Plakophilin-1 (PKP-1) is an armadillo family protein critical for desmosomal adhesion and epidermal integrity. In the autoimmune skin-blistering disease pemphigus vulgaris (PV), autoantibodies (IgG) target the desmosomal cadherin desmoglein 3 (Dsg3) and compromise keratinocyte cell-cell adhesion. Here, we report that enhanced expression of PKP-1 protects keratinocytes from PV IgG-induced loss of cell-cell adhesion. PKP-1 prevents loss of Dsg3 and other desmosomal proteins from cell-cell borders and prevents alterations in desmosome ultrastructure in keratinocytes treated with PV IgG. Using a series of Dsg3 chimeras and deletion constructs, we find that PKP-1 clusters Dsg3 with the desmosomal plaque protein desmoplakin in a manner dependent on the plakoglobin-binding domain of the Dsg3 tail. Furthermore, PKP-1 expression transforms desmosome adhesion from a calcium-dependent to a calcium-independent and hyperadhesive state. These results demonstrate that manipulating the expression of a single desmosomal plaque protein can block the pathogenic effects of PV IgG on keratinocyte adhesion.
Background and Purpose The molecular identity of calcium-activated chloride channels (CaCCs) in vascular endothelial cells remains unknown. This study sought to identify whether anoctamin-1 (Ano1, also known as TMEM16A) functions as a CaCC and whether hypoxia alters the biophysical properties of Ano1 in mouse cardiac vascular endothelial cells (CVECs). Experimental Approach Western blot, quantitative real-time PCR, confocal imaging analysis and patch-clamp analysis combined with pharmacological approaches were used to determine whether Ano1 was expressed and functioned as CaCC in CVECs. Key Results Ano1 was expressed in CVECs. The biophysical properties of the current generated in the CVECs, including the Ca2+ and voltage dependence, outward rectification, anion selectivity and the pharmacological profile, are similar to those described for CaCCs. The density of ICl(Ca) detected in CVECs was significantly inhibited by T16A inh-A01, an Ano1 inhibitor, and a pore-targeting, specific anti-Ano1 antibody, and was markedly decreased in Ano1 gene knockdown CVECs. The density of ICl(Ca) was significantly potentiated in CVECs exposed to hypoxia, and this hypoxia-induced increase in the density of ICl(Ca) was inhibited by T16Ainh-A01 or anti-Ano1 antibody. Hypoxia also increased the current density of ICl(Ca) in Ano1 gene knockdown CVECs. Conclusions and Implications Ano1 formed CaCC in CVECs of neonatal mice. Hypoxia enhances Ano1-mediated ICl(Ca) density via increasing its expression, altering the ratio of its splicing variants, sensitivity to membrane voltage and to Ca 2+. Ano1 may play a role in the pathophysiological processes during ischaemia in heart, and therefore, Ano1 might be a potential therapeutic target to prevent ischaemic damage.
Bestrophins are a newly identified family of Cl− channels. Mutations in the founding member of the family, human bestrophin-1 (hBest1), are responsible for a form of early onset macular degeneration called Best vitelliform macular dystrophy. The link between dysfunction of hBest1 and macular degeneration remains unknown. Because retinal pigmented epithelium (RPE) cells may be subjected to varying osmotic pressure due to light-dependent changes in the ionic composition of the subretinal space and because RPE cells may undergo large volume changes during phagocytosis of shed photoreceptor discs, we investigated whether bestrophin currents were affected by cell volume. When hBest1 and mBest2 were overexpressed in HEK 293, HeLa, and ARPE-19 cells, a new Ca2+-activated Cl− current appeared. This current was very sensitive to cell volume. A 20% increase in extracellular osmolarity caused cell shrinkage and a ∼70–80% reduction in bestrophin current. Decreases in extracellular osmolarity increased the bestrophin currents slightly, but this was difficult to quantify due to simultaneous activation of endogenous volume-regulated anion channel (VRAC) current. To determine whether a similar current was present in mouse RPE cells, the effect of hyperosmotic solutions on isolated mouse RPE cells was examined. Mouse RPE cells exhibited an endogenous Cl− current that resembled the expressed hBest1 in that it was decreased by hypertonic solution. We conclude that bestrophins are volume sensitive and that they could play a novel role in cell volume regulation of RPE cells.
Across their dendritic trees, neurons distribute thousands of protein species that are necessary for maintaining synaptic function and plasticity and that need to be produced continuously and trafficked to their final destination. As each dendritic branchpoint splits the protein flow, increasing branchpoints decreases the total protein number downstream. Consequently, a neuron needs to produce more proteins to maintain a minimal protein number at distal synapses. Combining in vitro experiments and a theoretical framework, we show that proteins that diffuse within the cell plasma membrane are, on average, 35% more effective at reaching downstream locations than proteins that diffuse in the cytoplasm. This advantage emerges from a bias for forward motion at branchpoints when proteins diffuse within the plasma membrane. Using 3D electron microscopy (EM) data, we show that pyramidal branching statistics and the diffusion lengths of common proteins fall into a region that minimizes the overall protein need.
8-oxoG is one of the most common and mutagenic DNA base lesions caused by oxidative damage. However, it has not been possible to study the replication of a known 8-oxoG base in vivo in order to determine the accuracy of its replication, the influence of various components on that accuracy, and the extent to which an 8-oxoG might present a barrier to replication. We have been able to place a single 8-oxoG into the Saccharomyces cerevisiae chromosome in a defined location using single-strand oligonucleotide transformation and to study its replication in a fully normal chromosome context. During replication, 8-oxoG is recognized as a lesion and triggers a switch to translesion synthesis by Pol η, which replicates 8-oxoG with an accuracy (insertion of a C opposite the 8-oxoG) of approximately 94%. In the absence of Pol η, template switching to the newly synthesized sister chromatid is observed at least one third of the time; replication of the 8-oxoG in the absence of Pol η is less than 40% accurate. The mismatch repair (MMR) system plays an important role in 8-oxoG replication. Template switching is blocked by MMR and replication accuracy even in the absence of Pol η is approximately 95% when MMR is active. These findings indicate that in light of the overlapping mechanisms by which errors in 8-oxoG replication can be avoided in the cell, the mutagenic threat of 8-oxoG is due more to its abundance than the effect of a single lesion. In addition, the methods used here should be applicable to the study of any lesion that can be stably incorporated into synthetic oligonucleotides.
by
Laura Bryant;
Dong Li;
Samuel G. Cox;
Dylan Marchione;
Evan F. Joiner;
Khadija Wilson;
Kevin Janssen;
Pearl Lee;
Michael E. March;
Divya Nair;
Elliott Sherr;
Brieana Fregeau;
Klaas J. Wierenga;
Alexandrea Wadley;
Grazia M. S. Mancini;
Nina Powell-Hamilton;
Jiddeke van de Kamp;
Theresa Grebe;
Juanita Neira;
Elizabeth J. Bhoj
Although somatic mutations in Histone 3.3 (H3.3) are well-studied drivers of oncogenesis, the role of germline mutations remains unreported. We analyze 46 patients bearing de novo germline mutations in histone 3 family 3A (H3F3A) or H3F3B with progressive neurologic dysfunction and congenital anomalies without malignancies. Molecular modeling of all 37 variants demonstrated clear disruptions in interactions with DNA, other histones, and histone chaperone proteins. Patient histone posttranslational modifications (PTMs) analysis revealed notably aberrant local PTM patterns distinct from the somatic lysine mutations that cause global PTM dysregulation. RNA sequencing on patient cells demonstrated up-regulated gene expression related to mitosis and cell division, and cellular assays confirmed an increased proliferative capacity. A zebrafish model showed craniofacial anomalies and a defect in Foxd3-derived glia. These data suggest that the mechanism of germline mutations are distinct from cancer-associated somatic histone mutations but may converge on control of cell proliferation.
The bed nucleus of the stria terminalis (BNST) is known to play a critical role in mediating the behavioural and autonomic responses to stressors. The oval nucleus of the BNST (BNSTov) contains cell bodies that synthesise the stress hormone corticotropin-releasing factor (CRF). Although afferent fibres originating from the BNSTov have been shown to innervate several key structures of the neuroendocrine and central autonomic system, the question remains as to whether some of these fibres are CRF-positive. To directly address this question, we injected a 'floxed' anterograde tracer (rAAV5/EF1a-DIO-mCherry) into the BNSTov of CRFp3.0CreGFP transgenic mice, which express a green fluorescent protein (GFP) under the control of the CRF promoter. Serial sections were then analysed for the presence of double-labelled fibres in potential projection sites. To determine whether CRF neurons in the rat BNSTov send comparable projections, we infused rat BNSTov with an adeno-associated viral vector (AAV) in which the human synapsin promoter drives enhanced GFP expression. We then used CRF immunoreactivity to examine double-labelled fluorescent fibres and axon terminals in projection sites from brain sections of the AAV-infused rats. We have observed several terminal fields in the mouse and rat brain with double-labelled fibres in the Dorsal raphe nucleus (DRD), the paraventricular nucleus of the hypothalamus and, to a lesser extent, in the ventral tegmental area. We found double-labelled terminal boutons in the nucleus accumbens shell, prelimbic cortex and posterior basolateral nucleus of the amygdala. The most intense double-labelling was found in midbrain, including substantia nigra pars compacta, red nucleus, periaqueductal grey and pontine nuclei, as well as DRD. The results of the present study indicate that CRF neurons are the output neurons of the BNSTov and they send projections not only to the centres of neuroendocrine and autonomic regulation, but also regions modulating reward and motivation, vigilance and motor function, as well as affective behaviour.
Advances in single-cell biotechnology have increasingly revealed interactions of cells with their surroundings, suggesting a cellular society at the microscale. Similarities between cells and humans across multiple hierarchical levels have quantitative inference potential for reaching insights about phenotypic interactions that lead to morphological forms across multiple scales of cellular organization, namely cells, tissues and organs. Here, the functional and structural comparisons between how cells and individuals fundamentally socialize to give rise to the spatial organization are investigated. Integrative experimental cell interaction assays and computational predictive methods shape the understanding of societal perspective in the determination of the cellular interactions that create spatially coordinated forms in biological systems. Emerging quantifiable models from a simpler biological microworld such as bacterial interactions and single-cell organisms are explored, providing a route to model spatio-temporal patterning of morphological structures in humans. This analogical reasoning framework sheds light on structural patterning principles as a result of biological interactions across the cellular scale and up.
by
Robert Rulach;
David Ball;
Kevin K. M. Chua;
Max Dahele;
Dirk De Ruysscher;
Kevin Franks;
Daniel Gomez;
Matthias Guckenberger;
Gerard G. Hanna;
Drew Moghanaki;
Alexander V. Louie;
David A. Palma;
Clive Peedell;
Ahmed Salem;
Shankar Siva;
Gregory M. M. Videtic;
Anthony J. Chalmers;
Stephen Harrow
Purpose
Thoracic reirradiation for non-small cell lung cancer with curative intent is potentially associated with severe toxicity. There are limited prospective data on the best method to deliver this treatment. We sought to develop expert consensus guidance on the safe practice of treating non-small cell lung cancer with radiation therapy in the setting of prior thoracic irradiation.
Methods and Materials
Twenty-one thoracic radiation oncologists were invited to participate in an international Delphi consensus process. Guideline statements were developed and refined during 4 rounds on the definition of reirradiation, selection of appropriate patients, pretreatment assessments, planning of radiation therapy, and cumulative dose constraints. Consensus was achieved once ≥75% of respondents agreed with a statement. Statements that did not reach consensus in the initial survey rounds were revised based on respondents’ comments and re-presented in subsequent rounds.
Results
Fifteen radiation oncologists participated in the 4 surveys between September 2019 and March 2020. The first 3 rounds had a 100% response rate, and the final round was completed by 93% of participants. Thirty-three out of 77 statements across all rounds achieved consensus. Key recommendations are as follows: (1) appropriate patients should have a good performance status and can have locally relapsed disease or second primary cancers, and there are no absolute lung function values that preclude reirradiation; (2) a full diagnostic workup should be performed in patients with suspected local recurrence and; (3) any reirradiation should be delivered using optimal image guidance and highly conformal techniques. In addition, consensus cumulative dose for the organs at risk in the thorax are described.
Conclusions
These consensus statements provide practical guidance on appropriate patient selection for reirradiation, appropriate radiation therapy techniques, and cumulative dose constraints.
The availability of effective vaccines has had the most profound positive effect on improving the quality of public health by preventing infectious diseases. Despite many successful vaccines, there are still old and new emerging pathogens against which there is no vaccine available. A better understanding of how vaccines work for providing protection will help to improve current vaccines as well as to develop effective vaccines against pathogens for which we do not have a proper means to control. Recent studies have focused on innate immunity as the first line of host defense and its role in inducing adaptive immunity; such studies have been an intense area of research, which will reveal the immunological mechanisms how vaccines work for protection.
Toll-like receptors (TLRs), a family of receptors for pathogen-associated molecular patterns on cells of the innate immune system, play a critical role in detecting and responding to microbial infections. Importantly, the innate immune system modulates the quantity and quality of longterm T and B cell memory and protective immune responses to pathogens. Limited studies suggest that vaccines which mimic natural infection and/or the structure of pathogens seem to be effective in inducing long-term protective immunity. A better understanding of the similarities and differences of the molecular and cellular events in host responses to vaccination and pathogen infection would enable the rationale for design of novel preventive measures against many challenging pathogens.