Optimal conditions to use Pfu exo(-) DNA polymerase for highly efficient ligation-mediated polymerase chain reaction protocols

Ligation-Mediated Polymerase Chain Reaction (LMPCR) is the most sensitive sequencing technique available to map single-stranded DNA breaks at the nucleotide level of resolution using genomic DNA. LMPCR has been adapted to map DNA damage and reveal DNA-protein interactions inside living cells. However, the sequence context (GC content), the global break frequency and the current combination of DNA polymerases used in LMPCR affect the quality of the results. In this study, we developed and optimized an LMPCR protocol adapted for Pyrococcus furiosus exo(-) DNA polymerase (Pfu exo(-)). The relative efficiency of Pfu exo(-) was compared to T7-modified DNA polymerase (Sequenase 2.0) at the primer extension step and to Thermus aquaticus DNA polymerase (Taq) at the PCR amplification step of LMPCR. At all break frequencies tested, Pfu exo(-) proved to be more efficient than Sequenase 2.0. During both primer extension and PCR amplification steps, the ratio of DNA molecules per unit of DNA polymerase was the main determinant of the efficiency of Pfu exo(-), while the efficiency of Taq was less affected by this ratio. Substitution of NaCl for KCl in the PCR reaction buffer of Taq strikingly improved the efficiency of the DNA polymerase. Pfu exo(-) was clearly more efficient than Taq to specifically amplify extremely GC-rich genomic DNA sequences. Our results show that a combination of Pfu exo(-) at the primer extension step and Taq at the PCR amplification step is ideal for in vivo DNA analysis and DNA damage mapping using LMPCR.     

Characteristics of children admitted to hospital with acute SARS-CoV-2 infection in Canada in 2020

Background:Risk factors for severe outcomes of SARS-CoV-2 infection are not well established in children. We sought to describe pediatric hospital admissions associated with SARS-CoV-2 infection in Canada and identify risk factors for more severe disease.Methods:We conducted a national prospective study using the infrastructure of the Canadian Paediatric Surveillance Program (CPSP). Cases involving children who were admitted to hospital with microbiologically confirmed SARS-CoV-2 infection were reported from Apr. 8 to Dec. 31 2020, through weekly online questionnaires distributed to the CPSP network of more than 2800 pediatricians. We categorized hospital admissions as related to COVID-19, incidental, or for social or infection control reasons and determined risk factors for disease severity in hospital.Results:Among 264 hospital admissions involving children with SARS-CoV-2 infection during the 9-month study period, 150 (56.8%) admissions were related to COVID-19 and 100 (37.9%) were incidental infections (admissions for other reasons and found to be positive for SARS-CoV-2 on screening). Infants (37.3%) and adolescents (29.6%) represented most cases. Among hospital admissions related to COVID-19, 52 (34.7%) had critical disease, 42 (28.0%) of whom required any form of respiratory or hemodynamic support, and 59 (39.3%) had at least 1 underlying comorbidity. Children with obesity, chronic neurologic conditions or chronic lung disease other than asthma were more likely to have severe or critical COVID-19.Interpretation:Among children who were admitted to hospital with SARS-CoV-2 infection in Canada during the early COVID-19 pandemic period, incidental SARS-CoV-2 infection was common. In children admitted with acute COVID-19, obesity and neurologic and respiratory comorbidities were associated with more severe disease.

As of Dec. 31, 2020, Canada had 581 427 confirmed cases of SARS-CoV-2 infection.¹ Similar to other countries, most confirmed infections were in adults, in part because of initial testing policies that targeted older and at-risk populations, as well as prolonged societal containment measures to minimize children’s risk of exposure. Although fewer SARS-CoV-2 infections in children were reported relative to adults during Canada’s first waves of the pandemic,² recent surges in pediatric cases across North America have challenged the notion that children are infected at a lower frequency than adults.^3,4 However, the severity of infection in children appears to be substantially lower, with fewer overall hospital admissions reported and substantially lower mortality compared with adults.^5,6Although risk factors for more severe outcomes of COVID-19 have been well described in adults,⁷ similar risks are less well described in children.⁸ Experience with other viral respiratory infections, including influenza and respiratory syncytial virus, has shown that patient-level factors can increase risk for severe disease in children.^9,10 Understanding populations at risk for severe disease is essential for developing evidence-informed testing strategies, recommendations around reducing exposure (including guidance informing in-person schooling) and potential prioritization of SARS-CoV-2 vaccines in children.To date, few published data have characterized admissions to hospital with SARS-CoV-2 infection among children in Canada. We sought to describe pediatric hospital admissions associated with acute SARS-CoV-2 infection in Canada and identify risk factors for severe disease among children admitted to hospital.     

Increased Stiffness in Aged Skeletal Muscle Impairs Muscle Progenitor Cell Proliferative Activity

Background

Skeletal muscle aging is associated with a decreased regenerative potential due to the loss of function of endogenous stem cells or myogenic progenitor cells (MPCs). Aged skeletal muscle is characterized by the deposition of extracellular matrix (ECM), which in turn influences the biomechanical properties of myofibers by increasing their stiffness. Since the stiffness of the MPC microenvironment directly impacts MPC function, we hypothesized that the increase in muscle stiffness that occurs with aging impairs the behavior of MPCs, ultimately leading to a decrease in regenerative potential.

Results

We showed that freshly isolated individual myofibers from aged mouse muscles contain fewer MPCs overall than myofibers from adult muscles, with fewer quiescent MPCs and more proliferative and differentiating MPCs. We observed alterations in cultured MPC behavior in aged animals, where the proliferation and differentiation of MPCs were lower and higher, respectively. These alterations were not linked to the intrinsic properties of aged myofibers, as shown by the similar values for the cumulative population-doubling values and fusion indexes. However, atomic force microscopy (AFM) indentation experiments revealed a nearly 4-fold increase in the stiffness of the MPC microenvironment. We further showed that the increase in stiffness is associated with alterations to muscle ECM, including the accumulation of collagen, which was correlated with higher hydroxyproline and advanced glycation end-product content. Lastly, we recapitulated the impaired MPC behavior observed in aging using a hydrogel substrate that mimics the stiffness of myofibers.

Conclusions

These findings provide novel evidence that the low regenerative potential of aged skeletal muscle is independent of intrinsic MPC properties but is related to the increase in the stiffness of the MPC microenvironment.     

Natural killer cells and natural killer T cells in Lyme arthritis

Introduction

Natural killer (NK) and natural killer T (NKT) cells provide a first line of defense against infection. However, these cells have not yet been examined in patients with Lyme arthritis, a late disease manifestation. Lyme arthritis usually resolves with antibiotic treatment. However, some patients have persistent arthritis after spirochetal killing, which may result from excessive inflammation, immune dysregulation and infection-induced autoimmunity.

Methods

We determined the frequencies and phenotypes of NK cells and invariant NKT (iNKT) cells in paired peripheral blood (PB) and synovial fluid (SF) samples from eight patients with antibiotic-responsive arthritis and fifteen patients with antibiotic-refractory arthritis using flow cytometry and cytokine analyses.

Results

In antibiotic-responsive patients, who were seen during active infection, high frequencies of CD56bright NK cells were found in SF, the inflammatory site, compared with PB (P <0.001); at both sites, a high percentage of cells expressed the activation receptor NKG2D and the chaperone CD94, a low percentage expressed inhibitory killer immunoglobulin-like receptors (KIR), and a high percentage produced IFN-γ. In antibiotic-refractory patients, who were usually evaluated near the conclusion of antibiotics when few if any live spirochetes remained, the phenotype of CD56bright cells in SF was similar to that in patients with antibiotic-responsive arthritis, but the frequency of these cells was significantly less (P = 0.05), and the frequencies of CD56dim NK cells tended to be higher. However, unlike typical NKdim cells, these cells produced large amounts of IFN-γ, suggesting that they were not serving a cytotoxic function. Lastly, iNKT cell frequencies in the SF of antibiotic-responsive patients were significantly greater compared with that of antibiotic-refractory patients where these cells were often absent (P = 0.003).

Conclusions

In patients with antibiotic-responsive arthritis, the high percentage of activated, IFN-γ-producing CD56bright NK cells in SF and the presence of iNKT cells suggest that these cells still have a role in spirochetal killing late in the illness. In patients with antibiotic-refractory arthritis, the frequencies of IFN-γ-producing CD56bright and CD56dim NK cells remained high in SF, even after spirochetal killing, suggesting that these cells contribute to excessive inflammation and immune dysregulation in joints, and iNKT cells, which may have immunomodulatory effects, were often absent.     

Impact of genotyping on outcome of prostatic biopsies: a multicenter prospective study

Single nucleotide polymorphisms (SNPs) have been associated with prostate cancer (PCa) risk and tumor aggressiveness in retrospective studies. To assess the value of genotyping in a clinical setting, we evaluated the correlation between three genotypes (rs1447295 and rs6983267[8q24] and rs4054823[17p12]) and prostatic biopsy outcome prospectively in a French population of Caucasian men. Five hundred ninety-eight patients with prostatic-specific antigen (PSA) >4 ng/mL or abnormal digital rectal examination (DRE) participated in this prospective, multicenter study. Age, familial history of PCa, body mass index (BMI), data of DRE, International Prostate Symptom Score (I-PSS) score, PSA value and prostatic volume were collected prospectively before prostatic biopsy. Correlation between genotypes and biopsy outcome (positive or negative) and Gleason score (≤6 or >6) were studied by univariate and multivariable analysis. rs1447295 and rs6983267 risk variants were found to be associated with the presence of PCa in univariate analysis. rs6983267 genotype remained significantly linked to a positive biopsy (odds ratio [OR] = 1.66, 95% confidence interval [CI]: 1.06-2.59, P = 0.026) in multivariable analysis, but rs1447295 genotype did not (OR = 1.47, 95% CI: 0.89-2.43, P = 0.13).When biopsy outcome was stratified according to Gleason score, risk variants of rs1447295 were associated with aggressive disease (Gleason score ≥7) in univariate and multivariable analysis (OR = 2.05 95% CI: 1.10-3.79, P = 0.023). rs6983267 GG genotype was not related to aggressiveness. The results did not reach significance concerning rs4054823 for any analysis. This inaugural prospective evaluation thus confirmed potential usefulness of genotyping PCa assessment. Ongoing clinical evaluation of larger panels of SNPs will detail the actual impact of genetic markers on clinical practice.     

The genetics of vitamin C loss in vertebrates

Vitamin C (ascorbic acid) plays important roles as an anti-oxidant and in collagen synthesis. These important roles, and the relatively large amounts of vitamin C required daily, likely explain why most vertebrate species are able to synthesize this compound. Surprisingly, many species, such as teleost fishes, anthropoid primates, guinea pigs, as well as some bat and Passeriformes bird species, have lost the capacity to synthesize it. Here, we review the genetic bases behind the repeated losses in the ability to synthesize vitamin C as well as their implications. In all cases so far studied, the inability to synthesize vitamin C is due to mutations in the L-gulono-γ-lactone oxidase (GLO) gene which codes for the enzyme responsible for catalyzing the last step of vitamin C biosynthesis. The bias for mutations in this particular gene is likely due to the fact that losing it only affects vitamin C production. Whereas the GLO gene mutations in fish, anthropoid primates and guinea pigs are irreversible, some of the GLO pseudogenes found in bat species have been shown to be reactivated during evolution. The same phenomenon is thought to have occurred in some Passeriformes bird species. Interestingly, these GLO gene losses and reactivations are unrelated to the diet of the species involved. This suggests that losing the ability to make vitamin C is a neutral trait.