaves HMGB1 to inactive its alarmin activity. C1 proteases also degrade many nucleolar autoantigens including nucleolin, a major GAR/RGG-containing autoantigen and alarmin. It appears that the different nuclear regions are intrinsically autoimmunogenic by containing autoantigens and alarmins. However, the extracellular complement C1 complex function to dampen nuclear autoimmunogenecity by degrading these nuclear proteins.

Diagnostic performance of convolutional neural networks for dental sexual dimorphism.

Convolutional neural networks (CNN) led to important solutions in the field of Computer Vision. More recently, forensic sciences benefited from the resources of artificial intelligence, especially in procedures that normally require operator-dependent steps. Forensic tools for sexual dimorphism based on morphological dental traits are available but have limited performance. This study aimed to test the application of a machine learning setup to distinguish females and males using dentomaxillofacial features from a radiographic dataset. The sample consisted of panoramic radiographs (n = 4003) of individuals in the age interval of 6 and 22.9 years. Image annotation was performed with V7 software (V7labs, London, UK). From Scratch (FS) and Transfer Learning (TL) CNN architectures were compared, and diagnostic accuracy tests were used. TL (82%) performed better than FS (71%). The correct classifications of females and males aged ≥ 15 years were 87% and 84%, respectively. For females and males < 15 years, the correct classifications were 80% and 83%, respectively. The Area Under the Curve (AUC) from Receiver-operating Characteristic (ROC) curves showed high classification accuracy between 0.87 and 0.91. The radio-diagnostic use of CNN for sexual dimorphism showed positive outcomes and promising forensic applications to the field of dental human identification.

Development of a Community-Based e-Health Program for Older Adults With Chronic Diseases: Pilot Pre-Post Study.

BACKGROUND: Chronic diseases may impact older adults' health outcomes, health care costs, and quality of life. Self-management is expected to encourage individuals to make autonomous decisions, adhere to treatment plans, deal with emotional and social consequences, and provide choices for healthy lifestyle. New eHealth solutions significantly increase the health literacy and empower patients in self-management of chronic conditions. OBJECTIVE: This study aims to develop a Community-Based e-Health Program (CeHP) for older adults with chronic diseases and conduct a pilot evaluation. METHODS: A pilot study with a 2-group pre- and posttest repeated measures design was adopted. Community-dwelling older adults with chronic diseases were recruited from senior activity centers in Singapore. A systematic 3-step process of developing CeHP was coupled with a smart-device application. The development of the CeHP intervention consists of theoretical framework, client-centric participatory action research process, content validity assessment, and pilot testing. Self-reported survey questionnaires and health outcomes were measured before and after the CeHP. The instruments used were the Self-care of Chronic Illness Inventory (SCCII), Healthy Aging Instrument (HAI), Short-Form Health Literacy Scale, 12 Items (HLS-SF 12), Patient Empowerment Scale (PES), and Social Support Questionnaire, 6 items. The following health outcomes were measured: Montreal Cognitive Assessment, Symbol Digit Modalities Test, total cholesterol (TC), high-density lipoproteins, low-density lipoproteins/very-low-density lipoproteins (LDL/VLDL), fasting glucose, glycated hemoglobin (HbA1c), and BMI. RESULTS: The CeHP consists of health education, monitoring, and an advisory system for older adults to manage their chronic conditions. It is an 8-week intensive program, including face-to-face and eHealth (Care4Senior App) sessions. Care4Senior App covers health education topics focusing on the management of hypertension, hyperlipidemia, and diabetes, brain health, healthy diet, lifestyle modification, medication adherence, exercise, and mindfulness practice. Content validity assessment indicated that the content of the CeHP is valid, with a content validity index (CVI) ranging 0.86-1 and a scale-CVI of 1. Eight participants in the CeHP group and 4 in the control group completed both baseline and post intervention assessments. Participants in the CeHP group showed improvements in fasting glucose, HbA1c, TC, LDL/VLDL, BMI, SCCII indices (Maintenance, Monitoring, and Management), HAI, and PES scores post intervention, although these changes were not significant. For the participants in the control group, the scores for SCCII (management and confidence) and HLS-SF 12 decreased post intervention. CONCLUSIONS: The CeHP is feasible, and it engages and empowers community-dwelling older adults to manage their chronic conditions. The rigorous process of program development and pilot evaluation provided valid evidence to expand the CeHP to a larger-scale implementation to encourage self-management, reduce debilitating complications of poorly controlled chronic diseases, promote healthy longevity and social support, and reduce health care costs.

The GAR/RGG motif defines a family of nuclear alarmins.

The nucleus is the target of autoantibodies in many diseases, which suggests intrinsic nuclear adjuvants that confer its high autoimmunogenicity. Nucleolin (NCL) is one abundant nucleolar autoantigen in systemic lupus erythematosus (SLE) patients and, in lupus-prone mice, it elicits autoantibodies early. With purified NCL, we observed that it was a potent alarmin that activated monocytes, macrophages and dendritic cells and it was a ligand for TLR2 and TLR4. NCL released by necrotic cells also exhibited alarmin activity. The NCL alarmin activity resides in its glycine/arginine-rich (GAR/RGG) motif and can be displayed by synthetic GAR/RGG peptides. Two more GAR/RGG-containing nucleolar proteins, fibrillarin (FBRL) and GAR1, were also confirmed to be novel alarmins. Therefore, the GAR/RGG alarmin motif predicts a family of nucleolar alarmins. The apparent prevalence of nucleolar alarmins suggests their positive contribution to tissue homeostasis by inducing self-limiting tissue inflammation with autoimmunity only occurring when surveillance is broken down.

The linker histone H1.2 is a novel component of the nucleolar organizer regions.

The nucleoli accumulate rRNA genes and are the sites of rRNA synthesis and rRNA assembly into ribosomes. During mitosis, nucleoli dissociate, but nucleolar remnants remain on the rRNA gene loci, forming distinct nucleolar organizer regions (NORs). Little is known about the composition and structure of NORs, but upstream binding factor (UBF) has been established as its master organizer. In this study, we sought to establish new proteins in NORs. Using UBF-Sepharose to isolate UBF-binding proteins, we identified histone H1.2 as a candidate partner but were puzzled by this observation, given that UBF is known to be located predominantly in nucleoli, whereas H1.2 distributed broadly among the chromatins in interphase nuclei. We then examined cells undergoing mitosis and saw that both H1.2 and UBF were recruited into NORs in this state, reconciling the results of our UBF pulldowns. Inhibiting rRNA synthesis in interphase nuclei also induced NOR-like structures containing both UBF and H1.2. When chromosomes were isolated and spread on coverslips, NORs appeared separated from the chromosomes containing both UBF and H1.2. After chromosomes were fragmented by homogenization, intact NORs remained visible. Results collectively suggest that NORs are independent structures and that the linker histone H1.2 is a novel component of this structure.

A Method for Extracting the Nuclear Scaffold from the Chromatin Network.

Each cell contains many large DNA polymers packed in a nucleus of approx. 10 µm in diameter. With histones, these DNA polymers are known to form chromatins. How chromatins further compact in the nucleus is unclear but it inevitably depends on an extensive non-chromatin nuclear scaffold. Imaging of endogenous chromatin network and the complementary scaffold that support this network has not been achieved but biochemical and proteomic investigations of the scaffold can still provide important insights into this chromatin-organizing network. However, this demands highly inclusive and reproducible extraction of the nuclear scaffold. We have recently developed a simple protocol for releasing the scaffold components from chromatins. The inclusiveness of the extract was testified by the observation that, upon its extraction from the nuclei, the remaining nuclear chromatins were liberated into extended and often parallel chromatin fibers. Basically, this protocol includes the generation of pure nuclei, treatment of the nuclei with Triton X-100 to generate envelope-depleted nuclei (TxN), and extraction of the nuclei at 500 mM NaCl in a sucrose-containing buffer. This combined extract of TxN is known as TxNE.

Broad Susceptibility of Nucleolar Proteins and Autoantigens to Complement C1 Protease Degradation.

Anti-nuclear autoantibodies, which frequently target the nucleoli, are pathogenic hallmarks of systemic lupus erythematosus (SLE). Although the causes of these Abs remain broad and ill-defined, a genetic deficiency in C1 complex (C1qC1r2C1s2) or C4 is able to induce these Abs. Considering a recent finding that, in dead cells, nucleoli were targeted by C1q and two nucleolar autoantigens were degraded by C1r/C1s proteases, we considered that C1 could help protect against antinuclear autoimmunity by broadly degrading nucleolar proteins or autoantigens. Nucleoli were isolated to homogeneity and structurally defined. After C1 treatment, cleaved nucleolar proteins were identified by proteomic two-dimensional fluorescence difference gel electrophoresis and mass spectrometry, and further verified by Western blotting using specific Abs. The extent of nucleolar autoantigen degradation upon C1 treatment was estimated using SLE patient autoantibodies. The isolated nucleoli were broadly reactive with SLE patient autoantibodies. These nucleoli lacked significant autoproteolysis, but many nucleolar proteins and autoantigens were degraded by C1 proteases; >20 nucleolar proteins were identified as C1 cleavable. These were further validated by Western blotting using specific Abs. The broad autoantigenicity of the nucleoli may attribute to their poor autoproteolysis, causing autologous immune stimulation upon necrotic exposure. However, C1q targets at these nucleoli to cause C1 protease activation and the cleavage of many nucleolar proteins or autoantigens. This may represent one important surveillance mechanism against antinuclear autoimmunity because C1 genetic deficiency causes anti-nuclear autoantibodies and SLE disease.

C1q protein binds to the apoptotic nucleolus and causes C1 protease degradation of nucleolar proteins.

In infection, complement C1q recognizes pathogen-congregated antibodies and elicits complement activation. Among endogenous ligands, C1q binds to DNA and apoptotic cells, but whether C1q binds to nuclear DNA in apoptotic cells remains to be investigated. With UV irradiation-induced apoptosis, C1q initially bound to peripheral cellular regions in early apoptotic cells. By 6 h, binding concentrated in the nuclei to the nucleolus but not the chromatins. When nucleoli were isolated from non-apoptotic cells, C1q also bound to these structures. In vivo, C1q exists as the C1 complex (C1qC1r2C1s2), and C1q binding to ligands activates the C1r/C1s proteases. Incubation of nucleoli with C1 caused degradation of the nucleolar proteins nucleolin and nucleophosmin 1. This was inhibited by the C1 inhibitor. The nucleoli are abundant with autoantigens. C1q binding and C1r/C1s degradation of nucleolar antigens during cell apoptosis potentially reduces autoimmunity. These findings help us to understand why genetic C1q and C1r/C1s deficiencies cause systemic lupus erythematosus.

Transcriptional Factor PU.1 Regulates Decidual C1q Expression in Early Pregnancy in Human.

C1q is the first recognition subcomponent of the complement classical pathway, which in addition to being synthesized in the liver, is also expressed by macrophages and dendritic cells (DCs). Trophoblast invasion during early placentation results in accumulation of debris that triggers the complement system. Hence, both early and late components of the classical pathway are widely distributed in the placenta and decidua. In addition, C1q has recently been shown to significantly contribute to feto-maternal tolerance, trophoblast migration, and spiral artery remodeling, although the exact mechanism remains unknown. Pregnancy in mice, genetically deficient in C1q, mirrors symptoms similar to that of human preeclampsia. Thus, regulated complement activation has been proposed as an essential requirement for normal successful pregnancy. Little is known about the molecular pathways that regulate C1q expression in pregnancy. PU.1, an Ets-family transcription factor, is required for the development of hematopoietic myeloid lineage immune cells, and its expression is tissue-specific. Recently, PU.1 has been shown to regulate C1q gene expression in DCs and macrophages. Here, we have examined if PU.1 transcription factor regulates decidual C1q expression. We used immune-histochemical analysis, PCR, and immunostaining to localize and study the gene expression of PU.1 transcription factor in early human decidua. PU.1 was highly expressed at gene and protein level in early human decidual cells including trophoblast and stromal cells. Surprisingly, nuclear as well as cytoplasmic PU.1 expression was observed. Decidual cells with predominantly nuclear PU.1 expression had higher C1q expression. It is likely that nuclear and cytoplasmic PU.1 localization has a role to play in early pregnancy via regulating C1q expression in the decidua during implantation.

Complement C1q production by osteoclasts and its regulation of osteoclast development.

C1q deficiency is the strongest known risk factor for SLE (systemic lupus erythematosus) but its endogenous cellular origin remains limitedly understood. In the present study we investigate the production of C1q by both cultured and endogenous bone osteoclasts. Blood monocytes were cultured with RANKL (receptor activator of nuclear factor κB ligand) and M-CSF (macrophage colony-stimulating factor) to generate osteoclasts and these cells expressed C1Q mRNA and also secreted C1q protein. Intracellular C1q was detectable in developing osteoclasts at day 3 by Western blotting and was also detectable by flow cytometry. By immunofluorescence microscopy, C1q was preferentially detected in immature osteoclasts. By multiple detection methods, C1q expression was markedly increased after IFNγ (interferon γ) treatment. By immunohistochemistry, C1q was also detected in endogenous bone osteoclasts. When osteoclasts were cultured on immobilized C1q, these cells exhibited 2-7-fold increases in the expression of signature osteoclast genes [TRAP (tartrate-resistant acid phosphatase), cathepsin K, calcitonin receptor, carbonic anhydrase II and NFATc1 (nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1)], suggesting an osteoclastogenic capability. This is the first report of C1q production by osteoclasts. Its ability to enhance osteoclast development implies reduced osteoclastogenesis in patients with SLE as they often experience decreased C1q levels. This is consistent with the non-erosive nature of lupus arthritis.

MHC class II-associated invariant chain (Ii) modulates dendritic cells-derived microvesicles (DCMV)-mediated activation of microglia.

Dendritic cells (DC) - the sentinels of the immune system - play an important role in the maintenance of tolerance and induction of immunity. However, in autoimmune diseases, DC initiate the diseases by presenting self antigens to autoreactive T cells, causing the immune system to mount a response against the body. An example is multiple sclerosis (MS) and its corresponding animal model, experimental autoimmune encephalomyelitis (EAE). During inflammation of the central nervous system (CNS), DC are recruited to activate autoreactive T cells. Microglia - resident mononuclear phagocytes of the brain - also play a role in the pathogenesis of the disease. In this study, we demonstrated that microvesicles derived from DC (DCMV) induced the activation of NF-κB in microglia. Furthermore, MHC class II-associated invariant chain (Ii), also known as CD74, was specifically recruited to DCMV and interestingly, was able to enhance the DCMV-mediated activation of NF-κB in microglia. Thus, this study emphasizes the role of microvesicles and Ii in the communication between DC and microglia.