Specifically, it became increasingly clear that the choice of cell types and culture method both constitute important parameters that affect the predictive power of test systems. In this review, we present current 3D culture paradigms for hepatotoxicity tests and critically evaluate their utility and performance for DILI prediction. In addition, we highlight possibilities of these emerging platforms for mechanistic evaluations of selected drug candidates and present current research directions towards the further improvement of preclinical liver safety tests. We conclude that organotypic and microphysiological liver systems have provided an important step towards more reliable DILI prediction. Furthermore, we expect that the increasing availability of comprehensive benchmarking studies will facilitate model dissemination that might eventually result in their regulatory acceptance.Drug-induced liver injury (DILI) is a major concern for the pharmaceutical industry and constitutes one of the most important reasons for the termination of promising drug development projects. Reliable prediction of DILI liability in preclinical stages is difficult, as current experimental model systems do not accurately reflect the molecular phenotype and functionality of the human liver. As a result, multiple drugs that passed preclinical safety evaluations failed due to liver toxicity in clinical trials or postmarketing stages in recent years. To improve the selection of molecules that are taken forward into the clinics, the development of more predictive  Drug-induced liver injury (DILI) constitutes a rare life-threatening adverse drug reaction (ADR). While it only occurs in 4–19 per 100,000 individuals, it is nevertheless the most common cause for acute liver failure in the US and Europe . Of the https://www.ich.org/products/guidelines/safety/article/safety-guidelines.html), whereas evaluation of DILI liability is not explicitly required. In 2010, however, EMEA has released a white paper to assess the risk of DILI during preclinical stages , and these developments can be interpreted as the intention of regulators to formalize specific guidance for DILI in the near future.In light of this preamble, it is clear that the assessment of potential safety liabilities is an essential element of preclinical drug development in order to reduce the risk of expensive late project failures. Importantly, projects with preclinical safety signals are often closed in the clinic due to safety issues, emphasizing the need to thoroughly evaluate and act upon preclinical safety profiles . The manIn Vitro Cytotoxicity (MEIC) project  during the first 24 h of culture  as the gold standard cell model for predictive toxicology. While freshly isolated PHH closely resemble hepatocytes  culture . Signifi culture . Further culture .To overcome these problems, PHH can be cultured in sandwich configuration in which 2D cultured hepatocytes are overlaid with an additional layer of Matrigel. In this culture paradigm PHH retain their morphology and polarity, resulting in physiologically relevant expression levels of transporters and the formation of functional bile canaliculi networks . While lin vitro models for the prediction of DILI, including spheroid models, micropatterned co-cultures (MPCC), bioreactors, and liver-on-a-chip devices, and critically discuss their strengths and weaknesses. In the last part of the review, we highlight emerging applications beyond the evaluation of hepatotoxicity for which organotypic liver models promise to provide conceptual advancements over conventional 2D cultures.In an attempt to improve phenotypic maintenance of liver cells in culture, a vast number of 3D culture methods have been developed during recent years . In the in vivo microenvironment, including the phenotypes of and interactions with relevant non-parenchymal cells (NPCs). Additionally, viability and molecular signatures of the cultured cells should be stable. As such, it is crucial that the rapid dedifferentiation observed within hours in 2D monolayer PHH cultures is prevented  and hormone levels, most importantly insulin. Furthermore, while the use of serum might be supporting overall cell health, its use can complicate result interpretation due to incomplete information regarding its active constituents. Similar arguments apply to the use of media with proprietary non-disclosed formulations.via mechanosensing pathways  . In addipathways . MoreoveScaffold-Free Spheroids in Perfused Stirred-Tank Bioreactors. The earliest reports of spheroid hepatocyte culture used newborn rat hepatocytes on poly-HEMA or proteoglycan-coated ULA surfaces and demonstrated that such spheroids were viable for multiple weeks and functionally superior compared to 2D monolayer culture   . Moreoveculture)  and 3D cculture) . Importa enzymes . Of note enzymes .Compared to HepG2, HepaRG cells exhibit higher metabolic activity . HepaRG in vivo in mice, thus providing a useful experimental tool for the prediction of drug-induced liver fibrosis.In addition to being used as a model for hepatocellular toxicity, Leite and colleagues recently demonstrated that HepaRG-stellate cell co-cultures can be used to detect stellate cell activation and drug-induced liver fibrosis of allyl alcohol and methotrexate . Furtherin vivo, whereas cultures from the same donors rapidly deteriorated in 2D monolayer or sandwich culture  leakage as readout . In addiRecently, two studies evaluated the predictive power of PHH spheroids offered by InSphero AG in media with proprietary composition to detect hepatic liabilities of selected compounds. Proctor et al. analyzed 110 drugs (69 DILI positive and 41 DILI negative) at concentrations up to 100× the therapeutic exposure levels for 2 weeks in undisclosed media formulations and reported sensitivity and specificity of 59% and 80%, respectively . The sammax  to evaluate the toxicity of 123 drugs (70 DILI positive and 53 DILI negative) . Of thesin vitro model for studies of human liver function and drug toxicity. To date, various differentiation protocols have been presented. While the overall differentiation state of HLCs has been improved, current protocols still fall short from producing cells that closely resemble the molecular phenotype of mature PHH (HLCs can be obtained by differentiation from human induced pluripotent stem cells (hiPS-Hep) and have been discussed as a promising alternative ture PHH . Importaet al. found that HepG2 spheroids were much more sensitive than HLC spheroids for 10/23 evaluated compounds following a single 72-h exposure  scaffolds have received growing attention in the context of hepatic spheroids, as they facilitate the deposition of essential components of the hepatic microenvironment, including collagens and fibronectin . Alginat50 values (approximately 40 mM), however, were still substantially higher than in scaffold-free spheroid culture or in vivo (approximately 1 mM). In contrast, HepG2 spheroids in ECM-based hydrogel ceased to proliferate and acquired a more differentiated phenotype, indicated by gradually increased expression of albumin, hepatic transcription factors, phase I and II drug metabolism enzymes, and transporters up to day 28, whereas culture in sandwich configuration or on porous polystyrene scaffold did not result in phenotypic improvements . Functional parameters, such as the activities of CYP2A6 and CYP3A4, as well as glucuronidation and sulfation reactions were drastically decreased to 5–25% of PHH levels. The system successfully distinguished between four toxic and four nontoxic compounds  using glutathione and ATP levels, as well as albumin secretion and urea synthesis as endpoints . In addiompounds .In hollow fiber bioreactors, cells are cultured in tubular modules, each permeated by a network of intratubular capillaries, which allow perfusion of medium and gases .10 cells in a cell compartment of 800-ml volume, which renders such approaches incompatible with parallel drug testing  in HepaRG bioreactor cultures was 95% lower than in PHH bioreactors, whereas CYP3A4 levels were found to be similar  agonist AZD6610 differed in HepaRG bioreactors, whereas they were similar between PHH and in vivo  than in spheroids and MPCC  and acetaminophen  was similar to previous reports in sandwich cultured PHH  . Notablyured PHH , and thuDraper recently presented a liver-on-a-chip system made of oxygen permeable thermoplastic that consists of 96 units, each containing sandwich cultured hepatocytes maintained in recirculating microfluidic conditions . The autAn interesting device was developed by Schepers and colleagues in which spheroidal hiPS-Hep aggregates are entrapped in perfusable C-shaped features . Jin andIn addition, a plethora of other liver chips have been presented. However, these models are only in their infancy and no comprehensive characterization data have been presented with regards to molecular hepatic phenotypes or predictive power for DILI predictions. For further details on hepatic chip platforms, we refer the interested reader to recent reviews on the topic .3D bioprinting encompasses a variety of methodologically distinct modalities that can create complex biological tissue structures by depositing cells and extracellular factors with high spatial and temporal precision. Droplet-based bioprinting (DBB), also termed inkjet bioprinting, refers to the precise deposition of cells dispensed in small droplets . AdvantaExtrusion-based bioprinting (EBB) results in the deposition of continuous filaments of desired shapes, differing from the individual droplets of inkjet-based bioprinters, and constitutes arguably the most widely used bioprinting modality (Ozbolat and Hospodiuk). This method is highly versatile and compatible with a wide range of bioink properties. Furthermore, it currently constitutes the only modality compatible with the printing of scaffold-free cell systems, such as spheroids . Howeverin vivo when transplanted into recipient mice in a liver injury model, suggesting the utility of such approaches for regenerative medicine applications  constitutes the least prevalent bioprinting modality, primarily due to its high cost, difficult handling, and lack of commercially available off-the-shelf solutions. However, due to its high resolution and printing speed coupled with low cell stress during the printing process, LAB constitutes a promising platform for tissue engineering. LAB allows bioprinting of structures mimicking the microscale hexagonal architecture of the human liver in which cells display improved molecular marker signatures compared to conventional 2D monolayer culture . FurtherBioink is a key component of the bioprinting process that should offer mechanical support for the printed structure while mimicking the natural microenvironment of the encapsulated cells. Recent years have seen tremendous progress in bioink development . For hepin vitro opens a multitude of promising liver-related applications. However, while promising proof-of-concept data have been presented, the added value for the prediction of drug metabolism and toxicity in bioprinted liver models compared to other 3D culture models has not yet been demonstrated.Promising data have been presented for DILI tests using bioprinted liver models. Specifically, bioprinted livers reproduced hepatotoxicity of acetaminophen at clinically relevant concentrations and successfully distinguished trovafloxacin from its nontoxic structural analogue levofloxacin . FurtherWhile a large number of studies using organotypic and microphysiological liver models have been presented that provide proof-of-concept for the detection of hepatotoxicity, we are only aware of 11 studies that have screened a larger (> 10) number of compounds , the analyzed test compounds were highly dissimilar. This is particularly important with regards to the definition of human hepatotoxicity. Most studies focused on compounds with specific hepatotoxic liability, whereas few others used drugs that were generally cytotoxic, such as anthracyclines, taxanes, or colchicine, which complicates model comparisons . Analogo50 values <1 mM. Heterogenous results were obtained for HepaRG spheroids. Whereas some studies reported very high sensitivity .The compound that was most commonly used for the evaluation of hepatotoxicity was acetaminophen. Therapeutic exposure levels of acetaminophen are 139 µM, whereas overdose plasma concentrations higher than 0.7 mM require immediate intervention . Notably50 = 4.5 mM), indicating that this culture paradigm might be specifically useful for the cultivation of stem cell-derived cells. However, acute exposure of PHH-MPCC did not result in relevant toxicity (TC50 = 35 mM). Liver-on-a-chip studies using acetaminophen as a benchmark have only recently been published and exhibit modest sensitivity (TC50 = 2–5.59 mM). Importantly, model sensitivity to acetaminophen increased overall with increasing exposure time up to 2 weeks, whereas acetaminophen hepatotoxicity is clinically most relevant in the context of acute acetaminophen poisoning, which occurs within hours. Acetaminophen depletes the cellular reduced glutathione (GSH) stores and excessive necrosis only manifests once GSH levels drop below 40%  and non-rodent (minipig and rhesus monkey) animal models cultured as spheroids can recapitulate species-specific toxicity at clinically relevant concentrations . The sysHepatic biotransformation of drugs can generate circulating metabolites, which may possess a safety profile different from the parent molecule. Importantly, the panorama of generated metabolites can differ both quantitatively and qualitatively across species. Thus, the identification and safety assessment of metabolites that are found at disproportionate levels in humans compared to animals constitute an important facet of preclinical toxicology. Current regulatory guidance defines disproportionate metabolites that mandate safety evaluation as metabolites that constitute >10% of circulating drug levels in humans and that are either not detected in animals or for which animal exposure is at least 50% lower . HoweverN-glucuronide and salbutamol-4-O-sulfate, which have not been detected in previous systems. Similarly, PHH MPCC identified 77% (43/56) of the human relevant metabolites of 27 drugs after 7 days of incubation, whereas hepatocyte suspension cultures (55%), S9 fractions (46%), and liver microsomes (39%) detected considerably less , by the Strategic Research Programme in Diabetes at Karolinska Institutet, by the Lennart Philipson Foundation, and by the Harald and Greta Jeansson Foundation. No writing assistance was utilized in the production of this manuscript.VML is co-founder and owner of HepaPredict AB.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This is due to the presence of specialized cells thatgenerate electrical impulses that propagate throughout the heart tissue, quickly andefficiently. This electrical impulse starts at the sinoatrial node (SAN) andpropagates sequentially to atrioventricular node (AVN), subsequently beingtransmitted to the ventricles via specialized conduction pathways. The electricalsignals are conducted from cell to cell through a cardiomyocyte permeability controlsystem formed by proteins called connexins, and connexin-43 is the type found in theheart and is associated with the formation of so-called gap junctions. By providingthe single electrical connection between the atria and the ventricles, AVN plays anessential role in the dynamics of cardiac contraction. Clinically, when the PRinterval is observed in the electrocardiographic recordings, we can correlate theelectrical impulse conduction time from its generation in the SAN to the delay inthe AVN region, which is called "decremental conduction".3 iron recycling in the spleen andliver,4 and participate inthe process of synaptic maturation in the healthy brain.5 Such non-canonical activities emphasize thefunctional diversity of macrophages and their ability to perform specific tasks inthe various tissues, in addition to host defence.6 In cardiac tissue, macrophages are intrinsic components ofthe myocardium in normal functioning, where they appear as spindle cellsintercalated between cardiomyocytes, fibroblasts and endothelial cells.7Over the years, several studies have described the macrophages as cells of phagocyticfunctions that would exclusively act in the immune system protecting the organismagainst pathogens. However, more recently this paradigm was mainly questioned aboutthe origin of macrophages. Several studies have provided evidence that asubpopulation of macrophages, which originated from embryonic development and do notcome from the bloodstream, reside and proliferate in virtually all body tissues andapparently act specifically on each organ. For example, resident macrophages ofadipose tissue contribute to the regulation of thermogenesis,8 Hulsmans et al.9 observed that mice that had their macrophage faunaweakened, had bradycardia and irregular beats. It is known that connexin-43 ispredominant in ventricles of humans and that its reduction promotes bradycardiaand AV block,8 thus, inobserving specialized cells in non-muscular electrical conduction, they foundthat macrophages are electrically coupled to cardiomyocytes and that theseresident macrophages facilitate electrical conduction through the AV node.Cardiac function depends on the appropriate moment of contraction in severaldistinct regions, as well as the heart rate.9 whose causein humans is still unknown.10Another intriguing point is that cardiac macrophages have a resting membranepotential of -35 mV on average and depolarize in synchrony with cardiomyocytes.This makes the membrane potential at the rest of the cardiomyocytes morepositive and according to the results obtained by computational simulation,accelerate both depolarization and repolarization phases.9 The cardioprotective role ofcardiac resident macrophages can go beyond the modulation of theelectrophysiological properties of the coupled cardiomyocytes. The perivascularlocalization of cardiac macrophages makes them uniquely positioned to interpretsystemic signals in the bloodstream.10Such conducting cells interleave with macrophages expressing connexin-43 formingadditional gap junctions between cardiomyocytes . The inv11 demonstratedthat inflammation caused by type I diabetes causes resident macrophages tosecrete interleukin 1β (IL-1β), acting in a paracrine manner,increasing oxidative stress in the surrounding cells and destabilizing theelectrical activity of cardiomyocytes provoking lethal ventricular arrhythmias.Moreover, atherosclerotic lesions are currently understood as inducers ofimportant inflammatory processes, which comprise components of the innate andacquired immune systems. Clinical data showed that increased leukocyte count,interleukin-6 (IL-6), tumour necrosis factor (TNF) and IL-1β were at riskof more severe cardiovascular events.Monnerat et al.14In fact, IL-6 is locally regulated during the coronary occlusion process inpatients with acute myocardial infarction with ST-segment elevation.15 Research has shown that sepsisis associated with an increased risk of acute and fatal coronary disease, butits cause is still a matter of debate, and acute coronary disease prevention maybe an important consideration in post-sepsis medical care.17Thus, a possible bias is one in which macrophages contribute to the arrhythmiccomplications of infectious, atherosclerotic and septicemic diseases, in whichtheir inflammatory responses may interfere with their role in modulatingelectrical conduction of the cardiomyocyte.18 Several studies have demonstrated thatpathological cardiac hypertrophy and fibrosis in heart failure are accompaniedby a systemic inflammatory response, infiltration and activation of cells of theimmune system.19 In view ofthis, immunotherapies for cardiovascular diseases are on the rise.Despite significant advances in prevention and treatment, cardiovascular diseases(CVD) continue to be the most common cause of death in the world. In fact,severe heart failure is more prevalent than cancer.20 Another study using CANTOS reinforces this idea andprovides strong evidence that the modulation of the IL-6-induced signalingpathway induced by IL-1α is associated with reduced rates ofcardiovascular changes and mortality.13The first cardiovascular immunotherapy was developed for the treatment ofhypercholesterolemia and its positive results paved the way for the clinicalevaluation of anti-inflammatory immunotherapy directed to interleukin 1β.CANTOS (Canakinumab Anti-Inflammatory Thrombosis Outcomes Study) has shown thatsubcutaneous injections of canakinumab (ACZ885), a human monoclonal antibodythat selectively neutralizes IL-1β, significantly reduced levels ofsystemic inflammatory biomarkers in patients after acute myocardial infarction,reducing risk cardiovascular disease in patients with previous heart attack andinflammatory atherosclerosis.in situ could be a reliable formof therapeutic strategy that could be applied to ensure normal cardiac rhythm inpatients with signs of arrhythmia.21 However, whatwe know so far is that resident macrophages act as "masters", orchestrating theheart rate.It is clear that further studies should be performed to address the actualinvolvement of resident macrophages in heart diseases. If alterations inmacrophages' function are linked to these clinical conditions, immunotherapywith macrophage reprogramming


Long noncoding RNAs (lncRNAs) are emerging as important regulators of numerous biological processes, especially in cancer development. Aberrantly expressed and specifically located in tumor cells, they exert distinct functions in different cancers via regulating multiple downstream targets such as chromatins, RNAs, and proteins. Differentiation antagonizing non-protein coding RNA (DANCR) is a cytoplasmic lncRNA that generally works as a tumor promoter. Mechanically, DANCR promotes the functions of vital components in the oncogene network by sponging their corresponding microRNAs or by interacting with various regulating proteins. DANCR's distinct expression in tumor cells and collective involvement in pro-tumor pathways make it a promising therapeutic target for broad cancer treatment. Herein, we summarize the functions and molecular mechanism of DANCR in human cancers. Furthermore, we introduce the use of CRISPR/Cas9, antisense oligonucleotides and small interfering RNAs as well as viral, lipid, or exosomal vectors for onco-lncRNA targeted treatment. Conclusively, DANCR is a considerable promoter of cancers with a bright prospect in targeted therapy. Approximately 75% of the human genome can be transcribed into RNAs, yet no more than 2% encodes proteins . Non-codDue to the overwhelming quantity of lncRNAs in the human genome and the lack of methods for predicting their functions, massive work is still needed to better understand the lncRNAs. LncRNA differentiation antagonizing non-protein coding RNA (DANCR) can serve as a paradigm for lncRNA investigation. DANCR is a typical oncogenic lncRNA which exerts multiple functions in cancer development. It has also demonstrated its value in cancer therapy as a promising target .This article summarized the functional properties and mechanisms of DANCR in tumor progression. The general targeted therapies against onco-lncRNAs were introduced and the specific methods to target DANCR were discussed. This article can provide referential value for latter onco-lncRNA investigations and insights into the clinical treatment against cancers caused by aberrant expression and regulation of lncRNAs.DANCR also named as Anti-Differentiation Non-coding RNA (ANCR), Small nucleolar RNA host gene protein 13(SNHG13), upregulating transcripts 2 (AGU2) is an oncogenic lncRNA located on chromosome 4 . It was The discovery that DANCR over-expressed in colorectal cancer promoted tumor progression and predicted poor prognosis introduced the study of DANCR into the field of cancer . DANCR cIn the wake of the deepening of the research, DANCR was found to be aberrantly expressed in numerous cancers and exerted multiple regulating functions. The underlying mechanisms were also being constantly elucidated.DANCR is a typical oncogenic lncRNA, overexpressed in various tumor cells. It can regulate the progression of numerous cancers by modulating the expression or activity of downstream targets and consequently promoting hallmarks of cancer including cell proliferation, cell motility, tumor angiogenesis, and cell viability.In hepatocellular carcinoma, it is observed that down-regulated DANCR expression can boost cell apoptosis and cell cycle block in G1 which underlies that DANCR can promote tumor cell proliferation by inhibiting cell apoptosis and facilitating growth inhibition evasion . DANCR oCell motility refers to the metastasis and invasion properties of cells and malignant cells to vital organs and it is the major cause of cancer deaths . DANCR cDANCR promotes the motility of nasopharyngeal carcinoma cells by stabilizing hypoxia inducible factor-1α, the subunit of Hypoxia inducible factor 1 (HIF-1) which are transcription factors that can activate several hypoxia-responsive genes in response to hypoxia microenvironment and promote the metastasis and invasion of cancer cells , 25. MatTumor angiogenesis is one of the hallmarks of cancer which provide essential nutrients and oxygen to proliferating cancer cells . DANCR cThe innate or acquired chemo-resistance of tumor cells is a main problem in the clinical cancer therapy. DANCR promotes the docetaxel resistance in prostate cancer cells by mediating the upregulation of multidrug resistance associated protein (MDR) and p-glycoprotein (P-gp), which bump many chemotherapeutic drugs out of the cells and limit the intracellular drug dosage to sub-therapeutic levels , 35. DANGenerally, DANCR exerts multiple regulatory functions on tumor progression of numerous cancers, revealing its role as an important pro-tumor regulator . And notLncRNAs perform their multiple regulatory functions on tumors by interacting with numerous downstream targets . The dowEZH2 is a common target protein of DANCR. Mechanically, EZH2 enables the silencing of gene expression by catalyzing histone H3 trimethylation at lysine 27 (H3K27me3) in target gene promoters . DANCR cMicroRNAs are small regulating RNAs (−22 nt in length) derived from the short hairpin regions of RNA transcripts and they induce cleavage and suppression of translation via complementary binding to target RNAs . DANCR cIn this section, the role of DANCR in particular cancers is discussed so as to clarify the molecular mechanisms of DANCR in a more specific way and instruct the therapy of DANCR aberrantly-expressed cancers .DANCR acts as a miRNA sponge in colorectal cancer (CRC) cells, which is the most common role that DANCR plays in cancer development. Harboring the same miR-577 binding site, DANCR and heat shock protein 27 (HSP27) mRNA competitively bind to miR-577. The overexpression of DANCR in CRC cells alleviates the degradation impact of miR-577 on HSP27, which contributes to cell proliferation and metastasis .DANCR activated by SALL-4   mediatesHIF-1α, generally known as the core regulator of the hypoxic response, has been validated as a critical positive regulator of nasopharyngeal carcinoma (NPC) cell metastasis. DANCR can combine the NF90 (nuclear factor 90)/NF45 (nuclear factor 45) complex with its AU-rich elements (AREs) in 3′ region, which stabilizes HIF-1α mRNA and upgrades the motility of NPC cells .In triple-negative breast cancer (TNBC), DANCR functions by mediating protein assembly and modification. DANCR can bind with the phosphorylation site of RXRA, recurring glycogen synthase kinase 3 beta (GSK3β) to sponge RXRA and promote RXRA ser phosphorylation. The structural change in RXRA suppresses its interaction with the promoter of phosphatidylinositol-4,5-biophosphate 3-kinase catalytic subunit alpha (PIK3CA), thereby enhancing PIK3CA in both mRNA and protein levels. Consequently, protein PIK3CA activates the P13K/AKT pathway, leading to the proliferation and tumor growth of TNBC .Bladder cancer (BC) whose proper diagnosis and efficient treatment are current challenges in urology, is the most frequent malignancy of the urinary tract . The folSimilar to CRC, oncogenic DANCR within hepatocellular carcinoma (HC) has also been found to have multiple regulating targets. KLF12 (Krueppel-like factor 12) can be positively regulated by DANCR through miR-216a-5p sponging, leading to malignant progression . DANCR cWe can conclude that DANCR can regulate the signaling pathways by alleviating miRNA's inhibitory functions on target RNA, combining with proteins and guiding them to modulate the expression or the stability of RNAs or facilitating the integration and subsequent modifying of proteins. Moreover, DANCR can manipulate more than one target in cancer and join in multiple pathways. As we can see, due to the diverse mechanisms of DANCR and limited research on it, it is hard to foresee the unknown mechanisms and functions of DANCR in cancers. However, lncRNA's collective involvement in the pathways of numerous cancers does make it a promising target for cancer therapy.The potency of lncRNA-based cancer therapy has been pointed out in previous years. Targeting lncRNAs for cancer treatment is a novel and promising therapy due to their collective therapeutic efficacy, high specificity, and few side effects . The posThe CRISPR/Cas9 technology can be used to target the endogenous loci of lncRNAs and restrict their transcription . The CRIin vivo. Combined with their relatively high charge and hydrophilicity (Antisense oligonucleotides (ASOs) can be used in lncRNA-targeted therapy. They are synthetic and modified oligonucleotide single strands that can complementarily bind to certain sequences of their target RNAs. ASOs exert their inhibiting effect mainly by recruiting ribonuclease H (Rnase H) to degrade the targets. They comprise complementary DNA single strands to form DNA/RNA duplexes for Rnase H recognition and later cleavage . Vulnerahilicity , all thehilicity  that invhilicity . In thishilicity . Additiohilicity . Howeverhilicity , which ihilicity . Uniformhilicity . Among ahilicity . LNA canhilicity . The cenhilicity . Both mihilicity . And to hilicity .We can also use small interfering RNAs (siRNAs) to target lncRNAs. In 2018 patisiran became the first approved RNAi drug to remedy hereditary transthyretin amyloidosis . SiRNAs Viral vectors were one of the earliest engineered nanoscale delivery systems used for transporting RNA products to targeted tissues. Viral vectors are known for their efficient transfer of codes to the cell interior and capability for escaping immunosurveillance by infected cells . In tumoCompared to viral vectors, non-viral vectors are of lower transfection efficiency . Howeverin vivo  finin vivo . HEK293Tin vivo ; (b) train vivo ; (c) isoAlvarez-Erviti et al. pioneered the practice of applying engineered exosomes to deliver siRNA. They build neuronal cell-targeted exosomes and use them to pass through the blood-brain barrier and treat Alzheimer's disease . A recenTo date, researches on targeting DANCR for cancer therapy remains limited. A previous study presented that the relative enrichment of the enzymes responsible for RNA degradation vary between cellular compartments, so the location of lncRNA can impact the suppressing efficacy of the molecular drugs on it. Comparatively, ASO is more capable of clearing the nuclear lncRNAs while RNAi have a better suppressive effect on lncRNAs in cytoplasm . ReferriThe review has shown the vital research value of DANCR. DANCR is also a critical oncogenic regulator which presents an increasingly important status in cancer study. It can regulate hallmarks of various cancers, indicate their progression and clinical outcomes and serve as a novel target for cancer targeted treatment. Researches on DANCR remain limited and there is an urgent need for further study on this critical onco-lncRNA.The recent progress on RNA interaction identification method includes the refined variants of immunoprecipitation techniques  and new high-throughput RNA interactome analysis methods  . WithoutS-JJ conceived, wrote the manuscript, and completed the figures and tables. M-ZJ revised the manuscript. W-LJ and B-RX conceived, organized, and edited the text.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


ZPBP) gene was significantly hypomethylated in the LLG promoterregion, and its expression was significantly upregulated in uterine tissue.Thus, the methylation status of ZPBP gene was identified as apotential indicator of litter size. Furthermore, we verified its negativecorrelation with litter size traits (TNB and NBA) in whole blood samples from172 Berkshire sows as a blood-based biomarker by a porcinemethylation-specific restriction enzyme polymerase chain reaction (PMP)assay. The results suggest that the methylation status of the ZPBPgene can serve as a valuable epigenetic biomarker for hyperprolific sows.In pigs, litter size is typically defined as the total number of piglets born(TNB) or the number of piglets born alive (NBA). Increasing pig litter sizeis of great economic interest as a means to increase productivity. Thecapacity of the uterus is a critical component of litter size and may play acentral role in prolificacy. In this study, we investigatedlitter-size-related epigenetic markers in uterine tissue from Berkshire pigswith smaller litter size groups (SLGs) and larger litter size groups (LLGs)using genome-wide bisulfite sequencing (GWBS). A total of 3269 differentiallymethylated regions (DMRs) were identified: 1566 were hypermethylated and 1703hypomethylated in LLG compared to SLG. The zona pellucida binding protein( Litter size is typically defined as thetotal number of piglets born (TNB) or the number of piglets born alive (NBA).Litter size is controlled by many factors, such as ovulation rate (number ofovulated eggs), number of corpora lutea, fertilization rate, uterinecapacity, and prenatal survival . Fetalsurvival is primarily determined by the uterine capacity of the dam, whichcan be defined in terms of the relative surface area of placental endometrialattachment required to support the nutrient requirements of an individualfetus throughout gestation . Therefore, uterine traitscan greatly influence litter size. In addition, the lowheritability of litter size (5 %–10 %) suggests that proximateenvironmental variables may contribute significantly to variation in it .PRKG2, CLCA4, and PCK1 genes play important rolesin improving litter size by increasing nutrition supply through theplacenta.DNA methylation is among the main epigenetic mechanisms and playssignificant roles in gene silencing , tissue differentiation , cellulardevelopment , X-chromosome inactivation, and genetic imprinting . Importantly, DNA methylation is both stably heritable andfully reversible. DNA methylation may reflect interactions between geneticand environmental factors in the development and reproduction of pigs. Inparticular, when DNA methylation occurs in a gene promoter, it typicallyacts to repress gene transcription. Several studies have suggested acorrelation between differentially methylated regions (DMRs) near promoterregions and gene expression changes . Furthermore, these types of dynamic changesin DNA methylation tend to occur during embryonic development as cellsdifferentiate or become reprogrammed . Many recent studies have examined thegenome-wide methylation profiles of livestock phenotypes associated withdisease resistance, milk production, and reproduction . DNAmethylation affects the expression of many genes that are critical toreproductive traits . Hwang et al. (2017) recently identifiedDMRs and differentially expressed genes (DEGs) associated with litter sizein pig placentas and suggested that the The objective of this study was to use epigenetic approaches to examineuterine tissues of Berkshire pigs with smaller and larger litter sizes,using genome-wide bisulfite sequencing (GWBS) technology. Our findings willprovide useful knowledge and a clearer understanding of the reproductivephenotypes of individual pigs and could help in selecting sows with highfecundity for breeding.22.1All animal experiments were approved by the Gyeongnam National University ofScience and Technology Institutional Animal Care and Use Committee (permitno. 2105-5). All Berkshire sows used in this study were reared under the sameenvironmental conditions  and providedwith the same commercial diet and water ad libitum. To analyze DNAprofiling according to litter size, animals were divided into smaller littersize groups .Genomic DNA (gDNA) was isolated from three endometrium tissues per groupusing a DNeasy Tissue Kit  and pooled for GWBSanalyses. gDNA  and MspI (NEB), a pair of methylation-sensitiveisoschizomers that have the same recognition site (CC|GG). An undigested gDNA assay is aPCR-based methylation method that uses methylation-sensitive restrictionenzymes to determine DNA methylation status. To verify the result of GWBS,whole blood samples were collected from Berkshire sows to isolate gDNA usinga Wizard Genomic DNA Purification Kit  accordingto the manufacturer's instructions and digested with 2.3ZPBP) and thegene encoding peptidylprolyl isomerase A (PPIA) , using appropriate primer pairs (Table 1). Amplification wasperformed using a Perkin Elmer 9700 system  under the following conditions: 95 Reverse-transcription PCR (RT-PCR) analysis was performed to detect gene expression. Total RNA was extracted fromthree uterine tissues from each group using the TRIzol Reagent  and then reverse-transcribed into cDNAusing Superscript II Reverse Transcriptase .cDNA was then subjected to RT-PCR to evaluate the relative gene expressionlevels of the zona pellucida binding protein (2.4ZPBP methylation status and sow litter sizetraits (TNB and NBA). All statistical analyses were conducted using SPSSsoftware .Comparisons between groups were performed using 33.1ZPBP was found to be strongly related to fecundity by GOenrichment analysis. This DMG was mainly observed at promoter regions  and was hypomethylated inLLG (Table 4).GWBS was performed on gDNA from pooled uterus samples . In particular, DNA methylation ingene promoters is strongly associated with gene silencing . Recently, studieshave been conducted to identify the genome-wide methylation profiles of farmanimals . Some studies have described DNA methylation for the pig uterus; however, few have reported uterine genome-wide methylationpatterns. GWBS, which allows unbiased genome-wide DNA methylation profiling,has been used to investigate prolificacy-related DNA methylation inunprecedented detail .In the current study, we used GWBS to investigate the DNA methylationprofiles of the genome in uterine tissues of high- and low-prolificacy pigs to explore the relationships between DNA methylation and litter size traits.3.2\opttitle{Confirmation of the methylation status of the \textit{ZPBP} gene in whole blood byPMP assay}ZPBP gene as a blood-based biomarkerfor increased litter size, DNA methylation patterns of the ZPBP gene wereverified in whole blood samples collected from three sows, used to obtainuterus tissues in each group by PMP assay. As shown in Fig. 2, we confirmedthat the ZPBP gene was hypomethylated in LLG. This result was consistent withthe results of GWBS analysis, and will allow early prediction of littersizes in prepubertal gilts from blood samples without slaughter.To assess the prognostic capability of the 3.3\opttitle{Gene expression of hypomethylated \textit{ZPBP} in the promoterregion}ZPBP with hypomethylated promoter,RT-PCR was performed on uterine tissues of three Berkshire sows from eachgroup. As shown in Fig. 4, ZPBP gene expression was significantly upregulated inLLG, as expected. Promoter methylation generally impedes the binding oftranscription factors and in a second stage leads to chromatincondensation, with long-term repression of gene expression. Therefore, this result supports the hypothesisthat ZPBP overexpression in uterine tissue is due to promoter hypomethylation.Next, to investigate the gene expression of As the DNA methylation status of promoter regions could affect geneexpression through changes in chromatin structure or transcriptionefficiency , we compared thegenome-wide methylation patterns of high- and low-prolificacy pigs toidentify DMGs that might affect prolificacy traits such as litter size.3.4\opttitle{Verification of the relationship between methylation status of the\textit{ZPBP} gene and litter size traits}ZPBPgene and litter size traits in Berkshire sows  and that sows with hypomethylatedZPBP had high fecundity. Thus, the ZPBP gene can act as anepigenetic marker for early prediction of litter size in Berkshire pigs.We verified the relationship between methylation status of the ZPBP, which localizes to the acrosomal membraneand likely interacts with multiple acrosomal matrix proteins, was named forits function, binding to the oocyte ZP protein following the acrosomereaction . ZPBP mainly acts in acrosomecompaction and sperm morphogenesis during spermiogenesis. Most research onZPBP has focused on its location in the acrosome of the sperm and itsfunction in sperm–oocyte interactions during fertilization . However, Campbell et al. (2006) reportedthat murine ZPBP was a luminal epithelium-specific gene with20-fold or higher expression in the uterine luminal epithelium than in thestroma–glandular epithelium. Implantation is essential for theestablishment of normal pregnancy and is initiated by a physical interactionbetween the trophoblast and the apical surface of the luminal epithelium,followed rapidly by adhesion and then by penetration through the luminalepithelium to the underlying stroma, which responds by decidualization. In other words, the uterineluminal epithelium plays a critical role in implantation. ZPBP is a serineprotease (serine protease 38) with ZP-binding properties that were initiallyidentified in porcine epididymal sperm . Serineproteases are characterized by the presence of serine as a nucleophilicamino acid at the active site of the enzyme . Someserine proteases are detectable in the uterus and are involved in femalereproduction, especially in oocyte development, ovulation, implantation, anddecidualization . Therefore, webelieve that ZPBP might play a role in female reproduction, includingimplantation.Zona pellucida (ZP) is a filamentous matrix of well-structured andglycosylated glycoproteins surrounding the oocyte that acts as amorphological criterion for oocyte selection. This matrix is formed of threeproteins encoded by three different genes: ZP1, ZP2, and ZP3 . 4ZPBP gene in its promoter region in LLG; its expressionwas upregulated in uterine tissues. We also verified that the methylationstatus of the ZPBP gene was significantly negatively associated with litter sizetraits in the larger pig population. Our results demonstrate that this genecan be used as a biomarker for hyperprolific sows and will likely contributeto improving reproductive capacity.This study reports the DNA methylation patterns of porcine uterine tissues,which are associated with litter size. We identified DMRs and detectedhypomethylation of the


Delivery of imaging reagents and drugs to tumors is essential for cancer diagnosis and therapy. In addition to therapeutic and diagnostic functionalities, peptides have potential benefits such as biocompatibility, ease to synthesize, smaller size, by-passing off-target side effects, and achieving the beneficial effects with lower-administered dosages. A particular type of peptide known as cell penetrating peptides (CPP) have been predominantly studied during last twenty years as they are not only capable to translocate themselves across membranes but also allow carrier drugs to translocate across plasma membrane, by different mechanisms depending on the CPP. This is of great potential importance in drug delivery systems, as the ability to pass across membranes is crucial to many drug delivery systems. In spite of significant progress in design and application of CPP, more investigations are required to further improve their delivery to tumors, with reduced side-effect and enhanced therapeutic efficacy. In this review, we emphasis on current advancements in preclinical and clinical trials based on using CPP for more efficient delivery of anti-cancer drugs and imaging reagents to cancer tissues and individual cells associated with them. We discuss the evolution of the CPPs-based strategies for targeted delivery, their current status and strengths, along with summarizing the role of CPPs in targeted drug delivery. We also discuss some recently reported diagnostic applications of engineered protease-responsive substrates and activable imaging complexes. We highlight the recent clinical trial data by providing a road map for better design of the CPPs for future preclinical and clinical applications. Despite significant advances in anti-cancer therapies such as chemo-, radio-, and hormone therapies, surgical resection combined with chemo-radiotherapy remains the standard approach for fighting malignant cancers . HoweverPeptides have emerged as alternatives for mAbs, because of their reduced non-specific toxic effects, rapid renal clearance, small size, high specificity, and efficient delivery to tumor . They haCPPs are the most exploited cargos for efficient intracellular delivery of nucleic acids , proteinIn this review, we will emphasize the benefits and challenges of using theranostic (therapeutic and diagnostic) CPPs for tumor detection and treatment, by focusing more on clinical translation criteria. We will discuss the importance of CPP design and selection criteria based on specific microenvironment characteristics of their targeted tumors. Addressing these factors will enable more efficient drug delivery to tumor tissues, followed by internalization of these peptides into their individual cancer cells. Most common CPPs such as MPG peptides, Pep peptides, and TAT peptides will be addressed in detail. We will also review recent diagnostic and therapeutic approaches, designed based on interaction of peptides with different types of highly-expressed cancer-specific proteases . Finally, we will provide various examples for effective use of CPPs for diagnostic applications such as cancer imaging or development of diagnostic assays, considering the recent clinical trials.CPPs are short sequence of 4–30 peptides that assist cellular uptake of various cargoes ranging from nanoparticles size to large fragments of nucleotide. Among several criteria that have been proposed over different time, CPPs can be classified as cationic, amphipathic, and hydrophobic peptides on basis of their physical–chemical properties. The cationic peptides have positive net charges and they mainly have arginines and lysines strands while amphipathic CPPs contain hydrophibic polar amino acid such as lysine and arginine as well as hydrophobic nonpolar types of amino acids such as leucine, isoleucine, alanine and valine. On the other hand, hydrophobic CPPs mainly comprise hydrophobic nonpolar residues, due to which they play an important role in cellular internalization.Various physiochemical properties such as types, concentration, size of CPPs or CPP conjugated cargoes play an important role in influencing their cellular uptake. Presently CPP mediated cargo delivery is hampered by lack of their cell specificity and mode of their delivery is not well understood. CPP can use two different routes to enter the cell named as energy-independent direct penetration of the plasma membrane and energy dependent endocytosis mechanism, which are summarize in Figure Energy independent direct penetration happens at low temperatures which involve multiple entry routes that are initially based on the internalization of CPP in the cell through pore formation and membrane destabilization by three different type of models named as barrel-stave pore model, carpet-like model and toroidal pore model , 17. In Although energy independent direct penetration was initially first anticipated as the principal route to enter the cell but further evidences suggest that energy dependent endocytosis is the main route of entry for internalization for many CPPs . InternaDrosophila melanogaster antennapedia. Hence, the first CPP peptide developed was named as antennapedia peptide (Antp) Galacto-RGD, [18F]Fluciclatide, [18F]RGD-K5, 18F-FPPRGD2, [18F]Alfatide, [68Ga]NOTA-PRGD2, and 99mTc-3PRGD2 [18F]Galacto-RGD, was the first RGD based PET tracer that was tried in human showing specific binding and rapid renal clearance in cancer patients. Phase I clinical trial of [18F]Fluciclatide with tumors was visible on the PET images in breast cancer patients [18F]FPPRGD2 was first dimeric RGD peptide approved by FDA that was used for human trial demonstrating positive results in glioblastomamultiforme (GBM) FPme (GBM) .47–57 [Several preclinical assessments with CPP-conjugated imaging molecules and drugs have provided encouraging results for cancer imaging and therapy. CPPs have also been used in several pre-clinical trialfor the treatment of oncological diseases . Major e47–57 . Wild-tyDTS-108, water-soluble compound containing chemotherapeutic agent SN38 are shown to have anti-tumor affect in colon, lungs and breast cancer . Amphipa+ solid tumors which are resistance to conventional method of treatments and therapies. p28 also shows antitumor activity and minimal toxicity with no immunogenicity and was well tolerated. Importantly, this was also highly successful among those patients having recalcitrant disease who had earlier rejected prior treatments. Significant improvement in the survival of p53-positive advanced solid tumors was observed after treatment for thrice per week for total duration of four weeks. The outcomes of this clinical trial strongly indicate that CPPs can be used clinically in cancer therapy [Azurin-derived CPP was recently used in a clinical trial (phase I) to treat refractory tumors. p28 is a 28-amino-acid peptide, which once penetrated in nucleus of cancer cell bind with tumor suppressor protein P53 and inhibits p53 ubiquitination that can reduce CDK2 and cyclin A1 level and stop tumor growth in the G2/M cell cycle stage, by cell apoptosis due to inhibiting proteasomal degradation. These Phase I clinical trials focused on safety, pharmacokinetics, maximum tolerance dose and efficacy of p28 in patients with p53 therapy , 91. Add therapy .Some latest findings have been focused on dissecting the role of p28 in treating young patients with repeated or progressive high grade glioma. In these phase-I clinical trial studies, p28 was injected 3x/week for 4 weeks and repeated every 6 weeks in pediatric patients with refractory glioma, to assess the toxicity and best dose of p28. This phase established that p28 is quite satisfactory tolerated in children with repeated high-grade glioma at the adult suggested phase II dose . AnotherCPPs have also been used in clinical trial as a transporter for cancer specific drug carriers. For instance, BR2 (a 17-amino acid long cell penetrating peptide) was fused with scfv and targeted towards K-ras mutated HCT116 cells. Br2-scFv complex was shown to cause increase rate of apoptosis in K-ras mutated cells without causing toxicity to normal cells. These outcomes indicate that BR2 has a huge potential to be used as a cancer specific drug carrier in clinics . Indeed in-vitro and in-vivo studies, no CPP or CPP-drug conjugate has received FDA approval for cancer therapeutics so far. Therefore, additional studies are required to conclusively address obstacles between preclinical efforts and FDA approval criteria, considering significant potentials of CPP class of peptides in the clinics.CPPs can not only be exploited as therapeutic molecules that can cross cell membrane, but also used as promising tools for delivery of diagnostic as well as therapeutic cargos including genes, nanocarriers, anti-cancer drugs and imaging agents inside cells. Conjugation of these imaging agents such as fluorophores to CPP enables efficient detection of the diseased tissues for diagnosis purposes. Early diagnosis of tumors and improved targeted drug delivery is required to increase the effectiveness of cancer therapy and depends on identification and development of tumor-targeting agents. Use of CPPs forcancer drug delivery is considered as unique and most promising approaches for treating cancers and has profound potential to change the field of cancer theranostics in the near future. Although promising results have been reported for It is crucial to explore novel approaches for delivery of CPP and prodrugs not only to the desired tissue or organ, but also inside the targeted cells, to achieve efficient treatment. Advancements in the processing and design of antagonistic peptides with extraordinary tumor-penetrating properties will play a substantial role in forthcoming cancer treatments. CPP can also be used as vector for intracellular delivery of transcription factor . TherefoIn recent time, several other strategies including modulating the switch between dormant CPP and active CPP is also gaining attention, thus allowing inactivated CPP to reactivation of CPP on basis of change in pH, temperature and enzymes . Targeti


Giant cell arteritis (GCA) or temporal arteritis (TA) is a granulomatous inflammation of medium to large-sized arteries. It may have a diverse presentation. The most common presenting symptoms of GCA are fever, malaise, unilateral headache, jaw claudication, polymyalgia rheumatica (PMR) and ophthalmoplegia. Most severe sequelae of GCA could be blindness. We report a case of a 65-year-old Caucasian male who presented for the third time with recurrent episodes of diplopia. Neurologic exam showed bilateral cranial nerve (CN) VI palsy, slightly worse on the right than the left side. Other focal neurological deficits were absent. GCA was considered and biopsy of the temporal artery was performed which showed necrotizing pan-arteritis, consistent with GCA. The patient was empirically treated with intravenous (IV) methylprednisolone while awaiting the biopsy results which resulted in the resolution of the symptoms. As far as we know, this is the second case in the literature about the bilateral sixth CN involvement in the background of GCA. Abducens nerve palsy results in lateral rectus paresis and presents as binocular horizontal diplopia. It has multiple etiologies including trauma, vasculopathic lesion, inflammation, tumor, demyelinating disease, and sub-arachnoid hemorrhage . OphthalPrevalence of GCA is 15 to 30 per 100,000 patients with most affected patients being 50 years and older. Most cases are seen in Caucasians with north European descents or North Americans, however, GCA may also affect Asians and other races . We descA 65-year-old right-handed Caucasian male presented with recurrent episodes of diplopia. Prior two episodes were brief and self-resolving; however, the current episode started two months ago and progressively worsened. He started perceiving objects brighter in the right eye. He also reported malaise, shoulder pain bilaterally, and approximately 15 lbs weight loss in the last few months. Exam showed bilateral CN VI palsy (right more than left) without any other focal neurological finding. Myasthenia gravis was earlier suspected and was ruled out clinically by negative blood tests and electromyography. Endocrine workup including thyroid panel was unremarkable. Computerized tomography (CT) angiogram of the head and neck did not show any flow limiting vessel stenosis. Magnetic resonance imaging (MRI) of the brain did not show any diffusion restriction. Cerebrospinal fluid (CSF) analysis for infectious and inflammatory processes and serum autoimmune workup was negative. Occult malignancy was excluded by a whole body CT. Erythrocyte sedimentation rate (ESR) and c reactive protein (CRP) was remarkably elevated. Due to concerns of generalized malaise, weight loss, and elevated inflammatory markers in an elderly individual; TA was considered. A patient was empirically started on 1 g IV methylprednisolone (solumedrol) for three days followed by a gradual taper while waiting for the temporal artery biopsy results. Temporal artery biopsy showed necrotizing pan-arteritis consistent with GCA  and visual symptoms , 3-9. InOcular CN involvement upon presentation, without the absence of other typical symptoms, suggesting GCA may lead to a complex process of differential diagnosis. Differential diagnosis in our case included myasthenia gravis, thyroid dysfunction, mass lesion inside the brain compressing the sixth CN, occult malignancy, and autoimmune inflammatory process. The American College of Rheumatology (ACR) had established the criteria in 1990 for the diagnosis of GCA which is shown in Table We did not wait for the biopsy results to begin the treatment as GCA is an ophthalmologic emergency with poor prognosis and irreversible visual loss being the most dreaded complication. One study that aimed at monitoring the visual symptoms improvement in GCA patients with corticosteroid therapy showed that 96% of GCA patients with visual loss did not improve even after corticosteroid therapy . In our GCA carries a broad range of presentation. Cases of GCA affecting different CNs have been reported before and include unilateral abducens nerve , bilaterGCA is a necrotizing and granulomatous inflammation of the medium and large size blood vessels. Untreated, it can result in permanent blindness. Though headache is a common association, it sometimes may be absent. Our case emphasizes the fact that GCA can present in an atypical way and that physicians should have a high level of suspicion as a prompt institution of treatment is necessary to prevent potential permanent blindness.


Trigeminal neuropathic pain is a chronic pain condition caused by damage or inflammation of the trigeminal nerve or its branches, with both peripheral and central nervous system dysfunction contributing to the disorder. Trigeminal pain conditions present with diagnostic and therapeutic challenges to healthcare providers and often require multiple therapeutic approaches for pain reduction. This review will provide the overview of pathophysiology in peripheral and central nociceptive circuits that are involved in neuropathic pain conditions involving the trigeminal nerve and the current therapeutics that are used to treat these disorders. Recent advances in treatment of trigeminal pain, including novel therapeutics that target ion channels and receptors, gene therapy and monoclonal antibodies that have shown great promise in preclinical studies and clinical trials will also be described. Trigeminal neuropathic pain, or trigeminal neuralgia, is the pain caused by damage or injury of the somatosensory nervous system originating from the regions of the face as well as within and around the mouth. The presentation of this type of pain may range from dental pain, which is the most common inflammatory pain in the region, to temporomandibular joint disorders (TMDs), trigeminal neuralgia, myofascial pain, headache, neuritis, and idiopathic pain conditions. Neuropathic trigeminal pain can be either episodic or continuous. These conditions are usually a result of injury or disease of one or more nerve roots of the trigeminal ganglion. Possible causes include nerve trauma , compresPain sensation in the orofacial region is carried by the trigeminal pathway to the brain. Noxious stimuli such as pinching, prick, hot or cold may activate mechanical and/or thermal nociceptors which are the free nerve endings of the trigeminal sensory afferents. These sensory nerve fibers are myelinated Aδ-fibers and non-myelinated C-fibers whose cell bodies sit in the trigeminal ganglion. Signals are transmitted centrally through these afferent fibers to the trigeminal spinal caudalis (Vc) nucleus of the brain stem where they synapse with second-order neurons that project to the somatosensory and limbic cortices via the thalamus . ThroughIn some cases, injury or inflammation of orofacial tissues that are innervated by the trigeminal nerve can alter the activity of trigeminal afferent neurons resulting in hyperexcitability, ectopic firing, increased sensitivity to noxious stimuli  and innocuous stimuli . Peripheral sensitization is characterized by a decreased threshold for activation of transducer channels and an increased firing rate of the sensory neurons leading to hyperalgesia . SensitiFollowing peripheral nerve-injury, inflammatory mediators including prostaglandin E2 (PGE2), cytokines TNFα, IL-1β, and IL-6 and neurotrophic factors such as brain-derived neurotrophic factor (BDNF) are secreted by activated immune cells including macrophages, neutrophils and mast cells at the site of tissue injury or inflammation  Figure . These iNeuropeptides are a diverse group of molecules that play an important role in pain transmission and modulation. Studies have shown that expression and release of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) increase in the trigeminal nerve and trigeminal ganglion (TG) in response to peripheral nerve-injury  Figure . In receA) and sustained currents (IK) of voltage-gated potassium (K+) channels [h) [2+) channel activation at central terminals [Transmission of sensory information from the periphery to the central nervous system requires coordinated activity of many ion channels and receptors which allow transduction of signals, action potential generation and neurotransmitter release at the synapse. Ion channel dysfunction at all levels of transmission can result in pathological signaling changes, which may contribute to neuropathic pain pathology. Expression and activity of a number of ion channels within pain pathways have been shown to change during the development of trigeminal neuropathic pain. Some of these ion channels contribute to hyperexcitability of TG neurons, including increased activity of Nav1.8 sodium channels due to increased expression ,35, a rechannels , changesnels [h)  and an ierminals ,39,40. CTrigeminal afferents express a number of different types of nociceptors  and expression of many of these receptors has been shown to increase in trigeminal neuropathic pain models ,42 Figu. TransieOverexpression of sodium channels in TG neurons is associated with an increase in neuronal excitability . Voltageh currents also play an important role in the hyperexcitability observed in sensitized peripheral trigeminal afferents. In nerve-injury model of neuropathic pain, Ih current density as well as the rate of activation were increased in TG neurons [h attenuated mechanical allodynia and reduced ectopic discharges following nerve-injury [Damage to trigeminal afferents through chronic constriction in animal models of nerve-injury leads to down-regulation of large-conductance, calcium-activated potassium channels (BK channels), resulting in neuronal hyperexcitability . BK chan neurons . In linee-injury .2+ channels [When a neuron is activated, the transmitted signals induce an influx of calcium ions at the terminal and triggers release of neurotransmitters. In pathological pain conditions, the excitatory glutamatergic neurons synapses are strengthened increasing the excitability of nociceptive neurons and facilitating the nociceptive transmission to higher brain regions ,59. Nocichannels ,59. Theschannels ,61,62. Bchannels ,64. Simichannels . A recenchannels .Interactions between neurons and glia in the trigeminal ganglion are thought to contribute to the development of neuropathic pain following nerve-injury or inflammation . Satellih currents reduced ectopic discharges from Aδ fibers and completely blocked ectopic discharges from Aβ fibers and alleviated allodynia in rat model of nerve-injury induced neuropathic pain [Another peripheral mechanism of trigeminal neuropathic pain that has been described is the cross-excitation from injured nerve fiber to an adjacent intact nerve fiber, which induces allodynia and hyperalgesia in rat models of neuropathic pain . Similarhic pain . This suProlonged peripheral nociceptive sensitization may lead to central pathological changes in the trigeminal ascending pain pathways by inducing neuroplastic changes and hyperexcitability of central neurons, and through changes to the pain modulation control by higher brain centers.Inflammatory mediators in the central nervous system (CNS) play a critical role in sensitization of central nociceptive neurons. Glial cells, namely microglia and astrocytes, proliferate and are activated in response to peripheral nerve injury ,80 Figu. ActivatOne of the key functions of astrocytes is to clear excess synaptic glutamate and enzymatically convert it to glutamine with glutamine synthetase. Activated astrocytes release glutamine which is taken up by the primary afferent terminals to replenish the supply of the excitatory neurotransmitter glutamate. More glutamate can then be released at the synaptic cleft allowing increased excitability of the neuron . Chiang Anatomical and functional reorganization of the central pain pathways of the spinal cord have been established in neuropathic pain models ,85,86. HA receptor or specific subunits of this receptor have shown potential as an analgesic strategy. Two recent studies reported that positive modulation of α6 GABAA receptors subunit potently inhibited trigeminal neuropathic pain. The first report by Vasovic et al. (2019), showed that deuterated pyrazoloquinolinone compound DK-I-56-1 inhibited the development and reduced the established trigeminal neuropathic pain in a rat model of chronic constriction injury of the infraorbital nerve. Moreover, the inhibition was long-lasting and the drug at this therapeutic dose of 10 mg/kg administered intraperitoneally did not affect mouse behavior [A receptors subunit, ameliorated capsaicin-induced vascular effects in an animal model of migraine, indicating that α6 GABAARs have potential as anti-migraine agents [A receptor either by its selective antagonist bicuculline or agonist muscimol did not show straightforward effect. In a naive rat, intracisternal administration of bicuculline produced mechanical allodynia while in an inflammatory pain models prepared by subcutaneous injection of interleukin 1 beta (IL-1β), the intracisternal administration of bicuculline produced anti-allodynic effect [A receptor antagonist, which previously has been shown to produce mechanical allodynia when given intrathecally in naïve animal [A receptor agonist muscimol depressed mechanically evoked response of Vc nociceptive neurons after intrathecal application in sham rats but not in IANX rats. The negative effect in the IANX rats was due to overall reduction in the number of GABAergic neurons as well as reduced expression of KCC2 in the Vc nucleus which may functionally change the GABAergic signaling from inhibitory to excitatory following inferior alveolar nerve transection [Targeting the GABAbehavior . In a see agents . Even thc effect . It is ee animal . Howevere animal . The ante animal . It is ne animal . Similarnsection ,97. B receptor. Subcutaneous injection of the GABAB receptor agonist baclofen alleviates the mechanical allodynia-like behavior in a nerve injury rat model of neuropathic pain [+ channels [Another approach to potentiate inhibition is to activate the GABAhic pain . It has channels .Synaptically released GABA is constantly removed from the synaptic cleft by GABA transporters, namely GAT1 and GAT3, which are present on astrocytes, thus regulating the concentration of inhibitory neurotransmitter at the synaptic junction. In one study it was shown that the expression of GAT-1 and GAT-3 transporters are increased in the spinal trigeminal nucleus in rats with inflammatory pain induced by carrageenan injection. This reduced inhibition of trigeminothalamic neurons in the spinal trigeminal nucleus may contribute to hyperalgesia following carrageenan injections in this rat model . These fA receptor-mediated signaling [Neuroplastic changes occur not only in the sensory ascending pain pathway but also in the descending modulatory pathways ,107,108 ignaling . Modulatignaling ,112. Druignaling ,114. In ignaling ,115,116.Trigeminal neuropathic pain is a debilitating chronic pain condition occurring after peripheral trigeminal nerve injury or inflammation, with symptoms that include hyperalgesia and mechanical allodynia. There are many potential mechanisms underlying this condition—ranging from cross-excitation of intact nerve by injured nerve, neuro-glial interactions, wiring changes in the CNS, loss of inhibitory control, change in ion channel function, and sensitization by inflammatory mediators. Thus, these symptoms arise because of the defects in pain processing pathway rather than noxious stimuli and deficits in the peripheral and central nervous system function contribute to the development and maintenance of these conditions. Much of our understanding of neuropathic pain processing comes from experiments in spinal cord dorsal horn, but comparatively fewer studies have been conducted in trigeminal pathway. There is still a huge knowledge gap in the central neuroplastic changes that occur after trigeminal nerve injury, which remains a major challenge to tackle in the coming years. Defects in several types of ion channels, cell types including both neurons and glia as well as neuronal circuits have been shown to be involved in processing the nociceptive information and are therefore potential therapeutic targets for the treatment of trigeminal neuropathic/inflammatory pain .


Gap junctions comprise arrays of intercellular channels formed by connexin proteins and provide for the direct communication between adjacent cells. This type of intercellular communication permits the coordination of cellular activities and plays key roles in the control of cell growth and differentiation and in the maintenance of tissue homoeostasis. After more than 50 years, deciphering the links among connexins, gap junctions and cancer, researchers are now beginning to translate this knowledge to the clinic. The emergence of new strategies for connexin targeting, combined with an improved understanding of the molecular bases underlying the dysregulation of connexins during cancer development, offers novel opportunities for clinical applications. However, different connexin isoforms have diverse channel-dependent and -independent functions that are tissue and stage specific. This can elicit both pro- and anti-tumorigenic effects that engender significant challenges in the path towards personalised medicine. Here, we review the current understanding of the role of connexins and gap junctions in cancer, with particular focus on the recent progress made in determining their prognostic and therapeutic potential. Connexins are integral membrane proteins that form channels between adjacent cells and thereby permit the bidirectional cytosolic exchange of ions, metabolites and secondary messengers  < ~ 1200 Da) Fig.  [1]. The200 Da FiThe human connexin protein family contains 21 members, of which the most widely studied is connexin 43 (Cx43) , 6. ThisThe idea that GJIC regulates tumour growth was proposed by Loewenstein and Kanno more than 50 years ago in a seminal ex vivo study, in which they demonstrated the loss of electrical coupling occurring in rat liver tumours . Since t2+, IP3 and cAMP [In accordance with the notion that connexins might act as tumour suppressors, the ectopic expression of connexins in cancer cells often partly restores growth control (e.g. refs. –25) and and cAMP –34. Moreand cAMP .There is increasing evidence that connexins can suppress the growth of cancer cells through channel-independent mechanisms , 36–39  biology. Malignant glioma stem cells (GSCs) express very low levels of Cx43, and GSC stemness is reduced by its transfection with Cx43 or treatment with peptides containing sequences of Cx43 that interact with c-Src (Cx43 mimetic peptides) , 55. A rOverall, connexins can be both anti- and pro-tumorigenic, acting via processes that include the regulation of CSCs, and this depends on the connexin isoform and tissue type. Understanding this complexity is the key to the development of new and efficient therapies.https://www.proteinatlas.org/pathology) includes an open-access database (retrieved from The Cancer Genome Atlas (TCGA)) containing correlation analyses based on mRNA expression levels with respect to the clinical outcome for 17 major cancer types and almost 8000 cancer patients [P < 0.001) in any of these cancer types are summarised in Table GJA1 (encoding Cx43) is featured in the list of 20 genes most significantly associated with an unfavourable prognosis in stomach cancer. Indeed, for most connexins and cancer types, high levels of connexin mRNA expression are associated with a poor prognosis. It is also notable that the mRNA expression of a given connexin-encoding gene can be both favourable and unfavourable, depending on the type of tumour  suggests that high levels of Cx43 mRNA are associated with good and poor prognoses in oestrogen receptor-positive and -negative breast cancer subtypes, respectively [A number of studies suggest that connexins can be independent tumour markers predicting both better and worse prognoses. Further insights into this seeming contradiction have been provided by recent analytical tools for public databases. The Pathology Atlas within the Human Protein Atlas database (patients . Those mectively .Table 1SGJB4) mRNA expression was shown to be increased in lung tumours, with the levels in blood buffy coat samples serving as a biomarker for diagnosis and poor prognosis [Several specific studies have also reported a correlation between connexin mRNA levels and survival or phenotypic features. For instance, high levels of Cx43 mRNA in glioma tumours have been correlated with poor survival . Expressrognosis . MechaniP < 0.001) [As shown in Table < 0.001) . Microar< 0.001) . At the < 0.001) . In agre< 0.001) .https://www.intogen.org or COSMIC http://cancer.sanger.ac.uk/cosmic) suggest relatively low mutation rates in connexin-encoding genes and, based on genetic data, none of the 21 connexin genes are classified as tumour suppressors or oncogenes. Nevertheless, individual genetic studies merit follow-up. For example, the C1019T polymorphism in GJA4 (encoding Cx37) is associated with Helicobacter pylori infection in patients with gastric cancer [GJB6 (encoding Cx30) was deleted in 25.8% of the 751 analysed tumours and was mutated in 15.8% of 158 tumours [GJB6 deletions (517 tumours from TCGA and 67 tumours from REMBRANDT) or GJB6 mRNA expression (in patients from TCGA). However, at the protein level, high Cx30 expression adversely influenced the survival , which can significantly influence the prediction of the clinical outcome.The complex and multifunctional role of connexins in cancer provides a wide spectrum of therapeutic opportunities and challenges. Increasing numbers of drugs, peptides and RNAi approaches are available to inhibit or enhance GJIC, hemichannel activity or connexin protein-signalling activity. Below, we highlight a selection of in vivo studies demonstrating that the cancer phenotype is altered as a direct consequence of such an experimental targeting of connexins.The restoration of GJIC and/or connexin expression has long been a potential therapeutic strategy. GJIC can be augmented either by enhancing the permeability of existing gap junctions or by increasing the expression of connexins and thereby elevating the number of open gap junction channels . Many fuhttps://clinicaltrials.gov/ct2/show/NCT02429570?id=NCT02429570&rank=1&load=cart). Recently, a novel role of Cx43-based gap junctions was also identified in breast cancer bone metastasis [However, because connexins under specific conditions can facilitate malignant features, in particular, metastasis or growth of metastases , there atastasis . Gap juntastasis .Thus, many of the studies reporting the inhibitory effects on tumour growth upon inhibition of GJIC mechanistically relate this effect to a loss of heterologous GJIC between tumour cells and cells in their microenvironment  can mimic the anti-oncogenic effect of Cx43 in glioma cells such as primary GSCs [Connexins interact with a wide variety of proteins that, independent of channel activity, can affect cancer cell phenotype, including cell growth, migration and differentiation  can also transmit through GJIC, inducing a bystander effect [The bystander effect is important in a wide range of circumstances other than suicide gene therapy. Notably, some chemotherapeutic drugs or radiation-induced metabolites such as DNA damage-induced sensing/signalling molecules or cytoplasmic irradiation responders  can inhibit the growth of tumour cells was shown in the mid 1980s by Loewenstein and colleagues . This phSalmonella bacteria induces Cx43 expression, which enhances the antigen presentation into infiltrating dendritic cells, activating an antitumour response [Tumour cells deliver antigenic peptides through gap junctions to dendritic cells, and this cross-presentation is associated with enhanced immune-mediated tumour elimination. Presentation of antigenic peptides between melanoma cells and dendritic cells increases the melanoma-specific T-cell response . The infresponse . Cx43-meresponse , 145. GJresponse . These sresponse ) are of response . Connexiresponse .Angiogenesis, the formation of new capillaries from pre-existing blood vessels, is required for cancer progression . Overexp40Gap27 that binds to the extracellular loop of Cx40 and blocks channel activity [Endothelial connexins also play important roles in tumour angiogenesis. The knockdown of endothelial Cx37, Cx40 or Cx43 or the pharmacologic inhibition of GJIC diminishes the angiogenic sprouting of endothelial cells in in vitro studies . A recenactivity .Because endothelial and myoendothelial gap junctions are fundamentally important for a coordinated vessel response over longer distances , 153, a There is also evidence of direct communication between tumour cells and endothelial cells Fig. . Recent As mentioned, Cx26 may facilitate extravasation into the endothelium during metastasis , and Cx4Connexins can also elicit pro-tumourigenic effects in brain tumours. Cx43 expression and heterologous GJIC between malignant glioma cells and reactive astrocytes enhance cell invasion into the brain parenchyma –163, whiv-src causes Cx43 tyrosine phosphorylation and reduced Cx43 levels [An estimated 10–12% of human cancers worldwide are caused by viruses and 4.5% are caused by the so-called “high-risk” human papillomaviruses (HPVs) . Rous sa3 levels . Similar3 levels . Human c3 levels . These dIn 1969, McNutt and Weinstein recognised that gap junction plaques are lost in cervical cancers . SubsequUpon infection, viral DNA is detected by cyclic GMP-AMP synthase, which synthesises the STING activator cGAMP to induce an antiviral state through intercellular transmission. Connexins are responsible for the bystander immunity to viruses , and so Tunnelling nanotubes (TNTs) are thin actin-based membrane bridges that connect cells over distances of up to several cell diameters . These sExtracellular vesicles (EVs), which constitute microvesicles, apoptotic bodies and exosomes, are membrane-based structures that can carry and deliver bioactive molecules, including proteins and nucleic acids, from one cell to another. This form of cell-to-cell communication can occur over very long distances and efficiently cross the blood–brain barrier. EVs released by tumour cells and cancer-associated fibroblasts affect cancer progression by transferring molecules influencing tumour initiation, angiogenesis, metastasis and drug resistance . Functiohttps://clinicaltrials.gov/ct2/show/NCT02429570?id=NCT02429570&rank=1&load=cart). Nevertheless, there are significant challenges that remain to be addressed. The fact that connexins can be both anti- and pro-tumorigenic is of particular concern. For example, would enhancing GJIC or connexin expression in a primary tumour run a risk of more efficient metastatic spread or growth? Does inhibition of GJIC in patients with metastasis increase the risk of further tumour dissemination or re-activated tumour growth in sites harbouring dormant tumour cells? Currently, this clinical problem can only be addressed by hypothetical risk assessment and thus further research, including an extensive use of in vivo models and a careful follow-up of ongoing clinical trials, is required.Substantial knowledge on how gap junctions contribute to cancer has accumulated since the seminal work by Loewenstein and Kanno demonstrated a loss of electrical coupling in liver cancer more than 50 years ago . ConnexiCurrently, only a few of the 21 connexin isoforms have been characterised in terms of their role in cancer. Additional studies are necessary to elucidate the GJIC-dependent and -independent mechanisms by which the various connexins positively or negatively affect cell growth, differentiation, invasion and other important cancer-associated features. In this context, more specific tools will be required to target the different functions of connexins. Further efforts need to be devoted to the identification of more specific connexin inhibitors. The use of specific peptides and peptidomimetics have shown a great promise towards this. However, their side effects must be carefully addressed  due to the critical functions of several connexins in various organs. Another important challenge will be to dissect the molecular basis of the regulation of the various connexin isoforms at the transcriptional, translational and post-translational levels and to define how the dysregulation of these processes contributes to aberrant levels or subcellular localisation of connexins during various stages of cancer progression. The identification of solutions to these research challenges will set the stage for new diagnostic and therapeutic advances.


During chronic kidney disease, the progressive deterioration of renal function induces several biological/clinical dysfunctions, including enhancement of synthesis of inflammation/oxidative stress mediators. Impaired renal function is an independent cardiovascular risk factor; indeed, cardiovascular complications dominate the landscape of both chronic kidney disease and end-stage renal disease. The aim of this study is to explore the correlation between the global oxidative balance in hemodialysis patients and both inflammatory markers and cardiovascular events. Using photometric tests, this study explored plasmatic oxidative balance in 97 hemodialysis patients compared to a healthy population. In the hemodialysis patients, we showed that oxidative stress values were significantly lower than in controls while effectiveness in the antioxidant barrier was significantly increased in the hemodialysis group. Furthermore, we highlighted a strong correlation between oxidative index and blood levels of C-reactive protein. When patients were divided into two groups based on previous cardiovascular events, we found that subjects with previous cardiovascular events had higher values of both oxidative stress and antioxidant barrier than patients without cardiovascular events. Our results indicated that in hemodialysis patients, the clinical and prognostic significance of oxidative status is very different from general population. As cardiovascular complications represent a strong negative factor for survival of hemodialysis patients, the research of new cardiovascular risk biomarkers in these patients takes on particular importance in order to translate them into clinical practice/primary care. Chronic kidney disease (CKD) is a major public health problem worldwide, and its main consequences include loss of renal function leading to end-stage renal disease (ESRD), increased risk of cardiovascular disease (CVD), significant increase in morbidity and mortality, and a decrease in health-related quality of life –3. A recDuring CKD, the progressive deterioration of kidney function has important systemic effects due to its central role in body homeostasis and induces several biological and clinical dysfunctions including alteration in cellular energetic metabolism, change in nitrogen input/output, protein malnutrition, resistance to insulin, and considerable enhancement of synthesis of inflammation/oxidative stress mediators. Inflammatory markers such as C-reactive protein and cytokines increase with renal function deterioration suggesting that CKD is a low-grade inflammatory process  comparabImpaired renal function is an independent cardiovascular risk factor; indeed, cardiovascular complications dominate the landscape of both CKD and ESRD . The comThe aim of this study is to explore the global oxidative balance in ESRD hemodialysis (HE) patients compared to a healthy population. Since inflammatory stress and cardiovascular complications represent two very frequent conditions in hemodialysis subjects, the correlation between the oxidative parameters and both inflammatory markers and cardiovascular events was also investigated.The study involved 97 patients older than 18 years of both sexes with ESRD on regular hemodialysis therapy for at least 6 months  recruited from the Department of Nephrology, Dialysis and Transplantation, Kidney and Transplantation Research Center, Annunziata Hospital of Cosenza , between August 2017 and December 2017. The hemodialysis prescription consisted of 4 hourly, thrice weekly sessions, using polysulfone membrane and bicarbonate-buffered dialysate. The blood flow rate was 300 mL/min and low molecular weight heparin was used as anticoagulant. Hemodialysis adequacy was determined by using Kt/V formula. 2), while blood pressure was measured in the sitting position using an automatic sphygmomanometer (Microlife BP A2). The biochemical measurements included glucose, total cholesterol, triglyceride (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), HbA1c, uric acid (UA), and creatinine levels. Blood tests also included the evaluation of prooxidant, and antioxidant status. Exclusion criteria included a family history of ESRD or an estimated glomerular filtration rate (eGFR) lower than 60 mL/min/1.73 m2, calculated by CKD-EPI equation [As controls for patient group, a recruitment campaign focused on the staff of the University of Calabria within the frame of the prevention and protection program adopted by the same university. In particular, 95 healthy volunteers age- and sex- matched with the ESRD group  were recruited between August 2017 and December 2017. They underwent through a structured interview during which sociodemographic, anthropometric, and clinic information was collected. The lifestyle was evaluated using self-administered questionnaires, which included items on smoking, exercise habits, and drinking habits. Anthropometric measurements included measurements of weight and height. Body mass index (BMI) was calculated as weight (kg)/height (mequation .All subjects (patients and controls) were studied in the morning and in a fasting state. Blood samples were taken from the antecubital vein and immediately centrifuged (2500 g for 15 min at 4°C), and the plasma obtained was stored at 4°C until measurements (maximum 6 hours of venous blood collection). Standard blood tests were performed at the clinical laboratory of Annunziata Hospital, Cosenza; prooxidant and antioxidant status were determined at the Analysis and Research on Oxidative Stress Laboratory (LARSO) of the University of Calabria.μmol/L of the reduced ferric ions.Oxidative stress and biological antioxidant potential determination were performed by using photometric measurement kits and a free radical analyzer system provided with spectrophotometric device reader . The d-ROM test helps to determine the oxidant ability of a plasma sample measuring the presence of reactive oxygen metabolite derivatives, in particular, hydroperoxides (oxidative index). Results are expressed in Carratelli units . The BAP test provides an overall measure of the biological antioxidant potential measuring the blood concentration of antioxidants  capable of reducing the iron from ferric to the ferrous form (antioxidant barrier). Results are expressed in t-test. The correlation between continuous variables was assessed using Pearson's coefficient. Data have been analyzed using R statistical language (http://www.r-project.org/).Differences between groups were examined using the independent sample Our study population consists of 97 hemodialysis patients and 95 healthy volunteers as control group. P value = 0.002). Moreover, ESRD patients also showed a greater effectiveness in the antioxidant barrier than CTR subjects . The relationship between oxidative index and antioxidant barrier values showed a borderline correlation  had higher values of both oxidative stress and antioxidant barrier than patients without cardiovascular events  were not related to both oxidative index and antioxidant barrier efficacy. Concerning smoking habits, it has not been possible to analyze any correlations given the small number of smoking subjects. Interestingly, when HD patients were stratified according to previous CVD events, we detected that subjects with previous acute myocardial infarction (CVD+, Our research focused on the evaluation of the overall redox state on a significant hemodialysis population. Our results clearly showed that the hemodialysis patients presented oxidative stress values significantly lower and antioxidant barrier effectiveness significantly higher compared to healthy controls. Despite the good plasmatic redox status found in all hemodialysis patients, we highlight a strong correlation between oxidative index and C-reactive protein blood levels. In addition, we reported that, in our cohort of hemodialysis patients, subjects with previous acute myocardial infarction had higher values of both oxidative stress and antioxidant barrier respect to patients without cardiovascular events. It is noteworthy that these results have been realized using noninvasive method to detect total plasma redox balance, which is of particular importance when analyzing the frail hemodialysis patients. Indeed, to detect plasmatic redox status, we utilized two simple methods widely used in recent years, the d-ROM and the BAP tests –20. The Several authors reported a profound imbalance between oxidants and antioxidants in CKD . The balIn our cohort of hemodialysis patients, we detected oxidative stress values significantly lower compared to healthy controls. As index of oxidative stress, we did not analyze the oxidative damage of single macromolecules but the plasmatic oxidant ability by measuring the presence of reactive oxygen metabolite derivatives, in particular, hydroperoxides. Actually, the most direct approach for lipid peroxidation evaluation is the quantification of the primary products, hydroperoxides, rather than the secondary or end products derived from hydroperoxides such as malondialdehyde. Plasmatic hydroperoxides are a sensitive and specific index that the presence of oxidative stress in vivo providing a global assessment of the imbalance induced by the alteration of the individual parameters . Indeed,Concerning the antioxidant systems, literature data are really complex to discern. Most of the studies in CKD patients have analyzed expression/activity of antioxidant enzymes with conflicting results , 27. In CKD is a low-grade, chronic, systemic inflammation with considerable enhancement of synthesis of inflammation mediators such as C-reactive protein and cytokines . LiteratIn recent decades, numerous studies and meta-analyses have highlighted that impaired renal function is an independent cardiovascular risk factor; indeed, cardiovascular complications dominate the landscape of both CKD and ESRD . TraditiOur results suggest that in HD patients, the clinical and prognostic significance of oxidative status associated with cardiovascular risk factors is very different from the general population. Although a direct causality cannot be inferred from such kind of correlative investigations, our data provide an important contribution to understanding the cross-talk between oxidative imbalance and cardiovascular risk in CKD also representing a point of departure to address further investigation. As cardiovascular complications represent a strong negative factor for hemodialysis subject survival, the preservation of an optimal global redox balance through effective methods may be considered as a potential target for therapies aimed at preventing cardiovascular complications during CKD progression.


Understanding the etiology of human papillomavirus (HPV)-driven oropharyngeal squamous cell carcinoma (OPSCC) is becoming a matter of increasing necessity and urgency as the incidence of HPV(+) OPSCC continues to rise and now exceeds that of HPV-associated cervical cancers . HPV(+) Treatment for advanced head and neck cancer includes radiation and platinum-based chemotherapy, which are associated with dose-related adverse side effects including acute toxicities like mucositis and loss of taste, as well as long-term problems of dysphagia, osteoradionecrosis, xerostomia, muscle fibrosis, and lymphedema. These side effects can lead to downstream infections, difficulty eating, and increased hospitalizations, which decrease a patient's quality of life. Limiting these side effects is especially important in HPV(+) patients, who present at a younger age. Despite their improved cure rates, 20-30% of HPV(+) tumors will recur and upon recurrence, effective treatment options are limited.Given toxicities associated with aggressive therapy, recent studies have focused to improve treatment of HPV(+) OPSCC. The most common strategies include limiting platinum-based chemotherapies and decreasing radiation fields or dosage. At best, these strategies can minimize therapeutic side effects, but at the risk of decreased cure rates. Our group has taken a different approach with two major points of focus. The first has been to identify vulnerabilities of HPV-associated OPSCC that may lead to more targeted toward HPV(+) tumors treatments. The second has been to identify reliable biomarkers that predict which patients can benefit from and safely undergo treatment de-escalation. We hope that these research directions will not only decrease side effects while maintaining efficacy, but also provide treatment for those with recurrent or resistant HPV(+) OPSCC.E6 and E7, which have been linked to hypermethylation and invasiveness in cervical cancer [MMPs) 1 and 10 that degrade the extracellular matrix to allow cancer cells to spread and have been shown to be critical for carcinomas invasion. Consistent with downregulation of MMPs, we found that xenografted HPV(+) OPSCC had reduced metastatic potential after 5-azaC treatment [Recently, we became interested in 5-azacytidine (5-azaC), a DNA-demethylating agent, following the discovery that HPV(+) tumors have a hypermethylated genome . PreviouThe ability of 5-azaC to activate interferon (IFN) response and induce a local production of T cells attracting cytokines in some HPV(+) head and neck cancer cells was another important finding of our study, suggesting a therapeutic potential of a combination of 5-azaC with anti PDL1/PD1 immunotherapy. Together, our data proposes the potential of 5-azaC in the treatment of HPV(+) OPSCC and prevention of metastasis  OPSCC patients with improved prognosis who may be candidates for de-escalated therapy. Our analysis of The Cancer Genome Atlas (TCGA) head and neck cancer database revealed nearly mutually exclusive inactivating mutations in TRAF3 and CYLD genes in 28% of HPV(+) specimens [Development of less toxic treatment options and identification of a patient population that may be best for testing these therapies are key to improving patient morbidity and mortality in HPV(+) OPSCC. As more clinicians and researchers recognize this distinct subgroup of tumors and more is discovered about HPV-driven carcinogenesis, the better equipped we will be to prevent and treat HPV(+) head and neck cancer patients.