Dopamine is widely associated with reward, motivation, and reinforcement learning. Research on dopamine has emphasized its contribution to compulsive behaviors, such as addiction and overeating, with less examination of its potential role in behavioral flexibility in normal, non-pathological states. In the study reviewed here, we investigated the effect of increased tonic dopamine in a two-lever homecage operant paradigm where the relative value of the levers was dynamic, requiring the mice to constantly monitor reward outcome and adapt their behavior. The data were fit to a temporal difference learning model that showed that mice with elevated dopamine exhibited less coupling between reward history and behavioral choice. This work suggests a way to integrate motivational and learning theories of dopamine into a single formal model where tonic dopamine regulates the expression of prior reward learning by controlling the degree to which learned reward values bias behavioral choice. Here I place these results in a broader context of dopamine's role in instrumental learning and suggest a novel hypothesis that tonic dopamine regulates thrift, the degree to which an animal needs to exploit its prior reward learning to maximize return on energy expenditure. Our data suggest that increased dopamine decreases thriftiness, facilitating energy expenditure, and permitting greater exploration. Conversely, this implies that decreased dopamine increases thriftiness, favoring the exploitation of prior reward learning, and diminishing exploration. This perspective provides a different window onto the role dopamine may play in behavioral flexibility and its failure, compulsive behavior. Law of Effect that states:Thorndike  first arOf several responses made to the same situation those which are accompanied or closely followed by satisfaction to the animal will, other things being equal, be more firmly connected with the situation, so that, when it recurs, they will be more likely to recur… the greater the satisfaction, the greater the strengthening…”“In his studies, Thorndike  placed cwhat is learned – an association between stimuli in the environment and the behavior such that when those stimuli are presented, the behavior is more likely to be emitted, (b) how learning occurs – a positive outcome increases the strength of that association increasing its impact on future behavior. Thorndike's formulation articulates the basic concept of instrumental learning: a positive outcome following an emitted behavior (response) strengthens the association between stimuli present during the behavior and the particular response to those stimuli that yielded “satisfaction”  on behavioral choice: “… enabling organisms to overcome obstacles or work-related response costs that separate them from significant stimuli.” Though they propose very different mechanisms, in both theories, dopamine effectively increases motivation , increasing the likelihood of reward pursuit – either by energizing behavior (reducing cost barriers) in Salamone's view or inducing “wanting” (increasing incentive) in Berridge's.The third broad perspective is the motivational view, where dopamine is viewed as serving an activational function, invigorating goal pursuit Robbins, , includicould do all these things. The challenge is to discern how these potential aspects of dopamine function may be integrated. Here we will focus on integrating the reinforcement learning and motivational views into a single framework.Though these different perspectives are often viewed as competing hypotheses and inspire controversy, there is nothing inherently mutually exclusive between them. It is conceivable that dopamine The potential problem with stimulus-response learning as a mechanism controlling behavior is a lack of flexibility: organisms could become enslaved to their prior reward history, simply emitting learned, reinforced responses to stimuli. Indeed, behaviorists in the early to middle twentieth century saw principles of S-R learning as a way to shape and control human behavior Skinner, , 1971. Twithin the system, a mechanism that prevents an organism from becoming a slavish automaton to arbitrary experiences of reward.The primary check on slavish S-R responding is generally believed to be a cognitive, deliberative system that exerts executive control, inhibiting and intervening in the automatic responses that might otherwise be generated by reinforced stimulus-response associations. This is reflected in theories of addiction where not only do drug-reinforced S-R associations predominate in an addict's behavioral control, but there is a concomitant failure of executive control , adaptation requires a degree of freedom from responding strictly controlled by learned S-R associations. Always choosing the response that has, in the past, yielded the greatest reward – known as a greedy strategy – is insufficiently flexible. Reward is essentially information about the environment. This information, in turn, assigns values to stimuli, actions, and their associations, which represents an understanding of the world. The challenge for an organism is in arriving at the best understanding of the world: how does an organism know that it “got it right” or that the environment has not changed recently? Thus, new information can be valuable  that arise from accumulating reward experience. More rewarding stimuli and actions will obtain greater value. These values, in term, bias action choices in favor of more rewarding options. The values are updated with new reward experience. The striatum has been implicated as a primary substrate for reinforcement learning where it is believed to play a role in action selection  with millisecond temporal resolution appropriate to signal discrete events, such as cues and rewards  on behavior, suggesting a potential way to integrate tonic dopamine and its putative motivational effects into reinforcement learning models as a mechanism regulating the beta, or explore-exploit, parameter  if so, what is the direction of dopamine's modulation of beta? Prior literature would suggest that increased tonic dopamine would increase the impact of reward on behavioral choice. Thus, we might expect that hyperdopaminergic mice will show increased beta and be more controlled by reward history.Given that dopamine is widely associated with enhanced pursuit of reward and believed to underlie compulsive behavioral disorders such as addiction, we asked whether increased tonic dopamine would impair behavioral flexibility  and one was always expensive where the cost incremented across the experiment (FR20-250). Which lever was which, however, randomly changed every 20–40 min. As a consequence, the mice had to monitor their on-going reward and constantly update the value of each lever based on recent returns. To assess the role of tonic dopamine, we used mice with a knock-down in the dopamine transporter (DATkd) that results in increased extracellular dopamine and an increased rate of tonic activity  interacting processes mediated by different neural substrates. Thus, in our studies, enhanced dopamine resulted in decreased exploitation (decreased coupling between reward history and choice), allowing exploration. The degree to which the behavioral strategy of equally sampling two familiar levers represents exploration is unclear. In the remainder of the review, we argue that dopamine favors exploration; however, we view this primarily as permissive. We remain agnostic as to what mechanisms direct exploration  how much energy do I have to expend and (2) how thrifty do I need to be in using available energy . The balance between exploration and exploitation can be thought of, energetically, as regulating thriftiness. In a reward/energy rich environment, there is no need to conserve or be frugal with energy expenditure. In such an environment, increasing exploration and energy expended in foraging (or other behaviors) represents an adaptive decrease in thriftiness. In contrast, in reward/energy poor environments, frugality in energy expenditure is critical. Thus, in environments with a low average rate of reward , exploiting prior experience to increase thriftiness and maximize return on expenditures is adaptive Figure . From ththrift, the degree to which an organism needs to exploit its prior knowledge to maximize return on energy investment.Importantly, this is not to suggest that dopamine is a sole regulator of the balance between exploration and exploitation. Elegant work by Aston-Jones and Cohen  has suggdegree to which one's behavior is ruled by the law of effect. This freedom, however, is not arbitrary or absolute. Instead, it arises in response to the richness of the environment – fundamentally, the energy environment – which we suggest is conveyed by tonic dopamine.The foregoing discussion suggests that organisms need not become slavish automatons responding to arbitrary reward history, even without elaborate cognitive, executive controls. Viewing Thorndike's  basic inreduced dopamine function (Di Chiara et al., Most views of dopamine and reward argue that dopamine increases the yoke between reward and behavioral choice. The perspective outlined here suggests that dopamine regulates the yoke between energy availability, or the richness of the environment, and the thriftiness of behavioral energy expenditure. Ultimately, this view may help resolve paradoxes in the dopamine field. For example, it is paradoxical that obesity has been associated with The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Escherichia coli and Salmonella enteritidis. Moreover, lysis efficiency, as measured by the OD600 value, reached 1.0 (109 CFU), avoiding the bottleneck problem observed with other bacterial lysis procedures, which results in a low concentration of bacteria in suspension, and consequent low production of bacterial ghosts. Our results may provide a promising avenue for the development of bacterial ghost vaccines.Bacterial ghosts that are generated using the regulated PhiX174 lysis gene E offer a new avenue for the study of inactivated vaccines. Here, we constructed a library of mutant gene E using a gene-shuffling technique. After screening and recombination with the prokaryotic non-fusion expression vector pBV220, two lysis plasmids were selected. Among which, a novel mutant E gene (named mE), consisting of a 74-bp non-encoding sequence at 5'-end and a 201-bp gene ΔE, significantly increased the lysis effect on prokaryotic  The lysis gene E of coliphage PhiX174 encodes a 91-amino acid (aa) residue protein of approximately 10 kDa that mediates inhibition of peptidoglycan biosynthesis of Gram-negative bacteria membrane. BGs retE. coli strain DH5α, lysis efficiency was 98%. Using parameters from previous studies, the optical density (OD) value at 600 nm of the bacterial suspension was maintained at 0.4 (106 CFU) by adding chloramphenicol (50 μg/mL) to inhibit bacterial proliferation [In a previous study, we constructed a lysis plasmid pBV-E from the PhiX174 lysis gene E and the plasmid pBV220. Upon transformation of E. coli and Salmonella enteriditis.To address these technical problems, the current study focused on the function of the bacteriophage PhiX174 lysis gene E. Mutations were introduced by gene shuffling, and after screening and recombination with the pBV220 vector, novel mutants of lysis E gene (named mEs) were selected and the lysis plasmid pBV-mEs were characterized. Plasmids pBV-mEs may be a useful tool for producing BGs of Gram-negative bacteria such as E. coli strain JL09 and S. enteriditis strain DH09 were isolated and identified in our lab. Bacteriophage PhiX174 was purchased from NEB . E. coli strain DH5α was maintained in our laboratory.Plasmid pBV220 was a kind gift from Professor Gui-hong Zhang of South China Agricultural University. It is a prokaryotic non-fusion expression vector with multiple cloning sites (MCS) adjacent with Shine-Dalgarno (SD) sequence, and a high-copy number plasmid with a gene for ampicillin resistance and a λPL/PR-cI857 temperature-sensitive system that can express target gene inserted in MCS at 42°C Figure . E. coliGAA TTC ATG GTA CGC TGG ACT TTG TGG-3'(EcoR I restriction site was underlined), downstream primer LysisE-L: 5'-AGG GGA TCC GAG CTC TCA CTC CTT CCG-3'(BamH I restriction site was underline). Gene E was amplified using PhiX174 double-stranded DNA as template, in a 50 μL reaction with amplification cycle parameters: 5 min at 95°C; 30 cycles of 30 s at 94°C, 30 s at 59°C, 1 min at 72°C, 5 min at 4°C. PCR products were resolved on 0.9% agarose gels, and the 300-bp gene E fragment was extracted with a gel extraction kit .Primers were designed according to the sequence of PhiX174 lysis gene E [DNA shuffling was carried out according to Evans  with minEcoR I- and BamH I-digested fragments of mutant gene E were inserted into pBV220 and transformed into DH5α competent cells, which were spread on LB plates containing ampicillin. Single colonies were picked and screened by colony PCR with primers LysisE-U and LysisE-L. Positive clones were cultured at 37°C and sent to Invitrogen Inc. (Shanghai) for sequencing. Expression of lysis gene E or mutants of E was induced at 42°C. After bacteriolysis, the bacterial suspension was diluted to an appropriate concentration and spread on LB containing ampicillin. After 37°C for 12 h, colonies were counted and the results expressed as CFU/ml. The lysis rate was calculated using the following formula: lysis rate = (1- CFU after induction/CFU before induction) × 100%. Plasmids with high lysis rates were selected and sequenced, and those with the highest lysis rate were electroporated into the clinically isolated bacterial strains E. coli JL09 and S. enteriditis DH09. Analysis, and calculation and comparison of lysis rates used the protocol above. The corresponding bacterial strain harboring pBV220 was used as control.Purified g for 10 min, before fixing with 2.5% glutaraldehyde (pH 7.2) for 1.5 h at 4°C. Samples were washed three times for 10 min with 0.01 M phosphate-buffered saline (PBS). Dehydration steps were 10 min each in 50%, 70%, 80%, and 90% ethanol, a 1:1 mixture of 100% ethanol and tert-butanol, and pure tert-butanol. After lyophilization for approximately 4 h, samples were placed on the microscope platform, coated with a layer of heavy metal and examined.Sample preparation for SEM  was by centrifugation of a induced bacterial suspension at 3000 × Sample preparation for TEM  was as above, except fixing was for 2-3 h. Samples were washed as above, and fixed with 2% osmic acid for 1.5 h, followed by three washings of 10 min each with 0.1 M PBS. Dehydration steps were 10 min each in 50%, 70%, 80%, 90%, and 100% ethanol. Samples were immersed in 1:1, 1:2, or 1:3 mixtures of SPI-Pon 812 resin (emicron) and acetone, before immersing overnight in 100% 812 resin. Resin-soaked sample blocks were polymerized at 70°C for 48 h, and samples sectioned and stained with uranyl acetate and lead citrate, before microscopy.The size of the PCR-amplified lysis gene E fragment was 276 bp, as expected Figure . The purEcoR I and Bam H I and inserted into the vector pBV220, and transformed into E. coli DH5α. Selection on LB containing ampicillin generated a lysis plasmid library of gene E mutants.Gel-purified mutants fragments were digested with EcoR I- and BamH I sites, the plasmid pBV-ΔE had lysis rate of 56.427%. and No. 4 (p4) had the highest lysis rates, at 99.999%. No lysis was observed in control group pBV220. Sequencing and comparison indicated that p3 and p4 share 100% identity with each other and share 98% identity with pBV-E and other four non-lysis plasmids Figure . A singl%.Figure  Thus, thThe results of lysis kinetics experiments showed that pBV-mE had very high lysis efficiency against DH5α cells. Effective lysis occurred at all tested OD values , and progressed rapidly, with lysis completed within 2 h Figure .E. coli and S. enteriditis. Comparison of SEM and TEM photographs of the cell surface of E. coli and S. enteriditis before and after lysis showed that BGs retained the basic cell morphology of the bacterial cells, but displayed obvious surface wrinkles from the loss of intracellular contents. Arrow c in Figure S. enteriditis BGs, located mainly at the cell poles. Ejection of the intracellular contents from the lysis tunnels can be seen, consistent with a previous report [Plasmid pBV-mE had the same lysis effect on both s report , althougGenetic inactivation of Gram-negative bacteria by inducing a regulated bacteriophage PhiX174 lysis gene E provides a new avenue for inactivated vaccine research . E-proteE. coli strain with previously reported constructs with a single pR or pL promoter [600 of 1.0 (109cfu) are sufficiently concentrated for vaccination, and the observed 99.999% lysis efficiency of pBV-mE was higher than another lysis plasmid, pBV-E (98%) that we previously generated [In this study, a 275-bp mE gene was selected from a library of mutant lysis gene E generated by DNA shuffling. The mutant contained a 74-bp non-encoding sequence and a 201-bp lysis gene ΔE. A lysis plasmid containing the mE gene driven by dual pR and pL promoters significantly increased lysis efficiency over the 95% efficiency in the same promoter . The lysE. coli and Salmonella showed good lysability, the lysis efficiency of protein mE was significantly higher than lysis protein E in both bacteria. Previous studies showed that the N-terminal 51 aa of PhiX174 lysis protein E are necessary for lysis activity [in vivo and the loss of lysis function. The initiation of cell division, rather than any specific functions of the septotome has been shown to play a pivotal role in protein E-mediated Gram-negative bacteriolysis [While both activity  and the activity . Anotheractivity . The C-tactivity . It was riolysis .It is interesting that our results differ from those of previous studies. For plasmid p1 and p6 with observed enhancement of lysis effect, single deletion of A from the 5'-end of E gene converted the first 75-bp encoding sequence to 74-bp non-encoding region, which resulted in the loss of first 25 aa in wild-type E protein. Considering the length of the mE protein is sufficient for crossing the membrane, we propose that lysis activity is not located only within the first 29 aa of protein E, but rather that sequences downstream of aa 25 also have lysis activity. However, so far as now, the detailed mechanism of E-mediated lysis is still obscure, let alone the mE protein. Therefore, we would work on the assumption as follows.It should be pointed out that, after sequence comparison, we found the similarity of this 74-bp sequence with many well-known viral enhancers Figure . MoreoveIn conclusion, the results from our study could indicate a potentially new avenue to solve two key problems in BG research and vaccine production: low bacterial concentration and low BG production. More work is needed to confirm the above assumptions about mE gene. Identification and characterization of 74-bp enhancer or enhance-like or promoter-like element is carrying out.The authors declare that they have no competing interests.SYY did laboratory testing, analyzed the test results, wrote and edited the manuscript. WP and WS did laboratory testing and co-wrote the manuscript. LY, HFL, HLZ, CLW, YHC and YZL did laboratory testing. SGL is the leader of the study group and organized the overall project and helped edit the manuscript. All the authors read and approve the final manuscript.


Polyurethane nanofibers with different concentration of CaCl2 were electrospun, and their bioactivity evaluation was conducted by incubating in biomimetic simulated body fluid (SBF) solution. The morphology, structure and thermal properties of the polyurethane/CaCl2 composite nanofibers were characterized by means of scanning electron microscopy (SEM), field-emission scanning electron microscopy, energy dispersive X-ray spectroscopy, X-ray diffraction, Fourier transform infrared spectroscopy and thermogravimetry. SEM images revealed that the CaCl2 salt incorporated homogeneously to form well-oriented nanofibers with smooth surface and uniform diameters along their lengths. The SBF incubation test confirmed the formation of apatite-like materials, exhibiting enhanced bioactive behavior of the polyurethane/CaCl2 composite nanofibers. This study demonstrated that the electrospun polyurethane containing CaCl2 composite nanofibers enhanced the in vitro bioactivity and supports the growth of apatite-like materials.Polyurethane nanofibers containing calcium chloride (CaCl It is interesting to note that the use of CaCl2 salt is salient to entrap the cells and make them grow faster [2 incorporation in the polyurethane nanofibers during electrospinning to enhance the bioactivity behavior of the composites.Electrospinning of biologically significant polymers has increased dramatically since the electrospun membranes were identified as a candidate for guided tissue regeneration applications -4. In paw faster . Calcium2, we synthesized polyurethane nanofiber mats with different concentrations of CaCl2 salt. The hydroxyapatite was coated by the biomimetic method on as-spun nanofibers by incubating in the simulated body fluid (SBF) solution for different timings to observe the formation of apatite-like materials. We performed extensive analysis to confirm the formation of apatite-like materials on the electrospun polyurethane/CaCl2 composite nanofibers, and we proposed a likely process for the mineralization of hydroxyapatite after incubating in the SBF solution.In this study, in order to obtain the role of CaCl2  was used to prepare nanofiber mats. The polymer solution with CaCl2 was kept for 12 h to dissolve completely in the solvent. Adding more amount of CaCl2 in the solution did not dissolve properly, which resulted in the non-electrospinnability of the polymer solution. Therefore, we optimized the CaCl2 content (up to 2 wt%) in the polymer solution to obtain a complete mixing. A high voltage power supply  of 22 kV to the syringe micro-tip was supplied to electrospin the nanofibers. The polymer solution was fed to the 5-ml syringe with a plastic micro-tip. The tip-to-collector distance was kept at 15 cm. During electrospinning, the drum was rotated at a constant speed by a DC motor to collect the developing nanofibers. All experiments were performed at room temperature.Polyurethane  was purchased from Cardio Tech, Japan. 2-butanone (MEK) and NN-dimethylformamide (DMF)  were used as solvents without further purification. Polyurethane solution with 10 wt% was prepared by dissolving in MEK and DMF. Polyurethane with 0, 1, 1.5 and 2 wt% of CaCl2 composite nanofibers in SBF solution was observed by using scanning electron microscopy . The SEM images were obtained for different durations  of incubation in SBF to monitor the mineralization of apatite-like materials on the nanofiber surfaces. The chemical composition of the polyurethane/CaCl2 composite nanofibers was analyzed by using energy dispersive X-ray (EDX) spectrometer attached to the field-emission scanning electron microscope (FE-SEM). Structural characterization was carried out by X-ray diffraction in a Rigaku X-ray diffractometer (XRD) operated with Cu-Kα radiation (λ = 1.540 Å). The bonding configurations of the samples were characterized by means of Fourier transform infrared (FTIR) spectroscopy. Thermogravimetric analysis  was carried out for the electrospun mats under nitrogen ambient with a flow rate of 20 ml/min. The samples were heated from 30 to 600°C at a rate of 10°C/min, and the differential thermogravimetry graph was recorded.The in vitro bioactivity analysis was performed by using SBF solution. The recipe for the preparation of SBF was adopted from the method reported elsewhere . The SBF2 salt was mixed in the polyurethane polymer solution and then fabricated as composite nanofibers. Figure 2 salt with 0, 1, 1.5 and 2 wt%. These as-spun polyurethane nanofibers exhibited a smooth surface and uniform diameters along their lengths. By varying the experimental parameters such as, applied voltage, syringe micro-tip distance, solvent concentration, amount of salt content and flow rate, one can obtain highly uniform ultrafine nanofibers with different pore sizes. As shown in Figures 1b12 salt in the polymer solution. This kind of phenomenon is in good agreement with those of our previous reports [2 can decrease the fiber diameter and also forming ultrafine mesh–shaped nanofibers in between the main fibers. The density of these mesh-like nanofibers was increased monotonically with increasing CaCl2 concentration from 1 to 2 wt%. The diameter of these ultrafine nanofibers (~8–20 nm) was of one order lower than those of the main fibers (~200–400 nm), which resulted in a large surface area-to-volume ratio and interconnected porosity. These features are considered to be of particular interest as they mimic the extracellular matrix as present in the natural tissue and have been shown to induce a significant increase in protein absorption and cell adhesion, compared to their micro-size counterparts [2 on the formation of apatite nucleation in the SBF solution, different concentrations of CaCl2 incorporated in the polyurethane nanofibers were synthesized.The CaCl reports , which cibers ~8– nm was o2 salt with 0, 1, 1.5 and 2 wt%. As shown in Figure 2 salt. By adding CaCl2 salt in polyurethane nanofibers, their compositions were increased dramatically with increasing concentration as shown in Figure 2 salt with polyurethane nanofibers was confirmed by the EDX spectra.The chemical composition of the as-spun nanofiber mats was analyzed by EDX spectrometer attached with FE-SEM. Figure 2 incorporation in polyurethane nanofibers for the formation of apatite-like materials after the incubation in SBF solutions, SEM analysis was performed. Figure 2 salt with 0, 1, 1.5 and 2 wt% after immersion in SBF solution for 2 h and 7 days, respectively. As expected, the pristine polyurethane nanofibers did not show any formation of apatite-like materials as shown in Figure 2 concentration. As shown in Figure 2 incorporated polyurethane nanofibers than on the pristine one. Generally, the apatite-like material formation does not occur spontaneously on most of the synthetic polymers without pretreatment to activate their surfaces [2 can be incorporated easily by electrospinning process and revealed excellent ability of apatite formation on its surface after soaking in SBF solution. The formation of apatite-like materials can be attributed to the partial dissolution of CaCl2 salt and the subsequent release of calcium ion. The exposure of calcium on the polyurethane nanofibers surfaces can provide nucleation sites for apatite-like material formation and favors further enhanced growth. To confirm the formation of apatite-like materials in the polymer mats after immersion in SBF solution, we performed the SEM-EDX analysis. Figure 2 salt after incubation in SBF solution for 7 days. EDX data clearly confirmed the presence of Ca and P in the polymer mat.To observe the role of CaClsurfaces ,23. In s2 composite nanofibers before and after incubation in SBF solution. Figure 2 concentrations before and after soaking in SBF solution. A broad peak at about 2θ = 19° was exhibited in all the samples, which correspond to the characteristic diffraction pattern for the polyurethane polymer. This diffraction peak appears to be very broad due to the amorphous nature of polyurethane. Besides, a feeble peak, which corresponds to the (222) CaCl2 plane, was observed at 24.5°. The diffraction patterns of all samples after 7-day incubation in SBF solution showed two clear peaks at 31.8 and 46.5° corresponding to (211) and (222) main reflection planes, which coincided well with the standard data for hydroxyapatite (JCPDS No. 09-0432). The XRD data clearly confirmed that the apatite-like materials were also formed on pristine polyurethane nanofibers. However, the intensity of the peak was increased with increasing CaCl2 concentration. The diffraction peaks of polyurethane/CaCl2 composite nanofibers after incubation in SBF were observed to be sharper and stronger suggesting that the apatite-like materials were formed with higher levels of crystallinity. The XRD data confirmed the apatite-like materials formed in polyurethane/CaCl2 composite nanofiber, which is in consistent with SEM data.The X-ray diffraction pattern was used to identify the crystallographic structure of the polyurethane/CaCl2 composite nanofibers. Figure 2 before and after incubation in SBF solution. These data clearly revealed the presence of the various vibrational modes corresponding to phosphates and hydroxyl groups. As shown in this figure, the characteristics transmittance bands of the phosphate ion, which appear from 600 to 1,500 cm-1, can be observed in the polyurethane/CaCl2 composite nanofibers. Within this range, the following main peaks at 817 cm-1 (ν1), 1,059 cm-1 (ν2) and 1,098 cm-1 (ν3), 555 and 603 cm-1 (ν4) were assigned to PO43-. And the broad transmittance at 3,356 cm-1 was attributed to the–OH stretching. The intensity of–OH band was observed to be lower for the pristine polyurethane than those of the samples containing CaCl2 owing to the formation of apatite-like materials. In addition, the bands corresponding to CO32- was observed at the wavenumbers 817, 1,409 and 1,537 cm-1, owing to a small amount of CO32- ions present in the apatite structure [2 concentration.FTIR spectroscopy was performed to investigate the functional groups present in the polyurethane/CaCltructure ,25. Comp2 composite nanofibers upon incubation in SBF solution. Figure 2 salt for before and after incubation in SBF solution. The TGA results showed that the polyurethane nanofibers were decomposed in a single step. The onset decomposition temperature of polyurethane nanofibers was 425°C. As expected, the induction of biological apatite material over nanofibers surfaces would result in an increased residual weight during TGA analyses. It was observed that the polyurethane/CaCl2 composite nanofibers after incubation in SBF solution showed high residual weights than those without incubation. The residual weight dramatically increased with increasing CaCl2 concentration in the polyurethane nanofibers as shown in solid lines in Figure 2 concentration in polyurethane nanofibers. The weight loss dramatically increased for the sample with CaCl2 content of 2 wt% in which the CaCl2 acted as a heterogeneous nucleating agent. Similar kind of phenomenon was also recently reported on electrospun composite consisting of hydroxyapatite/β-tricalcium phosphate and poly(ε-caprolactone) as a potential scaffold for bone tissue engineering application [2 in polyurethane nanofibers accelerates the formation of apatite aggregation during incubation in SBF solution. The TGA data are in good agreement with SEM results in which the apatite materials were formed on the nanofibers surfaces upon incubation in SBF solution.TGA analyses were performed to confirm the formation of apatite-like materials in the polyurethane/CaCllication . The pre2 composite nanofibers during immersion in SBF. The presence of calcium in polyurethane nanofibers accelerates the formation of apatite aggregation during incubation in the SBF solution. Development of the apatite-like materials on the polyurethane/CaCl2 composite nanofiber surface depends on the presence of nucleation sites and sufficient concentration of ionic species necessary to form the apatite. Relatively high specific surface area of the rugged calcium ions on the surfaces of the polyurethane/CaCl2 composite nanofibers provides the nucleation sites for apatite in SBF compared with pristine polyurethane nanofibers, results in more energetic nucleation sites. When a nucleus forms on the calcium ions, the contact area between nanofiber surface and nucleus is small enough such that a preferred orientation growth habit is active and initiates faster growth. Consequently, a large number of apatite nuclei are formed on the polyurethane/CaCl2 composite nanofibers surface. Then, the ions necessary for the growth can diffuse to the nucleation site from all directions and provide many nucleation sites resulting in spherical bunches of apatite-like material growth [see Figure 2 composite nanofibers, the bone-like apatite crystals appear to be formed via intermediate products of calcium phosphate. The result demonstrated the fast nucleation of apatite-like materials with increasing concentration of CaCl2 in polyurethane composite nanofibers, which can be utilized for the guided bone regeneration membrane.Figure 2 were successfully produced by electrospinning technique. These as-spun nanofibers exhibited a smooth surface and uniform diameters without any beads along their lengths. Addition of CaCl2 salt resulted in the formation of mesh-like ultrafine nanofibers in between the main fibers. The formation of apatite-like materials was spontaneously commenced, and their size was monotonically increased with increasing CaCl2 concentration after incubation in SBF solution. Good attachment and fast proliferation of apatite-like materials was observed in SEM analysis after incubation in SBF solution. The EDX and XRD data clearly confirmed the presence of apatite-like materials in the polyurethane/CaCl2 composite nanofibers. The results of FTIR and TGA analyses were also revealed the formation of plenty of apatite-like materials on the surfaces of polyurethane/CaCl2 composite nanofibers. Because of good attachment and formation behaviors, there is a potential to utilize polyurethane/CaCl2 composite nanofibers scaffold for the bone regeneration applications.Polyurethane nanofibers containing different concentrations of CaCl


The electrical properties of self-assembled organic crystalline nanofibers are studied by integrating these on field-effect transistor platforms using both top and bottom contact configurations. In the staggered geometries, where the nanofibers are sandwiched between the gate and the source-drain electrodes, a better electrical conduction is observed when compared to the coplanar geometry where the nanofibers are placed over the gate and the source-drain electrodes. Qualitatively different output characteristics were observed for top and bottom contact devices reflecting the significantly different contact resistances. Bottom contact devices are dominated by contact effects, while the top contact device characteristics are determined by the nanofiber bulk properties. It is found that the contact resistance is lower for crystalline nanofibers when compared to amorphous thin films. These results shed light on the charge injection and transport properties for such organic nanostructures and thus constitute a significant step forward toward a nanofiber-based light-emitting device. Inorganic semiconducting crystalline nanowires made from, e.g., Si or III-V materials have been the focus of much research due to the ability to synthesize these in large numbers with well-defined properties, which has led to the demonstration of nanowire field-effect transistors [In the last decade, much attention has been given to one-dimensional nanostructures due to their intriguing physics and in particular their application potential within for example electronics and optoelectronics -3. Inorgnsistors ,5, multinsistors , lasers nsistors , photo dnsistors ,9, and snsistors ,11.p6P) can self-assemble into crystalline nanofibers structures that emit polarized, blue light upon UV excitation [Today, however, the interest in alternative materials to the more conventional inorganic semiconductors is increasing. One example is organic materials based on small molecules, which similarly can be self-assembled into crystalline nanostructures. This can be done either from solution ,13 or bycitation , and it citation .A remaining challenge, however, is the integration of such organic nanofibers into the necessary surrounding circuitry such as metal electrodes for electrical biasing. Essentially, two different strategies can be used: (1) an in situ growth approach, in which the nanostructure is self-assembled directly on the device platform to establish the required electrical connections, and (2) a controlled transfer approach, in which pre-fabricated nanostructures are transferred to a device substrate for electrical wiring. Both strategies have been demonstrated on a wafer scale for inorganic nanowires ,23, and p6P nanofibers implemented in different field-effect transistor (FET) configurations. The p6P nanofibers were first grown on a special growth substrate for epitaxial growth and then transferred to a silicon-based transistor platform. We have recently demonstrated in details how fast and large-scale transfer of organic nanofibers from the growth substrate onto a device platform enables an easy fabrication of a large number of devices  without damaging the morphology and optical properties of the fragile p6P nanofibers. Since the electrical characteristics of organic FETs are known to depend on the exact transistor geometry [In this study, we report results from a study of the electrical properties of geometry , we havegeometry ,27. The geometry .-6 mbar) on top of the transferred and annealed nanofibers through a nanostencil [The type 1 devices, which had a bottom contact/bottom gate  configuration exhibits a lower output current than the staggered geometries due to a high contact resistance associated with the high injection barrier to the organic material [Figure material . In the material .The TC/BG configuration exhibits the highest output current. We propose that this is due to the smaller contact resistance between the nanofibers and the electrodes due to deposition of the electrodes under vacuum, which prevents water residues in the nanofiber-electrode interface in contrast to the bottom contact devices where the nanofiber-electrode interface is created under humid conditions during the transfer. As suggested by Bao and co-workers , moisturThe symmetric characteristics of the TC/BG device as opposed to the asymmetric behavior of the bottom connected devices can be observed in the inset of Figure In Figure p6P film on similar transistor platforms. Around eight times more material was used to form the films compared to the material used to grow the nanofibers. The higher current density for the p6P nanofibers in comparison with the film must be consequence of the crystallinity of the nanofibers, i.e., p6P nanofibers have a long range order compared with thin films which is believed to favor a high charge-carrier mobility as a result of the π-conjugated coupling between the packed molecules [Figure olecules  ) [p6P nanofibers on different transistor configurations to elaborate on the switching behavior.From Figure logIds))  were obtS) for the nanofibers on BC/BG, BC/TG, and TC/BG configurations were found to be 13.7, 9.5, and 7.5 V/decade, respectively. The TC/BG configuration exhibits the lowest subthreshold swing being almost half that of the BC/BG device. For comparison, Klauk et al. [2 as the gate dielectric and found a subthreshold swing of only 0.7 V/decade. Our results is around a decade above this, however, this is not unexpected since the p6P mobility is significantly below that found in pentacene [From the data in Figure k et al.  have stuentacene ,38 and sp6P nanofibers with amorphous thin films shows that both materials exhibit p-type behavior but the fibers conduct significantly better owing to their better crystallinity.In this study, we have for the first time demonstrated integration of transferred organic nanofibers on different field-effect transistor platform configurations, which have been electrically characterized to reveal the significant differences in electrical performance between the different configurations. The coplanar device geometry has a high contact resistance and consequently a poor conduction compared to the staggered geometries. Within the staggered geometries, the top contact geometry shows superior performance to the bottom contact geometry presumably due to a cleaner interface between the contact and the organic material and due to metal penetration into the organic material during contact deposition. The better electrical connection of the top contacts results in the nanofiber transistor output characteristics being dominated by the nanofiber bulk as opposed to the bottom contact devices which exhibit injection limited behavior. A direct comparison of the crystalline Such electrical contacted organic nanostructures can have a range of applications, notably as nanoscale organic light emitters. These can be realized in similar field-effect transistor configurations and are therefore an obvious next subject to be studied. The performance of such organic transistors is influenced by a range of factors and optimization can therefore be pursued for example using other gate dielectrics , electrop6P molecules under high vacuum conditions (p < 10-8 mbar) onto a heated muscovite mica substrate, which was cleaved in air before being immediately transferred to the vacuum chamber. During deposition (rate 0.1 Å · s-1), the substrate temperature was kept at 463 K. This enables the surface diffusion of the molecules and molecular clusters, which then agglomerate and form long, surface-bound, mutually parallel nanofibers with macroscopic lengths (up to millimeters), and nanoscopic cross sections  [p6P deposition were around 40 and 250 nm, respectively, as determined by atomic force microscopy.The nanofibers were prepared by vapor deposition of ometers) . The herThe integration of the nanofibers onto the device platform took place via a special transfer technique, the details of which will be reported elsewhere . In short, the mica substrate with the nanofibers was fixed on the sidewall of a transparent cylinder with an appropriate diameter. The transparency of the cylinder helps to align the nanofibers to the device substrate and also to visualize when the mica and the device substrate are in contact to perform the transfer process. The device substrate was placed on a soft rubber platform to avoid compressing the nanofibers during the transfer, and the nanofibers were transferred by rolling the cylinder with the nanofibers onto the device substrates under conditions of high humidity. After transfer, the chips were annealed at 80°C for 20 min. This procedure was adopted to remove the water adsorbed during the transferring process.2 and realized first by HF etching through the SiO2 layer followed by reactive ion etching 1 μm into the silicon to give a total platform height of 1.2 μm. On each receiver platform, two contact pads (390 μm × 180 μm) were prepared by photolithography, metal deposition (2 nm Ti/30 nm Au) and lift-off. We prepared two different types of substrates to be able to prepare both bottom contact (BC) and top contact (TC) devices. The TC device substrates were ready for nanofiber transfer after the preparation of the contact pads, while the BC substrates were processed additionally with one more sequence of photolithography, metal deposition (2 nm Ti/30 nm Au), and lift-off to form small, closely spaced electrodes, which were connected to the large contact pads, and onto which the nanofibers could be connected to span the gap. Gold was chosen as the electrode material due to its inertness and due to its high work function (5.1 eV) that promotes hole injection into the nanofibers.Silicon-based device substrates were used for integrating the nanofibers with source, drain, and gate electrodes to form a field-effect transistor configuration. The substrates included elevated platforms that were used as receiver platforms for the nanofibers in the subsequent nanofiber transfer step. These platforms, which had a size of 1000 μm × 200 μm, were lithographically patterned on a highly doped silicon substrate with 200 nm thermally grown SiOp6P nanofibers had changed and the nanofibers had a pronounced green appearance as opposed to the clear blue color of "perfect" nanofibers. We attribute this to the generation of defects in the nanofibers, which are known to give rise to peaks in the green part of the spectrum [p6P nanofibers' electrical characteristics and that the original p6P spectrum is also preserved after coating [The nanostencils were prepared from a 525 μm thick silicon wafer coated with a 100 nm low-stress silicon nitride (SiN) layer. The electrode pattern was realized in the frontside SiN layer by photolithography and reactive ion etching, and the membranes were released by photolithography and etching from the wafer backside in KOH solution (28 wt% KOH concentration at 80°C for approx. 9 h). A thin layer of photoresist was applied on the wafer frontside to protect the fragile membranes before dicing. After the initial tests of electrode deposition onto the nanofibers through the nanostencils, it was observed that the photoluminescence spectrum of the spectrum . This co coating . For thep6P thin film [p6P molecules directly onto the device substrates at room temperature resulting in a structure-less film.In addition to the nanofiber devices, hin film  devices The completed devices were inspected using white light microscopy, fluorescence microscopy (excitation wavelength of 365 nm), and scanning electron microscopy. The nanofiber dimensions were determined by tapping mode atomic force microscopy, and the field-effect transistor characteristics were recorded with a probe station and a labview-controlled characterization system based on a data acquisition card and voltage and current amplifiers.BC/BG: bottom contact/bottom gate; BC/TG: bottom contact/top gate; FET: field-effect transistor; OLEFETs: organic light-emitting field-effect transistors; TC/BG: top contact/bottom gate.The authors declare that they have no competing interests.LT was involved in growing of the nanofibers and developing the FET substrates, made the transfer technique, transferred the nanofibers, performed the electrical measurements, contributed in the interpretation of data and drafted the manuscript. JKH developed the project, contributed developing the FET substrates, analyzing the data, drafting the manuscript and revised it, and have given final approval of the version to be published. KTH helped in growing of the nanofibers and developing the FET substrates. HGR revised it for important intellectual content.


Soluble Epoxide Hydrolase (sEH) is a key enzyme in the metabolism and termination of action of epoxyeicosatrienoic acids, derivatives of arachidonic acid, which are protective against ischemic stroke. Mice lacking sEH globally are protected from injury following stroke; however, little is known about the role of endothelial sEH in brain ischemia. We generated transgenic mice with endothelial-specific expression of human sEH (Tie2-hsEH), and assessed the effect of transgenic overexpression of endothelial sEH on endothelium-dependent vascular reactivity and ischemic injury following middle cerebral artery occlusion (MCAO). Compared to wild-type, male Tie2-hsEH mice exhibited impaired vasodilation in response to stimulation with 1 µM acetylcholine as assessed by laser-Doppler perfusion monitoring in an in-vivo cranial window preparation. No difference in infarct size was observed between wild-type and Tie2-hsEH male mice. In females, however, Tie2-hsEH mice sustained larger infarcts in striatum, but not cortex, compared to wild-type mice. Sex difference in ischemic injury was maintained in the cortex of Tie2-hsEH mice. In the striatum, expression of Tie2-hsEH resulted in a sex difference, with larger infarct in females than males. These findings demonstrate that transgenic expression of sEH in endothelium results in impaired endothelium-dependent vasodilation in the cerebral circulation, and that females are more susceptible to enhanced ischemic damage as a result of increased endothelial sEH than males, especially in end-arteriolar striatal region. Epoxyeicosatrienoic acids (EETs) produced in brain play an important role in cerebral blood flow regulation Stroke is a major cause of morbidity and mortality worldwide, with lower incidence in premenopausal women compared to men of the same age. However, the risk of stroke in women greatly increases following menopause We have shown previously that sEH deletion is protective against ischemic injury in vivo, and also that increased sEH activity is detrimental to neurons in vitro following oxygen-glucose deprivation (OGD) The investigation conformed to the Association for Assessment and Accreditation of Laboratory Animal Care AAALAC Accreditation (approved November 2010) and the Office of Laboratory Animal Welfare . The Oregon Health and Science University Animal Care and Use Committee approved all experimentsTransgenic mice expressing Tie2- driven human sEH in endothelial cells on a C57BL/6 genetic background (Tie2-hsEH) have been recently described Mice were subjected to transient focal cerebral ischemia by use of the intraluminal MCAO technique Infarct size was measured 24 hours following MCAO in 2 mm thick coronal sections using 2,3,5-triphenyltetrazolium chloride (TTC) staining. Sections were incubated for 15 minutes in 1.2% TTC in saline at 37°C and fixed in formalin for 24 hours. Sections  were imaged, infarcted (unstained) and viable tissue (stained) areas were measured using MCID software (InterFocus) and integrated across all 5 sections. To discount the influence of edema, infarct size was calculated indirectly by subtracting the uninjured area in the ipsilateral hemisphere from the contralateral hemisphere, then expressing infarct volume as a percentage of the contralateral hemisphere.Anesthesia was induced as above. The head of the mouse was secured in a stereotaxic apparatus with 2% isoflurane in oxygen-enriched air by face mask. The scalp and connective tissue over the parietal cranial bone were removed. Rectal temperature was maintained at 36+/−0.5°C by water pad. An area with 1 mm diameter, 2 mm posterior and 2.5 mm lateral to Bregma, was marked for monitoring by laser-Doppler. A small drill hole was made superior  to the laser-Doppler probe site to expose the dura. The dura was pierced with care not to damage the epidural or pial vessels. A 33-gauge needle connected to a microperfusion pump with PE10 tubing was advanced 1 mm subdurally in a lateral direction (inflow). Another drill hole was made inferior  to the probe site, and the dura was incised for passive drainage of the superfused fluid (ouflow). Cerebrovascular cortical perfusion was monitored using a laser-Doppler flow probe with a tip diameter of 1 mm . The probe was placed in a location over the thinned skull that was devoid of large visible blood vessels. The probe position was kept fixed for the entire experiment, and background lighting was minimized and kept constant. For experiments investigating cortical blood flow in response to the endothelium- dependent dilator acetylcholine, subdural superfusion with artificial cerebrospinal fluid (CSF) started 5 minutes after completion of surgery and continued for 20 minutes at a constant rate of 3 µl/min. Following CSF perfusion acetylcholine (1 µM) was superfused for 90 minutes. Artificial CSF and acetylcholine were prepared daily and the solution was pre-warmed to 37°C.Wild-type (WT) and transgenic mouse tissues were prepared and stained as described previously Expression of human sEH in mouse brain was confirmed after RNA purification of brain tissue with the RNAeasy Mini kit , conversion to cDNA with High Capacity cDNA Archive kit,  and real-time PCR using TaqMan probes to murine GAPDH (Mm 99999915_g1) and human EPHX2 (Hs 00157403_m1) (Applied Biosystems).Plasma DHET levels were determined by liquid chromatography, tandem mass spectroscopy (LC/MS/MS) as described previously t-test, while a 2-way ANOVA with post hoc Holm Sidak test was used for comparisons between multiple groups. The criterion for statistical significance was set at P<0.05. All values are reported as mean +/−S.E.M.Differences between two groups were evaluated with a P<0.05). Medium collected from aortic endothelial cell cultures from Tie2-hsEH mice also had lower arachidonic acid and linoleic acid epoxide:diol ratios  as measured by LC-MS/MS, both of which serve as readouts of increased sEH function (P<0.05).Mice carrying the Tie2-hsEH transgene expressed human sEH transcript and protein in the brain . Immunolfunction  .Endothelium- dependent vasodilation was impaired in mice carrying the Tie2-hsEH transgene compared to wild-type controls . CorticaP>0.05).Since increased endothelial expression of sEH impaired ACh- stimulated vasodilation at baseline, we next investigated whether this endothelial dysfunction is deleterious following MCAO in Tie2-hsEH compared to WT mice. Male mice were subjected to 60 minutes of MCAO and infarct volume was assessed following 24 hours of reperfusion. Infarct volume was assessed histologically by TTC labeling . In maleP<0.05, P<0.05); this difference persisted in the Tie2-hsEH mice . In the striatum, while there was no significant sex difference in infarct size in the WT mice, a sex difference was revealed in mice over-expressing hsEH, with females exhibiting larger infarct volumes than males . To ensure that the insult sustained was comparable between groups and that this observed difference is not due to varying levels of occlusion, cerebral blood flow was measured during and immediately after MCAO  Endothelial over-expression of sEH impairs ACh- induced increase in cerebral blood flow in males. (2) Endothelial over-expression of sEH does not exacerbate ischemic damage following MCAO in males, however (3) females are susceptible to increased ischemic damage following MCAO due to increased sEH expression in brain endothelium, especially in end-arteriolar striatal region. (4) In the cortex, endothelial over-expression of sEH does not impact the observed sex difference in infarct size following MCAO, however (5) in the striatum while there is no sex difference in wild type mice, endothelial over-expression of sEH renders females more susceptible to ischemic damage than males.We have previously demonstrated a role for sEH in mediating vascular function. EETs are known vasodilators; it would follow that knocking out sEH, which hydrolyzes EETs would increase their concentration. Mice with global deletion of the sEH gene display higher cerebral blood flow levels during MCAO compared to WT controls Endothelial response to acetylcholine has been used as a benchmark for measuring endothelial dysfunction Male mice expressing the Tie2-hsEH transgene did not exhibit larger infarct sizes following MCAO. Although perhaps surprising, this can be explained by the fact that sEH is already highly expressed in males, and therefore higher levels may not be deleterious in terms of ischemic injury As discussed earlier, sEH expression in the brain is sexually dimorphic. In whole brain extracts, males exhibit higher protein levels than females; this is reflected in higher 14,15-DHET levels in males than females It is important to note that this study explores the effect of genetic manipulation of sEH specifically in the endothelium, however the effects are still within the context of the whole brain. Other cell types within the brain, including perivascular astrocytes and neurons have been shown to produce both EETs and sEH. In the human brain, sEH is expressed throughout the brain in neurons, oligodendrocytes, astrocytes, endothelial cells and also smooth muscle cells of arterioles In summary, the current study demonstrates a role for endothelial sEH in the regulation of cerebral blood flow. We also confirm the sexual dimorphism observed in endothelial sEH function, with increased sEH expression being detrimental to females but not males in terms of infarct size following MCAO. These findings suggest that endothelial sEH is a key regulator of cerebrovascular endothelial function and an important contributor to the sexually dimorphic response to cerebral ischemic injury.


We present TRAVIS , a free program package for analyzing and visualizing Monte Carlo and molecular dynamics trajectories . The aim


CSMD1. The effect of deleting CSMD1 on mouse behavior is unknown. The present study utilized mice with a mutant Csmd1 allele in which the first exon had been ablated (KO mice). All Csmd1 transcripts that included the first exon were absent in the brains of KO mice, but there was persistent expression of at least one other transcript that does not include the first exon. Wild type (WT), heterozygous (HET), and KO mice were assessed using several well-established behavioral paradigms that model aspects of schizophrenia. Csmd1 KO mice did not differ from wild-type littermates for sensorimotor gating (measured as prepulse inhibition), social interaction, anhedonia (measured by sucrose preference), or sensitivity to the locomotor stimulant effects of the dopaminergic agent d-amphetamine. These data demonstrate that loss of Csmd1 transcripts that include the first exon does not alter multiple well-established behaviors that model aspects of schizophrenia. The SNP most strongly associated with schizophrenia in humans is between exons 3 and 4; therefore, ablation of exon 1 appeared to be a logical animal model. Nevertheless, future studies should consider alternative mouse models including gain-of-function mutations, and loss-of-function mutations that target alternative transcripts of Csmd1.Schizophrenia is a debilitating psychotic disorder that affects up to 1.5% of the population worldwide. Two recent studies in humans identified genome-wide significant associations between schizophrenia and single-nucleotide polymorphisms (SNPs) in an intron of  Schizophrenia is a psychotic disorder characterized by positive symptoms  and negative symptoms  The exact etiology of schizophrenia is unknown, but human studies have demonstrated significant brain abnormalities, including decreased brain volume, reduced activity of the frontal and temporal lobes, and aberrant neural connectivity CSMD1, which encodes CUB and Sushi multiple domains 1 CSMD1It is clear that schizophrenia has a genetic component, as schizophrenia tends to aggregate in families, and there is an approximately three-fold increase in concordance among monozygotic twins compared to dizygotic twins CSMD1 and various diseases, including numerous cancers CSMD1 has also been associated with other diseases of the central nervous system (CNS), including multiple sclerosis CSMD1's function and its mechanism in disease. In fact, only two studies have investigated CSMD1's cellular function. One study demonstrated that CSMD1 has tumor suppressive properties in vitroCsmd1 expression in the CNS of rats, particularly in the neural growth cone during development Several human genetic studies have also identified associations between Csmd1 allele in which the first exon had been ablated. We tested these mice for behaviors modeling aspects of schizophrenia.In the present study, we characterized mice carrying a mutant Csmd1 knock-out (KO) mice were obtained from Taconic Farms (TF0137) and were originally created by Lexicon pharmaceuticals tm1LexCsmd1/Mmucd. Mice from our colony are now available from MMRRC (http://www.mmrrc.org/) with the stock number 32236. Mice for this study were generated by breeding heterozygous (HET) males and females and testing littermate offspring, which should control for the poorly-defined genetic background. All experiments were approved by the IACUC at the University of Chicago.Constitutive Actb, exons 1–2 or exon 70 of Csmd1. Csmd1 expression was normalized to Actb and reported as fold change versus WT.RNA was extracted from whole brains of adult male mice  using the RNeasy kit with DNase digestion (Qiagen). cDNA was generated by reverse transcription  using oligo dT primers (Invitrogen). cDNA was used as a template in qPCR using SYBR reagents (Applied Biosystems). Each sample was run in duplicate, and the values were averaged for each individual before statistical analysis. Primers targeted PPI was measured as described previously prepulse/SRpulse]), where SRprepulse is the average startle amplitude for prepulse trials, and SRpulse is the average startle amplitude for pulse-alone trials in the second and third testing blocks. The ‘no stimulus’ trials were used to identify technical problems but were not used to calculate any of the phenotypes assessed in this study.The startle response measure (“startle”) was the average startle amplitude for all of the pulse-alone trials and is expressed in arbitrary units. PPI at each intensity was calculated using the formula %PPI = 100%− were tested for socialization using a modified SI test Anhedonia was assessed using the SP test d-amphetamine using a three-day paradigm as described previously d-amphetamine HCl (Sigma-Aldrich). Immediately after injection, mice were placed in the open field for 1 hour. Locomotor data were collected and processed using the manufacturer's software (AccuScan Instruments). Group sizes for each dose were as follows: 2 mg/kg: KO = 12; HET = 16, WT = 6; 4 mg/kg: KO = 14, HET = 27, WT = 16, 8 mg/kg: KO = 25, HET = 29, WT = 17.Male and female mice were tested for locomotor response to d-amphetamine-induced locomotor activity, we used doses of d-amphetamine and genotype as between-groups factors. For the d-amphetamine data, we also examined time as a within-groups factor, but it did not interact with genotype, so we collapsed across time and used total distance traveled as the dependent variable. We also considered change from activity at baseline (day 2), but all results were similar, so here we present the data as activity on day 3. Post-hoc tests were performed using Newman–Keuls. In all cases P<0.05 was considered statistically significant. In studies that included male and female mice, we did not observe any interactions when sex was included as a factor (e.g. sex*genotype); therefore, both sexes were pooled for all final analyses.All statistical tests were performed with StatView (SAS Institute). One-way ANOVAs with genotype as the factor were used except in the following cases. For PPI, we used genotype as a between-groups factor and %PPI as a within-group factor. For SI, we used genotype as a between-groups factor and target presence as a within-group factor. For Csmd1 is a large gene that spans over 1.6 Mb and has 70 exons , the first three of which begin with exon 1  with two different primer sets, one specific for exons 1–2  and the other for exon 70 (included in transcripts Csmd1-3 and -4). Compared to WT mice, KO mice displayed a 99.4% reduction in expression of exons 1–2 of Csmd1, suggesting that the KO allele functioned as expected , social interaction, anhedonia (sucrose preference), and sensitivity to a dopaminergic challenge (d-amphetamine induced locomotor response).Using P = 0.78; P<0.001); however, there was no significant main effect of genotype on PPI , nor was there a significant interaction between genotype and prepulse intensity . Thus, Csmd1 KO mice had normal sensorimotor gating.We assessed sensorimotor gating by measuring PPI of the startle response. PPI refers to the ability of a weak, initial stimulus (prepulse) to inhibit the response to a subsequent startling stimulus P<0.001; P = 0.77), nor was there a significant interaction between genotype and target presence . Thus, we did not identify any deficits in social behavior in Csmd1 KO mice.Schizophrenic patients display negative symptoms including reduced social behavior, which we modeled using the SI test P = 0.70; P = 0.54; Csmd1 KO mice.Another negative symptom associated with schizophrenia is anhedonia, which we modeled using the SP test d-amphetamine. Increased locomotor activation from d-amphetamine is thought to model the positive symptoms of schizophrenia d-amphetamine compared to healthy controls P<0.0001; P = 0.96), nor was there an interaction between genotype and dose . Thus, we did not detect altered sensitivity to d-amphetamine in Csmd1 KO mice.We then assessed the psychostimulant response to the dopamine-releasing drug CSMD1 in schizophrenia Csmd1 in behaviors modeling aspects of schizophrenia in mice. To do so, we obtained mice with a null allele of Csmd1 and assessed them for behaviors using well-established tests that investigate different domains affected by schizophrenia, including sensorimotor gating (PPI), social behavior (SI), hedonic behavior (SP), and psychostimulant response to d-amphetamine. We did not observe differences between genotypes in any of these tests. We did not examine learning and memory paradigms, which may be a useful future direction.Human genetic studies have implicated Csmd1 is not essential for a range of behaviors thought to model key aspects of schizophrenia in mice. However, while the KO mice we studied had reduced expression of three transcripts, Csmd1-1, Csmd1-2, and Csmd1-3, there was residual expression of Csmd1-4, because this transcript does not utilize Exon 1  differ between mouse and human. Our study has focused on the four UCSC transcripts in mouse, three of which begin at exon 1. In contrast, while there are also four UCSC CSMD1 transcripts in human, only one of them begins at exon 1. The SNP most strongly associated with schizophrenia in the human GWAS was rs10503253, which lies in the intron between exons 3 and 4. Thus, ablation of exon 1-containing transcript models loss-of-function mutations that include the linkage disequilibrium block identified by the human GWAS results. The human association signal is almost 1 Mb from the start site of the shorter, non-exon 1-containing human transcripts, which suggests that they are less likely to be related to the observed genetic association at rs10503253. Nevertheless, in the absence of any data describing the functional meaning of the human association, it is not clear how best to model this association in mice. Therefore, future studies of alternative Csmd1 transcripts in mice may be warranted. Another point to consider is that the first human GWAS study that implicated CSMD1 actually found stronger evidence implicating CSMD2 than CSMD1Is ablation of exon 1-containing transcripts the best mouse model given the available human GWAS data? While it is difficult to fully address this question without knowing the functional differences underlying the human association signal, some speculation is warranted. The expressed transcripts reported as UCSC Genes (Csmd1 in mice should consider other alleles of the gene, including gain-of-function mutations and alterative transcripts. Additional insight into the functional consequences of the SNPs implicated by human GWAS may also help in designing future mouse models.In summary, we did not identify any differences between WT, HET, and KO mice for a range of phenotypes that model key aspects of schizophrenia. The fact that we utilized mice that only ablated transcripts including the first exon is an important limitation of our study. Future studies of


A new process impurity was detected during the HPLC analysis of Tizanidine hydrochloride (I) batches. The impurity (II) was isolated by preparative HPLC and characterized by NMR and Mass spectral analysis as 5-S-ethyl-N--2,1,3-benzothiadiazol-4-amine hydrochloride. Tizanidine hydrochloride (I), chemically known as 5-chloro-N--2,1,3-benzothiadiazol-4-amine hydrochloride, is a centrally acting alpha2-adrenergic agonist and is used to treat the spasms, cramping and tightness of muscles caused by medical problems such as multiple sclerosis, spastic diplegia, back pain or certain other injuries of the spine or central nervous system.viz. 4-amino-5-chloro-2,1,3-benzothiadiazole (related compound A), N-acetyltizanidine (related compound B) and 1-acetylimidazolodine-2-thione (related compound C) listed in USP monograph in II, which were attributed to an ethyl group. The relatively downfield shift of methylene was due to its attachment to a hetero atom. The presence of only two aromatic methines  together with the absence of isotopic pattern for chlorine in the mass spectrum suggested that S-ethyl group was present in place of chlorine. During the conversion of tizanidine to the corresponding HCl salt, II was also expected to form a HCl salt, which was confirmed by silver nitrate test. Thus, the structure of impurity (II) was established as 5-S-ethyl-N--2, 1,3-benzothiadiazol-4-amine hydrochloride  isothiouronium bromide (III) to tizanidine hydrochloride (I) using ethylenediamine/p-toluenesulfonic acid in toluene/water[The unknown process impurity (II) was obtained as a yellow powder having a purity of 94% by HPLC. The ESI-MS gave a molecular ion peak at  spectra  of procechloride . The str.28 t, J=.3 Hz, 3H


Laforin, encoded by a gene that is mutated in Lafora Disease , is a modular protein composed of a carbohydrate-binding module and a dual-specificity phosphatase domain. Laforin is the founding member of the glucan-phosphatase family and regulates the levels of phosphate present in glycogen. Multiple reports have described the capability of laforin to form dimers, although the function of these dimers and their relationship with LD remains unclear. Recent evidence suggests that laforin dimerization depends on redox conditions, suggesting that disulfide bonds are involved in laforin dimerization. Using site-directed mutagenesis we constructed laforin mutants in which individual cysteine residues were replaced by serine and then tested the ability of each protein to dimerize using recombinant protein as well as a mammalian cell culture assay. Laforin-Cys329Ser was the only Cys/Ser mutant unable to form dimers in both assays. We also generated a laforin truncation lacking the last three amino acids, laforin-Cys329X, and this truncation also failed to dimerize. Interestingly, laforin-Cys329Ser and laforin-Cys329X were able to bind glucans, and maintained wild type phosphatase activity against both exogenous and biologically relevant substrates. Furthermore, laforin-Cys329Ser was fully capable of participating in the ubiquitination process driven by a laforin-malin complex. These results suggest that dimerization is not required for laforin phosphatase activity, glucan binding, or for the formation of a functional laforin-malin complex. Cumulatively, these results suggest that cysteine 329 is specifically involved in the dimerization process of laforin. Therefore, the C329S mutant constitutes a valuable tool to analyze the physiological implications of laforin’s oligomerization. EPM2A gene. Mutations in EPM2A result in a rare, progressive myoclonus epilepsy called Lafora disease  Laforin is a 331 amino acid protein encoded by the EPM2B, in approximately half of the cases EPM2B encodes malin, an E3-ubiquitin ligase of the RING type. Data from multiple labs demonstrate that malin and laforin physically interact and that this interaction is required for ubiquitination of laforin itself The complexity of LD molecular etiology increased with the discovery of another affected locus, in vitroLaforin is classified in the family of atypical Dual Specificity Phosphatases , which belong to the larger Protein Tyrosine Phosphatase (PTP) superfamily in vitro and in vivo and it was postulated that dimerization was a functional feature of the protein that regulated its phosphatase activity To date, no crystal structure of laforin has been determined and thus the specific properties and relative positions of the N-terminal CBM and the C-terminal DSP domain remain uncertain. Interestingly, laforin was found to dimerize both These results have accomplished two complementary goals: first, we determined that Cys329 is necessary for laforin oligomerization; and second, we have obtained an exclusively monomeric form of laforin. This monomeric form of laforin can now be utilized as a valuable tool to elucidate the specific function of laforin dimers in the pathophysiology of Lafora disease as well as being utilized in crystallographic studies to determine the tertiary structure of this protein.For the creation of a multiple amino acid sequence analysis, sequence of human laforin was used as query for the retrieval of entries from the UniprotKB database  Human laforin DNA was amplified using pEG202-laforin Escherichia coli DH5α was used as the host strain for plasmid constructions. E. coli BL21 (RIL) was used for protein production. They were grown in LB  medium supplemented with 50 g/L ampicillin and 30 g/L chloramphenicol. Transformants were grown at 37°C until the absorbance at 600 nm reached a value of around 0.3. IPTG was then added to a final concentration of 0.1 mM, and cultures were maintained at 37°C for 3 h. Cells were harvested and resuspended in 20 mL of sonication buffer . Cells were disrupted by sonication and the fusion proteins purified by passing the extracts through columns containing 1 mL bed volume of amylose resin (N.E. Biolabs). Recombinant proteins were eluted from the column with 500 mM maltose. Gel filtration chromatography was performed using an AKTA Purifier with a HiLoad 16/60 Superdex 200 10/300 GL size exclusion column .In vitro phosphatase assays were performed using 4 µg of recombinant proteins diluted in phosphatase buffer , in the presence of 0.5 mM 3-O-methyl fluorescein phosphate . Reactions were carried out at 37°C in a final volume of 200 µl in 96-well ELISA plates. Phosphatase activity was measured for 3 hours as absorbance at 490 nm. One Unit of phosphatase activity is defined as the one unit of change in the absorbance at 490 nm per min of assay. Values are expressed as specific activity (Units/mg of protein).α ura3 leu2 trp1 lexA::lacZ lexA::HIS3lexA::ADE2] Yeast THY-AP4 strain . Cells were lysed by repeated passage through a 25-gauge needle. To analyze proteins under non-denaturing, non-reducing conditions, cell extracts (25 µg) were diluted in SDS- and DTT-free loading buffer  Human embryonic kidney (HEK293) cells were grown in DMEM (Lonza) supplemented with 100 units/mL penicillin, 100 µg/mL streptomycin, 2 mM glutamine, 10% inactivated fetal bovine serum (GIBCO). 1.5×102P2O7]. Cell lysates were then centrifuged at 13,000×g for 15 min at 4°C. Laforin-malin complexes were immunoprecipitated from supernatants  with anti-myc polyclonal antibody  and visualized by immunoblotting using anti-myc or anti-HA  antibodies.HEK293 cells were transfected with pCMV-HA-malin and pCMV-myc-laforin or pCMV-myc-laforin-C329S, or with the corresponding empty plasmid. Cells were scraped on ice in lysis buffer [50 mM Tris-HCl pH 8; 10 mM KCl, 50 mM EDTA; 15% glycerol, 1% nonidet P-40 (NP-40), complete protease inhibitor cocktail , 1 mM PMSF, 50 mM NaF, 2 mM NaVO4 and 5 mM NaTo study ubiquitination in intact cells, HEK293 cells were transfected with plasmids pCMV-6xHisUbiq , plasmids encoding laforin and the E3-ubiquitin ligase malin and pCMVmyc plasmids encoding the protein of interest, using the Lipofectamine 2000 reagent , according to the manufactureŕs instructions. After 36 hours of transfection, cells were lysed in buffer A . 1 mg of protein of a clarified extract  were incubated in 100 µl TALON column  in the presence of 10 mM imidazole, for 3 hours at room temperature on a rocking platform, to purify His-tagged proteins. The column was then successively washed with 2 mL each of buffer B (buffer A plus 10 mM imidazole), buffer C  and four more times with buffer C adjusted to pH 6.0. Bound proteins were eluted with 50 µl of 2× Laemmli’s sample buffer and analyzed by Western blotting using appropriate antibodies.Values are given as means ± SD of at least three independent experiments. Differences between phosphatase activities of wt laforin and mutant forms are analyzed by two-tailed student’s t-test. The significance has been considered at *p<0.05, **p<0.01 and ***p<0.001, as indicated in each case. Data for protein purification, gel electrophoresis, and ubiquitination is representative of at least three independent determinations.Nematostella vectensis and the protozoan Toxoplasma gondii. Based on the previously described phylogeny, amino acid sequences of laforin orthologs from several vertebrate organisms, one cnidarian, and one protozoon (see Methods for a list of entries) were selected and aligned using ClustalW. The resulting alignment shows a high degree of conservation, especially in the DSP domain. Human laforin contains nine cysteine residues, three of them located in the CBM  and six in the DSP   Figure 1E. coli with no epitope tag and recombinant mutant proteins were purified by affinity purification using an amylose resin. This methodology presents two advantages: first, it avoids the necessity of adding any tag in the purified protein, due to the natural capacity of laforin to bind carbohydrates; and second, it serves as a test of CBM functionality in each laforin mutant. All mutant proteins were successfully purified using this strategy with the exception of C278S. In this case, we were unable to obtain any recombinant protein from E. coli. In fact, laforin-C278S did not even express in E. coli and we did not observe laforin-C278S in either the soluble or the pellet fraction from the bacterial extracts. We also attempted to express laforin-C278S in a yeast model but again, we were unable to recover laforin-C278S from this system either. For this reason, the mutant form of laforin C278S was not further studied in this work. The rest of the seven mutants resulted in soluble proteins that all bound the amylose resin.Next, we generated homology models (see Methods) to estimate the location and solvent accessibility of each cysteine residue. The cysteine residues located in the CBM appear located in a surface exposed site and far from the residues involved in carbohydrate binding . Purified recombinant protein samples were analyzed by this method, Western blotted, and detected using anti-laforin antibody , a prototypical protein phosphatase that does not bind amylopectin After determining that laforin-C329X behaved similar to laforin-C329S with respect to dimerization, we then sought to determine the biochemical properties of laforin-C329X. First, we tested if laforin-C329X could bind glucans. We utilized a previously established glucan-binding assay for these experiments These results confirm that mutation of Cys329 abrogates the propensity of laforin to form dimers. This finding prompted us to assess the effects of mutating Cys329 using a mammalian cell model in order to define the implications of laforin oligomerization in a more biologically relevant system.As discussed in the introduction, laforin forms a complex with the E3 ubiquitin ligase malin that ubiquitinates multiple substrates involved in glycogen metabolism. We previously defined that laforin dimerization was not required for the interaction between laforin and malin 6-tagged ubiquitin plasmid. Ubiquitinated proteins from crude extracts were purified using a metal affinity resin as previously described First, we proceeded to test if laforin-C329S was still able to physically interact with malin. We analyzed this interaction by yeast two-hybrid analysis and found no significant differences between the interaction of laforin WT and C329S with malin Click here for additional data file.Table S2Primers used for mutagenesis analysis. Mutagenesis was performed by PCR reactions combining laforin terminal primers (upper panel) with internal primers harboring each mutation (down panel). Sequences are written in 5′–3′ sense.(DOCX)Click here for additional data file.


Optimality models of evolution, which ignore genetic details and focus on natural selection, are widely used but sometimes criticized as oversimplifications. Their utility for quantitatively predicting phenotypic evolution can be tested experimentally. One such model predicts optimal bacteriophage lysis interval, how long a virus should produce progeny before lysing its host bacterium to release them. The genetic basis of this life history trait is well studied in many easily propagated phages, making it possible to test the model across a variety of environments and taxa.We adapted two related small single-stranded DNA phages, ΦX174 and ST-1, to various conditions. The model predicted the evolution of the lysis interval in response to host density and other environmental factors. In all cases the initial phages lysed later than predicted. The ΦX174 lysis interval did not evolve detectably when the phage was adapted to normal hosts, indicating complete failure of optimality predictions. ΦX174 grown on slyD-defective hosts which initially entirely prevented lysis readily recovered to a lysis interval similar to that attained on normal hosts. Finally, the lysis interval still evolved to the same endpoint when the environment was altered to delay optimal lysis interval. ST-1 lysis interval evolved to be ~2 min shorter, qualitatively in accord with predictions. However, there were no changes in the single known lysis gene. Part of ST-1's total lysis time evolution consisted of an earlier start to progeny production, an unpredicted phenotypic response outside the boundaries of the optimality model.The consistent failure of the optimality model suggests that constraint and genetic details affect quantitative and even qualitative success of optimality predictions. Several features of ST-1 adaptation show that lysis time is best understood as an output of multiple traits, rather than in isolation. Models that describe evolutionary processes must strike a balance between simplicity and detail. At one end of the spectrum are optimality models, which predict phenotypic evolution based on natural selection in the context of ecology, often neglecting genetics except in the form of simple 'trade-offs' between phenotypic alternatives. The appeal of such models is in their apparent generality, enabling prediction across a wide variety of organisms and environments with minimal input about genetic parameters. The weakness of optimality models is argued to lie in their neglect of genetic details, the very matrix on which the evolutionary process rests . If thesThere is a long history of disagreement about the importance of genetic details in understanding phenotypic evolution . There sHere we use experimental evolution of bacteriophage to test an optimality model for a life history trait, lysis time. Environmental conditions are easily controlled when growing bacteriophages, making it simple to parameterize and test quantitative predictions about optimality in reasonably well-understood genetic systems. The lysis time model has been tested on three phages, λ, T7, and T4 -17, but Most bacteriophages lyse their hosts to enable progeny to escape into the environment Figure . Lysis oA simple optimality model predicts which lysis interval will maximize fitness simply as a function of the intrinsic growth rate of the phage population ,23. The E , when functional phage progeny begin to accumulate inside the cell) should evolve to equal the inverse of r . ,23. The e Figure . Since tThe simple model presented here ignores the effects of non-genetic lysis variation. Lysis asynchrony  is important in ΦX174 -like phages and affects the optimal lysis interval. A more complete model that accounts for this parameter was used for optimality predictions throughout (see equations 2 and 3 in Methods for details).This model can be explored with experimental evolution -17,25. AOnly two assumptions about lysis mechanisms are required by the optimality model; both are met in ΦX174 bacteriophage. First, average lysis time is evolvable, i.e. mutations exist that can change it ,26. SecoE) is directly implicated in lysis /t, where Nt is the total number of phage in the flask at time t hours, corrected for dilutions over multiple transfers. The intrinsic rate of increase per minute, r, is simply ln(2)/60 times the fitness in db/hr, but db/hr is easier to comprehend.Viral fitness was measured under conditions essentially identical to those used for adaptation. The rate of increase was determined by growth at low phage/cell ratios (not exceeding 0.1 by the end of transfer) across five consecutive transfers, using phage titers measured from the end of the second flask to the end of the fifth [see 17 for details]. Fitness is reported as doublings per hour, calculated as .Phage genome sequences used dideoxy chain termination reactions with ABI Big Dye Terminator and were analyzed with an ABI3100 automated sequencer. Sequence files were checked and compared with DNA Star Lasergene Seqman software (version 5.05). All sequencing was from polymerase chain reaction products, except the wild-type ST-1, which was sequenced from genomic DNA. The entire phage genomes from the end of ST-1 phase I adaptation, ST-1GQ149088]; homologues to all ΦX174 genes were found. ST-1's genome is most similar to the ΦK and α3 genomes, which are ΦX174-like but members of a distinct clade [The full genome of ST-1 was sequenced and is reported as part of this study [GenBank: ct clade . The genct clade .The simple optimality model for lysis presented in the background can be extended to account for the effects of lysis time variance, which is important for ΦX174 and may play a role in other phages as well . IfE the invariant eclipse time), and the lysis interval (L - E) is assumed to vary in a gamma distribution with variance σ2 between individual infections, the following equation(where is obtained . This car), which is useful when trying to determine how much higher the fitness would be if lysis interval changed.Another equation allows calculation of expected phage fitness  numerically. This parameter-based fitness estimation, which usually differed from direct measurements of fitness, was used to approximate the fitness effect of setting lysis time equal to the optimum.L - E - X = 0 , we used empirical data to simulate a normal distribution for eclipse time, post-infection lysis time, lysis variance, and r (growth rate), parameterized with the means and variances calculated from the data. Numbers were randomly drawn from these distributions, with all numbers less than zero rejected as biologically implausible. Lysis variance and r from the distributions were used to solve for X by equation (3). This was repeated 5000 times, recording the proportion of times L - E - X had the opposite sign from the mean deviation from the optimum. The result multiplied by two is the 2-tailed probability that the means were identical. The significance of all other differences was determined by t-tests assuming unequal variances [To test whether ariances -63.The authors declare that they have no competing interests.LC performed the adaptations, sequencing, the majority of the phenotypic assays, and helped draft the manuscript. RH performed calculations of predicted optima, phage release assays with preadsorption, adsorption assays, and drafted the manuscript. All authors read and approved the final manuscript.+ (solid line) and ST-1K-12 (dotted line)Release curves for ST-1. (A) Samples treated with lysis solution containing 2 mg/ml lysozyme and chloroform, used to estimate eclipse. (B) Untreated samples, used to estimate lysis and lysis interval.Click here for file


Intraoral ossifying fibromas have been described in the literature since the late 1940s. Peripheral ossifying fibroma (POF) is usually a fibroma of the gingival which shows areas of calcification or ossification. It is a nonneoplastic enlargement of gingiva. Due to its clinical and histopathological similarities, some POFs are believed to develop initially as a pyogenic granuloma that undergoes fibrous maturation and subsequent calcification. It has been suggested that POF represents a separate clinical entity rather than a transitional form of pyogenic granuloma or irritation fibroma. This paper describes a case report of a 60-year-old female patient reported with growth on gingiva in the upper left front region of mouth three years ago. Many types of localized reactive lesions are seen on the gingiva, including focal fibrous hyperplasia, pyogenic granuloma, peripheral giant cell granuloma, and peripheral ossifying fibroma (POF) –3. SynonA 60-year-old female patient reported with the chief complaint of painless growth on the gingiva in the upper left front region of mouth three years ago. It had progressed gradually to increase in size and attained the present size. Growth was associated with bleeding on brushing occasionally. The patient did not give any history of trauma.Intraoral examination revealed a solitary, sessile growth present on the residual ridge of the missing 23 in the interdental space between 22 and 24, extending mesiodistally from the distal aspect of 22 up to mesial aspect of the 24 region .Superoinferiorly extending superiorly from marginal gingiva of 22 and 24 and inferiorly from 1-2 cm below the incisor line . Antero-It was of the same color of the adjacent gingiva with the mesial half being more reddish in color. The growth was oval in shape and approximately 2.5 × 3 cm in size in greatest dimensions with well-defined borders. The surface of the growth was lobulated. No secondary changes were seen related to ulceration and fungation. The growth was firm and nontender on palpation. It was nonfluctuant, nonreducible, and noncompressible with mild bleeding on probing.The clinical differential diagnoses for the growth were pyogenic granuloma, traumatic fibroma, peripheral giant cell granuloma, and peripheral ossifying fibroma, and provisional diagnosis of pyogenic granuloma with respect to the 22, 23, and 24 regions was made for the gingival growth.Radiological investigations—intraoral periapical radiograph of left maxillary anterior region revealed the presence of 22 and 24 and missing 23. Evidence of faint, irregular radiopacity was noticed in the missing 23 region, suggestive of soft tissue mass, and mesial tipping of 22 and distal tipping of 24 were noticed .Maxillary occlusal radiograph reveals the presence of 11, 12, 22, 24, and 25. Irregular radiopacity is seen interspersed in the soft tissue shadow. The density of which is almost similar to the bone, signifying the presence of ossification .Growth was excised conservatively. The excised tissue was oval, 2.5 × 3 cm in size, pale white in color, and firm in consistency. Tissue was sent for histopathological examination. Adjacent teeth were scaled to remove the local irritants. The patient was recalled after one week for review .Histologically, the specimen showed parakeratinized stratified squamous epithelium overlying the connective tissue stroma. Epithelium showed hyperplasia in some areas. Connective tissue stroma consisted of highly cellular mass of proliferating fibroblasts intermingled with fibrillar tissue. Fibrous connective tissue also consisted of large and small trabeculae of bone and some dystrophic calcifications. Based on history, clinical presentation, and radiological and histopathological examination, final diagnosis of peripheral ossifying fibroma with respect to the 22, 23, and 24 regions was put forth. Menzel first described the lesion ossifying fibroma in 1872, but its terminology was given by Montgomery in 1927 [Though the etiopathogenesis of POF is uncertain, origin from cells of periodontal ligament has been suggested. The reasons for considering periodontal ligament origin include excessive occurrence of POF in the gingival interdental papilla, the proximity of the gingival to periodontal ligament, the presence of oxytalan fibres within the mineralized matrix of some lesion, and the fibrocellular response in periodontal ligament , 8. MigrIn vast majority of cases, there is no apparent underlying bone involvement visible on the roentgenogram. However, superficial erosion of bone is noted occasionally , 8, 9.Peripheral ossifying fibroma has to be differentiated from traumatic fibroma, peripheral giant cell granuloma, pyogenic granuloma, and peripheral odontogenic fibroma. Peripheral odontogenic fibroma is an uncommon neoplasm that is believed to arise from odontogenic epithelial rests in periodontal ligament or attached gingiva itself. Traumatic fibroma occur on buccal mucosa along the bite line. Pyogenic granuloma presents as soft, friable nodule, small in size that bleeds with tendency to hemorrhage and may or may not occasionally or do not show calcifications but tooth displacement and resorption of alveolar bone are not observed. Peripheral giant cell granuloma has clinical features similar to POF however POF lacks the purple or blue discoloration commonly associated with peripheral giant cell granuloma and radiographically shows flecks of calcification .It is possible to histologically differentiate PGCG and peripheral odontogenic fibroma from POF as PGCG contains giant cells, whereas peripheral odontogenic fibroma contains odontogenic epithelium and dysplastic dentine; all the features are not seen in POF .Treatment includes local surgical excision and oral prophylaxis . FollowuPOF being one of the commonest solitary swelling in the oral cavity is many times clinically diagnosed as pyogenic granuloma. Radiological and histopathological examination is required for confirmation of diagnosis.


The versatility of the papers accepted to the topic is surprisingly broad. Three trends became evident, presumably reflecting the most vivid interests in the field: (1) the “Brain slice studies have played an important role in gaining an understanding of fundamental neuronal, synaptic, and network traits but this preparation has never been free from serious limitations. Among them repeatedly mentioned are: absence of blood flow, diffusion peculiarities, inadequate oxygenation/metabolic support, and neuronal damage due to the slicing procedure  that increases chances of ketogenesis (while decreasing glycolytic flux) is shown to have favorable effects on metabolic profiles, thus exerting neuroprotective properties (Zilberter, Together, the articles comprising this Research Topic depict a complex intricately organized system with representative studies at both the macro- and micro-levels (e.g., involving molecular signaling pathways; Venkateswaran et al.,


To the Editor: The bacterium Vibrio vulnificus is a marine flora saprophyte that can cause necrotic skin infection and septicemia in humans who eat shellfish. Symptoms of septicemia  have been described mostly in Florida and Japan among persons who ate raw filter-feeding shellfish when seawater temperatures are >20°C .Patient 1 was a 51-year-old man with fever, muscle pains, bleeding gums, and a history of alcohol abuse; within 48 hours after symptom onset, he died of septic shock, with diffuse ecchymoses and purpura. Patient 2 was a 67-year-old woman with no known concurrent conditions who was admitted to the hospital with chills, diarrhea, and vomiting; septic shock developed, with painful erythematous plaques on the lower limbs becoming foamy, confluent, and necrotic. Patient 3 was a 74-year-old woman with untreated lupus who was hospitalized with lower-limb edema, hypotension, hypothermia, and erythematous skin lesions. All 3 patients received cephalosporins but died of multiple organ failure within 12 hours after hospital admission.2-enriched atmosphere. V. vulnificus was identified through the Vitek2 system (bioMérieux) and confirmed by using the Api 20E system (bioMérieux).Peripheral blood aerobic–anaerobic samples were taken from all patients, stored in BacT/Alert FA vials , and incubated in the BacT/Alert 3D system (bioMérieux). Curved mobile gram-negative bacilli were isolated from blood samples cultured on conventional media without additional salt within 24 h after incubation at 37°C in a 5% COStrains were sent to the Centre National de Reference des Vibrions et du Choléra, , which by PCR confirmed the gene encoding virulence-associated hemolysin, a species-specific marker . These specific conditions may have favored higher sea surface temperatures, lower salinity, increased turbidity, and subsequent multiplication of V. vulnificus in seawater.The literature mentions few cases of V. vulnificus infections. Local health authorities issued criteria for defining suspected cases of V. vulnificus infection and recommendations for early medical care of patients with clinical symptoms. Methods of detecting the bacterium in human and animal health laboratories were improved, particularly by the systematic use of selective media in the event of suspected clinical V. vulnificus infection and standardized reporting of V. vulnificus isolation. Preventive measures, such as improving microbial surveillance and warning consumers about risks associated with eating raw seafood, are essential to help reduce the risk for V. vulnificus–induced illness.A range of projects were implemented to train practitioners to recognize potential