Nicotinamide phosphoribosyl transferase in mammary gland epithelial cells is required for nicotinamide mononucleotide production in mouse milk.

Tissue-specific deficiency of nicotinamide phosphoribosyl transferase (NAMPT), the rate-limiting enzyme of the nicotinamide adenine dinucleotide (NAD+)-salvage pathway, causes a decrease of NAD+ in the tissue, resulting in functional abnormalities. The NAD+-salvage pathway is drastically activated in the mammary gland during lactation, but the significance of this has not been established. To investigate the impact of NAD+ perturbation in the mammary gland, we generated two new lines of mammary gland epithelial-cell-specific Nampt-knockout mice (MGKO). LC-MS/MS analyses confirmed that the levels of NAD+ and its precursor nicotinamide mononucleotide (NMN) were significantly increased in lactating mammary glands. We found that murine milk contained a remarkably high level of NMN. MGKO exhibited a significant decrease in tissue NAD+ and milk NMN levels in the mammary gland during lactation periods. Despite the decline in NAD+ levels, the mammary glands of MGKO appeared to develop normally. Transcriptome analysis revealed that the gene profiles of MGKO were indistinguishable from those of their wild-type counterparts, except for Nampt. Although the NMN levels in milk from MGKO were decreased, the metabolomic profile of milk was otherwise unaltered. The mammary gland also contains adipocytes, but adipocyte-specific deficiency of Nampt did not affect mammary gland NAD+ metabolism or mammary gland development. These results demonstrate that the NAD+ -salvage pathway is activated in mammary epithelial cells during lactation and suggest that this activation is required for production of milk NMN rather than mammary gland development. Our MGKO mice could be a suitable model for exploring the potential roles of NMN in milk.

Impact of Multiplex Polymerase Chain Reaction Test in Patients With Meningitis or Encephalitis.

Background: The objective of this study was to evaluate the impact of the FilmArray meningitis/encephalitis panel (FAME) on length of stay (LOS) and duration of antimicrobial treatment in children and adults in a Japanese community hospital. Methods: This retrospective cohort study was conducted in Japan between January 2016 and December 2022. We included hospitalized patients with cerebrospinal fluid (CSF) samples and those aged <2 months or who had 5 or more white blood cells/µL in the CSF. To compare the days of therapy (DOT) and LOS between the pre-FAME and FAME periods, multivariate Poisson regression analyses were conducted without an offset term. Results: The number of cases undergoing pathogen-specific polymerase chain reaction increased from 3.7% in the pre-FAME period to 57.5% in the FAME period (P < .001). The pathogen identification rate also increased during the FAME period, from 0.4% to 18.7% (P < .001). While the antibacterial DOT was not statistically different between the 2 periods (adjusted rate ratio [aRR], 1.06 [95% confidence interval {CI}, 1.00-1.13]; P = .063]), the antiviral DOT was significantly shorter in the FAME period (aRR, 0.80 [95% CI, .71-.89]; P < .001). Conclusions: This study revealed a significant reduction in antiviral use during the FAME period, whereas LOS and antibacterial use did not decrease. Given the possibility of factors (eg, the COVID-19 pandemic) affecting the epidemiology of meningitis and encephalitis, the indications and impact of the FAME test should be evaluated with continuous monitoring of the epidemiology of meningitis and encephalitis and its clinical impact.

Human sphingomyelin synthase 1 generates diacylglycerol in the presence and absence of ceramide via multiple enzymatic activities.

Sphingomyelin (SM) synthase 1 (SMS1), which is involved in lipodystrophy, deafness, and thrombasthenia, generates diacylglycerol (DG) and SM using phosphatidylcholine (PC) and ceramide as substrates. Here, we found that SMS1 possesses DG-generating activities via hydrolysis of PC and phosphatidylethanolamine (PE) in the absence of ceramide and ceramide phosphoethanolamine synthase (CPES) activity. In the presence of the same concentration (4.7 mol%) of PC and ceramide, the amounts of DG produced by SMS and PC-phospholipase C (PLC) activities of SMS1 were approximately 65% and 35% of total DG production, respectively. PC-PLC activity showed substrate selectivity for saturated and/or monounsaturated fatty acid-containing PC species. A PC-PLC/SMS inhibitor, D609, inhibited only SMS activity. Mn2+ inhibited only PC-PLC activity. Intriguingly, DG attenuated SMS/CPES activities. Our study indicates that SMS1 is a unique enzyme with PC-PLC/PE-PLC/SMS/CPES activities.

Feto-maternal cholesterol transport regulated by ß-Klotho-FGF15 axis is essential for fetal growth.

ß-Klotho (ß-KL) is indispensable to regulate lipid, glucose, and energy metabolism in adult animals. ß-KL is highly expressed in the yolk sac, but its role in the developmental stages has not been established. We hypothesized that ß-KL is required for metabolic regulation in the embryo and aimed to clarify the role of ß-KL during development. Here, we show that ß-KL regulates feto-maternal cholesterol transport through the yolk sac by mediating FGF 15 signaling, and also that impairment of the ß-KL-FGF15 axis causes fetal growth restriction (FGR). Embryos of ß- kl knockout (ß-kl-/-) mice were morphologically normal but exhibited FGR before placental maturation. The body weight of ß-kl-/- mice remained lower after birth. ß-KL deletion reduced cholesterol supply from the maternal blood and led to lipid shortage in the embryos. These phenotypes were similar to those of embryos lacking FGF15, indicating that ß-KL-FGF15 axis is essential for growth and lipid regulation in the embryonic stages. Our findings suggest that lipid abnormalities in early gestation provoke FGR, leading to reduced body size in later life.

Nuclear Magnetic Resonance Detection of Hydrogen Bond Network in a Proton Pump Rhodopsin RxR and Its Alteration during the Cyclic Photoreaction.

Hydrogen bond formation and deformation are crucial for the structural construction and functional expression of biomolecules. However, direct observation of exchangeable hydrogens, especially for oxygen-bound hydrogens, relevant to hydrogen bonds is challenging for current structural analysis approaches. Using solution-state NMR spectroscopy, this study detected the functionally important exchangeable hydrogens (i.e., Y49-ηOH and Y178-ηOH) involved in the pentagonal hydrogen bond network in the active site of R. xylanophilus rhodopsin (RxR), which functions as a light-driven proton pump. Moreover, utilization of the original light-irradiation NMR approach allowed us to detect and characterize the late photointermediate state (i.e., O-state) of RxR and revealed that hydrogen bonds relevant to Y49 and Y178 are still maintained during the photointermediate state. In contrast, the hydrogen bond between W75-εNH and D205-γCOO- is strengthened and stabilizes the O-state.

Diacylglycerol kinase ζ interacts with sphingomyelin synthase 1 and sphingomyelin synthase-related protein via different regions.

We previously reported that diacylglycerol (DG) kinase (DGK) δ interacts with DG-generating sphingomyelin synthase (SMS)-related protein (SMSr), but not SMS1 or SMS2, via their sterile α motif domains (SAMDs). However, it remains unclear whether other DGK isozymes interact with SMSs. Here, we found that DGKζ, which does not contain SAMD, interacts with SMSr and SMS1, but not SMS2. Deletion mutant analyses demonstrated that SAMD in the N-terminal cytosolic region of SMSr binds to the N-terminal half catalytic domain of DGKζ. However, the C-terminal cytosolic region of SMS1 interacts with the catalytic domain of DGKζ. Taken together, these results indicate that DGKζ associates with SMSr and SMS1 in different manners and suggest that they compose new DG signaling pathways.

Impact of the COVID-19 pandemic and multiplex polymerase chain reaction test on outpatient antibiotic prescriptions for pediatric respiratory infection.

This study aimed to evaluate the impact of the prolonged COVID-19 pandemic on outpatient antibiotic prescriptions for pediatric respiratory infections at an acute care hospital in Japan in order to direct future pediatric outpatient antibiotic stewardship. The impact of the COVID-19 pandemic and the FilmArray Respiratory Panel (RP) on outpatient antibiotic prescriptions was assessed from January 2019 to December 2021 using an interrupted time series analysis of children <20 years. The overall antimicrobial prescription rate decreased from 38.7% to 22.4% from the pre-pandemic period to the pandemic. The pandemic (relative risk [RR] level, 0.97 [0.58-1.61]; P = 0.90; RR slope, 1.05 [0.95-1.17] per month; P = 0.310) and FilmArray RP (RR level, 0.90 [0.46-1.75]; P = 0.75; RR slope, 0.95 [0.85-1.06] per month; P = 0.330) had no significant effect on the monthly antibiotic prescription rates. The COVID-19 pandemic was not significantly related to the antibiotic prescription rate, suggesting that it did not impact physicians' behavior toward antibiotic prescriptions. Replacing rapid antigen tests with the FilmArray RP introduced on December 1, 2020, did not affect the magnitude of the reduction in antibiotic prescription rate for pediatric respiratory infections.

Exponential increase of the gestational-age-specific incidence of preeclampsia onset (COPE study): a multicenter retrospective cohort study in women with maternal check-ups at <20 weeks of gestation in Japan.

According to the 2004 Japanese definition, early-onset (EO) preeclampsia (PE) is defined as PE occurring at <32 weeks of gestation. This was based on the presence of "dual peaks" (30-31 and 34-35 weeks) in the prevalence of severe forms of hypertension. In contrast, the international definition adopted a cutoff of 34 weeks based on the consensus. Our aim was to investigate whether there were "dual peaks" in the gestational-age-specific incidence or prevalence of PE onset in pregnant women who underwent maternal check-ups at <20 weeks of gestation in a multicenter retrospective cohort study. Diagnoses of PE and superimposed preeclampsia (SPE) were based on the new Japanese definition. A total of 26,567 pregnant women with singleton pregnancy were investigated. The best fitting equations for the distribution of the onset of gestational-age-specific incidence (hazard) rates of PE/SPE, PE, and PE with severe hypertension (a systolic blood pressure ≥160 and/or a diastolic blood pressure ≥110 mmHg) were investigated using the curve estimation function in SPSS. PE/SPE occurred in 1.83% of the patients. EO-PE/SPE with onset at <32 and <34 weeks of gestation and preterm PE/SPE occurred in 0.38, 0.56, and 1.07% of the patients, respectively. Gestational-age-specific incidence rates of PE/SPE, PE, and PE with severe hypertension showed exponential increases, with very high R2 values (0.975, 0.976, and 0.964, respectively). There were no "dual peaks" in the prevalence rates of women with SPE/PE, PE, and PE with severe hypertension. In conclusion, the absence of "dual peaks" refutes the previous rationale of EO-PE being defined as PE at <32 weeks of gestation. Further studies to determine an appropriate definition of EO-PE/SPE are needed.

Diacylglycerol kinase η regulates cell proliferation and its levels are elevated by glucocorticoids in undifferentiated neuroblastoma cells.

Knockout mice of diacylglycerol kinase (DGK) η, which has been repeatedly suggested to be associated with bipolar disorder (BPD) by genome-wide association studies, exhibited abnormal behaviors similar to the manic phase of BPD. Chronic stress is also linked to changes in mood symptoms, including BPD. In the present study, we analyzed the effects of the glucocorticoid stress hormones, triamcinolone acetonide (TAA) and dexamethasone (DEX), on DGKη protein levels in neuroblastoma cell lines, Neuro-2a and SH-SY5Y. The protein levels of DGKη were significantly increased in the undifferentiated Neuro-2a and SH-SY5Y cells by TAA and DEX, but not in the differentiated neuroblastoma cells. To assess the functions of DGKη in undifferentiated neuroblastoma cells, we established DGKη-deficient SH-SY5Y cells using the clustered regularly interspaced palindromic repeat/caspase 9 system. Notably, proliferation of DGKη-deficient SH-SY5Y cells was markedly attenuated, concomitant with the decrease in levels of phosphorylated extracellular signal-regulated kinase. Taken together, these results suggest that DGKη levels are increased in undifferentiated neuroblastoma cells by glucocorticoid stress hormones and regulate cell proliferation.

Difficulty in predicting intra-abdominal adhesion before cesarean section: A case report.

Severe adhesions between the bladder and uterus necessitated an atypical incision in the cesarean section of a woman with endometriosis. This could not be predicted with pre-surgery MRI. No methods in the literature are able to predict adhesions with true certainty; it is therefore still difficult to diagnose intra-abdominal adhesions.

Burden of Pediatric Central Nervous System Infection and Cost-Benefit Simulation of Multiplex Polymerase Chain Reaction in Japan.

To investigate the clinical use of multiplex polymerase chain reaction (mPCR) in Japan, epidemiological and clinical data for central nervous infections are needed. Here, we report on the epidemiology and economic burden of central nervous system infections and a simulation of the cost-benefit analysis of the Filmarray® Meningitis/Encephalitis (FAME) test for possible clinical use in Japan. We performed FAME tests on samples from 27 patients with pleocytosis aged between 0 and 20 years seen in six community hospitals in Nara and Osaka prefectures. All clinical management procedures were performed without knowledge of the mPCR test results. We analyzed the clinical data and calculated the required reduction in average length of stay for the FAME test to be cost-beneficial. Among the 27 cases, the FAME test revealed causal pathogens in 13 cases (48.1%). The average medical and social costs per case were ¥299,118 ($2,719.2) and ¥171,768 ($1,561.5), respectively. The minimal needed reduction in average length of stay for the FAME test to be cost-beneficial was 0.32- 0.86 days per meningitis case. The result can be informative for evaluating the cost-effectiveness of the clinical use of the FAME test in Japan.

The Epidemiology of Admission-Requiring Pediatric Respiratory Infections in a Japanese Community Hospital Using Multiplex PCR.

Respiratory tract infections (RTIs) are the most common diseases globally among children. This study aimed to assess the epidemiology of admission-requiring pediatric RTI cases and evaluate the effect of the pathogen type on the length of hospital stay (LOS) using the FilmArray® respiratory panel, a multiplex PCR test. The age-specific distribution and seasonality of viruses were investigated between March 26, 2018 and April 12, 2019. Multivariable linear regression analyses were performed to evaluate the effect of pathogen type and coinfection on LOS. Among 153 hospitalized RTI patients, respiratory syncytial virus was the leading cause of hospitalization in infants < 12 months of age (27.7%). Human metapneumovirus and parainfluenza virus were also major causes of hospitalization in patients aged 2-3 years (22.6% and 22.6%, respectively). In the multivariable linear regression model excluding rhinovirus/enterovirus, there was a significant association between viral coinfection and longer LOS (p = 0.012), while single viral infection of any type was not positively correlated with LOS. This study revealed the epidemiology of admission-requiring pediatric RTIs.

The impact analysis of a multiplex PCR respiratory panel for hospitalized pediatric respiratory infections in Japan.

BACKGROUND: Rapid molecular diagnosis of infections has contributed to timely treatments and antimicrobial stewardship. However, the benefit and cost-effectiveness vary in each country or community because they have different standard practices and health care systems. In Japan, rapid antigen tests (RATs) have been frequently used for pediatric respiratory infections. We investigated the impact and cost-effectiveness of a multiplex PCR (mPCR) respiratory panel for pediatric respiratory infections in a Japanese community hospital. METHODS: We replaced RATs with an mPCR respiratory panel (FilmArray®) for admitted pediatric respiratory infections on March 26, 2018. We compared the days of antimicrobial therapy (DOT) and length of stay (LOS) during the mPCR period (March 2018 to April 2019) with those of the RAT period (March 2012 to March 2018). RESULTS: During the RAT and mPCR periods, 1132 and 149 patients were analyzed. The DOT/case was 12.82 vs 8.56 (p < 0.001), and the LOS was 8.18 vs 6.83 days (p = 0.032) in the RAT and mPCR groups, respectively. The total costs during admissions were ∖258,824 ($2331.7) and ∖243,841 ($2196.8)/case, respectively. Pathogen detection rates were 30.2% vs 87.2% (p < 0.001). CONCLUSION: Compared to conventional RATs, the mPCR test contributed to a reduction in the DOT and LOS in a Japanese community hospital for admission-requiring pediatric respiratory infections. However, a proper stewardship program is essential to further reduce the unnecessary usage of antimicrobials.

Methyl-selective isotope labeling using α-ketoisovalerate for the yeast Pichia pastoris recombinant protein expression system.

Methyl-detected NMR spectroscopy is a useful tool for investigating the structures and interactions of large macromolecules such as membrane proteins. The procedures for preparation of methyl-specific isotopically-labeled proteins were established for the Escherichia coli (E. coli) expression system, but typically it is not feasible to express eukaryotic proteins using E. coli. The Pichia pastoris (P. pastoris) expression system is the most common yeast expression system, and is known to be superior to the E. coli system for the expression of mammalian proteins, including secretory and membrane proteins. However, this system has not yet been optimized for methyl-specific isotope labeling, especially for Val/Leu-methyl specific isotope incorporation. To overcome this difficulty, we explored various culture conditions for the yeast cells to efficiently uptake Val/Leu precursors. Among the searched conditions, we found that the cultivation pH has a critical effect on Val/Leu precursor uptake. At an acidic cultivation pH, the uptake of the Val/Leu precursor was increased, and methyl groups of Val and Leu in the synthesized recombinant protein yielded intense 1H-13C correlation signals. Based on these results, we present optimized protocols for the Val/Leu-methyl-selective 13C incorporation by the P. pastoris expression system.

Identification of methylated tubulin through analysis of methylated lysine.

Post-translational modifications to tubulin such as acetylation and detyrosination play important roles in microtubule functions. Methylation is an important post-translational modification; however, to date, few methylated tubulins have been identified. In the present study, we developed a method for analyzing methylated lysine with the aim of identifying methylated tubulin. This method involves four steps: (1) acid hydrolysis of tubulin into amino acids, (2) selective extraction of methylated lysine using a monolithic-silica disk-packed spin column, (3) fluorescence derivatization of methylated lysine with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), and (4) separation of NBD-methylated lysine on a column consisting of C18, cation and anion ligand, and fluorescence detection. Using the newly developed method, the dimethylation of lysine in tubulin was identified. This new method could be applied to searches for other methylated proteins.

Inhibition by non-steroidal anti-inflammatory drugs of compound action potentials in frog sciatic nerve fibers.

AIMS: Although antinociception produced by non-steroidal anti-inflammatory drugs (NSAIDs) is partly attributed to nerve conduction inhibition, this has not been thoroughly examined yet. The aim of the present study was to reveal quantitatively how various types of NSAIDs affect compound action potentials (CAPs), a measure of nerve conduction. MAIN METHODS: CAPs were recorded from the frog sciatic nerve by using the air-gap method. KEY FINDINGS: Soaking the sciatic nerve with acetic acid-based NSAIDs (diclofenac and aceclofenac) reduced the peak amplitude of CAP in a concentration-dependent manner; their IC50 values were 0.94 and 0.47 mM, respectively. Other acetic acid-based NSAIDs (indomethacin, acemetacin and etodolac) also inhibited CAPs [the extent of inhibition: some 40% (1 mM), 40% (0.5 mM) and 15% (1 mM), respectively], except for sulindac and felbinac at 1 mM that had no effects on CAP peak amplitudes. A similar inhibition was produced by fenamic acid-based NSAIDs [tolfenamic acid (IC50 = 0.29 mM), meclofenamic acid (0.19 mM), flufenamic acid (0.22 mM) and mefenamic acid] which are similar in chemical structure to diclofenac and aceclofenac; their derivatives (2,6-dichlorodiphenylamine and N-phenylanthranilic acid) also inhibited. On the other hand, salicylic acid-based (aspirin), propionic acid-based (ketoprofen, naproxen, ibuprofen, loxoprofen and flurbiprofen) and enolic acid-based (meloxicam and piroxicam) NSAIDs had no effects on CAP peak amplitudes. SIGNIFICANCE: At least a part of antinociception produced by NSAIDs used as a dermatological drug to alleviate pain may be attributed to their inhibitory effects on nerve conduction, which depend on the chemical structures of NSAIDs.

Spatiotemporal quantification of subcellular ATP levels in a single HeLa cell during changes in morphology.

The quantitative relationship between change in cell shape and ATP consumption is an unsolved problem in cell biology. In this study, a simultaneous imaging and image processing analysis allowed us to observe and quantify these relationships under physiological conditions, for the first time. We focused on two marginal regions of cells: the microtubule-rich 'lamella' and the actin-rich 'peripheral structure'. Simultaneous imaging and correlation analysis revealed that microtubule dynamics cause lamellar shape change accompanying an increase in ATP level. Also, image processing and spatiotemporal quantification enabled to visualize a chronological change of the relationships between the protrusion length and ATP levels, and it suggested they are influencing each other. Furthermore, inhibition of microtubule dynamics diminished motility in the peripheral structure and the range of fluctuation of ATP level in the lamella. This work clearly demonstrates that cellular motility and morphology are regulated by ATP-related cooperative function between microtubule and actin dynamics.