Intracellular Mechanisms Research Articles

Protein Networks Associated with Native Metabotropic Glutamate 1 Receptors (mGlu1) in the Mouse Cerebellum.

The metabotropic glutamate receptor 1 (mGlu1) plays a pivotal role in synaptic transmission and neuronal plasticity. Despite the fact that several interacting proteins involved in the mGlu1 subcellular trafficking and intracellular transduction mechanisms have been identified, the protein network associated with this receptor in specific brain areas remains largely unknown. To identify novel mGlu1-associated protein complexes in the mouse cerebellum, we used an unbiased tissue-specific proteomic approach, namely co-immunoprecipitation followed by liquid chromatography/tandem mass spectrometry analysis. Many well-known protein complexes as well as novel interactors were identified, including G-proteins, Homer, δ2 glutamate receptor, 14-3-3 proteins, and Na/K-ATPases. A novel putative interactor, KCTD12, was further investigated. Reverse co-immunoprecipitation with anti-KCTD12 antibodies revealed mGlu1 in wild-type but not in KCTD12-knock-out homogenates. Freeze-fracture replica immunogold labeling co-localization experiments showed that KCTD12 and mGlu1 are present in the same nanodomain in Purkinje cell spines, although at a distance that suggests that this interaction is mediated through interposed proteins. Consistently, mGlu1 could not be co-immunoprecipitated with KCTD12 from a recombinant mammalian cell line co-expressing the two proteins. The possibility that this interaction was mediated via GABAB receptors was excluded by showing that mGlu1 and KCTD12 still co-immunoprecipitated from GABAB receptor knock-out tissue. In conclusion, this study identifies tissue-specific mGlu1-associated protein clusters including KCTD12 at Purkinje cell synapses.

CyclosporinA Derivative as Therapeutic Candidate for Alport Syndrome by Inducing Mutant Type IV Collagen Secretion.

Type IV collagen α3,4,5 (α345(IV)) is an obligate trimer that is secreted to form a collagen network, which is the structural foundation of basement membrane. Mutation in one of the genes (COL4A3, A4, A5) encoding these proteins underlies the progressive genetic nephropathy Alport syndrome (AS) due to deficiency in trimerization and/or secretion of the α345(IV) trimer. Thus, improving mutant α345(IV) trimerization and secretion could be a good therapeutic approach for AS. Using the nanoluciferase-based platform that we previously developed to detect α345(IV) formation and secretion in HEK293T cells, we screened libraries of natural product extracts and compounds to find a candidate compound capable of increasing mutant α345(IV) secretion. The screening of >13,000 extracts and >600 compounds revealed that cyclosporin A (CsA) increased the secretion of mutant α345(IV)-G1244D. To elucidate the mechanism of the effect of CsA, we evaluated CsA derivatives with different ability to bind to calcineurin (Cn) and cyclophilin (Cyp). Alisporivir (ALV), which binds to Cyp but not to Cn, increased the trimer secretion of mutant α345(IV). Knockdown studies on Cyps showed that PPIF/CypD was involved in the trimer secretion-enhancing activity of CsA and ALV. We confirmed that other α345(IV) mutants are also responsive to CsA and ALV. CsA was previously reported to improve proteinuria in AS patients, but due to its nephrotoxic effect, CsA is not recommended for treatment in AS patients. Our data raise the possibility that ALV could be a safer option than CsA. This study provides a novel therapeutic candidate for AS with an innovative mechanism of action, and reveals an aspect of the intracellular regulatory mechanism of α345(IV) that was previously unexplored.

The role of circulating miR-19b miRNA in predicting the outcome of COVID-19

Background. MicroRNAs are short (20–22 nucleotides) non-coding RNAs that can posttranscriptionally regulate gene expression and are considered a regulator of the innate immunity system. Previously, many papers were published on the prediction of the interaction of the single-stranded (+)RNA virus SARS-CoV-2 with human microRNAs, as well as on the profile of circulating microRNAs in patients with COVID-19 of varying severity. However, no works are analyzing the possible contribution of miRNAs circulating in blood plasma to the severity of COVID-19.The aim. To study the features of the blood plasma microRNA profile of patients with different severity of the new coronavirus infection COVID-19 and to evaluate the possibility of microRNA interaction with the SARS-CoV-2 genome.Materials and methods. The results of NGS sequencing of plasma miRNAs of 3 recovered and 8 deceased patients with a highly severe form of COVID-19 were studied. Differentially presented microRNAs were determined using bioinformatics methods, and their binding sites with the SARS-CoV-2 genome were predicted.Results. This study demonstrates that in patients who have recovered from a highly severe form of COVID-19, the level of hsa-miR-19b-3p in the blood plasma is significantly increased. This microRNA makes up about 1.5 % of all circulating microRNAs and can bind to SARS-CoV-2 regions encoding proteins that suppress intracellular immunity mechanisms (NSP3, NSP9). In addition, this miRNA can stimulate the functional activity and proliferation of cytotoxic T-lymphocytes, one of the critical components of acquired cellular immunity against SARS-CoV-2.Conclusion. The results of the study can be used in the development of antiviral drugs based on RNA interference, as well as in the development of predictive test systems to optimize the tactics of treating patients with COVID-19.

Membrane Permeability Is Required for the Vasodilatory Effect of Carbonic Anhydrase Inhibitors in Porcine Retinal Arteries.

It has been demonstrated previously that a variety of carbonic anhydrase inhibitors (CAIs) can induce vasodilation in pre-contracted retinal arteriolar segments although with different efficacy and potency. Since the CAIs tested so far are able to permeate cell membranes and inhibit both intracellular and extracellular isoforms of the enzyme, it is not clear whether extra- or intracellular isoforms or mechanisms are mediating their vasodilatory effects. By means of small wire myography, we have tested the effects of four new CAIs on wall tension in pre-contracted retinal arteriolar segments that demonstrably do not enter cell membranes but have high affinity to both cytosolic and membrane-bound isoforms of CA. At concentrations between 10-6 M to 10-3 M, none of the four membrane impermeant CAIs had any significant effect on arteriolar wall tension, while the membrane permeant CAI benzolamide (10-3 M) fully dilated all arteriolar segments tested. This suggests that CAI act as vasodilators through cellular mechanisms located in the cytoplasm of vascular cells.

RORγt-Raftlin1 complex regulates the pathogenicity of Th17 cells and intestinal inflammation.

Abstract Th17 cells that produce IL-17 are pathogenic in many human diseases, including inflammatory bowel disease (IBD), but are, paradoxically, essential for maintaining the integrity of the intestinal barrier in a non-inflammatory state. However, the intracellular mechanisms that regulate distinct transcriptional profiles and functional diversity of Th17 cells remain unclear. We have found that Raftlin1, a lipid raft protein, is specifically upregulated in and forms a complex with RORγt via its conserved LLNSL motif in pathogenic Th17 cells. Disruption of the RORγt-Raftlin1 complex in Raftlin1 ΔLLNSLknock-in mice resulted in attenuated pathogenic Th17 cells elicited by Citrobacter rodentium; however, there was no effect on nonpathogenic Th17 cells induced by commensal segmented filamentous bacteria (SFB). Mechanistically, we found that Raftlin1 recruits distinct phospholipids to RORγt and promotes the pathogenicity of Th17 cells. Thus, we have identified a fundamental mechanism that drives the pathogenic function of Th17 cells, which could provide a platform for new therapeutic strategies to dampen Th17-mediated inflammatory diseases. This work was supported by funds from the National Institutes of Health (R01-DK115668, R01-AI155786) and Cancer Prevention Research Institute of Texas (RP190527) and a stimulus grant from the Harold C. Simmons Comprehensive Cancer Center, UT Southwestern Medical Center.

Divergent electrophysiology between two cardiac vagal motor populations despite similar chronotropic effects

Cardiac vagal motor output is generated by brainstem cardiac vagal motorneurons (CVNs) that send axonal projections, through the vagus nerve, to cardiac ganglia. CVNs have a well-established cardioinhibitory influence through reductions in heart rate (HR) and studies using vagotomy confirm cardiac vagal drive originates in CVNs. CVNs can be found in two brainstem regions: nucleus ambiguus (NA) and dorsal motor nucleus of the vagus (DMV). Although the cardioinhibitory role of CVNs in NA (CVN NA ) is widely accepted, effects of CVNs in DMV (CVN DMV ) on HR regulation is controversial. The present study aims to provide evidence of CVN DMV existence and identify the unique role DMV plays in HR regulation. We hypothesized that CVN DMV cause robust bradycardias and associate with unique electrophysiological properties making them hyperexcitable compared to CVN NA . Experiments were performed on adult (9-11 week old) male mice. Using a transgenic mouse line where light activated channelrhodopsin (ChR) is expressed in choline acetyltransferase neurons, activation of ChR in DMV using fiber optic probes resulted in a 359±43 bpm bradycardia (n=8) in awake behaving animals. DMV-mediated ChR-dependent bradycardia remained after i.p. administration of atenolol (256±31 bpm). However, i.p. scopolamine-methyl nitrate abolished the ability of ChR activation of DMV to impact HR (12±2 bpm, p=0.0002). ChR stimulation of NA resulted in a similar bradycardia (447±6 bpm; p<0.0001) that was also blocked by scopolamine-methyl nitrate (9±7 bpm, p=0.005). Retrograde tracing from cardiac tissue using rhodamine and cholera-toxin B (CTB) confirmed CVNs in DMV (50±15 labeled neurons, n=6; CTB: 12±4 labeled neurons, n=8). However, the number of CVN DMV was significantly lower than CVN NA (rhodamine: 116±24 labeled neurons, n=6; CTB: 83.33±9.548 labeled neurons, n=6, p<0.0001 for both). Unilateral vagotomy eliminated tracer presence in DMV ipsilateral (2±1 neurons) compared to contralateral (32±8 neurons; n=4; p=0.048). Using whole cell patch clamp, CVN DMV demonstrated spontaneous action potential firing in vitro (0.7±0.3 Hz, n=14 neurons/10 mice) whereas CVN NA were devoid of spontaneous firing (0.0±0.0 Hz, n=10 neurons/9 mice). CVN DMV (-58.6±2.7 Hz, n=14 neurons/10 mice) and CVN NA (-62.2±1.8 mV, n=10 neurons/9 mice, p=0.3) demonstrated similar resting membrane potential. However, CVN DMV (492.0±22.0 MΩ, n=14 neurons/10 mice) had higher input resistance compared to CVN NA (158.2±10.6 MΩ, n=10 neurons/9 mice, p<0.0001). Taken together, these data support the hypothesis that DMV contains a population of CVNs capable of producing cardioinhibitory actions with unique hyperexcitability compared to CVN NA . Future studies will examine both intracellular mechanism(s) of these differences, but also unique microcircuit pathways which utilize CVN DMV , and not CVN NA . NIH T32 HL007446 for MS; NIH R01HL157366 to CRB This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Supraphysiological levels of testosterone induces endothelial injury via increasing generation of reactive oxygen species and ERK1/2 activation

Introduction: High levels of testosterone (Testo) is associated with higher cardiovascular risk by positively modulate the generation of reactive oxygen species (ROS) and inflammatory response. Excessive ROS generation by NADPH oxidases (NOX, major source of ROS in the vasculature) leads to endothelial dysfunction, apoptosis, and inflammation. However, the intracellular mechanisms whereby supraphysiological levels of Testo promote cardiovascular damage are still ill defined. We sought to determine whether supraphysiological levels of Testo induces endothelial cell dysfunction via ROS generation and ERK1/2 activation. Methods: To study whether Testo present the same effects independent on endothelial cell type, we used Primary Human Mesenteric vascular Endothelial Cells (HMEC) and Human Umbilical Vein Endothelial Cells (HUVEC). Cells were stimulated with Testo (10 −7 M, for different time points) in the presence or absence of superoxide dismutase mimetic (Tempol, 10 -4 M, for 30 min). NOX activity, gene expression, and protein expression were determined pharmacological assays and immunoblotting, respectively. Results: Testo increased superoxide anion (O 2 − ) generation in HMEC and HUVEC at short (10 min) stimulation times, which was abolished by superoxide dismutase (SOD) mimetic [(% of control) Ctrl: 100.0 ± 0.0 n=5 vs Testo: 363.2 ± 19.77 n=6 vs. Tempol+Testo: 156.7 ± 12.12 n=6, *P<0.05]. Furthermore, long-term treatment (24 h) of endothelial cells with Testo increased Nox4 expression at gene (2.44-Fold change) and protein levels [(% of control) Ctrl: 100.0 ± 0.0 n=4 vs. Testo: 157.3 ± 4.49 n=4, *P<0.05]. Testo did not affect Nox1, Nox2, and Nox5 expressions. Furthermore, Testo increased ERK1/2 phosphorylation at 10 minutes and promoted endothelial cell inflammation at 8h, characterized by increased ICAM-1(6.49-Fold changes) and VCAM-1 [(1.42-Fold changes) gene expressions. To confirm that Testo-induced ROS production is leading to endothelial injury via ERK1/2, we treated endothelial cells with Tempol prior Testo treatment. Interestingly, superoxide dismutase mimetic prevented ERK1/2 phosphorylation induced by Testo [(% of control) Testo: 161.2 ± 11.39 n=5 vs. Tempol+Testo: 91.45 ± 9.61 n=5, *P<0.05]. Conclusion: These data indicate that supraphysiological levels of testosterone induce endothelial dysfunction via ROS generation and inflammatory response. Our data helps understanding the intracellular mechanisms that justify the cardiovascular damage caused by supraphysiological doses of testosterone and places antioxidant therapy as a possible pharmacological approach to treat patients with dysregulated testosterone levels. 2022/00103-3, São Paulo Research Foundation (FAPESP). NHLBI-R00 (R00HL14013903), AHA-CDA (CDA857268), Vascular Medicine Institute, the Hemophilia Center of Western Pennsylvania Vitalant, and Children's Hospital of Pittsburgh of the UPMC Health System. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

TGFβ trained immunity in mast cells: do inflammasomes matter?

Abstract Mast cell (MC) contributions to solid cancer microenvironments are controversial and seem tissue dependent. TGFβ is a potent MC chemokine abundantly expressed by high-grade cancers. Previous work reported that TGFβ alone induces biologically relevant expression of IL-6 from MC, which is potentially important for reciprocal paracrine interactions with cancer cells and other leukocytes. Therefore, the objective of the present study was to examine MC in an immunocompetent, high-grade model of breast cancer. Using the 4T1 system in BALB/c mice, we compared mast cell infiltration into primary tumors of wild-type versus 4T1 cell-specific knockouts of IL-6. We also hypothesized that TGFβ institutes a training effect on MC like other metabolically driven mechanisms involving the NLRP3 inflammasome. Using a mixture of flow cytometric and qPCR assays we explored potential MC training in the context of TGFβ. Results: Contrary to our prediction, MC infiltration into IL-6KO tumors was significantly increased compared to WT. Studies of classic intracellular training mechanisms in response to TGFβ were mixed. Glucose uptake, glycolytic capacity, mitochondrial potential, and ASC speck formation were unaffected by TGFβ, but IL-6 expression increased as previously seen. Additionally, we were unable to detect substantive NLRP3 expression in MC regardless of condition. Despite these results, we were surprised to observe robust increases in expression of IL-1β, IL-18, and caspase-1 with TGFβ stimulus. We conclude that MC are actively recruited to 4T1 tumors dependent on IL-6 (and TGFβ), MC produce select cytokines in response to TGFβ, yet the mechanism explaining this does not involve the NLRP3 inflammasome and remains unresolved.

Molecular Chameleon Carriers for Nucleic Acid Delivery: The Sweet Spot between Lipoplexes and Polyplexes.

Taking advantage of effective intracellular delivery mechanisms of both cationizable lipids and polymers, highly potent double pH-responsive nucleic acid carriers are generated by combining at least two lipo amino fatty acids (LAFs) as hydrophobic cationizable motifs with hydrophilic cationizable aminoethylene units into novel sequence-defined molecules. The pH-dependent tunable polarity of the LAF is successfully implemented by inserting a central tertiary amine, which disrupts the hydrophobic character once protonated, resulting in pH-dependent structural and physical changes. This "molecular chameleon character" turns out to be advantageous for dynamic nucleic acid delivery via lipopolyplexes. By screening different topologies (blocks, bundles, T-shapes, U-shapes), LAF types, and LAF/aminoethylene ratios, highly potent pDNA, mRNA, and siRNA carriers are identified, which are up to several 100-fold more efficient than previous carrier generations and characterized by very fast transfection kinetics. mRNA lipopolyplexes maintain high transfection activity in cell culture even in the presence of ≥90% serum at an ultra-low mRNA dose of 3 picogram (≈2 nanoparticles/cell), and thus are comparable in potency to viral nanoparticles. Importantly, they show great in vivo performance with high expression levels especially in spleen, tumor, lungs, and liver upon intravenous administration of 1-3µg luciferase-encoding mRNA in mice.

α7 Nicotinic acetylcholine receptor potentiation downregulates chemotherapy-induced inflammatory overactivation by overlapping intracellular mechanisms.

We studied, using a combination of animal andcellular models, the glial mechanisms underlying the anti-neuropathic and anti-inflammatory properties of PAM-2 [(E)-3-furan-2-yl-N-p-tolyl-acrylamide], a positive allosteric modulator of α7 nicotinic acetylcholine receptors (nAChRs). In mice, PAM-2 decreased the inflammatory process induced by the combination of oxaliplatin (OXA), a chemotherapeutic agent, and interleukin-1β (IL-1β), a pro-inflammatory molecule. In the brain and spinal cord of treated animals, PAM-2 reduced pro-inflammatory cytokines/chemokines by mechanisms involving mRNA downregulation of factors in the toll-like receptor 4 (TLR4)/nuclear factor (NF)-κB pathway, and increased the precursor of brain-derived neurotrophic factor (proBDNF). To determine the molecular mechanisms underlying the anti-inflammatory activity of PAM-2, both human C20 microglia and normal human astrocytes (NHA) were used. The results showed that PAM-2-induced potentiation of glial α7 nAChRs decreases OXA/IL-1β-induced overexpression of inflammatory molecules by different mechanisms, including mRNA downregulation of factors in the NF-κB pathway (in microglia and astrocyte) and ERK (only in microglia). The OXA/IL-1β-mediated reduction in proBDNF was prevented by PAM-2 in microglia, but not in astrocytes. Our findings also indicate that OXA/IL-1β-induced organic cation transporter 1 (OCT1) expression is decreased by PAM-2, suggesting that decreased OXA influx may be involved in the protective effects of PAM-2. The α7-selective antagonist methyllycaconitine blocked the most important effects mediated by PAM-2 at both animal and cellular levels, supporting a mechanism involving α7 nAChRs. In conclusion, glial α7 nAChR stimulation/potentiation downregulates neuroinflammatory targets, and thereby remains a promising therapeutic option for cancer chemotherapy-induced neuroinflammation and neuropathic pain.

Folpet promotes apoptosis of bovine mammary epithelial cells via disruption of redox homeostasis and activation of MAPK cascades.

Folpet, a phthalimide fungicide, is an agrochemical used to prevent fungal diseases in several crops. The toxicity of folpet has been demonstrated in Cyprinus carpio, pigs, and the human respiratory system. However, despite the possibilities of ingestion of folpet through feed, detrimental influences of folpet on dairy cattle have not been documented. Thus, this study aimed to record the harmful effects of folpet on the bovine mammary system and milk production using mammary epithelial cells (MAC-T cells), which play an essential role in the maintenance of yield and quality of milk production. In this study, we first confirmed that folpet exhibited cytotoxicity against MAC-T cells in both 2D and 3D cultures. Folpet treatment caused apoptosis, dysregulated intracellular calcium levels, and mitochondrial membrane potential, leading to cell death. We further demonstrated the induction of oxidative stress upon folpet treatment by assessing reactive oxygen species (ROS) content and lipid peroxidation in MAC-T cells. ROS generation following folpet treatment induced activation of MAPK cascades, including ERK1/2, JNK, and p38 signaling. This is the first report highlighting the detrimental impacts of folpet on bovine mammary glands and, consequently, the dairy industry by elucidating intracellular mechanisms using MAC-T cells.

Bifidobacterium bifidum prevents the IL-1B induced increase in intestinal permeability by a novel mechanism: TLR-2 dependent activation of PPAR-gamma and inhibition of NF-kB signaling pathway

Background: Bifidobacterium bifidum (BB) is one of the most common probiotic bacteria strains that have been shown to have protective effects against intestinal inflammation, primarily by enhancing the intestinal epithelial tight junction (TJ) barrier function. IL-1β contributes to the development of intestinal inflammation in inflammatory bowel disease (IBD) in part by causing an increase in intestinal permeability. The IL-1β induced increase in intestinal epithelial TJ permeability is mediated by an NF-κB-dependent activation of myosin light chain kinase (MLCK) gene and disruption of the intestinal TJ barrier. The peroxisome proliferator-activated receptor gamma (PPAR-γ) is an important nuclear receptor in enterocytes which is known to have anti-inflammatory activity by interfering with NF-κB activation. However, the role of BB/PPAR-γ (and the possible mechanism involved) in protecting against IL-1β increase in intestinal permeability remain unclear. Aims: The major purpose of this study was to delineate the protective effect of BB against the IL-1β induced increase in intestinal TJ permeability and the intracellular mechanism involved. Methods: Filter-grown Caco-2 monolayers ( in vitro) and recycling intestinal perfusion of live mice ( in vivo) were used to assess intestinal permeability by using a paracellular marker (dextran-10kDa). Results: IL-1β caused a rapid activation of NF-κB and NF-κB and MLCK-dependent increase in intestinal epithelial TJ permeability in vitro and in vivo. A specific strain of BB (1x108 CFU/ml) referred to as BB1 strain inhibited the IL-1β increase in intestinal TJ permeability while strain BB4 had no effect. Other BB strains examined (5 total strains including BB4) did not inhibit the IL-1β increase in intestinal TJ permeability, suggesting that the BB enhancement of Caco-2 TJ barrier function was strain-specific. BB1, not BB4 enhancement of the TJ barrier was associated with an increase in TLR-2 expression, and PPAR-γ activation; BB1 also inhibited NF-kB activation and MLCK activity in Caco-2 monolayers and in mouse enterocyte. The inhibitory effect of BB on 1L-1β induced increase intestinal permeability, NF-κB activation, MLCK expression and activity was blocked by siRNA-induced knockdown of TLR-2 or PPAR-γ in Caco-2 monolayers. We also generated Villin-cre intestinal epithelial specific PPAR-γ knockout mice to study the involvement of enterocytes TLR-2/PPAR-γ on BB1 effect. BB1 did not inhibit the IL-1β-induced activation of NF-κB, increase in MLCK expression and increase in mouse intestinal permeability in TLR-2 or PPAR-γ intestinal deficient mice. Conclusion: These studies provide a novel insight into the BB the protective mechanism against the IL-1β-induced increase in intestinal TJ permeability in -vitro and in-vivo. Our data show that BB protects against the IL-1β induced increase in intestinal TJ permeability by a novel mechanism involving TLR-2/PPAR-g mediated inhibition of NF-kB and MLCK gene activatiation. NIH funding This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mathematical Modelling of Neutrophil Responses to Aspergillus

Abstract Invasive aspergillosis is a dangerous infection commonly affecting immunocompromised patients. The spores of the fungal pathogen, Aspergillus, are found ubiquitously in the environment and are inhaled daily by all humans. These spores are cleared quickly and effectively healthy hosts without developing infection. On the other hand, patients with impaired immunity, can develop an invasive pneumonia with poor outcomes. Neutrophils are essential to the host defense against aspergillosis, both in direct fungal killing and as modulators of immunity against the pathogen. To better understand this role, we conducted a transcriptomic time series comparing the responses of human neutrophils to Aspergillus. Differential gene expression analysis as well as gene enrichment analysis indicated an up regulation of number of transcriptional markers and pathways including NFkB, NR4A, TNF, as well as neutrophil degranulation, cytokine-mediated signaling, chemokine signaling, and TREM1 signaling among others. Combining this data with known mechanisms and pathways from the literature, we built a mathematical model describing the intracellular pathways which determine the neutrophil’s response after detecting Aspergillus. Very few mathematical models have been built modeling intracellular mechanisms of a neutrophil and none that we know of which relates to the interaction between neutrophils and this organism. We intend to use our model to make predictions on the effects of altering different pathways within the cell. Moreover we intend to use this model in a larger systems model describing the immune system response to invasive pulmonary aspergillosis. Supported by: R01AI135128, U01EB024501, Keck Foundation Medical Research Grant 994413.

Renal proximal tubule cells: power and finesse

The proximal tubule is the high-capacity reabsorptive powerhouse of the kidney. Two papers in recent issues of the JCI highlight mechanisms of more delicate effects of the proximal tubule. Yoon et al. demonstrated the intracellular mechanism by which parathyroid hormone (PTH) increases production of 1,25-vitamin D. Activation of PTH receptor 1/cAMP/PKA signaling inhibited salt-inducible kinase 2 (SIK2) and SIK3, which increased CYB27B1 transcription and 1,25-vitamin D production. Replication of these effects with small-molecule SIK inhibitors suggests possible therapeutic applications for patients with disorders characterized by 1,25-vitamin D deficiency. Zhou et al. discovered that proximal tubule glycolysis acts as a phosphate sensor that regulates fibroblast growth factor 23 production in bone. They described several kidney-specific metabolic modifications that enabled glycolysis to be deployed as a phosphate sensor. The provocative results raise intriguing questions with implications for patients with disorders of phosphate homeostasis, including chronic kidney disease.

Delineating the twin role of autophagy in lung cancer.

Autophagy represents an intracellular defense mechanism equipped within each eukaryotic cells to enable them to cope with variety of physical, chemical, and biological stresses. This mechanism helps to restore the homeostasis and preserve the cellular integrity and function of the cells. In these conditions, such as hypoxia, nutrient deprivation, inhibition of protein synthesis or microbial attack, the process of autophagy is upregulated to maintain cellular homeostasis. The role of autophagy in cancer is an intriguing topic which needs further exploration. This process of autophagy has been many times referred as a double-edged sword in the process of tumorigenesis. In the initial stages, it may act as a tumor suppressor and enable to quench the damaged organelles and harmful molecules generated. In more advanced stages, autophagy has been shown to act as a tumor-promoting system as it may help the cancer cells to cope better with stressful microenvironments. Besides this, autophagy has been associated with development of resistance to anticancer drugs as well as promoting the immune evasion in cancer cells, representing a serious obstacle in cancer treatment and its outcome. Also, autophagy is associated with hallmarks of cancer that may lead to activation of invasion and metastasis. The information on this twin role needs further exploration and deeper understanding of the pathways involved. In this review, we discuss the various aspects of autophagy during tumor development, from early to late stages of tumor growth. Both the protective role of autophagy in preventing tumor growth and the underlying mechanisms adopted with evidence from past studies have been detailed. Further, the role of autophagy in conferring resistance to distinct lung cancer treatment and immune shielding properties has also been discussed. This is essential for further improving on treatment outcome and success rates.

Therapeutic Applications of Mesenchymal Stem Cell Loaded with Gold Nanoparticles for Regenerative Medicine.

In the present study, the various concentrations of AuNP (1.25, 2.5, 5, 10 ppm) were prepared to investigate the biocompatibility, biological performances and cell uptake efficiency via Wharton's jelly mesenchymal stem cells and rat model. The pure AuNP, AuNP combined with Col (AuNP-Col) and FITC conjugated AuNP-Col (AuNP-Col-FITC) were characterized by Ultraviolet-visible spectroscopy (UV-Vis), Fourier-transform infrared spectroscopy (FTIR) and Dynamic Light Scattering (DLS) assays. For in vitro examinations, we explored whether the Wharton's jelly MSCs had better viability, higher CXCR4 expression, greater migration distance and lower apoptotic-related proteins expression with AuNP 1.25 and 2.5 ppm treatments. Furthermore, we considered whether the treatments of 1.25 and 2.5 ppm AuNP could induce the CXCR4 knocked down Wharton's jelly MSCs to express CXCR4 and reduce the expression level of apoptotic proteins. We also treated the Wharton's jelly MSCs with AuNP-Col to investigate the intracellular uptake mechanisms. The evidence demonstrated the cells uptake AuNP-Col through clathrin-mediated endocytosis and the vacuolar-type H+-ATPase pathway with good stability inside the cells to avoid lysosomal degradation as well as better uptake efficiency. Additionally, the results from in vivo examinations elucidated the 2.5 ppm of AuNP attenuated foreign body responses and had better retention efficacy with tissue integrity in animal model. In conclusion, the evidence demonstrates that AuNP shows promise as a biosafe nanodrug delivery system for development of regenerative medicine coupled with Wharton's jelly MSCs.

Anti-Schmallenberg Virus Activities of Type I/III Interferons-Induced Mx1 GTPases from Different Mammalian Species.

Mx proteins are key factors of the innate intracellular defense mechanisms that act against viruses induced by type I/III interferons. The family Peribunyaviridae includes many viruses of veterinary importance, either because infection results in clinical disease or because animals serve as reservoirs for arthropod vectors. According to the evolutionary arms race hypothesis, evolutionary pressures should have led to the selection of the most appropriate Mx1 antiviral isoforms to resist these infections. Although human, mouse, bat, rat, and cotton rat Mx isoforms have been shown to inhibit different members of the Peribunyaviridae, the possible antiviral function of the Mx isoforms from domestic animals against bunyaviral infections has, to our knowledge, never been studied. Herein, we investigated the anti-Schmallenberg virus activity of bovine, canine, equine, and porcine Mx1 proteins. We concluded that Mx1 has a strong, dose-dependent anti-Schmallenberg activity in these four mammalian species.

Coordination in functional traits of Populus euphratica leaves under salt stress

Leaf functional traits allow plant survival and maintain their ecosystem function. Salinity affects leaf functional traits, but coordination among leaf functional traits is poorly known and may depend on salt severity. To increase our understanding of the coordination of leaf functional traits under salt stress, we determined hydraulic, gas exchange, and physiological and biochemical parameters in Populus euphratica Oliv. (P. euphratica) grown under salinity treatments, as well as gas exchange parameters under different CO2 concentrations. We found that P. euphratica can reinforce its hydraulic capacity by increasing the water transfer efficiency of both its leaves and stems when a salinity threat occurs for a specific duration of stress. Its stems were more adaptable than leaves. The economic and hydraulic traits of P. euphratica leaves were consistent during the middle stages of salt stress, but inconsistent during the onset and late stages of salt stress. There was almost no biochemical limitation under severe salinity conditions, and CO2 enrichment of P. euphratica had a greater effect on leaf economic traits. The mechanism of toxic ion exclusion based on water availability and intracellular mechanisms in leaves contributed to salt tolerance when P. euphratica was exposed to salinity stress. There was also a coordination mechanism for the plants during increasing salt stress. The leaf intracellular traits of P. euphratica can coordinate with the leaf economic and hydraulic traits and form a defense mechanism to reduce salt damage and guarantee growth under saline conditions. In conclusion, P. euphratica, the main constructional species of riparian forests, adapts to saline environments by adjustment and coordination of leaf functional traits, ensuring survival. These results provide a scientific basis for riparian forest restoration.

Protective Effects of a Red Grape Juice Extract against Bisphenol A-Induced Toxicity in Human Umbilical Vein Endothelial Cells.

Human exposure to bisphenol A (BPA) occurs through the ingestion of contaminated food and water, thus leading to endothelial dysfunction, the first signal of atherosclerosis. Vitis vinifera L. (grape) juice is well known for its health-promoting properties, due to its numerous bioactive compounds among which are polyphenols. The aim of this study was to evaluate the protective effect of a red grape juice extract (RGJe) against the endothelial damage induced by BPA in human umbilical vein endothelial cells (HUVECs) as an in vitro model of endothelial dysfunction. Our results showed that RGJe treatment counteracted BPA-induced cell death and apoptosis in HUVECs, blocking caspase 3 and modulating p53, Bax, and Bcl-2. Moreover, RGJe demonstrated antioxidant properties in abiotic tests and in vitro, where it reduced BPA-induced reactive oxygen species as well as restored mitochondrial membrane potential, DNA integrity, and nitric oxide levels. Furthermore, RGJe reduced the increase of chemokines (IL-8, IL-1β, and MCP-1) and adhesion molecules (VCAM-1, ICAM-1, and E-selectin), caused by BPA exposure, involved in the primary phase of atheromatous plaque formation. Overall, our results suggest that RGJe prevents BPA-induced vascular damage modulating specific intracellular mechanisms, along with protecting cells, owing to its antioxidant capability.

Parturition is Not the Mirror Image of Implantation: The Role of Progesterone

There has been an abundance of evidence from clinical, animal, and in vitro studies that progesterone (P4) is important for establishing, maintaining, and terminating a pregnancy. P4 exerts its primary action via its two receptors: progesterone receptor A (PGR-A) and B (PGR-B). Analyses of transcriptomeand cistrome genome have unearthed novel members and modifiers of the P4signaling pathway. The increase in serum P4 levels and down-regulation of PGR-B are important in the development of pinopodes, thus marking implantation. Additionally, it promotes the quiescent myometrial cell phenotype, and the inhibition of its production in myometrial cells induces labor and is the key physiologic initiator of parturition.Through genomic and non-genomic intracellular mechanisms involving these PGR isoforms, various physiologic states can be determined such as the quiescent and contractile myometrium during pregnancy.