Diversity and dynamics of bacteria at the Chrysomya megacephala pupal stage revealed by third-generation sequencing.

Characterization of the microbial community is essential for understanding the symbiotic relationships between microbes and host insects. Chrysomya megacephala is a vital resource, a forensic insect, a pollinator, and a vector for enteric bacteria, protozoa, helminths, and viruses. However, research on its microbial community is incomprehensive, particularly at the pupal stage, which comprises approximately half of the entire larval development stage and is important entomological evidence in forensic medicine. For the first time, this study investigated the bacterial communities of C. megacephala pupae at different ages using third-generation sequencing technology. The results showed that C. megacephala has a diverse and dynamic bacterial community. Cluster analysis at ≥ 97% similarity produced 154 operational taxonomic units (OTUs) that belonged to 10 different phyla and were distributed into 15 classes, 28 orders, 50 families, 88 genera, and 130 species. Overall, the number of bacterial OTUs increased with the development of pupae, and the relative abundance of Wolbachia in the Day5 group was significantly lower than that in the other groups. Within the pupal stage, Proteobacteria, Firmicutes, and Bacteroidetes were the dominant phyla of bacteria. At the genus level, Wolbachia and Ignatzschineria coexisted, a rarely known feature. In addition, we found Erysipelothrix rhusiopathiae, the etiological agent of swine erysipelas, which is rarely identified in insects. This study enriches the understanding of the microbial community of C. megacephala and provides a reference for better utilization and control of C. megacephala.

Identification of Common Sarcosaprophagous Flies in the Yangtze River Delta by COâ Gene. / 利用COâ åŸºå› é‰´åˆ«é•¿ä¸‰è§’åœ°åŒºå¸¸è§å—œå°¸æ€§è‡ç±».

OBJECTIVES: To identify the common sarcosaprophagous flies in the Yangtze River Delta based on mitochondrial cytochrome c oxidase subunit Ⅰ(COⅠ) gene sequence and verify the reliability of this method. METHODS: Seven common genetically stable sarcosaprophagous flies in three families and six genera were collected from large domestic pig carcasses placed in the field and cultured in the laboratory for many generations. The whole genome DNA was extracted and the COⅠ gene fragment was amplified. The forward and reverse sequencing was followed by splicing. The base composition of the amplified fragment and the rate of interspecific evolutionary divergence were analyzed by software such as Mega 7.0.26. The phylogenetic tree of COⅠ gene sequence of common necrophagous flies in the Yangtze River Delta was established by neighbor joining (NJ) method and unweighted pair-group method with arithmetic means (UPGMA) method. RESULTS: The average base composition of different flies was A(30.14%), T(38.23%), C(15.98%), G(15.65%). The rate of interspecific evolutionary divergence ranged from 2.2% to 15.3%, the lowest rate was between Chrysomya megacephala and Chrysomya pinguis, the highest rate was between Muscina stabulans and Boettcherisca peregrina. CONCLUSIONS: COⅠ gene can be used to identify the common necrophagous flies in the Yangtze River Delta.

Research Progress on Developmental Biology of Sarcosaprophagous Insects. / å—œå°¸æ€§æ˜†è™«å‘è‚²ç”Ÿç‰©å­¦ç ”ç©¶è¿›å±•.

Forensic entomology provides a feasible way to estimate postmortem interval (PMI), of which the growth and development of sarcosaprophagous insects is the most widely used indicator in forensic practice. Over the years, forensic entomologists have carried out a large number of studies on the development biology of sarcosaprophagous insects. This paper illustrates the main factors that affect the development of sarcosaprophagous insects, including temperature, humidity, light, food types and poisons. The development indicators of sarcosaprophagous insects were reviewed from the perspectives of morphology, differential gene expression and biochemical characteristics. It is emphasized that future research of development biology on sarcosaprophagous insects should fully absorb and integrate the methods of artificial intelligence and omics, and the research object also needs further expansion in order to establish a more objective and more accurate PMI estimation method.

LncRNA EIF3J-AS1 enhanced esophageal cancer invasion via regulating AKT1 expression through sponging miR-373-3p.

Esophageal cancer (ECa) remains a major cause of mortality across the globe. The expression of EIF3J-AS1 is altered in a plethora of tumors, but its role in ECa development and progression are undefined. Here, we show that EIF3J-AS1 is up-regulated in ECa and that its expression correlates with advanced TNM stage (P = 0.014), invasion depth (P = 0.001), positive lymph node metastasis (P < 0.001) and poor survival (OS: P = 0.0059; DFS: P = 0.0037) in ECa. Functional experiments showed that knockdown EIF3J-AS1 inhibited ECa growth and metastasis through in vitro and in vivo experiments. Regarding the mechanism, EIF3J-AS1/miR-373-3p/AKT1 established the ceRNA network involved in the modulation of cell progression of ECa cells. Overall, EIF3J-AS1 may exhibit an oncogenic function in ECa via acting as a sponge for miR-373-3p to up-regulate AKT1 mRNA level, and may serve as a potential therapeutic target and a prognostic biomarker for ECa patients.

Quantitative Statistics and Identification of Tool-Marks.

This study was designed to establish a feature identification method of tool-mark 2D data. A uniform local binary pattern histogram operator was developed to extract the tool-mark features, and the random forest algorithm was adopted to identify these. The presented method was used to conduct five groups of experiments with a 2D dataset of known matched and nonmatched tool-marks made by bolt clippers, cutting pliers, and screwdrivers. The experimental results show that the proposed method achieved a high rate of identification of the tool-mark samples generated under identical conditions. The proposed method effectively overcomes the disadvantage of unstable illumination of 2D tool-mark image data and avoids the difficulty in mark inspection caused by manually preset parameters in the existing methods, thus reducing the uncertainty of inspected results.

Estimating the age of Lucilia illustris during the intrapuparial period using two approaches: Morphological changes and differential gene expression.

Lucilia illustris (Meigen, 1826) (Diptera: Calliphoridae) is a cosmopolitan species of fly that has forensic and medical significance. However, there is no relevant study regarding the determination of the age of this species during the intrapuparial period. In this study, we investigated the changes in both morphology and differential gene expression during intrapuparial development, with an aim to estimate the age of L. illustris during the intrapuparial stage. The overall intrapuparial morphological changes of L. illustris were divided into 12 substages. Structures such as the compound eyes, mouthparts, antennae, thorax, legs, wings, and abdomen, each capable of indicating age during the intrapuparial stage, were observed in detail, and the developmental progression of each of these structures was divided into six to eight stages. We recorded the time range over which each substage or structure appeared. The differential expression of the three genes 15_2, actin, and tbp previously identified for predicting the timing of intrapuparial development was measured during L. illustris metamorphosis. The expression of these genes was quantified by real-time PCR, and the results revealed that these genes can be used to estimate the age of L. illustris during the intrapuparial period, as they exhibit regular changes and temperature dependence. This study provides an important basis for estimating the minimum postmortem interval (PMImin) in forensic entomology according to changes in intrapuparial development and differential gene expression. Furthermore, combination of the two approaches can generate a more precise PMImin than either approach alone.

Insect succession on remains of human and animals in Shenzhen, China.

Most forensic entomological succession studies have been carried out using pig or rabbit carcasses; however, there have been few studies on the differences between insect succession patterns on human cadavers and on animal carcasses. In order to clarify the differences between decomposition and insect succession patterns of human cadavers and animal carcasses, one 49.5kg human cadaver, two large pig carcasses (45 and 48kg), two small pig carcasses (23 and 25kg) and two rabbit carcasses (both 1.75kg) were placed in the same field conditions in Shenzhen, China for a comparative study on August, 2013. The results indicated that: (1) The duration from fresh to skeletonization is in order of human cadaver>large pig carcasses>small pig carcasses>rabbit carcasses; (2) insect assemblages (including developmental stages) are more complex on larger carcasses, in order of human cadaver=large pig carcasses>small pig carcasses>rabbit carcasses; (3) the developmental rates of the same forensically important fly species on all carcasses are consistent; (4) all identified species of Calliphoridae can complete development of one generation on human cadaver, and both large and small pig carcasses, while on rabbit carcasses, only a subset of the Calliphoridae species can finish development of one generation; (5) beetles can generate offspring on human cadaver, and both large and small pig carcasses, while they do not generate offspring on rabbit carcasses. This study provides useful comparative data for decomposition and insect succession pattern of human cadaver with animal carcasses.

Development of the green bottle fly Lucilia illustris at constant temperatures.

Lucilia illustris (Meigen 1826) (Diptera: Calliphoridae) is a cosmopolitan species that commonly colonizes carcasses and occasionally acts as parasites of humans or livestock, making it an insect of significant importance in forensic, medical, and veterinary entomology. However, only a few studies have documented the development of L. illustris. Here, we studied the developmental duration and larval body length changes of L. illustris under nine constant temperatures ranging from 15.0 to 35.0°C. Using these results, we generated an isomorphen diagram, thermal summation model, and isomegalen diagram for L. illustris. Simulation equations of the variation in the larval body length with time after hatching and variation in time after hatching with the body length were also obtained. L. illustris could complete its life cycle in 15.0-32.5°C, while its development was incomplete at 35.0°C, where the pupae failed to transform into adults. The development duration was 955.5±16.9, 625.7±16.9, 509.3±18.3, 410.0±17.0, 346.7±12.2, 290.2±6.7, 257.1±8.9, and 234.8±3.2h at 15.0, 17.5, 20.0, 22.5, 25.0, 27.5, 30.0, and 32.5°C, respectively. The developmental threshold temperature and thermal constant were 9.30±0.19°C and 5367.2±98.3°Ch, respectively. These results provide an important basis for the use of L. illustris development-based estimation of the minimum postmortem interval (PMImin) in forensic entomology.

Study on the pupal morphogenesis of Chrysomya rufifacies (Macquart) (Diptera: Calliphoridae) for postmortem interval estimation.

Chrysomya rufifacies (Macquart) is one of the most common species of blow flies at the scene of death in Southern China. Pupae are useful in postmortem interval (PMI) estimation due to their sedentary nature and longer duration of association with the corpse. However, to determine the age of a pupa is more difficult than that of a larva, due to the fact that morphological changes are rarely visible during pupal development. In this study, eggs of C. rufifacies were reared in climatic chambers under four different constant temperatures (20, 24, 28 and 32°C each±1°C, respectively) with same rearing conditions such as foodstuff, substrate, photoperiod and relative humidity. Ten duplicate pupae were sampled at 8-h intervals from prepupae to emergence under the different constant temperatures, respectively. The pupae were sampled, killed, fixed, dissected and with the puparium removed, the external morphological changes of the pupae were observed, recorded and photographed. The morphological characters of C. rufifacies pupae were described. Based on the visible external morphological characters during pupal morphogenesis at 28°C±1°C, the developmental period of C. rufifacies was divided into nine developmental stages and recorded in detailed description. Based on above-mentioned nine developmental stages, some visible external morphological characters were selected as indications for developmental stages. These indications mapped to 8-h sampling intervals at the four different constant temperatures were also described in this study. It is demonstrated that generally the duration of each developmental stage of C. rufifacies pupae is inversely correlated to appropriate developmental temperatures. This study provides relatively systematic pupal developmental data of C. rufifacies for the estimation of PMI. In addition, further work may improve by focus on other environmental factors, histological analysis, more thorough external examination by shortening sampling intervals, PAE (the Pupal Age Estimator) method and parasitic insects of C. rufifacies.

[The community succession of sarcosaphagous insects on pig carcasses in summer indoor and outdoor environment in Shenzhen area].

OBJECTIVE: To explore the growing development and community succession of main sarcosaphagous insects on pig carcasses in summer indoor and outdoor environment in Shenzhen area and to estimate the postmortem interval (PMI). METHODS: From early May to August in 2013, in Forensic Medical Examination Center of Shenzhen Public Security Bureau, the main insect species and the decomposition process were observed in two adult pig carcasses of simulative indoor and outdoor environment. The different decomposition stages and the community succession of insects were recorded. RESULTS: The indoor and outdoor pig carcasses showed skeleton 412.5 and 325 hours after death, respectively. The main species of flies on pig carcasses were Chrysomya megacephala, Chrysomya rufifacies and Chrysomya chani. The main species of beetles were Crecphilus maxillosus, Necrobia ruficollis, Saprinus splendens and Dermestes maculatu. The dominant species of flies in the outdoor pig carcasses obviously produced the second generations due to the effect of mass rainfall, nor in the indoor pig carcasses. CONCLUSION: There are regular patterns on the community succession of insects on pig carcasses in summer indoor and outdoor environment in Shenzhen area. The activity patterns of seven typical insects and their larva show important value for estimating PMI.

Ginsenoside Rd attenuates mitochondrial permeability transition and cytochrome C release in isolated spinal cord mitochondria: involvement of kinase-mediated pathways.

Ginsenoside Rd (Rd), one of the main active ingredients in Panax ginseng, has multifunctional activity via different mechanisms and neuroprotective effects that are exerted probably via its antioxidant or free radical scavenger action. However, the effects of Rd on spinal cord mitochondrial dysfunction and underlying mechanisms are still obscure. In this study, we sought to investigate the in vitro effects of Rd on mitochondrial integrity and redox balance in isolated spinal cord mitochondria. We verified that Ca2+ dissipated the membrane potential, provoked mitochondrial swelling and decreased NAD(P)H matrix content, which were all attenuated by Rd pretreatment in a dose-dependent manner. In contrast, Rd was not able to inhibit Ca2+ induced mitochondrial hydrogen peroxide generation. The results of Western blot showed that Rd significantly increased the expression of p-Akt and p-ERK, but had no effects on phosphorylation of PKC and p38. In addition, Rd treatment significantly attenuated Ca2+ induced cytochrome c release, which was partly reversed by antagonists of Akt and ERK, but not p-38 inhibitor. The effects of bisindolylmaleimide, a PKC inhibitor, on Rd-induced inhibition of cytochrome c release seem to be at the level of its own detrimental activity on mitochondrial function. Furthermore, we also found that pretreatment with Rd in vivo (10 and 50 mg/kg) protected spinal cord mitochondria against Ca2+ induced mitochondrial membrane potential dissipation and cytochrome c release. It is concluded that Rd regulate mitochondrial permeability transition pore formation and cytochrome c release through protein kinases dependent mechanism involving activation of intramitochondrial Akt and ERK pathways.

The molecular mechanisms of Tanshinone IIA on the apoptosis and arrest of human esophageal carcinoma cells.

OBJECTIVE: To explore the possible mechanisms of Tanshinone IIA (TanIIA) on esophageal carcinoma cell lines. METHODS: Two human esophageal carcinoma cell lines (EC-1 cells and ECa-109 cells) were treated with different concentrations of TanIIA. Cell proliferation was measured by CCK-8, colony-forming efficiency was calculated, cell cycle and apoptosis were measured, and changes in cell cycle- and apoptosis-related gene expression were measured by Western blotting. RESULTS: The CCK-8 and colony formation assay indicated that TanIIA inhibited the cell proliferation of human esophageal cancer cells (IC50 below 1 µg/mL) at 48 h. Hoechst 33258 and flow cytometry showed that TanIIA induced apoptosis in both esophageal cancer cell lines. Flow cytometry showed that TanIIA arrested cell cycle in S phase and G2/M phase. Western blotting analysis showed that Akt1 and its phosphorylation were inhibited, the Bax/Bcl-2 ratio increased, and both caspase-9 and caspase-3 were activated after treatment with 1.3 µg/mL TanIIA at 48 h. Meanwhile, p53 and p21 protein levels increased, whereas cyclin B1, CDC2, and CDC2 phosphorylation were inhibited. CONCLUSION: TanIIA inhibits the growth of esophageal cancer cells and induces apoptosis in a time-dependent and concentration-dependent manner, possibly by affecting cell cycle- and apoptosis-related signaling pathways.

Liquiritigenin prevents Staphylococcus aureus-mediated lung cell injury via inhibiting the production of α-hemolysin.

Staphylococcus aureus is a significant Gram-positive bacterium that is associated with a broad spectrum of diseases ranging from minor skin infections to lethal pneumonia, endocarditis, and toxinoses. α-Hemolysin is one of the most important exotoxins that contribute to the pathogenesis of S. aureus infections. Liquiritigenin is one of the most significant active components in licorice. In this study, hemolysis, western blot, and real-time reverse transcription-PCR assays were performed to investigate the impact of liquiritigenin on the production of S. aureus α-hemolysin. The results showed that low concentrations of liquiritigenin remarkably decreased S. aureus α-hemolysin production in a dose-dependent manner. Using live/dead cell staining and lactate dehydrogenase assays, we found that liquiritigenin could protect human lung cells (A549) from α-hemolysin-mediated injury. The data indicated that this compound could potentially be useful in developing drugs aiming at staphylococcal α-hemolysin.

Scutellarein inhibits hypoxia- and moderately-high glucose-induced proliferation and VEGF expression in human retinal endothelial cells.

AIM: This study was designed to examine the effect of scutellarein on high glucose- and hypoxia-stimulated proliferation of human retinal endothelial cells (HREC). METHODS: HREC were cultured under normal glucose (NG), moderate, and high glucose (NG supplemented with 10 or 25 mmol/L D-glucose) and/or hypoxic (cobalt chloride treated) conditions. Cell proliferation was evaluated by a cell counting kit. The expression of vascular endothelial growth factor (VEGF) was assessed by Western blot analysis. RESULTS: The proliferation of HREC was significantly elevated in response to moderately-high glucose and hypoxic conditions. The combination of high glucose and hypoxia did not have any additive effects on cell proliferation. Consistent with the proliferation data, the expression of VEGF was also upregulated under both moderately-high glucose and hypoxic conditions. The treatment with scutellarein (1x10(-11) to 1x10(-5) mol/L) significantly inhibited high glucose- or hypoxia-induced cell proliferation and VEGF expression. CONCLUSION: Both hypoxia and moderately-high glucose were potent stimuli for cell proliferation and VEGF expression in HREC without any significant additive effects. Scutellarein is capable of inhibiting the proliferation of HREC, which is possibly related to its ability to suppress the VEGF expression.

[A preliminary study on estimation of postmortem interval according to beta-actin mRNA stability in rat].

OBJECTIVE: In order to find a new parameter to estimate the postmortem interval, beta-actin mRNA in lung and thoracic muscle of rats was detected at different time point postmortem. METHODS: Rats were killed by neck dislocation and left in a temperature controlling system at 21 degrees C for 12 days postmortem. Total RNA in lung and thoracic muscle at different time point was extracted and beta-actin mRNA expression was examined by RT-PCR. Semi-quantification analysis of the image of electrophoresis was performed to confirm the changes of beta-actin mRNA expression. RESULTS: beta-actin mRNA in lung of rats still could be detected at 12 days postmortem, but-disappeared in thoracic muscle at 8 days postmortem. CONCLUSION: The expression of beta-actin mRNA in lung and thoracic muscle could be a new parameter for estimation of PMI.