AM-18002, a derivative of natural anmindenol A, enhances radiosensitivity in mouse breast cancer cells.

Natural anmindenol A isolated from the marine-derived bacteria Streptomyces sp. caused potent inhibition of inducible nitric oxide synthase without any significant cytotoxicity. This compound consists of a structurally unique 3,10-dialkylbenzofulvene skeleton. We previously synthesized and screened the novel derivatives of anmindenol A and identified AM-18002, an anmindenol A derivative, as a promising anticancer agent. The combination of AM-18002 and ionizing radiation (IR) improved anticancer effects, which were exerted by promoting apoptosis and inhibiting the proliferation of FM3A mouse breast cancer cells. AM-18002 increased the production of reactive oxygen species (ROS) and was more effective in inducing DNA damage. AM-18002 treatment was found to inhibit the expansion of myeloid-derived suppressor cells (MDSC), cancer cell migration and invasion, and STAT3 phosphorylation. The AM-18002 and IR combination synergistically induced cancer cell death, and AM-18002 acted as a potent anticancer agent by increasing ROS generation and blocking MDSC-mediated STAT3 activation in breast cancer cells.

Factors Involved in Removing the Non-Structural Protein of Foot-and-Mouth Disease Virus by Chloroform and Scale-Up Production of High-Purity Vaccine Antigens.

Foot-and-mouth disease (FMD) is an economically important and highly infectious viral disease, predominantly controlled by vaccination. The removal of non-structural proteins (NSPs) is very important in the process of FMD vaccine production, because vaccinated and naturally infected animals can be distinguished by the presence of NSP antibodies in the FMD serological surveillance. A previous study reported that 3AB protein, a representative of NSPs, was removed by chloroform treatment. Therefore, in this study, the causes of 3AB removal and factors affecting the effect of chloroform were investigated. As a result, the effectiveness of chloroform differed depending on the virus production medium and was eliminated by detergents. In addition, it was found that 3AB protein removal by chloroform is due to the transmembrane domain of the N-terminal region (59-76 amino acid domain). Further, industrial applicability was verified by applying the chloroform treatment process to scale-up FMD vaccine antigen production. A novel downstream process using ultrafiltration instead of polyethylene glycol precipitation for high-purity FMD vaccine antigen production was established. This result will contribute toward simplifying the conventional process of manufacturing FMD vaccine antigens and ultimately reducing the time and cost of vaccine production.

G9a Knockdown Suppresses Cancer Aggressiveness by Facilitating Smad Protein Phosphorylation through Increasing BMP5 Expression in Luminal A Type Breast Cancer.

Epigenetic abnormalities affect tumor progression, as well as gene expression and function. Among the diverse epigenetic modulators, the histone methyltransferase G9a has been focused on due to its role in accelerating tumorigenesis and metastasis. Although epigenetic dysregulation is closely related to tumor progression, reports regarding the relationship between G9a and its possible downstream factors regulating breast tumor growth are scarce. Therefore, we aimed to verify the role of G9a and its presumable downstream regulators during malignant progression of breast cancer. G9a-depleted MCF7 and T47D breast cancer cells exhibited suppressed motility, including migration and invasion, and an improved response to ionizing radiation. To identify the possible key factors underlying these effects, microarray analysis was performed, and a TGF-ß superfamily member, BMP5, was selected as a prominent target gene. It was found that BMP5 expression was markedly increased by G9a knockdown. Moreover, reduction in the migration/invasion ability of MCF7 and T47D breast cancer cells was induced by BMP5. Interestingly, a G9a-depletion-mediated increase in BMP5 expression induced the phosphorylation of Smad proteins, which are the intracellular signaling mediators of BMP5. Accordingly, we concluded that the observed antitumor effects may be based on the G9a-depletion-mediated increase in BMP5 expression and the consequent facilitation of Smad protein phosphorylation.

Increasing Transfection Efficiency of Lipoplexes by Modulating Complexation Solution for Transient Gene Expression.

Transient gene expression is a suitable tool for the production of biopharmaceutical candidates in the early stage of development and provides a simple and rapid alternative to the generation of stable cell line. In this study, an efficient transient gene expression methodology using DC-Chol/DOPE cationic liposomes and pDNA in Chinese hamster ovary suspension cells was established through screening of diverse lipoplex formation conditions. We modulated properties of both the liposome formation and pDNA solution, together called complexation solutions. Protein expression and cellular cytotoxicity were evaluated following transfection over the cell cultivation period to select the optimal complexation solution. Changes in hydrodynamic size, polydispersity index, and ζ potential of the liposomes and lipoplexes were analyzed depending on the various pH ranges of the complexation solutions using dynamic light scattering. The transfer of lipoplexes to the cytosol and their conformation were traced using fluorescence analysis until the early period of transfection. As a result, up to 1785 mg/L and 191 mg/L of human Fc protein and immunoglobulin G (bevacizumab), respectively, were successfully produced using acidic liposome formation and alkaline pDNA solutions. We expect that this lipoplex formation in acidic and alkaline complexation solutions could be an effective methodology for a promising gene delivery strategy.

Enhanced Soft 3D Reconstruction Method with an Iterative Matching Cost Update Using Object Surface Consensus.

In this paper, we propose a multi-view stereo matching method, EnSoft3D (Enhanced Soft 3D Reconstruction) to obtain dense and high-quality depth images. Multi-view stereo is one of the high-interest research areas and has wide applications. Motivated by the Soft3D reconstruction method, we introduce a new multi-view stereo matching scheme. The original Soft3D method is introduced for novel view synthesis, while occlusion-aware depth is also reconstructed by integrating the matching costs of the Plane Sweep Stereo (PSS) and soft visibility volumes. However, the Soft3D method has an inherent limitation because the erroneous PSS matching costs are not updated. To overcome this limitation, the proposed scheme introduces an update process of the PSS matching costs. From the object surface consensus volume, an inverse consensus kernel is derived, and the PSS matching costs are iteratively updated using the kernel. The proposed EnSoft3D method reconstructs a highly accurate 3D depth image because both the multi-view matching cost and soft visibility are updated simultaneously. The performance of the proposed method is evaluated by using structured and unstructured benchmark datasets. Disparity error is measured to verify 3D reconstruction accuracy, and both PSNR and SSIM are measured to verify the simultaneous enhancement of view synthesis.

Production of a Foot-and-Mouth Disease Vaccine Antigen Using Suspension-Adapted BHK-21 Cells in a Bioreactor.

The baby hamster kidney-21 (BHK-21) cell line is a continuous cell line used to propagate foot-and-mouth disease (FMD) virus for vaccine manufacturing. BHK-21 cells are anchorage-dependent, although suspension cultures would enable rapid growth in bioreactors, large-scale virus propagation, and cost-effective vaccine production with serum-free medium. Here, we report the successful adaptation of adherent BHK-21 cells to growth in suspension to a viable cell density of 7.65 × 106 cells/mL on day 3 in serum-free culture medium. The suspension-adapted BHK-21 cells showed lower adhesion to five types of extracellular matrix proteins than adherent BHK-21 cells, which contributed to the suspension culture. In addition, a chemically defined medium (selected by screening various prototype media) led to increased FMD virus production yields in the batch culture, even at a cell density of only 3.5 × 106 cells/mL. The suspension BHK-21 cell culture could be expanded to a 200 L bioreactor from a 20 mL flask, which resulted in a comparable FMD virus titer. This platform technology improved virus productivity, indicating its potential for enhancing FMD vaccine production.

Inhibitory effect of ginsenoside Rg3 on cancer stemness and mesenchymal transition in breast cancer via regulation of myeloid-derived suppressor cells.

Ginsenoside Rg3 (Rg3) has been studied in several cancer models and is suggested to act through various pharmacological effects. We investigated the anticancer properties of Rg3 through myeloid-derived suppressor cell (MDSC) modulation in FM3A mouse mammary carcinoma cells. The effects of Rg3 on MDSCs and consequent changes in cancer stem-like cells (CSCs) and epithelial-mesenchymal transition (EMT) were evaluated by diverse methods. MDSCs promoted cancer by enhancing breast cancer stemness and promoting EMT. Rg3 at a dose without obvious cytotoxicity downregulated MDSCs and repressed MDSC-induced cancer stemness and EMT. Mechanistic investigations suggested that these inhibitory effects of Rg3 on MDSCs and corresponding cancer progression depend upon suppression of the STAT3-dependent pathway, tumor-derived cytokines, and the NOTCH signaling pathway. In a mouse model, MDSCs accelerated tumor progression, and Rg3 delayed tumor growth, which is consistent with the results of in vitro experiments. These results indicated that Rg3 could effectively inhibit the progression of breast cancer. The anticancer effect of Rg3 might be partially due to its downregulation of MDSCs and consequent repression of cancer stemness and EMT in breast cancer. Hence, we suggest the regulation of MDSCs through Rg3 treatment as an effective therapeutic strategy for breast cancer patients.

Extrinsic Calibration between Camera and LiDAR Sensors by Matching Multiple 3D Planes.

This paper proposes a simple extrinsic calibration method for a multi-sensor system which consists of six image cameras and a 16-channel 3D LiDAR sensor using a planar chessboard. The six cameras are mounted on a specially designed hexagonal plate to capture omnidirectional images and the LiDAR sensor is mounted on the top of the plates to capture 3D points in 360 degrees. Considering each camera-LiDAR combination as an independent multi-sensor unit, the rotation and translation between the two sensor coordinates are calibrated. The 2D chessboard corners in the camera image are reprojected into 3D space to fit to a 3D plane with respect to the camera coordinate system. The corresponding 3D point data that scan the chessboard are used to fit to another 3D plane with respect to the LiDAR coordinate system. The rotation matrix is calculated by aligning normal vectors of the corresponding planes. In addition, an arbitrary point on the 3D camera plane is projected to a 3D point on the LiDAR plane, and the distance between the two points are iteratively minimized to estimate the translation matrix. At least three or more planes are used to find accurate external parameters between the coordinate systems. Finally, the estimated transformation is refined using the distance between all chessboard 3D points and the LiDAR plane. In the experiments, quantitative error analysis is done using a simulation tool and real test sequences are also used for calibration consistency analysis.

Deep vector-based convolutional neural network approach for automatic recognition of colonies of induced pluripotent stem cells.

Pluripotent stem cells can potentially be used in clinical applications as a model for studying disease progress. This tracking of disease-causing events in cells requires constant assessment of the quality of stem cells. Existing approaches are inadequate for robust and automated differentiation of stem cell colonies. In this study, we developed a new model of vector-based convolutional neural network (V-CNN) with respect to extracted features of the induced pluripotent stem cell (iPSC) colony for distinguishing colony characteristics. A transfer function from the feature vectors to the virtual image was generated at the front of the CNN in order for classification of feature vectors of healthy and unhealthy colonies. The robustness of the proposed V-CNN model in distinguishing colonies was compared with that of the competitive support vector machine (SVM) classifier based on morphological, textural, and combined features. Additionally, five-fold cross-validation was used to investigate the performance of the V-CNN model. The precision, recall, and F-measure values of the V-CNN model were comparatively higher than those of the SVM classifier, with a range of 87-93%, indicating fewer false positives and false negative rates. Furthermore, for determining the quality of colonies, the V-CNN model showed higher accuracy values based on morphological (95.5%), textural (91.0%), and combined (93.2%) features than those estimated with the SVM classifier (86.7, 83.3, and 83.4%, respectively). Similarly, the accuracy of the feature sets using five-fold cross-validation was above 90% for the V-CNN model, whereas that yielded by the SVM model was in the range of 75-77%. We thus concluded that the proposed V-CNN model outperforms the conventional SVM classifier, which strongly suggests that it as a reliable framework for robust colony classification of iPSCs. It can also serve as a cost-effective quality recognition tool during culture and other experimental procedures.

Real-Time External Respiratory Motion Measuring Technique Using an RGB-D Camera and Principal Component Analysis.

Accurate tracking and modeling of internal and external respiratory motion in the thoracic and abdominal regions of a human body is a highly discussed topic in external beam radiotherapy treatment. Errors in target/normal tissue delineation and dose calculation and the increment of the healthy tissues being exposed to high radiation doses are some of the unsolicited problems caused due to inaccurate tracking of the respiratory motion. Many related works have been introduced for respiratory motion modeling, but a majority of them highly depend on radiography/fluoroscopy imaging, wearable markers or surgical node implanting techniques. We, in this article, propose a new respiratory motion tracking approach by exploiting the advantages of an RGB-D camera. First, we create a patient-specific respiratory motion model using principal component analysis (PCA) removing the spatial and temporal noise of the input depth data. Then, this model is utilized for real-time external respiratory motion measurement with high accuracy. Additionally, we introduce a marker-based depth frame registration technique to limit the measuring area into an anatomically consistent region that helps to handle the patient movements during the treatment. We achieved a 0.97 correlation comparing to a spirometer and 0.53 mm average error considering a laser line scanning result as the ground truth. As future work, we will use this accurate measurement of external respiratory motion to generate a correlated motion model that describes the movements of internal tumors.

Multi-Layer SnSe Nanoflake Field-Effect Transistors with Low-Resistance Au Ohmic Contacts.

We report p-type tin monoselenide (SnSe) single crystals, grown in double-sealed quartz ampoules using a modified Bridgman technique at 920 °C. X-ray powder diffraction (XRD) and energy dispersive X-ray spectroscopy (EDX) measurements clearly confirm that the grown SnSe consists of single-crystal SnSe. Electrical transport of multi-layer SnSe nanoflakes, which were prepared by exfoliation from bulk single crystals, was conducted using back-gated field-effect transistor (FET) structures with Au and Ti contacts on SiO2/Si substrates, revealing that multi-layer SnSe nanoflakes exhibit p-type semiconductor characteristics owing to the Sn vacancies on the surfaces of SnSe nanoflakes. In addition, a strong carrier screening effect was observed in 70-90-nm-thick SnSe nanoflake FETs. Furthermore, the effect of the metal contacts to multi-layer SnSe nanoflake-based FETs is also discussed with two different metals, such as Ti/Au and Au contacts.

Synergistically enhanced selective intracellular uptake of anticancer drug carrier comprising folic acid-conjugated hydrogels containing magnetite nanoparticles.

Targeted drug delivery has long been extensively researched since drug delivery and release at the diseased site with minimum dosage realizes the effective therapy without adverse side effects. In this work, to achieve enhanced intracellular uptake of anticancer drug carriers for efficient chemo-therapy, we have designed targeted multifunctional anticancer drug carrier hydrogels. Temperature-responsive poly(N-isopropylacrylamide) (PNIPAm) hydrogel core containing superparamagnetic magnetite nanoparticles (MNP) were prepared using precipitation polymerization, and further polymerized with amine-functionalized copolymer shell to facilitate the conjugation of targeting ligand. Then, folic acid, specific targeting ligand for cervical cancer cell line (HeLa), was conjugated on the hydrogel surface, yielding the ligand conjugated hybrid hydrogels. We revealed that enhanced intracellular uptake by HeLa cells in vitro was enabled by both magnetic attraction and receptor-mediated endocytosis, which were contributed by MNP and folic acid, respectively. Furthermore, site-specific uptake of the developed carrier was confirmed by incubating with several other cell lines. Based on synergistically enhanced intracellular uptake, efficient cytotoxicity and apoptotic activity of HeLa cells incubated with anticancer drug loaded hybrid hydrogels were successfully achieved. The developed dual-targeted hybrid hydrogels are expected to provide a platform for the next generation intelligent drug delivery systems.

Distributional Variations in the Quantitative Cortical and Trabecular Bone Radiographic Measurements of Mandible, between Male and Female Populations of Korea, and its Utilization.

It is important to investigate the irregularities in aging-associated changes in bone, between men and women for bone strength and osteoporosis. The purpose of this study was to characterize the changes and associations of mandibular cortical and trabecular bone measures of men and women based on age and to the evaluation of cortical shape categories, in a large Korean population. Panoramic radiographs of 1047 subjects (603 women and 444 men) aged between 15 to 90 years were used. Mandibular cortical width (MCW), mandibular cortical index (MCI), and fractal dimensions (FD) of the molar, premolar, and anterior regions of the mandibular trabecular bone were measured. Study subjects were grouped into six 10-years age groups. A local linear regression smoothing with bootstrap resampling for robust fitting of data was used to estimate the relationship between radiographic mandibular variables and age groups as well as genders. The mean age of women (49.56 ± 19.5 years) was significantly higher than that of men (45.57 ± 19.6 years). The MCW of men and women (3.17mm and 2.91mm, respectively, p < 0.0001) was strongly associated with age and MCI. Indeed, trabecular measures also correlated with age in men (r > -0.140, p = 0.003), though not as strongly as in women (r > -0.210, p < 0.0001). In men aged over 55 years, only MCW was significantly associated (r = -0.412, p < 0.0001). Furthermore, by comparison of mandibular variables from different age groups and MCI categories, the results suggest that MCW was detected to be strongly associated in both men and women for the detection of bone strength and osteoporosis. The FD measures revealed relatively higher association with age among women than men, but not as strong as MCW.

Automatic detection of osteoporosis based on hybrid genetic swarm fuzzy classifier approaches.

OBJECTIVES: This study proposed a new automated screening system based on a hybrid genetic swarm fuzzy (GSF) classifier using digital dental panoramic radiographs to diagnose females with a low bone mineral density (BMD) or osteoporosis. METHODS: The geometrical attributes of both the mandibular cortical bone and trabecular bone were acquired using previously developed software. Designing an automated system for osteoporosis screening involved partitioning of the input attributes to generate an initial membership function (MF) and a rule set (RS), classification using a fuzzy inference system and optimization of the generated MF and RS using the genetic swarm algorithm. Fivefold cross-validation (5-FCV) was used to estimate the classification accuracy of the hybrid GSF classifier. The performance of the hybrid GSF classifier has been further compared with that of individual genetic algorithm and particle swarm optimization fuzzy classifiers. RESULTS: Proposed hybrid GSF classifier in identifying low BMD or osteoporosis at the lumbar spine and femoral neck BMD was evaluated. The sensitivity, specificity and accuracy of the hybrid GSF with optimized MF and RS in identifying females with a low BMD were 95.3%, 94.7% and 96.01%, respectively, at the lumbar spine and 99.1%, 98.4% and 98.9%, respectively, at the femoral neck BMD. The diagnostic performance of the proposed system with femoral neck BMD was 0.986 with a confidence interval of 0.942-0.998. The highest mean accuracy using 5-FCV was 97.9% with femoral neck BMD. CONCLUSIONS: The combination of high accuracy along with its interpretation ability makes this proposed automatic system using hybrid GSF classifier capable of identifying a large proportion of undetected low BMD or osteoporosis at its early stage.

Fast and scalable approximate spectral matching for higher order graph matching.

This paper presents a fast and efficient computational approach to higher order spectral graph matching. Exploiting the redundancy in a tensor representing the affinity between feature points, we approximate the affinity tensor with the linear combination of Kronecker products between bases and index tensors. The bases and index tensors are highly compressed representations of the approximated affinity tensor, requiring much smaller memory than in previous methods, which store the full affinity tensor. We compute the principal eigenvector of the approximated affinity tensor using the small bases and index tensors without explicitly storing the approximated tensor. To compensate for the loss of matching accuracy by the approximation, we also adopt and incorporate a marginalization scheme that maps a higher order tensor to matrix as well as a one-to-one mapping constraint into the eigenvector computation process. The experimental results show that the proposed method is faster and requires smaller memory than the existing methods with little or no loss of accuracy.

Simultaneous quantification of total and conjugated ubiquitin levels in a single immunoblot.

Ubiquitin (Ub) is a posttranslational modifier, and total Ub (UbT) is always in dynamic equilibrium among free Ub (UbF), activated Ub (UbA), and conjugated Ub (UbC) in the forms of mono-Ub, thioester-bond-linked Ub, and peptide-bond-linked Ub, respectively. In this study, we developed a simple method to simultaneously determine the levels of UbT, UbF+UbA, and UbC in a single immunoblot and demonstrated its reliability and reproducibility by determining [UbT], [UbF+UbA], and [UbC] in various mouse tissues and cultured cells.

Downregulation of ubiquitin level via knockdown of polyubiquitin gene Ubb as potential cancer therapeutic intervention.

Ubiquitin is involved in almost every cellular process, and it is also known to be a stress-inducible protein. Based on previous reports that many types of cancer display an elevated level of ubiquitin, we hypothesized that this increased amount of ubiquitin is essential for the growth of cancer cells and that, consequently, the downregulation of ubiquitin may be a potential anti-cancer treatment. We first found that the level of ubiquitin can be effectively downregulated via knockdown of a polyubiquitin gene, Ubb, with siRNA (Ubb-KD) and then demonstrated its anti-cancer effects in several cancer cell lines and xenograft mice. Ubb-KD resulted in the attenuation of TNFα-induced NF-κB activation, the stabilization of the tumor suppressor p53, and stress-sensitization. Taken together, downregulation of ubiquitin through Ubb-KD is a potential anti-cancer treatment by inhibiting ubiquitination at multiple sites related to oncogenic pathways and by weakening the ability of cancer cells to overcome increased stress.

PICOT increases cardiac contractility by inhibiting PKCζ activity.

Protein kinase C (PKC)-interacting cousin of thioredoxin (PICOT) has distinct anti-hypertrophic and inotropic functions. We have previously shown that PICOT exerts its anti-hypertrophic effect by inhibiting calcineurin-NFAT signaling through its C-terminal glutaredoxin domain. However, the mechanism underlying the inotropic effect of PICOT is unknown. The results of protein pull-down experiments showed that PICOT directly binds to the catalytic domain of PKCζ through its N-terminal thioredoxin-like domain. Purified PICOT protein inhibited the kinase activity of PKCζ in vitro, which indicated that PICOT is an endogenous inhibitor of PKCζ. The inhibition of PKCζ activity with a PKCζ-specific pseudosubstrate peptide inhibitor was sufficient to increase the cardiac contractility in vitro and ex vivo. Overexpression of PICOT or inhibition of PKCζ activity down-regulated PKCα activity, which led to the elevation of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) 2a activity, concomitant with the increased phosphorylation of phospholamban (PLB). Overexpression of PICOT or inhibition of PKCζ activity also down-regulated protein phosphatase (PP) 2A activity, which subsequently resulted in the increased phosphorylation of troponin (Tn) I and T, key myofilament proteins associated with the regulation of contractility. PICOT appeared to inhibit PP2A activity through the disruption of the functional PKCζ/PP2A complex. In contrast to the overexpression of PICOT or inhibition of PKCζ, reduced PICOT expression resulted in up-regulation of PKCα and PP2A activities, followed by decreased phosphorylation of PLB, and TnI and T, respectively, supporting the physiological relevance of these events. Transgene- or adeno-associated virus (AAV)-mediated overexpression of PICOT restored the impaired contractility and prevented further morphological and functional deterioration of the failing hearts. Taken together, the results of the present study suggest that PICOT exerts its inotropic effect by negatively regulating PKCα and PP2A activities through the inhibition of PKCζ activity. This finding provides a novel insight into the regulation of cardiac contractility.

Effect of ring torsion on intramolecular vibrational redistribution dynamics of 1,1'-binaphthyl and 2,2'-binaphthyl.

The role of ring torsion in the enhancement of intramolecular vibrational energy redistribution (IVR) in aromatic molecules was investigated by conducting excitation and dispersed fluorescence spectroscopy of 1,1'-binaphthyl (1,1'-BN) and 2,2'-BN. The dispersed fluorescence spectra of 1,1'-BN in the origin region of S(1)-S(0) were well resolved, which presented 25-27 cm(-1) gaps of torsional mode in the ground state. The overall profile of the dispersed spectra of 1,1'-BN is similar to that of naphthalene. In contrast, the spectra of 2,2'-BN were not resolved due to the multitude of the active torsional modes. In both cases, dissipative IVR was observed to take place with a relatively small excess vibrational energy: 237.5 cm(-1) for 1,1'-BN and 658 cm(-1) for 2,2'-BN, which clearly shows that ring torsion efficiently enhances the IVR rate. Ab initio and density functional theory calculations with medium-sized basis sets showed that the torsional potential of 1,1'-BN has a very flat minimum over the range of torsional angles from ca. 60° to 120°, whereas that of 2,2'-BN showed two well-defined potential minima at ca. 40° and 140°, in resemblance to the case of biphenyl. In this work, we propose that aromatic molecules be classified into "strong" and "weak" torsional hindrance cases: molecules with strong hindrance case show shorter torsional progressions and more effective IVR dynamics than do those with weak hindrance.

Magnetic imaging of a supercooling glass transition in a weakly disordered ferromagnet.

Spin glasses are founded in the frustration and randomness of microscopic magnetic interactions. They are non-ergodic systems where replica symmetry is broken. Although magnetic glassy behaviour has been observed in many colossal magnetoresistive manganites, there is no consensus that they are spin glasses. Here, an intriguing glass transition in (La,Pr,Ca)MnO3 is imaged using a variable-temperature magnetic force microscope. In contrast to the speculated spin-glass picture, our results show that the observed static magnetic configuration seen below the glass-transition temperature arises from the cooperative freezing of the first-order antiferromagnetic (charge ordered) to ferromagnetic transition. Our data also suggest that accommodation strain is important in the kinetics of the phase transition. This cooperative freezing idea has been applied to structural glasses including window glasses and supercooled liquids, and may be applicable across many systems to any first-order phase transition occurring on a complex free-energy landscape.