Protein-lipid acyl chain interactions: Depth-dependent changes of segmental mobility of phospholipid in contact with bacteriorhodopsin.

Boundary lipids surrounding membrane proteins play an essential role in protein function and structure. These protein-lipid interactions are mainly divided into electrostatic interactions between the polar amino acids of proteins and polar heads of phospholipids, and hydrophobic interactions between protein transmembrane sites and phospholipid acyl chains. Our previous report (Kawatake et al., Biochim. Biophys. Acta 1858 [2016] 2106-2115) covered a method for selectively analyzing boundary lipid interactions and showed differences in membrane protein-peripheral lipid interactions due to differences in their head group. Interactions in the hydrophobic acyl chains of phospholipids are relatively consistent among proteins, but the details of these interactions have not been elucidated. In this study, we reconstituted bacteriorhodopsin as a model protein into phospholipid membranes labeled with 2H and 13C for solid-state NMR measurement to investigate the depth-dependent effect of the head group structure on the lipid bilayer. The results showed that the position of the phospholipid near the carbonyl carbon was affected by the head group in terms of selectivity for protein surfaces, whereas in the deep interior of the bilayer near the leaflet interface, there was little difference between the head groups, indicating that the dependence of their interactions on the head group was much reduced.

Automatic breast carcinoma detection in histopathological micrographs based on Single Shot Multibox Detector.

Background: A diagnosis with histological classification by pathologists is very important for appropriate treatments to improve the prognosis of patients with breast cancer. However, the number of pathologists is limited, and assisting the pathological diagnosis by artificial intelligence becomes very important. Here, we presented an automatic breast lesions detection model using microscopic histopathological images based on a Single Shot Multibox Detector (SSD) for the first time and evaluated its significance in assisting the diagnosis. Methods: We built the data set and trained the SSD model with 1361 microscopic images and evaluated using 315 images. Pathologists and medical students diagnosed the images with or without the assistance of the model to investigate the significance of our model in assisting the diagnosis. Results: The model achieved 88.3% and 90.5% diagnostic accuracies in 3-class (benign, non-invasive carcinoma, or invasive carcinoma) or 2-class (benign or malignant) classification tasks, respectively, and the mean intersection over union was 0.59. Medical students achieved a remarkably higher diagnostic accuracy score (average 84.7%) with the assistance of the model compared to those without assistance (average 67.4%). Some people diagnosed images in a short time using the assistance of the model (shorten by average 6.4 min) while others required a longer time (extended by 7.2 min). Conclusion: We presented the automatic breast lesions detection method at high speed using histopathological micrographs. The present system may conveniently support the histological diagnosis by pathologists in laboratories.

Inhibition of current perception thresholds in A-delta and C fibers through somatosensory stimulation of the body surface.

Somatosensory stimulation of the body surface, such as through tactile and noxious stimulation, is widely known to inhibit pain. However, no studies have measured the threshold changes due to somatosensory stimulation of each nerve fiber (Aß, Aδ, and C) separately. We examined the changes in the current perception thresholds of Aδ, C, and Aß fibers induced by non-noxious and noxious somatosensory stimulation of the body surface. The current stimuli were sinusoidal waves at frequencies of 2000 Hz, 250 Hz, and 5 Hz, which selectively stimulated the Aß, Aδ, and C fibers, respectively. In the case of non-noxious stimulation, lightly rubbing the dorsal side of the forearm with a brush showed no significant physiological or clinical changes in the current perception thresholds of the Aδ, and C fibers; a significant increase was observed only in the Aß fibers. However, applying noxious stimulation to the body surface through hand immersion in cold water increased pain thresholds in both the Aδ and C fibers, and sensory threshold of the Aß fibers; changes in tactile thresholds were not significant. Inhibition of sensory information by nociceptive inputs may selectively suppress nociceptive stimuli.

Prevention of bone loss and improvement of pain-related behavior in hind limb-unloaded mice by administration of teriparatide and bisphosphonate.

OBJECTIVES: There are few reports on the comparison between teriparatide (PTH) and bisphosphonate (BP) in terms of osteoporosis pain-related behavior and immunohistochemical findings. The aims of this study were to evaluate skeletal pain associated with osteoporosis and to examine the inhibitory effect of PTH and BP on pain and bone loss in hind limb-unloaded (HU) mice. The mechanism of osteoporotic pain in HU mice was evaluated by examining pain-related behavior and immunohistochemical findings. The effects of PTH and alendronate (ALN), a potent osteoclast inhibitor, on these parameters were also assessed. METHODS: Eight-week-old male ddY mice were tail-suspended for 2 weeks and assigned to four groups: hind limb-loaded (HL) mice with only tail suspension treated with vehicle; HU mice with tail suspension treated with vehicle; HU mice treated with PTH; and HU mice treated with ALN. Starting immediately after reloading, vehicle, PTH, or ALN was injected subcutaneously. After a 2-week treatment, mechanical sensitivity was examined using von Frey filaments. Bilateral hind limbs were removed for micro-computed tomography, immunohistochemical analysis, and messenger RNA (mRNA) expression analysis. RESULTS: HU mice with tail suspension developed bone loss and mechanical hyperalgesia in the hind limbs. The HU mice showed an increased osteoclasts and sclerostin-positive cells in the hind limb bone. Furthermore, PTH and ALN both prevented HU-induced bone loss and mechanical hyperalgesia in the osteoporotic animal models. Histological examination of the hind limb bone revealed that, similar to ALN, PTH inhibited the osteoclasts and sclerostin-positive cells. The mRNA levels of TNFα and IL-6 tended to decrease with ALN or PTH treatment compared with those without any treatment. CONCLUSIONS: Treatment with PTH as well as BP prevented bone loss, mechanical hyperalgesia, osteoclast increase, and osteocyte increase. Similar to BP, the inhibitory effect of PTH on osteoclasts might contribute to the improvement of skeletal pain.

CTLA-4Ig Improves Hyperalgesia in a Mouse Model of Osteoporosis.

This study aimed to evaluate skeletal pain associated with osteoporosis and to examine the inhibitory effects of cytotoxic T lymphocyte-associated antigen-4Ig (CTLA-4Ig) administration in ovariectomized (OVX) mice. Eight-week-old female ddY mice were assigned to three groups: sham-operated mice (SHAM) treated with vehicle, OVX mice treated with vehicle (OVX), and OVX mice treated with CTLA-4Ig (CTLA-4Ig). Vehicle or CTLA-4Ig was injected intraperitoneally, starting immediately after surgery. After 4 weeks of treatment, mechanical sensitivity was examined, and the bilateral hind limbs were removed and evaluated by micro-computed tomography, immunohistochemical analyses, and messenger RNA expression analysis. Ovariectomy induced bone loss and mechanical hyperalgesia in the hindlimbs. CTLA-4Ig treatment prevented bone loss in the hindlimbs compared to vehicle administration in the OVX group. Moreover, mechanical hyperalgesia was significantly decreased in the CTLA-4Ig treatment group in comparison to the OVX group. The expression levels of tumor necrosis factor-α (TNF-α) and sclerostin (SOST), as well as the number of osteoclasts, were increased, and the expression level of Wnt-10b was decreased in the OVX group compared with the SHAM group, whereas these parameters were improved in the CTLA-4Ig group compared with the OVX group. The novelty of this research is that CTLA-4Ig administration prevented bone loss and mechanical hyperalgesia induced by ovariectomy in the hindlimbs.

Functional Block of Interleukin-6 Reduces a Bone Pain Marker but Not Bone Loss in Hindlimb-Unloaded Mice.

Interleukin-6 (IL-6) is widely accepted to stimulate osteoclasts. Our aim in this study was to examine whether the inhibitory effect of IL-6 on bone loss and skeletal pain associated with osteoporosis in hindlimb-unloaded (HU) mice in comparison with bisphosphonate. Eight-week-old male ddY mice were tail suspended for 2 weeks. Starting immediately after reload, vehicle (HU group), alendronate (HU-ALN group), or anti-IL-6 receptor antibody (HU-IL-6i group) was injected subcutaneously. After a 2-week treatment, pain-related behavior was examined using von Frey filaments. The bilateral distal femoral and proximal tibial metaphyses were analyzed three-dimensionally with micro-computed tomography. Calcitonin gene-related peptide (CGRP) expressions in dorsal root ganglion (DRG) neurons innervating the hindlimbs were examined using immunohistochemistry. HU mice with tail suspension developed bone loss. The HU mice showed mechanical hyperalgesia in the hindlimbs and increased CGRP immunoreactive neurons in the L3-5 DRG. Treatment with IL-6i and ALN prevented HU-induced mechanical hyperalgesia and upregulation of CGRP expressions in DRG neurons. Furthermore, ALN but not IL-6i prevented HU-induced bone loss. In summary, treatment with IL-6i prevented mechanical hyperalgesia in hindlimbs and suppressed CGRP expressions in DRG neurons of osteoporotic models. The novelty of this research suggests that IL-6 is one of the causes of immobility-induced osteoporotic pain regardless improvement of bone loss.

Induction of secondary metabolite production by hygromycin B and identification of the 1233A biosynthetic gene cluster with a self-resistance gene.

We found that the protein synthesis inhibitor hygromycin B induced the production of secondary metabolites, including lucilactaene, NG-391, fusarubin, 1233A, and 1233B, in the filamentous fungus, Fusarium sp. RK97-94. We identified the biosynthetic gene cluster for 1233A, an HMG-CoA synthase inhibitor. The biosynthetic gene cluster consisted of four genes, one of which was involved in conferring self-resistance to 1233A.

Biosynthetic gene cluster identification and biological activity of lucilactaene from Fusarium sp. RK97-94.

We identified the biosynthetic gene cluster for lucilactaene, a cell cycle inhibitor from a filamentous fungus Fusarium sp. RK 97-94. The luc1 knockout strain accumulated demethylated analogs, indicating the involvement of Luc1 methyltransferase in lucilactaene biosynthesis. Lucilactaene showed potent antimalarial activity. Our data suggested that methylation and ether ring formation are essential for its potent antimalarial activity.

Teriparatide improves pain-related behavior and prevents bone loss in ovariectomized mice.

PURPOSE: The aim of this study was to examine the inhibitory effect of teriparatide (TPTD) on pain and on bone loss in ovariectomized (OVX) mice. The mechanism of osteoporotic pain in OVX mice was evaluated through an examination of pain-related behavior as well as immunohistochemical examinations. METHODS: Eight-week-old female ddY mice were OVX and assigned to one of three groups: (1) OVX mice treated with vehicle (OVX), (2) OVX mice treated with teriparatide (OVX-TPTD), or (3) SHAM-operated mice treated with vehicle (SHAM). Starting immediately after surgery, vehicle or TPTD was injected subcutaneously. After a 4-week treatment, mechanical sensitivity was tested using von Frey filaments. The proximal tibial metaphyses were analyzed three-dimensionally by microcomputed tomography (µCT). Calcitonin gene-related peptide (CGRP) and transient receptor potential channel vanilloid 1 (TRPV1) expressions in L3-5 dorsal root ganglion (DRG) neurons were examined using immunohistochemistry. RESULTS: Ovariectomy induced bone loss and mechanical hyperalgesia in the hind limbs with upregulation of CGRP and TRPV1 expressions in DRG neurons innervating the hind limbs. Bone loss was prevented more effectively in the OVX-TPTD mice than in the OVX mice. Furthermore, mechanical hyperalgesia and upregulation of CGRP and TRPV1 expressions were significantly lower in the OVX-TPTD mice than in the OVX mice. CONCLUSION: TPTD treatment prevented ovariectomy-induced bone loss and ovariectomy-induced mechanical hyperalgesia in hind limbs, and it suppressed CGRP and TRPV1 expressions in DRG neurons. These results suggest that TPTD is useful for the treatment of osteoporotic pain in postmenopausal women.

Outbreak of aseptic meningitis caused by echovirus 30 in Kushiro, Japan in 2017.

BACKGROUND: Echovirus 30 (E30) is one of the most common causative agents for aseptic meningitis. OBJECTIVES: In the autumn of 2017, there was an outbreak caused by E30 in Kushiro, Hokkaido, Japan. The aim of this study was to characterize this outbreak. STUDY DESIGN: Fifty-nine patients were admitted to the Department of Pediatrics, Kushiro Red Cross Hospital (KRCH) with clinical diagnosis of aseptic meningitis. Among those, 36 patients were finally diagnosed as E30-associated aseptic meningitis by the detection of viral RNA using reverse transcription-polymerase chain reaction (RT-PCR) and/or the evidence of more than four-fold rise in neutralizing antibody (NA) titers in the convalescent phase relative to those in the acute phase. We investigated these 36 confirmed cases. RESULTS: The median age was 6 years (range: 6 months-14 years). The positive signs and symptoms were as follows: fever (100%), headache (94%), vomiting (92%), jolt accentuation (77%), neck stiffness (74%), Kernig sign (29%), and abdominal pain (28%). The median cerebrospinal fluid (CSF) white cell count, neutrophil count, and lymphocyte count were 222/µL (range: 3-1434/µL), 144/µL (range: 1-1269/µL), and 85/µL (range: 2-354/µL), respectively. Although the detected viral genes demonstrated same cluster, they were different from E30 strains observed in Japan between 2010 and 2014. CONCLUSION: We mainly showed clinical and virological features of the E30-associated aseptic meningitis outbreak that occurred in Kushiro. To prevent further spread of E30 infection, continuous surveillance of enterovirus (EV) circulation and standard precautions are considered essential.

Interleukin-6 Inhibitor Suppresses Hyperalgesia Without Improvement in Osteoporosis in a Mouse Pain Model of Osteoporosis.

The aim of this study was to evaluate skeletal pain associated with osteoporosis and examine the inhibitory effect of interleukin-6 (IL-6) on pain in ovariectomized (OVX) mice. The mechanism of osteoporotic pain in OVX mice was evaluated by examining pain-related behavior and immunohistochemistry. The effects of IL-6 receptor inhibitor (IL-6i) on these parameters were also assessed. Eight-week-old female ddY mice were ovariectomized and assigned to three groups: OVX mice treated with vehicle (OVX); OVX mice treated with alendronate (OVX-ALN); and OVX mice treated with anti-IL-6 receptor (anti-IL-6R) antibody (OVX-IL6i). Sham-operated mice were treated with vehicle. Immediately after surgery, vehicle, ALN, or anti-IL-6R antibody was injected subcutaneously. After a 4-week treatment, mechanical sensitivity was examined using von Frey filaments. The bilateral distal femoral metaphyses and proximal tibial metaphyses were analyzed three-dimensionally with micro-computed tomography. Calcitonin gene-related peptide (CGRP) expression in L3-L5 dorsal root ganglion (DRG) neurons was examined using immunohistochemistry. Ovariectomy induced bone loss and mechanical hyperalgesia in the hindlimbs with upregulation of CGRP expression in the DRG neurons innervating the hindlimbs. ALN treatment prevented bone loss, but anti-IL-6R antibody treatment had no effect on bone morphometry compared with that of the OVX group. However, mechanical hyperalgesia and CGRP expression were significantly decreased in the OVX-IL6i and OVX-ALN groups compared with those in the OVX group. Although anti-IL-6R antibody treatment had no effect on ovariectomy-induced bone loss, the treatment prevented ovariectomy-induced mechanical hyperalgesia in the hindlimbs and suppressed CGRP expression in DRG neurons. The results suggest that IL-6 is one of the causes of postmenopausal osteoporotic pain, and anti-IL-6R antibody might preserve bone health and decrease osteoporotic pain.

The effects of bisphosphonate on pain-related behavior and immunohistochemical analyses in hindlimb-unloaded mice.

OBJECTIVE: The aim of this study was to evaluate skeletal pain associated with immobility-induced osteoporosis and to examine the inhibitory effect of bisphosphonate (BP) administration on pain in hindlimb-unloaded (HU) mice. METHODS: The mechanism of osteoporotic pain in HU mice was evaluated through an examination of pain-related behavior, as well as immunohistochemical findings. In addition, the effects of alendronate (ALN), a potent osteoclast inhibitor, on these parameters were assessed. RESULTS: HU mice with tail suspension developed bone loss and mechanical hyperalgesia in the hindlimbs. The HU mice showed an increase in the number of calcitonin gene-related peptide (CGRP)-immunoreactive neurons and in transient receptor potential channel vanilloid subfamily member 1 (TRPV1)-immunoreactive neurons in the dorsal root ganglions (DRGs) innervating the hindlimbs. Furthermore, administration of ALN prevented HU-induced bone loss, mechanical hyperalgesia, and upregulation of CGRP and TRPV1 expressions in DRG neurons of immobility-induced osteoporotic animal models. CONCLUSIONS: HU mice appear to be a useful model for immobility-induced osteoporotic pain and hindlimb-unloading-induced bone loss, as well as upregulation of CGRP and TRPV1 expressions in DRG neurons, and BP treatment prevented bone loss and mechanical hyperalgesia. The inhibitory effect of BP on osteoclast function might contribute to improving osteoporosis-related pain.

Interleukin-6 receptor inhibitor suppresses bone metastases in a breast cancer cell line.

BACKGROUND: Interleukin-6 (IL-6) is a potent inflammatory cytokine that appears to play a key role in cancer growth and metastasis. In the present study, the effects of IL-6 receptor (IL-6R) on breast cancer aggressiveness and bone metastases were investigated. METHODS: MDA-MB-231 (MDA-231) cells were treated in the presence or absence of anti-human IL-6 receptor (IL-6R) monoclonal antibody and examined with respect to cell survival. The expressions of signal transducer and activator of transcription 3 (Stat3), vascular endothelial growth factor (VEGF), and receptor activator of NF-κB (RANK) were analyzed by SDS-PAGE and immunoblotting. MDA-231 cells were injected into the left ventricle of mice, and then anti-human IL-6R monoclonal antibody or saline was administered intraperitoneally for 28 days. After 28 days, the incidence of bone metastases was evaluated in the hind limbs by radiography and histology. RESULTS: Anti-human IL-6R monoclonal antibody reduced bone metastases in an animal model injected with MDA-231 cells on radiological and histomorphometric analyses. The mechanism of bone metastasis inhibition involved inhibited cell proliferation and decreased expressions of phospho-Stat3, VEGF, and RANK in MDA-231 cells. CONCLUSIONS: The results of the present study suggest that inhibition of IL-6 signaling may become a preventive therapeutic option for breast cancer and bone metastases.

Alendronate inhibits hyperalgesia and suppresses neuropeptide markers of pain in a mouse model of osteoporosis.

BACKGROUND: Chronic back pain is one of the most important complications of postmenopausal osteoporosis. The aim of this study was to evaluate skeletal pain associated with osteoporosis and to examine the inhibitory effect of bisphosphonates (BPs) on pain in ovariectomized (OVX) mice. The mechanism of osteoporotic pain in OVX mice was evaluated through an examination of pain-related behavior, as well as immunohistochemical findings. In addition, the effects of alendronate (ALN), a potent osteoclast inhibitor, on these parameters were assessed. METHODS: 8-week-old female ddY mice were ovariectomized and assigned to 3 groups: SHAM-operated mice treated with vehicle (SHAM; n = 8); OVX mice treated with vehicle (OVX-V; n = 8); and OVX mice treated with ALN (OVX-ALN; n = 8). Starting immediately after surgery, vehicle or 40 µg/kg ALN was injected subcutaneously twice a week for 4 weeks. The bilateral distal femoral metaphyses and proximal tibial metaphyses were analyzed three-dimensionally by µCT. Mechanical sensitivity was tested using von Frey filaments. Transient receptor potential channel vanilloid 1 (TRPV1) and calcitonin gene-related peptide (CGRP) expressions in L3-5 dorsal root ganglion (DRG) neurons were examined immunohistochemically. RESULTS: Ovariectomy induced bone loss and mechanical hyperalgesia in hindlimbs with upregulation of TRPV1 and CGRP expressions in DRG neurons innervating hindlimbs. ALN prevented bone loss and mechanical hyperalgesia in ovariectomized mouse hindlimbs, and it suppressed upregulation of pain markers. CONCLUSIONS: ALN prevented ovariectomy-induced bone loss and mechanical hyperalgesia in hindlimbs, and it suppressed TRPV1 and CGRP expressions in DRG neurons. The results suggest that bone resorption with upregulation of TRPV1 and CGRP expressions is one of the causes of postmenopausal osteoporotic pain.

Anti-tumor necrosis factor therapy inhibits lung metastasis in an osteosarcoma cell line.

BACKGROUND: Osteosarcoma is the most common primary malignancy of bone, and patients often develop pulmonary metastases. In a previous study, tumor necrosis factor (TNF)-α treatment of human osteosarcoma cells increases their metastatic ability in an animal model. TNF-α can act as a tumor necrosis factor and also as a tumor-promoting factor. In the present study, the effect of a TNF-α inhibitor on osteosarcoma aggressiveness and pulmonary metastases was investigated in vitro and in vivo. METHODS: The effect of infliximab, a TNF-α inhibitor, on a metastatic osteosarcoma 143B cell growth and motility was investigated in vitro. An orthotopic xenograft model of 143B cell growth and spontaneous metastasis in SCID mice was used to assess the in vivo effect of infliximab. RESULTS: Infliximab greatly reduced cell motility and pulmonary metastases in 143B cells. The mechanism of pulmonary metastasis inhibition involved decreased expression of CXC chemokine receptor 4 (CXCR4), Rho (small GTPase protein), and its effector. CONCLUSIONS: These results suggest a novel role for TNF-α inhibition in the reduction or prevention of pulmonary metastases of osteosarcoma in this animal model. TNF-α inhibition may become a preventive therapeutic option for the pulmonary metastases of osteosarcoma.