High-throughput genetic engineering tools for regulating gene expression in a microbial cell factory.

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.

Neuroprotective Effects of a Wnt Antagonist in Quinolinic Acid-Induced Excitotoxicity in N18D3 Cells.

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.

Synthetic fused sRNA for the simultaneous repression of multiple genes.

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%).

Biosensing Applications Using Nanostructure-Based Localized Surface Plasmon Resonance Sensors.

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.

Flow Behaviors of Polymer Colloids and Curing Resins Affect Pore Diameters and Heights of Periodic Porous Polymer Films to Direct Their Surface and Optical Characteristics.

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.

Pituitary Adenylate Cyclase-activating Polypeptide Inhibits Pacemaker Activity of Colonic Interstitial Cells of Cajal.

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.

Spontaneous electrical activity of cultured interstitial cells of cajal from mouse urinary bladder.

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.

Transplantation of neural differentiated human mesenchymal stem cells into the cochlea of an auditory-neuropathy guinea pig model.

The aim of this study was to determine the effects of transplanted neural differentiated human mesenchymal stem cells (hMSCs) in a guinea pig model of auditory neuropathy. In this study, hMSCs were pretreated with a neural-induction protocol and transplanted into the scala tympani of the guinea pig cochlea 7 days after ouabain injury. A control model was made by injection of Hanks balanced salt solution alone into the scala tympani of the guinea pig cochlea 7 days after ouabain injury. We established the auditory neuropathy guinea pig model using 1 mM ouabain application to the round window niche. After application of ouabain to the round window niche, degeneration of most spiral ganglion neurons (SGNs) without the loss of hair cells within the organ of Corti and increasing the auditory brain responses (ABR) threshold were found. After transplantation of neural differentiated hMSCs, the number of SGNs was increased, and some of the SGNs expressed immunoreactivity with human nuclear antibody under confocal laser scanning microscopy. ABR results showed mild hearing recovery after transplantation. Based on an auditory neuropathy animal model, these findings suggest that it may be possible to replace degenerated SGNs by grafting stem cells into the scala tympani.

Recent advances in tuning the expression and regulation of genes for constructing microbial cell factories.

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.

Receptor tyrosine and MAP kinase are involved in effects of H(2)O(2) on interstitial cells of Cajal in murine intestine.

Hydrogen peroxide (H(2)O(2)) is involved in intestinal motility through changes of smooth muscle activity. However, there is no report as to the modulatory effects of H(2)O(2) on interstitial cells of Cajal (ICC). We investigated the H(2)O(2) effects and signal transductions to determine whether the intestinal motility can be modulated through ICC. We performed whole-cell patch clamp in cultured ICC from murine intestine and molecular analyses. H(2)O(2) hyperpolarized the membrane and inhibited pacemaker currents. These effects were inhibited by glibenclamide, an inhibitor of ATP-sensitive K+ (K(ATP)) channels. The free-radical scavenger catalase inhibited the H(2)O(2)-induced effects. MAFP and AACOCF3 (a cytosolic phospholipase A2 inhibitors) or SC-560 and NS-398 (a selective COX-1 and 2 inhibitor) or AH6809 (an EP2 receptor antagonist) inhibited the H(2)O(2)-induced effects. PD98059 (a mitogen activated/ERK-activating protein kinase inhibitor) inhibited the H(2)O(2)-induced effects, though SB-203580 (a p38 MAPK inhibitor) or a JNK inhibitor did not affect. H(2)O(2)-induced effects could not be inhibited by LY-294002 (an inhibitor of PI3-kinases), calphostin C (a protein kinase C inhibitor) or SQ-22536 (an adenylate cyclase inhibitor). Adenoviral infection analysis revealed H2O2 stimulated tyrosine kinase activity and AG 1478 (an antagonist of epidermal growth factor receptor tyrosine kinase) inhibited the H(2)O(2)-induced effects. These results suggest H(2)O(2) can modulate ICC pacemaker activity and this occur by the activation of K(ATP) channels through PGE(2) production via receptor tyrosine kinase-dependent MAP kinase activation.

Functional neural differentiation of human adipose tissue-derived stem cells using bFGF and forskolin.

BACKGROUND: Adult mesenchymal stem cells (MSCs) derived from adipose tissue have the capacity to differentiate into mesenchymal as well as endodermal and ectodermal cell lineage in vitro. We characterized the multipotent ability of human adipose tissue-derived stem cells (hADSCs) as MSCs and investigated the neural differentiation potential of these cells. RESULTS: Human ADSCs from earlobe fat maintained self-renewing capacity and differentiated into adipocytes, osteoblasts, or chondrocytes under specific culture conditions. Following neural induction with bFGF and forskolin, hADSCs were differentiated into various types of neural cells including neurons and glia in vitro. In neural differentiated-hADSCs (NI-hADSCs), the immunoreactivities for neural stem cell marker (nestin), neuronal markers (Tuj1, MAP2, NFL, NFM, NFH, NSE, and NeuN), astrocyte marker (GFAP), and oligodendrocyte marker (CNPase) were significantly increased than in the primary hADSCs. RT-PCR analysis demonstrated that the mRNA levels encoding for ABCG2, nestin, Tuj1, MAP2, NFL, NFM, NSE, GAP43, SNAP25, GFAP, and CNPase were also highly increased in NI-hADSCs. Moreover, NI-hADSCs acquired neuron-like functions characterized by the display of voltage-dependent tetrodotoxin (TTX)-sensitive sodium currents, outward potassium currents, and prominent negative resting membrane potentials under whole-cell patch clamp recordings. Further examination by RT-PCR showed that NI-hADSCs expressed high level of ionic channel genes for sodium (SCN5A), potassium (MaxiK, Kv4.2, and EAG2), and calcium channels (CACNA1C and CACNA1G), which were expressed constitutively in the primary hADSCs. In addition, we demonstrated that Kv4.3 and Eag1, potassium channel genes, and NE-Na, a TTX-sensitive sodium channel gene, were highly induced following neural differentiation. CONCLUSIONS: These combined results indicate that hADSCs have the same self-renewing capacity and multipotency as stem cells, and can be differentiated into functional neurons using bFGF and forskolin.

Endothelial function in lacunar infarction: a comparison of lacunar infarction, cerebral atherosclerosis and control group.

BACKGROUND: There are conflicting evidences on endothelial function in lacunar infarction. This may be attributed to the effects of risk factors on the vascular smooth muscle. To test endothelial function only in patients with lacunar infarction, we evaluated the endothelium-dependent and -independent vasodilatation of the brachial artery. METHODS: We enrolled consecutive patients with lacunar infarction defined by clinical characteristics and MRI findings. The control group included age- and sex-matched patients with hypertension who do not have any history of clinical stroke, coronary artery disease or peripheral vascular disease. Endothelial function was evaluated using flow-mediated dilatation (FMD) and nitrogen-mediated dilatation (NMD) of the brachial artery. FMD and NMD were examined by an experienced vascular sonographer using a high-resolution ultrasound. Intracranial stenosis was defined as flow gap or >50% reduction in vessel diameter on MRA. RESULTS: FMD was 6.6 +/- 4.5% in the lacunar infarction group and 12.2 +/- 4.6% in the control group (p = 0.000). NMD was 14.3 +/- 4.9% in the lacunar infarction group and 13.8 +/- 4.9% in the control group (p = 0.37). FMD in patients with lacunar infarction and intracranial arterial stenosis was 6.4 +/- 3.9%, and FMD in patients with lacunar infarction was only 6.9 +/- 5.5%. In the control group, it was 12.2 +/- 4.6%. CONCLUSION: FMD was low in patients with lacunar infarction. NMD was similar between the lacunar infarction group and the control group. These results are suggestive of pure endothelial dysfunction in lacunar infarction. Endothelial dysfunction was as severe in lacunar infarction as in intracranial arterial stenosis.

Calcitonin gene-related peptide suppresses pacemaker currents by nitric oxide/cGMP-dependent activation of ATP-sensitive K(+) channels in cultured interstitial cells of Cajal from the mouse small intestine.

The effects of calcitonin gene-related peptide (CGRP) on pacemaker currents in cultured interstitial cells of Cajal (ICC) from the mouse small intestine were investigated using the whole-cell patch clamp technique at 30 degrees . Under voltage clamping at a holding potential of -70 mV, CGRP decreased the amplitude and frequency of pacemaker currents and activated outward resting currents. These effects were blocked by intracellular GDPbetaS, a G-protein inhibitor and glibenclamide, a specific ATP-sensitive K(+) channels blocker. During current clamping, CGRP hyperpolarized the membrane and this effect was antagonized by glibenclamide. Pretreatment with SQ-22536 (an adenylate cyclase inhibitor) or naproxen (a cyclooxygenase inhibitor) did not block the CGRP-induced effects, whereas pretreatment with ODQ (a guanylate cyclase inhibitor) or L-NAME (an inhibitor of nitric oxide synthase) did. In conclusion, CGRP inhibits pacemaker currents in ICC by generating nitric oxide via G-protein activation and so activating ATP-sensitive K(+) channels. Nitric oxide- and guanylate cyclase- dependent pathways are involved in these effects.

Tracking of neural stem cells in rats with intracerebral hemorrhage by the use of 3T MRI.

OBJECTIVE: To access the feasibility of clinically available 3T MRI to detect the migration of labeled neural stem cells (NSCs) in intracerebral hemorrhage (ICH) in a rat model. MATERIALS AND METHODS: The ethics committee of our institution approved this study. ICH was induced by the injection of collagenase type IV into the right striatum of ten Sprague-Dawley rats. Human NSCs conjugated with Feridex (super-paramagnetic iron oxide: SPIO) were transplanted into the left striatum one week after ICH induction. MRI was performed on a 3T scanner during the first, second, third, fourth, and sixth weeks post-transplantation. MRI was obtained using coronal T2- and T2*-weighted sequences. Two rats were sacrificed every week after in vivo MRI in order to analyze the histological findings. RESULTS: ICH in the right striatum was detected by MRI one and two weeks after transplantation without migration of the NSCs. There was no migration of the NSCs as seen on the histological findings one week after transplantation. The histological findings two weeks after transplantation showed a small number of NSCs along the corpus callosum. On MRI three weeks after transplantation, there was a hypointense line along the corpus callosum and decreased signal intensity in the right periventricular region. Histological findings three weeks after transplantation confirmed the presence of the hypointense line representing SPIO-labeled NSCs. MRI four and six weeks after transplantation showed a hypointense spot in the right periventricular region. The histological findings four and six weeks after transplantation showed the presence of prominent NSCs in the right periventricular region. CONCLUSION: 3T MRI can detect the migration of NSCs in rats with ICH along the corpus callosum. Therefore, 3T MRI could be feasible for detecting the migration of NSCs in the clinical setting of stem cell therapy.

Impact of marital status on outcomes following ST-segment elevation myocardial infarction.

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.

Effects of (-)-epigallocatechin-3-gallate on the activity of substantia nigra dopaminergic neurons.

Despite many studies on the biological and pharmacological properties of (-)-epigallocatechin-3-gallate (EGCG), an active component of green tea, information on neuronal modulation by EGCG is limited. This study was designed to investigate the effects of EGCG on the electrical activity of rat substantia nigra dopaminergic neurons using whole-cell patch clamp recordings. The spike frequency was increased to 6.33+/-0.23 (p<0.05) and 7.15+/-0.29 (p<0.05) by 5 and 10 microM EGCG, respectively, from the control level of 5.49+/-0.19 spikes/second, respectively (n=18). The resting membrane potential of the cells was decreased to -45.66+/-0.45 and -43.99+/-0.87 (p<0.05), by 5 and 10 microM EGCG, respectively, from -47.82+/-0.57 mV. The amplitude of afterhyperpolarization was decreased to 12.73+/-0.45 (p<0.05) and 11.60+/-0.57 (p<0.05) by 5 and 10 microM EGCG, respectively, from 13.80+/-0.31 mV. The neuronal activity of dopaminergic neurons is closely linked to dopamine release. When neurons switch from a single-spike firing to bursts of action potentials, the release of dopamine increases. The above experimental results suggest that EGCG increases the neuronal activity via inhibition of calcium-dependent potassium currents underlying the afterhyperpolarization, and it could act as a facilitating factor that elicits NMDA-dependent bursts of action potentials like apamin or bicuculline methiodide.

Inhibition of pacemaker currents by nitric oxide via activation of ATP-sensitive K+ channels in cultured interstitial cells of Cajal from the mouse small intestine.

We investigated the role of nitric oxide (NO) in pacemaker activity and signal mechanisms in cultured interstitial cells of Cajal (ICC) of the mouse small intestine using whole cell patch-clamp techniques at 30 degrees C. ICC generated pacemaker potential in the current clamp mode and pacemaker currents at a holding potential of -70 mV. (+/-)-S-nitroso-N-acetylpenicillamine (SNAP; a NO donor) produced membrane hyperpolarization and inhibited the amplitude and frequency of the pacemaker currents, and increased resting currents in the outward direction. These effects were blocked by the use of glibenclamide (an ATP-sensitive K+ channel blocker), but not by the use of 5-hydroxydecanoic acid (a mitochondrial ATP-sensitive K+ channel blocker). Pretreatment with ODQ (a guanylate cyclase inhibitor) almost blocked the NO-induced effects. The use of cell-permeable 8-bromo-cyclic GMP also mimicked the action of SNAP. However, the use of KT-5823 (a protein kinase G inhibitor) did not block the NO-induced effects. Spontaneous [Ca2+]i oscillations in ICC were inhibited by the treatment of SNAP, as seen in recordings of intracellular Ca2+ ([Ca2+]i). These results suggest that NO inhibits pacemaker activity by the activation of ATP-sensitive K+ channels via a cyclic GMP dependent mechanism in ICC, and the activation of ATP-sensitive K+ channels mediates the inhibition of spontaneous [Ca2+]i oscillations.

Non-canonical Wnt mediated neurogenic differentiation of human bone marrow-derived mesenchymal stem cells.

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.

Transplantation of human adipose tissue-derived stem cells for repair of injured spiral ganglion neurons in deaf guinea pigs.

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.

Comparison of Three Tests to Distinguish Platelet Reactivity in Patients with Renal Impairment during Dual Antiplatelet Therapy.

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.