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With the rapid advances in biotechnological tools and strategies, microbial cell factory-constructing strategies have been established for the production of value-added compounds. However, optimizing the tradeoff between the biomass, yield, and titer remains a challenge in microbial production. Gene regulation is necessary to optimize and control metabolic fluxes in microorganisms for high-production performance. Various high-throughput genetic engineering tools have been developed for achieving rational gene regulation and genetic perturbation, diversifying the cellular phenotype and enhancing bioproduction performance. In this paper, we review the current high-throughput genetic engineering tools for gene regulation. In particular, technological approaches used in a diverse range of genetic tools for constructing microbial cell factories are introduced, and representative applications of these tools are presented. Finally, the prospects for high-throughput genetic engineering tools for gene regulation are discussed.
Subject(s)
Biotechnology , Metabolic Engineering , Gene Expression Regulation , Biomass , Gene ExpressionABSTRACT
Efficient control over multiple gene expression still presents a major challenge. Synthetic sRNA enables targeted gene expression control in trans without directly modifying the chromosome, but its use to simultaneously target multiple genes can often cause cell growth defects because of the need for additional energy for transcription and lowering of their repression efficiency by limiting the amount of Hfq protein. To address these limitations, we present fusion sRNA (fsRNA) that simultaneously regulates the translation of multiple genes efficiently. It is constructed by linking the mRNA-binding modules for multiple targeted genes in one sRNA scaffold via one-pot generation using overlap extension PCR. The repression capacity of fsRNA was demonstrated by the construction of sRNAs to target four endogenous genes: caiF, hybG, ytfR and minD in Escherichia coli. Their cross-reactivity and the effect on cell growth were also investigated. As practical applications, we applied fsRNA to violacein- and protocatechuic acid-producing strains, resulting in increases of 13% violacein and 81% protocatechuic acid, respectively. The developed fsRNA-mediated multiple gene expression regulation system thus enables rapid and efficient development of optimised cell factories for valuable chemicals without cell growth defects and limiting cellular resources.Key points⢠Synthetic fusion sRNA (fsRNA)-based system was constructed for the repression of multiple target genes.⢠fsRNA repressed multiple genes by only expressing a single sRNA while minimising the cellular burden.⢠The application of fsRNA showed the increased production titers of violacein (13%) and protocatechuic acid (81%).
Subject(s)
Escherichia coli Proteins , RNA, Small Untranslated , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Gene Expression Regulation, Bacterial , Host Factor 1 Protein/genetics , Molecular Chaperones/metabolism , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , RNA, Small Untranslated/geneticsABSTRACT
Excessive stimulation of the quinolinic acid induces neuronal cell death and is implicated in developing several neurodegenerative diseases. This study investigated whether a Wnt5a antagonist plays a neuroprotective role by regulating the Wnt pathway, activating cellular signaling mechanisms, including MAP kinase and ERK, and acting on the antiapoptotic and the proapoptotic genes in N18D3 neural cells. The cells were pretreated with a Wnt5a antagonist Box5, for one hour and then exposed to quinolinic acid (QUIN), an NMDA receptor agonist for 24 hours. An MTT assay and DAPI staining were used to evaluate cell viability and apoptosis, respectively, demonstrating that Box5 protected the cells from apoptotic death. In addition, a gene expression analysis revealed that Box5 prevented the QUIN-induced expression of the pro-apoptotic genes, BAD and BAX, and increased that of the anti-apoptotic genes, Bcl-xL, BCL2, and BCLW. Further examination of potential cell signaling candidates involved in this neuroprotective effect showed that the immunoreactivity of ERK was significantly increased in the cells treated with Box5. These results suggest that the neuroprotective mechanism of Box5 against QUIN-induced excitotoxic cell death involves the regulation of ERK and modulation of cell survival and death genes through decreasing the Wnt pathway, specifically Wnt5a.
Subject(s)
Neuroprotective Agents , Wnt Signaling Pathway , Apoptosis , Cell Death , Neuroprotective Agents/pharmacology , Quinolinic Acid/toxicity , Animals , Mice , Cell Line , Wnt Signaling Pathway/drug effectsABSTRACT
Localized surface plasmon resonance (LSPR)-based biosensors have recently garnered increasing attention due to their potential to allow label-free, portable, low-cost, and real-time monitoring of diverse analytes. Recent developments in this technology have focused on biochemical markers in clinical and environmental settings coupled with advances in nanostructure technology. Therefore, this review focuses on the recent advances in LSPR-based biosensor technology for the detection of diverse chemicals and biomolecules. Moreover, we also provide recent examples of sensing strategies based on diverse nanostructure platforms, in addition to their advantages and limitations. Finally, this review discusses potential strategies for the development of biosensors with enhanced sensing performance.
ABSTRACT
To overcome environmental problems caused by the use of fossil resources, microbial cell factories have become a promising technique for the sustainable and eco-friendly development of valuable products from renewable resources. Constructing microbial cell factories with high titers, yields, and productivity requires a balance between growth and production; to this end, tuning gene expression and regulation is necessary to optimise and precisely control complicated metabolic fluxes. In this article, we review the current trends and advances in tuning gene expression and regulation and consider their engineering at each of the three stages of gene regulation: genomic, mRNA, and protein. In particular, the technological approaches utilised in a diverse range of genetic-engineering-based tools for the construction of microbial cell factories are reviewed and representative applications of these strategies are presented. Finally, the prospects for strategies and systems for tuning gene expression and regulation are discussed.
Subject(s)
Metabolic Engineering , Synthetic BiologyABSTRACT
Manipulation of both pore diameters and heights of two-dimensional periodic porous polymer films is important to extensively control their characteristics. However, except for using different sized colloid templates in replication methods, an effective method that tunes these factors has rarely been reported. We found that both parameters are controllable by adjusting the flow behaviors of polystyrene colloids and curing resin precursors during the preparation of phenolic resin and poly(dimethylsiloxane) periodic porous films by embedding their precursors into colloidal crystal monolayers. We adjust the flow behaviors by either varying film preparation temperatures (≥glass transition temperature of polystyrene) or using the precursors mixed with different amounts of solvents that renders the colloids viscous. Consequently, the pore diameters and film heights change by 36-56 and 56-84%, respectively. Such modulation results in the change in height to diameter ratios and the areal fractions of resins at air-film interfaces, thereby significantly changing the water contact angles on these surfaces and their photonic characteristics. This straightforward method does not require additional steps, differently sized colloids, or different amounts of precursors for these parameter controls.
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BACKGROUND: Mood disorders, depression, and loneliness are established risk factors for thrombotic occlusions. Social relationships in general, and marital status in particular may play a role in predicting cardiovascular outcomes and survival after ST-segment elevation myocardial infarction (STEMI), but the evidence is inconclusive especially in Asians. METHODS: The Korean patients presented with STEMI (n=980) constituted married (n=780); or widowed, divorced, or single (WDS, n=200) groups. After the matching for age, and gender, the groups were matched 1:1, with each group containing 172 patients. Clinical characteristics and STEMI prognosis such as major adverse cardiovascular events (MACE) and death at 1year, in married versus WDS patients were collected, and retrospectively analyzed. RESULTS: Overall, the total of 70 non-fatal MACE and 51 deaths occurred. At 1-year, the WDS patients exhibited significantly more MACE (44 vs.26; p=0.016), deaths (32 vs. 19; p=0.049) and shorter time to MACE occurrence (p=0.018), compared to the married patients. There were no differences in revascularization, cerebral infarction, cerebral bleeding, major bleeding, coronary artery bypass graft, early mortality and the overall survival between groups. CONCLUSION: Marital status may be linked to 1-year MACE including survival following STEMI, while being married may improve vascular outcomes compared to WDS in Korean patients. Further larger cohort or/and uniformed national registry studies are required to validate these data, and expand the evidence beyond East Asians.
Subject(s)
Marital Status , ST Elevation Myocardial Infarction/therapy , Aged , Aged, 80 and over , Female , Humans , Male , Mental Health , Middle Aged , Pilot Projects , Republic of Korea/epidemiology , Retrospective Studies , ST Elevation Myocardial Infarction/diagnosis , ST Elevation Myocardial Infarction/mortality , ST Elevation Myocardial Infarction/psychology , Time Factors , Treatment OutcomeABSTRACT
Bone marrow-derived mesenchymal stem cells (BM-MSCs), which are characterized by multipotency and self-renewal, are responsible for tissue regeneration and repair. We have previously reported in adipose tissue-derived MSCs that only Wnt5a is enhanced at neurogenic differentiation, and the mechanism of differentiation is dependent on the Wnt5a/JNK pathway; however, the role of Wnt/MAPK pathway is yet to be investigated in neurogenic differentiation in BM-MSCs. We compared the transcriptional expression of Wnt in neurogenic induced-hBM-MSCs (NI-hBM-MSCs) with that in primary hBM-MSCs, using RT-PCR, qPCR, and western blotting. Although the expression of Wnt1 and Wnt2 was unchanged, the expression of Wnt4, Wnt5a, and Wnt11 increased after neurogenic differentiation. In addition, only the expression of frizzled class receptor (Fzd) 3 gene was increased, but not of most of the Fzds and Wnt ligands in NI-hBM-MSCs. Interestingly, Wnt4, Wnt5a, and Wnt11 gene expressions significantly increased in NI-hBM-MSCs by qPCR. In addition, the protein expression level of Wnt4 and Wnt5a, but not Wnt3, increased after neurogenic induction. Furthermore, the expressions of phosphorylated-GSK-3ß, ERK1/2, and PKC decreased; however, JNK was activated after neurogenic differentiation. Thus, non-canonical Wnts, i.e., Wnt4, Wnt5a, and Wnt11, regulate neurogenic differentiation through Fzd3 activation and the increase in downstream targets of JNK, which is one of the non-canonical pathways, in hBM-MSCs.
Subject(s)
Cell Differentiation , Mesenchymal Stem Cells/metabolism , Neurons/metabolism , Wnt Proteins/metabolism , Cells, Cultured , Frizzled Receptors/metabolism , Gene Expression , Humans , Wnt Signaling Pathway , Wnt-5a Protein/metabolism , Wnt4 Protein/metabolismABSTRACT
Excessive noise, ototoxic drugs, infections, autoimmune diseases, and aging can cause loss of spiral ganglion neurons, leading to permanent sensorineural hearing loss in mammals. Stem cells have been confirmed to be able to differentiate into spiral ganglion neurons. Little has been reported on adipose tissue-derived stem cells (ADSCs) for repair of injured spiral ganglion neurons. In this study, we hypothesized that transplantation of neural induced-human ADSCs (NI-hADSCs) can repair the injured spiral ganglion neurons in guinea pigs with neomycin-induced sensorineural hearing loss. NI-hADSCs were induced with culture medium containing basic fibroblast growth factor and forskolin and then injected to the injured cochleae. Guinea pigs that received injection of Hanks' balanced salt solution into the cochleae were used as controls. Hematoxylin-eosin staining showed that at 8 weeks after cell transplantation, the number of surviving spiral ganglion neurons in the cell transplantation group was significantly increased than that in the control group. Also at 8 weeks after cell transplantation, immunohistochemical staining showed that a greater number of NI-hADSCs in the spiral ganglions were detected in the cell transplantation group than in the control group, and these NI-hADSCs expressed neuronal markers neurofilament protein and microtubule-associated protein 2. Within 8 weeks after cell transplantation, the guinea pigs in the cell transplantation group had a gradually decreased auditory brainstem response threshold, while those in the control group had almost no response to 80 dB of clicks or pure tone burst. These findings suggest that a large amount of NI-hADSCs migrated to the spiral ganglions, survived for a period of time, repaired the injured spiral ganglion cells, and thereby contributed to the recovery of sensorineural hearing loss in guinea pigs.
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BACKGROUND: Clopidogrel and aspirin combination remains a cornerstone for modern dual antiplatelet therapy (DAPT) following coronary stenting. Although monitoring is not currently recommended, certain high-risk cohorts may benefit from tailoring antiplatelet options to reduce thrombotic or/and hemorrhagic risks. Patients with diminished estimated glomerular filtration rate (eGFR) are prone to both vascular occlusions and bleeding events in whom monitoring may be especially advantageous. We compared the residual platelet reactivity assessed by 3 conventional tests during the maintenance antiplatelet therapy dependent on eGFR. METHODS: Post-stenting patients (n = 701) receiving aspirin 100 mg/daily and clopidogrel 75 mg/daily were prospectively enrolled in the cross-sectional single-center study. Patients were dichotomized into 5 groups: eGFR >90, 60-89, 30-59, <30 ml/min/1.73 m2, and dialysis. Platelet reactivity by VerifyNow™, light transmittance aggregometry (LTA), and Multiplate analyzer by multiple electrode platelet aggregometry (MEA) assays together with eGFR calculations were done simultaneously at 1 month after coronary stenting. RESULTS: VerifyNow assay distinguished residual platelet reactivity dependent on eGFR deterioration (191 ± 72 vs. 216 ± 78 vs. 248 ± 80 vs. 264 ± 70 vs. 317 ± 96 PRU; p < 0.001). In contrast, LTA (34.3 ± 18.1 vs. 34.7 ± 18.1 vs. 38.0 ± 16.6 vs. 33.0 ± 17.3 vs. 34.1 ± 29.3%; p = 0.242), or MEA (37.2 ± 19.6 vs. 33.8 ± 18.4 vs. 38.6 ± 21.4 vs. 36.5 ± 20.5 vs. 38.3 ± 28.3 AU/min; p = 0.086) failed to triage platelet reactivity in renal patients. Agreement among assays to identify patients with impaired platelet reactivity and eGFR during antiplatelet therapy was low. The multivariable regression analyses confirmed the VerifyNow advantage, since the differences in the platelet reactivity were highly significant for all renal impairment (RI) groups. In contrast, LTA did not distinguish RI patients, and for the MEA, only RI5 (dialysis) cohort exhibit borderline significant decline of residual platelet reactivity. CONCLUSION: Among 3 assays, VerifyNow was capable to reliably triage residual platelet reactivity in post-stenting DAPT patients dependent on the gradual decline of eGFR during therapy with clopidogrel and aspirin. These data should be confirmed in a large validation longitudinal trial, and may justify future platelet activity monitoring for potential regimen/dose adjustment in high-risk patients. The clinical implications of these data are still unclear, but may give an indication as to whether or when DAPT dose adjustment will become a reality.
Subject(s)
Kidney Diseases/blood , Kidney Diseases/drug therapy , Platelet Aggregation Inhibitors/therapeutic use , Platelet Function Tests , Aged , Aspirin/adverse effects , Aspirin/therapeutic use , Clopidogrel , Cross-Sectional Studies , Female , Glomerular Filtration Rate , Hemorrhage/prevention & control , Humans , Male , Middle Aged , Platelet Aggregation/drug effects , Prospective Studies , Thrombosis/prevention & control , Ticlopidine/administration & dosage , Ticlopidine/analogs & derivatives , Ticlopidine/therapeutic useABSTRACT
This study aimed to investigate the effect of pituitary adenylate cyclase-activating peptide (PACAP) on the pacemaker activity of interstitial cells of Cajal (ICC) in mouse colon and to identify the underlying mechanisms of PACAP action. Spontaneous pacemaker activity of colonic ICC and the effects of PACAP were studied using electrophysiological recordings. Exogenously applied PACAP induced hyperpolarization of the cell membrane and inhibited pacemaker frequency in a dose-dependent manner (from 0.1 nM to 100 nM). To investigate cyclic AMP (cAMP) involvement in the effects of PACAP on ICC, SQ-22536 (an inhibitor of adenylate cyclase) and cell-permeable 8-bromo-cAMP were used. SQ-22536 decreased the frequency of pacemaker potentials, and cell-permeable 8-bromo-cAMP increased the frequency of pacemaker potentials. The effects of SQ-22536 on pacemaker potential frequency and membrane hyperpolarization were rescued by co-treatment with glibenclamide (an ATP-sensitive K(+) channel blocker). However, neither N (G)-nitro-L-arginine methyl ester (L-NAME, a competitive inhibitor of NO synthase) nor 1H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ, an inhibitor of guanylate cyclase) had any effect on PACAP-induced activity. In conclusion, this study describes the effects of PACAP on ICC in the mouse colon. PACAP inhibited the pacemaker activity of ICC by acting through ATP-sensitive K(+) channels. These results provide evidence of a physiological role for PACAP in regulating gastrointestinal (GI) motility through the modulation of ICC activity.
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OBJECTIVES: In mammals, cochlear hair cell loss is irreversible and may result in a permanent sensorineural hearing loss. Secondary to this hair cell loss, a progressive loss of spiral ganglion neurons (SGNs) is presented. In this study, we have investigated the effects of neural-induced human mesenchymal stem cells (NI-hMSCs) from human bone marrow on sensory neuronal regeneration from neomycin treated deafened guinea pig cochleae. METHODS: HMSCs were isolated from the bone marrow which was obtained from the mastoid process during mastoidectomy for ear surgery. Following neural induction with basic fibroblast growth factor and forskolin, we studied the several neural marker and performed electrophysiological analysis. NI-hMSCs were transplanted into the neomycin treated deafened guinea pig cochlea. Engraftment of NI-hMSCs was evaluated immunohistologically at 8 weeks after transplantation. RESULTS: Following neural differentiation, hMSCs expressed high levels of neural markers, ionic channel markers, which are important in neural function, and tetrodotoxin-sensitive voltage-dependent sodium currents. After transplantation into the scala tympani of damaged cochlea, NI-hMSCs-injected animals exhibited a significant increase in the number of SGNs compared to Hanks balanced salt solution-injected animals. Transplanted NI-hMSCs were found within the perilymphatic space, the organ of Corti, along the cochlear nerve fibers, and in the spiral ganglion. Furthermore, the grafted NI-hMSCs migrated into the spiral ganglion where they expressed the neuron-specific marker, NeuN. CONCLUSION: The results show the potential of NI-hMSCs to give rise to replace the lost cochlear cells in hearing loss mammals.
ABSTRACT
Stem cells are a powerful resource for cell-based transplantation therapies, but understanding of stem cell differentiation at the molecular level is not clear yet. We hypothesized that the Wnt pathway controls stem cell maintenance and neural differentiation. We have characterized the transcriptional expression of Wnt during the neural differentiation of hADSCs. After neural induction, the expressions of Wnt2, Wnt4, and Wnt11 were decreased, but the expression of Wnt5a was increased compared with primary hADSCs in RT-PCR analysis. In addition, the expression levels of most Fzds and LRP5/6 ligand were decreased, but not Fzd3 and Fzd5. Furthermore, Dvl1 and RYK expression levels were downregulated in NI-hADSCs. There were no changes in the expression of ß-catenin and GSK3ß. Interestingly, Wnt5a expression was highly increased in NI-hADSCs by real time RT-PCR analysis and western blot. Wnt5a level was upregulated after neural differentiation and Wnt3, Dvl2, and Naked1 levels were downregulated. Finally, we found that the JNK expression was increased after neural induction and ERK level was decreased. Thus, this study shows for the first time how a single Wnt5a ligand can activate the neural differentiation pathway through the activation of Wnt5a/JNK pathway by binding Fzd3 and Fzd5 and directing Axin/GSK-3ß in hADSCs.
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We investigated the presence of ß3-adrenoceptor and its functional effects on pacemaker potentials in colonic interstitial cells of Cajal (ICCs) from mice. The whole-cell patch clamp technique was used to record pacemaker potentials in cultured ICCs and reverse transcription polymerase chain reaction (RT-PCR) was performed to detect the mRNA transcript levels ß-adrenoceptors. The ß3-adrenoceptor agonist, BRL37344, reduced the frequency of pacemaker potentials in a concentration-dependent manner. The inhibitory effects of BRL37344 were blocked by the pretreatment of propranolol, a nonspecific ß-adrenoceptor antagonist, but not by the selective ß1-adrenoceptor antagonist atenolol and the selective ß2-adrenoceptor antagonist butoxamine. ß3-adrenoceptor antagonists SR59230A and L748337 blocked the inhibitory effects of BRL37344. RT-PCR revealed mRNA transcripts of ß1- and ß3-adrenoceptor, but not ß2-adrenoceptor, in c-kit- and Ano-1-positive colonic ICCs. The K(+) channel blockers tetraethylammonium, apamin, and glibenclamide did not block the effects of BRL37344. N(ω)-Nitro-l-arginine methyl ester hydrochloride (L-NAME), an NO synthase inhibitor, and chelerythrine, a protein kinase C inhibitor, also did not block the effects of BRL37344. Noradrenaline mimicked the effects of BRL37344 in colonic ICCs. However, the inhibitory effects of noradrenaline on pacemaker potentials were blocked only by pretreatment with atenolol but not by butoxamine, SR59230A, or L748337. In small intestinal ICCs, BRL37344 had no effect on pacemaker potentials and mRNA transcripts of ß1-and ß2-adrenoceptor, but not ß3-adrenoceptor were detected. These results suggest that ß3-adrenoceptors are present in colonic ICCs and may play a role in regulating gastrointestinal motility by the inhibition of pacemaker potentials.
Subject(s)
Biological Clocks/physiology , Colon/cytology , Interstitial Cells of Cajal/physiology , Receptors, Adrenergic, beta-3/physiology , Adrenergic beta-Agonists/pharmacology , Adrenergic beta-Antagonists/pharmacology , Aminophenols/pharmacology , Animals , Atenolol/pharmacology , Benzophenanthridines/pharmacology , Biological Clocks/drug effects , Butoxamine/pharmacology , Calcium/metabolism , Colon/drug effects , Dose-Response Relationship, Drug , Ethanolamines/antagonists & inhibitors , Ethanolamines/pharmacology , Female , Interstitial Cells of Cajal/drug effects , Interstitial Cells of Cajal/metabolism , Intestine, Small/drug effects , Intestine, Small/metabolism , Intestine, Small/physiology , Male , Membrane Potentials/drug effects , Membrane Potentials/physiology , Mice , NG-Nitroarginine Methyl Ester/pharmacology , Norepinephrine/pharmacology , Potassium Channel Blockers/pharmacology , Propanolamines/pharmacology , Propranolol/pharmacology , Receptors, Adrenergic, beta-3/biosynthesis , Sulfonamides/pharmacologyABSTRACT
PURPOSE: To investigate whether carnosine can increase retinal ganglion cell (RGC) survival in ischemic mouse retina. METHODS: Retinal ischemia was induced by constant elevation of intraocular pressure (100-110 mmHg) for 60 min in C57BL/6 J mice pretreated with carnosine (1000 mg/kg) or saline. Hypoxia inducing factor-1 alpha (HIF-1α), glial fibrillary acidic protein (GFAP), and dynamin-related protein-1 (Drp-1) expressions were assessed at 6, 12, and 24 h after retinal ischemia. Bax and Bcl-2 expressions were also analyzed at 12 h after retinal ischemia. RGC survival was assessed by retrograde FluoroGold labeling at 2 weeks after retinal ischemia. RESULTS: The expression of HIF-1α, GFAP, and Drp-1 was increased within 24 h after ischemic injury. Carnosine treatment effectively decreased the elevated expression of HIF-1α, GFAP, and Drp-1 in ischemic mouse retina. In ischemic retina treated with carnosine, Bax expression was decreased, whereas Bcl-2 expression was increased compared with ischemic retina treated with saline. Carnosine treatment also protected against RGC loss in ischemia mouse retina. CONCLUSIONS: Our findings showed that carnosine treatment significantly decreased RGC loss through decreased expression of HIF-1α, GFAP, Drp-1, and Bax, and increased expression of Bcl-2 in ischemic mouse retina. We suggest that carnosine can be an effective endogenous neuroprotective molecule in the prevention of RGC loss in ischemic retina.
Subject(s)
Carnosine/therapeutic use , Neuroprotective Agents/therapeutic use , Reperfusion Injury/drug therapy , Retinal Diseases/drug therapy , Retinal Ganglion Cells/drug effects , Animals , Biomarkers/metabolism , Blotting, Western , Carnosine/administration & dosage , Cell Count , Cell Survival/drug effects , Dynamins/metabolism , Fluorescent Antibody Technique, Indirect , Glial Fibrillary Acidic Protein , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Injections, Intraperitoneal , Mice , Mice, Inbred C57BL , Nerve Tissue Proteins/metabolism , Neuroprotective Agents/administration & dosage , Proto-Oncogene Proteins c-bcl-2/metabolism , Retinal Diseases/metabolism , Retinal Ganglion Cells/metabolism , bcl-2-Associated X Protein/metabolismABSTRACT
OBJECTIVE: The purpose of this study was to establish a minimally invasive and reproducible protocol for estimating the gastrointestinal (GI) transit time in mice using barium and radiopaque markers. MATERIALS AND METHODS: Twenty 5- to 6-week-old Balb/C female mice weighing 19-21 g were used. The animals were divided into three groups: two groups that received loperamide and a control group. The control group (n = 10) animals were administered physiological saline (1.5 mL/kg) orally. The loperamide group I (n = 10) and group II (n = 10) animals were administered 5 mg/kg and 10 mg/kg loperamide orally, respectively. Thirty minutes after receiving the saline or loperamide, the mice was administered 80 µL of barium solution and six iron balls (0.5 mm) via the mouth and the upper esophagus by gavage, respectively. Afterwards, the mice were continuously monitored with fluoroscopic imaging in order to evaluate the swallowing of the barium solution and markers. Serial fluoroscopic images were obtained at 5- or 10-min intervals until all markers had been excreted from the anal canal. For analysis, the GI transit times were subdivided into intestinal transit times (ITTs) and colon transit times (CTTs). RESULTS: The mean ITT was significantly longer in the loperamide groups than in the control group (p < 0.05). The mean ITT in loperamide group II (174.5 ± 32.3) was significantly longer than in loperamide group I (133.2 ± 24.2 minute) (p < 0.05). The mean CTT was significantly longer in loperamide group II than in the control group (p < 0.05). Also, no animal succumbed to death after the experimental procedure. CONCLUSION: The protocol for our study using radiopaque markers and barium is reproducible and minimally invasive in determining the GI transit time of the mouse model.
Subject(s)
Gastrointestinal Transit/physiology , Analysis of Variance , Animals , Barium Sulfate/pharmacology , Contrast Media/administration & dosage , Female , Fluoroscopy , Iron , Loperamide/administration & dosage , Mice , Mice, Inbred BALB C , Microscopy, Electron, Scanning , Prostheses and Implants , Reproducibility of Results , Sodium Chloride/administration & dosage , Surface PropertiesABSTRACT
Interstitial cells of Cajal (ICCs) from the urinary bladder regulate detrusor smooth muscle activities. We cultured ICCs from the urinary bladder of mice and performed patch clamp and intracellular Ca(2+) ([Ca(2+)]i) imaging to investigate whether cultured ICCs can be a valuable tool for cellular functional studies. The cultured ICCs displayed two types of spontaneous electrical activities which are similar to those recorded in intact bladder tissues. Spontaneous electrical activities of cultured ICCs were nifedipine-sensitive. Carbachol and ATP, both excitatory neurotransmitters in the urinary bladder, depolarized the membrane and increased the frequency of spike potentials. Carbachol increased [Ca(2+)]i oscillations and basal Ca(2+) levels, which were blocked by atropine. These results suggest that cultured ICCs from the urinary bladder retain rhythmic phenotypes similar to the spontaneous electrical activities recorded from the intact urinary bladder. Therefore, we suggest that cultured ICCs from the urinary bladder may be useful for cellular and molecular studies of ICCs.
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BACKGROUND AND OBJECTIVES: The aim of this study was to confirm the predictive cut-off values for P2Y12 reaction units (PRU) and aspirin reaction units (ARU) and to evaluate the clinical impact of VerifyNow® assays. SUBJECTS AND METHODS: From November 2007 to October 2009, 186 eligible patients were prospectively recruited. Post-treatment platelet reactivity was measured by VerifyNow® assays within 12 to 24 hours after intervention, followed by standard dual maintenance dose therapy for 1 year. All patients had scheduled clinical follow-ups at 1, 3, 6, and 12 months. RESULTS: The rate of low responders to clopidogrel, aspirin, and both drugs were 41.4%, 10.2%, and 3.8%, respectively. The predictive factors for low responsiveness to clopidogrel (PRU ≥240) were female sex, age, and non-use of cilostazol medication in our univariate analysis and age ≥65 years and non-use cilostazol in the multivariate analysis. The predictors of low responsiveness to aspirin (ARU ≥550) were male sex and age in both univariate and multivariate analyses. There was no significant difference in the clinical event rate with a cut-off value of PRU ≥240 or ARU ≥550 for 30 days and 1-year (p>0.05). CONCLUSION: Hyporesponsiveness to antiplatelet agents (namely aspirin and clopidogrel) was identified in about half of the patients. The cut-off point of PRU ≥240 or ARU ≥550 did not confer predictive value for 30-day or 1-year clinical event rates in patients who had undergone coronary intervention with drug-eluting stents.
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BACKGROUND: The aim of this study was to evaluate myocardial function in patients with non-hypertensive metabolic syndrome. METHODS: We selected metabolic syndrome patients (n = 42) without evidence of hypertension and compared them to age-matched control individuals (n = 20). All patients were evaluated by two-dimensional and tissue Doppler echocardiography including tissue Doppler derived strain and strain rate measurements. RESULTS: There were no significant differences between the two groups in mitral E and A inflow velocities or the E/A ratio. However, systolic and early diastolic myocardial velocities, and strain rate were significantly lower in patients with metabolic syndrome than in the control group (all p < 0.05). Multiple stepwise regression analyses revealed that age, waist circumference, and systolic blood pressure were independently associated with peak systolic myocardial velocity. CONCLUSION: These results indicate that metabolic syndrome patients without hypertension may have decrease of myocardial systolic and early diastolic velocities on tissue Doppler imaging, even if they appear to have normal systolic and diastolic function on conventional echocardiography.
ABSTRACT
Sphingosine-1-phosphate (S1P) is emerging as a new class of second messenger involved in cellular proliferation, differentiation, and apoptosis and is implicated in diverse physiological functions. Despite many studies on the biological functions of S1P, however, little is known about its role in neuronal differentiation. By use of reverse transcription-polymerase chain reaction and immunostaining, this study aimed to explore whether S1P can differentiate neuroblastoma cells into neural cells. After incubation with 1 uM or 10 uM S1P, the number of neurite-bearing cells increased. Furthermore, the neuroblastoma cells revealed immunoreactivity for neural-specific markers such as GAP43, NFH, and SYP by immunostaining. The expression of NFH, MAP2, SYP, NeuroD1, and SYT mRNA, which is specific for neurons, was increased as shown by RT-PCR studies. The results of this study suggest that that S1P can induce neuronal differentiation and may be a good candidate for the treatment of neurodegenerative diseases.