Yet despite this, very little is known regarding the impact of such genomic abnormalities upon the functional biology of the tumor cell. We discuss here how recent advances in quantitative proteomic techniques are now providing new insights into the functional biology of these aggressive tumors, as illustrated by the use of proteomics to bridge the gap between the genotype and phenotype in the case of c-MYC-amplified/associated medulloblastoma. These integrated proteogenomic approaches now provide a new platform for understanding cancer biology by providing a functional context to frame genomic abnormalities.Brain tumors are the leading cause of cancer-related deaths in children, and medulloblastoma is the most prevalent malignant childhood/pediatric brain tumor. Providing effective treatment for these cancers, with minimal damage to the still-developing brain, remains one of the greatest challenges faced by clinicians. Understanding the diverse events driving tumor formation, maintenance, progression, and recurrence is necessary for identifying novel targeted therapeutics and improving survival of patients with this disease. Genomic copy number alteration data, together with clinical studies, identifies  Medulloblastoma (MB) is the most common pediatric malignant brain tumor and one of the leading causes of brain cancer deaths in children. Current therapy for high-risk medulloblastoma involves aggressive treatments that often leave survivors with significant neurological and intellectual disabilities due to the effects of these nonspecific cytotoxic therapies on the developing brain . Extensic-MYC copy number amplifications primarily confined to Group 3 tumors [Group 3 MB remain poorly understood despite accounting for over a quarter of medulloblastoma cases and displaying significant recurrence and mortality rates (around 50% across multiple studies) ,6. Altho3 tumors . The pre3 tumors .c-MYC amplification is driving tumor aggressiveness. Increased c-MYC mRNA transcripts are observed in Group 3 tumors compared to the SHH and Group 4 subgroups, yet there is no difference in comparison to tumors of the wingless (WNT) subgroup  when we compare MYC-amplified and nonamplified tumors within Group 3 tumors in humans  is also identified in Group 3 MB [OTX2 and c-MYC are frequently coexpressed at high levels in medulloblastoma and regulate many of the same genes, indicating there might be a functional interaction between these two genes [OTX2 has recently been shown to repress differentiation, increase proliferation, and upregulate c-MYC in medulloblastoma cells [Difficulties making inferences from genomic abnormality to cancer phenotype remain problematic for all types of cancer. With regard to medulloblastoma, it is unclear how subgroup . Unlike ications ,11 and isubgroup  tends to reviews  and remam proper  as opposm proper ,17. We dn humans , suggestoup 3 MB . Moreovewo genes . OTX2 isma cells ,19,20.c-MYC in medulloblastoma is further complicated by RNA-Seq studies showing persistent gene fusions involving the 5′ end of PVT1, a noncoding gene, which frequently coamplifies with c-MYC [PVT1 fusions, which are proposed to arise as a result of chromothripsis [PVT1 is non-protein-coding, it is a host gene for four microRNAs, miR-1204–miR1207. Intriguingly, earlier studies have implicated miR-1204, which is expressed at a higher level in c-MYC-amplified Group 3 tumors compared to the adjacent miR-1205 and miR-1206, as a candidate oncogene that enhances oncogenesis in combination with MYC [The tumorigenic role of th c-MYC . In thesthripsis . Althougwith MYC ,22.MYC amplification drives an aggressive phenotype in the permissive context provided by the cell of origin and in concert with other genomic events such as OTX2 amplification and PVT1 fusion.Taken together, the evidence supports a model in which MYC-driven animal models of medulloblastoma has provided strong support for the role of c-MYC over-expression in the tumorigenesis of Group 3 MB. Initial independent animal models involving the induced overexpression of c-MYC in cerebellar stem cells or granule precursor cells have clearly shown that the resultant tumors not only display highly aggressive large cell anaplastic histology, but also are transcriptionally similar to Group 3 medulloblastoma [MYC is not only required for tumor initiation, but also necessary for tumor maintenance. Inactivation of c-MYC causes rapid tumor regression in mouse model, indicating that targeting c-MYC itself might be an effective therapeutic strategy for this disease [The development of blastoma ,17. Thesc-MYC function in Group 3 MBs is that those mentioned above have all required the loss of Trp53 function to generate c-MYC associated medulloblastoma phenotypes. This is particularly significant as Trp53 mutations are not common in human Group 3 MB, and their absence was proposed to be indicative of other significant cooperative genetic or molecular events that inhibit Trp53 function, potentiating tumor initiation [MYC family amplifications and Trp53 pathway defects both emerged at relapse may provide support for this cooperativity [MYC-driven tumors without disrupted Trp53 function are being developed and show homology with human Group 3 MB [MYC plays a different tumorigenic role in these various models and whether they all result in similar tumor behavior.An important caveat to the development of initial animal models for studying itiation . An intrrativity  and may oup 3 MB . Furtherc-MYC as a significant contributor to the phenotype and maintenance of aggressive Group 3 MB, the exact mechanisms of its contribution remain incompletely understood. This is further complicated by the fact that c-MYC regulates a variety of pathways, often making it difficult to determine which molecular events are driving oncogenic behavior or are simply downstream passenger events. There is a growing consensus among clinicians that the key molecular mechanisms driving the aggressive recurrence of these tumors need to be clarified in order to rationally improve therapy for these high-risk patients [In summary, although the data support patients . Thus, Mc-MYC-amplified Group 3 medulloblastomas.Significant developments are being made in proteomic techniques and these are now providing clinicians with unique insights into commonly studied cancers . ResearcProtein profiling in cancer has advanced tremendously from early techniques, almost half a century ago, when starch gel electrophoresis was being used to determine globulin expression profiles in serum from patients with myeloma and macroglobulinemia . PossiblMajor concerns regarding proteomic techniques stem from the limited depth and reproducibility of some of the early MS technologies. However, there have been significant contemporary multi-laboratory collaborative studies, sponsored by the U.S. National Cancer Institute (NCI) and the Human Proteome Organization (HUPO), which have examined the sources of irreproducibility in MS-based proteomics ,36. Thes13C instead of 12C, an 15N instead of 14N, or 2H instead of H. When incorporated into a peptide, the heavy amino acid leads to a known mass shift  compared to the peptide with the normal (light) version of the amino acid [SILAC was first described almost 10 years ago and is one of the most popular peptide labeling techniques to date. The general concept is that relative and absolute quantification of a sample of interest can be performed when comparing its MS intensity with that of a labeled peptide standard introduced into the sample. To create the standard, peptides are metabolically labeled in culture by introducing the isotope label into every protein during cell growth and division, generating a labeled standard for every protein. This is done by growing parallel populations of the same cell line, one in media containing a “light”  amino acid and the other in media containing a “heavy” amino acid. The heavy amino acid can contain a ino acid . In a siino acid , to idenino acid , and to ino acid ,41.An extension of the SILAC protocol has been the creation of a reference standard from a mixture of multiple SILAC-labeled cell lines (termed super SILAC) that better serves for quantifying proteins  in complc-MYC function in Group 3 medulloblastoma [c-MYC amplified versus nonamplified tumors were observed, suggesting significant metabolic pathway differences between these groups [Early proteomic profiling studies in medulloblastoma focused on biomarker identification in cerebrospinal fluid (CSF) samples from pediatric patients ,42. Possblastoma . Taking e groups .c-MYC is known to regulate the expression of these proteins [c-MYC-driven tumors and may help to explain the metastatic propensity of these tumor subtypes.Our proteomic results showing increased expression of ribonucleoproteins (HNRNPs) associated with spliceosome activity  is supported by earlier work looking for c-MYC-dependent proteins in the medulloblastoma cell line D425Med . Furtherproteins . Importac-MYC overexpressing tumors, and may therefore provide interesting insights into the factors regulating tumorigenesis [MicroRNAs (miRNAs) are essential regulatory factors in the development of the central nervous system and differentially expressed across different medulloblastoma subtypes ,51. EarlOCT4 and KLF4 [A recent study conducted a specific analysis of proteins, miRNAs, and genes to characterize human medulloblastoma stem-like cells (hMB-SLCs) . These hand KLF4 . Similarand KLF4 .PVT1–MYC gene fusions in Group 3 medulloblastomas [Emerging studies clearly highlight the differential expression of miRNAs in different medulloblastoma subgroups, and proteomic analysis is providing key insights into how these factors may be regulating specific oncogenic pathways. These are particularly important when dealing with genomic aberrations that occur in non-gene-coding regions, such as seen with c-MYC had higher DNA/RNA levels of this oncogene within exosomes compared to non-c-MYC-amplified cells [Exosomes are membrane-derived extracellular vesicles that are shown to influence various cellular interactions. Intriguingly, tumor cells produce exosomes that can significantly influence a number of cancer-related processes such as metastatic invasion, drug resistance, angiogenesis and cellular growth . Exosomeed cells . It is sed cells .Proteomics is a powerful tool to identify the functional role for exosomes in medulloblastoma. Initial studies characterized the structure and expression of exosomal proteins from the medulloblastoma cell line D283MED, and further demonstrated increased tumor cell proliferation and migration when these exosomes were added to medulloblastoma cell lines . The traIt is abundantly clear that multiple post-transcriptional factors significantly restrict direct correlations between genomic aberrations and resultant oncogenic behavior. In addition to this, exciting new research now reveals the significance of extracellular vesicular release (exosomes) in modulating cancer cell behavior, either through altering transcriptional pathways or directly through proteomic signaling mechanisms. In this regard, proteomics is shedding new light into the influence of miRNAs and the other exosomal components on tumor cell behavior and response to injury.Quantitative proteomics has undergone rapid technological improvement over the past decade. The majority of this growth has occurred in the context of tightly controlled experimental variables and has not yet been extensively tested in the arena of clinical tissue samples or biofluids. Applied proteomics carries the promise of visualizing the functional compartment of the cell and thus the interface of cellular biology and cellular bioprocesses helping to focus on the link between genetic changes and functional phenotypes and to identify potential therapeutic targets. Additionally, this compartment is rich in therapeutic opportunities. For example, studies of protein modifications, such as phosphoproteomics offer the ability to understand active signaling matrices and compartment-based proteomics  can identify antigens for targeted antibody- and cell-based immunotherapies. This approach provides a platform for understanding the biology of other cancers by providing a functional context to frame genomic abnormalities.Currently, the proteomics field struggles with some significant challenges such as the lack of bona fide housekeeping proteins for intercellular normalization, the dynamic range of detection necessary to quantitate the entire proteome across its naturally occurring abundance range, and the lack of sensitivity necessary to tackle intercellular tumor heterogeneity or rare biomarker detection . In cancer research, genomic discovery platforms are remarkably capable, yet the data they provide is removed from the functional biology they ultimately control. This fact creates a challenge in contextualizing the massive yield of findings. Viewing the genomic landscape through the lens of proteomics may provide the necessary context by identifying which genomic aberrations are translated to the functional compartment of the cell, thus helping to discern the essential genomic features of a tumor. A rapid expansion of applied quantitative proteomic capabilities is anticipated in the near future with implications for biological investigation, drug target identification, and biomarker discovery.


Steroids are a group of anti-inflammatory drugs, commonly used to treat ocular and systemic conditions. Unmonitored use of steroids especially in eye drop formulations is common in situations when it is easily available over-the-counter, resulting in undesirable side effects.Among the ocular side effects, cataract and glaucoma are common. Steroid-induced ocular hypertension was reported in 1950, when long-term use of systemic steroid was shown to increase the intraocular pressure (IOP). Chronic administration of steroids in any form with raised IOP can cause optic neuropathy resulting in steroid-induced glaucoma.This review describes the pathophysiology and epidemiology of steroid-induced glaucoma, recognition of side effects, and principles of management. The purpose is to familiarize all clinicians with the potential dangers of administering steroids without monitoring the eye and the dangers of irreversible blind -ness in some instances of habitual self-prescription by patients. Phulke S, Kaushik S, Kaur S, Pandav SS. Steroid-induced Glaucoma: An Avoidable Irreversible Blindness. J Curr Glaucoma Pract 2017;11(2):67-72. There are many instances where one can avoid the use of steroids and switch over to nonsteroidal/anti-inflammatory alternatives and where it is not possible, monitoring the IOP is essential irrespective of the dose and duration of the steroid use. The ocular hypertensive response is fairly reversible and if intervened at the right time can prevent vision threatening complication which is especially important in children.Corticosteroids are a class of anti-inflammatory drugs commonly used to treat various ocular and systemic conditions. The use of steroids can lead to significant ocular side effects. Intraocular pressure (IOP) elevation following steroid use is well- documented.Steroid-induced glaucoma is a form of open angle glaucoma. The precise mechanism for IOP elevation after steroid intake is not very clear, but primarily it occurs due to reduced facility of aqueous outflow. The following are proposed theories for steroid-induced raised IOP:4 Steroid causes stabilization of lysosomal membranes and accumulation of polymerized glycosamino-glycans (GAGs) in the trabecular meshwork. These polymerized GAGs become hydrated, producing "biologic edema" and increased outflow resistance.56 Ultrastructurally, in steroid-induced glaucoma, there is accumulation of basement membrane like material staining for type IV collagen.78Glucocorticoids also increases the expression of extracellular matrix protein fibronectin, GAGs, elastin, and laminin within the trabecular meshwork cells which leads to increased trabecular meshwork resistance.9Corticosteroids cause inhibition of phagocytotic properties of endothelial cells lining the trabecular meshwork which leads to accumulation of aqueous debris.10 Zhang et al11 have suggested that FKBP06-binding immunophilin FKBP51 mediates nuclear transport of the human glucocorticoid receptor beta, suggesting that this may play a role in increased glucocorticoid responsiveness. Glucocorticoids have been shown to alter the trabecu -lar meshwork cell morphology by causing an increase in nuclear size and DNA content.12 Recent case series has shown that trabecular outflow obstruction can occur due to crystalline steroid particles after receiving intravitreal triamcinolone ace-tonide (IVTA) injections for diabetic macular edema.13 Glucocorticoid decreases the synthesis of prostaglandin, which regulates the aqueous outflow.Genetic influence: Several genes have been found to be associated with both protective and damaging glucocorticoid-treated trabecular meshwork cells. Glucocorticoids may exert their effect by increased expression of MYOC [Trabecular meshwork-induced glucocorticoid response (TIGR)] gene at locus GLC1A.13 However, recent studies in monkeys failed to show a significant link between myocilin gene and steroid-induced ocular hypertension.14 Other than myocilin gene alpha1 antichymotrypsin, pigment epithelium-derived factor, cornea-derived transcript 6, prostaglandin D2 synthetase, growth arrest specific 1, decorin, insulin-like growth factor binding protein 2, ferritin light chain, and fibulin-1C are other genes which have been postulated to play a role in steroid responsiveness but more studies are required to confirm their role in steroid responsiveness.171819Steroid-induced IOP elevation can occur in any age group. Most studies describing steroid-induced glaucoma have focused on adults. Children are also known to have a severe ocular hypertensive response to topical steroids when compared to adults and significant IOP elevation has also been reported in infants treated with nasal and inhalational steroids.20 Increasing trends of steroid-induced glaucoma in children over the last few years have been reported, probably signifying increasing use of unmonitored steroid use.21In a previously reported study, the authors found steroid-induced glaucoma to account for one-fourth of all acquired glaucomas in children.22 and Armaly,25 they categorized the general population into high, intermediate and low response according to their pressure response to topical steroid drops 26 Normal individuals classified as high steroid responders are more likely to develop POAG. First degree relatives of POAG patients are also considered to have a higher risk of being a steroid responder. – Approximately 30% of glaucoma suspects and 90% of POAG might have an ocular hypertensive response to a 4-week course of topical dexametha-sone 0.1% .29 or eyes with history of penetrating keratoplasty or refractive surgeries like photorefractive keratectomy,30 laser in situ keratomileusis (LASIK),31 and Descement’s Stripping Endothelial Keratoplasty (DSEK).32 Steroid-induced glaucoma after refractive surgery is masked by falsely low IOP measurement due to thin central corneal thickness, ocular rigidity changes, corneal edema, and fluid accumulation beneath the LASIK flap3334 High myopia Very young (<10 years of age) and older adults 35 or connective tissue disease 36 Diabetes mellitus37 or traumatic angle recession38 Eyes with pigment dispersion syndrome39 Endogenous hypercortisolism. Exogenous corticosteroids are more likely to cause IOP elevation. However, endogenous corticosteroids can also cause this condition, as enumerated in Topical therapy: IOP rise after steroid therapy occurs more frequently with topical administration than systemic administration. The IOP rise may occur with drops or ointment applied directly to the eye or over the skin of the eyelids.4041Periocular therapy: Periocular injections of repository corticosteroid is the most dangerous form of steroid because of their prolonged duration of action.42 Intraocular pressure elevation may occur in response to subconjunctival, subtenon, or retrobulbar injection of steroids.43 A patient’s response to earlier topical steroid therapy does not predict their response to periocular steroid.44Intravitreal therapy: Intraocular pressure elevation with intravitreal depot steroid has been reported in a large percentage of patients. Ozurdex is a slow release intravitreal implant of dexamethasone used in the treatment of macular edema secondary to vein occlusions and for the treatment of uveitis. Previous studies have indicated that >10 mm Hg IOP increase from baseline occurs in 12.6% of patients after the first treatment and 15.4% after the second dose of Ozurdex. As dexamethasone-induced ocular hypertension is transient and it is a more water-soluble steroid than triamcinolone or fluocinolone acetonide, IOP may be better controlled without causing any ocular complica-tions.45 Intravitreal injection of triamcinolone on the contrary can increase IOP by several mm Hg in about 50% of patients, within 2 to 4 weeks after the start of treatment and even may rise more rapidly in some eyes .4647 Smithen reported that among the nonglaucoma patients treated with intravitreal triam-cinolone, 60% of patients with baseline IOP >15 mm Hg showed a rise in IOP following injection compared to 22% of those who had baseline IOP <15 mm Hg.4849 However, if the pressure rises, this response does not correlate with the dosage or duration of treatment.50 Systemic therapy of steroids is the least likely route to causing IOP elevation.Another routes: There exists a strong association between inhaled corticosteroid and the presence of ocular hypertension and the risk increased with higher dose and more puffs in patients with family history of glaucoma.5152 However, the pressure-inducing potency is related to the dosage of the drug used .53In general, the pressure-inducing effect of steroids is directly proportional to its anti-inflammatory potency.In steroid responsive patients, IOP elevation usually develops within the first few weeks of steroid administration. However, it can be elevated within an hour or many years after chronic steroid use. After steroid is discontinued, IOP usually normalizes within 1 to 4 weeks.54 Long-term study by Bonini et al have reported 2% incidence of steroid-induced glaucoma in VKC.55Patients usually present with very few symptoms like that of POAG. Signs and symptoms vary with the age of the patients. Infants may present with features of primary congenital glaucoma like watering, blepharospasm and photophobia. Teenagers present like developmental or juvenile open angle glaucoma. Adult patients present with raised IOP, normal and open angles on gonioscopy, optic disk cupping, and visual field defects. Other ocular findings are increased corneal thickness, corneal ulcers, and posterior subcapsular cataract. Steroid-induced glaucoma in vernal keratoconjunctivitis (VKC) is a common complication as patients require long-term treatment and steroids are often used to provide relief of symptoms. Primary open angle glaucoma Normal tension glaucoma Juvenile open angle glaucoma Uveitic glaucoma Glaucomatocyclitic crises56 Discontinuation of the use of the steroid is the first line of management. In the majority of cases, steroid-induced acute rise of IOP typically normalizes within days of stopping the steroid and chronic forms take 1 to 4 weeks. In rare cases, IOP remains elevated, for which antiglaucoma medications or surgery may become necessary. The duration of steroid therapy also appears to influence the reversibility of the IOP elevation.57 Fluorometholone 0.25% is also less likely to increase IOP in steroid respond-ers, but increased concentration does not appear to enhance the drug’s anti-inflammatory effect.58 Sometimes steroids cannot be withheld in view of sys -temic need of patients, in such cases corticosteroids like betamethasone, prednisolone, and dexametha-sone can be substituted with nonadrenal steroids like rimexolone, loteprednol etabonate, fluorometholone, and medrysone. These drugs have useful anti-inflammatory properties with significantly less IOP elevation effect. Fluorometholone 0.1% is more effective in treating anterior segment ocular inflammation; however, significant IOP elevation has also been observed with this drug. Use of repository steroid is particularly dangerous due to their prolonged duration of action. It is better to inject this depot in the inferior quadrant and in anterior location to preserve the superior site for future glaucoma filtering surgeries, if needed in future.4446 If it is not possible to excise them, filtering surgery is advised to control IOP. Glaucoma due to intravitreal triamcinolone injections is medically managed, although role of pars plana vitrectomy has also been documented.59 Depot steroid-induced IOP elevation can be effectively controlled after excision.63 Another treatment modality could be substituting with steroid-sparing agents or topical non-steroidal anti-inflammatory drugs (NSAIDs) like flurbipro-fen, bromfenac, ketorolac, and nepafenac. These NSAIDs primarily act as cyclooxygenase inhibitors, may be effective in anterior segment ocular inflammation.Glaucoma therapy: When medical management fails to control the IOP, selective lasers or argon laser trabeculoplasty can be tried in, particularly in eyes treated with intravitreal triamcinolone.6465 Anterior subtenon injection of anecortave acetate, which is a synthetic derivative of cortisol used in the treatment of age-related macular degeneration,66 has shown to be effective in reducing IOP in these patients.67 Surgery like trabeculotomy,68 trabeculectomy with or without antimetabolites or glaucoma drainage device are reserved for medically uncontrolled or intractable glaucoma.Steroid-induced glaucoma is an iatrogenic disease, which can be prevented. Increased use of steroids for almost any ocular as well as extraocular pathology has led to their overuse. An increasing amount of cases with steroid-induced glaucoma reported in the literature have made this a global issue. There is an enormous disability caused by steroid-induced glaucoma in patients of all age groups. Cheap and easily available dexamethasone eye drop over the counter forms the primary prescription of many ophthalmologists and chemists. Use of such easily available steroids reduces the symptoms of the primary pathology, leading to a sense of betterment, which usually results in patients continuing to use steroids unmonitored over long periods of time. Steroid-induced glaucoma is a significant health problem in the pediatric age group, but it responds well to the withdrawal of steroids and medical treatment. The most effective way of treating this ocular hypertensive response appears to be the cessation of the steroid therapy. Self-medication should be avoided and discouraged under all circumstances. Wherever possible, other alternatives like immunosuppressant should be used where the risk of steroid-induced ocular hypertension or glaucoma is suspected.Steroid-induced glaucoma is an iatrogenic and preventable disease. The unwarranted and irrational use of steroids especially in developing countries by local medical practitioners as well as unmonitored self-use by patients themselves points to a lack of awareness about the disease. Prevention of steroid-induced glaucoma can be achieved with a few simple precautions. Identification of risk factors  and monitoring for ocular hypertension can decrease the development of irreversible glaucomatous optic neuropathy. For discouraging self-medication, monitoring for IOP after prescription of steroids in any form and prompt management is essential. These practical and safe guidelines for the use of steroids should be followed by all doctors.


Chemoresistance is the main challenge for the recurrent ovarian cancer therapy and responsible for treatment failure and unfavorable clinical outcome. Understanding mechanisms of chemoresistance in ovarian cancer would help to predict disease progression, develop new therapies and personalize systemic therapy. In the last decade, accumulating evidence demonstrates that epithelial-mesenchymal transition and cancer stem cells play important roles in ovarian cancer chemoresistance and metastasis. Treatment of epithelial-mesenchymal transition and cancer stem cells holds promise for improving current ovarian cancer therapies and prolonging the survival of recurrent ovarian cancer patients in the future. In this review, we focus on the role of epithelial-mesenchymal transition and cancer stem cells in ovarian cancer chemoresistance and explore the therapeutic implications for developing epithelial-mesenchymal transition and cancer stem cells associated therapies for future ovarian cancer treatment. Ovarian cancer (OC) is the leading cause of mortality from cancers of female reproductive tract in the world. Epithelial ovarian cancer (EOC) accounts for nearly 90% of all ovarian malignant diseases. EOCs fall into four main subtypes: serous, mucinous, endometrioid, and clear cell. In 2012, OC accounted for 151,900 deaths worldwide and there were 238,700 patients diagnosed with OC, according to the latest GLOBOCAN estimates [For the early stage OC patients, surgery can completely remove tumor and then give patients a full recovery. For the advanced stage OC patients, cytoreductive surgery followed by platinum/taxane acts as a standard therapy, resulting in a 75% high initial response rate . AlthougChemoresistance in cancer chemotherapy is a complicated process and affected by many factors. Although the exact mechanisms of OC chemoresistance and metastasis are still unclear, accumulating evidence supports that epithelial-mesenchymal transition (EMT) and cancer stem cells (CSCs) play critical roles in the development of resistance to chemotherapy, tumor relapse and metastasis of OC patients –7. UnderEMT is a process by which epithelial cells assume mesenchymal characteristics, facilitating migration through the extracellular matrix and settlement in areas of new organ formation during embryogenesis. This cell switch is known to be integral in development, wound healing and stem cell behavior, and contributes pathologically to fibrosis and cancer progression . EMT occDuring cancer progression, EMT appears to promote dissemination of cells from the tumor mass  and faciin vitro can induce chemoresistance and EMT. It was reported that continuous exposure to increasing doses of paclitaxel induced chemoresistance as well as EMT, and enhanced metastasis potential in EOC cells  [Increasing evidence demonstrates that there is a close relationship between EMT and chemoresistance in OC. Treatment with chemotherapeutics  SKOV-3) . Similar SKOV-3) . After t SKOV-3) .EMT markers and transcription factors are in correlation with chemoresistance in OC. In one study, up-regulation of EMT-related transcription factors Snail, Slug, Twist2 and Zeb2 in gene level and Snail, Slug, Vimentin in protein level was found in cisplatin resistant EOC cell line A2780-cis compared with cisplatin sensitive EOC cell line A2780 using gene expression and proteomic analysis, respectively . Using 1The potential mechanisms of EMT in OC chemoresistance are still not fully uncovered. Accumulating evidence from preclinical and human tissue studies indicates that several important signaling pathways may contribute to OC chemoresistance via EMT, resulting in tumor metastasis and recurrence after chemotherapy. Different EMT-related signaling pathways associated with OC chemoresistance are summarized in Table n = 72), it was demonstrated that patients without complete response (CR) to paclitaxel-based chemotherapy had lower miR-200c levels than patients with CR, additionally, low miR-200 family  expression had a trend toward poor PFS [In addition to signaling pathways, microRNAs (miRNAs) also play a significant role in EMT in OC chemoresistance, among which miR-200 family is the most important one. The aberrant expression of miR-200 family  in OC and its involvement in EMT were well-demonstrated , illustrpoor PFS . In a repoor PFS . Followipoor PFS . The putAll findings suggest that EMT is closely linked with OC metastasis, chemoresistance and progression, which is regulated by different signaling pathways and microRNAs, and that identification of main signaling pathways and new miRNA associated with EMT is undoubtedly an important step to develop new modalities to prevent metastatic, recurrent OC and improve the clinical outcomes of OC patients.Due to serious side effects of chemotherapy, targeted therapy plays a very important role in the current OC treatment. Accumulating evidence indicates that targeting EMT is a new developing research area and holds promise for chemoresistant OC patients in the future.in vitro and inhibit cisplatin-induced migration in zebrafish embryos in vivo [in vitro and intra-peritoneal mouse xenografts in vivo [Developing cancer cell signaling pathway treatment is an important area for EMT-targeting therapy. In OVCA433 EOC cell line and tumors collected from advanced-stage EOC patients, ERK2 signaling pathway was found to be critical to cisplatin-induced EMT and CSC phenotypes. It was reported that the ERK inhibitor U0126 could block cisplatin-induced ERK2 activation, and sensitize the residual cells to increase cell death in response to cisplatin  in vivo . Hyperac in vivo .in vitro and in vivo. Most importantly, both epigenetic modifiers increased the sensitivity of the highly chemoresistant EOC cell line HEY to cisplatin, suggesting their promising application in OC chemoresistance treatment [Epigenetic aberrations, including DNA methylation, histone modifications, and miRNA dysregulation also contribute to the EMT in OC. Therefore, reversing these changes has significant benefits as alternatives to traditional cytotoxic therapy. For example, epigenetic silencing of SFRP5 was observed to lead to oncogenic activation of the Wnt pathway and contribute to OC chemoresistance through the Twist-mediated EMT and AKT2 signaling, and the overexpression of SFRP5 inhibited EMT, down-regulated AKT2 and sensitized OC cells to chemotherapy, indicating the treatment promise of target reactivation of epigenetically silenced gene . Trichosreatment .in vitro and in vivo [n = 486) and Bagnoli (n = 130) datasets [miRNAs have also shown their treatment promise in OC. Zhu et al demonstrated that miR-186 was detected to be associated with EMT and cisplatin-resistance in EOC cell lines  and human EOC tissues (cisplatin-sensitive tumors vs cisplatin-resistant tumors), and the introduction of miR-186 to EOC cells led to reduction of Twist1 as well as EMT, and rendered the cells more sensitive to cisplatin  in vivo . Similardatasets . As a reIn terms of the application of EMT marker targeting, Chiu et al illustrated that FOXM1 was a critical regulator of the EMT, stemness, and chemoresistance in EOC cells, and the combination of FOXM1 inhibitor thiostrepton and cisplatin inhibited the expression of EMT related markers  in chemoresistant A2780CP70 cells and growth of ovarian xenograft tumors in a subcutaneous (s.c) mouse model, indicating the promising application of EMT targeted therapy for future EOC treatment .in vitro using EOC cell lines , and sensitized orthotopically implanted ovarian tumors to a low dose of cisplatin in vivo on athymic nude female mice [Some other small molecular inhibitors have also demonstrated the ability to reverse EMT and potentially increase chemosensitivity in OC. Using a gold nanoparticles (AuNPs), Xiong et al reversed cisplatin-induced chemoresistance by inhibiting EMT ale mice . TargetiAll findings support that targeting EMT is a bright research area to prevent OC chemoresistance and it is worthwhile developing combination approaches in the future to improve current modalities. However, we need to realize that although these agents can reverse OC chemoresistance for combination therapy, they have very broad activities or targeting signaling pathways that are not limited to the EMT targeting. Therefore, when choosing these agents for combination therapy, we should maximise their anti-EMT effects and avoid their side effects.Although there exists a long-lasting debate regarding the origin of CSCs, it is widely accepted that tumors are composed of phenotypically and functionally heterogeneous cells and CSCs are only a small subset of tumors cells. The stochastic model, hierarchy model and the dedifferentiation model are the three major theories as to how CSCs arises.The earlier CSC model is a static one. According to stochastic model, tumor cells are biologically equivalent but behave variably due to stochastic influences (intrinsic and extrinsic factors). The core of this theory is that behaviors of tumor cells cannot be predicted and every tumor cell is thought to have the potential to behave the activity of CSCs . The hieAccumulating evidence indicates that CSCs have close relationship with OC progression, metastasis, therapeutic resistance and tumor recurrence. The concept of CSCs has opened new areas of research in carcinogenesis, but has more immediate translational potential of uncovering new treatment targets.Ovarian CSCs (OCSCs) have been isolated from established OC cell lines, ascites, and primary and metastatic tumors –45. Theyin vivo, indicating that both CD44 and CD117 positive cells are OCSCs . Besides, they also found that CD44+/CD117+ cells had increased chemoresistance to taxane and platinum-based chemotherapy as well as the ability to self-propagate [+ cells were enriched in the ascites of OC patients, and the isolated CD44+ cells in mouse xenograftes gave rise to tumors with both CD44+ and CD44− cells, suggesting that those CD44+ cells can differentiate and self-renew with the feature of cancer-initiating cells, in addition, CD44+ EOC cells were demonstrated to be more resistant to paclitaxel and carboplatin compared with CD44− cells.[CD44 is a cell-surface glycoprotein involved in cell-cell interactions, cell adhesion and migration. The multiple protein isoforms are encoded via a single gene by alternative splicing and are further modified by a range of post-translational modifications (PTMs) . CD44 waropagate . Similar4− cells..+ OC cells appeared to be highly tumorigenic than CD117− OC cells as it only took approximately 103 cells to be able to self-renew, differentiate, and regenerate tumor in mouse models, and CD117 expression was statistically correlated with resistance to conventional chemotherapy [CD117, also known as c-Kit or stem cell growth factor receptor, is a proto-oncogene encoded by the KIT gene. It is a type of tyrosine kinase receptor involved in cell signal transduction and has been shown to be involved in apoptosis, cell differentiation, proliferation, and cell adhesion . CD117 wotherapy . Anotherotherapy , furtherin vitro cell lines, in vivo animal xeongrafts and human primary tumor experiments. It was reported that CD133+ A2780, PEO1 EOC cells generate both CD133+ and CD133− daughter cells, exhibiting enhanced resistance to platinum-based therapy in vitro and sorted CD133+ OC cells from A2780 line formed more aggressive tumor xenografts at a lower inoculum than their CD133− progenies on BALB/cAnNCr-nu/nu mice [+ cells from human primary OC tissues have an increased tumorigenic capacity and are capable of recapitulating the original heterogeneous tumor in NOD/SCID mice compared with CD133− cells [CD133, a pentaspan membrane glycoprotein, has been identified as a CSC marker for various cancers . In EOC,/nu mice . Curley 3− cells .− cells, CD24+ cells possesses stem-cell like characters with relative quiescence, self-renewal, differentiation, chemoresistance in vitro, and a xenograft injection of 5000 CD24+ cells produced tumors in nude mice, while injection of an equal number of CD24− cells failed to do so [+ OC cells were enriched for cancer-initiating cells, and CD24+ cells have increased tumor sphere-forming capacity and play a role in tumor migration and metastasis [+/CD24− phenotype in OC cells demonstrateCSC-like properties of enhanced differentiation, invasion, and resistance to chemotherapy and correlation to clinical endpoints with increased risk of recurrence and shorter PFS duration in patients with OC [− cells, if expanded from a singly sorted cell, may give rise to cells containing all of the markers, including CD24 [CD24, the cell surface protein, is highly expressed in many human cancers . It was to do so . Using atastasis . However with OC . Gunjal ing CD24 . Based o+CD133+ CSCs isolated from human ovarian tumors were sufficient to initiate tumors in mice and that the presence of ALDH+CD133+ cells in debulked primary tumor specimens correlated with reduced disease-free survival (DFS) and OS in OC patients, indicating that both ALDH and CD133 can be used as a functionally significant set of markers to identify OCSCs [+ CSCs might have increased Nrf2-induced antioxidant scavengers, which lower ROS level relevant to chemoresistance in ovarian clear cell carcinoma [ALDH enzymes belongs to a family of evolutionarily conserved enzymes comprised of 19 isoforms that are localized in the cytoplasm, mitochondria or nucleus. Generally, ALDHs are generated by a wide variety of metabolic processes and are responsible for oxidizing aldehydes to carboxylic acids . ALDH wafy OCSCs . Liu et fy OCSCs . Similararcinoma . In anotarcinoma . These d+/CD24+/EpCAM+/E-cadherin− subpopulation of cells from EOC cells (SKOV-3 and OVCAR-5) using flow cytometry, and found that this small population of cells (less than 1%) has increased colony formation and shorter tumor-free intervals on a mouse s.c xenograft model in vivo after limited dilution, showing initiating tumor cell properties, and is resistant to both doxorubicin and cispation therapies [EpCAM , is a glycosylated, 30~40 kDa type I transmembrane protein, which was reported to be expressed on essentially all human adenocarcinoma, and is part of the signature of CICs in numerous solid tumors as well as normal stem cells . EpCAM hherapies . By evalherapies . These rUsing existing CSC makers to define OCSC is an important step to uncover OC chemoresistant mechanisms, find useful therapeutic targets and develop new treatment modalities to cure metastatic, recurrent OC. However, none of these current CSC markers are exclusively expressed by OC tissues, highlighting that it is imperative to use combinatorial makers or delineate more specific markers and techniques to detect OCSCs.® DyeCycleTM Violet [In addition to cell surface OCSC markers, side population (SP) cells also play an important role in identification of OCSCs. SP cells can be separated by using the fluorescent DNA-binding dye such as VybrantM Violet , and HoeM Violet , dependiM Violet . The SP M Violet .Br) overlapping population is a promising approach to isolate highly purified CSCs/CICs from OC cells and demonstrated this population exhibited higher tumor-initiating ability than that of SP cells or ALDH Br cells alone, enabling initiation of tumor with as few as 100 cells and showing higher sphere-forming ability, cisplatin resistance, adipocyte differentiation ability and expression of SOX2 than those of SP/ALDHLow, MP (main population)/ALDHBr and MP/ALDHLow cells [The studies from different groups have identified and isolated SP of OC cells with typical “CSC phenotype”  , 73–74. ow cells . SP popuow cells  Using Hoow cells . These rDue to the importance of CSCs and the limitation of the current techniques to effectively detect CSCs, some new approaches are required to separate SP cells possessing CSCs properties for an in-depth investigation. All putative OCSC markers, origin and tumorigenicity in mice are shown in Table The CSC theory has offered a potential explanation for the relapse and resistance that occur in many tumors including OC after therapy . CSCs pr+ cells from OC cell lines  and patient ascites samples increased chemoresistance, and that patient ascites samples with > 25% CD44+ cells had significantly decreased median PFS  as well as propensity to recur  [in vitro and in vivo experiments, they also observed the overexpression of CD44 in chemoresistant cell lines , and the knockdown of CD44 by shRNA could significantly increase the sensitivity of OC cells (OVCAR8) to paclitaxel [Meng and colleagues demonstrated that the increased numbers of CD44= 0.003) . Through= 0.003) . Similarclitaxel .+ and higher grade ovarian tumors, advanced stage disease, and decreased response to chemotherapy in 400 OC primary tissue samples, demonstrating that CD133+ tumors are associated with decreased OS and shorter disease free interval [+ cells were found to be correlated with decreased PFS [Zhang et al found associations between CD133interval . In a meinterval . Using ainterval . Based ointerval . It was ased PFS . Zhu et ased PFS . These d+CD133+ OCSC-like cells from human primary ovarian tumors showed increased chemoresistance compared to their parental cells and the presence of ALDH+CD133+ cells in debulked primary tumors correlated with reduced disease-free and OS in OC patients [in vitro and in vivo [Increasing number of studies have demonstrated that CSCs may confer growth advantage and metastasis in chemoresistant ovarian tumors , 45. Silpatients . Cole etpatients . Abubake in vivo . We haveThe mechanism of CSCs in OC chemoresistance and recurrence is complex and not fully understood. It is possible that decreased chemotherapy responsiveness of CSCs may be partly due to the slow proliferation rate, cell cycle arrest, the high expression of ATP transporters, efficient DNA protection and repair mechanisms, the activation of some CSC-related signaling pathways, inactivation of cell death pathways, and the inherent epigenetic aberrations. The putative mechanisms of CSC in OC chemoresistance are shown in Figure + CSC cells were relatively quiescent and enriched at the S phase, leading to the chemotherapeutic resistance to cisplatin treatment compared with CD24− OC cells from human ovarian tumor specimens [It was reported that conventional chemotherapeutic treatment, which usually targets fast-dividing cells and acts in a cell-cycle specific manner, entitles CSCs the survival advantage because of their slow proliferation rate . Gao et pecimens . Abubakepecimens .CSCs are known to possess highly elaborated efflux systems for cytotoxic agents, of which ABC family of membrane transporters are the most important ones. In return, there is strong collective evidence that increased expression and the activity of ABC family of membrane transporters, especially ABCG2, also correlates with cancer stem-like phenotype . Ricci ein vivo [SP is also found to be related to OC chemoresistance via ABCG2. Kruger et al. demonstrated that SP cells from two murine carcinoma cell lines exhibited up-regulation of ABCG2 and stem cell markers Wnt-1 and Sca-1, increased resistance to chemotherapy, increased efflux of chemotherapeutic agents and increased ability to generate tumors in vivo .in vivo and in vitro [Moreover, up-regulated DNA protection and repair and inactivation of apoptosis may also be responsible for chemoresistance in OCSCs. Srivastava et al reported that an elevated expression of DNA polymerase η (Pol η) was observed in OCSCs isolated from both OC cell lines and primary tumors, and down-regulation of Pol η enhanced the cisplatin-induced apoptosis in CSCs both in vitro , indicatin vitro .In summary, there are many mechanisms involved in the OCSC-associated chemoresistance. Research efforts should be put in uncovering these mechanisms in the future in order to pave the way for OCSCs targeting therapy.CSCs are implicated in cancer metastasis, recurrence, and therapeutic resistance. Targeting CSCs may possess many advantages by eradicating the root of tumor and managing their malignant behaviors. However, the data related with effective targeting strategies for CSCs are still limited until now. The current CSC targeting therapy in OC are mainly focused on the employment of OCSCs markers and the signaling pathways related to CSCs.Imatinib (Gleevec), a clinical drug that blocks Abelson cytoplasmic tyrosine kinase (ABL), CD177, and PDGFR, demonstrated its inhibition potency on OCSCs. In this study, the combination of imatinib with cisplatin/paclitaxel showed higher efficiency against the regeneration of drug-resistant OCSCs compared to imatinib or cisplatin/paclitaxel alone , indicatin vitro and animal xenograft models in vivo, leading to a reduced tumor burden [In addition, some signaling pathway inhibitors preferentially targeting CSCs are also useful in reversing chemoresistance. VS-5584, a dual inhibitor of mTORC1/2 and class I PI3-Kinase, was reported to be up to 30-fold more potent in inhibiting the proliferation and survival of CSCs compared with non-CSC in OC and other solid tumor cells, and most importantly, VS-5584 was also able to significantly repress tumor regrowth after chemotherapy in an immunodeficient OC mouse model . In anotr burden . Ibrutinr burden . Furtherr burden . We haveHOXA10, therefore decreasing tumor initiating capacity and re-sensitizing CSCs to platinum. Moreover, the maintenance treatment with SGI-110 after carboplatin was also shown to inhibit CSC growth and decrease tumor progression in animal models in vivo [+ EOC cells, including their tumor-initiating capacity, resensitized these OCSCs to platinum, and induced re-expression of a differentiation-associated gene in mRNA level (HOXA10 mRNA) [Some emerging novel strategies have also demonstrated anti-CSC effects for improving OC chemoresistance. Donahoe et al demonstrated that mullerian inhibiting substance (MIS mimetic SP600125) preferentially inhibited chemotherapy-enriched CSC-like cells in human EOC cell lines including OVCAR-5, IGROV-1 and SKOV-3 and provided a new treatment paradigm . SGI-110 in vivo . Wang et10 mRNA) .in vitro. Furthermore, combination treatment using cisplatin and SR-T100 was more effective in inhibiting the tumor growth of A2780CP70 cells in mouse xenografts than either therapeutic alone, suggesting SR-T100 may have potential against chemoresistant OC cells [Wu et al recently reported that solanum incanum extract (SR-T100), containing the active ingredient solamargine, can down-regulate the expression of stem cell markers, including ALDH1, Notch1, and FoxM1, and reduce sphere formation in OC cells, increase the sensitivity of chemoresistant OC cells (A2780CP70) to cisplatin and paclitaxel OC cells . Our groOC cells –104. AptCSC markers are very dynamic and plastic during cancer progression. CSC phenotypes are regulated by tumor microenvironment. When designing a CSC targeting therapy, the CSC plasticity should be taken into consideration. It is important to determine the CSC expression at different stages of cancer. In addition, targeting several CSC markers may achieve a better clinical result than only targeting one CSC marker.Chemotherapy remains an important modality for advanced OC treatment with ongoing efforts towards designing new treatment modalities and techniques, which continue to improve the survival and quality of life in OC patients. However, chemoresistance and tumor recurrence are the major challenges for the current treatment failure. It is increasingly clear that EMT and CSCs play important roles in OC metastasis and progression and are related with chemoresistance, and targeting EMT and CSCs or their corresponding pathways by gene therapy, antisense therapy, specific inhibitor, aptamer-guided therapy or other methods may enhance the chemosensitivity of OC. The recent advances in EMT and CSCs have unlocked a new avenue for chemosensitivity research. Elucidating the role of EMT and CSCs in the cancer cells' response to chemotherapy will enhance our understanding of OC recurrence after chemodrug treatment, and may direct research towards novel and specific chemosensitizers that target EMT or CSCs. We expect that there will be increased understanding of the intrinsic and extrinsic factors that control the plasticity and maintenance of the EMT/CSC state  in OC research.Since EMT and CSCs have both been implicated in tumourigenesis and chemoresistance, it is critical to examine both populations and determine their phenotypic expression in human OC recurrent tissues in order to develop strategies to target these populations using targeting therapy. Therefore, further evaluation of EMT/CSC markers in OC tissues and investigation on how these cells can be targeted by novel treatment modalities might lead to more effective eradication of OC relapse.EMT and CSC markers could be also used as important biomarkers for chemotherapy response and prognosis. If these biomarkers for predicting the treatment response of individual OC patient and potential targets for chemosensitization are further validated, it will achieve a more favorable therapeutic ratio in clinics. In addition, future personalized medicine for OC therapy will be developed from EMT and CSC research.Studying the mechanisms of OC chemoresistance and metastasis is important for novel therapies. Chemoresistant mouse models or other contemporary models such as patients-derived xenografts (PDXs) or explanted human tissues from chemoresistant OC patients should be considered for new drug screening and testing. Combination of chemotherapy with novel agents targeting EMT/CSCs for improvement of chemosensitivity has demonstrated promise in preclinical studies on OC cell lines and animal models. However, only very limited data could be found in Phase I trials. These combination approaches should be further explored in clinical trials to validate their clinical effects in OC therapy.


Alphaviruses are arthropod-borne viruses and are predominantly transmitted via mosquito vectors. This vector preference by alphaviruses raises the important question of the determinants that contribute to vector competence. There are several tissue barriers of the mosquito that the virus must overcome in order to establish a productive infection. Of importance are the midgut, basal lamina and the salivary glands. Infection of the salivary glands is crucial for virus transmission during the mosquito’s subsequent bloodfeed. Other factors that may contribute to vector competence include the microflora and parasites present in the mosquito, environmental conditions, the molecular determinants of the virus to adapt to the vector, as well as the effect of co-infection with other viruses. Though mosquito innate immunity is a contributing factor to vector competence, it will not be discussed in this review. Detailed understanding of these factors will be instrumental in minimising transmission of alphaviral diseases. The term arbovirus collectively describes viruses which are transmitted by arthropod vectors, and include alphaviruses such as chikungunya (CHIKV), o’nyong-nyong (ONNV), Ross River (RRV) and Barmah Forest (BFV) viruses. Arboviruses require a host for replication  and a vector (mainly mosquitoes) for transmission to other susceptible organisms . In the Aedes species mosquitoes, and human outbreaks are uncommon . Sa. Sa15]. The mosquito’s potential to cause disease can be estimated via mathematical models of vectorial capacity ,19,20. TThough vectorial capacity and vector competence are both terms used to describe the vector’s potential to cause disease, the ability of the mosquito to become infected and successfully transmit the virus following ingestion of a bloodmeal is known as vector efficiency or vector competence ,13,15. TAfter a bloodmeal from an infectious individual, the ingested virus must be able to replicate in the mosquito midgut epithelium, and subsequently the salivary glands, in order for successive infection to a vertebrate host  12,21].,21.12,21Aedes aegypti infected with CHIKV intrathoracially. Using immunofluorescence and electron microscopy techniques, they observed that the virions were associated with the basal lamina in sugar-fed mosquitoes and were unable to infect the midgut epithelium [Once in the midgut epithelium, the virus replicates and escapes into the haemocoel, by crossing the midgut escape barrier (MEB) into the basal lamina ,21,25,26ithelium . In mosqithelium .Once the virus crosses the MEB, it enters the haemocoel, an open body cavity in which haemolymph circulates. Haemocytes are the immune cells present in the mosquito’s open circulatory system and together with haemolymph circulation, are crucial components for viral dissemination . Within Anopheles stephensi is surrounded by basal lamina that prevent binding of virus to cell surface receptors, thus acting as a barrier to prevent infection [Aedes aegypti as early as four days post-infection [Aedes albopictus [Aedes aegypti as soon as two days after oral exposure to the virus. However, a minimum of one week is required for virus to be released and detected in saliva, indicating absence of salivary gland escape barrier and presence of salivary infection barrier [Salivary glands are a crucial and final physical barrier that is important for effective transmission of arboviruses. The ability of the virus to traverse to the salivary glands varies depending on the virus strain and mosquito species. The salivary gland of nfection . The timnfection . Sindbisbopictus . When a bopictus ,35,36. Fbopictus ,37. The bopictus ,30. The bopictus  and viri barrier .Mosquitoes are frequently infected with parasites that may play a role in alphavirus-mosquito interactions . MicrofiAedes mosquitoes were allowed to feed on gerbils co-infected with Brugia malayi microfilaria and Eastern equine encephalistis virus (EEEV). The authors found decreasing levels of microfilarial enhancement of EEEV in Aedes taeniorhynchus, Aedes aegypti and Aedes triseriatus respectively and hypothesised that this may be due to differences in the amount of blood leaked into the hemocoel upon microfilarial penetration [Brugia malayi microfilaria used, the level of microfilarial enhancement was greater compared to the earlier study with EEEV [Although microfilarial enhancement appears to be dose-dependent, the levels of enhancement also varies among mosquito species . In a stetration . Consequetration . Despiteith EEEV . The mosith EEEV .Brugia malayi microfilaria, allowing it to be transported across the midgut epithelial barrier and suggesting this as the main mechanism for microfilarial enhancement [In a recent study done by the same group, it was found that EEEV and VEEV binds to ancement . Compariancement . Anotherancement .Wolbachia bacteria which has been extensively studied for the potential use in mosquito and virus transmission control. This bacteria is naturally present in several mosquito species which are vectors of human pathogenic viruses such as Aedes albopictus, also known for its wide geographical distribution [Aedes aegypti has not been found to be naturally infected with Wolbachia [Aedes aegypti can be infected with Wolbachia, despite the lack of evidence of natural infection [Aedes aegypti infected with the wMel strain of Wolbachia have been shown to have reduced vector competence for CHIKV [wMel Wolbachia infection did not affect the lifespan of the mosquitoes, suggesting that this is a possible means of minimising CHIKV transmission [Aedes aegypti from Singapore found reduced infections of CHIKV in wMel-carrying mosquitoes as compared to wild-type mosquitoes [wMel-carrying mosquitoes in this study were found to have CHIKV replicating in their midguts, the number of mosquitoes positive for CHIKV in their salivary glands was minimal, suggesting the role of wMel in preventing CHIKV dissemination to the salivary glands and blocking virus transmission [Wolbachia clades wAlbA and wAlbB in Aedes albopictus from several different geographical regions in Malaysia [Wolbachia colonisation did not inhibit CHIKV replication, and hypothesised that this may perhaps explain how this mosquito strain is able to serve as a competent vector for CHIKV [Wolbachia, which have lower vector competence, could displace the existing Wolbachia populations found in the mosquitoes [Another example of parasites that play roles in alphavirus infection is the ribution . Interesolbachia ,43,44. Nnfection . Aedes aor CHIKV . The autor CHIKV . They alsmission . A similMalaysia . Upon fuor CHIKV . Howeversquitoes .Wolbachia-related literature published, the mechanisms of how Wolbachia colonisation in mosquitoes could result in reduced vector competence for viruses remains poorly understood. Wolbachia density in mosquitoes may possibly be correlated to its antiviral properties. Non-native Wolbachia strains have also been shown to inhibit virus infection better compared to native Wolbachia strains. An example of this is demonstrated in a recent study by Schultz et al., whereby the wStri strain (native to the leafhopper Laodelphax striatella) was more effective than wAlbB  in Zika virus (ZIKV) inhibition in C710  [Wolbachia strains [wAlbB grows to higher density per cell in Aedes aegypti compared to Aedes albopictus, its native host [wAlbA- or wAlbB-infected Aedes albopictus mosquitoes in the field study by Ahmad et al. [Wolbachia densities and dengue virus (DENV) loads present in various tissues of Wolbachia-infected and uninfected mosquitoes but were unable to find any correlation between Wolbachia densities and DENV inhibition. The authors also found that DENV inhibition by Wolbachia was greatest at early stages of virus infection. This is consistent with findings by Rainey et al., who investigated the molecular aspects of Wolbachia-based Semliki SFV inhibition in Jw18 (Drosophila melanogaster cell line), and found that virus replication was inhibited as early as 7 h post infection [Wolbachia were found to be constitutively active as the authors were unable to detect any significant changes in Wolbachia gene expression upon virus infection [Wolbachia, suggesting that the antiviral effects observed were attributed to Wolbachia and not mediated by the host’s antiviral response [Wolbachia such as host immune priming and competition for host resources are discussed in a recent review by Terradas and McGraw [Aedes albopicus and Culex pipiens mosquitoes [Despite the abundance of ll line) . Indeed, strains ,46,47,48ive host . The phed et al. . Howeverd McGraw . Additiod McGraw . This issquitoes .Wolbachia bacteria, there are other key players in the mosquito gut microflora that influence virus infection as well. Of interest is a recent and comprehensive report by Angleró-Rodríguez et al., which describes the first fungus that is found naturally associated with the mosquito gut and is involved in virus infection [Talaromyces sp. fungus (designated Tsp_PR) was found in the midgut of field Aedes sp. mosquitoes and its identity confirmed through sequence analysis [Tsp_PR spores or secretome resulted in increased DENV replication in the midgut [Tsp_PR was replaced with another fungus strain previously isolated from field mosquitoes, Penicillium chrysogenum, no effect on DENV infection was found [Tsp_PR also did not have any effect on the bacteria flora present in the midgut, neither did it affect mosquito longevity [Tsp_PR secretome on mosquito transcriptional activity and found that many down-regulated genes were involved in bloodmeal digestion, which may explain the enhanced DENV infection observed [Though we have thus far introduced microfilariae and nfection . The Talanalysis . The inte midgut . Howevere midgut . Interesas found . The preongevity . The autobserved .Wolbachia to virus infection and often, the possible roles of the other bacteria present in the mosquito gut microflora is neglected. An example of another bacteria which displays anti-pathogenic properties is Csp_P, a Chromobacterium sp. isolated from field-caught Aedes aegypti mosquitoes [Anopheles gambiae as well [Plasmodium falciparum and DENV infection in mosquitoes and it secretes molecules with anti-pathogenic activity [Many studies focus on characterising the effect of squitoes . This ba as well . It is aactivity . Howeveractivity .Alphaviruses can be genetically diverse, which contribute to phenotypic plasticity and adaptability to dynamic environments, including new host species . For exaAlphaviruses are also able to effectively employ a motif mimicry-based strategy to productively infect and adapt to both their target hosts and vectors. Here, hijacking of host and/or vector cellular and functional processes through viral motif mimicry is modulated by small, linear and functionally constrained poly-amino acid motifs (approximately 3 to 10 amino acid residues in length) called Short Linear Motifs (SLiMs) . While sThe Indian Ocean island CHIKV epidemics of 2005–2006 provided the best example of how point mutations and/or convergent evolution within alphaviral SLiMs can influence viral adaptation to a specific mosquito vector . This CHAedes albopictus, thus improving viral dissemination and the transmission to suckling mice experimentally without affecting viral fitness in Aedes aegypti [Aedes albopictus is widely present and is actively displacing indigenous Aedes aegypti populations. This suggests that by altering the vector specificity or preference for CHIKV strains, the (E1-A226V) mutation also influenced both the vectorial capacity and vector competence of the Aedes albopictus population resulting in CHIKV strains carrying this mutation achieving what is widely recognized today as a unique spatial global distribution. Apart from the discovery of a point mutation or a convergent evolutionary event that occurred at position 226 of the E1 glycoprotein, very little is known regarding the precise molecular mechanisms by which CHIKV fitness  was increased in Aedes albopictus. The identities of the SLiM residues flanking this E1 envelope glycoprotein mutational site (226) are also unknown. Extensive analyses revealed that this mutation (E1-A226V) was directly responsible for an increase in CHIKV infectivity for  aegypti ,67. QuitAedes albopictus. It has been shown that the E1-226V residue is located at regions of fusion with host cell membranes [Aedes albopictus, the E1-226V variant has a greater capacity to cross the midgut barrier and infect local cells compared to the E1-226A variant [Aedes albopictus, suggesting that these are two independent phenotypic effects of the E1-226 mutation [Follow-up studies initially suggested that the E1-A226V mutation caused an increase in the dependency on cholesterol during the virus-host cell fusion ,68. Thisembranes . Further variant . Additio variant . Furthermutation .Aedes albopictus is common [Aedes albopictus [Aedes aegypti instead of Aedes albopictus in areas where they are sympatric [Aedes albopictus-fitted ECSA and IOL strains settle in the area [The evolutionary constraints imposed by epistatic interactions between residues 226 and 98 of the E1 glycoprotein may be the reason why the E1-A226V mutation only appears in East-South-Central African (ECSA) CHIKV strains and not in the Asian CHIKV strains circulating in areas where s common . Certainbopictus . There iympatric . Howeverthe area .Aedes albopictus [Aedes albopictus [Aedes aegypti as compared to its effect on Aedes albopictus [Aedes albopictus [Aedes albopictus as a major vector in areas where it is abundant. For instance, in Europe and North America where Aedes albopictus mosquitoes are rapidly spreading, there is heightened concern about the epidemic potential of mutant CHIKV strains [“Second-step” adaptive mutations have also been described in E1-A226V CHIKV strains that further potentiates viral replication in bopictus . The mosbopictus , facilitbopictus . The E2-bopictus . In addibopictus . Positivbopictus . These l strains .Aedes albopictus and Aedes aegypti but with no indication of the underlying molecular determinants driving increased competence. In a French study, researchers showed that Aedes albopictus was more competent for CHIKV compared to the DENV-2 virus [Aedes aegypti as compared to DENV [Aedes vigilax (Skuse) and Aedes notoscriptus mosquito populations [Several studies have also reported increased competence for CHIKV as compared to DENV in both -2 virus . Further-2 virus . A simil to DENV . Whetherulations ,82,83, fAnopheles gambiae rose from 0 to 63.5% [Aedes aegypti, the main vector of the virus [A. aegypti remains unknown. However, studies with the alphavirus SINV, have shown that deletions in the carboxy-terminus rendered mutants defective in initiating a productive infection, generating plaques in mosquito cells at only 1–2% the efficiency of the parental virus [By contrast, the molecular determinants that drive distinct mosquito vector adaptations in ONNV were recently located within the non-structural protein 3 (nsP3) region of the virus . The nsPto 63.5% . These ahe virus . How thial virus . Althougal virus . It is cCulex tarsalis decreased as a function of increasing temperature [Aedes vigilax kept at 18 °C [Aedes albopictus increased at lower rearing temperatures [Predicting the effects of climatic variables, such as temperature, on vector competence for alphaviruses has been challenging. Very little is currently known regarding how alphavirus-vector interactions are modulated. In particular, the role of climatic variables such as temperature. Temperature is an important biotic variable that directly affects the mosquito biting rate, fecundity , egg to adult survival, mosquito development rate, adult lifespan, the extrinsic incubation rate, and infection and transmission probabilities . Higher perature . This stperature . Similarat 18 °C . Anotherat 18 °C . Westbroeratures .Aedes aegypti larval rearing temperature and intraspecific larval competition on vector competence for SINV and found a direct relationship between Aedes aegypti larval density and SINV infection and dissemination rates at low temperature (20 °C) and an inverse relationship between larval density and SINV infection rate at high temperature (30 °C) [Aedes antipodeus, was shown to be efficiently infected with BFV and RRV at low extrinsic incubation temperatures simulating winter conditions (16 °C) where the mosquito vector was able to transmit each of the viruses after 21 days or earlier [Aedes antipodeus mosquito vector populations should be monitored as they are potentially competent vectors for RRV and BFV transmission in New Zealand [Culex tarsalis Coquillett mosquitoes. Furthermore, Reeves et al. evaluated the effect of temperature changes on the survival of this mosquito vector species and revealed that, (i) daily mortality of adult vectors increased by 1% for each 1 °C increase in temperature; (ii) only 5% of Culex tarsalis survived for eight or more days at 25 °C, the time required for extrinsic incubation of WEEV; (iii) increasing temperatures from 18 to 25 °C resulted in shortened extrinsic incubation times for WEEV, and that; (iv) WEEV infection and transmission decreased at 32 °C [The environmental temperature may affect mosquito physiology, thereby affecting viral replication and transmission. Muturi et al. evaluated the effect of  (30 °C) . In anot earlier . It has  Zealand . In the at 32 °C . This deAedes albopictus cell lines  persistently infected with the HR strain of SINV were able to prevent replication of both homologous (various strains of SINV) and heterologous alphaviruses  [Aedes albopictus cell lines [Aedes albopictus cells persistently-infected with SINV failed to replicate superinfecting SINV [Aedes albopictus cells efficiently excluded several heterologous alphaviruses  if the cells were superinfected at early times after primary SINV infection, but excluded them much less efficiently if superinfection occurred after long-term persistent infection [One of the most likely outcomes of viral co-infection is a phenomenon known as superinfection exclusion, where primary infection with a specific viral strain prevents secondary infections with either the same or other genetically distinct viral strains. Karpf et al. reported that three and RRV) . Though ll lines . Broadlying SINV . Howevering SINV . A follonfection . The intnfection ,108,109.nfection ,108,109,nfection ,108,109.Aedes genus mosquitoes [Aedes aegypti to transmit CHIKV, DENV-2 or ZIKV, individually and as double and triple co-infections [Aedes aegypti was susceptible to infection for all the combinations tested and was also able to transmit the viruses simultaneously. Moreover, the rates of infection, dissemination and transmission were not significantly affected by co-infection of these viruses [Aedes albopictus was shown to be amenable to oral co-infection with both DENV and CHIKV E1-226V [Aedes albopictus and this vector was noted to retain the ability to deliver infectious particles of both viruses in a single bite via saliva [Aedes albopictus carrying a DENV primary infection. The identification of SLiMs may be helpful in elucidating the mechanisms behind superinfection. For instance, the tripeptide Arginine-Glycine-Asparagine RGD motif is a SLiM found in the envelope proteins of alphaviruses and flaviviruses [Aedes aegypti and Aedes albopictus [Aedes albopictus C6/36 cell line characterized the co-infection dynamics of DENV-3 and CHIKV (ECSA genotype) and concluded that; (i) higher titres of DENV-3 resulted in competitive suppression of the replication of CHIKV and; (ii) DENV-3 and CHIKV replications depend on virus titre rather than on serial infection [Virus co-infection could also occur between virus families and several studies have presented the impact of alphavirus and flavivirus co-infection on the vector competence of squitoes ,111. Forfections . They fo viruses . In a re E1-226V . Herein,a saliva . Furtheriviruses . This SLiviruses . This suiviruses . Additiobopictus . In thisbopictus . The autbopictus . In contnfection . There iWolbachia infection is a potential vector control measure, different genetic strains may be required for different regions due to the variable mosquito populations in various geographical regions. Moreover, the Wolbachia strain must be compatible with the existing commensal microflora present in the mosquito midgut, and should not have any detrimental effects on mosquito longevity. Additionally, the constant evolution of the virus, Wolbachia, and mosquito vector makes it difficult to determine how long the effects of Wolbachia population displacement will last. Other targets for vector control will have to be determined and hence, further understanding of the complex interactions that affect vector competence is required.Many alphaviruses are medically important, and emerging global pathogens due to the presence of mosquito vectors and human populations. Though different regions are hosts to different mosquito vectors, it has been shown that alphaviruses are capable of successfully adapting to their chosen vectors. Additionally, many factors that contribute to vector competence come into play, resulting in increased vector competence of mosquito vectors previously less susceptible to virus infection. There are many challenges in the control of mosquito populations and though


The effects of chemogenetics on axon regeneration following peripheral nerve transection and repair were studied in mice expressing a Cre-dependent excitatory designer receptor exclusively activated by designer drugs (DREADD) and Cre-recombinase/yellow fluorescent protein (YFP) in a subset of motor and sensory neurons and cortical motoneurons (SLICK-A). Sciatic nerves were cut and repaired and mice were treated either once, at the time of injury, or five days per week for two weeks with clozapine N-oxide (CNO) , or were untreated controls. Two weeks after injury, the lengths of YFP+ axon profiles were measured in nerves harvested from euthanized animals. Compared to untreated controls, regenerating axon lengths were not significantly longer in mice treated only once with CNO, but they were more than three times longer in mice receiving CNO repeatedly. Based on results of retrograde labeling experiments, axons of more sensory and motor neurons had regenerated successfully in mice receiving multiple CNO treatments than animals receiving only one treatment or no treatments. The increase in numbers of labeled sensory, but not motor neurons could be accounted for by increases in the proportion of retrogradely labeled neurons also expressing the DREADD. Chemogenetic increases in neuronal excitability represent a potent and innovative treatment to promote peripheral nerve regeneration. Injuries to peripheral nerves are common. Nearly 200,000 new traumatic peripheral nerve injuries occur in the US each year ,2. AlthoChemogenetics has emerged as a means of effecting neuronal excitability, and potentially activity, using a pharmacologic approach. The basis of chemogenetics is designer receptors exclusively activated by designer drugs (DREADDs) . These aIn this study, we evaluated the effects of excitatory DREADD activation in mice in which hM3Dq was expressed genetically in both sensory and motor neurons. We bred mice that express an excitatory DREADD (hM3Dq) in a Cre-dependent manner with a double transgenic mouse known as SLICK (Single neuron labeling with inducible Cre-mediated knockout) . SeveralMice expressing a Cre-dependent Gq DREADD   were breTo induce Cre recombinase expression, 6–8 weeks old animals expressing both the SLICK-A and hM3Dq genotypes (referred to here as SLICK-Gq) were treated with tamoxifen , using a protocol that we have described in detail previously . BrieflyAll surgical procedures conducted using animals were approved by the Institutional Animal Care and Use Committee of Emory University (Protocol No. 2003261) and conformed to the Guidelines for the Use of Animals in Research of the Society for Neuroscience. All surgical procedures were performed under aseptic conditions. Numbers of mice in each group were chosen based on an a priori power sample size estimate, using a power of 0.8, α = 0.05, and the inter-animal variability observed in our previous studies .For experiments using axon profile lengths as an outcome measure, we used a nerve grafting method we developed several years ago . In TamoN = 5 each). In two control groups, nerves were cut and repaired but were untreated. One group contained tamoxifen-treated SLICK-Gq mice and the other SLICK-A mice that did not contain the hM3Dq transgene. A third group of tamoxifen-treated SLICK-Gq mice received a single treatment with the designer drug, clozapine N-oxide (CNO)  applied once, immediately after nerve repair had been completed. The fourth group of tamoxifen-treated SLICK-Gq mice received the same dose of CNO for a total of ten times over two weeks. Treatment was begun immediately following nerve repair surgery and continued five days per week for two weeks. The two week time course of this protocol was chosen to be similar that of the exercise protocols that we have used previously to enhance axon regeneration after nerve injury [Operated mice were studied in five treatment groups , post-hoc paired comparisons (Tukey-Kramer) were performed.Optical sections were made at 10 micrometer thickness through the entire extent of the harvested nerves using a confocal microscope , and theIn addition, we evaluated the extent of branching of regenerating axons in the different groups by comparing the numbers of regenerating axon profiles encountered. In SLICK-A mice, we have shown previously  that a cN = 20) were anesthetized and the sciatic nerve exposed, as described above. The intact nerves were secured on a small rectangle of SILASTIC film using fibrin glue. Once secured to the film, the nerve was cut with sharp scissors near the center of the film. This attachment of nerves to the film prevented the withdrawal of its cut segments and helped to align them. A second application of fibrin glue was then applied to secure the cut nerve segments. Deep tissues were then approximated with absorbable 5-0 sutures, and the skin was closed with non-absorbable 5-0 suture. Mice were studied in five treatment groups: untreated SLICK-A mice not expressing hM3Dq, tamoxifen-treated SLICK-Gq mice treated once with CNO, tamoxifen-treated SLICK-Gq mice treated ten times with CNO, and SLICK-A mice not expressing hM3Dq treated either once or ten times with CNO. Four nerve injuries were studied in each group.To determine the number of neurons whose axons had regenerated successfully and re-innervated the gastrocnemius muscle, retrograde labeling experiments were performed four weeks after nerve transection and repair. Mice    was injected into two sites within the lateral gastrocnemius (GAST) muscle using a 30 g needle attached to a Hamilton microliter syringe. Each muscle thus received a total 1 µL of CTB 555 solution. After each injection, the needle remained in place for 30 s prior to withdrawal, and sterile gauze was applied to the needle site for 30 s to minimize potential tracer leakage. The surgical site was washed three times with sterile saline solution. The wounds were closed, and the animals were returned to their cages. All nerve repair surgeries and tracing experiments were performed bilaterally.After 3–4 days, to allow for retrograde transport of the tracer, animals were euthanized and perfused, as described above. The lumbar spinal cords and fourth lumbar dorsal root ganglia (DRGs) were harvested and cryoprotected. Serial 20 micrometer thick cryostat sections were mounted onto subbed slides and cover-slipped with Vectashield. For counting labeled motoneurons, images of each section were obtained using filter settings appropriate for YFP and Alexafluor 555 and saved to disc. Motoneurons were identified as retrogradely-labeled if the red (CTB 555) label filled the cell soma (with a clear region of the nucleus present) and extended into its proximal dendrites. Care was taken to identify any fragments of each labeled neuron on adjacent serial sections to avoid double counting. Identified neurons that also contained YFP were scored as double-labeled. Mean numbers of tracer-labeled and double-labeled neurons were compared between the five treatment groups using ANOVA, with post hoc testing, where appropriate.For analysis of labeled DRG neurons, we studied every third serial section from the fourth lumbar ganglion. Analysis was restricted to every third serial section to avoid spurious double counting of small ganglion cells. Cells were identified as containing tracer only or tracer plus YFP, as above. Counts of labeled DRG neurons were compared between the same five treatment groups, as described above.N = 5 for each group) are shown as cumulative histograms in p < 0.01) to the right of the controls (No CNO) as well as that from mice treated only once (1 × CNO). No significant shift was found from controls in the 1 × CNO group. In tamoxifen-treated SLICK-Gq mice, peripheral nerves were cut and repaired with grafts harvested from non-fluorescent wild-type littermates A. They wN = 5) median axon profile lengths from these mice are shown in 3,16 = 32.60, p < 0.01). Based on post-hoc (Tukey-Kramer HSD) analysis, a single treatment with CNO did not result in significantly increased median axon profile lengths relative to either of the control groups. However, treatments with CNO ten times over the two-week period resulted in a greater than three-fold increase in median axon profile lengths, relative to controls. Thus multiple CNO treatments of tamoxifen-treated SLICK-Gq mice resulted in a striking enhancement of axon regeneration.Average  numbers of YFP+ regenerating axon profiles encountered in mice in the same experimental groups are shown in N = 4 for all groups) of single and double labeled motoneurons are shown in The number of motoneurons whose axons had regenerated successfully was determined using retrograde transport of fluorescent tracers injected into the GAST muscles four weeks after sciatic nerve transection and repair. Because the SLICK-A mice used in this study express YFP in only a subset of motoneurons and in all Cre-expressing cells (see above), we were able to differentiate motoneurons containing hM3Dq from those that did not. We counted motoneurons that contained only the retrograde label A, red an4,15 = 16.34, p < 0.01). Using post-hoc paired testing, significant (p < 0.05) differences were found between the group of mice expressing the Gq DREADD that had been treated ten times with CNO and all other groups, including mice treated with CNO but not expressing hM3Dq. Differences in the mean number of retrogradely labeled motoneurons between the other four groups were not significant. The same was true if the motoneurons marked only by the fluorescent retrograde tracer was considered. The omnibus test of a one-way ANOVA was significant  and in post-hoc paired testing only paired comparisons involving the mice expressing DREADDs and treated ten times with CNO were significant. However, the number of cells that contained both the retrograde tracer and YFP did not differ significantly among any of the treatment groups . Thus, repeated treatments with CNO promoted the successful regeneration of axons of motoneurons in tamoxifen-treated SLICK-Gq mice, irrespective of whether those motoneurons expressed the DREADD target of the treatment.The total number of retrogradely labeled motoneurons found four weeks after sciatic nerve transection and repair was more than three times larger in tamoxifen-treated SLICK-Gq mice that received ten treatments with CNO than in any of the other groups. The omnibus test of a one-way ANOVA was significant  of single and double labeled DRG neurons are shown in 4,15 = 12.14, p < 0.01). Using post-hoc paired testing, significantly more labeled DRG neurons were found in tamoxifen treated SLICK-Gq mice treated with CNO than any of the other three groups, whether the mice were treated once or treated ten times. No significant effect of CNO treatment was found in mice that did not express hM3Dq. Similar numbers of L4 DRG neurons regenerated successfully in these mice as in SLICK-Gq mice not treated with CNO. The proportion of all labeled DRG neurons that were double labeled in mice treated ten times with CNO was significantly  greater than in all of the other groups . Sensory neurons containing YFP were assumed to express hM3Dq. Examples of double labeled sensory neurons are shown in r groups C. The goal of this study was to evaluate the use of chemogenetics as a means of enhancing sensory and motor axon regeneration after peripheral nerve transection injury using neuroanatomical outcome measures. In mice in which an excitatory DREADD (hM3Dq) and YFP were expressed in a Cre-dependent manner, we evaluated the lengths and numbers of regenerating axons after sciatic nerve injury and the participation and success of regeneration of axons of motor and sensory neurons. The main finding was that repeated Gq DREADD activation by the designer drug, clozapine-N-oxide (CNO) resulted in significant enhancement of axon regeneration. Lengths of profiles of regenerating axons in mice treated ten times over a two week period with CNO were nearly three times longer than controls, or mice treated only once with CNO, and comparable to those we had shown in similar mice using post-injury exercise as a therapy . Axons oThe simplest explanation for this chemogenetic enhancement of regeneration is that activation of the Gq DREADD using CNO resulted in an increase in the excitation of the injured neurons, leading to enhanced elongation of regenerating axons—a form of activity-dependent enhancement. In a previous study, we found that background EMG activity was not increased by CNO treatment of rats induced to express hM3Dq in motoneurons , leadingThe enhanced regeneration of motor axons found in the mice treated daily with CNO might not be as easily explained. Although not significantly greater, in these animals, 50% more motoneurons were retrogradely labeled than in any of the other groups studied. We interpret this finding to mean that axons of more motoneurons had regenerated successfully in the mice treated daily with CNO than in the other groups. Thus, the daily CNO treatments enhanced the regeneration of axons substantially more in motoneurons that did not contain YFP, and were thus presumed not to express the hM3Dq, than in motoneurons that did express hM3Dq. One interpretation of this finding might be that the CNO treatments produced an increased excitation of motoneurons irrespective of their Gq DREADD content. Given the well-documented in vivo metabolism of CNO to n-desmethylclozapine ,10,11, sAn alternative explanation might be that the daily CNO treatments increased the excitation of other neuronal systems that express both Cre recombinase and hM3Dq, which, could contribute to the excitation of both Gq positive and Gq negative motoneurons. One such system is the corticospinal tract. It is known that in the SLICK-A mice used in this study, neurons in lamina V of the motor cortex and axons in the corticospinal tract express YFP, and tamoxifen-inducible Cre recombinase . Thus, wChemogenetic activation of excitatory DREADDs in the SLICK-A mouse resulted in enhanced regeneration of both sensory and motor axons following peripheral nerve injury.