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Insulinomas are rare, benign and functional tumors that coincidentally may become overt during pregnancy or in the post-partum period. As the general symptoms of a pregnancy might cover the clinical presentation, diagnosing remains challenging. We present one additional case of a post-partum insulinoma, combined with a systematic review of the literature to sum up relevant details in diagnosis and treatment. A systematic request of Pubmed/Medline was conducted using the following terms: "insulinoma AND pregnancy" and "insulinoma" for a second request of ClinicalTrials.gov. All publications concerning pregnant or post-partum women with insulinoma were included. Thirty-six cases could be identified for analysis. Each publication was reviewed for demographic, diagnostic and therapeutic data. The most frequent clinical signs were unconsciousness and neurological symptoms. 64.9% were diagnosed during early pregnancy and 35.1% post-partum. 91.9% underwent surgery with a third resected during pregnancy without severe influence on fetal or maternal outcome. Three patients died of metastatic disease or misdiagnosing, two of them miscarried. Insulinoma in pregnancy is rare but should be considered in case of unclear hyperinsulinemic hypoglycemia. Surgery can be performed during the second trimester or post-partum with promising outcome.

Hypoglycaemia related to exercise and lack of confidence to exercise, are common in T1DM. An online educational exercise tool (ExT1D) was tested to determine whether these parameters can be improved.Thirty two adults with T1DM (50%M, age 35.8 ± 9.5 yr diabetes duration 12.3 ± 9.9 yr, median HbA1c 7.1%[ICR 6.4-7.7] NGSPU) exercising ≥ 60 min/week enrolled in a RCT utilising ExT1D, with partial cross-over design. The primary end-point was Exercise-related hypoglycaemia (ErH) number corrected for exercise session number, with ErH defined as CGM episodes 4.0 mM occurring within 24 h of exercise. Secondary RCT endpoints were total ErH duration, and ErH duration/episode. A pre-defined longitudinal analysis with each subject compared with their baseline was also undertaken, for the three ErH parameters, and using fear of hypoglycaemia questionnaires.In the RCT a 50% lower median ErH number (P = 0.6) (37% lower ErH number per exercise session (P = 0.06, NS primary endpoint) occurred in the Intervention vs Control group. A 49% lower ErH duration per episode (P = 0.2), and 80% less ErH duration (P = 0.3), were also observed in the Intervention vs Control group. In the longitudinal study, ErH number reduced by 43% (P = 0.088), ErH duration per episode by 52% (P = 0.157) and total duration of ErH fell by 71% (P = 0.015). Confidence to prevent glucose lowering by exercise also improved (P = 0.039). Post-hoc analysis showed those with the greatest ErH events at baseline benefited most. Fructosamine and HbA1c levels were unchanged from baseline.ExT1D can reduce exercise-related hypoglycaemia and provide greater confidence to exercise.

Regulatory T cells (Tregs) play a pivotal role in regulating immune responses and are a significant source of the anti-inflammatory cytokine IL-10 in the intestine. Defects in Tregs function lead to the development of systemic and intestinal inflammation. Using a single-cell BTB-ZF transcription factor expression analysis, we identified a distinct subset of Tregs. These cells have an activated phenotype (CD62Llo, CD44hi) and constitutively express the Il10. Moreover, these Tregs accumulate in the intestine and expand in number following DSS induced colitis. The targeted deletion of the defining transcription factor impaired these cells' ability to secrete IL-10, leading to significant disruptions in mucosal architecture and the development of intestinal inflammation following treatment with dextran sodium sulfate (DSS). Thus, the conditional knockout (cKO) mice developed much more severe symptoms leading to the death of 60% of them. In contrast, all wild type mice fully recovered. Notably, adoptive transfer of this Treg subset was sufficient to prevent the death of cKO mice. In comparison, the transfer of cKO Tregs was not able to rescue the mice. Collectively, our data show that we have identified a distinct subset of Tregs and determined a specific member of the BTB-ZF family that controls their function. This rare population of T cells has potent immunosuppressive abilities and, we propose, rather than acutely producing IL-10 in response to activation, these cells maintain intestinal homeostasis through continuous secretion of IL-10. Our findings imply that analogous to NKT cells, some thymic-derived Tregs have innate-like effector functions that are not dependent upon differentiation in the periphery.

Myosin light chain kinase (MLCK) phosphorylates S19 of the myosin regulatory light chain (RLC), which is required to activate myosin's ATPase activity and contraction. Smooth muscles are known to display plasticity in response to factors such as inflammation, developmental stage, or stress, which lead to differential expression of nonmuscle and smooth muscle isoforms. Here, we compare steady-state kinetics parameters for phosphorylation of different MLCK substrates: (1) nonmuscle RLC, (2) smooth muscle RLC, and heavy meromyosin subfragments of (3) nonmuscle myosin IIB, and (4) smooth muscle myosin II. We show that MLCK has a ~2-fold higher kcat for both smooth muscle myosin II substrates compared with nonmuscle myosin IIB substrates, whereas Km values were very similar. Myosin light chain kinase has a 1.6-fold and 1.5-fold higher specificity (kcat /Km ) for smooth versus nonmuscle-free RLC and heavy meromyosin, respectively, suggesting that differences in specificity are dictated by RLC sequences. Of the 10 non-identical RLC residues, we ruled out 7 as possible underlying causes of different MLCK kinetics. The remaining 3 residues were found to be surface exposed in the N-terminal half of the RLC, consistent with their importance in substrate recognition. These data are consistent with prior deletion/chimera studies and significantly add to understanding of MLCK myosin interactions. SIGNIFICANCE OF THE STUDY Phosphorylation of nonmuscle and smooth muscle myosin by myosin light chain kinase (MLCK) is required for activation of myosin's ATPase activity. In smooth muscles, nonmuscle myosin coexists with smooth muscle myosin, but the two myosins have very different chemo-mechanical properties relating to their ability to maintain force. Differences in specificity of MLCK for different myosin isoforms had not been previously investigated. We show that the MLCK prefers smooth muscle myosin by a significant factor. These data suggest that nonmuscle myosin is phosphorylated more slowly than smooth muscle myosin during a contraction cycle.

Thousands of Hymenopteran endoparasitoids have developed a unique symbiotic relationship with viruses named polydnavirus (PDVs). These viruses immunocompromise the lepidopteran host allowing the survival of the wasp eggs. In a previous work, we have shown the horizontal transfer of some polydnaviral genes into the genome of the Lepidoptera, Spodoptera exigua. One of these genes, BV2-5 (named gasmin) interferes with actin polymerization, negatively affecting the multiplication of baculovirus in cell culture. In this work, we have focused in the study of the effect of Gasmin expression on different aspects of the baculovirus production. In addition, and since actin polymerization is crucial for phagocytosis, we have studied the effect of Gasmin expression on the larval interaction with bacterial pathogens. Over-expression of Gasmin on hemocytes significantly reduces their capacity to phagocytize the pathogenic bacteria Bacillus thuringiensis. According to these results, gasmin domestication negatively affects baculovirus replication, but increases larvae susceptibility to bacterial infections as pay off. Although the effect of Gasmin on the insect interaction with other pathogens or parasitoids remain unknown, the opposite effects described here could shape the biological history of this species based on the abundance of certain type of pathogens as suggested by the presence of truncated forms of this protein in several regions of the world.

Summary Trigeminal neuralgia (TN) is a common, debilitating neuropathic face pain syndrome often resistant to therapy. The familial clustering of TN cases suggests that genetic factors play a role in disease pathogenesis. However, no unbiased, large-scale genomic study of TN has been performed to date. Analysis of 290 whole exome-sequenced TN probands, including 20 multiplex kindreds and 70 parent-offspring trios, revealed enrichment of rare, damaging variants in GABA receptor-binding genes in cases. Mice engineered with a TN-associated de novo mutation (p.Cys188Trp) in the GABAA receptor Cl− channel γ-1 subunit (GABRG1) exhibited trigeminal mechanical allodynia and face pain behavior. Other TN probands harbored rare damaging variants in Na+ and Ca+ channels, including a significant variant burden in the α-1H subunit of the voltage-gated Ca2+ channel Cav3.2 (CACNA1H). These results provide exome-level insight into TN and implicate genetically encoded impairment of GABA signaling and neuronal ion transport in TN pathogenesis., Graphical Abstract, Highlights • Genomic analysis of trigeminal neuralgia (TN) using exome sequencing • Rare mutations in GABA signaling and ion transport genes are enriched in TN cases • Generation of a genetic TN mouse model engineered with a patient-specific mutation, Neuroscience; Structural Biology; Genomics

For studies requiring accurate conception-timing, reliable, efficient methods of detecting oestrus reduce time and costs, whilst improving welfare. Standard methods use vaginal cytology to stage cycle, and breeders are paired-up using approximately five proven females with proven males to achieve at least one conception on a specific day. We describe an alternative, fast, consistent, non-invasive method of timed-mating using detection of lordosis behaviour in Wistar and Lister-Hooded rats that used unproven females with high success rates. Rats under reverse lighting had body masses recorded pre-mating, day (d) 3–4, d8, d10 and d18 of pregnancy. Using only the presence of the oestrus dance to time-mate females for 24 hours, 89% of Wistar and 88% of Lister-Hooded rats successfully conceived. We did not observe behavioural oestrus in Sprague-Dawleys without males being present. Significant body mass increases following mating distinguished pregnant from non-pregnant rats, as early as d4 of pregnancy (10% ± 1.0 increase cf. 3% ± 1.2). The pattern of increases throughout gestation was similar for all pregnant rats until late pregnancy, when there were smaller increases for primi- and multiparous rats (32% ± 2.5; 25% ± 2.4), whereas nulliparous rats had highest gains (38% ± 1.5). This method demonstrated a distinct refinement of the previous timed-mating common practice used, as disturbance of females was minimised. Only the number required of nulli-, primi- or multiparous rats were mated, and body mass increases validated pregnancy status. This new breeding management method is now established practice for two strains of rat and has resulted in a reduction in animal use.

Several expressed sequence tags (ESTs) with homology to chitin deacetylase-like protein (CDA) were selected from a group of Helicoverpa armigera genes whose expression changed after infection with H. armigera single nucleopolyhedrovirus (HearNPV). Some of these ESTs coded for a midgut protein containing a chitin deacetylase domain (CDAD). The expressed protein, HaCDA5a, did not show chitin deacetylase activity, but it showed a strong affinity for binding to chitin. Sequence analysis showed the lack of any chitin binding domain, described for all currently known peritrophic membrane (PM) proteins. HaCDA5a has previously been detected in the H. armigera PM. Such localization, together with its downregulation after pathogen infection, led us to hypothesize that this protein might be responsible for the homeostasis of the PM structure and that, by reduction of its expression, the insect may reduce PM permeability, decreasing the entrance of baculovirus. To test this hypothesis, we constructed a recombinant nucleopolyhedrovirus to express HaCDA5a in insect cells and tested its influence on PM permeability as well as the influence of HaCDA5a expression on the performance of the baculovirus. The experiments showed that HaCDA5a increased PM permeability, in a concentration-dependent manner. Bioassays on Spodoptera frugiperda and Spodoptera exigua larvae revealed that NPV expressing HaCDA5a was more infective than its parental virus. However, no difference in virulence was observed when the viruses were injected intrahemocoelically. These findings support the downregulation of a midgut-specific CDA-like protein as a possible mechanism used by H. armigera to reduce susceptibility to baculovirus by decreasing PM permeability.

Insect innate immunity relies on numerous soluble and membrane-bound receptors, named pattern recognition proteins (PRPs), which enable the insect to recognize pathogen-associated molecular patterns. C-type lectins are among the best-studied PRPs and constitute the most diverse family of animal lectins. Here we have characterized two groups of Spodoptera exigua C-type lectins that differ in their phylogeny, domain architecture, and expression pattern. One group includes C-type lectins with similar characteristics to other lepidopteran lectins, and a second group includes bracoviral-related lectins (bracovirus-like lectins, Se-BLLs) recently acquired by horizontal gene transfer. Subsequently, we have investigated the potential role of some selected lectins in the susceptibility to Junonia coenia densovirus (JcDV). For this purpose, three of the bracoviral-related lectins were expressed, purified, and their effect on the densovirus infection to two different Spodoptera species was assessed. The results showed that Se-BLL3 specifically reduce the mortality of Spodoptera frugiperda larvae caused by JcDV. In contrast, no such effect was observed with S. exigua larvae. In a previous work, we have also shown that Se-BLL2 increased the tolerance of S. exigua larvae to baculovirus infection. Taken together, these results confirm the implication of two different C-type lectins in antiviral response and reflect the biological relevance of the acquisition of bracoviral genes in Spodoptera spp.

Scientific access to spaceflight and especially the International Space Station has revealed that physiological adaptation to spaceflight is accompanied or enabled by changes in gene expression that significantly alter the transcriptome of cells in spaceflight. A wide range of experiments have shown that plant physiological adaptation to spaceflight involves gene expression changes that alter cell wall and other metabolisms. However, while transcriptome profiling aptly illuminates changes in gene expression that accompany spaceflight adaptation, mutation analysis is required to illuminate key elements required for that adaptation. Here we report how transcriptome profiling was used to gain insight into the spaceflight adaptation role of Altered response to gravity 1 (Arg1), a gene known to affect gravity responses in plants on Earth. The study compared expression profiles of cultured lines of Arabidopsis thaliana derived from wild-type (WT) cultivar Col-0 to profiles from a knock-out line deficient in the gene encoding ARG1 (ARG1 KO), both on the ground and in space. The cell lines were launched on SpaceX CRS-2 as part of the Cellular Expression Logic (CEL) experiment of the BRIC-17 spaceflight mission. The cultured cell lines were grown within 60 mm Petri plates in Petri Dish Fixation Units (PDFUs) that were housed within the Biological Research In Canisters (BRIC) hardware. Spaceflight samples were fixed on orbit. Differentially expressed genes were identified between the two environments (spaceflight and comparable ground controls) and the two genotypes (WT and ARG1 KO). Each genotype engaged unique genes during physiological adaptation to the spaceflight environment, with little overlap. Most of the genes altered in expression in spaceflight in WT cells were found to be Arg1-dependent, suggesting a major role for that gene in the physiological adaptation of undifferentiated cells to spaceflight. Key Words: ARG1-Spaceflight-Gene expression-Physiological adaptation-BRIC. Astrobiology 17, 1077-1111.

Experimentation on the International Space Station has reached the stage where repeated and nuanced transcriptome studies are beginning to illuminate the structural and metabolic differences between plants grown in space compared to plants on the Earth. Genes that are important in establishing the spaceflight responses are being identified, their roles in spaceflight physiological adaptation are increasingly understood, and the fact that different genotypes adapt differently is recognized. However, the basic question of whether these spaceflight responses are actually required for survival has yet to be posed, and the fundamental notion that spaceflight responses may be non-adaptive has yet to be explored. Therefore the experiments presented here were designed to ask if portions of the plant spaceflight response can be genetically removed without causing loss of spaceflight survival and without causing increased stress responses. The CARA experiment compared the spaceflight transcriptome responses in the root tips of two Arabidopsis ecotypes, Col-0 and WS, as well as that of a PhyD mutant of Col-0. When grown with the ambient light of the ISS, phyD plants displayed a significantly reduced spaceflight transcriptome response compared to Col-0, suggesting that altering the activity of a single gene can actually improve spaceflight adaptation by reducing the transcriptome cost of physiological adaptation. The WS genotype showed an even simpler spaceflight transcriptome response in the ambient light of the ISS, more broadly indicating that the plant genotype can be manipulated to reduce the cost of spaceflight adaptation, as measured by transcriptional response. These differential genotypic responses suggest that genetic manipulation could further reduce, or perhaps eliminate the metabolic cost of spaceflight adaptation. When plants were germinated and then left in the dark on the ISS, the WS genotype actually mounted a larger transcriptome response than Col-0, suggesting that the in-space light environment affects physiological adaptation, which implies that manipulating the local habitat can also substantially impact the metabolic cost of spaceflight adaptation.

The use of zebrafish (Danio rerio) as an animal model for experimental studies of stress has increased rapidly over the years. Although many physiologic and behavioral characteristics associated with stress have been defined in zebrafish, the effects of stress on hematologic parameters have not been described. The purpose of our study was to induce a rise in endogenous cortisol through various acute and chronic stressors and compare the effects of these stressors on peripheral WBC populations. Acutely stressed fish underwent dorsal or full-body exposure to air for 3 min, repeated every 30 min over the course of 90 min. Chronically stressed fish underwent exposure to stressors twice daily over a period of 5 d. After the last stressful event, fish were euthanized, and whole blood and plasma were obtained. A drop of whole blood was used to create a blood smear, which was subsequently stained with a modified Wright-Giemsa stain and a 50-WBC differential count determined. Plasma cortisol levels were determined by using a commercially available ELISA. Endogenous cortisol concentrations were significantly higher in both stressed groups as compared with control fish. Acutely stressed fish demonstrated significant lymphopenia, monocytosis, and neutrophilia, compared with unstressed, control fish. Chronic stress induced lymphopenia and monocytosis but no significant changes in relative neutrophil populations in zebrafish. The changes in both stressed groups most likely are due to increases in endogenous cortisol concentrations and represent the first description of a stress leukogram in zebrafish.

Biological insecticides based on Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) can efficiently control S. exigua larvae on field and greenhouse crops in many parts of the world. Spanish wild populations and laboratory colonies of S. exigua are infected by two iflaviruses (SeIV-1 and SeIV-2). Here we evaluated the effect of iflavirus co-infection on the insecticidal characteristics of SeMNPV occlusion bodies (OBs). Overall, iflavirus co-inoculation consistently reduced median lethal concentrations (LC50) for SeMNPV OBs compared to larvae infected with SeMNPV alone. However, the speed of kill of SeMNPV was similar in the presence or absence of the iflaviruses. A reduction of the weight gain (27%) associated with iflavirus infection resulted in a 30% reduction in total OB production per larva. Adult survivors of SeMNPV OB inoculation were examined for covert infection. SeMNPV DNA was found to be present at a high prevalence in all SeIV-1 and SeIV-2 co-infection treatments. Interestingly, co-inoculation of SeMNPV with SeIV-2 alone or in mixtures with SeIV-1 resulted in a significant increase in the SeMNPV load of sublethally infected adults, suggesting a role for SeIV-2 in vertical transmission or reactivation of sublethal SeMNPV infections. In conclusion, iflaviruses are not desirable in insect colonies used for large scale baculovirus production, as they may result in diminished larval growth, reduced OB production and, depending on their host-range, potential risks to non-target Lepidoptera., This study was funded by the Spanish Ministerio de Ciencia e Innovacio´n, project number AGL2011-30352-C02-01. AC received a student scholarship from the Ministerio de Economı´a y Competitividad.

Controlled hypobaria presents biology with an environment that is never encountered in terrestrial ecology, yet the apparent components of hypobaria are stresses typical of terrestrial ecosystems. High altitude, for example, presents terrestrial hypobaria always with hypoxia as a component stress, since the relative partial pressure of O2 is constant in the atmosphere. Laboratory-controlled hypobaria, however, allows the dissection of pressure effects away from the effects typically associated with altitude, in particular hypoxia, as the partial pressure of O2 can be varied. In this study, whole transcriptomes of plants grown in ambient (97 kPa/pO2 = 21 kPa) atmospheric conditions were compared to those of plants transferred to five different atmospheres of varying pressure and oxygen composition for 24 h: 50 kPa/pO2 = 10 kPa, 25 kPa/pO2 = 5 kPa, 50 kPa/pO2 = 21 kPa, 25 kPa/pO2 = 21 kPa, or 97 kPa/pO2 = 5 kPa. The plants exposed to these environments were 10 day old Arabidopsis seedlings grown vertically on hydrated nutrient plates. In addition, 5 day old plants were also exposed for 24 h to the 50 kPa and ambient environments to evaluate age-dependent responses. The gene expression profiles from roots and shoots showed that the hypobaric response contained more complex gene regulation than simple hypoxia, and that adding back oxygen to normoxic conditions did not completely alleviate gene expression changes in hypobaric responses.

Aminopeptidase N (APN) isoforms from Lepidoptera are known for their involvement in the mode of action of insecticidal Cry proteins from Bacillus thuringiensis. These enzymes belong to a protein family with at least eight different members that are expressed simultaneously in the midgut of lepidopteran larvae. Here, we focus on the characterization of the APNs from Ostrinia nubilalis (OnAPNs) to identify potential Cry receptors. We expressed OnAPNs in insect cells using a baculovirus system and analyzed their enzymatic activity by probing substrate specificity and inhibitor susceptibility. The interaction with Cry1Ab and Cry1Fa proteins (both found in transgenic insect-resistant maize) was evaluated by ligand blot assays and immunocytochemistry. Ligand blots of brush border membrane proteins showed that both Cry proteins bound mainly to a 150 kDa-band, in which OnAPNs were greatly represented. Binding analysis of Cry proteins to the cell-expressed OnAPNs showed that OnAPN1 interacted with both Cry1Ab and Cry1Fa, whereas OnAPN3a and OnAPN8 only bound to Cry1Fa. Two isoforms, OnAPN2 and OnAPN3b, did not interact with any of these two proteins. This work provides the first evidence of a differential role of OnAPN isoforms in the mode of action of Cry proteins in O. nubilalis.

The consumption of probiotics has become increasingly popular as a means to try to improve health and well-being. Not only are probiotics considered beneficial to digestive health, but increasing evidence suggests direct and indirect interactions between gut microbiota (GM) and the central nervous system (CNS). Here, adult zebrafish were supplemented with Lactobacillus plantarum to determine the effects of probiotic treatment on structural and functional changes of the GM, as well as host neurological and behavioral changes. L. plantarum administration altered the β-diversity of the GM while leaving the major core architecture intact. These minor structural changes were accompanied by significant enrichment of several predicted metabolic pathways. In addition to GM modifications, L. plantarum treatment also significantly reduced anxiety-related behavior and altered GABAergic and serotonergic signaling in the brain. Lastly, L. plantarum supplementation provided protection against stress-induced dysbiosis of the GM. These results underscore the influence commensal microbes have on physiological function in the host and demonstrate bidirectional communication between the GM and the host.

Teams of myosin motors carry out intracellular transport and contract the actin cytoskeleton. To fully understand the behavior of multi-myosin ensembles we need to know the properties of individual myosins and the mode of interaction between them. Current models of the interactions within the myosin complex treat the actin filament as a stiff rod, not contributing to the regulation of collective myosin dynamics. Here, we present data suggesting that force transduction through the actin filament is an important element of interaction within myosin-6 ensembles in vitro. Multiple myosin-6s coordinate their steps if they are separated by a short (and therefore high-force bearing) segment of actin. The measurements were performed using Fluorescence Interference Contrast Microscopy (FLIC) to measure small changes in the height of fluorescently labeled actin. Using FLIC, we assign the positions of myosins in a gliding filament assay geometry and measure their attachment time to actin. We also identify actin segments that are buckled or under tension. We show that myosin-6 holds actin about 10 nm above the surface. However, due to asynchronous myosin stepping, frequent buckles up to about 60 nm high appear. The buckle lifetime decreases as the distance between the myosin-6s is reduced, a sign of inter-motor coordination. Our data are consistent with coordinated stepping of closely spaced myosins, but uncoordinated motility with widely separated myosins where buckles can form. These features would be expected to operate on myosins in the cell, where motor spacing may vary considerably depending on the target organelle.SIGNIFICANCE STATEMENTMyosins are molecular motors that carry out intracellular transport. Interactions between the myosins are crucial for understanding their function. Using Fluorescence Interference Contrast (FLIC) microscopy we characterized the interaction between multiple myosin-6 motors immobilized to the surface of a slide and pulling the same actin filament. Our results point towards coordination of myosin steps as a mechanism governing the behavior of a multi-myosin complex. We also demonstrated the unique application of FLIC microscopy for highly parallel identification and measurement of single myosin motors in a gliding filament format. These features of FLIC enable a robust study of collective myosin dynamics.

Background Skewing root patterns provide key insights into root growth strategies and mechanisms that produce root architectures. Roots exhibit skewing and waving when grown on a tilted, impenetrable surface. The genetics guiding these morphologies have been examined, revealing that some Arabidopsis ecotypes skew and wave (e.g. WS), while others skew insignificantly but still wave (e.g. Col-0). The underlying molecular mechanisms of skewing and waving remain unclear. In this study, transcriptome data were derived from two Arabidopsis ecotypes, WS and Col-0, under three tilted growth conditions in order to identify candidate genes involved in skewing. Results This work identifies a number of genes that are likely involved in skewing, using growth conditions that differentially affect skewing and waving. Comparing the gene expression profiles of WS and Col-0 in different tilted growth conditions identified 11 candidate genes as potentially involved in the control of skewing. These 11 genes are involved in several different cellular processes, including sugar transport, salt signaling, cell wall organization, and hormone signaling. Conclusions This study identified 11 genes whose change in expression level is associated with root skewing behavior. These genes are involved in signaling and perception, rather than the physical restructuring of root. Future work is needed to elucidate the potential role of these candidate genes during root skewing. Electronic supplementary material The online version of this article (doi:10.1186/s12870-017-0975-9) contains supplementary material, which is available to authorized users.

The baculovirus expression vector system (BEVS) has been widely used to produce a large number of recombinant proteins, and is becoming one of the most powerful, robust, and cost-effective systems for the production of eukaryotic proteins. Nevertheless, as in any other protein expression system, it is important to improve the production capabilities of this vector. The orf46 viral gene was identified among the most highly abundant sequences in the transcriptome of Spodoptera exigua larvae infected with its native baculovirus, the S. exigua multiple nucleopolyhedrovirus (SeMNPV). Different sequences upstream of the orf46 gene were cloned, and their promoter activities were tested by the expression of the GFP reporter gene using the Autographa californica nucleopolyhedrovirus (AcMNPV) vector system in different insect cell lines (Sf21, Se301, and Hi5) and in larvae from S. exigua and Trichoplusia ni. The strongest promoter activity was defined by a 120 nt sequence upstream of the ATG start codon for the orf46 gene. On average, GFP expression under this new promoter was more than two fold higher than the expression obtained with the standard polyhedrin (pph) promoter. Additionally, the orf46 promoter was also tested in combination with the pph promoter, revealing an additive effect over the pph promoter activity. In conclusion, this new characterized promoter represents an excellent alternative to the most commonly used baculovirus promoters for the efficient expression of recombinant proteins using the BEVS.

Myosin light chain kinase (MLCK) phosphorylates S19 of the myosin regulatory light chain (RLC), which is required to activate myosin's ATPase activity and contraction. Smooth muscles are known to display plasticity in response to factors such as inflammation, developmental stage, or stress, which lead to differential expression of nonmuscle and smooth muscle isoforms. Here, we compare steady-state kinetics parameters for phosphorylation of different MLCK substrates: (1) nonmuscle RLC, (2) smooth muscle RLC, and heavy meromyosin subfragments of (3) nonmuscle myosin IIB, and (4) smooth muscle myosin II. We show that MLCK has a ~2-fold higher kcat for both smooth muscle myosin II substrates compared with nonmuscle myosin IIB substrates, whereas Km values were very similar. Myosin light chain kinase has a 1.6-fold and 1.5-fold higher specificity (kcat/Km) for smooth versus nonmuscle-free RLC and heavy meromyosin, respectively, suggesting that differences in specificity are dictated by RLC sequences. Of the 10 non-identical RLC residues, we ruled out 7 as possible underlying causes of different MLCK kinetics. The remaining 3 residues were found to be surface exposed in the N-terminal half of the RLC, consistent with their importance in substrate recognition. These data are consistent with prior deletion/chimera studies and significantly add to understanding of MLCK myosin interactions.

Virus transmission and the prevalence of infection depend on multiple factors, including the interaction with other viral pathogens infecting the same host. In this study, active replication of an iflavirus, Spodoptera exigua iflavirus 1 (order Picornavirales) was observed in the offspring of insects that survived following inoculation with a pathogenic baculovirus, Spodoptera exigua multiple nucleopolyhedrovirus. Tracking the origin of the iflavirus suggested the association of this virus with the occlusion bodies of the baculovirus. Here we investigated the effect of this association on the stability and infectivity of both viruses. A reduction in baculovirus pathogenicity, without affecting its infectivity and productivity, was observed when associated with the iflavirus. In contrast, viral association increased the infectivity of the iflavirus and its resistance to ultraviolet radiation and high temperature, two of the main factors affecting virus stability in the field. In addition, electron microscopy analysis revealed the presence of particles resembling iflavirus virions inside the occlusion bodies of the baculovirus, suggesting the possible co-occlusion of both viruses. Results reported here are indicative of facultative phoresis of a virus and suggest that virus–virus interactions may be more common than currently recognized, and may be influential in the ecology of baculovirus and host populations and in consequence in the use of baculoviruses as biological insecticides., This study received financial support from the Spanish Ministry for Science and Technology (AGL2011-30352-C02 and AGL2014-57752-C2). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Rhytidectomy has been performed for over 100 years, but only more recently has it become generally accepted by society. Recent improvements in access to information has produced more public awareness of the possible aesthetic enhancements. Additionally, there has been a trend for the younger patient to seek cosmetic surgery, often requesting procedures with minimal incisions and less downtime. The mini-lift, with its decreased operating time and expense, rapid recovery, as well as more limited incisions and tissue elevation, is an ideal procedure for patients with a smaller degree of cheek and neck laxity. We describe our concept of the mini-lift and address what results can be expected.

Analysis of the transcriptome of Spodoptera exigua larvae revealed the presence of several ESTs with homology to virus of the order Picornavirales and with the highest similarity to Infectious flacherie virus (Iflaviridae) that infects Bombyx mori larvae. Iflaviridae is a recently defined family of insect-infecting viruses that consist of positive single strand RNA genomes translated into a single polyprotein of around 3000 amino acids long. Using the sequence information derived from the obtained ESTs, we have completed the genomic sequence of this virus. The novel S. exigua iflavirus (SeIV-1) has a genome of 10.3 kb and codes for a 3222 aa polyprotein. Expression analysis has revealed the presence of the virus in all tissues tested and insect stages, being more abundant in the midgut of the larvae. High infectivity of this virus against S. exigua has been demonstrated after observing the presence of this virus in different colonies that were reared in the same chamber with the virus-infected colony, despite no evidence of pathological effects. Further study of viral covert infections of SeIV-1 could lead to a better understanding of its pathological effect as well as any possible interaction with other microbial pathogens used for the control of this pest.

The spaceflight environment presents unique challenges to terrestrial biology, including but not limited to the direct effects of gravity. As we near the end of the Space Shuttle era, there remain fundamental questions about the response and adaptation of plants to orbital spaceflight conditions. We address a key baseline question of whether gene expression changes are induced by the orbital environment, and then we ask whether undifferentiated cells, cells presumably lacking the typical gravity response mechanisms, perceive spaceflight. Arabidopsis seedlings and undifferentiated cultured Arabidopsis cells were launched in April, 2010, as part of the BRIC-16 flight experiment on STS-131. Biologically replicated DNA microarray and averaged RNA digital transcript profiling revealed several hundred genes in seedlings and cell cultures that were significantly affected by launch and spaceflight. The response was moderate in seedlings; only a few genes were induced by more than 7-fold, and the overall intrinsic expression level for most differentially expressed genes was low. In contrast, cell cultures displayed a more dramatic response, with dozens of genes showing this level of differential expression, a list comprised primarily of heat shock–related and stress-related genes. This baseline transcriptome profiling of seedlings and cultured cells confirms the fundamental hypothesis that survival of the spaceflight environment requires adaptive changes that are both governed and displayed by alterations in gene expression. The comparison of intact plants with cultures of undifferentiated cells confirms a second hypothesis: undifferentiated cells can detect spaceflight in the absence of specialized tissue or organized developmental structures known to detect gravity. Key Words: Tissue culture—Microgravity—Low Earth orbit—Space Shuttle—Microarray. Astrobiology 12, 40–56.

Molecular biology experiments on the International Space Station (ISS) continue to face challenges of sample harvesting and sample return to earth for post flight analysis; however, the use of Kennedy Space Center Fixation Tubes filled with RNALater has proven to be a robust solution to many of these challenges. While it is clear that one direction of future spaceflight experimentation may be towards enhanced on-orbit analytical capabilities, the rapid progress of earth-bound analytical capacity dictates that facile return of molecular biology samples from the ISS will continue to be a mainstay of space life sciences research and flight operations. In this paper we present a case study of the successful performance of KFTs and RNALater over a broad set of operational conditions of ascent configuration, on-orbit experiment use, on-orbit storage and sample return configurations that are unique to ISS current operations and constraints. We also provide observations on performance limits and discuss deployment opportunities and scenarios that are consistent with continued successful ISS molecular biology experimentation.

A patient presenting to a facial plastic surgeon with lower eyelid aging often has accompanying midface descent. Many surgical options exist to address these deformities. Blepharoplasty techniques include both the transcutaneous skin-muscle flap as well as the transconjunctival approach. The midface can be addressed via a multitude of techniques, including percutaneous suspension of the malar fat pad, deep plane dissections, subperiosteal and endoscopic midface lifts, and transorbital approaches. Patients who have some midface ptosis but do not meet the standard criteria for formal midface lifting, or who want to avoid the extended recovery associated with such techniques, could benefit from less invasive procedures. We propose the extended skin muscle blepharoplasty to address midface descent and lower lid aging to patients as an alternative to formal midface lifting. The extended skin muscle blepharoplasty is an excellent technique for patients with orbicularis hypertrophy, skin redundancy, a tear trough deformity, and premalar ptosis, as it addresses each of these deformities with minimal downtime. Results for patients with mild but visible midface descent are gratifying.

Nematology , 2010, Vol. 12(1), 157-160 Short communication Species-specific polymerase chain reaction primers for simple detection of Bursaphelenchus fraudulentus (Nematoda: Parasitaphelenchidae) Anna F I L I P I A K 1 , ∗ , Agata J AKUBOW SKA 1 , 2 and Marek T OMALAK 1 Bursaphelenchus fraudulentus Ruhm, 1956 (Nema- toda: Parasitaphelenchidae) is a tree-inhabiting species frequently found in Europe (Ruhm, 1956; Schauer-Blume & Sturhan, 1989; Tomalak, 2004; Carletti et al ., 2005) and Asia (Braasch et al ., 2001). It is primarily found in dy- ing or dead deciduous trees (Ruhm, 1956; Schauer-Blume & Sturhan, 1989), although occasionally reported from conifers (Braasch et al ., 2001). Within the genus the spe- cific morphological characters (i.e. , vulval flap, shape of spicules, position of caudal papillae and presence of four incisures in the lateral fields) place B. fraudulentus into a distinct xylophilus -group which comprises eight other, morphologically similar, species, including the quarantine pest B. xylophilus (Gu et al ., 2008). Reliable methods of taxonomic identification of these species are therefore of particular interest to plant quarantine services. Several molecular techniques have been developed and used for identification of Bursaphelenchus spp. (Wang et al. , 1999; Matsunaga & Togashi, 2004; Burgermeister et al ., 2005; Castagnone et al. , 2005; Leal et al ., 2005, 2007) with ITS-RLFP analysis the most widely used in research and quarantine practice. An alternative method, based on a simple PCR amplification with primers specific for B. xylophilus and B. mucronatus (Matsunaga & Togashi, 2004), enables rapid and precise species identification, even from a single nematode (Filipiak et al ., 2007). Both native European species from the xylophilus - group, i.e. , B. mucronatus and B. fraudulentus , are rela- tively common and harmless to trees. They can be mor- phologically difficult to distinguish from each other and from the quarantine pest B. xylophilus. The provision of PCR primers specific for the DNA of B. fraudulentus , in addition to those already existing for B. xylophilus and B. 1 Department of Biological Pest Control and Quarantine, Institute of Plant Protection, Wladyslawa Wegorka 20, Poznan, 60-318, Poland 2 Department of Genetics, University of Valencia, Dr. Moliner 50, 46-100, Burjassot, Spain ∗ Corresponding author, e-mail: A.Filipiak@ior.poznan.pl Received: 22 October 2008; revised: 26 June 2009 Accepted for publication: 29 June 2009 Keywords: Bursaphelenchus xylophilus -group, diagnostics, molecular. mucronatus (Matsunaga & Togashi, 2004), enhances the utility of this method. The main objectives of our research were to sequence the ITS regions of the B. fraudulentus genome and to design specific primers for PCR amplifica- tion. Eight isolates of B. fraudulentus , two of B. mucrona- tus and one of B. xylophilus were examined. The Aus- trian (Osterreich), Russian (DE10w), German (Helmstedt and H26), and Hungarian (Ungarn) isolates of B. fraudu- lentus and the isolate of B. xylophilus (China) were ob- tained from the nematode collection of the Federal Re- search Centre for Cultivated Plants (formerly Federal Bi- ological Research Centre for Agriculture and Forestry – BBA), Braunschweig, Germany (Dr T. Schroder). All the remaining nematode populations were isolated from in- fested trees in Poland. The populations of B. fraudulentus originated from Poznan (PL-01 and PL-05) and Kornik (PL-04) and those of B. mucronatus from Nowy Tomysl (NTo-01) and Slawa Slaska (SlS-01). Their taxonomic identification was originally based on morphological and morphometric characteristics and subsequently confirmed by ITS-RLFP analysis according to the protocol described by Burgermeister et al. (2005). Prior to examination, all isolates were reared on Botrytis cinerea /malt agar (4.5%) at 25 ◦ C for ca 2 weeks. Propagated nematodes were col- lected by the Baermann funnel method and stored in 10 μ l H 2 O at − 20 ◦ C until use. Extraction of DNA was performed according to the method described by Iwahori et al. (1998), with mi- nor modifications to the composition of the lysis buffer: 100 mM Tris, pH 8.5, 100 mM NaCl, 50 mM EDTA, 1% SDS, 1% β -mercaptoethanol, 100 μ g ml − 1 proteinase K per 100 μ l buffer. Before incubation, the mixture was © Koninklijke Brill NV, Leiden, 2010 DOI:10.1163/138855409X12469543767319 Also available online - www.brill.nl/nemy 157

Insect nucleopolyhedroviruses (NPVs) are studied widely as agents for biological control, as expression vectors for the production of heterologous proteins, and as transduction vectors for gene therapy applications. Most of these applications rely on the existence of cell lines that allow in vitro multiplication of the virus. The influence of pH in the medium culture on the multiplication of SeMNPV, HearSNPV and AcMNPV in different cell culture lines was investigated. The study showed a strong influence of the medium pH on the virus multiplication with the best results at pH 6.5, about half pH unit above the pH of insect culture media used most commonly. Additional experiments using a recombinant AcMNPV, expressing the green fluorescent protein, suggested that the enhanced virus multiplication at pH 6.5 is due mainly to a facilitated entry of the budded virions into the cells.