The efficacy and safety of venetoclax therapy in elderly patients with relapsed,refractory chronic lymphocytic leukaemia

Elderly chronic lymphocytic leukaemia (CLL) patients treated outside of trials have notably greater toxicity with the Bruton's tyrosine kinase inhibitor ibrutinib compared to younger patients. It is not known whether the same holds true for the B-cell lymphoma 2 inhibitor venetoclax. We provide a comprehensive analysis of key safety measures and efficacy in 342 patients comparing age categories ≥75 and <75 years treated in the relapsed, refractory non-trial setting. We demonstrate that venetoclax has equivalent efficacy and safety in relapsed/refractory CLL patients who are elderly, the majority of whom are previous ibrutinib-exposed and therefore may otherwise have few clear therapeutic options.     

From confluent human iPS cells to self-forming neural retina and retinal pigmented epithelium

Progress in retinal-cell therapy derived from human pluripotent stem cells currently faces technical challenges that require the development of easy and standardized protocols. Here, we developed a simple retinal differentiation method, based on confluent human induced pluripotent stem cells (hiPSC), bypassing embryoid body formation and the use of exogenous molecules, coating, or Matrigel. In 2 wk, we generated both retinal pigmented epithelial cells and self-forming neural retina (NR)-like structures containing retinal progenitor cells (RPCs). We report sequential differentiation from RPCs to the seven neuroretinal cell types in maturated NR-like structures as floating cultures, thereby revealing the multipotency of RPCs generated from integration-free hiPSCs. Furthermore, Notch pathway inhibition boosted the generation of photoreceptor precursor cells, crucial in establishing cell therapy strategies. This innovative process proposed here provides a readily efficient and scalable approach to produce retinal cells for regenerative medicine and for drug-screening purposes, as well as an in vitro model of human retinal development and disease.Irreversible blindness caused by retinal diseases, such as inherited retinopathies, age-related macular degeneration (AMD), or glaucoma, is mainly due to the impairment or loss of function of photoreceptor cells, supporting retinal pigmented epithelium (RPE) or retinal ganglion cells (RGCs). Rescuing the degenerated retina is a major challenge for which specific cell replacement is one of the most promising approaches (1, 2). Pluripotent stem cells, like human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs), have the ability to be expanded indefinitely in culture and could be used as an unlimited source of retinal cells for the treatment of retinal degenerative diseases (3, 4). Several publications have indicated that hESCs and hiPSCs can be differentiated into RPE cells spontaneously after fibroblast growth factor (FGF) 2 removal (5–7) or by different floating aggregate methods (8–11). Concerning neural retinal cells, a growing body of convergent data has demonstrated the ability of hESCs or hiPSCs to be committed into the neural retinal lineage and further differentiated into cells expressing photoreceptor markers (12–15). Recent innovative approaches using 3D cultures from embryoid bodies (EBs) of hESCs or hiPSCs allowed the self-formation of optic cup (OC) structures (16) or the generation of optic vesicle (OV)-like structures (17), depending on the addition of exogenous molecules and different substrates used. These protocols require multiple steps and trained handling, which are not always compatible with the manufacturing process for therapeutic approach or drug screening that need a large-scale production of cells of interest. Therefore, very simple and reliable approaches minimizing the use of exogenous molecules should be developed to generate hESCs or hiPSC-derived retinal cells.In the present study, we report a new retinal differentiation process using confluent hiPSCs, without cell clumps or EB formation and in the absence of Matrigel or serum. We demonstrate that integration-free hiPSCs derived from adult human dermal fibroblasts (AHDFs) cultured in proneural medium can simultaneously generate RPE cells and self-forming neural retinal (NR)-like structures within 2 wk and that, when switched to floating cultures, structures containing retinal progenitor cells (RPCs) can differentiate into all retinal cell types, including RGCs and precursors of photoreceptors, needed for therapeutic applications.     

A novel tissue culture model for evaluating the effect of aging on stem cell fate in adult microvascular networks

GeroScience - In vitro models of angiogenesis are valuable tools for understanding the underlying mechanisms of pathological conditions and for the preclinical evaluation of therapies. Our...     

Dynamic knee valgus in competitive alpine skiers: Observation from youth to elite and influence of biological maturation

Numerous studies investigated the association between dynamic knee valgus and injury risk in post-pubertal and elite athletes; however, normative reference scores for competitive alpine skiers and observations on the development process throughout and beyond athletes' growth spurt are lacking. Thus, the aim of this study was to describe the dynamic knee valgus of competitive alpine skiers during drop jump landings (DJ) and single-leg squats (SLS) with respect to sex, sportive level, and biological maturation. Thirty-seven elite and 104 youth competitive alpine skiers around the growth spurt (U15) were examined for their maximal medial knee displacement (MKD) during DJ and SLS by a marker-based 3D motion analysis evaluating dynamic knee valgus. Additionally, skiers' age, anthropometry and biological maturation were assessed. MKD of youth and elite alpine skiers during DJ was comparable and did not improve with increasing training age. Female U15 skiers (on average further matured) had significantly larger MKD values during DJ than male U15 skiers (less matured) (P < .01). Moreover, MKD during DJ was directly associated with the athlete's individual biological maturation status. MKD values obtained from DJ significantly differed from those obtained during SLS (P < .01). The gender-specific difference in MKD values during DJ and their relationship with maturity offset highlight the fundamental changes to the neuromuscular control system during the growth spurt. Thus, biological maturation needs to be considered as a confounding factor for knee valgus screening. Caution is required when evaluating MKD by using high- and low-dynamic tasks, as corresponding information can differ.     

A repulsion mechanism explains magnesium permeation and selectivity in CorA

Magnesium (Mg²⁺) plays a central role in biology, regulating the activity of many enzymes and stabilizing the structure of key macromolecules. In bacteria, CorA is the primary source of Mg²⁺ uptake and is self-regulated by intracellular Mg²⁺. Using a gating mutant at the divalent ion binding site, we were able to characterize CorA selectivity and permeation properties to both monovalent and divalent cations under perfused two-electrode voltage clamp. The present data demonstrate that under physiological conditions, CorA is a multioccupancy Mg²⁺-selective channel, fully excluding monovalent cations, and Ca²⁺, whereas in absence of Mg²⁺, CorA is essentially nonselective, displaying only mild preference against other divalents (Ca²⁺ > Mn²⁺ > Co²⁺ > Mg²⁺ > Ni²⁺). Selectivity against monovalent cations takes place via Mg²⁺ binding at a high-affinity site, formed by the Gly-Met-Asn signature sequence (Gly312 and Asn314) at the extracellular side of the pore. This mechanism is reminiscent of repulsion models proposed for Ca²⁺ channel selectivity despite differences in sequence and overall structure.Among biological divalent cations, Mg²⁺ is not only the most abundant, but also plays an essential role in a wealth of cellular processes, including enzymatic reactions, and the stability of nucleic acids and biological membranes (1). Although the biological importance of Mg²⁺ is well established, the molecular entities and mechanisms that govern its cellular homeostasis are not well understood. In bacteria, Mg²⁺ influx is primarily catalyzed by members of the CorA family of divalent ion transport systems (2, 3). The X-ray structure of CorA has provided an excellent template toward a molecular understanding of the mechanisms underlying Mg²⁺ influx (4–7). However, although CorA has been crystallized in a wide range of conditions, so far all available CorA structures seem to correspond to nonconductive conformations, which obviously limits the basic mechanistic insights regarding Mg²⁺ selectivity and translocation that can be derived from these high-resolution structures. Computational analyses, together with NMR, X-ray absorption, and Raman spectroscopy studies, have established that Mg²⁺ holds to its first hydration shell much more tightly than any other physiological cation (8–11); this implies that any Mg²⁺-selective transport system must either compensate for the high hydration energy (and accommodate the invariable octahedral geometry of this hexacoordinated ion) or establish a selectivity mechanism able to discriminate a hydrated or partially hydrated Mg²⁺ ion from monovalent and other divalent cations.Several hypotheses have been postulated to explain CorA’s function, including its role as a Mg²⁺ -selective channel (12), a Co²⁺ transporter (13), and even as an exporter of divalent cations (14). However, detailed mechanistic evaluation of CorA’s functional properties has been limited by the resolution of existing functional assays (15). Mg²⁺ transport through CorA depends on the combination of three parameters: (i) number of open gates, (ii) the electrical potential across the membrane, and (iii) the Mg²⁺ driving force, none of which can be properly controlled with sufficient time-resolution in in vivo experiments. Although a prokaryotic membrane protein, we have been able to heterologously express CorA in Xenopus oocytes, which, in combination with standard electrophysiological approaches, allowed us to measure CorA-catalyzed divalent macroscopic currents under a variety of ionic conditions. Crystallographic studies have suggested that intracellular Mg²⁺ act as the main regulator of CorA gating under physiological conditions (6). That Mg²⁺ acts as both a gating ligand and charge carrier ultimately complicates functional studies of CorA permeation and selectivity properties. To circumvent this issue we used a mutation at the divalent cation sensor that abolishes CorA Mg²⁺-dependent gating (Fig. 1A). This construct is ideally suited to evaluate ion permeation because it stabilizes steady-state currents by inhibiting the divalent ion-driven negative-feedback loop that defines CorA gating. Our results demonstrate that CorA is a bona fide multioccupancy ion channel, and that its divalent cation permeation and tight selectivity against monovalent cations can be explained on the basis of a block and repulsion mechanism, where the canonical “signature sequence” Gly-Met-Asn (GMN) plays a central role.Open in a separate windowFig. 1.CorA-driven Mg²⁺ currents recorded from TEVC. (A) The divalent cation sensor is highlighted on a cartoon representation of CorA crystal structure. Residues Asp89 and Asp253 are shown as purple sticks (B). The membrane potential (Vm) is clamped and held at −60 mV. The external solution is exchanged between two isosmotic buffers: one containing no monovalent or divalent cation (colored in gray on the horizontal bar), and one containing 20 mM Mg²⁺ (noted Mg²⁺). A representative trace recorded on a CorA–WT-expressing oocyte is shown in teal, and control oocyte trace is shown in gray and D253K in purple. The horizontal dotted line indicates the 0 A current level. (C) Representative traces of CorA D253K mutant in TEVC. The voltage pulse protocol is shown on top of the current traces. The dotted line represents the 0 current levels. (D) The corresponding I/V relationships recorded at different external Mg²⁺ concentrations are shown. The GHK-flux equation fits are displayed as solid lines, and experimental values are dots. (E) Mg²⁺ current recorded at −60 mV under external solution perfusion. The external solution is changed stepwise and the corresponding solution exchange protocol is superimposed to the trace. (F) Mean values (±SD) of several traces (n ≥ 5) were recorded and normalized to the maximum current. The values were plotted against the external [Mg²⁺] and fitted with a single-site binding curve.     

The nature of protein folding pathways

How do proteins fold, and why do they fold in that way? This Perspective integrates earlier and more recent advances over the 50-y history of the protein folding problem, emphasizing unambiguously clear structural information. Experimental results show that, contrary to prior belief, proteins are multistate rather than two-state objects. They are composed of separately cooperative foldon building blocks that can be seen to repeatedly unfold and refold as units even under native conditions. Similarly, foldons are lost as units when proteins are destabilized to produce partially unfolded equilibrium molten globules. In kinetic folding, the inherently cooperative nature of foldons predisposes the thermally driven amino acid-level search to form an initial foldon and subsequent foldons in later assisted searches. The small size of foldon units, ∼20 residues, resolves the Levinthal time-scale search problem. These microscopic-level search processes can be identified with the disordered multitrack search envisioned in the “new view” model for protein folding. Emergent macroscopic foldon–foldon interactions then collectively provide the structural guidance and free energy bias for the ordered addition of foldons in a stepwise pathway that sequentially builds the native protein. These conclusions reconcile the seemingly opposed new view and defined pathway models; the two models account for different stages of the protein folding process. Additionally, these observations answer the “how” and the “why” questions. The protein folding pathway depends on the same foldon units and foldon–foldon interactions that construct the native structure.