Tobacco Hornworm (Manduca Sexta) Caterpillarhemolymph Modulates Reactive Oxygen Species and Calcium Generation in Tomato Protoplasts

Biophysical Journal

2021 Feb 01

Gandhi, A;Kariyat, R;Sahoo, N;
| DOI: 10.1016/j.bpj.2020.11.560

Plants have been at war with herbivorous insects for millions of years and have developed a set of highly regulated defense strategies to sense herbivore attack using chemical cues known as herbivore-associated elicitors (HAEs), including oral secretions, ovipositional fluids, and frass. These HAEs induce a series of signaling cascades, which ultimately provide induced defenses against them. Despite the existing HAEs and their role in plant defense induction, our knowledge of other herbivore generated HAEs in plant-herbivore interactions are limited. in this study, we demonstrate that ‘‘hemolymph’’ from Manduca sexta, caterpillarsalso induce plant defense signaling cascade and thereby act as an HAE.Using a dye-based imaging technique, our study showed that the application of crude M. sexta hemolymph potently increased Reactive Oxygen Species (ROS) productionin isolated tomato protoplasts. The addition of antioxidant NAC (N-acetyl-L-cysteine) antagonized hemolymph-induced ROS generation, indicating that M. sexta hemolymph is a ROS inducer in isolated protoplasts. Furthermore, incubating the protoplasts with Calcium (Ca2þ)chelator, BAPTA-AM efficiently abolished the hemolymph-induced ROS production, suggesting possible crosstalk between Ca2þ and ROS signaling. interestingly, the application of crude M. sexta hemolymph dramatically increased Ca2þ in tomato protoplasts. Also, hemolymph-mediated ROS and Ca2þ increase was inhibited in the absence of extracellular Ca2þ. Taken together, our study demonstrates that ‘‘hemolymph’’ from Manduca sexta can directly modulate intracellular ROS and Ca2þ production and possibly regulate defenses against insect herbivores by acting as an HAE.

Chloroquine diphosphate suppresses liver cancer via inducing apoptosis in Wistar rats using interventional therapy

Oncology Letters

2021 Jan 26

Hao, X;Li, W;
| DOI: 10.3892/ol.2021.12494

Liver cancer ranks as the second leading cause of cancer‑associated mortality worldwide. To date, neither current ablation therapy nor chemotherapy are considered ideal in improving the outcome of liver cancer. Therefore, more effective therapies for treating this devastating disease are urgently required. Interventional therapy has been used for numerous years in the treatment of different types of cancer, and is characterized by the direct delivery of anticancer drugs into the tumor. It has been reported that antimalarial chloroquine diphosphate (CQ) exerts effective anticancer activity against several types of cancer. However, its effect on liver cancer remains unclear. Therefore, in the present study, 2D monolayer cell culture and 3D spheroid _in vitro_ models, and a rat model, were utilized to investigate the effect of CQ on liver cancer. CQ demonstrated an effective anticancer effect on HepG2 cells and 3D liver spheroids. Furthermore, the drug significantly inhibited cell growth and viability in the 2D and 3D _in vitro_ models. The CQ‑based intervention treatment effectively attenuated tumor size and weight, increased food intake and consumption of drinking water, and improved body weight and survival rate of rats in the in vivo model. In addition, treatment with CQ potently increased the expression levels of the apoptosis‑related genes. Taken together, the findings of the present study may provide a novel insight into the development of safe and effective treatments for liver cancer.

Circuits between infected macrophages and T cells in SARS-CoV-2 pneumonia

Nature

2021 Jan 11

Grant, RA;Morales-Nebreda, L;Markov, NS;Swaminathan, S;Querrey, M;Guzman, ER;Abbott, DA;Donnelly, HK;Donayre, A;Goldberg, IA;Klug, ZM;Borkowski, N;Lu, Z;Kihshen, H;Politanska, Y;Sichizya, L;Kang, M;Shilatifard, A;Qi, C;Lomasney, JW;Argento, AC;Kruser, JM;Malsin, ES;Pickens, CO;Smith, SB;Walter, JM;Pawlowski, AE;Schneider, D;Nannapaneni, P;Abdala-Valencia, H;Bharat, A;Gottardi, CJ;Budinger, GRS;Misharin, AV;Singer, BD;Wunderink, RG;NU SCRIPT Study Investigators, ;
PMID: 33429418 | DOI: 10.1038/s41586-020-03148-w

Some patients infected with Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) develop severe pneumonia and the acute respiratory distress syndrome (ARDS)1. Distinct clinical features in these patients have led to speculation that the immune response to virus in the SARS-CoV-2-infected alveolus differs from other types of pneumonia2. We collected bronchoalveolar lavage fluid samples from 88 patients with SARS-CoV-2-induced respiratory failure and 211 patients with known or suspected pneumonia from other pathogens and subjected them to flow cytometry and bulk transcriptomic profiling. We performed single-cell RNA-seq on 10 bronchoalveolar lavage fluid samples collected from patients with severe COVID-19 within 48 hours of intubation. In the majority of patients with SARS-CoV-2 infection, the alveolar space was persistently enriched in T cells and monocytes. Bulk and single-cell transcriptomic profiling suggested that SARS-CoV-2 infects alveolar macrophages, which in turn respond by producing T cell chemoattractants. These T cells produce interferon-gamma to induce inflammatory cytokine release from alveolar macrophages and further promote T cell activation. Collectively, our results suggest that SARS-CoV-2 causes a slowly unfolding, spatially limited alveolitis in which alveolar macrophages harboring SARS-CoV-2 and T cells form a positive feedback loop that drives persistent alveolar inflammation.

Endogenous Opioid Signaling in the Mouse Retina Modulates Pupillary Light Reflex

International journal of molecular sciences

2021 Jan 08

Cleymaet, AM;Berezin, CT;Vigh, J;
PMID: 33429857 | DOI: 10.3390/ijms22020554

Opioid peptides and their receptors are expressed in the mammalian retina; however, little is known about how they might affect visual processing. The melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs), which mediate important non-image-forming visual processes such as the pupillary light reflex (PLR), express β-endorphin-preferring, µ-opioid receptors (MORs). The objective of the present study was to elucidate if opioids, endogenous or exogenous, modulate pupillary light reflex (PLR) via MORs expressed by ipRGCs. MOR-selective agonist [D-Ala2, MePhe4, Gly-ol5]-enkephalin (DAMGO) or antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) was administered via intravitreal injection. PLR was recorded in response to light stimuli of various intensities. DAMGO eliminated PLR evoked by light with intensities below melanopsin activation threshold but not that evoked by bright blue irradiance that activated melanopsin signaling, although in the latter case, DAMGO markedly slowed pupil constriction. CTAP or genetic ablation of MORs in ipRGCs slightly enhanced dim-light-evoked PLR but not that evoked by a bright blue stimulus. Our results suggest that endogenous opioid signaling in the retina contributes to the regulation of PLR. The slowing of bright light-evoked PLR by DAMGO is consistent with the observation that systemically applied opioids accumulate in the vitreous and that patients receiving chronic opioid treatment have slow PLR.

Dose-dependent response to infection with SARS-CoV-2 in the ferret model and evidence of protective immunity

Nature communications

2021 Jan 04

Ryan, KA;Bewley, KR;Fotheringham, SA;Slack, GS;Brown, P;Hall, Y;Wand, NI;Marriott, AC;Cavell, BE;Tree, JA;Allen, L;Aram, MJ;Bean, TJ;Brunt, E;Buttigieg, KR;Carter, DP;Cobb, R;Coombes, NS;Findlay-Wilson, SJ;Godwin, KJ;Gooch, KE;Gouriet, J;Halkerston, R;Harris, DJ;Hender, TH;Humphries, HE;Hunter, L;Ho, CMK;Kennard, CL;Leung, S;Longet, S;Ngabo, D;Osman, KL;Paterson, J;Penn, EJ;Pullan, ST;Rayner, E;Skinner, O;Steeds, K;Taylor, I;Tipton, T;Thomas, S;Turner, C;Watson, RJ;Wiblin, NR;Charlton, S;Hallis, B;Hiscox, JA;Funnell, S;Dennis, MJ;Whittaker, CJ;Catton, MG;Druce, J;Salguero, FJ;Carroll, MW;
PMID: 33398055 | DOI: 10.1038/s41467-020-20439-y

There is a vital need for authentic COVID-19 animal models to enable the pre-clinical evaluation of candidate vaccines and therapeutics. Here we report a dose titration study of SARS-CoV-2 in the ferret model. After a high (5 × 106 pfu) and medium (5 × 104 pfu) dose of virus is delivered, intranasally, viral RNA shedding in the upper respiratory tract (URT) is observed in 6/6 animals, however, only 1/6 ferrets show similar signs after low dose (5 × 102 pfu) challenge. Following sequential culls pathological signs of mild multifocal bronchopneumonia in approximately 5-15% of the lung is seen on day 3, in high and medium dosed groups. Ferrets re-challenged, after virus shedding ceased, are fully protected from acute lung pathology. The endpoints of URT viral RNA replication & distinct lung pathology are observed most consistently in the high dose group. This ferret model of SARS-CoV-2 infection presents a mild clinical disease.

lncRNA MEG3 Promotes Osteogenic Differentiation of Tendon Stem Cells Via the miR-129-5p/TCF4/β-Catenin Axis and thus Contributes to Trauma-Induced Heterotopic Ossification

Stem cell reviews and reports

2023 Jun 07

Liu, H;Sun, Z;Luo, G;Hu, Y;Ruan, H;Tu, B;Li, J;Fan, C;
PMID: 37284914 | DOI: 10.1007/s12015-023-10562-w

Heterotopic ossification (HO) is one of the most intractable conditions following injury to the musculoskeletal system. In recent years, much attention has been paid to the role of lncRNA in musculoskeletal disorders, but its role in HO was still unclear. Therefore, this study attempted to determine the role of lncRNA MEG3 in the formation of post-traumatic HO and further explore the underlying mechanisms.On the basis of high-throughput sequencing and qPCR validation, elevated expression of the lncRNA MEG3 was shown during traumatic HO formation. Accordingly, in vitro experiments demonstrated that lncRNA MEG3 promoted aberrant osteogenic differentiation of tendon-derived stem cells (TDSCs). Mechanical exploration through RNA pulldown, luciferase reporter gene assay and RNA immunoprecipitation assay identified the direct binding relationship between miR-129-5p and MEG3, or miR-129-5p and TCF4. Further rescue experiments confirmed the miR-129-5p/TCF4/β-catenin axis to be downstream molecular cascade responsible for the osteogenic-motivating effects of MEG3 on the TDSCs. Finally, experiments in a mouse burn/tenotomy model corroborated the promoting effects of MEG3 on the formation of HO through the miR-129-5p/TCF4/β-catenin axis.Our study demonstrated that the lncRNA MEG3 promoted osteogenic differentiation of TDSCs and thus the formation of heterotopic ossification, which could be a potential therapeutic target.

A Selective Peptidomimetic Modulator Of Cav2.2 (N-Type) Voltage-Gated Calcium Channels For Chronic Pain

The Journal of Pain

2023 Apr 01

Gomez, K;Santiago, U;Calderon-Rivera, A;Duran, P;Loya-Lopez, S;Ran, D;Perez-Miller, S;Handoko, H;Arora, P;Patek, M;King, T;Hu, H;Camacho, C;Khanna, R;
| DOI: 10.1016/j.jpain.2023.02.106

Transmembrane Cav2.2 (N-type) voltage-gated calcium channels are genetically and pharmacologically validated pain targets. Clinical block of Cav2.2 (e.g., with Prialt) or indirect modulation (e.g., with gabapentinoids) mitigates chronic pain but is constrained by side effects. The cytosolic auxiliary subunit collapsin response mediator protein 2 (CRMP2) targets Cav2.2 to the sensory neuron membrane and regulates their function. A CRMP2-derived peptide (CBD3) uncouples the Cav2.2-CRMP2 interaction to inhibit calcium influx, transmitter release and pain. Homology-guided mutagenesis of CBD3 revealed an antinociceptive core in the N-terminal A1RSR4. Here, we developed and applied a novel molecular dynamics approach to identify the Cav2.2 recognition motif of the core CBD3 peptide as the A1R2 dipeptide and used its presenting motif to design pharmacophore models to screen 27 million compounds in the open access server ZincPharmer. Of 200 curated hits, 77 compounds were assessed using depolarization‐evoked calcium influx in rat dorsal root ganglion (DRG) neurons. Nine compounds were tested using electrophysiology and one compound (CBD3063) was evaluated biochemically, electrophysiologically, and behaviorally in models of experimental pain. CBD3063 reduced membrane Cav2.2 expression and currents, uncoupled the Cav2.2-CRMP2 interaction, inhibited neuronal excitability, decreased spinal cord transmission, induced analgesia in naïve rats and reversed mechanical allodynia in rats with spared nerve injury. These results identify CBD3063, as a selective, first-in-class, CRMP2-based peptidomimetic, which allosterically regulates Cav2.2 to achieve analgesia.

Understanding spatiotemporal coupling of gene expression using single molecule RNA imaging technologies

Transcription

2023 Apr 12

Gerber, A;van Otterdijk, S;Bruggeman, FJ;Tutucci, E;
PMID: 37050882 | DOI: 10.1080/21541264.2023.2199669

Across all kingdoms of life, gene regulatory mechanisms underlie cellular adaptation to ever-changing environments. Regulation of gene expression adjusts protein synthesis and, in turn, cellular growth. Messenger RNAs are key molecules in the process of gene expression. Our ability to quantitatively measure mRNA expression in single cells has improved tremendously over the past decades. This revealed an unexpected coordination between the steps that control the life of an mRNA, from transcription to degradation. Here, we provide an overview of the state-of-the-art imaging approaches for measurement and quantitative understanding of gene expression, starting from the early visualizations of single genes by electron microscopy to current fluorescence-based approaches in single cells, including live-cell RNA-imaging approaches to FISH-based spatial transcriptomics across model organisms. We also highlight how these methods have shaped our current understanding of the spatiotemporal coupling between transcriptional and post-transcriptional events in prokaryotes. We conclude by discussing future challenges of this multidisciplinary field.Abbreviations: mRNA: messenger RNA; rRNA: ribosomal rDNA; tRNA: transfer RNA; sRNA: small RNA; FISH: fluorescence in situ hybridization; RNP: ribonucleoprotein; smFISH: single RNA molecule FISH; smiFISH: single molecule inexpensive FISH; HCR-FISH: Hybridization Chain-Reaction-FISH; RCA: Rolling Circle Amplification; seqFISH: Sequential FISH; MERFISH: Multiplexed error robust FISH; UTR: Untranslated region; RBP: RNA binding protein; FP: fluorescent protein; eGFP: enhanced GFP, MCP: MS2 coat protein; PCP: PP7 coat protein; MB: Molecular beacons; sgRNA: single guide RNA.

Ribonuclease inhibitor 1 (RNH1) deficiency cause congenital cataracts and global developmental delay with infection-induced psychomotor regression and anemia

European journal of human genetics : EJHG

2023 Mar 20

Hedberg-Oldfors, C;Mitra, S;Molinaro, A;Visuttijai, K;Fogelstrand, L;Oldfors, A;Sterky, FH;Darin, N;
PMID: 36935417 | DOI: 10.1038/s41431-023-01327-7

Ribonuclease inhibitor 1, also known as angiogenin inhibitor 1, encoded by RNH1, is a ubiquitously expressed leucine-rich repeat protein, which is highly conserved in mammalian species. Inactivation of rnh1 in mice causes an embryonically lethal anemia, but the exact biological function of RNH1 in humans remains unknown and no human genetic disease has so far been associated with RNH1. Here, we describe a family with two out of seven siblings affected by a disease characterized by congenital cataract, global developmental delay, myopathy and psychomotor deterioration, seizures and periodic anemia associated with upper respiratory tract infections. A homozygous splice-site variant (c.615-2A > C) in RNH1 segregated with the disease. Sequencing of RNA derived from patient fibroblasts and cDNA analysis of skeletal muscle mRNA showed aberrant splicing with skipping of exon 7. Western blot analysis revealed a total lack of the RNH1 protein. Functional analysis revealed that patient fibroblasts were more sensitive to RNase A exposure, and this phenotype was reversed by transduction with a lentivirus expressing RNH1 to complement patient cells. Our results demonstrate that loss-of-function of RNH1 in humans is associated with a multiorgan developmental disease with recessive inheritance. It may be speculated that the infection-induced deterioration resulted from an increased susceptibility toward extracellular RNases and/or other inflammatory responses normally kept in place by the RNase inhibitor RNH1.

Towards multiomic analysis of oral mucosal pathologies

Seminars in immunopathology

2023 Jan 01

Einhaus, J;Han, X;Feyaerts, D;Sunwoo, J;Gaudilliere, B;Ahmad, SH;Aghaeepour, N;Bruckman, K;Ojcius, D;Schürch, CM;Gaudilliere, DK;
PMID: 36790488 | DOI: 10.1007/s00281-022-00982-0

Oral mucosal pathologies comprise an array of diseases with worldwide prevalence and medical relevance. Affecting a confined space with crucial physiological and social functions, oral pathologies can be mutilating and drastically reduce quality of life. Despite their relevance, treatment for these diseases is often far from curative and remains vastly understudied. While multiple factors are involved in the pathogenesis of oral mucosal pathologies, the host's immune system plays a major role in the development, maintenance, and resolution of these diseases. Consequently, a precise understanding of immunological mechanisms implicated in oral mucosal pathologies is critical (1) to identify accurate, mechanistic biomarkers of clinical outcomes; (2) to develop targeted immunotherapeutic strategies; and (3) to individualize prevention and treatment approaches. Here, we review key elements of the immune system's role in oral mucosal pathologies that hold promise to overcome limitations in current diagnostic and therapeutic approaches. We emphasize recent and ongoing multiomic and single-cell approaches that enable an integrative view of these pathophysiological processes and thereby provide unifying and clinically relevant biological signatures.

A DLG1-ARHGAP31-CDC42 axis is essential for the intestinal stem cell response to fluctuating niche Wnt signaling

Cell stem cell

2023 Jan 05

Castillo-Azofeifa, D;Wald, T;Reyes, EA;Gallagher, A;Schanin, J;Vlachos, S;Lamarche-Vane, N;Bomidi, C;Blutt, S;Estes, MK;Nystul, T;Klein, OD;
PMID: 36640764 | DOI: 10.1016/j.stem.2022.12.008

A central factor in the maintenance of tissue integrity is the response of stem cells to variations in the levels of niche signals. In the gut, intestinal stem cells (ISCs) depend on Wnt ligands for self-renewal and proliferation. Transient increases in Wnt signaling promote regeneration after injury or in inflammatory bowel diseases, whereas constitutive activation of this pathway leads to colorectal cancer. Here, we report that Discs large 1 (Dlg1), although dispensable for polarity and cellular turnover during intestinal homeostasis, is required for ISC survival in the context of increased Wnt signaling. RNA sequencing (RNA-seq) and genetic mouse models demonstrated that DLG1 regulates the cellular response to increased canonical Wnt ligands. This occurs via the transcriptional regulation of Arhgap31, a GTPase-activating protein that deactivates CDC42, an effector of the non-canonical Wnt pathway. These findings reveal a DLG1-ARHGAP31-CDC42 axis that is essential for the ISC response to increased niche Wnt signaling.

Hedgehog signaling regulates Wolffian duct development through the primary cilium

Biology of reproduction

2022 Dec 15

Alves, MBR;Girardet, L;Augière, C;Moon, KH;Lavoie-Ouellet, C;Bernet, A;Soulet, D;Calvo, E;Teves, ME;Beauparlant, CJ;Droit, A;Bastien, A;Robert, C;Bok, J;Hinton, BT;Belleannée, C;
PMID: 36525341 | DOI: 10.1093/biolre/ioac210

Primary cilia play pivotal roles in embryonic patterning and organogenesis through transduction of the Hedgehog signaling pathway (Hh). While mutations in Hh morphogens impair the development of the gonads and trigger male infertility, the contribution of Hh and primary cilia in the development of male reproductive ductules, including the epididymis, remains unknown. From a Pax2Cre; Ift88fl/fl knock-out mouse model, we found that primary cilia deletion is associated with imbalanced Hh signaling and morphometric changes in the Wolffian duct (WD), the embryonic precursor of the epididymis. Similar effects were observed following pharmacological blockade of primary cilia formation and Hh modulation on WD organotypic cultures. The expression of genes involved in extracellular matrix (ECM), mesenchymal-epithelial transition, canonical Hh, and WD development was significantly altered after treatments. Altogether, we identified the primary cilia-dependent Hh signaling as a master regulator of genes involved in WD development. This provides new insights regarding the etiology of sexual differentiation and male infertility issues.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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