Folding Pathways of a Knotted Protein with a Realistic Atomistic Force Field

We report on atomistic simulation of the folding of a natively-knotted protein, MJ0366, based on a realistic force field. To the best of our knowledge this is the first reported effort where a realistic force field is used to investigate the folding pathways of a protein with complex native topology. By using the dominant-reaction pathway scheme we collected about 30 successful folding trajectories for the 82-amino acid long trefoil-knotted protein. Despite the dissimilarity of their initial unfolded configuration, these trajectories reach the natively-knotted state through a remarkably similar succession of steps. In particular it is found that knotting occurs essentially through a threading mechanism, involving the passage of the C-terminal through an open region created by the formation of the native -sheet at an earlier stage. The dominance of the knotting by threading mechanism is not observed in MJ0366 folding simulations using simplified, native-centric models. This points to a previously underappreciated role of concerted amino acid interactions, including non-native ones, in aiding the appropriate order of contact formation to achieve knotting.     

Matefin/SUN-1 Phosphorylation Is Part of a Surveillance Mechanism to Coordinate Chromosome Synapsis and Recombination with Meiotic Progression and Chromosome Movement

Faithful chromosome segregation during meiosis I depends on the establishment of a crossover between homologous chromosomes. This requires induction of DNA double-strand breaks (DSBs), alignment of homologs, homolog association by synapsis, and repair of DSBs via homologous recombination. The success of these events requires coordination between chromosomal events and meiotic progression. The conserved SUN/KASH nuclear envelope bridge establishes transient linkages between chromosome ends and cytoskeletal forces during meiosis. In Caenorhabditis elegans, this bridge is essential for bringing homologs together and preventing nonhomologous synapsis. Chromosome movement takes place during synapsis and recombination. Concomitant with the onset of chromosome movement, SUN-1 clusters at chromosome ends associated with the nuclear envelope, and it is phosphorylated in a chk-2- and plk-2-dependent manner. Identification of all SUN-1 phosphomodifications at its nuclear N terminus allowed us to address their role in prophase I. Failures in recombination and synapsis led to persistent phosphorylations, which are required to elicit a delay in progression. Unfinished meiotic tasks elicited sustained recruitment of PLK-2 to chromosome ends in a SUN-1 phosphorylation–dependent manner that is required for continued chromosome movement and characteristic of a zygotene arrest. Furthermore, SUN-1 phosphorylation supported efficient synapsis. We propose that signals emanating from a failure to successfully finish meiotic tasks are integrated at the nuclear periphery to regulate chromosome end–led movement and meiotic progression. The single unsynapsed X chromosome in male meiosis is precluded from inducing a progression delay, and we found it was devoid of a population of phosphorylated SUN-1. This suggests that SUN-1 phosphorylation is critical to delaying meiosis in response to perturbed synapsis. SUN-1 may be an integral part of a checkpoint system to monitor establishment of the obligate crossover, inducible only in leptotene/zygotene. Unrepaired DSBs and unsynapsed chromosomes maintain this checkpoint, but a crossover intermediate is necessary to shut it down.     

Chemokine Receptors CCR6 and CXCR3 Are Necessary for CD4+ T Cell Mediated Ocular Surface Disease in Experimental Dry Eye Disease

CD4⁺ T cells are essential to pathogenesis of ocular surface disease in dry eye. Two subtypes of CD4⁺ T cells, Th1 and Th17 cells, function concurrently in dry eye to mediate disease. This occurs in spite of the cross-regulation of IFN-γ and IL-17A, the prototypical cytokines Th1 and Th17 cells, respectively. Essential to an effective immune response are chemokines that direct and summon lymphocytes to specific tissues. T cell trafficking has been extensively studied in other models, but this is the first study to examine the role of chemokine receptors in ocular immune responses. Here, we demonstrate that the chemokine receptors, CCR6 and CXCR3, which are expressed on Th17 and Th1 cells, respectively, are required for the pathogenesis of dry eye disease, as CCR6KO and CXCR3KO mice do not develop disease under desiccating stress. CD4⁺ T cells from CCR6KO and CXCR3KO mice exposed to desiccating stress (DS) do not migrate to the ocular surface, but remain in the superficial cervical lymph nodes. In agreement with this, CD4⁺ T cells from CCR6 and CXCR3 deficient donors exposed to DS, when adoptively transferred to T cell deficient recipients manifest minimal signs of dry eye disease, including significantly less T cell infiltration, goblet cell loss, and expression of inflammatory cytokine and matrix metalloproteinase expression compared to wild-type donors. These findings highlight the important interaction of chemokine receptors on T cells and chemokine ligand expression on epithelial cells of the cornea and conjunctiva in dry eye pathogenesis and reveal potential new therapeutic targets for dry eye disease.     

Sputum analysis: Non‐invasive early lung cancer detection

Lung cancer is the leading cause of cancer‐related deaths over the world, characterized by a very high mortality rate. Molecular technique development tries to focus on early detection of cancers by studying molecular alterations that characterize cancer cells. Worldwide lung cancer research has focused on an ever‐increasing number of molecular elements of carcinogenesis at genetic, epigenetic and protein levels. The non‐invasiveness is the characteristic that all clinical trials on cancer detection should have. Abnormal chest imaging and/or non‐specific symptoms are initial signals of lung cancer that appear in an advanced stage of disease. This fact represents the cause of the low 5‐year survival rate: over 90% of patients dying within 5 years of diagnosis. Since smokers have higher quantity of sputum containing exfoliated cells from the bronchial tree, and the sputum represents the most easily accessible biological fluid and its collection is non‐invasive, analysis of this sample represents a good area of research in early lung cancer diagnosis. Continued cigarette smoking is the cause of chronic obstructive pulmonary disease (COPD), with an estimated attributable risk factor exceeding 80% in smoking affected individuals. Lung cancer is found in 40–70% of patients with COPD, particularly in severe disease, and it is a common cause of death in these patients. A large prospective trial of almost half a million non‐smokers showed as lung cancer is also common in patients with COPD who have never smoked. This review describes issues related to early lung cancer screening using non‐invasive methods. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.     

Mechanism of calsequestrin regulation of single cardiac ryanodine receptor in normal and pathological conditions

Release of Ca²⁺ from the sarcoplasmic reticulum (SR) drives contractile function of cardiac myocytes. Luminal Ca²⁺ regulation of SR Ca²⁺ release is fundamental not only in physiology but also in physiopathology because abnormal luminal Ca²⁺ regulation is known to lead to arrhythmias, catecholaminergic polymorphic ventricular tachycardia (CPVT), and/or sudden cardiac arrest, as inferred from animal model studies. Luminal Ca²⁺ regulates ryanodine receptor (RyR)2-mediated SR Ca²⁺ release through mechanisms localized inside the SR; one of these involves luminal Ca²⁺ interacting with calsequestrin (CASQ), triadin, and/or junctin to regulate RyR2 function.CASQ2-RyR2 regulation was examined at the single RyR2 channel level. Single RyR2s were incorporated into planar lipid bilayers by the fusion of native SR vesicles isolated from either wild-type (WT), CASQ2 knockout (KO), or R33Q-CASQ2 knock-in (KI) mice. KO and KI mice have CPVT-like phenotypes. We show that CASQ2(WT) action on RyR2 function (either activation or inhibition) was strongly influenced by the presence of cytosolic MgATP. Function of the reconstituted CASQ2(WT)–RyR2 complex was unaffected by changes in luminal free [Ca²⁺] (from 0.1 to 1 mM). The inhibition exerted by CASQ2(WT) association with the RyR2 determined a reduction in cytosolic Ca²⁺ activation sensitivity. RyR2s from KO mice were significantly more sensitive to cytosolic Ca²⁺ activation and had significantly longer mean open times than RyR2s from WT mice. Sensitivity of RyR2s from KI mice was in between that of RyR2 channels from KO and WT mice. Enhanced cytosolic RyR2 Ca²⁺ sensitivity and longer RyR2 open times likely explain the CPVT-like phenotype of both KO and KI mice.     

Interspecific relationships in co‐occurring populations of social parasites and their host ants

Myrmica ant colonies host numerous insect species, including the larvae of Maculinea butterflies and Microdon myrmicae hoverflies. Little is known about the interspecific relationships among these social parasites and their host ants occurring in sympatric populations. We investigated communities of social parasites to assess the strategies allowing them to share the same pool of resources (i.e. Myrmica colonies). The present study was carried out at five sites inhabited by different social parasite communities, each comprising varying proportions of Maculinea teleius, Maculinea nausithous, Maculinea alcon, and Microdon myrmicae. We investigated their spatial distributions, host segregation, the degree of chemical similarity between social parasites and hosts, and temporal overlaps in colony resource exploitation. Spatial segregation among social parasites was found in two populations and it arises from microhabitat preferences and biological interactions. Local conditions can drive selection on one social parasite to use a Myrmica host species that is not exploited by other social parasites. Myrmica scabrinodis and Myrmica rubra nests infested by larvae of two social parasite species were found and the most common co‐occurrence was between Ma. teleius and Mi. myrmicae. The successful coexistence of these two species derives from their exploitation of the host colony resources at different times of the year. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 699–709.     

An Automated Live Imaging Platform for Studying Merozoite Egress-Invasion in Malaria Cultures

Most cases of severe and fatal malaria are caused by the intraerythrocytic asexual reproduction cycle of Plasmodium falciparum. One of the most intriguing and least understood stages in this cycle is the brief preinvasion period during which dynamic merozoite–red-cell interactions align the merozoite apex in preparation for penetration. Studies of the molecular mechanisms involved in this process face formidable technical challenges, requiring multiple observations of merozoite egress-invasion sequences in live cultures under controlled experimental conditions, using high-resolution microscopy and a variety of fluorescent imaging tools. Here we describe a first successful step in the development of a fully automated, robotic imaging platform to enable such studies. Schizont-enriched live cultures of P. falciparum were set up on an inverted stage microscope with software-controlled motorized functions. By applying a variety of imaging filters and selection criteria, we identified infected red cells that were likely to rupture imminently, and recorded their coordinates. We developed a video-image analysis to detect and automatically record merozoite egress events in 100% of the 40 egress-invasion sequences recorded in this study. We observed a substantial polymorphism of the dynamic condition of pre-egress infected cells, probably reflecting asynchronies in the diversity of confluent processes leading to merozoite release.     

Beyond Motor Scheme: A Supramodal Distributed Representation in the Action-Observation Network

The representation of actions within the action-observation network is thought to rely on a distributed functional organization. Furthermore, recent findings indicate that the action-observation network encodes not merely the observed motor act, but rather a representation that is independent from a specific sensory modality or sensory experience. In the present study, we wished to determine to what extent this distributed and ‘more abstract’ representation of action is truly supramodal, i.e. shares a common coding across sensory modalities. To this aim, a pattern recognition approach was employed to analyze neural responses in sighted and congenitally blind subjects during visual and/or auditory presentation of hand-made actions. Multivoxel pattern analyses-based classifiers discriminated action from non-action stimuli across sensory conditions (visual and auditory) and experimental groups (blind and sighted). Moreover, these classifiers labeled as ‘action’ the pattern of neural responses evoked during actual motor execution. Interestingly, discriminative information for the action/non action classification was located in a bilateral, but left-prevalent, network that strongly overlaps with brain regions known to form the action-observation network and the human mirror system. The ability to identify action features with a multivoxel pattern analyses-based classifier in both sighted and blind individuals and independently from the sensory modality conveying the stimuli clearly supports the hypothesis of a supramodal, distributed functional representation of actions, mainly within the action-observation network.     

Ocular-Motor Profile and Effects of Memantine in a Familial Form of Adult Cerebellar Ataxia with Slow Saccades and Square Wave Saccadic Intrusions

Fixation instability due to saccadic intrusions is a feature of autosomal recessive spinocerebellar ataxias, and includes square wave intrusions (SWI) and macrosaccadic oscillations (MSO). A recent report suggested that the non-competitive antagonist of NMDA receptors, memantine, could decrease MSO and improve fixation in patients with spinocerebellar ataxia with saccadic intrusions (SCASI). We similarly tested two sisters, respectively of 58 and 60 years, with an unrecognized form of recessive, adult-onset cerebellar ataxia, peripheral neuropathy and slow saccades, who showed prominent SWI and also complained with difficulty in reading. We tested horizontal visually guided saccades (10°–18°) and three minutes of steady fixation in each patient and in thirty healthy controls. Both patients showed a significant reduction of peak and mean velocity compared with control subjects. Large SWI interrupting steady fixation were prominent during steady fixation and especially following visually guided saccades. Eye movements were recorded before and during the treatment with memantine, 20 mg/daily for 6 months. The treatment with memantine reduced both the magnitude and frequency of SWI (the former significantly), but did not modified neurological conditions or saccade parameters. Thus, our report suggests that memantine may have some general suppressive effect on saccadic intrusions, including both SWI and MSO, thereby restoring the capacity of reading and visual attention in these and in other recessive forms of ataxia, including Friedreich’s, in which saccadic intrusions are prominent.