CoRAL accurately resolves extrachromosomal DNA genome structures with long-read sequencing.

Extrachromosomal DNA (ecDNA) is a central mechanism for focal oncogene amplification in cancer, occurring in ∼15% of early-stage cancers and ∼30% of late-stage cancers. ecDNAs drive tumor formation, evolution, and drug resistance by dynamically modulating oncogene copy number and rewiring gene-regulatory networks. Elucidating the genomic architecture of ecDNA amplifications is critical for understanding tumor pathology and developing more effective therapies. Paired-end short-read (Illumina) sequencing and mapping have been utilized to represent ecDNA amplifications using a breakpoint graph, in which the inferred architecture of ecDNA is encoded as a cycle in the graph. Traversals of breakpoint graphs have been used to successfully predict ecDNA presence in cancer samples. However, short-read technologies are intrinsically limited in the identification of breakpoints, phasing together complex rearrangements and internal duplications, and deconvolution of cell-to-cell heterogeneity of ecDNA structures. Long-read technologies, such as from Oxford Nanopore Technologies, have the potential to improve inference as the longer reads are better at mapping structural variants and are more likely to span rearranged or duplicated regions. Here, we propose Complete Reconstruction of Amplifications with Long reads (CoRAL) for reconstructing ecDNA architectures using long-read data. CoRAL reconstructs likely cyclic architectures using quadratic programming that simultaneously optimizes parsimony of reconstruction, explained copy number, and consistency of long-read mapping. CoRAL substantially improves reconstructions in extensive simulations and 10 data sets from previously characterized cell lines compared with previous short- and long-read-based tools. As long-read usage becomes widespread, we anticipate that CoRAL will be a valuable tool for profiling the landscape and evolution of focal amplifications in tumors.

Dietary calcium intake and the risk of stroke: Meta-analysis of cohort studies.

BACKGROUND AND AIMS: Prospective cohorts are inconsistent regarding the association between dietary calcium intake and the risk of stroke. The aim was to perform a meta-analysis to determine whether an association exists between them in cohort studies. METHODS AND RESULTS: Relevant studies were identified by searching PubMed, EMBASE and Web of Science databases that published before December 2022. Prospective cohort studies that provided relative risk (RR) estimates with 95% confidence intervals (CIs) for the association were included. Study-specific risk estimates were combined by using a random effects model. Eighteen prospective studies, including 19,557 stroke cases among 882,181 participants, were pooled in the meta-analysis. We observed a nonlinear association between calcium intake and risk of stroke (Pnonlinearity < 0.003). Compared with the lowest value of zero assumed as the reference, the RRs (95% CI) of stroke across levels of calcium intake were 0.95 (0.92, 0.98) for 200 mg/day, 0.94 (0.90, 0.98) for 300 mg/day, 0.95 (0.90, 0.99) for 500 mg/day, 0.98 (0.93, 1.03) for 700 mg/day, and 1.04 (0.97, 1.11) for 1000 mg/day. The stratified analyses by geographic region showed nonlinear associations and indicated that the protective effect was observed in Asian countries (Pnonlinearity = 0.001) but not in non-Asian regions (Pnonlinearity = 0.047). CONCLUSION: This meta-analysis suggests that dietary calcium intake might play an effective role in the prevention of stroke, especially in Asian countries. Future research among Asia population should attempt to establish whether this association is causal. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42022357710.

Rapid differential detection of subtype H1 and H3 swine influenza viruses using a TaqMan-MGB-based duplex one-step real-time RT-PCR assay.

Swine influenza is an economically important respiratory disease in swine, but it also constantly poses a threat to human health. Therefore, developing rapid, sensitive, and efficient detection methods for swine influenza virus (SIV) is important. By aligning the haemagglutinin (HA) gene sequences of SIVs circulating in China over a 10-year period, an H1 primer-probe set targeting both Eurasian avian-like H1N1 (EA H1N1) and pandemic 2009 H1N1 ((H1N1)pdm09) lineages plus a H3 primer-probe set targeting the prevalent human-like H3N2 (HL H3N2) subtype were designed. Subsequently, a TaqMan-MGB-based duplex one-step real-time RT-PCR (RT-qPCR) assay was established and evaluated. The duplex RT-qPCR has a detection limit of 5 copies/µL of HA plasmid for EA H1N1, (H1N1)pdm09, and HL H3N2 subtype SIVs, and its overall detection sensitivity of 100% and specificity of 91.67% matches that of traditional virus isolation through chicken embryo inoculation using experimentally infected mouse lung samples. The method showed high repeatability both within run and between runs, and there was no cross-reactivity against several other porcine viruses that are commonly circulating in China. Furthermore, the duplex RT-qPCR method revealed a higher prevalence of subtype H1 than subtype H3 in 166 nasal swabs from pigs collected from one slaughterhouse between October and December 2019. This assay could be very helpful in the rapid differential detection and routine surveillance of EA H1N1, (H1N1)pdm09, and HL H3N2 SIVs in China.

G1-like M and PB2 genes are preferentially incorporated into H7N9 progeny virions during genetic reassortment.

BACKGROUND: Genotype S H9N2 viruses have become predominant in poultry in China since 2010. These viruses frequently donate their whole internal gene segments to other emerging influenza A subtypes such as the novel H7N9, H5N6, and H10N8 viruses. We recently reported that the PB2 and M genes of the genotype S H9N2 virus, which are derived from the G1-like virus, enhance the fitness of H5Nx and H7N9 avian influenza viruses in chickens and mice. However, whether the G1-like PB2 and M genes are preferentially incorporated into progeny virions during virus reassortment remains unclear; whether the G1-like PB2 and M genes from different subtypes are differentially incorporated into new virion progeny remains unknown. RESULTS: We conducted a reassortment experiment with the use of a H7N9 virus as the backbone and found that G1-like M/PB2 genes were preferentially incorporated in progeny virions over F/98-like M/PB2 genes. Importantly, the preference varied among G1-like M/PB2 genes of different subtypes. When competing with F/98-like M/PB2 genes during reassortment, both the M and PB2 genes from the H7N9 virus GD15 showed an advantage, whereas only the PB2 gene from the H9N2 virus CZ73 and the M gene from the H9N2 virus AH320 displayed the advantage. CONCLUSION: Our findings highlight the preferential and variable advantages of H9N2-derived G1-like M and PB2 genes in incorporating them into H7N9 progeny virions over SH14-derived F/98-like M/PB2 genes.

Early administration of amino acids with different doses in low birth weight premature infants.

BACKGROUND: The reasonable use of amino acids (AAs) in parenteral nutrition (PN) is very critical to the growth and development of premature infants. However, the appropriate dose of AAs has not been determined. Our study was designed to investigate the clinical effect of two different doses of AAs in PN for low birth weight premature infants. MATERIALS AND METHODS: This randomized controlled study included 191 preterm infants who admitted to the neonatal intensive care unit of the First Affiliated Hospital of Nanjing Medical University from June 2015 to December 2016 and they were randomly divided into Group 1 (n = 81) and Group 2 (n = 110). In Group 1, the starting dose of AAs dose was 1.0-1.5 g/kg/day, which was increased by 0.5 g/kg with the maximum dose at 3.5 g/kg/day. In Group 2, the starting dose of AAs was 1.8-2.5 g/kg/day and was increased by 1.0 g/kg with the maximum dose at 4.0-4.5 g/kg/day. We analyzed the clinical characteristics, body weight, body length, total calorie intake, nonprotein calorie intake, total protein intake, liver and kidney function, and complications of the two groups of preterm infants. RESULTS: The start of enteral feeding and the recovery of birth weight in Group 2 were earlier than those in Group 1 (3.83 ± 3.15 day vs. 5.53 ± 5.63 day, P = 0.016 and 6.36 ± 4.88 day vs. 8.48 ± 9.27 day, P = 0.043, respectively). The duration of PN and the time before total enteral nutrition were shorter in Group 2 than in Group 1 (16.46 ± 10.33 day vs. 21.41 ± 18.00 day, P = 0.029 and 15.47 ± 10.54 day vs. 19.47 ± 14.57 day, P = 0.038; respectively). The duration of mechanical ventilation (1.12 ± 2.62 day vs. 3.31 ± 8.13 day, P = 0.028) in Group 2 was shorter than that in Group 1. CONCLUSION: High doses of AAs in the early PN for preterm infants facilitate the promotion of early growth and development, advance recovery of birth weight, reduce the duration of PN, and reduce respiratory support without increasing the incidence of complications.

CoRAL accurately resolves extrachromosomal DNA genome structures with long-read sequencing.

Extrachromosomal DNA (ecDNA) is a central mechanism for focal oncogene amplification in cancer, occurring in approximately 15% of early stage cancers and 30% of late-stage cancers. EcDNAs drive tumor formation, evolution, and drug resistance by dynamically modulating oncogene copy-number and rewiring gene-regulatory networks. Elucidating the genomic architecture of ecDNA amplifications is critical for understanding tumor pathology and developing more effective therapies. Paired-end short-read (Illumina) sequencing and mapping have been utilized to represent ecDNA amplifications using a breakpoint graph, where the inferred architecture of ecDNA is encoded as a cycle in the graph. Traversals of breakpoint graph have been used to successfully predict ecDNA presence in cancer samples. However, short-read technologies are intrinsically limited in the identification of breakpoints, phasing together of complex rearrangements and internal duplications, and deconvolution of cell-to-cell heterogeneity of ecDNA structures. Long-read technologies, such as from Oxford Nanopore Technologies, have the potential to improve inference as the longer reads are better at mapping structural variants and are more likely to span rearranged or duplicated regions. Here, we propose CoRAL (Complete Reconstruction of Amplifications with Long reads), for reconstructing ecDNA architectures using long-read data. CoRAL reconstructs likely cyclic architectures using quadratic programming that simultaneously optimizes parsimony of reconstruction, explained copy number, and consistency of long-read mapping. CoRAL substantially improves reconstructions in extensive simulations and 9 datasets from previously-characterized cell-lines as compared to previous short-read-based tools. As long-read usage becomes wide-spread, we anticipate that CoRAL will be a valuable tool for profiling the landscape and evolution of focal amplifications in tumors.

Metabolomic analysis of serum short-chain fatty acid concentrations in a mouse of MPTP-induced Parkinson's disease after dietary supplementation with branched-chain amino acids.

The gut microbiota and microbial metabolites influence the enteric nervous system and the central nervous system via the microbial-gut-brain axis. Increasing body of evidence suggests that disturbances in the metabolism of peripheral branched-chain amino acids (BCAAs) can contribute to the development of neurodegenerative diseases through neuroinflammatory signaling. Preliminary research has shown that longitudinal changes in serum amino acid levels in mouse models of Parkinson's disease (PD) are negatively correlated with disease progression. Therefore, the aim of the present study was to determine the changes in serum levels of short-chain fatty acids (SCFAs) in a mouse model of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD after dietary BCAA supplementation. In our research, gas chromatography-mass spectrometry was used to detect serum SCFA concentrations. The data were then analyzed with principal component analysis and orthogonal partial least squares discriminant analysis. Finally, the correlations of serum SCFA levels with gut and motor function in MPTP-induced PD mice were explored. Propionic acid, acetic acid, butyric acid, and isobutyric acid concentrations were elevated in MPTP + H-BCAA mice compared with MPTP mice. Propionic acid concentration was increased the most, while the isovaleric acid concentration was decreased. Propionic acid concentration was positively correlated with fecal weight and water content and negatively correlated with the pole-climbing duration. In conclusion, these results not only suggest that propionic acid may be a potential biomarker for PD, but also indicate the possibility that PD may be treated by altering circulating levels of SCFA.

Effects of inflammatory factors on mesenchymal stem cells and their role in the promotion of tumor angiogenesis in colon cancer.

Mesenchymal stem cells (MSCs), which are modulated by cytokines present in the tumor microenvironment, play an important role in tumor progression. It is well documented that inflammation is an important part of the tumor microenvironment, so we investigated whether stimulation of MSCs by inflammatory cytokines would contribute to their ability to promote tumor growth. We first showed that MSCs could increase C26 colon cancer growth in mice. This growth-promoting effect was further accelerated when the MSCs were pre-stimulated by inflammatory factors IFN-γ and TNF-α. At the same time, we demonstrated that MSCs pre-stimulated by both inflammatory factors could promote tumor angiogenesis in vivo to a greater degree than untreated MSCs or MSCs pre-stimulated by either IFN-γ or TNF-α alone. A hen egg test-chorioallantoic membrane (HET-CAM) assay showed that treatment of MSC-conditioned medium can promote chorioallantoic membrane angiogenesis in vitro, especially treatment with conditioned medium of MSCs pretreated with IFN-γ and TNF-α together. This mechanism of promoting angiogenesis appears to take place via an increase in the expression of vascular endothelial growth factor (VEGF), which itself takes place through an increase in signaling in the hypoxia-inducible factor 1α (HIF-1α)-dependent pathway. Inhibition of HIF-1α in MSCs by siRNA was found to effectively reduce the ability of MSC to affect the growth of colon cancer in vivo in the inflammatory microenviroment. These results indicate that MSCs stimulated by inflammatory cytokines such as IFN-γ and TNF-α in the tumor microenvironment express higher levels of VEGF via the HIF-1α signaling pathway and that these MSCs then enhance tumor angiogenesis, finally leading to colon cancer growth in mice.

Association of obesity with headache among US children and adolescents: Evidence from NHANES 1999-2004.

Background: Children and adolescents increasingly commonly suffer from obesity and headache. It has been confirmed that there is an association between obesity and headache in adults; however, evidence of such an association in paediatric populations is still controversial. Therefore, this study examined the relationship between obesity and headache among children and adolescents in the US. Methods: The cross-sectional data of 3948 participants were obtained from the National Health and Nutrition Examination Survey 1999-2004. Weighted logistic regression models were applied to investigate the association between obesity and headache. Subgroup analysis stratified by sex and age was performed to explore the potential difference in the association of paediatric obesity with headache. The performance of paediatric obesity on headache was assessed by receiver operating characteristic (ROC) curve. Results: The present study involved 3948 participants, of whom 713 (18.1%) had headache. Compared to those without headache, participants with headache tended to be girls and adolescents, have less calcium intake, and have higher levels of body mass index (BMI), C-reactive protein (CRP), serum ferritin and triglycerides (TGs) (all P < 0.05). After fully adjusting for potential confounders, the ORs with 95% CIs for headache were 1.03 (0.58-1.54) and 1.25 (0.68-2.30) for overweight and obese participants in comparison with normal-weight controls, respectively, implying no association of paediatric obesity with headache independent of other potential confounding factors. In addition, although higher odds of headache were noted in girls and adolescents (aged 10-17 years), no statistically significant difference was found across any subgroups. The area under the ROC (AUC) of paediatric obesity on headache was 0.634. Conclusions: In summary, our study indicated that obesity is not associated with headache among US children and adolescents. Further prospective studies with larger sample size are needed to validate our findings.

Immunosuppressive effect of bone marrow-derived mesenchymal stem cells in inflammatory microenvironment favours the growth of B16 melanoma cells.

Mesenchymal stem cells (MSCs) are studied for their potential clinical use in regenerative medicine, tissue engineering and tumour therapy. However, the therapeutic application of MSCs in tumour therapy still remains limited unless the immunosuppressive role of MSCs for tumour growth in vivo is better understood. In this study, we investigated the mechanism of MSCs favouring tumour escape from immunologic surveillance in inflammatory microenvironment. We first compared the promotive capacity of bone marrow-derived MSCs on B16 melanoma cells growth in vivo, pre-incubated or not with the inflammatory cytokines interferon (IFN)-γ and tumour necrosis factor (TNF)-α. We showed that the development of B16 melanoma cells is faster when co-injected with MSCs pre-incubated with IFN-γ and TNF-α compared with control groups. Moreover, tumour incidence increases obviously in allogeneic recipients when B16 melanoma cells were co-injected with MSCs pre-incubated with IFN-γ and TNF-α. We then demonstrated that the immunosuppressive function of MSCs was elicited by IFN-γ and TNF-α. These cytokine combinations provoke the expression of inducible nitric oxide synthase (iNOS) by MSCs. The impulsive effect of MSCs treated with inflammatory cytokines on B16 melanoma cells in vivo can be reversed by inhibitor or short interfering RNA of iNOS. Our results suggest that the MSCs in tumour inflammatory microenvironment may be elicited of immunosuppressive function, which will help tumour to escape from the immunity surveillance.