Disk-shaped 316L stainless steel parts with various diameters and heights were additively manufactured using a direct metal laser sintering (DMLS) technique. Neutron diffraction was used to profile ...the residual stresses in the samples before and after removal of the build plate and support structures. Moreover, distortion level of the parts before and after the removal was quantified using a coordinate measuring machine (CMM). Large tensile in-plane stresses (up to ≈ 400MPa) were measured near the as-built disk top surfaces, where the stress magnitude decreased from the disk center to the edges. The stress gradient was steeper for the disks with smaller diameters and heights. Following the removal of the build plate and support structures, the magnitude of the in-plane residual stresses decreased dramatically (up to 330MPa) whereas the axial stress magnitude did not change significantly. The stress relaxation caused the disks to distort, where the distortion metric was higher for the disks with smaller diameters and heights. The distribution of the residual stresses revealed a marked breakdown of self-similarity in their distribution even comparing disk-shaped samples that were fabricated under identical printing parameters; the stress field profiles were not linearly scaled as a function of height and diameter.
The NFκB and MAPK signaling pathways are critical components of innate immunity that orchestrate appropriate immune responses to control and eradicate pathogens. Their activation results in the ...induction of proinflammatory mediators, such as TNFα a potent bioactive molecule commonly secreted by recruited inflammatory cells, allowing for paracrine signaling at the site of an infection. In this study we identified a novel mechanism by which the opportunistic pathogen Porphyromonas gingivalis dampens innate immune responses by disruption of kinase signaling and degradation of inflammatory mediators. The intracellular immune kinases RIPK1, TAK1, and AKT were selectively degraded by the P. gingivalis lysine-specific gingipain (Kgp) in human endothelial cells, which correlated with dysregulated innate immune signaling. Kgp was also observed to attenuate endothelial responsiveness to TNFα, resulting in a reduction in signal flux through AKT, ERK and NFκB pathways, as well as a decrease in downstream proinflammatory mRNA induction of cytokines, chemokines and adhesion molecules. A deficiency in Kgp activity negated decreases to host cell kinase protein levels and responsiveness to TNFα. Given the essential role of kinase signaling in immune responses, these findings highlight a unique mechanism of pathogen-induced immune dysregulation through inhibition of cell activation, paracrine signaling, and dampened cellular proinflammatory responses.
Insects with restricted diets rely on obligate microbes to fulfil nutritional requirements essential for biological function. Tsetse flies, vectors of African trypanosome parasites, feed exclusively ...on vertebrate blood and harbour the obligate endosymbiont Wigglesworthia glossinidia. Without Wigglesworthia, tsetse are unable to reproduce. These symbionts are sheltered within specialized cells (bacteriocytes) that form the midgut-associated bacteriome organ. To decipher the core functions of this symbiosis essential for tsetse's survival, we performed dual-RNA-seq analysis of the bacteriome, coupled with metabolomic analysis of bacteriome and haemolymph collected from normal and symbiont-cured (sterile) females. Bacteriocytes produce immune regulatory peptidoglycan recognition protein (pgrp-lb) that protects Wigglesworthia, and a multivitamin transporter (smvt) that can aid in nutrient dissemination. Wigglesworthia overexpress a molecular chaperone (GroEL) to augment their translational/transport machinery and biosynthesize an abundance of B vitamins (specifically B1-, B2-, B3- and B6-associated metabolites) to supplement the host's nutritionally deficient diet. The absence of Wigglesworthia's contributions disrupts multiple metabolic pathways impacting carbohydrate and amino acid metabolism. These disruptions affect the dependent downstream processes of nucleotide biosynthesis and metabolism and biosynthesis of S-adenosyl methionine (SAM), an essential cofactor. This holistic fundamental knowledge of the symbiotic dialogue highlights new biological targets for the development of innovative vector control methods.
Abstract
Background
Gonorrhea, caused by the bacterium Neisseria gonorrhoeae, is a globally prevalent sexually transmitted infection. The dynamics of gonococcal population biology have been poorly ...defined due to a lack of resolution in strain typing methods.
Methods
In this study, we assess how the core genome can be used to improve our understanding of gonococcal population structure compared with current typing schemes.
Results
A total of 1668 loci were identified as core to the gonococcal genome. These were organized into a core genome multilocus sequence typing scheme (N gonorrhoeae cgMLST v1.0). A clustering algorithm using a threshold of 400 allelic differences between isolates resolved gonococci into discrete and stable core genome groups, some of which persisted for multiple decades. These groups were associated with antimicrobial genotypes and non-overlapping NG-STAR and NG-MAST sequence types. The MLST-STs were more widely distributed among core genome groups.
Conclusions
Clustering with cgMLST identified globally distributed, persistent, gonococcal lineages improving understanding of the population biology of gonococci and revealing its population structure. These findings have implications for the emergence of antimicrobial resistance in gonococci and how this is associated with lineages, some of which are more predisposed to developing antimicrobial resistance than others.
Availability of gonococcal whole genome sequence data enhances our understanding of the population biology of this increasingly antimicrobial-resistant human pathogen. Here, genes core to the gonococcus are defined, which can be exploited to track emergence and transmission.
Since their detection in 2013, Aedes aegypti has become a widespread urban pest in California. The availability of cryptic larval breeding sites in residential areas and resistance to insecticides ...pose significant challenges to control efforts. Resistance to pyrethroids is largely attributed to mutations in the voltage gated sodium channels (VGSC), the pyrethroid site of action. However, past studies have indicated that VGSC mutations may not be entirely predictive of the observed resistance phenotype.
To investigate the frequencies of VGSC mutations and the relationship with pyrethroid insecticide resistance in California, we sampled Ae. aegypti from four locations in the Central Valley, and the Greater Los Angeles area. Mosquitoes from each location were subjected to an individual pyrethrum bottle bioassay to determine knockdown times. A subset of assayed mosquitoes from each location was then analyzed to determine the composition of 5 single nucleotide polymorphism (SNP) loci within the VGSC gene.
The distribution of knockdown times for each of the five Californian populations sampled was non-parametric with potentially bimodal distributions. One group succumbs to insecticidal effects around 35-45 min and the second group lasts up to and beyond the termination of the assay (120+ min). We detected 5 polymorphic VGSC SNPs within the sampled California populations. One is potentially new and alternatively spliced (I915K), and four are documented and associated with resistance: F1534C, V1016I, V410L and S723T. The Central Valley populations (Clovis, Dinuba, Sanger and Kingsburg) are fairly homogenous with only 5% of the mosquitoes showing heterozygosity at any given position. In the Greater LA mosquitoes, 55% had at least one susceptible allele at any of the five SNP loci. The known resistance allele F1534C was detected in almost all sampled mosquitoes (99.4%). We also observe significant heterogeneity in the knockdown phenotypes of individuals with the identical VGSC haplotypes suggesting the presence of additional undefined resistance mechanisms.
Resistance associated VGSC SNPs are prevalent, particularly in the Central Valley. Interestingly, among mosquitoes carrying all 4 resistance associated SNPs, we observe significant heterogeneity in bottle bioassay profiles suggesting that other mechanisms are important to the individual resistance of Ae. aegypti in California.
Metallic powder reuse presents attractive economic and environmental advantages for direct metal laser sintering (DMLS). However, continuous recycling of powder raises concerns of powder quality and ...sintered part performance, and complicates process validation. Efforts to examine the mechanical response of parts built with reused feedstocks are increasingly common in the technical literature, but none have optimized process parameters in DMLS to control for changes in material properties. In this paper, titanium powder reuse was investigated with the objective of optimizing the additive manufacturing (AM) process for reuse. Virgin Ti-6Al-4V powder was cycled a total of eight times through conditions representative of industrial DMLS machines. A full 23-factorial design of experiments (DOE) approach was employed to identify how parameter settings affect mechanical behavior, and include reuse as a process variable. The independent factors (laser power, laser speed, and hatch distance) did not significantly affect mechanical properties; however, measurements of ductility were found to be influenced by some interaction between the factors. These results were attributed to the narrow operating envelope which was required for successfully sintered specimens. Density and chemistry measurements further demonstrated no significant change with respect to reuse. The findings suggest that titanium powder can be reused up to eight times without any noticeable loss in strength or ductility.
•No important changes in the mechanical properties of Ti-6Al-4V printed parts are noted after 8 reuse cycles on DMLS.•The same DMLS printer parameters can be used for 8 reuse cycles the reuse cycles without compromising part quality.•The density of the printed parts remains constant for all reuses.•The thermal cycles and manipulation of the powder do not affect the chemical composition of the part after 8 reuse cycles.
Tsetse flies (Glossina spp.) transmit parasitic African trypanosomes (Trypanosoma spp.), including Trypanosoma congolense, which causes animal African trypanosomiasis (AAT). AAT detrimentally affects ...agricultural activities in sub-Saharan Africa and has negative impacts on the livelihood and nutrient availability for the affected communities. After tsetse ingests an infectious blood meal, T. congolense sequentially colonizes the fly's gut and proboscis (PB) organs before being transmitted to new mammalian hosts during subsequent feedings. Despite the importance of PB in blood feeding and disease transmission, little is known about its molecular composition, function and response to trypanosome infection. To bridge this gap, we used RNA-seq analysis to determine its molecular characteristics and responses to trypanosome infection. By comparing the PB transcriptome to whole head and midgut transcriptomes, we identified 668 PB-enriched transcripts that encoded proteins associated with muscle tissue, organ development, chemosensation and chitin-cuticle structure development. Moreover, transcripts encoding putative mechanoreceptors that monitor blood flow during tsetse feeding and interact with trypanosomes were also expressed in the PB. Microscopic analysis of the PB revealed cellular structures associated with muscles and cells. Infection with T. congolense resulted in increased and decreased expression of 38 and 88 transcripts, respectively. Twelve of these differentially expressed transcripts were PB-enriched. Among the transcripts induced upon infection were those encoding putative proteins associated with cell division function(s), suggesting enhanced tissue renewal, while those suppressed were associated with metabolic processes, extracellular matrix and ATP-binding as well as immunity. These results suggest that PB is a muscular organ with chemosensory and mechanosensory capabilities. The mechanoreceptors may be point of PB-trypanosomes interactions. T. congolense infection resulted in reduced metabolic and immune capacity of the PB. The molecular knowledge on the composition and putative functions of PB forms the foundation to identify new targets to disrupt tsetse's ability to feed and parasite transmission.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Tsetse flies are important vectors of human and animal trypanosomiasis. Ability to reduce tsetse populations is an effective means of disease control. Lactation is an essential component of tsetse's ...viviparous reproductive physiology and requires a dramatic increase in the expression and synthesis of milk proteins by the milk gland organ in order to nurture larval growth. In between each gonotrophic cycle, tsetse ceases milk production and milk gland tubules undergo a nearly two-fold reduction in width (involution). In this study, we examined the role autophagy plays during tsetse fly milk gland involution and reproductive output. Autophagy genes show elevated expression in tissues associated with lactation, immediately before or within two hours post-parturition, and decline at 24-48h post-parturition. This expression pattern is inversely correlated with that of the milk gland proteins (lactation-specific protein coding genes) and the autophagy inhibitor fk506-bp1. Increased expression of Drosophila inhibitor of apoptosis 1, diap1, was also observed in the milk gland during involution, when it likely prevents apoptosis of milk gland cells. RNAi-mediated knockdown of autophagy related gene 8a (atg8a) prevented rapid milk gland autophagy during involution, prolonging gestation, and reducing fecundity in the subsequent gonotrophic cycle. The resultant inhibition of autophagy reduced the recovery of stored lipids during the dry (non-lactating) periods by 15-20%. Ecdysone application, similar to levels that occur immediately before birth, induced autophagy, and increased milk gland involution even before abortion. This suggests that the ecdysteroid peak immediately preceding parturition likely triggers milk gland autophagy. Population modeling reveals that a delay in involution would yield a negative population growth rate. This study indicates that milk gland autophagy during involution is critical to restore nutrient reserves and allow efficient transition between pregnancy cycles. Targeting post-birth phases of reproduction could be utilized as a novel mechanism to suppress tsetse populations and reduce trypanosomiasis.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Female mosquitoes are effective disease vectors, because they take blood from vertebrate hosts to obtain nutrients for egg development. Amino acid signaling via the target of rapamycin (TOR) pathway ...has been identified as a key requirement for the activation of egg development after a blood meal. We report the characterization of the TOR kinase and one of its major downstream targets, S6 kinase, of the yellow fever mosquito Aedes aegypti during egg development in adult females. Both TOR and S6K mRNA are expressed at high levels in the ovaries and in lower levels in fat body and other tissues. After a blood meal, the subcellular localization of TOR shifts from the cytoplasm to the plasma membrane of fat body cells. By detecting phosphothreonine 388 of mosquito S6 kinase, we show that TOR activity strongly increases in fat body and ovaries after a blood meal in vivo. Furthermore, phosphorylation of S6 kinase increases in in vitro cultured fat bodies after stimulation with amino acids. This increase is sensitive to the TOR inhibitor rapamycin in a concentration-dependent manner but not to the phosphatidylinositol 3-kinase/phosphatidylinositol 3-kinase-related kinase inhibitor LY294002, the MAPK inhibitor PD98059, or the translational inhibitor cycloheximide. RNA interference-mediated reduction of S6 kinase strongly inhibits the amino acid-induced up-regulation of the major yolk protein vitellogenin in vitro and effectively disrupts egg development after a blood meal in vivo. Our data show that TOR-dependent activation of S6 kinase is a central step in the transduction of nutritional information during egg development in mosquitoes.
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