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Despite advancements in therapeutic monoclonal antibodies (mAbs) and cell line engineering, separating host cell proteins (HCPs) from mAbs during downstream purification remains challenging. Therefore, in this study, we developed a novel multimodal chromatography (MMC) resin to enhance HCP removal during mAb polishing processes. We evaluated the impact of both ligand structure and pore size of the MMC resin by purifying a post-protein A chromatography solution in flow-through mode. We observed that the efficiency of HCP clearance depended on the hydrophobic moiety structure of the ligand and predicted the mAb purification capability of MMC through linear salt-gradient elution experiments involving a mixture of transferrin, bovine serum albumin (BSA), and pepsin. Our findings revealed that the prototype immobilized 1,12-dodecanediamine via the formyl group exhibited the best performance attributed to its long alkyl chain. Furthermore, an investigation of effects of base bead pore size on HCP capacity using cellulose base beads of five different pore sizes showed that larger pore resin base beads had the highest HCP removal capacity. Specifically, MMC resins with a pore diameter exceeding 440 nm reduced the HCP level by three orders of magnitude under high mAb loading conditions (> 1000 mg/mL-resin). The MMC resin developed in this study, along with the insights gained into ligand structure and pore size, not only enhances mAb polishing efficiency but also contributes to improving downstream processes in mAb biopharmaceutical production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Background: ZNF384-fusion (Z-fusion) genes were recently identified in B-cell acute lymphoblastic leukemia (B-ALL) and are frequent in Japanese adult patients. The frequency is about 20% in those with Philadelphia chromosome-negative B-ALL. ZNF384 is a transcription factor and Z-fusion proteins have increased transcriptional activity; however, the detailed mechanisms of leukemogenesis of Z-fusion proteins have yet to be clarified., Methods: We established three transfectants of cell lines expressing different types of Z-fusion proteins, and analyzed their gene expression profile (GEP) by RNA-seq. We also analyzed the GEP of clinical ALL samples using our previous RNA-seq data of 323 Japanese ALL patients. We selected upregulated genes in both Z-fusion gene-expressing transfectants and Z-fusion gene-positive ALL samples, and investigated the binding of Z-fusion proteins to regulatory regions of the candidate genes by ChIP-qPCR., Results: We selected six commonly upregulated genes. After the investigation by ChIP-qPCR, we finally identified CREB5 and RGS1 as direct and common target genes. RGS1 is an inhibitor of CXCL12-CXCR4 signaling that is required for the homing of hematopoietic progenitor cells to the bone marrow microenvironment and development of B cells. Consistent with this, Z-fusion gene transfectants showed impaired migration toward CXCL12., Conclusions: We identified CREB5 and RGS1 as direct and common transcriptional targets of Z-fusion proteins. The present results provide novel insight into the aberrant transcriptional regulation by Z-fusion proteins., (© 2024 The Author(s). Cancer Medicine published by John Wiley & Sons Ltd.)

Ghrelin is a peptide hormone with various important physiological functions. The unique feature of ghrelin is its serine 3 acyl-modification, which is essential for ghrelin activity. The major form of ghrelin is modified with n-octanoic acid (C8:0) by ghrelin O-acyltransferase. Various acyl modifications have been reported in different species. However, the underlying mechanism by which ghrelin is modified with various fatty acids remains to be elucidated. Herein, we report the purification of bovine, porcine, and equine ghrelins. The major active form of bovine ghrelin was a 27-amino acid peptide with an n-octanoyl (C8:0) modification at Ser3. The major active form of porcine and equine ghrelin was a 28-amino acid peptide. However, porcine ghrelin was modified with n-octanol (C8:0), whereas equine ghrelin was modified with n-butanol (C4:0) at Ser3. This study indicates the existence of structural divergence in ghrelin and suggests that it is necessary to measure the minor and major forms of ghrelin to fully understand its physiology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ida, Tominaga, Iwamoto, Kurogi, Okura, Shimada, Kato, Kuwano, Ode, Nagata, Kitamura, Yazawa, Sato-Hashimoto, Yasuda, Miyazato, Shiimura, Sato and Kojima.)

Exercise-induced increases in shear rate (SR) acutely improve peripheral endothelial function, but the presence of this mechanism in cerebral arteries remains unclear. Thus, we evaluated shear-mediated dilation of the internal carotid artery (ICA), which is an index of cerebrovascular endothelial function, before and after exercise. Shear-mediated dilation was measured with 30 s of hypercapnia in 16 young adults before and 10 min after 30 min of sitting rest (CON) or three cycling exercises on four separate days. The target exercise intensity was 80% of oxygen uptake at the ventilatory threshold. To manipulate the ICA SR during exercise, participants breathed spontaneously (Ex SB , SR increase) or hyperventilated without (Ex HV , no increase in SR) or with ([Formula: see text], restoration of SR increase) addition of CO 2 to inspiratory air. Shear-mediated dilation was calculated as a percent increase in diameter from baseline. Doppler ultrasound measures ICA velocity and diameter. The CON trial revealed that 30 min of sitting did not alter shear-mediated dilation (4.34 ± 1.37% to 3.44 ± 1.23%, P = 0.052). ICA dilation after exercise compared with preexercise levels increased in the Ex SB trial (3.32 ± 1.37% to 4.74 ± 1.84%, P < 0.01), remained unchanged in the Ex HV trial (4.07 ± 1.55% to 3.21 ± 1.48%, P = 0.07), but was elevated in the [Formula: see text] trial (3.35 ± 1.15% to 4.33 ± 2.12%, P = 0.04). Our results indicate that exercise-induced increases in cerebral shear may play a crucial role in improving cerebrovascular endothelial function after acute exercise in young adults. NEW & NOTEWORTHY We found that 30-min cycling (target intensity was 80% of the ventilatory threshold) with increasing shear of the internal carotid artery (ICA) enhanced transient hypercapnia-induced shear-mediated dilation of the ICA, reflecting improved cerebrovascular endothelial function. This enhancement of ICA dilation was diminished by suppressing the exercise-induced increase in ICA shear via hyperventilation. Our results indicate that increases in cerebral shear may be a key stimulus for improving cerebrovascular endothelial function after exercise in young adults.

In Japan, Japanese Black cattle, known for their exceptional meat quality owing to their abundant intramuscular fat, undergo a unique three-stage feeding system with varying concentrate ratios. There is limited research on physiological and rumen microbial changes in Japanese Black cattle during these stages. Therefore, this study aimed to examine Japanese Black steers in these three stages: early (T1, 12-14 months), middle (T2, 15-22 months), and late (T3, 23-30 months). The rumen bacteria of 21 cattle per phase was analyzed using 16S rRNA gene sequencing. Rumen bacterial diversity was significantly higher in T1, with a distinct distribution, than in T2 and T3. Specific phyla and genera were exclusive to each stage, reflecting the shifts in feed composition. Certain genera dominated each stage: T1 had Flexilinea, Streptococcus, Butyrivibrio, Selenomonas, and Kandleria; T2 had Bifidobacterium, Shuttleworthia, and Sharpea; and T3 had Acetitomaculum, Mycoplasma, Atopobium, and Howardella. Correlation analysis revealed significant associations between certain microbial populations and physiological parameters. These findings indicate that changes in energy content and feed composition are associated with physiological and ruminal alterations. This study may guide strategies to improve rumen health and productivity in Japanese Black cattle by modifying diets to specific fattening stages., (© 2024. The Author(s).)

Taurine, biosynthesized from methionine or cysteine in the liver, plays a crucial regulatory role in bile acid conjugation, antioxidant effects, and glucose and cholesterol metabolism. This may influence the metabolic changes associated with fat accumulation in beef cattle. However, the physiological role of taurine in this species has not been fully elucidated. In this study, we explored the physiological role of taurine in Japanese Black steers (Bos taurus) in different phases during the fattening period. To examine the correlation among plasma taurine concentrations, various physiological parameters, and genes related to taurine synthesis in the liver, we used biopsied liver tissues, blood samples, and rumen fluids collected from 21 steers at three different stages, i.e., early (T1; 13 mo of age), middle (T2; 20 mo of age), and late (T3; 28 mo of age) phases. Additionally, to investigate the regulatory mechanisms underlying the expression profile of taurine synthesis genes, primary bovine hepatocytes obtained from 4-wk-old Holstein calves were treated with palmitate, oleate, acetate, propionate, or β-hydroxybutyrate (BHBA). Plasma taurine and cholesterol concentrations significantly (P < 0.001) increased in the T2 phase, which is potentially attributable to increased energy intake and assimilation induced by increased intake of concentrated feed. Cysteine-sulfinic acid decarboxylase (CSAD) expression significantly increased (P < 0.01) in T2 than in other phases. The expression levels of cysteine dioxygenase type 1 (CDO1) and cholesterol 7 alpha-hydroxylase (CYP7A1) were significantly higher (P < 0.05) in T2 than in T3; moreover, the CDO1/glutamate-cysteine ligase catalytic subunit (GCLC) ratio was higher (P < 0.05) in T2 than in T1. Plasma taurine concentrations were positively correlated with plasma methionine (r = 0.51; P < 0.05) and total cholesterol (r = 0.56; P < 0.05) concentrations at T2. Relative CDO1 mRNA expression was upregulated in cultured bovine hepatocytes treated with oleate and propionate, whereas it was downregulated upon acetate treatment. These findings indicate that the increase in plasma taurine concentrations in the T2 phase is associated with changes in lipid and methionine metabolism in Japanese Black steers., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society of Animal Science.)

The quantification of amino acid and related metabolite levels is important for evaluating amino acid metabolism and function in animals. However, a useful quantitative method is not enough. In this study, we developed and validated tert-butyldimethylsilyl derivatization method using gas chromatography-mass spectrometry to quantify plasma levels of free amino acids and related metabolites in Japanese Black cattle. Of the 51 metabolites examined, 24, including 20 amino acids, one amine, and three keto acids, could be quantified. Compared with the trimethylsilyl derivatization method using gas chromatography-mass spectrometry, which has been used for untargeted metabolomic analysis, the present method had higher analytical reliability. This method is advantageous for assessing branched-chain amino acid (BCAA) metabolism because it enables the quantification of not only BCAA levels (valine, leucine, and isoleucine) but also their bioactive metabolite keto acid levels (2-ketoisovaleric acid, 2-ketoisocaproic acid, and 2-keto-3-methylvaleric acid) in the plasma. In addition, this method can quantify the plasma levels of not only tryptophan but also its bioactive metabolites kynurenine and serotonin. These results suggest that this quantitative method has the potential to further our understanding of amino acid metabolic processes and their functions in Japanese Black cattle., (© 2024 Japanese Society of Animal Science.)

Purpose: Postprandial hyperglycemia is assumed to have a negative impact on flow-mediated dilation (FMD), an index of endothelial function, and blood flow of the peripheral conduit arteries. This study aimed to determine whether the enhancement of postprandial hyperglycemia by skipping breakfast accelerates endothelial dysfunction and reduces the blood flow in the brachial artery in young men., Methods: Using a randomized cross-over design, ten healthy men completed two trials: with and without breakfast (Eating and Fasting trials, respectively). Venous blood sampling and brachial FMD tests were conducted before, 30, 60, 90, and 120 min after a 75-g oral glucose tolerance test (OGTT)., Results: Skipping breakfast boosted post-OGTT glucose levels than having breakfast (P = 0.01). The magnitude of the decrease in FMD via OGTT did not vary between trials (main effect of trial P = 0.55). Although brachial blood flow tended to decrease after OGTT in both trials (interaction and main effect of time P = 0.61 and P = 0.054, respectively), the decrease in blood flow following OGTT was greater in the Fasting trial than in the Eating trial (main effect of trial, mean difference =  - 15.8 mL/min [95%CI =  - 25.6 to - 6.0 mL/min], P < 0.01)., Conclusion: Skipping breakfast did not enhance the magnitude of the decrease in FMD following glucose loading, but did accelerate hyperglycemia-induced reduction in brachial blood flow. Current findings suggest that even missing one breakfast has negative impacts on the blood flow regulation of the peripheral conduit arteries in young men who habitually eat breakfast., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

This study investigated the physiological characteristics and carcass performance associated with residual methane emissions (RME), and the effects of bull differences on CH 4 -related traits in Japanese Black cattle. Enteric methane (CH 4 ) emissions from 156 Japanese Black cattle (111 heifers and 45 steers) were measured during early fattening using the sniffer method. Various physiological parameters were investigated to clarify the physiological traits between the high, middle, and low RME groups. CH 4 -related traits were examined to determine whether bull differences affected progeny CH 4 emissions. Ruminal butyrate and NH 3 concentrations were significantly higher in the high-RME group than in the low-RME group, whereas the propionate content was significantly higher in the low-RME group. Blood urea nitrogen, β-hydroxybutyric acid, and insulin concentrations were significantly higher, and blood amino acids were lower in the high-RME group than in the other groups. No significant differences were observed in the carcass traits and beef fat composition between RME groups. CH 4 -related traits were significantly different among bull herds. Our results show that CH 4 -related traits are heritable, wherein bull differences affect progeny CH 4 production capability, and that the above-mentioned rumen fermentations and blood metabolites could be used to evaluate enteric methanogenesis in Japanese Black cattle., (© 2024 Japanese Society of Animal Science.)

Adjuvant therapy for patients with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative, node-positive, early breast cancer (EBC) remains challenging. The prognostic significance of HER2-low positivity in these patients is not fully understood. In our retrospective study, we analyzed 647 patients with HR-positive, HER2-negative, node-positive EBC, stratifying them into three cohorts based on axillary lymph node involvement, tumor size, and characteristics. Cohort 1 included patients with either ≥ 4 positive axillary lymph nodes or 1-3 positive nodes with histological grade 3 or tumor size ≥ 5 cm. Cohort 2 consisted of patients with 1-3 positive nodes, histological grade < 3, tumor size < 5 cm, and Ki-67 ≥ 20%. Cohort 3 comprised patients with 1-3 positive nodes, histological grade < 3, tumor size < 5 cm, and Ki-67 < 20%. We compared invasive disease-free survival (IDFS) and distant relapse-free survival (DRFS) between HER2-low (IHC1+ or IHC2+/FISH-) and HER2-zero (IHC0) groups in each cohort. In cohort 1, HER2-low patients exhibited significantly better 5-year IDFS (84.2% vs. 73.6%, p = 0.0213) and DRFS (88.2% vs. 79.8%, p = 0.0154). However, no significant differences were observed in cohorts 2 and 3. Our findings suggest HER2-low positivity as a prognostic factor in HR-positive, HER2-negative, and node-positive EBC., (© 2023. The Author(s).)

Background: Histological grade (HG) has been used in the MonrachE trial to select patients with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative, node-positive high-risk early breast cancer (EBC). Although nuclear grade (NG) is widely used in Japan, it is still unclear whether replacing HG with NG can appropriately select high-risk patients., Methods: We retrospectively reviewed 647 patients with HR-positive, HER2-negative, node-positive EBC and classified them into the following four groups: group 1: ≥ 4 positive axillary lymph nodes (pALNs) or 1-3 pALNs and either grade 3 of both grading systems or tumors ≥ 5 cm; group 2: 1-3 pALNs, grade < 3, tumor < 5 cm, and Ki-67 ≥ 20%; group 3: 1-3 pALNs, grade < 3, tumor < 5 cm, and Ki-67 < 20%; and group 4: group 2 or 3 by HG classification but group 1 by NG classification. We compared invasive disease-free survival (IDFS) and distant relapse-free survival (DRFS) among the four groups using the Kaplan-Meier method with the log-rank test., Results: Group 1 had a significantly worse 5-year IDFS and DRFS than groups 2 and 3 (IDFS 80.8% vs. 89.5%, P = 0.0319, 80.8% vs. 95.5%, P = 0.002; DRFS 85.2% vs. 95.3%, P = 0.0025, 85.2% vs. 98.4%, P < 0.001, respectively). Group 4 also had a significantly worse 5-year IDFS (78.0%) and DRFS (83.6%) than groups 2 and 3., Conclusions: NG was useful for stratifying the risk of recurrence in patients with HR-positive, HER2-negative, node-positive EBC and was the appropriate risk assessment for patient groups not considered high-risk by HG classification., (© 2023. The Author(s).)

Handgrip exercise (HG), a small muscle exercise, improves cognitive function and is expected to provide a useful exercise mode to maintain cerebral health. However, the effect of HG on cerebral blood flow regulation is not fully understood. The present study aimed to examine the effect of acute HG on cerebral endothelial function as one of the essential cerebral blood flow regulatory functions. Thirteen healthy young participants performed interval HG, consisting of 4 sets of 2 min HG at 25% of maximum voluntary contraction with 3 min recovery between each set. Cognitive performance was evaluated before and at 5 and 60 min after interval HG using the Go/No-Go task (reaction time and accuracy). The diameter and blood velocity of the internal carotid artery (ICA) were measured using a duplex Doppler ultrasound system. To assess cerebral endothelial function, hypercapnia (30 s of hypercapnia stimulation, end-tidal partial pressure of CO 2 : +9 mmHg)-induced cerebrovascular flow-mediated dilatation (cFMD) was induced, calculated as relative peak dilatation from baseline diameter. The shear rate (SR) was calculated using the diameter and blood velocity of the ICA. As a result, cognitive performance improved only at 5 min after interval HG (reaction time, P = 0.008; accuracy, P = 0.186), whereas ICA SR during interval HG and cFMD after interval HG were unchanged (P = 0.313 and P = 0.440, respectively). These results suggest that enhancement in cerebral endothelial function is not an essential mechanism responsible for acute HG-induced cognitive improvement. NEW FINDINGS: What is the central question of this study? Does handgrip exercise, a small muscle exercise, improve cerebral endothelial function? What is the main finding and its importance? Acute interval isometric handgrip exercise (2 min of exercise at 25% maximum voluntary contraction, followed by 3 min of recovery, repeated for a total of 4 sets) did not improve cerebral endothelial function. Since the cerebrovascular shear rate did not change during exercise, it is possible that acute handgrip exercise is not sufficient stimulation to improve cerebral endothelial function., (© 2023 The Authors. Experimental Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

The photoelectron momentum microscope (PMM) in operation at BL6U, an undulator-based soft x-ray beamline at the UVSOR Synchrotron Facility, offers a new approach for μm-scale momentum-resolved photoelectron spectroscopy (MRPES). A key feature of the PMM is that it can very effectively reduce radiation-induced damage by directly projecting a single photoelectron constant energy contour in reciprocal space with a radius of a few Å-1 or real space with a radius of a few 100 μm onto a two-dimensional detector. This approach was applied to three-dimensional valence band structure E(k) and E(r) measurements ("stereography") as functions of photon energy (hν), its polarization (e), detection position (r), and temperature (T). In this study, we described some examples of possible measurement techniques using a soft x-ray PMM. We successfully applied this stereography technique to μm-scale MRPES to selectively visualize the single-domain band structure of twinned face-centered-cubic Ir thin films grown on Al2O3(0001) substrates. The photon energy dependence of the photoelectron intensity on the Au(111) surface state was measured in detail within the bulk Fermi surface. By changing the temperature of 1T-TaS2, we clarified the variations in the valence band dispersion associated with chiral charge-density-wave phase transitions. Finally, PMMs for valence band stereography with various electron analyzers were compared, and the advantages of each were discussed., (© 2023 Author(s). Published under an exclusive license by AIP Publishing.)

Gene aberrations of B-cell regulators and growth signal components such as the JAK-STAT pathway are frequently found in B-cell acute lymphoblastic leukemia (B-ALL). EBF1 is a B-cell regulator that regulates the expression of PAX5 and co-operates with PAX5 to regulate B-cell differentiation. Here, we analyzed the function of the fusion protein of EBF1 and JAK2, EBF1-JAK2 (E-J). E-J caused constitutive activation of JAK-STAT and MAPK pathways and induced autonomous cell growth in a cytokine-dependent cell line. E-J did not affect the transcriptional activity of EBF1 but inhibited that of PAX5. Both the physical interaction of E-J with PAX5 and kinase activity of E-J were required for E-J to inhibit PAX5 function, although the detailed mechanism of inhibition remains unclear. Importantly, gene set enrichment analysis using the results of our previous RNA-seq data of 323 primary BCR-ABL1-negative ALL samples demonstrated repression of the transcriptional target genes of PAX5 in E-J-positive ALL cells, which suggests that E-J also inhibited PAX5 function in ALL cells. Our results shed new light on the mechanisms of differentiation block by kinase fusion proteins., (© 2023. Japanese Society of Hematology.)

Purpose: The impact of progesterone receptor (PR) status on the prognosis of breast cancer after isolated locoregional recurrence (ILRR) remains unclear. This study evaluated the impact of clinicopathologic factors, including PR status of ILRR, on distant metastasis (DM) after ILRR., Methods: We retrospectively identified 306 patients with ILRR diagnosed at the National Cancer Center Hospital between 1993 and 2021 from the database. Cox proportional hazards analysis was performed to examine factors associated with DM after ILRR. We developed a risk prediction model based on the number of detected risk factors and estimated survival curves using the Kaplan-Meier method., Results: During a median follow-up time of 4.7 years after ILRR diagnosis, 86 patients developed DM, and 50 died. Multivariate analysis revealed that seven risk factors were associated with poor distant metastasis-free survival (DMFS): estrogen receptor-positive/PR-negative/human epidermal growth factor receptor 2-negative ILRR, short disease-free interval, recurrence site other than ipsilateral breast, no-resection of ILRR tumor, chemotherapy for the primary tumor, nodal stage in the primary tumor, and no endocrine therapy for ILRR. The predictive model classified patients into 4 groups based on the number of risk factors: low-, intermediate-, high-, and the highest-risk groups with 0 to 1, 2, 3 to 4, and 5 to 7 factors, respectively. This revealed significant variation in DMFS among the groups. A higher number of the risk factors was associated with poorer DMFS., Conclusion: Our prediction model, which considered the ILRR receptor status, may contribute to the development of a treatment strategy for ILRR., (© 2023. The Author(s).)

Excessive arterial pressure elevation induced by resistance exercise (RE) attenuates peripheral vasodilatory function, but its effect on cerebrovascular function is unknown. We aimed to evaluate the effect of different pressor responses to RE on hypercapnia-induced vasodilation of the internal carotid artery (ICA), an index of cerebrovascular function. To manipulate pressor responses to RE, 15 healthy young adults (11M/4F) performed two RE: high intensity with low repetitions (HL) and low intensity with high repetitions (LH) dynamic knee extension. ICA dilation, induced by 3 min of hypercapnia, was measured before and 10 min after RE using Doppler ultrasound. HL exercise elicited a greater pressor response than LH exercise. In relaxation phases of RE, ICA blood velocity increased in both HL and LH trials. However, ICA shear rate did not significantly increase in either trial (P = 0.06). Consequently, neither exercise altered post-exercise hypercapnia-induced ICA dilation (HL, 3.9 ± 1.9% to 5.1 ± 1.7%; LH, 4.6 ± 1.4% to 4.8 ± 1.8%; P > 0.05 for all). When viewed individually, the changes in ICA shear rate were positively correlated with changes in end-tidal partial pressure of carbon dioxide (P ET CO 2 ) (r = 0.46, P < 0.01) than with mean arterial pressure (r = 0.32, P = 0.02). These findings suggest that the effects of RE-induced pressor response on cerebrovascular function may be different from peripheral arteries. An increase in P ET CO 2 during the relaxation phase may play a more crucial role than elevated pressure in increasing cerebral shear during dynamic RE., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The present study aimed to examine the validity of a novel method to assess cerebrovascular carbon dioxide (CO 2 ) reactivity (CVR) that does not require a CO 2 inhalation challenge, e.g., for use in patients with respiratory disease or the elderly, etc. In twenty-one healthy participants, CVR responses to orthostatic stress (50° head-up tilt, HUT) were assessed using two methods: (1) the traditional CO 2 inhalation method, and (2) transfer function analysis (TFA) between middle cerebral artery blood velocity (MCA V) and predicted arterial partial pressure of CO 2 (PaCO 2 ) during spontaneous respiration. During HUT, MCA V steady-state (i.e., magnitude) and MCA V onset (i.e., time constant) responses to CO 2 inhalation were decreased ( p < 0.001) and increased ( p = 0.001), respectively, indicative of attenuated CVR. In contrast, TFA gain in the very low-frequency range (VLF, 0.005-0.024 Hz) was unchanged, while the TFA phase in the VLF approached zero during HUT (-0.38 ± 0.59 vs. 0.31 ± 0.78 radians, supine vs. HUT; p = 0.003), indicative of a shorter time (i.e., improved) response of CVR. These findings indicate that CVR metrics determined by TFA without a CO 2 inhalation do not track HUT-evoked reductions in CVR identified using CO 2 inhalation, suggesting that enhanced cerebral blood flow response to a change in CO 2 using CO 2 inhalation is necessary to assess CVR adequately.

The egg coat including mammalian zona pellucida (ZP) and the avian equivalent, i.e., inner-perivitelline layer (IPVL), is a specialized extracellular matrix being composed of the ZP glycoproteins and surrounds both pre-ovulatory oocytes and ovulated egg cells in vertebrates. The egg coat is well known for its potential importance in both the reproduction and early development, although the underlying molecular mechanisms remain to be fully elucidated. Interestingly, ZP3, one of the ZP-glycoprotein family members forming scaffolds of the egg-coat matrices with other ZP glycoproteins, exhibits extreme but distinctive microheterogeneity to form a large number of isoelectric-point isoforms at least in the chicken IPVL. In the present study, we performed three-dimensional confocal imaging and two-dimensional polyacrylamide-gel electrophoresis (2D-PAGE) of chicken IPVLs that were isolated from the ovarian follicles at different growth stages before ovulation. The results suggest that the relative proportions of the ZP3 isoforms are differentially altered during the structural maturation of the egg-coat matrices. Furthermore, tandem mass spectrometry (MS/MS) analyses and ZP1 binding assays against separated ZP3 isoforms demonstrated that each ZP3 isoform contains characteristic modifications, and there are large differences among ZP3 isoforms in the ZP1 binding affinities. These results suggest that the microheterogeneity of chicken ZP3 might be regulated to be associated with the formation of egg-coat matrices during the structural maturation of chicken IPVL. Our findings may provide new insights into molecular mechanisms of egg-coat assembly processes., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Okumura et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

CEBPA-IGH, a fusion gene of the immunoglobulin heavy-chain locus (IGH) and the CCAAT enhancer-binding protein α (C/EBPα) gene, is recurrently found in B-ALL cases and causes aberrant expression of C/EBPα, a master regulator of granulocyte differentiation, in B cells. Forced expression of C/EBPα in B cells was reported to cause loss of B-cell identity due to the inhibition of Pax5, a master regulator of B-cell differentiation; however, it is not known whether the same mechanism is applicable for B-ALL development by CEBPA-IGH. It is known that a full-length isoform of C/EBPα, p42, promotes myeloid differentiation, whereas its N-terminal truncated isoform, p30, inhibits myeloid differentiation through the inhibition of p42; however, the differential role between p42 and p30 in ALL development has not been clarified. In the present study, we examined the effect of the expression of p42 and p30 in B cells by performing RNA-seq of mRNA from LCL stably transfected with p42 or p30. Unexpectedly, suppression of PAX5 target genes was barely observed. Instead, both isoforms suppressed the target genes of MEF2 family members (MEF2s), other regulators of B-cell differentiation. Similarly, MEF2s target genes rather than PAX5 target genes were suppressed in CEBP-IGH-positive ALL (n = 8) compared with other B-ALL (n = 315). Furthermore, binding of both isoforms to MEF2s target genes and the reduction of surrounding histone acetylation were observed in ChIP-qPCR. Our data suggest that the inhibition of MEF2s by C/EBPα plays a role in the development of CEBPA-IGH-positive ALL and that both isoforms work co-operatively to achieve it., (© 2022 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Angiopoietin-like protein 3 (ANGPTL3) is expressed predominantly in the liver and plays a major role in regulating the circulating triglyceride and lipoprotein fraction concentrations by inhibiting lipoprotein lipase (LPL) activity. Given these physiological roles, ANGPTL3 may play an important role in metabolic changes related to fat accumulation during the fattening period in Japanese Black. This study aimed to reveal the physiological roles of hepatic ANGPTL3 in Japanese Black steers (Bos taurus) during the fattening period and investigate the regulatory effects of hepatic ANGPTL3. To investigate the gene expression and protein localization of ANGPTL3, 18 tissue samples were collected from tree male Holstein bull calves aged 7 wk. Biopsied liver tissues and blood samples were collected from 21 Japanese Black steers during the early (T1; 13 mo of age), middle (T2; 20 mo), and late fattening phases (T3; 28 mo). Relative mRNA expression, blood metabolite concentrations, hormone concentrations, growth, and carcass traits were analyzed. To identify the regulatory factors of hepatic ANGPTL3, primary bovine hepatocytes collected by two Holstein calves aged 7 wk were incubated with insulin, palmitate, oleate, propionate, acetate, or beta-hydroxybutyric acid (BHBA). The ANGPTL3 gene was most highly expressed in the liver, with minor expression in the renal cortex, lungs, reticulum, and jejunum in Holstein bull calves. In Japanese Black steers, relative ANGPTL3 mRNA expressions were less as fattening progressed, and blood triglyceride, total cholesterol, and nonesterified fatty acid (NEFA) concentrations increased. Relative ANGPTL8 and Liver X receptor alpha (LXRα) mRNA expressions decreased in late and middle fattening phases, respectively. Furthermore, relative ANGTPL3 mRNA expression was positively correlated with ANGPTL8 (r = 0.650; P < 0.01) and ANGPTL4 (r = 0.540; P < 0.05) in T3 and T1, respectively, and LXRα showed no correlation with ANGPTL3. Relative ANGTPL3 mRNA expression was negatively correlated with total cholesterol (r = -0.434; P < 0.05) and triglyceride (r = -0.645; P < 0.01) concentrations in T3 and T1, respectively; There was no significant correlation between ANGTPL3 and carcass traits. Relative ANGTPL3 mRNA expression in cultured bovine hepatocytes was downregulated in oleate treatment. Together, these findings suggest that ANGPTL3 downregulation in late fattening phases is associated with the changes in lipid metabolism., (© The Author(s) 2023. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The effects of crude protein (CP) and neutral detergent fiber (NDF) percentages in the diet of Japanese Black steers on rumen fluid properties, blood biochemical properties, and carcass characteristics were examined. Twelve 13-month-old Japanese Black steers were used for this study and slaughtered at 30 months of age. Steers were assigned to a control group (n = 6) and test group (n = 6) and were fed a concentrate containing 12.9%-13.9% CP and 26.5%-29.8% NDF or 9.1%-9.6% CP and 29.9%-31.2% NDF, respectively. Lipopolysaccharide activity levels in rumen fluid were lower in the test group than in the control group. Plasma urea nitrogen concentration and activities of aspartate aminotransferase and γ-glutamyltransferase remained lower in the test group than in the control group. In contrast, plasma vitamin A concentrations remained higher in the test group than in the control group. Carcass characteristics did not significantly differ between the two groups. These results suggest that dietary CP and NDF percentages in feed for Japanese Black steers older than 13 months of age affected rumen fluid properties and blood biochemical properties, indicating a reduced load on the liver with a small effect on carcass characteristics., (© 2023 Japanese Society of Animal Science.)

Intermittent (IH), as opposed to continuous hypoxia (CH), is thought to have beneficial effects on cardiovascular function and health. In the present study, we examined the acute effects of IH and CH (∼80% pulse oxygen saturation via 10% oxygen tank) on peripheral vascular function. Brachial artery flow-mediated dilation (FMD) was used to assess vascular function in 12 young adults (23 ± 5 yr; 8 M/4 F) before and after 50 min of IH (5 cycles; 4-min normoxia/6-min hypoxia per cycle), CH (20-min normoxia followed by 30-min hypoxia), or time control (50-min normoxia) interventions. Brachial artery diameter and velocity were measured using Doppler ultrasound to assess blood flow and shear rate. The total change in shear rate was greater during IH (634 ± 1,073·s -1 , P < 0.05) and CH (321 ± 833·s -1 , P = 0.05) than during time control (-412 ± 789·s -1 ). %FMD was reduced following time control (7.4 ± 1.2 to 5.9 ± 1.1%, P < 0.05) but was maintained following both hypoxia trials (IH: 7.2 ± 1.5 to 7.5 ± 1.5%, P = 0.52; CH: 6.9 ± 1.6 to 6.8 ± 1.4%, P = 0.73). Normalized %FMD for shear rate area under the curve (%FMDSR AUC ) was reduced following the time control trial (4.2 ± 1.4 to 3.7 ± 0.9%, P < 0.05) with no change observed with CH (4.0 ± 1.5 to 3.9 ± 1.4%, P = 0.71). However, %FMDSR AUC increased with IH (3.8 ± 1.1 to 4.5 ± 1.5%, P < 0.05). Our data suggest that acute exposure to hypoxia (both intermittently and continuously) offsets the decline in vascular function after brief inactivity. The potential beneficial effect of hypoxia on peripheral vascular function observed in the current study may be associated with enhanced brachial artery shear in response to the hypoxic challenge.

POEMS syndrome is a rare monoclonal plasma cell disorder, with unique symptoms distinct from those of other plasma cell neoplasms, including high serum VEGF levels. Because the prospective isolation of POEMS clones has not yet been successful, their real nature remains unclear. Herein, we performed single-cell RNA-Seq of BM plasma cells from patients with POEMS syndrome and identified POEMS clones that had Ig λ light chain (IGL) sequences (IGLV1-36, -40, -44, and -47) with amino acid changes specific to POEMS syndrome. The proportions of POEMS clones in plasma cells were markedly smaller than in patients with multiple myeloma (MM) and patients with monoclonal gammopathy of undetermined significance (MGUS). Single-cell transcriptomes revealed that POEMS clones were CD19+, CD138+, and MHC class IIlo, which allowed for their prospective isolation. POEMS clones expressed significantly lower levels of c-MYC and CCND1 than MM clones, accounting for their small size. VEGF mRNA was not upregulated in POEMS clones, directly indicating that VEGF is not produced by POEMS clones. These results reveal unique features of POEMS clones and enhance our understanding of the pathogenesis of POEMS syndrome.

Background: There are currently no scoring-type predictive models using only easily available pre- and intraoperative data developed for assessment of the risk of advanced axillary lymph node metastasis (ALNM) in patients with breast cancer with metastatic sentinel lymph nodes (SLNs). We aimed to develop and validate a scoring system using only pre- and intraoperative data to distinguish between non-advanced (≤ 3 lymph nodes) and advanced (> 3 lymph nodes) ALNM in patients with breast cancer with metastatic SLNs., Methods: We retrospectively identified 804 patients with breast cancer (cT1-3cN0) who had metastatic SLNs and had undergone axillary lymph node dissection (ALND). We evaluated the risk factors for advanced ALNM using logistic regression analysis and developed and validated a scoring system for the prediction of ALNM using training (n = 501) and validation (n = 303) cohorts, respectively. The predictive performance was assessed using the receiver operating characteristic (ROC) curve, area under the curve (AUC), and calibration plots., Results: Ultrasound findings of multiple suspicious lymph nodes, SLN macrometastasis, the ratio of metastatic SLNs to the total number of SLNs removed, and the number of metastatic SLNs were significant risk factors for advanced ALNM. Clinical tumor size and invasive lobular carcinoma were of borderline significance. The scoring system based on these six variables yielded high AUCs (0.90 [training] and 0.89 [validation]). The calibration plots of frequency compared to the predicted probability showed slopes of 1.00 (training) and 0.85 (validation), with goodness-of-fit for the model. When the cutoff score was set at 4, the negative predictive values (NPVs) of excluding patients with advanced ALNM were 96.8% (training) and 96.9% (validation). The AUC for predicting advanced ALNM using our scoring system was significantly higher than that predicted by a single independent predictor, such as the number of positive SLNs or the proportion of positive SLNs. Similarly, our scoring system also showed good discrimination and calibration ability when the analysis was restricted to patients with one or two SLN metastases., Conclusion: Our easy-to-use scoring system can exclude advanced ALNM with high NPVs. It may contribute to reducing the risk of undertreatment with adjuvant therapies in patients with metastatic SLNs, even if ALND is omitted., (© 2022. The Author(s).)

In this study, using enteric methane emissions, we investigated the metabolic characteristics of Japanese Black cattle. Their methane emissions were measured at early (age 13 months), middle (20 months), and late fattening phases (28 months). Cattle with the highest and lowest methane emissions were selected based on the residual methane emission values, and their liver transcriptome, blood metabolites, hormones, and rumen fermentation characteristics were analyzed. Blood β-hydroxybutyric acid and insulin levels were high, whereas blood amino acid levels were low in cattle with high methane emissions. Further, propionate and butyrate levels differed depending on the enteric methane emissions. Hepatic genes, such as SERPINI2, SLC7A5, ATP6, and RRAD, which were related to amino acid transport and glucose metabolism, were upregulated or downregulated during the late fattening phase. The above mentioned metabolites and liver transcriptomes could be used to evaluate enteric methanogenesis in Japanese Black cattle., (© 2022. The Author(s).)

Werner syndrome (WS) is a progeroid syndrome caused by mutations in the WRN gene, which encodes the RecQ type DNA helicase for the unwinding of unusual DNA structures and is implicated in DNA replication, DNA repair, and telomere maintenance. patients with WS are prone to develop malignant neoplasms, including hematological malignancies. However, the pathogenesis of WS-associated hematological malignancies remains uncharacterized. Here we investigated the somatic gene mutations in WS-associated myelodysplastic syndrome/acute myeloid leukemia (MDS/AML). Whole-exome sequencing (WES) of 4 patients with WS with MDS/AML revealed that all patients had somatic mutations in TP53 but no other recurrent mutations in MDS/AML. TP53 mutations were identified at low allele frequencies at more than one year before the MDS/AML stage. All 4 patients had complex chromosomal abnormalities including those that involved TP53. Targeted sequencing of nine patients with WS without apparent blood abnormalities did not detect recurrent mutations in MDS/AML except for a PPM1D mutation. These results suggest that patients with WS are apt to acquire TP53 mutations and/or chromosomal abnormalities involving TP53, rather than other MDS/AML-related mutations. TP53 mutations are frequently associated with prior exposure to chemotherapy; however, all four patients with WS with TP53 mutations/deletions had not received any prior chemotherapy, suggesting a pathogenic link between WRN mutations and p53 insufficiency. These results indicate that WS hematopoietic stem cells with WRN insufficiency acquire competitive fitness by inactivating p53, which may cause complex chromosomal abnormalities and the subsequent development of myeloid malignancies. These findings promote our understanding of the pathogenesis of myeloid malignancies associated with progeria., (Copyright © 2022 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

The genetic basis of leukemogenesis in adults with B-cell acute lymphoblastic leukemia (B-ALL) is largely unclear, and its clinical outcome remains unsatisfactory. This study aimed to advance the understanding of biological characteristics, improve disease stratification, and identify molecular targets of adult B-ALL. Adolescents and young adults (AYA) (15 to 39 years old, n = 193) and adults (40 to 64 years old, n = 161) with Philadelphia chromosome-negative (Ph-) B-ALL were included in this study. Integrated transcriptomic and genetic analyses were used to classify the cohort into defined subtypes. Of the 323 cases included in the RNA sequencing analysis, 278 (86.1%) were classified into 18 subtypes. The ZNF384 subtype (22.6%) was the most prevalent, with 2 novel subtypes (CDX2-high and IDH1/2-mut) identified among cases not assigned to the established subtypes. The CDX2-high subtype (3.4%) was characterized by high expression of CDX2 and recurrent gain of chromosome 1q. The IDH1/2-mut subtype (1.9%) was defined by IDH1 R132C or IDH2 R140Q mutations with specific transcriptional and high-methylation profiles. Both subtypes showed poor prognosis and were considered inferior prognostic factors independent of clinical parameters. Comparison with a previously reported pediatric B-ALL cohort (n = 1003) showed that the frequencies of these subtypes were significantly higher in AYA/adults than in children. We delineated the genetic and transcriptomic landscape of adult B-ALL and identified 2 novel subtypes that predict poor disease outcomes. Our findings highlight the age-dependent distribution of subtypes, which partially accounts for the prognostic differences between adult and pediatric B-ALL., (© 2022 by The American Society of Hematology.)

We investigated the physiological changes during the fattening period and production characteristics in Japanese Black steers bred and raised using the typical feeding system in Japan. Here, 21 Japanese Black steers aged 12 months were used, with experimental period divided into early (12-14 months of age), middle (15-22 months), and late fattening phases (23-30 months). The liver transcriptome, blood metabolites, hormones, and rumen fermentation characteristics were analyzed. Blood triglyceride and non-esterified fatty acid concentrations increased, whereas blood ketone levels decreased, with fattening phases. Blood insulin increased with fattening phases and was positively correlated with carcass weight and marbling in late fattening phases. Rumen fermentation characteristics showed high propionate levels and low butyrate levels in late fattening phases, likely due to increased energy intake. Genes related to glucose metabolism, such as SESN3, INSR, LEPR, and FOXO3, were down-regulated in late fattening phases. Genes related to lipid metabolism, such as FABP4, were up-regulated, whereas FADS1 and FADS2 were down-regulated. These findings suggest that the physiological changes resulted from changes in the energy content and composition of diets. Liver metabolism changed with changes in fat metabolism. Insulin was strongly associated with physiological changes and productivity in Japanese Black cattle., (© 2022. The Author(s).)

Clinical response predictions through image examinations after neoadjuvant chemotherapy (NAC) for breast cancer is important. The present study aimed to evaluate the utility of a novel imaging modality, positron-emission tomography/magnetic resonance imaging (PET/MRI), in predicting the pathological complete response (pCR) to NAC in patients with early breast cancer. A total of 74 patients underwent PET/MRI, mammography (MG), including tomosynthesis, and ultrasound (US) after NAC. The complete response was predicted using each modality and these outcomes were compared accordingly. In terms of PET/MRI, complete response (CR) was defined as the disappearance of 18F-fluorodeoxyglucose uptake and the absence of enhanced lesions with contrast enhanced MRI. In MG and US, undetectable lesions were considered as CR. The background and tumor characteristics of patients were also analyzed between the pCR and non-pCR cases. Overall, 18 (24.3%) of the 74 patients achieved pCR. The overall sensitivity and specificity of PET/MRI were 72.2 and 78.6%, respectively. Both the sensitivity in hormone receptor (HR)-positive cases and the specificity in HR-negative cases were 100%. HR-negative and human epidermal growth factor receptor 2 (HER2)-positive cases demonstrated a significant association with pCR compared with HR-positive cases and triple negative cases (P=0.017). Furthermore, patients with 'mass' type lesions evaluated by MRI before NAC experienced pCR with a higher frequency than those with 'non-mass' type lesions. There was a statistically significant difference between the two groups (P=0.018). In conclusion, PET/MRI is a different diagnostic approach that utilizes a multi-modality system. It demonstrates reasonable diagnostic accuracies of the responses of NAC with reference to hormonal subtypes in breast cancer., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Sekine et al.)