Cross-sectional multicentre study on the cohort of all the French junior lecturers in general practice

Background: General practice became an academic discipline quite recently in many western countries. In France, junior lecturer work is specified in a three-part mandate: medical work in general practice, teaching in the university, and research. Since 2007, 130 junior lecturers have been appointed in general practice. The aim of the creation of junior lecturer status was to align general practice with other specialties and to develop research and education in primary care.

Objectives: To describe the healthcare, teaching and research undertaken by junior lecturers in general practice, practising in October 2014.

Methods: A cross-sectional multicentre study using an online self-administered questionnaire on the cohort composed of all the junior lecturers in general practice with open questions and the qualitative analysis of written verbatim accounts.

Results: Of the 95 junior lecturers practising at the date of the study, 75 (79%) responded; average age 32 years; gender ratio (F/M) 2.4:1. They spent five, two and three half-days per week respectively in healthcare, teaching and research. The healthcare activity was predominantly carried out in the community (73%). Thirty-nine per cent worked as part of a multi-professional team taking on 50 consultations per week. Most of the educational work involved lecturing and mentoring students specializing in general practice (median 86?hours per year). Research output increased during the fellowship. Research topics were varied and relevant to the disciplinary field.

Conclusion: During the fellowship, the balancing, and even the reinforcement, of healthcare and research contributions were accompanied by a significant investment in educational provision.     

Lateral unicondylar knee arthroplasty (UKA): Contemporary indications,surgical technique,and results

Unicompartmental femoro-tibial osteoarthritis usually affects the medial compartment of the knee, but in 10 %, the lateral compartment is primarily involved. Femoral osteotomy is attractive to avoid TKA in younger patients with low-grade unicompartmental osteoarthritis and a valgus deformity. However, only limited functional results can be expected for patients with Ahlback grade 2 or greater osteoarthritis. Moreover, because of previous skin incisions and hardware removal, TKA after femoral osteotomy remains a complex procedure with poor functional results. Unicompartmental knee arthroplasty for both the medial and the lateral compartments has been performed since the 1970s. In a patient with involvement of only one compartment, a medial or a lateral UKA can provide a quicker recovery and enhanced function when compared to TKA. In addition, it preserves bone stock and can be “easily” revised by a TKA. Technical improvements, combined with strict patient selection, have resulted in ten year survivorships greater than 90 %. However, lateral UKA is technically more challenging than medial UKA due to the lower number of indications, as well as the functional anatomy of the lateral compartment. The goals of this article are to present up-to-date information concerning indications, patients’ selection, surgical technique and results of lateral compartment UKA.     

In vitro evolution of antibody affinity via insertional scanning mutagenesis of an entire antibody variable region

We report a systematic combinatorial exploration of affinity enhancement of antibodies by insertions and deletions (InDels). Transposon-based introduction of InDels via the method TRIAD (transposition-based random insertion and deletion mutagenesis) was used to generate large libraries with random in-frame InDels across the entire single-chain variable fragment gene that were further recombined and screened by ribosome display. Knowledge of potential insertion points from TRIAD libraries formed the basis of exploration of length and sequence diversity of novel insertions by insertional-scanning mutagenesis (InScaM). An overall 256-fold affinity improvement of an anti–IL-13 antibody BAK1 as a result of InDel mutagenesis and combination with known point mutations validates this approach, and suggests that the results of this InDel mutagenesis and conventional exploration of point mutations can synergize to generate antibodies with higher affinity.

Powerful selection technologies have made in vitro evolution of protein binders more efficient and paved the way for the use of tailor-made antibodies in therapy. After initial selections of antibody candidates with desired specificity, lead antibodies are typically improved by affinity maturation in multiple rounds of randomization and selection (1) to reach the subnanomolar affinities ideally required for targeting soluble ligands (2–4). This is usually attempted by introduction of point substitutions, either at random positions across the entire V-gene (5, 6) or in the complementary-determining regions (CDRs; e.g., by CDR walking mutagenesis) (7).In Nature, diversification of the primary antibody repertoire occurs by several mechanisms that generate variation in the regions forming the antigen-binding site, the CDRs, including considerable length variation (8–11) that is initially introduced by recombination of V(D)J gene segments. Length variations are concentrated in the CDR3 region (12), at the junctions of the joined segments, where additional diversity is produced by N- or P-nucleotide additions that can further extend the CDR3. The length of the CDRs considerably affects the topography of the combining site, as different shapes brought about by extension or shortening can form pockets, grooves, or fill space (13, 14).Following B cell stimulation by the antigen, further diversification of the antigen-binding interface is generated through somatic hypermutation (SHM) (15), involving mainly point mutagenesis that preferentially targets hotspots in the CDRs (16, 17). This process is initiated through deamination of cytosine to uracil by activation-induced cytidine deaminase (AID), leading to uracil:guanine mismatches (16). Upon removal of these uracil bases by base excision-repair enzymes, error-prone DNA polymerases are then recruited to fill in the gaps and introduce mutations around the position of the deaminated cytosines. Interestingly, up to 6% of the mutations generated by SHM are insertions and deletions (InDels) (18), which occur due to misalignment of repeated DNA sequences (19, 20). Thus, insertions occur by duplication, while deletions are brought about by removal of repeated sequences (21, 22).A small percentage of antibodies selected by in vivo SHM contain InDels in the CDRs 1 and 2 (1.6 to 6.5%) (21–24), while junctional diversity by N- or P-nucleotide additions in the CDR3 confounds the analysis of SHM-derived InDels, leading to an underestimation of the total percentage of affinity-improving InDels. In vitro-directed evolution has been unsuitable for introduction of InDels at random positions into an antibody gene, because of restrictions in the diversity of InDels that could be introduced (i.e., insertions by duplication in in vitro SHM) (22, 25). Rational (26) or computational (27) strategies have been successful at introducing InDels in a few, carefully chosen positions instead of random sampling. In contrast, an unusually high percentage of InDels with a functional role among in vivo affinity matured broadly neutralizing antibodies (bnAbs) to HIV-1 (28–30): ∼40% of the reported anti–HIV-1 bnAbs contain InDels that accumulate during in vivo SHM (28). Based on the frequent occurrence of InDels among multispecific, cross-reactive antibodies, one could infer that they provide a molecular solution for recognizing multiple targets by providing an altered interface (enlarged or tightened), possibly even involving conformational diversity (31). The accumulation of InDels in bnAbs has been attributed to extensive in vivo SHM, so that even positions that are rarely modified by SHM are also altered (17, 28).Insertions in the V-genes occur only by duplication of adjacent sequences (21, 22), so that the actual sequence diversity of the resulting insertions is limited because they repeat existing modules. To introduce more diversity in the inserted sequences, point mutations are required in subsequent rounds of SHM. However, since the CDRs can tolerate considerable length variation, it is likely that the antibody fold can accommodate a larger number of affinity-enhancing InDels compared to those observed in antibodies affinity-matured by SHM.Affinity gains by introduction of InDels have indeed been recognized (22, 25, 26, 32, 33) in in vitro-directed evolution, but often were by-products of campaigns focused on point mutations and not elicited systematically (32, 33). Only in mammalian cell surface display does the action of AID lead to InDels, just as AID brings about InDels in SHM in vivo (22, 25). In a seminal study by Bowers et al. (22), overexpression of AID enabled in vitro SHM of 53 antibodies against 21 antigens to identify InDels in multiple regions likely to improve binding, in particular to variable heavy domain (V_H) and variable light domain (V_L) CDR1, where 9 of 53 antibodies contained InDels. Despite the comprehensive nature of this study, AID-enabled insertions mirrored in vivo SHM and were therefore limited to direct duplication of adjacent sequences, not allowing the full exploration of length and sequence diversity in the insertions, and the low frequency of incorporation of in-frame InDels by AID (<0.1%) limited the combinatorial diversity explored. Finally, InDels have been introduced rationally based on structural analysis and natural length variation (26, 27). Taken together, only limited diversity of InDels in terms of length, position, and insert sequence across the variable domains has been explored thus far.Here we address this omission and explore libraries with in-frame InDels of different lengths and high diversity of inserted sequences at random positions across the entire antibody variable regions (Fig. 1). We applied a new transposon-based mutagenesis approach, dubbed TRIAD (transposition-based random insertion and deletion mutagenesis) (34) that introduces short in-frame insertions and deletions randomly across a gene (in sequences of steps following transposition that excise the transposon, religate the plasmid, and insert designed cassettes) (SI Appendix, Figs. S1 and S2). TRIAD was used here to build libraries with InDels at random positions across an entire single-chain variable fragment (scFv) gene. The antibody chosen for this campaign was the anti–IL-13 antibody BAK1 (35), a derivative of which, tralokinumab, is under clinical investigation for asthma (36). In addition, we built libraries that explore diversity in the different lengths of insertions in a semirandom approach, insertional-scanning mutagenesis (InScaM). These InDel libraries were starting points for antibody affinity evolution in vitro, leading to insertions in two loops that, together with two previously known point mutations, brought about a 256-fold affinity improvement. The observation of alternative routes to affinity maturation validate our strategy and suggest that InDel mutagenesis can complement existing approaches.Open in a separate windowFig. 1.Overview of the affinity maturation of the antibody BAK1 by transposon-based TRIAD and subsequent insertional scanning mutagenesis. TRIAD (Left) was applied to make libraries with deletions of one to three amino acids (step 1a) or single amino acid insertions (step 1b) at random positions across the scFv gene. These libraries were recombined (step 2) and four rounds of ribosome display selections for improved affinity to IL-13 were carried out by panning (step 3). The best binder was carrying an insertion in the V_L FWR3 (BAK1-INS1). Scanning (Right) was used to guide the design of libraries with different lengths of insertions at targeted positions. A fraction of the insertion library generated in step 1b (5,632 variants) was screened by HTRF to identify variants with insertions that retained binding to IL-13 (step 4). Based on sequencing analysis, regions able to tolerate single amino acid insertions were identified (Fig. 4) and the V_L CDR3 was chosen for targeted insertional mutagenesis. Libraries with zero to five amino acid insertions in targeted positions in the V_L CDR3 were constructed (step 5), followed by four rounds of phage display selections for improved affinity to IL-13 (step 6).     

Manual aiming in healthy aging: does proprioceptive acuity make the difference?

The present study examines whether non-active older adults are more dependent on visual information when executing aiming movements and whether age-related declines in proprioception play a mediating role herein. Young (N = 40) and older adults (N = 38) were divided into physically active and non-active subgroups based on self-reported sports participation levels. In experiment 1, participants executed wrist-aiming movements with and without visual feedback. In experiment 2, passive proprioceptive acuity was assessed using wrist motion detection and position matching tests. Results showed similar aiming accuracy across age groups both with and without visual feedback, but older adults exhibited longer movement times, prolonged homing-in phase, and made more corrective submovements. Passive proprioceptive acuity was significantly affected by physical activity level and age, with participants in the active group scoring better than their non-active peers. However, these declines did not predict performance changes on the aiming task. Taken together, our observations suggest that decline in proprioceptive acuity did not predict performance changes on the aiming task and older adults were able to compensate for their decreased motion and position sense when allowed sufficient time. In line with these observations, we proposed that older adults are able to compensate for their decline in proprioception by increasing their reliance on predictive models.     

What about physical contacts between epidermal keratinocytes and sensory neurons?

Recent studies have demonstrated that keratinocytes closely participate in sensory transduction, and therefore, intra‐epidermal free nerve endings are not exclusive transducers of pain. This discovery implies the existence of close afferent communication from keratinocytes to sensory neurons. Although reciprocal interactions between keratinocytes and intra‐epidermal free nerve endings via soluble mediators are well established, little attention has been paid to physical contacts between keratinocytes and intra‐epidermal free nerve endings. This review proposes to consider the ultrastructural and functional knowledge of these contacts, in both human skin biopsies and keratinocyte‐sensory neuron cocultures to speculate on the possible existence of synaptic contacts.     

What are the visuo-motor tendencies of omnidirectional scene free-viewing in virtual reality?

Central and peripheral vision during visual tasks have been extensively studied on two-dimensional screens, highlighting their perceptual and functional disparities. This study has two objectives: replicating on-screen gaze-contingent experiments removing central or peripheral field of view in virtual reality, and identifying visuo-motor biases specific to the exploration of 360 scenes with a wide field of view. Our results are useful for vision modelling, with applications in gaze position prediction (e.g., content compression and streaming). We ask how previous on-screen findings translate to conditions where observers can use their head to explore stimuli. We implemented a gaze-contingent paradigm to simulate loss of vision in virtual reality, participants could freely view omnidirectional natural scenes. This protocol allows the simulation of vision loss with an extended field of view (>80°) and studying the head''s contributions to visual attention. The time-course of visuo-motor variables in our pure free-viewing task reveals long fixations and short saccades during first seconds of exploration, contrary to literature in visual tasks guided by instructions. We show that the effect of vision loss is reflected primarily on eye movements, in a manner consistent with two-dimensional screens literature. We hypothesize that head movements mainly serve to explore the scenes during free-viewing, the presence of masks did not significantly impact head scanning behaviours. We present new fixational and saccadic visuo-motor tendencies in a 360° context that we hope will help in the creation of gaze prediction models dedicated to virtual reality.