Pharmacological control of angiogenesis by regulating phosphorylation of myosin light chain 2.

BACKGROUND: Control of angiogenesis is widely considered a therapeutic strategy, but reliable control methods are still under development. Phosphorylation of myosin light chain 2 (MLC2), which regulates actin-myosin interaction, is critical to the behavior of vascular endothelial cells (ECs) during angiogenesis. MLC2 is phosphorylated by MLC kinase (MLCK) and dephosphorylated by MLC phosphatase (MLCP) containing a catalytic subunit PP1. We investigated the potential role of MLC2 in the pharmacological control of angiogenesis. METHODS AND RESULTS: We exposed transgenic zebrafish Tg(fli1a:Myr-mCherry)ncv1 embryos to chemical inhibitors and observed vascular development. PP1 inhibition by tautomycetin increased length of intersegmental vessels (ISVs), whereas MLCK inhibition by ML7 decreased it; these effects were not accompanied by structural dysplasia. ROCK inhibition by Y-27632 also decreased vessel length. An in vitro angiogenesis model of human umbilical vein endothelial cells (HUVECs) showed that tautomycetin increased vascular cord formation, whereas ML7 and Y-27632 decreased it. These effects appear to be influenced by regulation of cell morphology rather than cell viability or motility. Actin co-localized with phosphorylated MLC2 (pMLC2) was abundant in vascular-like elongated-shaped ECs, but poor in non-elongated ECs. pMLC2 was associated with tightly arranged actin, but not with loosely arranged actin. Moreover, knockdown of MYL9 gene encoding MLC2 reduced total MLC2 and pMLC2 protein and inhibited angiogenesis in HUVECs. CONCLUSION: The present study found that MLC2 is a pivotal regulator of angiogenesis. MLC2 phosphorylation may be involved in the regulation of of cell morphogenesis and cell elongation. The functionally opposite inhibitors positively or negatively control angiogenesis, probably through the regulating EC morphology. These findings may provide a unique therapeutic target for angiogenesis.

Radiographic knee osteoarthritis severity has no impact on fall risk: the locomotive syndrome and health outcomes in the aizu cohort study (LOHAS): a cross-sectional study.

BACKGROUND: To investigate factors that have an impact on the risk of falls and determine whether radiographic knee osteoarthritis (KOA) is a factor involved in falls independent of knee pain, psychological factors, and physical function. METHODS: A cross-sectional analysis was conducted on 1083 subjects for the 2009 Locomotive Syndrome and Health Outcomes in the Aizu Cohort Study (LOHAS). A logistic regression analysis was performed to examine the relationship between radiographic KOA and fall history. RESULTS: Fall history was significantly associated with the severity of knee pain. Compared to subjects with no knee pain, the odds ratio (OR) was 1.53 times higher in the subjects with mild knee pain (95% confidence interval [CI]: 1.04-2.25), 1.69 times higher in those with moderate knee pain (95%CI: 1.03-2.79), and 2.98 times higher in those with severe knee pain (95%CI: 1.67-5.30). In subjects with depression, the OR was 1.91 (95%CI: 1.25-2.92), and in those with decreased mobility, the OR was 1.70 (95%CI: 1.08-2.69). Age, gender, knee crepitus, BMI, OLST, and sleeping pill use were not significantly associated with fall risk. In a multivariate analysis, radiographic KOA severity was not significantly associated with fall risk (OR 0.81, 95%CI 0.44-1.50 in mild OA; OR 1.10, 95%CI 0.57-2.14 in severe OA). CONCLUSION: Knee pain, decreased mobility, and depression, but not the radiographic KOA severity, were significantly associated with a fall risk. Regardless of the individual's radiographic KOA severity, the risk of falls may be reduced by treating his/her knee pain, mobility problems, and/or psychological factors.

A case of autoimmune pulmonary alveolar proteinosis during the course of treatment of rapidly progressive interstitial pneumonia associated with anti-MDA5 antibody-positive dermatomyositis.

BACKGROUND: Autoimmune pulmonary alveolar proteinosis (APAP) is a diffuse lung disease that causes abnormal accumulation of lipoproteins in the alveoli; however, its pathogenesis remains unclear. Recently, APAP cases have been reported during the course of dermatomyositis. The combination of these two diseases may be coincidental; however, it may have been overlooked because differentiating APAP from a flare-up of interstitial pneumonia associated with dermatomyositis is challenging. This didactic case demonstrates the need for early APAP scrutiny. CASE PRESENTATION: A 50-year-old woman was diagnosed with anti-melanoma differentiation-associated gene 5 (anti-MDA5) antibody-positive dermatitis and interstitial pneumonia in April 2021. The patient was treated with corticosteroids, tacrolimus, and cyclophosphamide pulse therapy for interstitial pneumonia complicated by MDA5 antibody-positive dermatitis, which improved the symptoms and interstitial pneumonia. Eight months after the start of treatment, a new interstitial shadow appeared that worsened. Therefore, three additional courses of cyclophosphamide pulse therapy were administered; however, the respiratory symptoms and interstitial shadows did not improve. Respiratory failure progressed, and 14 months after treatment initiation, bronchoscopy revealed turbid alveolar lavage fluid, numerous foamy macrophages, and numerous periodic acid-Schiff-positive unstructured materials. Blood test results revealed high anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antibody levels, leading to a diagnosis of APAP. The patient underwent whole-lung lavage, and the respiratory disturbance promptly improved. Anti-GM-CSF antibodies were measured from the cryopreserved serum samples collected at the time of diagnosis of anti-MDA5 antibody-positive dermatitis, and 10 months later, both values were significantly higher than normal. CONCLUSIONS: This is the first report of anti-MDA5 antibody-positive dermatomyositis complicated by interstitial pneumonia with APAP, which may develop during immunosuppressive therapy and be misdiagnosed as a re-exacerbation of interstitial pneumonia. In anti-MDA5 antibody-positive dermatomyositis, APAP comorbidity may have been overlooked, and early evaluation with bronchoalveolar lavage fluid and anti-GM-CSF antibody measurements should be considered, keeping the development of APAP in mind.

Investigation of walking speed and plantar pressure after chopart amputation.

BACKGROUND: In foot amputation, Chopart amputation is considered to have a high risk of deformity, and can result in poor function. We experienced a case in which Chopart amputation combined with tendon transfer and tendon lengthening was performed, and the patient was eventually able to walk independently with a foot prosthesis without experiencing deformity of the foot. We investigated walking speed and plantar pressure after Chopart amputation with and without a foot prosthesis. CASE: A 78-year-old man underwent Chopart amputation with tendon transfer and tendon lengthening. As a result, he was able to stand up and walk, both while bearing weight on the heel of the affected foot, but he was unable to push off the ground using that foot. When a foot prosthesis was introduced, the patient's walking speed increased from 0.6 m/s without the prosthesis to 0.8 m/s with the prosthesis, which was an increase of 33%. The plantar pressure at the stump decreased from 129.3 N/cm2 on average without the prosthesis to 51.6 N/cm2 with the prosthesis, which was a 59% decrease. Wearing a foot prosthesis improved the patient's walking speed and decreased plantar pressure at the amputation stump.

Gender differences on preoperative psychologic factors affecting acute postoperative pain in patients with lumbar spinal disorders.

BACKGROUND: Psychosexual factors are one of the preoperative factors influencing acute postoperative pain. Because of gender differences in pain, the preoperative factors that influence acute postoperative pain may also differ between males and females. However, there have been no reports on such factors in patients with spinal disorders that focused on gender differences. Therefore, the purpose of this study was to examine the preoperative factors that influence acute postoperative pain, focusing on gender differences. METHODS: The subjects were 75 males and 60 females admitted for surgery for lumbar spinal disorders. Preoperatively, the following were assessed: low back pain using the Numeric Rating Scale (NRS); anxiety and depression using the Japanese version of the Hospital Anxiety and Depression Scale (HADS); catastrophic thinking using the Pain Catastrophizing Scale (PCS); psychiatric problems using the Brief Scale for Psychiatric Problems in Orthopaedic Patients (BS-POP); and neurological assessments. Acute postoperative pain was also assessed using the NRS within 48 h, postoperatively. Based on these data, we analyzed gender differences in preoperative factors affecting acute postoperative pain in patients with lumbar spinal disorders. RESULTS: Postoperative NRS and preoperative PCS scores were higher in females compared to males. In the males, the coefficient of determination of the multiple regression equation was 0.088, and PCS (ß = 0.323, p = 0.015) was extracted as a significant factor. In the females, the coefficient of determination of the multiple regression equation was 0.075, and BS-POP (ß = 0.300, p = 0.019) was extracted as a significant factor. CONCLUSION: Preoperative factors influencing acute postoperative pain for patients with lumbar spinal disorders vary by gender. It was suggested that males should be screened using PCS. In females, on the other hand, PCS alone is not sufficient for evaluation. It was suggested that evaluation using BS-POP should be considered in addition to PCS.

Relationship between dysphagia and motor function in community-dwelling older people.

AIM: Dysphagia is a problem typically associated with aging. The aim was to investigate the relationship between dysphagia and motor function using a simple assessment method that can be performed in the community setting, and to promote the early detection and prevention of dysphagia. METHODS: Data from the Locomotive Syndrome and Health Outcome in Aizu Cohort Study (LOHAS) were used. Those aged ≥65 years were included. Motor function was assessed using a grip strength test, single limb standing test (SLS), and timed up and go test (TUG). Swallowing function was assessed using the Japanese version of the 10-item Eating Assessment Tool (EAT-10). The association between motor function and swallowing function was analyzed. RESULTS: In total, 1732 participants were included. In logistic regression modes in which grip strength, SLS, and TUG results were included separately, the odds ratio for dysphagia increased by 1.08 (P = 0.001) for each 1-kg decrease in grip strength, and increased by 1.15 (P < 0.001) for each 1-s increase in TUG time. No association was found for SLS. In the model in which grip strength and TUG were included simultaneously, the odds ratio for dysphagia increased by 1.06 (P = 0.01) in grip strength, and increased by 1.11 (P = 0.009) in TUG time. CONCLUSION: Our results suggest that skeletal muscle strength and dynamic balance function are associated with dysphagia in community-dwelling older people. Geriatr Gerontol Int 2023; 23: 603-608.

Effective treatment with mepolizumab in a patient with severe eosinophilic granulomatosis with polyangiitis complicated with small intestine perforation.

Eosinophilic granulomatosis with polyangiitis (EGPA) is systemic vasculitis caused by eosinophilia affecting small to medium-sized blood vessels, which damages the organs. Antineutrophil cytoplasmic antibody-associated vasculitis EGPA treatment guidelines added anti-interleukin-5 antibody mepolizumab to the standard treatment protocol for active-non-severe EGPA based on the MIRRA study. Nevertheless, the role of mepolizumab in treating patients with active severe EGPA has not been established. We treated a patient with EGPA complicated with small intestine perforation using steroid pulse intravenous, high-dose glucocorticoids, intravenous high-dose immunoglobulin therapy, and mepolizumab without immunosuppression agents; the patient went into remission, suggesting that mepolizumab is an effective therapeutic agent that could lead to remission in severe EGPA.

Chopart amputation with tendon balancing.

BACKGROUND: When foot necrosis occurs due to lower limb blood flow disorder caused by diabetes or peripheral arterial occlusion, many patients require lower limb amputation. The functional prognosis after lower limb amputation largely depends on whether the heel can be preserved. However, there are many reports that Chopart amputation causes varus and equinus deformity, and is functionally unfavorable. We herein report a case of Chopart amputation performed with muscle balancing. Postoperatively, the foot was not deformed and the patient was able to walk independently with a foot prosthesis. CASE: A 78-year-old man presented with ischemic necrosis of his right forefoot. The range of necrosis extended to the central part of the sole, so Chopart amputation was performed. In the operation, to prevent varus and equinus deformity, the Achilles tendon was lengthened, the tibialis anterior tendon was transferred through a tunnel created in the neck of talus, and the peroneus brevis tendon was transferred through a tunnel created in the anterior part of the calcaneus. At the final follow-up 7 years after the operation, no varus or equinus deformity was observed. The patient became able to stand up and walk on his heel without a prosthesis. In addition, step motion was possible by wearing a foot prosthesis.

Sustained antitumor response to lenvatinib with weekend-off and alternate-day administration in chemotherapy-refractory thymic carcinoma: a case report.

Lenvatinib is a multitargeted kinase inhibitor and maintaining its dose intensity has been shown to be beneficial in patients with thyroid and hepatocellular carcinomas. However, most patients require lenvatinib interruption and dose reduction due to the high incidence of adverse events (AEs). Lenvatinib was recently approved in Japan for patients with unresectable thymic carcinoma; however, real-world evidence of its clinical benefit is limited. Here, we report the case of chemotherapy-refractory thymic carcinoma in a patient who was administered a starting dose of lenvatinib using a 5-day on/2-day off (weekend-off) protocol, followed by alternate-day administration after fatigue onset derived from overt or subclinical hypothyroidism. Consequently, the patient exhibited a durable response to lenvatinib, with a 17-month progression-free survival without any severe or intolerable AEs. The present case suggests that maintaining lenvatinib dose intensity using such alternative administration regimens contributes to favorable clinical outcomes in thymic carcinoma.

Contributions of Resin Cast Etching to Visualising the Osteocyte Lacuno-Canalicular Network Architecture in Bone Biology and Tissue Engineering.

Recent years have witnessed an evolution of imaging technologies towards sophisticated approaches for visualising cells within their natural environment(s) and for investigating their interactions with other cells, with adjacent anatomical structures, and with implanted biomaterials. Resin cast etching (RCE) is an uncomplicated technique involving sequential acid etching and alkali digestion of resin embedded bone to observe the osteocyte lacuno-canalicular network using scanning electron microscopy. This review summarises the applicability of RCE to bone and the bone-implant interface. Quantitative parameters such as osteocyte size, osteocyte density, and number of canaliculi per osteocyte, and qualitative metrics including osteocyte shape, disturbances in the arrangement of osteocytes and canaliculi, and physical communication between osteocytes and implant surfaces can be investigated. Ageing, osteoporosis, long-term immobilisation, spinal cord injury, osteoarthritis, irradiation, and chronic kidney disease have been shown to impact osteocyte lacuno-canalicular network morphology. In addition to titanium, calcium phosphates, and bioactive glass, observation of direct connectivity between osteocytes and cobalt chromium provides new insights into the osseointegration potential of materials conventionally viewed as non-osseointegrating. Other applications include in vivo and in vitro testing of polymer-based tissue engineering scaffolds and tissue-engineered ossicles, validation of ectopic osteochondral defect models, ex vivo organ culture of whole bones, and observing the effects of gene dysfunction/deletion on the osteocyte lacuno-canalicular network. Without additional contrast staining, any resin embedded specimen (including clinical biopsies) can be used for RCE. The multitude of applications described here attest to the versatility of RCE for routine use within correlative analytical workflows, particularly in biomaterials science.

Durable response to afatinib rechallenge in a long-term survivor of non-small cell lung cancer harboring EGFR L858R and L747V mutations.

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors are standard therapeutic agents for non-small cell lung cancer (NSCLC) patients with major EGFR mutations such as exon 19 deletions and a L858R mutation, whereas treatment strategies for cases with uncommon EGFR mutations remain to be fully established. Here, we report a long-term (≥20 years from initial diagnosis) NSCLC survivor carrying EGFR L858R and L747V mutations. The patient received gefitinib monotherapy, systemic chemotherapy/chemoimmunotherapy, and local consolidative therapies for oligometastatic lesions, and responded to afatinib rechallenge with a progression-free survival of 12 months. The current case suggests that afatinib is effective in NSCLC patients with EGFR L858R and L747V mutations and that a therapeutic approach combining appropriately timed systemic therapies with local consolidative therapies for oligometastatic lesions improves long-term survival.

Differentiating Trypanosoma cruzi in a Host Mammalian Cell Imaged in Aqueous Liquid by Atmospheric Scanning Electron Microscopy.

Atmospheric Scanning Electron Microscopy (ASEM) is a powerful tool to observe a wet specimen at high resolution under atmospheric pressure. Here, we visualized a protozoan parasite Trypanosoma cruzi over the course of its infection cycle in the host mammalian cell. This is the first observation of intracellular parasite using a liquid-phase EM. Unlike regular SEM, aldehyde-fixed cell body of T. cruzi appears translucent, allowing the visualization of internal structures such as kinetoplast of trypomastigote and nucleus of amastigote. Plasma membrane of the host mammalian cell also appears translucent, which enabled direct observation of differentiating intracellular parasites and dynamic change of host cellular structures in their near-natural states. Various water-rich structures including micro- and macro- vesicles were visualized around T. cruzi. In addition, Correlative Light and Electron Microscopy exploiting open sample dish of ASEM allowed identification of parasite nucleus and transfected fluorescence-labeled parasites soon after internalization, while location of this morphological intermediate was otherwise obscure. Successful visualization of the differentiation of T. cruzi within the host cell demonstrated here opens up the possibility of using ASEM for observation of variety of intracellular parasites. IMPORTANCE Using Atmospheric Scanning Electron Microscopy (ASEM), we visualized interaction between infectious stage of Trypanosoma cruzi and completely intact host mammalian cell. Plasma membrane appears translucent under ASEM, which not only enables direct observation of T. cruzi within its host cell, but also reveals internal structures of the parasite itself. Sample deformation is minimal, since the specimen remains hydrated under atmospheric pressure at all times. This nature of ASEM, along with the open structure of ASEM sample dish, is suited for correlative light-electron microscopy, which can further be exploited in identification of fluorescent protein in the intracellular parasites.

Biofilm formation of Staphylococcus epidermidis imaged using atmospheric scanning electron microscopy.

Staphylococcus epidermidis are gram-positive bacteria that form a biofilm around implanted devices and develop an infection into a chronic state. Recently, it has been revealed that microvesicles have important roles in biofilm formation and intercellular communication among bacteria. However, biofilm formation of Staphylococcus epidermidis, and its relation to microvesicle secretion, is poorly understood because of the difficulty required to preserve the delicate water-rich morphology of biofilm for high-resolution observations. Here, we successfully imaged the microvesicles secreted from Staphylococcus epidermidis and the subsequent process of their integration into biofilm using liquid-phase imaging using atmospheric scanning electron microscopy (ASEM). In the biofilm, cells were connected by nanotube-like structures attached by microvesicles, and surrounded by extracellular polymeric substances. Cells cultured in the ASEM specimen holder were aldehyde-fixed and stained using positively charged nanogold labelling and/or using National Center for Microscopy and Imaging Research method. The samples immersed in aqueous radical scavenger glucose buffer were imaged by the inverted SEM of ASEM. Information regarding the morphologies of microvesicles, nanotube-like fibrils, and biofilm formed by Staphylococcus epidermidis is expected to be useful to elucidate the biological mechanism of biofilm formation and to develop a medicine against biofilms and their associated infections.

Biofilm Spreading by the Adhesin-Dependent Gliding Motility of Flavobacterium johnsoniae: 2. Role of Filamentous Extracellular Network and Cell-to-Cell Connections at the Biofilm Surface.

Flavobacterium johnsoniae forms a thin spreading colony on nutrient-poor agar using gliding motility. As reported in the first paper, WT cells in the colony were sparsely embedded in self-produced extracellular polymeric matrix (EPM), while sprB cells were densely packed in immature biofilm with less matrix. The colony surface is critical for antibiotic resistance and cell survival. We have now developed the Grid Stamp-Peel method whereby the colony surface is attached to a TEM grid for negative-staining microscopy. The images showed that the top of the spreading convex WT colonies was covered by EPM with few interspersed cells. Cells exposed near the colony edge made head-to-tail and/or side-to-side contact and sometimes connected via thin filaments. Nonspreading sprB and gldG and gldK colonies had a more uniform upper surface covered by different EPMs including vesicles and filaments. The EPM of sprB, gldG, and WT colonies contained filaments ~2 nm and ~5 nm in diameter; gldK colonies did not include the latter. Every cell near the edge of WT colonies had one or two dark spots, while cells inside WT colonies and cells in SprB-, GldG-, or GldK-deficient colonies did not. Together, our results suggest that the colony surface structure depends on the capability to expand biofilm.

Osteocytes as main responders to low-intensity pulsed ultrasound treatment during fracture healing.

Ultrasound stimulation is a type of mechanical stress, and low-intensity pulsed ultrasound (LIPUS) devices have been used clinically to promote fracture healing. However, it remains unclear which skeletal cells, in particular osteocytes or osteoblasts, primarily respond to LIPUS stimulation and how they contribute to fracture healing. To examine this, we utilized medaka, whose bone lacks osteocytes, and zebrafish, whose bone has osteocytes, as in vivo models. Fracture healing was accelerated by ultrasound stimulation in zebrafish, but not in medaka. To examine the molecular events induced by LIPUS stimulation in osteocytes, we performed RNA sequencing of a murine osteocytic cell line exposed to LIPUS. 179 genes reacted to LIPUS stimulation, and functional cluster analysis identified among them several molecular signatures related to immunity, secretion, and transcription. Notably, most of the isolated transcription-related genes were also modulated by LIPUS in vivo in zebrafish. However, expression levels of early growth response protein 1 and 2 (Egr1, 2), JunB, forkhead box Q1 (FoxQ1), and nuclear factor of activated T cells c1 (NFATc1) were not altered by LIPUS in medaka, suggesting that these genes are key transcriptional regulators of LIPUS-dependent fracture healing via osteocytes. We therefore show that bone-embedded osteocytes are necessary for LIPUS-induced promotion of fracture healing via transcriptional control of target genes, which presumably activates neighboring cells involved in fracture healing processes.

Biofilm Spreading by the Adhesin-Dependent Gliding Motility of Flavobacterium johnsoniae. 1. Internal Structure of the Biofilm.

The Gram-negative bacterium Flavobacterium johnsoniae employs gliding motility to move rapidly over solid surfaces. Gliding involves the movement of the adhesin SprB along the cell surface. F. johnsoniae spreads on nutrient-poor 1% agar-PY2, forming a thin film-like colony. We used electron microscopy and time-lapse fluorescence microscopy to investigate the structure of colonies formed by wild-type (WT) F. johnsoniae and by the sprB mutant (ΔsprB). In both cases, the bacteria were buried in the extracellular polymeric matrix (EPM) covering the top of the colony. In the spreading WT colonies, the EPM included a thick fiber framework and vesicles, revealing the formation of a biofilm, which is probably required for the spreading movement. Specific paths that were followed by bacterial clusters were observed at the leading edge of colonies, and abundant vesicle secretion and subsequent matrix formation were suggested. EPM-free channels were formed in upward biofilm protrusions, probably for cell migration. In the nonspreading ΔsprB colonies, cells were tightly packed in layers and the intercellular space was occupied by less matrix, indicating immature biofilm. This result suggests that SprB is not necessary for biofilm formation. We conclude that F. johnsoniae cells use gliding motility to spread and maturate biofilms.

Immune-related adverse events caused by treatment with pembrolizumab in a patient with lung cancer who infected influenza virus.

A 67-year-old man with stage IV B lung adenocarcinoma was treated with pembrolizumab. The patient was admitted to the hospital because of influenza on the day of the second cycle of pembrolizumab treatment. He was diagnosed with pneumonia and was treated with antiviral drugs and steroids. However, the patient eventually died. In this case, treatment with immune checkpoint inhibitors might have affected the immune response caused by influenza virus infection, that might have caused lung injury, which is an immune-related adverse event (irAE). Hence, it is important that, caution should be taken to prevent transmission of viral infection, and Therefore, it is important to prevent viral infections, but caution should also be paid to the possibility that infections may cause irAEs in patients with lung cancer.

Liquid-phase ASEM imaging of cellular and structural details in cartilage and bone formed during endochondral ossification: Keap1-deficient osteomalacia.

Chondrogenesis and angiogenesis drive endochondral ossification. Using the atmospheric scanning electron microscopy (ASEM) without decalcification and dehydration, we directly imaged angiogenesis-driven ossification at different developmental stages shortly after aldehyde fixation, using aqueous radical scavenger glucose solution to preserve water-rich structures. An embryonic day 15.5 mouse femur was fixed and stained with phosphotungstic acid (PTA), and blood vessel penetration into the hypertrophic chondrocyte zone was visualised. We observed a novel envelope between the perichondrium and proliferating chondrocytes, which was lined with spindle-shaped cells that could be borderline chondrocytes. At postnatal day (P)1, trabecular and cortical bone mineralisation was imaged without staining. Additional PTA staining visualised surrounding soft tissues; filamentous connections between osteoblast-like cells and osteocytes in cortical bone were interpreted as the osteocytic lacunar-canalicular system. By P10, resorption pits had formed on the tibial trabecular bone surface. The applicability of ASEM for pathological analysis was addressed using knockout mice of Keap1, an oxidative-stress sensor. In Keap1-/- femurs, we observed impaired calcification and angiogenesis of epiphyseal cartilage, suggesting impaired bone development. Overall, the quick ASEM method we developed revealed mineralisation and new structures in wet bone tissue at EM resolution and can be used to study mineralisation-associated phenomena of any hydrated tissue.

Colony spreading of the gliding bacterium Flavobacterium johnsoniae in the absence of the motility adhesin SprB.

Colony spreading of Flavobacterium johnsoniae is shown to include gliding motility using the cell surface adhesin SprB, and is drastically affected by agar and glucose concentrations. Wild-type (WT) and ΔsprB mutant cells formed nonspreading colonies on soft agar, but spreading dendritic colonies on soft agar containing glucose. In the presence of glucose, an initial cell growth-dependent phase was followed by a secondary SprB-independent, gliding motility-dependent phase. The branching pattern of a ΔsprB colony was less complex than the pattern formed by the WT. Mesoscopic and microstructural information was obtained by atmospheric scanning electron microscopy (ASEM) and transmission EM, respectively. In the growth-dependent phase of WT colonies, dendritic tips spread rapidly by the movement of individual cells. In the following SprB-independent phase, leading tips were extended outwards by the movement of dynamic windmill-like rolling centers, and the lipoproteins were expressed more abundantly. Dark spots in WT cells during the growth-dependent spreading phase were not observed in the SprB-independent phase. Various mutations showed that the lipoproteins and the motility machinery were necessary for SprB-independent spreading. Overall, SprB-independent colony spreading is influenced by the lipoproteins, some of which are involved in the gliding machinery, and medium conditions, which together determine the nutrient-seeking behavior.

Prediction of oral intake recovery for inpatients with aspiration pneumonia by videoendoscopic evaluation using the Hyodo-Komagane score in Japan.

OBJECTIVE: To verify the validity of the prediction of oral intake recovery for inpatients with aspiration pneumonia using the Hyodo-Komagane score. BACKGROUND: Patients admitted for treatment of aspiration pneumonia sometimes have difficulty in resuming oral intake due to decreased swallowing function. Predicting whether the swallowing function will recover enough to achieve oral ingestion at discharge is an important factor in developing a treatment strategy. No studies have investigated the prediction of oral intake recovery using videoendoscopic examination. METHODS: Subjects were 65 patients who were admitted to an acute care hospital for the treatment of aspiration pneumonia. The patients were divided into two groups, the oral feeding group and the tube feeding group, according to their oral intake status at discharge or transfer. Logistic regression analysis was performed using the condition that tube feeding was not required as an objective variable and the items with significant differences between the two groups as explanatory variables. Additionally, the receiver operating characteristic curve was used to identify patients who could take food orally at discharge. RESULTS: The odds ratios for the Hyodo-Komagane score and the pharyngeal clearance score were 1.485 and 3.379, respectively. When the cut-off values of the Hyodo-Komagane score and the pharyngeal clearance score were 6 and 1, the sensitivity was 0.88 and 0.91, and the specificity was 0.64 and 0.70, respectively. CONCLUSION: The Hyodo-Komagane score and especially the pharyngeal clearance score are useful indices to predict oral intake recovery for inpatients with aspiration pneumonia.