Drosophila Nociceptors Mediate Larval Aversion to Dry Surface Environments Utilizing Both the Painless TRP Channel and the DEG/ENaC Subunit, PPK1

A subset of sensory neurons embedded within the Drosophila larval body wall have been characterized as high-threshold polymodal nociceptors capable of responding to noxious heat and noxious mechanical stimulation. They are also sensitized by UV-induced tissue damage leading to both thermal hyperalgesia and allodynia very similar to that observed in vertebrate nociceptors. We show that the class IV multiple-dendritic(mdIV) nociceptors are also required for a normal larval aversion to locomotion on to a dry surface environment. Drosophila melanogaster larvae are acutely susceptible to desiccation displaying a strong aversion to locomotion on dry surfaces severely limiting the distance of movement away from a moist food source. Transgenic inactivation of mdIV nociceptor neurons resulted in larvae moving inappropriately into regions of low humidity at the top of the vial reflected as an increased overall pupation height and larval desiccation. This larval lethal desiccation phenotype was not observed in wild-type controls and was completely suppressed by growth in conditions of high humidity. Transgenic hyperactivation of mdIV nociceptors caused a reciprocal hypersensitivity to dry surfaces resulting in drastically decreased pupation height but did not induce the writhing nocifensive response previously associated with mdIV nociceptor activation by noxious heat or harsh mechanical stimuli. Larvae carrying mutations in either the Drosophila TRP channel, Painless, or the degenerin/epithelial sodium channel subunit Pickpocket1(PPK1), both expressed in mdIV nociceptors, showed the same inappropriate increased pupation height and lethal desiccation observed with mdIV nociceptor inactivation. Larval aversion to dry surfaces appears to utilize the same or overlapping sensory transduction pathways activated by noxious heat and harsh mechanical stimulation but with strikingly different sensitivities and disparate physiological responses

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Correlation of genotype-specific effects of transgenic mdIV nociceptor manipulation in a horizontal dry surface locomotion assay.

<p>(A) Schematic representation of the horizontal dry surface locomotion assay(see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032878#s4" target="_blank">Materials and Methods</a>). (B) Horizontal locomotion behavior parallels genotypic differences in PHI. Wandering stage larvae of the indicated genotypes were assayed in an alternative protocol for larval locomotion on a dry horizontal glass surface under constant humidity. Higher scores represent increased distance of movement away from a moist filter over a dry glass surface(see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032878#s4" target="_blank">Materials and Methods</a>). Paralleling results from determination of PHI, <i>ppk1-GAL4/UAS-PPK1[S551V]</i> larvae expressing a constitutively active PPK1 isoform preferred to avoid extensive locomotion over the dry surface. The wild-type control <i>ppk1-GAL4/+</i> larvae moved an intermediate distance and <i>ppk1</i> null mutant larvae(w¹¹¹⁸; DfA/DfB) moved a significantly increased distance over the dry glass surface before becoming stranded. When assayed under the same conditions except at high humidity levels(75%), both <i>ppk1-GAL4/UAS-PPK1[S551V]</i> and <i>ppk1-GAL4/+</i> larvae displayed significantly increased locomotion over the dry glass surface. Error bars represent SEM with n>15 for all values. **P<.01, ***P<.0001, one-way ANOVA with Tukey posttest.</p

Hypersensitive aversion to dry surfaces caused by transgenic hyperactivation of mdIV nociceptor neurons.

<p>(A) Hyperactivation of mdIV nociceptors by transgenic expression of the constitutively-active PPK1[S551V] isoform resulted in a drastic decrease in pupation height depicted as a downward deflection of Pupation Height Index. This hypersensitive aversion behavior was completely suppressed by high external humidity(75%). (B) Hyperactivation of mdIV nociceptors by transgenic expression of the low-threshold voltage-gated Na channel <i>UAS-NaChBac-EGFP</i> caused a similar decrease in pupation height that was suppressed by high external humidity(75%). (C) Hyperactivation of mdIV nociceptors by transgenic expression of the heat-activated TrpA1.K channel caused the same significant decrease in overall pupation height that was suppressed by growth at high humidity(75%). All vials contained 50 larvae. (DE) Larval surface preference assay. (D) Third-instar larvae of the indicated genotypes were given a choice for pupation on the glass vial sides or on a strip of wetted Whatman filter paper inserted vertically at the center of the vial(see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032878#s4" target="_blank">Materials and Methods</a>). A preference index was calculated to indicate a preference for pupation on the moistened filter paper as a deflection to the right and a preference for the glass surface as a deflection to the left. Larvae with hyperactivated mdIV nociceptors expressing the constitutively-active PPK1[S551V] isoform showed a strong preference for pupation on the moist filter paper surface. Inactivation of mdIV nociceptors in ppk1GAL4/UAS-shi[ts1] and <i>ppk1</i> null larvae caused an enhanced preference for pupation on the glass surface. (E) Schematic representation of the larval surface preference assay. Error bars represent SEM with n≥15 for all values. ***P<.0001, one-way ANOVA with Tukey posttest.</p

Control of pupation height behavior by the internal microenvironment of the culture vial.

<p>(A) Schematic representation of a <i>Drosophila</i> culture vial and the internal humidity gradient generated within the microenvironment of the plugged vial. Higher humidity at the bottom near the food surface is indicated as darker shading decreasing to low humidity at the top of the vial. Pupae are shown as black ovals within the designated zones indicated as height(cm) above the vial bottom used to calculate the pupation height index(PHI). PHI = ((#pupae>3 cm)-(#pupae<3 cm))/total # pupae. (BC) Pupation height behavior for ppk1-GAL4/+ control larvae represented by the indicated Pupation Height Index; (B) Pupation height is strongly influenced by external humidity. With constant larval density(50 larvae/vial), low external humidity(25%) causes larvae to pupate at sites lower in the vial indicated as a downward deflection. At higher humidity(50% and 75%), larvae choose to predominantly pupate at higher sites within the vial indicated as an upward deflection. (C) Pupation height is influenced by larval density. With vials grown at constant external humidity(50%), low larval density(25 larvae/vial) causes a low pupation height. Higher larval density causes a drastic increase in pupation height resulting from the altered microenvironment as larvae exit the food source. Error bars represent SEM with n≥15 for all values. ***P<.0001, one-way ANOVA with Tukey posttest.</p

Larval aversion to dry surfaces requires both the DEG/ENaC subunit PPK1 and the TRP channel Pain.

<p>(A) Schematic depiction summarizing opposing mdIV nociceptor gain-of-function and loss-of-function phenotypes. (B) <i>pain</i> mutant alleles showed increased pupation height phenotype that was rescued by transgenic expression of wild-type Pain protein. (C) Transgenic expression of the dominant-negative PPK1[E145X] isoform after 96 h AEL caused an increase in pupation height similar to that observed with mdIV nociceptor inactivation. (D) <i>ppk1</i> null mutant larvae displayed an increase in pupation height that was rescued by transgenic expression of wild-type PPK1 or the constitutively-active PPK1[S551V] isoform in mdIV nociceptor neurons. (E) Fraction of total larvae pupating in the top regions of the vial(>7 cm) was significantly increased in <i>ppk1</i> null mutants and rescued by mdIV-specific expression of wild-type PPK1. (F) Fraction of total larvae displaying the lethal desiccated larval phenotype was significantly increased in <i>ppk1</i> null mutant larvae. Larval desiccation was rescued by transgenic expression of wild-type PPK1 or by growth at high humidity(75%). All vials were shifted to 29°C/40% humidity at 96 h AEL unless otherwise indicated. Error bars represent SEM with n≥15 for all values. ***P<.0001, one-way ANOVA with Tukey posttest.</p

Pharmacokinetics and Pharmacodynamics of Antibacterials, Antifungals, and Antivirals Used Most Frequently in Neonates and Infants

Antimicrobials and antivirals are widely used in young infants and neonates. These patients have historically been largely excluded from clinical trials and, as a consequence, the pharmacokinetics and pharmacodynamics of commonly used antibacterials, antifungals, and antivirals are incompletely understood in this population. This review summarizes the current literature specific to neonates and infants regarding pharmacokinetic parameters and changes in neonatal development that affect antimicrobial and antiviral pharmacodynamics. Specific drug classes addressed include aminoglycosides, aminopenicillins, cephalosporins, glycopeptides, azole antifungals, echinocandins, polyenes, and guanosine analogs. Within each drug class, the pharmacodynamics, pharmacokinetics, and clinical implications and future directions for prototypical agents are discussed. β-Lactam antibacterial activity is maximized when the plasma concentration exceeds the minimum inhibitory concentration for a prolonged period, suggesting that more frequent dosing may optimize β-lactam therapy. Aminoglycosides are typically administered at longer intervals with larger doses in order to maximize exposure (i.e., area under the plasma concentration–time curve) with gestational age and weight strongly influencing the pharmacokinetic profile. Nonetheless, safety concerns necessitate therapeutic drug monitoring across the entire neonatal and young infant spectrum. Vancomycin, representing the glycopeptide class of antibacterials, has a long history of clinical utility, yet there is still uncertainty about the optimal pharmacodynamic index in neonates and young infants. The high degree of pharmacokinetic variability in this population makes therapeutic drug monitoring essential to ensure adequate therapeutic exposure. Among neonates treated with the triazole agent fluconazole, it has been speculated that loading doses may improve pharmacodynamic target attainment rates. The use of voriconazole necessitates therapeutic drug monitoring and dose adjustments for patients with hepatic dysfunction. Neonates treated with lipid-based formulations of the polyene amphotericin B may be at an increased risk of death, such that alternative antifungal agents should be considered for neonates with invasive fungal infections. Alternative antifungal agents such as micafungin and caspofungin also exhibit unique pharmacokinetic considerations in this population. Neonates rapidly eliminate micafungin and require nearly three times the normal adult dose to achieve comparable levels of systemic exposure. Conversely, peak caspofungin concentrations have been reported to be similar among neonates and adults. However, both of these drugs feature favorable safety profiles. Recent studies with acyclovir have suggested that current dosing regimens may not result in therapeutic central nervous system concentrations and more frequent dosing may be required for neonates at later postmenstrual ages. Though ganciclovir and valganciclovir demonstrate excellent activity against cytomegalovirus, they are associated with significant neutropenia. In summary, many pharmacokinetic and pharmacodynamic studies have been conducted in this vulnerable population; however, there are also substantial gaps in our knowledge that require further investigation. These studies will be invaluable in determining optimal neonatal dosing regimens that have the potential to improve clinical outcomes and decrease adverse effects associated with antimicrobial and antiviral treatments