<span style="font-family:Verdana;">The Japan Dementia Prevention Society conducted a questionnaire survey of dementia specialists conducted in August 2020. Regarding the worsening of symptoms in people...<span style="font-family:Verdana;">The Japan Dementia Prevention Society conducted a questionnaire survey of dementia specialists conducted in August 2020. Regarding the worsening of symptoms in people with dementia, 8% answered that they “accept more” and 32% answered that they “recognize a minority”. In the previous research report (Open Journal of Therapy and Rehabilitation Vol.8 No.3), we examined whether hand care therapy (HCT) as one of the passive horticultural therapy was effective in preventing dementia/MCI. Start of the test in August 2018 (I: BL) to February 2020 (VII: 18M) was, the use of HCT was to be effective in preventing dementia and MCI illness and slowing progression. Due to the COVID19 pandemic, VIII (24M) was evaluated in August 2020, and IX (27M) also measured ANS during activity. At that time, they divided into the Group E who had refrained from welfare facilities for the elderly due to the influence of COVID19 and the Group F who HCT continued. In the evaluation of VIII (24M), it was found that the cognitive function of the Group E was significantly reduced compared to VII, and Group F did not show a significant decrease. ADL decreased and depressive symptoms increase by continuing a life with less going out due to the influence of COVID 19 regardless of the use of the facility. It became clear that both Group E and Group F show good changes in ANS balance in the IX. Therefore, by utilizing horticultural therapy that the power of nature and plants in addition to utilizing welfare facilities for the elderly, cognitive impairment in COVID 19</span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">.</span></span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;"> It was expected to lead to prevention and prevention of negative psychology and behavior such as depressive views and depressive tendencies. In the previous research report (Open Journal of Therapy and Rehabilitation Vol.8 No.3), we examined whether hand care therapy (HCT) as one of passive horticultural therapy was effective in preventing dementia/MCI. In the trend from the start of the test in August 2018 (I: BL) to February 2020 (VII: 18M) was, the use of HCT was shown to be effective in preventing dementia and MCI illness and slowing progression. Due to the COVID19 pandemic, various activity restrictions have been implemented in Japan since March 2020, and the number of elderly people who do not receive long-term care or rehabilitation has increased. Therefore, in August and November 2020, when the spread of COVID19 was reduced, cognitive function evaluation was carried out. At that time, they divided into the elderly group (Group E) who had refrained from welfare facilities for the elderly due to the influence of COVID19 and the elderly group (Group F) who HCT continued.</span></span></span></span>展开更多
Osteocytes are mechanosensitive bone cells, but little is known about their effects on tumor cells in response to mechanical stimulation. We treated breast cancer cells with osteocyte-derived conditioned medium(CM) an...Osteocytes are mechanosensitive bone cells, but little is known about their effects on tumor cells in response to mechanical stimulation. We treated breast cancer cells with osteocyte-derived conditioned medium(CM) and fluid flow-treated conditioned medium(FFCM) with 0.25 Pa and 1 Pa shear stress. Notably, CM and FFCM at 0.25 Pa induced the mesenchymal-to-epithelial transition(MET), but FFCM at 1 Pa induced the epithelial-to-mesenchymal transition(EMT). This suggested that the effects of fluid flow on conditioned media depend on flow intensity. Fluorescence resonance energy transfer(FRET)-based evaluation of Src activity and vinculin molecular force showed that osteopontin was involved in EMT and MET switching. A mouse model of tumorinduced osteolysis was tested using dynamic tibia loadings of 1, 2, and 5 N. The low 1 N loading suppressed tumor-induced osteolysis, but this beneficial effect was lost and reversed with loads at 2 and 5 N, respectively. Changing the loading intensities in vivo also led to changes in serum TGFβ levels and the composition of tumor-associated volatile organic compounds in the urine.Collectively, this study demonstrated the critical role of intensity-dependent mechanotransduction and osteopontin in tumorosteocyte communication, indicating that a biophysical factor can tangibly alter the behaviors of tumor cells in the bone microenvironment.展开更多
Osteocytes are the most abundant cells in bone,which is a frequent site of breast cancer metastasis.Here,we focused on Wnt signaling and evaluated tumor-osteocyte interactions.In animal experiments,mammary tumor cells...Osteocytes are the most abundant cells in bone,which is a frequent site of breast cancer metastasis.Here,we focused on Wnt signaling and evaluated tumor-osteocyte interactions.In animal experiments,mammary tumor cells were inoculated into the mammary fat pad and tibia.The role of Lrp5-mediated Wnt signaling was examined by overexpressing and silencing Lrp5 in osteocytes and establishing a conditional knockout mouse model.The results revealed that administration of osteocytes or their conditioned medium(CM)inhibited tumor progression and osteolysis.Osteocytes overexpressing Lrp5 or β-catenin displayed strikingly elevated tumor-suppressive activity,accompanied by downregulation of tumor-promoting chemokines and upregulation of apoptosis-inducing and tumor-suppressing proteins such as p53.The antitumor effect was also observed with osteocyte-derived CM that was pretreated with a Wnt-activating compound.Notably,silencing Lrp5 in tumors inhibited tumor progression,while silencing Lrp5 in osteocytes in conditional knockout mice promoted tumor progression.Osteocytes exhibited elevated Lrp5 expression in response to tumor cells,implying that osteocytes protect bone through canonical Wnt signaling.Thus,our results suggest that the Lrp5/β-catenin axis activates tumor-promoting signaling in tumor cells but tumor-suppressive signaling in osteocytes.We envision that osteocytes with Wnt activation potentially offer a novel cell-based therapy for breast cancer and osteolytic bone metastasis.展开更多
Mechanical loading to the bone is known to be beneficial for bone homeostasis and for suppressing tumor-induced osteolysis in the loaded bone.However,whether loading to a weight-bearing hind limb can inhibit distant t...Mechanical loading to the bone is known to be beneficial for bone homeostasis and for suppressing tumor-induced osteolysis in the loaded bone.However,whether loading to a weight-bearing hind limb can inhibit distant tumor growth in the brain is unknown.We examined the possibility of bone-to-brain mechanotransduction using a mouse model of a brain tumor by focusing on the response to Lrp5-mediated Wnt signaling and dopamine in tumor cells.The results revealed that loading the tibia with elevated levels of tyrosine hydroxylase,a rate-limiting enzyme in dopamine synthesis,markedly reduced the progression of the brain tumors.The simultaneous application of fluphenazine(FP),an antipsychotic dopamine modulator,enhanced tumor suppression.Dopamine and FP exerted antitumor effects through the dopamine receptors DRD1 and DRD2,respectively.Notably,dopamine downregulated Lrp5 via DRD1 in tumor cells.A cytokine array analysis revealed that the reduction in CCN4 was critical for loading-driven,dopamine-mediated tumor suppression.The silencing of Lrp5 reduced CCN4,and the administration of CCN4 elevated oncogenic genes such as MMP9,Runx2,and Snail.In summary,this study demonstrates that mechanical loading regulates dopaminergic signaling and remotely suppresses brain tumors by inhibiting the Lrp5-CCN4 axis via DRD1,indicating the possibility of developing an adjuvant bone-mediated loading therapy.展开更多
Western blotting is a popular technique for examining expression levels of proteins using gel-based electrophoretic fractionation followed by blotting and antibody reactions. Although this is a mature technique, one o...Western blotting is a popular technique for examining expression levels of proteins using gel-based electrophoretic fractionation followed by blotting and antibody reactions. Although this is a mature technique, one of the major limitations is the need to prepare an individual electrophoretic gel for each of the protein species to be analyzed. Since most analyses require the detection of multiple protein species, a procedure that allows utilization of a single gel for detecting multiple protein species should significantly save time and resources. In this paper, we developed a novel multiprotein detection device, which enabled simultaneous detection of several proteins species from a single electrophoretic gel. In this device, a protein transfer unit utilized a multi-anode plate that generated a non-uniform voltage profile. This voltage profile enabled uniform transfer regardless of molecular mass of proteins. In vitro experiments using samples, isolated from boneforming osteoblast cells, showed that the expression levels of 5 - 7 different proteins were detectable in the presence and absence of mechanical stimulation that activated genes necessary for bone formation. The result supports the notion that through simultaneous detection of multiple protein species, the described device contributes to reduction in procedural time and sample amounts, as well as a removal of variations among multiple gels.展开更多
This study presents the development of an innovative artificial finger-like device that provides position specific mechanical loads at the end of the long bone and induces mechanotransduction in bone. Bone cells such ...This study presents the development of an innovative artificial finger-like device that provides position specific mechanical loads at the end of the long bone and induces mechanotransduction in bone. Bone cells such as osteoblasts are the mechanosensitive cells that regulate bone remodelling. When they receive gentle, periodic mechanical loads, new bone formation is promoted. The proposed device is an under-actuated multi-fingered artificial hand with 4 fingers, each having two phalanges. These fingers are connected by mechanical linkages and operated by a worm gearing mechanism. With the help of 3D printing technology, a prototype device was built mostly using plastic materials. The experimental validation results show that the device is capable of generating necessary forces at the desired frequencies, which are suitable for the stimulation of bone cells and the promotion of bone formation. It is recommended that the device be tested in a clinical study for confirming its safety and efficacy with patients.展开更多
Blotting is a common technique widely used for molecular analysis in life sciences. The Western blot, in particular, is a process of transferring protein samples from a polyacrylamide gel to a blotting membrane and de...Blotting is a common technique widely used for molecular analysis in life sciences. The Western blot, in particular, is a process of transferring protein samples from a polyacrylamide gel to a blotting membrane and detecting the levels of specific proteins through reactions with primary and secondary antibodies. The state-of-the-art of Western blotting usually generates one blotting membrane per gel. However, multiple copies of blots are useful in many applications. Two blotting copies from a single protein gel, for instance, can be used for identifying a total amount of proteins of interest as well as its specific subpopulation level such as a phosphorylated isoform. To achieve this multi-blotting operation from a single gel, we modified a blotting procedure and developed a novel blotting device. The device consisted of a multi-anode plate and a microcontroller. It was designed to generate a well-controlled electrophoretic voltage profile, which allowed a quasi-uniform transfer of proteins of any size. The prototype device was built and its operation procedure was described. The experimental results clearly supported the notion that the described device was able to achieve multiple blotting from a single gel and reduce time and cost for protein analysis.展开更多
BlotMan is a protein blotting device that allows generating multiple membranes from a single polyacrylamide gel. To transfer all proteins uniformly with the same efficiency regardless of protein size, BlotMan employs ...BlotMan is a protein blotting device that allows generating multiple membranes from a single polyacrylamide gel. To transfer all proteins uniformly with the same efficiency regardless of protein size, BlotMan employs pulse-width-modulated (PWM) voltage that applies a higher average voltage to a larger protein species. BlotMan can be controlled not only by its custom-made interface but also by a smart phone via Bluetooth technology. In this study, we examined effects of PWM signals (50%, 60%, and 80% duty cycle) on transfer efficiency and signal intensity in comparison to a constant voltage signal (100% duty cycle). The result revealed that in response to the same average voltage of 150 V, a lower duty cycle with a higher maximum voltage increased transfer efficiency as well as sharpness of transferred proteins. We validated BlotMan’s capability using a chondrosarcoma cell line (SW1353 cells) and a breast cancer cell line (MDA-MB231 cells) in response to antitumor chemical agents. BlotMan successfully generated 5 membranes from a single gel and detected 5 protein species such as c-Src, eukaryotic translation initiation factor 2 alpha (eIF2), phosphorylated eIF2, lamin B, and actin. Collectively, we demonstrated herein that BlotMan reduces an amount of protein samples by generating multiple membranes from a single gel and improving signal intensity with PWM voltage signals.展开更多
Cancer cells tend to develop resistance to chemotherapy and enhance aggressive-ness.A counterintuitive approach is to tame aggressiveness by an agent that acts opposite to chemotherapeutic agents.Based on this strateg...Cancer cells tend to develop resistance to chemotherapy and enhance aggressive-ness.A counterintuitive approach is to tame aggressiveness by an agent that acts opposite to chemotherapeutic agents.Based on this strategy,induced tumor-suppressing cells(iTSCs)have been generated from tumor cells and mesenchymal stem cells.Here,we examined the possi-bility of generating iTSCs from lymphocytes by activating PKA signaling for suppressing the pro-gression of osteosarcoma(OS).While lymphocyte-derived CM did not present anti-tumor capabilities,the activation of PKA converted them into iTSCs.Inhibiting PKA conversely gener-ated tumor-promotive secretomes.In a mouse model,PKA-activated CM suppressed tumorinduced bone destruction.Proteomics analysis revealed that moesin(MSN)and calreticulin(Calr),which are highly expressed intracellular proteins in many cancers,were enriched in PKA-activated CM,and they acted as extracellular tumor suppressors through CD44,CD47,and CD91.The study presented a unique option for cancer treatment by generating iTSCs that secret tumor-suppressive proteins such as MSN and Calr.We envision that identifying these tu-mor suppressors and predicting their binding partners such as CD44,which is an FDA-approved oncogenic target to be inhibited,may contribute to developing targeted protein therapy.展开更多
Weight-bearing bone is constantly adapting its structure and function to mechanical environments. Loading through routine exercises stimulates bone formation and prevents bone loss, but unloading through bed rest and ...Weight-bearing bone is constantly adapting its structure and function to mechanical environments. Loading through routine exercises stimulates bone formation and prevents bone loss, but unloading through bed rest and cast immobilization as well as exposure to weightlessness during spaceflight reduces its mass and strength. In order to elucidate the mechanism underlying unloading-driven bone adaptation, ground-based in vitro and in vivo analyses have been conducted using rotating cell culturing and hindlimb suspension. Focusing on gene expression studies in osteoblasts and hindlimb suspension studies, this minireview introduces our recent understanding on bone homeostasis under weightlessness in space. Most of the existing data indicate that unloading has the opposite effects to loading through common signaling pathways. However, a question remains as to whether any pathway unique to unloading (and not to loading) may exist.展开更多
Since the early days of manned spaceflight, hazardous effects of the space environment on living organisms have been disputed. With the continuous manning of the International Space Station, the planned Chinese space ...Since the early days of manned spaceflight, hazardous effects of the space environment on living organisms have been disputed. With the continuous manning of the International Space Station, the planned Chinese space station, and renewed interest in returning to the Moon and sending manned flights to Mars, identifying and addressing the potential outcomes of long-term space exposures is critically important.展开更多
Understanding the regulatory mechanism that controls the alteration of global gene expression patterns continues to be a challenging task in computational biology. We previously developed an ant algorithm, a biologica...Understanding the regulatory mechanism that controls the alteration of global gene expression patterns continues to be a challenging task in computational biology. We previously developed an ant algorithm, a biologically-inspired computational technique for microarray data, and predicted putative transcription-factor binding motifs (TFBMs) through mimicking interactive behaviors of natural ants. Here we extended the algorithm into a set of web-based software, Ant Modeler, and applied it to investigate the transcriptional mechanism underlying bone formation. Mechanical loading and administration of bone morphogenic proteins (BMPs) are two known treatments to strengthen bone. We addressed a question: Is there any TFBM that stimulates both "anabolic responses of mechanical loading" and "BMP-mediated osteogenic signaling"? Although there is no significant overlap among genes in the two responses, a comparative model-based analysis suggests that the two independent osteogenic processes employ common TFBMs, such as a stress responsive element and a motif for peroxisome proliferator-activated receptor (PPAR). The post-modeling in vitro analysis using mouse osteoblast cells supported involvements of the predicted TFBMs such as PPAR, Ikaros 3, and LMO2 in response to mechanical loading. Taken together, the results would be useful to derive a set of testable hypotheses and examine the role of specific regulators in complex transcriptional control of bone formation.展开更多
文摘<span style="font-family:Verdana;">The Japan Dementia Prevention Society conducted a questionnaire survey of dementia specialists conducted in August 2020. Regarding the worsening of symptoms in people with dementia, 8% answered that they “accept more” and 32% answered that they “recognize a minority”. In the previous research report (Open Journal of Therapy and Rehabilitation Vol.8 No.3), we examined whether hand care therapy (HCT) as one of the passive horticultural therapy was effective in preventing dementia/MCI. Start of the test in August 2018 (I: BL) to February 2020 (VII: 18M) was, the use of HCT was to be effective in preventing dementia and MCI illness and slowing progression. Due to the COVID19 pandemic, VIII (24M) was evaluated in August 2020, and IX (27M) also measured ANS during activity. At that time, they divided into the Group E who had refrained from welfare facilities for the elderly due to the influence of COVID19 and the Group F who HCT continued. In the evaluation of VIII (24M), it was found that the cognitive function of the Group E was significantly reduced compared to VII, and Group F did not show a significant decrease. ADL decreased and depressive symptoms increase by continuing a life with less going out due to the influence of COVID 19 regardless of the use of the facility. It became clear that both Group E and Group F show good changes in ANS balance in the IX. Therefore, by utilizing horticultural therapy that the power of nature and plants in addition to utilizing welfare facilities for the elderly, cognitive impairment in COVID 19</span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">.</span></span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;"> It was expected to lead to prevention and prevention of negative psychology and behavior such as depressive views and depressive tendencies. In the previous research report (Open Journal of Therapy and Rehabilitation Vol.8 No.3), we examined whether hand care therapy (HCT) as one of passive horticultural therapy was effective in preventing dementia/MCI. In the trend from the start of the test in August 2018 (I: BL) to February 2020 (VII: 18M) was, the use of HCT was shown to be effective in preventing dementia and MCI illness and slowing progression. Due to the COVID19 pandemic, various activity restrictions have been implemented in Japan since March 2020, and the number of elderly people who do not receive long-term care or rehabilitation has increased. Therefore, in August and November 2020, when the spread of COVID19 was reduced, cognitive function evaluation was carried out. At that time, they divided into the elderly group (Group E) who had refrained from welfare facilities for the elderly due to the influence of COVID19 and the elderly group (Group F) who HCT continued.</span></span></span></span>
基金supported in part by funds from the breast cancer advocacy group 100 Voices of Hope, as well as by NIHR01 AR52144, R03 CA238555 (HY) and R01 AR053237 (AGR).NIHR01 AR52144,R03 CA238555 (HY) and R01 AR053237 (AGR)100 Voices of Hope (HY)。
文摘Osteocytes are mechanosensitive bone cells, but little is known about their effects on tumor cells in response to mechanical stimulation. We treated breast cancer cells with osteocyte-derived conditioned medium(CM) and fluid flow-treated conditioned medium(FFCM) with 0.25 Pa and 1 Pa shear stress. Notably, CM and FFCM at 0.25 Pa induced the mesenchymal-to-epithelial transition(MET), but FFCM at 1 Pa induced the epithelial-to-mesenchymal transition(EMT). This suggested that the effects of fluid flow on conditioned media depend on flow intensity. Fluorescence resonance energy transfer(FRET)-based evaluation of Src activity and vinculin molecular force showed that osteopontin was involved in EMT and MET switching. A mouse model of tumorinduced osteolysis was tested using dynamic tibia loadings of 1, 2, and 5 N. The low 1 N loading suppressed tumor-induced osteolysis, but this beneficial effect was lost and reversed with loads at 2 and 5 N, respectively. Changing the loading intensities in vivo also led to changes in serum TGFβ levels and the composition of tumor-associated volatile organic compounds in the urine.Collectively, this study demonstrated the critical role of intensity-dependent mechanotransduction and osteopontin in tumorosteocyte communication, indicating that a biophysical factor can tangibly alter the behaviors of tumor cells in the bone microenvironment.
基金This study was supported in part by funds from a breast cancer advocacy group,100 Voices of Hope(H.Y.)the Indiana University Precision Health Initiative(H.N.)NIH R01AR52144(H.Y.),R03CA238555(H.Y.),and R01AR053237(A.R.).
文摘Osteocytes are the most abundant cells in bone,which is a frequent site of breast cancer metastasis.Here,we focused on Wnt signaling and evaluated tumor-osteocyte interactions.In animal experiments,mammary tumor cells were inoculated into the mammary fat pad and tibia.The role of Lrp5-mediated Wnt signaling was examined by overexpressing and silencing Lrp5 in osteocytes and establishing a conditional knockout mouse model.The results revealed that administration of osteocytes or their conditioned medium(CM)inhibited tumor progression and osteolysis.Osteocytes overexpressing Lrp5 or β-catenin displayed strikingly elevated tumor-suppressive activity,accompanied by downregulation of tumor-promoting chemokines and upregulation of apoptosis-inducing and tumor-suppressing proteins such as p53.The antitumor effect was also observed with osteocyte-derived CM that was pretreated with a Wnt-activating compound.Notably,silencing Lrp5 in tumors inhibited tumor progression,while silencing Lrp5 in osteocytes in conditional knockout mice promoted tumor progression.Osteocytes exhibited elevated Lrp5 expression in response to tumor cells,implying that osteocytes protect bone through canonical Wnt signaling.Thus,our results suggest that the Lrp5/β-catenin axis activates tumor-promoting signaling in tumor cells but tumor-suppressive signaling in osteocytes.We envision that osteocytes with Wnt activation potentially offer a novel cell-based therapy for breast cancer and osteolytic bone metastasis.
基金supported in part by funds from the NIH(R01 AR052144 and R03 CA238555 to H.Y.).
文摘Mechanical loading to the bone is known to be beneficial for bone homeostasis and for suppressing tumor-induced osteolysis in the loaded bone.However,whether loading to a weight-bearing hind limb can inhibit distant tumor growth in the brain is unknown.We examined the possibility of bone-to-brain mechanotransduction using a mouse model of a brain tumor by focusing on the response to Lrp5-mediated Wnt signaling and dopamine in tumor cells.The results revealed that loading the tibia with elevated levels of tyrosine hydroxylase,a rate-limiting enzyme in dopamine synthesis,markedly reduced the progression of the brain tumors.The simultaneous application of fluphenazine(FP),an antipsychotic dopamine modulator,enhanced tumor suppression.Dopamine and FP exerted antitumor effects through the dopamine receptors DRD1 and DRD2,respectively.Notably,dopamine downregulated Lrp5 via DRD1 in tumor cells.A cytokine array analysis revealed that the reduction in CCN4 was critical for loading-driven,dopamine-mediated tumor suppression.The silencing of Lrp5 reduced CCN4,and the administration of CCN4 elevated oncogenic genes such as MMP9,Runx2,and Snail.In summary,this study demonstrates that mechanical loading regulates dopaminergic signaling and remotely suppresses brain tumors by inhibiting the Lrp5-CCN4 axis via DRD1,indicating the possibility of developing an adjuvant bone-mediated loading therapy.
文摘Western blotting is a popular technique for examining expression levels of proteins using gel-based electrophoretic fractionation followed by blotting and antibody reactions. Although this is a mature technique, one of the major limitations is the need to prepare an individual electrophoretic gel for each of the protein species to be analyzed. Since most analyses require the detection of multiple protein species, a procedure that allows utilization of a single gel for detecting multiple protein species should significantly save time and resources. In this paper, we developed a novel multiprotein detection device, which enabled simultaneous detection of several proteins species from a single electrophoretic gel. In this device, a protein transfer unit utilized a multi-anode plate that generated a non-uniform voltage profile. This voltage profile enabled uniform transfer regardless of molecular mass of proteins. In vitro experiments using samples, isolated from boneforming osteoblast cells, showed that the expression levels of 5 - 7 different proteins were detectable in the presence and absence of mechanical stimulation that activated genes necessary for bone formation. The result supports the notion that through simultaneous detection of multiple protein species, the described device contributes to reduction in procedural time and sample amounts, as well as a removal of variations among multiple gels.
文摘This study presents the development of an innovative artificial finger-like device that provides position specific mechanical loads at the end of the long bone and induces mechanotransduction in bone. Bone cells such as osteoblasts are the mechanosensitive cells that regulate bone remodelling. When they receive gentle, periodic mechanical loads, new bone formation is promoted. The proposed device is an under-actuated multi-fingered artificial hand with 4 fingers, each having two phalanges. These fingers are connected by mechanical linkages and operated by a worm gearing mechanism. With the help of 3D printing technology, a prototype device was built mostly using plastic materials. The experimental validation results show that the device is capable of generating necessary forces at the desired frequencies, which are suitable for the stimulation of bone cells and the promotion of bone formation. It is recommended that the device be tested in a clinical study for confirming its safety and efficacy with patients.
文摘Blotting is a common technique widely used for molecular analysis in life sciences. The Western blot, in particular, is a process of transferring protein samples from a polyacrylamide gel to a blotting membrane and detecting the levels of specific proteins through reactions with primary and secondary antibodies. The state-of-the-art of Western blotting usually generates one blotting membrane per gel. However, multiple copies of blots are useful in many applications. Two blotting copies from a single protein gel, for instance, can be used for identifying a total amount of proteins of interest as well as its specific subpopulation level such as a phosphorylated isoform. To achieve this multi-blotting operation from a single gel, we modified a blotting procedure and developed a novel blotting device. The device consisted of a multi-anode plate and a microcontroller. It was designed to generate a well-controlled electrophoretic voltage profile, which allowed a quasi-uniform transfer of proteins of any size. The prototype device was built and its operation procedure was described. The experimental results clearly supported the notion that the described device was able to achieve multiple blotting from a single gel and reduce time and cost for protein analysis.
文摘BlotMan is a protein blotting device that allows generating multiple membranes from a single polyacrylamide gel. To transfer all proteins uniformly with the same efficiency regardless of protein size, BlotMan employs pulse-width-modulated (PWM) voltage that applies a higher average voltage to a larger protein species. BlotMan can be controlled not only by its custom-made interface but also by a smart phone via Bluetooth technology. In this study, we examined effects of PWM signals (50%, 60%, and 80% duty cycle) on transfer efficiency and signal intensity in comparison to a constant voltage signal (100% duty cycle). The result revealed that in response to the same average voltage of 150 V, a lower duty cycle with a higher maximum voltage increased transfer efficiency as well as sharpness of transferred proteins. We validated BlotMan’s capability using a chondrosarcoma cell line (SW1353 cells) and a breast cancer cell line (MDA-MB231 cells) in response to antitumor chemical agents. BlotMan successfully generated 5 membranes from a single gel and detected 5 protein species such as c-Src, eukaryotic translation initiation factor 2 alpha (eIF2), phosphorylated eIF2, lamin B, and actin. Collectively, we demonstrated herein that BlotMan reduces an amount of protein samples by generating multiple membranes from a single gel and improving signal intensity with PWM voltage signals.
基金supported by The Biomechanics and Bio-materials Research Center at Indiana University-Purdue University Indianapolis,USA(No.2201-01)The NIH/Eunice Kennedy Shriver NICHD,USA(No.P50HD090215)+2 种基金The NIH/NCI Cancer Center Support Grant,USA(No.P30CA082709)The Tyler Trent Cancer Research Endowment for the Riley Hospital for Children IU-Health,USAThe Indiana University Grand ChallengeePrecision Health Initiative,USA.
文摘Cancer cells tend to develop resistance to chemotherapy and enhance aggressive-ness.A counterintuitive approach is to tame aggressiveness by an agent that acts opposite to chemotherapeutic agents.Based on this strategy,induced tumor-suppressing cells(iTSCs)have been generated from tumor cells and mesenchymal stem cells.Here,we examined the possi-bility of generating iTSCs from lymphocytes by activating PKA signaling for suppressing the pro-gression of osteosarcoma(OS).While lymphocyte-derived CM did not present anti-tumor capabilities,the activation of PKA converted them into iTSCs.Inhibiting PKA conversely gener-ated tumor-promotive secretomes.In a mouse model,PKA-activated CM suppressed tumorinduced bone destruction.Proteomics analysis revealed that moesin(MSN)and calreticulin(Calr),which are highly expressed intracellular proteins in many cancers,were enriched in PKA-activated CM,and they acted as extracellular tumor suppressors through CD44,CD47,and CD91.The study presented a unique option for cancer treatment by generating iTSCs that secret tumor-suppressive proteins such as MSN and Calr.We envision that identifying these tu-mor suppressors and predicting their binding partners such as CD44,which is an FDA-approved oncogenic target to be inhibited,may contribute to developing targeted protein therapy.
文摘Weight-bearing bone is constantly adapting its structure and function to mechanical environments. Loading through routine exercises stimulates bone formation and prevents bone loss, but unloading through bed rest and cast immobilization as well as exposure to weightlessness during spaceflight reduces its mass and strength. In order to elucidate the mechanism underlying unloading-driven bone adaptation, ground-based in vitro and in vivo analyses have been conducted using rotating cell culturing and hindlimb suspension. Focusing on gene expression studies in osteoblasts and hindlimb suspension studies, this minireview introduces our recent understanding on bone homeostasis under weightlessness in space. Most of the existing data indicate that unloading has the opposite effects to loading through common signaling pathways. However, a question remains as to whether any pathway unique to unloading (and not to loading) may exist.
文摘Since the early days of manned spaceflight, hazardous effects of the space environment on living organisms have been disputed. With the continuous manning of the International Space Station, the planned Chinese space station, and renewed interest in returning to the Moon and sending manned flights to Mars, identifying and addressing the potential outcomes of long-term space exposures is critically important.
文摘Understanding the regulatory mechanism that controls the alteration of global gene expression patterns continues to be a challenging task in computational biology. We previously developed an ant algorithm, a biologically-inspired computational technique for microarray data, and predicted putative transcription-factor binding motifs (TFBMs) through mimicking interactive behaviors of natural ants. Here we extended the algorithm into a set of web-based software, Ant Modeler, and applied it to investigate the transcriptional mechanism underlying bone formation. Mechanical loading and administration of bone morphogenic proteins (BMPs) are two known treatments to strengthen bone. We addressed a question: Is there any TFBM that stimulates both "anabolic responses of mechanical loading" and "BMP-mediated osteogenic signaling"? Although there is no significant overlap among genes in the two responses, a comparative model-based analysis suggests that the two independent osteogenic processes employ common TFBMs, such as a stress responsive element and a motif for peroxisome proliferator-activated receptor (PPAR). The post-modeling in vitro analysis using mouse osteoblast cells supported involvements of the predicted TFBMs such as PPAR, Ikaros 3, and LMO2 in response to mechanical loading. Taken together, the results would be useful to derive a set of testable hypotheses and examine the role of specific regulators in complex transcriptional control of bone formation.