Iron-sulphur protein catalysed [4+2] cycloadditions in natural product biosynthesis.

To the best of our knowledge, enzymes that catalyse intramolecular Diels-Alder ([4+2] cycloaddition) reactions are frequently reported in natural product biosynthesis; however, no native enzymes utilising Lewis acid catalysis have been reported. Verticilactam is a representative member of polycyclic macrolactams, presumably produced by spontaneous cycloaddition. We report that the intramolecular [4+2] cycloadditions can be significantly accelerated by ferredoxins (Fds), a class of small iron-sulphur (Fe-S) proteins. Through iron atom substitution by Lewis acidic gallium (Ga) iron and computational calculations, we confirm that the ubiquitous Fe-S cluster efficiently functions as Lewis acid to accelerate the tandem [4+2] cycloaddition and Michael addition reactions by lowering free energy barriers. Our work highlights Nature's ingenious strategy to generate complex molecule structures using the ubiquitous Fe-S protein. Furthermore, our study sheds light on the future design of Fd as a versatile Lewis acid catalyst for [4+2] cycloaddition reactions.

Hypersensitive Reaction of Oxidized Regenerated Cellulose Powder in Total Knee Arthroplasty.

The powdered form of oxidized regenerated cellulose (ORC powder) is a widely used biodegradable hemostatic material in the field of surgery. There are several reports of its effectiveness and safety; however, excessive foreign body reactions remain a concern for surgeons in total knee arthroplasty (TKA). A 70-year-old woman who underwent unilateral TKA using ORC powder to control perioperative blood loss exhibited a skin rash around her operated knee at six days postoperatively. These reactions were potentially hypersensitive to ORC powder. After receiving antiallergic medication for 18 days, the skin rash disappeared. Although there are several reports on the safety of ORC powder, inadequate intraoperative lavage of the product may induce hypersensitive reactions such as skin rash.

Longevity control by supersulfide-mediated mitochondrial respiration and regulation of protein quality.

Supersulfides, which are defined as sulfur species with catenated sulfur atoms, are increasingly being investigated in biology. We recently identified pyridoxal phosphate (PLP)-dependent biosynthesis of cysteine persulfide (CysSSH) and related supersulfides by cysteinyl-tRNA synthetase (CARS). Here, we investigated the physiological role of CysSSH in budding yeast (Saccharomyces cerevisiae) by generating a PLP-binding site mutation K109A in CRS1 (the yeast ortholog of CARS), which decreased the synthesis of CysSSH and related supersulfides and also led to reduced chronological aging, effects that were associated with an increased endoplasmic reticulum stress response and impaired mitochondrial bioenergetics. Reduced chronological aging in the K109A mutant could be rescued by using exogenous supersulfide donors. Our findings indicate important roles for CARS in the production and metabolism of supersulfides-to mediate mitochondrial function and to regulate longevity.

Metallothionein Cup1 attenuates nitrosative stress in the yeast Saccharomyces cerevisiae.

Metallothionein (MT), which is a small metal-binding protein with cysteine-rich motifs, functions in the detoxification of heavy metals in a variety of organisms. Even though previous studies suggest that MT is involved in the tolerance mechanisms against nitrosative stress induced by toxic levels of nitric oxide (NO) in mammalian cells, the physiological functions of MT in relation to NO have not been fully understood. In this study, we analyzed the functions of MT in nitrosative stress tolerance in the yeast Saccharomyces cerevisiae. Our phenotypic analyses showed that deletion or overexpression of the MT-encoding gene, CUP1, led to higher sensitivity or tolerance to nitrosative stress in S. cerevisiae cells, respectively. We further examined whether the yeast MT Cup1 in the cell-free lysate scavenges NO. These results showed that the cell-free lysate containing a higher level of Cup1 degraded NO more efficiently. On the other hand, the transcription level of CUP1 was not affected by nitrosative stress treatment. Our findings suggest that the yeast MT Cup1 contributes to nitrosative stress tolerance, possibly as a constitutive rather than an inducible defense mechanism.

Clinical three-dimensional anatomy of the femur considering navigation-aided surgery of total knee arthroplasty in Japanese patients.

PURPOSE: Few studies exist regarding sagittal alignment describing femur morphology in navigation-aided surgery. This study investigated the three-dimensional (3D) sagittal femoral alignment of the whole femur. METHODS: Seventy-three consecutive patients (59 females, 14 males, mean age: 76.1 years), yielding 140 femurs, were included in this study. A computed-tomography-based patient-specific 3D femur model was used to define a mechanical axis-based reference plane. Proximal and distal femoral axis angles (PFA, DFA) to the reference plane were measured in 3D using custom software. PFA and DFA represent the proximal and distal inclination of the femoral anatomical axis in sagittal plane, respectively. RESULTS: PFA (10.6 ± 1.5°) was greater than DFA (2.6 ± 1.6°; P < 0.0001). DFA in females (2.3 ± 1.4°) was smaller than in males (3.9 ± 1.7°; P < 0.0001). CONCLUSION: This is the first report of measurement of femoral sagittal alignment related to both 3D anatomy and decision making of femoral flexion angle using navigation surgery for total knee arthroplasty. This report shows a robust DFA measurement that could be used as a template for femoral implants flexion angle when performing both conventional and navigated total knee arthroplasty.

Recovery from the damage of cranial radiation modulated by memantine, an NMDA receptor antagonist, combined with hyperbaric oxygen therapy.

BACKGROUND: Radiotherapy is an important treatment option for central nervous system malignancies. However, cranial radiation induces hippocampal dysfunction and white matter injury; this leads to cognitive dysfunction, and results in a reduced quality of life in patients. Excitatory glutamate signaling through N-methyl-d-aspartate receptors (NMDARs) plays a central role both in hippocampal neurogenesis and in the myelination of oligodendrocytes in the cerebrum. METHODS: We provide a method for quantifying neurogenesis in human subjects in live brain during cancer therapy. Neuroimaging using originally created behavioral tasks was employed to examine human hippocampal memory pathway in patients with brain disorders. RESULTS: Treatment with memantine, a non-competitive NMDAR antagonist, reversed impairment in hippocampal pattern separation networks as detected by functional magnetic resonance imaging. Hyperbaric preconditioning of the patients just before radiotherapy with memantine mostly reversed white matter injury as detected by whole brain analysis with Tract-Based Spatial Statics. Neuromodulation combined with the administration of hyperbaric oxygen therapy and memantine during radiotherapy facilitated the restoration of hippocampal function and white matter integrity, and improved higher cognitive function in patients receiving cranial radiation. CONCLUSIONS: The method described herein, for diagnosis of hippocampal dysfunction, and therapeutic intervention can be utilized to restore some of the cognitive decline experienced by patients who have received cranial radiation. The underlying mechanism of restoration is the production of new neurons, which enhances functionality in pattern separation networks in the hippocampi, resulting in an increase in cognitive score, and restoration of microstructural integrity of white matter tracts revealed by Tract-Based Spatial Statics Analysis.

Immunostimulatory effects of a subcritical water extract of Ganoderma.

Ganoderma, a medicinal mushroom with various physiological activities, has been extensively investigated regarding its effectiveness. The aim of the present study was to examine the effects of a subcritical water extract of Ganoderma (SWEG) on the immune system. The use of subcritical water with a higher temperature and pressure than hot water allows efficient elution of components from natural products. As an evaluation of the effectiveness of SWEG, a cell proliferation and a cell differentiation test were carried out using A-6 cells, a model of hematopoietic stem cells. Furthermore, an oral administration test in mice was conducted to examine the effects of SWEG on the number and function of immune cells. As a result, SWEG was revealed to promote both self-renewal and differentiation into immune cells such as T cells and natural killer (NK) cells in experiments with A-6 cells. These results were not obtained in experiments using hot water extract of Ganoderma lucidum and Ganoderma sinense. The oral administration test in mice demonstrated that SWEG increased hematopoietic precursor cells, immature B cells, and NK cells in the bone marrow, and T cells in the thymus. In addition, SWEG enhanced the immune functions in the spleen by promoting granzyme B expression and NK cell activity. SWEG was demonstrated to be a food material that acts on HSCs and regulates immunity in vivo.

Resting energy expenditure depends on energy intake during weight loss in people with obesity: a retrospective cohort study

Abstract Objective: Resting energy expenditure (REE) decreases if there is reduced energy intake and body weight (BW). The decrease in REE could make it difficult for patients with obesity to maintain decreased BW. This study aimed to investigate the correlation among changes in REE, energy intake, and BW during the weight loss process in patients with obesity. Materials and methods: We conducted a retrospective cohort study of patients hospitalized for the treatment of obesity in Japan. Patients received fully controlled diet during hospitalization and performed exercises if able. REE was measured once a week using a hand-held indirect calorimetry. Energy intake was determined by actual dietary intake. Results: Of 44 inpatients with obesity, 17 were included in the analysis. Their BW decreased significantly after 1 week (−4.7 ± 2.0 kg, P < 0.001) and 2 weeks (−5.7 ± 2.2 kg, P < 0.001). The change in REE after 1 and 2 weeks was positively correlated with the energy intake/energy expenditure ratio (r = 0.66, P = 0.004 at 1 week, r = 0.71, P = 0.002 at 2 weeks). Using a regression equation (y = 0.5257x - 43.579), if the energy intake/energy expenditure ratio within the second week was 82.9%, the REE after 2 weeks was similar to the baseline level. There was no significant correlation between the change in REE and BW. Conclusions: Our data suggest that changes in REE depend on energy intake/energy expenditure ratio and that the decrease in REE can be minimized by matching energy intake to energy expenditure, even during the weight loss process.

Inflammation in VTA Caused by HFD Induces Activation of Dopaminergic Neurons Accompanied by Binge-like Eating.

Binge eating is a characteristic symptom observed in obese individuals that is related to dysfunction of dopaminergic neurons (DNs). Intermittent administration of a high-fat diet (HFD) is reported to induce binge-like eating, but the underlying mechanisms remain unclear. We generated dopaminergic neuron specific IKKß deficient mice (KO) to examine the effects of inflammation in DNs on binge-like eating under inflammatory conditions associated with HFD. After administration of HFD for 4 weeks, mice were fasted for 24 h, and then the consumption of HFD was measured for 2 h. We also evaluated that the mRNA expressions of inflammatory cytokines, glial markers, and dopamine signaling-related genes in the ventral tegmental area (VTA) and striatum. Moreover, insulin was administered intraventricularly to assess downstream signaling. The consumption of HFD was significantly reduced, and the phosphorylation of AKT in the VTA was significantly increased in female KO compared to wild-type (WT) mice. Analyses of mRNA expressions revealed that DNs activity and inflammation in the VTA were significantly decreased in female KO mice. Thus, our data suggest that HFD-induced inflammation with glial cell activation in the VTA affects DNs function and causes abnormal eating behaviors accompanied by insulin resistance in the VTA of female mice.

The Cdc25/Ras/cAMP-dependent protein kinase A signaling pathway regulates proline utilization in wine yeast Saccharomyces cerevisiae under a wine fermentation model.

Proline is a predominant amino acid in grape must, but it is poorly utilized by the yeast Saccharomyces cerevisiae in wine-making processes. This sometimes leads to a nitrogen deficiency during fermentation and proline accumulation in wine. In this study, we clarified that a glucose response is involved in an inhibitory mechanism of proline utilization in yeast. Our genetic screen showed that strains with a loss-of-function mutation on the CDC25 gene can utilize proline even under fermentation conditions. Cdc25 is a regulator of the glucose response consisting of the Ras/cAMP-dependent protein kinase A (PKA) pathway. Moreover, we found that activation of the Ras/PKA pathway is necessary for the inhibitory mechanism of proline utilization. The present data revealed that crosstalk exists between the carbon and proline metabolisms. Our study could hold promise for the development of wine yeast strains that can efficiently assimilate proline during the fermentation processes.

Increased Risk of Thyroid Dysfunction by PD-1 and CTLA-4 Blockade in Patients Without Thyroid Autoantibodies at Baseline.

BACKGROUND: Previous studies showed that although the risk of thyroid dysfunction [thyroid immune-related adverse events (irAEs)] induced by anti-programmed cell death-1 antibodies (PD-1-Ab) was as low as 2% to 7% in patients negative for anti-thyroid antibodies (ATAs) at baseline, it was much higher (30%-50%) in patients positive for ATAs. However, whether a similar increase occurs with combination therapy using PD-1-Ab plus anti-cytotoxic T-lymphocyte antigen-4 antibody (CTLA-4-Ab) is unknown. METHODS: A total of 451 patients with malignancies treated with PD-1-Ab, CTLA-4-Ab, or a combination of PD-1-Ab and CTLA-4-Ab (PD-1/CTLA-4-Abs) were evaluated for ATAs at baseline and for thyroid function every 6 weeks for 24 weeks after treatment initiation and then observed until the last clinical visit. RESULTS: Of the 451 patients, 51 developed thyroid irAEs after immunotherapy [41 of 416 (9.9%) treated with PD-1-Ab, 0 of 8 (0%) treated with CTLA-4-Ab, and 10 of 27 (37.0%) treated with PD-1/CTLA-4-Abs]. The cumulative incidence of thyroid irAEs was significantly higher in patients who were positive vs negative for ATAs at baseline after both PD-1-Ab [28/87 (32.2%) vs 13/329 (4.0%), P < 0.001] and PD-1/CTLA-4-Abs [6/10 (60.0%) vs 4/17 (23.5%), P < 0.05] treatments. The risk of thyroid irAEs induced by PD-1/CTLA-4Abs, which was significantly higher than that induced by PD-1-Ab, in patients negative for ATAs at baseline was not statistically different from that induced by PD-1-Ab in patients positive for ATAs at baseline. CONCLUSIONS: This study showed that the incidence of thyroid irAEs was high and not negligible after PD-1/CTLA-4-Abs treatment even in patients negative for ATAs at baseline.

Enhancement of lysine biosynthesis confers high-temperature stress tolerance to Escherichia coli cells.

Lysine, a nutritionally important amino acid, is involved in adaptation and tolerance to environmental stresses in various organisms. Previous studies reported that lysine accumulation occurs in response to stress and that lysine supplementation enhances stress tolerance; however, the effect of lysine biosynthesis enhancement on stress tolerance has yet to be elucidated. In this study, we confirmed that lysine supplementation to the culture medium increased intracellular lysine content and improved cell growth of Escherichia coli at high temperature (42.5 °C). Lysine-overproducing strains were then isolated from the lysine analogue S-adenosylmethionine-resistant mutants by conventional mutagenesis and exhibited higher tolerance to high-temperature stress than the wild-type strain. We identified novel amino acid substitutions Gly474Asp and Cys554Tyr on ThrA, a bifunctional aspartate kinase/homoserine dehydrogenase (AK/HSDH), in the lysine-overproducing mutants. Interestingly, the Gly474Asp and Cys554Tyr variants of ThrA induced lysine accumulation and conferred high-temperature stress tolerance to E. coli cells. Enzymatic analysis revealed that the Gly474Asp substitution in ThrA reduced HSDH activity, suggesting that the intracellular level of aspartate semialdehyde, which is a substrate for HSDH and an intermediate for lysine biosynthesis, is elevated by the loss of HSDH activity and converted to lysine in E. coli. The present study demonstrated that both lysine supplementation and lysine biosynthesis enhancement improved the high-temperature stress tolerance of E. coli cells. Our findings suggest that lysine-overproducing strains have the potential as stress-tolerant microorganisms and can be applied to robust host cells for microbial production of useful compounds. KEY POINTS: • Lysine supplementation improved the growth of E. coli cells at high temperature. • The G474D and C554Y variant ThrA increased lysine productivity in E. coli cells. • The G474D substitution in ThrA reduced homoserine dehydrogenase activity. • E. coli cells that overproduce lysine exhibited high-temperature stress tolerance.

Cysteine residues in the fourth zinc finger are important for activation of the nitric oxide-inducible transcription factor Fzf1 in the yeast Saccharomyces cerevisiae.

Nitric oxide (NO) is a ubiquitous signaling molecule in various organisms. In the yeast Saccharomyces cerevisiae, NO functions in both cell protection and cell death, depending on its concentration. Thus, it is important for yeast cells to strictly regulate NO concentration. The transcription factor Fzf1, containing five zinc fingers, is reportedly important for NO homeostasis by regulating the expression of the YHB1 gene, which encodes NO dioxygenase. However, the mechanism by which NO activates Fzf1 is still unclear. In this study, we showed that NO activated Fzf1 specifically at the protein level by RT-qPCR and Western blotting. Our further transcriptional analyses indicated that cysteine residues in the fourth zinc finger (ZF4) are required for the NO-responsive activation of Fzf1. Additionally, the present results suggest that ZF4 is important for the protein stability of Fzf1. From these results, we proposed possible mechanisms underlying Fzf1 activation.

Examination of refractory discoid lateral meniscus injury.

BACKGROUND: Lateral discoid meniscus (LDM) should be treated and preserved with saucerization and/or suture repair. However, repair of the meniscal hoop structure is sometimes difficult due to displacement or large defects. In this study, we aimed to examine tear patterns based on the Ahn classification in those requiring meniscal repair and those undergoing subtotal meniscectomy. METHODS: Twenty-three patients were evaluated (mean age, 27.4 years; mean follow-up period, 2.5 years). The following were evaluated: displacement morphology based on the Ahn classification, site of tear under arthroscopy, morphology, surgical procedure, Lysholm score at final postoperative follow-up, and clinical outcome of meniscus using Barrett's criteria. RESULT: There were 16 knees without displacement (saucerization with suture repair, 13 knees; subtotal meniscectomy, 3 knees) and 10 knees with displacement (reduction with suture repair, 3 knees; subtotal meniscectomy, 7 knees). Subtotal meniscectomy was performed more often in cases with dislocation, especially in the central shift type as defined by the Ahn classification. The mean Lysholm score was 65.0 points preoperatively and 95.3 points postoperatively. Twenty-three knees (88%) were postoperatively categorized under the Barrett's criteria as healing and 3 knees (12%) were categorized as non-healing. The number of non-healing cases that underwent subtotal meniscectomy was relatively small (1 of 10 knees), and the short-term results were not poor. CONCLUSION: Localized peripheral longitudinal tears tended to be repairable even with displacement, while peripheral tears covering the entire meniscus or with severe defects/tears in the body of the meniscus tended to be difficult to repair, leading to subtotal meniscectomy.

CD4+ T cells are essential for the development of destructive thyroiditis induced by anti-PD-1 antibody in thyroglobulin-immunized mice.

Immune-related adverse events induced by anti-programmed cell death-1 antibodies (PD-1-Ab), including destructive thyroiditis (thyroid-irAE), are thought to be caused by activated T cells. However, the T cell subsets that are directly responsible for damaging self-organs remain unclear. To clarify which T cell subsets are involved in the development of thyroid-irAE, a mouse model of thyroid-irAE was analyzed. PD-1-Ab administration 2.5 months after immunization with thyroglobulin caused destructive thyroiditis. Thyroiditis was completely prevented by previous depletion of CD4+ T cells and partially prevented by depleting CD8+ T cells. The frequencies of central and effector memory CD4+ T cell subsets and the secretion of interferon-γ after stimulation with thyroglobulin were increased in the cervical lymph nodes of mice with thyroid-irAE compared with controls. Histopathological analysis revealed infiltration of CD4+ T cells expressing granzyme B in thyroid glands and major histocompatibility complex class II expression on thyrocytes in mice with thyroid-irAE. Adoptive transfer of CD4+ T cells from cervical lymph nodes in mice with thyroid-irAE caused destruction of thyroid follicular architecture in the irradiated recipient mice. Flow cytometric analyses showed that the frequencies of central and effector memory CD4+ T cells expressing the cytotoxic marker CD27 were higher in peripheral blood mononuclear cells collected from patients with thyroid-irAE induced by PD-1-Ab versus those without. These data suggest a critical role for cytotoxic memory CD4+ T cells activated by PD-1-Ab in the pathogenesis of thyroid-irAE.

Anti-pituitary antibodies and susceptible human leukocyte antigen alleles as predictive biomarkers for pituitary dysfunction induced by immune checkpoint inhibitors.

BACKGROUND: Pituitary dysfunction is a life-threatening immune-related adverse event (irAE) induced by immune checkpoint inhibitors (ICIs). To date, it is not possible to identify patients who may develop pituitary irAEs prior to ICI treatment. The aim of this study was to characterize the predisposition for ICI-induced pituitary irAEs by analyzing anti-pituitary antibodies (APAs) and human leukocyte antigens (HLAs). METHODS: In this case-control study, APAs and HLA alleles were analyzed in 62 patients (17 who developed ICI-induced isolated adrenocorticotropic hormone deficiency (ICI-IAD), 5 who developed ICI-induced hypophysitis (ICI-H) and 40 who did not develop pituitary irAEs) treated with ICIs between November 2, 2015, and March 31, 2020, at Nagoya University Hospital. The main outcome measures in this study were the association between the development of pituitary irAEs with APAs at baseline and after treatment and HLA alleles. RESULTS: Eleven of 17 (64.7%) patients who developed ICI-IAD had APAs at baseline, whereas APAs were positive only in 1 of 40 (2.5%) control patients. Although APAs were negative at baseline in all patients who developed ICI-H, they had become positive before the onset of ICI-H in 3 of 4 patients several weeks after ipilimumab administration. At the onset of ICI-IAD and ICI-H, APAs were positive in 15 of 17 (88.2%) and 4 of 5 (80%) patients, respectively. The prevalence of HLA-Cw12, HLA-DR15, HLA-DQ7, and HLA-DPw9 was significantly higher in patients with ICI-IAD, whereas that of HLA-Cw12 and HLA-DR15 was significantly higher in patients with ICI-H than in controls. CONCLUSIONS: This study showed distinct and overlapped patterns of APAs and HLA alleles between ICI-IAD and ICI-H. Our findings also showed that positive APAs at baseline and after treatment, together with susceptible HLA alleles, could become predictive biomarkers for ICI-IAD and ICI-H, respectively. TRIAL REGISTRATION NUMBER: UMIN000019024.

High-fat Feeding Causes Inflammation and Insulin Resistance in the Ventral Tegmental Area in Mice.

The reward system plays an important role in the pathogenesis of not only drug addiction, but also diet-induced obesity. Recent studies have shown that insulin and leptin receptor signaling in the ventral tegmental area (VTA) regulate energy homeostasis and that their dysregulation is responsible for obesity and altered food preferences. Although a high-fat diet (HFD) induces inflammation that leads to insulin and leptin resistance in the brain, it remains unclear whether HFD induces inflammation in the VTA. In the present study, we placed male mice on a chow diet or HFD for 3, 7, and 28 days and evaluated the mRNA expression of inflammatory cytokines and microglial activation markers in the VTA. The HFD group showed significantly elevated mRNA expressions of IL1ß at 3 days; tumor necrosis factor-alpha (TNFα), IL1ß, IL6, Iba1, and CD11b at 7 days; and TNFα, IL1ß, Iba1, and CD11b at 28 days. The changes in TNFα were also confirmed in immunohistochemical analysis. Next, after administration of chow or HFD for 7 days, we selected mice with equal weights in both groups. In experiments using these mice, Akt phosphorylation in the VTA was significantly decreased after intracerebroventricular injection of insulin, whereas no change in STAT3 phosphorylation was found with leptin. Taken together, these results suggest that HFD induces inflammation at least partly associated with microglial activation in the VTA leading to insulin resistance, independently of the energy balance. Our data provide new insight into the pathophysiology of obesity caused by a dysfunctional reward system under HFD conditions.

Preventive Effect of the Japanese Traditional Herbal Medicine Boiogito on Posttraumatic Osteoarthritis in Rats.

BACKGROUND: Considering the anti-inflammatory properties of the Japanese traditional Kampo medicine Boiogito (BO), we aimed to investigate the therapeutic effect of BO to prevent the development of knee osteoarthritis (KOA) in rats with surgically induced KOA. METHODS: Destabilization of the medial meniscus (DMM) was performed to induce osteoarthritis in the right knees of 12-week-old Wistar rats under general anesthesia. The rats were orally administered 3% BO in standard powder chow for 4 weeks after surgery (controls: n = 6; sham group: n = 6; DMM group: n = 5; DMM + BO group: n = 5). During this period, the rotarod test was performed to monitor locomotive function. After 4 weeks, histological assessment was performed on the right knee. RESULTS: Oral administration of BO improved locomotive function in the rotarod test. Walking time on postoperative days 1, 14, or later was significantly longer in the DMM + BO group than in the DMM group. Histologically, the DMM group showed significant progression of KOA, which, in the DMM + BO group, was strongly suppressed, as assessed by the Osteoarthritis Research Society International score. CONCLUSIONS: Our results showed that oral administration of BO had a clinically preventive effect on early stage posttraumatic KOA.

Endoplasmic reticulum chaperone BiP/GRP78 knockdown leads to autophagy and cell death of arginine vasopressin neurons in mice.

The immunoglobulin heavy chain binding protein (BiP), also referred to as 78-kDa glucose-regulated protein (GRP78), is a pivotal endoplasmic reticulum (ER) chaperone which modulates the unfolded protein response under ER stress. Our previous studies showed that BiP is expressed in arginine vasopressin (AVP) neurons under non-stress conditions and that BiP expression is upregulated in proportion to the increased AVP expression under dehydration. To clarify the role of BiP in AVP neurons, we used a viral approach in combination with shRNA interference for BiP knockdown in mouse AVP neurons. Injection of a recombinant adeno-associated virus equipped with a mouse AVP promoter and BiP shRNA cassette provided specific BiP knockdown in AVP neurons of the supraoptic (SON) and paraventricular nuclei (PVN) in mice. AVP neuron-specific BiP knockdown led to ER stress and AVP neuronal loss in the SON and PVN, resulting in increased urine volume due to lack of AVP secretion. Immunoelectron microscopy of AVP neurons revealed that autophagy was activated through the process of AVP neuronal loss, whereas no obvious features characteristic of apoptosis were observed. Pharmacological inhibition of autophagy by chloroquine exacerbated the AVP neuronal loss due to BiP knockdown, indicating a protective role of autophagy in AVP neurons under ER stress. In summary, our results demonstrate that BiP is essential for the AVP neuron system.

Generation of four induced pluripotent stem cell lines (FHUi003-A, FHUi003-B, FHUi004-A and FHUi004-B) from two affected individuals of a familial neurohypophyseal diabetes insipidus family.

Four disease-specific induced pluripotent stem cell (iPSC) lines were respectively derived from peripheral blood mononuclear cells of two affected individuals in a family affected by familial neurohypophyseal diabetes insipidus carrying the c.314G>C mutation. The expression of pluripotency markers (NANOG, OCT4, and SOX2), maintenance of a normal karyotype, absence of episomal vectors used for iPSC generation, and presence of the original pathogenic mutation were confirmed for each iPSC line. The ability to differentiate into three germ layers was confirmed by a teratoma formation assay. These iPSC lines can help in disease recapitulation in vitro using organoids and elucidation of disease mechanisms.