Intestinal epithelial cells of Japanese flounder (Paralichthys olivaceus) as an in vitro model for studying intestine immune function based on transcriptome analysis.

Japanese flounder (Paralichthys olivaceus) is an economically crucial marine species, but diseases like hemorrhagic septicemia caused by Edwardsiella tarda have resulted in significant economic losses. E. tarda infects various hosts, and its pathogenicity in fish is not fully understood. Lipopolysaccharides (LPS) are components of the outer membrane of Gram-negative bacteria and are representative of typical PAMP molecules that cause activation of the immune system. The PoIEC cell line is a newly established intestinal epithelial cell line from P. olivaceus. In order to investigate whether it can be used as an in vitro model for studying the pathogenesis of E. tarda and LPS stimulation, we conducted RNA-seq experiments for the PoIECs model of E. tarda infection and LPS stimulation. In this study, transcriptome sequencing was carried out in the PoIEC cell line after treatment with LPS and E. tarda. A total of 62.52G of high-quality data from transcriptome sequencing results were obtained in nine libraries, of which an average of 87.96% data could be aligned to the P. olivaceus genome. Data analysis showed that 283 and 414 differentially expressed genes (DEGs) in the LPS versus Control (LPS-vs-Con) and E. tarda versus Control groups (Et-vs-Con), respectively, of which 60 DEGs were shared in two comparation groups. The GO terms were predominantly enriched in the extracellular space, inflammatory response, and cytokine activity in the LPS-vs-Con group, whereas GO terms were predominantly enriched in nucleus and positive regulation of transcription by RNA polymerase II in the Et-vs-Con group. KEGG analysis revealed that three immune-related pathways were co-enriched in both comparison groups, including the Toll-like receptor signaling pathway, C-type lectin receptor signaling pathway, and Cytokine-cytokine receptor interaction. Five genes were randomly screened to confirm the validity and accuracy of the transcriptome data. These results suggest that PoIEC cell line can be an ideal in vitro model for studies of marine fish gut immunity and pathogenesis of Edwardsiellosis.

Design and fabrication of a differential-pressure optical fiber grating sensor for monitoring the flow rate of fluid.

A fiber Bragg grating (FBG) flow sensor is designed and fabricated, in which two FBGs are fixed on the front and other side of the metal diaphragm, and differential pressure is used to monitor the flow rate of fluid. The temperature sensitivity of these two FBGs is 0.030 and 0.029 nm/°C, which is almost the same, suggesting that the influence of temperature on the flow measurement can be effectively eliminated. The static pressure sensitivity of these two FBGs can be up to 86.7 nm/MPa and 68.6 nm/MPa, respectively; accordingly, the static pressure sensitivity of the sensor overall is 155.3 nm/MPa. Furthermore, the flow rate sensitivity is 0.00029 L/s. This FBG flow sensor exhibits high sensitivity, high accuracy, and a low start-up flow rate. Furthermore, the cross effect between the temperature and strain on the sensing sensitivity is eliminated, which makes this FBG flow sensor suitable for real-time monitoring of the trace flow rate in oil and gas wells.

Oxidant-Free Electrochemical Direct Oxidative Benzyl Alcohols to Benzyl Aldehydes Using Three-Dimensional Printing PPAR Polyoxometalate.

The oxidation of benzyl alcohols is an important reaction in organic synthesis. Traditional methods for benzyl alcohol oxidation have not been widely utilized due to the use of significant amounts of precious metals and environmentally unfriendly reagents. In recent years, electrocatalytic oxidation has gained significant attention, particularly electrochemical anodic oxidation, which offers a sustainable alternative for oxidation without the need for external oxidants or reducing agents. Here, a copper monosubstituted phosphotungstate-based polyacrylate resins (Cu-LPOMs@PPAR) catalyst has been fabricated with immobilization and recyclability using 3D printing technology that can be successfully applied in the electrocatalytic oxidation of benzyl alcohol to benzaldehyde, achieving atom economy and reducing pollution. In this protocol, we obtain benzaldehyde in good yields with excellent functional group toleration under metal-free and oxidant-free conditions. This strategy could provide a new avenue for heterogeneous catalysts in application for enhancing the efficiency and selectivity of electrocatalytic oxidation processes.

Development and validation of a forensic multiplex InDel assay: The AGCU InDel 60 kit.

Marker sets based on insertion/deletion polymorphisms (InDels) combine the characteristics of both short tandem repeats (STRs) and single nucleotide polymorphisms and have served as effective complementary or stand-alone systems for human identification in forensics. We developed a novel multiplex amplification detection system, designated the AGCU InDel 60 kit, containing 57 autosomal InDels, 2 Y-chromosomal InDels, and the amelogenin locus and validated the kit in a series of studies, which included tests of the PCR conditions; tests for sensitivity, species specificity, reproducibility, stability, and mock case samples; degradation studies; and a population study. The results indicated that the AGCU InDel 60 kit was accurate, specific, reproducible, stable, and robust. Complete DNA profiles were obtained even with 125 pg of human DNA. In tests of artificially degraded samples, we found that the number of alleles detected by the validated kit was considerably greater than that detected by the STR-based AGCU 21+1 kit, even as the degree of degradation increased. Additionally, 564 unrelated individuals from three Han groups were investigated using this novel system, and the values of combined power of discrimination and combined power of exclusion were not less than 1-4.9026 × 10-24 and 1-3.1123 × 10-5 , respectively. Thus, the results indicated that the novel kit was more powerful than the previous version of the InDel kit (the AGCU InDel 50 kit). Our results suggest that the AGCU InDel 60 kit can serve as an efficient tool for human forensics and a supplementary kit for population genetics research.

A smartphone-adaptable fluorescent sensing tag for non-contact and visual monitoring of the freshness of fish.

Fish-based food products play important roles in our daily diet. The related food safety is vitally essential for human health, thus it is very necessary to screen the freshness of fish-based foods. In this work, we presented a ratiometric fluorescent probe PTCN for the determination of cadaverine, a metabolic biomarker of the spoilage of fish. PTCN displayed a ratiometric fluorescence response towards cadaverine with good specificity, high sensitivity (LOD = 46 nM) and ultra-fast response (<15 s), and thus has been successfully utilized to determine cadaverine from the spoilage of fish. PTCN was fabricated into cheap and portable sensing tags, which can visually detect gaseous cadaverine with obvious fluorescence color transformation from red to green and a low detection limit (8.65 ppm). Moreover, the PTCN tags were used as smart fluorescent tags for non-contact and visual monitoring of cadaverine in fish. Furthermore, the ratiometric fluorescence signals were utilized to create a smartphone-adaptable digital sensing profile for indicating cadaverine in fish products.

Versatile Superatom Complex Nanocluster for the Construction of Framework Materials.

Accurately controlling the assembly of nanometer-sized building blocks presents an important but significant challenge for the construction of functional framework materials, which requires the development of highly stable versatile nanosized assembly modules with multiple coordination sites. In this study, [Ag23(SAdm)12]3+ (Ag23, in which SAdm = 1-adamantanethiol, i.e., C10H15S), a chiral superatom complex nanocluster, was synthesized and assembled into various topologies. We constructed two kinds of framework materials, i.e., superatom complex inorganic framework (SCIF) and superatom complex organic framework (SCOF) materials, including [Ag23(SAdm)12](SbF6)2X (Ag23-1; X = Cl-/SbF6-, a SCIF), [Ag23(SAdm)12](SbF6)3 (Ag23-2, a SCIF), [Ag23(SAdm)12](SbF6)3(bpy)3 (Ag23-bpy, a SCOF, in which bpy = 4,4'-bipyridine, i.e., C10H8N2), and [Ag23(SAdm)12](SbF6)3(dpbz)3 (Ag23-dpbz, a SCOF, in which dpbz = 1,4-bis(4-pyridyl)benzene, i.e., C16H12N2), owing to strong interactions between the versatile Ag23 and the inorganic and organic linkers. Ag23-1, Ag23-2, and Ag23-bpy exhibit two superstructures with interpenetrating frameworks and adamantane-like, hexagonal, and cubic topologies, while Ag23-dpbz displays three superstructures with interpenetrating frameworks and cubic topologies. Ag23-dpbz exhibits the largest specific surface area as well as the strongest photoluminescence and electrochemiluminescence signals owing to its dense network arrangement. This work contributes to the construction of nanocluster-based framework materials and helps to elucidate the effect of the assembly mode on the material properties and functionalities.

Applying resonance Rayleigh scattering and spectrofluorimetric techniques for the selective determination of neomycin sulfate using Congo red as a probe: Applications for wastewater analysis.

Two simple, sensitive, and low-cost fluorescence spectroscopy methods for neomycin (NEO) detection were developed. Both methods were based on the interaction between NEO and Congo red (CR) in acidic buffer medium to form an ion-association complex. The quenching effect of the formed ion-association complex on the fluorescence of CR at 421 nm is a basic principle of fluorescence analysis, whilst the resonance Rayleigh scattering (RRS) method was used to enhance the resonance Rayleigh scattering spectrum at 384 nm by adding NEO. Experimental conditions such as pH, temperature, reaction time, CR concentration, and the ionic strength of the two methods were investigated and optimized. In addition, the effect of common coexisting substances on the method was tested and the results showed good selectivity. The composition of ion-association complexes, the reaction mechanism, and reasons for the enhanced intensity of RRS are discussed. Under optimum conditions, the responses of the fluorescence spectrophotometry and RRS methods showed good linearity with NEO concentrations in the range 0.2-3.0 µg ml-1 and 0.1-3.0 µg ml-1 , respectively. The detection limits of fluorescence spectrophotometry and RRS spectroscopy techniques were 0.02 µg ml-1 and 0.01 µg ml-1 , respectively. Finally, the two methods were applied to the analysis of wastewater and the results were satisfactory.

Prevalence and Factors Associated with Post-Endoscopic Retrograde Cholangiopancreatography Pancreatitis in Children.

BACKGROUND: Pancreatitis is the most common complication of endoscopic retrograde cholangiopancreatography (ERCP). AIM: To assess the prevalence and factors associated with post-ERCP pancreatitis (PEP) in a Chinese pediatric population. METHODS: Sixty-six children who underwent ERCP between March 2018 and March 2019 at Shanghai Children's Medical Center were retrospectively recruited for the study. Clinical data, including demographics, indications, comorbidities, and procedural data, were reviewed to identify the prevalence and factors associated with PEP. RESULTS: Ninety-two ERCPs were performed on 66 pediatric patients aged from 8 months to 14 years. The indications for ERCP were chronic pancreatitis (49, 53.2%), pancreaticobiliary maljunction (19, 20.7%), pancreas divisum (19, 20.7%), and pancreatic pseudocyst (5, 5.4%). All ERCPs were performed for therapeutic purposes. PEP was identified in 19 (20.7%) patients; there were ten mild cases, eight moderate cases, and one severe case. The univariate analysis revealed that a history of chronic pancreatitis was negatively associated with PEP (P = 0.033), and sphincterotomy was positively associated with PEP (P = 0.01). The multivariate analysis showed that sphincterotomy was a risk factor for PEP (P = 0.017, OR 4.17; 95% CI, 1.29, 13.54). CONCLUSIONS: Our data revealed a high prevalence of PEP in a Chinese pediatric population. Chronic pancreatitis was a protective factor, and sphincterotomy was a risk factor for PEP development.

A Single Fluorescent Chemosensor for Simultaneous Discriminative Detection of Gaseous Phosgene and a Nerve Agent Mimic.

A fluorescent chemosensor has been developed for discriminative detection of phosgene and a nerve agent mimic diethyl chlorophosphate (DCP), which was comprised of an anthracene-carboxyimide fluorophore and o-phenylenediamine (OPD) reaction site. Upon phosphorylation of OPD, the chemosensor displays an obvious fluorescence turn-on response toward DPC at 588 nm with instant response and a low detection limit (88 nM). By contrast, the chemosensor exhibits a colorimetric and fluorescence enhancement response at 500 nm toward phosgene with fast response (<2 min), high selectivity, and a low detection limit (72 nM). Furthermore, chemosensor-loaded test membrane was fabricated for real-time, portable and efficient discriminative detection of trace amounts of gaseous phosgene and DCP vapor with different optical responses.

6-gingerol ameliorates age-related hepatic steatosis: Association with regulating lipogenesis, fatty acid oxidation, oxidative stress and mitochondrial dysfunction.

The prevalence of NAFLD increases with age. As the main active ingredient of ginger, 6-gingerol significantly improves lipid metabolism abnormalities in adult rodents. However, few studies have reported its effect on age-related NAFLD. This study was to investigate the effects of 6-gingerol on age-related hepatic steatosis and its potential targets. As expected, 6-gingerol dramatically normalized the hepatic triglyceride content, plasma insulin and HOMA-IR index of ageing rats. Mechanistically, 6-gingerol affected lipid metabolism by increasing ß-oxidation and decreasing lipogenesis through activation of PPARα and CPT1α and inhibition of DGAT-2. Furthermore, 6-gingerol reversed the decreases in citrate, Cs and ATP, lessened the damage caused by ROS, and upregulated mitochondrial marker enzymes NOX, SDH, and SIRT3 in the ageing liver, indicating its ability to strengthen mitochondrial function. Our results showed 6-gingerol exerted a positive effect on insulin sensitivity by regulating Akt. In conclusion, the hepatic anti-steatotic effect of 6-gingerol is associated with inhibition of de novo lipogenesis, upregulation of fatty acid oxidation, reduction in oxidative stress and synergistic enhancement of mitochondrial function.

Exploratory laparoscopy combined with pathological examination in the diagnosis of obscure gastrointestinal bleeding in a child: a case report.

BACKGROUND: The diagnosis of obscure gastrointestinal bleeding (OGIB) which is defined as bleeding of unknown origin of the small bowel by routine evaluation in childhood is a challenge. CASE PRESENTATION: Here we report a one-year-old Chinese girl who was suspected with idiopathic pulmonary haemosiderosis (IPH) and referred to our department for further diagnosis. Finally she was diagnosed with vascular malformations (VM) by exploratory laparoscopy combined with pathological examination. CONCLUSIONS: Children OGIB could be easily misdiagnosed in the beginning, and OGIB children with active ongoing bleeding may benefit from proceeding directly to exploratory laparoscopy, followed by pathological confirmation of the diagnosis.

Megakaryocytic Smad4 Regulates Platelet Function through Syk and ROCK2 Expression.

Smad4, a key transcription factor in the transforming growth factor-ß signaling pathway, is involved in a variety of cell physiologic and pathologic processes. Here, we characterized megakaryocyte/platelet-specific Smad4 deficiency in mice to elucidate its effect on platelet function. We found that megakaryocyte/platelet-specific loss of Smad4 caused mild thrombocytopenia and significantly extended first occlusion time and tail bleeding time in mice. Smad4-deficient platelets showed reduced agonist-induced platelet aggregation. Further studies showed that a severe defect was seen in integrin αIIbß3-mediated bidirectional (inside-out and outside-in) signaling in Smad4-deficient platelets, as evidenced by reduced fibrinogen binding and α-granule secretion, suppressed platelet spreading and clot retraction. Microarray analysis showed that the expression levels of multiple genes were altered in Smad4-deficient platelets. Among these genes, spleen tyrosine kinase (Syk) and Rho-associated coiled-coil containing protein kinase 2 (ROCK2) were downregulated several times as confirmed by quantitative reverse-transcription polymerase chain reaction and immunoblotting. Further research showed that Smad4 directly regulates ROCK2 transcription but indirectly regulates Syk. Megakaryocyte/platelet-specific Smad4 deficiency caused decreased expression levels of Syk and ROCK2 in platelets. These results suggest potential links among Smad4 deficiency, attenuated Syk, and ROCK2 expression and defective platelet activation.

Response of genes involved in lipid metabolism in rat epididymal white adipose tissue to different fasting conditions after long-term fructose consumption.

There has been much concern regarding the dietary fructose contributes to the development of metabolic syndrome. High-fructose diet changes the expression of genes involved in lipid metabolism. Levels of a number of hepatic lipogenic enzymes are increased by a high-carbohydrate diet in fasted-refed model rats/mice. Both the white adipose tissue (WAT) and the liver play a key role in the maintenance of nutrient homeostasis. Here, the aim of this study was to analyze the expression of key genes related to lipid metabolism in epididymal WAT (eWAT) in response to different fasting condition after long-term chronic fructose consumption. Rats were fed standard chow supplemented with 10% w/v fructose solution for 5 weeks, and killed after chow-fasting and fructose withdrawal (fasting) or chow-fasting and continued fructose (fructose alone) for 14 h. Blood parameters and the expression of genes involved in fatty acid synthesis (ChREBP, SREBP-1c, FAS, SCD1), triglyceride biosynthesis (DGAT-1, DGAT-2) and lipid mobilization (ATGL, HSL) in eWAT were analyzed. In addition, mRNA levels of PPAR-γ, CD36 and LPL were also detected. As expected, fructose alone increased the mRNA expression of FAS, SCD1, and correspondingly decreased ATGL and HSL mRNA levels. However, ChREBP, DGAT-2, ATGL and HSL mRNA levels restored near to normal while FAS and SCD1 tend to basic level under fasting condition. The mRNA expression of SREBP-1c, PPAR-γ and LPL did not changed at any situations but CD36 mRNA decreased remarkably in fructose alone group. In conclusion, these findings demonstrate that genes involved in lipid metabolism in rat eWAT are varied in response to different fasting conditions after long-term fructose consumption.

Long-term fructose consumption prolongs hepatic stearoyl-CoA desaturase 1 activity independent of upstream regulation in rats.

Dietary fructose is considered a risk factor for metabolic disorders, such as fatty liver disease. However, the mechanism underlying the effects of fructose is not well characterized. We investigated the hepatic expression of key regulatory genes related to lipid metabolism following fructose feeding under well-defined conditions. Rats were fed standard chow supplemented with 10% w/v fructose solution for 5 weeks, and killed after chow-fasting and fructose withdrawal (fasting) or chow-fasting and continued fructose (fructose alone) for 14 h. Hepatic deposition of triglycerides was found in rats from both groups. As expected, fructose alone increased mRNA levels of lipogenesis-related genes and correspondingly decreased mRNA levels of lipid oxidative genes in the liver. Interesting, hepatic levels of stearoyl-CoA desaturase (SCD)1 mRNA remained elevated under fructose withdrawn conditions, although expression levels of other genes, including two key transcription factors (carbohydrate response element binding protein (ChREBP) and sterol regulatory element-binding protein (SREBP)-1c) fell to normal levels, indicating that long-term fructose intake increased SCD1 activity, independent of upstream regulatory genes, such as ChREBP and SREBP-1c. In conclusion, SCD1 overexpression in fatty liver disease is not affected by fasting after long-term fructose consumption in rats. Regulation of SCD1 plays an important role in fructose-induced hepatic steatosis.

A practical chromogenic and fluorogenic dual-mode sensing platform for rapid quantification of sulfite in food.

Sulfur dioxide (SO2) and its derivatives (HSO3- and SO32-) are widely used in food-processing. Whereas excessive consumption of sulfur dioxide and its derivatives (>0.70 mg·kg-1day-1) severely endangers human health. In this work, we rationally constructed a practical dual-mode probe (dicyanomethylene)-1-methyl-1,4-dihydroquinolin-2-yl)vinyl)-1-methylquinolinium (QMN), which underwent a specific 1, 4-Michael addition with sulfite to afford a noticeable color change from pale yellow to red along with a high-contrast fluorescence turn-on response at 598 nm. QMN has the advantages of rapid response, high signal-to-noise ratio, excellent selectivity, good water-solubility, large Stokes shift and low detection limit (LOD = 31.9 nM). QMN has been successfully used to on-site visually determine sulfite in a diversity of foods with satisfactory recoveries (91.33-111.33 %) and high accuracy (93.74-98.71 %). Furthermore, a portable smartphone-based fluorescence sensing platform was fabricated for on-site determination of sulfite in food with good performance.

A highly sensitive fluorescence probe for on-site detection of nerve agent mimic diethylchlorophosphonate DCP.

Nerve agents are the most toxic chemical warfare agents that pose severe threat to human health and public security. In this work, we developed a novel fluorescent probe NZNN based on naphthylimide and o-phenylenediamine to detect nerve agent mimic diethylchlorophosphonate (DCP). DCP underwent a specific nucleophilic reaction with the o-phenylenediamine group of NZNN to produce a significant fluorescence turn-on response with high selectivity, exceptional linearity, bright fluorescence, rapid response (<6 s) and a low detection limit (30.1 nM). Furthermore, a portable sensing device was fabricated for real-time detection of DCP vapor with excellent performance. This portable and sensitive device is favorable for monitoring environmental pollution and defense against chemical warfare agents.

Forensic characteristics and genetic structure of the Chinese Tibetan population revealed by 38 X-chromosomal InDel loci.

With the increasing importance of X-chromosome (Chr-X) genotyping in kinship identification, the exploitation of X chromosome genetic marker multiplex kits is increasing. The Human X-InDels amplification kit is a novel developed system which contained 38 X-chromosomal Insertion/deletion markers (X-InDels) and Amelogenin. Herein, we investigated the genetic diversity of the 38 X-InDels in the Tibetan ethnic minority (n = 792) from seven regions and evaluated the application potential of this novel panel. The rs16368 was the least variable locus, whereas the most polymorphic locus was the rs59605609 in Tibetan population. We confirmed three linkage groups with the haplotype diversities ranged from 0.5032 to 0.5976. The overall combined power of discrimination (PD) in males and females were 0.999999999582066 and 0.999999999999993, respectively. And the overall combined mean exclusion chance (MEC) values were not lower than 0.999125526990159. In addition, we explored the genetic relationships among the Tibetans in seven different regions via series of population comparison analyses, finding that the genetic relationship between the Ngari Tibetan and Chamdo Tibetan was the farthest, which was consistent with geographical distribution.

A proof-of-principle study: The potential application of MiniHap biomarkers in ancestry inference based on the QNome nanopore sequencing.

Haplotyped SNPs convey forensic-related information, and microhaplotypes (MHs), as the most representative of this kind of marker, have proved the potential value for human forensics. In recent years, nanopore sequencing technology has developed rapidly, with its outstanding ability to sequence long continuous DNA fragments and obtain phase information, making the detection of longer haplotype marker possible. In this proof-of-principle study, we proposed a new type of forensic marker, MiniHap, based on five or more SNPs within a molecular distance less than 800 bp, and investigated the haplotype data of 56 selected MiniHaps in five Chinese populations using the QNome nanopore sequencing. The sequencing performance, allele (haplotype) frequencies, forensic parameters, effective number of alleles (Ae), and informativeness (In) were subsequently calculated. In addition, we performed principal component analysis (PCA), phylogenetic tree, and structure analysis to investigate the population genetic relationships and ancestry components among the five investigated populations and 26 worldwide populations. MiniHap-04 exhibited remarkable forensic efficacy, with 148 haplotypes reported and the Ae was 66.9268. In addition, the power of discrimination (PD) was 0.9934, the probability of exclusion (PE) was 0.9898, and the In value was 0.7893. Of the 56 loci, 85.71% had PD values above 0.85, 66.07% had PE values above 0.54, 67.86% had Ae values over 7.0%, and 55.36% were with In values above 0.2 across all samples, indicating that most of the MiniHaps are suitable for individual identification, paternity testing, mixture deconvolution, and ancestry inference. Moreover, the results of PCA, phylogenetic tree and structure analysis demonstrated that this MiniHap panel had the competency in continental population ancestry inference, but the differentiation within intracontinental/linguistically restricted subpopulations was not ideal. Such findings suggested that the QNome device for MiniHap detection was feasible and this novel marker has the potential in ancestry inference. Yet, the establishment of a more comprehensive database with sufficient reference population data remains necessary to screen more suitable MiniHaps.

Ligand-protected metal nanoclusters as low-loss, highly polarized emitters for optical waveguides.

Photoluminescent molecules and nanomaterials have potential applications as active waveguides, but such a use has often been limited by high optical losses and complex fabrication processes. We explored ligand-protected metal nanoclusters (LPMNCs), which can have strong, stable, and tunable emission, as waveguides. Two alloy LPMNCs, Pt1Ag18 and AuxAg19-x (7 ≤ x ≤ 9), were synthesized and structurally determined. Crystals of both exhibited excellent optical waveguide performance, with optical loss coefficients of 5.26 × 10-3 and 7.77 × 10-3 decibels per micrometer, respectively, lower than those demonstrated by most inorganic, organic, and hybrid materials. The crystal packing and molecular orientation of the Pt1Ag18 compound led to an extremely high polarization ratio of 0.91. Aggregation enhanced the quantum yields of Pt1Ag18 and AuxAg19-x LPMNCs by 115- and 1.5-fold, respectively. This photonic cluster with low loss and high polarization provides a generalizable and versatile platform for active waveguides and polarizable materials.

CmVCall: An automated and adjustable nanopore analysis pipeline for heteroplasmy detection of the control region in human mitochondrial genome.

Genetic associations between human mitochondrial DNA (mtDNA) heteroplasmy and mitochondrial diseases, aging, and cancer have been elaborated, contributing a lot to the further understanding of mtDNA polymorphic spectrum in anthropology, population, and forensic genetics. In the past decade, heteroplasmy detection using Sanger sequencing and next generation sequencing (NGS) was hampered by the former's inefficiency and the latter's inherent bias due to amplification and mapping of short reads, respectively. Nanopore sequencing stands out for its ability to yield long contiguous segments of DNA, providing a new insight into heterogeneity authentication. In addition to MinION from Oxford Nanopore Technologies, an alternative nanopore sequencer QNome (Qitan Technology) has also been applied to various biological research and the forensic applicability of this platform has been proved recently. In this study, we evaluated the performance of four commonly used variant callers in the heterogeneity authentication of the control region of human mtDNA based on simulations of different ratios generated by mixing QNome nanopore sequencing reads of two synthetic sequences. Then, an open-source and python-based nanopore analytics pipeline, CmVCall was developed and incorporated multiple programs including reads filtering, removal of nuclear mitochondrial sequences (NUMTs), alignment, optional 'Correction' mode, and heterogeneity identification. CmVCall can achieve high precision, accuracy, and recall of 100%, 99.9%, and 92.3% with a 5% heteroplasmy level in 'Correction' mode. Moreover, blood, saliva, and hair shaft samples from monozygotic (MZ) twins were used for heterogeneity evaluation and comparison with the NGS data. Results of MZ twin samples showed that CmVCall could identify more point heteroplasmy sites, revealing significant levels of inter- and intra-individual mtDNA polymorphism. In conclusion, we believe that this analysis pipeline will lay a solid foundation for the development of a comprehensive nanopore analysis pipeline targeting the whole mitochondrial genome.