Kliza and Husnjak). In this Research Topic, we present a salient collection of original research, methods and review articles that cover novel, promising and recent trends in the ubiquitin(-like) field.Since the pioneering work of Hershko, Ciechanover and their colleagues 40 years ago, our understanding of one of the most complex and widespread signaling networks in biology has evolved greatly through genetic, proteomic, biochemical, and cell biological studies  ubiquitination—a transient and spatially regulated modification, complexifying their detection and quantification.Protein ubiquitination is a powerful post-translational modulator (PTM) as it controls almost every process in cells. To accomplish this, various ubiquitin (Ub) modifications adopt distinct conformations, utilizing what is commonly referred to as the “Ub code”, leading to different cellular functions. Modification by ubiquitin of a target protein is tightly controlled by the action of hundreds of regulatory enzymes employed in specific combinations involving three main steps: activation (E1 enzymes), conjugation (E2 enzymes), and ligation (E3 enzymes). To counterbalance ubiquitination, it can be removed from substrate proteins by deubiquitinating enzymes (DUBs). Modification of a substrate protein can occur by a single Ub moiety on a single target lysine (monoubiquitination) or on multiple lysines (multi-monoubiquitination). Additionally, after a single Ub is transferred to the substrate protein, any of the eight amino groups of the initial substrate-conjugated ubiquitin  can be modified with another Ub molecule, yielding polyubiquitin chains of variable linkage type, length, and configuration (homo- vs. heterotypic Ub chains). Though the functional significance of cellular Ub modifications, such as Lys48- and Lys63-linked polyUb chains, are largely known, the biological significance of other homotypic polyUb chains, collectively referred to as atypical Ub chains, is still far from being fully understood. While Keiten-Schmitz et al. review the role of SUMO chains in chromatin dynamics and genome stability networks, whereas Fernández et al. describe ISGylation as well as strategies targeting this PTM for therapeutic applications. Intriguingly, unlike most PTMs such as acetylation and phosphorylation, ubiquitin itself can be highly customized through further post-translational modification by other PTMs, thereby expanding the Ub code for distinct cellular outcomes which either alter the originally encrypted message or encode a completely new one. The cross-functionality introduced through additional post-translational modification of Ub molecules by UbL proteins, rendering hybrid Ub/UbL chains, is discussed by Perez Berrocal et al..Next to Ub, a vast number of ubiquitin-like (UbL) proteins  can also be attached to proteins increasing the complexity and fine-tuning cellular responses even further. Reits and Sap). During the last few years defects in the ubiquitin-proteasome system (UPS)—a central player in protein quality control facilitating elimination of misfolded or otherwise aberrant proteins—including hampered E3 ligase activity, have been the focus of many studies. Understanding the mechanism of action, as well as identifying which substrates are regulated by a given E3 ligase could provide invaluable knowledge toward the development of therapeutic strategies. Garcia-Barcena et al. review the generation and usage of E3 mutants, thereby highlighting the complexity of this family of enzymes. As the E3 enzymes exhibit different substrate specificity, determining which E3 enzyme maps to which substrate is crucial for our understanding of this complex intrinsic network. Salas-Lloret et al. describe an improved TULIP2 methodology facilitating mass spectrometry toward the identification of E3-substrate networks.Considering the impact of ubiquitination on the regulation of a vast array of fundamental biological processes, dysregulation of this intrinsic process gives rise to numerous diseases ranging from autoimmunity, cancer, and neurodegenerative diseases such as Alzheimer and Huntington's disease  of activity modulators. In addition, the general functional status of the UPS in cells can be examined using reporter substrates as discussed by Gierisch et al.. More recently technologies have been established that induce targeted protein degradation by chimeric small molecules as reviewed by Naito et al.. These technologies, such as Proteolysis Targeting Chimeras (PROTACs), hijack the cellular machinery for ubiquitination thereby subjecting the ubiquitinated proteins to proteasomal degradation. This promoted several drug development research programs as proteins which had previously been regarded as “undruggable” by traditional small molecule therapies can now be degraded by inducing selective intracellular proteolysis.While inhibition of the UPS has proven promising in the treatment of cancer, stimulation of the proteasome has been proposed as a potential therapeutic strategy for neurodegenerative disorders. In the pursuit for novel therapeutics and intervention points, robust assays and tools are key toward understanding and identification of specific components of the UPS. van Kruijsbergen et al. describe a strategy and the encountered challenges toward the development of site-specific ubiquitin antibodies. Together with advances in synthetic strategies for ubiquitin generation, enabling the development of a plethora of ubiquitin activity-based probes (ABPs) and assay reagents, the study of enzymes involved in the complex system of ubiquitination is now within reach. ABPs react covalently at the active site on the enzyme, and thus represent a powerful method to report on specific enzyme activity and to evaluate cellular and physiological enzyme dynamics and function. Taylor and McGouran present the developments made in the “traditional” ubiquitin based ABPs, whereas Conole et al. discuss recent developments in cell-permeable small molecule ABPs. With these advances in the ABP field, activity based protein profiling has emerged as a powerful technique to study these important enzymes as exemplified by the work of Pinto-Fernández et al.. Here, they combined advanced mass spectrometry technology with propargylic-based ubiquitin ABPs to reveal the proportion of active cellular DUBs adding another layer of information in addition to their endogenous expression levels.With an increased focus on the development of novel therapeutics of ubiquitin(-like) system components, characterization of their dynamics is imminent. Understanding the mechanisms of ubiquitin regulation requires the generation of antibodies or alternative reagents that detect ubiquitin in a site-specific manner. Hameed et al. report on the synthesis of diUb chains, fully 15N-labeled on the distal  Ub and demonstrate their applicability for gaining insights into linkage-selective ubiquitin recognition of a unique UBD.Further understanding of the ubiquitin signaling pathway includes the knowhow on how different polyUb chains are recognized by interacting proteins. Often these interacting proteins contain a specific ubiquitin-binding domain (UBD) that bind specifically to polyUb chains, thereby rendering different cellular outcomes. We believe that our Probing the Ubiquitin Landscape Research Topic demonstrates the multidisciplinary nature of research in the ubiquitin field. Advancement in the field is dependent on increased understanding through interconnected research strategies, thereby allowing development of therapeutics as exemplified in this body of work.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Gout is a common metabolic disease with growing burden, caused by monosodium urate (MSU) microcrystal deposition. In situ and chemical-specific histological identification of MSU is crucial in the diagnosis and management of gout, yet it remains inaccessible for current histological methods.Methods: Stimulated Raman scattering (SRS) microscopy was utilized to image MSU based on its fingerprint Raman spectra. We first tested SRS for the diagnosis capability of gout and the differentiation power from pseudogout with rat models of acute gout arthritis, calcium pyrophosphate deposition disease (CPDD) and comorbidity. Then, human synovial fluid and surgical specimens (n=120) were were imaged with SRS to obtain the histopathology of MSU and collagen fibers. Finally, quantitative SRS analysis was performed in gout tissue of different physiological phases (n=120) to correlate with traditional histopathology including H&E and immunohistochemistry staining.Results: We demonstrated that SRS is capable of early diagnosis of gout, rapid detection of MSU in synovial fluid and fresh unprocessed surgical tissues, and accurate differentiation of gout from pseudogout in various pathophysiological conditions. Furthermore, quantitative SRS analysis revealed the optical characteristics of MSU deposition at different pathophysiological stages, which were found to matched well with corresponding immunofluorescence histochemistry features.Conclusion: Our work demonstrated the potential of SRS microscopy for rapid intraoperative diagnosis of gout and may facilitate future fundamental researches of MSU-based diseases. Gout is a crystal-deposition disease caused by uric acid disturbance, and has attracted growing interests of clinicians and researchers due to its rising prevalence and disease burden The first-line strategy for the management of chronic gout is to reduce MSU deposition by medication or surgeries Although crystal identifications were performed mostly in synovial fluid 3-105 enhanced Raman signal due to the coherent stimulated emission process Raman scattering spectroscopy provides intrinsic vibrational fingerprints of chemical bonds for specific detection and analysis of biomolecules. Although spontaneous Raman spectroscopy of MSU in gout and calcium pyrophosphate dihydrate (CPPD) in pseudogout have been investigated in previous studies In this report, we demonstrated the feasibility of SRS to specifically image MSU depositions in gout tissues in situ based on its fingerprint Raman peaks. Simultaneous imaging of MSU and collagen fibers has been realized with the combination of SRS and second harmonic generation (SHG) microscopy. By evaluating various MSU depositions in both rat model and human surgical specimens, we were able to show the early diagnosis of gout, and differentiation of gout from pseudogout based on the distinct Raman features of MSU and CPPD. Furthermore, comparing with H&E and immunohistochemical staining, our quantitative and statistically results showed that MSU contents moderately correlate with the expression level of inflammatory cytokines, which was consistent with the previous studies on the structure of tophi and neutrophil extracellular traps (NETs) -1 was identified as the spectral “marker” for MSU, not only because it has the highest intensity, but also for its clean background, i.e. no background spectra from the tissue, purine or pyrimidine derivatives would effectively interfere with the MSU spectra  of CPPD could be used to differentiate from MSU by either the 630 cm-1 peak or the nearby peaks at 1011 cm-1 and 1066 cm-1   A 29, 31. of view .-1 mode in the tissues of gout rats , first metatarsal joint (n=1) and ankle joint (n=2). All recruited patients were diagnosed as gout from the existence of large amount of MSU deposits in the tissues  for SRS imaging without further processing. We tested samples from multiple joint sources, including the elbow  of 212 µm×212 µm from the three groups, and measured the dependence of various parameters on the distance to the tophi center. The area percentage of MSU was quantified by calculating the mean percentage of pixels in the MSU channel of SRS FOVs. It can be seen that the extent of MSU formation increases monotonically from the 20 mm distance towards the core (0 mm) Figure A. On theThese observations indicate that SRS may provide a convenient means to quantitatively measure MSU in terms of area, concentration, content and crystallization ratio. Their spatial distributions were found to be consistent with the general recognition of the pathophysiological process of tophi. The depositions of MSU tend to recruit a large number of neutrophils in acute phase, which then ingest MSU crystals, activate inflammasomes and produce large number of pro-inflammatory factors through the IL-1β-dominated signaling pathway Finally, we analyzed the correlations between SRS and immunofluorescence intensities to quantify the distribution pattern of IL-1β and TNF-α expression levels in the three groups. As mentioned above, proinflammatory factors are expressed differently in the non-cellular and cellular regions. Cytokines are degraded by NETs in the MSU deposition region, while the collagen region is dominated by fibroblasts and plasma cells. Previous studies have suggested that the pathophysiological process in the coronal zone is similar to that of an acute flare phase We have demonstrated the advantages of SRS microscopy for histological identification of gout without tissue fixation or staining. The results were verified in various types of tissues, including synovial fluid, synovium, tendon and tophi from both rat models and different joints of human. Sensitive and chemical specific detection of MSU crystals were realized, even in cases where traditional histopathology might have failed, as shown in the early diagnosis results of rat acute gouty arthritis model. In previous studies on rat gouty arthritis model, histopathological examinations tend to show the absence of MSU depositions due to limited dose of MSU and metablism by uricase It is also worth comparing SRS with another popular Raman enhancing technique known as surface-enhanced Raman scattering (SERS). SERS has the unique capability of detecting low concentration molecules with extremely high sensitivity, and has been investigated in gout diagnosis, with SERS nanoparticles probing uric acid in human body fluids include synovial fluid or tear fluid in vivo and ex vivo, by point-checking the resection cavities in vivo when combined with arthroscopy to provide adequate quantitative information of MSU depositions intraoperatively, which could offer guidance to optimize surgical strategies and assist decision making, especially on resection margins with maximum removal of MSU deposition and effective preservation of normal structures. Moreover, SRS microscopy integrated with deep-learning algorithms could potentially realize rapid and accurate intraoperative gouty diagnosis, which is among our further work plans The finding that SRS could provide intraoperative and differential diagnosis of gout and pseudogout has important clinical value. Intraoperatively, arthroscopy observed that the MSU is not only enveloped at the first metatarsophalangeal joint, elbow joint and the finger joint, but also deposited at the atypical areas (such as the Os trigonum or the Achilles tendon etc.) with different shapes and sizes. Our study confirmed that SRS is able to diagnose gout in a variety of different joints and demonstrate the distribution relationship between MSU deposition and surrounding tissues A good consistency has been reached between traditional histopathology and MSU channel of SRS microscopy. For refractory gout, surgery often remains a last resort of debulking tophi, yet it still suffers from complications caused by inaccuracy  due to the lack of real-time evaluation methods  in vivo process of MSU crystallization and the interactions with immune system in animal models may also become possible.Our study quantified the characteristics of MSU deposition in tophi and correlated it with the level of inflammation. Researches on the interactions between various physicochemical and biological factors  on the solubility, nucleation and growth of MSU have attracted increasing attentions In summary, we have demonstrated that SRS microscopy could provide label-free and chemical specific detection of MSU, enabling early detection and rapid diagnosis of gout in various types of tissues and joints. Exploiting the vibrational fingerprints of MSU and CPPD, SRS is able to precisely differentiate gout from pseudogout. Furthermore, quantitative SRS measurements of MSU in gout tissues were found to correlate well with the pathophysiological process of tophi. Our study may open up new opportunities for intraoperative diagnosis of gout, as well as fundamental biomedical researches on the formation and development of MSU in various MSU-based diseases.n=2). We then collected intraoperative specimens of tophi or synovial tissue from different patients (n=4), All processes were approved by the Ethics Committee of Huashan Hospital with informed written consent (KY2020-060). The patients  were diagnosed with hyperuricemia for more than five years and had limited mobility due to a joint mass and met surgical indication after evaluation. Intraoperative specimens were collected from the elbow (n=1), first metatarsophalangeal (n=1) and ankle (n=2). In the part of quantitative diagnosis, two intact enveloped tophi were divided into three groups according to the distance from the center of tophi . For each group, four specimens were dissected with equal size (3 mm×3 mm×3 mm). The specimens (n=12) were fixed in 4% paraformaldehyde solution for 24 hours and embedded in OCT-Freeze medium. For each specimen, 10 groups of sections were made, each consisting of three consecutive 8-μm-thick sections. For each group of sections, three consecutive sections were then imaged by SRS microscopy, and optical microscope after H&E staining or immunofluorescence double-label staining  with red plate compensator  were used for the present study. Experimental procedures passed a review by the Animal Welfare and Ethics Group, Department of Experimental Animal Science; Shanghai Medical College of Fudan University, Shanghai, China . The MSU crystal  was prepared by PH titration of uric acid according to the method proposed by previous study p illuminates the sample and the signal is generated at the Stokes and anti-Stokes frequency, respectively, due to inelastic scattering. In SRS, however, due to stimulated emission, two laser beams at ωp and ωs coincide on the sample. When the difference frequency, Δω= ωp-ωs matches a particular molecular vibrational frequency Ω, amplification of the Raman signal is achieved and SRS boosts vibrational excitation by a factor of 107-1) of MSU in spontaneous Raman spectral  of CPPD. Typically, 801-nm pump laser was used to imaging lipid/protein. The Stokes beam was intensity modulated by an electro-optical modulator (EOM) at 10 MHz and aligned with pump beam through a dichroic mirror . The SRS signal intensity of MSU in images is rely on polarization of laser beams , a charge-coupled device camera , and a microscope  with a 40X objective. A 633 nm helium-neon laser beam excited spontaneous Raman scattering of MSU and CPPD standard samples. In order to get Raman spectral of lipid and protein, we measured the Raman spectral of OA (Oleic acid) and BSA (bovine serum oleate) which can represent the distribution of lipid and protein in human body. Moreover, quartz cover slips were used to cover MSU crystal which can depress autofluorescence generated by normal glass.All steps of image process used ImageJ. For gouty tissues, firstly, we let SHG images subtract SRS images to extract the collagen fiber channel because both MSU crystal and collagen fibers contribute to SHG signal. Secondly, we can merge the MSU (green) and collagen fiber (red) channel processed previously into a two-color image so that the distribution of MSU and collagen fibers was shown clearly. The image process of CPDD model is similar to gouty tissues. For large-scale tissues imaging, Olympus software has an automatic mosaic imaging program and we form a full-sized image by merge each FOVs with custom written MATLAB program. Quantitative analysis of SRS and SHG images were performed with ImageJ. Firstly, we performed “adjust” and “threshold” function to set the threshold value of intensity profile to get proper MSU SRS signal and depress background. Average SRS intensity of MSU was calculated to analysis the density of MSU in each FOV because of the linear dependence of SRS intensity on chemical concentration The synovium from patients and rats were processed for H&E staining and immunofluorescence staining as previous described Using an immunofluorescence average optical density value (AO) analysis assay, TNF-α/IL-1β expression was quantified. At least 5 200 - fold fields were randomly selected in each group to be photographed. It was insured that the organization filled the entire field of view and the background light is the same for each photo. Image-pro Plus 6.0 software  was used to convert green/red fluorescence monochrome photos into black and white images, and then the unified standard for judging all positive photos was determined. The integral optical density (IOD) and pixel AREA of tissues of positive images were obtained by analyzing each photo. Average optical density value was calculated, and AO=IOD/AREA. The higher the AO value indicated the higher the positive expression level.Statistical analyses were performed by using SPSS 13.0 and GraphPad Prism 8.0.1 software. All datasets were tested for normality for t-test, and if the normality test failed, the Mann Whitney rank-sum test was used for intra-group comparison. For the comparison of SRS signal intensity, area and fluorescence AO, data were assessed for normality for one-way ANOVA, and Kruskal-Wallis test followed by Dunn's multiple comparisons test was used if the normality test failed. For correlation analysis, data were assessed for normality for Pearson r test, and Spearman r test was used if the normality test failed. Results are expressed as mean ± s.e.m. or median quartile. P < 0.05 is considered as significant.Supplementary figures.Click here for additional data file.


Zika virus (ZIKV) acquired a special relevance due to the pandemic that occurred in the Americas in 2015, when an important number of fetal microcephaly cases occurred. Since then, numerous studies have tried to elucidate the pathogenic mechanisms and the potential therapeutic approaches to combat the virus. Cellular and animal models have proved to be a basic resource for this research, with the more recent addition of organoids as a more realistic and physiological 3D culture for the study of ZIKV. Nanotechnology can also offer a promising therapeutic tool, as the nanoparticles developed by this field can penetrate cells and deliver a wide array of drugs in a very specific and controlled way inside the cells. These two state-of-the-art scientific tools clearly provide a very relevant resource for the study of ZIKV, and will help researchers find an effective treatment or vaccine against the virus. Zika virus (ZIKV) is a member of the Flaviviridae virus family and the Flavivirus genus, together with other well-known viruses, such as West Nile, dengue (DENV), and yellow fever viruses . It has ZIKV is a single-stranded RNA virus that possesses a genome of around 11 kb, which is translated into a big polyprotein encoding 10 viral proteins. Three of these proteins are structural proteins aimed at assembling the viral particles (capsid (C), membrane (M) and envelope (E) proteins), whereas the other seven are nonstructural proteins used by the virus to orchestrate viral replication, avoid host defenses and use the cell machinery to its advantage ,8. The vAedes aegypti, which feeds almost exclusively on human blood [The main transmission route for infection is through a mosquito vector, namely mosquitoes from the Aedes genus, especially an blood ,13. Howean blood ,13,14,15The chosen cellular targets of ZIKV are neural-type cells, preferentially neural progenitor cells (NPCs), oligodendrocyte progenitor cells (OPCs) and glial precursors, though there exists some extent of mature neural cell infection too, such as neurons and glial cells ,17,18,19The life cycle of ZIKV is very similar to that of other viruses from the Flavivirus Genus. The E protein is in charge of initiating the fusion of the mature virions to the host cell membrane, followed by endocytosis into the cell cytoplasm. The viral RNA genome then gets released into the cytoplasm and is translated into the single long polyprotein on the endoplasmic reticulum (ER) membrane. Subsequently, the viral polyprotein gets cleaved to the distinctive 10 proteins . Maturation of the virions occurs in the trans-Golgi network, where they undergo cleavage of the spiky protein prM-E heterodimer complexes by the protease furin, thus giving rise to the separate M and E proteins, and decreasing the virion particle diameter from ~600 Å to ~500 Å. Maturation is followed by release of the mature virions from the host cell to the extracellular milieu by exocytosis, which is when the pr peptide gets cleaved and released due to the pH returning to a more neutral value ,9,27. InA very relevant difference of ZIKV with respect to other members of the Flaviviridae family relies on the fact that ZIKV is able to target and provoke neurological symptoms, mostly in fetuses and infants as they have a big population of neural progenitors which is chosen as a target by the virus . Most ZIThere is currently no vaccine or approved small-molecule based drug to prevent or efficiently treat ZIKV infection . SeveralGiven the actual limitations on vaccine design against ZIKV infection, passive vaccination by using monoclonal neutralizing antibodies (MAbs) is consequently one of the most thoroughly studied molecules to try and treat the infection. However, most of MAbs tested to date have shown a weak response against ZIKV, which, again, could create the risk of developing ADE related to ZIKV infection ,39, as wFinally, a huge variety of drug compounds have been tested both in silico and in vitro to date , with diA drug that has been recently arising interest in the fight against ZIKV infection is sofosbuvir. Sofosbuvir is a molecule approved for the treatment of hepatitis C virus (HCV) infection that targets and inhibits RNA-dependent RNA polymerase (RdRp). It is usually combined with ledipasvir and is considered a class B drug, thus not being safe for pregnant women. It has shown a promising antiviral effect against ZIKV in vitro and in vivo, as most flaviviruses share a very similar RdRp domain, but it has not been tested on humans in clinical trials. However, given that they this drug is not suitable for pregnant women, its potential usefulness would be restricted to preventing chronic persistence of ZIKV in men and non-pregnant women or used as a prophylactic on high-risk people expending short periods of time in endemic areas of ZIKV infection ,48 Figu. As therSince the big outbreak in 2015–2016, many cellular and animal models have been used to study and develop drugs and vaccines against ZIKV infection, to understand its pathogenic mechanisms, transmission routes, life cycle and tropism, among biological aspects of the virus . In geneA wide array of human primary cells and cells lines have been used to better understand the pathogenic mechanisms and tropism of ZIKV. Primary cell infection has been proved for neonatal keratinocytes and dendritic cells (DCs), adult DCs, Sertoli cells, amniotic epithelial cells (AmepCs), trophoblast progenitor cells, placental fibroblasts, cytotrophoblasts (CTBs) and human endothelial stromal cells, astrocytes and microglial cells .Aedes aegypti- and albopictus-derived cell lines [With regard to cell lines, diverse human cell lines, as well as nonhuman primate cell lines and small animals cells lines have been tested to date with different applications and aims, from studying the replication cycle of ZIKV to testing new potential drugs ,52. Withll lines ,54.Animal models have been developed to study the effects on the different risk populations. Thus, some strains focused on ZIKV infection on adults and neonates, others aimed at studying the virus effect during pregnancy, while some others targeted the study of sexual transmission, which is quite unique within the Flavivirus Genus ,56.−/−Ifnar1 C57BL/6 (IFN-α/β receptor-deficient mice) [−/−Irf3;−/−Irf5;−/−Irf7 C57BL/6 (mice deficient in three regulatory factors of IFN genes) [−/−Stat2 C57BL/6 (deficient in a transcription factor that regulates induction of IFN-stimulated genes) [−/−Ifnar1 C57BL/6 and −/−Irf3;−/−Irf5;−/−Irf7 C57BL/6 mice models showed symptoms of ZIKV infection after inoculating a similar amount of ZIKV injected by the mosquito. Symptoms observed included hunching, limb weakness, ruffled fur and paralysis, with infection even getting lethality depending on the dose and location of inoculation [−/−Ifnar1 mice that were crossed with C57BL/6 mice also showed fetal development problems leading to fetal demise due to infection of the placenta and the fetus brain [−/−Ifnar1 mice were also used by other authors to study which cells in the CNS were more susceptible to ZIKV invasion. Astrocytes were demonstrated to be the most susceptible cell type to infection, while the cerebral cortex neurons were the most resistant ones [Mice models are one of the most used ones for the study of ZIKV life cycle, tropism, transmission route and effectiveness of drugs as well as vaccines. Most mice models generated are based on the concept that genetically altering the type I IFN pathway genes makes mice very susceptible to the Flaviviridae Family (as well as other Arboviruses) infections. This pathway is crucial to defend mammals against RNA viruses by upregulating many IFN-stimulated genes (ISGs), to try and stop viral replication and spread ,58,59. Tnt mice) ,61, Irf3N genes) , Stat2−/d genes) , A129 (ld genes) ,64 and Aculation . Interesant ones . The othant ones ,63,64. Tant ones . AG129 hant ones . InteresImmunosuppression of the immune system by dexamethasone in BALB/c mice is another method to generate useful mice models for the study of ZIKV impact. Inoculation of ZIKV in this model resulted in the appearance of some symptoms mimicking the human pathology: viremia, mild weight loss and detection of ZIKV in several tissues .-/-Ifnar1 C57BL/6 mice infected with ZIKV, using either subcutaneous or intraperitoneal inoculation displayed inflammation and injury in different tissues of the male reproductive tract, and ZIKV was detected in various cell types. In addition, severe damage on the testis was observed as late as two months after ZIKV infection, which would suggest that ZIKV infection could potentially cause male infertility [-/-Rag1 mice, which possesses small lymph nodes with no mature B or T cells, caused a persistent infection in the mice, and ZIKV was detected in several cell types of the male reproductive tract, including Sertoli cells, spermatogonia or spermatocytes, among others. ZIKV infection in this model also showed a high risk for the mice to become infertile [-/-, Mavs-/-, Irf3-/-, Irf7-/-Tlr7 mice was not lethal but allowed the detection of the virus in vaginal fluids [-/-Ifnar1 mice [-/-Ifnar1 mice were inoculated subcutaneously with ZIKV, observing placental infection, as well as fetal resorption, fetal brain injury and neural cell death when using subcutaneous inoculation, and even more severe consequences for the fetus when using the intravaginal inoculation [Mice models can also aid in the study on how ZIKV is sexually transmitted, as well as how it affects pregnancy and fetal development. Diverse mice models have been used to study the sexual transmission route of ZIKV. Male ertility ,70. Inocnfertile . Female l fluids , while i-/- mice . With re-/- mice . In the -/- mice . Intrave-/- mice . Immunoc-/- mice . By theiculation .Stat2-deficient hamsters and guinea pigs have also been used to study ZIKV pathogeny, resulting in a mild representation of the disease [ disease ,76. Chic disease .Nonhuman primates have also been used as evident animal models for the study of the virus life cycle and pathogenesis, as ZIKV also has them as hosts and reservoirs. Subcutaneous inoculation in either rhesus or cynomolgus macaques provoked mild clinical disease with viraemia and persistence of the virus in several tissues of the body, including the CNS, the lymph nodes or the female or male genital tract ,79,80. IOrganoids are 3D cell cultures that develop and acquire morphological and functional characteristics like those of the organs they try to resemble, given the appropriate conditions to do so . OrganoiWith regard to ZIKV infection, brain organoid models have been used to study the pathogenic mechanisms of the infection . CompariBrain organoids generated by using a mini-biorreactor were used to assess the impact of ZIKV infection at different stages of pregnancy. Early-stage organoids simulating the first trimester of human pregnancy were infected and the cells affected by the virus analyzed. It was demonstrated that the virus had a preference to infect NPCs, though it could also target IPCs, immature neurons and even astrocytes. When infecting more complex and aged organoids, more closely resembling the second trimester of pregnancy, specific infection of SOX2+ cells, including HOPX+ outer radial glia cells (oRGs) was observed. All these findings correlated with an increase in cell apoptosis and reduced proliferation, and mimicked some of the features observed in human fetal microcephaly . Using bThough not yet generated, vaginal and placental organoids, as well as testes organoids, would also be very interesting and useful, as ZIKV is the only virus in the flavivirus genus that is able to infect the body through sexual transmission, with transmission from the male to the female being the most usual direction of infection ,56 Figu.The use of skin organoids would also aid in the study on how the virus enters the body through the mosquito bite . Skin exNanotechnology is the branch of science aiming to generate compounds at the nanoscale range, i.e., 1–100 nM [However, these compounds have also shown a promising potential for infectious diseases, including viral infections like those caused by ZIKV. Given the fact that no vaccine or molecule tested to date has been completely effective or curative against the virus, nanotechnology could offer an alternative approach for the treatment of ZIKV infection, as well as for the prevention and elimination of the mosquito vector . Though In summary, there is a lot that is still unknown about ZIKV, and the occurrence of more prominent epidemics caused by other pathogenic microorganisms has slowed down the advance in ZIKV related research. The cellular and animal models available have shed light on how the virus acts, why it acquired a more aggressive nature in the 2015 epidemics and how to potentially combat it more effectively, but there is still a lot of progress to be made in the development of an effective vaccines, and both organoid cultures and nanotechnology compounds can pave the way to achieve them.


Pluripotent stem cells (PSCs) are characterized by their unique capacity for both unlimited self-renewal and their potential to differentiate to all cell lineages contained within the three primary germ layers. While once considered a distinct cellular state, it is becoming clear that pluripotency is in fact a continuum of cellular states, all capable of self-renewal and differentiation, yet with distinct metabolic, mitochondrial and epigenetic features dependent on gestational stage. In this review we focus on two of the most clearly defined states: “naïve” and “primed” PSCs. Like other rapidly dividing cells, PSCs have a high demand for anabolic precursors necessary to replicate their genome, cytoplasm and organelles, while concurrently consuming energy in the form of ATP. This requirement for both anabolic and catabolic processes sufficient to supply a highly adapted cell cycle in the context of reduced oxygen availability, distinguishes PSCs from their differentiated progeny. During early embryogenesis PSCs adapt their substrate preference to match the bioenergetic requirements of each specific developmental stage. This is reflected in different mitochondrial morphologies, membrane potentials, electron transport chain (ETC) compositions, and utilization of glycolysis. Additionally, metabolites produced in PSCs can directly influence epigenetic and transcriptional programs, which in turn can affect self-renewal characteristics. Thus, our understanding of the role of metabolism in PSC fate has expanded from anabolism and catabolism to include governance of the pluripotent epigenetic landscape. Understanding the roles of metabolism and the factors influencing metabolic pathways in naïve and primed pluripotent states provide a platform for understanding the drivers of cell fate during development. This review highlights the roles of the major metabolic pathways in the acquisition and maintenance of the different states of pluripotency. Pluripotency describes the developmental capacity of a cell to form the three primary germ layers, which in turn can give rise to all cell types of the adult body. As such it does not represent a single fixed stage, but rather a gradient of cellular phenotypes from the “naïve” pluripotent stem cells (PSCs) of the inner cell mass (ICM) of the early blastocyst, to the “primed” PSCs of the early post-implantation epiblast . Upon utin vivo, several stages can be stably recapitulated in vitro (in vitro without LIF when in the presence of fibroblast growth factor (FGF) and activin A  derived from the ICM of the human blastocyst resemble a primed rather than naïve state  Figure . This alGiven ethical concerns and restrictions in deriving human ESC lines, combined with the fact that hESCs can only yield allogenic options for cell-based therapies; nuclear reprogramming has paved the way for the generation of patient derived induced pluripotent stem cells (iPSCs) . This teDistinct developmental stages are associated with specific patterns of metabolic activity, and growing evidence supports the concept that early metabolic changes are critical for regulating epigenetics and establishing cellular identity. Cells constantly modulate their metabolic state in response to nutrient availability, extracellular signals and reprogramming/differentiation cues, which induces changes in the concentrations of key intermediary metabolites that can serve as cofactors for the epigenetic regulation of gene transcription, including histone methylation and acetylation. This crosstalk between intermediary metabolism and epigenetics may in part account for how metabolic pathways can contribute to stem cell fate determination. In the context of pluripotency, studies have focused on several key metabolites, including S-adenosylmethionine (SAM), alpha-ketoglutarate (αKG) and acetyl-CoA, that sit at the nexus between intermediary metabolism and epigenetics. For example, methylation of discrete residues on histones play a fundamental role in regulating ESCs differentiation and pluripotent state. PSCs have chromatin characterized by “bivalent” domains, which are regulatory regions containing both an activating histone modification, histone H3 lysine 4 trimethylation (H3K4me3), and a repressive modification, histone H3 lysine 27 trimethylation (H3K27me3), which poise developmental genes to maintain their repression in the absence of differentiation signals, yet allow timely activation . Indeed,13C-labeling studies that confirm the utilization of glucose-derived carbons in the manufacturing of nucleotides and amino acids such as serine  oxaloacetate by pyruvate carboxylase to anapleurotically replenish TCA cycle intermediates that are being utilized for anabolic pathways. In PSCs, pyruvate is largely metabolized to lactate, with the remaining being catabolized in the TCA cycle, in contrast to differentiated cells where pyruvate is mainly catabolized. Multiple enzymes responsible for pyruvate flux into the TCA cycle are differentially regulated in naïve versus primed PSCs, such as pyruvate dehydrogenase kinase (PDK)  . For exase (PDK) . It was se (PDK) . Interes pathway . In summAB) is sufficient to induce a stable naïve-like state in hPSCs  to supply the electron transport chain (ETC) and ATP synthesis. Complementing its role in energy generation, the TCA cycle also maintains a balance between anapleurosis, the reactions to replenish TCA intermediates using predominantly pyruvate and glutamine as substrates, and cataplerosis, whereby partially oxidized intermediates can be extracted to serve as building blocks for anabolic processes including lipid, amino acid and nucleotide biosynthesis, as well as post-translational modification of proteins , which is highly dependent on the metabolic requirements of a specific cell state. FAO consists of active transport of medium and long chain fatty acids into the mitochondria through a regulated carnitine palmitoyl transferase system, with subsequent oxidation by the chain-length specific enzymes of beta-oxidation to generate acetyl-CoA that feeds into the TCA cycle and NADH that donates its electrons to the ETC. In contrast, de novo fatty acid biosynthesis requires substrates from multiple metabolic pathways, including acetyl-CoA, reducing factors and ATP, in order to build essential fatty acids. Despite the essential role of lipids in cellular homeostasis, the impact of lipids on PSC maintenance and self-renewal remains relatively unexplored in comparison with other metabolic pathways. Recent studies have begun to examine the impact of lipid availability on hPSCs metabolism and function  and anabolism  ratio. Indeed, this metabolic and epigenetic remodeling also increased the propensity for neuroectodermal differentiation, which is consistent with the observation that fatty acid synthase dependent-de novo lipogenesis is essential for neural stem cell proliferation and neurogenesis  and folate  and acetyl-CoA carboxylase 1 (ACC1) (1 (ACC1) , and sup1 (ACC1) . Long-ch1 (ACC1) . AdditioAlthough significant advances have been made in nuclear reprogramming technology, incomplete reprogramming can generate iPSCs that have transcriptional, epigenetic, and metabolic memory of their parental source . While tCollectively, these studies demonstrate that complex and coordinated remodeling of energy metabolism is critical to drive efficient nuclear reprogramming, in part thought the resetting of the epigenetic landscape. While this appears to be the case in nuclear reprogramming, early metabolic remodeling may also play an important role in other cell state transitions, including from naive to primed pluripotency and release from pluripotency along lineage specific differentiation.Over the past decade, significant advances have been made in our understanding of the metabolic requirements of stem cells and the important role that energy metabolism plays in regulating stem cell function and fate. An emerging concept supports metabolism as not simply a homeostatic system that matches energy supply with energetic demands, but a critical early step during transition between cellular states. This concept is backed by two observations: a) metabolic changes are often some of the earliest to occur during cell fate transitions and b) there is growing appreciation that metabolic pathways directly contribute to the epigenetic remodeling of the cell. This realization has placed emphasis on understanding the microenvironment in which these cells are cultured, as numerous studies have now shown that modifying availability of a single metabolic substrate can dramatically impact stem cell identity. As the field increasingly dissects the finer metabolic distinctions between pluripotent states and discovers how these interact with regulators of stem cell fate, it will be interesting to see if manipulation of substrate supply and energy metabolism may help to enable the derivation and culture of other PSC states, such as totipotent stem cells or formative PSCs. In addition, modulation of energy metabolism may ultimately be harnessed for translational and clinical applications, not only to support manufacturing and lineage specification of PSCs for regenerative therapies, but also to promote innate regenerative capacity and augment current cell-based therapies.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Soft tissue sarcomas (STS) are heterogeneous rare malignancies comprising ~1% of all solid cancers in adults and including more than 70 histological and molecular subtypes with different pathological and clinical development characteristics. Over the last two decades, the increased knowledge of the new molecular and genomic mechanisms of different STS histotypes allowed for a reclassification of these tumors and consequently to the development of novel chemotherapeutic agents. Generally, surgery, in combination with radiotherapy only in selected cases of localized disease, represents the most common treatment of primary STS, whereas the principal treatment modality for locally advanced or metastatic disease is first-line chemotherapy. The principal treatment for the preponderance of STS patients is usually an anthracycline (epirubicin and doxorubicin) in monotherapy or in combination with other drug novel chemotherapeutic agents. However, survival for treated patients with metastatic disease is poor, and a 2-years survival rate is about 30%. In this scenario, Pharmacogenomics (PGx) biomarkers that can predict drug response play an important role in the improvement of molecular diagnostics in clinical routines and contribute to elucidating the genetic basis for the differences in treatment efficacy and toxicity among STS patients. This review focuses on recent insight in the PGx biomarkers that have been described to modulate responsiveness and toxicity parameters of conventional and new chemotherapeutics drugs in several STS histotypes. Soft tissue sarcomas (STS) are heterogeneous rare malignancies representing of about 1% of all solid tumors in adult and including more than 70 histological and molecular subtypes with a multiplicity of pathogenic and clinical development features . STSs orOver the last two decades, the increased knowledge of the new primary molecular and genomic mechanisms of different STS histotypes allowed to a reclassification of these tumors and consequently to the discovery of innovative chemotherapeutic agents . OverallDespite the prominent progress in discovering genetic aberrations and their functions in STS, the major therapeutic modality for most local recurrence and metastatic sarcomas remains cytotoxic chemotherapy. Generally, surgery, in combination with radiotherapy only in selected cases of localized disease, represents the most common treatment of primary STS, whereas the principal treatment modality for locally advanced, or metastatic disease is chemotherapy. First-line drug protocol for the preponderance of STS patients is usually an anthracycline (epirubicin and doxorubicin) alone or in combination with another drug , 10. HowTherefore, there is an urgent need to develop novel treatments and find biomarkers that can help physicians to identify patients who are possible good responders or resistant to specific therapies and predict individual predisposition to toxicity reactions associated with therapies.Differences in pharmacological response to drugs represent the most common cause of patient morbidity and mortality. From this specific point of view, pharmacogenomics (PGx) biomarkers that can predict drug response play an important role in the improvement of molecular diagnostics in clinical routines and contribute to elucidating the genetic basis for the differences in treatment efficacy and toxicity among patients. Moreover, PGx markers predicting efficacy or risk to develop adverse drug reactions (ADRs) are commonly positioned in transporters, drug-metabolizing enzyme genes, drug targets, or HLA alleles.Genetic variability harboring in the germline genome of the patient can influence systemic pharmacokinetics and pharmacodynamics of the treatments, acting as prognostic biomarkers for drug-induced toxicity and treatment efficacy. Instead, the aberrations in cancer somatic genome mostly function as drug targets and they can be used to select treatment or to be predictive of response to treatment .Very penetrant predisposed mutations and frequent genetic variants particularly single-nucleotide polymorphisms (SNPs) which heredity pass between the generations represent the mostly germline variations that are considered as useful biomarkers for ADR and drug response. Contrary, due to exposure to chemotherapeutics that likely act through damage to DNA, cells could acquire randomly somatic mutations that are potentially used as drug targets , 13.Thanks to significant improvements in biotechnology and bioinformatics knowledge, genomic research quickly advanced from investigations based on modifications at the single gene level to studies on the whole-genome scale using extensive genotyping, and Next Generation Sequencing (NGS) methods. These new methodologies considerably decrease sequencing times and costs and allow early detection of disorders and identification of pharmacogenomics markers to customize treatments , 14.Candidate gene methods are performed to recognize most of the germline variations while genome-wide association (GWAS) approach is archived sequencing up to a large number of SNPs. On the other hand, somatic mutations from cancer genomes are analyzed through NGS technique that uses the concurrent sequencing of a huge number of DNA parts to create an enormous pool of genomic arrangement information. This procedure allowed genotyping a selected number of the gene of interest (gene panel), the complete exome or the whole genome.In this review, we outline recent studies on PGx biomarkers that have been described to modulate responsiveness and toxicity parameters of conventional and new option chemotherapeutics drugs in several STS histotypes , 2.Several germline biomarkers could impact on effectiveness of therapies and survival in STS patients and may be useful to stratify patients liable to develop treatment-associated toxicities.Ecteinascidia turbinate.One of the new therapeutic alternatives among the few options of STS treatments is trabectedin (Yondelis) a marine-derived compound extracted from the Caribbean Sea squirt In phase III clinical trial in advanced leiomyosarcoma and liposarcoma patients showing progression disease after anthracycline-based chemotherapy, trabectedin significantly increases disease control respect to conventional dacarbazine treatment .Italiano et colleagues have pointed out the relationship of precise haplotypes associated with trabectedin sensitivity to specific SNPs within the BRCA1 gene  of the mutated BRCA2 gene. These analyses corroborate the assumption that different DNA repair defects existing in tumors positively conditioned the response to trabectedin and that BRCAness malignant genotype is significant in influencing the effectiveness of treatment including trabectedin .VEGFRA2, VEGFA, and Solute Carrier Organic Anion Transporter Family Member 1B3 (SLCO1B3) display a correlation of these SNPs with PFS in patients with advanced GIST receiving imatinib  are the most prevalent tumors of the gastrointestinal tract origin from mesenchymal lineage . Mutatioimatinib . GeneticKloth and colleagues. In this study, PFS and OS in 127 patients with advanced GIST treated with sunitinib were associated with 49 SNPs involved in the pharmacokinetic and pharmacodynamic pathway of this TKI. More specifically, PFS was significantly extended in carriers rs1056878 (TT genotype) in Cytochrome p450 oxidoreductase (POR). Otherwise, the presence in patients carrying the T-allele in SLCO1B3 rs4149117, the CCC-CCC alleles in SLC22A5 haplotype, and the GC-GC alleles in the IL4 R haplotype were predictive for OS -mediated angiogenesis and by directly blocking PDGFRs, fibroblast growth factor receptors (FGFRs), and KIT , 30. Ther damage .Genetic analysis of STS shows low mutational load including predominantly by copy number changes . Whole-eTP53 mutations were shown to have significant association with a longer PFS respect to TP53 wild-type. Predictors factors of pazopanib effectiveness and toxicity in STS patients are associated also with modulation of cytokines and circulating angiogenic factors in serum  , revealsin serum . Indeed,Gemcitabine, in monotherapy or combined with docetaxel, has been usually approved in leiomyosarcoma  and angiIntracellular uptake of prodrug gemcitabine into tumoral cells takes place through a transmembrane protein human equilibrative nucleoside transporter 1 (hENT1) . A recenp < 0.01) and longer median survival . Interestingly, patients with decreased level of mRNA BRCA1 showed a better median PFS  and a progression-free at 6-months (PFS-6) after treatment . Moreover, a significant correlation between increased responses to trabectedin treatment with high expression level of ERCC5/XPG complex was also observed in patients showing an improvement in term of disease control rate , median PFS , and PFS after 6 months after trabectedin therapy . These data support the hypothesis of a direct association between DNA damage repair system functionality and responsiveness to trabectedin, differently from other DNA interacting agents.Not only germline variants as discussed before but also somatic alterations in the homologous repair system are reported to be responsible for a deeper and longer activity of trabectedin in STS patients where drug response is inversely correlated with the BRCA1 mRNA levels . In thisIn myxofibrosarcoma, a common adult STS characterized by a high local recurrence rate and infiltrative growth pattern surgery combined with neoadjuvant or adjuvant radiotherapy represent the standard care in localized disease –42. HoweGenotyping analysis in patient-derived MFS primary cultures demonstrated the promising role of surface glycoprotein CD109, a negative regulator of transforming growth factor-beta (TGF-β) pathway in the differential diagnosis of more aggressive high-grade myxofibrosarcoma identifying this marker as a possible therapeutic target . MoreoveRB1, CDKN2A, CDKN2B, CCND1, and CDK6) with a worse overall survival as well as TP53 alteration and KRAS amplification. Thus, considering as PGx markers in a specific subset of these tumors genetic alterations in the Rb pathway, comprising CCND1 or CDK6 amplification, these data will contribute to knowledge for the use of novel therapeutic approaches such as CDK4/6 inhibitors.In an extensive integrated genetic and epigenetic study of 99 myxofibrosarcoma performed by WES, RNA sequencing, and methylation analysis, a large number of driver genes were identified as potential drug targets and molecular prognostic factors in this STS histotype . This stBesides genetic factors, epigenetic modifications of DNA together with miRNA regulation of gene expression have been linked to differences in drug response, through regulation key drug-metabolizing genes or increasing expression of drug efflux transporters –49.Halichondria okadai. The inhibition of tubulin by eribulin induces G2/M cell-cycle arrest, disruption of mitotic spindles, and, finally, apoptosis. Patient-derived primary coltures of adipocytic and undifferentiated pleomorphic sarcoma demonstrated high sensitivity to eribulin (The role of these biomarkers in mediating chemotherapy efficacy was underlined in eribulin-based therapies in STS patients. Eribulin mesylate is a microtubule inhibitor equivalent to halichondrin B derivate from the marine sponge eribulin , 51. Moreribulin –54. miRNeribulin .Pharmacogenomics studies of anti-cancer drugs in STS play an important role in identifying patients avoiding adverse events, and optimizing drug dose. The aim of these investigations is to take advantage of personalized chemotherapies regarding cancer treatment and prevention. Development in NGS technologies has been open a new opportunity for characterizing the genomic landscape of these tumors, together with the possibility of applying the genetic diagnostic tests relevant in cost-benefit analysis. However, due to the several rare STS histological subtypes harboring specific fusion genes , certainFinally, future studies in this field should be considered in terms of identification and validation of drug-sensitivity test systems for routine use that include known specific PGx markers in common clinical management.CC contributed to references collection of the study. CG contributed to the writing of this manuscript. All authors approved the final version.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.


DNA damage response (DDR) and apoptosis are reported to be involved in the pathogenesis of many neurodegenerative diseases including polyglutamine (polyQ) disorders, such as Spinocerebellar ataxia type 3 (SCA3) and Huntington's disease (HD). Consistently, an increasing body of studies provide compelling evidence for the crucial roles of ATX3, whose polyQ expansion is defined as the cause of SCA3, in the maintenance of genome integrity and regulation of apoptosis. The polyQ expansion in ATX3 seems to affect its physiological functions in these distinct pathways. These advances have expanded our understanding of the relationship between ATX3's cellular functions and the underlying molecular mechanism of SCA3. Interestingly, dysregulated DDR pathways also contribute to the pathogenesis of other neurodegenerative disorder such as HD, which presents a common molecular mechanism yet distinct in detail among different diseases. In this review, we provide a comprehensive overview of the current studies about the physiological roles of ATX3 in DDR and related apoptosis, highlighting the crosslinks between these impaired pathways and the pathogenesis of SCA3. Moreover, whether these mechanisms are shared in other neurodegenerative diseases are analyzed. Finally, the preclinical studies targeting DDR and related apoptosis for treatment of polyQ disorders including SCA3 and HD are also summarized and discussed. Dominant inheritance of mutant Ataxin-3 (ATX3) leads to neurodegenerative disorder Machado-Joseph disease , with abnormal expansion of its C terminal polyglutamine (polyQ) repeats up to 55–87 in comparison to 10–51 in healthy individuals. The polyQ expansion length correlates positively with the disease severity and inversely with the age of disease onset  derived from normal metabolism, or be exogenous such as ultraviolet (UV) from sunlight , Parkinson's disease (PD), and HD  called MJD1 or ATX3 which contains an unstable CAG repeat  domains, with the most common isoform found in human brain having 3 UIM domains  or single-strand breaks (SSBs). Except in routine cellular processes such as DNA replication, meiosis, and V(D)J recombination, DSBs are generally highly deleterious lesions arising from exposure to ionizing radiation (IR). DSBs can induce chromosomal rearrangement such as deletion, translocation, or amplification. DSBs are repaired primarily by homologous recombination (HR) and non-homologous end joining (NHEJ). The error-free HR repair operates in S and G2 phase, with sister chromatids available as repair templates, while error-prone NHEJ is active throughout the cell cycle. Failure to repair DSBs can trigger permanent growth arrest and finally cell death  , and further facilitates the loading of other DNA damage repair proteins, to promote DNA repair and checkpoint signaling. Previous studies revealed that DNA damage-induced SUMOylation, an ubiquitin-like modifier, recruits the SUMO-targeted ubiquitin ligase (STUBL) RNF4 to the sites of DNA lesions, which promotes the ubiquitin-dependent extraction of MDC1 and RPA, and thus facilitates DSB repair via NHEJ and HR, respectively   mutant  and DSBs, is essential for genome integrity maintenance and cell survival in eukaryotic cells. Chk1 can phosphorylate its downstream effectors to regulate various cellular pathways such as cell cycle checkpoint, DNA repair or cell death if the damage is too severe to be repaired  transcription factors are reported to be associated with the regulation of cell cycle arrest, and protection against cell death induced by oxidative stress  lines and pons tissue from SCA3 patients  is a central kinase of DNA damage response. Phosphorylated H2AX mediated by ATM serves as a platform to recruit other DNA damage repair proteins (Ciccia and Elledge, Drosophila, and PNKP overexpression significantly rescues cell toxicity induced by mutant HTT (Gao et al., Recently, both mutant ATX3 and mutant HTT were reported to impair PNKP activity, disrupting strand break repair and transcription. Consistently, there is a higher level of DNA breaks in transcribed genes in affected brain regions of SCA3 patients and mice, and brains of HD patients and mice also show DNA damage accumulation as compared to control (Gao et al., In addition, elevated ADP-ribose levels after DNA damage are observed in PNKP-patient cells (Hoch et al., Apoptosis is regarded as the dominant mechanism underlying neurodegeneration in PD (Kountouras et al., It was previously reported that p53 is involved in the pathogenesis of HD (Steffan et al., Although significant advances have been made in establishing normal functions of ATX3 and etiology of SCA3, the underlying molecular mechanism of SCA3 pathogenesis is still urging for sussing out. The impaired functions of ATX3-interacting proteins, sequestered in the polyQ aggregates, is thought to associate with cellular toxicity and neurodegeneration in SCA3 (Paulson et al., Given that the post-mitotic status of neurons and high level of oxygen metabolism in brain, efficient DDR is absolutely essential for neuronal function and survival. ROS are reported to be responsible for various neurological disorders such as AD and PD. Although the underlying mechanism that contributes to selective neuronal death and pathological changes remains to be investigated, it is thought that the role of ROS in the pathogenesis of these disorders is associated with proteins including a-synuclein, DJ-1, Amyloid β and tau protein (Jiang et al., DDR is also an important pathway in cancer genesis. Sharing the same pathway, it seemed that neurodegeneration and cancer have a subtle linkage, and this relationship alerts us that we need to cautiously assess the cancer risk if we plan to use drugs targeting DDR to slow down the progress of neurodegenerative diseases. For example, p53 is abnormally activated in SCA3, inhibiting the function of p53 may alleviate the degeneration disease, whereas bring about high risk of cancer. Therefore, although many preclinical studies indicate the efficiency of DDR and apoptosis modulation in improving the pathology of neurodegenerative diseases including SCA3, further investigation is undoubtedly needed to confirm target specificity and minimize side effects before their employment for therapeutic intervention. We hope that future studies regarding SCA3 and neurodegenerative diseases will provide effective solutions for clinical therapy.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Pregnane X receptor  is a member of the ligand-activated nuclear receptor superfamily. This receptor is promiscuous in its activation profile and is responsive to a broad array of both endobiotic and xenobiotic ligands. PXR is involved in pivotal cellular detoxification processes to include the regulation of genes that encode key drug-metabolizing cytochrome-P450 enzymes, oxidative stress response, as well as enzymes that drive steroid and bile acid metabolism. While PXR clearly has important regulatory roles in the liver and gastrointestinal tract, this nuclear receptor also has biological functions in breast tissue. In this review, we highlight current knowledge of PXR’s role in mammary tumor carcinogenesis. The elevated level of PXR expression in cancerous breast tissue suggests a likely interface between aberrant cell division and xeno-protection in cancer cells. Moreover, PXR itself exerts positive effect on the cell cycle, thereby predisposing tumor cells to unchecked proliferation. Activation of PXR also plays a key role in regulating apoptosis, as well as in acquired resistance to chemotherapeutic agents. The repressive role of PXR in regulating inflammatory mediators along with the existence of genetic polymorphisms within the sequence of the PXR gene may predispose individuals to developing breast cancer. Further investigations into the role that PXR plays in driving tumorigenesis are needed. Pregnane receptor (PXR) NR1I2), termed steroid and xenobiotic receptor (SXR) in humans, is a nuclear receptor that acts as a xenobiotic and endobiotic sensor that recognizes a large variety of hydrophobic ligands I2, terme. First dPXR activity is governed by the coordinated efforts of ligands, other nuclear receptors and cofactors, as well as epigenetic and post-translational modifications such as phosphorylation, acetylation, ubiquitination, and SUMOlytion. The DNA-binding activity of PXR is activated by a wide variety of endobiotic compounds including steroid hormones and bile acids such as estrogen or lithocholic acid, respectively. PXR is also activated by a plethora of exogenous compounds including xenobiotic chemicals, food ingredients, and a wide variety of herbal remedies and pharmaceutical agents. The net effect of canonical ligand-mediated PXR activation in mammals is the coordinate regulation of a pathway of genes that encode pivotal hepatic and intestinal proteins which are involved in the coordinate uptake, biotransformation, and eventual excretion of a myriad of specific—and potentially toxic—metabolites in feces or urine .The activation of PXR regulates the expression of hepatic uptake drug and bile acid transporter proteins including organic anion transporting polypeptide protein 2 (Oatp2) and organic cation transporter 1 (Oct1) ,21,22. MGiven the breadth of compounds that activate PXR and its role in the coordination between multiple biological processes, it makes sense that there would be crosstalk between PXR and other nuclear receptors including farnesoid X receptor (FXR), constitutive androstane receptor (CAR), peroxisome proliferator-activated receptor alpha (PPARα), liver X receptor (LXR) and androgen receptor . In addiThe primarily anti-apoptotic role of PXR in the cell cycle and cell proliferation has been evidenced through research on hepatocytes. It has long been established that PXR activation in hepatocytes can induce liver regeneration and even lead to hepatomegaly. PXR was specifically found to do this through an interaction with YAP . Shizu eThe relationship of p53 and PXR is likely also important in PXRs role in cancer. The tumor suppressor p53 is essential for inducing cell cycle arrest in DNA damaged cells and is often referred to as the ‘guardian of the genome’. Without activated p53, the cell cycle goes unchecked predisposing it to malignancy. Recent studies have demonstrated that PXR exerts an inhibitory effect on p53 through reducing p53 transactivation along with the expression of target genes involved in apoptosis and cell cycle arrest . AlternaBAG3, BIRC2, and MCL-1—were subsequently found to be elevated while proapoptotic genes BAK1 and p53 were decreased. Similar results were observed in normal mice colon epithelium with activation of PXR leading to decreased deoxycholic acid-induced apoptosis and increased sensitization to colon cancer [Another study provided further evidence for the antiapoptotic function of PXR independent of its drug detoxifying effects through the upregulation of antiapoptotic proteins and downregulation of proapoptotic proteins. In the study, PXR-producing colon cancer cells were exposed to deoxycholic acid, a component of bile acid capable of inducing apoptosis in colon epithelium, both in the presence and absence of a PXR agonist. Expression of antiapoptotic genes—n cancer . While Pn cancer . PXR knon cancer .Acquired resistance to chemotherapies in tumor cells is a complex process, with multiple genes likely involved. Changes in gene expression of multidrug resistant-associated proteins is thought to play a major role in the transition from responsive to resistant cancers. Several downstream target genes of PXR may be involved in this transition, including phase I metabolic enzymes, CYP enzymes, phase II metabolic enzymes, glucuronyltransferase (UGT) and phase III drug transporters, multidrug resistance protein 1 (MRP1), and breast cancer resistance protein (BCRP) ,45.Although PXR is expressed mainly in the liver, intestine, and colon tissues, it has been found to be expressed in normal breast tissue, and at even higher levels in breast cancer tissue ,38,39,43The drug uptake transporter organic anion transporter polypeptide 1A2 (OATP1A2), which mediates cellular uptake of estrogen metabolites, has also been documented as a downstream target of PXR. OATP1A2 was first identified through its characterization of enhanced expression in breast cancer as compared to healthy breast tissues . Meyer zCancer cell motility leading to metastasis, along with angiogenesis, are major components of cancer progression and areas in which PXR may be involved. PXR, when activated, has been shown to be sufficient at enhancing neoplastic characteristics including: cell growth, invasion, and metastasis through activation of Fibroblast Growth Factor 19 (FGF19) gene expression in colon cancer. Shown in both human colon tumor cell lines and in primary human colon cancer tissue xenografted into immunodeficient mice, the role of PXR activation through FGF19 induction mediates colon cancer cell proliferation and migration . PXR’s iAnother important factor that contributes to cancer growth and spread is angiogenesis. This process is primarily mediated by vascular endothelial growth factor (VEGF) and nitric oxide (NO). This process works first by the release of VEGF, which then activates the Nuclear Factor Kappa B (NF-κB) pathway leading to an increase in nitric oxide synthase and thus nitric oxide. Nitric oxide then stimulates angiogenesis of new blood vessels needed by cancer cells to thrive . EspositHistorically immune responses, such as inflammation, were thought to help eradicate tumors but evidence is growing that antitumoral responses put pressure on the tumor to evade immune destruction. The intersections between inflammation and cancer pathogenesis has shown the functionally important tumor-promoting effects that immune cells in the innate system have on cancer progression . It has Cancer-induced inflammation is present in at least 60% of patients with advanced cancer and should be considered as a source of variability in the clearance of anticancer drugs . SpecifiRecent studies have found a link between PXR and the proinflammatory mediator NF-κB. These studies actually demonstrated an inverse relationship with activated PXR inhibiting NF-κB signaling. It was also found that NF-κB target gene expression was upregulated in PXR knockout mice leading to a proinflammatory state . NF-κB wGenetic polymorphisims within the sequence of PXR, including several SNPs within the 3′UTR, have been suggested to correlate with an increased predisposition to breast cancer by influencing its expression and/or function ,71. AddiA number of SNPs within genes involved in the endobiotic metabolism of estrogen have been suggested to alter breast cancer susceptibility, through modification of circulating estrogen levels. A recent meta-analysis was performed on 10 studied SNPs in PXR and the susceptibility to overall cancer . Two of Revathidevi et al. (2016) screened the 3′ UTR of PXR in 96 breast cancer patients to identify polymorphisms that impact PXR expression levels . They foMDR1 gene, whereas in liver it does not [PXR is expressed and is biologically functional in breast tissue, and a preponderance of evidence suggests that it likely plays a key role in acquired chemoresistance. PXR effects on tissue growth, metastasis, and apoptosis are very likely a tissue-specific phenomenon, whereby in one tissue it may act as a growth proliferator  but in others  it could potentially have an opposite or variable overall effect. There is good precedence for this notion as in enterocytes, PXR activation regulates the drug-inducible expression of the does not ,26,76. Tdoes not ,78.PXR has been shown to be moderately expressed in both ER+ and ER- breast tumors , while oFurthermore, the idea of PXR functionality in breast cancer initiation and breast cancer development is an area that needs further investigation. Differences in the role of PXR based on tumor maturity would most likely be due to the presence or absence of co-factors and target pathways. Additionally, it is likely that modifications of PXR through post-transcriptional and -translational modifications—as well as splice variants and polymorphisms—are associated with breast cancer risk, development, and outcome. Further investigations into the role that PXR plays in context-specific physiological processes via target gene expression, the relevance of its interactions with additional pathways to alter cellular responses, as well as various regulatory mechanisms that may be altered due to structural modifications to PXR are needed.It is highly possible that PXR has dual roles in both growth and the inhibition of growth. For example, PXR in early cancer development could function in an opposite manner when compared in late cancer activation. This kind of behavior is seen with inflammasomes  ,81,82,83


For years there have been concerns whether the results of large-scale clinical trials that include limited specific patient populations can be applied to patients in real-world clinical practice. Therefore, it is crucially important to verify whether emerging evidences obtained from large-scale clinical trials on limited specific patient populations can be applied to patients at real-world clinical settings. Recent cardiovascular outcome trials with sodium-glucose cotransporter 2 (SGLT2) inhibitors showed a consistent risk reduction of approximately 30% for hospitalization for heart failure (HF), and the SGLT2 inhibitors had a great potential to be effective for prevention of HF in a wide variety of type 2 diabetes (T2D) patients independent of their history of HF or cardiovascular disease (CVD). Furthermore, the DAPA-HF trial also demonstrated that dapagliflozin proved clinically effective in patients with HF with reduced ejection fraction regardless of diabetes, suggesting its robust benefits in some specific patients with HF. According to these evidences, SGLT2 inhibitor is increasingly recognized as an emerging and promising option to reduce the risk of HF in patient with T2D. To use appropriately SGLT2 inhibitors for HF prevention in the real-world setting, it would be required to determine the optimal patient population who can receive better clinical benefits from SGLT2 inhibitors. In this commentary, based on the current understandings and lessons learned from the most recent studies, we discussed the importance of future research on the safety and efficacy of SGLT2 inhibitor in clinical situations of HF other than those examined in previous cardiovascular outcome trials. For years there have been concerns whether the results of large-scale clinical trials that include limited specific patient populations can be applied to patients in real-world clinical practice. When the clinical background and characteristics of a patient match those of a ‘specific’ patient population in a clinical trial it is essential to know whether the patient can achieve similar benefits to those observed in the trial, beyond consideration of the estimated number needed to treat. It is therefore important to verify whether clinical trial results can be applied to patients at a general population level by evaluating real-world data and using an appropriate estimation model. This is an important step when aligning daily medical care with guidelines that are formulated based mainly on the results of large-scale clinical trials.Recent cardiovascular outcome trials (CVOTs) on newer antidiabetic agents have increasingly resulted in major paradigm shifts in care aimed at preventing cardiovascular and renal complications in patients with type 2 diabetes (T2D). Among those agents, a sodium-glucose cotransporter 2 (SGLT2) inhibitor is one class of glucose-lowering agents, which acts uniquely via inhibition of glucose reabsorption in the renal proximal tubule. Yet, a meta-analysis of previous CVOTs for SGLT2 inhibitor highlighted the utility of SGLT2 inhibitor in preventing and/or delaying the development of heart failure (HF) in patients with type 2 diabetes (T2D) . More reA previous large observational study suggested that compared to other glucose-lowering drugs initiation of SGLT2 inhibitor was associated with a lower risk of death and HF in real-world patients with T2D regardless of pre-existing CVDs . RecentlIn 2019, the Dapagliflozin and Prevention of Adverse outcomes in Heart Failure (DAPA-HF) trial was the first to show that dapagliflozin treatment reduced the risk of hospitalization for HF and mortality specifically in patients with HF with reduced ejection fraction (HFrEF) regardless of T2D . In a reGiven these findings and the current therapeutic algorithm for symptomatic HFrEF patients in guidelines, the use of dapagliflozin may be useful in preventing and/or delaying the need for downstream therapies. Earlier treatment with dapagliflozin in patients with symptomatic HFrEF may have a positive impact on outcomes. Nonetheless, whether dapagliflozin would be an effective replacement in some clinical settings of up-titrated standard drugs  and considering additional therapies, such as cardiac device, is still uncertain. Additionally, clinical efficacy of SGLT2 inhibitor is unknown in several specific HF conditions, such as drug-naïve patients, electrical disorders-derived, severely reduced ejection fraction, New York Heart Association class IV, and acute decompensated situation, since those patients were generally excluded or were a minor population in CVOTs. Additionally, cardiologists should even expect the possible application of SGLT2 inhibitor in patients with HF with preserved ejection fraction.Taken together, SGLT2 inhibitor is increasingly recognized as an emerging and promising treatment option to reduce the risk of cardiovascular events, including HF, in patient with T2D . Due to


Human papillomaviruses are important pathogens responsible for approximately 5% of cancer as well as other important human diseases, but many aspects of the papillomavirus life cycle are poorly understood. To undergo genome replication, HPV DNA must traffic from the cell surface to the nucleus. Recent findings have revolutionized our understanding of HPV entry, showing that it requires numerous cellular proteins and proceeds via a series of intracellular membrane-bound vesicles that comprise the retrograde transport pathway. This paper reviews the evidence supporting this unique entry mechanism with a focus on the crucial step by which the incoming virus particle is transferred from the endosome into the retrograde pathway. This new understanding provides novel insights into basic cellular biology and suggests novel rational approaches to inhibit HPV infection. To reach the site of viral genome replication, most DNA viruses journey from the cell surface to the nucleus. This review summaries the unique entry mechanism used by papillomaviruses, with a focus on recent results that have clarified several key aspects of intracellular HPV trafficking during virus entry, and suggests potential therapeutic approaches. For comprehensive reviews on HPV entry, readers are referred to the excellent reviews by Campos, Sapp, and Banks and their colleagues ,2,3.The non-enveloped papillomavirus particle contains only two viral proteins, the major capsid protein L1 and minor capsid protein L2. Seventy-two pentamers of the L1 protein form the outer shell of the capsid, which also contains 12 to 72 molecules of the L2 protein and the ~8000 base pair double-stranded DNA viral genome bound to nucleosomes . The L1 Knockdown and inhibitor studies have identified numerous cell proteins required for HPV entry and trafficking, including but not limited to furin, kallikrein-8, ADAM17, cyclophilin B, SNX17, SNX27, γ-secretase, VPS4, obscurin-like1, TSG101,TRAPPC8, growth factor receptors, DCT, annexin A2/S100A10, and tetraspanins CD131 and CD63 ,27,28,29To comprehensively identify cellular proteins involved in HPV entry, we conducted a genome-wide siRNA screen. In this screen, ~20,000 different siRNA pools were transfected into HeLa cells in a microtiter plate format to repress individual cellular genes, and the ability of an HPV16 pseudovirus to express GFP from the encapsidated reporter plasmid was measured in each well of cells . The resThese genetic findings inspired a series of immunodetection experiments that detected incoming HPV pseudovirus in intracellular compartments comprising the retrograde pathway ,31. In aOne of the key factors required for HPV entry is retromer, a cytoplasmic protein complex comprised of three core subunits, VPS26, VPS29, and VPS35  34]. Th. Th34]. Inspection of the amino acid sequence of the HPV capsid proteins identified a short hydrophobic sequence near the C terminus of the L2 protein, phenylalanine-tyrosine-leucine (FYL), that is conserved in all HPV L2 proteins and resembles a consensus retromer binding motif found in some cellular retromer cargos  42,43].,43.42,43Pull-down experiments with biotinylated peptides and recombinant fusion proteins purified from bacteria demonstrated that the L2 FYL sequence (as well as a nearby non-conserved YYML hydrophobic sequence) does in fact bind directly to retromer . ImportaAll HPV and most animal papillomaviruses contain FYL or a similar sequence near the C terminus end of L2, implying that retromer-mediated intracellular trafficking arose early in evolution of this virus family. However, a few animal papillomaviruses deeply rooted in the papillomavirus phylogenetic tree do not contain an obvious retromer binding site. It is not known if these viruses have a retromer binding site that is unrecognizable or if they utilize a different entry mechanism. Retromer function is normally regulated by various cellular proteins including sorting nexins and the Rab7 small GTPases ,49. KnocThe identification of HPV as a direct cargo of retromer poses a conundrum, because the endocytosed virus is in the lumen of the endosome, separated from retromer by the endosomal membrane. Several lines of published information suggest a possible resolution of this challenge. Sapp and colleagues reported that the C terminus of L2 contains a “membrane destabilizing sequence” required for HPV entry , and CamProtrusion of L2 through the endosome membrane is an attractive solution to the problem posed by the localization of the capsid and retromer in different cellular compartments, but insertion of proteins through membranes poses a significant energy barrier. In fact, most transmembrane proteins are inserted into membranes during translation via interactions between an N-terminal signal peptide and the complex translocation machinery at the ER membrane. In contrast, HPV is a non-enveloped virus particle lacking membranes or classic transmembrane proteins and enters cells as a virus particle comprised solely of mature soluble proteins. To determine if the C terminus of L2 actually extends through the endosome membrane into the cytoplasm, a split green fluorescent protein (GFP) assay was performed in which an inactive segment of GFP known as GFP11 was fused to the C terminus of the L2 protein, and the rest of GFP (GFP1-10) was expressed in the cytoplasm . The twoAlthough these experiments established that L2 protrudes into the cytoplasm, they did not address the mechanism of protrusion. The C-terminal segment of L2 near the retromer binding sites contains a highly conserved sequence of basic amino acids that resembles a class of protein segments known as cationic cell-penetrating peptides (CPPs)  53]. CP. CP53]. Based on the sequence similarity between the basic segment of L2 and cationic CPPs, we hypothesized that the L2 basic segment might act as a CPP to deliver the L2 protein not across the plasma membrane but rather across the endosome membrane into the cytoplasm so that it could bind retromer and other HPV entry factors. Standard membrane translocation assays with the L2 basic sequence as a free-standing peptide or fused to GFP showed that it indeed has cell penetrating activity . Mutant The experiments summarized above provide important new insights into not only the HPV entry process, but also cell biology in general. The biological roles of CPPs have been largely a mystery for decades. Because most prior studies focused exclusively on searching (unsuccessfully) for natural examples in which CPPs transferred secreted proteins across the plasma membrane into cells, other activities of CPPs may have been missed. In contrast to this supposed canonical CPP activity, the HPV CPP performs a very different role. Namely, it transfers a segment of a protein from one intracellular compartment into another. In addition, after membrane passage, the L2 N-terminal transmembrane domain may anchor L2 in the endosome membrane, so that the L2 protein adopts a transmembrane existence . Thus, aMost CPPs studied to date are either artificial sequences or are excised from larger proteins with no known natural cell-penetrating activity. We speculate that many of these sequences display cell-penetrating behavior because of their underlying chemistry but in fact do not necessarily serve this function in vivo when linked to their host proteins. If this is the case, these sequences have not been selected by evolution for membrane passage activity. In contrast, all of the more than 300 sequenced papillomaviruses of both humans and animals have a C-terminal, basic L2 segment that presumably acts as a CPP during virus entry. Thus, this viral membrane-penetration activity has persisted for hundreds of millions of years of papillomavirus evolution. CPPs in extant L2 proteins therefore represent the results of the long evolutionary history of this virus group. Because they act in nature to transfer protein segments across membranes, papillomavirus CPPs are professional CPPs, in contrast to most CPPs studied to date, which may be amateurs. We also note that the rate-limiting step for CPP action in studied cases is not cell uptake but endosomal escape, precisely the natural activity of the papillomavirus CPPs. Although all papillomavirus CPPs are rich in basic amino acids, the precise sequences differ among different virus types. The CPP sequences found in multiple papillomavirus types  may be more active or have other favorable properties compared to CPPs present in only one or a few papillomavirus types. Similarly, the sequences flanking the core basic CPP, which are conserved but not identical among different papillomavirus types, may have been selected to enhance or fine-tune the activity of their associated CPP, for example to set the preferred pH or temperature of membrane protrusion. Continued analysis of papillomavirus CPPs and their flanking sequences will determine whether they are optimal for cargo translocation for practical uses. Papillomavirus particles are stable outside of their host cell. In the virus particle, the L2 protein is thought to be largely buried under the shell of L1 pentamers, but there is evidence that both the N terminus and C terminus of L2 are accessible on the surface of the particle before it encounters cells or soon thereafter ,55,56. BPapillomavirus entry is blocked by pharmacologic inhibition of the cellular protease γ-secretase or by knockdown of any of the four γ-secretase subunits, all of which are transmembrane proteins ,24,31. WProphylactic vaccination will be the mainstay of HPV prevention efforts in coming years, but there is still need for alternative preventive approaches, because most people are not vaccinated and many pathogenic types of HPV (primarily cutaneous types) are not in current vaccine formulations. Can our newfound understanding of HPV entry suggest potential anti-viral strategies?The retromer-mediated model of HPV retrograde entry suggests that agents that bind retromer would titrate retromer away from the virus and inhibit infection. Accordingly, we hypothesized that flooding the cytoplasm with peptides containing retromer binding sites would compete with L2 in the incoming virion for retromer binding and thereby inhibit HPV trafficking. As noted above, the limitation with most peptide therapeutics is inefficient delivery of the peptide into the cytoplasm, but the presence of the L2 CPP adjacent to the retromer binding sites provides a simple solution to this problem. We synthesized a short peptide containing the HPV16 L2 retromer binding sites, the HPV16 or HPV31 L2 CPP, and flanking sequences. When this peptide is added to the culture medium, the peptide enters cells, binds retromer and displaces it from incoming virus, and aborts infection by several pathogenic HPV types . As predThe genetic and biochemical analysis of retromer action during HPV entry also suggests that compounds that inhibit Rab7, TBC1D5, or retromer itself may have activity against HPV. Because retromer evolved to transport cellular, not viral, proteins, compounds that block retromer action are also likely to inhibit transport of cellular cargos. In fact, trafficking of cellular cargos is affected by the peptides discussed above as well as by expression of dominant-negative Rab7 (unpublished and ). HoweveRecent study of HPV entry and trafficking and the elaboration of the retrograde model of HPV entry have revolutionized our understanding of this process and suggested novel preventive approaches. Although the broad outlines and some of the fascinating details of this process have emerged, there is still much to learn. Further study of HPV entry will reveal additional interesting aspects of this complex process and may lead to the development of clinically viable strategies to combat HPV infection. These studies will also continue to provide important new insights into fundamental cellular processes such as the action of retromer and CPPs and other aspects of intracellular protein trafficking. As has historically been the case, viruses will serve as valuable tools to understand biology.Pengwei Zhang and Daniel DiMaio are inventors on a patent application related to this work.