Clarithromycin overcomes stromal cell-mediated drug resistance against proteasome inhibitors in myeloma cells via autophagy flux blockage leading to high NOXA expression.

We previously reported that macrolide antibiotics, such as clarithromycin (CAM), blocked autophagy flux, and simultaneous proteasome and autophagy inhibition by bortezomib (BTZ) plus CAM resulted in enhanced apoptosis induction in multiple myeloma (MM) cells via increased endoplasmic reticulum (ER) stress loading. However, in actual therapeutic settings, cell adhesion-mediated drug resistance between bone marrow stromal cells (BMSC) and MM cells has been known to be a barrier to treatment. To investigate whether CAM could enhance BTZ-induced cytotoxicity in MM cells under direct cell adhesion with BMSC, we established a co-culture system of EGFP-labeled MM cells with BMSC. The cytotoxic effect of BTZ on MM cells was diminished by its interaction with BMSC; however, the attenuated cytotoxicity was recovered by the co-administration of CAM, which upregulates ER stress loading and NOXA expression. Knockout of NOXA in MM cells canceled the enhanced cell death by CAM, indicating that NOXA is a key molecule for cell death induction by the co-administration of CAM. Since NOXA is degraded by autophagy as well as proteasomes, blocking autophagy with CAM resulted in the sustained upregulation of NOXA in MM cells co-cultured with BMSC in the presence of BTZ. Our data suggest that BMSC-associated BTZ resistance is mediated by the attenuation of ER stress loading. However, the addition of CAM overcomes BMSC-associated resistance via upregulation of NOXA by concomitantly blocking autophagy-mediated NOXA degradation and transcriptional activation of NOXA by ER stress loading.

Macrophage depletion using clodronate liposomes reveals latent dysfunction of the hematopoietic microenvironment associated with persistently imbalanced M1/M2 macrophage polarization in a mouse model of hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH), a hyperinflammatory syndrome, is caused by the incessant activation of lymphocytes and macrophages, resulting in damage to organs, including hematopoietic organs. Recently, we demonstrated that repeated lipopolysaccharide (LPS) treatment induces HLH-like features in senescence-accelerated (SAMP1/TA-1) mice but not in senescence-resistant control (SAMR1) mice. Hematopoietic failure in LPS-treated SAMP1/TA-1 mice was attributed to hematopoietic microenvironment dysfunction, concomitant with severely imbalanced M1 and M2 macrophage polarization. Macrophages are a major component of the bone marrow (BM) hematopoietic microenvironment. Clodronate liposomes are useful tools for in vivo macrophage depletion. In this study, we depleted macrophages using clodronate liposomes to determine their role in the hematopoietic microenvironment in SAMP1/TA-1 and SAMR1 mice. Under clodronate liposome treatment, the response between SAMR1 and SAMP1/TA-1 mice differed as follows: (1) increase in the number of activated M1 and M2 macrophages derived from newly generated macrophages and M2-dominant and imbalanced M1 and M2 macrophage polarization in the BM and spleen; (2) severe anemia and thrombocytopenia; (3) high mortality rate; (4) decrease in erythroid progenitors and B cell progenitors in the BM; and (5) decrease in the mRNA expression of erythroid-positive regulators such as erythropoietin and increase in that of erythroid- and B lymphoid-negative regulators such as interferon-γ in the BM. Depletion of residual macrophages in SAMP1/TA-1 mice impaired hematopoietic homeostasis, particularly erythropoiesis and B lymphopoiesis, owing to functional impairment of the hematopoietic microenvironment accompanied by persistently imbalanced M1/M2 polarization. Thus, macrophages play a vital role in regulating the hematopoietic microenvironment to maintain homeostasis.

Size Comparison of the Cadaveric Anterior Cruciate Ligament Midsubstance Cross-Sectional Area and the Cross-Sectional Area of Semitendinosus Double-Bundle Anterior Cruciate Ligament Reconstruction Autografts in Surgery.

The purpose of this study was to compare the cadaveric midsubstance cross-sectional anterior cruciate ligament (ACL) area and the cross-sectional semitendinosus (ST) double-bundle ACL autograft area in surgery. Thirty-nine nonpaired formalin-fixed cadaveric knees and 39 subjects undergoing ST double-bundle ACL reconstruction were included in this study. After soft tissue resection, cadaveric knees were flexed at 90 degrees, and the tangential line of the femoral posterior condyles was marked and sliced on the ACL midsubstance. The cross-sectional ACL area was measured using Image J software. In the patients undergoing ACL surgery, the harvested ST was cut and divided into anteromedial (AM) bundle and posterolateral (PL) bundle. Each graft edge diameter was measured by a sizing tube, and the cross-sectional graft area was calculated: (AM diameter/2)2 × 3.14 + (PL diameter/2)2 × 3.14. Statistical analysis was performed for the comparison of the cross-sectional area between the cadaveric ACL midsubstance and the ST double-bundle ACL autografts. The cadaveric midsubstance cross-sectional ACL area was 49.0 ± 16.3 mm2. The cross-sectional ST double-bundle autografts area was 52.8 ± 7.6 mm2. The ST double-bundle autograft area showed no significant difference when compared with the midsubstance cross-sectional ACL area. ST double-bundle autografts were shown to be capable of reproducing the midsubstance cross-sectional ACL area.

Imbalanced M1 and M2 Macrophage Polarization in Bone Marrow Provokes Impairment of the Hematopoietic Microenvironment in a Mouse Model of Hemophagocytic Lymphohistiocytosis.

Lipopolysaccharide (LPS) treatment induced hemophagocytic lymphohistiocytosis in senescence-accelerated mice (SAMP1/TA-1), but not in senescence-resistant control mice (SAMR1). SAMP1/TA-1 treated with LPS exhibited functional impairment of the hematopoietic microenvironment, which disrupted the dynamics of hematopoiesis. Macrophages are a major component of the bone marrow (BM) hematopoietic microenvironment, which regulates hematopoiesis. Qualitative and quantitative changes in activated macrophages in LPS-treated SAMP1/TA-1 are thought to contribute to the functional deterioration of the hematopoietic microenvironment. Thus, we examined the polarization of pro-inflammatory (M1) and anti-inflammatory (M2) macrophages, and the dynamics of macrophage production in the BM of SAMP1/TA-1 and SAMR1 after LPS treatment. After LPS treatment, the proportions of M1 and M2 macrophages and the numbers of macrophage progenitor (CFU-M) cells increased in both SAMP1/TA-1 and SAMR1. However, compared to the SAMR1, the increase in the M1 macrophage proportion was prolonged, and the increase in the M2 macrophage proportion was delayed. The increase in the number of CFU-M cells was prolonged in SAMP1/TA-1 after LPS treatment. In addition, the levels of transcripts encoding an M1 macrophage-inducing cytokine (interferon-γ) and macrophage colony-stimulating factor were markedly increased, and the increases in the levels of transcripts encoding M2 macrophage-inducing cytokines (interleukin (IL)-4, IL-10, and IL-13) were delayed in SAMP1/TA-1 when compared to SAMR1. Our results suggest that LPS treatment led to the severely imbalanced polarization of activated M1/M2 macrophages accompanied by a prolonged increase in macrophage production in the BM of SAMP1/TA-1, which led to the impairment of the hematopoietic microenvironment, and disrupted the dynamics of hematopoiesis.

Comparing effects of microgravity and amyotrophic lateral sclerosis in the mouse ventral lumbar spinal cord.

Microgravity (MG) exposure and motor neuron diseases, such as amyotrophic lateral sclerosis (ALS), lead to motor deficits, including muscle atrophy and loss of neuronal activity. Abnormalities in motor neurons and muscles caused by MG exposure can be recovered by subsequent ground exercise. In contrast, the degeneration that occurs in ALS is irreversible. A common phenotype between MG exposure and ALS pathology is motor system abnormality, but the causes may be different. In this study, to elucidate the motor system that is affected by each condition, we investigated the effects of MG and the human SOD1 ALS mutation on gene expression in various cell types of the mouse ventral lumbar spinal cord, which is rich in motor neurons innervating the lower limb. To identify cell types affected by MG or ALS pathogenesis, we analyzed differentially expressed genes with known cell-type markers, which were determined from previous single-cell studies of the spinal cord in MG-exposed and SOD1G93A mice, an ALS mouse model. Differentially expressed genes were observed in MG mice in various spinal cord cell types, including neurons, microglia, astrocytes, oligodendrocytes, oligodendrocyte precursor cells, meningeal cells/Schwann cells, and vascular cells. We also examined neuronal populations in the spinal cord. Gene expression in putative excitatory and inhibitory neurons changed more than that in cholinergic motor neurons of the spinal cord in both MG and SOD1G93A mice. Many putative neuron types, especially visceral motor neurons, and axon initial segments (AIS) were affected in MG mice. In contrast, the effect on neurons and AIS in SOD1G93A mice was slight at P30 but progressed with aging. Interestingly, changes in dopaminergic system-related genes were specifically altered in the spinal cord of MG mice. These results indicate that MG and ALS pathology in various cell types contribute to motor neuron degeneration. Furthermore, there were more alterations in neurons in MG-exposed mice than in SOD1G93A mice. A large number of differentially expressed genes (DEGs) in MG mice represent more than SOD1G93A mice with ALS pathology. Elucidation of MG pathogenesis may provide more insight into the pathophysiology of neurodegenerative diseases.

The Anterior Piriform Cortex and Predator Odor Responses: Modulation by Inhibitory Circuits.

Rodents acquire more information from the sense of smell than humans because they have a nearly fourfold greater variety of olfactory receptors. They use olfactory information not only for obtaining food, but also for detecting environmental dangers. Predator-derived odor compounds provoke instinctive fear and stress reactions in animals. Inbred lines of experimental animals react in an innate stereotypical manner to predators even without prior exposure. Predator odors have also been used in models of various neuropsychiatric disorders, including post-traumatic stress disorder following a life-threatening event. Although several brain regions have been reported to be involved in predator odor-induced stress responses, in this mini review, we focus on the functional role of inhibitory neural circuits, especially in the anterior piriform cortex (APC). We also discuss the changes in these neural circuits following innate reactions to odor exposure. Furthermore, based on the three types of modulation of the stress response observed by our group using the synthetic fox odorant 2,5-dihydro-2,4,5-trimethylthiazoline, we describe how the APC interacts with other brain regions to regulate the stress response. Finally, we discuss the potential therapeutic application of odors in the treatment of stress-related disorders. A clearer understanding of the odor-stress response is needed to allow targeted modulation of the monoaminergic system and of the intracerebral inhibitory networks. It would be improved the quality of life of those who have stress-related conditions.

Neurovascular unit pathology is observed very early in disease progression in the mutant SOD1G93A mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disease characterized by motor neuron degeneration that causes neuromuscular denervation, resulting in muscle weakness and atrophy. Work over the decades using ALS mouse models has revealed that while initial pathology may occur within motor neurons, disease pathology is cell non-autologous. Impairment of the blood-spinal cord barrier (BSCB) occurs before motor neuron frank degeneration; however, precisely when the early pathogenesis of the neurovascular units occurs is not fully understood. Here we examine changes in morphology of neurovascular units, associated gene and protein expression in the lumbar spinal cord of SOD1G93A, and wild-type mice and correlate results with previous reports of early pathological events. Using RNA-sequencing and immunolabeling, we also show that both the neurovascular units and the vasculature of the SOD1G93A lumbar spinal cord present important modifications throughout the disease. Genes relevant for the neurovascular unit and immune cells were differentially expressed in the SOD1G93A ventral lumbar spinal cord compared to wild-type. A reduction in capillary density and tight junction (TJ) with overt BSCB breakdown was observed in the SOD1G93A lumbar spinal cord and ultrastructural observation revealed intact TJ. Additionally, thickened basement membrane, increased pericytes, and string vessels were observed. These alterations in neurovascular units and the vasculature are observed prior to reports of initial neuromuscular junction denervation. The identification of early pathogenesis may be critical to develop diagnostic tests and development of novel treatment strategies that target these early events.

The hdeD Gene Represses the Expression of Flagellum Biosynthesis via LrhA in Escherichia coli K-12.

Escherichia coli survives under acid stress conditions by the glutamic acid-dependent acid resistance (GAD) system, which enzymatically decreases intracellular protons. We found a linkage between GAD and flagellar systems in E. coli. The hdeD gene, one of the GAD cluster genes, encodes an uncharacterized membrane protein. A reporter assay showed that the hdeD promoter was induced in a GadE-dependent manner when grown in the M9 glycerol medium. Transcriptome analysis revealed that most of the transcripts were from genes involved in flagellum synthesis, and cell motility increased not only in the hdeD-deficient mutant but also in the gadE-deficient mutant. Defects in both the hdeD and gadE increased the intracellular level of FliA, an alternative sigma factor for flagellum synthesis, activated by the master regulator FlhDC. The promoter activity of the lrhA gene, which encodes repressor for the flhDC operon, was found to decrease in both the hdeD- and gadE-deficient mutants. Transmission electron microscopy showed that the number of flagellar filaments on the hdeD-, gadE-, and lrhA-deficient cells increased, and all three mutants showed higher motility than the parent strain. Thus, HdeD in the GAD system activates the lrhA promoter, resulting in a decrease in flagellar filaments in E. coli cells. We speculated that the synthesis of HdeD, stimulated in E. coli exposed to acid stress, could control the flagellum biosynthesis by sensing slight changes in pH at the cytoplasmic membrane. This could help in saving energy through termination of flagellum biosynthesis and improve bacterial survival efficiency within the animal digestive system. IMPORTANCE E. coli cells encounter various environments from the mouth down to the intestines within the host animals. The pH of gastric juice is lower than 2.0, and the bacterial must quickly respond and adapt to the following environmental changes before reaching the intestines. The quick response plays a role in cellular survival in the population, whereas adaptation may contribute to species survival. The GAD and flagellar systems are important for response to low pH in E. coli. Here, we identified the novel inner membrane regulator HdeD, encoding in the GAD cluster, to repress the synthesis of flagella. These insights provide a deeper understanding of how the bacteria enter the animal digestive system, survive, and form colonies in the intestines.

Age-related exacerbation of hematopoietic organ damage induced by systemic hyper-inflammation in senescence-accelerated mice.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening systemic hyper-inflammatory disorder. The mortality of HLH is higher in the elderly than in young adults. Senescence-accelerated mice (SAMP1/TA-1) exhibit characteristic accelerated aging after 30 weeks of age, and HLH-like features, including hematopoietic organ damage, are seen after lipopolysaccharide (LPS) treatment. Thus, SAMP1/TA-1 is a useful model of hematological pathophysiology in the elderly with HLH. In this study, dosing of SAMP1/TA-1 mice with LPS revealed that the suppression of myelopoiesis and B-lymphopoiesis was more severe in aged mice than in young mice. The bone marrow (BM) expression of genes encoding positive regulators of myelopoiesis (G-CSF, GM-CSF, and IL-6) and of those encoding negative regulators of B cell lymphopoiesis (TNF-α) increased in both groups, while the expression of genes encoding positive-regulators of B cell lymphopoiesis (IL-7, SDF-1, and SCF) decreased. The expression of the GM-CSF-encoding transcript was lower in aged mice than in young animals. The production of GM-CSF by cultured stromal cells after LPS treatment was also lower in aged mice than in young mice. The accumulation of the TNF-α-encoding transcript and the depletion of the IL-7-encoding transcript were prolonged in aged mice compared to young animals. LPS dosing led to a prolonged increase in the proportion of BM M1 macrophages in aged mice compared to young animals. The expression of the gene encoding p16INK4a and the proportion of ß-galactosidase- and phosphorylated ribosomal protein S6-positive cells were increased in cultured stromal cells from aged mice compared to those from young animals, while the proportion of Ki67-positive cells was decreased in stromal cells from aged mice. Thus, age-related deterioration of stromal cells probably causes the suppression of hematopoiesis in aged mice. This age-related latent organ dysfunction may be exacerbated in elderly people with HLH, resulting in poor prognosis.

Isolation and Characterization of a Novel Phage SaGU1 that Infects Staphylococcus aureus Clinical Isolates from Patients with Atopic Dermatitis.

The bacterium Staphylococcus aureus, which colonizes healthy human skin, may cause diseases, such as atopic dermatitis (AD). Treatment for such AD cases involves antibiotic use; however, alternate treatments are preferred owing to the development of antimicrobial resistance. This study aimed to characterize the novel bacteriophage SaGU1 as a potential agent for phage therapy to treat S. aureus infections. SaGU1 that infects S. aureus strains previously isolated from the skin of patients with AD was screened from sewage samples in Gifu, Japan. Its genome was sequenced and analyzed using bioinformatics tools, and the morphology, lytic activity, stability, and host range of the phage were determined. The SaGU1 genome was 140,909 bp with an average GC content of 30.2%. The viral chromosome contained 225 putative protein-coding genes and four tRNA genes, carrying neither toxic nor antibiotic resistance genes. Electron microscopy analysis revealed that SaGU1 belongs to the Myoviridae family. Stability tests showed that SaGU1 was heat-stable under physiological and acidic conditions. Host range testing revealed that SaGU1 can infect a broad range of S. aureus clinical isolates present on the skin of AD patients, whereas it did not kill strains of Staphylococcus epidermidis, which are symbiotic resident bacteria on human skin. Hence, our data suggest that SaGU1 is a potential candidate for developing a phage therapy to treat AD caused by pathogenic S. aureus.

A newly developed age estimation method based on CpG methylation of teeth-derived DNA using real-time methylation-specific PCR.

Age estimation of unidentified bodies is important in forensic medicine and crime scenes. There is accumulating evidence that DNA methylation in the human genome isolated from body fluids changes with age. Most of the data have been obtained by pyrosequencing. In the forensic field, a simple, quick, and economical method is required to evaluate the age of various types of samples. In this study, an age estimation method based on methylation levels of DNA extracted from teeth using real-time methylation-specific PCR (MSP) was developed. The CpG island in the upstream region of ELOVL2, which is known as a validated biomarker in blood samples, was selected as a target site. The CpG methylation levels highly correlated with age (r = 0.843, n = 29). Age-related increase in DNA methylation levels was not affected by sex differences. In addition, the simple regression model based on methylation status of the CpG island exhibited moderate accuracy with a mean absolute deviation between chronological age and predicted age of 8.94 years. The results imply that real-time MSP can be a new tool to perform age prediction of unidentified bodies in forensic scenes.

Kinetics of leukemic cells in 3D culture with stromal cells and with arginine deprivation stress.

Previously, we established a three-dimensional (3D) bone marrow culture system that maintains normal hematopoiesis, including prolongation of hematopoietic stem cell proliferation and differentiation. To analyze the role of bone marrow stromal cells that compose the microenvironment, the growth of a leukemic cell line (K562) in the 3D condition and with arginine deprivation stress was compared with two-dimensional stromal cell monolayers (2D) and suspension cultures without stromal cells (stroma (-)). Arginine is essential for the proliferation and differentiation of erythrocytes. The proliferation and differentiation of K562 cells cultured in the 3D system were stabilized compared with cells in 2D or stroma (-). Furthermore, the number of K562 cells in the G0/G1 phase in 3D was increased significantly compared with cells grown in 2D or stroma (-). Interestingly, the mRNA expression of various hematopoietic growth factors of stromal cells in 3D was not different from 2D, even though supportive activity on K562 cell growth was observed in the arginine deprivation condition. Thus, the hematopoietic microenvironment involves multi-dimensional and complex systems including biochemical and physiochemical factors that regulate quiescence, proliferation, activation, and differentiation of normal hematopoietic cells and cloned leukemic cells. Our 3D culture system may be a valuable new tool for investigating leukemic cell-stromal cell interactions in vitro.

The location of the femoral ACL footprint center is different depending on the Blumensaat's line morphology.

PURPOSE: The purpose of this study was to evaluate the difference in the center point of the femoral ACL footprint according to the morphological variations of the Blumensaat's line. METHODS: Fifty-nine non-paired human cadaver knees were used. The ACL was cut in the middle, and the femoral bone was cut at the most proximal point of the femoral notch. Digital images were evaluated using the Image J software. The periphery of the femoral ACL footprint was outlined and the center point was measured automatically. Following Iriuchishima's classification, the morphology of the Blumensaat's line was classified into straight and hill types (small and large hill types). The center of the femoral ACL footprint and hilltop placement were evaluated using the quadrant method. A quadrant grid was placed uniformly, irregardless of hill existence, and not including the articular cartilage. A correlation analysis was performed between the center point of the femoral ACL footprint and hilltop placement. RESULTS: The straight type consisted of 19 knees, and the hill type 40 knees (small hill type 13 knees and large hill type 27 knees). The center of the femoral ACL footprint (shallow-deep/high-low) in the straight and hill type knees was 33.7/47.6%, and 37.2/50.3%, respectively. In the hill type, the ACL footprint center was significantly more shallow when compared to the straight type. Significant correlation was observed between the center point of the femoral ACL footprint and hilltop placement of the Blumensaat's line. CONCLUSION: The center point of the femoral ACL footprint was significantly more shallow in the hill type knees when compared to the straight type. For clinical relevance, considering that the location of the femoral ACL footprint center is different depending on the Blumensaat's line morphology, to perform accurate ACL reconstruction, femoral ACL tunnel placement should be made based on Blumensaat's line morphological variations.

Dynamics of hematopoiesis is disrupted by impaired hematopoietic microenvironment in a mouse model of hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening systemic hyperinflammatory disorder. We found recently that repeated lipopolysaccharide (LPS) treatment induces HLH-like features in senescence-accelerated mice (SAMP1/TA-1) but not in senescence-resistant control mice (SAMR1). In this study, we analyzed the dynamics of hematopoiesis in this mouse model of HLH. When treated repeatedly with LPS, the numbers of myeloid progenitor cells (CFU-GM) and B-lymphoid progenitor cells (CFU-preB) in the bone marrow (BM) rapidly decreased after each treatment in both strains. The number of CFU-GM in SAMP1/TA-1 and SAMR1, and of CFU-preB in SAMR1, returned to pretreatment levels by 7 days after each treatment. However, the recovery in the number of CFU-preB in SAMP1/TA-1 was limited. In both strains, the BM expression of genes encoding positive regulators of myelopoiesis (granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), and interleukin (IL)-6), and negative regulators of B lymphopoiesis (tumor necrosis factor (TNF)-α) was increased. The expression of genes encoding positive regulators of B lymphopoiesis (stromal-cell derived factor (SDF)-1, IL-7, and stem cell factor (SCF)) was persistently decreased in SAMP1/TA-1 but not in SAMR1. Expression of the gene encoding p16INK4a and the proportion of ß-galactosidase-positive cells were increased in cultured stromal cells obtained from LPS-treated SAMP1/TA-1 but not in those from LPS-treated SAMR1. LPS treatment induced qualitative changes in stromal cells, which comprise the microenvironment supporting appropriate hematopoiesis, in SAMP1/TA-1; these stromal cell changes are inferred to disrupt the dynamics of hematopoiesis. Thus, hematopoietic tissue is one of the organs that suffer life-threatening damage in HLH.

The evaluation of the distance between the popliteus tendon and the lateral collateral ligament footprint and the implant in Total knee Arthroplasty using a 3-dimensional template.

BACKGROUND: The popliteus tendon (PT) or lateral collateral ligament (LCL) stabilizes the postero-lateral aspects of the knees. When surgeons perform total knee arthroplasty (TKA), PT and LCL iatrogenic injuries are a risk because the femoral attachments are relatively close to the femoral bone resection area. The purpose of this study was to evaluate the distance between the PT or LCL footprint and the TKA implant using a 3D template system and to evaluate any significant differences according to the implant model. METHODS: Eighteen non-paired formalin fixed cadaveric lower limbs were used (average age: 80.3). Whole length lower limbs were resected from the pelvis. All the surrounding soft tissue except the PT, knee ligaments and meniscus were removed from the limb. Careful dissection of the PT and LCL was performed, and the femoral footprints were detected. Each footprint periphery was marked with a 1.5 mm K-wire. Computed tomography (CT) scanning of the whole lower limb was then performed. The CT data was analyzed with a 3D template system. This simulation models for TKA were the Journey II BCS and the Persona PS. The area of each footprint, and the length between the most distal and posterior point of the lateral femoral condyle and the edge of each footprint were measured. Matching the implant model to the CT image of the femur, the shortest length between each footprint and the bone resection area were calculated. RESULTS: PT and LCL footprint were detected in all knees. The area of the PT and LCL footprints was 38.7 ± 17.7 mm2 and 58.0 ± 24.6 mm2, respectively. The length between the most distal and posterior point of the lateral femoral condyle and the edge of the PT footprint was 10.3 ± 2.4 mm and 14.2 ± 2.8 mm, respectively. The length between most distal and most posterior point of the lateral femoral condyle and the edge of the LCL footprint was 16.3 ± 2.3 mm and 15.5 ± 3.3 mm, respectively. Under TKA simulation, the shortest length between the PT footprint and the femoral bone resection area for the Journey II BCS and the Persona PS was 4.3 ± 2.5 mm and 3.2 ± 2.9 mm, respectively. The shortest length between the LCL footprint and the femoral bone resection area for the Journey II BCS and the Persona PS was 7.2 ± 2.3 mm and 5.6 ± 2.1 mm, respectively. The PT attachment was damaged by the bone resection of the Journey II BCS and the Persona PS TKA in 3 and 9 knees, respectively. CONCLUSION: The PT and LCL femoral attachments existed close to the femoral bone resection area of the TKA. To prevent postero-lateral instability in TKA, careful attention is needed to avoid damage to the PT and LCL during surgical procedures.

Abemaciclib induces atypical cell death in cancer cells characterized by formation of cytoplasmic vacuoles derived from lysosomes.

In the cell cycle, the G1 /S transition is controlled by the cyclin-dependent kinase (CDK) 4/6-cyclin D complex. Constitutive activation of CDK4/6 dysregulates G1 /S transition, leading to oncogenic transformation. We found that 3 CDK4/6 inhibitors, abemaciclib, ribociclib, and palbociclib, exerted a cytocidal effect as well as a cytostatic effect at the G1 phase in cancer cell lines, including A549 human non-small cell lung cancer cells. Among these inhibitors, abemaciclib exhibited the most potent cytotoxic effect. The cell-death phenotype induced by abemaciclib, which entailed formation of multiple cytoplasmic vacuoles, was not consistent with apoptosis or necroptosis. Abemaciclib blocked autophagic flux, resulting in accumulation of autophagosomes, however vacuole formation and cell death induced by abemaciclib were independent of autophagy. In addition, methuosis, a cell-death phenotype characterized by vacuole formation induced by excessive macropinocytosis, was excluded because the vacuoles did not incorporate fluorescent dextran. Of note, both formation of vacuoles and induction of cell death in response to abemaciclib were inhibited by vacuolar-type ATPase (V-ATPase) inhibitors such as bafilomycin A1 and concanamycin A. Live-cell imaging revealed that the abemaciclib-induced vacuoles were derived from lysosomes that expanded following acidification. Transmission electron microscopy revealed that these vacuoles contained undigested debris and remnants of organelles. Cycloheximide chase assay revealed that lysosomal turnover was blocked by abemaciclib. Furthermore, mTORC1 inhibition along with partial lysosomal membrane permeabilization occurred after abemaciclib treatment. Together, these results indicate that, in cancer cells, abemaciclib induces a unique form of cell death accompanied by swollen and dysfunctional lysosomes.

Morphology of the resident's ridge, and the cortical thickness in the lateral wall of the femoral intercondylar notch correlate with the morphological variations of the Blumensaat's line.

PURPOSE: The purpose of this study was to reveal the morphological correlation between the lateral wall of femoral intercondylar notch and the Blumensaat's line. METHODS: Forty-one non-paired human cadaveric knees were included in this study (23 female, 18 male: median age 83). Knees were resected, and 3 dimensional computed tomography (3D-CT) was performed. In the axial CT image, bony protrusion (resident's ridge) and cortical thickness in the lateral wall of the femoral intercondylar notch were detected. The length between the top of the ridge, or the most anterior, middle, and most posterior border of cortical thickness and posterior femoral condylar line was measured. Following Iriuchishima's classification, the morphology of the Blumensaat's line was classified into straight and hill types (small and large hill types). In the hill types, the length between the hilltop and the posterior border of the Blumensaat's line or the posterior border of the femoral condyle was evaluated. Statistical correlation was calculated between the top of the ridge location, cortical thickness location in the notch, and hilltop location. RESULTS: There were 7 straight type knees and 34 hill type knees (9 small hill type knees and 25 large hill type knees). Only the hill types of knees were evaluated. The top of the ridge, anterior margin, middle, and posterior border of cortical thickness in the lateral wall of the femoral intercondylar notch existed at 61.8 ± 4.6%, 58.3 ± 12.3%, 42.1 ± 7.9%, and 25.5 ± 5.4% from the posterior condylar line, respectively. The hilltop existed at 24.9 ± 5.9% and 30.7 ± 5.0%, from the posterior border of the Blumensaat's line and from the posterior border of the femoral condyle, respectively. Significant correlation was observed between resident's ridge top, cortical thickness location and hilltop location. CONCLUSION: In all cadaveric knees, cortical thickness was detected in the lateral wall of the femoral intercondylar notch. The resident's ridge and cortical thickness location had significant correlation with the hill location in the Blumensaat's line, indicating a continuation of the cortical bone from the posterior cortex of the femoral shaft via the hilltop of the Blumensaat's line to the cortical thickness in the lateral wall of the femoral intercondylar notch. For clinical relevance, hilltop location in the Blumensaat's line is a new bony landmark in anterior cruciate ligament surgery.

Simultaneous activities in both mirror-image glomerular maps in the olfactory bulb may have an important role in stress-related neuronal responses in mice.

In the mouse olfactory bulb (OB), odor input from the olfactory epithelium innervates topographically to form odorant maps, which are mirror-image arrangements of glomerular clusters with domain organization. However, the functional role of the mirror-image representation in the OB remains unknown. Predator odors induce stress responses, and the dorsal domain of the dorsolateral wall of the olfactory bulb (dlOB) is known to be involved in this process. However, it remains unclear whether the activities in the medial wall of the OB (mOB), the other mirror half, are also involved in stress responses. Therefore, in this study, we investigated whether the mOB and dlOB are required for the induction of stress responses using lesioning or electrical stimulation. Although there were no significant differences in the number of activated neurons in the bed nucleus of the stria terminalis, posterior piriform cortex or amygdalo-piriform transition area, fewer activated neurons were observed in the anterior piriform cortex (APC) following lesion of both the mOB and dlOB combined. No changes were observed in the density of activated cells in any examined brain region following stimulation of either the mOB or dlOB alone. However, activated neurons in the APC were significantly more numerous following simultaneous stimulation of the mOB and dlOB. Collectively, our results suggest that simultaneous activation in both the mOB and dlOB is needed to induce APC neural activities that produce stress-like behavior. These findings provide insight into olfactory information processing, and may also help in the development of therapies for odor-induced stress behaviors.

The flexible linker of the secreted FliK ruler is required for export switching of the flagellar protein export apparatus.

The hook length of the flagellum is controlled to about 55 nm in Salmonella. The flagellar type III protein export apparatus secretes FliK to determine hook length during hook assembly and changes its substrate specificity from the hook protein to the filament protein when the hook length has reached about 55 nm. Salmonella FliK consists of an N-terminal domain (FliKN, residues 1-207), a C-terminal domain (FliKC, residues 268-405) and a flexible linker (FliKL, residues 208-267) connecting these two domains. FliKN is a ruler to measure hook length. FliKC binds to a transmembrane export gate protein FlhB to undergo the export switching. FliKL not only acts as part of the ruler but also contributes to this switching event, but it remains unknown how. Here we report that FliKL is required for efficient interaction of FliKC with FlhB. Deletions in FliKL not only shortened hook length according to the size of deletions but also caused a loose length control. Deletion of residues 206-265 significantly reduced the binding affinity of FliKC for FlhB, thereby producing much longer hooks. We propose that an appropriate length of FliKL is required for efficient interaction of FliKC with FlhB.

Flagellar Hook/Needle Length Control and Secretion Control in Type III Secretion Systems.

The flagellum is a motile organ, and the needle complex is a type III secretion apparatus for pathogenesis. There are more similarities than differences between the two structures at the molecular level. Here I focus on the hook and the needle and discuss their length control mechanism. The hook is a substructure of the flagellum and the needle is a part of the needle complex. Both structures are tubular structures that have a central channel for protein secretion. Their lengths are controlled by an intriguing mechanism involving a ruler protein and a switchable gate of the protein secretion system. A model for length control is proposed.