Over the last two decades, several tissue clearing methodologies have been established that render tissues optically transparent and allow imaging of unsectioned tissues of significant volumes, thus improving the capacity to study the relationships between cell and 3D tissue architecture. Despite these technical advances, the important unsolved challenges that these methods face include complexity, time, consistency of tissue size before and after clearing, and ability to immunolabel various antibodies in cleared tissue. Here, we established very simple and fast tissue clearing protocol, FxClear, which involves acrylamide-free electrophoretic tissue clearing (ETC). By removal of the acrylamide infusion step, we were able to achieve fast reaction time, smaller tissue expansion, and higher immunoreactivity. Especially, immunoreactivity and fluorescence intensity were increased in FxClear-processed tissues compared to un-cleared tissues. Our protocol may be suitable for small-sized biopsy samples for 3D pathological examinations.
Acrylamide ; Antibodies ; Biopsy ; Fluorescence ; Immunohistochemistry ; Methods ; Reaction Time ; Tissue Engineering ; Tissue Expansion

Rheb (Ras homolog enriched in the brain) is a small GTPase protein that plays an important role in cell signaling for development of the neocortex through modulation of mTORC1 (mammalian-target-of-rapamycin-complex-1) activity. mTORC1 is known to control various biological processes including axonal growth in forming complexes at the lysosomal membrane compartment. As such, anchoring of Rheb on the lysosomal membrane via the farnesylation of Rheb at its cysteine residue (C180) is required for its promotion of mTOR activity. To test the significance of Rheb farnesylation, we overexpressed a farnesylation mutant form of Rheb, Rheb C180S, in primary rat hippocampal neurons and also in mouse embryonic neurons using in utero electroporation. Interestingly, we found that Rheb C180S maintained promotional effect of axonal elongation similar to the wild-type Rheb in both test systems. On the other hand, Rheb C180S failed to exhibit the multiple axon-promoting effect which is found in wild-type Rheb. The levels of phospho-4EBP1, a downstream target of mTORC1, were surprisingly increased in Rheb C180S transfected neurons, despite the levels of phosphorylated mTOR being significantly decreased compared to control vector transfectants. A specific mTORC1 inhibitor, rapamycin, also could not completely abolish axon elongation characteristics of Rheb C180S in transfected cells. Our data suggests that Rheb in a non-membrane compartment can promote the axonal elongation via phosphorylation of 4EBP1 and through an mTORC1-independent pathway.
Animals ; Axons ; Biological Processes ; Cysteine ; Electroporation ; GTP Phosphohydrolases ; Hand ; Membranes ; Mice ; Neocortex ; Neurons ; Phosphorylation ; Prenylation ; Protein Prenylation ; Rats ; Sirolimus ; TOR Serine-Threonine Kinases

Adenovirus is an important etiologic agent in the pediatric population with fever and respiratory disease, but in newborn period, it has been known rarely to cause illness. But, its tendency of dissemination in immunocompromized host makes the neonate a high risk group of severe adenoviral infection. A newborn girl who presented with pneumonia, pulmonary hemorrhage, hepatitis, and disseminated intravascular coagulation continued to deteriorate in spite of the antibiotic therapy and maximal ventilatory support. The autopsy revealed adenoviral infection in the lung by electron microscopy. Adenoviral infection should be also suspected when a newborn infant has pneumonia, hepatitis, neurologic symptoms, and disseminated intravascular coagulation and especially his or her bacterial culture results are negative.
Adenoviridae Infections* ; Adenoviridae* ; Autopsy ; Disseminated Intravascular Coagulation ; Female ; Fever ; Hemorrhage ; Hepatitis ; Humans ; Infant, Newborn* ; Lung ; Microscopy, Electron ; Neurologic Manifestations ; Pneumonia

The mammographic or physical evaluation of breast parenchymal abnormalities in the presence of radiopaque implants or silicone injections is difficult; mammography often fails to discriminate between breast cancer and a radiopaque dense breast. Although the sonographic evaluation of a palpable mass in an augmented breast may be impaired by the presence of posterior acoustic shadowing, the modality may be helpful in evaluating such masses, and dynamic contrast-enhanced MR imaging facilitates differentiation. FDG-PET is a possible alternative diagnostic tool for the evaluation of parenchymal lesions under these circumstances, but its drawbacks are its high cost and limited availability. The sensitivity and specificity of both FDG-PET and MRI can be improved by correlating their findings with those of radiological studies, and through an awareness of potential false-negative and false-positive causes.
Acoustics ; Breast Neoplasms* ; Breast* ; Foreign Bodies* ; Granuloma, Foreign-Body* ; Magnetic Resonance Imaging* ; Mammography* ; Sensitivity and Specificity ; Shadowing (Histology) ; Silicones ; Ultrasonography

Mycoplasma pneumoniae is a common cause of respiratory tract infections. And M. pneumoniae infection frequently manifests with extrapulmonary symptoms such as central nervous system complications, skin or mucosal involvement, and gastrointestinal problems. However, cardiac complications associated with M. pneumoniae are rarely reported. We report the case of a 47-month-old girl who died of fulminant myocarditis associated with M. pneumoniae pneumonia.
Central Nervous System ; Mycoplasma ; Mycoplasma pneumoniae ; Myocarditis ; Pneumonia ; Pneumonia, Mycoplasma ; Preschool Child ; Respiratory Tract Infections ; Skin

With the increasing use of culture-expanded mesenchymal stromal cells (MSCs) for cell therapies, factors that regulate the cellular characteristics of MSCs have been of major interest. Oxygen concentration has been shown to influence the functions of MSCs, as well as other normal and malignant stem cells. However, the underlying mechanisms of hypoxic responses and the precise role of hypoxia-inducible factor-1alpha (Hif-1alpha), the master regulatory protein of hypoxia, in MSCs remain unclear, due to the limited span of Hif-1alpha stabilization and the complex network of hypoxic responses. In this study, to further define the significance of Hif-1alpha in MSC function during their self-renewal and terminal differentiation, we established adult bone marrow (BM)-derived MSCs that are able to sustain high level expression of ubiquitin-resistant Hif-1alpha during such long-term biological processes. Using this model, we show that the stabilization of Hif-1alpha proteins exerts a selective influence on colony-forming mesenchymal progenitors promoting their self-renewal and proliferation, without affecting the proliferation of the MSC mass population. Moreover, Hif-1alpha stabilization in MSCs led to the induction of pluripotent genes (oct-4 and klf-4) and the inhibition of their terminal differentiation into osteogenic and adipogenic lineages. These results provide insights into the previously unrecognized roles of Hif-1alpha proteins in maintaining the primitive state of primary MSCs and on the cellular heterogeneities in hypoxic responses among MSC populations.
*Cell Differentiation ; Cell Proliferation ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics/*metabolism ; Kruppel-Like Transcription Factors/genetics/metabolism ; Mesenchymal Stromal Cells/cytology/*metabolism/physiology ; Octamer Transcription Factor-3/genetics/metabolism ; Protein Stability

PURPOSE: This study was performed for the purpose of finding causative organisms and clinical features of septic arthritis or acute osteomyelitis in children. METHODS: The study involved a retrospective review of the medical records of 63 microbiologically confirmed cases of acute pyogenic arthritis and osteomyelitis. All of the cases were brought about by community-associated infections and managed at the Seoul National University Children's Hospital or Seoul National University Bundang Hospital from June 2003 to July 2009. RESULTS: The median age of all cases was 60 months and there were 35 males and 28 females. Major involved joints included the hip joint (15 cases), knee joint (7 cases), shoulder joint (4 cases), and elbow joint (4 cases). Also, major involved bones included the femur (20 cases), tibia (13 cases), humerus (7 cases), and radius (7 cases). Staphylococcus aureus was the most commonly identified causative organism, accounting for 49 cases (77.8%). Of the 49 isolates of S. aureus, methicillin-resistant S. aureus (MRSA) accounted for 8 cases (16.3%). Group B streptococcus spp. (GBS) and Salmonella spp. accounted for 3 cases, respectively. Nafcillin or cefazolin was often prescribed as an initial empirical antibiotic. There were 9 cases that were managed by a regimen that included vancomycin as the first choice. Fifty four cases (85.7%) recovered without any complications. Methicillin-sensitive S. aureus (MSSA), responsible for 41 cases, caused chronic complications in 3 cases. Of 8 cases caused by MRSA, 1 case showed chronic complication. There were no fatal cases. CONCLUSION: S. aureus remains the most common organism causing acute pyogenic arthritis and osteomyelitis of childhood acquired in the community. The high prevalence of methicillin resistance among S. aureus should be considered carefully in the selection of initial empirical antibiotics.
Accounting ; Anti-Bacterial Agents ; Arthritis ; Arthritis, Infectious ; Cefazolin ; Child ; Elbow Joint ; Female ; Femur ; Hip Joint ; Humans ; Humerus ; Joints ; Knee Joint ; Male ; Medical Records ; Methicillin Resistance ; Methicillin-Resistant Staphylococcus aureus ; Nafcillin ; Osteoarthritis ; Osteomyelitis ; Prevalence ; Radius ; Retrospective Studies ; Salmonella ; Shoulder Joint ; Staphylococcus aureus ; Streptococcus ; Tibia ; Vancomycin