Age-Dependent Effects of Catechol-O-Methyltransferase (COMT) Gene Val158Met Polymorphism on Language Function in Developing Children.

The genetic basis controlling language development remains elusive. Previous studies of the catechol-O-methyltransferase (COMT) Val158Met genotype and cognition have focused on prefrontally guided executive functions involving dopamine. However, COMT may further influence posterior cortical regions implicated in language perception. We investigated whether COMT influences language ability and cortical language processing involving the posterior language regions in 246 children aged 6-10 years. We assessed language ability using a language test and cortical responses recorded during language processing using a word repetition task and functional near-infrared spectroscopy. The COMT genotype had significant effects on language performance and processing. Importantly, Met carriers outperformed Val homozygotes in language ability during the early elementary school years (6-8 years), whereas Val homozygotes exhibited significant language development during the later elementary school years. Both genotype groups exhibited equal language performance at approximately 10 years of age. Val homozygotes exhibited significantly less cortical activation compared with Met carriers during word processing, particularly at older ages. These findings regarding dopamine transmission efficacy may be explained by a hypothetical inverted U-shaped curve. Our findings indicate that the effects of the COMT genotype on language ability and cortical language processing may change in a narrow age window of 6-10 years.

Effects of sex and proficiency in second language processing as revealed by a large-scale fNIRS study of school-aged children.

Previous neuroimaging studies in adults have revealed that first and second languages (L1/L2) share similar neural substrates, and that proficiency is a major determinant of the neural organization of L2 in the lexical-semantic and syntactic domains. However, little is known about neural substrates of children in the phonological domain, or about sex differences. Here, we conducted a large-scale study (n = 484) of school-aged children using functional near-infrared spectroscopy and a word repetition task, which requires a great extent of phonological processing. We investigated cortical activation during word processing, emphasizing sex differences, to clarify similarities and differences between L1 and L2, and proficiency-related differences during early L2 learning. L1 and L2 shared similar neural substrates with decreased activation in L2 compared to L1 in the posterior superior/middle temporal and angular/supramarginal gyri for both sexes. Significant sex differences were found in cortical activation within language areas during high-frequency word but not during low-frequency word processing. During high-frequency word processing, widely distributed areas including the angular/supramarginal gyri were activated in boys, while more restricted areas, excluding the angular/supramarginal gyri were activated in girls. Significant sex differences were also found in L2 proficiency-related activation: activation significantly increased with proficiency in boys, whereas no proficiency-related differences were found in girls. Importantly, cortical sex differences emerged with proficiency. Based on previous research, the present results indicate that sex differences are acquired or enlarged during language development through different cognitive strategies between sexes, possibly reflecting their different memory functions.

Genetic diversity of feline morbilliviruses isolated in Japan.

Feline morbillivirus (FmoPV) is an emerging virus in domestic cats and considered to be associated with tubulointerstitial nephritis. Although FmoPV was first described in China in 2012, there has been no report of the isolation of this virus in other countries. In this report, we describe the isolation and characterization of FmoPV from domestic cats in Japan. By using reverse transcription (RT)-PCR, we found that three of 13 urine samples from cats brought to veterinary hospitals were positive for FmoPV. FmoPV strains SS1 to SS3 were isolated from the RT-PCR-positive urine samples. Crandell-Rees feline kidney (CRFK) cells exposed to FmoPV showed cytopathic effects with syncytia formation, and FmoPV N protein was detected by indirect immunofluorescence assays. In addition, pleomorphic virus particles with apparent glycoprotein envelope spikes were observed by electron microscopy. By sequence analysis of FmoPV H and L genes, we found that FmoPVs showed genetic diversity; however, signatures of positive selection were not identified.

Sound to language: different cortical processing for first and second languages in elementary school children as revealed by a large-scale study using fNIRS.

A large-scale study of 484 elementary school children (6-10 years) performing word repetition tasks in their native language (L1-Japanese) and a second language (L2-English) was conducted using functional near-infrared spectroscopy. Three factors presumably associated with cortical activation, language (L1/L2), word frequency (high/low), and hemisphere (left/right), were investigated. L1 words elicited significantly greater brain activation than L2 words, regardless of semantic knowledge, particularly in the superior/middle temporal and inferior parietal regions (angular/supramarginal gyri). The greater L1-elicited activation in these regions suggests that they are phonological loci, reflecting processes tuned to the phonology of the native language, while phonologically unfamiliar L2 words were processed like nonword auditory stimuli. The activation was bilateral in the auditory and superior/middle temporal regions. Hemispheric asymmetry was observed in the inferior frontal region (right dominant), and in the inferior parietal region with interactions: low-frequency words elicited more right-hemispheric activation (particularly in the supramarginal gyrus), while high-frequency words elicited more left-hemispheric activation (particularly in the angular gyrus). The present results reveal the strong involvement of a bilateral language network in children's brains depending more on right-hemispheric processing while acquiring unfamiliar/low-frequency words. A right-to-left shift in laterality should occur in the inferior parietal region, as lexical knowledge increases irrespective of language.

Neural correlates of foreign-language learning in childhood: a 3-year longitudinal ERP study.

A foreign language (a language not spoken in one's community) is difficult to master completely. Early introduction of foreign-language (FL) education during childhood is becoming a standard in many countries. However, the neural process of child FL learning still remains largely unknown. We longitudinally followed 322 school-age children with diverse FL proficiency for three consecutive years, and acquired children's ERP responses to FL words that were semantically congruous or incongruous with the preceding picture context. As FL proficiency increased, various ERP components previously reported in mother-tongue (L1) acquisition (such as a broad negativity, an N400, and a late positive component) appeared sequentially, critically in an identical order to L1 acquisition. This finding was supported not only by cross-sectional analyses of children at different proficiency levels but also by longitudinal analyses of the same children over time. Our data are consistent with the hypothesis that FL learning in childhood reproduces identical developmental stages in an identical order to L1 acquisition, suggesting that the nature of the child's brain itself may determine the normal course of FL learning. Future research should test the generalizability of the results in other aspects of language such as syntax.

Second-language instinct and instruction effects: nature and nurture in second-language acquisition.

Adults seem to have greater difficulties than children in acquiring a second language (L2) because of the alleged "window of opportunity" around puberty. Postpuberty Japanese participants learned a new English rule with simplex sentences during one month of instruction, and then they were tested on "uninstructed complex sentences" as well as "instructed simplex sentences." The behavioral data show that they can acquire more knowledge than is instructed, suggesting the interweaving of nature (universal principles of grammar, UG) and nurture (instruction) in L2 acquisition. The comparison in the "uninstructed complex sentences" between post-instruction and pre-instruction using functional magnetic resonance imaging reveals a significant activation in Broca's area. Thus, this study provides new insight into Broca's area, where nature and nurture cooperate to produce L2 learners' rich linguistic knowledge. It also shows neural plasticity of adult L2 acquisition, arguing against a critical period hypothesis, at least in the domain of UG.

Sex differences in pain-induced modulation of corticotropin-releasing hormone neurons in the dorsolateral part of the stria terminalis in mice.

We earlier reported female-biased, sex-specific involvement of the dorsolateral bed nucleus of the stria terminalis (dl BST) in the formalin-induced pain response in rats. The present study investigated pain effects on mice behaviors. Because the dl BST is densely populated with corticotropin-releasing hormone (CRH) neurons, we examined sex differences in these parameters for the dl BST CRH neurons in male and female mice of a mouse line for which the CRH gene promoter (corticotropin-releasing factor [CRF]-Venus ΔNeo) controls the expression of the modified yellow fluorescent protein (Venus). Approximately 92% of Venus-positive cells in the dl BST were also CRH mRNA-positive, irrespective of sex. Therefore, the cells identified using Venus fluorescence were regarded as CRH neurons. A female-biased sex difference was observed in pain-induced behaviors during the interphase (5-15 min after formalin injection) but not during the later phase (phase 2, 15-60 min) in wild-type mice. In CRF-Venus ΔNeo mice, a female-biased difference was observed in either the earlier phase (phase 1, 0-5 min) or the interphase, but not in phase 2. Patch-clamp recordings taken using an acute BST slice obtained from a CRF-Venus ΔNeo mouse after formalin injection showed miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs). Remarkably, the mEPSCs frequency was higher in the Venus-expressing cells of formalin-injected female mice than in vehicle-treated female mice. Male mice showed no increase in mEPSC frequency by formalin injection. Formalin injection had no effect on mEPSC or mIPSC amplitudes in either sex. Pain-induced changes in mEPSC frequency in putative CRH neurons were phase-dependent. Results show that excitatory synaptic inputs to BST CRH neurons are temporally enhanced along with behavioral sex differences in pain response, suggesting that pain signals alter the BST CRH neurons excitability in a sex-dependent manner.

MMP9 secreted from mononuclear cell quality and quantity culture mediates STAT3 phosphorylation and fibroblast migration in wounds.

INTRODUCTION: Intractable ulcers may ultimately lead to amputation. To promote wound healing, researchers developed a serum-free ex vivo peripheral blood mononuclear cell quality and quantity culture (MNC-QQc) as a source for cell therapy. In mice, pigs, and even humans, cell therapy with MNC-QQc reportedly yields a high regenerative efficacy. However, the mechanism of wound healing by MNC-QQc cells remains largely unknown. Hence, using an in vitro wound healing model, this study aimed to investigate MNC-QQc cells and the migratory potential of dermal fibroblasts. METHODS: After separation from a 50 mL blood sample from healthy individuals, mononuclear cells were cultured for 7 days in a serum-free ex vivo expansion system with five different cytokines (MNC-QQc method). The effects of MNC-QQc cells on human dermal fibroblast migration were observed by scratch assay. An angiogenesis array screened the MNC-QQc cell supernatant for proteins related to wound healing. Finally, fibroblast migration was confirmed by observing the intracellular signal transduction pathways via Western blot. RESULTS: The migration of fibroblasts co-cultured with MNC-QQc cells increased by matrix metallopeptidase-9 (MMP9) secretion, as suggested by the angiogenesis array. Furthermore, the phosphorylation of signal transducer and activator of transcription 3 (STAT3) in fibroblast/MNC-QQc cell co-culture and fibroblast culture with added recombinant human MMP9 protein increased. When fibroblasts were cultured with either an MMP9 inhibitor or a STAT3 inhibitor, both fibroblast migration and STAT3 phosphorylation were significantly suppressed. CONCLUSIONS: MNC-QQc cells promote wound healing by the secretion of MMP9, which induces fibroblast migration via the STAT3 signaling pathway.

Ex vivo conditioning of peripheral blood mononuclear cells of diabetic patients promotes vasculogenic wound healing.

The quality and quantity of endothelial progenitor cells (EPCs) are impaired in patients with diabetes mellitus patients, leading to reduced tissue repair during autologous EPC therapy. This study aimed to address the limitations of the previously described serum-free Quantity and Quality Control Culture System (QQc) using CD34+ cells by investigating the therapeutic potential of a novel mononuclear cell (MNC)-QQ. MNCs were isolated from 50 mL of peripheral blood of patients with diabetes mellitus and healthy volunteers (n = 13 each) and subjected to QQc for 7 days in serum-free expansion media with VEGF, Flt-3 ligand, TPO, IL-6, and SCF. The vascular regeneration capability of MNC-QQ cells pre- or post-QQc was evaluated with an EPC colony-forming assay, FACS, EPC culture, tube formation assay, and quantitative real time PCR. For in vivo assessment, 1 × 104 pre- and post-MNC-QQc cells from diabetic donors were injected into a murine wound-healing model using Balb/c nude mice. The percentage of wound closure and angio-vasculogenesis was then assessed. This study revealed vasculogenic, anti-inflammatory, and wound-healing effects of MNC-QQ therapy in both in vitro and in vivo models. This system addresses the low efficiency and efficacy of the current naïve MNC therapy for wound-healing in diabetic patients. As this technique requires a simple blood draw, isolation, and peripheral blood MNC suspension culture for only a week, it can be used as a simple and effective outpatient-based vascular and regenerative therapy for patients with diabetes mellitus.

Unconscious and Distinctive Control of Vocal Pitch and Timbre During Altered Auditory Feedback.

Vocal control plays a critical role in smooth social communication. Speakers constantly monitor auditory feedback (AF) and make adjustments when their voices deviate from their intentions. Previous studies have shown that when certain acoustic features of the AF are artificially altered, speakers compensate for this alteration in the opposite direction. However, little is known about how the vocal control system implements compensations for alterations of different acoustic features, and associates them with subjective consciousness. The present study investigated whether compensations for the fundamental frequency (F0), which corresponds to perceived pitch, and formants, which contribute to perceived timbre, can be performed unconsciously and independently. Forty native Japanese speakers received two types of altered AF during vowel production that involved shifts of either only the formant frequencies (formant modification; Fm) or both the pitch and formant frequencies (pitch + formant modification; PFm). For each type, three levels of shift (slight, medium, and severe) in both directions (increase or decrease) were used. After the experiment, participants were tested for whether they had perceived a change in the F0 and/or formants. The results showed that (i) only formants were compensated for in the Fm condition, while both the F0 and formants were compensated for in the PFm condition; (ii) the F0 compensation exhibited greater precision than the formant compensation in PFm; and (iii) compensation occurred even when participants misperceived or could not explicitly perceive the alteration in AF. These findings indicate that non-experts can compensate for both formant and F0 modifications in the AF during vocal production, even when the modifications are not explicitly or correctly perceived, which provides further evidence for a dissociation between conscious perception and action in vocal control. We propose that such unconscious control of voice production may enhance rapid adaptation to changing speech environments and facilitate mutual communication.

The cAMP response element-binding protein in the bed nucleus of the stria terminalis modulates the formalin-induced pain behavior in the female rat.

Abstract Differences in male and female responses to pain are widely recognized in many species, including humans, but the cerebral mechanisms that generate these responses are unknown. Using the formalin test, we confirmed that proestrus female rats showed nociceptive behavior, modulated by estrogen that was distinct from male rats, particularly during the interphase period. We then explored the brain areas, which were involved in the female pattern of nociceptive behavior. We found that, after a formalin injection and at the time corresponding to the behavioral interphase, the number of phosphorylated cAMP response element-binding protein (pCREB)-immunoreactive neurons observed by immunocytochemistry increased in the dorsolateral division of the bed nucleus of the stria terminalis (BSTLD) in female but not male rats. There were no significant sex differences in pCREB expression following formalin in any region other than the BSTLD. The increased pCREB in female rats was eliminated after an ovariectomy and restored with 17beta-estradiol treatment. Neither an orchidectomy nor 17beta-estradiol treatment affected the pCREB response in male rats. The increase in pCREB expression in the BSTLD in female rats after formalin injection was confirmed with immunoblotting. To determine the role of CREB in the BSTLD, adenovirus-mediated expression of a dominant-negative form of CREB (mCREB) was carried out. The nociceptive behavior during interphase was significantly attenuated by injection of virus carrying mCREB into the BSTLD in female rats but not in male rats. These results suggest a novel role for CREB in the BSTLD as a modulator of the pain response in a female-specific, estrogen-dependent manner.

Carboxymethylation of CRMP2 is associated with decreased Schwann cell myelination efficiency.

Pyridoxal, an active form of vitamin B6, is known to inhibit formation of advanced glycation end-products and protect tissues from diabetic complications. Here we identified that pyridoxal is a required component for establishing Schwann cell myelination in our Schwann cell-dorsal root ganglion neuron co-culture system. When the co-culture was maintained without pyridoxal, carboxymethylation of collapsin response mediator protein 2 (CRMP2) became detectable. Carboxymethylation decreased the affinity of CRMP2 to bind with microtubules, indicating that carboxymethylation affected CRMP2 function. These results suggest that carboxymethylation of CRMP2 may be an indicator of dysfunction caused by glycation which is observed in pathological conditions, including diabetic neuropathy.

Quality and Quantity-Cultured Murine Endothelial Progenitor Cells Increase Vascularization and Decrease Fibrosis in the Fat Graft.

BACKGROUND: Fat grafting has become a valuable technique for soft-tissue reconstruction; however, long-lasting success depends on several determinants. An early blood supply to the transplanted adipocytes is important to prevent ischemia. The recently developed quality and quantity (QQ) culture increases the vasculogenic potential of endothelial progenitor cells. The authors used a murine fat grafting model to address the hypothesis that QQ-cultured endothelial progenitor cells stimulate the establishment of a blood vessel network and increase graft success. METHODS: c-KitSca-1Lin (KSL) cells were isolated as endothelial progenitor cell precursors from C57BL/6 mice. Adipose tissue was grafted with QQ-cultured KSL cells (QQKSL group), uncultured KSL cells (KSL group), adipose-derived stem cells (ASC group), and a combination (QQKSL+ASC group), and compared to a control group. Five and 10 weeks later, grafts were weighed, histologic and immunohistochemical parameters were evaluated, and gene expression was quantified by quantitative polymerase chain reaction. RESULTS: The highest vessel density was observed in the combined QQKSL+ASC group (68.0 ± 4.3/mm; p < 0.001) and the QQKSL group (53.9 ± 3.0/mm; p < 0.001). QQKSL cells were engrafted in proximity to the graft vasculature. QQKSL cells decreased the fibrosis percentage (13.8 ± 1.8 percent; p < 0.05). The combined QQKSL+ASC group (22.4 ± 1.8/mm; p < 0.001) showed the fewest local inflammation units. A significant up-regulation of platelet-derived growth factor and adiponectin expression was observed in the QQKSL group and QQKSL+ASC group. Graft weight persistence was not significantly different between groups. CONCLUSIONS: Supplementing fat grafts with quality and quantity-cultured endothelial progenitor cells improves graft quality by stimulating vascularization. The increased vessel density is associated with less fibrosis, less inflammation, and better adipose tissue integrity. Enriching fat grafts with QQ-cultured endothelial progenitor cells is a potential solution to their clinical shortcomings.

Keloid patients have higher peripheral blood endothelial progenitor cell counts and CD34+ cells with normal vasculogenic and angiogenic function that overexpress vascular endothelial growth factor and interleukin-8.

BACKGROUND: One suggested reason for aberrant wound healing in keloid scars is chronic inflammation of the dermis. We hypothesized that excessive blood vessel formation and high capillary density in keloid tissue is caused by dysfunction of endothelial progenitor cells. METHODS: We compared the number of circulating endothelial progenitor cells and vasculogenic and angiogenic capacity, as well as secretory function, of circulating CD34+ cells in keloid patients and healthy individuals. RESULTS: Compared to mononuclear cell cultures from healthy donors, cultures of peripheral blood mononuclear cells obtained from keloid patients showed a more than twofold increase in the number of peripheral blood EPCs (fibronectin-adhering cells that phagocytized acetylated low-density lipoprotein and bound Ulex europaeus agglutinin-I lectin). However, there was no difference in colony-forming ability and participation in in vitro angiogenesis between circulating CD34+ cells isolated from keloid patients and healthy individuals. This means that circulating CD34+ /endothelial progenitor cells in keloid patients have normal vasculogenic and angiogenic function. However, CD34+ cells derived from keloid patients demonstrated a more than sevenfold expression of the interleukin-8 gene and a more than fivefold expression of the vascular endothelial growth factor gene than CD34+ cells derived from healthy individuals. CONCLUSIONS: These results support the role of vascular endothelial growth factor and interleukin-8 in increased recruitment of endothelial progenitor cells in keloid patients.

Phencyclidine-induced cognitive deficits in mice are improved by subsequent subchronic administration of the antibiotic drug minocycline.

BACKGROUND: The N-methyl-d-aspartate (NMDA) receptor antagonist phencyclidine (PCP)-induced cognitive deficits have been used as an animal model for schizophrenia. This study was undertaken to determine whether the antibiotic drug minocycline could improve PCP-induced cognitive deficits in mice. METHODS: Saline (10 ml/kg/day, s.c., once daily on day 1-5, 8-12) or PCP (10 mg/kg/day, s.c., once daily on day 1-5, 8-12) were administered to mice for 10 days. Subsequently, vehicle (10 ml/kg/day, i.p.) or minocycline (4.0 or 40 mg/kg/day, i.p.) was injected for 14 consecutive days. One day after the final injection, a novel object recognition test was performed. RESULTS: PCP-induced cognitive deficits in mice were significantly improved by subsequent subchronic (14 days) administration of minocycline (40 mg/kg), but not minocycline (4.0 mg/kg). CONCLUSIONS: This study suggests that minocycline could be a potential therapeutic drug for cognitive deficits in schizophrenic patients.

Phencyclidine-induced cognitive deficits in mice are improved by subsequent subchronic administration of the antipsychotic drug perospirone: role of serotonin 5-HT1A receptors.

Accumulating evidence suggests that the serotonin 5-HT(1A) receptor may play a role in the pathophysiology of schizophrenia. The present study was undertaken to examine the effects of perospirone, an atypical antipsychotic drug with 5-HT(1A) receptor agonism, on cognitive deficits in mice after repeated administration of the NMDA receptor antagonist phencyclidine (PCP). Subsequent subchronic (14 days) administration of perospirone (1.0, 3.0, or 10 mg/kg) significantly attenuated PCP (10 mg/kg)-induced cognitive deficits in mice, in a dose-dependent manner. The effects of perospirone (10 mg/kg) were significantly antagonized by co-administration of the selective 5-HT(1A) receptor antagonist WAY100635 (1.0 mg/kg). Furthermore, hypothermia by the 5-HT(1A) receptor agonist 8-OH DPAT (0.25 mg/kg) was significantly attenuated in mice treated with PCP. Moreover, a receptor binding assay using [(3)H]WAY100635 revealed that levels of 5-HT(1A) receptors in the hippocampus, but not in the frontal cortex, of PCP-treated mice were significantly lower than those of saline-treated mice. These findings suggest that repeated PCP administration alters 5-HT(1A) receptor function in the mouse brain, and that subsequent subchronic administration of perospirone ameliorates PCP-induced cognitive deficits via 5-HT(1A) receptors. Therefore, perospirone could be a potential therapy for the cognitive deficits observed in schizophrenic patients.

Effectiveness of endothelial progenitor cell culture under microgravity for improved angiogenic potential.

Endothelial progenitor cell (EPC) transplantation is beneficial for ischemic diseases such as critical limb ischemia and ischemic heart disease. The scarcity of functional EPCs in adults is a limiting factor for EPC transplantation therapy. The quality and quantity culture (QQc) system is an effective ex vivo method for enhancing the number and angiogenic potential of EPCs. Further, microgravity environments have been shown to enhance the functional potential of stem cells. We therefore hypothesized that cells cultured with QQc under microgravity may have enhanced functionality. We cultured human peripheral blood mononuclear cells using QQc under normal (E), microgravity (MG), or microgravity followed by normal (ME) conditions and found that ME resulted in the most significant increase in CD34+ and double positive Dil-Ac-LDL-FITC-Ulex-Lectin cells, both EPC markers. Furthermore, angiogenic potential was determined by an EPC-colony forming assay. While numbers of primitive EPC-colony forming units (pEPC-CFU) did not change, numbers of definitive EPC-CFU colonies increased most under ME conditions. Gene-expression profiling also identified increases in angiogenic factors, including vascular endothelial growth factor, under MG and ME conditions. Thus, QQc along with ME conditions could be an efficient system for significantly enhancing the number and angiogenic potential of EPCs.

Explicit Performance in Girls and Implicit Processing in Boys: A Simultaneous fNIRS-ERP Study on Second Language Syntactic Learning in Young Adolescents.

Learning a second language (L2) proceeds with individual approaches to proficiency in the language. Individual differences including sex, as well as working memory (WM) function appear to have strong effects on behavioral performance and cortical responses in L2 processing. Thus, by considering sex and WM capacity, we examined neural responses during L2 sentence processing as a function of L2 proficiency in young adolescents. In behavioral tests, girls significantly outperformed boys in L2 tests assessing proficiency and grammatical knowledge, and in a reading span test (RST) assessing WM capacity. Girls, but not boys, showed significant correlations between L2 tests and RST scores. Using functional near-infrared spectroscopy (fNIRS) and event-related potential (ERP) simultaneously, we measured cortical responses while participants listened to syntactically correct and incorrect sentences. ERP data revealed a grammaticality effect only in boys in the early time window (100-300 ms), implicated in phrase structure processing. In fNIRS data, while boys had significantly increased activation in the left prefrontal region implicated in syntactic processing, girls had increased activation in the posterior language-related region involved in phonology, semantics, and sentence processing with proficiency. Presumably, boys implicitly focused on rule-based syntactic processing, whereas girls made full use of linguistic knowledge and WM function. The present results provide important fundamental data for learning and teaching in L2 education.

Short-term fasting decreases excitatory synaptic inputs to ventromedial tuberoinfundibular dopaminergic neurons and attenuates their activity in male mice.

Tuberoinfundibular dopaminergic (TIDA) neurons in the arcuate nucleus (ARC) of the hypothalamus play a role in inhibiting prolactin (PRL) secretion from the anterior pituitary. PRL is involved in a variety of behaviors, including feeding. Consequently, we hypothesized that fasting might reduce the activity of TIDA neurons, which might alter PRL secretion. However, direct examinations of TIDA neuron activity are difficult. Recently, transgenic mice were generated that expressed green fluorescent protein (GFP) under the control of the rat tyrosine hydroxylase gene. We first determined that GFP in the dorsomedial ARC was a reliable marker of TIDA neurons. Then, we performed electrophysiology and immunocytochemistry in GFP-labeled TIDA neurons to examine whether different feeding conditions could change their activity. Eight-week-old male mice were fed or fasted for 24 h. After sacrifice, we prepared acutely isolated brain slices for conducting whole-cell voltage-clamp recordings. TIDA neurons were identified with fluorescence microscopy. The mean amplitude of miniature excitatory postsynaptic currents (mEPSCs) was significantly reduced in fasting mice compared to fed mice, but different feeding conditions did not affect the mean mEPSC intervals. This result suggested that fasting reduced the number of excitatory synaptic inputs to TIDA neurons. To determine whether a reduction in excitatory synaptic inputs would cause a reduction in TIDA neuron activity, we examined the effect of 24-h fasting on c-Fos expression in the ARC. We found that fasting significantly reduced the number of Fos-positive TIDA neurons. In addition, serum PRL levels were significantly increased. Taken together, the present findings suggested that short-term fasting attenuated TIDA neuron activity.

Quality-Quantity Control Culture Enhances Vasculogenesis and Wound Healing Efficacy of Human Diabetic Peripheral Blood CD34+ Cells.

Autologous endothelial progenitor cell (EPC) therapy is commonly used to stimulate angiogenesis in ischemic repair and wound healing. However, low total numbers and functional deficits of EPCs make autologous EPC therapy ineffective in diabetes. Currently, no known ex vivo culture techniques can expand and/or ameliorate the functional deficits of EPCs for clinical usage. Recently, we showed that a quality-quantity culture (QQc) system restores the vasculogenic and wound-healing efficacy of murine diabetic EPCs. To validate these results and elucidate the mechanism in a translational study, we evaluated the efficacy of this QQc system to restore the vasculogenic potential of diabetic human peripheral blood (PB) CD34+ cells. CD34+ cells purified from PB of diabetic and healthy patients were subjected to QQc. Gene expression, vascular regeneration, and expression of cytokines and paracrine mediators were analyzed. Pre- or post-QQc diabetic human PB-CD34+ cells were transplanted into wounded BALB/c nude mice and streptozotocin-induced diabetic mice to assess functional efficacy. Post-QQc diabetic human PB-CD34+ cell therapy significantly accelerated wound closure, re-epithelialization, and angiogenesis. The higher therapeutic efficacy of post-QQc diabetic human PB-CD34+ cells was attributed to increased differentiation ability of diabetic CD34+ cells, direct vasculogenesis, and enhanced expression of angiogenic factors and wound-healing genes. Thus, QQc can significantly enhance the therapeutic efficacy of human PB-CD34+ cells in diabetic wounds, overcoming the inherent limitation of autologous cell therapy in diabetic patients, and could be useful for treatment of not only wounds but also other ischemic diseases. Stem Cells Translational Medicine 2018;7:428-438.