Mechanism of peroxidasin inactivation in hyperglycemia: Heme damage by reactive oxygen species.

Diabetic complications present a serious health problem. Functional damage to proteins due to post-translational modifications by glycoxidation reactions is a known factor contributing to pathology. Extracellular proteins are especially vulnerable to diabetic damage because robust antioxidant defenses are lacking outside the cell. We investigated glucose-induced inactivation of peroxidasin (PXDN), a heme protein catalyzing sulfilimine crosslinking of collagen IV that reinforce the basement membranes (BM). Experiments using physiological diabetic glucose levels were carried out to exclude several potential mechanisms of PXDN inactivation i.e., direct adduction of glucose, reactive carbonyl damage, steric hindrance, and osmotic stress. Further experiments established that PXDN activity was inhibited via heme degradation by reactive oxygen species. Activity of another extracellular heme protein, myeloperoxidase, was unaffected by glucose because its heme was resistant to glucose-induced oxidative degradation. Our findings point to specific mechanisms which may compromise BM structure and stability in diabetes and suggest potential modes of protection.

Collagen IV of basement membranes: III. Chloride pressure is a primordial innovation that drives and maintains the assembly of scaffolds.

Collagen IV scaffold is a primordial innovation enabling the assembly of a fundamental architectural unit of epithelial tissues-a basement membrane attached to polarized cells. A family of six α-chains (α1 to α6) coassemble into three distinct protomers that form supramolecular scaffolds, noted as collagen IVα121, collagen IVα345, and collagen IVα121-α556. Chloride ions play a pivotal role in scaffold assembly, based on studies of NC1 hexamers from mammalian tissues. First, Cl- activates a molecular switch within trimeric NC1 domains that initiates protomer oligomerization, forming an NC1 hexamer between adjoining protomers. Second, Cl- stabilizes the hexamer structure. Whether this Cl--dependent mechanism is of fundamental importance in animal evolution is unknown. Here, we developed a simple in vitro method of SDS-PAGE to determine the role of solution Cl- in hexamer stability. Hexamers were characterized from 34 animal species across 15 major phyla, including the basal Cnidarian and Ctenophora phyla. We found that solution Cl- stabilized the quaternary hexamer structure across all phyla except Ctenophora, Ecdysozoa, and Rotifera. Further analysis of hexamers from peroxidasin knockout mice, a model for decreasing hexamer crosslinks, showed that solution Cl- also stabilized the hexamer surface conformation. The presence of sufficient chloride concentration in solution or "chloride pressure" dynamically maintains the native form of the hexamer. Collectively, our findings revealed that chloride pressure on the outside of cells is a primordial innovation that drives and maintains the quaternary and conformational structure of NC1 hexamers of collagen IV scaffolds.

Identification of brominated proteins in renal extracellular matrix: Potential interactions with peroxidasin.

Peroxidasin (PXDN) is an extracellular peroxidase, which generates hypobromous acid to form sulfilimine cross-links within collagen IV networks. We have previously demonstrated that mouse and human renal basement membranes (BM) are enriched in bromine due to PXDN-dependent post-translational bromination of protein tyrosine residues. The goal of the present study was identification of specific brominated sites within renal BM. A comprehensive analysis of brominated proteome of mouse glomerular matrix had been performed using liquid chromatography-tandem mass spectrometry. We found that out of over 200 identified proteins, only three were detectably brominated, each containing a single distinct brominated tyrosine site i.e., Tyr-1485 in collagen IV α2 chain, Tyr-292 in TINAGL1 and Tyr-664 in nidogen-2. To explain this highly selective bromination, we proposed that these proteins interact with PXDN within the glomerular matrix. Experiments using purified proteins demonstrated that both TINAGL1 and nidogen-2 can compete with PXDN for binding to collagen IV and that TINAGL1 can directly interact with PXDN. We propose that a protein complex, including PXDN, TINAGL1, nidogen-2 and collagen IV, may exist in renal BM.

Protective Properties of Spheroidal Taxifolin Form in Streptozotocin-Induced Diabetic Rats.

One of the key factors in the pathogenesis of diabetes and its complications is oxidative stress. To inhibit this process, antioxidants may be helpful. Herein, we focused on the protective properties of taxifolin spheroidal form (TS) in the streptozotocin rat model of diabetes mellitus. After 4 weeks of treatment with TS, the fasting blood glucose level of the diabetic animals decreased by 12% compared with the level right after the injection of streptozotocin. While the feed intake in the untreated diabetic rats increased by 5.3% compared with the healthy group, the TS-treated group showed a pronounced 15.3% decrease. Therapeutic administration of TS has a protective effect on the pancreas and the liver against the cytotoxic action of streptozotocin. The plasma antioxidant capacity of all diabetic groups appeared to be approximately 15% lower than in healthy rats with no significant difference between the TS-treated and untreated diabetic animals. Apparently, this can be attributed to taxifolin and plasma proteins binding. These data demonstrate the potential of TS in antidiabetic therapy.

Long-Term Prognosis after Coronary Artery Bypass Grafting: The Impact of Arterial Stiffness and Multifocal Atherosclerosis.

The aim of the study was to study the effect of arterial stiffness and multifocal atherosclerosis on the 10-year prognosis of patients after coronary artery bypass grafting. Methods. Patients with coronary artery disease (n = 274) who underwent coronary artery bypass grafting (CABG), in whom cardio-ankle vascular index (CAVI) was assessed using the VaSera VS-1000 device and the presence of peripheral atherosclerosis in Doppler ultrasound. Groups were distinguished with normal CAVI (<9.0, n = 163) and pathological CAVI (≥9.0, n = 111). To assess the prognosis, coronary and non-coronary death, myocardial infarction, acute cerebrovascular accident/transient ischemic attack, repeated CABG, percutaneous coronary intervention, carotid endarterectomy, peripheral arterial surgery, pacemaker implantation were analyzed. Results. During the observation period, mortality was 27.7%. A fatal outcome from all causes was in 37 (22.7%) patients in the group with normal CAVI and in 39 (35.14%) in the group with pathological CAVI (p = 0.023). Death from cardiac causes was more common in the group with CAVI ≥ 9.0­in 25 cases (22.52%) than in the group with CAVI < 9.0­in 19 (11.6%, p = 0.016). The combined endpoint in patients with pathological CAVI was detected in 66 (59.46%) cases, with normal CAVI values­in 76 (46.63%) cases (p = 0.03). The presence of diabetes mellitus, multifocal atherosclerosis (p = 0.004), pathological CAVI (p = 0.063), and male gender were independent predictors of death at 10-year follow-up after CABG. The presence of multifocal atherosclerosis and pathological CAVI during the preoperative examination of patients were independent predictors of the combined endpoint development. Findings. Patients with coronary artery disease with pathological CAVI before CABG were more likely to experience adverse events and death in the long-term follow-up than patients with normal CAVI. Further studies are needed to investigate the possibility of correcting pathological CAVI after CABG after secondary prevention and the possible impact of this correction on prognosis.

Mitochondrial Fus1/Tusc2 and cellular Ca2+ homeostasis: tumor suppressor, anti-inflammatory and anti-aging implications.

FUS1/TUSC2 (FUSion1/TUmor Suppressor Candidate 2) is a tumor suppressor gene (TSG) originally described as a member of the TSG cluster from human 3p21.3 chromosomal region frequently deleted in lung cancer. Its role as a TSG in lung, breast, bone, and other cancers was demonstrated by several groups, but molecular mechanisms of its activities are starting to unveil lately. They suggest that Fus1-dependent mechanisms are relevant in etiologies of diseases beyond cancer, such as chronic inflammation, bacterial and viral infections, premature aging, and geriatric diseases. Here, we revisit the discovery of FUS1 gene in the context of tumor initiation and progression, and review 20 years of research into FUS1 functions and its molecular, structural, and biological aspects that have led to its use in clinical trials and gene therapy. We present a data-driven view on how interactions of Fus1 with the mitochondrial Ca2+ (mitoCa2+) transport machinery maintain cellular Ca2+ homeostasis and control cell apoptosis and senescence. This Fus1-mediated cellular homeostasis is at the crux of tumor suppressor, anti-inflammatory and anti-aging activities.

Assessment of Arterial Stiffness by Cardio-Ankle Vascular Index for Prediction of Five-Year Cardiovascular Events After Coronary Artery Bypass Surgery.

The study aim was to investigate the possibility of cardiovascular complications development predicting during a five-year follow-up of patients after coronary artery bypass grafting (CABG) using the cardio-ankle vascular index (CAVI) assessment. Methods: Three hundred and fifty-six patients after elective CABG were enrolled in the study. Prior to surgery, arterial stiffness was assessed in all patients using CAVI. The follow-up was performed five years after the surgery, information was obtained on 238 patients, who were divided into two groups: patients with pathological (≥9.0, n = 88), and normal (<9.0, n = 150) CAVI. Results: Pathological CAVI (≥9.0) was detected in 33% patients before CABG, in stepwise analyses only age and left atrium dimensions statistically significantly predicted CAVI. In patients with pathological CAVI the combined endpoint (major adverse cardiovascular events and hospitalization) and cardiovascular death developed more often in a five-year follow-up after CABG compared with normal CAVI (48.86% versus 34.9%, p = 0.034 and 4.55% versus 0.67%, p = 0.049, respectively). Pathological CAVI (p = 0.021) and the number of coronary bypass grafts (p = 0.023) were independent factors associated with the combined endpoint. Conclusions: Patients with pathological CAVI before CABG surgery are more likely to develop cardiovascular complications and cardiovascular death within a subsequent five-year follow-up. Evaluation of CAVI after CABG in dynamics deserves further study, it is important for monitoring the effects of secondary prevention and the possibility of influencing the prognosis.

Collagen IV Exploits a Cl- Step Gradient for Scaffold Assembly.

Collagen molecules are crucial extracellular players in animal tissue development and in functions ranging from ultrafiltration to organism locomotion. Among the 28 types of collagen found in human, type IV collagen stands out as a primordial type found in all species of the animal kingdom. Collagen IV forms smart scaffolds for basement membranes, sheet-like acellular structures that isolate, coordinate, and direct cells during morphogenesis. Collagen IV is also involved in multiple functions in developed tissues. As part of the basement membrane, collagen IV scaffolds provide mechanical strength, spatially tether extracellular macromolecules and directly signal to cells via receptor binding sites. Proper assembly and structure of the scaffolds are critical for development and function of multiple types of basement membranes. Within last 5 years it was established that Cl- concentration is a key factor for initiating collagen IV scaffold assembly. The biological role of Cl- in multiple physiological processes and detailed mechanisms for its signaling and structural impacts are well established. Cl- gradients are generated across the plasma and intracellular organelle membranes. As collagen IV molecules are secreted outside the cell, they experience a switch from low to high Cl- concentration. This transition works as a trigger for collagen IV scaffold assembly. Within the scaffold, collagen IV remains to be a Cl- sensor as its structural integrity continues to depend on Cl- concentration. Here, we review recent findings and set future directions for studies on the role of Cl- in type IV collagen assembly, function, and disease.

Molecular Beam Epitaxy of Layered Group III Metal Chalcogenides on GaAs(001) Substrates.

Development of molecular beam epitaxy (MBE) of two-dimensional (2D) layered materials is an inevitable step in realizing novel devices based on 2D materials and heterostructures. However, due to existence of numerous polytypes and occurrence of additional phases, the synthesis of 2D films remains a difficult task. This paper reports on MBE growth of GaSe, InSe, and GaTe layers and related heterostructures on GaAs(001) substrates by using a Se valve cracking cell and group III metal effusion cells. The sophisticated self-consistent analysis of X-ray diffraction, transmission electron microscopy, and Raman spectroscopy data was used to establish the correlation between growth conditions, formed polytypes and additional phases, surface morphology and crystalline structure of the III-VI 2D layers. The photoluminescence and Raman spectra of the grown films are discussed in detail to confirm or correct the structural findings. The requirement of a high growth temperature for the fabrication of optically active 2D layers was confirmed for all materials. However, this also facilitated the strong diffusion of group III metals in III-VI and III-VI/II-VI heterostructures. In particular, the strong In diffusion into the underlying ZnSe layers was observed in ZnSe/InSe/ZnSe quantum well structures, and the Ga diffusion into the top InSe layer grown at ~450 °C was confirmed by the Raman data in the InSe/GaSe heterostructures. The results on fabrication of the GaSe/GaTe quantum well structures are presented as well, although the choice of optimum growth temperatures to make them optically active is still a challenge.

Design and Characterization of a Sharp GaAs/Zn(Mn)Se Heterovalent Interface: A Sub-Nanometer Scale View.

The distribution of magnetic impurities (Mn) across a GaAs/Zn(Mn)Se heterovalent interface is investigated combining three experimental techniques: Cross-Section Scanning Tunnel Microscopy (X-STM), Atom Probe Tomography (APT), and Secondary Ions Mass Spectroscopy (SIMS). This unique combination allowed us to probe the Mn distribution with excellent sensitivity and sub-nanometer resolution. Our results show that the diffusion of Mn impurities in GaAs is strongly suppressed; conversely, Mn atoms are subject to a substantial redistribution in the ZnSe layer, which is affected by the growth conditions and the presence of an annealing step. These results show that it is possible to fabricate a sharp interface between a magnetic semiconductor (Zn(Mn)Se) and high quality GaAs, with low dopant concentration and good optical properties.

Peroxidasin-mediated bromine enrichment of basement membranes.

Bromine and peroxidasin (an extracellular peroxidase) are essential for generating sulfilimine cross-links between a methionine and a hydroxylysine within collagen IV, a basement membrane protein. The sulfilimine cross-links increase the structural integrity of basement membranes. The formation of sulfilimine cross-links depends on the ability of peroxidasin to use bromide and hydrogen peroxide substrates to produce hypobromous acid (HOBr). Once a sulfilimine cross-link is created, bromide is released into the extracellular space and becomes available for reutilization. Whether the HOBr generated by peroxidasin is used very selectively for creating sulfilimine cross-links or whether it also causes oxidative damage to bystander molecules (e.g., generating bromotyrosine residues in basement membrane proteins) is unclear. To examine this issue, we used nanoscale secondary ion mass spectrometry (NanoSIMS) imaging to define the distribution of bromine in mammalian tissues. We observed striking enrichment of bromine (79Br, 81Br) in basement membranes of normal human and mouse kidneys. In peroxidasin knockout mice, bromine enrichment of basement membranes of kidneys was reduced by ∼85%. Proteomic studies revealed bromination of tyrosine-1485 in the NC1 domain of α2 collagen IV from kidneys of wild-type mice; the same tyrosine was brominated in collagen IV from human kidney. Bromination of tyrosine-1485 was reduced by >90% in kidneys of peroxidasin knockout mice. Thus, in addition to promoting sulfilimine cross-links in collagen IV, peroxidasin can also brominate a bystander tyrosine. Also, the fact that bromine enrichment is largely confined to basement membranes implies that peroxidasin activity is largely restricted to basement membranes in mammalian tissues.

Prediction of influenza peaks in Russian cities: Comparing the accuracy of two SEIR models.

This paper is dedicated to the application of two types of SEIR models to the influenza outbreak peak prediction in Russian cities. The first one is a continuous SEIR model described by a system of ordinary differential equations. The second one is a discrete model formulated as a set of difference equations, which was used in the Baroyan-Rvachev modeling framework for the influenza outbreak prediction in the Soviet Union. The outbreak peak day and height predictions were performed by calibrating both models to varied-size samples of long-term data on ARI incidence in Moscow, Saint Petersburg, and Novosibirsk. The accuracy of the modeling predictions on incomplete data was compared with a number of other peak forecasting methods tested on the same dataset. The drawbacks of the described prediction approach and possible ways to overcome them are discussed.

MYB fusions and CD markers as tools for authentication and purification of cancer stem cells from salivary adenoid cystic carcinoma.

Cancer stem cells (CSC) are considered the major cause of aggressive tumor behavior, recurrence, metastases, and resistance to radiation, making them an attractive therapeutic target. However, isolation of CSC from tumor tissue and their characterization are challenging due to uncertainty about their molecular markers and conditions for their propagation. Adenoid cystic carcinoma (ACC), which arises predominantly in the salivary glands, is a slow-growing but relentless tumor that frequently invades nerves and metastasizes. New effective treatment approaches for ACC have not emerged over the last 40years. Previously, based on a highly conserved SOX10 gene signature that we identified in the majority of ACC tumors, we suggested the existence in ACC of SOX10+ cells with neural stem properties and corroborated this hypothesis via isolation from ACC tissue a novel population of CSC, termed ACC-CSC. These cells activated NOTCH1 signaling and co-expressed SOX10 and other ACC-intrinsic neural crest stem cell markers with CD133, a CSC cell surface marker, suggesting that ACC is driven by a previously uncharacterized population of SOX10+/CD133+ cells with neural stem cell properties. Here, we authenticated ACC identity of our primary cultures by demonstrating that most of them harbor MYB-NFIB fusions, which are found in 86% of ACC. We demonstrated using CyTOF, a novel mass cytometry technology, that these cells express high ß-catenin and STAT3 levels and are marked by CD24 and CD44. Finally, to streamline development of ACC cell lines, we developed RT-PCR tests for distinguishing mouse and human cells and used immunomagnetic cell sorting to eliminate mouse cells from long-term cell cultures. Overall, this study describes a new population of CSC that activates signaling pathways associated with poor prognosis, validates their ACC identity, and optimizes approaches that can be used for purification of ACC-CSC and generation of cell lines.

Chromosomal abnormalities and molecular landscape of metastasizing mucinous salivary adenocarcinoma.

BACKGROUND: Mucinous adenocarcinoma of the salivary gland (MAC) is a lethal cancer with unknown molecular etiology and a high propensity to lymph node metastasis. Mostly due to its orphan status, MAC remains one of the least explored cancers that lacks cell lines and mouse models that could help translational and pre-clinical studies. Surgery with or without radiation remains the only treatment modality but poor overall survival (10-year, 44%) underscores the urgent need for mechanism-based therapies. METHODS: We developed the first patient-derived xenograft (PDX) model for pre-clinical MAC studies and a cell line that produces aggressively growing tumors after subcutaneous injection into nude mice. We performed cytogenetic, exome, and proteomic profiling of MAC to identify driving mutations, therapeutic targets, and pathways involved in aggressive cancers based on TCGA database mining and GEO analysis. RESULTS: We identified in MAC KRAS (G13D) and TP53 (R213X) mutations that have been previously reported as drivers in a variety of highly aggressive cancers. Somatic mutations were also found in KDM6A, KMT2D, and other genes frequently mutated in colorectal and other cancers: FAT1, NBEA, RELN, RLP1B, and ZFHX3. Proteomic analysis of MAC implied epigenetic up-regulation of a genetic program involved in proliferation and cancer stem cell maintenance. CONCLUSION: Genomic and proteomic analyses provided the first insight into potential molecular drivers of MAC metastases pointing at common mechanisms of CSC propagation in aggressive cancers. The in vitro/in vivo models that we created should aid in the development and validation of new treatment strategies against MAC.

Expression Profiling of Clinical Specimens Supports the Existence of Neural Progenitor-Like Stem Cells in Basal Breast Cancers.

BACKGROUND: We previously characterized in salivary adenoid cystic carcinoma (ACC) a novel population of cancer stem cells (CSCs) marked by coexpression of 2 stemness genes, sex-determining region Y (SRY)-related HMG box-containing factor 10 (SOX10) and CD133. We also reported that in ACC and basal-like breast carcinoma (BBC), a triple-negative breast cancer subtype, expression of SOX10 similarly demarcates a highly conserved gene signature enriched with neural stem cell genes. On the basis of these findings, we hypothesized that BBC might be likewise driven by SOX10-positive (SOX10+)/CD133+ cells with neural stem cell properties. MATERIALS AND METHODS: To validate our hypothesis on clinical data, we used a novel approach to meta-analysis that merges gene expression data from independent breast cancer studies and ranks genes according to statistical significance of their coexpression with the gene of interest. Genes that showed strong association with CD133/PROM1 as well as SOX10 were validated across different platforms and data sets and analyzed for enrichment with genes involved in neurogenesis. RESULTS: We identified in clinical breast cancer data sets a highly conserved SOX10/PROM1 gene signature that contains neural stem cell markers common for Schwann cells, ACC, BBC, and melanoma. Identification of tripartite motif-containing 2 (TRIM2), TRIM29, MPZL2, potassium calcium-activated channel subfamily N member 4 (KCNN4), and V-set domain containing T cell activation inhibitor 1 (VTCN1)/B7 homolog 4 (B7H4) within this signature provides insight into molecular mechanisms of CSC maintenance. CONCLUSION: Our results suggest that BBC is driven by SOX10+/CD133+ cells that express neural stem cell-specific markers and share molecular similarities with CSCs of neural crest origin. Our study provides clinically relevant information on possible drivers of these cells that might facilitate development of CSC-targeting therapies against this cancer distinguished with poor prognosis and resistance to conventional therapies.

High-Output-Power Ultraviolet Light Source from Quasi-2D GaN Quantum Structure.

Quasi-2D GaN layers inserted in an AlGaN matrix are proposed as a novel active region to develop a high-output-power UV light source. Such a structure is successfully achieved by precise control in molecular beam epitaxy and shows an amazing output power of ≈160 mW at 285 nm with a pulsed electron-beam excitation. This device is promising and competitive in non-line-of-sight communications or the sterilization field.

NOTCH1 and SOX10 are Essential for Proliferation and Radiation Resistance of Cancer Stem-Like Cells in Adenoid Cystic Carcinoma.

PURPOSE: Although the existence of cancer stem cells (CSC) in adenoid cystic carcinoma (ACC) has been proposed, lack of assays for their propagation and uncertainty about molecular markers prevented their characterization. Our objective was to isolate CSC from ACC and provide insight into signaling pathways that support their propagation. EXPERIMENTAL DESIGN: To isolate CSC from ACC and characterize them, we used ROCK inhibitor-supplemented cell culture, immunomagnetic cell sorting, andin vitro/in vivoassays for CSC viability and tumorigenicity. RESULTS: We identified in ACC CD133-positive CSC that expressed NOTCH1 and SOX10, formed spheroids, and initiated tumors in nude mice. CD133(+)ACC cells produced activated NOTCH1 (N1ICD) and generated CD133(-)cells that expressed JAG1 as well as neural differentiation factors NR2F1, NR2F2, and p27Kip1. Knockdowns ofNOTCH1, SOX10, and their common effectorFABP7had negative effects on each other, inhibited spheroidogenesis, and induced cell death pointing at their essential roles in CSC maintenance. Downstream effects ofFABP7knockdown included suppression of a broad spectrum of genes involved in proliferation, ribosome biogenesis, and metabolism. Among proliferation-linked NOTCH1/FABP7 targets, we identified SKP2 and its substrate p27Kip1. A γ-secretase inhibitor, DAPT, selectively depleted CD133(+)cells, suppressed N1ICD and SKP2, induced p27Kip1, inhibited ACC growthin vivo, and sensitized CD133(+)cells to radiation. CONCLUSIONS: These results establish in the majority of ACC the presence of a previously uncharacterized population of CD133(+)cells with neural stem properties, which are driven by SOX10, NOTCH1, and FABP7. Sensitivity of these cells to Notch inhibition and their dependence on SKP2 offer new opportunities for targeted ACC therapies.

Clinical and molecular insights into adenoid cystic carcinoma: Neural crest-like stemness as a target.

OBJECTIVES: This review surveys trialed therapies and molecular defects in adenoid cystic carcinoma (ACC), with an emphasis on neural crest-like stemness characteristics of newly discovered cancer stem cells (CSCs) and therapies that may target these CSCs. DATA SOURCES: Articles available on Pubmed or OVID MEDLINE databases and unpublished data. REVIEW METHODS: Systematic review of articles pertaining to ACC and neural crest-like stem cells. RESULTS: Adenoid cystic carcinoma of the salivary gland is a slowly growing but relentless cancer that is prone to nerve invasion and metastases. A lack of understanding of molecular etiology and absence of targetable drivers has limited therapy for patients with ACC to surgery and radiation. Currently, no curative treatments are available for patients with metastatic disease, which highlights the need for effective new therapies. Research in this area has been inhibited by the lack of validated cell lines and a paucity of clinically useful markers. The ACC research environment has recently improved, thanks to the introduction of novel tools, technologies, approaches, and models. Improved understanding of ACC suggests that neural crest-like stemness is a major target in this rare tumor. New cell culture techniques and patient-derived xenografts provide tools for preclinical testing. CONCLUSION: Preclinical research has not identified effective targets in ACC, as confirmed by the large number of failed clinical trials. New molecular data suggest that drivers of neural crest-like stemness may be required for maintenance of ACC; as such, CSCs are a target for therapy of ACC.

Line mixing effects in isotropic Raman spectra of pure N2: a classical trajectory study.

Line mixing effects in the Q branch of pure N2 isotropic Raman scattering are studied at room temperature using a classical trajectory method. It is the first study using an extended modified version of Gordon's classical theory of impact broadening and shift of rovibrational lines. The whole relaxation matrix is calculated using an exact 3D classical trajectory method for binary collisions of rigid N2 molecules employing the most up-to-date intermolecular potential energy surface (PES). A simple symmetrizing procedure is employed to improve off-diagonal cross-sections to make them obeying exactly the principle of detailed balance. The adequacy of the results is confirmed by the sum rule. The comparison is made with available experimental data as well as with benchmark fully quantum close coupling [F. Thibault, C. Boulet, and Q. Ma, J. Chem. Phys. 140, 044303 (2014)] and refined semi-classical Robert-Bonamy [C. Boulet, Q. Ma, and F. Thibault, J. Chem. Phys. 140, 084310 (2014)] results. All calculations (classical, quantum, and semi-classical) were made using the same PES. The agreement between classical and quantum relaxation matrices is excellent, opening the way to the analysis of more complex molecular systems.

Fus1/Tusc2 is a novel regulator of mitochondrial calcium handling, Ca2+-coupled mitochondrial processes, and Ca2+-dependent NFAT and NF-κB pathways in CD4+ T cells.

AIMS: Fus1 has been established as mitochondrial tumor suppressor, immunomodulator, and antioxidant protein, but molecular mechanism of these activities remained to be identified. Based on putative calcium-binding and myristoyl-binding domains that we identified in Fus1, we explored our hypothesis that Fus1 regulates mitochondrial calcium handling and calcium-coupled processes. RESULTS: Fus1 loss resulted in reduced rate of mitochondrial calcium uptake in calcium-loaded epithelial cells, splenocytes, and activated CD4(+) T cells. The reduced rate of mitochondrial calcium uptake in Fus1-deficient cells correlated with cytosolic calcium increase and dysregulation of calcium-coupled mitochondrial parameters, such as reactive oxygen species production, ΔµH(+), mitochondrial permeability transition pore opening, and GSH content. Inhibition of calcium efflux via mitochondria, Na(+)/Ca(2+) exchanger significantly improved the mitochondrial calcium uptake in Fus1(-/-) cells. Ex vivo analysis of activated CD4(+) T cells showed Fus1-dependent changes in calcium-regulated processes, such as surface expression of CD4 and PD1/PD-L1, proliferation, and Th polarization. Fus1(-/-) T cells showed increased basal expression of calcium-dependent NF-κB and NFAT targets but were unable to fully activate these pathways after stimulation. INNOVATION: Our results establish Fus1 as one of the few identified regulators of mitochondrial calcium handling. Our data support the idea that alterations in mitochondrial calcium dynamics could lead to the disruption of metabolic coupling in mitochondria that, in turn, may result in multiple cellular and systemic abnormalities. CONCLUSION: Our findings suggest that Fus1 achieves its protective role in inflammation, autoimmunity, and cancer via the regulation of mitochondrial calcium and calcium-coupled parameters.