Somatic nuclear mitochondrial DNA insertions are prevalent in the human brain and accumulate over time in fibroblasts.

The transfer of mitochondrial DNA into the nuclear genomes of eukaryotes (Numts) has been linked to lifespan in nonhuman species and recently demonstrated to occur in rare instances from one human generation to the next. Here, we investigated numtogenesis dynamics in humans in 2 ways. First, we quantified Numts in 1,187 postmortem brain and blood samples from different individuals. Compared to circulating immune cells (n = 389), postmitotic brain tissue (n = 798) contained more Numts, consistent with their potential somatic accumulation. Within brain samples, we observed a 5.5-fold enrichment of somatic Numt insertions in the dorsolateral prefrontal cortex (DLPFC) compared to cerebellum samples, suggesting that brain Numts arose spontaneously during development or across the lifespan. Moreover, an increase in the number of brain Numts was linked to earlier mortality. The brains of individuals with no cognitive impairment (NCI) who died at younger ages carried approximately 2 more Numts per decade of life lost than those who lived longer. Second, we tested the dynamic transfer of Numts using a repeated-measures whole-genome sequencing design in a human fibroblast model that recapitulates several molecular hallmarks of aging. These longitudinal experiments revealed a gradual accumulation of 1 Numt every ~13 days. Numtogenesis was independent of large-scale genomic instability and unlikely driven by cell clonality. Targeted pharmacological perturbations including chronic glucocorticoid signaling or impairing mitochondrial oxidative phosphorylation (OxPhos) only modestly increased the rate of numtogenesis, whereas patient-derived SURF1-mutant cells exhibiting mtDNA instability accumulated Numts 4.7-fold faster than healthy donors. Combined, our data document spontaneous numtogenesis in human cells and demonstrate an association between brain cortical somatic Numts and human lifespan. These findings open the possibility that mito-nuclear horizontal gene transfer among human postmitotic tissues produces functionally relevant human Numts over timescales shorter than previously assumed.

Dual mitigation of immunosuppression combined with photothermal inhibition for highly effective primary tumor and metastases therapy.

T-cell based immune response can attack cancer cells formidably when certain immune checkpoint (e.g., PD-1/PD-L1) is blocked. Unfortunately, PD-1/PD-L1 blockade only provoke limited immune response because the differentiation of tumor-reactive T lymphocytes is often suppressed by TGF-ß pathway. Namely, the combating cancer weapon is weakened. In this study, other than employing photothermal therapy (PTT) to eliminate the primary tumor, we also aimed to expose in situ tumor-associated antigens and exert immune response for metastases inhibition. This enhanced immunotherapeutic strategy is achieved by IR780/SB-505124 based nanoliposomes (Nano-IR-SB@Lip). Upon administration, TGF-ß pathway is inhibited by SB to drive effector T cells into a responsive state and reduce the infiltration of Treg cells, eventually greatly enhancing the weapon against cancer. In the meantime, the immunosuppressive "protection" of tumor cells is also neutralized by blocking PD-1/PD-L1 immune checkpoint. By virtue of inherent characteristics of IR780, Nano-IR-SB@Lip can selectively accumulate, penetrate deeply in tumor tissues, and preferentially retain in mitochondria. The above features are of critical importance to tumor therapy. Thus, highly effective cancer immunotherapy is implemented via selective accumulation/deep penetration of Nano-IR-SB@Lip in tumor, achieving PTT induced immunogenic cell death and dual mitigation of immunosuppression strategy (TGF-ß inhibition/PD-1/PD-L1 blockade), which is a promising therapeutic modality for cancer.

Hypoxia modulation by dual-drug nanoparticles for enhanced synergistic sonodynamic and starvation therapy.

BACKGROUND: Sonodynamic therapy (SDT) is an emerging non-invasive therapeutic technique. SDT-based cancer therapy strategies are presently underway, and it may be perceived as a promising approach to improve the efficiency of anti-cancer treatment. In this work, multifunctional theranostic nanoparticles (NPs) were synthesized for synergistic starvation therapy and SDT by loading glucose oxidase (GOx, termed G) and 5,10,15,20-tetrakis (4-chlorophenyl) porphyrin) Cl (T (p-Cl) PPMnCl, termed PMnC) in Poly (lactic-co-glycolic) acid (PLGA) NPs (designated as MG@P NPs). RESULTS: On account of the peroxidase-like activity of PMnC, MG@P NPs can catalyze hydrogen peroxide (H2O2) in tumor regions to produce oxygen (O2), thus enhancing synergistic therapeutic effects by accelerating the decomposition of glucose and promoting the production of cytotoxic singlet oxygen (1O2) induced by ultrasound (US) irradiation. Furthermore, the NPs can also serve as excellent photoacoustic (PA)/magnetic resonance (MR) imaging contrast agents, effectuating imaging-guided cancer treatment. CONCLUSION: Multifunctional MG@P NPs can effectuate the synergistic amplification effect of cancer starvation therapy and SDT by hypoxia modulation, and act as contrast agents to enhance MR/PA dual-modal imaging. Consequently, MG@P NPs might be a promising nano-platform for highly efficient cancer theranostics.

Comprehensive identification of somatic nucleotide variants in human brain tissue.

BACKGROUND: Post-zygotic mutations incurred during DNA replication, DNA repair, and other cellular processes lead to somatic mosaicism. Somatic mosaicism is an established cause of various diseases, including cancers. However, detecting mosaic variants in DNA from non-cancerous somatic tissues poses significant challenges, particularly if the variants only are present in a small fraction of cells. RESULTS: Here, the Brain Somatic Mosaicism Network conducts a coordinated, multi-institutional study to examine the ability of existing methods to detect simulated somatic single-nucleotide variants (SNVs) in DNA mixing experiments, generate multiple replicates of whole-genome sequencing data from the dorsolateral prefrontal cortex, other brain regions, dura mater, and dural fibroblasts of a single neurotypical individual, devise strategies to discover somatic SNVs, and apply various approaches to validate somatic SNVs. These efforts lead to the identification of 43 bona fide somatic SNVs that range in variant allele fractions from ~ 0.005 to ~ 0.28. Guided by these results, we devise best practices for calling mosaic SNVs from 250× whole-genome sequencing data in the accessible portion of the human genome that achieve 90% specificity and sensitivity. Finally, we demonstrate that analysis of multiple bulk DNA samples from a single individual allows the reconstruction of early developmental cell lineage trees. CONCLUSIONS: This study provides a unified set of best practices to detect somatic SNVs in non-cancerous tissues. The data and methods are freely available to the scientific community and should serve as a guide to assess the contributions of somatic SNVs to neuropsychiatric diseases.

Treatment Effect of Low-Intensity Pulsed Ultrasound on Benzene- and Cyclophosphamide-Induced Aplastic Anemia in Rabbits.

BACKGROUND: Transplantation and immunosuppressive therapies are the available treatments for aplastic anemia; however, each strategy has its advantages and disadvantages. OBJECTIVE: The aim of this study was to find a new strategy for aplastic anemia treatment. DESIGN: This was an experimental and comparative study. METHODS: The aplastic anemia model was established by injecting rabbits with benzene and cyclophosphamide. The rabbits with aplastic anemia were divided into low-intensity pulsed ultrasound (LIPUS) and control groups. The distal femoral metaphysis of rabbits in the LIPUS group was treated with ultrasound for 30 days (20 min/d), whereas the control group received a sham treatment. Diarrhea, mortality, and blood cell count were evaluated. The levels of forkhead box P3, interleukin 17, interleukin 4, and interferon gamma were measured using an enzyme-linked immunosorbent assay. Bone marrow hyperplasia was observed by hematoxylin-eosin staining and scanning electron microscopy. RESULTS: The numbers of red blood cells (RBCs), white blood cells (WBCs), and platelets (PLTs) were lower, the amount of hematopoietic tissue was lower, and the amount of adipose tissue was higher in the rabbit aplastic anemia model than in the normal rabbits. The numbers of RBCs, WBCs, and PLTs increased after LIPUS treatment. The interleukin 17 level decreased, whereas the forkhead box P3 level increased. The amount of hematopoietic tissue increased, whereas the amount of adipose tissue decreased. LIMITATIONS: The number of hematopoietic stem cells could not be evaluated. CONCLUSIONS: LIPUS improved the hematopoietic microenvironment, accelerated the reconstruction of bone marrow cells, and increased the quantity and quality of RBCs, WBCs, and PLTs in the peripheral blood. Hence, it can serve as a novel treatment strategy for aplastic anemia in the future.

Synergies of accelerating differentiation of bone marrow mesenchymal stem cells induced by low intensity pulsed ultrasound, osteogenic and endothelial inductive agent.

In terms to investigate the effect of low-intensity pulsed ultrasound (LIPUS) for differentiation of bone marrow mesenchymal stem cells (BMSCs) and the feasibility of simultaneously inducing into osteoblasts and vascular endothelial cells within the cell culture medium in which two inductive agents are added at the same time with or without LIPUS. Cells were divided into a non-induced group, an osteoblast-induced group, a vascular endothelial-induced group, and a bidirectional differentiation-induced group. Each group was further subdivided into LIPUS and non-LIPUS groups. The cell proliferation in each group was measured by MTT assay. Cell morphological and ultrastructural changes were observed by inverted phase contrast microscopy and transmission electron microscopy. The differentiation of BMSCs was detected by confocal microscopy, flow cytometry and quantitative RT-PCR. Results demonstrated that both osteoblast and vascular endothelial cell differentiation markers were expressed in the bidirectional differentiation induction group and early osteogenesis and angiogenesis appeared. The cell proliferation, differentiation rate and expression of osteocalcin and vWF in the LIPUS groups were all significantly higher than those in the corresponding non-LIPUS group (p < .05), suggesting LIPUS treatment can promote the differentiation efficiency and rate of BMSCs, especially in the bidirectional differentiation induction group. This study suggests the combination of LIPUS and dual-inducing agents could induce and accelerate simultaneous differentiation of BMSCs to osteoblasts and vascular endothelial cells. These findings indicate the method could be applied to research on generating vascularized bone tissue with a shape and function that mimics natural bone to accelerate early osteogenesis and angiogenesis.

Low-Intensity Pulsed Ultrasound Relieves Mild and Severe Myelosuppression Induced by Cyclophosphamide in Rabbits.

OBJECTIVE: This study aimed to investigate the effect of low-intensity pulsed ultrasound (LIPUS) on cyclophosphamide (CTX)-induced rabbit myelosuppression. METHODS: Rabbits (n = 90) were randomly divided into a mild myelosuppression group (n = 40), a severe myelosuppression group (n = 40), and a normal control group (group Cu28 ; n = 10). The mild and severe myelosuppression models were established by daily ear vein injection of 15- and 40-mg/kg CTX for 4 continuous days, respectively. Then they were randomly divided into LIPUS groups (Au and Bu ) and control groups (Ac and Bc ). LIPUS was applied once per day for 20 minutes for 7 (Au7 and Bu7 ) and 28 (Au28 and Bu28 ) days. Physical conditions, mortality, blood cell counts, and bone marrow proliferation were calculated. Erythropoietin interleukin 3, and granulocyte-macrophage colony-stimulating factor levels were measured by an enzyme-linked immunosorbent assay. Flow cytometry was used to detect the granulocyte phagocytosis rate. Hematoxylin-eosin staining was performed to analyze changes of skin and muscle. RESULTS: Compared with the control group, LIPUS improved the number of peripheral blood cells (P < .05) and bone marrow nucleated cells and reduced the mortality of rabbits with myelosuppression of different degrees. Long-term treatment for 28 days had no effect on the levels of erythropoietin, interleukin 3, and granulocyte-macrophage colony-stimulating factor and granulocyte phagocytosis (P > .05). The parts of the skin where LIPUS was applied did not show any burning marks, and the muscle tissue in the path of LIPUS acoustic channels showed no obvious pathologic changes. CONCLUSIONS: Low-intensity pulsed ultrasound is a safe and effective method to relieve CTX-induced myelosuppression.

knnAUC: an open-source R package for detecting nonlinear dependence between one continuous variable and one binary variable.

BACKGROUND: Testing the dependence of two variables is one of the fundamental tasks in statistics. In this work, we developed an open-source R package (knnAUC) for detecting nonlinear dependence between one continuous variable X and one binary dependent variables Y (0 or 1). RESULTS: We addressed this problem by using knnAUC (k-nearest neighbors AUC test, the R package is available at https://sourceforge.net/projects/knnauc/ ). In the knnAUC software framework, we first resampled a dataset to get the training and testing dataset according to the sample ratio (from 0 to 1), and then constructed a k-nearest neighbors algorithm classifier to get the yhat estimator (the probability of y = 1) of testy (the true label of testing dataset). Finally, we calculated the AUC (area under the curve of receiver operating characteristic) estimator and tested whether the AUC estimator is greater than 0.5. To evaluate the advantages of knnAUC compared to seven other popular methods, we performed extensive simulations to explore the relationships between eight different methods and compared the false positive rates and statistical power using both simulated and real datasets (Chronic hepatitis B datasets and kidney cancer RNA-seq datasets). CONCLUSIONS: We concluded that knnAUC is an efficient R package to test non-linear dependence between one continuous variable and one binary dependent variable especially in computational biology area.

Treatment effect of low intensity pulsed ultrasound on leukopenia induced by cyclophosphamide in rabbits.

OBJECTIVE: This study aims to examine the effects of low intensity pulsed ultrasound (LIPUS) on leukopenia induced by cyclophosphamide in a rabbit model. METHODS: The leukopenia model in New Zealand rabbit was established by injecting cyclophosphamide into the ear vein. Forty leukopenia model rabbits were randomly allocated to control group (n = 20) and LIPUS group (n = 20). LIPUS group underwent 20 minutes of daily ultrasound treatment at femoral metaphysis for 7 days while control group received sham treatment. Diarrhea rate, mortality and blood cell count were calculated. IgA, IgG and IgM levels were measured by ELISA. Flow cytometry was used to detect CD44, CD49d, and PU.1. HE staining was performed to analyze bone marrow hyperplasia and changes of skin and muscle. RESULTS: LIPUS treatment significantly promoted the proliferation of bone marrow nucleated cells, increased the number of WBC, IgA, IgG and IgM in the peripheral blood, and reduced the diarrhea rate and mortality. The irradiated skin and muscle tissues showed no obvious damages. LIPUS treatment promoted the migration of hematopoietic cells to peripheral blood by decreasing the expression of CD49d and CD44 on the surface of CD34 positive cells. It also promoted the differentiation of hematopoietic stem cells into granulocytes and lymphocytes by decreasing the expression of PU.1. CONCLUSION: LIPUS can be used as a safe and effective clinical treatment for cyclophosphamide induced leukopenia.

Predictive model for inflammation grades of chronic hepatitis B: Large-scale analysis of clinical parameters and gene expressions.

BACKGROUND: Liver biopsy is the gold standard to assess pathological features (eg inflammation grades) for hepatitis B virus-infected patients although it is invasive and traumatic; meanwhile, several gene profiles of chronic hepatitis B (CHB) have been separately described in relatively small hepatitis B virus (HBV)-infected samples. We aimed to analyse correlations among inflammation grades, gene expressions and clinical parameters (serum alanine amino transaminase, aspartate amino transaminase and HBV-DNA) in large-scale CHB samples and to predict inflammation grades by using clinical parameters and/or gene expressions. METHODS: We analysed gene expressions with three clinical parameters in 122 CHB samples by an improved regression model. Principal component analysis and machine-learning methods including Random Forest, K-nearest neighbour and support vector machine were used for analysis and further diagnosis models. Six normal samples were conducted to validate the predictive model. RESULTS: Significant genes related to clinical parameters were found enriching in the immune system, interferon-stimulated, regulation of cytokine production, anti-apoptosis, and etc. A panel of these genes with clinical parameters can effectively predict binary classifications of inflammation grade (area under the ROC curve [AUC]: 0.88, 95% confidence interval [CI]: 0.77-0.93), validated by normal samples. A panel with only clinical parameters was also valuable (AUC: 0.78, 95% CI: 0.65-0.86), indicating that liquid biopsy method for detecting the pathology of CHB is possible. CONCLUSIONS: This is the first study to systematically elucidate the relationships among gene expressions, clinical parameters and pathological inflammation grades in CHB, and to build models predicting inflammation grades by gene expressions and/or clinical parameters as well.

Combination of low-intensity pulsed ultrasound and C3H10T1/2 cells promotes bone-defect healing.

AIMS: We systematically investigated the effect of combined use of low-intensity pulsed ultrasound (LIPUS) and bone mesenchymal stem cells C3H10T1/2 on bone-defect healing. METHODS: C3H10T1/2 cells were first induced into a stationary phase by incubation with low fetal bovine serum (5 ml/l) for five days and then sonicated with LIPUS for ten minutes once every day for five consecutive days. The same LIPUS treatment combined with C3H10T1/2 cells, which were incubated in regular fetal bovine serum (10 ml/l) were used to aid femoral fracture healing in Sprague-Dawley rats during four consecutive weeks. C3H10T1/2 cell proliferation activity was detected by MTT assay. Cell-cycle changes were determined, and cell proliferation index was calculated using flow cytometry. Bone reparation was evaluated by X-ray imaging and hematoxylin and eosin (H&E) staining during the healing process. RESULTS: LIPUS promoted C3H10T1/2 cell proliferation, the mechanism of which was possibly the up-regulation of Bmi-1 gene expression. At the end of week two after combined use of LIPUS and C3H10T1/2, the femoral gap was reduced on X-ray images. According to H&E staining results, new bone had homogeneous and similar density compared with normal surrounding bone after combined use of LIPUS and C3H10T1/2. At the end of week four, bone defects could not be observed by X-ray in all four groups and repaired bone substance in all four groups could be observed by H&E staining. CONCLUSIONS: LIPUS treatment effectively promotes C3H10T1/2 cells to enter the growth/split phase from the stationary phase. This process enhances cell proliferation, which consequently promotes bone-defect healing.

Correlation between frequency of non-allelic homologous recombination and homology properties: evidence from homology-mediated CNV mutations in the human genome.

Non-allelic homologous recombination (NAHR) is one of the key mechanisms of DNA rearrangement. NAHR occurring between direct homologous repeats can generate genomic copy number variation (CNV) and make significant contributions to both genome evolution and human diseases such as cancer. Intriguingly, previous observations on the rare CNVs at certain genomic disorder loci suggested that NAHR frequency could be dependent on homology properties. However, such a correlation remains unclear at the other NAHR-mediated CNV loci, especially the common CNVs in human populations. Different from the rare CNVs associated with genomic disorders, it is challenging to identify de novo NAHR events at common CNV loci. Therefore, our previously proposed statistic M was employed in estimating relative mutation rate for the NAHR-mediated CNVs in human populations. By utilizing generalized regression neural network and principal component analysis in studying 4330 CNVs ascertained in 3 HapMap populations, we identified the CNVs mediated by NAHR between paired segmental duplications (SDs) and further revealed the correlations between SD properties and NAHR probability. SD length and inter-SD distance were shown to make major contributions to the occurrence of NAHR, whereas chromosomal position and sequence similarity of paired SDs are also involved in NAHR. An integrated effect of SD properties on NAHR frequency was revealed for the common CNVs in human populations. These observations can be well explained by ectopic synapsis in NAHR together with our proposed model of chromosomal compression/extension/looping (CCEL) for homology mis-pairing. Our findings showed the important roles of SDs in NAHR and human genomic evolution.

Genome-wide CNV analysis in mouse induced pluripotent stem cells reveals dosage effect of pluripotent factors on genome integrity.

BACKGROUND: Induced pluripotent stem cells (iPSCs) derived from somatic cells have enormous potential for clinical applications. Notably, it was recently reported that reprogramming from somatic cells to iPSCs can induce genomic copy number variation (CNV), which is one of the major genetic causes of human diseases. However it was unclear if this genome instability is dependent on reprogramming methods and/or the genetic background of donor cells. Furthermore, genome-wide CNV analysis is technically challenging and CNV data need to be interpreted with care. RESULTS: In order to carefully investigate the possible CNV instability during somatic reprogramming, we performed genome-wide CNV analyses with 41 mouse iPSC lines generated from the same parental donor; therefore, the donor's genetic background can be controlled. Different reprogramming factor combinations and dosages were used for investigating potential method-dependent effects on genome integrity. We detected 63 iPSC CNVs using high-resolution comparative genomic hybridization. Intriguingly, CNV rates were negatively associated with the dosages of classic factor(s). Furthermore, the use of high-performance engineered factors led to less CNVs than the classic factor(s) of the same dosage. CONCLUSION: Our observations suggest that sufficient reprogramming force can protect the genome from CNV instability during the reprogramming process.

Increased genome instability in human DNA segments with self-chains: homology-induced structural variations via replicative mechanisms.

Environmental factors including ionizing radiation and chemical agents have been known to be able to induce DNA rearrangements and cause genomic structural variations (SVs); however, the roles of intrinsic characteristics of the human genome, such as regional genome architecture, in SV formation and the potential mechanisms underlying genomic instability remain to be further elucidated. Recently, locus-specific observations showed that 'self-chain' (SC), a group of short low-copy repeats (LCRs) in the human genome, can induce autism-associated SV mutations of the MECP2 and NRXN1 genes. In this study, we conducted a genome-wide analysis to investigate SCs and their potential roles in genomic SV formation. Utilizing a vast amount of human SV data, we observed a significant biased distribution of human germline SV breakpoints to SC regions. Notably, the breakpoint distribution pattern is different between SV types across deletion, duplication, inversion and insertion. Our observations were coincident with a mechanism of SC-induced DNA replicative errors, whereas SC may sporadically be used as substrates of nonallelic homologous recombination (NAHR). This contention was further supported by our consistent findings in somatic SV mutations of cancer genomes, suggesting a general mechanism of SC-induced genome instability in human germ and somatic cells.

Association of polymorphisms in the solute carrier organic anion transporter family member 1B1 gene with essential hypertension in the Uyghur population.

Solute carrier organic anion transporter family member 1B1 (SLCO1B1) is an important hepatic uptake transporter that can transport a wide variety of endogenous compounds, including thyroid hormones and prostaglandin E2. Dysregulation of thyroid hormones and prostaglandin E2 plays a role in the development of hypertension, suggesting that SLCO1B1 might contribute to the aetiology of essential hypertension (EH). In this study, we selected five single nucleotide polymorphisms (SNPs) at the SLCO1B1 gene promoter or coding regions and performed a case-control association study involving 731 unrelated Uyghur subjects, including 374 hypertensive and 357 normotensive individuals, to investigate the potential genetic contribution of SLCO1B1 to the aetiology of EH. Of the five polymorphisms, only one (i.e., rs4149014) showed correlation with EH. The minor allele of SNP rs4149014 at the SLCO1B1 promoter showed association with increased risk for EH (adjusted OR 1.88; 95% CI 1.36-2.60; P= 1.22 × 10(-4)). This study provides preliminary genetic evidence for the role of variant of SLCO1B1 in the susceptibility to human EH in Uyghurs.