The HPV DNA is more frequently found unintegrated in the cancers of the oropharynx compared to those that arise in the cervix, and may include novel HPV-human DNA hybrids episomes. Here, we review current understanding of HPV biology in the oropharynx and discuss the cell-based systems being used to model the HPV life cycle in tonsil keratinocytes and how they can be used to inform on HPV-driven neoplastic progression in the oropharynx.Most human papillomavirus (HPV) positive head and neck cancers arise in the tonsil crypts; deep invaginations at the tonsil surface that are lined with reticulated epithelium infiltrated by immune cells. As in cervical HPV infections, HPV16 is the most prevalent high-risk type in the oropharyngeal cancers, and a genital-oral route of infection is most likely. However, the natural history of HPV-driven oropharyngeal pathogenesis is an enigma, although there is evidence that it is different to that of cervical disease. It is not known if the virus establishes a productive or abortive infection in keratinocytes of the tonsil crypt, or if viral infections progress to cancer This article is part of the theme issue ‘Silent cancer agents: multi-disciplinary modelling of human DNA oncoviruses’. IntriThere is much interest in patients with HPV-OPSCC because they have overall increased survival compared to those with HPV-negative disease, and thus it has been proposed that this group of younger patients could benefit from deintensification of therapy to reduce the longer term toxicities in anticipation of long-term survival ,16. HoweThe high-risk (HR) HPV types found in cervical cancers are also present in OPSCC and HPV16 is the most prevalent HR type in both types of tumour . HoweverChromosomal integration of HPV genomes is a common feature of cervical cancers and at this body site is linked to a poorer clinical outcome. Integration of the viral DNA most often disrupts the E1 and E2 genes and deregulates virus gene transcription, leading to maintained and often high expression of the HPV oncoproteins E6 and E7 in the tumours. However, in HPV-OPSCC the primary form of HPV DNA is non-integrated episomes and viral DNA integrants are less frequent than some earlier studies indicated; most likely owing to a combination of the use of different methodologies to detect episomes and/or integrants, and ambiguities in data interpretation –29. As iPIK3CA, EP300, PTEN, FBXW7 and TGFBR2. However, several other genes found to be commonly mutated in HPV-OPSCC, including ZNF750, FGFR3, CASZ1, CYLD and RIPK4 have not been identified in cervical cancer [Recent analysis of the mutational landscape of HPV-positive (predominantly OPSCC) in comparison to HPV-negative  squamous cell carcinomas provides evidence that the mutation burden in HPV-positive tumours is indicative of apoliprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) cytidine deaminase editing . Interesl cancer , indicat(a)The mucosa of the tongue base and tonsils  is invaginated and forms crypts lined with reticulated epithelium . It is wAnalysis of non-malignant tonsil tissue for indications of HPV infection suggest that HPV infection at this site is rare, even when the reticulated epithelia was sampled . This raMucosal squamous epithelia at different anatomical sites that are susceptible to HPV infection are at different risks of cancer development. The uterine cervix has a nearly 100% association with HR HPV whereas at other sites including anus, vagina/vulva and oropharynx, only a subset of the tumours are HPV-associated. This differential risk between mucosa sites could be explained by the nature of the target cell for HPV infection, which influences disease outcome. In the cervix, a small and discrete population of non-stratified cuboidal cells of embryonic origin in the metaplastic transformation zone at the squamocolumnar junction (SCJ) are highly susceptible to HPV infection and from which the majority of neoplasms arise . The SCJCervical cancer pathogenesis is characterized by a well-defined premalignant phase of low-grade to high-grade intraepithelial neoplasias (CIN1 to 3) that develop over many years, but dysplastic lesions have rarely been reported in the oropharynx. This, combined with lack of detection of HPV productive infections at this anatomical site, the dominance of HPV16-positivity and the difference in integration rates between the two sites suggests that the natural history of HPV-driven disease in the oropharynx is very different to the cervix, but as yet unexplained .2.One approach that has proved very successful in understanding the interaction between HR HPV types and cervical keratinocytes has been by the analysis of cells isolated from low-grade cervical neoplasias. The most highly researched are HPV16-positive W12 cells  and the (a)Transgenic mouse models engineered to express the HPV16 oncoproteins E6 and E7 in squamous epithelia and treated with chemical carcinogens have been very informative of the role of the viral oncoproteins to the development of skin, cervical, and head and neck (tongue and oesophagus) carcinogenesis –60. Thes(b)ex-vivo model maintains tissue cytoarchitecture, including the major lymphocyte subtypes and follicular dendritic network, and would enable HPV infection to be interrogated in the presence of a physiological immunological landscape.One approach that as yet has not been exploited for investigating HPV-tonsil interactions is the use of tonsil explants. Fresh human tonsils dissected into small blocks can be cultured on collagen rafts at the liquid–air interface . Tonsil (c)a).Another approach and one that has been used extensively in the papillomavirus field is the generation of ‘HPV infected’ epithelium equivalents from primary keratinocytes grown in three-dimensional (3D) organotypic raft culture. Keratinocytes harbouring the viral genomes are seeded onto a dermal equivalent formed from rat tail type 1 collagen embedded with fibroblasts (mouse 3T3-J2 fibroblasts are routinely used in the HPV replication model). After a short period to allow expansion of the keratinocytes into a confluent monolayer on the collagen plugs, they are lifted onto a solid support and cultured at the air-liquid interface for a period of about two weeks—the time it takes for a basal keratinocyte to fully differentiate. This involves feeding the rafts from underneath with serum-containing medium that is free of epidermal growth factor, providing nutrients and allowing cytokines and growth factors produced from the fibroblasts to be delivered to the keratinocytes. The ‘raft’ system enables analysis of the spatial and temporal organization of the virus life cycle—from early events to viral progeny assembly a.FigureEstablishment of HPV genome-containing keratinocytes has largely relied upon transfection of HR HPV genomes along with a drug selectable marker into primary keratinocytes, followed by a brief period of drug selection to remove non-transfected cells . The lifSeveral groups including our own, are using primary tonsil keratinocytes to investigate HPV biology at this site . Because(i)One important question is why the tonsil crypt is more susceptible to HPV transformation than the surface squamous epithelium. A very interesting study isolated tonsil progenitor cells; cells expressing high levels of cell surface antigens CD44 and nerve growth factor receptor (NGFR) from the crypt and the surface epithelium . In surf(ii)HPV16 does replicate in ‘unsorted’ primary tonsil keratinocytes following transfection of complete viral genomes or infection with HPV16 pseudovirions ,70,71. U(iii)The rarity of other HR HPV types in oropharyngeal tissues might be because these types are unable to infect these tissues or they differ in replication potential. However, HPV18 is very effective in extending the lifespan of tonsil keratinocytes and the cells support HPV18 productive events upon 3D culture ( figures).Figure(iv)b,c). However, tonsil surface and crypt epithelia undergo alternative differentiation programmes and in particular, there are marked differences in their keratin profiles [When grown in organotypic raft culture, keratinocytes from squamous epithelia of different body sites exhibit an intrinsic potential for tissue-specific differentiation. Thus, keratinocytes isolated from neonate foreskin tissue form a cornified structure with a strong keratinized corneum, whereas tonsil keratinocytes recapitulate the structure of non-keratinizing epithelium b,c. Howeprofiles . Cytokerprofiles . Anotherprofiles .a). However, intense CK8 expression occurred in superficial cells of isogenic HPV16 and HPV18 tonsil epithelium equivalents, with low-level staining in basal keratinocytes (a). This was specific to the site of origin of the keratinocytes because very low CK8 staining occurred in rafts of foreskin keratinocytes harbouring the HR HPV episomes (a). In the tonsil keratinocyte rafts, CK7 was restricted to the occasional superficial cell, but in HPV16 and HPV18 genome-containing isogenic lines, the levels of this cytokeratin were markedly increased in cells of the upper intermediate and superficial layers corresponding to the productive phase of the HPV life cycle (b). A similar effect on CK7 expression did not occur in HPV18-positive foreskin keratinocyte equivalents (b). These findings provide evidence that HR HPV alters the expression pattern of tonsil crypt-specific differentiation markers, a phenomenon that appears to be unique to this anatomical site. HPV is known to delay differentiation in the cervix to support virus replication, but in the tonsil perhaps the virus is able to reprogramme the keratinocyte to express the differentiation characteristics of the tonsil crypt. Since CK7 has been implicated in a translational mechanism of HPV mRNAs [So, do the tonsil epithelium equivalents show CK8 and CK7 expression patterns of the surface or the crypt, and does HPV replication affect the expression profiles of these cytokeratins? Our HR HPV tonsil keratinocytes models are beginning to answer these questions. Immunostained organotypic rafts of primary human tonsil keratinocytes using anti-CK8 antibodies showed patchy CK8 staining; it was largely absent from areas of full stratification, but in areas that were not full thickness, some basal/parabasal cells were positive for this cytokeratin a. Howeveinocytes a. This wepisomes a. In thefe cycle b. A simiivalents b. These PV mRNAs , this mi(v)E2 gene [Patients with HPV-positive oropharyngeal cancer often present with more advanced disease compared to those with HPV-negative OPSCC, but they have a better prognosis and overall survival, although recurrence is still a concern ,75. So wE2 gene ,30. IdenHPV DNA integration is far less frequent in HPV OPSCC than in the cervix; but in addition to integrated and unintegrated forms of the viral DNA, a third form of a viral DNA-human hybrid has been proposed . WhetherIntriguingly, in our models of HPV replication in primary tonsil keratinocytes, in the three donors investigated to date, HPV18 is maintained as an episome for between 40 and 70 population doublings after which there is a marked decrease in episomal forms of the viral genome as determined by Southern blotting. Whereas in the same three donors, HPV16 DNA either integrated very early upon establishment of the cell lines (in one donor), and in the two other donors remained episomal in extended cell culture (greater than 90 population doublings to date). While the donor sample size is too small to conclude that HPV16 is less likely to integrate than HPV18 in primary tonsil keratinocytes, these models are useful to investigate differences in HPV16 and HPV18 biology and virus–host interactions in isogenic tonsil keratinocyte backgrounds grown in short and long-term cell culture under non-stress conditions .3.It is a puzzle that despite the recognition of an association with HR HPV and OPSCC, infection at these sites in non-malignant tissue has not been conclusively found. Primary keratinocytes isolated from tonsil tissue are not refractory to HPV16 infection and are able to support the production of infectious progeny, but are the primary tonsil keratinocytes truly representative of the target cell for the virus? If stem cells in the crypt are the natural target and oral HPV infections may represent a source of virus to infect the tonsil, then these cells might be less permissive for HPV replication, leading to deregulated expression of E6 and E7. Since in the anogenital tract, the phenotype of the HR-HPV target cell is a determinant of carcinogenic risk, the molecular definition of the target cell of these viruses in the tonsil is needed .The reason for the prevalence of HPV16 in HPV-OPSCC is also unclear; but primary tonsil keratinocytes support the infectious cycle of HPV18, that is rarely found in tonsil cancers, so simply that this HPV type is less able to infect or replicate in the tonsil cells seems an unlikely explanation of why we see less HPV18-positive tonsil cancers. Perhaps the nature of the interaction between these viruses and the immune system in this lymphoepithelial tissue is different between the various HPV types, and this contributes to the different outcomes of infection. Further studies to understand the effects of HPV infection and replication in tonsil keratinocytes upon innate signalling and other arms of the immune system are needed. With regards to this topic, RNA-Seq of gingivial keratinocytes immortalised with the telomerase enzyme hTERT and HPV16 genomes showed downregulation of the interferon-regulated innate signalling complex, interferon-stimulated gene factor 3 (ISGF3) and a number of the gene targets of this complex . AlthougInvestigation of the biology of the HPV life cycle in primary tonsils is one level of understanding of the virus–host interaction. However, since the tonsil is a lymphoidal tissue with significant infiltration of lymphoidal cells into the tonsil crypt, future studies might advance these models by co-culturing keratinocytes with immune components in two-dimensional and 3D culture systems.in vivo [While cell lines have been established from cervical neoplasias and have been of huge importance in understanding the natural history of cervical HPV pathogenesis, equivalents from the oropharynx have not been established. This might be because at this anatomical site the natural history of HPV infection is very different to the cervix as either a lengthy premalignant stage does not occur , or the in vivo . Our isoPerforming global gene expression analysis on organotypic raft cultures of primary cervical keratinocytes harbouring HPV16 episomes revealed a number of host genes modulated by the virus  that had not been observed in HPV16-positive foreskin based models . The bioNeither should studies be limited only to HPV16, but include models harbouring HR HPV types found less frequently in HPV-OPSCC which might aid identification of the molecular reasons for the different pathogenicity in tonsil tissue.It is commonly assumed that papillomaviruses evolve slowly, but next-generation sequencing of HPV DNA isolated from lesions is challenging this assumption and in fact inter-host HPV genotype variability might be high, although conservation of E7 has been demonstrated in the cervix ,80,81. TIn summary, it is becoming recognized that virus-driven oropharyngeal disease is different to cervical pathogenesis: thus the refinement and enhancement of tonsil keratinocyte-based models will contribute to a greater understanding of HPV disease at this anatomical site.


Bradykinin has important physiological actions related to the regulation of blood vessel tone and renal function, and protection from ischemia reperfusion injury. However, bradykinin also contributes to pathological states such as angioedema and inflammation. Bradykinin is metabolized by many different peptidases that play a major role in the control of bradykinin levels. Peptidase inhibitor therapies such as angiotensin converting enzyme (ACE) and neprilysin inhibitors increase bradykinin levels, and the challenge for such therapies is to achieve the beneficial cardiovascular and renal effects without the adverse consequences such as angioedema that may result from increased bradykinin levels. Neprilysin also metabolizes natriuretic peptides. However, despite the potential therapeutic benefit of increased natriuretic peptide and bradykinin levels, neprilysin inhibitor therapy has only modest efficacy in essential hypertension and heart failure. Initial attempts to combine neprilysin inhibition with inhibition of the renin angiotensin system led to the development of omapatrilat, a drug that combines ACE and neprilysin inhibition. However, omapatrilat produced an unacceptably high incidence of angioedema in patients with hypertension (2.17%) in comparison with the ACE inhibitor enalapril (0.68%), although angioedema incidence was less in patients with heart failure with reduced ejection fraction (HFrEF) treated with omapatrilat (0.8%), and not different from that for enalapril therapy (0.5%). More recently, LCZ696, a drug that combines angiotensin receptor blockade and neprilysin inhibition, was approved for the treatment of HFrEF. The approval of LCZ696 therapy for HFrEF represents the first approval of long-term neprilysin inhibitor administration. While angioedema incidence was acceptably low in HFrEF patients receiving LCZ696 therapy (0.45%), it remains to be seen whether LCZ696 therapy for other conditions such as hypertension is also accompanied by an acceptable incidence of angioedema. Despite decreasing incidence, cardiovascular disease remains a major cause of premature morbidity and mortality , and theNeprilysin, also known as neutral endopeptidase 24.11, common acute lymphoblastic leukemia antigen (CALLA), and cluster of differentiation cell surface molecule 10 (CD10), is a member of the neprilysin (M13) family of metallopeptidases. The neprilysin family also includes the neprilysin homolog membrane metalloendopeptidase-like 1 (NEP2) , endothe0-bradykinin) from both high and low molecular weight kininogens  receptor and the type 2 (B2) receptor. The B2 receptor normally predominates, whereas B1 receptors are induced by tissue injury. Bradykinin and kallidin are more potent on the B2 receptor, whereas the carboxypeptidase N (kininase I) metabolites bradykinin-(1-8) and Lys0-bradykinin-(1-8) are also bioactive and more potent on B1 receptors  deletion and mutation, study of the effects of neprilysin inhibitor administration on physiological bradykinin levels, and study of the metabolism of exogenous  bradykinin levels and the effect of neprilysin and other peptidase inhibitors on the metabolism of exogenous bradykinin. The effect of inhibition of an enzyme on bradykinin levels depends not only on the specific enzyme's contribution to bradykinin metabolism, relative to other enzymes, but also the baseline degradation rate for bradykinin. This is best illustrated by bradykinin metabolism by the pulmonary circulation, where bradykinin is degraded with approximately 99% efficiency -thiorphan) on bradykinin levels in Sprague Dawley rats -salt hypertensive rats, spontaneously hypertensive rats and renovascular hypertensive rats . TogetheKm (Michaelis constant) of the enzyme for bradykinin degradation , an enzyme with a low Km, such as angiotensin converting enzyme (ACE)  Table , may havare high .Km than ACE  was the predominant bradykinin metabolite when bradykinin was infused into human subjects , indicatCE Table  played aCE Table , 77, thetabolism .Km is the study of bradykinin metabolism by the isolated perfused rat mesenteric arterial bed  into the perfusate, consistent with 3H-bradykinin-(1-7) formation in the interstitial compartment of the heart by neprilysin-mediated cleavage of 3H-bradykinin  inhibitors to demonstrate a role for bradykinin in mediating the effects of neprilysin inhibitors. Two different mechanisms may account for the potentiation of bradykinin receptor-mediated actions by neprilysin inhibitors Figure . Firstly effects . BradykiIcatibant prevented the diuretic and natriuretic effects of neprilysin inhibition in normal Sprague Dawley rats –96. BradIcatibant prevented neprilysin inhibitor-induced reduction in ischemia-reperfusion injury in the rat heart , and nepDespite the potential therapeutic benefits of increased natriuretic peptide and bradykinin levels, neprilysin inhibitor therapy has only modest efficacy in essential hypertension and heart failure, which might be due in part to the inhibition of neprilysin metabolism of the vasoconstrictors angiotensin II and endothelin 1, and the increased plasma angiotensin II, endothelin 1 and noradrenaline levels that accompany neprilysin inhibitor therapy . TherefoThe potential consequences of combined neprilysin and ACE inhibition were examined in the rat tracheal plasma extravasation assay Table . WhereasP = 0.13). However, the protocol of the PARADIGM–HF study might have resulted in a lower incidence of angioedema in the trial population than might occur in patients naive to LCZ696 therapy. The exclusion criteria for the PARADIGM-HF study included a history of angioedema during treatment with an ACE inhibitor or ARB, and 78 and 22% of participants, respectively, were previously treated with an ACE inhibitor or ARB. Additionally, the study involved a run-in period before randomization during which participants received at least 2 weeks of enalapril therapy, followed by 4–6 weeks of LCZ696 therapy.There is currently no information on the effects of LCZ696, sacubitril or LBQ657 on bradykinin levels. However, several lines of evidence indicate a role for bradykinin in the therapeutic benefits of LCZ696 therapy, and also the angioedema associated with this therapy. Whereas ARBs produce angioedema with an incidence approximately half that of ACE inhibitor therapy in patients without heart failure , 110, LC2 receptor in the actions of ARBs, possibly mediated by AT2 receptor stimulation by the increased angiotensin II levels that accompany ARB therapy (Losartan increases bradykinin levels approximately 2-fold in arterial blood of patients with hypertension , similar therapy –124. How therapy , and at  therapy , a lower therapy .i of 2.3 nmol/L for neprilysin inhibition by LBQ657 (Km for bradykinin than NEP (Table In contrast to the plasma transudation seen with combined neprilysin and ACE inhibition in the rat tracheal plasma transudation model Table , no trany LBQ657 , and theEP Table .LCZ696 therapy may therefore potentiate bradykinin-mediated actions by several mechanisms Figure , the incTissue levels of bradykinin are higher than circulating levels and the contribution of neprilysin to bradykinin degradation is specific to the tissue and the tissue compartment. Bradykinin is a likely contributor to the therapeutic benefits of neprilysin inhibitor therapy, particularly the renal and cardioprotective effects. However, bradykinin is also an important contributor to angioedema that may result from peptidase inhibitor therapy, including neprilysin inhibitor therapy, particularly when neprilysin inhibition is combined with ACE inhibitor therapy. LBQ657 inhibits not only neprilysin but also ACE, NEP2, and ECE-2. Although angioedema incidence was acceptable, and similar for LCZ696 and enalapril therapy in HFrEF patients, it remains to be seen whether LCZ696 therapy for other conditions such as hypertension is also accompanied by an acceptable incidence of angioedema.The author confirms being the sole contributor of this work and has approved it for publication.The author declares received research funding from Servier Laboratories, Bayer Pharmaceutical Company, Solvay Pharmaceutical Company and Novartis Institutes for Biomedical Sciences, Inc.


Colorectal cancer is the third most common malignancy in men and the second most common cancer in women. Despite the success of screening programs and the development of adjuvant therapies, the global burden of colorectal cancer is expected to increase by 60% to more than 2.2 million new cases and 1.1 million deaths by 2030. In recent years, a great effort has been made to demonstrate the utility of protein kinase inhibitors for cancer treatment. Considering this heterogeneous disease is defined by mutations that activate different Receptor Tyrosine Kinases (RTKs) and affect downstream components of RTK-activated transduction pathways, in this review we analyze the potential utility of different kinase inhibitors for colorectal cancer treatment. Bowel cancer, colon cancer and rectal cancer have many features in common and are often grouped together as colorectal cancer (CRC), the third most common cancer in men and the second most common cancer in women worldwide. CRC represented approximately 10% of all incident cancer cases and caused 8.5% of all cancer deaths in the year 2012 [In CRC patients, metastasis is the main cause of cancer-related mortality . Most caHowever, despite the success of CRC screening programs, approximately 25% of patients present a metastatic disease at time of diagnosis and, during the course of the disease, 40% of them will develop metastases  mainly tThus, although diagnostic and therapeutic advances have allowed a decrease in CRC incidence rates (6.1% in year 2018), the latest Global Cancer Incidence, Mortality and Prevalence (GLOBOCAN 2018) database still shows a significantly increased mortality rate (9.2%) and an estimate of 1,096,601 new cases and 551,269 deaths worldwide in 2018 , which jColorectal tumors diagnosed at an early stage are usually successfully handled with first line treatments such as surgery, radiotherapy or conventional chemotherapy. In these patients, 5-year relative survival rate is 88–92% while for patients with stage IIIC this percentage decreases to 58–72% [Despite the great impact of conventional chemotherapy in cancer treatment, their nonspecific toxicity against rapidly dividing cells and the acquisition of secondary resistances remain a major constraint on achieving optimal results. In this respect, the great progress achieved in the field of molecular oncology has allowed for the development of highly selective treatments designed to induce cancer cell death by interfering with specific genes or proteins involved in cell growth or resistance to apoptosis. Apart from their enhanced tumor selectivity, these highly effective treatments also cause less side effects than conventional treatments, which has turned them into an invaluable tool for cancer treatment.Molecular profiling techniques including Sanger sequencing, immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), quantitative polymerase chain reaction (qPCR) and Next-Generation Sequencing (NGS) are clinically used to detect alterations at gene sequence level such as DNA mutations, copy number variations and gene fusions across the genome that would be valuable as response biomarkers to different treatment approaches.Despite the limitations of these rapidly developing technologies, along with bioinformatics they are significantly contributing to improving our understanding of the molecular basis of cancer , which iMolecular profiling has allowed CRC stratification and a better understanding of this heterogeneous and usually lethal disease. Based on independent molecular classification systems, gene expression profiling studies and analytical approaches , an inteAPC) and TP53 tumor suppressor genes, KRAS (Kirsten Rat Sarcoma Oncogene 2), SMAD4 and PIK3CA .On the other hand, the recent molecular pathological classification system proposed by the Cancer Genome Atlas Project is based on a wide-ranging genomic and transcriptomic characterization study using array-based and sequencing technologies , which cBoth classification systems point out the presence of altered kinases in different CRC subtypes which, despite their novelty, have clinical implications and show great potential as predictive biomarkers for the efficacy of conventional and targeted treatments , deserviProtein kinases (PK) catalyze the transfer of phosphate, diphosphate, nucleotidyl residues and other groups to a receptor molecule . The humProtein kinases have also been classically classified based on their location in the cell or in accordance to sequence comparison of their structurally conserved protein domain containing the catalytic core. These classification systems are widely accepted and are commonly used in current scientific literature .The transfer of the gamma phosphate from a nucleotide phosphate, normally an ATP or GTP molecule, to one or more amino-acid residues in the side chain of substrate protein usually results in a conformational change of the targeted protein which affects its function, cellular location or association with other proteins. These reversible reactions, mediated by protein kinases and their agonist phosphatases and often translated as a on or off switch , triggerAccordingly, over the past decade, many studies have proven the role of protein kinases in human tumorigenesis and cancer progression and have validated their use as targets for cancer treatment  Table 1Table 1. Apart from genetic mutations, cancer can result from the aberrant functioning of signal transduction pathways and the alteration of normal cell mechanisms controlling gene expression. Given the role of protein kinases and phosphatases as master regulators of cell signaling and the involvement of dysregulated or mutated kinases in human tumorigenesis and cancer progression ,29, thesThe designing process of selective small kinase inhibitors is based on the conserved structure and sequence of a kinase catalytic core, in which protein crystal structures, computational molecular modeling and docking studies have been of vital importance .Kinase catalytic cores also share a similar structural fold , which iThe structure of multiple protein kinase catalytic subunits has been solved in recent years , and hasAccording to their action mechanism, kinase inhibitors are classified into two main groups: Type-I small kinase inhibitors, which are designed to compete for the primary ATP-binding domain of kinases catalytic core in the active state, and type-II inhibitors, that additionally bind to an allosteric pocket adjacent to the ATP-binding site in the inactive state  Figure . Although type-II is more selective than type-I inhibitors , both tyAdvances in the molecular biology of cancer have led to the identification of mutated or dysregulated protein kinases involved in molecular events related to cancer development and progression regardless of tumor origin in the body. These highly relevant findings may have immediate clinical implications, since different protein kinase inhibitors that were firstly designed for the treatment of a different malignancy would be useful for CRC treatment.CRC is a heterogeneous disease defined by mutations that activate different Receptor Tyrosine Kinases (RTKs) and that also affect downstream components of RTK-activated transduction pathways .KRAS and PIK3CA mutations in non-hypermutated CRC tumors [KRAS and PKI3CA mutations [KRAS mutants are present in every molecular subtype, they would be more prevalent among CMS3 CRC (68%) [These alterations have already been reported in CRC molecular classification systems. The Cancer Genome Atlas Network system has described C tumors . In lineutations . NotablyRC (68%) . ApproxiRC (68%) . To dateRC (68%) .RTKs are cell-surface transmembrane receptor kinases composed of an extracellular ligand binding domain, a single transmembrane helix and a cytoplasmic region containing the protein tyrosine kinase activity with C-terminal regulatory regions.RTK activation leads to autophosphorylation in the tyrosine kinase domain causing a conformational change that allows intracellular ligands docking and the activation of signal transduction pathways . Thus, RSimilar to RTK, Receptor Serine/Threonine Kinases (RSTK) are transmembrane proteins, localized to the plasma membrane, containing extracellular ligand-binding domains and cytoplasmic kinase domains. On the basis of primary amino acid sequence comparison, RSTK are classified into two main subfamilies (Type I and II RSTK), both of which act as signaling receptors for members of the TGF-β superfamily of secreted polypeptides . Table Upon ligand binding, a cell-surface complex of type I and type II receptors is formed in which type II receptors phosphorylate the kinase domain of type I partners and allow the binding and phosphorylation of some SMAD proteins , which aAmong these receptors, TGFBRs have a great relevance in CRC, since a large proportion of these tumors display mutational inactivation of the TGF-β pathway along with an enhanced TGF-production . ProvideGenomic instability together with improved cell survival over time increases the probability that tumor cells will acquire new mutations affecting additional kinases. For this reason, and given the tight interconnection between receptor kinases and down-stream transduction pathways, the effectiveness of receptor-kinase inhibitors as monotherapy would be compromised and would make necessary the use of combination therapies targeting down-stream kinases. PIK3CA mutations and PTEN (Phosphatase and Tensin homologue deleted on chromosome 10) mutations and deletions which, combined, are found in about 40% of large bowel tumors [As in other types of cancer, constitutive activation of central survival pathways involving MAPK or protein kinase B (PKB/AKT) is usually found in CRC cells . Other cl tumors .MAPK kinases (MAPKs) belong to a large family of serine-threonine kinases and constitute the major signaling pathway from the cell surface to pro-survival transcriptional responses within the nucleus including cell growth, proliferation, differentiation, development, transformation, migration or death . The conMAPKs are involved in three major signal transduction pathways :The MAPK/ERK pathway, also known as the RAS/RAF/MEK/ERK pathway, is located downstream of many growth-factor receptors usually overexpressed or activated in CRC, and upstream several key transcription factors and proto-oncogenes . Indeed,Membrane receptor activation triggers RAS  phosphorylation and the subsequent consecutive activation of downstream MAP3K (RAF), MAP2K (MEK) and MAPK1 (ERK) kinases. The activation of this enzymatic cascade is of importance in the activation of transcription factors such as Myc. The stress-activated protein kinases/c-Jun Nh(2)-terminal kinase (SAPK/JNK) signaling pathway, which can be activated in response to cellular stress and extracellular ligands  like cytMAPK14 (p38 MAPK) pathway: despite the anti-proliferative and tumor suppressor activity of MAPK14 in some tissues , these kUnder normal conditions, MAPK pathway is tightly regulated by phosphatases and by bidirectional communication with other pathways, such as PI3K/AKT . PIK3CA) [The PI3K/AKT cascade is an intracellular signal transduction pathway, involved in apoptosis inhibition and cell growth and proliferation promotion , which iPIK3CA) .Upon extracellular stimuli , the inactive form of PI3K in resting cells is recruited to the inner surface of the plasma membrane, where its activity is modulated by RAS and SRC family kinases . ActivatExperimental data and computational simulations have shown that there is a dynamic and complex link between MAPK/RAS and PI3K/AKT pathways at different stages of signal propagation . In shorKRAS and PIK3CA mutations are not mutually exclusive, the constitutive activation at physiological concentration of growth factors of both the MAPK and PI3K/AKT pathways would be a selective advantage [Last but not least, provided that dvantage  for cellPTEN, is a tumor-suppressor phosphatase involved in the inhibition of cellular proliferation, survival and growth by inactivating PI3K-dependent signaling . PTEN isUnder the selective pressure caused by the immune system or chemotherapeutic agents, deregulated kinases that cause apoptosis inhibition and cell proliferation represent a survival advantage over other cells that, at the same time, implies an addictive effect to kinase signaling and other oncogenic pathways ,121. AccAs the use of single kinase inhibitors has only demonstrated modest clinical benefits , over thHowever, despite the encouraging results that multi-kinase inhibitors have achieved in the treatment of different types of cancer, preclinical studies in the field of CRC are showing contradictory results and just one multi-kinase inhibitor, Regorafenib, has been approved for advanced CRC treatment and other malignancies with a common altered kinase .NTRK gene fusion without a known acquired resistance mutation [In this regard, FDA approval of pembrolizumab  in 2017 as the first drug for the treatment of adult and pediatric patients with a common biomarker , has beemutation . LarotreDespite the promising prospects of kinase inhibition in CRC, results of preclinical and clinical studies are not always proving as satisfactory as expected. Indeed, less than 20% of patients with metastatic CRC respond to clinically available targeted drugs when used as monotherapy , justifyStudies carried out in this regard show that, given the existence of complex cross-talks among different kinase cascades, targeting one pathway would cause an imbalance between interacting kinases and the activation of compensatory signaling resulting in apoptosis evasion . For thiOn the other hand, given the genomic instability of cancer cells, there is a high probability that the receptor kinase is altered by mutations and causes resistance to selective inhibitors , which wThe aim of EGFR-mAbs is to prevent the activation of down-stream transduction pathways, such as MAPK/RAS and PI3K/AKT, as key components of cell proliferation and survival in EGFR-dependent cells . For thiSince oncogenic mutations in genes encoding key downstream effectors within EGFR-signaling pathway are responsible for primary intrinsic resistance and reduced efficacy of EGFR-mAbs , furtherKRAS proto-oncogene, which is mutated in approximately 32–40% of CRC tumors [KRAS has become a predictor of resistance to EGFR-mAbs and has allowed for the implementation of personalized medicine in CRC patients according to their genotype (KRAS-wild or mutated) [BRAF, which is also found to be mutated in 9–15% [KRAS mutations, there is still insufficient evidence to demonstrate the value of BRAF mutations as predictive biomarker of survival benefit from anti-EGFR mAbs in CRC [RAS is an important effector of EGFR signaling mainly, but not exclusively, through RAF, MAPK and also PI3K pathways . KRAS prC tumors ,109, hasmutated) ,147. In in 9–15% ,109 of Cin 9–15% , limitedin 9–15% , and wits in CRC . KRAS, NRAS or BRAF genes [The ineffectiveness of EGFR-antibodies in CRC patients with mutations in the AF genes , requireAF genes .Within this context, a throughout preliminary study of patients along with the search of predictive biomarkers based in additional molecular alterations in CRC patients that could affect the efficacy of kinase inhibitors have signaled the emergence of a new era in which patients that would benefit from different kinase inhibitors are previously selected.Protein kinase inhibitors have emerged as a promising therapeutic option for the treatment of cancer patients including those with advanced CRC. However, the complex cross-talks among different kinase cascades along with the existing heterogeneity at the kinome level and the high probability of mutations, are the cause of resistance to selective inhibitors and have proved to be a major impediment to achieving the desired results. Although there is still the need of further research to better understand the complexity of kinases pathways and their role in CRC in order to design rational combination therapies that allow enhanced response and minimal resistance to treatment, numerous studies have already demonstrated the effectiveness of combination therapies and multi-kinase inhibitors for cancer treatment in both in vitro and in vivo.Although the currently available options for CRC patients are still limited, the great advances achieved during the last decade in the field of molecular oncology have become a turning point by making it possible to treat patients with different tumors but with a common gene mutation affecting a receptor kinase. Given the importance of receptor kinases in CRC tumorigenesis and progression, this new approach will serve to considerably enhance the number of therapeutic options for progressing CRC patients in the near future.Nevertheless, there is still a need to investigate which kinases would be valuable as predictive biomarkers as well as their role in CRC so that new kinase inhibitors could be developed in the future. Finally, it is worth highlighting the potential of CRC stratification systems based on CRC molecular profiling as promising tools to predict patient response, implementation of CRC subtype-based interventions or clinical trial design.All of this leads us to personalized cancer medicine based on genetic profiling of tumors that targets an individual’s unique mutational profile in which a preliminary study and selection of CRC patients that would better benefit from each combination treatment would be required.


Alzheimer’s disease (AD) is the most common form of dementia and affects over 45 million people worldwide. Both type-2-diabetes (T2D), a metabolic condition associated with aging, and disrupted sleep are implicated in the pathogenesis of AD, but how sleep and metabolism interact to affect AD progression remains unclear. In the healthy brain, sleep/wake cycles are a well-coordinated interaction between metabolic and neuronal activity, but when disrupted, are associated with a myriad of health-related issues, including metabolic syndrome, cardiovascular disease, T2D, and AD. Therefore, this review will explore our current understanding of the relationship between metabolism, sleep, and AD-related pathology to identify the causes and consequences of disease progression in AD. Moreover, sleep disturbances and metabolic dysfunction could serve as potential therapeutic targets to mitigate the increased risk of AD in individuals with T2D or offer a novel approach for treating AD. Alzheimer’s disease (AD) is severe neurodegenerative disorder characterized by the accumulation of extracellular amyloid plaques and intracellular neurofibrillary tau tangles (NFT), with detectable pathology occurring 15–20 years prior to the onset of clinical symptoms [as reviewed in The relationship between metabolic activity and neuronal activity is central to both healthy brain function as well as the pathogenesis of AD. First, neuroimaging studies confirmed that prior to the hypometabolism typically described in preclinical stages of AD, brain regions most vulnerable to Aβ and tau accumulation are uniquely reliant on glucose for normal brain function and display increased glucose consumption at a young age . AerobicAPOE4 targeted replacement mice, a genetic knock-in model of AD  are closely tied to changes in neuronal activity. However, it is still unclear how alterations in glucose availability and glycemic variability can modify the relationship between neuronal and metabolic activity to influence AD-related pathology and dementia. Elevated blood glucose levels, or hyperglycemia, are associated with an increased prevalence of dementia and AD, faster progression from mild cognitive impairment (MCI) to AD, and increased rates of Aβ accumulation . Similarel of AD , where nIndividuals with T2D have a 2–4-fold increased risk of developing AD . AlthougSleep is a conserved behavior that occurs in two distinct stages – non-rapid eye movement (NREM) and rapid eye movement (REM). NREM sleep is characterized by large, slow waves that propagate across the entire cortex, producing synchronized periods of neuronal activation and silence . It is hGlucose and lactate also show consistent patterns across the sleep/wake cycle closely related to neuronal activity. Glucose concentrations in the brain increase during both NREM sleep, which is thought to aid in restoration and replenishment of glucose stores, and waking, indicative of increased metabolic functioning. While glucose cannot accurately measure sleep propensity because of these increases across both states, it is useful as a biomarker for cerebral metabolism . LactateIn the periphery, blood glucose is homeostatically regulated to maintain euglycemia and prevent extreme fluctuations. Glucose tolerance reflects both hepatic gluconeogenesis and glucose utilization and it is closely related to insulin release from pancreatic beta cells, insulin sensitivity of peripheral tissues, or the ability of insulin to inhibit glucose production and increase glucose utilization. With decreased insulin sensitivity or insulin resistance, the body is unable to effectively combat hyperglycemia, allowing for consistent states of high blood glucose . Blood gDisrupted sleep is commonly observed in individuals with symptomatic AD  and can 1C levels, a long-term biomarker of blood glucose levels (Poor sleep quality and quantity is also a common feature of individuals with T2D , and cane levels .It is worth noting that the circadian system controls the timing of sleep, as mutations in circadian clock genes which alter clock function are associated with sleep phenotypes in mice and humans . FragmenAs previously discussed, treatment of OSA with CPAP to promote increased sleep efficiency can improve glucose tolerance and overall metabolic health  as well This review outlines the interactions between sleep, altered glucose metabolism, and Alzheimer’s disease and suggests the emergence of a feedforward cascade where sleep dysfunction and metabolic impairment contribute to the progression of AD pathology . SimilarCC and SM conceived of the idea and equally contributed to the writing of the manuscript.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.


Women with gestational diabetes mellitus (GDM) have a 30% to 70% risk for developing type 2 diabetes and are at increased risk for cardiovascular disease. Little is known about how anthropometric changes in the first postpartum year modify cardiometabolic risk factors.We randomly assigned women in the Balance After Baby study to an intervention group consisting of participation in a web-based lifestyle program or to a control group in which no program was offered. We measured weight, height, waist circumference, blood pressure, lipids, insulin, adiponectin, interleukin-6, and high-sensitivity C-reactive protein, and we conducted 2-hour oral glucose tolerance tests at 6 weeks, 6 months, and 12 months postpartum. We evaluated whether women assigned to the intervention had improved cardiometabolic risk markers compared with the control group. We then conducted a post-hoc analysis, pooling the 2 groups to compare changes in weight and waist circumference with changes in cardiometabolic risk factors.Women in the intervention group did not significantly improve cardiometabolic risk markers compared with women in the control group. We noted a large overlap of weight change and change in waist circumference between groups. In our post-hoc analysis pooling groups, changes in diabetes and cardiovascular risk factors were significantly correlated with changes in weight and waist circumference. The strongest associations were observed for fasting insulin, HOMA, and fasting glucose.Anthropometric changes in weight and waist circumference in women with recent GDM may affect cardiometabolic risk factors, even in the first postpartum year. Our study demonstrates the importance of the postpartum year as an opportunity to decrease future risk for diabetes and cardiovascular disease in women with a history of GDM. Several longer term studies demonstrated that weight loss decreases the risk for conversion to type 2 diabetes in women with a history of gestational diabetes.This report shows that changes in weight and waist circumference in the postpartum year among women with recent GDM are significantly correlated with changes in cardiometabolic risk factors. This study adds further evidence to the potential for the postpartum year to serve as a window of opportunity to decrease future risk of cardiometabolic disease. The incidence of gestational diabetes mellitus (GDM) in US women is from 3% to 9%, depending on the population and the diagnostic criteria used . The incIn 2011 the American Heart Association published updated guidelines to include a history of GDM as a CVD risk factor when screening parous women , and theIn the Balance after Baby study we adapted the Diabetes Prevention Program into a web-based lifestyle intervention designed to decrease postpartum weight retention in women with recent GDM . Women rFor this analysis we first sought to determine the effect of the web-based lifestyle intervention program on markers of cardiometabolic risk by analyzing the change by group assignment. Given the small sample size and substantial overlap in change in postpartum weight and waist circumference, we then pooled the sample to conduct post-hoc analyses to explore the effect of postpartum weight loss and changes in waist circumference on markers of cardiometabolic risk, irrespective of study group assignment. We sought to determine whether anthropometric changes in the first postpartum year were significantly associated with changes in cardiovascular, metabolic, and inflammatory markers.2) of 24 or higher (≥22 for Asian participants) and less than 50. Women were excluded if they had a history of type 2 diabetes or bariatric surgery or if they were taking medications known to affect glucose metabolism or weight. Women were also excluded if they delivered before 32 weeks gestation or if they had net weight loss during pregnancy.The Balance after Baby randomized controlled trial (no. NCT01158131) was a prospective intervention study of 75 women with recent GDM to examine the effect of a web-based lifestyle intervention on postpartum weight loss. Feasibility and efficacy of the trial have been reported . BrieflyQualifying participants attended an initial study visit at approximately 6 weeks postpartum for baseline measurements, and measures conducted at baseline were repeated at approximately 6 months and approximately 12 months postpartum. Women diagnosed with type 2 diabetes  at the 6-month visit completed the 12-month study visit excluding the OGTT. For the pooled analysis, we restricted the sample to women who had baseline measurements of anthropometry, blood pressure, and biomarkers, as well as anthropometric measurements, blood pressure, and a fasting blood sample at 6 months and/or 12 months.1c), fasting glucose, 75 g 2-hour OGTTs, fasting insulin, adiponectin, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, fasting triglycerides, blood pressure, and the inflammatory markers high-sensitivity C-reactive protein (hsCRP) and interleukin-6 (IL-6). We requested that women not perform exercise nor breastfeed during the OGTT. We calculated BMI and HOMA-IR ( vs 1.4 kg ;  groups) . We note groups) . The womWe observed no significant difference in change in waist circumference between women assigned to the intervention group and women assigned to the control group, although it trended in the same direction as weight and BMI at both 6 and 12 months. Overall fasting insulin and HOMA-IR tended to increase over time in both groups, and fasting lipids decreased over time in both groups. HsCRP also decreased in both groups over time. In the sensitivity analysis, including a variable in the mixed-effects regression models to represent breastfeeding status over time did not substantively change the findings.In the pooled analysis, irrespective of group assignment, a positive change in weight was significantly associated with changes in most diabetes risk markers . Some ofChange in waist circumference was also significantly correlated with most diabetes risk markers, including fasting glucose and fasting insulin . A changWe noted significant changes between the intervention and control groups for change in weight between baseline and 6 and 12 months postpartum, as well as the change from prepregnancy weight. In this population of women with recent GDM, a return to prepregnancy weight is clinically significant with respect to the prevention of type 2 diabetes and cardiovascular disease (Overall, changes in weight were more often significantly correlated with cardiometabolic risk factors than changes in waist circumference. The strongest associations were seen for fasting insulin, HOMA, and fasting glucose. These findings illustrate that changes in weight and waist circumference in women with recent GDM have a significant relationship with diabetes risk factors, even in the first postpartum year. Although risk factors for cardiovascular disease had fewer significant correlations, significant relationships were noted between triglycerides and change in weight, as well as changes in weight and waist circumference and change in hsCRP.Our findings demonstrated the strongest relationships between change in weight and markers of insulin sensitivity, including fasting glucose and insulin. The impact of weight loss on insulin sensitivity is well established. Studies demonstrate that postpartum insulin sensitivity is one of the strongest predictors of progression to type 2 diabetes following GDM in the first 5 years following the index pregnancy . Other sWe did not find a significant association between 2-hour glucose and weight change in the pooled analysis. This may be because there was a substantial percentage of women with 2-hour glucose values lower than their baseline fasting glucose values. It has been hypothesized that these paradoxically low glucose values may be a precursor to insulin resistance . This maMarkers of cardiovascular risk have been less studied in the early postpartum period among women with a history of GDM. In one prospective study of 305 women, of whom approximately 30% had GDM, Kew and colleagues reported that women who gained weight between 3 and 12 months postpartum tended to have worsening of certain cardiometabolic risk factors, including blood pressure, HOMA-IR, adiponectin, and LDL cholesterol . They diFewer data are available on the relationship between changes in waist circumference and cardiometabolic risk factors. Among studies in general populations, findings are mixed on whether changes in weight or waist circumference are a more powerful predictor of changes in cardiometabolic risk factors –26. AlthLimitations of this study include its being a single-site study with a small sample size and the use of multiple correlations to analyze the relationships between weight change and cardiometabolic markers. The pooled analysis was a post-hoc analysis and therefore should be considered hypothesis-generating. Our data set also contained missing values at some points.Studies show that pregnancy weight retained beyond 6 to 12 months postpartum is more likely to be retained long-term and is a powerful independent risk factor for future obesity , which h


Recent approvals of oligonucleotide analogue drugs to alter gene expression have been welcomed by patient communities but not universally supported. These compounds represent a class of drugs that are designed to target a specific gene transcript, and they include a number of chemical entities to evoke different antisense mechanisms, depending upon the disease aetiology. To date, oligonucleotide therapeutics that are in the clinic or at advanced stages of translation target rare diseases, posing challenges to clinical trial design, recruitment and evaluation and requiring new evaluation paradigms. This review discusses the currently available and emerging therapeutics that alter exon selection through an effect on pre-mRNA splicing and explores emerging concerns over safety and efficacy. Although modification of synthetic nucleic acids destined for therapeutic application is common practice to protect against nuclease degradation and to influence drug function, such modifications may also confer unexpected physicochemical and biological properties. Negatively charged oligonucleotides have a strong propensity to bind extra- and intra-cellular proteins, whereas those analogues with a neutral backbone show inefficient cellular uptake but excellent safety profiles. In addition, the potential for incorporation of chemically modified nucleic acid monomers, yielded by nuclease degradation of exogenous oligonucleotides, into biomolecules has been raised and the possibility not entirely discounted. We conclude with a commentary on the ongoing efforts to develop novel antisense compounds and enhance oligonucleotide delivery in order to further improve efficacy and accelerate implementation of antisense therapeutics for human disease. Although only a small number of antisense drugs are in the clinic4 or undergoing translation at this time, ongoing efforts to improve oligonucleotide drug delivery and efficacy and to better understand antisense mechanisms will drive further advances in the field.The integrity of all gene expression is reliant on RNA, and the property of nucleic acids that permits complementary base pairing has long been an attractive option to alter gene expression by modulating RNA structure, function or abundance to treat human disease. Advances in the field have been predicated upon development of synthetic nucleic acid analogues . Phosphorothioate oligonucleotides carry a strong negative charge, allowing binding to various proteins and complex formation with cationic liposomes for efficient transfectionin vitro. Other nucleobase and backbone modifications can confer specific characteristics and mechanisms of action, and include peptide nucleic acids and phosphorodiamidate morpholino oligomers (PMOs)   that car (PMOs) .6. The concept of altering splice site selection to modify disease-causing transcripts was validatedin vitro over 25 years ago7 and requires the design and application of an RNA analogue that targets motifs participating in exon selection and retention in the mature mRNA. Antisense targeting of splice sites or motifs necessary for exon selection can influence endogenous alternative splicing, block aberrant splicing, exclude a cryptic exon or an exon carrying a disease-causing mutation, or restore the open reading frame around a frame-shifting deletion, whereas targeting splicing silencers can enhance selection of an exon, otherwise compromised by nucleotide changes 11.Eteplirsen (Exondys51), Sarepta Therapeutics, Cambridge, MA, USA) is an antisense PMO that targets splice enhancer motifs in theDMD pre-mRNA to exclude exon 51 and restore the dystrophin mRNA reading frame, disrupted by most deletions beginning at exon 52 or ending at exon 50.Eteplirsen received accelerated approval from the US Food and Drug Administration (FDA) in September 2016 but is not yet approved in Europe. The drugnusinersen  for the treatment of SMA is a 2′-O-methoxyethyl antisense oligonucleotide (AO) on a phosphorothioate backbone.Nusinersen targets a splice silencer (ISS-N1) in survival motor neuron 2 (SMN2) intron 7 and promotes exon 7 selection and retention during pre-mRNA splicing12 and was approved by the FDA in December 2016. The roughly 25-year history of antisense drug development parallels that of an increasing interest in therapeutics for rare diseases. Of patients with any of the more than 6,000 rare diseases described to date, only a few groups of patients benefit from an effective treatment. It is perhaps fitting that the first splice intervention antisense therapeutics to reach the clinic address two of the more common and debilitating of the life-limiting inherited rare diseases of childhood. This review focuses on the development and current status of AO drugs that alter exon selection and exert a therapeutic effect by restoring or altering protein structure and function.At this time, two mRNA splice-modulating drugs are reported to have delivered therapeutic benefit to a subset of patients with Duchenne muscular dystrophy (DMD)7. This was followed by parallel programs to develop RNA therapeutics to alter exon selection during processing of the dystrophin (DMD) pre-mRNA and generate internally truncated but functional dystrophin isoforms that reflect variants found in the less severe allelic disorder, Becker muscular dystrophy  that also targets dystrophin exon 51 and received accelerated approval from the FDA in 2016. The approval ofeteplirsen evoked lively discussion21 largely because of the small study group; use of a trial structure that had the placebo cohort transition to treatment after 6 months, when the difference in ambulation, the primary endpoint, reached significance; and the low  levels of muscle dystrophin induced by the treatment10. In the aftermath of FDA review and concern regarding the efficacy ofeteplirsen, much attention focused on dystrophin quantitation and clinical trial outcome measures. However, seven years after initiation of Sarepta Therapeutics study 201 and completion of the extension study 202 (long-term safety and efficacy), the trial participants receivingeteplirsen continue to show functional benefits, respiratory function decline is half that expected from the natural history of the disease22, and no treatment-related serious adverse events have been reported. Class II evidence of increased dystrophin expression over baseline dystrophin was presented23, and although the treatment regimen is costly  showed increased event-free survival and significant improvements in motor function, not seen in patients with SMA type 111, prompting early termination of the study26. Comprehensive review and analysis ofnusinersen treatment in SMA by the Canadian Agency for Drugs and Technologies in Health include commentary on the cost-benefit 27.Nusinersen is now available in many European countries, the USA, Canada, Australia, New Zealand and Japan and is reported to deliver clinical benefit in both SMA type 1 and type 2 patients28.SMA is a recessive disease characterized by deficiency of SMN protein that leads to the selective loss of spinal motor neurons and skeletal muscle paralysis, affecting the trunk and limbs. SMA is the most common genetic cause of infant mortality with a prevalence of 1 in about 11,000 live births and carrier frequency of 1 in 40 to 67, depending on ethnicitycasimersen (SRP-4045) and 53golodirsen (SRP-4053) by Sarepta Therapeutics (https://www.sarepta.com/pipeline/exon-skipping-duchenne); an unrelated dystrophin exon 53 program30; exon-skipping strategies to treat dysferlinopathies  by excluding exons 37 and 38 in aDysf mutant mouse model; targeting cryptic exons activated by deep intronic mutations causing choroideremia31, Leber congenital amaurosis32 and USH2A-associated retinal degeneration33; correction of mis-splicing of harmonium in USH1C and exon inclusion to address a common splice variant causing adult-onset Pompe disease34. Two different exon-skipping strategies to treat spinocerebellar ataxia type 3 were recently reported. The pathogenic expanded polyglutamine repeat can be removed by skipping exon 1035; however, in an innovative strategy, exons 8 and 9 were skipped in order to prevent proteolytic cleavage and generation of toxic protein fragments36.Pre-clinical and clinical studies on novel splice-modulating experimental drugs are gaining momentum.  may well begin a new era for splice-modifying therapies, particularly if tissue or cell targeting can be achieved, perhaps leading to reduced dosages.Although development of novel molecular therapeutics is eagerly anticipated by patients and families and has instigated efforts to find new targets and intervention opportunities, current limitations to broader applicability need to be overcome, primarily inefficient delivery to target tissues. The charge-neutral PMOs have an excellent safety profile but are rapidly cleared from the circulation, showing poor uptake into cells. Pre-clinical development of exon-skipping PMOs in a mouse muscular dystrophy model showed moderate restoration of skeletal muscle dystrophin but minimal uptake in the myocardium47) and independent effects. Phosphorothioate oligonucleotides administeredin vivo bind to serum and cellular proteins48  that are reported to cause hepatotoxicity due to off-target RNA hybridization and downregulation54. In an elegant study, Dieckmannet al.54 showed that hybridization-dependent toxicity of RNase H1-competent phosphorothioate oligonucleotides can be mitigated by limiting the affinity of the oligonucleotide and improving target specificity.Oligonucleotide backbone and sugar modifications influence resistance to nucleases, mechanism of action, tissue distribution and binding to various proteins but can also cause hybridization-dependent  for the treatment of homozygous hypercholesteremia56.The propensity for phosphorothioate oligonucleotides to cause injection site reactionset al.57 examined cells transfected with phosphorothioate oligonucleotides with various 2′ modifications. They found that these compounds bind a large number of intracellular proteins and can replace the architectural long non-coding RNANEAT1 in subnuclear bodies called paraspeckles. The repertoire of proteins bound by phosphorothioate AOs and the effects of protein depletion or altered cellular localization are influenced by any 2′ modification58. The most widely used modification, 2′-O-methoxyethyl, was found to have an acceptable tolerability profile and consistent behaviour between sequences3, whereas 2′-fluoro-modified phosphorothioate oligonucleotides disturb nuclear biology, impair cell proliferation and cause loss of cellular proteins59.Although the hepatotoxicity may be attributable in part to off-target hybridization, the exact cause of the injection site reactions remains unknown. ShenO-methyl phosphorothioate AOs with nuclear proteins and report backbone-specific effects of modified oligonucleotides on subnuclear organelles, altered distribution of nuclear proteins, the appearance of novel large structured nuclear inclusions, modification of RNA processing and marked disturbance of the transcriptome in cultured cells. We speculate that some side effects and adverse events reported after clinical evaluation of phosphorothioate nucleic acid drugs may be mediated, at least in part, by non-specific interaction of nuclear components with the phosphorothioate backbone60. The sequence-independent effects of phosphorothioate compounds indeed may enhance or confound apparent antisense effects61 as a consequence of extensive disturbance of nuclear proteins and organelles, particularly those critical for RNA processing and splicing. Strategies to reduce cellular protein binding by phosphorothioate AOs and mitigate toxic effects, thereby improving the therapeutic profile of these molecules by backbone62 and 2′-O-modifications63 are important steps in guiding AO design. Other considerations that may warrant comprehensive investigation are whether phosphorothioate nucleotide degradation products are incorporated into cellular nucleic acids, a question that has been raised64, and whether the immune activation by intra-cerebroventricular injection of a 2′-O-methyl phosphorothioate AO reported in mice50 should be of concern in humans who receive similar compounds.We explored the interaction of 2′-In an era of promising gene replacement and gene correction strategies, exploiting novel recombinant viruses that can be selected and tailored for tropism to specific tissues, the rationale for pursuing transcript-modifying therapies might be questioned. Many of these emerging molecular and gene therapeutics target rare and life-limiting diseases caused by multiple gene variants that may show variable progression and phenotypes within one patient group and, in the event that more than one treatment modality is investigated, compete for the same restricted patient population in recruiting clinical trial participants.However, consider the situation where different treatment modalities could be used in tandem. For example, an antisense strategy might be the only option following vector gene delivery if the virus results in seroconversion and precludes re-delivery. PMOs designed to alter exon selection are specific to the target transcript and, to the best of our knowledge, have not been reported to cause off-target effects on splicing and have no effect when delivered to cells lacking the target transcript. In the case of cryptic exon or aberrant splice site usage, antisense-mediated strategies can generate a normal product, unlikely to be immunogenic, that is expressed under endogenous, tissue/cell/developmental regulation. Gain-of-function mutations may be particularly amenable to splice interventions, whereas for loss-of-function mutations, splice variants can be designed to encode products that may exhibit reduced function but could ameliorate otherwise severe disease.eteplirsen andnusinersen and from the failure ofdrispersen will inform the development of emerging and future molecular medicines.We conclude by acknowledging the contribution of the patients and the combined efforts of chemists, researchers and clinicians and those in the biopharmaceutical industry who championed these drugs and the insightful regulators who have yielded two splice interventions, now available to patients. Although there is general agreement that better drug uptake and greater efficacy are desirable, these drugs, by all accounts, are altering the course of previously untreatable disease. There is potential for numerous therapeutics in the “splice modulating” class. The lessons learnt over the course of translatingAO, antisense oligonucleotide; DMD, Duchenne muscular dystrophy; FDA, US Food and Drug Administration; PMO, phosphorodiamidate morpholino oligomer; SMA, spinal muscular atrophy; SMN, survival motor neuron


Patient-initiated partner notification (PN) following the diagnosis of a sexually transmitted infection is a critical component of disease control in men who have sex with men (MSM) sexual networks. Both printed and internet-based technologies offer potential tools to enhance traditional partner notification approaches among MSM in resource-limited settings.This randomized controlled trial aimed to evaluate the effect of 2 different PN technologies on notification outcomes following syphilis diagnosis among MSM in Peru: a Web-based notification system and patient-delivered partner referral cards.During 2012-2014, we screened 1625 MSM from Lima, Peru, for syphilis infection and enrolled 370 MSM with symptomatic primary or secondary syphilis (n=58) or asymptomatic latent syphilis diagnosed by serology . Prior to enrollment, potential participants used a computer-based self-interviewing system to enumerate their recent sexual partnerships and provide details of their 3 most recent partners. Eligible participants were randomly assigned to one of 4 intervention arms: (1) counseling and patient-initiated Web-based PN (n=95), (2) counseling with Web-based partner notification and partner referral cards (n=84), (3) counseling and partner referral cards (n=97), and (4) simple partner notification counseling . Self-reported partner notification was assessed after 14 days among 354 participants who returned for the follow-up assessment.P=.003 and OR 1.68, 95% CI 1.01-2.79; P=.045, respectively). The proportion of partners notified was also higher in both Web-based partner notification  and referral card  arms than in the control arm .The median age of enrolled participants was 27  years, with a median of 2 partners (IQR 1-5) reported in the past month. Compared with those who received only counseling (arm 4), MSM provided with access to Web-based partner notification (arms 1 and 2) or printed partner referral cards (arms 2 and 3) were more likely to have notified one or more of their sexual partners  Initially developed in the 1930s as a technique for syphilis control in the United States, notification of sexual partners following the diagnosis of a sexually transmitted infection (STI) remains central to efforts aimed at controlling the spread of HIV and other STIs in at-risk populations ,3. PartnIn the field of PN, 3 different types of notification are commonly recognized: (1) provider-initiated notification, in which professional counselors and newly diagnosed individuals work together to identify and notify recent partners; (2) patient-directed notification, in which the index case is encouraged to independently notify their partners after a brief counseling intervention; and (3) contract notification, in which the responsibility for notification shifts from patient to provider after a specified time period . While pTo characterize patient-initiated notification systems, Ferreira et al differentiated between simple and enhanced patient referral . In the Previously, printed partner referral cards have been used in various circumstances and appear to be effective in promoting notification within heterosexual partnerships ,20. HoweData the on the use of internet-based PN systems is mixed. Surveys of STI clinic patients in the United States and Australia, as well as MSM internet users in the United States, have found high levels of acceptability for Web-based notification systems -26. In ETo address this gap in knowledge, we assessed the effect of new and traditional PN technologies on self-reported PN outcomes among MSM recently diagnosed with syphilis infection in Lima, Peru. While the Peruvian Ministry of Health maintains detailed guidelines for managing sexual partners of individuals with syphilis , these services are delivered inconsistently in practice ,36. The Between November, 2012 and July, 2014, we conducted a four-arm, factorial RCT (NCT01720641) to assess the effect of new PN tools on notification outcomes among MSM in Lima, Peru, with untreated syphilis infection. The 4 arms included were (1) printed referral cards; (2) Web-based PN; (3) both printed referral cards and Web-based notification; and (4) control (standard of care) counseling procedures.La Cueva ”).Potential participants were recruited from both community and HIV or STI clinic sites by the staff of the Asociacion Civil Impacta Salud y Educacion . Both men and transgender women (TW) who reported anal intercourse with at least one male or transgender female partner in the previous 6 months were invited to participate in an STI screening protocol. Participants in the screening study completed a computer-assisted self-administered (CASI) survey that addressed demographic characteristics, history of HIV and STIs, alcohol and drug use, and sexual network characteristics, as well as attitudes, beliefs, and perceived community norms regarding PN for HIV and STIs. The survey also asked for detailed characteristics of participants’ 3 most recent sexual partners, including each partner’s gender and sexual identity, sexual practices performed with the partner during the last encounter, and the likelihood of notifying the partner in the event of an STI diagnosis. In order to assist with future recall of partner data, participants were asked to identify each of these 3 partners with a nickname or other identifying characteristic . Following the clinical examination, participants’ blood samples were collected to test for syphilis infection using the rapid plasma reagin (RPR) assay  with microhemagglutination assay for Treponema pallidum antibodies (MHA-TP) confirmation  and serial dilution of positive RPR titers. Although all participants were offered free HIV testing, it was not required as a condition of enrollment. The results of laboratory assays were provided within 2 weeks of screening.Participants with syphilis infection were treated according to the stage of infection, as determined by a study physician following the review of participants’ history of syphilis infection, antibiotic treatment, and RPR titer(s). Cases of symptomatic primary, secondary, and early latent syphilis infection were treated with a single intramuscular injection of 4.2 million units penicillin G benzathine. Conversely, cases of asymptomatic late latent infection were treated with 3 weekly injections of 2.4 million IU penicillin G benzathine. However, participants with newly diagnosed HIV infection were referred to local HIV treatment centers for the initiation of free antiretroviral therapy provided by the Peruvian Ministry of Health.We enrolled MSM and TW diagnosed with untreated syphilis . Participants with symptomatic evidence of primary or secondary infection were enrolled at the initial screening visit, while participants with asymptomatic infection diagnosed by serology were enrolled after receiving the results of their RPR or MHA-TP testing. All participants provided signed informed consent for a study of, “If and how men with an STI inform their recent sexual partners of their diagnosis.” Next, we assigned participants in a 1:1:1:1 fashion to one of the 4 study arms according to a predefined 400-subject randomization scheme generated by the first author at the website (www.random.org).Each randomization envelope contained an assignment to one of the following 4 arms: (1) Web-based PN, (2) referral cards, (3) combined referral cards and Web-based PN, or (4) control. All allocation assignments were concealed in sealed, opaque, sequentially numbered envelopes that were opened in a numerical order by the study counselor at the point of randomization. To ensure visual and physical consistency of sealed randomization envelopes and to maintain concealment prior to allocation, each envelope was filled with a written study arm assignment, 1 PN counseling script, 5 partner referral cards or blank sheets of paper of the same color and consistency as the referral cards, and 1 Web-based PN access card or a blank note card of the same color and consistency. No deviation from the sequential allocation order or wasting of randomization envelopes was reported.Randomization envelopes for all 4 arms included a standardized script that was read verbatim by the counselor. The counseling script advised the participants about the importance of notifying their recent partner of their STI diagnosis and informed them of the availability of free testing and treatment resources at the study website, as well as at other area health centers. Participants were also reminded that their safety was paramount and so they should not attempt to notify any partner who might react with violence or abuse.Participants in the control arm did not receive any additional counseling or PN tools. Participants in the referral cards arm were provided with 5 printed cards to be delivered to a maximum of 5 of their recent sexual partners. Each card contained information about the symptoms and sequelae of syphilis infection, as well as the locations and operating hours of local sites offering free or low-cost HIV and STI testing services. Participants in the Web-based PN arm were read a brief script describing the Web-based notification resources available at  created by YTH, a nonprofit organization designed to use technology to promote youth health and wellness. A Spanish language “Peru” section of the inSPOT website was created specifically for this study and not publicized outside of the trial. Website content was not modified after initiation of the trial. In addition to providing anonymous PN messaging services, the Peru section provided information on testing and treatment resources available in major metropolitan areas of the country. Participants in the Web-based referral arms were provided with a note card indicating the website address. Participants in the combination referral cards/Web-based PN arm were provided with both printed referral cards and access to the inSPOT website using the methods described above.Participants in all four arms were asked to return to the clinic in 14-21 days for a follow-up evaluation. The prespecified primary endpoint was self-reported PN. At the follow-up visit, participants completed a brief CASI survey to assess how many of their recent partners (from the 30 days before screening) had been notified, as well as whether each of their 3 most recent partners had been notified and received antibiotic treatment. Participants were reminded of the total number of sexual partners that they had reported at the baseline visit and asked to quantify how many of these partners had been notified. To assist with recall of data for the 3 most recent partners, participants were reminded of the nickname or other identifying characteristic they had assigned each partner, as well as partners’ gender and sexual identity. Furthermore, survey questions asked whether each partner had been notified and used a 4-point Likert scale to assess the participants’ degree of certainty for whether the partner had received the notification message, whether the partner had sought HIV or STI testing, and whether the partner had received any STI treatment. Operational statistics on the use of the inSPOT.org website during the study period were collected by the YTH staff.Sample size calculations were based on previous observational studies of Peruvian men and women diagnosed with HIV or STI ,39. AssuP values were two-sided. All statistical analyses were conducted in Stata 14.1 .For the preplanned primary analysis, we recategorized the study arms as follows: (1) Web-based PN (arms 1 and 2); (2) referral cards (arms 2 and 3); and (3) control (arm 4). This approach allowed us to maximize the use of limited resources by assessing two different PN tools within a single clinical trial design, although it was not powered to assess for a synergistic interaction between the interventions ,41. DescPeruvian Instituto Nacional de Salud before the initiation of any activities. All participants underwent separate informed consent procedures for the screening and RCT protocols and provided written informed consent for each protocol. The clinical trial was registered with clinicaltrials.gov (Protocol Number NCT01720641).All study procedures were reviewed and approved by the University of California, Los Angeles Office for Human Research Participant Protection  and the Asociacion Civil Impacta Comite de Bioetica (Certificate #0052-2012-CE) and were registered with the Between November 2012 and June 2014, we screened 1625 individuals, of whom 537 were eligible for enrollment based on results of the physical examination or syphilis serology .We noted signs and symptoms of primary syphilis in 36 individuals, whereas secondary infection was identified in 22 others. Of 479 MSM and TW with latent infection and positive RPR and MHA-TP assays, 74 were diagnosed with a previously treated infection that did not require additional treatment, 64 did not return for their results, and 29 were enrolled in a concomitant trial of expedited partner therapy for gonorrhea or chlamydia infection . We enroThe median age of participants was 30 years in the control group and 27 years in the other two arms . The majAt 14-21 Days Follow-up, the proportion of subjects who reported notifying any recent sexual partners of their diagnosis was significantly lower in the Control arm  than in both the Referral Cards  and the Web-based PN  arms .After excluding individuals who denied having any sexual partners in the 30 days before enrollment, the strength of the correlation between the intervention arm and the PN outcomes increased further .P<.001) and Web-based PN  interventions than among those in the control arm  and stable male partners (P=.01). Although the frequency of notification for casual male partners was significantly higher in both intervention arms, these differences did not attain statistical significance (P=.16 and P=.54).The proportion of participants’ 3 most recent partners who had been notified was significantly higher among those assigned to the referral cards . No episodes of interpersonal violence or partner abuse were reported by study participants in any of the arms.In this study, both new Web-based and traditional printed-media notification tools resulted in significant increases in self-reported PN outcomes compared with simple counseling. Peruvian MSM with newly diagnosed syphilis who were randomly assigned to receive printed referral cards and/or access to a Web-based notification system were more likely to report that they had notified at least one recent sexual partner and to report a larger proportion of recent sex partners. While the greatest impact on notification outcomes was observed among stable partners, smaller increases in notification were also observed with casual partners, particularly among participants randomly assigned to receive access to the Web-based PN system. These findings provide important data to guide global STI control efforts and suggest the potential importance of simple, inexpensive methods to promote patient-directed PN by MSM in resource-limited settings.To the best of our knowledge, this study is the first to provide RCT evidence of the effectiveness of patient-controlled, Web-based notification systems among MSM. The odds of notifying a recent sexual partner of a potential syphilis exposure were significantly higher among men who received access to the PN website. These results are consistent with findings from our formative research and imply an absolute increase of 30% in the proportion of stable male partners notified and 15% among casual male partners ,43.Although extensive research has been conducted on the effect of printed referral cards for PN, this study is one of the first to evaluate their use within MSM partnerships in Latin America. As with Web-based systems, participants randomly assigned to receive printed partner referral cards exhibited a higher likelihood of notifying at least one partner and a considerably larger proportion of all their recent partners. The observed effect of referral card distribution was highest among stable male partners, with only a minimal effect on the number of casual male partners notified. These findings suggest that notification tools, such as partner referral cards, could be the most valuable in the context of established partnerships, where face-to-face communication is more likely, as opposed to casual sexual contacts where direct, in-person communication is often rare or nonexistent. Taken together, our data support the routine use of these simple, inexpensive tools as resources to enhance patient-driven notification in LMIC settings or other contexts where provider-guided notification programs are unavailable.In contrast to the substantial impact of both intervention tools on the frequency of notification and the proportion of partners notified, data on more distal outcomes in the notification cascade, such as partner testing and STI treatment, were inconclusive. The assessment of outcomes limited to participants’ 3 most recent sexual partners revealed a nonsignificant increase in the participant-reported notification and confirmation of notification, but minor, or even negative, differences in the frequency of partner STI testing and treatment (as reported by participants). As this study was designed to promote and assess only the initial step of notification, the small number of partners included in this section of the analysis and the use of generalized estimating equation modeling to control for multiple observations per participant is likely to have limited our power to detect statistically significant differences. In addition, the dissipation of the intervention effect observed as we progressed through the partner management cascade might reflect the participants’ inability to follow up on their notification messages, particularly the ones sent to casual partners through an anonymous, Web-based system. The lack of an observed effect on downstream outcomes indicates the need for additional interventions to support partner management outcomes throughout the partner management cascade, beginning with notification and culminating with a linkage to and retention in HIV or STI care . While tThere are several limitations to be considered when interpreting our findings. First, the fact that our data are based on participants’ self-report, without independent confirmation by sexual partners, increases the possibility that observed improvements in notification might have been due to social desirability bias in reporting. However, this bias would have likely affected all arms of the study, resulting in a type II error in favor of the null hypothesis and thereby underestimated the true impact of these notification tools. In order to minimize the possibility of desirability bias, we enrolled participants using standardized scripts informing them only that they were invited to a study of, “If and how men with an STI inform their recent sexual partners of their diagnosis,” without specifying the different intervention tools being evaluated. In addition, all participants, regardless of the intervention arm, were read the same standardized counseling script advising on the importance of notification and the availability of local testing and treatment resources. Due to the lack of independent partner confirmation, data on the more distal outcomes in the prevention cascade  are less reliable and require further investigation with directly confirmed outcomes to be validated. As we did not survey partners on notification outcomes, we were unable assess the cross-contamination between study arms and determine whether individual partners received notification from multiple sources. Similarly, given the relatively small sample size of this study, secondary evaluations of participant- and partner-level factors that might have modified the effect of PN technologies, including the presence or absence of biological symptoms, new diagnosis of HIV coinfection, and differences in the gender and sexual identities of participants and their partners, are beyond the scope of the data presented. Finally, as few TW were enrolled in this study and few cisgender female partners were reported, we did not have sufficient data to draw any conclusions regarding notification by or to MSM and these other groups. Despite these limitations, this study provides important preliminary data to support research into new methods for PN following STI diagnosis in resource-limited settings.This study provides critical clinical trial evidence to support the effectiveness of both new and traditional notification technologies to support patient-directed PN among MSM with syphilis in Latin America. Regarding the global resurgence in the syphilis incidence within MSM sexual networks, accompanied by endemic levels of transmission among MSM in Latin America, these tools offer simple, inexpensive resources that can dramatically affect the frequency of PN following syphilis diagnosis. While provider-initiated notification by dedicated health professionals is highly effective in promoting PN, testing, and treatment, several resource-limited health systems lack the workforce to implement provider-based notification systems. As a result, strategies to support patient-initiated notification are critical to controlling the disseminated syphilis epidemics that exist among MSM and their male, female, and transgender partners in Latin America. Additional research is needed to explore partner responses to patient-initiated notification messages and develop effective interventions to support testing and treatment outcomes throughout the partner management cascade. Both Web-based notification systems and printed partner referral cards offer simple, effective tools to support the first step in PN, testing, and linkage to care cascade and can fill a unique and essential niche for global HIV and STI prevention efforts.


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