Redefining the bacteriophage mv4 site-specific recombination system and the sequence specificity of its attB and core-attP sites.

Through their involvement in the integration and excision of a large number of mobile genetic elements, such as phages and integrative and conjugative elements (ICEs), site-specific recombination systems based on heterobivalent tyrosine recombinases play a major role in genome dynamics and evolution. However, despite hundreds of these systems having been identified in genome databases, very few have been described in detail, with none from phages that infect Bacillota (formerly Firmicutes). In this study, we reanalyzed the recombination module of Lactobacillus delbrueckii subsp. bulgaricus phage mv4, previously considered atypical compared with classical systems. Our results reveal that mv4 integrase is a 369 aa protein with all the structural hallmarks of recombinases from the Tn916 family and that it cooperatively interacts with its recombination sites. Using randomized DNA libraries, NGS sequencing, and other molecular approaches, we show that the 21-bp core-attP and attB sites have structural similarities to classical systems only if considering the nucleotide degeneracy, with two 7-bp inverted regions corresponding to mv4Int core-binding sites surrounding a 7-bp strand-exchange region. We also examined the different compositional constraints in the core-binding regions, which define the sequence space of permissible recombination sites.

A subclass of the IS1202 family of bacterial insertion sequences targets XerCD recombination sites.

We describe here a new family of IS which are related to IS1202, originally isolated from Streptococcus pneumoniae in the mid-1990s and previously tagged as an emerging IS family in the ISfinder database. Members of this family have impacted some important properties of their hosts. We describe here another potentially important property of certain family members: specific targeting of xrs recombination sites. The family could be divided into three subgroups based on their transposase sequences and the length on the target repeats (DR) they generate on insertion: subgroup IS1202 (24-29 bp); ISTde1 (15-18 bp); and ISAba32 (5-6 bp). Members of the ISAba32 subgroup were repeatedly found abutting Xer recombinase recombination sites (xrs), separated by an intervening copy of a DR. These xrs sites, present in multiple copies in a number of Acinetobacter plasmids flanking antibiotic resistance genes, were proposed to form a new type of mobile genetic element using the chromosomally-encoded XerCD recombinase for mobility. Transposase alignments identified subgroup-specific indels which may be responsible for the differences in the transposition properties of the three subgroups (i.e. DR length and target specificity). We propose that this collection of IS be classed as a new insertion sequence family: the IS1202 family composed of three subgroups, only one of which specifically targets plasmid-borne xrs. We discuss the implications of xrs targeting for gene mobility.

DNA Segregation in Enterobacteria.

DNA segregation ensures that cell offspring receive at least one copy of each DNA molecule, or replicon, after their replication. This important cellular process includes different phases leading to the physical separation of the replicons and their movement toward the future daughter cells. Here, we review these phases and processes in enterobacteria with emphasis on the molecular mechanisms at play and their controls.

Level of knowledge on conservative management of adolescent idiopathic scoliosis among undergraduate students in healthcare: A scoping review.

BACKGROUND: Adolescent Idiopathic Scoliosis (AIS) is a three-dimensional deformity of the spine associated with pain and aesthetic changes. Various health care professionals may be led to evaluate and/or manage adolescents presenting AIS. There is no compiling of the studies evaluating the level of knowledge on AIS conservative management among undergraduate students in healthcare. OBJECTIVE: To identify and map the current studies evaluating the level of knowledge of health profession students on AIS conservative management. DESIGN: Scoping review. METHODS: The search strategy was conducted in Scopus database. Three concepts were included: "Adolescent idiopathic scoliosis", "Knowledge" and "Undergraduate students studying in a healthcare field". Studies identification included (1) duplicates removing, (2) title and abstract screening, and (3) full-text screening. The quality of the included studies was assessed. Studies' characteristics were extracted, and results were summarized. RESULTS: Searches yielded the identification of 245 citations. After duplicates removal and abstract screening, three full-text articles were identified. Following full-text review, two full-text articles were finally included. Both studies evaluated students in physiotherapy, but using distinct questionnaires. Both studies reported an unsatisfactory level of knowledge. CONCLUSION: Knowledge of future health professionals about the conservative management of AIS has been barely evaluated. Therefore, no conclusion can be drawn regarding the level of knowledge of undergraduate health professions' students on AIS conservative management. The development a standardized questionnaire to adequately assess this knowledge across institutions and professions is required.

Long-term male-specific chronic pain via telomere- and p53­mediated spinal cord cellular senescence.

Mice with experimental nerve damage can display long­lasting neuropathic pain behavior. We show here that 4 months and later after nerve injury, male but not female mice displayed telomere length (TL) reduction and p53­mediated cellular senescence in the spinal cord, resulting in maintenance of pain and associated with decreased lifespan. Nerve injury increased the number of p53­positive spinal cord neurons, astrocytes, and microglia, but only in microglia was the increase male­specific, matching a robust sex specificity of TL reduction in this cell type, which has been previously implicated in male­specific pain processing. Pain hypersensitivity was reversed by repeated intrathecal administration of a p53­specific senolytic peptide, only in male mice and only many months after injury. Analysis of UK Biobank data revealed sex-specific relevance of this pathway in humans, featuring male­specific genetic association of the human p53 locus (TP53) with chronic pain and a male-specific effect of chronic pain on mortality. Our findings demonstrate the existence of a biological mechanism maintaining pain behavior, at least in males, occurring much later than the time span of virtually all extant preclinical studies.

Single-Molecule Sandwich Aptasensing on Nanoarrays by Tethered Particle Motion Analysis.

High-throughput single-molecule techniques are expected to challenge the demand for rapid, simple, and sensitive detection methods in health and environmental fields. Based on a single-DNA-molecule biochip for the parallelization of tethered particle motion analyses by videomicroscopy coupled to image analysis and its smart combination with aptamers, we successfully developed an aptasensor enabling the detection of single target molecules by a sandwich assay. One aptamer is grafted to the nanoparticles tethered to the surface by a long DNA molecule bearing the second aptamer in its middle. The detection and quantification of the target are direct. The recognition of the target by a pair of aptamers leads to a looped configuration of the DNA-particle complex associated with a restricted motion of the particles, which is monitored in real time. An analytical range extending over 3 orders of magnitude of target concentration with a limit of detection in the picomolar range was obtained for thrombin.

[COVID-19 crisis management during the first three waves in a large dialysis organisation: Feedback from NephroCare France]. / Gestion de la crise COVID-19 dans une organisation multicentrique de dialyse au cours des trois premières vagues : retour d'expérience de NephroCare France.

At the start of the COVID crisis, NephroCare operated 40 dialysis units in 7 regions, with 2,740 hemodialysis patients. The national COVID-19 crisis team implemented early the necessary measures to ensure the safety of dialysis patients and caregivers in the context of the pandemic. These measures were mostly traditional, but some were specific to our organization. They were modified during the 3 successive waves. The first wave mainly impacted NephroCare Ile-de-France which recorded 75% of the contaminations with an impact on the dialysis parameters of non-COVID patients which was not found during the second wave due to reduced stress (34% of contaminations) and a better management of COVID+ patients. The effectiveness of the measures put in place is suggested by the absence of PCR+ in asymptomatic patients and the perfect adequacy of the anti-SARS-CoV2 antibodies with the diagnosis of COVID in one severely impacted Ile-de-France unit, opposite to literature reporting significant rates of positive PCR or serology in asymptomatic patients. In addition, the contamination rate was calculated below the national rate reported by the Biomedicine Agency. The third wave was marked by the implementation of the anti-SARS-CoV2 vaccination with a proportion of vaccinated patients not different from national data and a decrease in COVID cases at the end of the third wave while the national incidence remained stable on the period. In conclusion, this experience of facing an unprecedented serious situation showed the responsiveness of the organization, significant innovations and the efficacy of the implemented measures.

Devices Used to Measure Force-Time Characteristics of Spinal Manipulations and Mobilizations: A Mixed-Methods Scoping Review on Metrologic Properties and Factors Influencing Use.

Background: Spinal manipulations (SMT) and mobilizations (MOB) are interventions commonly performed by many health care providers to manage musculoskeletal conditions. The clinical effects of these interventions are believed to be, at least in part, associated with their force-time characteristics. Numerous devices have been developed to measure the force-time characteristics of these modalities. The use of a device may be facilitated or limited by different factors such as its metrologic properties. Objectives: This mixed-method scoping review aimed to characterize the metrologic properties of devices used to measure SMT/MOB force-time characteristics and to determine which factors may facilitate or limit the use of such devices within the context of research, education and clinical practice. Methods: This study followed the Joanna Briggs Institute's framework. The literature search strategy included four concepts: (1) devices, (2) measurement of SMT or MOB force-time characteristics on humans, (3) factors facilitating or limiting the use of devices, and (4) metrologic properties. Two reviewers independently reviewed titles, abstracts and full articles to determine inclusion. To be included, studies had to report on a device metrologic property (e.g., reliability, accuracy) and/or discuss factors that may facilitate or limit the use of the device within the context of research, education or clinical practice. Metrologic properties were extracted per device. Limiting and facilitating factors were extracted and themes were identified. Results: From the 8,998 studies initially retrieved, 46 studies were finally included. Ten devices measuring SMT/MOB force-time characteristics at the clinician-patient interface and six measuring them at patient-table interfaces were identified. Between zero and eight metrologic properties were reported per device: measurement error (defined as validity, accuracy, fidelity, or calibration), reliability/repeatability, coupling/crosstalk effect, linearity/correlation, sensitivity, variability, drift, and calibration. From the results, five themes related to the facilitating and limiting factors were developed: user-friendliness and versatility, metrologic/intrinsic properties, cost and durability, technique application, and feedback. Conclusion: Various devices are available to measure SMT/MOB force-time characteristics. Metrologic properties were reported for most devices, but terminology standardization is lacking. The usefulness of a device in a particular context should be determined considering the metrologic properties as well as other potential facilitating and limiting factors.

Post-replicative pairing of sister ter regions in Escherichia coli involves multiple activities of MatP.

The ter region of the bacterial chromosome, where replication terminates, is the last to be segregated before cell division in Escherichia coli. Delayed segregation is controlled by the MatP protein, which binds to specific sites (matS) within ter, and interacts with other proteins such as ZapB. Here, we investigate the role of MatP by combining short-time mobility analyses of the ter locus with biochemical approaches. We find that ter mobility is similar to that of a non ter locus, except when sister ter loci are paired after replication. This effect depends on MatP, the persistence of catenanes, and ZapB. We characterise MatP/DNA complexes and conclude that MatP binds DNA as a tetramer, but bridging matS sites in a DNA-rich environment remains infrequent. We propose that tetramerisation of MatP links matS sites with ZapB and/or with non-specific DNA to promote optimal pairing of sister ter regions until cell division.

Dynamical control of denaturation bubble nucleation in supercoiled DNA minicircles.

We examine the behavior of supercoiled DNA minicircles containing between 200 and 400 base-pairs, also named microDNA, in which supercoiling favors thermally assisted DNA denaturation bubbles of nanometer size and controls their lifetime. Mesoscopic modeling and accelerated dynamics simulations allow us to overcome the limitations of atomistic simulations encountered in such systems, and offer detailed insight into the thermodynamic and dynamical properties associated with the nucleation and closure mechanisms of long-lived thermally assisted denaturation bubbles which do not stem from bending- or torque-driven stress. Suitable tuning of the degree of supercoiling and size of specifically designed microDNA is observed to lead to the control of opening characteristic times in the millisecond range, and closure characteristic times ranging over well distinct timescales, from microseconds to several minutes. We discuss how our results can be seen as a dynamical bandwidth which might enhance selectivity for specific DNA binding proteins.

An improved Xer-cise technology for the generation of multiple unmarked mutants in Mycobacteria.

Xer-cise is a technique using antibiotic resistance cassettes flanked by dif sites allowing spontaneous and accurate excision from bacterial chromosomes with a high frequency through the action of the cellular recombinase XerCD. Here, we report a significant improvement of Xer-cise in Mycobacteria. Zeocin resistance cassettes flanked by variants of the natural Mycobacterium tuberculosis dif site were constructed and shown to be effective tools to construct multiple unmarked mutations in M. tuberculosis and in the model species Mycobacterium smegmatis. The dif site variants harbor mutations in the central region and can therefore not recombine with the wild-type or other variants, resulting in mutants of increased genetic stability. The herein described method should be generalizable to virtually any transformable bacterial species.

How does temperature impact the conformation of single DNA molecules below melting temperature?

The double stranded DNA molecule undergoes drastic structural changes during biological processes such as transcription during which it opens locally under the action of RNA polymerases. Local spontaneous denaturation could contribute to this mechanism by promoting it. Supporting this idea, different biophysical studies have found an unexpected increase in the flexibility of DNA molecules with various sequences as a function of the temperature, which would be consistent with the formation of a growing number of locally denatured sequences. Here, we take advantage of our capacity to detect subtle changes occurring on DNA by using high throughput tethered particle motion to question the existence of bubbles in double stranded DNA under physiological salt conditions through their conformational impact on DNA molecules ranging from several hundreds to thousands of base pairs. Our results strikingly differ from previously published ones, as we do not detect any unexpected change in DNA flexibility below melting temperature. Instead, we measure a bending modulus that remains stable with temperature as expected for intact double stranded DNA.

Zscan4 Inhibits Maintenance DNA Methylation to Facilitate Telomere Elongation in Mouse Embryonic Stem Cells.

Proper telomere length is essential for embryonic stem cell (ESC) self-renewal and pluripotency. Mouse ESCs (mESCs) sporadically convert to a transient totipotent state similar to that of two-cell (2C) embryos to recover shortened telomeres. Zscan4, which exhibits a burst of expression in 2C-like mESCs, is required for telomere extension in these cells. However, the mechanism by which Zscan4 extends telomeres remains elusive. Here, we show that Zscan4 facilitates telomere elongation by inducing global DNA demethylation through downregulation of Uhrf1 and Dnmt1, major components of the maintenance DNA methylation machinery. Mechanistically, Zscan4 recruits Uhrf1 and Dnmt1 and promotes their degradation, which depends on the E3 ubiquitin ligase activity of Uhrf1. Blocking DNA demethylation prevents telomere elongation associated with Zscan4 expression, suggesting that DNA demethylation mediates the effect of Zscan4. Our results define a molecular pathway that contributes to the maintenance of telomere length homeostasis in mESCs.

SMARCD2 subunit of SWI/SNF chromatin-remodeling complexes mediates granulopoiesis through a CEBPɛ dependent mechanism.

Recent studies suggest that individual subunits of chromatin-remodeling complexes produce biologically specific meaning in different cell types through combinatorial assembly. Here we show that granulocyte development requires SMARCD2, a subunit of ATP-dependent SWI/SNF (BAF) chromatin-remodeling complexes. Smarcd2-deficient mice fail to generate functionally mature neutrophils and eosinophils, a phenotype reminiscent of neutrophil-specific granule deficiency (SGD) in humans, for which loss-of-function mutations in CEBPE (encoding CEBPɛ) have been reported. SMARCD2-containing SWI/SNF complexes are necessary for CEBPɛ transcription factor recruitment to the promoter of neutrophilic secondary granule genes and for granulocyte differentiation. The homologous SMARCD1 protein (63% identical at the amino acid level) cannot replace the role of SMARCD2 in granulocyte development. We find that SMARCD2 functional specificity is conferred by its divergent coiled-coil 1 and SWIB domains. Strikingly, both CEBPE and SMARCD2 loss-of-function mutations identified in patients with SGD abolish the interaction with SWI/SNF and thereby secondary granule gene expression, thus providing a molecular basis for this disease.

Protein kinase CK2 controls T-cell polarization through dendritic cell activation in response to contact sensitizers.

Allergic contact dermatitis (ACD) represents a severe health problem with increasing worldwide prevalence. It is a T-cell-mediated inflammatory skin disease caused by chemicals present in the daily or professional environment. NiSO4 and 2,4-dinitrochlorobenzene (DNCB) are 2 chemicals involved in ACD. These contact sensitizers are known to induce an up-regulation of phenotypic markers and cytokine secretion in dendritic cells (DCs; professional APCs), leading to the generation of CD8+ Tc1/Tc17 and CD4+ Th1/Th17 effector T cells. In the present study, using a peptide array approach, we identified protein kinase CK2 as a novel kinase involved in the activation of human monocyte-derived DCs (MoDCs) in response to NiSO4 and DNCB. Inhibition of CK2 activity in MoDCs led to an altered mature phenotype with lower expression of CD54, PDL-1, CD86, and CD40 in response to NiSO4 or DNCB. CK2 activity also regulated proinflammatory cytokine production, such as TNF-α, IL-1ß, and IL-23 in MoDCs. Moreover, in a DC/T cell coculture model in an allogeneic setup, CK2 activity in MoDCs played a major role in Th1 polarization in response to NiSO4 and DNCB. CK2 inhibition in MoDCs led to an enhanced Th2 polarization in the absence of contact sensitizer stimulation.

FtsK translocation permits discrimination between an endogenous and an imported Xer/dif recombination complex.

In bacteria, the FtsK/Xer/dif (chromosome dimer resolution site) system is essential for faithful vertical genetic transmission, ensuring the resolution of chromosome dimers during their segregation to daughter cells. This system is also targeted by mobile genetic elements that integrate into chromosomal dif sites. A central question is thus how Xer/dif recombination is tuned to both act in chromosome segregation and stably maintain mobile elements. To explore this question, we focused on pathogenic Neisseria species harboring a genomic island in their dif sites. We show that the FtsK DNA translocase acts differentially at the recombination sites flanking the genomic island. It stops at one Xer/dif complex, activating recombination, but it does not stop on the other site, thus dismantling it. FtsK translocation thus permits cis discrimination between an endogenous and an imported Xer/dif recombination complex.

The Ability of American Football Helmets to Manage Linear Acceleration With Repeated High-Energy Impacts.

CONTEXT: Football players can receive up to 1400 head impacts per season, averaging 6.3 impacts per practice and 14.3 impacts per game. A decrease in the capacity of a helmet to manage linear acceleration with multiple impacts could increase the risk of traumatic brain injury. OBJECTIVE: To investigate the ability of football helmets to manage linear acceleration with multiple high-energy impacts. DESIGN: Descriptive laboratory study. SETTING: Laboratory. MAIN OUTCOME MEASURE(S): We collected linear-acceleration data for 100 impacts at 6 locations on 4 helmets of different models currently used in football. Impacts 11 to 20 were compared with impacts 91 to 100 for each of the 6 locations. RESULTS: Linear acceleration was greater after multiple impacts (91-100) than after the first few impacts (11-20) for the front, front-boss, rear, and top locations. However, these differences are not clinically relevant as they do not affect the risk for head injury. CONCLUSIONS: American football helmet performance deteriorated with multiple impacts, but this is unlikely to be a factor in head-injury causation during a game or over a season.

An intact putative mouse telomerase essential N-terminal domain is necessary for proper telomere maintenance.

BACKGROUND INFORMATION: Naturally occurring telomerase reverse transcriptase (TERT) isoforms may regulate telomerase activity, and possibly function independently of telomeres to modulate embryonic stem (ES) cell self-renewal and differentiation. RESULTS: We report the characterisation of two novel mouse TERT (mTERT) splice variants, Ins-i1[1-102] (Insi1 for short) and Del-e12[1-40] (Dele12 for short) that have not been previously described. Insi1 represents an in-frame insertion of nucleotides 1-102 from intron 1, encoding a 34 amino acid insertion at amino acid 73. Based on known functions of this region in human and Tetrahymena TERTs, the insertion interrupts the RNA interaction domain 1 implicated in low-affinity RNA binding and the telomerase essential N-terminal domain implicated in DNA substrate interactions. Dele12 contains a 40 nucleotide deletion of exon 12 which generates a premature stop codon, and possible protein lacking the C-terminus. We found Insi1 expressed in adult mouse brain and kidney and Dele12 expressed in adult mouse ovary. Dele12 was inactive in vitro and in mTERT(-/-) ES cells and Insi1 retained 26-48% of telomerase activity reconstituted by wild-type mTERT in vitro and in mTERT(-/-) ES cells. The Insi1 variant exhibited reduced DNA substrate binding in vitro and both variants exhibited a reduction in binding the telomerase RNA, mTR, when expressed in mTERT(-/-) ES cells. Stable expression of Dele12 in the mouse fibroblast CB17 cell line inhibited telomerase activity and slowed cell growth, suggesting a potential dominant-negative effect. Levels of signal-free ends, representing short telomeres, and end-to-end fusions were higher in mTERT(-/-) ES cells expressing mTERT-Insi1 and mTERT-Dele12, compared with levels observed in mTERT(-/-) ES cells expressing wild-type mTERT. In addition, in mTERT(-/-) cells expressing mTERT-Insi1, we observed chromosomes that were products of repeated breakage-bridge-fusion cycles and other telomere dysfunction-related aberrations. CONCLUSION AND SIGNIFICANCE: An intact mTERT N-terminus which contributes to mTR binding, DNA binding and telomerase activity is necessary for elongation of short telomeres and the maintenance of functional telomeres. It is reasonable to speculate that relative levels of mTERT-Insi1 may regulate telomere function in specific tissues.

Platinum(II) phenanthroimidazole G-quadruplex ligand induces selective telomere shortening in A549 cancer cells.

Telomere maintenance, achieved by the binding of protective shelterin capping proteins to telomeres and by either telomerase or a recombination-based alternative lengthening of telomere (ALT) mechanism, is critical for cell proliferation and survival. Extensive telomere shortening or loss of telomere integrity activates DNA damage checkpoints, leading to cell senescence or death. Although telomerase upregulation is an attractive target for anti-cancer therapy, the lag associated with telomere shortening and the potential activation of ALT pose a challenge. An alternative approach is to modify telomere interactions with binding proteins (telomere uncapping). G-quadruplex ligands stabilize structures generated from single-stranded G-rich 3'-telomere end (G-quadruplex) folding, which in principle, cannot be elongated by telomerase, thus leading to telomere shortening. Ligands can also mediate rapid anti-proliferative effects by telomere uncapping. We previously reported that the G-quadruplex ligand, phenylphenanthroimidazole ethylenediamine platinum(II) (PIP), inhibits telomerase activity in vitro[47]. In the current study, a long-term seeding assay showed that PIP significantly inhibited the seeding capacity of A549 lung cancer cells and to a lesser extent primary MRC5 fibroblast cells. Importantly, treatment with PIP caused a significant dose- and time-dependent decrease in average telomere length of A549 but not MRC5 cells. Moreover, cell cycle analysis revealed a significant increase in G1 arrest upon treatment of A549 cells, but not MRC5 cells. Both apoptosis and cellular senescence may contribute to the anti-proliferative effects of PIP. Our studies validate the development of novel and specific therapeutic ligands targeting telomeric G-quadruplex structures in cancer cells.

A novel somatic mutation in ACD induces telomere lengthening and apoptosis resistance in leukemia cells.

BACKGROUND: The identification of oncogenic driver mutations has largely relied on the assumption that genes that exhibit more mutations than expected by chance are more likely to play an active role in tumorigenesis. Major cancer sequencing initiatives have therefore focused on recurrent mutations that are more likely to be drivers. However, in specific genetic contexts, low frequency mutations may also be capable of participating in oncogenic processes. Reliable strategies for identifying these rare or even patient-specific (private) mutations are needed in order to elucidate more personalized approaches to cancer diagnosis and treatment. METHODS: Here we performed whole-exome sequencing on three cases of childhood pre-B acute lymphoblastic leukemia (cALL), representing three cytogenetically-defined subgroups (high hyperdiploid, t(12;21) translocation, and cytogenetically normal). We applied a data reduction strategy to identify both common and rare/private somatic events with high functional potential. Top-ranked candidate mutations were subsequently validated at high sequencing depth on an independent platform and in vitro expression assays were performed to evaluate the impact of identified mutations on cell growth and survival. RESULTS: We identified 6 putatively damaging non-synonymous somatic mutations among the three cALL patients. Three of these mutations were well-characterized common cALL mutations involved in constitutive activation of the mitogen-activated protein kinase pathway (FLT3 p.D835Y, NRAS p.G13D, BRAF p.G466A). The remaining three patient-specific mutations (ACD p.G223V, DOT1L p.V114F, HCFC1 p.Y103H) were novel mutations previously undescribed in public cancer databases. Cytotoxicity assays demonstrated a protective effect of the ACD p.G223V mutation against apoptosis in leukemia cells. ACD plays a key role in protecting telomeres and recruiting telomerase. Using a telomere restriction fragment assay, we also showed that this novel mutation in ACD leads to increased telomere length in leukemia cells. CONCLUSION: This study identified ACD as a novel gene involved in cALL and points to a functional role for ACD in enhancing leukemia cell survival. These results highlight the importance of rare/private somatic mutations in understanding cALL etiology, even within well-characterized molecular subgroups.