Modulation of cognition and neuronal plasticity in gain- and loss-of-function mouse models of the schizophrenia risk gene Tcf4.

The transcription factor TCF4 was confirmed in several large genome-wide association studies as one of the most significant schizophrenia (SZ) susceptibility genes. Transgenic mice moderately overexpressing Tcf4 in forebrain (Tcf4tg) display deficits in fear memory and sensorimotor gating. As second hit, we exposed Tcf4tg animals to isolation rearing (IR), chronic social defeat (SD), enriched environment (EE), or handling control (HC) conditions and examined mice with heterozygous deletion of the exon 4 (Tcf4Ex4δ+/-) to unravel gene-dosage effects. We applied multivariate statistics for behavioral profiling and demonstrate that IR and SD cause strong cognitive deficits of Tcf4tg mice, whereas EE masked the genetic vulnerability. We observed enhanced long-term depression in Tcf4tg mice and enhanced long-term potentiation in Tcf4Ex4δ+/- mice indicating specific gene-dosage effects. Tcf4tg mice showed higher density of immature spines during development as assessed by STED nanoscopy and proteomic analyses of synaptosomes revealed concurrently increased levels of proteins involved in synaptic function and metabolic pathways. We conclude that environmental stress and Tcf4 misexpression precipitate cognitive deficits in 2-hit mouse models of relevance for schizophrenia.

Conservation of Hox gene clusters in the self-fertilizing fish Kryptolebias marmoratus (Cyprinodontiformes; Rivulidae).

In this study, whole Hox gene clusters in the self-fertilizing mangrove killifish Kryptolebias marmoratus (Cyprinodontiformes; Rivulidae), a unique hermaphroditic vertebrate in which both sex organs are functional at the same time, were identified from whole genome and transcriptome sequences. The aim was to increase the understanding of the evolutionary status of conservation of this Hox gene cluster across fish species.

Changes in nasal airflow and heat transfer correlate with symptom improvement after surgery for nasal obstruction.

Surgeries to correct nasal airway obstruction (NAO) often have less than desirable outcomes, partly due to the absence of an objective tool to select the most appropriate surgical approach for each patient. Computational fluid dynamics (CFD) models can be used to investigate nasal airflow, but variables need to be identified that can detect surgical changes and correlate with patient symptoms. CFD models were constructed from pre- and post-surgery computed tomography scans for 10 NAO patients showing no evidence of nasal cycling. Steady-state inspiratory airflow, nasal resistance, wall shear stress, and heat flux were computed for the main nasal cavity from nostrils to posterior nasal septum both bilaterally and unilaterally. Paired t-tests indicated that all CFD variables were significantly changed by surgery when calculated on the most obstructed side, and that airflow, nasal resistance, and heat flux were significantly changed bilaterally as well. Moderate linear correlations with patient-reported symptoms were found for airflow, heat flux, unilateral allocation of airflow, and unilateral nasal resistance as a fraction of bilateral nasal resistance when calculated on the most obstructed nasal side, suggesting that these variables may be useful for evaluating the efficacy of nasal surgery objectively. Similarity in the strengths of these correlations suggests that patient-reported symptoms may represent a constellation of effects and that these variables should be tracked concurrently during future virtual surgery planning.

Deviated nasal septum hinders intranasal sprays: a computer simulation study.

BACKGROUND: This study investigates how deviated nasal septum affects the quantity and distribution of spray particles, and examines the effects of inspiratory airflow and head position on particle transport. METHODS: Deposition of spray particles was analysed using a three-dimensional computational fluid dynamics model created from a computed tomography scan of a human nose with leftward septal deviation and a right inferior turbinate hypertrophy. Five simulations were conducted using FluentTM software, with particle sizes ranging from 20-110 μm, a spray speed of 3 m/s, plume angle of 68(deg), and with steady state inspiratory airflow either present (15.7 L/min) or absent at varying head positions. RESULTS: With inspiratory airflow present, posterior deposition on the obstructed side was approximately four times less than the contralateral side, regardless of head position, and was statistically significant. When airflow was absent, predicted deposition beyond the nasal valve on the left and right sides were between 16% and 69% lower and positively influenced by a dependent head position. CONCLUSION: Simulations predicted that septal deviation significantly diminished drug delivery on the obstructed side. Furthermore, increased particle penetration was associated with presence of nasal airflow. Head position is an important factor in particle deposition patterns when inspiratory airflow is absent.

Augmenting neurotransmitter release by enhancing the apparent Ca2+ affinity of synaptotagmin 1.

Synaptotagmin 1 likely acts as a Ca2+ sensor in neurotransmitter release by Ca2+-binding to its two C2 domains. This notion was strongly supported by the observation that a mutation in the C2A domain causes parallel decreases in the apparent Ca2+ affinity of synaptotagmin 1 and in the Ca2+ sensitivity of release. However, this study was based on a single loss-of-function mutation. We now show that tryptophan substitutions in the synaptotagmin 1 C2 domains act as gain-of-function mutations to increase the apparent Ca2+ affinity of synaptotagmin 1. The same substitutions, when introduced into synaptotagmin 1 expressed in neurons, enhance the Ca2+ sensitivity of release. Mutations in the two C2 domains lead to comparable and additive effects in release. Our results thus show that the apparent Ca2+ sensitivity of release is dictated by the apparent Ca2+ affinity of synaptotagmin 1 in both directions, and that Ca2+ binding to both C2 domains contributes to Ca2+ triggering of release.

An essential role for vesicular glutamate transporter 1 (VGLUT1) in postnatal development and control of quantal size.

Quantal neurotransmitter release at excitatory synapses depends on glutamate import into synaptic vesicles by vesicular glutamate transporters (VGLUTs). Of the three known transporters, VGLUT1 and VGLUT2 are expressed prominently in the adult brain, but during the first two weeks of postnatal development, VGLUT2 expression predominates. Targeted deletion of VGLUT1 in mice causes lethality in the third postnatal week. Glutamatergic neurotransmission is drastically reduced in neurons from VGLUT1-deficient mice, with a specific reduction in quantal size. The remaining activity correlates with the expression of VGLUT2. This reduction in glutamatergic neurotransmission can be rescued and enhanced with overexpression of VGLUT1. These results show that the expression level of VGLUTs determines the amount of glutamate that is loaded into vesicles and released and thereby regulates the efficacy of neurotransmission.

Proteolytic enzyme conjugated to SC-glucan as an enzymatic transdermal drug penetration enhancer.

The objective of this study was to investigate the effect of papain, a proteolytic enzyme, on the percutaneous absorption of drugs. To guarantee the enzyme stability during the skin penetration, papain was modified by the conjugation to SC-glucan. The enhancing activity of drug penetration was evaluated using antipyrine and indomethacin as hydrophilic and hydrophobic model drugs, respectively. The SC-glucan-papain conjugate was found to be very effective for facilitating the percutaneous absorption of antipyrine. Microscopic observations showed that the thickness of stratum corneum and viable epidermis was increased by the treatment of the SC-glucan-papain conjugate. Moreover, it induced phase separation, lacuna formation, and lamellar disruption within the stratum corneum interstices. These structural changes by the SC-glucan-papain conjugate are likely to be induced from hydrolysis of extensive crosslinking of corneocyte envelopes and intracellular proteins. However, the SC-glucan-papain conjugate showed no skin irritation according to the Draize test, which may be due to the difficulty of the SC-glucan-papain conjugate in penetrating into the skin.

Homologous expression of the lipase and ABC transporter gene cluster, tliDEFA, enhances lipase secretion in Pseudomonas spp.

The ABC transporter TliDEF was found to be an efficient secretory apparatus for extracellular lipase TliA in Pseudomonas fluorescens. For the enhanced secretion of the lipase, we tried to coexpress tliA and tliDEF in various Pseudomonas species. Whereas the coexpression of tliA and tliDEF was required for the lipase secretion in P. fragi, the expression of tliA was sufficient for the lipase secretion in P. fluorescens, P. syringae, and P. putida, indicating the existence of compatible ABC transporter in these species. However, P. fluorescens harboring tliDEFA secreted much more lipase than P. fluorescens harboring only tliA, but the tliDEF was functional only at temperatures below 30 degrees C. The recombinant P. fluorescens overexpressing tliDEFA showed the highest secretion level, 217 U/ml. OD (optical density) (28 microg/ml. OD) of lipase in Luria-Bertani medium under microaerated conditions. With the increase of aeration, the lipase production was decreased and the lipase seemed to be degraded as the cells entered the cell death phase. These results demonstrate that P. fluorescens can be used as a host system for the secretory production of the lipase using the ABC transporter, thus producing lipase in over 14% of the total protein.

Identification of differentiation-associated brain-specific phosphate transporter as a second vesicular glutamate transporter (VGLUT2).

Glutamate is the major excitatory neurotransmitter in mammalian CNS. In the presynaptic nerve terminal, glutamate is stored in synaptic vesicles and released by exocytosis. Previously, it has been shown that a transport protein originally identified as a brain-specific Na(+)-dependent inorganic phosphate transporter I (BNPI) functions as vesicular glutamate transporter and thus has been renamed VGLUT1. Recently, a protein highly homologous to VGLUT1, "differentiation-associated BNPI" (DNPI), has been discovered. Northern blot and in situ hybridization analyses indicate that DNPI mRNA is expressed in some brain regions in which VGLUT1 mRNA is not expressed. We now show that DNPI functions as vesicular glutamate transporter with properties very similar to VGLUT1 and propose to rename the protein VGLUT2. VGLUT2 is highly enriched in synaptic vesicles. Furthermore, VGLUT2 resides on a vesicle population that is distinct from vesicles containing the vesicular GABA transporter or VGLUT1, showing that the expression of VGLUT1 and VGLUT2 do not overlap. When VGLUT2 was expressed in BON cells, membrane fractions displayed ATP-dependent, carbonyl cyanide p-trifluoromethoxyphenylhydrazone-sensitive glutamate uptake. Overexpression of VGLUT2 in cultured autaptic GABAergic neurons yielded postsynaptic currents that were insensitive to the GABA(A) receptor antagonist bicuculline but blocked by the AMPA-receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfonyl-benzo[F]quinoxaline. Thus, expression of VGLUT2 suffices to cause GABAergic neurons to release glutamate in addition to GABA in a manner very similar to that reported previously for VGLUT1.

Experimental trauma to the malar eminence: fracture biomechanics and injury patterns.

OBJECTIVES: To document patterns of facial fractures after trauma to the malar eminence and to elucidate biomechanical factors relevant to the injury patterns. STUDY DESIGN AND SETTING: Studies were conducted on 14 cadaver heads. Study variables included impact velocity, contact area, impact force, and zygomatic skin thickness. Bony fractures and clinical injury patterns were documented. A fracture severity rating scale was devised and statistically correlated to the study variables using regression ANOVA analysis. RESULTS: A broad spectrum of facial fracture patterns was found. Skin thickness and surface area did not correlate with fracture severity (P = 0.67, P = 0.83, respectively). Impact force demonstrated a trend toward significance (P = 0.14). Velocity was most correlative with fracture severity (P = 0.07). A critical threshold velocity (3.5 m/s) was found to correlate with the most severe fracture patterns. CONCLUSIONS: A broad spectrum of facial fracture patterns was demonstrated after experimental trauma to the malar eminence. Contact surface area and zygomatic skin thickness were not found to be significant factors in fracture severity. Velocity, rather than impact force, was most correlative with fracture severity. The most severe fracture patterns were elicited by velocities above 3.5 m/s.

Developmental changes in P2X purinoceptors on glycinergic presynaptic nerve terminals projecting to rat substantia gelatinosa neurones.

1. In mechanically dissociated rat spinal cord substantia gelatinosa (SG) neurones attached with native presynaptic nerve endings, glycinergic miniature inhibitory postsynaptic currents (mIPSCs) were recorded using nystatin perforated patch recording mode under voltage-clamp conditions. Under these conditions, it was tested whether the changes in P2X receptor subtype on the glycinergic presynaptic nerve terminals occur during postnatal development. 2. ATP facilitated glycinergic mIPSC frequency in a concentration-dependent manner through all developmental stages tested, whereas alphabeta-methylene-ATP (alphabeta-me-ATP) was only effective at later developmental stages. 3. alphabeta-me-ATP-elicited mIPSC frequency facilitation was completely occluded in the Ca2+-free external solution, but it was not affected by adding 10(-4) M Cd2+. 4. alphabeta-me-ATP still facilitated mIPSC frequency even in the presence of 10(-6) M thapsigargin, a Ca2+ pump blocker. 5. In later developmental stages, ATP-elicited presynaptic or postsynaptic responses were reversibly blocked by 10(-5) M pyridoxal-5-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), but only partially blocked by 10(-7) M 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP). However, alphabeta-me-ATP-elicited presynaptic or postsynaptic responses were completely and reversibly blocked by either 10(-5) M PPADS or 10(-7) M TNP-ATP. 6. alphabeta-me-ATP significantly reduced the evoked glycinergic IPSC amplitude in postnatal 28-30 day neurones, whereas it had no effect in 10-12 day neurones. 7. It was concluded that alphabeta-me-ATP-sensitive P2X receptors were functionally expressed on the glycinergic presynaptic nerve terminals projecting to SG neurones in later developmental stages. Such developmental changes of presynaptic P2X receptor subtypes might contribute to synaptic plasticity such as the regulation of neuronal excitability and the fine controlling of the pain signal in spinal dorsal horn neurones.

Antagonizing effect of protein kinase C activation on the mu-opioid agonist-induced inhibition of high voltage-activated calcium current in rat periaqueductal gray neuron.

Opioids have been thought to induce analgesia by activating the descending pain control system, especially at the level of periaqueductal gray, and regulate the neurotransmitter release through the inhibition of calcium channel. In the present study, the modulatory effects of protein kinase C and protein kinase A on the mu-opioid agonist-induced inhibition of the high-voltage activated calcium current were examined in the acutely dissociated rat periaqueductal gray neurons with the nystatin-perforated patch-clamp technique. Among 505 neurons tested, the barium current passing through the high-voltage activated calcium channels of 172 neurons (34%) were inhibited by 32+/-3% with the application of an mu-opioid agonist, [D-Ala(2),N-MePhe(4),Gly(5)-ol]-enkephalin (DAMGO, 1 microM). The barium currents itself and the DAMGO-induced inhibitory effects were not affected by the application of either an adenylate cyclase activator (forskolin, 1 microM) or a protein kinase inhibitor (staurosporin, 10 nM) for 2 min. The DAMGO inhibition was completely and irreversibly antagonized by the application of a protein kinase C activator, phorbol-12-myristate-13-acetate (PMA, 1 microM) for 2 min without any alteration of the barium current itself. However, the antagonizing effect of PMA was completely abolished by the application of 10 nM staurosporin for 2 min. After then, PMA did not show the antagonizing effect any more. Inversely, when staurosporin was applied before PMA, the antagonizing effect of PMA was also not shown. These results demonstrate that the mu-opioid agonist-induced inhibition of the periaqueductal gray neuronal high-voltage activated calcium current can be antagonized by protein kinase C activation. This finding may provide us a significant clue to understand the action mechanism of opioid-induced analgesia in the periaqueductal gray.

Histaminergic modulation of GABAergic transmission in rat ventromedial hypothalamic neurones.

1. The ventromedial nucleus of the hypothalamus (VMH) is a key nucleus in the homeostatic regulation of neuroendocrine and behavioural functions. In mechanically dissociated rat VMH neurones with attached native presynaptic nerve endings, GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded using the nystatin perforated patch recording mode under voltage-clamp conditions. 2. Histamine reversibly inhibited the sIPSC frequency in a concentration-dependent manner without affecting the mean current amplitude. The selective histamine receptor type 3 (H(3)) agonist imetit (100 nM) mimicked this effect and it was completely abolished by the selective H(3) receptor antagonists clobenpropit (3 microM) and thioperamide (10 microM). 3. The GTP-binding protein inhibitor N-ethylmaleimide (10 microM) removed the histaminergic inhibition of GABAergic sIPSCs. 4. Elimination of external Ca(2+) reduced the GABAergic sIPSC frequency without affecting the distribution of current amplitudes. In this condition, the inhibitory effect of imetit on the sIPSC frequency completely disappeared, suggesting that the histaminergic inhibition requires extracellular Ca(2+). 5. The P/Q-type Ca(2+) channel blocker omega-agatoxin IVA (300 nM) attenuated the histaminergic inhibition of the GABAergic sIPSC frequency, but neither the N-type Ca(2+) channel blocker omega-conotoxin GVIA (3 microM) nor the L-type Ca(2+) channel blocker nicardipine (3 microM) was effective. 6. Activation of adenylyl cyclase with forskolin (10 microM) had no effect on histaminergic inhibition of the sIPSCs. 7. In conclusion, histamine inhibits spontaneous GABA release from presynaptic nerve terminals projecting to VMH neurones by inhibiting presynaptic P/Q-type Ca(2+) channels via a G-protein coupled to H(3) receptors and this may modulate the excitability of VMH neurones.

Enhancement of stability and activity of phospholipase A(1) in organic solvents by directed evolution.

We attempted to apply the directed evolution approach to enhancing enzyme properties in the presence of organic solvents, in which enzyme stability and activity were often drastically reduced. Stability and catalytic activity of phospholipase A(1) in the presence of an organic solvent were enhanced by error-prone polymerase chain reaction (PCR) and DNA shuffling followed by a filter-based visual screening. Three mutants (SA8, SA17 and SA20) were isolated on indicator plates (i.e., 1% phosphatidylcholine gels containing 30% dimethyl sulfoxide (DMSO)) after a second mutant library was treated in 50% DMSO for 36 h. The half-life values of the three mutants exhibited an approximately 4-fold increase. The three mutants also exhibited increased stability in all organic solvents tested compared with the wild-type enzyme. Thus, an enzyme variant having superior catalytic efficiency in most of the organic solvents could be obtained by using any solvent suitable for designing the efficient screening system, regardless of the properties of the particular solvent.

Evaluation on the adsorption capabilities of new chemically modified polymeric adsorbents with protoporphyrin IX.

A chemically modified polymeric adsorbent was synthesized to evaluate the availability as an adsorbent for solid-phase extraction (SPE) of phenol and chlorophenols. Commercially available Amberlite XAD-2 and XAD-4 resins were modified with macrocyclic protoporphyrin IX (PPIX) through the ketone linkage. Adsorption isotherms were obtained by batch experiments and the data were fitted to the Freundlich equation to calculate the adsorption parameters. Breakthrough volumes were measured by column experiments. Physical properties such as surface area, average pore diameter and micropore volume of resins were measured to correlate with the adsorption characteristics. As a result, adsorption capacity was increased for the chemically modified resins and it can be concluded that the increase of pi-pi interaction due to the introduction of the porphyrin molecule is the major factor for the increase of the adsorption capacity.

Functional interaction of the active zone proteins Munc13-1 and RIM1 in synaptic vesicle priming.

Synaptic neurotransmitter release is restricted to active zones, where the processes of synaptic vesicle tethering, priming to fusion competence, and Ca2+-triggered fusion are taking place in a highly coordinated manner. We show that the active zone components Munc13-1, an essential vesicle priming protein, and RIM1, a Rab3 effector with a putative role in vesicle tethering, interact functionally. Disruption of this interaction causes a loss of fusion-competent synaptic vesicles, creating a phenocopy of Munc13-1-deficient neurons. RIM1 binding and vesicle priming are mediated by two distinct structural modules of Munc13-1. The Munc13-1/RIM1 interaction may create a functional link between synaptic vesicle tethering and priming, or it may regulate the priming reaction itself, thereby determining the number of fusion-competent vesicles.

Single-stage adult laryngotracheal reconstruction without stenting.

OBJECTIVES/HYPOTHESIS: To present the indications, surgical technique, and results of single-stage laryngotracheal reconstruction (SSLTR) without stenting for laryngotracheal stenosis (LTS) in adults. Various open surgical techniques have been previously described for LTS in adults; however, these techniques usually involve placement of intraluminal stents. The practice of early extubation without stenting is common for pediatric SSLTR. The success of this technique in the pediatric population has led to a trial of the same technique in selected cases of adult LTS. STUDY DESIGN: Retrospective review. METHODS: A retrospective review was conducted on 15 patients with glottic, subglottic, or tracheal stenosis or a combination of these, who underwent SSLTR with composite nasal septal grafts or costal cartilage grafts without stenting. RESULTS: All patients were extubated or decannulated 1 to 7 days after surgery. Three of the 15 patients had no further procedures. Three patients had a second SSLTR to repair stenosis at a different level with no further difficulties. Eight patients had additional endoscopic airway procedures after extubation or decannulation, and one patient died in the immediate postoperative period. All 14 surviving patients are decannulated and well at the time of writing. CONCLUSION: For LTS in selected adult cases, SSLTR without stenting is a viable option. Indications, surgical technique, and complications are presented.

Substance P abolishes the facilitatory effect of ATP on spontaneous glycine release in neurons of the trigeminal nucleus pars caudalis.

Glycine release was facilitated by the activation of presynaptic ATP receptors (P(2X)-type) in a preparation of dissociated trigeminal nucleus pars caudalis neurons in which the native synaptic boutons were preserved. The action of ATP was completely blocked by substance P (SP) without alteration of the miniature IPSC (mIPSC) amplitude distribution. SP itself had no effect on mIPSC frequency or amplitude. The inhibitory effect of SP on ATP action was blocked by CP99994, indicating that the SP receptors are of the neurokinin-1 type. The ATP-induced facilitation of the mIPSC frequency was unaffected by Cd(2+). Moreover, SP did not inhibit the increase in mIPSC frequency induced high K(+) application, suggesting that SP did not modulate voltage-dependent calcium channels or subsequent steps in the release process. KT5720 and phorbol 12-myristate 13-acetate did not block SP action, indicating that neither the cAMP-protein kinase A nor the protein kinase C pathway mediates the SP effects. However, in the presence of N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide (W-7), SP was no longer able to inhibit the ATP-induced stimulation of mIPSC frequency. 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine also suppressed the SP action, suggesting that SP modulates P(2X) receptors via a Ca(2+)/calmodulin-dependent protein kinase II-mediated pathway. In conventional whole-cell mode, the presence of W-7 in the patch pipette did not affect the SP inhibitory action. Thus, SP is not likely to be generating its modulation through the production of a retrograde signal (involving calmodulin) from the postsynaptic cell to the presynaptic boutons. These results are the first demonstration of the modulation of one presynaptic receptor by another. Because SP inhibits the ATP stimulation of glycine release, SP may play a significant role in hyperalgesia or chronic pain.