Somatic mutations in aging and disease.

Time always leaves its mark, and our genome is no exception. Mutations in the genome of somatic cells were first hypothesized to be the cause of aging in the 1950s, shortly after the molecular structure of DNA had been described. Somatic mutation theories of aging are based on the fact that mutations in DNA as the ultimate template for all cellular functions are irreversible. However, it took until the 1990s to develop the methods to test if DNA mutations accumulate with age in different organs and tissues and estimate the severity of the problem. By now, numerous studies have documented the accumulation of somatic mutations with age in normal cells and tissues of mice, humans, and other animals, showing clock-like mutational signatures that provide information on the underlying causes of the mutations. In this review, we will first briefly discuss the recent advances in next-generation sequencing that now allow quantitative analysis of somatic mutations. Second, we will provide evidence that the mutation rate differs between cell types, with a focus on differences between germline and somatic mutation rate. Third, we will discuss somatic mutational signatures as measures of aging, environmental exposure, and activities of DNA repair processes. Fourth, we will explain the concept of clonally amplified somatic mutations, with a focus on clonal hematopoiesis. Fifth, we will briefly discuss somatic mutations in the transcriptome and in our other genome, i.e., the genome of mitochondria. We will end with a brief discussion of a possible causal contribution of somatic mutations to the aging process.

Age-related somatic mutation burden in human tissues.

The genome of multicellular organisms carries the hereditary information necessary for the development of all organs and tissues and to maintain function in adulthood. To ensure the genetic stability of the species, genomes are protected against changes in sequence information. However, genomes are not static. De novo mutations in germline cells are passed on to offspring and generate the variation needed in evolution. Moreover, postzygotic mutations occur in all somatic cells during development and aging. These somatic mutations remain limited to the individual, generating tissues that are genome mosaics. Insight into such mutations and their consequences has been limited due to their extremely low abundance, with most mutations unique for each cell. Recent advances in sequencing, including whole genome sequencing at the single-cell level, have now led to the first insights into somatic mutation burdens in human tissues. Here, we will first briefly describe the latest methodology for somatic mutation analysis, then review our current knowledge of somatic mutation burden in human tissues and, finally, briefly discuss the possible functional impact of somatic mutations on the aging process and age-related diseases, including cancer and diseases other than cancer.

[Comparison of the efficacy of different venous intubation in venous-arterial extracorporeal membrane oxygenation assisted lung transplantation].

OBJECTIVE: To compare the curative effects of different venous cannulas and drainage to improve patient's whole body oxygenation during the auxiliary process of venous-arterial extracorporeal membrane oxygenation (VA-ECMO) in lung transplantation. METHODS: From December 2016 to December 2019, 12 patients who were assisted by VA-ECMO in one lung transplantation in People's Hospital of Henan Province were selected as the research objects. According to the number of side holes of venous cannulas, they were divided into two groups: one group with few side holes and other group with multiple side holes. The differences in blood gas indexes among the right radial artery, left radial artery, and right internal jugular vein before and after assistance were compared, and the assistance effect was evaluated. RESULTS: The arterial partial pressure of oxygen (PaO2) of blood gas indexes of the right and left radial arteries in both groups were significantly higher than that before assistance [mmHg (1 mmHg = 0.133 kPa): right and left radial artery in few side holes group: 79.5±4.2 vs. 48.3±3.8 and 88.1±3.5 vs. 48.3±3.8; right and left radial artery in multiple side holes group: 67.7±5.9 vs. 48.7±3.2 and 84.0±3.8 vs. 48.7±3.2, all P < 0.05]. The arterial partial pressure of carbon dioxide (PaCO2) of blood gas index was significantly lower than that before assistance (mmHg: 44.2±2.6 vs. 71.7±4.4 for the right radial artery and 44.7±1.4 vs. 71.7±4.4 for the left radial artery in the group with few side holes; 46.2±2.1 vs. 71.2±3.5 for the right radial artery and 44.1±1.9 vs. 71.2±3.5 for the left radial artery in the group with multiple side holes, all P < 0.05). The partial pressure of oxygen in venous blood (PvO2) of blood gas index of ECMO system in the group with few side holes was significantly lower than that of the multiport side holes group (mmHg: 56.4±3.2 vs. 88.7±1.5, P < 0.01), and the partial pressure of carbon dioxide in venous blood (PvCO2) was significantly higher than that of multiport side holes group (mmHg: 63.6±3.7 vs. 44.2±1.7, P < 0.01). CONCLUSIONS: When VA-ECMO is used in lung transplantation, the superior vena cava blood flow can be fully drained by using intravenous cannula with few side holes. It can effectively improve the oxygenation of the upper body of lung transplant patients, avoid the dilemma of hypoxemia in the upper body and hyperxemia in the lower body, provide more effective assistance to patients undergoing single lung transplantation, and is more meaningful for improving the oxygenation status of the whole body in patients undergoing single lung transplantation.

Suramin interacts with the positively charged region surrounding the 5-fold axis of the EV-A71 capsid and inhibits multiple enterovirus A.

Suramin was previously shown to bind to the EV-A71 capsid through its naphthalenetrisulfonic acid groups, thereby reducing virus-cell binding and inhibiting viral replication. Here, we identify VP1-145 as the critical amino acid that accounts for the differential sensitivity of EVA-71 viruses to suramin. A single Q or G to E substitution at VP1-145 results in an approximately 30-fold shift of IC50 or IC90 values reproducing the inhibition profile observed with field isolates expressing either the 145Q or E mutation. Our data support the conclusion that suramin binds to the positively charged region surrounding the 5-fold axis of the capsid and consequently blocks the virus attachment and entry into host cells. In order to assess the antiviral-spectrum of suramin, we analyzed 18 representative enteroviruses: A (n = 7), B (n = 5), C (n = 5) and D (n = 1). We show that suramin potency is restricted to enterovirus A species. Clinical development of suramin is further supported by pharmacokinetic data demonstrating bioactive plasma levels after a single dose intramuscular administration in macaques. Altogether, our findings support the clinical development of suramin as a novel entry inhibitor for the treatment of enterovirus A infections.

The approved pediatric drug suramin identified as a clinical candidate for the treatment of EV71 infection-suramin inhibits EV71 infection in vitro and in vivo.

Enterovirus 71 (EV71) causes severe central nervous system infections, leading to cardiopulmonary complications and death in young children. There is an urgent unmet medical need for new pharmaceutical agents to control EV71 infections. Using a multidisciplinary approach, we found that the approved pediatric antiparasitic drug suramin blocked EV71 infectivity by a novel mechanism of action that involves binding of the naphtalentrisulonic acid group of suramin to the viral capsid. Moreover, we demonstrate that when suramin is used in vivo at doses equivalent to or lower than the highest dose already used in humans, it significantly decreased mortality in mice challenged with a lethal dose of EV71 and peak viral load in adult rhesus monkeys. Thus, suramin inhibits EV71 infection by neutralizing virus particles prior to cell attachment. Consequently, these findings identify suramin as a clinical candidate for further development as a therapeutic or prophylactic treatment for severe EV71 infection.

Seroepidemiology of Enterovirus 71 infection prior to the 2011 season in children in Shanghai.

BACKGROUND: In 2010, China experienced the largest outbreak on record of Enterovirus 71 (EV71)-associated Hand Foot and Mouth Disease (HFMD) with more than 1.7 million cases, 27,000 patients with severe neurological complications and 905 deaths. Understanding of the seroprevalence of neutralizing antibodies (NAb) against EV71 and their protective role against HFMD in children is crucial for the implementation of future therapeutic and prophylactic intervention. OBJECTIVES: To correlate the prevalence of NAb against EV71 genotype C4a in children prior to the 2011 epidemic season with severe EV71-associated HFMD disease during the subsequent 2011 epidemic season. STUDY DESIGN: 614 sera samples were collected from children without HFMD. EV71 NAb were tested by a quantitative PCR assay. Samples with NAb ≥1:8 were scored as positive. RESULTS: 122 (19.9%) of 614 sera were EV71-seropositive. The NAb seroprevalence was highest in infants 0-5 months of age (28.6%) and lowest in children 1-1.9 years of age (13.4%). 64.1% of severe EV71-associated HFMD occurred in children 1-2.9 years. CONCLUSIONS: Despite the large 2010 outbreak, the overall seroprevalence of EV71 in children is relatively low. The seropositive rate of EV71 NAb prior to the 2011 season was inversely correlated with the number of EV71-infected severe cases in 2011. Loss of maternal antibodies in infants and lack of acquired anti-EV71 immunity are responsible for increased proportion of severe HFMD in the 1-2 years age group. Our data suggest that future vaccination campaigns should be initiated as early as 6 months.

Molecular monitoring of causative viruses in child acute respiratory infection in endemo-epidemic situations in Shanghai.

BACKGROUND: Numerous viruses are responsible for respiratory infections; however, both their distribution and genetic diversity, in a limited area and a population subgroup, have been studied only rarely during a sustained period of time. METHODS: A 2-year surveillance program of children presenting with acute respiratory infections (ARIs) was carried out to characterize the viral etiology and to assess whether using gene amplification and sequencing could be a reliable approach to monitor virus introduction and spread in a population subgroup. RESULTS: Using multiplex RT-PCR, 15 different respiratory viruses were detected within the 486 nasopharyngeal positive samples collected among 817 children aged <9-year old who presented with ARI during October 2006 to September 2008. A single virus was detected in 373 patients (45.7%), and two to four viruses in 113 patients (13.8%). The most frequent causative viruses were respiratory syncytial virus (RSV) (24.7%), human bocavirus (24.5%), and human rhinovirus (HRV) (15%). RSV was more prevalent in winter and among young infants. Cases of seasonal influenza A and B viruses were reported mainly in January and August. An increase in adenovirus infection was observed during the spring of the second year of the study. Sequence analyses showed multiple introductions of different virus subtypes and identified a high prevalence of the newly defined HRV-C species. A higher viral incidence was observed during the winter of 2008, which was unusually cold. CONCLUSIONS: This study supports the usefulness of multiplex RT-PCR for virus detection and co-infection, and for implementation of a molecular monitoring system for endemic and epidemic viral respiratory infections.

Shedding of the Pandemic Swine-Origin Influenza A Virus (H1N1) after Oseltamivir Administration.

We analyzed the virus shedding of an oseltamivir-treated patient who had been infected with the pandemic swine-origin influenza A (H1N1) virus which had an oseltamivir-sensitive neuraminidase. The virus was isolated from the pharyngeal swabs of the patient using MDCK cells, and the virus genome RNA was detected in the same samples both by real-time RT-PCR and RT-PCR. The virus was isolated until 44 h after oseltamivir administration although the virus genome was detected until one day after oseltamivir treatment was stopped. Due to their high sensitivity, RT-PCR and real-time RT-PCR may cause misdiagnosis by detection of viral genome which does not infect, and classical virus isolation and clinical symptoms are recommended for the evaluation of oseltamivir treatment.

Correlation between bocavirus infection and humoral response, and co-infection with other respiratory viruses in children with acute respiratory infection.

BACKGROUND: Human bocavirus (HBoV), a recently discovered virus, is prevalent among children with respiratory tract infection throughout the world. Co-infection was frequently found in HBoV-positive patients. Thus, whether HBoV is responsible for the respiratory disease is still arguable. OBJECTIVES: A comprehensive study was carried out to integrate clinical and virological prevalence in HBoV-positive outpatient children, and to determine genetic and serologic characteristics of HBoV in Shanghai, China. STUDY DESIGN: Nasal/throat swabs and sera were obtained over a 2-year period from 817 children with respiratory tract infection to examine the presence of HBoV and its co-infection. The seroepidemiology of HBoV was studied by ELISA and Western blot against the capsid protein VP2-based fragment. Persistence of HBoV was also analyzed in 12 pairs of return-visit cases. RESULTS: HBoV was identified in 96 samples (11.8%). The co-infection rate with other respiratory viruses was 51%. IgM was detected in 55.7% of HBoV RT-PCR-positive patients, and in 72.7% of those who had high viral genome load. In addition, persistent viral DNA positivity was detected in 10 of 12 HBoV-positive cases tested, an average of 14 days later, and one child was still HBoV-positive after 31 days. CONCLUSION: HBoV was found frequently in children with respiratory tract symptoms associated with other respiratory viruses, and persisted in the respiratory tract and in serum and urine. The presence of IgM was significantly more prevalent in viremic patients and those diagnosed with high load of HBoV DNA in nasal/throat swabs.

Evidence of recombination and genetic diversity in human rhinoviruses in children with acute respiratory infection.

BACKGROUND: Human rhinoviruses (HRVs) are a highly prevalent cause of acute respiratory infection in children. They are classified into at least three species, HRV-A, HRV-B and HRV-C, which are characterized by sequencing the 5' untranslated region (UTR) or the VP4/VP2 region of the genome. Given the increased interest for novel HRV strain identification and their worldwide distribution, we have carried out clinical and molecular diagnosis of HRV strains in a 2-year study of children with acute respiratory infection visiting one district hospital in Shanghai. METHODOLOGY/FINDINGS: We cloned and sequenced a 924-nt fragment that covered part of the 5'UTR and the VP4/VP2 capsid genes. Sixty-four HRV-infected outpatients were diagnosed amongst 827 children with acute low respiratory tract infection. Two samples were co-infected with HRV-A and HRV-B or HRV-C. By comparative analysis of the VP4/VP2 sequences of the 66 HRVs, we showed a high diversity of strains in HRV-A and HRV-B species, and a prevalence of 51.5% of strains that belonged to the recently identified HRV-C species. When analyzing a fragment of the 5' UTR, we characterized at least two subspecies of HRV-C: HRV-Cc, which clustered differently from HRV-A and HRV-B, and HRV-Ca, which resulted from previous recombination in this region with sequences related to HRV-A. The full-length sequence of one strain of each HRV-Ca and HRV-Cc subspecies was obtained for comparative analysis. We confirmed the close relationship of their structural proteins but showed apparent additional recombination events in the 2A gene and 3'UTR of the HRV-Ca strain. Double or triple infections with HRV-C and respiratory syncytial virus and/or bocavirus were diagnosed in 33.3% of the HRV-infected patients, but no correlation with severity of clinical outcome was observed. CONCLUSION: Our study showed a high diversity of HRV strains that cause bronchitis and pneumonia in children. A predominance of HRV-C over HRV-A and HRV-B was observed, and two subspecies of HRV-C were identified, the diversity of which seemed to be related to recombination with former HRV-A strains. None of the HRV-C strains appeared to have a higher clinical impact than HRV-A or HRV-B on respiratory compromise.

Simultaneous detection of respiratory viruses in children with acute respiratory infection using two different multiplex reverse transcription-PCR assays.

A 4-tube multiplex RT-PCR (mRT-PCR), which showed higher sensitivity over conventional methods, was previously developed for the diagnosis of 14 viral pathogens of the respiratory tract. Herein the mRT-PCR was compared to the commercial Luminex mPCR-microsphere flow cytometry assay (Resplex II) which allows the detection of 12 different viruses. Eleven different viruses were identified in 91 nasopharyngeal swabs of children with acute respiratory infection, influenza A (IAV) and B, respiratory syncytial virus (RSV), human rhinovirus (hRhV), human echovirus, parainfluenza viruses (PIV) 1, 2, 3 and 4, human metapneumovirus (hMPV), and human coronavirus NL63. The results of the two techniques showed 53 and 40 positive patients by the Resplex II assay and mRT-PCR, respectively, with a concordance in 35 positive and 33 negative patients (74.7%). Individual RT-PCR tests were performed to control viruses not simultaneously detected by the two multiplex assays. The major virus misdiagnosed by mRT-PCR was IAV whereas the major viruses misdiagnosed by Resplex II were PIV1, 3 and 4. The mRT-PCR remains a simple, rapid, and specific assay for the specific detection of respiratory viruses, and can be easily implemented with standards in clinical laboratories at a low cost.

design of multiplexed detection assays for identification of avian influenza a virus subtypes pathogenic to humans by SmartCycler real-time reverse transcription-PCR.

Influenza A virus (IAV) epidemics are the result of human-to-human or poultry-to-human transmission. Tracking seasonal outbreaks of IAV and other avian influenza virus (AIV) subtypes that can infect humans, aquatic and migratory birds, poultry, and pigs is essential for epidemiological surveillance and outbreak alerts. In this study, we performed four real-time reverse transcription-PCR (rRT-PCR) assays for identification of the IAV M and hemagglutinin (HA) genes from six known AIVs infecting pigs, birds, and humans. IAV M1 gene-positive samples tested by single-step rRT-PCR and a fluorogenic Sybr green I detection system were further processed for H5 subtype identification by using two-primer-set multiplex and Sybr green I rRT-PCR assays. H5 subtype-negative samples were then tested with either a TaqMan assay for subtypes H1 and H3 or a TaqMan assay for subtypes H2, H7, and H9 and a beacon multiplex rRT-PCR identification assay. The four-tube strategy was able to detect 10 RNA copies of the HA genes of subtypes H1, H2, H3, H5, and H7 and 100 RNA copies of the HA gene of subtype H9. At least six H5 clades of H5N1 viruses isolated in Southeast Asia and China were detected by that test. Using rRT-PCR assays for the M1 and HA genes in 202 nasopharyngeal swab specimens from children with acute respiratory infections, we identified a total of 39 samples positive for the IAV M1 gene and subtypes H1 and H3. When performed with a portable SmartCycler instrument, the assays offer an efficient, flexible, and reliable platform for investigations of IAV and AIV in remote hospitals and in the field.