Splice Variants of Activation Induced Deaminase (AID) Do Not Affect the Efficiency of Class Switch Recombination in Murine CH12F3 Cells

<div><p>Activation Induced Deaminase (AID) triggers the antigen-driven antibody diversification processes through its ability to edit DNA. AID dependent DNA damage is also the cause of genetic alterations often found in mature B cell tumors. A number of splice variants of AID have been identified, for which a role in the modulation of its activity has been hypothesized. We have thus tested two of these splice variants, which we find catalytically inactive, for their ability to modulate the activity of endogenous AID in CH12F3 cells, a murine lymphoma cell line in which Class Switch Recombination (CSR) can be induced. In contrast to full-length AID, neither these splice variants or a catalytically impaired AID mutant affect the efficiency of Class Switch Recombination. Thus, while a role for these splice variants at the RNA level remains possible, it is unlikely that they exert any regulatory effect on the function of AID.</p></div

Schematic representation of the splice variants of AID and their activity in bacteria.

<p>(A) The exonic structure of the splice isoforms is shown. The position of functional features of the full-length AID (AID-FL) is indicated: the catalytic domain, the cytoplasmic retention signal (CRS; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121719#pone.0121719.ref020" target="_blank">20</a>]) and the nuclear export signal (NES; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121719#pone.0121719.ref016" target="_blank">16</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121719#pone.0121719.ref019" target="_blank">19</a>]). The coding sequence appears in grey and the retained intron 3 is indicated (AID-ivs3) by the angle-striped pattern. The asterisks indicate the AID isoforms tested in the study. (B) Western blot analysis showing the expression levels of AID-FL, AID-ΔE4, AID-ivs3, or an empty plasmid in KL16 bacteria after induction with IPTG. Equal amounts of protein lysates (10 μg) were loaded on SDS-PAGE. The apparent molecular weights from prestained protein ladder are shown on left. The arrows indicate the expected molecular weight of the AID isoforms. The asterisk indicates an unspecific band. The asterisk indicates an unspecific band. (C) Rifampicin assay using the various AID isoforms. Only the revertants resistant to rifampicin can grow. While AID induces a mutator phenotype (<i>P</i><10⁻³ by Dunn’s multiple comparison test), the tested splice variants display levels of revertants similar to the negative control (empty plasmid). The mutation rate is calculated after normalization with Ampicillin resistant viable colonies. The median value is indicated.</p

Expression of the AID variants in HEK293T cells.

<p>Western blot analysis of HEK293T cells transiently transfected with the various constructs (AID-FL, AID-ΔE4, AID-ivs3, AID^E58A). Equal amounts of protein lysates (30 μg) were loaded on SDS-PAGE. ß-actin was used as a loading control. The apparent molecular weights from prestained protein ladder are shown on left. The arrows indicate the expected molecular weight of the AID isoforms. The asterisks indicate unspecific bands.</p

Effects of common gram&#8209;negative pathogens causing male genitourinary&#8209;tract infections on human sperm functions

Male genitourinary tract (MGT) bacterial infections are considered responsible for 15% of male infertility, but the mechanisms underlying decreased semen quality are poorly known. We evaluated in vitro the effect of strains of Gram-negative uropathogenic species (two E.coli strains, three K. pneumoniae strains, P. aeruginosa and E. cloacae) on motility, viability, mitochondrial oxidative status, DNA fragmentation and caspase activity of human spermatozoa. All strains, except P. aeruginosa, reduced significantly sperm motility, with variable effects. Sperm Immobilizing Factor (SIF) was largely responsible for deteriorating effects on sperm motility of E. coli strains since they were completely reverted by knockout of SIF coding recX gene. Sequence alignment for RecX showed the presence of high homologous sequences in K. pneumoniae and E. cloacae but not in P. aeruginosa. These results suggest that, in addition to E.coli, other common uropathogenic Gram-negative bacteria affect sperm motility through RecX products. In addition to sperm motility, the E. coli strain ATCC 35218 also affected sperm viability, and induced caspase activity, oxidative stress and DNA fragmentation suggesting an interspecies variability in the amount and/or type of the produced spermatotoxic factors. In general, our results highlight the need for a careful evaluation of semen infections in the diagnostic process of the infertile man

Hypervirulent Klebsiella pneumoniae Strains Modulate Human Dendritic Cell Functions and Affect TH1/TH17 Response

Hypervirulent Klebsiella pneumoniae (Hv-Kp) strains have emerged as pathogens causing life-threatening, invasive disease even in immunocompetent hosts. Systemic dissemination usually occurs following perturbations of the gut microbiota and is facilitated by Hv-Kp resistance to phagocytosis and complement activity. Hv-Kp are usually associated with K1 or K2 capsular types, produce several iron uptake systems (e.g., aerobactin and salmochelin) and are often but not invariably, capsular material hyper-producers (hypermucoviscous phenotype: HMV). Whether Hv-Kp escape the immune response at mucosal site is unknown. In this work, we studied the effects of Hv-Kp on human dendritic cells (DCs), central players of the IL-23/IL-17 and IL-12/IFN-&gamma; axis at mucosal sites, essential for pathogen clearance. Four Hv-Kp and HMV strains were selected and their activity on DC maturation and cytokine production was compared to that of non-virulent Kp strains with classic or HMV phenotypes. While the maturation process was equally induced by all Kp strains, significant differences between virulent and non-virulent strains were found in the expression of genes for cytokines involved in T-cell activation and differentiation. The non-virulent KP04C62 and the classic Kp, KPC157 induced high expression of TH1 (IL-12p70 and TNF&alpha;) and TH17 cytokines (IL-23, IL-1&beta; and IL-6), while Hv-Kp poorly activated these cytokine genes. Moreover, conditioned media from DCs cultured with non-virulent Kp, either classical or hypercapsulated, induced the activation of IL-17 and IFN-&gamma; genes in preactivated CD4+-cells suggesting their TH17/TH1 differentiation. Conditioned media from Hv-Kp poorly activated IL-17 and IFN-&gamma; genes. In summary, our data indicate that Hv-Kp interfere with DC functions and T-cell differentiation and suggest that the escape from the IL-23/IL-17 and IL-12/IFN-&gamma; axes may contribute to pathogen dissemination in immunocompetent hosts

COVID-19 annual update: a narrative review

Abstract Three and a half years after the pandemic outbreak, now that WHO has formally declared that the emergency is over, COVID-19 is still a significant global issue. Here, we focus on recent developments in genetic and genomic research on COVID-19, and we give an outlook on state-of-the-art therapeutical approaches, as the pandemic is gradually transitioning to an endemic situation. The sequencing and characterization of rare alleles in different populations has made it possible to identify numerous genes that affect either susceptibility to COVID-19 or the severity of the disease. These findings provide a beginning to new avenues and pan-ethnic therapeutic approaches, as well as to potential genetic screening protocols. The causative virus, SARS-CoV-2, is still in the spotlight, but novel threatening virus could appear anywhere at any time. Therefore, continued vigilance and further research is warranted. We also note emphatically that to prevent future pandemics and other world-wide health crises, it is imperative to capitalize on what we have learnt from COVID-19: specifically, regarding its origins, the world’s response, and insufficient preparedness. This requires unprecedented international collaboration and timely data sharing for the coordination of effective response and the rapid implementation of containment measures