Novel chimeric antigen receptor-expressing T cells targeting the malignant mesothelioma-specific antigen sialylated HEG1.

The selection of highly specific target antigens is critical for the development of clinically efficient and safe chimeric antigen receptors (CARs). In search of diagnostic marker for malignant mesothelioma (MM), we have established SKM9-2 monoclonal antibody (mAb) which recognizes a MM-specific molecule, sialylated Protein HEG homolog 1 (HEG1), with high specificity and sensitivity. In this study, to develop a novel therapeutic approach against MM, we generated SKM9-2 mAb-derived CARs that included the CD28 (SKM-28z) or 4-1BB (SKM-BBz) costimulatory domain. SKM-28z CAR-T cells showed continuous growth and enhanced Tim-3, LAG-3, and PD-1 expression in vitro, which might be induced by tonic signaling caused by self-activation; however, these phenotypes were not observed in SKM-BBz CAR-T cells. In addition, SKM-BBz CAR-T cells exhibited slightly stronger in vitro killing activity against MM cell lines than SKM-28z CAR-T cells. More importantly, only SKM-BBz CAR-T cells, but not SKM-28z CAR-T cells, significantly inhibited tumor growth in vivo in a MM cell line xenograft mouse model. Gene expression profiling and reporter assays revealed differential signaling pathway activation; in particular, SKM-BBz CAR-T cells exhibited enhanced NF-kB signaling and reduced NFAT activation. In addition, SKM-BBz CAR-T cells showed upregulation of early memory markers, such as TCF7 and CCR7, as well as downregulation of pro-apoptotic proteins, such as BAK1 and BID, which may be associated with phenotypical and functional differences between SKM-BBz and SKM-28z CAR-T cells. In conclusion, we developed novel SKM9-2-derived CAR-T cells with the 4-1BB costimulatory domain, which could provide a promising therapeutic approach against refractory MM.

In vivo cytosolic H2O2 changes and Ca2+ homeostasis in mouse skeletal muscle.

Hydrogen peroxide (H2O2) and calcium ions (Ca2+) are functional regulators of skeletal muscle contraction and metabolism. Although H2O2 is one of the activators of the type-1 ryanodine receptor (RyR1) in the Ca2+ release channel, the interdependence between H2O2 and Ca2+ dynamics remains unclear. This study tested the following hypotheses using an in vivo model of mouse tibialis anterior (TA) skeletal muscle. 1) Under resting conditions, elevated cytosolic H2O2 concentration ([H2O2]cyto) leads to a concentration-dependent increase in cytosolic Ca2+ concentration ([Ca2+]cyto) through its effect on RyR1; and 2) in hypoxia (cardiac arrest) and muscle contractions (electrical stimulation), increased [H2O2]cyto induces Ca2+ accumulation. Cytosolic H2O2 (HyPer7) and Ca2+ (Fura-2) dynamics were resolved by TA bioimaging in young C57BL/6J male mice under four conditions: 1) elevated exogenous H2O2; 2) cardiac arrest; 3) twitch (1 Hz, 60 s) contractions; and 4) tetanic (30 s) contractions. Exogenous H2O2 (0.1-100 mM) induced a concentration-dependent increase in [H2O2]cyto (+55% at 0.1 mM; +280% at 100 mM) and an increase in [Ca2+]cyto (+3% at 1.0 mM; +8% at 10 mM). This increase in [Ca2+]cyto was inhibited by pharmacological inhibition of RyR1 by dantrolene. Cardiac arrest-induced hypoxia increased [H2O2]cyto (+33%) and [Ca2+]cyto (+20%) 50 min postcardiac arrest. Compared with the exogenous 1.0 mM H2O2 condition, [H2O2]cyto after tetanic muscle contractions rose less than one-tenth as much, whereas [Ca2+]cyto was 4.7-fold higher. In conclusion, substantial increases in [H2O2]cyto levels evoke only modest Ca2+ accumulation via their effect on the sarcoplasmic reticulum RyR1. On the other hand, contrary to hypoxia secondary to cardiac arrest, increases in [H2O2]cyto from muscle contractions are small, indicating that H2O2 generation is unlikely to be a primary factor driving the significant Ca2+ accumulation after, especially tetanic, muscle contractions.NEW & NOTEWORTHY We developed an in vivo mouse myocyte H2O2 imaging model during exogenous H2O2 loading, ischemic hypoxia induced by cardiac arrest, and muscle contractions. In this study, the interrelationship between cytosolic H2O2 levels and Ca2+ homeostasis during muscle contraction and hypoxic conditions was revealed. These results contribute to the elucidation of the mechanisms of muscle fatigue and exercise adaptation.

Cooling of male rat skeletal muscle during endurance-like contraction attenuates contraction-induced PGC-1α mRNA expression.

This study aimed to determine effects of cooling on contraction-induced peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and vascular endothelial growth factor (VEGF) gene expression, phosphorylations of its related protein kinases, and metabolic responses. Male rats were separated into two groups; room temperature (RT) or ice-treated (COLD) on the right tibialis anterior (TA). The TA was contracted isometrically using nerve electrical stimulation (1-s stimulation × 30 contractions, with 1-s intervals, for 10 sets with 1-min intervals). The TA was treated before the contraction and during 1-min intervals with an ice pack for the COLD group and a water pack at RT for the RT group. The muscle temperature of the COLD group decreased to 19.42 ± 0.44°C (p < 0.0001, -36.4%) compared with the RT group after the experimental protocol. An increase in mRNA expression level of PGC-1α, not VEGF, after muscle contractions was significantly lower in the COLD group than in the RT group (p < 0.0001, -63.0%). An increase in phosphorylated AMP-activated kinase (AMPK) (p = 0.0037, -28.8%) and a decrease in glycogen concentration (p = 0.0231, +106.3%) after muscle contraction were also significantly inhibited by cooling. Collectively, muscle cooling attenuated the post-contraction increases in PGC-1α mRNA expression coinciding with decreases in AMPK phosphorylation and glycogen degradation.

Cmah deficiency blunts cellular senescence in adipose tissues and improves whole-body glucose metabolism in aged mice.

AIM: Cytidine monophosphate-N-acetylneuraminic acid (Neu5Ac) hydroxylase (Cmah) is an enzyme, which converts Neu5Ac to the sialic acid Neu5Gc. Neu5Gc is thought to increase inflammatory cytokines, which are, in part, produced in senescent cells of adipose tissues. Cellular senescence in adipose tissues induces whole-body aging and impaired glucose metabolism. Therefore, we hypothesized that Cmah deficiency would prevent cellular senescence in adipose tissues and impaired glucose metabolism. METHODS: Wild-type (WT) and Cmah knockout (KO) mice aged 24-25 months were used. Whole-body metabolism was assessed using a metabolic gas analysis system. We measured blood glucose and insulin concentrations after oral glucose administration. The size of the lipid droplets in the liver was quantified. Markers of cellular senescence and senescence-associated secretory phenotypes were measured in adipose tissues. RESULTS: Cmah KO had significantly increased VO2 and energy expenditure (P < 0.01). Unlike glucose, the insulin concentration after oral glucose administration was significantly lower in the Cmah KO group than in the WT group (P < 0.001). Lipid droplets in the liver were significantly lower in the Cmah KO group than in the WT group (P < 0.05). The markers of cellular senescence and senescence-associated secretory phenotypes in the adipose tissues were significantly lower in the Cmah KO group than in the WT group (P < 0.05). CONCLUSIONS: Cmah deficiency blunted cellular senescence in adipose tissues and improved whole-body glucose metabolism. These characteristics in aged Cmah KO mice might be associated with higher energy expenditure. Geriatr Gerontol Int 2023; 23: 958-964.

Chronic repetitive cooling and caffeine-induced intracellular Ca2+ elevation differentially impact adaptations in slow- and fast-twitch rat skeletal muscles.

Intracellular Ca2+ concentration ([Ca2+]i) is considered important in the regulation of skeletal muscle mass. This study tested the hypothesis that chronic repeated cooling and/or caffeine ingestion would acutely increase [Ca2+]i and hypertrophy muscles potentially in a fiber-type-dependent manner. Control rats and those fed caffeine were subjected to repeated bidiurnal treatments of percutaneous icing, under anesthesia, to reduce the muscle temperature below ∼5°C. The predominantly fast-twitch tibialis anterior (TA) and slow-twitch soleus (SOL) muscles were evaluated after 28 days of intervention. The [Ca2+]i elevating response to icing was enhanced by caffeine loading only in the SOL muscle, with the response present across a significantly higher temperature range than in the TA muscle under caffeine-loading conditions. In both the TA and SOL muscles, myofiber cross-sectional area (CSA) was decreased by chronic caffeine treatment (mean reductions of 10.5% and 20.4%, respectively). However, in the TA, but not the SOL, CSA was restored by icing (+15.4 ± 4.3% vs. noniced, P < 0.01). In the SOL, but not TA, icing + caffeine increased myofiber number (20.5 ± 6.7%, P < 0.05) and satellite cell density (2.5 ± 0.3-fold) in cross sections. These contrasting muscle responses to cooling and caffeine may reflect fiber-type-specific [Ca2+]i responses and/or differential responses to elevated [Ca2+]i.

Validation of EZH2 Inhibitor Efficiency in Anaplastic Thyroid Carcinoma Cell Lines.

BACKGROUND/AIM: The prognosis of anaplastic thyroid carcinoma (ATC) is poor, and there is currently no established treatment to improve its outcome. We previously reported that enhancer of zeste homolog 2 (EZH2) was highly expressed in ATC, and may be a therapeutic target; however, the effects of EZH2 on ATC growth currently remain unknown. MATERIALS AND METHODS: We investigated the effects of an EZH2 inhibitor (DZNep) on four ATC cell lines (8305C, KTA1, TTA1 and TTA2). We performed a gene panel analysis of all ATC cell lines to identify differences in DZNep sensitivity between the cell lines. To investigate the effects of DZNep on the recovery of differentiation, we assessed changes in thyroid differentiation markers (TDMs) before and after the DZNep treatment using PCR. RESULTS: EZH2 was expressed in all ATC cell lines. The cell-reducing effects of DZNep were detected in all ATC cell lines, and were the strongest in KTA1 cells followed by TTA2 cells. The TTA1 and 8305C cell lines, which showed weak cell-reducing effects, had TP53 mutations. No changes in TDMs were observed in any ATC cell line. CONCLUSION: DZNep, an EZH2 inhibitor, exerted suppressive effects on the growth of ATC cell lines and has potential as a therapeutic strategy; however, its effects may be attenuated in ATC with TP53 mutations.

Evaluation of X-ray and carbon-ion beam irradiation with chemotherapy for the treatment of cervical adenocarcinoma cells in 2D and 3D cultures.

BACKGROUND: Cervical cancer is the second most common cancer in women and causes more than 250,000 deaths worldwide. Among these, the incidence of cervical adenocarcinomas is increasing. Cervical adenocarcinoma is not only difficult to detect and prevent in the early stages with screening, but it is also resistant to chemotherapy and radiotherapy, and its prognosis worsens significantly as the disease progresses. Furthermore, when recurrence or metastasis is observed, treatment options are limited and there is no curative treatment. Recently, heavy-particle radiotherapy has attracted attention owing to its high tumor control and minimal damage to normal tissues. In addition, heavy particle irradiation is effective for cancer stem cells and hypoxic regions, which are difficult to treat. METHODS: In this study, we cultured cervical adenocarcinoma cell lines (HeLa and HCA-1) in two-dimensional (2D) or three-dimensional (3D) spheroid cultures and evaluated the effects of X-ray and carbon-ion (C-ion) beams. RESULTS: X-ray irradiation decreased the cell viability in a dose-dependent manner in 2D cultures, whereas this effect was attenuated in 3D spheroid cultures. In contrast, C-ion irradiation demonstrated the same antitumor effect in 3D spheroid cultures as in 2D cultures. In 3D spheroid cultures, X-rays and anticancer drugs are attenuated because of hypoxia inside the spheroids. However, the impact of the C-ion beam was almost the same as that of the 2D culture, because heavy-particle irradiation was not affected by hypoxia. CONCLUSION: These results suggest that heavy-particle radiotherapy may be a new therapeutic strategy for overcoming the resistance of cervical adenocarcinoma to treatment.

Branched-chain amino acid supplementation suppresses the detraining-induced reduction of mitochondrial content in mouse skeletal muscle.

Exercise training enhances oxidative capacity whereas detraining reduces mitochondrial content in skeletal muscle. The strategy to suppress the detraining-induced reduction of mitochondrial content has not been fully elucidated. As previous studies reported that branched-chain amino acid (BCAA) ingestion increased mitochondrial content in skeletal muscle, we evaluated whether BCAA supplementation could suppress the detraining-induced reduction of mitochondrial content. Six-week-old male Institute of Cancer Research (ICR) mice were randomly divided into four groups as follows: control (Con), endurance training (Tr), detraining (DeTr), and detraining with BCAA supplementation (DeTr + BCAA). Mice in Tr, DeTr, and DeTr + BCAA performed treadmill running exercises [20-30 m/min, 60 min, 5 times/week, 4 weeks]. Then, mice in DeTr and DeTr + BCAA were administered with water or BCAA [0.6 mg/g of body weight, twice daily] for 2 weeks of detraining. In whole skeletal muscle, mitochondrial enzyme activities and protein content were decreased after 2 weeks of detraining, but the reduction was suppressed by BCAA supplementation. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) protein content, a master regulator of mitochondrial biogenesis, was decreased by detraining irrespective of BCAA ingestion. Regarding mitochondrial degradation, BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), a mitophagy-related protein, was significantly higher in the Tr group than in the DeTr + BCAA group, but not different from in the DeTr group. With respect to mitochondrial quality, BCAA ingestion did not affect oxygen consumption rate (OCR) and reactive oxygen species (ROS) production in isolated mitochondria. Our findings suggest that BCAA ingestion suppresses the detraining-induced reduction of mitochondrial content partly through inhibiting mitophagy.

TROP-2, Nectin-4, GPNMB, and B7-H3 Are Potentially Therapeutic Targets for Anaplastic Thyroid Carcinoma.

BACKGROUND: Anaplastic thyroid carcinoma (ATC) is a highly aggressive thyroid tumor with a poor prognosis. However, there are limited choices for ATC treatment. Recently, the effectiveness of antibody-drug conjugates has been demonstrated in various carcinomas. Whether the targets of antibody-drug conjugates are expressed in anaplastic thyroid carcinoma remains unclear. METHODS: Fifty-four patients with ATC were enrolled in this study. Tissue microarrays were constructed using the archives of formalin-fixed paraffin-embedded tissue blocks. All sections were stained with the following antibody-drug conjugate targets: human epidermal growth factor receptor 2 (HER2), nectin-4, trophoblast cell surface antigen 2 (TROP-2), glycoprotein non-metastatic B (GPNMB), and B7-H3. RESULTS: HER2 was negative in all tissues, whereas GPNMB and B7-H3 were expressed in most ATC tissues. TROP-2 and nectin-4 were expressed in 65% and 59% of ATC tissues, respectively. TROP-2 was expressed at significantly higher levels in ATC undifferentiated from papillary thyroid carcinoma than in ATC undifferentiated from follicular thyroid carcinoma and de novo ATC. In contrast, nectin-4 expression was markedly higher in patients with de novo ATC than in those with papillary and follicular thyroid carcinoma. CONCLUSIONS: TROP-2 and nectin-4 are potential therapeutic targets for ATC undifferentiated from papillary thyroid carcinoma and de novo ATC, respectively. GPNMB and B7-H3 potential for treating all types of ATC.

Effect of endurance training and PGC-1α overexpression on calculated lactate production volume during exercise based on blood lactate concentration.

Lactate production is an important clue for understanding metabolic and signal responses to exercise but its measurement is difficult. Therefore, this study aimed (1) to develop a method of calculating lactate production volume during exercise based on blood lactate concentration and compare the effects between endurance exercise training (EX) and PGC-1α overexpression (OE), (2) to elucidate which proteins and enzymes contribute to changes in lactate production due to EX and muscle PGC-1α OE, and (3) to elucidate the relationship between lactate production volume and signaling phosphorylations involved in mitochondrial biogenesis. EX and PGC-1α OE decreased muscle lactate production volume at the absolute same-intensity exercise, but only PGC-1α OE increased lactate production volume at the relative same-intensity exercise. Multiple linear regression revealed that phosphofructokinase, monocarboxylate transporter (MCT)1, MCT4, and citrate synthase equally contribute to the lactate production volume at high-intensity exercise within physiological adaptations, such as EX, not PGC-1α OE. We found that an exercise intensity-dependent increase in the lactate production volume was associated with a decrease in glycogen concentration and an increase in P-AMPK/T-AMPK. This suggested that the calculated lactate production volume was appropriate and reflected metabolic and signal responses but further modifications are needed for the translation to humans.

Aboveground biomass increments over 26 years (1993-2019) in an old-growth cool-temperate forest in northern Japan.

Assessing long-term changes in the biomass of old-growth forests with consideration of climate effects is essential for understanding forest ecosystem functions under a changing climate. Long-term biomass changes are the result of accumulated short-term changes, which can be affected by endogenous processes such as gap filling in small-scale canopy openings. Here, we used 26 years (1993-2019) of repeated tree census data in an old-growth, cool-temperate, mixed deciduous forest that contains three topographic units (riparian, denuded slope, and terrace) in northern Japan to document decadal changes in aboveground biomass (AGB) and their processes in relation to endogenous processes and climatic factors. AGB increased steadily over the 26 years in all topographic units, but different tree species contributed to the increase among the topographic units. AGB gain within each topographic unit exceeded AGB loss via tree mortality in most of the measurement periods despite substantial temporal variation in AGB loss. At the local scale, variations in AGB gain were partially explained by compensating growth of trees around canopy gaps. Climate affected the local-scale AGB gain: the gain was larger in the measurement periods with higher mean air temperature during the current summer but smaller in those with higher mean air temperature during the previous autumn, synchronously in all topographic units. The influences of decadal summer and autumn warming on AGB growth appeared to be counteracting, suggesting that the observed steady AGB increase in KRRF is not fully explained by the warming. Future studies should consider global and regional environmental factors such as elevated CO2 concentrations and nitrogen deposition, and include cool-temperate forests with a broader temperature range to improve our understanding on biomass accumulation in this type of forests under climate change.

Machine learning-based image analysis for accelerating the diagnosis of complicated preneoplastic and neoplastic ductal lesions in breast biopsy tissues.

PURPOSE: Diagnosis of breast preneoplastic and neoplastic lesions is difficult due to their similar morphology in breast biopsy specimens. To diagnose these lesions, pathologists perform immunohistochemical analysis and consult with expert breast pathologists. These additional examinations are time-consuming and expensive. Artificial intelligence (AI)-based image analysis has recently improved, and may help in ordinal pathological diagnosis. Here, we showed the significance of machine learning-based image analysis of breast preneoplastic and neoplastic lesions for facilitating high-throughput diagnosis. METHODS: Images were obtained from normal mammary glands, hyperplastic lesions, preneoplastic lesions and neoplastic lesions, such as usual ductal hyperplasia (UDH), columnar cell lesion (CCL), ductal carcinoma in situ (DCIS), and DCIS with comedo necrosis (comedo DCIS) in breast biopsy specimens. The original enhanced convoluted neural network (CNN) system was used for analyzing the pathological images. RESULTS: The AI-based image analysis provided the following area under the curve values (AUC): normal lesion versus DCIS, 0.9902; DCIS versus comedo DCIS, 0.9942; normal lesion versus CCL, 0.9786; and UDH versus DCIS, 1.000. Multiple comparison analysis showed precision and recall scores similar to those of single comparison analysis. Based on the gradient-weighted class activation mapping (Grad-CAM) used to visualize the important regions reflecting the result of CNN analysis, the ratio of stromal tissue in the whole weighted area was significantly higher in UDH and CCL than that in DCIS. CONCLUSIONS: These analyses may provide a more accurate and rapid pathological diagnosis of patients. Moreover, Grad-CAM identifies uncharted important histological characteristics for newer pathological findings and targets of research for understanding diseases.

Short-Term Calorie Restriction Maintains Plasma Insulin Concentrations along with a Reduction in Hepatic Insulin-Degrading Enzyme Levels in db/db Mice.

Maintaining blood insulin levels is important for patients with diabetes because insulin secretion capacity declines with the development of the disease. Calorie restriction (CR) is effective for the improvement of glucose tolerance, but it is not clear whether CR can maintain insulin levels in the late stage of diabetes. We examined the effect of CR on whole-body glucose tolerance and fasting blood insulin concentrations in the late stage of diabetes. Male db/db mice were subjected to either a standard laboratory diet ad libitum for 3 weeks (dbdb group) or 40% CR (dbdb+CR group). CR significantly decreased body mass and epididymal fat weight. Glucose tolerance and fasting glucose levels were significantly improved with 3-week CR. Fasting insulin concentrations were decreased in the dbdb group but were maintained in the dbdb+CR group. CR significantly reduced insulin-degrading enzyme (IDE) levels in the liver, and hepatic IDE levels were significantly positively and negatively correlated with plasma glucose concentrations (area under the curve) after glucose administration and after fasting insulin concentrations, respectively. Therefore, 3-week CR maintained blood insulin levels and improved glucose tolerance with decreased hepatic IDE levels in an animal model of late-stage diabetes.

In vivo Ca2+ dynamics during cooling after eccentric contractions in rat skeletal muscle.

The effect of cooling on in vivo intracellular calcium ion concentration [Ca2+]i after eccentric contractions (ECs) remains to be determined. We tested the hypothesis that cryotherapy following ECs promotes an increased [Ca2+]i and induces greater muscle damage in two muscles with substantial IIb and IIx fiber populations. The thin spinotrapezius (SPINO) muscles of Wistar rats were used for in vivo [Ca2+]i imaging, and tibialis anterior (TA) muscles provided greater fidelity and repeatability of contractile function measurements. SPINO [Ca2+]i was estimated using fura 2-AM and the magnitude, location, and temporal profile of [Ca2+]i determined as the temperature near the muscle surface post-ECs was decreased from 30°C (control) to 20°C or 10°C. Subsequently, in the TA, the effect of post-ECs cooling to 10°C on muscle contractile performance was determined at 1 and 2 days after ECs. TA muscle samples were examined by hematoxylin and eosin staining to assess damage. In SPINO, reducing the muscle temperature from 30°C to 10°C post-ECs resulted in a 3.7-fold increase in the spread of high [Ca2+]i sites generated by ECs (P < 0.05). These high [Ca2+]i sites demonstrated partial reversibility when rewarmed to 30°C. Dantrolene, a ryanodine receptor Ca2+ release inhibitor, reduced the presence of high [Ca2+] sites at 10°C. In the TA, cooling exacerbated ECs-induced muscle strength deficits via enhanced muscle fiber damage (P < 0.05). By demonstrating that cooling post-ECs potentiates [Ca2+]i derangements, this in vivo approach supports a putative mechanistic basis for how postexercise cryotherapy might augment muscle fiber damage and decrease subsequent exercise performance.

Trans-omic Analysis Reveals ROS-Dependent Pentose Phosphate Pathway Activation after High-Frequency Electrical Stimulation in C2C12 Myotubes.

Skeletal muscle adaptation is mediated by cooperative regulation of metabolism, signal transduction, and gene expression. However, the global regulatory mechanism remains unclear. To address this issue, we performed electrical pulse stimulation (EPS) in differentiated C2C12 myotubes at low and high frequency, carried out metabolome and transcriptome analyses, and investigated phosphorylation status of signaling molecules. EPS triggered extensive and specific changes in metabolites, signaling phosphorylation, and gene expression during and after EPS in a frequency-dependent manner. We constructed trans-omic network by integrating these data and found selective activation of the pentose phosphate pathway including metabolites, upstream signaling molecules, and gene expression of metabolic enzymes after high-frequency EPS. We experimentally validated that activation of these molecules after high-frequency EPS was dependent on reactive oxygen species (ROS). Thus, the trans-omic analysis revealed ROS-dependent activation in signal transduction, metabolome, and transcriptome after high-frequency EPS in C2C12 myotubes, shedding light on possible mechanisms of muscle adaptation.

Single-Cell Information Analysis Reveals That Skeletal Muscles Incorporate Cell-to-Cell Variability as Information Not Noise.

Cell-to-cell variability in signal transduction in biological systems is often considered noise. However, intercellular variation (i.e., cell-to-cell variability) has the potential to enable individual cells to encode different information. Here, we show that intercellular variation increases information transmission of skeletal muscle. We analyze the responses of multiple cultured myotubes or isolated skeletal muscle fibers as a multiple-cell channel composed of single-cell channels. We find that the multiple-cell channel, which incorporates intercellular variation as information, not noise, transmitted more information in the presence of intercellular variation than in the absence according to the "response diversity effect," increasing in the gradualness of dose response by summing the cell-to-cell variable dose responses. We quantify the information transmission of human facial muscle contraction during intraoperative neurophysiological monitoring and find that information transmission of muscle contraction is comparable to that of a multiple-cell channel. Thus, our data indicate that intercellular variation can increase the information capacity of tissues.

NH2 -terminal fragment of ZF21 protein suppresses tumor invasion via inhibiting the interaction of ZF21 with FAK.

Cellular migration, coupled with the degradation of the extracellular matrix (ECM), is a key step in tumor invasion and represents a promising therapeutic target in malignant tumors. Focal adhesions (FAs) and invadopodia, which are distinct actin-based cellular structures, play key roles in cellular migration and ECM degradation, respectively. The molecular machinery coordinating the dynamics between FAs and invadopodia is not fully understood, although several lines of evidence suggest that the disassembly of FAs is an important step in triggering the formation of invadopodia. In a previous study, we identified the ZF21 protein as a regulator of both FA turnover and invadopodia-dependent ECM degradation. ZF21 interacts with multiple factors for FA turnover, including focal adhesion kinase (FAK), microtubules, m-Calpain, and Src homology region 2-containing protein tyrosine phosphatase 2 (SHP-2). In particular, the dephosphorylation of FAK by ZF21 is a key event in tumor invasion. However, the precise role of ZF21 binding to FAK remains unclear. We established a method to disrupt the interaction between ZF21 and FAK using the FAK-binding NH2 -terminal region of ZF21. Tumor cells expressing the ZF21-derived polypeptide had significantly decreased FA turnover, migration, invadopodia-dependent ECM degradation, and Matrigel invasion. Furthermore, the expression of the polypeptide inhibited an early step of experimental lung metastasis in mice. These findings indicate that the interaction of ZF21 with FAK is necessary for FA turnover as well as ECM degradation at the invadopodia. Thus, ZF21 is a potential regulator that coordinates the equilibrium between FA turnover and invadopodia activity by interacting with FAK.

Membrane type 1 matrix metalloproteinase regulates anaplastic thyroid carcinoma cell growth and invasion into the collagen matrix.

Anaplastic thyroid carcinoma (ATC) is one of the most aggressive cancer types; however, the molecular mechanism contributing to the aggressive characteristics remain unclear. Membrane type 1 matrix metalloproteinase (MT1-MMP) plays an important role in cancer invasion and has been associated with a poor prognosis in various malignant neoplasms. In this study, we investigated the relationship between MT1-MMP expression and the proliferation and invasion of ATC cells, along with the association with clinicopathologic factors in patients with ATC. Suppression of MT1-MMP reduced the proliferation and invasion of ATC cells, and suppressed ERK activity, indicating a role in cancer cell proliferation in collagen matrix culture conditions. The expression of MT1-MMP was detected in 29 of 34 (85.3%) surgical specimens from ATC patients. In addition, the expression of MT1-MMP in the tumor lesion was higher than that of normal and stromal tissues. Collectively, these results suggest that elevated MT1-MMP expression plays a role in the pathogenesis of ATC, which may promote its aggressive characteristics such as proliferation and invasion, highlighting a potential new therapeutic target.

Effects of electrical stimulation-induced resistance exercise training on white and brown adipose tissues and plasma meteorin-like concentration in rats.

Chronic endurance exercise training induces morphological and metabolic alterations including mitochondrial biogenesis in white adipose tissue (WAT) and brown adipose tissue (BAT) in rodents. A myokine called meteorin-like (Metrnl) is associated with morphological and metabolic adaptation and increased in blood after acute resistance exercise. However, the effects of chronic resistance exercise training (RT), which aims to increase muscle mass and strength, on WAT and BAT are unclear. Therefore, we aimed to clarify the effects of RT on morphological and metabolic parameters in WAT and BAT and on plasma Metrnl concentrations. We applied electrical stimulation to both legs of rats as RT three times a week for 4 weeks. RT reduced adipocyte size in subcutaneous WAT but induced no changes in mitochondrial and thermogenesis proteins. In BAT, peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) protein levels and mitochondrial content markers were significantly higher in the RT group compared with the control group. A significant positive correlation was found between the expression of PGC-1α in BAT and plasma Metrnl concentrations. These results suggest that plasma Metrnl is associated with PGC-1α and mitochondrial biogenesis in BAT. This study describes a potential role of RT in preventing metabolic diseases via altering WAT and BAT and increasing plasma Mertnl concentrations.

Sensitization of HT29 colorectal cancer cells to vemurafenib in three-dimensional collagen cultures.

The extracellular matrix to which cancer cells adhere affects cellular sensitivity to anticancer drugs. We sought to examine the changes in sensitivity of colorectal cancer cells carrying the BRAF V600E mutation to vemurafenib cultured in three-dimensional (3D) collagen-I gels, while also identifying the signaling pathways involved in these changes. HT29 colorectal cancer cells were cultured in conventional tissue culture (TC) plastic plates or in collagen-I gels. The HT29 cells demonstrated approximately 10-fold higher sensitivity to vemurafenib in 3D-collagen-I gels compared with those cultured on conventional TC plastic plates. Furthermore, in cells cultured on TC plastic, vemurafenib was found to augment tyrosine phosphorylation of focal adhesion kinase (FAK), while 3D-cultured cells expressed lower levels of FAK and vemurafenib did not affect its tyrosine phosphorylation, suggesting that FAK contributes to vemurafenib resistance. However, pharmacological inhibition of FAK did not sensitize the cells to vemurafenib. Also, the level of tyrosine-phosphorylated epidermal growth factor receptor (EGFR)/ERBB2 family proteins was found to be lower in cells cultured in 3D-collagen gel compared with those in cells cultured on TC plastic. Afatinib, an inhibitor of the EGFR/ERBB family of kinases, sensitized the cells to higher concentrations of vemurafenib, implying their participation in vemurafenib resistance. Adhesion to collagen-I gel but not to the collagen-I-coated plastic surface sensitized the cells, suggesting that the rigidity of the media rather than adherence to collagen-I may be important for cellular sensitivity to vemurafenib.