Dual function of PHF16 in reinstating homeostasis of murine intestinal epithelium after crypt regeneration.

Intestinal stem cells (ISCs) are highly vulnerable to damage, being in a constant state of proliferation. Reserve stem cells repair the intestinal epithelium following damage-induced ablation of ISCs. Here, we report that the epigenetic regulator plant homology domain (PHD) finger protein 16 (PHF16) restores homeostasis of the intestinal epithelium after initial damage-induced repair. In Phf16-/Y mice, revival stem cells (revSCs) showed defects in exiting the regenerative state, and intestinal crypt regeneration failed even though revSCs were still induced in response to tissue damage, as observed by single-cell RNA sequencing (scRNA-seq). Analysis of Phf16-/Y intestinal organoids by RNA sequencing (RNA-seq) and ATAC sequencing identified that PHF16 restores homeostasis of the intestinal epithelium by inducing retinoic acid receptor (RAR)/retinoic X receptor (RXR) target genes through HBO1-mediated histone H3K14 acetylation, while at the same time counteracting YAP/TAZ activity by ubiquitination of CDC73. Together, our findings demonstrate the importance of timely suppression of regenerative activity by PHF16 for the restoration of gut homeostasis after acute tissue injury.

Assessment of Blood Prostaglandin E2 Metabolite Levels among Infants Born Preterm with Patent Ductus Arteriosus: A Prospective Study.

OBJECTIVE: To assess the role of prostaglandin E2 (PGE2) by measuring blood prostaglandin E2 metabolite (PGEM) concentrations in preterm infants with patent ductus arteriosus (PDA). STUDY DESIGN: A prospective observational study of preterm infants born before 32 weeks of gestational age (GA) was performed in a single tertiary hospital in Japan. Blood samples were collected to measure serum concentrations of PGEM, ibuprofen (IBU), and cytokines. Multiple regression analyses assessed associations between blood PGEM levels and perinatal factors, development of hemodynamically significant PDA (hsPDA), and IBU treatment response of hsPDA. RESULTS: Seventy-nine infants (median GA 28 weeks) were enrolled in this study. Forty-seven received IBU for hsPDA treatment 1 d after birth in median. PDA closure occurred in 25 infants after a single IBU treatment. Serum PGEM concentrations were associated with histological chorioamnionitis (p <0.01), but not with GA, respiratory distress syndrome, or serum IL-6 concentrations. Serum PGEM concentrations decreased after initial IBU treatment; however, they were not associated with hsPDA development (p = 0.39). IBU concentrations correlated with IBU treatment response (aOR 1.29, p <0.01). However, pre-IBU serum PGEM levels and PGEM reduction ratio did not (p = 0.13, 0.15, respectively). CONCLUSIONS: Serum PGEM concentrations in preterm infants were associated with maternal histological chorioamnionitis, but not hsPDA development. IBU treatment response was associated with higher blood IBU concentrations, but not PGEM concentrations.

Effects of natural environments on drug contents in nails: comparison of drug residual rates between nails and hair to determine the drug-use history of corpses in unnatural death cases using micro-segmental analysis.

PURPOSE: We previously developed evaluation methods using micro-segmental analysis (MSA) to examine the effects of external environments on drug content in hair and nails. In this study, the effects of the natural environmental factors (water, temperature, humidity, light, and soil) on drug contents in nails were examined and compared with our previous experimental data on hair. METHODS: Four hay-fever medicines were used as model drugs (fexofenadine, epinastine, cetirizine, and desloratadine) to evaluate drug stability in the nails. Reference nails containing the four medicines were collected from patients with hay fever who ingested the medicines daily for four months. The nails were exposed to various natural environments for up to four months. RESULTS: The effects of temperature, humidity, and light on drug contents in the nails were comparatively small. Soil significantly decomposed the nail surfaces and decreased the drug content of the nails (up to 17 %). Water also decreased the drug content (up to 12 %), although no apparent changes in nail surfaces were observed. CONCLUSIONS: In comparison with hair data obtained under the same environmental conditions, light affected drugs in the hair rather than in nails, whereas water and soil greatly affected drugs in the nails rather than in hair. Although the disposition of drugs incorporated in the tissues differed between nails and hair, the analytes were detected in nails and hair strands left in severe natural environments. MSA could be useful for estimating drug-use histories and personal profiles using the nails and hair of a corpse.

Development of a simple estimation method of serum caffeine concentration using a point-of-care test kit for urinary caffeine.

PURPOSE: Serum caffeine concentration is an indicator of caffeine intoxication; however, it is difficult to measure it in most emergency departments. We developed a simple estimation method using a point-of-care test kit for urinary caffeine. METHODS: Caffeine-spiked human serum (100, 50, 25, and 10 µg/mL) was diluted 10-, 20-, 50-, and 100-fold with phosphate-buffered saline and applied to the kit. After 5 min incubation, the kit was scanned by a flatbed scanner and the membrane image was processed with ImageJ. RESULTS: When the 20-fold diluted serum was applied, serum samples with initial caffeine concentration ≤ 25 and ≥ 50 µg/mL were caffeine-negative and -positive, respectively. When the 100-fold diluted serum was applied, none of the caffeine-spiked serum samples gave positive results. Therefore, we proposed the following test procedure: (i) 20-fold diluted serum was initially tested and (ii) 100-fold diluted serum was additionally tested when the initial result was caffeine positive. Using this procedure, caffeine concentration is expected to be classified into three levels: ≤ 25, > 25- ≤ 100, and > 100 µg/mL, which almost correspond to no or mild, severe, and potentially fatal intoxication, respectively. The test procedure was validated using postmortem heart blood from two cases of fatal caffeine intoxication (caffeine concentration: 276 and 175 µg/mL) and two cases of other intoxication. CONCLUSIONS: Our developed method using point-of-care urinary caffeine test kits enabled simple estimation of serum caffeine concentration.

Revalidation of DNA Fragmentation Analyses for Human Sperm-Measurement Principles, Comparative Standards, Calibration Curve, Required Sensitivity, and Eligibility Criteria for Test Sperm.

(1) Background: Double-strand breaks (DSBs) in a single nucleus are usually measured using the sperm chromatin structure assay (SCSA), sperm chromatin dispersion (SCD) test, and comet assay (CA). Mono-dimensional single-cell pulsed-field gel electrophoresis (1D-SCPFGE) and angle-modulated two- dimensional single-cell pulsed-field gel electrophoresis (2D-SCPFGE) were developed to observe DNA fragmentation in separated motile sperm. (2) Methods: Comparative standards, calibration curves, required sensitivity levels, and eligibility criteria for test sperm were set up to validate the measurement principles of these tests. (3) Results: The conventional methods overlooked the interference of nucleoproteins in their measurements. In-gel proteolysis improves the measurement accuracies of 1D- and 2D-SCPFGE. Naked DNA is suitable for comparative standards and test specimens. Moreover, several dysfunctions that might induce DNA damage are observed in the separated motile sperm. Overall, the discussion highlights the need to revisit the conventional univariable analyses based on the SCSA, SCD test, and CA. (4) Conclusions: Human infertility is a complex syndrome, and the aim of quality control in intracytoplasmic sperm injection is to identify the underlying dysfunctions remaining in the separated motile sperm that render them ineligible for injection. Multivariable analyses with special consideration to confounding factors are necessary in future cohort studies.

Synthesis and analytical characterization of 1-(2-thienoyl)-6-allyl-nor-d-lysergic acid diethylamide (1T-AL-LAD).

Lysergic acid diethylamide (LSD) analogs have emerged as new psychoactive substances (NPS) since the mid-2010s, and new compounds continue to emerge for recreational use. Since the end of 2023, "1D-AL-LAD" appeared on X (formerly Twitter) and other websites. As for the compound "1D-LSD" (which also has "1D" in the name), several studies show that the ingredient of seized blotter paper printed "1D-LSD" was actually 1-(2-thienoyl)-LSD (1T-LSD). However, there are no reports of seizures of 1-(1,2-dimethylcyclobutanecarbonyl)-LSD (1D-LSD). Accordingly, it was considered that all or at least a certain percentage of "1D-AL-LAD (1-(1,2-dimethylcyclobutanecarbonyl)-6-allyl-nor-LSD)" is actually 1-(2-thienoyl)-6-allyl-nor-LSD (1T-AL-LAD). This compound is handled by a number of distributors as of April 2024; therefore, it should be characterized in advance if seized. In this study, 1T-AL-LAD was synthesized and characterized using nuclear magnetic resonance spectroscopy, Fourier transform-infrared spectroscopy, liquid chromatography/high-resolution mass spectrometry (LC/HRMS) and gas chromatography/MS (GC/MS). This compound was easily distinguished from previously reported lysergamides. There were some differences in the detectability of 1T-AL-LAD compared with other lysergamides using GC/MS and the fragmentation patterns in LC/HRMS. These differences can be reasonably explained. This information will be of help to determine this substance in seized materials should it emerge on the market.

Efficacy of very-low-dose oral food challenge in children with severe hen egg allergy: A retrospective, single-center case series.

BACKGROUND: To avoid complete elimination of hen eggs (HE) from diet, we introduced a very-low-dose (VLD) oral food challenge (OFC) in patients with severe HE allergy in 2019. Herein, we investigated the efficacy of VLD HE OFC for achieving the full dose OFC. METHODS: Patients with an overt allergic reaction to LD (1/32 HE [≤100 mg]) or less, egg white (EW) protein within 6 months were included. In the VLD group, patients not achieving full-dose OFC (1/2 HE: 1600 mg EW protein) within 2 years were excluded. We retrospectively compared the rate of passing a full-dose OFC between patients who underwent a LD OFC before 2019 (LD group) and those who underwent a VLD OFC (1/100 HE: 32 mg EW protein) after 2019 (VLD group). The period for passing the full-dose OFC was evaluated using Kaplan-Meier survival analysis. RESULTS: We enrolled 411 and 111 patients in the LD and VLD groups, respectively. The median age at OFC initiation was 2.2 [1.5-3.6] and 2.1 [1.4-3.2] years in the LD and VLD groups, respectively. EW- and ovomucoid-specific IgE levels were 38.3 (12.5-72.9) and 21.0 (8.3-46.2) kUA/L in the LD group and 49.8 [18.8-83.9] and 32.1 [15.6-67.8] kUA/L in the VLD group, respectively. Over 4 years, the LD and VLD groups passed the full-dose OFC at rates of 70 and 95%, respectively, with significant differences (log-rank test, P < 0.001). CONCLUSIONS: VLD HE OFC may contribute to passing a full-dose OFC in patients with severe HE allergies.

[PRACTICE AND TRAINING RELATED TO USING ADRENALINE AUTOINJECTORS FOR PARAMEDICS: FACTS AND ISSUES].

BACKGROUND: Although paramedics can use adrenaline autoinjectors (AAIs) during their duties, the actual conditions of their use and the challenges faced remain unclear. We investigated the actual situation and issues pertaining to creating an environment in which paramedics can operate AAIs more effectively. METHODS: A web-based survey was conducted among paramedics who participated in a web-based training session related to their latest knowledge on food allergies and emergency responses in 2022. The survey items included practice and training environments, practices of AAI administration, and regarding AAI administration. RESULTS: Seventy paramedics responded to the survey. Twenty-eight respondents (40%) had experienced cases in which they wished they had an AAI in their work to date, but only one had actually administered one. Thirty-four (49%) indicated that it would be good to have an AAI in the ambulance at all times; 48 (69%) were not concerned about the use of AAI, and the level of concern about its use was significantly related to length of service. The study also revealed that paramedics do not have an adequate training environment regarding AAI. CONCLUSION: Few paramedics have experience in administering AAI, although they are aware of the need for it. For more effective use of AAI, it is necessary to establish a training environment to familiarize paramedics with anaphylaxis and an environment that enables them to use AAI promptly in the field.

Metabolism of highly potent synthetic opioid nitazene analogs: N-ethyl-N-(1-glucuronyloxyethyl) metabolite formation and degradation to N-desethyl metabolites during enzymatic hydrolysis.

The metabolism of the highly potent synthetic opioids metonitazene, etonitazene, and protonitazene was investigated in fresh human hepatocytes. In the hydrolyzed culture medium, N-desethyl-, N,N-di-desethyl-, O-desalkyl-, N-desethyl-O-desalkyl-, N,N-di-desethyl-O-desalkyl-, and N-oxidated metabolites were detected as phase I metabolites, whereas in the unhydrolyzed culture medium, O-glucuronides of phase I metabolites with O-dealkylation were detected as phase II metabolites. The detected phase I metabolites were identified by comparing their analytical data with those of synthesized authentic standards. In contrast, phase II metabolites were identified by comparing their analytical data with those of the glucuronidated products formed by the incubation of the corresponding substrates with human liver microsomes in the presence of uridine diphosphate glucuronic acid. In addition to the aforementioned metabolites, some putative N-ethyl-N-(1-glucuronyloxyethyl) metabolites were detected in the unhydrolyzed culture medium. Purification and hydrolysis experiments revealed that N-ethyl-N-(1-glucuronyloxyethyl) metabolites formed the corresponding N-desethyl metabolites via unstable N-ethyl-N-(1-hydroxyethyl) metabolites during enzymatic hydrolysis.

Development of a method to evaluate the effects of external environments on drug stability in nails using micro-segmental analysis.

Nails can be used as an alternative to hair for examining past drug use. However, daily hand-and-nail care can eliminate the internal drugs. Therefore, we developed an evaluation method to examine the effects of the external environment on drug stability in nails using micro-segmental analysis. First, reference nails containing drugs were prepared by collecting fingernails from participants who had consumed hay-fever medicines continuously for 4 months. Next, the entire free edge of a reference nail was cut into halves at the centerline; one side was stored as an untreated block, and the other was treated with various hand/nail care products. Both nail blocks were washed and segmented at 0.5-mm intervals in the width direction. Each segment in the extraction solution was crushed with stainless-steel beads, sonicated, and soaked in the solution for 24 h. The analytes in extracts were quantified by LC-MS/MS, and the drug concentrations between the treated and untreated blocks were compared. The drug concentrations decreased slightly in nails treated with manicure and gel-nail products. The analytes in nails tended to be lower in water-rich products such as hand soap and hand cream than in oil-rich products such as nailcare oil and acetone-free remover. The developed method using micro-segmental analysis enabled the evaluation of the effects of various hand/nail care products on drug stability in a limited number of nails. This would also be useful for examining the effects of severe environments on drugs in nails collected from cases of unnatural death.

A novel bioinformatic approach reveals cooperation between Cancer/Testis genes in basal-like breast tumors.

Breast cancer is the most prevalent type of cancer in women worldwide. Within breast tumors, the basal-like subtype has the worst prognosis, prompting the need for new tools to understand, detect, and treat these tumors. Certain germline-restricted genes show aberrant expression in tumors and are known as Cancer/Testis genes; their misexpression has diagnostic and therapeutic applications. Here we designed a new bioinformatic approach to examine Cancer/Testis gene misexpression in breast tumors. We identify several new markers in Luminal and HER-2 positive tumors, some of which predict response to chemotherapy. We then use machine learning to identify the two Cancer/Testis genes most associated with basal-like breast tumors: HORMAD1 and CT83. We show that these genes are expressed by tumor cells and not by the microenvironment, and that they are not expressed by normal breast progenitors; in other words, their activation occurs de novo. We find these genes are epigenetically repressed by DNA methylation, and that their activation upon DNA demethylation is irreversible, providing a memory of past epigenetic disturbances. Simultaneous expression of both genes in breast cells in vitro has a synergistic effect that increases stemness and activates a transcriptional profile also observed in double-positive tumors. Therefore, we reveal a functional cooperation between Cancer/Testis genes in basal breast tumors; these findings have consequences for the understanding, diagnosis, and therapy of the breast tumors with the worst outcomes.

Epitope Mapping of an Anti-Mouse CD39 Monoclonal Antibody Using PA Scanning and RIEDL Scanning.

A cell-surface ectonucleotidase CD39 mediates the conversion of extracellular adenosine triphosphate into immunosuppressive adenosine with another ectonucleotidase CD73. The elevated adenosine in the tumor microenvironment attenuates antitumor immunity, which promotes tumor cell immunologic escape and progression. Anti-CD39 monoclonal antibodies (mAbs), which suppress the enzymatic activity, can be applied to antitumor therapy. Therefore, an understanding of the relationship between the inhibitory activity and epitope of mAbs is important. We previously established an anti-mouse CD39 (anti-mCD39) mAb, C39Mab-1 using the Cell-Based Immunization and Screening method. In this study, we determined the critical epitope of C39Mab-1 using flow cytometry. We performed the PA tag (12 amino acids [aa])-substituted analysis (named PA scanning) and RIEDL tag (5 aa)-substituted analysis (named RIEDL scanning) to determine the critical epitope of C39Mab-1 using flow cytometry. By the combination of PA scanning and RIEDL scanning, we identified the conformational epitope, spanning three segments of 275-279, 282-291, and 306-323 aa of mCD39. These analyses would contribute to the identification of the conformational epitope of membrane proteins.

An Efficient Method for Isolating and Purifying Nuclei from Mice Brain for Single-Molecule Imaging Using High-Speed Atomic Force Microscopy.

Nuclear pore complexes (NPCs) on the nuclear membrane surface have a crucial function in controlling the movement of small molecules and macromolecules between the cell nucleus and cytoplasm through their intricate core channel resembling a spiderweb with several layers. Currently, there are few methods available to accurately measure the dynamics of nuclear pores on the nuclear membranes at the nanoscale. The limitation of traditional optical imaging is due to diffraction, which prevents achieving the required resolution for observing a diverse array of organelles and proteins within cells. Super-resolution techniques have effectively addressed this constraint by enabling the observation of subcellular components on the nanoscale. Nevertheless, it is crucial to acknowledge that these methods often need the use of fixed samples. This also raises the question of how closely a static image represents the real intracellular dynamic system. High-speed atomic force microscopy (HS-AFM) is a unique technique used in the field of dynamic structural biology, enabling the study of individual molecules in motion close to their native states. Establishing a reliable and repeatable technique for imaging mammalian tissue at the nanoscale using HS-AFM remains challenging due to inadequate sample preparation. This study presents the rapid strainer microfiltration (RSM) protocol for directly preparing high-quality nuclei from the mouse brain. Subsequently, we promptly utilize HS-AFM real-time imaging and cinematography approaches to record the spatiotemporal of nuclear pore nano-dynamics from the mouse brain.

CDK4/6 inhibitor-induced bone marrow micronuclei might be caused by cell cycle arrest during erythropoiesis.

BACKGROUND: A micronucleus test is generally used to evaluate the genotoxic potential of chemicals. Exaggerated erythropoiesis, as occurs following bleeding, may induce an unexpected increase in micronucleus frequency. This false positive result would be typical in a genotoxicity study due to the enhanced progression of the cell cycle that restores decreased blood cells. The cyclin-dependent kinase (CDK) family is known to play an essential role in preventing genomic instability. Conversely, a selective CDK4/6 inhibitor PD0332991, clinically named Palbociclib, is reported to have genotoxic potential, shown by positive results in both in vitro and in vivo micronucleus studies. To clarify the mechanism by which cell cycle arrest induced by a CDK4/6 inhibitor increases micronucleus frequency, we investigated the positive results of the bone marrow micronucleus test conducted with PD0332991. RESULTS: Rats treated with PD0332991 exhibited increased micronucleus frequency in an in vivo bone marrow micronucleus test whereas it was not increased by treatment in human lymphoblastoid TK6 cells. In addition, all other genotoxicity tests including the Ames test and the comet assay showed negative results with PD0332991. Interestingly, PD0332991 treatment led to an increase in erythrocyte size in rats and affected the size distribution of erythrocytes, including the micronucleus. The mean corpuscular volume of reticulocytes (MCVr) in the PD0332991 treatment group was significantly increased compared to that of the vehicle control (83.8 fL in the PD0332991, and 71.6 fL in the vehicle control.). Further, the average micronucleated erythrocytes (MNE) size of the PD0332991 group and vehicle control was 8.2 and 7.3 µm, respectively. In the histogram, the vehicle control showed a monomodal distribution with a peak near 7.3 µm. In contrast, the PD0332991 group showed a bimodal distribution with peaks around 7.5 and 8.5 µm. Micronucleated erythrocytes in the PD0332991 group were significantly larger than those in the vehicle control. These results suggest that the increase in micronucleus frequency induced by the CDK4/6 inhibitor is not due to genotoxicity, but is attributable to disturbance of the cell cycle, differentiation, and enucleation of erythroblasts. CONCLUSIONS: It was suggested that the positive outcome of the in vivo bone marrow micronucleus test resulting from treatment with PD0332991 could not be attributed to its genotoxicity. Further studies to clarify the mechanism of action can contribute to the development of drug candidate compounds lacking intrinsic genotoxic effects.

Development of a Sensitive Anti-Mouse CD39 Monoclonal Antibody (C39Mab-1) for Flow Cytometry and Western Blot Analyses.

CD39 is involved in adenosine metabolism by converting extracellular ATP to adenosine. As extracellular adenosine plays a critical role in the immune suppression of the tumor microenvironment, the inhibition of CD39 activity by monoclonal antibodies (mAbs) is one of the important strategies for tumor therapy. This study developed specific and sensitive mAbs for mouse CD39 (mCD39) using the Cell-Based Immunization and Screening method. The established anti-mCD39 mAb, C39Mab-1 (rat IgG2a, kappa), reacted with mCD39-overexpressed Chinese hamster ovary-K1 (CHO/mCD39) by flow cytometry. The kinetic analysis using flow cytometry indicated that the dissociation constant of C39Mab-1 for CHO/mCD39 was 7.3 × 10-9 M. Furthermore, C39Mab-1 detected the lysate of CHO/mCD39 by western blot analysis. These results indicated that C39Mab-1 is useful for the detection of mCD39 in many functional studies.

Identifying a suspect powder as a cannabis concentrate through chemical analysis and DNA testing.

PURPOSE: Cannabis is regulated in many countries, and cannabis products are diversifying, which can hinder identification. Here, we report the seizure of a powder sample with a cannabis-like odor in a spice bottle labeled "nutmeg" and identification of the sample by chemical testing and cannabis DNA testing. METHODS: The sample was observed under a microscope, extracted with methanol, and analyzed by gas chromatography-mass spectrometry (GC-MS). The chemical profile of the seized powder was compared with that of nutmeg samples. Gas chromatography-flame ionization detection was used to estimate the total Δ9-tetrahydrocannabinol (Δ9-THC) concentration in the sample. A commercially available cannabis DNA testing kit was used to confirm the presence of cannabis plant DNA in the seized sample. RESULTS: The characteristics of cannabis in the seized powder were difficult to determine through microscopic observation alone. GC-MS analysis identified ß-caryophyllene (an aromatic component of cannabis) and five cannabinoids unique to cannabis, including Δ9-THC. No common compounds were identified in the seized powder or nutmeg samples. The total Δ9-THC concentration in the sample was very high (approximately 47% by weight). Cannabis DNA testing confirmed that the seized powder contained cannabis. CONCLUSIONS: The seized powder was found to be a processed product made from a finely pulverized resin-like cannabis concentrate. Our results indicate that combined chemical and DNA analysis should help identify cannabis-related samples in various forms.