Pathophysiological Cellular Processes Research Articles

Comparing thiol and selenol reactivity towards peroxynitrite by computer simulation

Peroxynitrite is a very reactive species implicated in a variety of pathophysiological cellular processes. Particularly, peroxynitrite-mediated oxidation of cellular thiol-containing compounds such as cysteine residues is a key process which has been extensively studied. Cysteine plays roles in many redox biochemistry pathways. In contrast, selenocysteine, the 21st amino acid, is only present in 25 human proteins. Investigating the molecular basis of selenocysteine's reactivity may provide insights into its unique role in these selenocysteine-containing proteins. The two-electron oxidation of thiols or selenols by peroxynitrite is a process that is carried out by the thiolate/selenate forms and peroxynitrous acid.In this work, we shed light on the molecular basis of the differential reactivity of both species towards peroxynitrite by means of state-of-the-art computer simulations. We performed electronic structure calculations of the reaction in the methanethiolate and methaneselenolate model systems with peroxynitrous acid at different levels of theory using an implicit solvent scheme. In addition, we employed a multi-scale quantum mechanics/molecular mechanics approach for obtaining free energy profiles of these chemical reactions in aqueous solution, which enabled the comparison between the simulations and the available experimental data. Our results suggest that the larger reactivity observed in the selenocysteine case at physiological pH is mainly due to the lower pKa, which affords a larger fraction of the reactive anionic species in these conditions, and in a second place to a slightly enhanced intrinsic reactivity of the selenate form due to its larger nucleophilicity.

Role of microRNA-363 during tumor progression and invasion.

Recent progresses in diagnostic and therapeutic methods have significantly improved prognosis in cancer patients. However, cancer is still considered as one of the main causes of human deaths in the world. Late diagnosis in advanced tumor stages can reduce the effectiveness of treatment methods and increase mortality rate of cancer patients. Therefore, investigating the molecular mechanisms of tumor progression can help to introduce the early diagnostic markers in these patients. MicroRNA (miRNAs) has an important role in regulation of pathophysiological cellular processes. Due to their high stability in body fluids, they are always used as the non-invasive markers in cancer patients. Since, miR-363 deregulation has been reported in a wide range of cancers, we discussed the role of miR-363 during tumor progression and metastasis. It has been reported that miR-363 has mainly a tumor suppressor function through the regulation of transcription factors, apoptosis, cell cycle, and structural proteins. MiR-363 also affected the tumor progression via regulation of various signaling pathways such as WNT, MAPK, TGF-β, NOTCH, and PI3K/AKT. Therefore, miR-363 can be introduced as a probable therapeutic target as well as a non-invasive diagnostic marker in cancer patients.

HDAC7: a promising target in cancer.

Histones have a vital function as components of nucleosomes, which serve as the fundamental building blocks of chromatin. Histone deacetylases (HDACs), which target histones, suppress gene transcription by compacting chromatin. This implies that HDACs have a strong connection to the suppression of gene transcription. Histone deacetylase 7 (HDAC7), a member of the histone deacetylase family, may participate in multiple cellular pathophysiological processes and activate relevant signaling pathways to facilitate the progression of different tumors by exerting deacetylation. In recent years, HDAC7 has been increasingly studied in the pathogenesis of tumors. Studies that are pertinent have indicated that it has a significant impact on the growth and metastasis of tumors, the formation of the vascular microenvironment, and the emergence of resistance to drugs. Therefore, HDAC7 could potentially function as a potent predictor for tumor prognosis and a promising target for mitigating drug resistance in tumors. This review primarily concentrates on elucidating the structure and function of HDAC7, its involvement in the development of various tumors, and its interplay with relevant signaling pathways. Meanwhile, we briefly discuss the research direction and prospect of HDAC7.

Exploring the role of non-coding RNAs in atrial septal defect pathogenesis: A systematic review.

Extensive research has recognized the significant roles of non-coding RNAs (ncRNAs) in various cellular pathophysiological processes and their association with diverse diseases, including atrial septal defect (ASD), one of the most prevalent congenital heart diseases. This systematic review aims to explore the intricate involvement and significance of ncRNAs in the pathogenesis and progression of ASD. Four databases (PubMed, Embase, Scopus, and the Web of Science) were searched systematically up to June 19, 2023, with no year restriction. The risk of bias assessment was evaluated using the Newcastle-Ottawa scale. The present systematic review included thirteen studies with a collective study population of 874 individuals diagnosed with ASD, 21 parents of ASD patients, and 22 pregnant women carrying ASD fetuses. Our analysis revealed evidence linking five long ncRNAs (STX18-AS1, HOTAIR, AA709223, BX478947, and Moshe) and several microRNAs (hsa-miR-19a, hsa-miR-19b, hsa-miR-375, hsa-miR-29c, miR-29, miR-143/145, miR-17-92, miR-106b-25, and miR-503/424, miR-9, miR-30a, miR-196a2, miR-139-5p, hsa-let-7a, hsa-let-7b, and hsa-miR-486) to ASD progression, corresponding to previous studies. NcRNAs play a crucial role in unraveling the underlying mechanisms of ASD, contributing to both biomarker discovery and therapeutic advancements. This systematic review sheds light on the mechanisms of action of key ncRNAs involved in ASD progression, providing valuable insights for future research in this field.

NAT10 regulates the LPS-induced inflammatory response via the NOX2-ROS-NF-κB pathway in macrophages

Periodontitis is a chronic osteolytic inflammatory disease resulting from complex dynamic interactions among bacterial pathogens and the host immune response. Macrophages play a vital role in the pathogenesis of periodontitis by triggering periodontal inflammation and inducing periodontium destruction. N-Acetyltransferase 10 (NAT10) is an acetyltransferase that has been shown to catalyse N4-acetylcytidine (ac4C) mRNA modification and is related to cellular pathophysiological processes, including the inflammatory immune response. Nevertheless, whether NAT10 regulates the inflammatory response of macrophages in periodontitis remains unclear. In this study, the expression of NAT10 in macrophages was found to decrease during LPS-induced inflammation. NAT10 knockdown significantly reduced the generation of inflammatory factors, while NAT10 overexpression had the opposite effect. RNA sequencing revealed that the differentially expressed genes were enriched in the NF-κB signalling pathway and oxidative stress. Both the NF-κB inhibitor Bay11-7082 and the ROS scavenger N-acetyl-L-cysteine (NAC) could reverse the upregulation of inflammatory factors. NAC inhibited the phosphorylation of NF-κB, but Bay11-7082 had no effect on the production of ROS in NAT10-overexpressing cells, suggesting that NAT10 activated the LPS-induced NF-κB signalling pathway by regulating ROS generation. Furthermore, the expression and stability of Nox2 was promoted after NAT10 overexpression, indicating that Nox2 may be a potential target of NAT10. In vivo, the NAT10 inhibitor Remodelin reduced macrophage infiltration and bone resorption in ligature-induced periodontitis mice. In summary, these results showed that NAT10 accelerated LPS-induced inflammation via the NOX2-ROS-NF-κB pathway in macrophages and that its inhibitor Remodelin might be of potential therapeutic significance in periodontitis treatment.

The Emerging, Multifaceted Role of WTAP in Cancer and Cancer Therapeutics.

Cancer is a grave and persistent illness, with the rates of both its occurrence and death toll increasing at an alarming pace. N6-methyladenosine (m6A), the most prevalent mRNA modification in eukaryotic organisms, is catalyzed by methyltransferases and has a significant impact on various aspects of cancer progression. WT1-associated protein (WTAP) is a crucial component of the m6A methyltransferase complex, catalyzing m6A methylation on RNA. It has been demonstrated to participate in numerous cellular pathophysiological processes, including X chromosome inactivation, cell proliferation, cell cycle regulation, and alternative splicing. A better understanding of the role of WTAP in cancer may render it a reliable factor for early diagnosis and prognosis, as well as a key therapeutic target for cancer treatment. It has been found that WTAP is closely related to tumor cell cycle regulation, metabolic regulation, autophagy, tumor immunity, ferroptosis, epithelial mesenchymal transformation (EMT), and drug resistance. In this review, we will focus on the latest advances in the biological functions of WTAP in cancer, and explore the prospects of its application in clinical diagnosis and therapy.

The Role of Circular RNAs in Ischemic Stroke.

Ischemic stroke (IS), a devastating condition characterized by intracranial artery stenosis and middle cerebral artery occlusion leading to insufficient oxygen supply to the brain, is a major cause of death and physical disability worldwide. Recent research has demonstrated the critical role of circular RNAs (circRNAs), a class of covalently enclosed noncoding RNAs that are widespread in eukaryotic cells, in regulating various physiological and pathophysiological cellular processes, including cell apoptosis, autophagy, synaptic plasticity, and neuroinflammation. In the past few years, circRNAs have attracted extensive attention in the field of IS research. This review summarizes the current understanding of the mechanisms underlying the involvement of circRNAs in IS development. A better understanding of circRNA-mediated pathogenic mechanisms in IS may pave the way for translating circRNA research into clinical practice, ultimately improving the clinical outcomes of IS patients.

Free radicals: Relationship to Human Diseases and Potential Therapeutic applications.

Reactive species are highly-reactive enzymatically, or non-enzymatically produced compounds with important roles in physiological and pathophysiological cellular processes. Although reactive species represent an extensively researched topic in biomedical sciences, many aspects of their roles and functions remain unclear. This review aims to systematically summarize findings regarding the biochemical characteristics of various types of reactive species and specify the localization and mechanisms of their production in cells. In addition, we discuss the specific roles of free radicals in cellular physiology, focusing on the current lines of research that aim to identify the reactive oxygen species-initiated cascades of reactions resulting in adaptive or pathological cellular responses. Finally, we present recent findings regarding the therapeutic modulations of intracellular levels of reactive oxygen species, which may have substantial significance in developing novel agents for treating several diseases.

Genetically encoded fluorescent biosensors for GPCR research.

G protein-coupled receptors (GPCRs) regulate a wide range of physiological and pathophysiological cellular processes, thus it is important to understand how GPCRs are activated and function in various cellular contexts. In particular, the activation process of GPCRs is dynamically regulated upon various extracellular stimuli, and emerging evidence suggests the subcellular functions of GPCRs at endosomes and other organelles. Therefore, precise monitoring of the GPCR activation process with high spatiotemporal resolution is required to investigate the underlying molecular mechanisms of GPCR functions. In this review, we will introduce genetically encoded fluorescent biosensors that can precisely monitor the real-time GPCR activation process in live cells. The process includes the binding of extracellular GPCR ligands, conformational change of GPCR, recruitment of G proteins or β-arrestin, GPCR internalization and trafficking, and the GPCR-related downstream signaling events. We will introduce fluorescent GPCR biosensors based on a variety of strategies such as fluorescent resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), circular permuted fluorescent protein (cpFP), and nanobody. We will discuss the pros and cons of these GPCR biosensors as well as their applications in GPCR research.

Sigma-1 Receptor: A Potential Therapeutic Target for Traumatic Brain Injury.

The sigma-1 receptor (Sig-1R) is a chaperone receptor that primarily resides at the mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) and acts as a dynamic pluripotent modulator regulating cellular pathophysiological processes. Multiple pharmacological studies have confirmed the beneficial effects of Sig-1R activation on cellular calcium homeostasis, excitotoxicity modulation, reactive oxygen species (ROS) clearance, and the structural and functional stability of the ER, mitochondria, and MAM. The Sig-1R is expressed broadly in cells of the central nervous system (CNS) and has been reported to be involved in various neurological disorders. Traumatic brain injury (TBI)-induced secondary injury involves complex and interrelated pathophysiological processes such as cellular apoptosis, glutamate excitotoxicity, inflammatory responses, endoplasmic reticulum stress, oxidative stress, and mitochondrial dysfunction. Thus, given the pluripotent modulation of the Sig-1R in diverse neurological disorders, we hypothesized that the Sig-1R may affect a series of pathophysiology after TBI. This review summarizes the current knowledge of the Sig-1R, its mechanistic role in various pathophysiological processes of multiple CNS diseases, and its potential therapeutic role in TBI.

Non-Coding RNAs: Emerging Therapeutic Targets in Spinal Cord Ischemia-Reperfusion Injury.

Paralysis or paraplegia caused by transient or permanent spinal cord ischemia–reperfusion injury (SCIRI) remains one of the most devastating post-operative complications after thoracoabdominal aortic surgery, even though perioperative strategies and surgical techniques continue to improve. Uncovering the molecular and cellular pathophysiological processes in SCIRI has become a top priority. Recently, the expression, function, and mechanism of non-coding RNAs (ncRNAs) in various diseases have drawn wide attention. Non-coding RNAs contain a variety of biological functions but do not code for proteins. Previous studies have shown that ncRNAs play a critical role in SCIRI. However, the character of ncRNAs in attenuating SCIRI has not been systematically summarized. This review article will be the first time to assemble the knowledge of ncRNAs regulating apoptosis, inflammation, autophagy, and oxidative stress to attenuate SCIRI. A better understanding of the functional significance of ncRNAs following SCIRI could help us to identify novel therapeutic targets and develop potential therapeutic strategies. All the current research about the function of nRNAs in SCIRI will be summarized one by one in this review.

Selectivity of the collagen-binding integrin inhibitors, TC-I-15 and obtustatin

Integrins are a family of 24 adhesion receptors which are both widely-expressed and important in many pathophysiological cellular processes, from embryonic development to cancer metastasis. Hence, integrin inhibitors are valuable research tools which may have promising therapeutic uses. Here, we focus on the four collagen-binding integrins α1β1, α2β1, α10β1 and α11β1. TC-I-15 is a small molecule inhibitor of α2β1 that inhibits platelet adhesion to collagen and thrombus deposition, and obtustatin is an α1β1-specific disintegrin that inhibits angiogenesis. Both inhibitors were applied in cellular adhesion studies, using synthetic collagen peptide coatings with selective affinity for the different collagen-binding integrins and testing the adhesion of C2C12 cells transfected with each. Obtustatin was found to be specific for α1β1, as described, whereas TC-I-15 is shown to be non-specific, since it inhibits both α1β1 and α11β1 as well as α2β1. TC-I-15 was 100-fold more potent against α2β1 binding to a lower-affinity collagen peptide, suggestive of a competitive mechanism. These results caution against the use of integrin inhibitors in a therapeutic or research setting without testing for cross-reactivity.

Discovery and characterization of a novel glucose-6-phosphate dehydrogenase (G6PD) inhibitor via high-throughput screening

Altered glucose-6-phosphate dehydrogenase (G6PD) status is influential in many cellular pathophysiological processes and diseases, making G6PD a potential target for cancer therapy. However, the available G6PD inhibitors are very limited and restricted. Here we developed a reducing equivalent nicotinamide adenine dinucleotide phosphate (NADPH) absorption photometry assay based on enzyme kinetics to characterize G6PD activity. In this way, we performed a high-throughput screening (HTS) to an in house library. And then we identified compound named Wedelolactone inhibiting G6PD strongly in a non-competitive, reversible way. In addition, we did the surface Plasmon Resonance (SPR) assay and indicated the KD between Wedelolactone and G6PD protein was 3.64 μM. Furthermore, our basic colony formation assay showed the inhibitory effect of Wedelolactone on the proliferation of ovarian cancer cells (IC50 ~ 10 µM). Thus, we provided a high-throughput screening assay to quickly and efficiently discover G6PD inhibitors, and identified Wedelolactone as a G6PD inhibitor, implying that Wedelolactone suppresses ovarian cancer partly through targeting G6PD.

Activation of dsRNA-Dependent Protein Kinase R by MicroRNA-378 Sustains Metabolic Inflammation in Hepatic Insulin Resistance.

MicroRNAs (miRNAs) are noncoding small RNAs that regulate various pathophysiological cellular processes. Here we reported that expression of the miR-378 family was significantly induced by metabolic inflammatory inducers, a high-fructose diet, and inflammatory cytokine TNFα. Hepatic miRNA profiling revealed that expression of miR-378a was highly upregulated which, in turn, targeted the 3'-UTR of PPARα mRNA, impaired mitochondrial fatty acid β-oxidation and induced mitochondrial and ER stress. More importantly, the upregulated miR-378a can directly bind to and activate the dsRNA-dependent protein kinase R (PKR) to sustain the metabolic stress. In vivo, genetic depletion of miR-378a prevented PKR activation, ameliorated inflammatory stress and insulin resistance. Counterbalancing the upregulated miR-378a using nanoparticles encapsulated with an anti-miR-378a oligonucleotide restored PPARα activity, inhibited PKR activation and ER stress, and improved insulin sensitivity in the fructose-fed mice. Conclusion: Our study delineated a novel mechanism of miRNA-378a in the pathogenesis of metabolic inflammation and insulin resistance through targeting metabolic signaling at both mRNA (e.g., PPARα) and protein (e.g., PKR) molecules. This novel finding of functional interaction between miRNAs (e.g., miR-378a) and cellular RNA binding protein(s) (e.g., PKR) is biologically significant as it greatly broadens the potential targets of miRNAs in cellular pathophysiological processes.

Activation of dsRNA-Dependent Protein Kinase R by miR-378 Sustains Metabolic Inflammation in Hepatic Insulin Resistance.

MicroRNAs (miRNAs) are noncoding small RNAs that regulate various pathophysiological cellular processes. Here, we report that expression of the miR-378 family was significantly induced by metabolic inflammatory inducers, a high-fructose diet, and inflammatory cytokine tumor necrosis factor-α. Hepatic miRNA profiling revealed that expression of miR-378a was highly upregulated, which, in turn, targeted the 3'-untranslated region of PPARα mRNA, impaired mitochondrial fatty acid β-oxidation, and induced mitochondrial and endoplasmic reticulum stress. More importantly, the upregulated miR-378a can directly bind to and activate the double-strand RNA (dsRNA)-dependent protein kinase R (PKR) to sustain the metabolic stress. In vivo, genetic depletion of miR-378a prevented PKR activation and ameliorated inflammatory stress and insulin resistance. Counterbalancing the upregulated miR-378a using nanoparticles encapsulated with an anti-miR-378a oligonucleotide restored PPARα activity, inhibited PKR activation and ER stress, and improved insulin sensitivity in fructose-fed mice. Our study delineated a novel mechanism of miR-378a in the pathogenesis of metabolic inflammation and insulin resistance through targeting metabolic signaling at both mRNA (e.g., PPARα) and protein (e.g., PKR) molecules. This novel finding of functional interaction between miRNAs (e.g., miR-378a) and cellular RNA binding proteins (e.g., PKR) is biologically significant because it greatly broadens the potential targets of miRNAs in cellular pathophysiological processes.

MicroRNA-21 ameliorates the impairment of autophagy in palatal wound healing.

Fibroblast injury and autophagy dysfunction have been shown to contribute to the persistence of oral wounds. Recently, microRNAs have emerged as vital regulators and fine tuners of various pathophysiological cellular processes that influence the wound healing process. This study explored the biological function and regulatory mechanism of miRNA-21 (miR-21) in the healing of oral wounds by interfering with autophagy. Healthy gingival cells derived from wild-type (WT) and from miR-21KO mice were characterized by immunocytofluorescence, and changes in wound healing were subsequently assessed using an in vitro scratch wound healing assay. The roles of critical proteins required for autophagy, autophagy related 5 (ATG5) and Bcl-2 interacting coiled-coil protein 1 (Beclin1) were evaluated by immunohistochemistry. Human gingival fibroblasts (HGFs) were transfected with a miR-21 mimic and a miR-negative control, and the relative expression of miR-21, ATG5, Beclin1 and LC3-I/II was characterized by qRT-PCR and Western blot. Pathological changes were observed in a palatal wound healing model using WT and miR-21KO mice. Immunohistochemistry was used to examine extracellular matrix (ECM) proteins and autophagy markers. Cell migration was delayed in gingiva-derived mesenchymal stem cells (GMSCs) from miR-21KO mice compared with WT mice. The expression of ATG5 and Beclin1 was significantly up-regulated in miR-21KO gingiva. Transfection of a miR-21 mimic into HGFs inhibited autophagy and up-regulated miR-21 expression. Knockdown of miR-21 suppressed the expression of fibronectin and CTGF, enhanced the autophagy effect of fibroblasts, suggesting that autophagy is involved in miR-21 regulated palatal wound healing. Taken together, these results suggest that miR-21 promotes oral wound healing by increasing ECM production through the inhibition of autophagy and facilitates clinical management of wound healing.

MiR-378a-3p Is Critical for Burkitt Lymphoma Cell Growth

Simple SummaryMicroRNAs (miRNAs) are small RNAs that regulate expression of specific target genes. We observed elevated levels of miR-378a-3p in Burkitt lymphoma (BL) and studied its role in the pathogenesis of BL. Inhibition of miR-378a-3p reduced growth of BL cells, confirming its significance in BL. Identification of BL specific target genes of miR-378a-3p revealed four candidates. For two of them, MNT and IRAK4, miR-378a-dependent regulation was confirmed at the protein level. Overexpression of MNT and IRAK4 in BL cell lines resulted in a similar effect as observed upon miR-378a-3p inhibition, suggesting their involvement in the growth regulatory role of miR-378a-3p.MicroRNAs (miRNAs) are small RNA molecules with important gene regulatory roles in normal and pathophysiological cellular processes. Burkitt lymphoma (BL) is an MYC-driven lymphoma of germinal center B (GC-B) cell origin. To gain further knowledge on the role of miRNAs in the pathogenesis of BL, we performed small RNA sequencing in BL cell lines and normal GC-B cells. This revealed 26 miRNAs with significantly different expression levels. For five miRNAs, the differential expression pattern was confirmed in primary BL tissues compared to GC-B cells. MiR-378a-3p was upregulated in BL, and its inhibition reduced the growth of multiple BL cell lines. RNA immunoprecipitation of Argonaute 2 followed by microarray analysis (Ago2-RIP-Chip) upon inhibition and ectopic overexpression of miR-378a-3p revealed 63 and 20 putative miR-378a-3p targets, respectively. Effective targeting by miR-378a-3p was confirmed by luciferase reporter assays for MAX Network Transcriptional Repressor (MNT), Forkhead Box P1 (FOXP1), Interleukin 1 Receptor Associated Kinase 4 (IRAK4), and lncRNA Just Proximal To XIST (JPX), and by Western blot for IRAK4 and MNT. Overexpression of IRAK4 and MNT phenocopied the effect of miR-378a-3p inhibition. In summary, we identified miR-378a-3p as a miRNA with an oncogenic role in BL and identified IRAK4 and MNT as miR-378a-3p target genes that are involved in its growth regulatory role.

Role of calcium-sensing receptor in cadmium-induced apoptosis of rat primary osteoblasts in vitro

Calcium is essential to various physiological and pathophysiological cellular processes. Calcium-sensing receptor (CasR), a seven-transmembrane-spanning protein that responds to changes in extracellular Ca2+, partly modulates calcium homeostasis, thereby influencing bone metabolism. In this study, we aimed to elucidate the role of CasR in Cd-induced calcium homeostasis disruption and OB apoptosis, and the underlying mechanisms. Cd treatment dramatically increased the protein expression of CasR and elevated the intracellular calcium concentration. Meanwhile, OBs apoptosis rate and caspase-dependent apoptosis protein levels, including cleaved caspase 3, cleaved caspase 9 and the ratio of Bax/Bcl-2 were increased. However, downregulation of CasR by CasR siRNA effectively suppressed the effects of Cd on theses phenomena. At the same time, we illustrated that CasR siRNA pretreatment blocked Cd-inhibited the phosphorylation of PKC and decreased Cd-induced the phosphorylation of PI3K/AKT.Our results suggested that CasR-mediated PKC and PI3K/AKT signaling pathways involve in calcium oscillation and apoptosis in OB caused by Cd maybe responsible for the bone homeostasis.

Dynamic control of hydrogel crosslinking via sortase-mediated reversible transpeptidation

Cell-laden hydrogels whose crosslinking density can be dynamically and reversibly tuned are highly sought-after for studying pathophysiological cellular fate processes, including embryogenesis, fibrosis, and tumorigenesis. Special efforts have focused on controlling network crosslinking in poly(ethylene glycol) (PEG) based hydrogels to evaluate the impact of matrix mechanics on cell proliferation, morphogenesis, and differentiation. In this study, we sought to design dynamic PEG-peptide hydrogels that permit cyclic/reversible stiffening and softening. This was achieved by utilizing reversible enzymatic reactions that afford specificity, biorthogonality, and predictable reaction kinetics. To that end, we prepared PEG-peptide conjugates to enable sortase A (SrtA) induced tunable hydrogel crosslinking independent of macromer contents. Uniquely, these hydrogels can be completely degraded by the same enzymatic reactions and the degradation rate can be tuned from hours to days. We further synthesized SrtA-sensitive peptide linker (i.e., KCLPRTGCK) for crosslinking with 8-arm PEG-norbornene (PEG8NB) via thiol-norbornene photocrosslinking. These hydrogels afford diverse softening paradigms through control of network structures during crosslinking or by adjusting enzymatic parameters during on-demand softening. Importantly, user-controlled hydrogel softening promoted spreading of human mesenchymal stem cells (hMSCs) in 3D. Finally, we designed a bis-cysteine-bearing linear peptide flanked with SrtA substrates at the peptide’s N- and C-termini (i.e., NH2-GGGCKGGGKCLPRTG-CONH2) to enable cyclic/reversible hydrogel stiffening/softening. We show that matrix stiffening and softening play a crucial role in growth and chemoresistance in pancreatic cancer cells. These results represent the first dynamic hydrogel platform that affords cyclic gel stiffening/softening based on reversible enzymatic reactions. More importantly, the chemical motifs that affords such reversible crosslinking were built-in on the linear peptide crosslinker without any post-synthesis modification. Statement of SignificanceCell-laden ‘dynamic’ hydrogels are typically designed to enable externally stimulated stiffening or softening of the hydrogel network. However, no enzymatic reaction has been used to reversibly control matrix crosslinking. The application of SrtA-mediated transpeptidation in crosslinking and post-gelation modification of biomimetic hydrogels is innovative because of the specificity of the reaction and reversible tunability of crosslinking kinetics. While SrtA has been previously used to crosslink and fully degrade hydrogels, matrix softening and reversible stiffening of cell-laden hydrogels has not been reported. By designing simple peptide substrates, this unique enzymatic reaction can be employed to form a primary network, to gradually soften hydrogels, or to reversibly stiffen hydrogels. As a result, this dynamic hydrogel platform can be used to answer important matrix-related biological questions that are otherwise difficult to address.

The C-C Chemokines CCL17 and CCL22 and Their Receptor CCR4 in CNS Autoimmunity.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS). It affects more than two million people worldwide, mainly young adults, and may lead to progressive neurological disability. Chemokines and their receptors have been shown to play critical roles in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), a murine disease model induced by active immunization with myelin proteins or transfer of encephalitogenic CD4+ T cells that recapitulates clinical and neuropathological features of MS. Chemokine ligand-receptor interactions orchestrate leukocyte trafficking and influence multiple pathophysiological cellular processes, including antigen presentation and cytokine production by dendritic cells (DCs). The C-C class chemokines 17 (CCL17) and 22 (CCL22) and their C-C chemokine receptor 4 (CCR4) have been shown to play an important role in homeostasis and inflammatory responses. Here, we provide an overview of the involvement of CCR4 and its ligands in CNS autoimmunity. We review key clinical studies of MS together with experimental studies in animals that have demonstrated functional roles of CCR4, CCL17, and CCL22 in EAE pathogenesis. Finally, we discuss the therapeutic potential of newly developed CCR4 antagonists and a humanized anti-CCR4 antibody for treatment of MS.