Complex Folding Research Articles

A novel therapeutic to enhance the immunogenicity of breast cancer cells.

119 Background: Breast cancer is a challenge for immunotherapy because of its inherent genetic heterogeneity and decreased immunogenicity. A possible solution is to generate dying breast cancer cells that operate like a vaccine to stimulate tumor-specific immunity. This is termed immunogenic cell death (ICD) and is characterized by a unique molecular signature, involving the release of molecules that attract and stimulate phagocytes and sensitize tumor cells to killing by natural killer (NK) cells. However, the current doses of drugs used to induce ICD may be too high to translate into clinically relevant regimens. Methods: Our group discovered a novel peptide, CT20p, that is effective at nanomolar concentrations and can be delivered to breast tumors using targeted nanoparticles. CT20p works by inhibiting a unique protein folding complex, called chaperonin-containing TCP-1 (CCT), which is highly expressed in cancer cells but not normal cells. Using triple negative breast cancer (TNBC) and normal cell lines as well as patient-derived tumor cells, we examined the biological consequences of CCT inhibition by CT20p in terms of the induction of endoplasmic reticulum (ER) stress, through the accumulation of unfolded proteins, and the release of the danger signals associate with ICD that could stimulate innate immunity. Results: Dying TNBC cells treated with CT20p displayed alterations in PERK, a mediator of the unfolded protein response (UPR), which resulted in the membrane translocation of calreticulin (Crt), an “eat me”’ signal and a key marker of ICD. CT20p-killed TNBC cells were more readily phagocytosed, and NK cells more effectively killed remaining cancer cells. In our experiments normal breast epithelial cells, macrophages or NK cells were unaffected by CT20p. Conclusions: Our study suggests that inhibition of CCT by CT20p generates dying cancer cells that stimulate innate immune responses, through the release of signals like Crt, to attract phagocytes and promote NK cell cytotoxicity. By enhancing the immunogenicity of breast cancer cells, CT20p could increase responsiveness to to checkpoint inhibitors or cellular immunotherapy, hence providing more complete protection from breast cancer recurrence and metastasis.

Using stratiform magnetic anomalies to map near-surface architecture: insights from the Amadeus Basin

ABSTRACTThe Amadeus Basin displays subtle magnetic anomalies that trace strata for considerable distance, highlighting complex folding patterns. Magnetic modelling techniques can be utilised on these stratiform anomalies to extrapolate the near-surface structure of the basin. However, because of the mathematical trade-off between the dip and magnetisation of bodies, the dips of the bodies cannot be known unless the magnetisation is also known. Normally it would be optimal to measure the magnetisation, but this is not always possible or feasible. In this study, we investigate the relationships between dip and magnetisation using an approach that would generally be considered a little backward, i.e. constraining magnetisation direction using geological data. Three study areas were chosen to investigate a number of stratigraphic horizons, the Waterhouse Range, Glen Helen and Ross River areas. Modelling results suggest that some layers primarily retain induced magnetisation, remanence is dominant in others, but both are present in most. Remanence is mainly associated with relatively oxidised units that contain only hematite (e.g. Arumbera Sandstone), and we have demonstrated that these magnetisations predate folding of the Ross River Syncline. In some cases, the anomalies represent redox zonation within units, e.g. the Pertatataka Formation near Glen Helen, where discrete magnetic layers correspond to thin grey (reduced, magnetite-rich) horizons interbedded with more prevalent red (oxidised, hematite-rich) horizons. We also found that where magnetised units are relatively thin and occur near the surface, their magnetic response is sharp, and in aeromagnetic data such adjacent anomalies commonly overlap to form a single anomaly, thus misrepresenting the magnetic field, and mis-mapping the actual magnetic horizons. While the magnetic properties of the causative bodies are variable, we have demonstrated that a better understanding of the magnetic properties of these magnetised horizons can be used to provide insights into the structure and tectonic history of the Amadeus Basin.

A Trimer Consisting of the Tubulin-specific Chaperone D (TBCD), Regulatory GTPase ARL2, and β-Tubulin Is Required for Maintaining the Microtubule Network

Microtubule dynamics involves the polymerization and depolymerization of tubulin dimers and is an essential and highly regulated process required for cell viability, architecture, and division. The regulation of the microtubule network also depends on the maintenance of a pool of αβ-tubulin heterodimers. These dimers are the end result of complex folding and assembly events, requiring the TCP1 Ring Complex (TriC or CCT) chaperonin and five tubulin-specific chaperones, tubulin binding cofactors A-E (TBCA-TBCE). However, models of the actions of these chaperones are incomplete or inconsistent. We previously purified TBCD from bovine tissues and showed that it tightly binds the small GTPase ARL2 but appears to be inactive. Here, in an effort to identify the functional form of TBCD and using non-denaturing gels and immunoblotting, we analyzed lysates from a number of mouse tissues and cell lines to identify the quaternary state(s) of TBCD and ARL2. We found that both proteins co-migrated in native gels in a complex of ∼200 kDa that also contained β-tubulin. Using human embryonic kidney cells enabled the purification of the TBCD·ARL2·β-tubulin trimer found in cell and tissue lysates as well as two other novel TBCD complexes. Characterization of ARL2 point mutants that disrupt binding to TBCD suggested that the ARL2-TBCD interaction is critical for proper maintenance of microtubule densities in cells. We conclude that the TBCD·ARL2·β-tubulin trimer represents a functional complex whose activity is fundamental to microtubule dynamics.

Dual time-resolved temperature-jump fluorescence and infrared spectroscopy for the study of fast protein dynamics

Time-resolved temperature-jump (T-jump) coupled with fluorescence and infrared (IR) spectroscopy is a powerful technique for monitoring protein dynamics. Although IR spectroscopy of the polypeptide amide I mode is more technically challenging, it offers complementary information because it directly probes changes in the protein backbone, whereas, fluorescence spectroscopy is sensitive to the environment of specific side chains. With the advent of widely tunable quantum cascade lasers (QCL) it is possible to efficiently probe multiple IR frequencies with high sensitivity and reproducibility. Here we describe a dual time-resolved T-jump fluorescence and IR spectrometer and its application to study protein folding dynamics. A Q-switched Ho:YAG laser provides the T-jump source for both time-resolved IR and fluorescence spectroscopy, which are probed by a QCL and Ti:Sapphire laser, respectively. The Ho:YAG laser simultaneously pumps the time-resolved IR and fluorescence spectrometers. The instrument has high sensitivity, with an IR absorbance detection limit of <0.2mOD and a fluorescence sensitivity of 2% of the overall fluorescence intensity. Using a computer controlled QCL to rapidly tune the IR frequency it is possible to create a T-jump induced difference spectrum from 50ns to 0.5ms. This study demonstrates the power of the dual time-resolved T-jump fluorescence and IR spectroscopy to resolve complex folding mechanisms by complementary IR absorbance and fluorescence measurements of protein dynamics.

Ensemble simulations: folding, unfolding and misfolding of a high-efficiency frameshifting RNA pseudoknot

Massive all-atom molecular dynamics simulations were conducted across a distributed computing network to study the folding, unfolding, misfolding and conformational plasticity of the high-efficiency frameshifting double mutant of the 26 nt potato leaf roll virus RNA pseudoknot. Our robust sampling, which included over 40 starting structures spanning the spectrum from the extended unfolded state to the native fold, yielded nearly 120 μs of cumulative sampling time. Conformational microstate transitions on the 1.0 ns to 10.0 μs timescales were observed, with post-equilibration sampling providing detailed representations of the conformational free energy landscape and the complex folding mechanism inherent to the pseudoknot motif. Herein, we identify and characterize two alternative native structures, three intermediate states, and numerous misfolded states, the latter of which have not previously been characterized via atomistic simulation techniques. While in line with previous thermodynamics-based models of a general RNA folding mechanism, our observations indicate that stem-strand-sequence-separation may serve as an alternative predictor of the order of stem formation during pseudoknot folding. Our results contradict a model of frameshifting based on structural rigidity and resistance to mechanical unfolding, and instead strongly support more recent studies in which conformational plasticity is identified as a determining factor in frameshifting efficiency.

When proteostasis goes bad: Protein aggregation in the cell.

Protein aggregation is a hallmark of the major neurodegenerative diseases including Alzheimer's, Parkinson's, Huntington's and motor neuron and is a symptom of a breakdown in the management of proteome foldedness. Indeed, it is remarkable that under normal conditions cells can keep their proteome in a highly crowded and confined space without uncontrollable aggregation. Proteins pose a particular challenge relative to other classes of biomolecules because upon synthesis they must typically follow a complex folding pathway to reach their functional conformation (native state). Non-native conformations, including the unfolded nascent chain, are highly prone to aberrant interactions, leading to aggregation. Here we review recent advances in knowledge of proteostasis, approaches to monitor proteostasis and the impact that protein aggregation has on biology. We also include discussion of the outstanding challenges. © 2017 IUBMB Life, 69(2):49-54, 2017.

PSMA-Targeted Theranostic Nanocarrier for Prostate Cancer.

Herein, we report the use of a theranostic nanocarrier (Folate-HBPE(CT20p)) to deliver a therapeutic peptide to prostate cancer tumors that express PSMA (folate hydrolase 1). The therapeutic peptide (CT20p) targets and inhibits the chaperonin-containing TCP-1 (CCT) protein-folding complex, is selectively cytotoxic to cancer cells, and is non-toxic to normal tissue. With the delivery of CT20p to prostate cancer cells via PSMA, a dual level of cancer specificity is achieved: (1) selective targeting to PSMA-expressing prostate tumors, and (2) specific cytotoxicity to cancer cells with minimal toxicity to normal cells. The PSMA-targeting theranostic nanocarrier can image PSMA-expressing cells and tumors when a near infrared dye is used as cargo. Meanwhile, it can be used to treat PSMA-expressing tumors when a therapeutic, such as the CT20p peptide, is encapsulated within the nanocarrier. Even when these PSMA-targeting nanocarriers are taken up by macrophages, minimal cell death is observed in these cells, in contrast with doxorubicin-based therapeutics that result in significant macrophage death. Incubation of PSMA-expressing prostate cancer cells with the Folate-HBPE(CT20p) nanocarriers induces considerable changes in cell morphology, reduction in the levels of integrin β1, and lower cell adhesion, eventually resulting in cell death. These results are relevant as integrin β1 plays a key role in prostate cancer invasion and metastatic potential. In addition, the use of the developed PSMA-targeting nanocarrier facilitates the selective in vivo delivery of CT20p to PSMA-positive tumor, inducing significant reduction in tumor size.

TOWARD REAL-TIME PHYSICALLY-BASED VOICE SIMULATION: AN EIGENMODE-BASED APPROACH.

While physically-based continuum models of voice production have potential applications in clinical intervention of voice disorders and personalized natural speech synthesis, their current use is limited due to the high computational cost associated with resolving the complex fluid-structure interaction during voice production process. The goal of this study is to summarize our recent efforts in developing a physically-based, computationally-efficient continuum model of voice production toward near real-time applications. The model uses an eigenmode-based formulation of the governing equations, in which vocal fold eigenmodes are used as building blocks to reconstruct more complex vocal fold vibration patterns. Simulations show that a reasonable accuracy in the fundamental frequency, vocal intensity, and selected spectral measures can be reached with the use of the first 100 vocal fold eigenmodes, thus significantly reducing the degrees of freedom of the governing equations (as compared to tens of thousands in finite element models) and computational time. It is expected that for applications in which absolute values are not as essential, even a smaller number of eigenmodes would be acceptable. Examples are provided to demonstrate the capability of the model in modeling large range of voice qualities, natural voice quality change over time, and speech production in general.

MA02.09 Long Non-Coding RNA Expression from Pseudogene Loci as a Novel Mechanism of Cancer Gene Regulation

The advent of next generation sequencing has led to the discovery of the functional importance of non-coding RNAs (ncRNAs) in a wide variety of cellular processes, and these genes can be exploited by tumors to drive the hallmarks of cancer. Pseudogenes are DNA sequences that are defunct relatives of their functional parent genes but retain high sequence homology. Long non-coding RNAs (lncRNAs) have been shown to regulate protein-coding genes; however, complex folding patterns make lncRNA function difficult to predict. Several lncRNAs expressed from pseudogene loci have been shown to regulate the protein-coding parent genes of these pseudogenes in trans due to sequence complementarity. The biological impact of this mechanism has not been investigated in lung adenocarcinoma (LUAD). We hypothesize that expression changes in lncRNAs expressed from pseudogene loci can affect the expression of corresponding protein-coding parent genes in trans, and that these events provide an alternative mechanism of cancer gene deregulation in LUAD tumorigenesis. We analyzed RNA-seq data from 50 LUAD with matched non-malignant tissue obtained from the TCGA for both protein-coding and non-coding gene expression. Significantly differentially expressed lncRNAs located within pseudogene loci were identified by sign-rank test (p<0.001). Mann Whitney U-tests were used to identify lncRNA-parent gene pairs which significantly correlated expression, and survival analysis was performed using a Cox proportional hazard model. Our analysis has identified 172 lncRNAs expressed from pseudogene loci that were significantly deregulated in LUAD. Remarkably, many of these lncRNAs were expressed from the loci of pseudogenes related to known cancer genes. One of these lncRNAs, CTD-2583A14.8, was expressed from a pseudogene to ubiquitin-conjugating enzyme E2C (UBE2C), which regulates tumor growth, apoptosis, and angiogenesis through phospho-ERK1/2. We find CTD-2583A14.8 as well as the UBE2C parent gene to be significantly upregulated in LUAD tumors compared to matched normal tissue. Furthermore, tumors with higher levels of CTD-2583A14.8 have significantly higher levels of UBE2C expression than tumors with low levels of CTD-2583A14.8, indicating that CTD-2583A14.8 may positively regulate UBE2C in trans. Here we show expression of lncRNAs within pseudogene loci is deregulated in LUAD, and can correlate with the expression of their protein-coding counterparts. Many of these genes associated with this putative lncRNA-pseudogene-protein-coding axis have previously been implicated in cancer. Therefore, this represents an alternative mechanism of cancer gene deregulation, and may represent novel therapeutic intervention points for the treatment of LUAD.

Decoding Single Molecule Time Traces with Dynamic Disorder.

Single molecule time trajectories of biomolecules provide glimpses into complex folding landscapes that are difficult to visualize using conventional ensemble measurements. Recent experiments and theoretical analyses have highlighted dynamic disorder in certain classes of biomolecules, whose dynamic pattern of conformational transitions is affected by slower transition dynamics of internal state hidden in a low dimensional projection. A systematic means to analyze such data is, however, currently not well developed. Here we report a new algorithm—Variational Bayes-double chain Markov model (VB-DCMM)—to analyze single molecule time trajectories that display dynamic disorder. The proposed analysis employing VB-DCMM allows us to detect the presence of dynamic disorder, if any, in each trajectory, identify the number of internal states, and estimate transition rates between the internal states as well as the rates of conformational transition within each internal state. Applying VB-DCMM algorithm to single molecule FRET data of H-DNA in 100 mM-Na+ solution, followed by data clustering, we show that at least 6 kinetic paths linking 4 distinct internal states are required to correctly interpret the duplex-triplex transitions of H-DNA.

Exploring the sub-salt play in the frontier Amadeus Basin - Insights from regional 2D seismic and potential field data

Early exploration of the frontier Amadeus Basin in the Northern Territory has been limited due to its size and remoteness, along with complexity of structuring and limited early success. The primary exploration targets of the southern Amadeus are the sub-salt and intra-salt plays of the Neoproterozoic lower Gillen-Heavitree petroleum system. Two wells have tested the sub-salt play, both flowing gas with high helium content, confirming the sealing capacity of the Gillen evaporates despite two significant orogenic events.In 2013, the first regional framework of 2D seismic was acquired over the southern Amadeus. In areas where seismic coverage is sparse, or of poor quality due to halotectonics, higher spatial density magnetic and gravity surveys have been used to interpolate trends between seismic and well control and create a high resolution depth-to-basement model. Alternate methods of seismic processing have also been trialled to improve sub-salt imaging.The newly acquired seismic and depth-to-basement model are revealing the architecture of the basin and provide a regional perspective of the sub-salt and intra-salt plays. With improved seismic imaging large sub-salt structures are emerging beneath the complex folds resulting from thin-skinned deformation.Learnings from the challenges associated with acquisition and processing, along with a better understanding of the complex halotectonics, will have significant implications for future exploration in the region.

Interference folding and orocline implications: A structural study of the Ponga Unit, Cantabrian orocline, northern Spain

The origin of the Cantabrian orocline of the Variscan orogen in NW Iberia remains a topic of debate. We present a structural study of the Ponga Unit, a Cambrian to Carboniferous tectonostratigraphic package within the West European Variscan belt foreland fold-and-thrust belt that lies within the core region of the orocline. Our primary goal was to determine if W-plunging folds of the fold-and-thrust belt are attributable to formation of the Cantabrian orocline, or if they reflect lateral ramps in the underlying Variscan thrust faults. The major lithologic units of the Ponga Unit are the rheologically competent Lower Ordovician Barrios quartzite, and the less-competent, Carboniferous Barcaliente limestone and Beleno shale and sandstone formation. Our mapping and structural analysis within the Ponga Unit focused on the Laviana, Rioseco, and Campo de Caso thrust sheets, and associated bounding thrusts. Over 800 structural orientation measurements were collected across the study area. These data, coupled with data compiled from regional geological maps, allow for analysis of the crustal structure. West-plunging folds of the Laviana, Rioseco, and Campo de Caso thrust sheets form kilometer-scale anticline-syncline pairs, producing a complex fold interference pattern that is characteristic of the Ponga Unit. Our analysis shows that: (1) the geometry of the W-plunging folds is inconsistent with a lateral ramp model; (2) the map pattern defines a mushroom-type fold interference pattern, indicating two distinct deformational events characterized by principal compressive stresses oriented at a high angle (perpendicular) to one another; and (3) paleomagnetic data from the study area are consistent with the secondary model of orocline formation and indicate that there was a short window of time between the end of Variscan orogenesis and the onset of oroclinal buckling. Our results indicate that early N-S–trending folds, which resulted from Variscan orogenesis, were refolded during a post-Variscan orogen-parallel compression event attributable to formation of the Cantabrian orocline.

Signatures of Multiple Mineralization Processes in the Archean Orogenic Gold Deposit of the Pampalo Mine, Hattu Schist Belt, Eastern Finland

The Hattu schist belt is an emerging gold-producing ore district in the western part of the Archean Karelian Province of the Fennoscandian Shield. The belt consists of 2.76 to 2.70 Ga tonalite, granodiorite, and leucogranite intruded into a mafic-felsic epiclastic-volcanic sequence of an only slightly older age. Complex and successive folding, shearing, and hydrothermal processes affected these rocks prior to the lower amphibolites facies peak metamorphism (550° ± 50°C; 3–5 kbar) at ca. 2.70 Ga. Orogenic gold deposits are hosted by the highly strained zones that developed during the Archean deformation of the belt. However, previous K-Ar and Rb-Sr geochronological studies indicated that a second tectonothermal overprint affected the Hattu schist belt between 1.7 and 1.8 Ga during the Svecofennian orogeny. Results of our field, mineralogical, textural, and Pb isotope studies suggest that ore deposition at Pampalo was initiated by hydrothermal processes at the time of the emplacement of feldspar porphyry and tonalite intrusions and dikes, at around 2.72 Ga. Albitization and quartz-tourmaline-biotite-muscovite veining characterize this hydrothermal activity. The major stage of gold ore deposition can be confined to the subsequent development of high-strain zones in an intermediate-felsic tuffaceous unit (mafic schist) characterized by biotite-carbonate-pyrite alteration. Ar-Ar studies revealed the complete resetting of the Ar-Ar system in muscovite and biotite due to the Svecofennian orogeny, with closure of the isotopic system at 1.81 Ga. Results of Pb isotope studies of hydrothermal K-feldspar, galena, and altaite by laser ablation-inductively coupled plasma-mass spectrometry indicate that K-feldspar alteration and remobilization of metals also took place during the Svecofennian reactivation of high-strain zones. The petrographic manifestation of this process is the replacement of albite by hydrothermal K-feldspar. Fluid inclusion data from hydrothermal K-feldspar suggest that carbonic-aqueous, low-salinity fluids interacted with the Archean ore at 350° to 400°C and 1.8 to 2.4 kbar during the Svecofennian overprint. Results of mass transfer calculations indicate that potassium gain correlates with increase of gold concentrations in the mafic schist and feldspar-porphyry units. Therefore, the Svecofennian overprint locally also enhanced the grade of gold mineralization, mostly along competency differences of the mafic schist and feldspar porphyry blocks/dikes. Later percolation of relatively low temperature (<300°C) saline basin fluids in some fractures of the crystalline basement also left their Pb isotope and fluid inclusion signatures on the mineralization while further modifying the composition of the ore. Occurrences of hydrothermal proto-ores, as well as fluid flow events along structures reactivated by overprinting orogenic processes, are not exceptional features in Archean orogenic gold provinces. This study shows that the combination of in situ Pb isotope studies of U-poor alteration minerals and Pb-rich ore minerals, together with evaluation of relationships between gold enrichment and hydrothermal alteration and fluid inclusion studies, can be very useful in determining the significance and conditions of overprinting processes, as well as their potential implications for genetic and exploration models.

YidC assists the stepwise and stochastic folding of membrane proteins.

How chaperones, insertases and translocases facilitate insertion and folding of complex cytoplasmic proteins into cellular membranes is not fully understood. Here, we utilize single-molecule force spectroscopy to observe YidC, a transmembrane chaperone/insertase, sculpting the folding trajectory of the polytopic α-helical membrane protein lactose permease (LacY). In the absence of YidC, unfolded LacY inserts individual structural segments into the membrane; however, misfolding dominates the process so that folding cannot be completed. YidC prevents LacY from misfolding by stabilizing the unfolded state from which LacY inserts structural segments stepwise into the membrane until folding is completed. During stepwise insertion, YidC and membrane together stabilize the transient folds. Remarkably, the order of insertion of structural segments is stochastic, thereby indicating that LacY can fold along variable pathways towards the native structure. Since YidC is essential in membrane protein biogenesis and LacY a paradigm for the major facilitator superfamily, our observations have general relevance.

Neotectonic transformation of Cenozoic fold structures in the northwestern Caucasus

The performed morphotectonic regionalization of the northwestern Caucasus shows that the fold structures directly expressed in the topography of the territory and continuing to evolve under the settings of contemporary lateral shortening predominate in the northwestern Caucasus. A map of fold structures expressed in the topography of the northwestern Caucasus is presented. The districts distinguished therein correspond to the largest regional tectonic units, the fold topography of which occurs at various stages of tectonic evolution from primary brachyanticlinal ridges of the Taman and Sochi districts to the complex fold–thrust and inversion fold ridges of the axial zone. Data on active newly formed fold and inversion structures are given. These inherited structures develop under the combined action of selective denudation, beddingplane upthrow faulting, and thrusting.

Single-molecule Force Spectroscopy Reveals the Calcium Dependence of the Alternative Conformations in the Native State of a βγ-Crystallin Protein

Although multidomain proteins predominate the proteome of all organisms and are expected to display complex folding behaviors and significantly greater structural dynamics as compared with single-domain proteins, their conformational heterogeneity and its impact on their interaction with ligands are poorly understood due to a lack of experimental techniques. The multidomain calcium-binding βγ-crystallin proteins are particularly important because their deterioration and misfolding/aggregation are associated with melanoma tumors and cataracts. Here we investigate the mechanical stability and conformational dynamics of a model calcium-binding βγ-crystallin protein, Protein S, and elaborate on its interactions with calcium. We ask whether domain interactions and calcium binding affect Protein S folding and potential structural heterogeneity. Our results from single-molecule force spectroscopy show that the N-terminal (but not the C-terminal) domain is in equilibrium with an alternative conformation in the absence of Ca(2+), which is mechanically stable in contrast to other proteins that were observed to sample a molten globule under similar conditions. Mutagenesis experiments and computer simulations reveal that the alternative conformation of the N-terminal domain is caused by structural instability produced by the high charge density of a calcium binding site. We find that this alternative conformation in the N-terminal domain is diminished in the presence of calcium and can also be partially eliminated with a hitherto unrecognized compensatory mechanism that uses the interaction of the C-terminal domain to neutralize the electronegative site. We find that up to 1% of all identified multidomain calcium-binding proteins contain a similarly highly charged site and therefore may exploit a similar compensatory mechanism to prevent structural instability in the absence of ligand.

Micro-to nano-scale characterisation of polyamide structures of the SW30HR RO membrane using advanced electron microscopy and stain tracers

The development of new reverse osmosis (RO) membranes with enhanced performance would benefit from a detailed knowledge of the membrane structures which participate in the filtration process. Here, we examined the hierarchical structures of the polyamide (PA) active layer of the SW30HR RO membrane. Scanning electron microscopy combined with focused ion beam milling (FIB-SEM) was used to obtain the 3-D reconstructions of membrane morphology with 5nm cross-sectional resolution (comparable with the resolution of low magnification TEM imaging in 2D) and 30nm slice thickness. The complex folding of the PA layer was examined in 3 dimensions, enabling the quantification of key structural properties of the PA layer, including the local thickness, volume, surface area and their derivatives. The PA layer was found to exhibit a much higher and convoluted surface area than that estimated via atomic force microscopy (AFM). Cross-sectional scanning transmission electron microscopy (STEM) was used to observe the distribution of a tracer stain under various conditions. The behaviour of stain in dry and wet PA indicated that the permeation pathways have a dynamic nature and are activated by water. High resolution STEM imaging of the stained PA nano-films revealed the presence of <1nm pore-like structures with a size compatible with free volume estimations by positron annihilation lifetime spectroscopy (PALS). This study presents a comprehensive map of the active PA layer across different length scales (from micro- to sub-nanometre) and mechanistic insight into their role in the permeation process.

Folding style controlled by intermediate decollement thickness change in the Lurestan region (NW of the Zagros fold-and-thrust belt), using analogue models

The basal and intermediate decollements play an important role in structural style of fold-and-thrust belts. The decollement units, or different mechanical stratigraphy within the rock units, are not uniform throughout the ZFTB and show a strong spatial variation. The Lurestan region with varied thickness of the intermediate decollement in its northern and southern parts is one of the most important parts of the Zagros fold-and-thrust belt, regarding its hydrocarbon exploration–extraction projects. Thickness variation of the intermediate decollement in different parts of the Lurestan region allows us to address its role on folding style. Based on scaled analogue modeling, this study outlines the impact of thickness and facies variation of sedimentary rocks in the northern and southern parts of this region on folding style. Two models simulated the mechanical stratigraphy and its consequent different folding styles of the northern and southern parts of the region. In the models, only thickness of the intermediate decollement (thick and thin) for the northern and southern parts of the Lurestan region was varied. Detached minor folds above the intermediate decollement were created in response to the presence of the thicker intermediate decollement, northern part of the study area, which consequently deformed complexly and disharmonically folded, in contrast to polyharmonic folding style in the section, compared to polyharmonic folding style in the southern part, where thin intermediate decollement exists. The model results documented that thickness variation of intermediate decollement levels could explain complex and different folding styles in natural examples which must be taken into account for hydrocarbon exploration throughout these areas.

Numerical Analyses of Transcranial Magnetic Stimulation Based on Individual Brain Models by Using a Scalar-Potential Finite-Difference Method

Recent studies show that transcranial magnetic stimulation (TMS) is effective for the treatment of neurological and psychiatric diseases and that the therapeutic effect varies significantly among patients. The distribution of the electric fields induced by TMS is affected by individual variability in the shape of the brain. Numerical analyses of the electric fields in each patient performed by using individual brain models are potentially valuable for maximizing the therapeutic effect if the calculated fields are correlated with the effect of TMS. However, most of the numerical simulations of TMS are carried out by using standard human models. In this paper, we develop a new TMS simulation system, which is able to calculate the electric field induced in the brain with consideration of a complex gyral folding pattern, based on a scalar-potential finite-difference (SPFD) method. An easy implementation and a high efficiency of calculation resulting in a reduced computation time are the advantages of the SPFD method. The simulation is first performed on a sphere conductor model and a cube conductor model with 0.75 mm voxels, and we evaluate its accuracy through comparison with a finite-element method simulation. Second, we perform the simulations using six individual brain models constructed by segmenting magnetic resonance images and calculate the intensity of the field induced in each subject when they are stimulated with the intensity of their resting motor threshold (RMT). As a result, we find that there is a diversity of not only the intensity of the RMT but also the induced electric field in their brain tissues; the difference between the maximum and minimum values of the induced field on the stimulation spot among the subjects is 97 V/m, while the average value of the induced field is 203 V/m. Finally, we discuss the potential of this software in practical use and in the research of TMS.

Analysis of the functional consequences of targeted exon deletion in COL7A1 reveals prospects for dystrophic epidermolysis bullosa therapy.

Genetically evoked deficiency of collagen VII causes dystrophic epidermolysis bullosa (DEB)-a debilitating disease characterized by chronic skin fragility and progressive fibrosis. Removal of exons carrying frame-disrupting mutations can reinstate protein expression in genetic diseases. The therapeutic potential of this approach is critically dependent on gene, protein, and disease intrinsic factors. Naturally occurring exon skipping in COL7A1, translating collagen VII, suggests that skipping of exons containing disease-causing mutations may be feasible for the treatment of DEB. However, despite a primarily in-frame arrangement of exons in the COL7A1 gene, no general conclusion of the aptitude of exon skipping for DEB can be drawn, since regulation of collagen VII functionality is complex involving folding, intra- and intermolecular interactions. To directly address this, we deleted two conceptually important exons located at both ends of COL7A1, exon 13, containing recurrent mutations, and exon 105, predicted to impact folding. The resulting recombinantly expressed proteins showed conserved functionality in biochemical and in vitro assays. Injected into DEB mice, the proteins promoted skin stability. By demonstrating functionality of internally deleted collagen VII variants, our study provides support of targeted exon deletion or skipping as a potential therapy to treat a large number of individuals with DEB.