Expansion of the osteocytic lacunar-canalicular system involved in pharmacological action of PTH revealed by AI-driven fluorescence morphometry in female rabbits.

Osteoporosis is an age-related disorder that is characterized by reduced bone mass. Its prevention and treatment are important healthcare issues for maintaining social activity in aged societies. Although bone fractures mostly occur at sites of weakened cortical bone, pathophysiological and pharmacological evaluations of bone mass have tended to be predominantly assessed in trabecular bone. To statistically characterize cortical bone remodeling, we originally established multimode fluorescence imaging and artificial intelligence (AI)-driven morphometric analyses in six-month-old female rabbits with well-defined cortical remodeling, similar to that in humans. We evaluated three distinct administration frequencies of teriparatide [TPTD; human parathyroid hormone, hPTH (1-34)]: once (1/w), twice (2/w), and seven times (7/w) a week, with the same total dose (140 µg/kg/week). Our analyses revealed significant expansions of the osteocytic lacunar-canalicular system and Haversian canals accompanied by the development of cortical porosity and endosteal naïve bone formation induced by a frequent administration regimen (7/w) of TPTD; however, once-weekly (1/w) and twice-weekly (2/w) administration of TPTD showed little effect. These findings demonstrate a clear contrast between the effects of frequent and infrequent administration of TPTD on cortical bone metabolism and suggest that osteocytic bone remodeling is involved in the pharmacological action of PTH.

Prescription trends in anti-seizure medications for adult patients with epilepsy in Japan: A retrospective cohort study using the database of health insurance claims between 2015 and 2019.

OBJECTIVE: To investigate whether newer anti-seizure medications (ASMs) are widely prescribed for a range of adult patients in Japan, including patients with previously and newly diagnosed epilepsy, or with focal and generalized epilepsies. METHODS: A retrospective cohort study was conducted using the Japanese insurance claims database including 8.4 million people to identify adults (≥16 years of age) with epilepsy diagnosis code identified between January 2015 and December 2018. Patients were included in the prevalent population if epilepsy was already diagnosed at baseline, and in the incident population if prior baseline data for at least 12 months included no epilepsy diagnosis code or ASM prescription. Patients were followed up from the month when the initial oral ASM was prescribed for up to 4 years until the end of 2019 as long as at least one ASM was prescribed. Proportions of prescribed oral ASMs were analyzed by population with epilepsy (prevalent vs. incident) and classification (focal vs. generalized). Anti-seizure medications were classified into older vs. newer ASMs according to the date of approval before and after 1990, respectively. RESULTS: A total of 24,691 patients fulfilled the eligibility criteria for the analysis. Of these, 21,046 and 3,645 were included in the prevalent and incident populations, respectively. The proportion of older ASMs significantly decreased, whereas the proportion of newer ASMs significantly increased (p < 0.0001) during the study period. This trend was more apparent in the population with incident epilepsy than in that with prevalent epilepsy, and was also apparent in the subgroup of focal epilepsy, but not in that of generalized epilepsy. Levetiracetam was the most frequently prescribed of the newer ASMs. CONCLUSION: Newer ASMs became more widely prescribed throughout the study period in populations with both prevalent and incident epilepsies, as well as the subpopulation with focal epilepsy. The advantages of newer ASMs such as better safety profiles may have led to the increasing proportions of prescriptions and newer ASMs may increase the treatment options for patients.

Removal of blood amyloid-ß with hemodialysis reduced brain amyloid-ß, confirmed by brain imaging: a case report.

The accumulation of amyloid-ß protein (Aß) in the brain signifies a major pathological change of Alzheimer's disease (AD). Extracorporeal blood Aß removal system (E-BARS) has been under development as a tool for enhancing the clearance of Aß from the brain. Previously, we revealed that dialyzers remove blood Aßs effectively, evoking substantial Aß influx into the blood during hemodialysis sessions as one form of blood Aß removal by E-BARS, and that postmortem brains of hemodialysis patients exhibited lower Aß accumulation. Here, we present a case report of a 77-year-old male patient with end-stage renal failure whose Aß accumulation in the brain declined by initiating and continuing hemodialysis for 6 months. This report suggests that blood Aß removal by E-BARS could be an effective therapeutic method for AD.

Administration frequency as well as dosage of PTH are associated with development of cortical porosity in ovariectomized rats.

To investigate whether the administration frequency of parathyroid hormone (PTH) is associated with the development of cortical porosity, this study established 15 dosage regimens of teriparatide [human PTH(1-34), TPTD] with four distinct concentrations and four distinct administration frequencies of TPTD to 16-week-old ovariectomized rats. Our analyses demonstrated that the bone mineral density, mechanical properties, and bone turnover were associated with the total amount of TPTD administered. Our observations further revealed that the cortical porosity was markedly developed as a result of an increased administration frequency with a lower concentration of total TPTD administration in our setting, although the highest concentration also induced cortical porosity. Deconvolution fluorescence tiling imaging on calcein-labeled undecalcified bone sections also demonstrated the development of cortical porosity to be closely associated with the bone site where periosteal bone formation took place. This site-specific cortical porosity involved intracortical bone resorption and an increased number and proximity of osteocytic lacunae, occasionally causing fused lacunae. Taken together, these findings suggested the involvement of local distinctions in the rate of bone growth that may be related to the site-specific mechanical properties in the development of cortical porosity induced by frequent and/or high doses of TPTD.

The Direct Comparison of Two Interferon-gamma Release Assays in the Tuberculosis Screening of Japanese Healthcare Workers.

Objective Two interferon-gamma release assays (IGRAs), the QuantiFERON-TB Gold In-Tube (QFT-GIT) and T-SPOT.TB (T-SPOT), are commercially available. The agreement between the two IGRAs in the screening of healthcare workers (HCWs) for latent tuberculosis is not well known. Methods The QFT-GIT and T-SPOT tests were performed for the baseline tuberculosis screening of 654 HCWs who worked at Mie University Hospital in Japan. The results of the two tests were directly compared. Results Nineteen (2.9%), 28 (4.3%) and 33 (5.0%) of the 654 HCWs were found to be positive by the QFT-GIT, T-SPOT, and the QFT-GIT and/or T-SPOT methods using cut-off values of 0.35 IU/mL (QFT-GIT) and 6 spots (T-SPOT). After excluding 4 cases with indeterminate results, there were 14 concordant positive (2.2%), 618 concordant negative (95.1%), and 18 discordant (2.8%) results using the cut-off values of 0.35 IU/mL (QFT-GIT) and 6 spots (T-SPOT). The agreement of the two IGRAs was 97.2% (κ=0.595). When cut-off values of 0.35 IU/mL (QFT-GIT) and 8 spots (T-SPOT) were applied, there were 11 concordant positive (1.7%), 626 concordant negative (96.3%), and 13 discordant (2.0%) results, with 98.0% agreement (κ=0.618). When the borderline criteria for the QFT-GIT (0.1 to <0.35 IU/mL) and T-SPOT (5-7 spots) were applied, there were 11 concordant positive (1.7%), 11 concordant borderline (1.7%), 586 concordant negative (90.2%), and 42 discordant (6.5%) results, with 93.5% agreement between the two methods (κ=0.538). Conclusion When standard cut-off values were used, the agreement between the two IGRAs in the tuberculosis screening of Japanese HCWs was moderate to high. Importantly, some HCWs showed discordant results, especially those whose results were in the borderline zones.

Increased cortical porosity is associated with daily, not weekly, administration of equivalent doses of teriparatide.

INTRODUCTION: The pharmacokinetic profile of parathyroid hormone (PTH) determines its effects on bone resorption and formation. When administered intermittently, anabolic effects are favored in comparison with the continuous treatment. Among the intermittent treatment regimens, lower frequency of administration may have a lower effect on bone remodeling. We therefore hypothesized that weekly administration of teriparatide will produce less increase in intracortical remodeling and porosity than reported using daily treatment. METHODS: We treated 17 female New Zealand white rabbits aged 6months for 1month with teriparatide [human PTH(1-34)] as follows. (i) Vehicle-treated Control (n=4); (ii) 20µg/kg daily (n=3); (iii) 40µg/kg daily (n=3); (iv) 140µg/kg weekly (n=3); (v) 280µg/kg weekly (n=4). Proximal femurs were imaged ex vivo using micro-CT (Scanco Viva CT-40) at 15µmvoxel size. Areas, pore size, and porosity were analyzed on the total, compact cortex (CC), and transitional zones in a 10mm length region of interest (ROI) starting at the midshaft using StrAx1.0. RESULTS: Compared to controls, the 20µg/kg daily was associated with 3.0% higher porosity in the transitional zone (p=0.09) while the 40µg/kg daily was associated with a higher porosity in the cortex (8.7%; p=0.04) and in the transitional zone (5.7%; p=0.007). The daily regimens were also associated with a greater proportion of porosity due to pores >15µm2; particularly in the transitional zone where 20 and 40µg/kg daily increased porosity 2 fold (p=0.06) and 5 fold (p=0.04) relative controls respectively. The 140 and 280µg/kg weekly were not associated with an increase in porosity. There was no difference in total, compact or transitional zone cross sectional areas between the groups. CONCLUSION: Effects of intermittent teriparatide depend on the dose and frequency of administration. Daily dosing, particularly the higher dose, but not weekly dosing, increased cortical porosity. Work is needed to investigate the effects of the regimens on bone formation.

Light-induced silencing of neural activity in Rosa26 knock-in mice conditionally expressing the microbial halorhodopsin eNpHR2.0.

Temporally precise inhibition of genetically defined cell populations in intact nervous systems has been enabled by the microbial halorhodopsin NpHR, a fast, light-activated chloride pump. Here, we report the generation of new mouse strains that express eNpHR2-EYFP fusion proteins after Cre- and/or Flp-mediated recombination to silence neural activity in vivo. In these mouse strains, Cre/Flp recombination induced a high-level of eNpHR2-EYFP expression. Slice whole-cell patch clamp experiments confirmed that eNpHR2-EYFP-expressing neurons could be optically hyperpolarized and inhibited from firing action potentials. Thus, these mouse strains offer powerful tools for light-induced silencing of neural activity in genetically defined cell populations.

In vivo evaluation of ΦC31 recombinase activity in transgenic mice.

Genome engineering strategies employing site-specific recombinases (SSRs) such as Cre, Flp and PhiC31, have become powerful tools to analyze gene function and manipulate neural network in vertebrates. In the present study, we evaluated the ability of PhiC31 phage integrase to induce genomic recombination in transgenic mice. PhiC31 is the integrase encoded by the Streptomyces bacteriophage that promotes recombination between heterotypic attP and attB sites. We generated transgenic mice that express codon-optimized PhiC31 (PhiC31o) in neural stem/progenitor cells or tyrosine hydroxylase (TH) expressing catecholaminergic neurons. PhiC31 was functional in these cells and capable of excising a transcriptional stop cassette flanked by PhiC31-specific attP/B recognition sites. PhiC31-ER(T2), a fusion protein of PhiC31o (without the nuclear localization signal) and the mutated ligand-binding domain of the human estrogen receptor, was able to induce recombination in neural stem/progenitor cells in a tamoxifen-dependent manner, but the recombination rate was less efficient than for PhiC31. Thus, PhiC31 integrase is functional in transgenic mice and is suitable for mosaic recombination in restricted cell populations.

A multifunctional teal-fluorescent Rosa26 reporter mouse line for Cre- and Flp-mediated recombination.

Reporters of Cre and/or Flp activity are important for defining the spatial and temporal extent of Cre/Flp-mediated recombination. Here, we describe R26-CAG-LF-mTFP1, a multifunctional fluorescent reporter mouse that strongly expresses mTFP1 (bright teal fluorescent protein) after Cre- and Flp-mediated recombination. To meet the need for single recombinase-mediated reporter expression, we generated derivatives of R26-CAG-LF-mTFP1. The germline excision of the Frt-flanked stop cassette in R26-CAG-LF-mTFP1 generated a Cre-dependent reporter (R26-CAG-LoxP-mTFP1). Similarly, R26-CAG-FRT-mTFP1, in which the loxP-flanked stop cassette was excised in the germline, requires only Flp to activate mTFP1 expression.

Molecular cloning and characterization of a novel gamma-CGTase from alkalophilic Bacillus sp.

We found a novel cyclodextrin glucanotransferase (CGTase) from alkalophilic Bacillus sp. G-825-6. The enzyme was expressed in the culture broth by recombinant Bacillus subtilis KN2 and was purified and characterized. The enzyme named CGTase825-6 showed 95% amino acid sequence identity with a known enzyme beta-/gamma-CGTase from Bacillus firmus/lentus 290-3. However, the product specificity of CGTase825-6 differed from that of beta-/gamma-CGTase. CGTase825-6 produced gamma-cyclodextrin (CD) as the main product, but degradation of gamma-CD was observed with prolonged reaction. The product specificity of the enzyme was positioned between gamma-CGTase produced by Bacillus clarkii 7364 and B. firmus/lentus 290-3 beta-/gamma-CGTase. It showed that the difference of product specificity was dependent on only 28 amino acid residues in 671 residues in CGTase825-6. We compared the amino acid sequence of CGTase825-6 and those of other CGTases and constructed a protein structure model of CGTase825-6. The comparison suggested that the diminished loop (Val138-Asp142) should provide subsite -8 for gamma-CD production and that Asp142 might have an important role in product specificity. CGTase825-6 should be a useful tool to produce gamma-CD and to study the differences of producing mechanisms between gamma-CD and beta-CD.