Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and tomato. The susceptibility of M. sexta larvae to a variety of entomopathogenic bacterial species^1-5, as well as the wealth of information available regarding the insect''s immune system^6-8, and the pending genome sequence⁹ make it a good model organism for use in studying host-microbe interactions during pathogenesis. In addition, M. sexta larvae are relatively large and easy to manipulate and maintain in the laboratory relative to other susceptible insect species. Their large size also facilitates efficient tissue/hemolymph extraction for analysis of the host response to infection.The method presented here describes the direct injection of bacteria into the hemocoel (blood cavity) of M. sexta larvae. This approach can be used to analyze and compare the virulence characteristics of various bacterial species, strains, or mutants by simply monitoring the time to insect death after injection. This method was developed to study the pathogenicity of Xenorhabdus and Photorhabdus species, which typically associate with nematode vectors as a means to gain entry into the insect. Entomopathogenic nematodes typically infect larvae via natural digestive or respiratory openings, and release their symbiotic bacterial contents into the insect hemolymph (blood) shortly thereafter¹⁰. The injection method described here bypasses the need for a nematode vector, thus uncoupling the effects of bacteria and nematode on the insect. This method allows for accurate enumeration of infectious material (cells or protein) within the inoculum, which is not possible using other existing methods for analyzing entomopathogenesis, including nicking¹¹ and oral toxicity assays¹². Also, oral toxicity assays address the virulence of secreted toxins introduced into the digestive system of larvae, whereas the direct injection method addresses the virulence of whole-cell inocula.The utility of the direct injection method as described here is to analyze bacterial pathogenesis by monitoring insect mortality. However, this method can easily be expanded for use in studying the effects of infection on the M. sexta immune system. The insect responds to infection via both humoral and cellular responses. The humoral response includes recognition of bacterial-associated patterns and subsequent production of various antimicrobial peptides⁷; the expression of genes encoding these peptides can be monitored subsequent to direct infection via RNA extraction and quantitative PCR¹³. The cellular response to infection involves nodulation, encapsulation, and phagocytosis of infectious agents by hemocytes⁶. To analyze these responses, injected insects can be dissected and visualized by microscopy^{13, 14}.     

Patterns and processes in the evolution of the eukaryotic endomembrane system

Abstract

The eukaryotic endomembrane system (ES) is served by hundreds of dedicated proteins. Experimental characterization of the ES-associated molecular machinery in several model eukaryotes complemented by a recent progress in phylogenomics and comparative genomics have revealed a conserved complex core of the machinery that appears to have been established before the last eukaryotic common ancestor (LECA). At the same time, modern eukaryotes exhibit a huge variation in the ES resulting from a multitude of evolutionary processes operating along the ever-branching paths from the LECA to its descendants. The most important source of evolutionary novelty in the ES functioning has undoubtedly been gene duplication followed by divergence of the gene copies, responsible not only for the pre-LECA establishment of many multi-paralog families of proteins in the very core of the ES-associated machinery, but also for post-LECA lineage-specific elaborations via family expansions and the origin of novel components. Extreme sequence divergence has obscured actual homologous relationships between potentially many components of the machinery, even between orthologous proteins, as illustrated by the yeast Vps51 subunit of the vesicle tethering complex GARP hypothesized here to be a highly modified ortholog of a conserved eukaryotic family typified by the zebrafish Fat-free (Ffr) protein. A dynamic evolution of many ES-associated proteins, especially those centred around RAB and ARF GTPases, seems to take place at the level of their domain architectures. Finally, reductive evolution and recurrent gene loss are emerging as pervasive factors shaping the ES in all phylogenetic lineages.     

Abstracts

ABSTRACT

The normal development of empathy has been proposed to be imperative to the healthy emotional and social functioning of youths. In contrast, compromised levels of empathy have been linked to an increased propensity to engage in antisocial behaviors, including animal cruelty. Previous findings have revealed parent attachment to be intrinsically linked to the development of empathy. This association has been shown to play a role in predicting the expression of various outcome behaviors, including both those which are prosocial in nature, and those which are antisocial, and potentially aggressive. This study examines these associations in a sample of 281 12- to 18-year-old students. The aims included the investigation of the direct predictive roles played by attachment and empathy for prosocial and antisocial behaviors directed at both humans and animals. We also investigated the mediating role played by empathy in these relationships. Attachment and empathy significantly predicted prosocial and antisocial behaviors, both individually, and in combination. Furthermore, empathy was found to serve a mediating role in the associations between attachment and: human-directed prosocial behavior, the humane treatment of animals, and animal cruelty. These findings expand upon existing literature by demonstrating that it is, at least partially, through empathy that attachment to parents predicts prosocial and antisocial behaviors during adolescence. This is in contrast to the direction of relationships implied by some previous findings and proposals, which have suggested that treating animals humanely fosters the normal development of empathy, for example. Notwithstanding the promising findings revealed by the current study, we recommend that replicating this research using a larger sample will assist in addressing the limited generalizability identified in the current study. It is further proposed that the use of a lie scale could limit the influence of social desirability responding. Future research is also needed to determine the direction of the demonstrated relationships.     

Linking behavioral states to landscape features for improved conservation management

1. A central theme for conservation is understanding how animals differentially use, and are affected by change in, the landscapes they inhabit. However, it has been challenging to develop conservation schemes for habitat‐specific behaviors.
2. Here we use behavioral change point analysis to identify behavioral states of golden eagles (Aquila chrysaetos) in the Sonoran and Mojave Deserts of the southwestern United States, and we identify, for each behavioral state, conservation‐relevant habitat associations.
3. We modeled behavior using 186,859 GPS points from 48 eagles and identified 2,851 distinct segments comprising four behavioral states. Altitude above ground level (AGL) best differentiated behavioral states, with two clusters of short‐distance movement behaviors characterized by low AGL (state 1 AGL = 14 m (median); state 2 AGL = 11 m) and two associated with longer‐distance movement behaviors and characterized by higher AGL (state 3 AGL = 108 m; state 4 AGL = 450 m).
4. Behaviors such as perching and low‐altitude hunting were associated with short‐distance movements in updraft‐poor environments, at higher elevations, and over steeper and more north‐facing terrain. In contrast, medium‐distance movements such as hunting and transiting were over gentle and south‐facing slopes. Long‐distance transiting occurred over the desert habitats that generate the best updraft.
5. This information can guide management of this species, and our approach provides a template for behavior‐specific habitat associations for other species of management concern.

     

Mantis Shrimp Navigate Home Using Celestial and Idiothetic Path Integration

Download : Download video (46MB)     

Triptolide prevents bone loss via suppressing osteoclastogenesis through inhibiting PI3K-AKT-NFATc1 pathway

Bone loss (osteopenia) is a common complication in human solid tumour. In addition, after surgical treatment of gynaecological tumour, osteoporosis often occurs due to the withdrawal of oestrogen. The major characteristic of osteoporosis is the low bone mass with micro-architectural deteriorated bone tissue. And the main cause is the overactivation of osteoclastogenesis, which is one of the most important therapeutic targets. Inflammation could induce the interaction of RANKL/RANK, which is the promoter of osteoclastogenesis. Triptolide is derived from the traditional Chinese herb lei gong teng, presented multiple biological effects, including anti-cancer, anti-inflammation and immunosuppression. We hypothesized that triptolide could inhibits osteoclastogenesis by suppressing inflammation activation. In this study, we confirmed that triptolide could suppress RANKL-induced osteoclastogenesis in bone marrow mononuclear cells (BMMCs) and RAW264.7 cells and inhibited the osteoclast bone resorption functions. PI3K-AKT-NFATc1 pathway is one of the most important downstream pathways of RANKL-induced osteogenesis. The experiments in vitro indicated that triptolide suppresses the activation of PI3K-AKT-NFATc1 pathway and the target point located at the upstream of AKT because both NFATc1 overexpression and AKT phosphorylation could ameliorate the triptolide suppression effects. The expression of MDM2 was elevated, which demonstrated the MDM-p53-induced cell death might contribute to the osteoclastogenesis suppression. Ovariectomy-induced bone loss and inflammation activation were also found to be ameliorated in the experiments in vivo. In summary, the new effect of anti-cancer drug triptolide was demonstrated to be anti-osteoclastogenesis, and we demonstrated triptolide might be a promising therapy for bone loss caused by tumour.     

Circulating adiposity-related microRNAs as predictors of the response to a low-fat diet in subjects with obesity

Recent studies have revealed the critical role of several microRNAs (miRNAs) in energy homeostasis and metabolic processes and suggest that circulating miRNAs can be used as early predictors of weight loss in the design of precision nutrition. Thus, the aim of this study was to investigate circulating adiposity-related miRNAs as biomarkers of the response to two specific weight loss dietary treatments. The expression of 86 miRNAs was investigated in plasma of 78 subjects with obesity randomized to two different diets [moderately high-protein diet (n = 38) and low-fat diet (n = 40)] and in 25 eutrophic controls (BMI ≤ 25 kg/m²). Bioinformatic analyses were performed to explore the target genes and biological pathways regulated by the dysregulated miRNAs. As results, 26 miRNAs were found differently expressed in eutrophic and volunteers with obesity. Moreover, 7 miRNAs (miR-130a-3p, miR-142-5p, miR-144-5p, miR-15a-5p, miR-22-3p, miR-221-3p and miR-29c-3p) were differentially expressed between responders and non-responders to a low-fat diet. Furthermore, after adjustment for basal glucose levels, 1-SD increase in miR-22-3p expression was associated with reduction in the risk of non-response to low-fat diet [OR = 0.181, 95% CI (0.084-0.947), P = .043]. Bioinformatic analyses evidenced that these 7 miRNAs regulate the expression of genes participating in important metabolic pathways. Conclusively, 7 circulating miRNAs related to adiposity could be used for predicting the response to a low-fat diet intervention prescribed to lose weight.     

加味八珍汤联合PFNA术治疗老年股骨转子间骨折患者的临床效果及对血液流变学指标的影响

目的:研究加味八珍汤联合股骨近端防旋髓内钉(Proximal Femoral Nail Antirotation,PFNA)对老年股骨转子间骨折患者的临床效果及对术后隐性失血和血液流变学的影响。方法:选择2014年3月至2017年3月我院收治的68例接受PFNA治疗的转子间骨折者,将其随机平均分为观察组(34例,术后口服加味八珍汤颗粒7天)、对照组(34例)。对比两组总失血量、隐性失血量、隐性失血比例、血液流变学指标(血浆粘度及全血黏度低、中、高切)、不良反应等。结果:⑴两组各剔除2例,94.12%(64/68)的受试者完成研究。⑵两组总失血量相当,差异无统计学意义(P0.05);与对照组相比,观察组隐性失血量及隐性失血发生率均显著降低(P0.05)。⑶治疗前,两组血浆粘度及低、中、高切全血黏度比较差异无统计学意义(P0.05);治疗后,两组血浆粘度及低、中、高切全血黏度均显著低于本组治疗前(P0.05),且观察组以上指标均明显低于对照组(P0.05)。⑷观察组发生肌间静脉血栓1例,无药物过敏及毒副反应;对照组发生1例深静脉血栓、4例肌间静脉血栓。观察组不良反应率为3.125%,显著低于对照组(15.625%,P0.05)。结论:加味八珍汤既可显著减少老年股骨转子间骨折患者PFNA术后隐性失血,又能改善血流动力学、减少并发症。     

Nonlinear analysis of heart rate variability for evaluating the growing pig stress response to an acute heat episode

Heart rate variability (HRV) is a proxy measure of autonomic function and can be used as an indicator of swine stress. While traditional linear measures are used to distinguish between stressed and unstressed treatments, inclusion of nonlinear HRV measures that evaluate data structure and organization shows promise for improving HRV interpretation. The objective of this study was to evaluate the inclusion of nonlinear HRV measures in response to an acute heat episode. Twenty 12- to 14-week-old growing pigs were individually housed for 7 days and acclimated to thermoneutral conditions (20.35°C ± 0.01°C; 67.6% ± 0.2% RH) before undergoing one of the two treatments: (1) thermoneutral control (TN; n = 10 pigs) or (2) acute heat stress (HS; n = 10 pigs; 32.6°C ± 0.1°C; 26.2% ± 0.1% RH). In Phase 1 of the experimental procedure (P1; 60 min), pigs underwent a baseline HRV measurement period in thermoneutral conditions before treatment [Phase 2; P2; 60 min once gastrointestinal temperature (T_g) reached 40.6°C], where HS pigs were exposed to heated conditions and TN pigs remained in thermoneutral conditions. After P2, all pigs were moved back to thermoneutral conditions (Phase 3; P3; 60 min). During each phase, T_g data were collected every 5 min and behavioural data were collected to evaluate the amount of time each pig spent in an active posture. Additionally, linear (time and frequency domain) and nonlinear [sample entropy (SampEn), de-trended fluctuation analysis, percentage recurrence, percentage determinism (%DET), mean diagonal line length in a recurrence plot] HRV measures were quantified. Heat stressed pigs exhibited greater T_g (P = 0.002) and spent less time in an active posture compared to TN pigs during P2 (P = 0.0003). Additionally, low frequency to high frequency ratio was greater in HS pigs during P3 compared to TN pigs (P = 0.02). SampEn was reduced in HS pigs during P2 (P = 0.01) and P3 (P = 0.03) compared to TN pigs. Heat stressed pigs exhibited greater %DET during P3 (P = 0.03) and tended to have greater %DET (P = 0.09) during P2 than TN pigs. No differences between treatments were detected for the remaining HRV measures. In conclusion, linear HRV measures were largely unchanged during P2. However, changes to SampEn and %DET suggest increased heat stress as a result of the acute heat episode. Future work should continue to evaluate the benefits of including nonlinear HRV measures in HRV analysis of swine heat stress.