The dynamin inhibitor dynasore inhibits bone resorption by rapidly disrupting actin rings of osteoclasts

The cytoskeletal organization of osteoclasts is required for bone resorption. Binding of dynamin with guanosine triphosphate (GTP) was previously suggested to be required for the organization of the actin cytoskeleton. However, the role of the GTPase activity of dynamin in the organization of the actin cytoskeleton as well as in the bone-resorbing activity of osteoclasts remains unclear. This study investigated the effects of dynasore, an inhibitor of the GTPase activity of dynamin, on the bone-resorbing activity of and actin ring formation in mouse osteoclasts in vitro and in vivo. Dynasore inhibited the formation of resorption pits in osteoclast cultures by suppressing actin ring formation and rapidly disrupting actin rings in osteoclasts. A time-lapse image analysis showed that dynasore shrank actin rings in osteoclasts within 30 min. The intraperitoneal administration of dynasore inhibited receptor activator of nuclear factor κB ligand (RANKL)-induced trabecular bone loss in mouse femurs. These in vitro and in vivo results suggest that the GTPase activity of dynamin is critical for the bone-resorbing activity of osteoclasts and that dynasore is a seed for the development of novel anti-resorbing agents.     

Repetitive transcranial magnetic stimulation of human MT+ reduces apparent motion perception

We investigated the effects of repetitive transcranial magnetic stimulation (rTMS) over the human cerebral cortex on apparent motion perception. Previous studies have shown that human extrastriate visual area MT+ (V5) processes not only real but also apparent motion. However, the functional relevance of MT+ on long-range apparent motion perception remains unclear. Here, we show direct evidence for the involvement of MT+ in apparent motion perception using rTMS, which is known to temporarily inhibit a localized region in the cerebral cortex. The results showed that apparent motion perception decreased after applying rTMS over MT+, but not after applying rTMS over the control region (inferior temporal gyrus). The decrease in performance caused by applying rTMS to MT+ suggests that MT+ is a causally responsible region for apparent motion perception, and thus, further supports the idea that MT+ plays a major role in the perception of motion.     

Large Na+ influx and high Na+, K+–ATPase activity in mitochondria-rich epithelial cells of the inner ear endolymphatic sac

Fluid in the mammalian endolymphatic sac (ES) is connected to the endolymph in the cochlea and the vestibule. Since the dominant ion in the ES is Na(+), it has been postulated that Na(+) transport is essential for regulating the endolymph pressure. This study focused on the cellular mechanism of Na(+) transport in ES epithelial cells. To evaluate the Na(+) transport capability of the ES epithelial cells, changes in intracellular Na(+) concentration ([Na(+)](i)) of individual ES cells were measured with sodium-binding benzofurzan isophthalate in a freshly dissected ES sheet and in dissociated ES cells in response to either the K(+)-free or ouabain-containing solution. Analysis of the [Na(+)](i) changes by the Na(+) load and mitochondrial staining with rhodamine 123 showed that the ES cells were classified into two groups; one exhibited an intensive [Na(+)](i) increase, higher Na(+), K(+)-ATPase activity, and intensive mitochondrial staining (mitochondria-rich cells), and the other exhibited a moderate [Na(+)](i) increase, lower Na(+), K(+)-ATPase activity, and moderate mitochondrial staining (filament-rich cells). These results suggest that mitochondria-rich ES epithelial cells (ca. 30% of ES cells) endowed with high Na(+) permeability and Na(+), K(+)-ATPase activity potentially contribute to the transport of Na(+) outside of the endolymphatic sac.     

Melatonin alterations and brain acetylcholine lesions in sleep disorders in Cockayne syndrome

Background: Cockayne syndrome (CS) is a genetic disorder caused by deficient nucleotide excision repair. Patients with CS exhibit progeroid features, developmental delay, and various neurological disorders; they are also known to suffer from sleep problems, which have never been investigated in detail. Objective: The aim of this study is to investigate the pathogenesis of sleep disorders in patients with CS. Methods: We performed a questionnaire survey of the families of patients with CS, enzyme-linked immunosorbent analyses of the melatonin metabolite, 6-sulphatoxymelatonin (6-SM), in the patients’ urine, and immunohistochemistry in the hypothalamus, the basal nucleus of Meynert (NbM), and the pedunculopontine tegmental nucleus (PPN) in four autopsy cases. Results: Sleep–wakefulness rhythms were disturbed in patients with CS, and these disturbances seemed to be related to a reduced urinary excretion of 6-SM. In addition, although the hypothalamic nuclei were comparatively preserved, acetylcholine neurons (AchNs) were severely decreased in the NbM and PPN. Conclusions: AchNs modulate both arousal and rapid eye movement sleep, and selective lesions of AchNs in the PPN and/or NbM in combination with disturbed melatonin metabolism might be involved in the sleep disorders in CS.     

Hepatectomy-Related Hypophosphatemia: A Novel Phosphaturic Factor in the Liver-Kidney Axis

Marked hypophosphatemia is common after major hepatic resection, but the pathophysiologic mechanism remains unknown. We used a partial hepatectomy (PH) rat model to investigate the molecular basis of hypophosphatemia. PH rats exhibited hypophosphatemia and hyperphosphaturia. In renal and intestinal brush-border membrane vesicles isolated from PH rats, Na⁺-dependent phosphate (Pi) uptake decreased by 50%–60%. PH rats also exhibited significantly decreased levels of renal and intestinal Na⁺-dependent Pi transporter proteins (NaPi-IIa [NaPi-4], NaPi-IIb, and NaPi-IIc). Parathyroid hormone was elevated at 6 hours after PH. Hyperphosphaturia persisted, however, even after thyroparathyroidectomy in PH rats. Moreover, DNA microarray data revealed elevated levels of nicotinamide phosphoribosyltransferase (Nampt) mRNA in the kidney after PH, and Nampt protein levels and total NAD concentration increased significantly in the proximal tubules. PH rats also exhibited markedly increased levels of the Nampt substrate, urinary nicotinamide (NAM), and NAM catabolites. In vitro analyses using opossum kidney cells revealed that NAM alone did not affect endogenous NaPi-4 levels. However, in cells overexpressing Nampt, the addition of NAM led to a marked decrease in cell surface expression of NaPi-4 that was blocked by treatment with FK866, a specific Nampt inhibitor. Furthermore, FK866-treated mice showed elevated renal Pi reabsorption and hypophosphaturia. These findings indicate that hepatectomy-induced hypophosphatemia is due to abnormal NAM metabolism, including Nampt activation in renal proximal tubular cells.Inorganic phosphate (Pi) absorption in the renal proximal tubules and small intestine is important for Pi homeostasis.¹ The Na⁺-dependent Pi (Na/Pi) transport system includes type IIa and type IIc Na/Pi transporters, which are localized in the apical membrane of the proximal tubular cells, and type IIb Na/Pi transporters, which are localized in the apical membrane of the intestinal epithelial cells.^1,2 Pi (re)absorption is regulated by the dietary Pi content, parathyroid hormone (PTH), and the active metabolite of vitamin D, 1α, 25-dihydroxyvitamin D3 [1,25(OH)₂D₃].³ Other phosphaturic hormones, termed phosphatonins, also control renal Pi handling.⁴ The discovery that fibroblast growth factor (FGF) 23, the first identified phosphatonin,⁵ originated from osteocytes established the concept of the bone-kidney axis.^6,7The incidence of liver transplantation has steadily increased and the incidence of partial hepatectomy (PH) has also consequently increased.⁸ Hypophosphatemia frequently occurs after liver resection.^9–11 Acute hypophosphatemia causes septicemia and is associated with a poor prognosis.^11,12 Acute hypophosphatemia is of considerable clinical relevance because many hepatectomized patients develop marked hypophosphatemia and, thus, large doses of Pi replacement are required to maintain metabolic homeostasis.¹³ Urinary Pi excretion is markedly increased in many patients. After hepatectomy, hypophosphatemia is associated with hyperphosphaturia.¹³For many years, the increased metabolic demand of the regenerating liver was considered the underlying pathologic mechanism of hypophosphatemia. The magnitude of Pi uptake by the recovering liver, however, cannot explain the severity of the resulting hypophosphatemia.¹¹ Hepatectomy-induced hypophosphatemia is associated with an increased renal fractional excretion index for Pi unrelated to intact FGF23, FGF7, or secreted frizzled-related protein 4 as a phosphaturic factor,¹⁴ indicating that other factors have a role in the pathogenesis of hypophosphatemia.Nicotinamide (NAM) inhibits intestinal and renal Na/Pi transport activity in normal rats.^15–17 Administration of NAM to rats produces a specific dose-dependent inhibition of Na/Pi transport across the renal brush-border membrane (BBM) and an increase in urinary Pi excretion.^16,17 NAM suppresses hyperphosphatemia in hemodialysis patients.¹⁸ Nicotinamide phosphoribosyltransferase (Nampt) catalyzes the first rate-limiting step in converting NAM to NAD,^19,20 which is essential for cellular metabolism, energy production, and DNA repair.^20–22 Nampt exists in two known forms: intracellular Nampt (iNampt) and secreted extracellular Nampt (eNampt).²³ eNampt also generates an intermediate product, nicotinamide mononucleotide (NMN).²³Our findings indicate that the acceleration of NAM metabolism through Nampt function in the kidney is involved in the hepatectomy-induced hypophosphatemia in rodent models. This study also suggests that NAM metabolism through the liver-kidney axis is important in Pi homeostasis.     

Effects of Heat Stress on Ocular Blood Flow During Exhaustive Exercise

The hypothesis that heat stress reduces the ocular blood flow response to exhaustive exercise was tested by measuring ocular blood flow, blood pressure, and end- tidal carbon dioxide partial pressure (P_ETCO₂) in 12 healthy males while they performed cycle ergometer exercise at 75% of the maximal heart rate at ambient temperatures of 20°C (control condition) and 35°C (heat condition), until exhaustion. The blood flows in the retinal and choroidal vasculature (RCV), the superior temporal retinal arteriole (STRA) and the superior nasal retinal arteriole (SNRA) were recorded at rest and at 6 and 16 min after the start of exercise period and at exhaustion [after 16 ± 2 min (mean ± SE) and 24 ± 3 min of exercise in the heat and control condition, respectively]. The mean arterial pressure at exhaustion was significantly lower in the heat condition than in the control condition at both 16 min and exhaustion. The degree of P_ETCO₂ reduction did not differ significantly between the two thermal conditions at either 16 min or exhaustion. The blood flow velocity in the RCV significantly increased from the resting baseline value at 6 min in both thermal conditions (32 ± 6% and 25 ± 5% at 20°C and 35°C, respectively). However, at 16 min the increase in RCV blood flow velocity had returned to the resting baseline level only in the heat condition. At exhaustion, the blood flows in the STRA and SNRA had decreased significantly from the resting baseline value in the heat condition (STRA: -19 ± 5% and SNRA: -30 ± 6%), and SNRA blood flow was lower than that in the control condition (-14 ± 6% vs -30 ± 6% at 20°C and 35°C, respectively), despite the finding that both thermal conditions induced the same reductions in P_ETCO₂ and vascular conductance. These findings suggested that the heat condition decreases or suppresses ocular blood flow via attenuation of pressor response during exhaustive exercise.

Key Points

• The ocular (retinal and choroidal) blood flow response to exhaustive exercise with heat stress is unknown.
• We hypothesized that the heat stress decreases ocular blood flow response to exhaustive exercise, since cerebral flow, which is regulated similarly to ocular flow, was reported to decrease during heat stress.
• To test this hypothesis, ocular blood flow was measured during exhaustive exercise at 20°C (control condition) and 35°C (heat condition).
• At exhaustion in the heat condition, the ocular flow response was suppressed or decreased with an attenuated pressor response.
• It is suggested that the heat condition decreases or suppresses the ocular blood flow to exhaustive exercise via attenuation of pressor response.

Key Words: Hyperthermia, exercise, healthy subjects, retinal circulation, choroidal circulation, laser-speckle flowgraphy     

Percutaneous autologous concentrated bone marrow grafting in the treatment for nonunion

The purpose of this study was to evaluate the clinical and radiographic treatment effects of percutaneous autologous concentrated bone marrow grafting in nonunion cases and to evaluate the effectiveness of this grafting procedure. We enrolled 17 cases those had atrophic changes due to continuous nonunion for over 9 months after injury and had undergone low-intensity pulsed ultrasound treatment for more than 3 months. The site of nonunion was the femur in 10 cases, the tibia in 5 cases, the humerus in 1 case, and the ulna in 1 case. They underwent percutaneous autologous concentrated bone marrow grafting and continued low-intensity pulsed ultrasound stimulation treatment after grafting. Patients were evaluated using the visual analogue scale for pain at immediately before the procedure, 3, 6, and 12 months after grafting. Plain radiographs of the affected site were taken and evaluated about the healing of the nonunion site at each clinical evaluation. As quantitative assessment, CT scans were undertaken before the procedure and 6 months after grafting. The visual analogue scale pain score was reduced consistently after grafting in all patients. About the healing at the nonunion site, 11 and 13 cases of bone union were observed at 6 and 12 months after grafting. The mean volume of callus formation based on CT images was 4,147 (262–27,392) mm³ total between grafting and 6 months. Percutaneous autologous concentrated bone marrow grafting is an effective procedure for the treatment of patients with nonunion.     

Citalopram and escitalopram plasma drug and metabolite concentrations: genome-wide associations

Aims

Citalopram (CT) and escitalopram (S-CT) are among the most widely prescribed selective serotonin reuptake inhibitors used to treat major depressive disorder (MDD). We applied a genome-wide association study to identify genetic factors that contribute to variation in plasma concentrations of CT or S-CT and their metabolites in MDD patients treated with CT or S-CT.

Methods

Our genome-wide association study was performed using samples from 435 MDD patients. Linear mixed models were used to account for within-subject correlations of longitudinal measures of plasma drug/metabolite concentrations (4 and 8 weeks after the initiation of drug therapy), and single-nucleotide polymorphisms (SNPs) were modelled as additive allelic effects.

Results

Genome-wide significant associations were observed for S-CT concentration with SNPs in or near the CYP2C19 gene on chromosome 10 (rs1074145, P = 4.1 × 10⁻⁹) and with S-didesmethylcitalopram concentration for SNPs near the CYP2D6 locus on chromosome 22 (rs1065852, P = 2.0 × 10⁻¹⁶), supporting the important role of these cytochrome P450 (CYP) enzymes in biotransformation of citalopram. After adjustment for the effect of CYP2C19 functional alleles, the analyses also identified novel loci that will require future replication and functional validation.

Conclusions

In vitro and in vivo studies have suggested that the biotransformation of CT to monodesmethylcitalopram and didesmethylcitalopram is mediated by CYP isozymes. The results of our genome-wide association study performed in MDD patients treated with CT or S-CT have confirmed those observations but also identified novel genomic loci that might play a role in variation in plasma levels of CT or its metabolites during the treatment of MDD patients with these selective serotonin reuptake inhibitors.     

Association between Grit Scales and adherence to regular hospital visits among Japanese patients with type 2 diabetes: Prospective observational study

This study examined the association between Grit Scales and adherence to a schedule of regular hospital visits among Japanese type 2 diabetes patients. Patients with type 2 diabetes who visited the outpatient clinic as new patients comprised the study’s participants. Self-administered Short Grit Scale data were obtained from 122 patients at the first consultation and were then observed for 1 year. As the results, 21 participants failed to attend the hospital. In a logistic regression analysis, the Grit Scale as a continuous variable was positively associated with adherence to regular clinical visits. Its odds ratio and 95% confidential interval was 9.68 and 2.87–32.65 (P = 0.0003). In conclusion, it is likely that the Grit Scale is closely associated with adherence to regular hospital visits among Japanese type 2 diabetes patients.     

Retrolaminar versus epidural block for postoperative analgesia after minor video-assisted thoracic surgery: a retrospective,matched, non-inferiority study

BackgroundThe role of thoracic epidural analgesia (TEA) for postoperative analgesia after video-assisted thoracic surgery (VATS) is still controversial. Some studies have reported the efficacy of ultrasound-guided retrolaminar block (RLB) for the postoperative management of pain after chest wall surgery. The purpose of this study was to compare the postoperative analgesic efficacy and adverse effects of ultrasound-guided RLB with those of TEA in patients undergoing minor VATS procedures.MethodsA total of 192 relevant records of patients were enrolled in this study. We reviewed electronic medical records of patients undergoing minor VATS procedures under general anesthesia. The primary outcome was the median differences in the numerical rating scale (NRS) scores during rest between the groups at the morning of postoperative day 1 (POD 1m). A propensity-matched analysis incorporating preoperative variables was used to compare the efficacy of postoperative analgesia in two groups.ResultsOverall, 94 patients were identified for analysis. Propensity score matching resulted in 47 patients in each group. There were no significant differences in the NRS scores between the two groups. The median differences in NRS scores during rest between the two groups at POD 1m were under 1, which indicates non-inferiority of RLB. There were no significant differences in the incidence of adverse effects and rescue dose of analgesic consumption between the two groups.ConclusionsThe analgesic effects of continuous ultrasound-guided RLB were non inferior to those of TEA for minor VATS procedures.