Development and Characterization of a Novel FVB-PrkdcR2140C Mouse Model for Adriamycin-Induced Nephropathy.

The Adriamycin (ADR) nephropathy model, which induces podocyte injury, is limited to certain mouse strains due to genetic susceptibilities, such as the PrkdcR2140C polymorphism. The FVB/N strain without the R2140C mutation resists ADR nephropathy. Meanwhile, a detailed analysis of the progression of ADR nephropathy in the FVB/N strain has yet to be conducted. Our research aimed to create a novel mouse model, the FVB-PrkdcR2140C, by introducing PrkdcR2140C into the FVB/NJcl (FVB) strain. Our study showed that FVB-PrkdcR2140C mice developed severe renal damage when exposed to ADR, as evidenced by significant albuminuria and tubular injury, exceeding the levels observed in C57BL/6J (B6)-PrkdcR2140C. This indicates that the FVB/N genetic background, in combination with the R2140C mutation, strongly predisposes mice to ADR nephropathy, highlighting the influence of genetic background on disease susceptibility. Using RNA sequencing and subsequent analysis, we identified several genes whose expression is altered in response to ADR nephropathy. In particular, Mmp7, Mmp10, and Mmp12 were highlighted for their differential expression between strains and their potential role in influencing the severity of kidney damage. Further genetic analysis should lead to identifying ADR nephropathy modifier gene(s), aiding in early diagnosis and providing novel approaches to kidney disease treatment and prevention.

Assessment of thiamylal sodium as a euthanasia drug in mice.

Euthanasia agents should rapidly induce death and loss of consciousness without causing pain or distress. Various methods exist for the euthanasia of laboratory animals, and injectable anesthetics, particularly barbiturate derivatives, are widely used due to the rapid onset of unconsciousness induced by these agents. Moreover, pharmaceutical-grade drugs should be used to eliminate undesirable side effects as much as possible. However, in Japan, the sale of pharmaceutical-grade pentobarbital sodium (PB) ended in 2019, and that of secobarbital sodium (SB) ended in 2023, leading to a demand for new pharmaceutical-grade injectable euthanasia drugs. This study evaluates thiamylal sodium (TM), a barbiturate derivative that is available domestically, as a euthanasia agent for mice. The results showed that when administered at dosages of 200 mg/kg or more, TM exhibited effects equivalent to those of PB and SB. In addition, the impact of TM administration on hematological characteristics was examined. In female mice administered TM, decreased blood chloride and calcium levels and increased aspartate aminotransferase and alanine aminotransferase levels, which are markers of liver damage, were observed. These findings suggest that high concentrations of TM may affect renal and liver function. This study revealed that TM is effective as a euthanasia agent at dosages of 200 mg/kg or more. However, considering the potential risks of renal and liver damage due to TM administration, it may be preferable to use alternative euthanasia drugs when these risks could affect the objectives or outcomes of the research.

Comparison of masticatory muscle activity between young adults and elderly participants using a novel standardized bite device.

OBJECTIVES: Standardized bite training is required to prevent oral hypofunction in elderly individuals. We aimed to compare masticatory muscle activity between 24 young adults and 16 pre-elderly individuals during a biting task using a novel standardized bite device (BD). METHODS: The BD was made of silicone rubber and included a high-force or low-force plate spring or no plate spring (dummy). The compressive stiffness of the material in each BD was measured using a texture analyzer. All participants performed a biting task 50-times at a rate of 1/s each using the three types of BDs on the right first molar. Electromyographic (EMG) activity was recorded bilaterally from the masseter and temporalis muscles. The variability of each biting training session was calculated as the coefficient of variance (CV) from the EMG activity during each biting task for each muscle. Masticatory muscle fatigue was assessed using a numerical rating scale (NRS). RESULTS: Compressive stiffness was significantly different between each BD (P < 0.001). The CV and NRS scores were not significantly different between the groups. The EMG activities during each bite task in all muscles were not significantly different for any measurement item between the age groups. The EMG activities of high- and low-force BDs in the right temporalis (ipsilateral) muscle were significantly higher than those of the dummy BD (P < 0.001). CONCLUSIONS: Compressive stiffness of the BD affected EMG activity only in the ipsilateral temporalis muscle, but not in the masseter or contralateral temporalis muscles, without any age effect.

Analysis of definite awake bruxism using a portable electromyography device.

PURPOSE: This study aimed to compare awake bruxism events between subjective and objective evaluations using a questionnaire survey and a modified portable electromyography (EMG) device, and to examine correlations between sleep quality and awake bruxism. METHODS: The Epworth Sleepiness Scale (ESS), Pittsburgh Sleep Quality Index (PSQI), and awareness of awake bruxism as clarified via interviews were conducted on 34 participants as subjective evaluations. The EMG device was used to record left temporal muscle activity for 6.5 h (from 09:00 to 15:30) and the number of awake bruxism episodes per hour. The participants were then classified into "bruxer" and "non-bruxer" groups based on the number of awake bruxism episodes. RESULTS: The mean number of awake bruxism episodes per hour was 33.6 ± 21.4, and 23% of the participants who reported having no awareness of awake bruxism in the interviews were defined as "bruxers" in the objective evaluations. In the bruxer group, positive correlations were found between the number of awake bruxism episodes and both ESS and PSQI scores. CONCLUSION: These findings suggest that objective measurements using a portable EMG device can increase the diagnostic accuracy for awake bruxism, and that sleep quality is a major risk factor for awake bruxism.

Effect of clinical experience and training with visual feedback on standardized palpation outcomes-Potential implications for assessment of jaw muscle sensitivity.

BACKGROUND: The evaluation of muscle pain and sensitivity by manual palpation is an important part of the clinical examination in patients with myalgia. However, the effects of clinical experience and visual feedback on palpation of the masticatory muscles with or without a palpometer are not known. OBJECTIVE: To estimate the effects of clinical experience and visual feedback on the accuracy of palpation in standardized settings. METHODS: Thirty-two dentists (age 35 ± 11 years) classified as either specialists (n = 16) or generalists (n = 16) participated in this experiment. All dentists were instructed to target force levels of 500- or 1000-gf, as determined on an electronic scale using either standardized palpometers or manual palpation (MP). All dentists participated in four different tests: MP, MP with visual feedback (MPVF), palpometer (PAL) and PAL with visual feedback (PALVF). Actual force values for each type of palpation from 0 to 2, 2 to 5 and 0 to 5 s were analysed by calculating target force level. RESULTS: The relative differences during 2-5 and 0-5 s with 1000 gf were significantly lower for generalists than for specialists (p < .05). In generalists and specialists, the coefficients of variation and the relative differences during 2-5 s were significantly lower for PAL and PALVF than for MP (p < .05). CONCLUSIONS: These findings suggest that the use of a palpometer, but not clinical experience with palpation of masticatory muscles, increases the accuracy of palpation, and ≥2 s of palpation with a palpometer is optimal for masticatory muscles.

Latissimus dorsi musculocutaneous flap grafting to the infected recipient site in a patient with irradiated locally advanced breast cancer and multiple lung metastases.

BACKGROUND: Musculocutaneous (MC) flaps are more resistant to infection than implants, but no clinical results have been reported so far about the grafting of MC flap to the overtly infected sites. CASE PRESENTATION: A 66-year-old woman had received radiotherapy, a total dose of 50 Gy, to her large mucinous breast cancer to control bleeding from the tumor and was referred to our hospital for further treatment. On her first visit to our hospital, her left breast showed radiation-induced total necrosis with Pseudomonas aeruginosa infection. Removal of the necrotic breast tissue resulted in direct exposure of the left ribs and intercostal muscles with intractable chest pain requiring analgesics. The presence of concomitant life-threatening multiple lung metastases made us change the treatment from letrozole and palbociclib to bevacizumab and paclitaxel, leading to marked regression of the lung metastases. To alleviate her chest pain and get local wound healing, we treated the patient with latissimus dorsi (LD)-MC flap grafting to the exposed chest wall after four months of taxane-containing chemotherapy. The patient has got marked pain relief immediately after the operation. Skin island of the grafted LD-MC flap showed no problems for 4 days just after the operation but gradually turned out to be edematous to ill-colored in the distal part of the skin island. Post-operative clinical outcome suggested that Pseudomonas aeruginosa infection might have had some adverse effect, e.g., microemboli, on MC flap blood flow. Partial necrosis of the LD-MC flap made the patient receive conservative wound management for a very long period of 11 months, finally leading to complete wound healing. After palliative surgery, the patient has been receiving fulvestrant and palbociclib for 14 months and doing well with good control of multiple lung metastases. CONCLUSIONS: Breast surgical oncologists should note that partial flap necrosis can occur when a LD-MC flap is grafted to the infected recipient site and consider the anti-coagulant therapy just after the operation to avoid the adverse effects of the infection.

Juvenile semi-wild fish have a higher metabolic rate than farmed fish.

Fish from commercially farmed stocks are often released into the natural environment to supplement wild populations. This practice is often applied to salmonid fish as they are an essential fishery resource and also used for recreational angling. However, farmed fish tend to show lower survival rates after release than wild fish. For this reason, the release of semi-wild fish is increasingly used in Japan; these fish are generated using female fish from domesticated stocks and male fish of wild origin. The survival rate of released semi-wild fish is higher than that of farmed fish, but the reason for this is unknown. This study compared the metabolism and swimming performance of semi-wild and farmed masu salmon (Oncorynchus masou). The analyses showed that resting metabolic rate (RMR), maximum metabolic rate (MMR) and swimming speeds that minimize energy costs of travel (optimal swimming speed) were higher in semi-wild fish than in farmed fish. Critical swimming speed did not differ significantly between the two groups of fish. Semi-wild fish with high RMR may have a social status advantage over farmed fish because a previous study reported that SMR, which is the value closest to basal metabolism significantly affects feeding motivation. This means that individuals with higher social status may be more motivated to feed. As RMR is proportional to food requirements, then release programs should be planned taking food resources at the release site into consideration.

BAP1 and YY1 regulate expression of death receptors in malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is a rare, aggressive, and incurable cancer arising from the mesothelial lining of the pleura, with few available treatment options. We recently reported that loss of function of the nuclear deubiquitinase BRCA1-associated protein 1 (BAP1), a frequent event in MPM, is associated with sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. As a potential underlying mechanism, here we report that BAP1 negatively regulates the expression of TRAIL receptors: death receptor 4 (DR4) and death receptor 5 (DR5). Using tissue microarrays of tumor samples from MPM patients, we found a strong inverse correlation between BAP1 and TRAIL receptor expression. BAP1 knockdown increased DR4 and DR5 expression, whereas overexpression of BAP1 had the opposite effect. Reporter assays confirmed wt-BAP1, but not catalytically inactive BAP1 mutant, reduced promoter activities of DR4 and DR5, suggesting deubiquitinase activity is required for the regulation of gene expression. Co-immunoprecipitation studies demonstrated direct binding of BAP1 to the transcription factor Ying Yang 1 (YY1), and chromatin immunoprecipitation assays revealed BAP1 and YY1 to be enriched in the promoter regions of DR4 and DR5. Knockdown of YY1 also increased DR4 and DR5 expression and sensitivity to TRAIL. These results suggest that BAP1 and YY1 cooperatively repress transcription of TRAIL receptors. Our finding that BAP1 directly regulates the extrinsic apoptotic pathway will provide new insights into the role of BAP1 in the development of MPM and other cancers with frequent BAP1 mutations.

The analysis of Period1 gene expression in vivo and in vitro using a micro PMT system.

To detect a small amount of Period1 (Per1) expression, we developed a micro-photomultiplier tube (µPMT) system which can be used both in vivo and in vitro. Using this system, we succeeded in detecting Per1 gene expression in the skin of freely moving mice over 240 times higher compared with that of the tissue contact optical sensor (TCS) as previously reported. For in vitro studies, we succeeded in detecting elevated Per1 expression by streptozotocin (STZ) treatment in the scalp hairs at an early stage of diabetes, when glucose content in the blood was still normal. In addition, we could detect elevated Per1 expression in a single whisker hair at the time of diabetes onset. These results show that our µPMT system responds to minute changes in gene expression in freely moving mice in vivo and in mice hair follicles in vitro. Furthermore, Per1 in the hair can be used for a marker of diabetic aggravation.

Membrane perturbation by lipidated Atg8 underlies autophagosome biogenesis.

Autophagosome biogenesis is an essential feature of autophagy. Lipidation of Atg8 plays a critical role in this process. Previous in vitro studies identified membrane tethering and hemi-fusion/fusion activities of Atg8, yet definitive roles in autophagosome biogenesis remained controversial. Here, we studied the effect of Atg8 lipidation on membrane structure. Lipidation of Saccharomyces cerevisiae Atg8 on nonspherical giant vesicles induced dramatic vesicle deformation into a sphere with an out-bud. Solution NMR spectroscopy of Atg8 lipidated on nanodiscs identified two aromatic membrane-facing residues that mediate membrane-area expansion and fragmentation of giant vesicles in vitro. These residues also contribute to the in vivo maintenance of fragmented vacuolar morphology under stress in fission yeast, a moonlighting function of Atg8. Furthermore, these aromatic residues are crucial for the formation of a sufficient number of autophagosomes and regulate autophagosome size. Together, these data demonstrate that Atg8 can cause membrane perturbations that underlie efficient autophagosome biogenesis.

Period1 gene expression in the olfactory bulb and liver of freely moving streptozotocin-treated diabetic mouse.

Clock genes express circadian rhythms in most organs. These rhythms are organized throughout the whole body, regulated by the suprachiasmatic nucleus (SCN) in the brain. Disturbance of these clock gene expression rhythms is a risk factor for diseases such as obesity. In the present study, to explore the role of clock genes in developing diabetes, we examined the effect of streptozotocin (STZ)-induced high glucose on Period1 (Per1) gene expression rhythm in the liver and the olfactory bub (OB) in the brain. We found a drastic increase of Per1 expression in both tissues after STZ injection while blood glucose content was low. After a rapid expression peak, Per1 expression showed no rhythm. Associated with an increase of glucose content, behavior became arrhythmic. Finally, we succeeded in detecting an increase of Per1 expression in mice hair follicles on day 1 after STZ administration, before the onset of symptoms. These results show that elevated Per1 expression by STZ plays an important role in the aggravation of diabetes.

Syntheses and antimicrobial activities of ogipeptin derivatives.

Novel cyclic peptide derivatives based on ogipeptins A, B, C, and D were synthesized. Starting with a mixture of ogipeptins A-D, a practical four-step synthetic procedure was followed to prepare novel derivatives with various kinds of acyl side chains. Among the 45 new synthetic derivatives identified, the antibacterial activities of compounds 8-3 and 8-38 were comparable with those of ogipeptin A. In in vitro nephrotoxicity screening using LLC-PK1 cells, compounds 8-3 and 8-38 showed significantly lower cytotoxicity (LD20 > 480 µM) than colistin (LD20 = 44.2 µM).

CAPS1 is involved in hippocampal synaptic plasticity and hippocampus-associated learning.

Calcium-dependent activator protein for secretion 1 (CAPS1) is a key molecule in vesicular exocytosis, probably in the priming step. However, CAPS1's role in synaptic plasticity and brain function is elusive. Herein, we showed that synaptic plasticity and learning behavior were impaired in forebrain and/or hippocampus-specific Caps1 conditional knockout (cKO) mice by means of molecular, physiological, and behavioral analyses. Neonatal Caps1 cKO mice showed a decrease in the number of docked vesicles in the hippocampal CA3 region, with no detectable changes in the distribution of other major exocytosis-related molecules. Additionally, long-term potentiation (LTP) was partially and severely impaired in the CA1 and CA3 regions, respectively. CA1 LTP was reinforced by repeated high-frequency stimuli, whereas CA3 LTP was completely abolished. Accordingly, hippocampus-associated learning was severely impaired in adeno-associated virus (AAV) infection-mediated postnatal Caps1 cKO mice. Collectively, our findings suggest that CAPS1 is a key protein involved in the cellular mechanisms underlying hippocampal synaptic release and plasticity, which is crucial for hippocampus-associated learning.

FEN1 endonuclease as a therapeutic target for human cancers with defects in homologous recombination.

Synthetic lethality strategies for cancer therapy exploit cancer-specific genetic defects to identify targets that are uniquely essential to the survival of tumor cells. Here we show RAD27/FEN1, which encodes flap endonuclease 1 (FEN1), a structure-specific nuclease with roles in DNA replication and repair, and has the greatest number of synthetic lethal interactions with Saccharomyces cerevisiae genome instability genes, is a druggable target for an inhibitor-based approach to kill cancers with defects in homologous recombination (HR). The vulnerability of cancers with HR defects to FEN1 loss was validated by studies showing that small-molecule FEN1 inhibitors and FEN1 small interfering RNAs (siRNAs) selectively killed BRCA1- and BRCA2-defective human cell lines. Furthermore, the differential sensitivity to FEN1 inhibition was recapitulated in mice, where a small-molecule FEN1 inhibitor reduced the growth of tumors established from drug-sensitive but not drug-resistant cancer cell lines. FEN1 inhibition induced a DNA damage response in both sensitive and resistant cell lines; however, sensitive cell lines were unable to recover and replicate DNA even when the inhibitor was removed. Although FEN1 inhibition activated caspase to higher levels in sensitive cells, this apoptotic response occurred in p53-defective cells and cell killing was not blocked by a pan-caspase inhibitor. These results suggest that FEN1 inhibitors have the potential for therapeutically targeting HR-defective cancers such as those resulting from BRCA1 and BRCA2 mutations, and other genetic defects.

Human ATG2B possesses a lipid transfer activity which is accelerated by negatively charged lipids and WIPI4.

Atg2 is one of the essential factors for autophagy. Recent advance of structural and biochemical study on yeast Atg2 proposed that Atg2 tethers the edge of the isolation membrane (IM) to the endoplasmic reticulum and mediates direct lipid transfer (LT) from ER to IM for IM expansion. In mammals, two Atg2 orthologs, ATG2A and ATG2B, participate in autophagic process. Here we showed that human ATG2B possesses the membrane tethering (MT) and LT activity that was promoted by negatively charged membranes and an Atg18 ortholog WIPI4. By contrast, negatively charged membranes reduced the yeast Atg2 activities in the absence of Atg18. These results suggest that the MT/LT activity of Atg2 is evolutionally conserved although their regulation differs among species.

Response variance prediction using transient statistical energy analysis.

Statistical energy analysis (SEA) is a prominent method for predicting the high frequency response of complex structures under steady loading where the structure is split into subsystems and the subsystem energies are calculated. Since at high frequencies, the dynamic response of nominally identical structures can differ greatly, methods have been developed to predict both the mean and variance of the energy in the subsystems of a system across an ensemble of systems. SEA can be extended to predict the transient response of a system, either to shock or time-varying inputs and is known as transient SEA, although this formulation has so far only been interested in the mean response. In this paper, a method for predicting the variance of the transient response is derived by considering how an individual realisation can deviate from the mean. A matrix differential equation for the covariance of the subsystem energies is derived which is driven by terms representing the variability in the system. These variance terms are provided by assuming that the natural frequencies in each subsystem conform to the Gaussian orthogonal ensemble. The accuracy of the method is investigated both numerically and experimentally using systems involving coupled plates and its limitations are discussed.

Retrospective response analysis of BAP1 expression to predict the clinical activity of systemic cytotoxic chemotherapy in mesothelioma.

Introduction BRCA1 associated protein-1 (BAP1) is a key tumor driver in mesothelioma and a potential biomarker predicting response to several targeted therapies in clinical testing. Whether it also modulates response to cytotoxic chemotherapy is undetermined. This study used retrospective response analysis of BAP1 expression in archival tumor biopsies taken from patients in the MS01 trial (NCT00075699). We aimed to determine if BAP1 expression correlated with overall survival within the three treatment arms in this trial, namely active symptom control (ASC); ASC plus mitomycin, vinblastine and cisplatin (MVP); and ASC plus vinorelbine. Materials and methods We used immunohistochemical analysis of tumor samples from the MS01 trial to identify subgroups with and without nuclear BAP1 expression. We performed correlative analysis of clinical characteristics (age at diagnosis, sex and histological subtype) and overall survival within treatment arms with nuclear BAP1 expression. Results 89 tumor samples from the 409 patients originally in the trial were available for analysis. Of these, 60 samples harbored a positive internal control, in the form of positive staining of inflammatory cells for BAP1, and were carried forward for analysis. Correlative analysis suggested no significant association between loss of nuclear BAP1 expression and age at diagnosis, sex and histological subtype. Kaplan Meier survival analysis revealed a small, though non-significant, overall survival disadvantage associated with BAP1 expression in tumors from patients treated with vinorelbine. Discussion This exploratory analysis suggests BAP1 expression may modify response to vinorelbine in MPM, possibly due to prevention of mitotic microtubule formation. We suggest ongoing and planned clinical studies of vinorelbine in MPM assess BAP1 expression as a predictive biomarker of response.

Loss of functional BAP1 augments sensitivity to TRAIL in cancer cells.

Malignant mesothelioma (MM) is poorly responsive to systemic cytotoxic chemotherapy and invariably fatal. Here we describe a screen of 94 drugs in 15 exome-sequenced MM lines and the discovery of a subset defined by loss of function of the nuclear deubiquitinase BRCA associated protein-1 (BAP1) that demonstrate heightened sensitivity to TRAIL (tumour necrosis factor-related apoptosis-inducing ligand). This association is observed across human early passage MM cultures, mouse xenografts and human tumour explants. We demonstrate that BAP1 deubiquitinase activity and its association with ASXL1 to form the Polycomb repressive deubiquitinase complex (PR-DUB) impacts TRAIL sensitivity implicating transcriptional modulation as an underlying mechanism. Death receptor agonists are well-tolerated anti-cancer agents demonstrating limited therapeutic benefit in trials without a targeting biomarker. We identify BAP1 loss-of-function mutations, which are frequent in MM, as a potential genomic stratification tool for TRAIL sensitivity with immediate and actionable therapeutic implications.

CAPS1 stabilizes the state of readily releasable synaptic vesicles to fusion competence at CA3-CA1 synapses in adult hippocampus.

Calcium-dependent activator protein for secretion 1 (CAPS1) regulates exocytosis of dense-core vesicles in neuroendocrine cells and of synaptic vesicles in neurons. However, the synaptic function of CAPS1 in the mature brain is unclear because Caps1 knockout (KO) results in neonatal death. Here, using forebrain-specific Caps1 conditional KO (cKO) mice, we demonstrate, for the first time, a critical role of CAPS1 in adult synapses. The amplitude of synaptic transmission at CA3-CA1 synapses was strongly reduced, and paired-pulse facilitation was significantly increased, in acute hippocampal slices from cKO mice compared with control mice, suggesting a perturbation in presynaptic function. Morphological analysis revealed an accumulation of synaptic vesicles in the presynapse without any overall morphological change. Interestingly, however, the percentage of docked vesicles was markedly decreased in the Caps1 cKO. Taken together, our findings suggest that CAPS1 stabilizes the state of readily releasable synaptic vesicles, thereby enhancing neurotransmitter release at hippocampal synapses.