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Hypertrophic scars (HTS) develop from an excessive synthesis of structural proteins like collagen and a decreased expression of proteoglycans such as decorin. Previous research has demonstrated that decorin expression is significantly down-regulated in HTS, deep dermal tissue, and thermally injured tissue, reducing its ability to regulate pro-fibrotic transforming growth factor-beta 1 (TGF-β1) and normal fibrillogenesis. However, treatment of HTS fibroblasts with interferon-alpha 2b (IFN-α2b) has been shown to reduce excessive collagen synthesis and improve HTS by reducing serum TGF-β1 levels. The expression of decorin isoforms in HTS is currently unknown and the effects of TGF-β1 and IFN-α2b on decorin, decorin isoform expression and type 1 collagen are of great interest to our group. Dermal fibroblasts were treated with TGF-β1 and/or IFN-α2b, for 48 h. The expression and secretion of decorin, decorin isoforms and type 1 collagen were quantified with reverse transcription-quantitative polymerase chain reaction, immunofluorescence staining and enzyme-linked immunosorbent assays. The mRNA expression of decorin and each isoform was significantly reduced in HTS fibroblasts relative to normal skin. TGF-β1 decreased the mRNA expression of decorin and decorin isoforms, whereas IFN-α2b showed the opposite effect. IFN-α2b significantly inhibited TGF-β1's effect on the mRNA expression of type I collagen alpha 1 in papillary dermal fibroblasts and overall showed relative effects of inhibiting TGF-β1. These data support that a further investigation into the structural and functional roles of decorin isoforms in HTS pathogenesis is warranted and that IFN-α2b is an important agent in reducing fibrotic outcomes., (© 2024 The Wound Healing Society.)

Deactivation of the medial prefrontal cortex (mPFC) has been broadly reported in both neuropathic pain models and human chronic pain patients. Several cellular mechanisms may contribute to the inhibition of mPFC activity, including enhanced GABAergic inhibition. The functional effect of GABA A (γ-aminobutyric acid type A)-receptor activation depends on the concentration of intracellular chloride in the postsynaptic neuron, which is mainly regulated by the activity of Na-K-2Cl cotransporter isoform 1 (NKCC1) and K-Cl cotransporter isoform 2 (KCC2), 2 potassium-chloride cotransporters that import and extrude chloride, respectively. Recent work has shown that the NKCC1-KCC2 ratio is affected in numerous pathological conditions, and we hypothesized that it may contribute to the alteration of mPFC function in neuropathic pain. We used quantitative in situ hybridization to assess the level of expression of NKCC1 and KCC2 in the mPFC of a mouse model of neuropathic pain (spared nerve injury), and we found that KCC2 transcript is increased in the mPFC of spared nerve injury mice while NKCC1 is not affected. Perforated patch recordings further showed that this results in the hypernegative reversal potential of the GABA A current in pyramidal neurons of the mPFC. Computational simulations suggested that this change in GABA A reversal potential is sufficient to significantly reduce the overall activity of the cortical network. Thus, our results identify a novel pathological modulation of GABA A function and a new mechanism by which mPFC function is inhibited in neuropathic pain. Our data also help explain previous findings showing that activation of mPFC interneurons has proalgesic effect in neuropathic, but not in control conditions. PERSPECTIVE: Chronic pain is associated with the presence of depolarizing GABA A current in the spinal cord, suggesting that pharmacological NKCC1 antagonism has analgesic effects. However, our results show that in neuropathic pain, GABA A current is actually hyperinhibitory in the mPFC, where it contributes to the mPFC functional deactivation. This suggests caution in the use of NKCC1 antagonism to treat pain., (Copyright © 2024 United States Association for the Study of Pain, Inc. Published by Elsevier Inc. All rights reserved.)

Numb is an evolutionarily conserved protein that regulates the differentiation of neuronal progenitor cells through unknown mechanisms. Numb has four alternative splice variants with different lengths of phosphotyrosine-binding (PTB) and proline-rich regions (PRR) domains. In this study, we demonstrated that Numb expression was increased in the primary cultures of rat cortical and hippocampal neurons over time in vitro, and Numb antisense inhibited neurite outgrowth. We verified that cells overexpressing short PTB (SPTB) or long PTB (LPTB) domains exhibited differentiation or proliferation, respectively. SPTB-mediated differentiation was related to the PRR domains, as cells expressing SPTB/LPRR had longer dendrites and more branched dendrites than cells expressing SPTB/SPRR. The differentiation of both cell types was completely blocked by the Ca 2+ chelator. Western blot analysis revealed the increased total protein expression of voltage-gated calcium channel (VGCC) subunit α1C and α1D in cells expressing SPTB and LPTB Numb. The increased expression of the VGCC β3 subunit was only observed in cells expressing SPTB Numb. Immunocytochemistry further showed that SPTB-mediated cell differentiation was associated with increased membrane expression of VGCC subunits α1C, α1D and β3, which corresponded to the higher Ca 2+ current (ICa) densities. Furthermore, we found that VGCC of cells transfected with SPTB/SPRR or SPTB/LPRR Numb isoforms exhibit steady-state inactivation (SSI) in both differentiated and undifferentiated phenotypes. A similar SSI of VGCC was observed in the differentiated cells transfected with SPTB/SPRR or SPTB/LPRR Numb isoforms, whereas a left shift SSI of VGCC in cells expressing SPTB/LPRR was detected in the undifferentiated cells. Collectively, these data indicate that SPTB domain is essential for neurite outgrowth involving in membrane expression of VGCC subunits, and LPRR plays a role in neuronal branching and the regulation of VGCC inactivation kinetics., Competing Interests: Declaration of Competing Interest The authors declared no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Background: In the tolerogenic liver, inadequate or ineffective interferon signaling fails to clear chronic HBV infection. Lambda IFNs (IFNL) bind the interferon lambda receptor-1 (IFNLR1) which dimerizes with IL10RB to induce transcription of antiviral interferon-stimulated genes (ISG). IFNLR1 is expressed on hepatocytes, but low expression may limit the strength and antiviral efficacy of IFNL signaling. Three IFNLR1 transcriptional variants are detected in hepatocytes whose role in regulation of IFNL signaling is unclear: a full-length and signaling-capable form (isoform 1), a form that lacks a portion of the intracellular JAK1 binding domain (isoform 2), and a secreted form (isoform 3), the latter two predicted to be signaling defective. We hypothesized that altering expression of IFNLR1 isoforms would differentially impact the hepatocellular response to IFNLs and HBV replication., Methods: Induced pluripotent stem-cell derived hepatocytes (iHeps) engineered to contain FLAG-tagged, doxycycline-inducible IFNLR1 isoform constructs were HBV-infected then treated with IFNL3 followed by assessment of gene expression, HBV replication, and cellular viability., Results: Minimal overexpression of IFNLR1 isoform 1 markedly augmented ISG expression, induced de novo proinflammatory gene expression, and enhanced inhibition of HBV replication after IFNL treatment without adversely affecting cell viability. In contrast, overexpression of IFNLR1 isoform 2 or 3 partially augmented IFNL-induced ISG expression but did not support proinflammatory gene expression and minimally impacted HBV replication., Conclusions: IFNLR1 isoforms differentially influence IFNL-induced gene expression and HBV replication in hepatocytes. Regulated IFNLR1 expression in vivo could limit the capacity of this pathway to counteract HBV replication., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Neuropeptide oxytocin appears to be involved in the formation of hippocampal circuitry, underlying social memory and behaviour. Recent studies point to the role of oxytocin in regulating the levels of nerve growth factors that could influence neurogenesis and neuritogenesis during the early stages of brain development. Therefore, the aim of the present study was to evaluate the early developmental effect of oxytocin administration (P2 and P3 days, two doses, 5 μg/pup, s.c.) on the expression of 1) brain-derived neurotrophic factor (BDNF) isoforms and 2) GABAergic and glutamatergic markers in the male rat hippocampus. Furthermore, we evaluated the branching of dendrites of primary hippocampal GABAergic and glutamatergic neurons in response to incubation with oxytocin (1 μM). We found that after oxytocin administration, levels of proBDNF increased on P5 and mBDNF on P7 in the CA1 hippocampal region. We also observed a reduction in the expression of glutamatergic marker (VGluT2) on P7 compared to P5 in control and oxytocin treated rats. During the early developmental stages (P5, P7, P9) the expression of GABAergic markers (Gad65 and Gad67) decreased regardless of oxytocin treatment. Incubation in a presence of oxytocin reduced branching of glutamatergic hippocampal neurons and the opposite stimulatory effect of oxytocin was observed in GABAergic neurons. These findings suggest that oxytocin affects neurotrophin isoforms in the male rat hippocampus in the early stages of development, which could explain changes in glutamatergic neurons and their morphology., Competing Interests: Declaration of Competing Interest Authors declare no conflict of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Regulation of RNA stability and translation by RNA-binding proteins (RBPs) is a crucial process altering gene expression. Musashi family of RBPs comprising Msi1 and Msi2 is known to control RNA stability and translation. However, despite the presence of MSI2 in the heart, its function remains largely unknown. Here, we aim to explore the cardiac functions of MSI2. We confirmed the presence of MSI2 in the adult mouse, rat heart, and neonatal rat cardiomyocytes. Furthermore, Msi2 was significantly enriched in the heart cardiomyocyte fraction. Next, using RNA-seq data and isoform-specific PCR primers, we identified Msi2 isoforms 1, 4, and 5, and two novel putative isoforms labeled as Msi2 6 and 7 to be expressed in the heart. Overexpression of Msi2 isoforms led to cardiac hypertrophy in cultured cardiomyocytes. Additionally, Msi2 exhibited a significant increase in a pressure-overload model of cardiac hypertrophy. We selected isoforms 4 and 7 to validate the hypertrophic effects due to their unique alternative splicing patterns. AAV9-mediated overexpression of Msi2 isoforms 4 and 7 in murine hearts led to cardiac hypertrophy, dilation, heart failure, and eventually early death, confirming a pathological function for Msi2. Using global proteomics, gene ontology, transmission electron microscopy, seahorse, and transmembrane potential measurement assays, increased MSI2 was found to cause mitochondrial dysfunction in the heart. Mechanistically, we identified Cluh and Smyd1 as direct downstream targets of Msi2. Overexpression of Cluh and Smyd1 inhibited Msi2-induced cardiac malfunction and mitochondrial dysfunction. Collectively, we show that Msi2 induces hypertrophy, mitochondrial dysfunction, and heart failure., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany.)

Background: Paraoxonase 2 (PON2) and neuronal uncoupling proteins (UCP4 and UCP5) possess antioxidant, anti-apoptotic activities and minimize accumulation of reactive oxygen species in mitochondria. While age and sex are risk factors for several disorders that are linked with oxidative stress, no study has explored the age- and sex-dependent expression of PON2 isoforms, UCP4 and UCP5 in primate brain or identified a drug to activate UCP4 and UCP5 in vivo. Preclinical studies suggest that the peroxisome proliferator-activated receptor gamma agonist, pioglitazone (PIO), can be neuroprotective, although the mechanism responsible is unclear. Our previous studies demonstrated that pioglitazone activates PON2 in primate brain and we hypothesized that pioglitazone also induces UCP4/5. This study was designed to elucidate the age- and sex-dependent expression of PON2 isoforms, UCP4 and UCP5, in addition to examining the impact of systemic PIO treatment on UCP4 and UCP5 expression in primate brain., Methods: Western blot technique was used to determine the age- and sex-dependent expression of UCP4 and UCP5 in substantia nigra and striatum of African green monkeys. In addition, we tested the impact of daily oral pioglitazone (5 mg/kg/day) or vehicle for 1 or 3 weeks on expression of UCP4 and UCP5 in substantia nigra and striatum in adult male monkeys. PIO levels in plasma and cerebrospinal fluid (CSF) were determined using LC-MS., Results: We found no sex-based difference in the expression of PON2 isoforms, UCP4 and UCP5 in striatum and substantia nigra of young monkeys. However, we discovered that adult female monkeys exhibit greater expression of PON2 isoforms than males in substantia nigra and striatum. Our data also revealed that adult male monkeys exhibit greater expression of UCP4 and UCP5 than females in substantia nigra but not in striatum. PIO increased UCP4 and UCP5 expression in substantia nigra and striatum at 1 week, but after 3 weeks of treatment this activation had subsided., Conclusions: Our findings demonstrate a sex-, age- and region-dependent profile to the expression of PON2, UCP4 and UCP5. These data establish a biochemical link between PPARγ, PON2, UCP4 and UCP5 in primate brain and demonstrate that PON2, UCP4 and UCP5 can be pharmacologically stimulated in vivo, revealing a novel mechanism for observed pioglitazone-induced neuroprotection. We anticipate that these outcomes will contribute to the development of novel neuroprotective treatments for Parkinson's disease and other CNS disorders., (© 2023. Society for Women's Health Research and BioMed Central Ltd.)

Cardiac ryanodine receptors (RyR2) release the Ca 2+ from intracellular stores that is essential for cardiac myocyte contraction. The ion channel opening is tightly regulated by intracellular factors, including the FK506 binding proteins, FKBP12 and FKBP12.6. The impact of these proteins on RyR2 activity and cardiac contraction is debated, with often apparently contradictory experimental results, particularly for FKBP12. The isoform that regulates RyR2 has generally been considered to be FKBP12.6, despite the fact that FKBP12 is the major isoform associated with RyR2 in some species and is bound in similar proportions to FKBP12.6 in others, including sheep and humans. Here, we show time- and concentration-dependent effects of adding FKBP12 to RyR2 channels that were partly depleted of FKBP12/12.6 during isolation. The added FKBP12 displaced most remaining endogenous FKBP12/12.6. The results suggest that FKBP12 activates RyR2 with high affinity and inhibits RyR2 with lower affinity, consistent with a model of negative cooperativity in FKBP12 binding to each of the four subunits in the RyR tetramer. The easy dissociation of some FKBP12/12.6 could dynamically alter RyR2 activity in response to changes in in vivo regulatory factors, indicating a significant role for FKBP12/12.6 in Ca 2+ signalling and cardiac function in healthy and diseased hearts. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

Bax-interacting factor-1 (Bif-1) is a multifunctional protein involved in apoptosis, autophagy, and mitochondrial morphology. However, the associations between Bif-1 and viruses are poorly understood. As discrete Bif-1 isoforms are selectively expressed and exert corresponding effects, we evaluated the effects of neuron-specific/ubiquitous Bif-1 isoforms on rabies virus (RABV) proliferation. First, infection with the RABV CVS-11 strain significantly altered Bif-1 expression in mouse neuroblastoma (N2a) cells, and Bif-1 knockdown in turn promoted RABV replication. Overexpression of neuron-specific Bif-1 isoforms (Bif-1b/c/e) suppressed RABV replication. Moreover, our study showed that Bif-1c colocalized with LC3 and partially alleviated the incomplete autophagic flux induced by RABV. Taken together, our data reveal that neuron-specific Bif-1 isoforms impair the RABV replication process by abolishing autophagosome accumulation and blocking autophagic flux induced by the RABV CVS-11 strain in N2a cells. IMPORTANCE Autophagy can be triggered by viral infection and replication. Autophagosomes are generated and affect RABV replication, which differs by viral strain and infected cell type. Bax-interacting factor-1 (Bif-1) mainly has a proapoptotic function but is also involved in autophagosome formation. However, the association between Bif-1-involved autophagy and RABV infection remains unclear. In this study, our data reveal that a neuron-specific Bif-1 isoform, Bif-1c, impaired viral replication by unchoking autophagosome accumulation induced by RABV in N2a cells to a certain extent. Our study reveals for the first time that Bif-1 is involved in modulating autophagic flux and plays a crucial role in RABV replication, establishing Bif-1 as a potential therapeutic target for rabies., Competing Interests: The authors declare no conflict of interest.

Aims: Human-induced pluripotent stem cell-cardiomyocytes (hiPSC-CMs) are widely used to study arrhythmia-associated mutations in ion channels. Among these, the cardiac sodium channel SCN5A undergoes foetal-to-adult isoform switching around birth. Conventional hiPSC-CM cultures, which are phenotypically foetal, have thus far been unable to capture mutations in adult gene isoforms. Here, we investigated whether tri-cellular cross-talk in a three-dimensional (3D) cardiac microtissue (MT) promoted post-natal SCN5A maturation in hiPSC-CMs., Methods and Results: We derived patient hiPSC-CMs carrying compound mutations in the adult SCN5A exon 6B and exon 4. Electrophysiological properties of patient hiPSC-CMs in monolayer were not altered by the exon 6B mutation compared with isogenic controls since it is not expressed; further, CRISPR/Cas9-mediated excision of the foetal exon 6A did not promote adult SCN5A expression. However, when hiPSC-CMs were matured in 3D cardiac MTs, SCN5A underwent isoform switch and the functional consequences of the mutation located in exon 6B were revealed. Up-regulation of the splicing factor muscleblind-like protein 1 (MBNL1) drove SCN5A post-natal maturation in microtissues since its overexpression in hiPSC-CMs was sufficient to promote exon 6B inclusion, whilst knocking-out MBNL1 failed to foster isoform switch., Conclusions: Our study shows that (i) the tri-cellular cardiac microtissues promote post-natal SCN5A isoform switch in hiPSC-CMs, (ii) adult splicing of SCN5A is driven by MBNL1 in these tissues, and (iii) this model can be used for examining post-natal cardiac arrhythmias due to mutations in the exon 6B., Translational Perspective: The cardiac sodium channel is essential for conducting the electrical impulse in the heart. Postnatal alternative splicing regulation causes mutual exclusive inclusion of fetal or adult exons of the corresponding gene, SCN5A. Typically, immature hiPSCCMs fall short in studying the effect of mutations located in the adult exon. We describe here that an innovative tri-cellular three-dimensional cardiac microtissue culture promotes hiPSC-CMs maturation through upregulation of MBNL1, thus revealing the effect of a pathogenic genetic variant located in the SCN5A adult exon. These results help advancing the use of hiPSC-CMs in studying adult heart disease and for developing personalized medicine applications., Competing Interests: Conflict of interest: C.L.M. is co-founder of Ncardia bv., (© The Author(s) 2022. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Brain-derived neurotrophic factor has functional mRNA isoforms, whose expression is assumed to mediate the beneficial effects of exercise in neuropsychiatric disorders. This study aims to reveal the mechanism of intensity-dependent effects of voluntary exercise, focusing on the expression of Bdnf mRNA isoforms in Hatano rats. Animals with different voluntary activity were housed in cages with a locked or unlocked wheel for 5 weeks. The expression levels of Bdnf isoforms and the corresponding coding sequences (CDS) were measured in the hippocampus using real-time polymerase chain reaction (PCR). We found that exercise increased the expression of Bdnf isoform containing exon 1 in the high-intensity-running strain and decreased the expressions of Bdnf exon 1, 3, 6, 7, 8, and 9a in mild-intensity-running animal. The expression of Bdnf CDS was increased by exercise in both strains. These results suggest that expressions of Bdnf isoforms depend on the intensities of voluntary exercise, but the involvement of subjects' genetic background could not be excluded. Our finding also implies that the bidirectional effects of exercise may not be mediated via the final product of Bdnf., (© 2023 The Authors. Neuropsychopharmacology Reports published by John Wiley & Sons Australia, Ltd on behalf of The Japanese Society of Neuropsychopharmacology.)

Purpose: Glioblastoma (GBM) is a malignant brain tumor with poor prognosis. Radioresistance is a major challenge in the treatment of brain tumors. The development of several types of tumors, including GBM, involves the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway. Upon activation, this pathway induces radioresistance. In this study, we investigated whether additional use of selective inhibitors of PI3K isoforms would enhance radiosensitivity in GBM., Materials and Methods: We evaluated whether radiation combined with PI3K isoform selective inhibitors can suppress radioresistance in GBM. Glioma 261 expressing luciferase (GL261- luc ) and LN229 were used to confirm the effect of combination of radiation and PI3K isoform inhibitors in vitro. Cell viability was confirmed by clonogenic assay, and inhibition of PI3K/AKT signaling activation was observed by Western blot. To confirm radiosensitivity, the expression of phospho-γ-H2AX was observed by immunofluorescence. In addition, to identify the effect of a combination of radiation and PI3K-α isoform inhibitor in vivo, an intracranial mouse model was established by implanting GL261- luc . Tumor growth was observed by IVIS imaging, and survival was analyzed using Kaplan-Meier survival curves., Results: Suppression of the PI3K/AKT signaling pathway increased radiosensitivity, and PI3K-α inhibition had similar effects on PI3K-pan inhibition in vitro. The combination of radiotherapy and PI3K-α isoform inhibitor suppressed tumor growth and extended survival in vivo., Conclusion: This study verified that PI3K-α isoform inhibition improves radiosensitivity, resulting in tumor growth suppression and extended survival in GBM mice., Competing Interests: The authors have no potential conflicts of interest to disclose., (© Copyright: Yonsei University College of Medicine 2023.)

In Sleepy (Sik3Slp) or Sik3S551A mice, deletion or mutation of inhibitory phosphorylation site serine551 from salt-inducible kinase 3 (SIK3) markedly increases daily non-rapid eye movement sleep (NREMS) amount, accompanied with constitutively elevated NREMS delta power density-a measure of sleep intensity. Multiple SLP/SIK3 isoforms are expressed in mouse brain neurons, however, their respective roles in sleep regulation remain to be elucidated. Here, we identified a new and most abundant short isoform of SLP/SIK3 and examined sleep phenotypes resulted from isoform-specific expression of SLP-short (S) and long (L) isoforms. Adeno-associated virus (AAV)-mediated adult brain chimeric (ABC)-expression of SLP-S in neurons, but not in astrocytes, significantly and constitutively elevates NREMS delta power, whereas slightly increases NREMS amount. The ability of SLP-S to regulate sleep quantity/intensity is abrogated by kinase-inactivating mutations, suggesting that the sleep-promoting activity of SLP-S is dependent on its kinase activity. In Sik3S551A-L knock-in mice, isoform-specific expression of SIK3S551A-L (or SLP-L) significantly increases NREMS amount with a modest effect on NREMS delta power. ABC-expression of SLP-S complements the sleep phenotypes of heterozygous Sik3S551A-L mice by further increasing NREMS amount and NREMS delta power to levels of Sik3Slp or Sik3S551A mice. Taken together, these results indicate that both SLP-L and SLP-S isoforms contribute critically to the increases of sleep quantity and intensity in Sik3Slp or Sik3S551A mice., (© The Author(s) 2022. Published by Oxford University Press on behalf of Sleep Research Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

EGFR inhibitor therapies have brought significant benefit to NSCLC patients. However, all patients gradually progress to acquired resistance via diverse mechanisms. Akt3 overexpression but not Akt1/2 is one of the found molecular events that mediate osimertinib ( 1 ) resistance in NSCLC patients. Here, we report 12l as the first bona fide isoform-selective Akt3 degrader which potently induced proteasomal degradation of the target both in vitro and in vivo , whereas its effects on Akt1/2 were minimal. Using 12l as a tool, non-canonical function of Akt3 was validated to contribute greatly to survival of 1 -resistant H1975OR NSCLC cells. Degrader 12l potently suppressed the growth of H1975OR as well as several NSCLC cell lines with low nanomolar IC 50 values and demonstrated promising in vivo antitumor efficacy in nude mice bearing H1975OR or PC9 NSCLC xenograft models. Selective degradation of Akt3 may be considered as a novel strategy for human cancer therapy.

Cellular plasticity lies at the core of cancer progression, metastasis, and resistance to treatment. Stemness and epithelial-mesenchymal plasticity in cancer are concepts that represent a cancer cell's ability to coopt and adapt normal developmental programs to promote survival and expansion. The cancer stem cell model states that a small subset of cancer cells with stem cell-like properties are responsible for driving tumorigenesis and metastasis while remaining especially resistant to common chemotherapeutic drugs. Epithelial-mesenchymal plasticity describes a cancer cell's ability to transition between epithelial and mesenchymal phenotypes which drives invasion and metastasis. Recent research supports the existence of stable epithelial/mesenchymal hybrid phenotypes which represent highly plastic states with cancer stem cell characteristics. The cell adhesion molecule CD44 is a widely accepted marker for cancer stem cells, and it lies at a functional intersection between signaling networks regulating both stemness and epithelial-mesenchymal plasticity. CD44 expression is complex, with alternative splicing producing many isoforms. Interestingly, not only does the pattern of isoform expression change during transitions between epithelial and mesenchymal phenotypes in cancer, but these isoforms have distinct effects on cell behavior including the promotion of metastasis and stemness. The role of CD44 both downstream and upstream of signaling pathways regulating epithelial-mesenchymal plasticity and stemness make this protein a valuable target for further research and therapeutic intervention., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Arsenic trioxide (ATO) and statins have been demonstrated to have anti-neoplastic properties; however, the data regarding their combination therapy is limited. Thus, we aimed to study the effects of ATO, Simvastatin and their combination in proliferation, apoptosis and pathological angiogenesis in prostate cancer cell lines. The human prostate cell lines were treated with different concentrations of Simvastatin and ATO alone and combined to find effective doses and IC50 values. In addition, the percentage of apoptotic cells was evaluated by annexin/PI staining, and mRNA expression levels of the apoptotic gene, including OPN isoforms and VEGF, were investigated using real-time PCR. Our data displayed that Simvastatin (12 and 8 μM in PC3 and LNCaP cell lines respectively), ATO (8 and 5 μM in PC3 and LNCaP cell lines respectively), and also their combination (12 μM Simvastatin and 8 μM ATO in PC3, 8 μM Simvastatin and 5 μM ATO in LNCaP cell lines respectively) significantly increased the percentage of apoptotic cells. Also, we showed that the combination therapy by Simvastatin and ATO increased cell apoptosis and inhibited cell proliferation, providing anti-proliferative and anti-angiogenic properties, possibly via downregulation of the expression of VEGF and OPN genes. These results provide new perceptions regarding the anticancer roles of ATO and statins' combination therapy in prostate cancer., (© 2022 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Hyperoxaluria is a risk factor for urolithiasis and can cause renal epithelial cell injury secondary to oxidative stress. Reactive oxygen species (ROS) produced during cell damage originate from different sources and play different roles. Here, we explored the potential sources of ROS production and investigated the role of ROS from various sources in oxalate-induced oxidative stress and cell injury in normal rat kidney-52 epithelial (NRK-52E) cells. Oxalate-induced injury was assessed by lactate dehydrogenase (LDH) release experiments. 2,7-dichlorodihydrofluorescein diacetate and mitoSOX Red were used to determine the intracellular and mitochondrial ROS (mtROS) production, respectively. The expression level of Nox4, Nox2, and p22 protein was detected by Western blotting to observe the effect of oxalate on nicotinamide adenine dinucleotide phosphate oxidase (NADPH) oxidase (Nox). Furthermore, a specific NADPH oxidase subtype inhibitor and targeted mitochondrial antioxidants were used to preliminarily identify the role of ROS from different sources in renal tubular epithelial cell injury induced by oxalate. We found that oxalate inhibited cell viability, induced LDH release, and prompted intracellular and mitochondrial ROS (mtROS) production. Oxalate also decreased the protein expression level of Nox4 and increased the protein expression level of p22. Mitochondria were also a source of ROS production. In addition, Nox2 inhibitor or mtROS scavenging prevented oxalate-induced cell injury, reversed by an inhibitor of Nox4/1. We concluded that ROS from different sources might play different roles in oxalate-induced renal tubular epithelial cell injury. We also identified new potential targets for preventing or alleviating oxalate-induced renal tubular epithelial cell injury., (© 2022. The Author(s).)

This study aimed at determining the effect of prenatal ethanol exposure (PEE) on the expression and activity of cytochrome P450 (CYP) isozymes at different life stages of male rat offspring. Pregnant Wistar rats were administered with ethanol (4 g/kg/d) intragastrically from gestational day (GD) 9-20. Male offspring's gene and activity of CYP isozymes were analyzed on GD 20 (only expression), postnatal day (PD) 84 and 196. Using aniline as probe, we compared the enzyme kinetics of hepatic CYP2E1 between two groups. Expression of CYP isozymes was examined in rat primary hepatocytes and human hepatic cell lines treated with ethanol or/and glucocorticoid. Gene level of Cyp1a2, 2b1, 2d1, 2e1, 3a1 and aryl hydrocarbon receptor were increased in PEE group on GD 20 and PD 84 and Cyp2e1 still exhibited an increasing trend on PD 196 compared with the control. PEE inhibited CYP2D1 and 2E1 activities in male offspring on PD 84. CYP activities in two groups became the same level on PD 196. PEE induced an opposite change in gene and protein level of hepatic CYP2E1 before and after birth. In consistent with lower protein level, aniline metabolism in PEE was weaker in liver microsome. Both single and combined use of ethanol or/and glucocorticoid increased CYPs expression in vitro. In conclusion, PEE programmed a higher gene and lower protein level of CYPs in male offspring, which dwindled with age. Impairment of protein levels and enzyme activities of CYPs may affect individual metabolism of endogenous and exogenous substances in early adulthood., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Curcumin is a yellow pigment in turmeric (Curcuma longa) with various physiological effects in the body. To elucidate the molecular mechanisms by which bioactive compounds exert their function, identification of their molecular targets is crucial. In this study, we show that curcumin activates G protein-coupled receptor 97 (GPR97). Curcumin dose-dependently activated serum-response element-, but not serum-response factor-response element-, nuclear factor of activated T-cell-response element-, or cAMP-response element-, mediated transcription in cells overexpressed with GPR97. The structure-activity relationship indicated that (i) the double-bonds of the central 7-carbon chain were essential for activation; (ii) a methoxy group on the aromatic ring was required for maximal activity; (iii) the addition of glucuronic acid moiety or a methoxy group to the aromatic ring, but not the methylation of the aromatic p-hydroxy group, eliminated the activity; (iv) the stability of curcumin would be related to receptor activation. Both mutant GPR97(T250A) lacking the cleavage at GPCR proteolysis site and mutant GPR97(ΔN) lacking the N-terminal extracellular region were activated by curcumin and its related compounds similar to wild-type GPR97. In contrast, the synthetic glucocorticoid beclomethasone dipropionate and l-Phe activated wild-type GPR97 and GPR97(T250A), but not GPR97(ΔN). Moreover, curcumin exerted an additive effect on the activation of wild-type GPR97 with beclomethasone dipropionate, but not with l-Phe. Taken together, these results indicate that curcumin activates GPR97 coupled to Gi/Go subunit, and suggest that curcumin and glucocorticoid activate GPR97 in a different manner., Competing Interests: Declaration of competing interest Authors have no conflicts of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Post-translational modifications (PTMs) diversify peptide structure and allow for greater flexibility within signaling networks. The cardiac neuromuscular system of the American lobster, Homarus americanus , is made up of a central pattern generator, the cardiac ganglion (CG), and peripheral cardiac muscle. Together, these components produce flexible output in response to peptidergic modulation. Here, we examined the role of PTMs in determining the effects of a cardioactive neuropeptide, myosuppressin (pQDLDHVFLRFamide), on the whole heart, the neuromuscular junction/muscle, the isolated CG, and the neurons of the CG. Mature myosuppressin and noncyclized myosuppressin (QDLDHVFLRFamide) elicited similar and significant changes in whole heart contraction amplitude and frequency, stimulated muscle contraction amplitude and the bursting pattern of the intact and ligatured neurons of the ganglion. In the whole heart, nonamidated myosuppressin (pQDLDHVFLRFG) elicited only a small decrease in frequency and amplitude. In the absence of motor neuron input, nonamidated myosuppressin did not cause any significant changes in the amplitude of stimulated contractions. In the intact CG, nonamidated myosuppressin elicited a small but significant decrease in burst duration. Further analysis revealed a correlation between the extent of modulation elicited by nonamidated myosuppressin in the whole heart and the isolated, intact CG. When the neurons of the CG were physically decoupled, nonamidated myosuppressin elicited highly variable responses. Taken together, these data suggest that amidation, but not cyclization, is critical in enabling this peptide to exert its effects on the cardiac neuromuscular system. NEW & NOTEWORTHY Myosuppressin (pQDLDHVFLRFamide), a well-characterized crustacean neuropeptide, and its noncyclized (QDLDHVFLRFamide) and nonamidated (pQDLDHVFLRFG) isoforms alter the output of the cardiac neuromuscular system of the American lobster, Homarus americanus. Mature myosuppressin and noncyclized myosuppressin elicited similar and significant changes across all levels of the isolated system, whereas responses to nonamidated myosuppressin were significantly different from other isoforms and were highly variable. These data support the diversity of peptide action as a function of peptide structure.

Type I interferons (IFN-I) exert pleiotropic biological effects during viral infections, balancing virus control versus immune-mediated pathologies, and have been successfully employed for the treatment of viral diseases. Humans express 12 IFN-alpha (α) subtypes, which activate downstream signaling cascades and result in distinct patterns of immune responses and differential antiviral responses. Inborn errors in IFN-I immunity and the presence of anti-IFN autoantibodies account for very severe courses of COVID-19; therefore, early administration of IFN-I may be protective against life-threatening disease. Here we comprehensively analyzed the antiviral activity of all IFNα subtypes against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to identify the underlying immune signatures and explore their therapeutic potential. Prophylaxis of primary human airway epithelial cells (hAEC) with different IFNα subtypes during SARS-CoV-2 infection uncovered distinct functional classes with high, intermediate, and low antiviral IFNs. In particular, IFNα5 showed superior antiviral activity against SARS-CoV-2 infection in vitro and in SARS-CoV-2-infected mice in vivo. Dose dependency studies further displayed additive effects upon coadministration with the broad antiviral drug remdesivir in cell culture. Transcriptomic analysis of IFN-treated hAEC revealed different transcriptional signatures, uncovering distinct, intersecting, and prototypical genes of individual IFNα subtypes. Global proteomic analyses systematically assessed the abundance of specific antiviral key effector molecules which are involved in IFN-I signaling pathways, negative regulation of viral processes, and immune effector processes for the potent antiviral IFNα5. Taken together, our data provide a systemic, multimodular definition of antiviral host responses mediated by defined IFN-I. This knowledge will support the development of novel therapeutic approaches against SARS-CoV-2., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

The most common host entry point of human adapted coronaviruses (CoV) including SARS-CoV-2 is through the initial colonization in the nostril and mouth region which is responsible for spread of the infection. Most recent studies suggest that the commercially available oral and nasal rinse products are effective in inhibiting the viral replication. However, the anti-viral mechanism of the active ingredients present in the oral rinses have not been studied. In the present study, we have assessed in vitro enzymatic inhibitory activity of active ingredients in the oral mouth rinse products: aloin A and B, chlorhexidine, eucalyptol, hexetidine, menthol, triclosan, methyl salicylate, sodium fluoride and povidone, against two important proteases of SARS-CoV-2 PLpro and 3CLpro. Our results indicate only aloin A and B effectively inhibited proteolytic activity of PLpro with an IC 50 of 13.16 and 16.08 μM. Interestingly, neither of the aloin isoforms inhibited 3CLpro enzymatic activity. Computational structural modelling of aloin A and B interaction with PLpro revealed that, both aloin isoforms form hydrogen bond with Tyr 268 of PLpro, which is critical for their proteolytic activity. Furthermore, 100 ns molecular dynamics (MD) simulation studies predicted that both aloin isoforms have strong interaction with Glu 167 , which is required for PLpro deubiquitination activity. Our results from the in vitro deubiquitinase inhibition assay show that aloin A and B isomers exhibit deubiquitination inhibitory activity with an IC 50 value of 15.68 and 17.51 µM, respectively. In conclusion, the isoforms of aloin inhibit both proteolytic and the deubiquitinating activity of SARS-CoV-2 PLpro, suggesting potential in inhibiting the replication of SARS-CoV-2 virus., (© 2022. The Author(s).)

Abstract: Over the past decade, histone deacetylases (HDACs) has been proven to manipulate development and exacerbation of cardiovascular diseases, including myocardial ischemia/reperfusion injury, cardiac hypertrophy, ventricular remodeling, and myocardial fibrosis. Inhibition of HDACs, especially class-I HDACs, is potent to the protection of ischemic myocardium after ischemia/reperfusion (I/R). Herein, we examine whether mocetinostat (MGCD0103, MOCE), a class-I selective HDAC inhibitor in phase-II clinical trial, shows cardioprotection under I/R in vivo and in vitro, if so, reveal its potential pharmacological mechanism to provide an experimental and theoretical basis for mocetinostat usage in a clinical setting. Human cardiac myocytes (HCMs) were exposed to hypoxia and reoxygenation (H/R), with or without mocetinostat treatment. H/R reduced mitochondrial membrane potential and induced HCMs apoptosis. Mocetinostat pretreatment reversed these H/R-induced mitochondrial damage and cellular apoptosis and upregulated CREB, p-CREB, and PGC-1α in HCMs during H/R. Transfection with small interfering RNA against PGC-1α or CREB abolished the protective effects of mocetinostat on cardiomyocytes undergoing H/R. In vivo, mocetinostat was demonstrated to protect myocardial injury posed by myocardial I/R via the activation of CREB and upregulation of PGC-1α. Mocetinostat (MGCD0103) can protect myocardium from I/R injury through mitochondrial protection mediated by CREB/PGC-1α pathway. Therefore, activation of the CREB/PGC-1α signaling pathway via the inhibition of Class-I HDACs may be a promising new therapeutic strategy for alleviating myocardial reperfusion injury., Competing Interests: The authors report no conflicts of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

This chapter is an introduction to phosphoinositide 3-kinases (PI3K), with class I PI3Ks as the central focus. First, the various PI3K isoforms in class I are presented with emphasis on their overall structure, subunits, subunit constitutive domains, domain-domain interactions, and functional relevance. This structural analysis is followed by a comprehensive history of seminal investigations into PI3K activity. Next, we highlight the divergent roles of the isoforms: PI3Kα, PI3Kβ, PI3Kδ, and PI3Kγ. This section details signaling pathways in which these PI3K isoforms are involved, including the key upstream regulators of PI3K activity and some downstream cellular effects. Nodes of the PI3K pathway are also presented. Inhibitors of some isoforms are discussed to give an overview of the basis of some immunotherapies that are being used to target cell signaling. Finally, the chapter ends with a discussion of the dysregulation of PI3Ks in diseases including APDS, asthma, arthritis, and oncogenic mutations., (© 2022. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

This study aimed to evaluate the interspecies difference in metabolism of mulberrin and examine the interaction between mulberrin and CYP enzymes or recombinant human uridine 5'-diphosphate (UDP)-glucuronosyltransferase (UGT) enzymes. Liver microsomes from human (HLMs), Beagle dog (DLMs), minipig (PLMs), monkey (MLMs), rabbit (RLMs), rat (RAMs), and mouse (MIMs) were used to investigate metabolic diversity among different species. Additionally, recombinant human supersomes were used to confirm that metabolic enzymes are involved in the biotransformation of mulberrin. We also evaluated the influence of mulberrin on protein expression by Western blot analysis. Mulberrin metabolism showed significant interspecies differences. We found four and two metabolites in phase I and II reaction systems, respectively. In phase I metabolism profiles of mulberrin for HLMs, PLMs and MLMs conformed to the classic Michaelis-Menten kinetics, RAMs and MIMs followed biphasic kinetics; phase II reaction of mulberrin in HLMs, DLMs, PLMs, MLMs, RLMs, RAMs and MIMs followed biphasic kinetics. UGT1A1 were the major CYP isoforms responsible for the metabolism of mulberrin. Mulberrin showed potent inhibitory effects against CYP3A4, CYP2C9, CYP2E1, UGT1A1, UGT1A3 and UGT2B7 with IC 50 values of 54.21, 9.93, 39.12, 3.84, 2.01, 16.36 µM, respectively. According to Western blot analysis, mulberrin can upregulate the protein expression of CYP2C19, and downregulate the expression levels of CYP3A5 and CYP2C9 in HepG2 cells as concentration increased. The interspecies comparisons can help find other species with metabolic pathways similar to those in humans for future in vivo studies.

Background: The mechanism of the perivascular adipose tissue (PVAT) anticontractile effect is well characterized in rodent visceral vascular beds; however, little is known about the mechanism of PVAT anticontractile function in subcutaneous vessels. In addition, we have previously shown that PVAT anticontractile function is nitric oxide synthase (NOS) dependent but have not investigated the roles of NOS isoforms., Objective: Here, we examined PVAT anticontractile function in the mouse gracilis artery, a subcutaneous fat depot, in lean control and obese mice and investigated the mechanism in comparison to a visceral depot., Method: Using the wire myograph, we generated responses to noradrenaline and electrical field stimulation in the presence of pharmacological tools targeting components of the known PVAT anticontractile mechanism. In addition, we performed ex vivo "fat transplants" in the organ bath., Results: The mechanism of PVAT anticontractile function is similar between subcutaneous and visceral PVAT depots. Both endothelial and neuronal NOS isoforms mediated the PVAT anticontractile effect. Loss of PVAT anticontractile function in obesity is independent of impaired vasoreactivity, and function can be restored in visceral PVAT by NOS activation., Conclusions: Targeting NOS isoforms may be useful in restoring PVAT anticontractile function in obesity, ameliorating increased vascular tone, and disease., (© 2022 The Author(s). Published by S. Karger AG, Basel.)

Chronic infection with Helicobacter pylori increases risk of gastric diseases including gastric cancer. Despite development of a robust immune response, H. pylori persists in the gastric niche. Progression of gastric inflammation to serious disease outcomes is associated with infection with H. pylori strains which encode the cag Type IV Secretion System (cag T4SS). The cag T4SS is responsible for translocating the oncogenic protein CagA into host cells and inducing pro-inflammatory and carcinogenic signaling cascades. Our previous work demonstrated that nutrient iron modulates the activity of the T4SS and biogenesis of T4SS pili. In response to H. pylori infection, the host produces a variety of antimicrobial molecules, including the iron-binding glycoprotein, lactoferrin. Our work shows that apo-lactoferrin exerts antimicrobial activity against H. pylori under iron-limited conditions, while holo-lactoferrin enhances bacterial growth. Culturing H. pylori in the presence of holo-lactoferrin prior to co-culture with gastric epithelial cells, results in repression of the cag T4SS activity. Concomitantly, a decrease in biogenesis of cag T4SS pili at the host-pathogen interface was observed under these culture conditions by high-resolution electron microscopy analyses. Taken together, these results indicate that acquisition of alternate sources of nutrient iron plays a role in regulating the pro-inflammatory activity of a bacterial secretion system and present novel therapeutic targets for the treatment of H. pylori-related disease., (© 2021 Wiley-VCH GmbH.)

Bothrops asper is one of the most important snake species in Central America, mainly because of its medical importance in countries like Ecuador, Panama and Costa Rica, where this species causes a high number of snakebite accidents. Several basic phospholipases A 2 (PLA 2 s) have been previously characterized from B. asper venom, but few studies have been carried out with its acidic isoforms. In addition, since snake venom is a rich source of bioactive substances, it is necessary to investigate the biotechnological potential of its components. In this context, this study aimed to carry out the biochemical characterization of PLA 2 isoforms isolated from B. asper venom and to evaluate the antiparasitic potential of these toxins. The venom and key fractions were subjected to different chromatographic steps, obtaining nine PLA 2 s, four acidic ones (BaspAc-I, BaspAc-II, BaspAc-III and BaspAc-IV) and five basic ones (BaspB-I, BaspB-II, BaspB-III, BaspB-IV and BaspB-V). The isoelectric points of the acidic PLA 2 s were also determined, which presented values ranging between 4.5 and 5. The findings indicated the isolation of five unpublished isoforms, four Asp49-PLA, corresponding to the group of acidic isoforms, and one Lys49-PLA 2 -like. Acidic PLA 2 s catalyzed the degradation of all substrates evaluated; however, for the basic PLA 2 s, there was a preference for phosphatidylglycerol and phosphatidic acid. The antiparasitic potential of the toxins was evaluated, and the acidic PLA 2 s demonstrated action against the epimastigote forms of T. cruzi and promastigote forms of L. infantum, while the basic PLA 2 s BaspB-II and BaspB-IV showed activity against P. falciparum. The results indicated an increase of up to 10 times in antiplasmodial activity, when the Asp49-PLA 2 and Lys49-PLA 2 were associated with one another, denoting synergistic action between these PLA 2 isoforms. These findings correspond to the first report of synergistic antiplasmodial action for svPLA 2 s, demonstrating that these molecules may be important targets in the search for new antiparasitic agents., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Mutations to the cholesterol transport protein apolipoprotein E (ApoE) have been identified as a major risk factor for the development of sporadic or late-onset Alzheimer's disease (AD), with the e4 allele representing an increased risk and the rare e2 allele having a reduced risk compared to the primary e3 form. The reasons behind the change in risk are not entirely understood, though ApoE4 has been connected to inflammation and toxicity in both the brain and the periphery. The goal of this study was to better understand how the ApoE isoforms (ApoE2/3/4) confer differential AD-related risk by assessing cell-specific ApoE-related neuroinflammatory and neurotoxic effects. We compared the effects of ApoE isoforms in vitro on human astrocytes, a human immortalized microglia cell line (HMC3), and the human neuroblastoma cell line SH-SY5Y. Cells were treated for 24 h with or without recombinant ApoE2, ApoE3, or ApoE4 (20 nM) and inflammation and toxicity markers assessed. Our results indicated the expression of inflammatory cytokines IL-1β, TNFα, and IL-6 in human astrocytes was increased in response to all ApoE isoforms, with ApoE4 evoking the highest level of cytokine expression. In response to ApoE2 or ApoE3, microglial cells showed reduced levels of microglial activation markers TREM2 and Clec7a, while ApoE4 induced increased levels of both markers. ApoE2 promoted neuron survival through increased BDNF release from astrocytes. In addition, ApoE2 promoted, while ApoE4 reduced, neuronal viability. Overall, these results suggest that ApoE4 acts on cells in the brain to promote inflammation and neuronal injury and that the deleterious effects of ApoE4 on these cells may, in part, contribute to its role as a risk factor for AD.

Transforming growth factor beta (TGF-β) plays an important role in the viral liver disease progression via controlling viral propagation and mediating inflammation-associated responses. However, the antiviral activities and mechanisms of TGF-β isoforms, including TGF-β1, TGF-β2 and TGF-β3, remain unclear. Here, we demonstrated that all of the three TGF-β isoforms were increased in Huh7.5 cells infected by hepatitis C virus (HCV), but in turn, the elevated TGF-β isoforms could inhibit HCV propagation with different potency in infectious HCV cell culture system. TGF-β isoforms suppressed HCV propagation through interrupting several different stages in the whole HCV life cycle, including virus entry and intracellular replication, in TGF-β/SMAD signalling pathway-dependent and TGF-β/SMAD signalling pathway-independent manners. TGF-β isoforms showed additional anti-HCV activities when combined with each other. However, the elevated TGF-β1 and TGF-β2, not TGF-β3, could also induce liver fibrosis with a high expression of type I collagen alpha-1 and α-smooth muscle actin in LX-2 cells. Our results showed a new insight into TGF-β isoforms in the HCV-related liver disease progression., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

N -Glycosylation represents an essential type of posttranslational modification for proteins. However, deciphering the functions of N -glycosylation remains a challenge due to the lack of analytical and biochemical methods to accurately differentiate the protein glycoforms with various intact glycans. Here we report our synthesis and evaluation of homogeneously glycosylated interleukin-17A (IL-17A), based on a synthetic approach combining solid-phase synthesis of (glyco)peptides, chemoenzymatic glycan modification on segments, and chemical ligations. The obtained homogeneous glycoproteins allow for the demonstration of the stabilizing role of N -glycans during the folding step. A comparison of three IL-17A glycoforms in a normal human dermal fibroblast (NHDF) assay reveals dose-dependent interleukin-6-inducing activities in all cases, wherein the glycoform with sialyl undecasaccharides displays much weaker stimulatory effect than that of the GlcNAc- or GlcNAc(β 1→4 )GlcNAc-modified proteins. Further surface plasmon resonance (SPR) and hydrogen/deuterium exchange mass spectroscopic experiments confirm that the evaluated complex type N -glycan impedes the binding between IL-17A and its receptor IL-17RA. This structure-activity relationship study on glycoproteins highlights the viability of applying the de novo approach to probe the roles of N -glycans.

Background: Epithelial-mesenchymal transition (EMT) is a key process in the onset and development of idiopathic pulmonary fibrosis (IPF) with unclear mechanisms. Our previous studies found that bleomycin and tunicamycin could induce ER stress and consequently trigger EMT accompanying with IL-32 overexpression. This study was aimed to investigate the effects of IL-32 on EMT and ER stress to elucidate the pathogenesis of IPF., Methods: Human lung adenocarcinoma A549 cells were treated with recombinant human (rh)IL-32, IL-32 siRNA and EMT inducer tunicamycin, or 4-phenylbutyric acid (4-PBA), respectively. Then the cell morphology was observed and the expression of ER-related markers and EMT-related markers were detected by RT-qPCR or western blotting., Results: Stimulation of A549 cells with rhIL-32 led to a morphological change from a pebble-like shape to an elongated shape in a portion of the cells, accompanied by down regulated expression of the epithelial cell marker E-cadherin and up regulated expression of the mesenchymal cell markers N-cadherin, Vimentin, and Zeb-1. However, these rhIL-32 induced changes were inhibited by the ER stress inhibitor 4-PBA. Suppression of IL-32 expression with siRNA inhibited TM-induced EMT. Further stimulation of the A549 cells with rhIL-32 demonstrated an increase in the expression of GRP78, although this increase was also inhibited by 4-PBA., Conclusions: These results suggest that IL-32 induces EMT in A549 cells by triggering ER stress, and IL-32 may be a novel marker for IPF.

Ovarian-derived inhibin A and inhibin B (heterodimers of common α- and differing β-subunits) are secreted throughout the menstrual cycle in a discordant pattern, with smaller follicles producing inhibin B, whereas the dominant follicle and corpus luteum produce inhibin A. The classical function for endocrine inhibins is to block signalling by activins (homodimers of β-subunits) in gonadotrope cells of the anterior pituitary and, thereby, inhibit the synthesis of FSH. Whether inhibin A and inhibin B have additional physiological functions is unknown, primarily because producing sufficient quantities of purified inhibins, in the absence of contaminating activins, for preclinical studies has proven extremely difficult. Here, we describe novel methodology to enhance inhibin A and inhibin B activity and to produce these ligands free of contaminating activins. Using computational modeling and targeted mutagenesis, we identified a point mutation in the activin β A-subunit, A347H, which completely disrupted activin dimerization and activity. Importantly, this β A-subunit mutation had minimal effect on inhibin A bioactivity. Mutation of the corresponding residue in the inhibin β B-subunit, G329E, similarly disrupted activin B synthesis/activity without affecting inhibin B production. Subsequently, we enhanced inhibin A potency by modifying the binding site for its co-receptor, betaglycan. Introducing a point mutation into the α-subunit (S344I) increased inhibin A potency ~12-fold. This study has identified a means to eliminate activin A/B interference during inhibin A/B production, and has facilitated the generation of potent inhibin A and inhibin B agonists for physiological exploration., (© Endocrine Society 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Several food-derived molecules, including proteins and peptides, can show bioactivities toward the promotion of well-being and disease prevention in humans. There is still a lack of information about the potential effects on immune and inflammatory responses in mammalian cells following the ingestion of seed storage proteins. This study, for the first time, describes the potential immunomodulation capacity of chenopodin, the major protein component of quinoa seeds. After characterizing the molecular features of the purified protein, we were able to separate two different forms of chenopodin, indicated as LcC (Low charge Chenopodin, 30% of total chenopodin) and HcC (High charge Chenopodin, 70% of total chenopodin). The biological effects of LcC and HcC were investigated by measuring NF-κB activation and IL-8 expression studies in undifferentiated Caco-2 cells. Inflammation was elicited using IL-1β. The results indicate that LcC and HcC show potential anti-inflammatory activities in an intestinal cell model, and that the proteins can act differently, depending on their structural features. Furthermore, the molecular mechanisms of action and the structural/functional relationships of the protein at the basis of the observed bioactivity were investigated using in silico analyses and structural predictions.

A treatment for intractable diseases is expected to be the replacement of damaged tissues with products from human induced pluripotent stem cells (hiPSCs). Target cell purification is a critical step for realizing hiPSC-based therapy. Here, we found that hiPSC-derived ocular cell types exhibited unique adhesion specificities and growth characteristics on distinct E8 fragments of laminin isoforms (LNE8s): hiPSC-derived corneal epithelial cells (iCECs) and other non-CECs rapidly adhered preferentially to LN332/411/511E8 and LN211E8, respectively, through differential expression of laminin-binding integrins. Furthermore, LN332E8 promoted epithelial cell proliferation but not that of the other eye-related cells, leading to non-CEC elimination by cell competition. Combining these features with magnetic sorting, highly pure iCEC sheets were fabricated. Thus, we established a simple method for isolating iCECs from various hiPSC-derived cells without using fluorescence-activated cell sorting. This study will facilitate efficient manufacture of iCEC sheets for corneal disease treatment and provide insights into target cell-specific scaffold selection., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Tau deposition in the brain is a pathological hallmark of many neurodegenerative disorders, including Alzheimer's disease (AD). During the course of these tauopathies, tau spreads throughout the brain via synaptically-connected pathways. Such propagation of pathology is thought to be mediated by tau species ("seeds") containing the microtubule binding region (MTBR) composed of either three repeat (3R) or four repeat (4R) isoforms. The tau MTBR also forms the core of the neuropathological filaments identified in AD brain and other tauopathies. Multiple approaches are being taken to limit tau pathology, including immunotherapy with anti-tau antibodies. Given its key structural role within fibrils, specifically targetting the MTBR with a therapeutic antibody to inhibit tau seeding and aggregation may be a promising strategy to provide disease-modifying treatment for AD and other tauopathies. Therefore, a monoclonal antibody generating campaign was initiated with focus on the MTBR. Herein we describe the pre-clinical generation and characterisation of E2814, a humanised, high affinity, IgG 1 antibody recognising the tau MTBR. E2814 and its murine precursor, 7G6, as revealed by epitope mapping, are antibodies bi-epitopic for 4R and mono-epitopic for 3R tau isoforms because they bind to sequence motif HVPGG. Functionally, both antibodies inhibited tau aggregation in vitro. They also immunodepleted a variety of MTBR-containing tau protein species. In an in vivo model of tau seeding and transmission, attenuation of deposition of sarkosyl-insoluble tau in brain could also be observed in response to antibody treatment. In AD brain, E2814 bound different types of tau filaments as shown by immunogold labelling and recognised pathological tau structures by immunohistochemical staining. Tau fragments containing HVPGG epitopes were also found to be elevated in AD brain compared to PSP or control. Taken together, the data reported here have led to E2814 being proposed for clinical development.

In the present work, the mechanisms involved in the recently reported antiviral activity of zebrafish C-reactive protein-like protein (CRP1-7) against the spring viraemia of carp rhabdovirus (SVCV) in fish are explored. The results neither indicate blocking of the attachment or the binding step of the viral replication cycle nor suggest the direct inhibition of G protein fusion activity or the stimulation of the host's interferon system. However, an antiviral state in the host is induced. Further results showed that the antiviral protection conferred by CRP1-7 was mainly due to the inhibition of autophagic processes. Thus, given the high affinity of CRPs for cholesterol and the recently described influence of the cholesterol balance in lipid rafts on autophagy, both methyl-β-cyclodextrin (a cholesterol-complexing agent) and 25-hydroxycholesterol (a cholesterol molecule with antiviral properties) were used to further describe CRP activity. All the tested compounds exerted antiviral activity by affecting autophagy in a similar manner. Further assays indicate that CRP reduces autophagy activity by initially disturbing the cholesterol ratios in the host cellular membranes, which in turn negatively affects the intracellular regulation of reactive oxygen species (ROS) and increases lysosomal pH as a consequence. Ultimately, here we propose that such pH changes exert an inhibitory direct effect on SVCV replication by disrupting the pH-dependent membrane-fusogenic ability of the viral glycoprotein G, which allows the release of the virus from endosomes into cytoplasm during its entry phase.

The mTOR/S6Ks signaling is one of the intracellular pathways important for metabolic control, acting both peripherally and centrally. In the hypothalamus, mTOR/S6Ks axis mediates the action of leptin and insulin and can modulate the expression of neuropeptides. We analyzed the role of different S6Ks isoforms in the hypothalamic regulation of metabolism. We observed decreased food intake and decreased expression of agouti-related peptide (AgRP) following intracerebroventricular (icv) injections of adenoviral-mediated overexpression of three different S6Ks isoforms. Moreover, mice overexpressing p70-S6K1 in undefined periventricular hypothalamic neurons presented changes in glucose metabolism, as an increase in gluconeogenesis. To further evaluate the hypothalamic role of a less-studied S6K isoform, p54-S6K2, we used a Cre-LoxP approach to specifically overexpress it in AgRP neurons. Our findings demonstrate the potential participation of S6K2 in AgRP neurons regulating feeding behavior.

Protein-based therapies are potential treatments for cancer, immunological, and cardiovascular diseases. However, effective delivery systems are needed because of their instability, immunogenicity, and so on. Crosslinked negatively charged heparin polysaccharide nanoparticle (HepNP) is proposed for protein delivery. HepNP can efficiently condense vascular endothelial growth factor (VEGF) because of the unique electronegative sulfonic acid and carboxyl domain of heparin. HepNP is then assembled with VEGF-C (Hep@VEGF-C) or VEGF-A (Hep@VEGF-A) protein for the therapy of myocardial infarction (MI) via intravenous (iv) injection. Hep@VEGF-A-mediated improvement of cardiac function by promoting angiogenesis is limited because of elevated vascular permeability, while Hep@VEGF-C effectively promotes lymphangiogenesis and reduces edema. On this basis, a graded delivery of VEGF-C (0.5-1 h post-MI) and VEGF-A (5 d post-MI) using HepNP is developed. At the dose ratio of 3:1 (Hep@VEGF-C vs Hep@VEGF-A), Hep@VEGF functional complexes substantially reduce the scar formation (≈-39%; p < 0.05) and improve cardiac function (≈+74%; p < 0.05). Such a HepNP delivery system provides a simple and effective therapeutic strategy for cardiovascular diseases by delivering functional proteins. Because of the unique binding ability of heparin with cytokines and growth factors, HepNP also has considerable application prospects in protein therapy for other serious diseases., (© 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Tachylectin-2, a 27-kDa protein consisting of a five-bladed β-propeller structure, is purified by three steps of chromatography, including dextran sulfate-Sepharose CL-6B, CM-Sepharose CL-6B, and Mono S. Three isolectins of tachylectin-2 including tachylectin-2a, -2b, and -2c are purified. These isolectins exhibit hemagglutinating activity against human A-type erythrocytes in a Ca 2+ -independent manner with tachylectin-2b showing the highest activity. Tachylectin-2b specifically agglutinates Staphylococcus saprophyticus KD. The tachylectin-2b-mediated hemagglutination is inhibited in the presence of GlcNAc and GalNAc. The association constants for GlcNAc and GalNAc are K a  = 1.95 × 10 4  M -1 and K a  = 1.11 × 10 3  M -1 , respectively. Ultracentrifugation analysis shows that tachylectin-2b is present in monomer form in solution.

Background: Human IL-32 is a polyfunctional cytokine that was initially reported to inhibit HIV-1 infection. However, recent data suggest that IL-32 may enhance HIV-1 replication by activating the HIV-1 primary targets, CD4 T-cells. Indeed, IL-32 is expressed in multiple isoforms, some of which are proinflammatory, whereas others are anti-inflammatory., Setting and Methods: Here, we aimed to determine the relative expression of IL-32 isoforms and to test their inflammatory nature and potential to induce HIV-1 production in latently infected cells from virologically suppressed HIV-infected individuals. IL-32 and other cytokines were quantified from plasma and supernatant of CD4 T-cells by ELISA. Transcripts of IL-32 isoforms were quantified by qRT-PCR in peripheral blood mononuclear cells. The impact of recombinant human IL-32 isoforms on HIV-1 transcription was assessed in CD4 T-cells from HIV-1cART individuals by qRT-PCR., Results: All IL-32 isoforms were significantly upregulated in HIV-1cART compared to HIV individuals with IL-32β representing the dominantly expressed isoform, mainly in T-cells and NK-cells. At the functional level, although IL-32γ induced typical proinflammatory cytokines (IL-6 and IFN-γ) in TCR-activated CD4 T-cells, IL-32α showed an anti-inflammatory profile by inducing IL-10 but not IL-6 or IFN-γ. However, IL-32β showed a dual phenotype by inducing both pro- and anti-inflammatory cytokines. Interestingly, consistent with its highly pro-inflammatory nature, IL-32γ, but not IL-32α or IL-32β, induced HIV-1 production in latently infected CD4 T-cells isolated from combined antiretroviral therapy-treated individuals., Conclusions: Our data report on the differential expression of IL-32 isoforms and highlight the potential role of IL-32, particularly the γ isoform, in fueling persistent inflammation and transcription of viral reservoir in HIV-1 infection.

Infections with HCV and HBV are serious worldwide health problems. Here, we report the anti-HCV and -HBV proficiency of Apis mellifera major royal-jelly protein (MRJP) 2 and its isoform X1. The efficiency of these proteins was evaluated in vitro and their safety was examined in vivo in comparison with Sofosbuvir (SOF) drug. Various in-silico methodologies were achieved for better understanding the antiviral mechanism of these MRJPs. Results proved their precluding ability to the viral receptors, CD81 and scavenger receptor class B type I (SR-B1). In addition, they targeted HCV-NS3/NS4A protease, HCV-NS5B polymerase, and HBV-polymerase (DNA-dependent DNA polymerase, and reverse transcriptase). Co-treatment with these proteins exerted different efficiencies toward CD81 and SR-B1 (synergistic), HBV-enzymes (antagonistic), and HCV-enzymes (either additive or synergistic). The studied proteins maximized their antiviral effect by their safety and superior potency to SOF. Collectively, these outcomes will shed the light on MRJP2 and its isoform X1 as two promising safe-inhibitors for both HCV and HBV., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The gastric secretory trefoil factor family (TFF) peptides xP1 and xP4 are the Xenopus laevis orthologs of mammalian TFF1 and TFF2, respectively. The aim of this study was to analyze the molecular forms of xP1 and xP4 in the X. laevis gastric mucosa by FPLC. xP1 mainly occurred in a monomeric low-molecular-mass form and only a minor subset is associated with the mucus fraction. The occurrence of monomeric xP1 is unexpected because of its odd number of cysteine residues. Probably a conserved acidic residue flanking Cys 55 allows monomeric secretion. Furthermore, Cys 55 is probably post-translationally modified. For the first time, we hypothesize that the free thiol of monomeric xP1-and probably also its mammalian ortholog TFF1-could have a protective scavenger function, e.g., for reactive oxygen/nitrogen species. In contrast, xP4 mainly occurs in a high-molecular-mass form and is non-covalently bound to a mucin similarly as TFF2. In vitro binding studies with radioactively labeled porcine TFF2 even showed binding to X. laevis gastric mucin. Thus, xP4 is expected to bind as a lectin to an evolutionary conserved sugar epitope of the X. laevis ortholog of mucin MUC6 creating a tight mucus barrier. Taken together, xP1 and xP4 appear to have different gastric protective functions.

The present work aimed to explore the innovative hypothesis that different transcript/protein variants of a pro-neurotrophin may generate different biological outcomes in a cellular system. Nerve growth factor (NGF) is important in the development and progression of neurodegenerative and cancer conditions. Mature NGF (mNGF) originates from a precursor, proNGF, produced in mouse in two major variants, proNGF-A and proNGF-B. Different receptors bind mNGF and proNGF, generating neurotrophic or neurotoxic outcomes. It is known that dysregulation in the proNGF/mNGF ratio and in NGF-receptors expression affects brain homeostasis. To date, however, the specific roles of the two major proNGF variants remain unexplored. Here we attempted a first characterization of the possible differential effects of proNGF-A and proNGF-B on viability, differentiation and endogenous ngf gene expression in the PC12 cell line. We also investigated the differential involvement of NGF receptors in the actions of proNGF. We found that native mouse mNGF, proNGF-A and proNGF-B elicited different effects on PC12 cell survival and differentiation. Only mNGF and proNGF-A promoted neurotrophic responses when all NGF receptors are exposed at the cell surface. Tropomyosine receptor kinase A (TrkA) blockade inhibited cell differentiation, regardless of which NGF was added to culture media. Only proNGF-A exerted a pro-survival effect when TrkA was inhibited. Conversely, proNGF-B exerted differentiative effects when the p75 neurotrophin receptor (p75 NTR ) was antagonized. Stimulation with NGF variants differentially regulated the autocrine production of distinct proNgf mRNA. Overall, our findings suggest that mNGF and proNGF-A may elicit similar neurotrophic effects, not necessarily linked to activation of the same NGF-receptor, while the action of proNGF-B may be determined by the NGF-receptors balance. Thus, the proposed involvement of proNGF/NGF on the development and progression of neurodegenerative and tumor conditions may depend on the NGF-receptors balance, on specific NGF trancript expression and on the proNGF protein variant ratio., (Copyright © 2019. Published by Elsevier Ltd.)

Identification of primary targets associated with phenotypes can facilitate exploration of the underlying molecular mechanisms of compounds and optimization of the structures of promising drugs. However, the literature reports limited effort to identify the target major isoform of a single known target gene. The majority of genes generate multiple transcripts that are translated into proteins that may carry out distinct and even opposing biological functions through alternative splicing. In addition, isoform expression is dynamic and varies depending on the developmental stage and cell type. To identify target major isoforms, we integrated a breast cancer type-specific isoform coexpression network with gene perturbation signatures in the MCF7 cell line in the Connectivity Map database using the 'shortest path' drug target prioritization method. We used a leukemia cancer network and differential expression data for drugs in the HL-60 cell line to test the robustness of the detection algorithm for target major isoforms. We further analyzed the properties of target major isoforms for each multi-isoform gene using pharmacogenomic datasets, proteomic data and the principal isoforms defined by the APPRIS and STRING datasets. Then, we tested our predictions for the most promising target major protein isoforms of DNMT1, MGEA5 and P4HB4 based on expression data and topological features in the coexpression network. Interestingly, these isoforms are not annotated as principal isoforms in APPRIS. Lastly, we tested the affinity of the target major isoform of MGEA5 for streptozocin through in silico docking. Our findings will pave the way for more effective and targeted therapies via studies of drug targets at the isoform level.

Neuromedin U (NMU) plays important roles in energy homeostasis in rodents and birds. Previously, our group has isolated four cDNAs encoding precursor proteins of NMU from the goldfish brain and gut, and it was assumed that these transcripts are produced by alternative splicing. We have also demonstrated that intracerebroventricular (ICV) injection of putative goldfish NMU inhibits food intake. However, as native goldfish NMU has not yet been identified, we attempted to purify it from goldfish brain and gut extracts. To assess NMU activity in fractions at each purification step, we measured changes in the intracellular concentrations of Ca 2+ using HEK293 cells expressing goldfish NMU-R1 or -R2. We isolated a 25-amino-acid peptide (NMU-25) from the brain and gut and found that its primary structure is similar to that of mammalian NMU. Another 21-amino-acid peptide (NMU-21) was purified from the brain, but not from the gut. Furthermore, a 9-amino-acid peptide (NMU-9) identical to the C-terminus of NMU-21 and -25 was also isolated from the brain and gut. Treatment with synthetic NMU-9, -21 and -25 dose-dependently increased the intracellular Ca 2+ concentration in mammalian cells expressing goldfish NMU-R1 and -R2. We also examined the effect of ICV-administered synthetic goldfish NMUs on goldfish food intake. NMU-25 inhibited food intake to the same degree as NMU-21. However, the inhibitory effect of NMU-9 was slightly weaker than those of NMU-21 and -25. These results indicate that several molecular forms of NMU exist in the goldfish brain and gut, and that all of them play physiological roles via NMU-R1 and NMU-R2., (Copyright © 2019. Published by Elsevier Inc.)

Clinical administration of Interferon α (IFNα) resulted in limited therapeutic success against some viral infections. Immune modulation of CD8 + T cell responses during IFNα therapy is believed to play a pivotal role in promoting viral clearance. However, these clinical studies primarily focused on IFNα subtype 2. To date, the immunomodulatory roles of the remaining 10-13 IFNα subtypes remains poorly understood, thereby precluding assessments of their potential for more effective treatments. Here, we report that virus-specific CD8 + T cell responses were influenced to various extents by individual IFNα subtypes. IFNα4, 6, and 9 had the strongest effects on CD8 + T cells, including antiproliferative effects, improved cytokine production and cytotoxicity. Interestingly, augmented cytokine responses were dependent on IFNα subtype stimulation of dendritic cells (DCs), while antiproliferative effects and cytotoxicity were mediated by IFNAR signaling in either CD8 + T cells or DCs. Thus, precise modulation of virus-specific CD8 + T cell responses may be feasible for specific antiviral immunotherapies through careful selection and administration of individual IFNα subtypes., (Copyright © 2019 Dickow, Francois, Kaiserling, Malyshkina, Drexler, Westendorf, Lang, Santiago, Dittmer and Sutter.)

Background: Atherosclerosis is an inflammatory process involving activation of monocytes recruited by various chemoattractant factors, among which lipoprotein(a) and its specific apolipoprotein apo(a). Lp(a) contains a specific apolipoprotein apo(a) which size is determined by a variable number of repeats of a specific structural domain, the kringle IV type 2 (IV-2). Lp(a) plasma concentration and apo(a) size is inversely correlated, and smaller apo(a) are major risk factors for coronary heart disease., Design and Methods: The aim of this study was to evaluate the effect of recombinant apo(a) isoforms (containing 10, 18 or 34 kringles) on monocytes interacting with type I collagen., Results: Apo(a) isoforms stimulated reactive oxygen species (ROS) and matrix metalloproteinase-9 (MMP-9) production by monocytes, and not modified monocytes adhesion on type I collagen. This effect was specific of apo(a) since no effect was observed in the presence of plasminogen and was inversely related to apo(a) size. The lysine analogue 6-aminohexanoic acid which blocks the lysine binding sites (LBS), and carboxypeptidase B (CpB) which cleaves carboxy-terminal lysine residues, abolished apo(a)-induced ROS and MMP-9 production, highlighting an effect mediated by apo(a) lysing-binding sites., Conclusions: These results indicate that activation of collagen-primed monocytes stimulated with apo(a) is a Kringle number-dependent effect and reinforce the hypothesis of a role for small size apo(a) isoforms in atherothrombosis.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen causing severe infections in hospitalized and immunosuppressed patients, particularly individuals affected by cystic fibrosis. Several clinically isolated P. aeruginosa strains were found to be resistant to three or more antimicrobial classes indicating the importance of identifying new antimicrobials active against this pathogen. Here, we characterized the antimicrobial activity and the action mechanisms against P. aeruginosa of two natural isoforms of the antimicrobial peptide cecropin B, both isolated from the silkworm Bombyx mori . These cecropin B isoforms differ in a single amino acid substitution within the active portion of the peptide, so that the glutamic acid of the E53 CecB variant is replaced by a glutamine in the Q53 CecB isoform. Both peptides showed a high antimicrobial and membranolytic activity against P. aeruginosa , with Q53 CecB displaying greater activity compared with the E53 CecB isoform. Biophysical analyses, live-cell NMR, and molecular-dynamic-simulation studies indicated that both peptides might act as membrane-interacting elements, which can disrupt outer-membrane organization, facilitating their translocation toward the inner membrane of the bacterial cell. Our data also suggest that the amino acid variation of the Q53 CecB isoform represents a critical factor in stabilizing the hydrophobic segment that interacts with the bacterial membrane, determining the highest antimicrobial activity of the whole peptide. Its high stability to pH and temperature variations, tolerance to high salt concentrations, and low toxicity against human cells make Q53 CecB a promising candidate in the development of CecB-derived compounds against P. aeruginosa .