Real-Time Signal Analysis with Wider Dynamic Range and Enhanced Sensitivity in Multiplex Colorimetric Immunoassays Using Encoded Hydrogel Microparticles.

The simultaneous quantification of multiple proteins is crucial for accurate medical diagnostics. A promising technology, the multiplex colorimetric immunoassay using encoded hydrogel microparticles, has garnered attention, due to its simplicity and multiplex capabilities. However, it encounters challenges related to its dynamic range, as it relies solely on the colorimetric signal analysis of encoded hydrogel microparticles at the specific time point (i.e., end-point analysis). This necessitates the precise determination of the optimal time point for the termination of the colorimetric reaction. In this study, we introduce real-time signal analysis to quantify proteins by observing the continuous colorimetric signal change within the encoded hydrogel microparticles. Real-time signal analysis measures the "slope", the rate of the colorimetric signal generation, by focusing on the kinetics of the accumulation of colorimetric products instead of the colorimetric signal that appears at the end point. By developing a deep learning-based automatic analysis program that automatically reads the code of the graphically encoded hydrogel microparticles and obtains the slope by continuously tracking the colorimetric signal, we achieved high accuracy and high throughput analysis. This technology has secured a dynamic range more than twice as wide as that of the conventional end-point signal analysis, simultaneously achieving a sensitivity that is 4-10 times higher. Finally, as a demonstration of application, we performed multiplex colorimetric immunoassays using real-time signal analysis covering a wide concentration range of protein targets associated with pre-eclampsia.

Nanoengineered Macrophages Armed with TLR7/8 Agonist Enhance Remodeling of Immunosuppressive Tumor Microenvironment.

Although adoptive cell-based therapy is illuminated as one of the promising approaches in cancer immunotherapy, it shows low antitumor efficacy because transferred cells adapt and alter toward a pro-tumoral phenotype in response to the tumor's immunosuppressive milieu. Herein, nanoengineered macrophages anchored with functional liposome armed with cholesterol-conjugated Toll-like receptor 7/8 agonist (masked TLR7/8a, m7/8a) are generated to overcome the shortcomings of current macrophage-based therapies and enhance the remodeling of the immunosuppressive tumor microenvironment (TME). The liposome-anchored macrophages (LAMΦ-m7/8a), are fabricated by anchoring dibenzocyclooctyne-modified liposome(m7/8a) onto azido-expressing macrophages via a bio-orthogonal click reaction, are continuously invigorated due to the slow internalization of liposome(m7/8a) and sustained activation. LAMΦ-m7/8a secreted ≈3 and 33-fold more IL-6 and TNF-α than conventional M1-MΦ, maintained the M1 phenotype, and phagocytosed tumor cells for up to 48 h in vitro. Both intratumoral and intravenous injections of LAMΦ-m7/8a induced effective antitumor efficacy when treated in combination with doxorubicin-loaded liposomes in 4T1-tumor bearing mice. It not only increases the infiltration of antigen-specific CD8+ T cells secreting granzyme B, IFN-γ, and TNF-α within the TME, but also reduces myeloid-derived suppressor cells. These results suggest that LAMΦ-m7/8a may provide a suitable alternative to next-generation cell-based therapy platform.

Pharmacometabolomics in Drug Disposition, Toxicity and Precision Medicine.

The precision medicine initiative has driven a substantial change in the way scientists and health care practitioners think about diagnosing and treating disease. While it has long been recognized that drug response is determined by the intersection of genetic, environmental and disease factors, improvements in technology have afforded precision medicine guided dosing of drugs to improve efficacy and reduce toxicity. Pharmacometabolomics aims to evaluate small molecule metabolites in plasma and/or urine to help evaluate mechanisms that predict and/or reflect drug efficacy and toxicity. In this mini review, we provide an overview of pharmacometabolomic approaches and methodologies. Relevant examples where metabolomic techniques have been used to better understand drug efficacy and toxicity in major depressive disorder and cancer chemotherapy are discussed. In addition, the utility of metabolomics in drug development and understanding drug metabolism, transport and pharmacokinetics is reviewed. Pharmacometabolomic approaches can help understand factors mediating drug disposition, efficacy and toxicity. While important advancements in this area have been made, their remain several challenges that must be overcome before this approach can be fully implemented into clinical drug therapy. Significance Statement Pharmacometabolomics has emerged as an approach to identify metabolites that allow for implementation of precision medicine approaches to pharmacotherapy. This review article provides an overview pharmacometabolomics including highlights of important examples.

Soluble ANPEP Released From Human Astrocytes as a Positive Regulator of Microglial Activation and Neuroinflammation: Brain Renin-Angiotensin System in Astrocyte-Microglia Crosstalk.

Astrocytes are major supportive glia and immune modulators in the brain; they are highly secretory in nature and interact with other cell types via their secreted proteomes. To understand how astrocytes communicate during neuroinflammation, we profiled the secretome of human astrocytes following stimulation with proinflammatory factors. A total of 149 proteins were significantly upregulated in stimulated astrocytes, and a bioinformatics analysis of the astrocyte secretome revealed that the brain renin-angiotensin system (RAS) is an important mechanism of astrocyte communication. We observed that the levels of soluble form of aminopeptidase N (sANPEP), an RAS component that converts angiotensin (Ang) III to Ang IV in a neuroinflammatory milieu, significantly increased in the astrocyte secretome. To elucidate the role of sANPEP and Ang IV in neuroinflammation, we first evaluated the expression of Ang IV receptors in human glial cells because Ang IV mediates biological effects through its receptors. The expression of angiotensin type 1 receptor was considerably upregulated in activated human microglial cells but not in human astrocytes. Moreover, interleukin-1ß release from human microglial cells was synergistically increased by cotreatment with sANPEP and its substrate, Ang III, suggesting the proinflammatory action of Ang IV generated by sANPEP. In a mouse neuroinflammation model, brain microglial activation and proinflammatory cytokine expression levels were increased by intracerebroventricular injection of sANPEP and attenuated by an enzymatic inhibitor and neutralizing antibody against sANPEP. Collectively, our results indicate that astrocytic sANPEP-induced increase in Ang IV exacerbates neuroinflammation by interacting with microglial proinflammatory receptor angiotensin type 1 receptor, highlighting an important role of indirect crosstalk between astrocytes and microglia through the brain RAS in neuroinflammation.

Impact of the Oral Administration of Polystyrene Microplastics on Hepatic Lipid, Glucose, and Amino Acid Metabolism in C57BL/6Korl and C57BL/6-Lepem1hwl/Korl Mice.

The impact of microplastics (MPs) on the metabolic functions of the liver is currently unclear and not completely understood. To investigate the effects of the administration of MPs on the hepatic metabolism of normal and obese mice, alterations in the lipid, glucose (Glu), and amino acid regulation pathways were analyzed in the liver and adipose tissues of C57BL/6Korl (wild type, WT) or C57BL/6-Lepem1hwl/Korl mice (leptin knockout, Lep KO) orally administered polystyrene (PS) MPs for 9 weeks. Significant alterations in the lipid accumulation, adipogenesis, lipogenesis, and lipolysis pathways were detected in the liver tissue of MP-treated WT and Lep KO mice compared to the vehicle-treated group. These alterations in their liver tissues were accompanied by an upregulation of the serum lipid profile, as well as alterations in the adipogenesis, lipogenesis, and lipolysis pathways in the adipose tissues of MP-treated WT and Lep KO mice. Specifically, the level of leptin was increased in the adipose tissues of MP-treated WT mice without any change in their food intake. Also, MP-induced disruptions in the glycogenolysis, Glu transporter type 4 (GLUT4)-5' AMP-activated protein kinase (AMPK) signaling pathway, levels of lipid intermediates, and the insulin resistance of the liver tissues of WT and Lep KO mice were observed. Furthermore, the levels of seven endogenous metabolites were remarkably changed in the serum of WT and Lep KO mice after MP administrations. Finally, the impact of the MP administration observed in both types of mice was further verified in differentiated 3T3-L1 adipocytes and HepG2 cells. Thus, these results suggest that the oral administration of MPs for 9 weeks may be associated with the disruption of lipid, Glu, and amino acid metabolism in the liver tissue of obese WT and Lep KO mice.

Cooperative Assembly of Self-Adjusting α-Helical Coiled Coils along the Length of an mRNA Chain to Form a Thermodynamically Stable Nanotube Carrier.

Although mRNA delivery technology is very promising, problems in safety and transport arise due to the intrinsically low thermodynamic stability of the current mRNA carriers. Considering that mRNAs are filamentous and a nanotube is one of the most thermodynamically stable shapes among nanoassemblies, a nanotube is one of the most stable supramolecular structures that can be assembled with mRNA. Here, we develop a nanotube-shaped filamentous mRNA delivery platform that shows exceptionally high thermodynamic stability. The key to the development of the mRNA nanotube is the design of self-adjusting supramolecular building blocks (SABs) that have two disparate properties, i.e., dynamic property and stiffness, in a single molecule. The counterbalance of the dynamic property and stiffness in SABs enables the coating of mRNA by winding its way through the flexible and irregular mRNA chain via cooperative interactions. SAB nanotubes with targeting ligands installed show a high uptake efficiency in mammalian cells and controllable gene expression behavior. Thus, the mRNA nanotube provides an enabling technology toward the development of safe and stable mRNA vaccines and therapeutics.

Directional fabrication and dissolution of larval and juvenile oyster shells under ocean acidification.

Biomineralization is one of the key biochemical processes in calcifying bivalve species such as oysters that is affected by ocean acidification (OA). Larval life stages of oysters are made of aragonite crystals whereas the adults are made of calcite and/or aragonite. Though both calcite and aragonite are crystal polymorphs of calcium carbonate, they have different mechanical properties and hence it is important to study the micro and nano structure of different life stages of oyster shells under OA to understand the mechanisms by which OA affects biomineralization ontogeny. Here, we have studied the larval and juvenile life stages of an economically and ecologically important estuarine oyster species, Crassostrea hongkongensis, under OA with focus over shell fabrication under OA (pHNBS 7.4). We also look at the effect of parental exposure to OA on larvae and juvenile microstructure. The micro and nanostructure characterization reveals directional fabrication of oyster shells, with more organized structure as biomineralization progresses. Under OA, both the larval and juvenile stages show directional dissolution, i.e. the earlier formed shell layers undergo dissolution at first, owing to longer exposure time. Despite dissolution, the micro and nanostructure of the shell remains unaffected under OA, irrespective of parental exposure history.

Epigenetic-associated phenotypic plasticity of the ocean acidification-acclimated edible oyster in the mariculture environment.

For marine invertebrates with a pelagic-benthic life cycle, larval exposure to ocean acidification (OA) can affect adult performance in response to another environmental stressor. This carry-over effect has the potential to alter phenotypic traits. However, the molecular mechanisms that mediate "OA"-triggered carry-over effects have not been explored despite such information being key to improving species fitness and management strategies for aquafarming. This study integrated the genome-wide DNA methylome and transcriptome to examine epigenetic modification-mediated carry-over OA impacts on phenotypic traits of the ecologically and commercially important oyster species Crassostrea hongkongensis under field conditions. Larvae of C. hongkongensis were exposed to control pH 8.0 and low pH 7.4 conditions, mimicking near future OA scenario in their habitat, before being outplanted as post-metamorphic juveniles at two mariculture field sites with contrasting environmental stressors for 9 months. The larval carry-over OA effect was found to have persistent impacts on the growth and survival trade-off traits on the outplanted juveniles, although the beneficial or adverse effect depended on the environmental conditions at the outplanted sites. Site-specific plasticity was demonstrated with a diverse DNA methylation-associated gene expression profile, with signal transduction and the endocrine system being the most common and highly enriched functions. Highly methylated exons prevailed in the key genes related to general metabolic and endocytic responses and these genes are evolutionarily conserved in various marine invertebrates in response to OA. These results suggest that oysters with prior larval exposure history to OA had the ability to trigger rapid local adaptive responses via epigenetic modification to cope with multiple stressors in the field.

The effect of micro-particle curcumin on chronic kidney disease progression: the MPAC-CKD randomized clinical trial.

BACKGROUND: Curcumin is a commonly used herbal supplement with anti-inflammatory and anti-fibrotic properties. Animal studies and small human trials suggest that curcumin reduces albuminuria in patients with chronic kidney disease (CKD). Micro-particle curcumin is a new, more bioavailable formulation of curcumin. METHODS: To determine whether micro-particle curcumin versus placebo slows the progression of albuminuric CKD we conducted a randomized, double-blind, placebo-controlled trial with 6-month follow-up. We included adults with albuminuria [a random urine albumin-to-creatinine ratio >30 mg/mmol (265 mg/g) or a 24-h urine collection with more than 300 mg of protein] and an estimated glomerular filtration rate (eGFR) between 15 and 60 mL/min/1.73 m2 within the 3 months before randomization. We randomly allocated participants 1:1 to receive micro-particle curcumin capsules (90 mg/day) or matching placebo for 6 months. After randomization, the co-primary outcomes were the changes in albuminuria and the eGFR. RESULTS: We enrolled 533 participants, but 4/265 participants in the curcumin group and 15/268 in the placebo group withdrew consent or became ineligible. The 6-month change in albuminuria did not differ significantly between the curcumin and placebo groups [geometric mean ratio 0.94, 97.5% confidence interval (CI) 0.82 to 1.08, P = .32]. Similarly, the 6-month change in eGFR did not differ between groups (mean between-group difference -0.22 mL/min/1.73 m2, 97.5% CI -1.38 to 0.95, P = .68). CONCLUSIONS: Ninety milligrams of micro-particle curcumin daily did not slow the progression of albuminuric CKD over 6 months. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02369549.

Metabolomic identification of predictive and early biomarkers of cisplatin-induced acute kidney injury in adult head and neck cancer patients.

AIM: Cisplatin causes acute kidney injury (AKI) in approximately one third of patients. Serum creatinine and urinary output are poor markers of cisplatin-induced AKI. Metabolomics was utilized to identify predictive or early diagnostic biomarkers of cisplatin-induced AKI. METHODS: Thirty-one adult head and neck cancer patients receiving cisplatin (dose ≥70 mg/m2 ) were recruited for metabolomics analysis. Urine and serum samples were collected prior to cisplatin (pre), 24-48 h after cisplatin (24-48 h) and 5-14 days (post) after cisplatin. Based on serum creatinine concentrations measured at the post timepoint, 11/31 patients were classified with clinical AKI. Untargeted metabolomics was performed using liquid chromatography-mass spectrometry (LC-MS). RESULTS: Metabolic discrimination was observed between "AKI" patients and "no AKI" patients at all timepoints. Urinary glycine, hippuric acid sulfate, 3-hydroxydecanedioc acid and suberate were significantly different between AKI patients and no AKI patients prior to cisplatin infusion. Urinary glycine and hippuric acid sulfate were lower (-2.22-fold and -8.85-fold), whereas 3-hydroxydecanedioc acid and suberate were higher (3.62-fold and 1.91-fold) in AKI patients relative to no AKI patients. Several urine and serum metabolites were found to be altered 24-48 h following cisplatin infusion, particularly metabolites involved with mitochondrial energetics. CONCLUSIONS: We propose glycine, hippuric acid sulfate, 3-hydroxydecanedioc acid and suberate as predictive biomarkers of predisposition to cisplatin-induced AKI. Metabolites indicative of mitochondrial dysfunction may serve as early markers of subclinical AKI.

Evaluation of ichroma™ COVID-19 interferon gamma release assay for detection of vaccine-induced immunity in healthcare workers.

OBJECTIVES: We compared the performance of a new interferon gamma release assay (IGRA) format assay, the ichroma™ COVID-19 IGRA (IGRA-SARS), with that of the widely used QuantiFERON SARS-CoV-2 ELISA kit (QFN-SARS) in vaccinated healthcare workers (HCWs). Additionally, we analyzed the long-term changes in IGRA results after the final vaccine dose. METHODS: A total of 383 specimens from 281 HCWs were enrolled in this study, and the results of SARS-IGRA and QFN-SARS assays were compared. In addition, we performed the receive operator curve analysis to estimate the optimal cut-off value for IGRA-SARS. RESULTS: For all specimens, IGRA-SARS and QFN-SARS showed 75.7% and 64.2% of the positive results, respectively. The absolute agreement between IGRA-SARS and QFN-SARS was 80.0%, and the Fleiss' κ value was 0.525, indicating moderate agreement. ROC curve analysis of the IGRA-SARS results showed a cut-off value of >0.254 IU/mL, which was consistent with the manufacturer's specifications. The positive rates of both IGRA assays decreased significantly after a postvaccination period of 6 months. CONCLUSIONS: IGRA-SARS showed acceptable performance in the detection of vaccine-induced immunity against COVID-19; however, harmonization of IGRA assays has not yet been achieved. Additionally, the significant decline of positive rates of IGRA after the last vaccination would support the necessity of booster vaccination after a postvaccination period of 6 months.

Neurogenic pulmonary edema and Takotsubo cardiomyopathy in aneurysmal subarachnoid hemorrhage.

PURPOSE: Neurogenic pulmonary edema (NPE) combined with Takotsubo cardiomyopathy (TCM) is a rare condition associated with aneurysmal subarachnoid hemorrhage (aSAH). Although several mechanisms have been proposed, the pathophysiology and management strategies are not yet fully established. We aimed to determine the radiological and clinical outcomes of patients with NPE and with TCM after aSAH to propose management strategies. METHODS: We analyzed the data of 564 patients with aSAH recorded at a single medical center from February 2015 to July 2022. This study retrospectively investigated the incidence and demographics of SAH combined with both NPE and TCM and the clinical outcomes of the patients. Correlating factors, independently associated with NPE-TCM, were also investigated. RESULTS: During the 7 years, 11 (2.0%) of 564 patients had NPE complicated with TCM after aSAH. Seven of 11 (63.6%) patients had poor-grade SAH (Hunt-Hess Grade 4 to 5). Three of 11 patients had a posterior circulation in the NPE-TCM group. The most prevalent treatment option was endovascular coil embolization, except for one case of clip. Long-term outcomes were favorable in 6 of 11 patients, and there was one case of mortality. Age, troponin I level, and alveolar-arterial oxygen gradient were correlating factors of NPE-TCM. CONCLUSION: Although NPE-TCM represents a rare complication associated with aSAH, achieving active resolution of underlying neurological causes through early and appropriate treatment may contribute to a favorable prognosis. Considering the limited incidence of SAH complicated with NPE-TCM, a multi-center study may be needed.

Depression or Anxiety According to Management Modalities in Patients With Unruptured Intracranial Aneurysms.

BACKGROUND: In the treatment of unruptured intracranial aneurysms, the risk was usually estimated by objective neurological sequelae. However, their effects on depression and anxiety are rare and remain controversial. We aimed to evaluate the risk of depression and anxiety in patients with unruptured intracranial aneurysm stratified by management strategies in a population-based, longitudinal cohort study. METHODS: Using the Korean National Health Insurance Research Database, 71 750 patients with unruptured intracranial aneurysms between 2008 and 2011 were identified and followed up until the end of 2020. The risk of depression and anxiety was compared among management strategies with respect to age, sex, and medical comorbidities. RESULTS: The Kaplan-Meier survival curves indicated that the treatment (clipping and endovascular treatment) group developed depression more frequently than the observation group (P<0.001). The adjusted hazard ratio was 1.11 (95% CI, 1.07-1.15) in the treatment group. According to the management modality, the Kaplan-Meier survival curves indicated that clipping and endovascular treatment groups developed depression more frequently than the observation group (P<0.0001). The adjusted hazard ratio was 1.15 (95% CI, 1.10-1.21) for clipping and 1.07 (95% CI, 1.02-1.12) for endovascular treatment. The depression risk was higher with advanced age (hazard ratio for 45-64 years, 1.37 [95% CI, 1.29-1.45] and hazard ratio for ≥65 years, 2.04 [95% CI, 1.92-2.17]). The risk for anxiety did not differ among the management modalities. CONCLUSIONS: In this study, the risk of depression was slightly greater after clipping surgery than endovascular treatment. Data on treatment-related, long-term psychological outcomes, such as depression, may aid decision-making for preventive treatment of asymptomatic unruptured intracranial aneurysm patients.

Transdural Revascularization by Multiple Burrhole After Erythropoietin in Stroke Patients With Cerebral Hypoperfusion: A Randomized Controlled Trial.

BACKGROUND: In patients with acute symptomatic stroke, reinforcement of transdural angiogenesis using multiple burr hole (MBH) procedures after EPO (erythropoietin) treatment has rarely been addressed. We aimed to investigate the efficacy and safety of cranial MBH procedures under local anesthesia for augmenting transdural revascularization after EPO treatment in patients with stroke with perfusion impairments. METHODS: This prospective, randomized, blinded-end point trial recruited patients with acute ischemic stroke with a perfusion impairment of grade ≥2 within 14 days of symptom onset, steno-occlusive mechanisms on imaging examinations, and absence of transdural collaterals on transfemoral cerebral angiography. Patients were randomly assigned to receive MBH + EPO or MBH alone. The primary and secondary outcomes were revascularization success (trans-hemispheric and trans-burr hole) at 6 months and adverse events, respectively. RESULTS: We evaluated 42 of the 44 targeted patients, with 2 patients lost to follow-up. The combined and MBH-only (n=21 each) groups showed no differences in demographic characteristics and baseline perfusion parameters. Significantly, more cases of trans-hemispheric (19/21 [90.5%] versus 12/21 [57.1%]) and trans-burr hole (42/58 [72.4%] versus 30/58 [51.7%]) revascularization and significant improvements in perfusion parameters were observed in the combined group relative to the MBH-only group. No differences in treatment-related complications were observed between groups. Even after adjustment for potential covariates, EPO usage was an independent factor of successful hemispheric revascularization in this study (odds ratio, 6.41 [95% CI, 1.08-38.02]). CONCLUSIONS: The combination of MBH and EPO is safe and feasible for reinforcing transdural revascularization in acute steno-occlusive patients with perfusion impairments. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT02603406.

Impact of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Vaccination and Pediatric Age on Delta Variant Household Transmission.

BACKGROUND: In Singapore, quarantine of all close contacts with entry and exit polymerase chain reaction testing enabled evaluation of the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination and pediatric age on transmission of the Delta variant. METHODS: This retrospective cohort study included all household close contacts between 1 March 2021 and 31 August 2021. RESULTS: Among 8470 Delta variant-exposed contacts linked to 2583 indices, full-vaccination of the index with BNT162b2 or mRNA-1273 was associated with reduction in acquisition by contacts (adjusted odds ratio [aOR], 0.56; 95% robust confidence interval [RCI], .44-.71 and aOR, 0.51; 95% RCI, .27-.96, respectively). Compared with young adults (aged 18-29 years), children (aged 0-11 years) were significantly more likely to transmit (aOR, 2.37; 95% RCI, 1.57-3.60) and acquire (aOR, 1.43; 95% RCI, 1.07-1.93) infection, vaccination considered. Longer duration from vaccination completion among contacts was associated with decline in protection against acquisition (first-month aOR, 0.42; 95% RCI, .33-.55; fifth-month aOR, 0.84; 95% RCI, .55-.98; P < .0001 for trend) and symptomatic disease (first-month aOR, 0.30; 95% RCI, .23-.41; fifth-month aOR, 0.62; 95% RCI, .38-1.02; P < .0001 for trend). Contacts immunized with mRNA-1273 had significant reduction in acquisition (aOR, 0.73; 95% RCI, .58-.91) compared with BNT162b2. CONCLUSIONS: Among household close contacts, vaccination prevented onward SARS-CoV-2 transmission and there was in-creased risk of SARS-CoV-2 acquisition and transmission among children compared with young adults. Time after completion of vaccination and vaccine type affected SARS-CoV-2 acquisition.

Identification of accession-specific variants and development of KASP markers for assessing the genetic makeup of Brassica rapa seeds.

BACKGROUND: Most crop seeds are F1 hybrids. Seed providers and plant breeders must be confident that the seed supplied to growers is of known, and uniform, genetic makeup. This requires maintenance of pure genotypes of the parental lines and testing to ensure the genetic purity of the F1 seed. Traditionally, seed purity has been assessed with a grow-out test (GOT) in the field, a time consuming and costly venture. Early in the last decade, seed testing with molecular markers was introduced as a replacement for GOT, and Kompetitive allele specific PCR (KASP) markers were recognized as promising tools for genetic testing of seeds. However, the markers available at that time could be inaccurate and applicable to only a small number of accessions or varieties due to the limited genetic information and reference genomes available. RESULTS: We identified 4,925,742 SNPs in 50 accessions of the Brasscia rapa core collection. From these, we identified 2,925 SNPs as accession-specific, considering properties of flanking region harboring accession-specific SNPs and genic region conservation among accessions by the Next Generation Sequencing (NGS) analysis. In total, 100 accession-specific markers were developed as accession-specific KASP markers. Based on the results of our validation experiments, the accession-specific markers successfully distinguised individuals from the mixed population including 50 target accessions from B. rapa core collection and the outgroup. Additionally, the marker set we developed here discriminated F1 hybrids and their parental lines with distinct clusters. CONCLUSIONS: This study provides efficient methods for developing KASP markers to distinguish individuals from the mixture comprised of breeding lines and germplasms from the resequencing data of Chinese cabbage (Brassica rapa spp. pekinensis).

Pseudo-Isolated α-Helix Platform for the Recognition of Deep and Narrow Targets.

Although interest in stabilized α-helical peptides as next-generation therapeutics for modulating biomolecular interfaces is increasing, peptides have limited functionality and stability due to their small size. In comparison, α-helical ligands based on proteins can make steric clash with targets due to their large size. Here, we report the design of a monomeric pseudo-isolated α-helix (mPIH) system in which proteins behave as if they are peptides. The designed proteins contain α-helix ligands that do not require any covalent chemical modification, do not have frayed ends, and importantly can make sterically favorable interactions similar to isolated peptides. An optimal mPIH showed a more than 100-fold increase in target selectivity, which might be related to the advantages in conformational selection due to the absence of frayed ends. The α-helical ligand in the mPIH displayed high thermal stability well above human body temperature and showed reversible and rapid folding/unfolding transitions. Thus, mPIH can become a promising protein-based platform for developing stabilized α-helix pharmaceuticals.

Dynamic regulation of lipid metabolism in the placenta of in vitro and in vivo models of gestational diabetes mellitus†.

The purpose of this study was to investigate lipid metabolism in the placenta of gestational diabetes mellitus individuals and to evaluate its effect on the fetus. We examined the expression of lipogenesis- and lipolysis-related proteins in the in vitro and in vivo gestational diabetes mellitus placenta models. The levels of sterol regulatory element binding protein-1c were increased, and fat accumulated more during early hyperglycemia, indicating that lipogenesis was stimulated. When hyperglycemia was further extended, lipolysis was activated due to the phosphorylation of hormone-sensitive lipase and expression of adipose triglyceride lipase. In the animal model of gestational diabetes mellitus and in the placenta of gestational diabetes mellitus patients during the extended stage of gestational diabetes mellitus, the expression of sterol regulatory element binding protein-1c decreased and the deposition of fat increased. Similar to the results obtained in the in vitro study, lipolysis was enhanced in the animal and human placenta of extended gestational diabetes mellitus. These results suggest that fat synthesis may be stimulated by lipogenesis in the placenta when the blood glucose level is high. Subsequently, the accumulated fat can be degraded by lipolysis and more fat and its metabolites can be delivered to the fetus when the gestational diabetes mellitus condition is extended at the late stage of gestation. Imbalanced fat metabolism in the placenta and fetus of gestational diabetes mellitus patients can cause metabolic complications in the fetus, including fetal macrosomia, obesity, and type 2 diabetes mellitus.

Admixture of divergent genomes facilitates hybridization across species in the family Brassicaceae.

Hybridization and polyploidization are pivotal to plant evolution. Genetic crosses between distantly related species are rare in nature due to reproductive barriers but how such hurdles can be overcome is largely unknown. Here we report the hybrid genome structure of xBrassicoraphanus, a synthetic allotetraploid of Brassica rapa and Raphanus sativus. We performed cytogenetic analysis and de novo genome assembly to examine chromosome behaviors and genome integrity in the hybrid. Transcriptome analysis was conducted to investigate expression of duplicated genes in conjunction with epigenome analysis to address whether genome admixture entails epigenetic reconfiguration. Allotetraploid xBrassicoraphanus retains both parental chromosomes without genome rearrangement. Meiotic synapsis formation and chromosome exchange are avoided between nonhomologous progenitor chromosomes. Reconfiguration of transcription network occurs, and less divergent cis-elements of duplicated genes are associated with convergent expression. Genome-wide DNA methylation asymmetry between progenitors is largely maintained but, notably, B. rapa-originated transposable elements are transcriptionally silenced in xBrassicoraphanus through gain of DNA methylation. Our results demonstrate that hybrid genome stabilization and transcription compatibility necessitate epigenome landscape adjustment and rewiring of cis-trans interactions. Overall, this study suggests that a certain extent of genome divergence facilitates hybridization across species, which may explain the great diversification and expansion of angiosperms during evolution.

Arsenicicoccus cauae sp. nov., isolated from the blood of a pediatric gastroenteritis patient.

A Gram-stain-positive coccus was isolated from the blood of a paediatric patient suffering from gastroenteritis. The taxonomic position of this catalase-positive, non-motile, non-spore-forming facultative anaerobe designated as strain MKL-02T was investigated using a polyphasic approach. Colonies grown on tryptic soy agar with 10 % sheep blood were circular, creamy yellow, and convex. Phylogenetic analysis based on 16S rRNA gene and whole-genome sequences revealed that this strain was most closely related to Arsenicicoccus bolidensis CCUG 47306T within the cluster of the genus Arsenicicoccus. Average nucleotide identity and digital DNA-DNA hybridization values between strain MKL-02T and A. bolidensis DSM 15745T, A. dermatophillus DSM 25571T and A. piscis DSM 22760T were 89.5 and 37.0 %, 79.6 and 22.4 %, and 75.9 and 21.0 %, respectively. The genomic size of strain MKL-02T was 3 423 857 bp with a 72.7 mol% G+C content. Growth was observed at 10-45 °C (optimum, 37-40 °C) and pH 6.0-10.0 (optimum, pH 7.0), in the presence of 0-10 % (w/v) NaCl (optimum, 0.5 %). Cells of strain MKL-02T were non-motile cocci and 0.50-0.60 µm long, as determined by transmission electron microscopy. The strain was catalase-positive and oxidase-negative. The major fatty acid type (>10 % of total) was C15 : 0. The polar lipid profile consisted of two unidentified phospholipids, three unidentified lipids and an unidentified aminophospholipid. The strain contained MK-8 (H4) as the predominant menaquinone. Based on phylogenetic and phenotypic considerations, it is proposed that strain MKL-02T be classified as a new species, named Arsenicicoccus cauae sp. nov. The type strain is MKL-02T (=NCCP 16967T=JCM 34624T).