DNA Extraction and Comparison between Old and Fresh Necrophilic Fly Samples.

A total of five samples of Chrysomya megacephala samples - three fresh samples, one sample stored in alcohol for 2 years, and one sample stored in dry sealed storage for 2 years protected from light only - were selected to investigate whether a blood DNA extraction kit could extract DNA from necrophilous flies and to determine whether alcohol could prolong the preservation of necrophilous flies' DNA. First, the blood DNA extraction kit was used to extract DNA from their thorax tissues. Then, the DNA purity and concentration were examined using a microplate reader and a fluorometer. Finally, PCR amplification and electrophoresis of the extracted DNA were done with necrophilic fly-specific primers located in the mitochondrial CO I gene sequence. The results showed that the DNA purity of all samples was greater than 2.0. The DNA concentration was observed to be of the following order: fresh samples > alcohol-preserved old samples > untreated, old samples. All samples had specific electrophoretic bands after PCR amplification. In conclusion, a blood DNA extraction kit can be used to extract DNA from necrophilic flies successfully, and the DNA concentration of fresh fly samples is greater than that of old fly samples. The flies can be stored in alcohol for a long time.

Geographical Distribution and Multimethod Species Identification of Forensically Important Necrophagous Flies on Hainan Island.

Forensic entomology offers unique advantages for the minimum postmortem interval (PMImin) estimation of decomposed corpses in forensic investigations. Accurate species identification and up-to-date locality information are essential. Hainan Island has a tropical rainforest climate and a vast territory. In this study, the community structure of necrophagous flies on Hainan Island was investigated in detail according to geographical environment. The results showed that the dominant species included C. megacephala, S. peregrina, C. rufifacies, S. misera, H. ligurriens, S. sericea, S. cinerea, S. dux, C. pinguis, and M. domestica. Furthermore, C. rufifacies and C. villeneuvi were found only in the high-altitude areas of Wuzhi Mountain, while S. cinerea was distributed only in coastal areas; the latter is a representative species of Hainan Island and has not been reported before. Furthermore, a GenBank database of forensically important flies was established, whilst a high-resolution melt (HRM) curve analysis was applied to identify the common species of Hainan Island for the first time. This study enriches the database of forensically important flies in tropical rainforest regions.

Extraction of Diatom DNA from Water Samples and Tissues.

Diatom testing is an essential auxiliary means in forensic practice to determine whether the corpse drowned in water and to infer the drowning location. Diatom testing is also an important research content in the field of the environment and plankton. The diatom molecular biology testing technology, which focuses on diatom DNA as the primary research object, is a new method of diatom testing. Diatom DNA extraction is the basis of diatom molecular testing. At present, the kits commonly used for diatom DNA extraction are expensive, which increases the cost of carrying out related research. Our laboratory improved the general whole blood genomic DNA rapid extraction kit and obtained a satisfactory diatom DNA extraction effect, thus providing an alternative economical and affordable DNA extraction solution based on glass beads for related research. The diatom DNA extracted using this protocol could satisfy many downstream applications, such as PCR and sequencing.

Synthesis and in vitro Anti-proliferative Activities on LNCaP, LS180 and MKN45 of Novel 20(R)-Panaxadiol Derivatives.

BACKGROUND: 20(R)-PD, a tetracyclic triterpenoid, is a non-natural saponin present in the form of protopanaxadiol. Because of its essential biological activities, especially anti-tumor activity, structural modification of 20(R)-PD and the development of innovative and novel 20(R)-PD derivatives with better anti-tumor activity are increasingly relevant. AIMS: 20(R)-Panaxadiol (20(R)-PD) can inhibit tumor proliferation. Three series of novel 20(R-PD derivatives were synthesized by modifying the A-ring. OBJECTIVE: The objective of this work was to synthesize and evaluate the in vitro anti-proliferative activities of 20(R)- PD derivatives in LNCaP, LS180, and MKN45 cancer cells. Structural modifications were performed at the C-3 position and A-ring. METHODS: The in vitro anti-proliferative activities of novel derivatives in LNCaP, LS180, and MKN45 cells were evaluated by the MTT assay. The effects of compounds 5 and C9 on apoptosis were determined by flow cytometry. RESULTS: Compounds 5, B2, C2, C4, C7, C8, C9, C10, and C11 exhibited good anti-proliferative activities in LNCaP, LS180, and MKN45 cells in vitro. The best anti-proliferative activity was observed for the C-series derivatives with the introduction of amino acids at the C-3 position. C9 exhibited good potent activity with an IC50 of 2.89 µM. CONCLUSION: Compound C9 is a potential candidate with potent anti-proliferative activity.

Autopsy results from a COVID-19 patient treated in a tropical area, and a review of the epidemiological history.

Since the start of the COVID-19 pandemic, there has been an urgent need to produce accurate and sensitive tests. However, there have been instances where a positive nucleic acid test turns negative after treatment, and then positive again. This case report describes such an instance from the tropical region of Hainan, China. The patient was a 61-year-old female who went to Hainan on vacation from Wuhan during the COVID-19 pandemic in 2020. Symptoms appeared 9 d after arriving in Hainan, and it was confirmed that the nucleic acid test was positive after 4 repeats. Her condition declined rapidly, her heart stopped beating, and she was admitted in a coma to the ICU. After treatment, the SARS-CoV-2 virus nucleic acid test of several nasopharyngeal swabs were negative, and tests on whole blood, anal swabs, and urine were also negative. Later, however, nucleic acid tests on a lower respiratory tract sputum swab and lower respiratory tract lavage fluid were positive. An autopsy examination was carried out 12 h after her death, and multi-organ secretions were extracted for nucleic acid testing. The SARS-CoV-2 virus nucleic acid was only detected in the swabs from the end of the bronchus, which was confirmed by the visualization of the coronavirus by electron microscopy. Autopsy confirmed that the damage was mainly concentrated in the lungs and immune organs and tissues throughout the body. Epidemiology indicated that none of the people she came into contact with after arriving in Hainan, including close contacts, were infected. This is in sharp contrast to the highly contagious virus in Wuhan in the temperate zone during the same period. This case report indicates: (1) The high temperatures in tropical areas may have an impact on the spread and harm of COVID-19, and (2) The reason why nucleic acid testing for COVID-19 was initially negative and then positive after treatment may be related to the survival of the SARS-CoV-2 virus in deep lung tissues.

Application Progress of High-Throughput Sequencing Technology in Forensic Diatom Detection.

Diatom detection is an important method for identifying drowning and throwing corpses after death and inferring the drowning sites in forensic examination of corpses in water. In recent years,high-throughput sequencing technology has achieved rapid development and has been widely used in research related to diatom taxonomic investigations. This paper reviews the research status and prospects of high-throughput sequencing technology and its application in forensic diatom detection.

Improved Glass Bead-Vortex Oscillation Method for DNA Extraction from Diatom.

OBJECTIVES: To examine the effect of improving diatom DNA extraction by glass bead - vortex oscillation method. METHODS: The DNeasy PowerSoil Pro kit was used as control, two plant DNA extraction kits with different principles (New Plant genomic DNA extraction kit and Plant DNA Isolation kit) and one whole blood DNA extraction kit (whole blood genomic DNA extraction kit) were selected to extract diatom DNA from lung tissue and water sample of the same drowning case. The combination of mass ratio of glass beads with different sizes and vortex oscillation time was designed, and the optimal DNA extraction conditions were selected with the addition of glass beads oscillation. The extracted products of the conventional group and the modified group were directly electrophoretic and detected by diatom specific PCR. Finally, all the extracts were quantified by qPCR, and the Ct values of different groups were statistically analyzed. RESULTS: When the frequency of vortex oscillation was 3 000 r/min, the optimal combination of DNA extraction was vortex oscillation for 4 min, and the mass ratio of large glass beads to small glass beads was 1∶1. The DNeasy PowerSoil Pro kit was used as a reference, and the Ct value of 10 mL water sample was greater than that of 0.5 g tissue. The Ct values of the other three kits used for plant DNA extraction decreased after the glass beads-vortex oscillation method was used, and the Ct values of the tissues before and after the improvement were statistically significant (P<0.05). The whole blood genomic DNA extraction kit used in this study could successfully extract diatom DNA, the extraction of water samples was close to DNeasy PowerSoil Pro kit, after the modified method was applied to tissue samples, the difference in Ct value was statistically significant (P<0.05). However, when the three kits were used to extract diatom DNA from water samples, Ct values before and after the improvement were only statistically significant in New Plant genomic DNA extraction kit group (P<0.05). CONCLUSIONS: The improved glass bead-vortex oscillation method can improve the extraction efficiency of diatom DNA from forensic materials, especially from tissue samples, by plant and blood DNA extraction kits.

Co-composting of fresh tobacco leaves and soil: an exploration on the utilization of fresh tobacco waste in farmland.

A large amount of fresh tobacco waste with high water content are produced in farmland, and it may cause environmental pollution if it is not properly treated. The fresh tobacco waste is not easily collected and transported, resulting in its centralized treatment is expensive. This study is to clarify whether it is feasible to treat fresh tobacco wastes by co-composting of them and soil in farmland and applied the obtained compost product into the soil instead of a part of tobacco-specific fertilizer. The results showed that, compared with that in original soil, the relative abundance of Pseudomonas, Azotobacter, and Coprinus of the co-composted products increased by roughly 244%, 323%, and 675%, respectively, and effective nitrogen and available potassium increased by roughly 157% and 132%, respectively. In addition, the nicotine content in co-composted products decreased dramatically compared with the discarded tobacco leaves. The application of the co-composted products and 20% fertilizer amount (15 g/plant) (YD5) exhibited the highest relative abundance of beneficial microbial communities in the soil and the best growth of tobacco plants. The co-composting of fresh tobacco waste and soil in farmland is an effective measure to treat the fresh tobacco waste, and its products increased beneficial microorganisms and stimulate the growth of tobacco plants by replacing an amount of the fertilizer.

A preliminary study on the distribution of necrophagous flies on Hainan Island, China.

To date, there have been no studies on necrophagous fly populations on Hainan Island in China. Thus, we investigated the species composition of necrophagous flies as well as their geographic distribution on Hainan Island for the first time. Ten sites in different climatic regions across the island were sampled for 7 days per location from November 1, 2018 to February 28, 2019 and from July 1, 2019 to October 31, 2019. Bottle traps made of 1.5 L soft plastic bottles were used to trap necrophagous flies. The collected individuals were identified to species. The specimens represented 5 families and 28 species. Chrysomya megacephala (Fabricius, 1794) was the most dominant species, followed by Chrysomya rufifacies (Macquart, 1843), Hemipyrellia ligurriens (Wiedemann, 1830), Boettcherisca peregrine (Robineau-Desvoidy, 1830), Parasarcophaga dux (Thomson, 1868), Parasarcophaga misera (Walker, 1849), Synthesiomyia nudiseta (Wulp, 1883), and Ophyra chalcogaster (Wiedemann, 1924). The largest collection of flies was obtained in the semiarid region. Species richness was highest in the subhumid region and was higher in summer than in winter, but there were exceptions, such as L. hainanensis (Fan,1965), Boettcherisca formosensis (Lopes, 1961), and Muscina stabulans (Fallen, 1817). Fannia pusio (Wiedemann, 1830), and Boettcherisca formosensis (Lopes, 1961) were newly recorded species on Hainan Island. Of the necrophagous flies collected during the study, we propose several predominant species based on the criteria of distribution, occurrence frequency, and resource preference. Our results not only investigate necrophagous flies on Hainan Island but also accumulate data for criminal investigations in the region.

Advances in Research on the Preparation and Biological Activity of Maslinic Acid.

Maslinic acid, a pentacyclic triterpene acid, is mainly isolated from olives. Maslinic acid and its derivatives exhibit a broad range of biological properties, such as anti-inflammatory, anticancer, anti-diabetic, antimicrobial, neuroprotective and hepatoprotective activities. In this minireview, the progress of research on maslinic acid with regard to its bioactivities, extraction, semisynthetic preparation and patents is reported. The relationships between the structure and the activity of maslinic acid and its derivatives are also discussed.

Advances in the chemistry, pharmacological diversity, and metabolism of 20(R)-ginseng saponins.

Ginseng has been used as a popular herbal medicine in East Asia for at least two millennia. However, 20(R)-ginseng saponins, one class of important rare ginsenosides, are rare in natural products. 20(R)-ginseng saponins are generally prepared by chemical epimerization and microbial transformation from 20(S)-isomers. The C20 configuration of 20(R)-ginseng saponins are usually determined by 13C NMR and X-ray single-crystal diffraction. 20(R)-ginseng saponins have antitumor, antioxidative, antifatigue, neuroprotective, and osteoclastogenesis inhibitory effects, among others. Owing to the chemical structure and pharmacological and stereoselective properties, 20(R)-ginseng saponins have attracted a great deal of attention in recent years. In this study, the discovery, identification, chemical epimerization, microbial transformation, pharmacological activities, and metabolism of 20(R)-ginseng saponins are summarized.

The Ong Be language-speaking population in Hainan Island: genetic diversity, phylogenetic characteristics and reflections on ethnicity.

The Ong Be language-speaking population (Lingao population) settled in the north-central coast of Hainan Island and has attracted little attention because of its small population size (about five hundred thousand) as well as its relative geographical isolation in linguistics, anthropology, and forensic genetics. The Lingao population selected "Han Chinese" as its ethnic component around the founding period of the PRC. Hence, we used the Goldeneye™ DNA ID System 20A (including 13 CODIS core loci and 6 expanded CODIS loci) to obtain Lingao population genotypes and to enable the publishing of relative forensic parameters; further, this data will allow the evaluation of the Lingao ethnic component from different perspectives. Genetic differences between the Lingao population and Han Chinese populations from north and south administrative divisions of China as well as genetic distinctions among official ethnic groups were also investigated by the principal component analysis (PCA). The phylogenic tree was investigated by the unweighted pair-group method with arithmetic averages (UPGMA). We analysed the genetic polymorphisms of 19 autosomal STR loci in 821 individuals from the Lingao population and observed a total of 269 alleles at 19 autosomal STR loci, with the corresponding allelic frequencies ranging from 0.0006 to 0.5780. The combined power of discrimination (CPD) and combined power of exclusion (CPE) for the 19 autosomal STR loci were 0.99999999999999999999998569 and 0.999999989, respectively. No evidence of deviation from the Hardy-Weinberg equilibrium (HWE) was identified and no linkage disequilibrium (LD) was observed. The results demonstrated that the Goldeneye™ DNA ID System 20A had highly genetic diversities in the 19 STR loci in the Lingao population for forensic applications. In addition, the Lingao population had relatively close genetic relationships with Guangxi Han and Hainan Li populations compared to other populations. However, from a historical and linguistic perspective, "Han Chinese" is probably not an accurate description of the Lingao population. In conclusion, it is neither accurate or appropriate to classify the Ong Be language-speaking population as "Han Chinese" for multiple reasons. The present study can increase our understanding of the genetic relationships between the Lingao population and other Chinese groups. Nonetheless, further genetic studies are still needed to explore the mysteries of the Ong Be language-speaking population.

Population data of 19 autosomal STR loci in the Li population from Hainan Province in southernmost China.

In the present study, population data of 19 autosomal STR loci included in the Goldeneye™ DNA ID System 20A in 653 Li individuals was obtained and population genetic relationships among 13 populations were investigated. MDS and phylogenetic analysis suggested that the Hainan Li population kept a close genetic relationship with the Chinese Han populations, especially for Southern Han populations (Guangdong Han, Sichuan Han, and Hunan Han). Our results indicated that the 19 autosomal STRs are highly discriminative and polymorphic in the Hainan Li population suitable for personal forensic identification and paternity testing.

A brief review of forensically important flesh flies (Diptera: Sarcophagidae).

Forensic entomology could provide valuable data for the minimum postmortem interval (PMImin) estimation and other relevant information, such as causes and circumstances of death. Some representatives of flesh flies are one of the dominant necrophagous insects during early stages of decomposition, demonstrating unique biological characteristics compared with other necrophagous flies. Moreover, they lead to global health concerns as carriers of various pathogenic micro-organisms, and dominantly result in the traumatic myiasis. Thus, sarcophagid flies are considered important in decomposition processes for PMImin estimation. However, the utility of sarcophagid flies has been seriously hampered by limited ecological, biological and taxonomic knowledge of them. The aim of this paper is to provide a brief review on the species, distribution and biological habit of forensically important sarcophagid flies. In addition, the relation between traumatic myiasis and flesh flies, molecular identification methods and developmental pattern of flesh flies are summarized.

The evaluation of forensic characteristics and the phylogenetic analysis of the Ong Be language-speaking population based on Y-STR.

The Ong Be language, an important branch of the Tai-Kadai language family, is one of the most distinctive languages on Hainan Island. Ong Be language speakers, who have lived in the Lingao district of Hainan Island for generations, were classified as Han Chinese in the early days of the establishment of the People's Republic of China but have distinct differences from the Han Chinese in language, lifestyle, customs and values and particularly in appearances and features. Currently, Y-chromosomal short tandem repeat (STR) haplotypes have been widely used in genetic applications, such as human forensics, historical investigations and genealogical research. In this study, 487 unrelated male individuals residing in the Lingao district volunteered, and their Y-STR haplotypes were investigated using the Yfiler and Yfiler Plus with 17 and 27 Y-STR loci, respectively. Furthermore, we combined our population data on the Lingao people with existing datasets from Asian nations (East, South and Southeast Asia) to explore the genetic variance and relationships with Han Chinese from different administrative regions in Northern and Southern China and Chinese ethnic minorities officially recognized by the PRC. Population comparisons demonstrated that the Lingao people had distant relationships with Asian nations at the national level and had relatively close genetic and linguistic relationships with Hainan Li and Guizhou Gelao, both of whom belong to the Tai-Kadai language family. The present results increase our understanding of the genetic relationships between the Lingao people and other groups, and further research in genetics and other areas is still needed to trace the origin of the Lingao people.

Identification of plasma biomarkers for diffuse axonal injury in rats by iTRAQ-coupled LC-MS/MS and bioinformatics analysis.

DAI is a serious and complex brain injury associated with significant morbidity and mortality. The lack of reliable objective diagnostic modalities for DAI delays administration of therapeutic interventions. Hence, identifying reliable biomarkers is urgently needed to enable early DAI diagnosis in the clinic. Herein, we established a rat model of DAI and applied an isobaric tags for a relative and absolute quantification (iTRAQ) coupled with nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) proteomics approach to screen differentially expressed plasma proteins associated with DAI. A total of 58 proteins were found to be significantly modulated in blood plasma samples of the injury group in at least one time point compared to controls. Bioinformatics analysis of the differentially expressed proteins revealed that the pathogenesis of axonal injury underlying DAI is multi-stage biological process involved. Two significantly changed proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and hemopexin (Hpx), were identified as potential diagnostic biomarkers for DAI, and were successfully confirmed by further western blot analysis. This proteomic profiling study not only identified novel plasma biomarkers that may facilitate the development of clinically diagnostic for DAI, but also provided enhanced understanding of the molecular mechanisms underlying DAI.

Identification of Forensically Important Blow Flies (Diptera: Calliphoridae) in China Based on COI.

Blow flies are among the most important insects in forensic entomology casework. Identification of blow fly species can be a time consuming and difficult task, especially at their early development stages. Present DNA-based technologies provide a promising identification method for these forensically important calliphorids. The cytochrome oxidase subunit I (COI) sequence has been applied as a suitable DNA marker in calliphorid identification for many years; however, limitation exists in using short sequence to determine genetically close species. In this study, COI long sequences were utilized in species-level identification. Seventy-two specimens were collected from 27 locations across 22 Chinese provinces, and unambiguously determined as 16 species under seven genera of Calliphoridae. Analysis of long mitochondrial COI sequence (1,021-1,382 bp) data from forensically relevant blow flies collected in the inland region of China provided a reliable marker for accurate identification. Our data provide genetic diversity and reference for global forensic-related blow fly species identification, and conductive meaning on future utilization of Chinese calliphorids used in forensic entomological practice.