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Many neurotransmitter receptors activate G proteins through exchange of GDP for GTP. The intermediate nucleotide-free state has eluded characterization, due largely to its inherent instability. Here we characterize a G protein variant associated with a rare neurological disorder in humans. Gα o K46E has a charge reversal that clashes with the phosphate groups of GDP and GTP. As anticipated, the purified protein binds poorly to guanine nucleotides yet retains wild-type affinity for G protein βγ subunits. In cells with physiological concentrations of nucleotide, Gα o K46E forms a stable complex with receptors and Gβγ, impeding effector activation. Further, we demonstrate that the mutant can be easily purified in complex with dopamine-bound D2 receptors, and use cryo-electron microscopy to determine the structure, including both domains of Gα o , without nucleotide or stabilizing nanobodies. These findings reveal the molecular basis for the first committed step of G protein activation, establish a mechanistic basis for a neurological disorder, provide a simplified strategy to determine receptor-G protein structures, and a method to detect high affinity agonist binding in cells., (© 2024. The Author(s).)

Our understanding of mitochondrial signaling in the nervous system has been limited by the technical challenge of analyzing mitochondrial function in vivo. In the transparent genetic model Caenorhabditis elegans, we were able to manipulate and measure mitochondrial reactive oxygen species (mitoROS) signaling of individual mitochondria as well as neuronal activity of single neurons in vivo. Using this approach, we provide evidence supporting a novel role for mitoROS signaling in dendrites of excitatory glutamatergic C. elegans interneurons. Specifically, we show that following neuronal activity, dendritic mitochondria take up calcium (Ca 2+ ) via the mitochondrial Ca 2+ uniporter (MCU-1) that results in an upregulation of mitoROS production. We also observed that mitochondria are positioned in close proximity to synaptic clusters of GLR-1, the C. elegans ortholog of the AMPA subtype of glutamate receptors that mediate neuronal excitation. We show that synaptic recruitment of GLR-1 is upregulated when MCU-1 function is pharmacologically or genetically impaired but is downregulated by mitoROS signaling. Thus, signaling from postsynaptic mitochondria may regulate excitatory synapse function to maintain neuronal homeostasis by preventing excitotoxicity and energy depletion., Competing Interests: RD, KK, ED, FH No competing interests declared, (© 2024, Doser et al.)

Protein kinase C (PKC) functions are essential for synaptic plasticity, learning, and memory. However, the roles of specific members of the PKC family in synaptic function, learning, and memory are poorly understood. Here, we investigated the role of individual PKC homologs for synaptic plasticity in Caenorhabditis elegans and found a differential role for pkc-2 and tpa-1 , but not pkc-1 and pkc-3 in associative olfactory learning and memory. More specifically we show that PKC-2 is essential for associative learning and TPA-1 for short-term associative memory (STAM). Using endogenous labeling and cell-specific rescues, we show that TPA-1 and PKC-2 are required in AVA for their functions. Previous studies demonstrated that olfactory learning and memory in C. elegans are tied to proper synaptic content and trafficking of AMPA-type ionotropic glutamate receptor homolog GLR-1 in the AVA command interneurons. Therefore, we quantified synaptic content, transport, and delivery of GLR-1 in AVA and showed that loss of pkc-2 and tpa-1 leads to decreased transport and delivery but only a subtle decrease in GLR-1 levels at synapses. AVA-specific expression of both PKC-2 and TPA-1 rescued these defects. Finally, genetic epistasis showed that PKC-2 and TPA-1 likely act in the same pathway to control GLR-1 transport and delivery, while regulating different aspects of olfactory learning and STAM. Thus, our data tie together cell-specific functions of 2 PKCs to neuronal and behavioral outcomes in C. elegans , enabling comparative approaches to understand the evolutionarily conserved role of PKC in synaptic plasticity, learning, and memory., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 the authors.)

Calcium (Ca 2+ ) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca 2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca 2+ dynamics in vivo, where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca 2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca 2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca 2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca 2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca 2+ handling regulates neuronal function and plasticity.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) that function as molecular switches for cellular growth and metabolism are activated by GTP and inactivated by GTP hydrolysis. In uveal melanoma, a conserved glutamine residue critical for GTP hydrolysis in the G protein α subunit is often mutated in Gα q or Gα 11 to either leucine or proline. In contrast, other glutamine mutations or mutations in other Gα subtypes are rare. To uncover the mechanism of the genetic selection and the functional role of this glutamine residue, we analyzed all possible substitutions of this residue in multiple Gα isoforms. Through cell-based measurements of activity, we showed that some mutants were further activated and inactivated by G protein-coupled receptors. Through biochemical, molecular dynamics, and nuclear magnetic resonance-based structural studies, we showed that the Gα mutants were functionally distinct and conformationally diverse, despite their shared inability to hydrolyze GTP. Thus, the catalytic glutamine residue contributes to functions beyond GTP hydrolysis, and these functions include subtype-specific, allosteric modulation of receptor-mediated subunit dissociation. We conclude that G proteins do not function as simple on-off switches. Rather, signaling emerges from an ensemble of active states, a subset of which are favored in disease and may be uniquely responsive to receptor-directed ligands.

Polypropylene meshes used in pelvic organ prolapse (POP) repair are hampered by complications. Most POP meshes are highly unstable after tensioning ex vivo, as evidenced by marked deformations (pore collapse and wrinkling) that result in altered structural properties and material burden. By intentionally introducing collapsed pores and wrinkles into a mesh that normally has open pores and remains relatively flat after implantation, we reproduce mesh complications in vivo. To do this, meshes were implanted onto the vagina of rhesus macaques in nondeformed (flat) vs deformed (pore collapse +/- wrinkles) configurations and placed on tension. Twelve weeks later, animals with deformed meshes had two complications, (1) mesh exposure through the vaginal epithelium, and (2) myofibroblast proliferation with fibrosis - a mechanism of pain. The overarching response to deformed mesh was vaginal thinning associated with accelerated apoptosis, reduced collagen content, increased proteolysis, deterioration of mechanical integrity, and loss of contractile function consistent with stress shielding - a precursor to mesh exposure. Regional differences were observed, however, with some areas demonstrating myofibroblast proliferation and matrix deposition. Variable mechanical cues imposed by deformed meshes likely induce these two disparate responses. Utilizing meshes associated with uniform stresses on the vagina by remaining flat with open pores after tensioning is critical to improving outcomes. STATEMENT OF SIGNIFICANCE: Pain and exposure are the two most reported complications associated with the use of polypropylene mesh in urogynecologic procedures. Most meshes have unstable geometries as evidenced by pore collapse and wrinkling after tensioning ex vivo, recapitulating what is observed in meshes excised from women with complications in vivo. We demonstrate that collapsed pores and wrinkling result in two distinct responses (1) mesh exposure associated with tissue degradation and atrophy and (2) myofibroblast proliferation and matrix deposition consistent with fibrosis, a tissue response associated with pain. In conclusion, mesh deformation leads to areas of tissue degradation and myofibroblast proliferation, the likely mechanisms of mesh exposure and pain, respectively. These data corroborate that mesh implantation in a flat configuration with open pores is a critical factor for reducing complications in mesh-augmented surgeries., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. We would like to draw the attention of the Editor to the following facts which may be considered as potential declaration of interests: Pamela A. Moalli reports equipment, drugs, or supplies was provided by Coloplast Corp. Katrina M. Knight reports financial support was provided by Magee-Womens Research Institute & Foundation. Pamela A. Moalli reports a relationship with Hologic Inc that includes: board membership. However, there has been no significant financial support for this work that could have influenced its outcome., (Copyright © 2022. Published by Elsevier Ltd.)

In Australia, destruction of overwintering pupae of Helicoverpa armigera (Hübner) and Helicoverpa punctigera (Wallengren) (Lepidoptera: Noctuidae) has been a key component of mandatory resistance management schemes to constrain development of resistance to Bt toxins in transgenic cotton. This has been accomplished by tillage ('pupae busting'), but it is expensive and can interfere with farming operations. Bisexual attract-and-kill technology based on plant volatile formulations offers a potential alternative in some circumstances. We discuss strategies for using such products and describe two trials in which three applications of an attract-and-kill formulation substantially reduced the numbers of Helicoverpa spp. moths and the numbers of potentially overwintering eggs they laid. One trial tested a curative strategy in which the last generation of moths emerging from transgenic cotton was targeted. The other tested a preventive strategy which aimed to reduce the numbers of eggs in the last generation. The preventive strategy reduced egg numbers by about 90% and is now included as an optional alternative to pupae busting in resistance management strategies for Australian cotton. It is limited to fields which have not been defoliated prior to 31 March and was developed to be used primarily in southern New South Wales. In the 2020-2021 cotton season, it was adopted on approximately 60% of the eligible cotton area. We describe the process whereby the strategy was developed in collaboration with the transgenic technology provider, supported by the cotton industry, and approved by the regulatory authority., (© The Author(s) 2022. Published by Oxford University Press on behalf of Entomological Society of America.)

Introduction and Hypothesis: We compared the impact of a mesh manufactured from the soft elastomer polydimethylsiloxane (PDMS) to that of a widely used lightweight polypropylene (PP) mesh. To achieve a similar overall device stiffness between meshes, the PDMS mesh was made with more material and therefore was heavier and less porous. We hypothesized that the soft polymer PDMS mesh, despite having more material, would have a similar impact on the vagina as the PP mesh., Methods: PDMS and PP meshes were implanted onto the vaginas of 20 rabbits via colpopexy. Ten rabbits served as sham. At 12 weeks, mesh-vagina complexes were explanted and assessed for contractile function, histomorphology, total collagen, and glycosaminoglycan content. Outcome measures were compared using one-way ANOVA and Kruskal-Wallis testing with appropriate post-hoc testing., Results: Relative to sham, vaginal contractility was reduced following the implantation of PP (p = 0.035) but not the softer PDMS (p = 0.495). PP had an overall greater negative impact on total collagen and glycosaminoglycan content, decreasing by 53% (p < 0.001) and 54% (p < 0.001) compared to reductions of 35% (p = 0.004 and p < 0.001) with PDMS. However, there were no significant differences in the contractility, collagen fiber thickness, total collagen, and glycosaminoglycan content between the two meshes., Conclusions: Despite having a substantially higher weight, PDMS had a similar impact on the vagina compared to a low-weight PP mesh, implicating soft polymers as potential alternatives to PP. The notion that heavyweight meshes are associated with a worse host response is not applicable when comparing across materials., (© 2021. The International Urogynecological Association.)

Background: Although falls are common and can cause serious injury to older adults, many health care facilities do not have falls prevention resources available. Falls prevention resources can reduce injury and mortality rates. Using the Centers for Disease Control and Prevention's (CDC) Stopping Elderly Accidents, Deaths & Injuries (STEADI) model, a falls risk clinic was implemented in a rural Indian Health Service (IHS) facility., Methods: A Fall Risk Questionnaire was created and implemented into the Provider's Electronic Health Records system interface to streamline provider screening and referral of patients who may be at risk for falls to a group falls risk reduction class., Results: Participants exhibited average improvements in the Timed Up and Go (6.8 s) (P = 0.0001), Five-Time Sit-to-Stand (5.1 s) (P = 0.0002), and Functional Reach (3.6 inches) (P = 1.0) tests as compared to their own baseline. Results were analyzed via paired t test. 71% of participants advanced out of an "increased risk for falls" category in at least one outcome measure. Of the participants to complete the clinic, all were successfully contacted and three (18%) reported one or more falls at the 90-day mark, of which one (6%) required a visit to the Emergency Department but did not require hospital admission., Conclusions: In regards to reducing falls in the community, per the CDC STEADI model, an integrated approach is best. All clinicians can play a part in reducing elder falls., (© 2021. The Author(s).)

This article describes the design and > 20 years of effective implementation of a proactive resistance-management plan for transgenic Bacillus thuringiensis (Bt) cotton that targets Helicoverpa armigera (Hübner) and Helicoverpa punctigera (Wallengren) in Australia, considering pest biology and ecology, insights from resistance-evolution modelling, and the importance of the human component to effective implementation. This is placed in the context of processes associated with adaptive resource management. Bt cotton has provided Australian cotton growers with technology to manage Helicoverpa species that previously challenged the industry's viability, while at the same time resulting in no detectable changes in the resistance allele frequency in field populations of either Helicoverpa species in eastern Australia. This is the most long-lived and successful global example of a proactive resistance management plan for an insect pest. Six key learnings important to the successful development and implementation of a proactive transgenic-crop resistance management plan are: the programme has to have a strong science base; there has to be broad stakeholder support at all levels; there has to be a strong implementation programme; the plan needs to be supported by auditing and enforced remediation of deviations from the mandated resistance management plan; A programme of rigorous and on-going resistance allele monitoring; an attitude of continuous improvement for all aspects of the resistance management plan. The lessons learnt from the deployment of Bt cotton in Australia are relevant globally and provide important guidelines for the deployment of transgenic crops for insect control wherever they are grown. © 2021 Society of Chemical Industry., (© 2021 Society of Chemical Industry.)

Cardiac dysfunction is a common phenotypic manifestation of primary mitochondrial disease with multiple nuclear and mitochondrial DNA pathogenic variants as a cause, including disorders of mitochondrial translation. To date, five patients have been described with pathogenic variants in MRPL44, encoding the ml44 protein which is part of the large subunit of the mitochondrial ribosome (mitoribosome). Three presented as infants with hypertrophic cardiomyopathy, mild lactic acidosis, and easy fatigue and muscle weakness, whereas two presented in adolescence with myopathy and neurological symptoms. We describe two infants who presented with cardiomyopathy from the neonatal period, failure to thrive, hypoglycemia and in one infant lactic acidosis. A decompensation of the cardiac function in the first year resulted in demise. Exome sequencing identified compound heterozygous variants in the MRPL44 gene including the known pathogenic variant c.467 T > G and two novel pathogenic variants. We document a combined respiratory chain enzyme deficiency with emphasis on complex I and IV, affecting heart muscle tissue more than skeletal muscle or fibroblasts. We show this to be caused by reduced mitochondrial DNA encoded protein synthesis affecting all subunits, and resulting in dysfunction of complex I and IV assembly. The degree of oxidative phosphorylation dysfunction correlated with the impairment of mitochondrial protein synthesis due to different pathogenic variants. These functional studies allow for improved understanding of the pathogenesis of MRPL44-associated mitochondrial disorder., Competing Interests: Declaration of Competing Interest JVH participates in clinical trials of mitochondrial disorders by Stealth Biotherapeutic, Inc. All other authors deny any real or apparent conflict of interest in the field of mitochondrial diseases., (Copyright © 2021 Elsevier Inc. All rights reserved.)

G proteins play a central role in signal transduction and pharmacology. Signaling is initiated by cell-surface receptors, which promote guanosine triphosphate (GTP) binding and dissociation of Gα from the Gβγ subunits. Structural studies have revealed the molecular basis of subunit association with receptors, RGS proteins, and downstream effectors. In contrast, the mechanism of subunit dissociation is poorly understood. We use cell signaling assays, molecular dynamics (MD) simulations, and biochemistry and structural analyses to identify a conserved network of amino acids that dictates subunit release. In the presence of the terminal phosphate of GTP, a glycine forms a polar network with an arginine and glutamate, putting torsional strain on the subunit binding interface. This "G-R-E motif" secures GTP and, through an allosteric link, discharges the Gβγ dimer. Replacement of network residues prevents subunit dissociation regardless of agonist or GTP binding. These findings reveal the molecular basis of the final committed step of G protein activation., Competing Interests: Declaration of interests R.H.J.O. is a co-inventor of the TRUPATH technology and could receive royalties. This relationship has been disclosed to and is under management by UNC-Chapel Hill., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Stress urinary incontinence carries a significant healthcare burden for women worldwide. Single incision slings are minimally invasive mesh devices designed to treat stress urinary incontinence. For prolapse repair, meshes with higher porosity and lower structural stiffness have been associated with improved outcomes., Objective: In this study, we compared the higher stiffness, lower porosity Altis sling with the lower stiffness, higher porosity Solyx sling in an ovine model. We hypothesized that SIS-B would have a negative impact on the host response., Study Design: A total of Altis and Solyx single incision slings were implanted suburethrally into sheep according to the manufacturer's instructions on minimal tension. The mesh-urethral-vaginal complex and adjacent ungrafted vagina (no mesh control) were harvested en bloc at 3 months. Masson's trichrome and picrosirius red staining of 6 μm thin sections was performed to measure interfiber distance and tissue integration. Smooth muscle contractility to a 120 mM KCl stimulus was performed in an organ bath to measure myofiber-driven contractions. Standard biochemical assays were used to quantify glycosaminoglycan, total collagen, and elastin content, and collagen subtypes. Bending stiffness was performed in response to a uniaxial force to define susceptibility to folding/buckling. Statistical analysis was performed using Mann-Whitney, Gabriel's pairwise post hoc, Wilcoxon matched-pairs, and chi-square tests., Results: The animals had similar ages (3-5 years), parity (multiparous), and weights (45-72 kg). Trichrome cross sections showed that the Altis sling buckled in a "C" or "S" shape in most samples (8 of 11), whereas buckling after Solyx sling implantation was observed in only a single sample (1 of 13; P=.004). Tissue integration, as measured by the presence of collagen or smooth muscle between the mesh fibers on trichrome 4× imaging, was increased in samples implanted with the Solyx sling compared with the Altis sling (P<.05). Total collagen content decreased significantly with both products when compared with the ungrafted vagina consistent with stress shielding. There was no difference in the 2 groups with regard to glycosaminoglycan or elastin content. The Altis sling mesh tissue complex demonstrated significantly higher amounts of both collagen types I and III than the Solyx sling-implanted tissue and the ungrafted control. Smooth muscle contractility in response to 120 mM KCl was decreased after implantation of both slings compared with the sham (P=.011 and P<.01), with no difference between mesh types (P=.099). Bending stiffness in the Altis sling was more than 4 times lower than in the Solyx, indicating an increased propensity to buckle (0.0186 vs 0.0883)., Conclusion: The structurally stiffer Altis sling had decreased tissue integration and increased propensity to buckle after implantation. Increased collagen types I and III after the implantation of this device suggests that these changes may be associated with a fibrotic response. In contrast, the Solyx sling largely maintained a flat configuration and had improved tissue integration. The deformation of the Altis sling is not an intended effect and is likely caused by its lower bending stiffness. Both meshes induced a decrease in collagen content and smooth muscle contractility similar to previous findings for prolapse meshes and consistent with stress shielding. The long-term impact of buckling warrants further investigation., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Genetic defects in mitochondrial DNA encoded tRNA genes impair mitochondrial translation with resultant defects in the mitochondrial respiratory chain and oxidative phosphorylation system. The phenotypic spectrum of disease seen in mitochondrial tRNA defects is variable and proving pathogenicity of new variants is challenging. Only three pathogenic variants have been described previously in the mitochondrial tRNA Tyr gene MT-TY, with the reported phenotypes consisting largely of adult onset myopathy and ptosis. We report a patient with a novel MT-TY acceptor stem variant m.5889A>G at high heteroplasmy in muscle, low in blood, and absent in the mother's blood. The phenotype consisted of a childhood-onset severe multi-system disorder characterized by a neurodegenerative course including ataxia and seizures, failure-to-thrive, combined myopathy and neuropathy, and hearing and vision loss. Brain imaging showed progressive atrophy and basal ganglia calcifications. Mitochondrial biomarkers lactate and GDF15 were increased. Functional studies showed a deficient activity of the respiratory chain enzyme complexes containing mtDNA-encoded subunits I, III and IV. There were decreased steady state levels of these mitochondrial complex proteins, and presence of incompletely assembled complex V forms in muscle. These changes are typical of a mitochondrial translational defect. These data support the pathogenicity of this novel variant. Careful review of variants in MT-TY additionally identified two other pathogenic variants, one likely pathogenic variant, nine variants of unknown significance, five likely benign and four benign variants., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Diagnosing complex V deficiencies caused by new variants in mitochondrial DNA is challenging due to the rarity, phenotypic diversity, and limited functional assessments. We describe a child with the m.9032T > C variant in MT-ATP6 encoding p.(Leu169Pro), with primary presentation of microcephaly, ataxia, hearing loss, and lactic acidosis. Functional studies reveal abnormal fragment F 1 of complex V on blue native gel electrophoresis. Respirometry showed excessively tight coupling through complex V depressing oxygen consumption upon ADP stimulation and an excessive increase following uncoupling, in the presence of upregulation of mitochondrial biogenesis. These data add evidence about pathogenicity and functional impact of this variant., (Copyright © 2020 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

Disorders of the white matter are genetically very heterogeneous including several genes involved in mitochondrial bioenergetics. Diagnosis of the underlying cause is aided by pattern recognition on neuroimaging and by next-generation sequencing. Recently, genetic changes in the complex I assembly factor NUBPL have been characterized by a consistent recognizable pattern of leukoencephalopathy affecting deep white matter including the corpus callosum and cerebellum. Here, we report twin boys with biallelic variants in NUBPL, an unreported c.351 G > A; p.(Met117Ile) and a previously reported pathological variant c. 693 + 1 G > A. Brain magnetic resonance imaging showed abnormal T2 hyperintense signal involving the periventricular white matter, external capsule, corpus callosum, and, prominently, the bilateral thalami. The neuroimaging pattern evolved over 18 months with marked diffuse white matter signal abnormality, volume loss, and new areas of signal abnormality in the cerebellar folia and vermis. Magnetic resonance spectroscopy showed elevated lactate. Functional studies in cultured fibroblasts confirmed pathogenicity of the genetic variants. Complex I activity of the respiratory chain was deficient spectrophotometrically and on blue native gel with in-gel activity staining. There was absent assembly and loss of proteins of the matrix arm of complex I when traced with an antibody to NDUFS2, and incomplete assembly of the membrane arm when traced with an NDUFB6 antibody. There was decreased NUBPL protein on Western blot in patient fibroblasts compared to controls. Compromised NUBPL activity impairs assembly of the matrix arm of complex I and produces a severe, rapidly-progressive leukoencephalopathy with thalamic involvement on MRI, further expanding the neuroimaging phenotype., Competing Interests: Declaration of Competing Interest JVH participates in clinical trials of mitochondrial disorders by Stealth Biotherapeutic, Inc. RPS participates in clinical trials of mitochondrial disorders by Bioelectron Therapeutics, Stealth Biotherapeutics, Inc. and a National Institutes of Health funded phase 3 trial of dichloroacetate in pyruvate decarboxylase deficiency., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Introduction and Hypothesis: New Zealand white rabbits are an inexpensive large-animal model. This study explored the rabbit as a model for mesh-augmented colpopexy using the intra-abdominal vagina. We hypothesized that polypropylene mesh would negatively impact rabbit vaginal smooth muscle (VSM) morphology and contractile function, similar to the nonhuman primate (NHP)-the established model for prolapse mesh evaluation., Methods: Restorelle was implanted onto the vagina of ten rabbits via lumbar colpopexy after a hysterectomy. Ten rabbits served as sham. Twelve weeks post-implantation, the vagina was excised and VSM morphology and vaginal contractility were assessed. Outcome measures were compared using independent samples t and Mann-Whitney U tests with a Bonferroni correction, where appropriate. Results from the rabbits were compared with published NHP data., Results: Animals had similar age, parity and BMI. VSM was 18% thinner after Restorelle implantation, P = 0.027. Vaginal contractility was 43% decreased in response to 120 mM KCl (P = 0.003), similar to the 46% reduction observed in the NHP vagina implanted with Restorelle (P = 0.027). Three meshes wrinkled in vivo, resulting in dramatic thinning of the underlying vagina in the area of the mesh causing a mesh exposure., Conclusions: Polypropylene mesh negatively impacts VSM morphology and vaginal contractility in the rabbit, similar to the NHP, suggesting that the rabbit may serve as an alternative large-animal model. The vaginal thinning and appearance of a mesh exposure in the area of a mesh wrinkle suggest the rabbit may also serve as a model for understanding the pathophysiology of mesh exposure.

Synthetic mesh for pelvic organ prolapse (POP) repair is associated with high complication rates. While current devices incorporate large pores (>1 mm), recent studies have shown that uniaxial loading of mesh reduces pore size, raising the risk for complications. However, it is difficult to translate uniaxial results to transvaginal meshes, as in vivo loading is multidirectional. Thus, the aim of this study was to (1) experimentally characterize deformation of pore diameters in a transvaginal mesh in response to clinically relevant multidirectional loading and (2) develop a computational model to simulate mesh behavior in response to in vivo loading conditions. Tension (2.5 N) was applied to each of mesh arm to simulate surgical implantation. Two loading conditions were assessed where the angle of the applied tension was altered and image analysis was used to quantify changes in pore dimensions. A computational model was developed and used to simulate pore behavior in response to these same loading conditions and the results were compared to experimental findings. For both conditions, between 26.4% and 56.6% of all pores were found to have diameters <1 mm. Significant reductions in pore diameter were noted in the inferior arms and between the two superior arms. The computational model identified the same regions, though the model generally underestimated pore deformation. This study demonstrates that multiaxial loading applied clinically has the potential to locally reduce porosity in transvaginal mesh, increasing the risk for complications. Computational simulations show potential of predicting this behavior for more complex loading conditions.

Pelvic organ prolapse (POP) meshes are exposed to predominately tensile loading conditions in vivo that can lead to pore collapse by 70-90%, decreasing overall porosity and providing a plausible mechanism for the contraction/shrinkage of mesh observed following implantation. To prevent pore collapse, we proposed to design synthetic meshes with a macrostructure that results in auxetic behavior, the pores expand laterally, instead of contracting when loaded. Such behavior can be achieved with a range of auxetic structures/geometries. This study utilized finite element analysis (FEA) to assess the behavior of mesh models with eight auxetic pore geometries subjected to uniaxial loading to evaluate their potential to allow for pore expansion while simultaneously providing resistance to tensile loading. Overall, substituting auxetic geometries for standard pore geometries yielded more pore expansion, but often at the expense of increased model elongation, with two of the eight auxetics not able to maintain pore expansion at higher levels of tension. Meshes with stable pore geometries that remain open with loading will afford the ingrowth of host tissue into the pores and improved integration of the mesh. Given the demonstrated ability of auxetic geometries to allow for pore size maintenance (and pore expansion), auxetically designed meshes have the potential to significantly impact surgical outcomes and decrease the likelihood of major mesh-related complications.

Background: Herbicide safening in cereals is linked to a rapid xenobiotic response (XR), involving the induction of glutathione transferases (GSTs). The XR is also invoked by oxidized fatty acids (oxylipins) released during plant stress, suggesting a link between these signalling agents and safening. To examine this relationship, a series of compounds modelled on the oxylipins 12-oxophytodienoic acid and phytoprostane 1, varying in lipophilicity and electrophilicity, were synthesized. Compounds were then tested for their ability to invoke the XR in Arabidopsis and protect rice seedlings exposed to the herbicide pretilachlor, as compared with the safener fenclorim., Results: Of the 21 compounds tested, three invoked the rapid GST induction associated with fenclorim. All compounds possessed two electrophilic carbon centres and a lipophilic group characteristic of both oxylipins and fenclorim. Minor effects observed in protecting rice seedlings from herbicide damage positively correlated with the XR, but did not provide functional safening., Conclusion: The design of safeners based on the characteristics of oxylipins proved successful in deriving compounds that invoke a rapid XR in Arabidopsis but not in providing classical safening in a cereal. The results further support a link between safener and oxylipin signalling, but also highlight species-dependent differences in the responses to these compounds. © 2018 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2018 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

Polymicrogyria is a malformation of cortical development. The aetiology of polymicrogyria remains poorly understood. Using whole-exome sequencing we found de novo heterozygous missense GRIN1 mutations in 2 of 57 parent-offspring trios with polymicrogyria. We found nine further de novo missense GRIN1 mutations in additional cortical malformation patients. Shared features in the patients were extensive bilateral polymicrogyria associated with severe developmental delay, postnatal microcephaly, cortical visual impairment and intractable epilepsy. GRIN1 encodes GluN1, the essential subunit of the N-methyl-d-aspartate receptor. The polymicrogyria-associated GRIN1 mutations tended to cluster in the S2 region (part of the ligand-binding domain of GluN1) or the adjacent M3 helix. These regions are rarely mutated in the normal population or in GRIN1 patients without polymicrogyria. Using two-electrode and whole-cell voltage-clamp analysis, we showed that the polymicrogyria-associated GRIN1 mutations significantly alter the in vitro activity of the receptor. Three of the mutations increased agonist potency while one reduced proton inhibition of the receptor. These results are striking because previous GRIN1 mutations have generally caused loss of function, and because N-methyl-d-aspartate receptor agonists have been used for many years to generate animal models of polymicrogyria. Overall, our results expand the phenotypic spectrum associated with GRIN1 mutations and highlight the important role of N-methyl-d-aspartate receptor signalling in the pathogenesis of polymicrogyria.

The type III secretion system (T3SS) is essential for the pathogenesis of many bacteria including Salmonella and Shigella, which together are responsible for millions of deaths worldwide each year. The structural component of the T3SS consists of the needle apparatus, which is assembled in part by the protein-protein interaction between the tip and the translocon. The atomic detail of the interaction between the tip and the translocon proteins is currently unknown. Here, we used NMR methods to identify that the N-terminal domain of the Salmonella SipB translocon protein interacts with the SipD tip protein at a surface at the distal region of the tip formed by the mixed α/β domain and a portion of its coiled-coil domain. Likewise, the Shigella IpaB translocon protein and the IpaD tip protein interact with each other using similar surfaces identified for the Salmonella homologs. Furthermore, removal of the extreme N-terminal residues of the translocon protein, previously thought to be important for the interaction, had little change on the binding surface. Finally, mutations at the binding surface of SipD reduced invasion of Salmonella into human intestinal epithelial cells. Together, these results reveal the binding surfaces involved in the tip-translocon protein-protein interaction and advance our understanding of the assembly of the T3SS needle apparatus. Proteins 2016; 84:1097-1107. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Introduction and Hypothesis: Parity is the leading risk factor for the development of pelvic organ prolapse. To assess the impact of pregnancy and delivery on vaginal tissue, researchers commonly use nonhuman primate (NHP) and rodent models. The purpose of this study was to evaluate the ewe as an alternative model by investigating the impact of parity on the ewe vaginal mechanical properties and collagen structure., Methods: Mechanical properties of 15 nulliparous and parous ewe vaginas were determined via uniaxial tensile tests. Collagen content was determined by hydroxyproline assay and collagen fiber thickness was analyzed using picrosirius red staining. Outcome measures were compared using Independent samples t or Mann-Whitney U tests. ANOVA (Gabriel's pairwise post-hoc test) or the Welch Alternative for the F-ratio (Games Howell post-hoc test) was used to compare data with previously published NHP and rodent data., Results: Vaginal tissue from the nulliparous ewe had a higher tangent modulus and tensile strength compared with the parous ewe (p < 0.025). The parous ewe vagina elongated 42 % more than the nulliparous ewe vagina (p = 0.015). No significant differences were observed in collagen structure among ewe vaginas. The tangent modulus of the nulliparous ewe vagina was not different from that of the NHP or rodent (p = 0.290). Additionally, the tangent moduli of the parous ewe and NHP vaginas did not differ (p = 0.773)., Conclusions: Parity has a negative impact on the mechanical properties of the ewe vagina, as also observed in the NHP. The ewe may serve as an alternative model for studying parity and ultimately prolapse development., Competing Interests: K.M. Knight, A. Nolfi, S. Palcsey, and W. Barone have no conflicts of interest to disclose. P.A. Moalli received a research grant from the National Institutes of Health, R01 HD-061811, and has a cooperative research agreement with ACell. S.D. Abramowitch received a research grant from the National Institutes of Health, R01 HD-061811, and has a cooperative research agreement with ACell.

Background: Due to large geographical distances, the telephone is central to enabling rural Australian communities to access care from their local health service. While there is a history of rural nurses providing care via the telephone, it has been a highly controversial practice that is not routinely documented and little is known about how the practice is governed. The lack of knowledge regarding governance extends to the role of Directors of Nursing as clinical leaders charged with the responsibility of ensuring practice safety, quality, regulation and risk management. The purpose of this study was to identify clinical governance processes related to managing telephone presentations, and to explore Directors of Nursing perceptions of processes and clinical practices related to the management of telephone presentations to health services in rural Victoria, Australia., Methods: Qualitative documentary analysis and semi structured interviews were used in the study to examine the content of health service policies and explore the perceptions of Directors of Nursing in eight rural health services regarding policy content and enactment when people telephone rural health services for care. Participants were purposively selected for their knowledge and leadership role in governance processes and clinical practice. Data from the interviews were analysed using framework analysis. The process of analysis resulted in the identification of five themes., Results: The majority of policies reviewed provided little guidance for managing telephone presentations. The Directors of Nursing perceived policy content and enactment to be largely inadequate. When organisational structures failed to provide appropriate governance for the context, the Directors of Nursing engaged in protective mechanisms to support rural nurses who manage telephone presentations., Conclusions: Rural Directors of Nursing employed intuitive behaviours to protect rural nurses practicing within a clinical governance context that is inadequate for the complexities of the environment. Protective mechanisms provided indicators of clinical leadership and governance effectiveness, which may assist rural nurse leaders to strengthen quality and safe care by unlocking the potential of intuitive behaviours. Kanter's theory of structural power provides a way of conceptualising these protective mechanisms, illustrating how rural nurse leaders enact power.

Objective: To characterize the neonatal and maternal outcomes of type 2 diabetic patients as compared with type 1 diabetic patients and nondiabetic controls., Study Design: We performed a retrospective cohort study reviewing perinatal outcomes of type 1 and type 2 diabetic patients and nondiabetic controls from July 2000 to August 2006. Analysis of variance, t testing and chi2 analysis were used to compare groups. Post hoc power analysis indicated 80% power was necessary to detect a 15% difference in composite poor neonatal outcomes., Results: A total of 64 type 2 and 64 type 1 diabetic patients were compared with 256 controls. Type 1 diabetic patients had higher incidences of composite poor neonatal outcome and congenital anomalies than did type 2 diabetic and control patients. Both diabetic groups had similarly higher incidences of cesarean delivery, preeclampsia, preterm delivery, polyhydramnios and macrosomia than did controls., Conclusion: Type 2 diabetic patients have a decreased incidence of adverse neonatal outcomes when compared with that of type 1 diabetic patients. No difference was observed between the diabetic groups in the incidence of a majority of the adverse maternal outcomes examined, however both diabetic groups had overall worse outcomes that did nondiabetic controls.

Plants respond to synthetic chemicals by eliciting a xenobiotic response (XR) that enhances the expression of detoxifying enzymes such as glutathione transferases (GSTs). In agrochemistry, the ability of safeners to induce an XR is used to increase herbicide detoxification in cereal crops. Based on the responsiveness of the model plant Arabidopsis thaliana to the rice safener fenclorim (4,6-dichloro-2-phenylpyrimidine), a series of related derivatives was prepared and tested for the ability to induce GSTs in cell suspension cultures. The XR in Arabidopsis could be divided into rapid and slow types depending on subtle variations in the reactivity (electrophilicity) and chemical structure of the derivatives. In a comparative microarray study, Arabidopsis cultures were treated with closely related compounds that elicited rapid (fenclorim) and slow (4-chloro-6-methyl-2-phenylpyrimidine) XRs. Both chemicals induced major changes in gene expression, including a coordinated suppression in cell wall biosynthesis and an up-regulation in detoxification pathways, whereas only fenclorim selectively induced sulfur and phenolic metabolism. These transcriptome studies suggested several linkages between the XR and oxidative and oxylipin signaling. Confirming links with abiotic stress signaling, suppression of glutathione content enhanced GST induction by fenclorim, whereas fatty acid desaturase mutants, which were unable to synthesize oxylipins, showed an attenuated XR. Examining the significance of these studies to agrochemistry, only those fenclorim derivatives that elicited a rapid XR proved effective in increasing herbicide tolerance (safening) in rice.

Objectives: We sought to determine whether unhealthy behaviors play a stress-buffering role in observed racial disparities in physical and mental health., Methods: We conducted logistic regressions by race on data from the first 2 waves of the Americans' Changing Lives Survey to determine whether unhealthy behaviors had buffering effects on the relationship between major stressors and chronic health conditions, and on the relationship between major stressors and meeting the criteria for major depression., Results: Among Whites, unhealthy behaviors strengthened the relationship between stressors and meeting major-depression criteria. Among Blacks, however, the relationship between stressors and meeting major-depression criteria was stronger among those who had not engaged in unhealthy behaviors than among those who had. Among both race groups there was a positive association between stressors and chronic health conditions. Among Blacks there was an additional positive association between number of unhealthy behaviors and number of chronic conditions., Conclusions: Those who live in chronically stressful environments often cope with stressors by engaging in unhealthy behaviors that may have protective mental-health effects. However, such unhealthy behaviors can combine with negative environmental conditions to eventually contribute to morbidity and mortality disparities among social groups.

Objective: Maternal serum alpha-fetoprotein (MSAFP) values are reported to be lower in type 1 diabetic patients, and a correction factor is often applied. We sought to determine whether type 2 diabetic patients require the same MSAFP adjustments as type 1 diabetic patients., Study Design: We performed a retrospective review of MSAFP levels from a university laboratory in type 1 and type 2 diabetic patients between July 2000 and August 2006, matched 1:2 with controls. Groups were compared using analysis of variance and Student t testing., Results: Seventy-seven type 1 and 75 type 2 diabetic patients were compared with 304 controls. Type 1 and type 2 diabetic patients differed significantly from each other and controls before corrections. Diabetic patients were similar to each other, but significantly lower than controls, after weight corrections. These differences were eliminated by a 10% correction factor., Conclusion: Type 1 and type 2 diabetic patients require both weight and diabetes corrections to adjust MSAFP values to nondiabetic levels.

Creontiades spp. (Hemiptera: Miridae) are sucking pests that attack buds, flowers and young pods in mungbeans, Vigna radiata (L.), causing these structures subsequently to abort. If left uncontrolled, mirids can cause 25-50% yield loss. Traditional industry practice has involved prophylactic applications of dimethoate to control mirids at budding and again a week later. The present trial was initiated to highlight the dangers of such a practice, in particular the risk of a subsequent Helicoverpa spp. lepidopteran pest outbreak. A single application of dimethoate halved the population of important natural enemies of Helicoverpa spp., and caused an above-threshold outbreak of Helicoverpa spp. within 11 days. This shows that even a moderate (e.g. 50%) reduction in natural enemies may be sufficient to increase Helicoverpa spp. populations in mungbeans. As a result, prophylactic sprays should not be used for the control of mirids in mungbeans, and dimethoate should be applied only when mirids are above the economic threshold. Indoxacarb was also tested to establish its effect on Helicoverpa spp., mirids and natural enemies. Indoxacarb showed potential for Helicoverpa spp. control and suppression of mirids and had little impact on natural enemies.

Current guidelines state that education is fundamental to help people with diabetes modify their lifestyle and prevent ill health and early death. However, many people with diabetes are not receiving adequate education. There is a widespread assumption that transferring knowledge will improve health outcomes but there is little empirical support for this assertion. Indeed, knowledge and behaviour are poorly correlated. Knowledge may be a necessary condition but is rarely a sufficient condition for behaviour change. Single interventions, cognitive or behavioural, have had disappointing results, unsurprisingly given the complexity of human behaviour. The most effective interventions are multifaceted and include education, behavioural and psychosocial elements, and target lifestyle change and factors such as self-efficacy and empowerment. We advocate that educational interventions should have multiple components. They should aim to improve patients' sense of self-efficacy and empowerment, and build attitudes towards diabetes that will support the lifestyle changes needed for successful self-management. These conclusions have implications for future research and clinical practice.

Purpose: Motivational interviewing (MI), a method of augmenting an individual's motivation to change problematic behaviours, is a patient-centred counselling style that seeks to help patients resolve ambivalence about behaviour change. MI has successfully been used in the field of addictions and has recently received increased interest as a means of promoting treatment adherence in physical health care settings. This systematic review is aimed to evaluate the effectiveness of MI interventions in physical health care settings., Methods: Electronic databases were searched for articles specifying the use of 'motivational interviewing' in physical health care settings between 1966 and April 2004. Fifty-one relevant abstracts were yielded and data was extracted from eight relevant selected studies., Results: Eight studies were identified in the fields of diabetes, asthma, hypertension, hyperlipidaemia, and heart disease. The majority of studies found positive results for effects of MI on psychological, physiological, and life-style change outcomes. Problems with research in this area include: small sample sizes, lack of power, use of disparate multiple outcomes, inadequate validation of questionnaires, poorly-defined therapy and training., Conclusions: While MI has high face validity across a number of domains in physical health care settings, the general quality of trials in this area is inadequate and therefore recommendations for its dissemination in this area cannot yet be made. More research into MI applied to health behaviour change is urgently required.

Background: More people than ever are turning to the Internet for health-related information, and recent studies indicate that the information patients find online directly affects the decisions they make about their health care. Little is known about the information needs or actual search behavior of people who use the Internet for health information., Objective: This study analyzes what people search for when they use a health-education Web site offering information about arthritis, orthopaedics, and sports-medicine topics. Additionally, it determines who is performing these searches: is it patients, friends or relatives of patients, or neither? Finally, it examines the similarities and differences among questions submitted by Web site visitors from different countries., Methods: Content analysis was performed on 793 free-text search queries submitted to a patient-education Web site owned and operated by the Department of Orthopaedics and Sports Medicine at the University of Washington Medical Center. The 793-query data set was coded into 3 schemes: (1) the purpose of the query, (2) the topic of the query, and (3) the relationship between the asker of the query and the patient. We determined the country from which each query was submitted by analyzing the Internet Protocol addresses associated with the queries., Results: The 5 most frequent reasons visitors searched the Web site were to seek: (1) information about a condition, (2) information about treatment, (3) information about symptoms, (4) advice about symptoms, and (5) advice about treatment. We were able to determine the relationship between the person submitting the query and the patient in question for 178 queries. Of these, the asker was the patient in 140 cases, and the asker was a friend or relative of the patient in 38 cases. The queries were submitted from 34 nations, with most coming from the United States, Australia, the United Kingdom, and Canada. When comparing questions submitted from the United States versus those from all other countries, the 3 most frequent types of questions were the same for both groups (and were the top 3 question types listed above)., Conclusions: These results provide the University of Washington Department of Orthopaedics and Sports Medicine, as well as other organizations that provide health-information Web sites, with data about what people around the world are seeking when they turn to the Internet for health information. If Web site managers can adapt their health-information Web sites in response to these findings, patients may be able to find and use Internet-based health information more successfully, enabling them to participate more actively in their health care.

The authors have previously described a model of in vivo tissue generation based on an implanted, microsurgically created vessel loop in a plastic chamber (volume, 0.45 ml) containing a poly(DL-lactic-co-glycolic acid) (PLGA) scaffold. Tissue grew spontaneously in association with an intense angiogenic sprouting from the loop and almost filled the chamber, resulting in a mean amount of tissue in chambers of 0.23 g with no added matrix scaffold and 0.33 g of tissue in PLGA-filled chambers after 4 weeks of incubation. The aim of the present study was to investigate whether a greater volume of tissue could be generated when the same-size vessel loop was inserted into a larger (1.9 ml) chamber. In four groups of five rats, an arteriovenous shunt sandwiched between two disks of PLGA, used as a scaffold for structural support, was placed inside a large polycarbonate growth chamber. Tissue and PLGA weight and volume, as well as histological characteristics of the newly formed tissue, were assessed at 2, 4, 6, and 8 weeks. Tissue weight and volume showed a strong linear correlation. Tissue weight increased progressively from 0.13 +/- 0.04 g at 2 weeks to 0.57 +/- 0.06 g at 6 weeks (p < 0.0005). PLGA weight decreased progressively from 0.89 +/- 0.07 g at 2 weeks to 0.20 +/- 0.09 g at 8 weeks (p < 0.0005). Histological examination of the specimens confirmed increased tissue growth and maturation over time. It is concluded that larger quantities of tissue can be grown over a longer period of time by using larger-size growth chambers.

In prepubertal girls with vaginal discharge, consideration of the etiology must be given to respiratory pathogens (Streptococcus pyogenes, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae, and Neisseria meningitidis), enteric pathogens (Escherichia coli, Shigella, and Yersinia), poor hygiene, foreign body, nonabsorbent undergarments, irritants, vulvar skin disease, anatomic abnormalities (double vagina with fistula, pelvic abscess, and ectopic ureter), and sexual abuse. Prepubertal girls, outside the newborn period, with suspected gonococcal infection should be strongly considered to be victims of sexual abuse, once congenital and other newborn acquired forms of gonorrhea are excluded. We present a case of a three-year-old female with vaginal discharge and fever with a clouded social history, disproportionate distress on physical exam, and initial laboratory gram stain suggestive of gonococcus.

In a multicenter study, responses to a combined vaccine containing standard diphtheria-tetanus-pertussis (DTP) and polyribosylribitol phosphate (PRP), the capsular polysaccharide of Haemophilus influenzae type b, were evaluated in 107 infants who received single doses at two, four, and six months of age and compared with those in 61 infants given single doses of DTP alone on the same schedule. Reaction rates were comparable in the two treatment groups. At seven months of age 61% of the subjects given the combined DTP-PRP vaccine and 8% and of those given DTP alone showed an antibody response to PRP, as defined as a twofold increase in titer over the lowest previous level. Among those given the DTP-PRP combined vaccine, 92% of the positive antibody responses occurred after the third dose. There was a variation in antibody response, possibly due to a difference in the vaccine lots administered.