Vinculin is an integral component of integrin adhesions, where it functions as a molecular clutch coupling intracellular contraction to the extracellular matrix. Quantitating its contribution to the ...reinforcement of newly forming adhesions, however, requires ultrasensitive cell force assays covering short time and low force ranges. Here, we have combined atomic force microscopy‐based single‐cell force spectroscopy (SCFS) and optical tweezers force spectroscopy to investigate the role of vinculin in reinforcement of individual nascent adhesions during the first 5 min of cell contact with fibronectin or vitronectin. At minimal adhesion times (5‐10 s), mouse embryonic fibroblast (MEF) wildtype (wt) and vinculin knock‐out (vin(−/−)) cells develop comparable adhesion forces on the scale of several individual integrin‐ligand bonds, confirming that vinculin is dispensable for adhesion initiation. In contrast, after 60 to 120 s, adhesion strength and traction reinforce quickly in wt cells, while remaining low in vin(−/−) cells. Re‐expression of full‐length vinculin or a constitutively active vinculin mutant (vinT12) in MEF vin(−/−) cells restored adhesion and traction with the same efficiency, while vinculin with a mutated talin‐binding head region (vinA50I) or missing the actin‐binding tail‐domain (vin880) was ineffective. Integrating total internal reflection fluorescence imaging into the SCFS setup furthermore enabled us to correlate vinculin‐green fluorescent protein (GFP) recruitment to nascent adhesion sites with the built‐up of vinculin‐dependent adhesion forces directly. Vinculin recruitment and cell adhesion reinforcement followed synchronous biphasic patterns, suggesting vinculin recruitment, but not activation, as the rate‐limiting step for adhesion reinforcement. Combining sensitive SCFS with fluorescence microscopy thus provides insight into the temporal sequence of vinculin‐dependent mechanical reinforcement in nascent integrin adhesions.
Vinculin is an integral component of integrin adhesions, where it functions as a molecular clutch coupling intracellular contraction to the extracellular matrix. Using a combination of AFM‐based single‐cell force spectroscopy (SCFS) and total internal reflection microscopy (TIRF) reveals biphasic, synchronous patterns of vinculin recruitment and nascent adhesion reinforcement. These results suggest vinculin recruitment, but not activation, as the rate‐limiting step for nascent adhesion reinforcement.
In natural environments, microbial communities must constantly adapt to stressful environmental conditions. The genetic and phenotypic mechanisms underlying the adaptive response of microbial ...communities to new (and often complex) environments can be tackled with a combination of experimental evolution and next generation sequencing. This combination allows to analyse the real-time evolution of microbial populations in response to imposed environmental factors or during the interaction with a host, by screening for phenotypic and genotypic changes over a multitude of identical experimental cycles. Experimental evolution (EE) coupled with comparative genomics has indeed facilitated the monitoring of bacterial genetic evolution and the understanding of adaptive evolution processes. Basically, EE studies had long been done on single strains, allowing to reveal the dynamics and genetic targets of natural selection and to uncover the correlation between genetic and phenotypic adaptive changes. However, species are always evolving in relation with other species and have to adapt not only to the environment itself but also to the biotic environment dynamically shaped by the other species. Nowadays, there is a growing interest to apply EE on microbial communities evolving under natural environments. In this paper, we provide a non-exhaustive review of microbial EE studies done with systems of increasing complexity (from single species, to synthetic communities and natural communities) and with a particular focus on studies between plants and plant-associated microorganisms. We highlight some of the mechanisms controlling the functioning of microbial species and their adaptive responses to environment changes and emphasize the importance of considering bacterial communities and complex environments in EE studies.
For children with cerebral malaria, mortality is high, and in survivors, long-term neurologic and cognitive dysfunctions are common. While specific clinical factors are associated with death or ...long-term neurocognitive morbidity in cerebral malaria, the association of EEG features with these outcomes, particularly neurocognitive outcomes, is less well characterized.
In this prospective cohort study of 149 children age 6 months to 12 years who survived cerebral malaria in Kampala, Uganda, we evaluated whether depth of coma, number of clinical seizures, or EEG features during hospitalization were associated with mortality during hospitalization, short-term and long-term neurologic deficits, or long-term cognitive outcomes (overall cognition, attention, memory) over the 2-year follow-up.
Higher Blantyre or Glasgow Coma Scores (BCS and GCS, respectively), higher background voltage, and presence of normal reactivity on EEG were each associated with lower mortality. Among clinical and EEG features, the presence of >4 seizures on admission had the best combination of negative and positive predictive values for neurologic deficits in follow-up. In multivariable modeling of cognitive outcomes, the number of seizures and specific EEG features showed independent association with better outcomes. In children younger than 5 years throughout the study, seizure number and presence of vertex sharp waves were independently associated with better posthospitalization cognitive performance, faster dominant frequency with better attention, and higher average background voltage and faster dominant background frequency with better associative memory. In children younger than 5 years at CM episode but 5 years or older at cognitive testing, seizure number, background dominant frequency, and the presence of vertex sharp waves were each associated with changes in cognition, seizure number and variability with attention, and seizure number with working memory.
In children with cerebral malaria, seizure number is strongly associated with the risk of long-term neurologic deficits, while seizure number and specific EEG features (average background voltage, dominant rhythm frequency, presence of vertex sharp waves, presence of variability) are independently associated with cognitive outcomes. Future studies should evaluate the predictive value of these findings.
Single-molecule force spectroscopy (SMFS) (1) measures the extension of a molecule when subjected to force. The folding of a protein can be explored by pulling on one terminus to unfold it; upon ...relaxation, it may refold toward its native states (2,3 ). Transmembrane proteins typically unfold stepwise as structural segments (which can consist of parts of single or multiple secondary structures) are extracted from the membrane (see the figure, top panel) (4 ) . Once extracted, the unfolded segment can insert back into the membrane and fold toward the native protein structure (5,6 ). Complex proteins extracted from the membrane may refold back into the membrane (5,6 ) but tend to misfold and require the assistance of chaperones, insertases, or both (7,8 ). Incremental folding steps of secondary structures are theorized to happen in the order of milliseconds (9 ) but have been undetectable because of the limited time resolution of SMFS-related techniques. On page 945 of this issue, Yu et al. ( 10 ) address this problem by applying a newly developed SMFS technique based on atomic force microscopy (AFM) that records the response of individual membrane proteins (bacteriorhodopsin) subjected to mechanical forces at microsecond resolution.
To report the symptomatic and functional outcomes in patients with major depressive disorder (MDD) during a 2-phase treatment trial and to estimate the value of early improvement after 2 weeks in ...predicting clinical response to escitalopram and subsequently to adjunctive treatment with aripiprazole.
Participants with MDD (N = 211) identified with the Montgomery-Asberg Depression Rating Scale (MADRS) and confirmed with the Mini-International Neuropsychiatric Interview were recruited from 6 outpatient centers across Canada (August 2013 through December 2016) and treated with open-label escitalopram (10-20 mg) for 8 weeks (Phase 1). Clinical and functional outcomes were evaluated using the MADRS, Quick Inventory of Depressive Symptomatology-Self-Rated (QIDS-SR), Sheehan Disability Scale (SDS), and Lam Employment Absence and Productivity Scale (LEAPS). Participants were evaluated at 8 and 16 weeks for clinical and functional response and remission. Phase 1 responders continued escitalopram while nonresponders received adjunctive aripiprazole (2-10 mg) for a further 8 weeks (Phase 2).
After Phase 1, MADRS response (≥ 50% decrease from baseline) and remission (score ≤ 10) were, respectively, 47% and 31%, and SDS response (score ≤ 12) and remission (score ≤ 6) were, respectively, 53% and 24%. Response to escitalopram was maintained in 91% of participants at week 16, while 61% of the adjunctive aripiprazole group achieved MADRS response during Phase 2. Response and remission rates with the QIDS-SR were lower than with the MADRS. The LEAPS demonstrated significant occupational improvement (P < .05). Early symptomatic improvement predicted outcomes with modest accuracy.
This study demonstrates comparable symptomatic and functional outcomes to those of other large practical-design studies. There was a high response rate with the adjunctive use of aripiprazole in escitalopram nonresponders. Given the limited value of early clinical improvement to predict outcome, integration of clinical and biological markers deserves further exploration.
ClinicalTrials.gov identifier: NCT01655706.
Wheat, one of the major crops in the world, has had a complex history that includes genomic hybridizations between
and
species and several domestication events, which resulted in various wild and ...domesticated species (especially
and
), many of them still existing today. The large body of information available on wheat-microbe interactions, however, was mostly obtained without considering the importance of wheat evolutionary history and its consequences for wheat microbial ecology. This review addresses our current understanding of the microbiome of wheat root and rhizosphere in light of the information available on pre- and post-domestication wheat history, including differences between wild and domesticated wheats, ancient and modern types of cultivars as well as individual cultivars within a given wheat species. This analysis highlighted two major trends. First, most data deal with the taxonomic diversity rather than the microbial functioning of root-associated wheat microbiota, with so far a bias toward bacteria and mycorrhizal fungi that will progressively attenuate thanks to the inclusion of markers encompassing other micro-eukaryotes and archaea. Second, the comparison of wheat genotypes has mostly focused on the comparison of
cultivars, sometimes with little consideration for their particular genetic and physiological traits. It is expected that the development of current sequencing technologies will enable to revisit the diversity of the wheat microbiome. This will provide a renewed opportunity to better understand the significance of wheat evolutionary history, and also to obtain the baseline information needed to develop microbiome-based breeding strategies for sustainable wheat farming.
Lithium‐ion batteries (LIBs) will play a crucial role in achieving decarbonization and reducing greenhouse gases. If the EU wants to be competitive in the global market of LIBs, it has to ensure a ...sustainable and secure supply of the raw materials needed for the manufacturing of these batteries. Limited understanding of how the battery material cycles are linked with raw materials supply chains may hinder policy measures targeting the set‐up of a domestic supply chain in the EU since no precise information on where to intervene will be available. The novelty of this work lies in a multilayer system approach developed to reveal interlinkages between the flows of five raw materials contained in LIBs (cobalt, lithium, manganese, natural graphite, and nickel) in the EU. This was achieved by aligning material system analysis datasets of raw materials contained in LIBs with datasets on stocks and flows of this type of batteries in the EU. The results demonstrate the EU's strong import dependency on LIBs and battery raw materials. The EU recycling of lithium and natural graphite is low/nonexistent hindering the sustainable supply of these materials. The results also show that the majority of battery materials are increasingly accumulated in use or hoarding stocks. The proposed approach is designed to help identify bottlenecks and possible solutions to increase the efficiency of the EU LIB system, which could go unnoticed if each material supply chain were examined individually. This study also highlights how the lessons learned can support EU resource‐management policies.
Cell–cell adhesion is a complex process that is involved in the tethering of cells, cell–cell communication, tissue formation, cell migration and the development and metastasis of tumors. Given the ...heterogeneous and complex nature of cell surfaces it has previously proved difficult to characterize individual cell–cell adhesion events. Force spectroscopy, using an atomic force microscope, is capable of resolving such individual cell–cell binding events, but has previously been limited in its application due to insufficient effective pulling distances. Extended pulling range is critical in studying cell–cell interactions due to the potential for large cell deformations. Here we describe an approach to such experiments, where the sample stage can be moved 100
μm in the
z-direction, by closed loop, linearized piezo elements. Such an approach enables an increase in pulling distance sufficient for the observation of long-distance cell-unbinding events without reducing the imaging capabilities of the atomic force microscope. The atomic force microscope head and the piezo-driven sample stage are installed on an inverted optical microscope fitted with a piezo-driven objective, to allow the monitoring of cell morphology by conventional light microscopy, concomitant with force spectroscopy measurements. We have used the example of the WM115 melanoma cell line binding to human umbilical vein endothelial cells to demonstrate the capabilities of this system and the necessity for such an extended pulling range when quantifying cell–cell adhesion events.
Extra-abdominal desmoid tumor fibromatosis (DTF) is a rare, locally aggressive soft tissue tumour. The best treatment modality for this patient cohort is still object of debate.
This paper aimed to ...(1) to compare the outcomes of DTF after different treatment modalities, (2) to assess prognostic factors for recurrence following surgical excision, and (3) to assess prognostic factors for progression during observation.
This was a retrospective multicenter study under the patronage of the European Musculoskeletal Oncology Society (EMSOS). All seven centres involved were tertiary referral centres for soft tissue tumours. Baseline demographic data was collected for all patients as well as data on the diagnosis, tumour characteristics, clinical features, treatment modalities and whether they had any predisposing factors for DTF.
Three hundred eighty-eight patients (240 female, 140 male) with a mean age of 37.6 (±18.8 SD, range: 3-85) were included in the study. Two hundred fifty-seven patients (66%) underwent surgical excision of ADF, 70 patients (18%) were observed without therapy, the residual patients had different conservative treatments. There were no significant differences in terms of tumour recurrence or progression between the different treatment groups. After surgical excision, younger age, recurrent disease and larger tumour size were risk factors for recurrence, while tumours around the shoulder girdle and painful lesions were at risk of progression in the observational group.
Local recurrence rate after surgery was similar to progression rates under observation. Hence, observation in DTF seems to be justified, considering surgery in case of dimensional progression in 2 consecutive controls (3 and 6 months) and in painful lesions, with particular attention to lesions around the shoulder girdle.
Clozapine (CLZ) is the most effective treatment for treatment-resistant schizophrenia (SCZ) patients, with potential added benefits of reduction in suicide risk and aggression. However, CLZ is also ...mainly underused due to its high risk for the potentially lethal side-effect of agranulocytosis as well as weight gain and related metabolic dysregulation. Pharmacogenetics promises to enable the prediction of patient treatment response and risk of adverse effects based on patients' genetics, paving the way toward individualized treatment.
This article reviews pharmacogenetics studies of CLZ response and side-effects with a focus on articles from January 2012 to February 2015, as an update to the previous reviews. Pharmacokinetic genes explored primarily include CYP1A2, while pharmacodynamic genes consisted of traditional pharmacogenetic targets such as brain-derived neurotrophic factor as well novel mitochondrial genes, NDUFS-1 and translocator protein.
Pharmacogenetics is a promising avenue for individualized medication of CLZ in SCZ, with several consistently replicated gene variants predicting CLZ response and side-effects. However, a large proportion of studies have yielded mixed results. Large-scale Genome-wide association studies (e.g., CRESTAR) and targeted gene studies with standardized designs (response measurements, treatment durations, plasma level monitoring) are required for further progress toward clinical translation. Additionally, in order to improve study quality, we recommend accounting for important confounders, including polypharmacy, baseline measurements, treatment duration, gender, and age at onset.
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