New Mitogenomes of the Polypedilum Generic Complex (Diptera: Chironomidae): Characterization and Phylogenetic Implications.

Mitochondrial genomics, as a useful marker for phylogenetics and systematics of organisms, are important for molecular biology studies. The phylogenetic relationships of the Polypedilum generic complex remains controversial, due to lack taxonomy and molecular information. In this study, we newly sequenced mitogenomes of 14 species of the Polypedilum generic complex. Coupled with three recently published sequences, we analyzed the nucleotide composition, sequence length, and evolutionary rate of this generic complex. The control region showed the highest AT content. The evolution rate of protein coding genes was as follows: ATP8 > ND6 > ND5 > ND3 > ND2 > ND4L > ND4 > COX1 > ND1 > CYTB > APT6 > COX2 > COX3. We reconstructed the phylogenetic relationships among the genera within the Polypedilum generic complex based on 19 mitochondrial genomes (seventeen ingroups and two outgroups), using Bayesian Inference (BI) and Maximum Likelihood (ML) methods for all databases. Phylogenetic analysis of 19 mitochondrial genomes demonstrated that the Endochironomus + Synendotendipes was sister to Phaenopsectra + Sergentia.

Identification of volatile organic compounds in muscle tissues of different species based on Headspace-Gas-Chromatography Ion-Mobility spectrometry.

Species identification of unknown biological samples is crucial for forensic applications, especially in cases of explosion, disaster accidents, and body mutilation after murdering, as well as poaching, illegal trade in endangered animals, and meat food fraud. In this study, we identified 60 volatile organic compounds (VOCs) in fresh skeletal muscle tissues of seven different animal species (cattle, sheep, pigs, rabbits, rats, chickens and carp) and a human dead body by headspace-gas-chromatography ion-mobility spectrometry (HS-GC-IMS), and compared their differences by retention time, drift time and molecular weight. The results showed that these VOCs formed different gallery plot fingerprints in the skeletal muscle tissues of the human dead body and seven animal species. Principal component analysis (PCA) showed significantly different fingerprints between these species, and these fingerprints maintained good stability between the species and within the same species. Some VOCs have high species specificity, while VOCs of human fresh muscle tissues from different individual sources have little difference, demonstrating that all tested muscle tissue samples could be distinguished based on different VOCs. HS-GC-IMS has proved to be a rapid, high-throughput, highly sensitive and specific species identification method, which can be used for forensic species identification in criminal cases and disaster accidents, as well as detection in the field of food safety, such as meat fraud and adulteration.

[Study on the Antioxidant Activity of Aloe-Emodin Metal Complex].

OBJECTIVE: To synthesize three kinds of metal complexes of aloe-emodin and compare the antioxidant activities of the ligands and the complexes. METHODS: Three kinds of aloe emodin metal complex, the aloe-emodin-iron (Ⅱ), the aloe-emodin-copper (Ⅱ) and the aloe-emodin-magnesium (Ⅱ) complexes, were synthesized by dissolving and stirring in anhydrous ethanol solvent, and their structures were characterized. The Fe 2+-H 2O 2-methylene blue method, the diphenyl bitter hydrazine radical method (DPPH method) and other assays were used to determine the clearance effect of ligands and complexes on superoxide radicals (O 2 －•), hydroxyl radicals (•OH) and phenyl bitter hydrazine radical (DPPH•). RESULTS: Three kinds of aloe emodin metal complex, the aloe-emodin-iron (Ⅱ), the aloe-emodin-copper (Ⅱ) and the aloe-emodin-magnesium (Ⅱ) complexes, were successfully synthesized. According to the results of structural characterization, we speculated that the aloe-emodin metal complexes were formed at the site between the two molecules of aloe-emodin and one molecule of metal ions (Fe 2+, Mg 2+, Cu 2+) via the 9 th carbonyl and 8 th hydroxyl groups of the aloe-emodin molecules. Both the complex and the ligand have clearance effects on three kinds of free radicals, and the complex showed stronger effects than its ligand ( P<0.05). CONCLUSION: Coordination of aloe-emodin with metal ions, such as Fe 2+, Cu 2+, and Mg 2+, could enhance the antioxidant activity of the ligand itself.

Haplotype analysis of 36 Y-STR loci in a Chinese Han population from Anhui Province, Eastern China.

We analyzed haplotypes for 36 Y chromosomal STRs (Y-STRs), including 27 Yfiler Plus loci and 9 additional STRs (DYS549, DYS643, DYS508, DYS447, DYS596, DYS444, DYS557, and DYS527a/b) in 2018 unrelated Chinese Han individuals from Anhui Province using DNATyperTM 36Y Kit. Phylogenetic analysis was performed to determine the genetic relationship of the Anhui Han population with other neighboring and/or linguistically close populations.

Identifying potential treatments of COVID-19 from Traditional Chinese Medicine (TCM) by using a data-driven approach.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese Medicine (TCM) has been widely used as an approach worldwide. Chinese Medicines (CMs) had been used to treat and prevent viral infection pneumonia diseases for thousands of years and had accumulated a large number of clinical experiences and effective prescriptions. AIM OF THE STUDY: This research aimed to systematically excavate the classical prescriptions of Chinese Medicine (CM), which have been used to prevent and treat Pestilence (Wenbing, Wenyi, Shiyi or Yibing) for long history in China, to obtain the potential prescriptions and ingredients to alternatively treat COVID-19. MATERIALS AND METHODS: We developed the screening system based on data mining, molecular docking and network pharmacology. Data mining and association network were used to mine the high-frequency herbs and formulas from ancient prescriptions. Virtual screening for the effective components of high frequency CMs and compatibility Chinese Medicine was explored by a molecular docking approach. Furthermore, network pharmacology method was used to preliminarily uncover the molecule mechanism. RESULTS: 574 prescriptions were obtained from 96,606 classical prescriptions with the key words to treat "Warm diseases (Wenbing)", "Pestilence (Wenyi or Yibing)" or "Epidemic diseases (Shiyi)". Meanwhile, 40 kinds of CMs, 36 CMs-pairs, 6 triple-CMs-groups existed with high frequency among the 574 prescriptions. Additionally, the key targets of SARS-COV-2, namely 3CL hydrolase (Mpro) and angiotensin-converting enzyme 2(ACE2), were used to dock the main ingredients from the 40 kinds by the LigandFitDock method. A total of 66 compounds components with higher frequency were docked with the COVID-19 targets, which were distributed in 26 kinds of CMs, among which Gancao (Glycyrrhizae Radix Et Rhizoma), HuangQin (Scutellariae Radix), Dahuang (Rhei Radix Et Rhizome) and Chaihu (Bupleuri Radix) contain more potential compounds. Network pharmacology results showed that Gancao (Glycyrrhizae Radix Et Rhizoma) and HuangQin (Scutellariae Radix) CMs-pairs could also interact with the targets involving in immune and inflammation diseases. CONCLUSIONS: These results we obtained probably provided potential candidate CMs formulas or active ingredients to overcome COVID-19. Prospectively, animal experiment and rigorous clinic studies are needed to confirm the potential preventive and treat effect of these CMs and compounds.

Toll­Like receptor 4 promotes the phosphorylation of CRMP2 via the activation of Rho­kinase in MCAO rats.

The mechanism associated with Toll­like receptor 4 (TLR4) in neurological injury remains unclear. The aim of the present study was to investigate the pathology of TLR4 in middle cerebral artery occlusion (MCAO)/reperfusion rat models via the regulation of collapsin response mediator protein 2 (CRMP2) phosphorylation. The modified neurological severity score (mNSS) was applied to assess neurological recovery. Immunofluorescence and western blotting were used to detect the protein expressions of TLR4, Rho­associated protein kinase 2 (ROCK­II) and CRMP2 following the intracerebroventricular administration of TLR4­specific agonist, lipopolysaccharide (LPS) and TLR4­neutralizing antibody, the ROCK­II specific inhibitor Y­27632 or LPS+Y­27632 30 min prior to MCAO. The expression levels of TLR4 and the phosphorylation of CRMP2 significantly increased in response to LPS­mediated induction and/or MCAO; however, they were reversed by treatment with LPS+TLR4­neutralizing antibody. Y­27632 decreased the expression of ROCK­II and phosphorylated (p)­CRMP2, and suppressed the increased ROCK­II and p­CRMP2 induced by LPS; however, no effect on the levels of TLR4 expression was observed. The neurological function as measured by mNSS score was reduced in the LPS group when compared with the MCAO group, whereas the LPS+Y­27632 group reversed the reduced neurological function at 7 and 14 days post­MCAO. The results of the present study suggested that TLR4 may promote the phosphorylation of CRMP2 via the activation of ROCK­II in MCAO rats, which further characterizes the pathological mechanism of TLR4 in stroke, and that modulation of TLR4 could be a potential target to limit secondary post­stroke brain damage.

Developmental Times of Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae) at Constant Temperatures and Applications in Forensic Entomology.

The characteristic life stages of infesting blowflies (Calliphoridae) such as Chrysomya megacephala (Fabricius) are powerful evidence for estimating the death time of a corpse, but an established reference of developmental times for local blowfly species is required. We determined the developmental rates of C. megacephala from southwest China at seven constant temperatures (16-34°C). Isomegalen and isomorphen diagrams were constructed based on the larval length and time for each developmental event (first ecdysis, second ecdysis, wandering, pupariation, and eclosion), at each temperature. A thermal summation model was constructed by estimating the developmental threshold temperature D0 and the thermal summation constant K. The thermal summation model indicated that, for complete development from egg hatching to eclosion, D0 = 9.07 ± 0.54°C and K = 3991.07 ± 187.26 h °C. This reference can increase the accuracy of estimations of postmortem intervals in China by predicting the growth of C. megacephala.

Cedrol induces autophagy and apoptotic cell death in A549 non-small cell lung carcinoma cells through the P13K/Akt signaling pathway, the loss of mitochondrial transmembrane potential and the generation of ROS.

The objective of the present study was to determine the anticancer effects of cedrol in A549 human non-small cell lung cancer cells by examining the effects of cedrol on apoptosis induction, the phosphatidylinositol 3'-kinase (PI3K)/Akt signaling pathway, autophagy, reactive oxygen species (ROS) generation and mitochondrial transmembrane potential (MTP). The anticancer effects of cedrol were examined using A549 human lung carcinoma cells as an in vitro model. Cell viability was determined using MTT and lactate dehydrogenase (LDH) assays, and an inverted phase contrast microscope was used to examine the morphological changes in these cells. Cedrol­triggered autophagy was confirmed by transmission electron microscopy (TEM) analysis of the cells, as well as by western blot analysis of microtubule-associated protein light-chain 3 (LC3)B expression. Intracellular ROS generation was measured by flow cytometry using 5-(6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate (CM-DCFH2-DA) staining and MTP was measured using flow cytometry. The results demonstrated that cedrol reduced cell viability and induced cell apoptosis in a dose-dependent manner. Mechanistic evaluations indicated that cedrol induced apoptosis by reducing the MTP and by decreasing the levels of phosphorylated (p-)PI3K and p-Akt. Cedrol induced autophagy, which was confirmed by TEM analysis, by increasing intracellular ROS formation in a concentration-dependent manner, which was almost completely reversed by N-acetyl-L-cysteine (NAC) and tocopherol. Taken together, these findings reveal that cedrol inhibits cell proliferation and induces apoptosis in A549 cells through mitochondrial and PI3K/Akt signaling pathways. Our findings also reveal that cedrol induced pro-death autophagy by increasing intracellular ROS production.

[Species Identification of Puparium Samples of Common Sarcosaphagous Flies Based on Molecular Marker Analysis].

OBJECTIVE: To investigate the application value of empty puparia in species identification of common sarcosaphagous flies. METHODS: Fifty-five samples of adult flies and their empty puparia were collected. All the samples were identified as 2 families, 6 genera and 8 species by morphological characteristics. The samples were divided into 3 groups according to their time period between eclosion and our analyses: less than 2 years (n = 23), 2-5 years (n = 20), and more than 5 years (n = 12). The mtDNA of each sample was extracted by CTAB method. The purity and concentration of DNA were tested. PCR products were amplified using two sets of primers. Two sequences of CO I gene (sequence I: 498 bp, sequence II : 841 bp) from each sample were compared to the sequences in GenBank using BLAST for species identification. RESULTS: The mtDNA was extracted successfully from all the samples. DNA concentration of adult chest muscle preserved less than or equal to 5 years and empty puparia preserved less than 2 years ranged from 1.0 to 3.0 µg/µl, and the value of A260/A280 ranged from 1.6 to 1.8. The purity and concentration was lower than 1.6 and 1.0 µg/µl, when the adult chest muscle and empty puparia preserved more than 5 years and 2 years, respectively. DNA concentration of the samples significantly decreased with the prolonged preservation time (P < 0.01). Two sequences of CO I gene was amplified in adult chest muscle and empty puparia which preserved less than 2 years. The success rates of amplification decreased with the prolonged preservation time, especially for the sequence II (P < 0.01). The morphological identification of 8 species did not match exactly with the results based on the COI gene, correct species identification occurred in 6 and 7 species out of 8 based on the two sequences, respectively, and their Max ident value exceeded 97% CONCLUSION: Empty puparium samples can be used to extract mtDNA and identify species.

Technical note: Development of Hemipyrellia ligurriens (Wiedemann) (Diptera: Calliphoridae) at constant temperatures: Applications in estimating postmortem interval.

Blowflies (Calliphoridae) are recognized as a powerful tool for estimating the minimum postmortem interval (PMImin). The times for blowflies to develop from oviposition to eclosion is mainly controlled by temperature, which can differ between even closely related species. Hemipyrellia ligurriens (Wiedemann) (Diptera: Calliphoridae) is a blowfly distributed throughout Asia and Australia. However, a systematic determination of the developmental times of H. ligurriens under constant temperature, necessary for estimating the PMImin, is lacking. Such an examination would broaden the forensic importance of the species. Thus, this study explored the growth curves of larval H. ligurriens at 7 constant temperatures (16, 19, 22, 25, 28, 31, and 34°C). Isomegalen and isomorphen diagrams were successfully constructed, depicting the time of larval length or developmental event, respectively, at different temperatures. A thermal summation model was also constructed via regression analysis, by estimating the developmental threshold temperature t and thermal summation constant K. The thermal summation model indicated that t at 8.3°C and K at 5747.5 degree-hours (°Ch) are required for complete development from oviposition to eclosion, and suggested an optimum temperature range of 16-28°C for the development of H. ligurriens. These data establish for the first time the temperature-dependent developmental time of H. ligurriens for forensic entomology application. The 3 developmental models are provided.

[Effect of diets with different fat levels on the body size and development of Lucilia sericata].

OBJECTIVE: To observe the effect of diets with different fat levels on the body size and development of Lucilia sericata. METHODS: Under the constant temperature of 28 degrees C, the larvae were reared on the diets containing 0% (G0), 10% (G1), 30% (G3), 50% (G5) and 80% (G8) fat tissues (fat/muscle ratio), respectively. Length and weight of larvae and pupae were measured at 12 h interval since 16 h after eclosion. Length of inter-medial cross vein (m-m) of adult left wing was measured. 10 samples were collected in each group. The developmental duration time, mortality and sex ratios of adults were recorded. RESULTS: The mean maximal larval length [(13.3 +/- 1.2), (12.0 +/- 1.1), (10.2 +/- 0.9) and (8.8 +/- 0.8) mm, respectively] and mean maximal larval weight [(72.8 +/- 6.1), (62.2 +/- 5.7), (47.2 +/- 4.3), and (34.9 +/- 5.7) mg] in G1, G3, G5 and G8 groups were significantly less than that of the G0 group [(14.8 +/- 1.3) mm and (80.4 +/- 8.1) mg](P < 0.01). The body size of pupae and adults was also significantly less than that of G0 group (P < 0.01). The total duration time of G5 and G8 groups [(293.3 +/- 22.2) and (285.2 +/- 24.6) h] were significantly shorter than that of G0 group [(312.8 +/- 20.1)h] (P < 0.01). The mortality of larvae [(32.6 +/- 5.6)% and (44.3 +/- 7.7)%] and pupae [(28.6 +/- 5.5)% and (43.5 +/- 6.2)%] of G5 and G8 group were also significantly higher than that of G0 group [(5.7 +/- 3.3)% and (4.5 +/- 1.9)%] (P < 0.01). There was no significant difference in sex ratio among the 5 groups (P > 0.05). CONCLUSION: The body size of larvae, pupae and adults of Lucilia sericata is smaller, the development time is shorter and mortality is higher when the food substrate contains more fat tissues.

Magnetic bead-based separation of sperm from buccal epithelial cells using a monoclonal antibody against MOSPD3.

Forensic DNA analysis of sexual assault evidence requires unambiguous differentiation of DNA profiles in mixed samples. To investigate the feasibility of magnetic bead-based separation of sperm from cell mixtures using a monoclonal antibody against MOSPD3 (motile sperm domain-containing protein 3), 30 cell samples were prepared by mixing 10(4) female buccal epithelial cells with sperm cells of varying densities (10(3), 10(4), or 10(5) cells/mL). Western blot and immunofluorescence assays showed that MOSPD3 was detectable on the membrane of sperm cells, but not in buccal epithelial cells. After biotinylated MOSPD3 antibody was incubated successively with the prepared cell mixtures and avidin-coated magnetic beads, microscopic observation revealed that each sperm cell was bound by two or more magnetic beads, in the head, neck, mid-piece, or flagellum. A full single-source short tandem repeat profile could be obtained in 80% of mixed samples containing 10(3) sperm cells/mL and in all samples containing ≥10(4) sperm cells/mL. For dried vaginal swab specimens, the rate of successful detection was 100% in both flocked and cotton swabs preserved for 1 day, 87.5% in flocked swabs and 40% in cotton swabs preserved for 3 days, and 40% in flocked swabs and 16.67% in cotton swabs preserved for 10 days. Our findings suggest that immunomagnetic bead-based separation is potentially a promising alternative to conventional methods for isolating sperm cells from mixed forensic samples.

[Effect of feeding on different tissues on larva development of Lucilia sericata].

OBJECTIVE: To observe the effect of feeding on different pig tissues on the development of Lucilia sericata larvae. METHODS: Under a constant temperature of 25 degrees C, about 200 larvae each were reared on four different substrates, i.e. pig's brain, liver, muscle and a mixture of minced pork muscle and fat (6:4). Length and weight of larvae and pupae were measured at 12 h interval 16 h after eclosion. The time of development, mortality, sex ratio of adults were recorded. RESULTS: Compared to the other groups, the larvae of liver and mixture groups grew slower, time of reaching maximum length and weight was delayed for 12-24 h. The duration of larva development of liver group [(284.0 +/- 12.6) h] was longer than that of brain group [(257.0 +/- 11.9) h], muscle group [(258.0 +/- 10.2) h] and mixture group [(260.0 +/- 9.8) h] (P < 0.05). The mean maximum larva length and weight in mixture group [(11.85 +/- 0.36) mm, (40.4 +/- 0.2) mg] and liver group [(12.01 +/- 0.43) mm, (42.8 +/- 0.4) mg] was statistically less than that of brain group and muscle group (P < 0.05). The pupal length and weight in mixture group [(7.81 +/- 0.60) mm, (38.4 +/- 2.4) mg] was less than that of other three groups (P < 0.05). The larval and pupal mortality of mixture group [(9.8 +/- 2.4)% and (10.3 +/- 1.8)%] was statistically higher than that of other three groups (P < 0.05). There was no significant difference in the sex ratio among the four groups (P > 0.05). CONCLUSION: The development duration of the larvae fed on liver tissue is longer than other groups, and the larvae body length and weight of liver group are less than other groups. The body length and weight of larvae and pupae fed on mixture diet are less than other groups with higher mortality.

[Effect of feeding on different tissues on larva development of Chrysomya megacephala (Diptera: Calliphoridae)].

OBJECTIVE: To observe the effect of feeding on different pig tissues on the development of Chrysomya megacephala larvae. METHODS: About 200 larvae each were reared on four different substrates, i.e. pig's brain, liver, muscle and a mixture of minced pork muscle and fat (7 : 3) at a constant temperature of 25 degrees C. Length and weight of larvae and pupae were measured at 12 h interval 16 h after eclosion. 10 larvae or pupae were collected each time. The time of development, mortality, and sex ratio of adults were recorded. RESULTS: Three replicated experiments showed that the larvae fed on liver grew slowly, time of reaching maximum length and weight was delayed for about 24-36 h, and the duration of larva development was longer than that of other groups (P<0.01). The mean maximal larval length in mixture group [(14.89 +/- 0.39) mm] was statistically shorter than that of brain group, muscle group and liver group, [(17.81 +/- 0.54), (16.94 +/- 0.43) and (17.14 +/- 0.27) mm, respectively] (P<0.01). The mean maximal larval weight in liver group [(73.5 +/- 6.8) mg] and mixture group [(63.0 +/- 5.4) mg] was statistically lighter than brain group [(91.2 +/- 7.5) mg] and muscle group [(86.3 +/- 7.3) mg] (P<0.01). The pupal length in mixture group was statistically shorter than that of other 3 groups (P<0.01). The pupal weight of mixture group and liver group was statistically lighter than that of brain group and muscle group (P<0.01). The larval and pupal mortality of mixture group [(9.8 +/- 3.1)% and (8.9 +/- 3.1)%] was statistically higher than that of brain group [(5.5 +/- 3.1)% and (4.6 +/- 1.5)%], muscle group [(4.7 +/- 2.2)% and (3.8 +/- 2.0)%] and liver group [(5.4 +/- 2.3)% and (4.8 +/- 1.7)%] (P<0.01). There was no significant difference in the sex ratio among the four groups (P>0.05). CONCLUSION: The development duration of the larvae fed on liver is longer than other groups. The body length and weight of larvae and pupae fed on mixture diet are less than other groups with higher mortality.