Preadipocytes potentiate melanoma progression and M2 macrophage polarization in the tumor microenvironment.

Melanoma, the deadliest skin cancer, originates from epidermal melanocytes. The influence of preadipocytes on melanoma is less understood. We co-cultured mouse melanoma B16 cells with 3T3L1 preadipocytes to form mixed spheroids and observed increased melanoma proliferation and growth compared to B16-only spheroids. Metastasis-related proteins YAP, TAZ, and PD-L1 levels were also higher in mixed spheroids. Treatment with exosome inhibitor GW4869 halted melanoma growth and reduced expression of these proteins, suggesting exosomal crosstalk between B16 and 3T3L1 cells. MiR-155 expression was significantly higher in mixed spheroids, and GW4869 reduced its levels. Additionally, co-culturing with Raw264.7 macrophage cells increased M2 markers IL-4 and CD206 in Raw264.7 cells, effects that were diminished by GW4869. These results indicate that preadipocytes may enhance melanoma progression and metastasis via exosomal interactions.

Protein purification, crystallization, and structure determination of transcription factor YhaJ in complex with DNT metabolites.

The microbial transcription factor YhaJ responds to 2,4-dinitrotoluene (DNT) derivatives. Here, we describe steps for overexpression and purification of the protein, characterization for the binding of a DNT derivative methylhydroquinone, and crystallization by using a random seeding technique. We then detail procedures for structure determination by employing the crystal-twin resolving processes. This protocol can also be performed using other DNT derivatives. For complete details on the use and execution of this protocol, please refer to Kim et al.1.

Structural basis of transcription factor YhaJ for DNT detection.

Detection of landmines without harming personnel is a global issue. The bacterial transcription factor YhaJ selectively detects metabolites of explosives, and it can be used as a key component of DNT biosensors. However, the wild-type YhaJ has a binding affinity that is not sufficient for the detection of trace amounts of explosives leaked from landmines buried in the soil. Here, we report crystal structures of the effector-binding domain of YhaJ in both the apo- and effector-bound forms. A structural comparison of the two forms revealed that the loop above the primary effector-binding site significantly switches its conformation upon effector binding. The primary effector-binding site involves hydrophobic and polar interactions, having specificity to hydroxyl-substituted benzene compounds. The structures explain the mechanism of activity-enhancing mutations and provide information for the rational engineering of YhaJ biosensors for the sensitive detection of explosives.

Applications of Bioinspired Platforms for Enhancing Immunomodulatory Function of Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSCs) have attracted scientific and medical interest due to their self-renewing properties, pluripotency, and paracrine function. However, one of the main limitations to the clinical application of MSCs is their loss of efficacy after transplantation in vivo. Various bioengineering technologies to provide stem cell niche-like conditions have the potential to overcome this limitation. Here, focusing on the stem cell niche microenvironment, studies to maximize the immunomodulatory potential of MSCs by controlling biomechanical stimuli, including shear stress, hydrostatic pressure, stretch, and biophysical cues, such as extracellular matrix mimetic substrates, are discussed. The application of biomechanical forces or biophysical cues to the stem cell microenvironment will be beneficial for enhancing the immunomodulatory function of MSCs during cultivation and overcoming the current limitations of MSC therapy.

Role of extrinsic physical cues in cancer progression.

The tumor microenvironment (TME) is a complex system composed of many cell types and an extracellular matrix (ECM). During tumorigenesis, cancer cells constantly interact with cellular components, biochemical cues, and the ECM in the TME, all of which make the environment favorable for cancer growth. Emerging evidence has revealed the importance of substrate elasticity and biomechanical forces in tumor progression and metastasis. However, the mechanisms underlying the cell response to mechanical signals-such as extrinsic mechanical forces and forces generated within the TME-are still relatively unknown. Moreover, having a deeper understanding of the mechanisms by which cancer cells sense mechanical forces and transmit signals to the cytoplasm would substantially help develop effective strategies for cancer treatment. This review provides an overview of biomechanical forces in the TME and the intracellular signaling pathways activated by mechanical cues as well as highlights the role of mechanotransductive pathways through mechanosensors that detect the altering biomechanical forces in the TME. as an adjuvant for cancer immunotherapy.[BMB Reports 2023; 56(5): 287-295].

A Dynamic Substrate Pool Revealed by cryo-EM of a Lipid-Preserved Respiratory Supercomplex.

Aims: Mitochondrial respiratory supercomplexes mediate redox electron transfer, generating a proton gradient for ATP synthesis. To provide structural information on the function of supercomplexes in physiologically relevant conditions, we conducted cryoelectron microscopy studies with supercomplexes in a lipid-preserving state. Results: Here, we present cryoelectron microscopy structures of bovine respiratory supercomplex I1III2IV1 by using a lipid-preserving sample preparation. The preparation greatly enhances the intercomplex quinone transfer activity. The structures reveal large intercomplex motions that result in different shapes and sizes of the intercomplex space between complexes I and III, forming a dynamic substrate pool. Biochemical and structural analyses indicated that intercomplex phospholipids mediate the intercomplex motions. An analysis of the different classes of focus-refined complex I showed that structural switches due to quinone reduction led to the formation of a novel channel that could transfer reduced quinones to the intercomplex substrate pool. Innovation and Conclusion: Our results indicate potential mechanism for the facilitated electron transfer involving a dynamic substrate pool and intercomplex movement by which supercomplexes play an active role in the regulation of metabolic flux and reactive oxygen species.

Graphene Oxide-Supported Microwell Grids for Preparing Cryo-EM Samples with Controlled Ice Thickness.

Cryogenic-electron microscopy (cryo-EM) is the preferred method to determine 3D structures of proteins and to study diverse material systems that intrinsically have radiation or air sensitivity. Current cryo-EM sample preparation methods provide limited control over the sample quality, which limits the efficiency and high throughput of 3D structure analysis. This is partly because it is difficult to control the thickness of the vitreous ice that embeds specimens, in the range of nanoscale, depending on the size and type of materials of interest. Thus, there is a need for fine regulation of the thickness of vitreous ice to deliver consistent high signal-to-noise ratios for low-contrast biological specimens. Herein, an advanced silicon-chip-based device is developed which has a regular array of micropatterned holes with a graphene oxide (GO) window on freestanding silicon nitride (Six Ny ). Accurately regulated depths of micropatterned holes enable precise control of vitreous ice thickness. Furthermore, GO window with affinity for biomolecules can facilitate concentration of the sample molecules at a higher level. Incorporation of micropatterned chips with a GO window enhances cryo-EM imaging for various nanoscale biological samples including human immunodeficiency viral particles, groEL tetradecamers, apoferritin octahedral, aldolase homotetramer complexes, and tau filaments, as well as inorganic materials.

Validation of a gyroscope-based wearable device for real-time position monitoring of patients in a hospital.

BACKGROUND: Monitoring patients' position is important, but there have been few studies related to validation. OBJECTIVE: The objective of this study was to assess the validity of position monitoring measured using a wearable device by comparing the device's measurements to a patient's actual position. METHODS: We constructed a wearable device with a three-axis gyroscope and applied it to 10 patients who were unable to change their position independently. We compared the actual angle of the position and the angle transmitted from the wearable device using a Bland-Altman plot and a receiver operating characteristic curve. RESULTS: We compared the actual angle of the position and the angle transmitted from the wearable device using a Bland-Altman plot, but it was difficult to observe statistical similarity. The angles transmitted from the wearable device in the lateral and supine positions showed significant differences. The cutoff value separating the lateral and supine positions was found to be 27.1∘ (sensitivity = 100%, specificity = 99.9%). CONCLUSIONS: Through our method, the measured values from the gyroscope-based wearable device did not accurately reflect the patient's actual position. However, the wearable device was able to distinguish the lateral position from the supine position.

The Usefulness of a Wearable Device in Daily Physical Activity Monitoring for the Hospitalized Patients Undergoing Lumbar Surgery.

OBJECTIVE: Functional outcomes have traditionally been evaluated and compared using subjective surveys, such as visual analog scores (VAS), the Oswestry disability index (ODI), and Short Form-36 (SF-36), to assess symptoms and quality of life. However, these surveys are limited by their subjective natures and inherent bias caused by differences in patient perceptions of symptoms. The Fitbit Charge® (Fitbit Inc., San Francisco, CA, USA) provides accurate and objective measures of physical activity. The use of this device in patients after laminectomy would provide objective physical measures that define ambulatory function, activity level, and degree of recovery. Therefore, the present study was conducted to identify relationships between the number of steps taken by patients per day and VAS pain scores, prognoses, and postoperative functional outcomes. METHODS: We prospectively investigated 22 consecutive patients that underwent laminectomy for spinal stenosis or a herniated lumbar disc between June 2015 and April 2016 by the same surgeon. When patients were admitted for surgery and first visited after surgery, preoperative and postoperative functional scores were recorded using VAS scores, ODI scores, and SF-36. The VAS scores and physical activities were recorded daily from postoperative day (POD) 1 to POD 7. The relationship between daily VAS scores and daily physical activities were investigated by simple correlation analysis and the relationship between mean number of steps taken and ODI scores after surgery was subjected to simple regression analysis. In addition, Wilcoxon's signed-rank test was used to investigate the significance of pre-to-postoperative differences in VAS, ODI, and SF-36 scores. RESULTS: Pre-to-postoperative VAS (p<0.001), ODI (p<0.001), SF-36 mental composite scores (p=0.009), and SF-36 physical composite scores (p<0.001) scores were found to be significantly different. Numbers of steps taken from POD 1 to POD 7 were negatively correlated with daily VAS scores (r=-0.981, p<0.001). In addition, the mean number of steps from POD 3 to POD 7 and the decrease in ODI conducted one month after surgery were statistically significant (p=0.029). CONCLUSION: Wearable devices are not only being used increasingly by consumers as lifestyle devices, but are also progressively being used in the medical area. This is the first study to demonstrate the usefulness of a wearable device for checking patient physical activity and predicting pain and prognosis after laminectomy. Based on our experience, the wearable device used to provide measures of physical activity in the present study has the potential to provide objective information on pain severity and prognosis.

The family-wide structure and function of human dual-specificity protein phosphatases.

Dual-specificity protein phosphatases (DUSPs), which dephosphorylate both phosphoserine/threonine and phosphotyrosine, play vital roles in immune activation, brain function and cell-growth signalling. A family-wide structural library of human DUSPs was constructed based on experimental structure determination supplemented with homology modelling. The catalytic domain of each individual DUSP has characteristic features in the active site and in surface-charge distribution, indicating substrate-interaction specificity. The active-site loop-to-strand switch occurs in a subtype-specific manner, indicating that the switch process is necessary for characteristic substrate interactions in the corresponding DUSPs. A comprehensive analysis of the activity-inhibition profile and active-site geometry of DUSPs revealed a novel role of the active-pocket structure in the substrate specificity of DUSPs. A structure-based analysis of redox responses indicated that the additional cysteine residues are important for the protection of enzyme activity. The family-wide structures of DUSPs form a basis for the understanding of phosphorylation-mediated signal transduction and the development of therapeutics.

Structure of the catalytic domain of protein tyrosine phosphatase sigma in the sulfenic acid form.

Protein tyrosine phosphatase sigma (PTPσ) plays a vital role in neural development. The extracellular domain of PTPσ binds to various proteoglycans, which control the activity of 2 intracellular PTP domains (D1 and D2). To understand the regulatory mechanism of PTPσ, we carried out structural and biochemical analyses of PTPσ D1D2. In the crystal structure analysis of a mutant form of D1D2 of PTPσ, we unexpectedly found that the catalytic cysteine of D1 is oxidized to cysteine sulfenic acid, while that of D2 remained in its reduced form, suggesting that D1 is more sensitive to oxidation than D2. This finding contrasts previous observations on PTPα. The cysteine sulfenic acid of D1 was further confirmed by immunoblot and mass spectrometric analyses. The stabilization of the cysteine sulfenic acid in the active site of PTP suggests that the formation of cysteine sulfenic acid may function as a stable intermediate during the redox-regulation of PTPs.