Protein Dynamic Landscape of Pig Embryos during Peri-Implantation Development.

Properly developed embryos are critical for successful embryo implantation. The dynamic landscape of proteins as executors of biological processes in pig peri-implantation embryos has not been reported so far. In this study, we collected pig embryos from days 9, 12, and 15 of pregnancy during the peri-implantation stage for a PASEF-based quantitative proteomic analysis. In total, approximately 8000 proteins were identified. These proteins were classified as stage-exclusive proteins and stage-specific proteins, respectively, based on their presence and dynamic abundance changes at each stage. Functional analysis showed that their roles are consistent with the physiological processes of corresponding stages, such as the biosynthesis of amino acids and peptides at P09, the regulation of actin cytoskeletal organization and complement activation at P12, and the vesicular transport at P15. Correlation analysis between mRNAs and proteins showed a general positive correlation between pig peri-implantation embryonic mRNAs and proteins. Cross-species comparisons with human early embryos identified some conserved proteins that may be important in regulating embryonic development, such as STAT3, AP2A1, and PFAS. Our study provides a comprehensive overview of the pig embryo proteome during implantation, fills gaps in relevant developmental studies, and identifies some important proteins that may serve as potential targets for future research.

Investigation of Babesia spp. and Theileria spp. in ticks from Western China and identification of a novel genotype of Babesia caballi.

Babesia spp. and Theileria spp. are tick-borne protozoan parasites with veterinary importance. In China, epidemiological and genetic investigations on many Babesia and Theileria species were still absent in many areas and many tick species. From Aug 2021 to May 2023, 645 ticks were collected from the body surface of domestic animals (camels, goats, sheep, and cattle) using tweezers in seven counties in three provinces including Xinjiang (Qitai, Mulei, Hutubi, and Shihezi counties), Chongqing (Youyang and Yunyang counties), and Qinghai (Huangzhong county). Three tick species were morphologically and molecularly identified (334 Hyalomma asiaticum from Xinjiang, 245 Rhipicephalus microplus from Chongqing, and 66 Haemaphysalis qinghaiensis from Qinghai). A total of three Babesia species and two Theileria species were detected targeting the 18S gene. The COI and cytb sequences were also recovered from Babesia strains for further identification. In R. microplus from Chongqing, Babesia bigemina, the agent of bovine babesiosis, was detected. Notably, in H. asiaticum ticks from Xinjiang, a putative novel genotype of Babesia caballi was identified (0.90%, 3/334), whose COI and cytb genes have as low as 85.82% and 90.64-90.91% nucleotide identities to currently available sequences. It is noteworthy whether the sequence differences of its cytb contribute to the drug resistance of this variant due to the involvement of cytb in the drug resistance of Babesia. In addition, Theileria orientalis and Theileria annulata were detected in R. microplus from Chongqing (12.20%, 31/245) and H. asiaticum from Xinjiang (1.50%, 5/334), respectively. These results suggest that these protozoan parasites may be circulating in domestic animals in these areas. The pathogenicity of the novel genotype of B. caballi also warrants further investigation.

Genome-wide association studies for loin muscle area, loin muscle depth and backfat thickness in DLY pigs.

This study aimed at identifying genes associated with loin muscle area (LMA), loin muscle depth (LMD) and backfat thickness (BFT). We performed single-trait and multi-trait genome-wide association studies (GWASs) after genotyping 685 Duroc × (Landrace × Yorkshire) (DLY) pigs using the Geneseek Porcine 50K SNP chip. In the single-trait GWASs, we identified two, eight and two significant SNPs associated with LMA, LMD and BFT, respectively, and searched genes within the 1 Mb region near the significant SNPs with relevant functions as candidate genes. Consequently, we identified one (DOCK5), three (PID1, PITX2, ELOVL6) and three (CCR1, PARP14, CASR) promising candidate genes for LMA, LMD and BFT, respectively. Moreover, the multi-trait GWAS identified four significant SNPs associated with the three traits. In conclusion, the GWAS analysis of LMA, LMD and BFT in a DLY pig population identified several associated SNPs and candidate genes, further deepening our understanding of the genetic basis of these traits, and they may be useful for marker-assisted selection to improve the three traits in DLY pigs.

Identification and characterization of circRNAs in peri-implantation endometrium between Yorkshire and Erhualian pigs.

BACKGROUND: One of the most critical periods for the loss of pig embryos is the 12th day of gestation when implantation begins. Recent studies have shown that non-coding RNAs (ncRNAs) play important regulatory roles during pregnancy. Circular RNAs (circRNAs) are a kind of ubiquitously expressed ncRNAs that can directly regulate the binding proteins or regulate the expression of target genes by adsorbing micro RNAs (miRNA). RESULTS: We used the Illumina Novaseq6,000 technology to analyze the circRNA expression profile in the endometrium of three Erhualian (EH12) and three Yorkshire (YK12) pigs on day 12 of gestation. Overall, a total of 22,108 circRNAs were identified. Of these, 4051 circRNAs were specific to EH12 and 5889 circRNAs were specific to YK12, indicating a high level of breed specificity. Further analysis showed that there were 641 significant differentially expressed circRNAs (SDEcircRNAs) in EH12 compared with YK12 (FDR < 0.05). Functional enrichment of differential circRNA host genes revealed many pathways and genes associated with reproduction and regulation of embryo development. Network analysis of circRNA-miRNA interactions further supported the idea that circRNAs act as sponges for miRNAs to regulate gene expression. The prediction of differential circRNA binding proteins further explored the potential regulatory pathways of circRNAs. Analysis of SDEcircRNAs suggested a possible reason for the difference in embryo survival between the two breeds at the peri-implantation stage. CONCLUSIONS: Together, these data suggest that circRNAs are abundantly expressed in the endometrium during the peri-implantation period in pigs and are important regulators of related genes. The results of this study will help to further understand the differences in molecular pathways between the two breeds during the critical implantation period of pregnancy, and will help to provide insight into the molecular mechanisms that contribute to the establishment of pregnancy and embryo loss in pigs.

Characterization of Complete Mitochondrial Genome and Phylogenetic Analysis of a Nocturnal Wasps-Provespa barthelemyi (Hymenoptera: Vespidae).

Genus Provespa contains nocturnal wasps mainly found in the southeastern region of Asia. There are no complete genome resources available of this genus, which hinders the study of its phylogenetic evolution and the origin of nocturnal behavior in the Vespidae family. Through high-throughput sequencing, we obtained the mitochondrial genome of Provespa barthelemyi (Hymenoptera: Vespidae), which is 17,721 base pairs in length and contains 13 protein-coding genes (PCGs), 22 tRNAs, and two rRNAs. We identified four gene rearrangement events of P. barthelemyi that frequently occur in the Vespidae family. We used Maximum Likelihood (ML) methodologies to construct a phylogenetic tree based on the sequenced mitochondrial genome and the available data of reported species belonging to Vespinae. Our findings confirmed the monophyly of Vespinae. Our study reports the first complete mitochondrial genome of Provespa and compares its characteristics with other mitochondrial genomes in the family Vespidae. This research should shed light on the phylogenetic relationships and ecological characteristics of the Vespidae family.

The Lnc-RNA APPAT Suppresses Human Aortic Smooth Muscle Cell Proliferation and Migration by Interacting With MiR-647 and FGF5 in Atherosclerosis.

PURPOSE: LncRNA-Atherosclerotic plaque pathogenesis-associated transcript (APPAT) could be detected in circulating blood and has been demonstrated to correlate with the development of atherosclerosis in our previous work. It could be a potential noninvasive biomarker for earlier diagnoses of clinical cardiovascular disease. Moreover, the expression of miR-647 increased in ox-LDL-treated vascular smooth muscle cells and peripheral blood of patients with coronary heart disease. A negative correlation between APPAT and miR-647 was confirmed, and FGF5 was screened as molecular target of miR-647. However, it is largely unclear how APPAT, miR-647, and FGF5 interact and function in disease development. Here, we aim to explore the underlying molecular mechanism in this progression. MATERIALS AND METHODS: APPAT, miR-647, and FGF5 expression levels were detected by quantitative reverse transcription polymerase chain reaction; cell proliferation was detected by EdU incorporation assay; cell migration was detected by wound-healing assay; the molecular interaction of APPAT/FGF5 with miR-647 was verified by dual-luciferase reporter assay; the western blot was performed to determine the gene expression at protein levels; subcellular localizations of APPAT and miR-647 were observed by fluorescence in situ hybridization; cytosolic and nucleus fractionation assay was performed to further detect the distribution of miR-647. RESULTS: APPAT and miR-647 have inverse effects on human aortic smooth muscle cells' (HASMCs) proliferation and migration. APPAT negatively regulated the cell activity, whereas miR-647 did it in a positive way (p<0.05). Three pairs of molecular interplay were found: mutual negative regulation between APPAT and miR-647, APPAT downregulated FGF5, miR-647 regulation on FGF5 (p<0.05). Subcellular location assay confirmed the molecular interaction of APPAT and miR-647. CONCLUSIONS: APPAT could suppress the migration and proliferation of ox-LDL-treated HASMCs via interacting with miR-647 and FGF5. We revealed a nontypical competing endogenous RNA mechanism of long noncoding RNA in the progression of atherosclerosis.

Comprehensive analysis of pre-mRNA alternative splicing regulated by m6A methylation in pig oxidative and glycolytic skeletal muscles.

BACKGROUND: Different types of skeletal myofibers exhibit distinct physiological and metabolic properties that are associated with meat quality traits in livestock. Alternative splicing (AS) of pre-mRNA can generate multiple transcripts from an individual gene by differential selection of splice sites. N6-methyladenosine (m6A) is the most abundant modification in mRNAs, but its regulation for AS in different muscles remains unknown.  RESULTS: We characterized AS events and m6A methylation pattern in pig oxidative and glycolytic muscles. A tota1 of 1294 differential AS events were identified, and differentially spliced genes were significantly enriched in processes related to different phenotypes between oxidative and glycolytic muscles. We constructed the regulatory network between splicing factors and corresponding differential AS events and identified NOVA1 and KHDRBS2 as key splicing factors. AS event was enriched in m6A-modified genes, and the methylation level was positively correlated with the number of AS events in genes. The dynamic change in m6A enrichment was associated with 115 differentially skipping exon (SE-DAS) events within 92 genes involving in various processes, including muscle contraction and myofibril assembly. We obtained 23.4% SE-DAS events (27/115) regulated by METTL3-meditaed m6A and experimentally validated the aberrant splicing of ZNF280D, PHE4DIP, and NEB. The inhibition of m6A methyltransferase METTL3 could induce the conversion of oxidative fiber to glycolytic fiber in PSCs. CONCLUSION: Our study suggested that m6A modification could contribute to significant difference in phenotypes between oxidative and glycolytic muscles by mediating the regulation of AS. These findings would provide novel insights into mechanisms underlying muscle fiber conversion.

Insights into gut microbiota communities of Poecilobdella manillensis, a prevalent Asian medicinal leech.

AIMS: Medicinal leeches (Annelida: Hirudinea) are fresh water ectoparasitic species which have been applied as traditional therapy. However, gut microbiota could bring high risks of opportunistic infections after leeching and arouses great interests. Here, gut bacterial and fungal communities of an Asian prevalent leech Poecilobdella manillensis were characterized and analysed through culture-independent sequencing. METHODS AND RESULTS: With high coverage in 18 samples (>0.999), a more complicated community was apparent after comparing with previous leech studies. A total of 779/939 OTUs of bacteria and fungi were detected from leech guts. The bacterial community was dominated by the phylum Bacteroidetes and Synergistetes. Genera Mucinivorans and Fretibacterium accounted mostly at the genus level, and genus Aeromonas showed an extremely low abundance (2.02%) on average. The fungal community was dominated by the phylum Ascomycota and Basidiomycota. At the genus level, the dominant OTUs included Mortierella, Geminibasidium and Fusarium. The analysis of core taxa included those above dominant genera and some low-abundance genera (>1%). The functional annotation of the bacterial community showed a close correlation with metabolism (34.8 ± 0.6%). Some fungal species were predicted as opportunistic human pathogens including Fusarium and Chaetomiaceae. CONCLUSIONS: The present study provides fundamental rationales for further studies of such issues as bacteria-fungi-host interactions, host fitness, potential pathogens, and infecting risks after leeching. It shall facilitate in-depth explorations on the safe utilization of leech therapy. SIGNIFICANCE AND IMPACT OF STUDY: Present paper is the first-ever exploration on microbiota of a prevalent Asian medicinal leech based on culture-independent technical. And it is also the first report of gut fungi community of medicinal leech. The diversity and composition of bacteria in P. manillensis was far different from that of the European leech. The main components and core OTUs indicate a particular gut environment of medicinal leech. Unknown bacterial and fungal species were also recovered from leech gut.

Global proteomic analysis of serum during early pregnancy in the pig using data-independent acquisition mass spectrometry with verification by parallel reaction monitoring.

CONTEXT: The current pregnancy diagnosis is generally not ideal in accuracy and efficiency, and the physiological process of early pregnancy in pig remains unclarified. AIMS: This study aimed to evaluate protein expression profiles and identify typical proteins of early pregnancy for more understanding of physiological processes. METHODS: Data-independent acquisition-based (DIA) quantitative proteomic analysiswas performed to compare the serum proteome profiles on days 0, 5, 12, 16, and 19 of gestation in Tibetan pig.Parallel reaction monitoring (PRM) was subsequently performed to verify relative expression level. KEY RESULTS: 396 proteins were detected, of which 113 differentially expressed proteins (DEPs) were identified. Functional annotation and pathway analysis indicated that the DEPs were mainly involved in catalytic activity, metabolic processes and the proteasome. Four candidate DEPs (talin 1, profilin, carbonic anhydrase, and HGF activator) showed consistent expression trends in both DIA and PRM approaches. CONCLUSIONS: The DIA based proteomic methods indicate the involvement of numerous serum proteins in early pregnancy physiological function in pigs. The combination of DIA-PRM based global proteomic analysis may provide insights for function study and pregnancy diagnosis biomarkers. IMPLICATIONS: The global proteomic analyses performed here have increased the knowledge of early pregnancy in Tibetan swine and provide potential methods for pregnancy detection.

Differential MicroRNA Expression in Porcine Endometrium Related to Spontaneous Embryo Loss during Early Pregnancy.

Litter size is an important indicator to measure the production capacity of commercial pigs. Spontaneous embryo loss is an essential factor in determining sow litter size. In early pregnancy, spontaneous embryo loss in porcine is as high as 20-30% during embryo implantation. However, the specific molecular mechanism underlying spontaneous embryo loss at the end of embryo implantation remains unknown. Therefore, we comprehensively used small RNA sequencing technology, bioinformatics analysis, and molecular experiments to determine the microRNA (miRNA) expression profile in the healthy and arresting embryo implantation site of porcine endometrium on day of gestation (DG) 28. A total of 464 miRNAs were identified in arresting endometrium (AE) and healthy endometrium (HE), and 139 differentially expressed miRNAs (DEMs) were screened. We combined the mRNA sequencing dataset from the SRA database to predict the target genes of these miRNAs. A quantitative real-time PCR assay identified the expression levels of miRNAs and mRNAs. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed on differentially expressed target genes of DEMs, mainly enriched in epithelial development and amino acids metabolism-related pathways. We performed fluorescence in situ hybridization (FISH) and the dual-luciferase report gene assay to confirm miRNA and predicted target gene binding. miR-205 may inhibit its expression by combining 3'-untranslated regions (3' UTR) of tubulointerstitial nephritis antigen-like 1 (TINAGL1). The resulting inhibition of angiogenesis in the maternal endometrium ultimately leads to the formation of arresting embryos during the implantation period. This study provides a reference for the effect of miRNA on the successful implantation of pig embryos in early gestation.

Ssc-miR-92b-3p Regulates Porcine Trophoblast Cell Proliferation and Migration via the PFKM Gene.

Embryo implantation, the pivotal stage of gestation, is fundamentally dependent on synchronous embryonic development and uterine receptivity. In the early gestation period, the uterus and conceptus secrete growth factors, cytokines, and hormones to promote implantation. Circulating exosomal miRNAs are potential indicators of normal or complicated gestation. Our previous study revealed that pregnant sows' serum exosomes had upregulated miR-92b-3p expression compared to non-pregnant sows, and that the expression level progressively increased during early gestation. The present study's findings indicate that, compared to the ninth day of the estrous cycle (C9), pregnant sows had upregulated miR-92b-3p expression in the endometrium and embryos during the implantation stage ranging from day 9 to day 15 of gestation. Additionally, our results demonstrate that miR-92b-3p promotes the proliferation and migration of Porcine Trophoblast Cells (PTr2). Dual-Luciferase Reporter (DLR) gene assay, real-time fluorescent quantitative PCR (RT-qPCR), and Western blotting (WB) confirmed the bioinformatics prediction that phosphofructokinase-M (PFKM) serves as a target gene of miR-92b-3p. Notably, interference of PFKM gene expression markedly promoted PTr2 proliferation and migration. Furthermore, mice with downregulated uterine miR-92b-3p expression had smaller rates of successful embryo implantation. In summary, miR-92b-3p putatively modulates embryo implantation by promoting PTr2 proliferation and migration via its target gene PFKM.

A meta-analysis of genome-wide association studies for average daily gain and lean meat percentage in two Duroc pig populations.

BACKGROUND: Average daily gain (ADG) and lean meat percentage (LMP) are the main production performance indicators of pigs. Nevertheless, the genetic architecture of ADG and LMP is still elusive. Here, we conducted genome-wide association studies (GWAS) and meta-analysis for ADG and LMP in 3770 American and 2090 Canadian Duroc pigs. RESULTS: In the American Duroc pigs, one novel pleiotropic quantitative trait locus (QTL) on Sus scrofa chromosome 1 (SSC1) was identified to be associated with ADG and LMP, which spans 2.53 Mb (from 159.66 to 162.19 Mb). In the Canadian Duroc pigs, two novel QTLs on SSC1 were detected for LMP, which were situated in 3.86 Mb (from 157.99 to 161.85 Mb) and 555 kb (from 37.63 to 38.19 Mb) regions. The meta-analysis identified ten and 20 additional SNPs for ADG and LMP, respectively. Finally, four genes (PHLPP1, STC1, DYRK1B, and PIK3C2A) were detected to be associated with ADG and/or LMP. Further bioinformatics analysis showed that the candidate genes for ADG are mainly involved in bone growth and development, whereas the candidate genes for LMP mainly participated in adipose tissue and muscle tissue growth and development. CONCLUSIONS: We performed GWAS and meta-analysis for ADG and LMP based on a large sample size consisting of two Duroc pig populations. One pleiotropic QTL that shared a 2.19 Mb haplotype block from 159.66 to 161.85 Mb on SSC1 was found to affect ADG and LMP in the two Duroc pig populations. Furthermore, the combination of single-population and meta-analysis of GWAS improved the efficiency of detecting additional SNPs for the analyzed traits. Our results provide new insights into the genetic architecture of ADG and LMP traits in pigs. Moreover, some significant SNPs associated with ADG and/or LMP in this study may be useful for marker-assisted selection in pig breeding.

Carbon nanotubes as electronic mediators combined with Bi2MoO6and g-C3N4to form Z-scheme heterojunctions to enhance visible light photocatalysis.

In this paper, Z-scheme Bi2MoO6/CNTs/g-C3N4composite photocatalysts were prepared through a simple hydrothermal method. The analysis was performed by XRD, FT-IR, SEM, EDS, TEM, HRTEM, XPS, BET, UV-Vis diffuse reflectance and PL spectrums. Various analyses show that CNTs not only act as excellent charge transfer bridges, but also enable a formation of the Z-scheme of charge transfer mechanism between Bi2MoO6and g-C3N4. This process not only effectively isolates electrons and holes, but also prolongs electron-hole pair lifetimes, resulting in a substantial improvement in the photocatalytic performance of the composite photocatalyst. Best photocatalytic degradation performance was shown by Bi2MoO6/CNTs/g-C3N4composite photocatalyst under simulated sunlight, while the composite photocatalyst still maintained extremely high degradation performance in cycling tests.

Dynamic transcriptome profiling exploring cold tolerance in forensically important blow fly, Aldrichina grahami (Diptera: Calliphoridae).

BACKGROUND: Aldrichina grahami (Diptera: Calliphoridae) is a forensically important fly, which has been widely applied to practical legal investigations. Unlike other necrophagous flies, A. grahami exhibits cold tolerance which helps to maintain its activity during low-temperature months, when other species are usually not active. Hence, A. grahami is considered an important forensic insect especially in cold seasons. In this study, we aim to explore the molecular mechanisms of cold tolerance of A. grahami through transcriptome. RESULTS: We collected eggs and larvae (first-instar, second-instar and third-instar) at three different temperatures (4 °C, 12 °C and 20 °C) and performed RNA-seq analyses. The differentially expressed genes (DEGs) associated with the cold-tolerance were screened out. The Venn analysis of DEGs from egg to third-instar larvae at three different temperatures showed there were 9 common genes. Candidate biological processes and genes were identified which refer to growth, and development of different temperatures, especially the chitin and cuticle metabolic process. The series-clusters showed crucial and unique trends when the temperature changed. Moreover, by comparing the results of growth and developmental transcriptomes from different temperatures, we found that DEGs belonging to the family of larval cuticle proteins (LCP), pupal cuticle protein (CUP), and heat shock proteins (HSP) have certain differences. CONCLUSIONS: This study identified functional genes and showed differences in the expression pattern of diverse temperatures. The DEGs series-clusters with increasing or decreasing trends were analyzed which may play an important role in cold-tolerance. Moreover, the findings in LCP, CUP and HSP showed more possible modulations in a cold environment. This work will provide valuable information for the future investigation of the molecular mechanism of cold tolerance in A. grahami.

Population Genomics Provide Insights into the Evolution and Adaptation of the Eastern Honey Bee (Apis cerana).

The mechanisms by which organisms adapt to variable environments are a fundamental question in evolutionary biology and are important to protect important species in response to a changing climate. An interesting candidate to study this question is the honey bee Apis cerana, a keystone pollinator with a wide distribution throughout a large variety of climates, that exhibits rapid dispersal. Here, we resequenced the genome of 180 A. cerana individuals from 18 populations throughout China. Using a population genomics approach, we observed considerable genetic variation in A. cerana. Patterns of genetic differentiation indicate high divergence at the subspecies level, and physical barriers rather than distance are the driving force for population divergence. Estimations of divergence time suggested that the main branches diverged between 300 and 500 Ka. Analyses of the population history revealed a substantial influence of the Earth's climate on the effective population size of A. cerana, as increased population sizes were observed during warmer periods. Further analyses identified candidate genes under natural selection that are potentially related to honey bee cognition, temperature adaptation, and olfactory. Based on our results, A. cerana may have great potential in response to climate change. Our study provides fundamental knowledge of the evolution and adaptation of A. cerana.

Study on tribological mechanism for multi-layer porous structure of diatom frustule.

Tribological mechanism of the diatom frustule with multi-layers of pores is studied with the liquid-solid interaction (FSI) method. Based on the reconstructed representative Coscinodiscus sp. frustule with two-layer porous structure, the tribological performances for the diatom frustule at its different pore diameter ratios, pore depth ratios, and velocities are solved through governing equations involved with FSI method. The numerical result shows that the existence of the two-layer porous structure of the diatom helps to reduce the friction between it and ambient water, and to increase its ability to resist the ambient water pressure. The two-layer porous structure effectively improve the tribological performances for the diatom frustule due to the change in the frustule velocity.

Generation of transgenic pigs by cytoplasmic injection of piggyBac transposase-based pmGENIE-3 plasmids.

The process of transgenesis involves the introduction of a foreign gene, the transgene, into the genome of an animal. Gene transfer by pronuclear microinjection (PNI) is the predominant method used to produce transgenic animals. However, this technique does not always result in germline transgenic offspring and has a low success rate for livestock. Alternate approaches, such as somatic cell nuclear transfer using transgenic fibroblasts, do not show an increase in efficiency compared to PNI, while viral-based transgenesis is hampered by issues regarding transgene size and biosafety considerations. We have recently described highly successful transgenesis experiments with mice using a piggyBac transposase-based vector, pmhyGENIE-3. This construct, a single and self-inactivating plasmid, contains all the transpositional elements necessary for successful gene transfer. In this series of experiments, our laboratories have implemented cytoplasmic injection (CTI) of pmGENIE-3 for transgene delivery into in vivo-fertilized pig zygotes. More than 8.00% of the injected embryos developed into transgenic animals containing monogenic and often single transgenes in their genome. However, the CTI technique was unsuccessful during the injection of in vitro-fertilized pig zygotes. In summary, here we have described a method that is not only easy to implement, but also demonstrated the highest efficiency rate for nonviral livestock transgenesis.

Multienzymatic disintegration of DNA-scaffolded magnetic nanoparticle assembly for malarial mitochondrial DNA detection.

Early diagnosis of malaria can prevent the spread of disease and save lives, which, however, remains challenging in remote and less developed regions. Here we report a portable and low-cost optomagnetic biosensor for rapid amplification and detection of malarial mitochondrial DNA. Bioresponsive magnetic nanoparticle assemblies are constructed by using nucleic acid scaffolds containing endonucleolytic DNAzymes and their substrates, which can be activated by the presence of target DNA and self-disintegrated to release magnetic nanoparticles for optomagnetic quantification. Specifically, target molecules can induce padlock probe ligation and subsequent one-pot homogeneous cascade reactions consisting of nicking-enhanced rolling circle amplification, DNAzyme-assisted nucleic acid recycling, and strand-displacement-driven disintegration of the magnetic assembly. With an optimized magnetic actuation process for reaction acceleration, a detection limit of 1 fM can be achieved by the proposed biosensor with a total assay time of ca. 90 min and a dynamic detection range spanning 3 orders of magnitude. The robustness of the system was validated by testing target molecules spiked in 5% serum samples. Clinical sample validation was conducted by testing malaria-positive clinical blood specimens, obtaining quantitative results concordant with qPCR measurements.

Intrapuparial stage aging and PMI estimation based on the developmental transcriptomes of forensically important Aldrichina grahami (Diptera: Calliphoridae) gene expression.

Background: The expression profiles of differentially expressed genes (DEGs) during pupal development have been demonstrated to be vital in age estimation of forensic entomological study. Here, using forensically important Aldrichina grahami (Diptera: Calliphoridae), we aimed to explore the potential of intrapuparial stage aging and postmortem interval (PMI) estimation based on characterization of successive developmental transcriptomes and gene expression patterns. Methods: We collected A. grahami pupae at 11 successive intrapuparial stages at 20 °C and used the RNA-seq technique to build the transcriptome profiles of their intrapuparial stages. The DEGs were identified during the different intrapuparial stages using comparative transcriptome analysis. The selected marker DEGs were classified and clustered for intrapuparial stage aging and PMI estimation and then further verified for transcriptome data validation. Ultimately, we categorized the overall gene expression levels as the dependent variable and the age of intrapuparial A. grahami as the independent variable to conduct nonlinear regression analysis. Results: We redefined the intrapuparial stages of A. grahami into five key successive substages (I, II, III, IV, and V), based on the overall gene expression patterns during pupal development. We screened 99 specific time-dependent expressed genes (stage-specific DEGs) to determine the different intrapuparial stages based on comparison of the gene expression levels during the 11 developmental intrapuparial stages of A. grahami. We observed that 55 DEGs showed persistent upregulation during the development of intrapuparial A. grahami. We then selected four DEGs (act79b, act88f, up and ninac) which presented consistent upregulation using RT-qPCR (quantitative real-time PCR) analysis, along with consideration of the maximum fold changes during the pupal development. We conducted nonlinear regression analysis to simulate the calculations of the relationships between the expression levels of the four selected DEGs and the developmental time of intrapuparial A. grahami and constructed fitting curves. The curves demonstrated that act79b and ninac showed continuous relatively increasing levels. Conclusions: This study redefined the intrapuparial stages of A. grahami based on expression profiles of developmental transcriptomes for the first time. The stage-specific DEGs and those with consistent tendencies of expression were found to have potential in age estimation of intrapuparial A. grahami and could be supplementary to a more accurate prediction of PMI.

HOFE: an interactive forensic entomological database.

The significance of entomological evidence in inferring the time, location and cause of death has been demonstrated both theoretically and practically. With the advancement of sequencing technologies, reports have emerged on necrophagous insects' nuclear genomes, transcriptomes, proteomes and mitochondrial genomes. However, within the field of forensic entomology, there is currently no available database that can integrate, store and share the resources of necrophagous insects. The absence of a database poses an inconvenience to the application of entomological evidence in judicial practice and hampers the development of the forensic entomology discipline. Given this, we have developed the Home Of Forensic Entomology database, encompassing 10 core functional modules: Home, Browse, Mitochondria, Proteome, JBrowse, Search, BLAST, Tools, Case base and Maps. Notably, the 'Tools' module enables multiple sequence alignment analysis (Muscle), homologous protein prediction (Genewise), primer design (Primer), large-scale genomic analysis (Lastz), Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis, as well as expression profiling (PCA Analysis, Hcluster and Correlation Heatmap). In addition, the present database also works as an interactive platform for researchers by sharing forensic entomological case reports and uploading data and material. This database provides potential visitors with a comprehensive function for multi-omics data analysis, offers substantial references to researchers and criminal scene investigators and facilitates the utilization of entomological evidence in court. Database URL: http://ihofe.com/.