[FLT3-ITD mutation-positive acute myeloid leukemia undergoing clonal transition with PTPN11 mutation at relapse].

A 28-year-old man was diagnosed with acute myelomonocytic leukemia. He achieved complete remission (CR) after two cycles of induction therapy. However, after consolidation therapy, bone marrow aspiration performed to prepare for allogeneic hematopoietic stem cell transplantation revealed disease relapse. Companion diagnostics confirmed the presence of the FLT3-ITD mutation. The patient received gilteritinib monotherapy and achieved CR. Subsequently, he underwent unrelated allogeneic bone marrow transplantation. One year after transplantation, the patient relapsed, and gilteritinib was resumed. However, the leukemia progressed, and panel sequencing using a next-generation sequencer showed that the FLT3-ITD mutation disappeared. A mutation in PTPN11, which regulates the RAS/MAPK signaling pathway, was also detected. Gilteritinib was discontinued, and the patient achieved CR with salvage chemotherapy. He underwent related haploidentical peripheral blood stem cell transplantation but died of relapse. This was a case in which genetic analysis revealed clonal transition and acquisition of resistance to treatment.

A case report of a truncated ABL1 mutation in 2 cases with Philadelphia chromosome-positive B cell precursor acute lymphoblastic leukemia.

Acquired point mutations in the ABL1 gene are widely recognized as a cause of Philadelphia chromosome-positive B cell precursor acute lymphoblastic leukemia (Ph+ B-ALL) that is resistant to tyrosine kinase inhibitors, whereas there are few reports about other types of the ABL1 mutation. Here, we report 2 cases of Ph+ B-ALL gaining a partial deletion type mutation of the ABL1 gene (Δ184-274 mutation), which resulted in truncation of the ABL1 molecule and loss of kinase activity. In both cases, the disease was refractory to multiple agents in the recurrent phase after allogeneic hematopoietic cell transplantation. This is a case report of a truncated ABL1 mutation in 2 patients with Ph+ B-ALL.

Elevation of Urinary Liver-Type Fatty Acid-Binding Protein Is a Harbinger of Poor Patient Prognosis after Allogeneic Stem Cell Transplantation.

Several recent studies have demonstrated that urinary levels of liver-type fatty acid-binding protein (L-FABP) can be used to stratify the prognosis of cardiac disease, cardiac intensive care unit admission, cirrhosis, and coronavirus disease 2019. Our initial prospective study revealed that urinary L-FABP (uL-FABP) was associated with a high probability of acute kidney injury after stem cell transplantation (SCT); however, the relevance of elevated uL-FABP to the prognosis of patients undergoing SCT remains to be determined. We aimed to investigate whether uL-FABP levels can be used to stratify patient prognosis after SCT. To achieve this aim, we conducted a new long-term follow-up study using data from patients enrolled in our preceding prospective cohort study. Patients were classified into high and low uL-FABP groups based on levels measured at baseline (ie, before initiating the conditioning regimen), using an uL-FABP cutoff of 8.4 µg/gCr, which was determined based on data from healthy adults. uL-FABP levels were also measured on days 0, 7, and 14 after SCT. Cox proportional hazard regression was used to examine the effects of each factor on survival outcomes, and Fine-Gray regression was used in the presence of competing risks. Multivariate analysis incorporating confounders was then performed for factors with P < .1 in univariate analysis. In total, 20 of 84 patients (23.8%), 57 of 84 patients (67.9%), 34 of 49 patients (69.4%), and 34 of 46 patients (73.9%) were classified into the high uL-FABP group at baseline and on days 0, 7, and 14, respectively. The 5-year overall survival (OS) rate was 23.9% in the high uL-FABP group and 68.9% in the low uL-FABP group. The multivariate analysis identified a high uL-FABP level at baseline as a significant prognostic factor for poor OS (hazard ratio [HR], 3.54; P = .002). The 5-year cumulative incidence rate for nonrelapse mortality (NRM) was 50.0% in the high uL-FABP group and 19.9% in the low uL-FABP group. In the multivariate analysis, high uL-FABP at baseline was a significant prognostic factor for NRM (HR, 3.37; P = .01). uL-FABP levels did not significantly stratify the cumulative incidence of relapse (HR, 2.13; P = .11). uL-FABP levels on days 0, 7, and 14 were not significant predictors of survival. High uL-FABP level before initiation of conditioning significantly influences OS and NRM following SCT, whereas a high uL-FABP level at any point after the conditioning regimen does not. Our results show that measuring uL-FABP level at baseline may be a simple way to predict survival in patients undergoing SCT.

Deep intronic founder mutations identified in the ERCC4/XPF gene are potential therapeutic targets for a high-frequency form of xeroderma pigmentosum.

Xeroderma pigmentosum (XP) is a genodermatosis defined by cutaneous photosensitivity with an increased risk of skin tumors because of DNA repair deficiency. The worldwide prevalence of XP is ~1 to 4 in million, with higher incidence in some countries and regions including Japan (1 in 22,000) and North Africa due to founder mutations and a high degree of consanguinity. Among XP, the complementation group F (XP-F), is a rare form (1% of worldwide XP); however, this is underdiagnosed, because the ERCC4/XPF gene is essential for fetal development and most of previously reported ERCC4/XPF pathogenic variants are hypomorphs causing relatively mild phenotypes. From the largest Japanese XP cohort study, we report 17 XP-F cases bearing two pathogenic variants, both identified in deep intronic regions of the ERCC4/XPF gene. The first variant, located in intron 1, is a Japanese founder mutation, which additionally accounts for ~10% of the entire Japanese XP cases (MAF = 0.00196), causing an aberrant pre-mRNA splicing due to a miss-binding of U1snRNA. The second mutation located in intron eight induces an alternative polyadenylation. Both mutations cause a reduction of the ERCC4/XPF gene expression, resulting in XP clinical manifestations. Most cases developed early-onset skin cancers, indicating that these variants need critical attention. We further demonstrate that antisense oligonucleotides designed for the mutations can restore the XPF protein expression and DNA repair capacity in the patients' cells. Collectively, these pathogenic variants can be potential therapeutic targets for XP.

Aeromicelle-A new form of liquid aerosol for delivering aqueous samples into a single-particle mass spectrometer.

For applying highly sensitive mass spectrometry to chemical analysis of aqueous samples, we have developed a novel technique using a new form of liquid droplets, which we call "aeromicelle" (AM), to deliver aqueous sample solutions directly into the vacuum region of a single-particle mass spectrometer in liquid form and conduct immediate mass analysis. AMs are generated by spraying an aqueous solution containing a surfactant at a concentration significantly lower than its critical micelle concentration (CMC). When the solution is sprayed, liquid droplets containing the surfactant are formed, which gradually dry in an air flow. Upon drying, the surfactant concentration in the droplet exceeds its CMC, and consequently, the surfactant molecules begin to cover the droplet surface. Finally, the surface is expected to be fully covered with surfactant molecules such as reverse micelles. The surface coverage helps suppress the evaporation of water, thereby enhancing the residence time of the liquid droplet. Our experimental results show that the AMs retained a liquid form for at least 100 s in air and survived even under vacuum conditions for further mass analysis: each AM delivered in the vacuum region of a single-particle mass spectrometer is ablated with an intense laser pulse and then, mass analyzed. Individual AMs generated from an aqueous solution containing CsCl were analyzed using a single-particle mass spectrometer. The Cs+ ion peak was observed even in AMs generated from the 10 nM solution. The number of Cs atoms in each AM was estimated to be approximately 7 × 103, which corresponds to 1.2 × 10-20 mol (12 zmol). Meanwhile, in the mass analysis of tyrosine, both positive and negative fragmentation ions from tyrosine in AMs were observed in the mass spectrum and 4.6 × 105 (760 zmol) tyrosine molecules were detected.

Comparison of the Upper and Lower Extremity and Trunk Muscle Masses between Children with Down Syndrome and Children with Typical Development.

PURPOSE: Comparison of not only the upper and lower extremity but also trunk muscle masses measured by means of an ultrasound imaging device between children with Down syndrome (DS) and children with typical development (TD). METHODS: The study included 35 children with TD (TD group) and 26 children with DS (DS group). The upper and lower extremity and trunk muscle thicknesses were measured using an ultrasound imaging device. RESULTS: The thicknesses of the rectus abdominis, obliquus externus and internus abdominis, rectus femoris, and short head of the biceps femoris muscles were significantly lower in the DS group than in the TD group. The thicknesses of the other upper and lower extremity and trunk muscles did not differ significantly between the groups. CONCLUSIONS: The results of this study demonstrated lower masses of trunk flexor and knee extensor and flexor muscles in children with DS compared to those in children with TD.

Comparison of the degree of pes planovalgus and muscle mass of the ankle joint and foot muscles between children with Down syndrome and children with typical development.

We aimed to compare the degree of pes planovalgus and muscle mass of the ankle joint and foot muscles between children with Down syndrome (DS) and children with typical development (TD). We also examined the association of the degree of pes planovalgus with muscle mass of the ankle joint and foot muscles in children with DS and children with TD. The subjects were 24 children with TD (control [CTR] group) and 23 children with DS (DS group). To assess pes planovalgus, the Arch Height Index (AHI) in the standing position was measured. Muscle thickness of the ankle joint and foot muscles was measured using an ultrasound imaging device. The AHI and thickness of the soleus and tibialis posterior muscles were significantly lower in the DS group than those in the CTR group. The thickness of the flexor digitorum longus muscle was significantly greater in the DS group than that in the CTR group. Stepwise regression analysis revealed that the thickness of the flexor digitorum longus muscle was a significant and independent factor of the AHI in children comprising the CTR and DS groups. The thickness of the flexor digitorum longus muscle increased with decreasing AHI. The results of this study suggest that the AHI and muscle mass of the soleus and tibialis posterior muscles decrease, while muscle mass of the flexor digitorum longus muscle increases in children with DS. The results also indicate that decreased AHI is associated with increased muscle mass of the flexor digitorum longus muscle in children.

Ubiquitination of DNA Damage-Stalled RNAPII Promotes Transcription-Coupled Repair.

Transcription-coupled nucleotide excision repair (TC-NER) is initiated by the stalling of elongating RNA polymerase II (RNAPIIo) at DNA lesions. The ubiquitination of RNAPIIo in response to DNA damage is an evolutionarily conserved event, but its function in mammals is unknown. Here, we identified a single DNA damage-induced ubiquitination site in RNAPII at RPB1-K1268, which regulates transcription recovery and DNA damage resistance. Mechanistically, RPB1-K1268 ubiquitination stimulates the association of the core-TFIIH complex with stalled RNAPIIo through a transfer mechanism that also involves UVSSA-K414 ubiquitination. We developed a strand-specific ChIP-seq method, which revealed RPB1-K1268 ubiquitination is important for repair and the resolution of transcriptional bottlenecks at DNA lesions. Finally, RPB1-K1268R knockin mice displayed a short life-span, premature aging, and neurodegeneration. Our results reveal RNAPII ubiquitination provides a two-tier protection mechanism by activating TC-NER and, in parallel, the processing of DNA damage-stalled RNAPIIo, which together prevent prolonged transcription arrest and protect against neurodegeneration.

Involvement of natriuretic peptide system in C2C12 myocytes.

The natriuretic peptide system, a key regulator of cGMP signaling, comprises three types of natriuretic peptides, osteocrin/musclin (OSTN), and their natriuretic peptide receptors. Although this system plays important roles in many organs, its physiological roles in skeletal muscle have not been clearly described. In the present study, we investigated the role of the natriuretic peptide system in C2C12 myocytes. All three natriuretic peptide receptors were expressed by cells differentiating from myoblasts to myotubes, and natriuretic peptide receptor B (NPR-B) transcripts were detected at the highest levels. Further, higher levels of cGMP were generated in response to stimulation with C-type natriuretic peptide (CNP) versus atrial natriuretic peptide (ANP), which reflected receptor expression levels. A cGMP analog downregulated the expression of a few ER stress-related genes. Furthermore, OSTN gene expression was strongly upregulated after 20 days of differentiation. Augmented gene expression was found to correlate closely with endoplasmic reticulum (ER) stress, and C/EBP [CCAAT-enhancer-binding protein] homologous protein (CHOP), which is known to be activated by ER stress, affected the expression of OSTN. Together, these results suggest a role for natriuretic peptide signaling in the ER stress response of myocytes.

SELEX-Based Screening of Exosome-Tropic RNA.

Cell-derived nanosized vesicles or exosomes are expected to become delivery carriers for functional RNAs, such as small interfering RNA (siRNA). A method to efficiently load functional RNAs into exosomes is required for the development of exosome-based delivery carriers of functional RNAs. However, there is no method to find exosome-tropic exogenous RNA sequences. In this study, we used a systematic evolution of ligands by exponential enrichment (SELEX) method to screen exosome-tropic RNAs that can be used to load functional RNAs into exosomes by conjugation. Pooled single stranded 80-base RNAs, each of which contains a randomized 40-base sequence, were transfected into B16-BL6 murine melanoma cells and exosomes were collected from the cells. RNAs extracted from the exosomes were subjected to next round of SELEX. Cloning and sequencing of RNAs in SELEX-screened RNA pools showed that 29 of 56 clones had a typical RNA sequence. The sequence found by SELEX was enriched in exosomes after transfection to B16-BL6 cells. The results show that the SELEX-based method can be used for screening of exosome-tropic RNAs.

Macrophage-dependent clearance of systemically administered B16BL6-derived exosomes from the blood circulation in mice.

Previous studies using B16BL6-derived exosomes labelled with gLuc-lactadherin (gLuc-LA), a fusion protein of Gaussia luciferase (a reporter protein) and lactadherin (an exosome-tropic protein), showed that the exosomes quickly disappeared from the systemic circulation after intravenous injection in mice. In the present study, the mechanism of rapid clearance of intravenously injected B16BL6 exosomes was investigated. gLuc-LA-labelled exosomes were obtained from supernatant of B16BL6 cells after transfection with a plasmid DNA encoding gLuc-LA. Labelling was stable when the exosomes were incubated in serum. By using B16BL6 exosomes labelled with PKH26, a lipophilic fluorescent dye, it was demonstrated that PKH26-labelled B16BL6 exosomes were taken up by macrophages in the liver and spleen but not in the lung, while PKH26-labelled exosomes were taken up by the endothelial cells in the lung. Subsequently, gLuc-LA-labelled B16BL6 exosomes were injected into macrophage-depleted mice prepared by injection with clodronate-containing liposomes. The clearance of the intravenously injected B16BL6 exosomes from the blood circulation was much slower in macrophage-depleted mice than that in untreated mice. These results indicate that macrophages play important roles in the clearance of intravenously injected B16BL6 exosomes from the systemic circulation.

Quantitative analysis of tissue distribution of the B16BL6-derived exosomes using a streptavidin-lactadherin fusion protein and iodine-125-labeled biotin derivative after intravenous injection in mice.

We previously succeeded in the visualization of tissue distribution of B16BL6 cells-derived exosomes by labeling with Gaussia luciferase (gLuc)-LA, a fusion protein of gLuc (a reporter protein) and lactadherin (LA; an exosome-tropic protein). However, total amount of B16BL6-derived exosomes delivered to each organ could not be evaluated because of the reduction of luminescent signal from gLuc-LA. The aim of the present study was to quantitatively evaluate the tissue distribution of B16BL6-derived exosomes. To this end, we labeled B16BL6-derived exosomes with iodine-125 ((125) I) based on streptavidin (SAV)-biotin system. A plasmid vector encoding fusion protein, SAV-LA, was constructed, and B16BL6 cells were transfected with the plasmid to obtain SAV-LA-coupled exosomes. SAV-LA-coupled exosomes were incubated with (3-(125) I-iodobenzoyl) norbiotinamide ((125) I-IBB) to obtain (125) I-labeled B16BL6 exosomes. After intravenous injection of (125) I-labeled B16BL6 exosomes into mice, radioactivity quickly disappeared from the blood circulation. At 4 h, 28%, 1.6%, and 7% of the injected radioactivity/organ was detected in the liver, spleen, and lung, respectively. These results indicate that (125) I-labeling of exosomes using SAV-biotin system is a useful method to quantitatively evaluate the amount of exogenously administered exosomes delivered to each organ and that the liver is the major organ in the clearance of exogenously administered B16BL6-derived exosomes.

Analytical performance of newly developed multiplex human papillomavirus genotyping assay using Luminex xMAP™ technology (Mebgen™ HPV Kit).

Regional differences in human papillomavirus (HPV) genotypes and the presence of mixed HPV infections may affect adversely the efficacy of the HPV vaccine. Therefore, a simple and high-throughput HPV genotyping system is required. Recently, a novel HPV genotyping kit (the Mebgen™ HPV kit) was developed. This kit uses multiplex PCR and Luminex xMAP™ technology to detect 13 types of high-risk HPVs and an internal control in a 96-well format. In the present study, the analytical performance of the kit was examined using HPV plasmid DNA. All 13 types of HPVs were detected with a minimum detection sensitivity of 250 copies/test, and highly specific signals were observed. HPV 16 plasmid was detected in samples containing mixtures with other HPV-type plasmids in ratios ranging from 1:1 to 1:1000. No cross reactivity was observed with DNA from 27 types of other infectious microbes. A clinical evaluation was carried out using cervical samples from 356 patients with persistent abnormal smears diagnosed at mass public health screenings for cervical cancer. The samples were preserved in Tacas™ medium until analysis. HPV was detected in 162 (45.5%) samples including 110 (67.9%) with single infections and 52 (32.1%) with multiple infections. The type distribution of the 13 high-risk HPVs was as follows: 28.4% HPV 16, 11.7% HPV 18, 6.8% HPV 31, 3.1% HPV 33, 3.7% HPV 35, 9.3% HPV 39, 1.9% HPV 45, 8.6% HPV 51, 37.0% HPV 52, 9.3% HPV 56, 16.7% HPV 58, 3.7% HPV 59, and 1.9% HPV 68. To evaluate sample stability over time, changes in the detection of HPV DNA derived from HeLa and SiHa cells were measured in 3 types of liquid-based cytology media. HPV DNA was detected in Tacas and Thinprep™ samples after storage at 4°C or 30°C for 4 weeks and within 1 week of collection in Surepath™ samples. These results suggest that this newly developed HPV genotyping kit is suitable for use in both clinical applications and large-scale epidemiological studies.

Deletion of hypoxia-inducible factor-1α in adipocytes enhances glucagon-like peptide-1 secretion and reduces adipose tissue inflammation.

It is known that obese adipose tissues are hypoxic and express hypoxia-inducible factor (HIF)-1α. Although some studies have shown that the expression of HIF-1α in adipocytes induces glucose intolerance, the mechanisms are still not clear. In this study, we examined its effects on the development of type 2 diabetes by using adipocyte-specific HIF-1α knockout (ahKO) mice. ahKO mice showed improved glucose tolerance compared with wild type (WT) mice. Macrophage infiltration and mRNA levels of monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor α (TNFα) were decreased in the epididymal adipose tissues of high fat diet induced obese ahKO mice. The results indicated that the obesity-induced adipose tissue inflammation was suppressed in ahKO mice. In addition, in the ahKO mice, serum insulin levels were increased under the free-feeding but not the fasting condition, indicating that postprandial insulin secretion was enhanced. Serum glucagon-like peptide-1 (GLP-1) levels were also increased in the ahKO mice. Interestingly, adiponectin, whose serum levels were increased in the obese ahKO mice compared with the obese WT mice, stimulated GLP-1 secretion from cultured intestinal L cells. Therefore, insulin secretion may have been enhanced through the adiponectin-GLP-1 pathway in the ahKO mice. Our results suggest that the deletion of HIF-1α in adipocytes improves glucose tolerance by enhancing insulin secretion through the GLP-1 pathway and by reducing macrophage infiltration and inflammation in adipose tissue.

[Clinical evaluation of a novel multiplex HPV genotyping reagent using the luminex xMAP technology].

Of the more than 100 strains of human papillomavirus (HPV) reported to date, 13 strains are clinically significant due to the highly associated risk of cervical cancer. HPV test reagents are currently being developed to aid detection of these strains. The present study compared a novel HPV genotyping reagent using a Luminex device, which enables simultaneous quantitation of multiple targets using fluorescent beads, and gene-specific multiplex polymerase chain reaction (Luminex method) with approved in vitro diagnostics. Subjects comprised 256 patients examined at our hospital for either secondary cervical cancer testing or follow-up (mean age, 40 years; age range, 20-85 years) and specimens were obtained using a cervical brush and lavage fluid. The HPV-positive rate with the Luminex method was 51.4%(132/256), of which single and multiple strain infections constituted 65.9% (87/132) and 34.1% (45/132), respectively. Correlation testing of 50 positive and 50 negative Luminex method specimens was conducted using the HPV DNA Hybrid Capture II (HCII), revealing high overall (97%[97/100]), positive(100% [47/47]) and negative(94.3% [50/53]) concordance rates. Correlation was also high for the Clinichip HPV (Clinichip), with overall, positive and negative concordance rates of 98% (98/100), 100%(48/48) and 96.2%(50/52), respectively. Furthermore, the detection concordance rate with the Clinichip was 95.8% (46/48). Sequencing of the seven specimens showing result discrepancies (HCII, n = 3; Clinichip, n = 4) confirmed concordance with the Luminex method results in all cases, indicating the validity of the present method. The present findings suggest that the Luminex method has sufficient response capability for clinical application.

Elimination kinetics of quinaprilat and perindoprilat in hypertensive patients with renal failure on haemodialysis.

Objective of the present study was to investigate the elimination kinetics of quinaprilat and perindoprilat, the active metabolites of angiotensin-converting enzyme (ACE) inhibitors quinapril and perindopril, in hypertensive patients with renal failure under haemodialysis to evaluate the appropriate duration of off-dose of these drugs before starting of low-density lipoprotein (LDL) apheresis. The informed consent was received from 12 hypertensive patients with renal failure, who were under haemodialysis (42 to 62 years). The patients received oral administration of quinapril (10 mg) or perindopril (2 mg) once a day for four weeks. First, to evaluate the dialyzability of each metabolite, blood samples were collected before and after haemodialysis one week after the repeated doses. Second, to evaluate the elimination kinetics of quinaprilat or perindoprilat, blood samples were collected at 24, 72, 120, 192 and 240 h after the final administration. Plasma concentrations of quinaprilat and perindoprilat were measured by high-performance liquid chromatography (HPLC) and radioimmunoassay, respectively. Pharmacokinetic parameters were determined by a model-dependent method. Values of haemodialysis clearance (CL(HD)) and extraction ratio (ER) were 51.5+/-30.2 ml/min and 0.35+/-0.21 for quinaprilat and 108.1+/-5.9 ml/min and 0.75+/-0.04 for perindoprilat, respectively. The terminal elimination half-lives of quinaprilat and perindoprilat were 60.7+/-2.1 and 79.9+/-14.0 h, respectively. The dialyzability of perindoprilat was much higher than that of quinaprilat probably due to low protein binding potency. The present study suggests that hypertensive patients receiving chronic therapy with quinapril or perindopril on haemodialysis should be withdrawn for at least 2 to 3 weeks before LDL apheresis.