Single-cell RNA sequencing data imputation using bi-level feature propagation.

Single-cell RNA sequencing (scRNA-seq) enables the exploration of cellular heterogeneity by analyzing gene expression profiles in complex tissues. However, scRNA-seq data often suffer from technical noise, dropout events and sparsity, hindering downstream analyses. Although existing works attempt to mitigate these issues by utilizing graph structures for data denoising, they involve the risk of propagating noise and fall short of fully leveraging the inherent data relationships, relying mainly on one of cell-cell or gene-gene associations and graphs constructed by initial noisy data. To this end, this study presents single-cell bilevel feature propagation (scBFP), two-step graph-based feature propagation method. It initially imputes zero values using non-zero values, ensuring that the imputation process does not affect the non-zero values due to dropout. Subsequently, it denoises the entire dataset by leveraging gene-gene and cell-cell relationships in the respective steps. Extensive experimental results on scRNA-seq data demonstrate the effectiveness of scBFP in various downstream tasks, uncovering valuable biological insights.

TLR5 agonist in combination with anti-PD-1 treatment enhances anti-tumor effect through M1/M2 macrophage polarization shift and CD8+ T cell priming.

Immune checkpoint inhibitors have revolutionized anti-tumor therapy, notably improving treatment responses in various tumors. However, many patients remain non-responsive and do not experience benefits. Given that Toll-like receptors (TLRs) can counteract tumor immune tolerance by stimulating both innate and adaptive immune responses, TLR agonists are being explored as potential immune adjuvants for cancer treatment. In this study, we assessed the potential of enhancing the efficacy of immune checkpoint inhibitors by activating innate immunity with a TLR5 agonist. In a mouse tumor model, combination therapy with TLR5 agonist and anti-PD-1 significantly inhibited tumor growth. The TLR5 agonist shifted the balance from M2-like to M1-like macrophages and upregulated the expression of co-stimulatory molecules in macrophages. Furthermore, TLR5 agonist promoted the activation and tumor infiltration of CD8+ T cells. As a result, the TLR5 agonist augmented the anti-tumor efficacy of anti-PD-1, suggesting its potential in modulating the tumor microenvironment to enhance the anti-tumor response. Our findings point toward the possibility of optimizing immune checkpoint inhibitor therapy using TLR5 agonists.

Multiplexable and Scalable Aqueous Synthesis Platform for Oleate-Based, Bilayer-Coated Gold Nanoparticles.

Despite gold-based nanomaterials having a unique role in nanomedicine, among other fields, synthesis limitations relating to reaction scale-up and control result in prohibitively high gold nanoparticle costs. In this work, a new preparation procedure for lipid bilayer-coated gold nanoparticles in water is presented, using sodium oleate as reductant and capping agent. The seed-free synthesis not only allows for size precision (8-30 nm) but also remarkable particle concentration (10 mm Au). These reaction efficiencies allow for multiplexing and reaction standardization in 96-well plates using conventional thermocyclers, in addition to simple particle purification via microcentrifugation. Such a multiplexing approach also enables detailed spectroscopic investigation of the nonlinear growth process and dynamic sodium oleate/oleic acid self-assembly. In addition to scalability (at gram-level), resulting gold nanoparticles are stable at physiological pH, in common cell culture media, and are autoclavable. To demonstrate the versatility and applicability of the reported method, a robust ligand exchange with thiolated polyethylene glycol analogues is also presented.

Deep single-cell RNA-seq data clustering with graph prototypical contrastive learning.

MOTIVATION: Single-cell RNA sequencing enables researchers to study cellular heterogeneity at single-cell level. To this end, identifying cell types of cells with clustering techniques becomes an important task for downstream analysis. However, challenges of scRNA-seq data such as pervasive dropout phenomena hinder obtaining robust clustering outputs. Although existing studies try to alleviate these problems, they fall short of fully leveraging the relationship information and mainly rely on reconstruction-based losses that highly depend on the data quality, which is sometimes noisy. RESULTS: This work proposes a graph-based prototypical contrastive learning method, named scGPCL. Specifically, scGPCL encodes the cell representations using Graph Neural Networks on cell-gene graph that captures the relational information inherent in scRNA-seq data and introduces prototypical contrastive learning to learn cell representations by pushing apart semantically dissimilar pairs and pulling together similar ones. Through extensive experiments on both simulated and real scRNA-seq data, we demonstrate the effectiveness and efficiency of scGPCL. AVAILABILITY AND IMPLEMENTATION: Code is available at https://github.com/Junseok0207/scGPCL.

HCC EV ECG score: An extracellular vesicle-based protein assay for detection of early-stage hepatocellular carcinoma.

BACKGROUND AND AIMS: The sensitivity of current surveillance methods for detecting early-stage hepatocellular carcinoma (HCC) is suboptimal. Extracellular vesicles (EVs) are promising circulating biomarkers for early cancer detection. In this study, we aim to develop an HCC EV-based surface protein assay for early detection of HCC. APPROACH AND RESULTS: Tissue microarray was used to evaluate four potential HCC-associated protein markers. An HCC EV surface protein assay, composed of covalent chemistry-mediated HCC EV purification and real-time immuno-polymerase chain reaction readouts, was developed and optimized for quantifying subpopulations of EVs. An HCC EV ECG score, calculated from the readouts of three HCC EV subpopulations ( E pCAM + CD63 + , C D147 + CD63 + , and G PC3 + CD63 + HCC EVs), was established for detecting early-stage HCC. A phase 2 biomarker study was conducted to evaluate the performance of ECG score in a training cohort ( n  = 106) and an independent validation cohort ( n  = 72).Overall, 99.7% of tissue microarray stained positive for at least one of the four HCC-associated protein markers (EpCAM, CD147, GPC3, and ASGPR1) that were subsequently validated in HCC EVs. In the training cohort, HCC EV ECG score demonstrated an area under the receiver operating curve (AUROC) of 0.95 (95% confidence interval [CI], 0.90-0.99) for distinguishing early-stage HCC from cirrhosis with a sensitivity of 91% and a specificity of 90%. The AUROCs of the HCC EV ECG score remained excellent in the validation cohort (0.93; 95% CI, 0.87-0.99) and in the subgroups by etiology (viral: 0.95; 95% CI, 0.90-1.00; nonviral: 0.94; 95% CI, 0.88-0.99). CONCLUSION: HCC EV ECG score demonstrated great potential for detecting early-stage HCC. It could augment current surveillance methods and improve patients' outcomes.

Multifunctional Photo-Cross-Linking Probes: From Target Protein Searching to Imaging Applications.

Despite advances in genome sequencing technology, the complete molecular interaction networks reflecting the biological functions of gene products have not been fully elucidated due to the lack of robust molecular interactome profiling techniques. Traditionally, molecular interactions have been investigated in vitro by measuring their affinity. However, such a reductionist approach comes with throughput constraints and does not depict an intact living cell environment. Therefore, molecular interactions in live cells must be captured to minimize false-positive results. The photo-cross-linking technique is a promising tool because the production of a temporally controlled reactive functional group can be induced using light exposure. Photoaffinity labeling is used in biochemistry and medicinal chemistry for bioconjugation, including drug and antibody conjugation, target protein identification of bioactive compounds, and fluorescent labeling of target proteins. This Account summarizes recent advances in multifunctional photo-cross-linkers for drug target identification and bioimaging. In addition to our group's contributions, we reviewed the most notable examples from the last few decades to provide a comprehensive overview of how this field is evolving. Based on cross-linking chemistry, photo-cross-linkers are classified as either (i) reactive intermediate-generating or (ii) electrophile-generating. Reactive intermediates generating photoaffinity tags have been extensively modified to target a molecule of interest using aryl azide, benzophenone, diazirine, diazo, and acyl silanes. These species are highly reactive and can form covalent bonds, irrespective of residue. Their short lifetime is ideal for the instant capture and labeling of biomolecules. Recently, photocaged electrophiles have been investigated to take advantage of their residue selectivity and relatively high yield for adduct formation with tetrazole, nitrobenzyl alcohol, o-nitrophenylethylene, pyrone, and pyrimidone. Multifunctional photo-cross-linkers for two parallel practical applications have been developed using both classes of photoactivatable groups. Unbiased target interactome profiling of small-molecule drugs requires a challenging structure-activity relationship study (SAR) step to retain the nature or biological activity of the lead compound, which led to the design of a multifunctional "minimalist tag" comprising a bio-orthogonal handle, a photoaffinity labeling group, and functional groups to load target molecules. In contrast, fluorogenic photo-cross-linking is advantageous for bioimaging because it does not require an additional bio-orthogonal reaction to introduce a fluorophore to the minimalist tag. Our group has made progress on minimalist tags and fluorogenic photo-cross-linkers through fruitful collaborations with other groups. The current range of photoactivation reactions and applications demonstrate that photoaffinity tags can be improved. We expect exciting days in the rational design of new multifunctional photo-cross-linkers, particularly clinically interesting versions used in photodynamic or photothermal therapy.

Differential Sensing Approach as a Pattern-based Discrimination for Biological Samples.

The differential sensing approach uses fingerprint patterning to distinguish uncharacterized biological samples. Inspired by natural sensory systems, an array of cross-reactive sensors generates unique response fingerprint depending on the samples. Until today, this array system has been developed using various materials, including the library of surface-charged nanoparticles and chemosensors. Many differential array systems have demonstrated accurate identification of bacterial species, viral subtypes, and cancer cells, as well as distinguishing disease states in blood or urine. This capability is particularly important for distinguishing between normal and abnormal states when specific marker molecules have not yet been identified, providing a powerful diagnostic tool. In this concept, we summarized representative outcomes of differential sensing applications for biological sample discrimination.

Fluorescent Phenotyping of Blood Cells Using a Differential Sensing Strategy: Differentiating Physiological Aging Stages and Neuro-Degenerative Disease Drugs.

Blood continually contributes to the maintenance of homeostasis of the body and contains information regarding the health state of an individual. However, current hematological analyses predominantly rely on a limited number of CD markers and morphological analysis. In this work, differentially sensitive fluorescent compounds based on TCF scaffolds are introduced that are designed for fluorescent phenotyping of blood. Depending on their structures, TCF compounds displayed varied responses to reactive oxygen species, biothiols, redox-related biomolecules, and hemoglobin, which are the primary influential factors within blood. Contrary to conventional CD marker-based analysis, this unbiased fluorescent phenotyping method produces diverse fingerprints of the health state. Precise discrimination of blood samples from 37 mice was demonstrated based on their developmental stages, ranging from 10 to 19 weeks of age. Additionally, this fluorescent phenotyping method enabled the differentiation between drugs with distinct targets, serving as a simple yet potent tool for pharmacological analysis to understand the mode of action of various drugs.

Risk compensation after COVID-19 vaccination: Evidence from vaccine rollout by exact birth date in South Korea.

We utilize the phased rollout of COVID-19 vaccines by exact birth date in South Korea as a natural experiment for testing risk compensation. People may resume face-to-face social activities following vaccination because they perceive lower risk of infection. Applying a regression discontinuity design based on birth date cutoffs for vaccine eligibility, we find no evidence of risk-compensating behaviors, as measured by large, high-frequency data from credit card and airline companies as well as survey data. We find some evidence of self-selection into vaccine take-up based on perception toward vaccine effectiveness and side effects, but the treatment effects do not differ between compliers and never-takers.

Mesenchymal stem cells preconditioned with a TLR5 agonist enhanced immunoregulatory effect through M2 macrophage polarization in a murine graft-versus-host disease model.

Graft-versus-host disease (GVHD) is a common complication following hematopoietic stem cell transplantation and can be life-threatening. Mesenchymal stem cells (MSCs), adult stem cells with immunomodulatory properties, have been used as therapeutic agents in a variety of ways and have demonstrated efficacy against acute GVHD (aGVHD); however, variability in MSC pro- and anti-inflammatory properties and the limitation that they only exhibit immunosuppressive effects at high levels of inflammation have prevented their widespread clinical use. The outcomes of GVHD treated with MSCs in the clinic have been variable, and the underlying mechanisms remain unclear. Therefore, the unique biological effects of Toll-like receptor 5 (TLR5) agonists led us to compare and validate the efficacy of MSCs primed with KMRC011, a TLR5 agonist. KMRC011 is a stimulant that induces the secretion of cytokines, which play an important role in immune regulation. In this study, we found that MSCs pretreated with KMRC011 increased the secretion of immunosuppressive cytokines indoleamine 2,3-dioxygenase (IDO) and cyclooxygenase-2 (COX2) and increased the expression of M2 macrophage polarizing cytokines macrophage colony-stimulating factor (M-CSF) and interleukin 10 (IL-10) in vitro. We investigated the immunosuppressive effects of TLR5 agonist (KMRC011)-primed MSCs on lymphocytes and their preventive and therapeutic effects on an in vivo mouse aGVHD model. In vitro experiments showed that KMRC011-primed MSCs had enhanced immunosuppressive effects on lymphocyte proliferation. In vivo experiments showed that KMRC011-primed MSCs ameliorated GVHD severity in a mouse model of induced GVHD disease. Finally, macrophages harvested from the spleens of mice treated with KMRC011-primed MSCs showed a significant increase in the anti-inflammatory M2 phenotype. Overall, the results suggest that KMRC011-primed MSCs attenuated GVHD severity in mice by polarizing macrophages to the M2 phenotype and increasing the proportion of anti-inflammatory cells, opening new horizons for GVHD treatment.

Repeated Infusions of Bone-Marrow-Derived Mesenchymal Stem Cells over 8 Weeks for Steroid-Refractory Chronic Graft-versus-Host Disease: A Prospective, Phase I/II Clinical Study.

Chronic graft-versus-host disease (cGVHD) is a long-term complication of allogeneic hematopoietic stem cell transplantation associated with poor quality of life and increased morbidity and mortality. Currently, there are several approved treatments for patients who do not respond to steroids, such as ruxolitinib. Nevertheless, a significant proportion of patients fail second-line treatment, indicating the need for novel approaches. Mesenchymal stem cells (MSCs) have been considered a potential treatment approach for steroid-refractory cGVHD. To evaluate the safety and efficacy of repeated infusions of MSCs, we administered intravenous MSCs every two weeks to ten patients with severe steroid-refractory cGVHD in a prospective phase I clinical trial. Each patient received a total of four doses, with each dose containing 1 × 106 cells/kg body weight from the same donor and same passage. Patients were assessed for their response to treatment using the 2014 National Institutes of Health (NIH) response criteria during each visit. Ten patients with diverse organ involvement were enrolled, collectively undergoing 40 infusions as planned. Remarkably, the MSC infusions were well tolerated without severe adverse events. Eight weeks after the initial MSC infusion, all ten patients showed partial responses characterized by the amelioration of clinical symptoms and enhancement of their quality of life. The overall response rate was 60%, with a complete response rate of 20% and a partial response (PR) rate of 40% at the last follow-up. Overall survival was 80%, with a median follow-up of 381 days. Two patients died due to relapse of their primary disease. Immunological analyses revealed a reduction in inflammatory markers, including Suppression of Tumorigenicity 2 (ST2), C-X-C motif chemokine ligand (CXCL)10, and Secreted phosphoprotein 1(SPP1), following the MSC treatment. Repeated MSC infusions proved to be both feasible and safe, and they may be an effective salvage therapy in patients with steroid-refractory cGVHD. Further large-scale clinical studies with long-term follow-up are needed in the future to determine the role of MSCs in cGVHD.

SARS-CoV-2-Specific T-Cell as a Potent Therapeutic Strategy against Immune Evasion of Emerging COVID-19 Variants.

Despite advances in vaccination and therapies for coronavirus disease, challenges remain due to reduced antibody longevity and the emergence of virulent variants like Omicron (BA.1) and its subvariants (BA.1.1, BA.2, BA.3, and BA.5). This study explored the potential of adoptive immunotherapy and harnessing the protective abilities using virus-specific T cells (VSTs). Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) VSTs were generated by stimulating donor-derived peripheral blood mononuclear cells with spike, nucleocapsid, and membrane protein peptide mixtures. Phenotypic characterization, including T-cell receptor (TCR) vß and pentamer analyses, was performed on the ex vivo-expanded cells. We infected human leukocyte antigen (HLA)-partially matched human Calu-3 cells with various authentic SARS-CoV-2 strains in a Biosafety Level 3 facility and co-cultured them with VSTs. VSTs exhibited a diverse TCR vß repertoire, confirming their ability to target a broad range of SARS-CoV-2 antigens from both the ancestral and mutant strains, including Omicron BA.1 and BA.5. These ex vivo-expanded cells exhibited robust cytotoxicity and low alloreactivity against HLA-partially matched SARS-CoV-2-infected cells. Their cytotoxic effects were consistent across variants, targeting conserved spike and nucleocapsid epitopes. Our findings suggest that third-party partial HLA-matching VSTs could counter immune-escape mechanisms posed by emerging variants of concern.

Disaggregation-Activated pan-COX Imaging Agents for Human Soft tissue Sarcoma.

Cancer stem cells are pivotal players in tumors initiation, growth, and metastasis. While several markers have been identified, there remain challenges particularly in heterogeneous malignancies like adult soft tissue sarcomas, where conventional markers are inherently overexpressed. Here, we designed BODIPY scaffold fluorescence probes (BD-IMC-1, BD-IMC-2) that activate via disaggregation targeting for cyclooxygenase (COX), a potential marker for CSCs in sarcoma in clinical pathology. Based on their structures, BD-IMC-1 showcased higher susceptibility to disaggregation compared to BD-IMC-2, consistent with their selective interaction with COX. Notably, the BD-IMC-1 revealed positive cooperativity binding to COX-2 at sub-micromolar ranges. Both probes showed significant fluorescence turn-on upon LPS or PMA triggered COX-2 upregulation in live RAW264.7, HeLa, and human sarcoma cell line (Saos-LM2) up to 2-fold increase with negligible toxicity. More importantly, the BD-IMC-1 demonstrated their practical imaging for COX-2 positive cells in paraffin-fixed human sarcoma tissue. Considering the fixed tissues are most practiced pathological sample, our finding suggests a potential of disaggregation activated chemosensor for clinical applications.

Rational Design of an Activatable Near-Infrared Fluorogenic Platform for In Vivo Orthotopic Tumor Imaging and Resection.

Rational and effective design of a universal near-infrared (NIR) light-absorbed platform employed to prepare diverse activatable NIR fluorogenic probes for in vivo imaging and the imaging-guided tumor resection remains less exploited but highly meaningful. Herein, mandelic acid with a core structure of 4-hydroxylbenzyl alcohol to link recognition unit, a fluorophore and a quencher was employed to prepare activatable probes. We exemplified ester as carboxylesterase (CE)-recognized unit, ferrocene as quencher and phenothiazinium as NIR fluorophore to afford fluorogenic probes termed NBS-Fe-CE and NBS-C-Fe-CE. These probes enabled the conversion toward CE with significant fluorescence increases and successfully discriminate CE activity in cells. NIR light enhances the tumor penetration and enable imaging-guided orthotopic tumor resection. This specific case demonstrated that this platform can be effectively used to construct diverse NIR probes for imaging analytes in biological systems.

NeuM: A Neuron-Selective Probe Incorporates into Live Neuronal Membranes via Enhanced Clathrin-Mediated Endocytosis in Primary Neurons.

The development of a small-molecule probe designed to selectively target neurons would enhance the exploration of intricate neuronal structures and functions. Among such probes, NeuO stands out as the pioneer and has gained significant traction in the field of research. Nevertheless, neither the mechanism behind neuron-selectivity nor the cellular localization has been determined. Here, we introduce NeuM, a derivative of NeuO, designed to target neuronal cell membranes. Furthermore, we elucidate the mechanism behind the selective neuronal membrane trafficking that distinguishes neurons. In an aqueous buffer, NeuM autonomously assembles into micellar structures, leading to the quenching of its fluorescence (Φ=0.001). Upon exposure to neurons, NeuM micelles were selectively internalized into neuronal endosomes via clathrin-mediated endocytosis. Through the endocytic recycling pathway, NeuM micelles integrate into neuronal membrane, dispersing fluorescent NeuM molecules in the membrane (Φ=0.61). Molecular dynamics simulations demonstrated that NeuM, in comparison to NeuO, possesses optimal lipophilicity and molecular length, facilitating its stable incorporation into phospholipid layers. The stable integration of NeuM within neuronal membrane allows the prolonged monitoring of neurons, as well as the visualization of intricate neuronal structures.

Cytarabine induces cachexia with lipid malabsorption via zippering the junctions of lacteal in murine small intestine.

Chemotherapy-induced cachexia causes severe metabolic abnormalities independently of cancer and reduces the therapeutic efficacy of chemotherapy. The underlying mechanism of chemotherapy-induced cachexia remains unclear. Here we investigated the cytarabine (CYT)-induced alteration in energy balance and its underlying mechanisms in mice. We compared energy balance-associated parameters among the three groups of mice: CON, CYT, and PF (pair-fed mice with the CYT group) that were intravenously administered vehicle or CYT. Weight gain, fat mass, skeletal muscle mass, grip strength, and nocturnal energy expenditure were significantly lowered in the CYT group than in the CON and PF groups. The CYT group demonstrated less energy intake than the CON group and higher respiratory quotient than the PF group, indicating that CYT induced cachexia independently from the anorexia-induced weight loss. Serum triglyceride was significantly lower in the CYT group than in the CON group, whereas the intestinal mucosal triglyceride levels and the lipid content within the small intestine enterocyte were higher after lipid loading in the CYT group than in the CON and PF groups, suggesting that CYT inhibited lipid uptake in the intestine. This was not associated with obvious intestinal damage. The CYT group showed increased zipper-like junctions of lymphatic endothelial vessel in duodenal villi compared to that in the CON and CYT groups, suggesting their imperative role in the CYT-induced inhibition of lipid uptake. CYT worsens cachexia independently of anorexia by inhibiting the intestinal lipid uptake, via the increased zipper-like junctions of lymphatic endothelial vessel.

Transcytosis-Inducing Multifunctional Albumin Nanomedicines with Deep Penetration Ability for Image-Guided Solid Tumor Treatment.

Transcytosis is an active transcellular transportation pathway that has garnered interest for overcoming the limited deep penetration of nanomedicines in solid tumors. In this study, a charge-convertible nanomedicine that facilitates deep penetration into solid tumors via transcytosis is designed. It is an albumin-based calcium phosphate nanomedicine loaded with IR820 (mAlb-820@CaP) for high-resolution photoacoustic imaging and enhanced photothermal therapy. Biomineralization on the surface stabilizes the albumin-IR820 complex during circulation and provides calcium ions (Ca2+ ) for tissue penetration on degradation in an acidic environment. pH-triggered transcytosis of the nanomedicine enabled by caveolae-mediated endocytosis and calcium ion-induced exocytosis in 2D cellular, 3D spheroid, and in vivo tumor models is demonstrated. Notably, the extravasation and penetration ability of the nanomedicine is observed in vivo using a high-resolution photoacoustic system, and nanomedicine shows the most potent photothermal antitumor effect in vivo. Overall, the strategy provides a versatile theragnosis platform for both noninvasive photoacoustic imaging and high therapeutic efficiency resulting from deep penetration of nanomedicine.

Superparamagnetic Iron Oxide Nanoparticles as Additives for Microwave-Based Sludge Prehydrolysis: A Perspective.

Wastewater treatment plants are critical for environmental pollution control. The role that they play in protecting the environment and public health is unquestionable; however, they produce massive quantities of excess sludge as a byproduct. One pragmatic approach to utilizing excess sludge is generating methane via anaerobic digestion. For this, a prehydrolysis step can significantly improve digestion by increasing biogas quality and quantity while decreasing final sludge volumes. One of the many prehydrolysis approaches is to deliver heat into sludge via microwave irradiation. Microwave-absorbing additives can be used to further enhance thermal degradation processes. However, the implications of such an approach include potential release of said additive materials into the environment via digested sludge. In this perspective, we present and discuss the potential of superparamagnetic iron oxide nanoparticles (SPIONs) as recoverable, hyperreactive microwave absorbers for sludge prehydrolysis. Due to their size and characteristics, SPIONs pack spin electrons within a single domain that can respond to the magnetic field without remanence magnetism. SPIONs have properties of both paramagnetic and ferromagnetic materials with little to no magnetic hysteresis, which can enable their rapid recovery from slurries, even in complicated reactor installations. Further, SPIONs are excellent microwave absorbers, which result in high local heat gradients. This perspective introduces the vision that SPION properties can be tuned for desirable dielectric heating and magnetic responses while maintaining material integrity to accomplish repeated use for microwave-enhanced pretreatment.

Risk Factors for the Occurrence and Severity of Vertebral Fractures in Inflammatory Bowel Disease Patients: A Nationwide Population-Based Cohort Study.

BACKGROUND: The risk of vertebral fractures is increased in inflammatory bowel disease (IBD) patients. However, whether the severity of vertebral fractures differs between IBD patients and the general population, or between patients with Crohn's disease (CD) and ulcerative colitis (UC), is unknown. METHODS: We investigated risk factors associated with the occurrence and severity of vertebral fractures in IBD patients using The National Healthcare Insurance Service (NHIS) database. We defined the patients who underwent vertebroplasty or kyphoplasty after being diagnosed with a vertebral fracture as having a severe vertebral fracture than those with only diagnosis codes. RESULTS: From 2008 to 2018, there were 33,778 patients with IBD (24,370 UC patients and 9,408 CD patients) and 101,265 patients in the reference population. The incidence rate ratio of vertebral fractures in the IBD patients was 1.27 per 1,000 person-years (95% confidence interval [CI], 1.26-1.27). The risk of vertebral fracture was higher in CD and UC patients than in the matched reference group (hazard ratio [HR], 1.59; 95% CI, 1.31-1.92; P < 0.001 and HR, 1.26; 95% CI, 1.14-1.41; P < 0.001, respectively). In a multivariate analysis, the occurrence of vertebral fracture was associated with CD (HR, 1.31; 95% CI, 1.08-1.59; P = 0.006), older age (CD: HR, 1.09; 95% CI, 1.08-1.09; P < 0.001 and UC: HR, 1.09; 95% CI, 1.08-1.09; P < 0.001), female sex (CD: HR, 1.81; 95% CI, 1.63-2.01; P < 0.001 and UC: HR, 2.02; 95% CI, 1.83-2.22; P < 0.001), high Charlson Comorbidity Index (CCI) score (CD: HR, 1.42; 95% CI, 1.23-1.63; P < 0.001 and UC: HR, 1.46; 95% CI, 1.29-1.65, P < 0.001), and long-term steroid use (CD: HR, 3.71; 95% CI, 2.84-3.37; P < 0.001 and UC: HR, 3.88; 95% CI, 3.07-4.91; P < 0.001). The severity of vertebral fractures was associated with IBD (CD: HR, 1.82; 95% CI, 1.17-2.83; P = 0.008 and UC: HR, 1.49; 95% CI, 1.17-1.89; P < 0.001) and older age (HR, 1.06; 95% CI, 1.05-1.07; P < 0.001). CONCLUSION: Vertebral fractures occur frequently and more severely in IBD patients, particularly those with CD. Therefore, we suggest monitoring of bone density, regular vitamin D supply, and reducing the use of corticosteroids to prevent vertebral fractures in IBD patients who are older, female, or have comorbidities.

Bioinformatic Expansion of Borosins Uncovers Trans-Acting Peptide Backbone N-Methyltransferases in Bacteria.

Backbone N-methylation is one of the prominent peptide modifications that can greatly enhance the pharmacological properties of a peptide. Naturally occurring backbone N-methylated peptides are produced via nonribosomal or ribosomal pathways, the latter of which was only recently identified in the borosin family of ribosomally synthesized and post-translationally modified peptides. Although previous bioinformatic analyses have revealed new putative genes for borosin biosynthesis, the natural scope of structural and biosynthetic diversity of the borosin family has not been thoroughly explored. Here, we report a comprehensive overview of the borosin family of peptide natural products. Using a genome mining approach, we identified more than 1400 new putative biosynthetic gene clusters for borosins and demonstrated that, unlike those previously reported, most of them are found in bacterial genomes and encode a precursor peptide unfused to its cognate methyltransferase enzyme. Biochemical analysis confirmed the backbone N-methylation of the precursor peptide in trans in eight enzyme-precursor pairs and revealed two novel types of enzyme-recognizing sequences in the precursor peptide. This work significantly expands the biosynthetic diversity of borosins and paves the way for the enzymatic production of diverse backbone N-methylated peptides.