The severity and prevalence of many diseases are known to differ between the sexes. Organ specific sex-biased gene expression may underpin these and other sexually dimorphic traits. To further our understanding of sex differences in transcriptional regulation, we performed meta-analyses of sex biased gene expression in multiple human tissues. We analyzed 22 publicly available human gene expression microarray data sets including over 2500 samples from 15 different tissues and 9 different organs. Briefly, by using an inverse-variance method we determined the effect size difference of gene expression between males and females. We found the greatest sex differences in gene expression in the brain, specifically in the anterior cingulate cortex, (1818 genes), followed by the heart (375 genes), kidney (224 genes), colon (218 genes), and thyroid (163 genes). More interestingly, we found different parts of the brain with varying numbers and identity of sex-biased genes, indicating that specific cortical regions may influence sexually dimorphic traits. The majority of sex-biased genes in other tissues such as the bladder, liver, lungs, and pancreas were on the sex chromosomes or involved in sex hormone production. On average in each tissue, 32% of autosomal genes that were expressed in a sex-biased fashion contained androgen or estrogen hormone response elements. Interestingly, across all tissues, we found approximately two-thirds of autosomal genes that were sex-biased were not under direct influence of sex hormones. To our knowledge this is the largest analysis of sex-biased gene expression in human tissues to date. We identified many sex-biased genes that were not under the direct influence of sex chromosome genes or sex hormones. These may provide targets for future development of sex-specific treatments for diseases. Differences in both disease severity, prevalence, symptoms, and age of onset vary greatly between males and females Morrow, . For exaMany studies neglect to account for sample sex in the design and analysis of their experiments  and using the inverse–variance method as previously described .Probes were re-annotated to Ensembl gene identifiers using biomaRt , we firstly downloaded the coordinates of AREs from JASPAR  binding sites within 10 kb upstream/downstream of sex-biased genes were analyzed using oPOSSUM-3 and the JASPAR vertebrate core profiles  < 0.05.Gene ontology (GO) analysis was performed using all human genes in the Database for Annotation, Visualization, and Integrated Discovery (DAVID) v6.7  made up 32% of all samples across all data sets  has been shown to have protective roles in pro-inflammatory responses , isolated systolic hypertension can be up to 14% more prevalent in females than males  that includes genes that regulate transcription, elongation and pre-mRNA splicing events  were expressed more highly in females. In addition, we also found STS [a gene involved in the production of estrogen precursors  and ribosomal proteins . These genes were also expressed more highly in males in other tissues such as the brain, heart, and kidney. It is worth mentioning that we also found other members of the RPL gene family to be more highly expressed in females in other tissues  as an enriched TF within the AMY, CB, FC, bladder, and lung. In addition, HNF1A and HNF1B were enriched in genes upregulated in both males and females within all tissues apart from the NC and DLPFC. HNF1A and HNF1B are homeobox TFs that are required for expression of specific liver genes  enrichment analysis using oPOSSUM-3 and the JASPAR core motifs  project  consistently classified as sex-biased in the 10-fold cross validation. However, in the original analysis we identified four autosomal genes to be sex-biased and upregulated in females  were from individuals who were post-reproductive age (average age = 47 years) which may not have captured the true extent of sex-biased gene expression that would otherwise be evident during early adulthood when sex hormones are at their peak production. Thus, using data from older individuals limited our ability to assess sex-biased gene expression in individuals of reproductive age. We also report a number of genes previously associated with diseases and disorders that were differentially expressed between sexes. RNA expression differences do not necessarily cause phenotypic variation, as there are multiple levels of gene and protein regulation that can occur post-transcription. Next-generation sequencing, as opposed to microarrays used in this study, would allow a more complete assessment of sex-dependent gene expression differences but there is currently more samples that have been analyzed using microarrays and therefore more statistical power can be achieved. Furthermore, on average, 64% of genes differentially expressed between sexes in each tissue had a magnitude log.5 Table . TherefoDespite the large amount of genomic data that was available for this study it was unfortunate not to consider clinical and lifestyle factors such as age, smoking status, sample heterogeneity and body mass index (BMI) which may potentially have an effect on gene expression. We were unable to correct for these potential confounding factors because, as detailed in Table Our analyses have revealed substantial differences in the transcriptional landscape between sexes across a range of human organs and tissues and highlight possible mechanisms by which gene expression may contribute to sexually dimorphic traits. Improved understanding of these is fundamental to understanding diseases with different prevalence between the sexes. Our data show that sex differences in gene expression vary widely across different tissues. We identified a consistent trend for genes known to regulate the immune system to be more highly expressed in females and those involved in energy production and growth were more highly expressed in males. These may be the result of different evolutionary pressures between the sexes. The brain demonstrates the largest differences in sex-biased gene expression with several sex-biased genes associated with specific brain disorders, providing insight into possible mechanisms for the association of sex-specific prevalence of certain brain disorders.Our findings also indicate that many sex biased genes within tissues are independent of sex chromosome genes or sex hormones. Approximately 32% of autosomal genes in each tissue contained an ARE or ERE, which suggests there are other mechanisms that underpin sex differences in gene expression. One potential mechanism is through epigenetic factors, such as chromatin modeling which has been suggested to have sex specific functional roles  Project Grant (GNT1059120) awarded to CR, CS, VC, and TB. CR is supported by a NHMRC Senior Research Fellowship GNT1020749. CS is supported by an Australian Research Council Future Fellowship (FT120100086). VC is supported by a NHMRC Senior Research Fellowship GNT1041918. SB is supported by an NHMRC-ARC Dementia Research Development Fellowship Grant (APP1111206). BM is supported by an Australian Post-graduate Award.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.


Intracerebral hemorrhage (ICH), a form of brain bleeding and minor subtype of stroke, leads to significant mortality and long-term disability. There are currently no validated approaches to promote functional recovery after ICH. Research in stroke recovery and rehabilitation has largely focused on ischemic stroke, but given the stark differences in the pathophysiology between ischemic and hemorrhagic stroke, it is possible that strategies to rehabilitate the brain in distinct stroke subtypes will be different. Here, we review our current understanding of recovery after primary intracerebral hemorrhage with the intent to provide a framework to promote novel, stroke-subtype specific approaches. In humans, primary ICH occurs because of a rupture of an intracerebral vessel, most often due to hypertension or cerebral amyloid angiopathy. Vessel rupture leads to mechanical tissue disruption (secondary to the jet of blood) and hematoma formation, followed by secondary injury processes, including disruption of the blood-brain barrier, expansion of perihematomal edema, inflammation, neuronal cell death, and chemical or cellular barriers to repair3. These secondary injury processes occur over hours to days after the primary injury and are attributable to toxins from lysed blood, including hemoglobin (Hgb), hemin (oxidized form of heme), and thrombi8. These toxins could affect cell survival or repair via interactions with a host of cell types, not only neurons.Intracerebral hemorrhage (ICH) accounts for about 15% of all strokes in the USA and Europe and leads to significant mortality and long-term disability despite advances in acute medical and surgical treatmentAlthough research in the field of stroke recovery and rehabilitation has grown, there have been very few studies specifically focusing on rehabilitation in ICH. In this review, we summarize the recent advances in our knowledge of the pathophysiology of primary ICH, how rehabilitation affects functional outcomes after ICH, and the mechanisms that may underlie the gains seen with rehabilitation in preclinical models and patients.9. The two most commonly used rodent models of ICH are autologous blood injection and bacterial collagenase infusion10. In the whole-blood injection model, lysed blood is injected directly into the brain parenchyma, with almost no spontaneous bleeding11. The bacterial collagenase model involves intraparenchymal infusion of bacterial collagenase, an enzyme that damages the basal lamina and causes bleeding that evolves and grows over 24 hours12. Models of hypertensive rodents in which ICH occurs spontaneously, which may more closely mimic what occurs in patients, have been less widely used. In reviewing the literature, one must consider that these preclinical models have different pathophysiological processes which may affect the course of recovery and response to rehabilitation; thus, the use of multiple models has been recommended to better understand post-ICH recovery in preclinical studies13.Preclinical models have provided important insights into the pathophysiological processes that occur after ICH, which differ from those seen after ischemic stroke14. In the collagenase model, there is markedly greater delayed cell death, leading to worse functional deficits even when the hematoma size is matched14. Historically, it was believed that Hgb or hemin induced toxicity following ICH by loading neurons with redox-active free iron leading to the formation of hydroxyl radicals via Fenton chemistry9. Indirect support for this model came from studies, not replicated by all groups, that iron chelators such as deferoxamine (DFO) improve functional recovery in pig15 and rodent16 models. More recent studies suggest that DFO and a more selective metal chelator, adaptaquin, act not by inhibiting Fenton chemistry but by inhibiting a specific family of iron-, 2-oxoglutarate-, and oxygen-dependent dioxygenases, the hypoxia-inducible factor proly hydroxylases (HIF PHDs)17. HIF PHDs are oxygen sensors and accordingly, with drugs such as adaptaquin, the brain can be fooled into thinking it is hypoxic when it is not. The consequence of turning on hypoxic adaptation in ICH with adaptaquin is to inhibit the synthesis of a cassette of genes  that are involved in mediating neuronal death. Accordingly iron, likely via its ability to act as a cofactor for the HIF PHDs, promotes the synthesis of ATF4-dependent genes that mediate neuronal death.After ICH, there is both immediate and ongoing cell death in both whole-blood and collagenase modelsOf note, ATF4 regulates the synthesis of a pseudokinase inhibitor of AKT, called tribbles homolog 3 (Trib3). Studies in non-neural cells have shown that Trib3 and ATF4 can dimerize to inhibit transcription by the pro-plasticity transcription factor, CREB (cAMP-responsive element-binding protein). Activators of CREB have been shown to improve learning and memory as well as functional recovery after stroke. Accordingly, iron’s ability to load the HIF PHDs could lead to the transcriptional induction of genes  that constrain neuronal survival and plasticity following hemorrhagic stroke. The model is consistent with data showing that adaptaquin, a small-molecule inhibitor of the HIF PHDs, improves functional recovery out to a month in collagenase and blood infusion models in mice and rats, respectively. Future studies will clarify the role of ATF4 in regenerative failure following ICH.Parthanatos19. By contrast, recent data suggest that cell death following ICH occurs via a ferroptotic pathway that also involves necroptosis20. Of note, inhibitors ofParthanatos have no effect on ICH-induced deathin vitro7, suggesting that the pathways of cell damage and, by extension, regenerative failure might be different.How do cells die following ICH in cellular and rodent models? Recent studies from two groups suggest that cell death in ICH is completely distinct from that found following ischemic stroke. In ischemic stroke, cell death occurs via a poly(ADP-ribose) polymerase-1 (PARP)-dependent pathway leading to AIF release from the mitochondria and21. Interestingly, activated microglia can take on pro-inflammatory (via classic activation) or anti-inflammatory  phenotypes, the latter associated with wound healing and repair23. A recent study demonstrated that, after ICH, microglia have dynamic changes in gene expression that result in a transition from an early, pro-inflammatory phenotype to an anti-inflammatory phenotype during the resolution phase of ICH24. Transforming growth factor-beta 1 (TGF-β1) was identified as the most likely mediator of this effect, and treatment with TGF-β1 improved functional outcomes in a murine model. Furthermore, early increases in TGF-β1 levels were also shown to be highly associated with improved 90-day outcomes in patients with ICH.Other studies have focused on the role of the immune system in secondary injury and repair. After ICH, there is a marked inflammatory reaction with activation of microglia and recruitment of leukocytes to the perihematomal region, leading to the production of inflammatory mediators, including cytokines, chemokines, and matrix metalloproteinases3. In a preclinical study, treatment with a PPARγ agonist, rosiglitazone, promoted hematoma resolution, reduced neuronal damage, downregulated the expression of pro-inflammatory genes , reduced oxidative stress, and was associated with improved neurological function25.Another area of research involves the transcription factor peroxisome proliferator-activated receptor gamma (PPARγ), which plays a significant role in phagocytosis and modulating inflammation and neuroprotective mechanisms29. Given the differences in pathophysiology between ischemic stroke and ICH, one could assume that the recovery outcomes or mechanisms would be dissimilar between the two stroke subtypes. Comparisons between recovery outcomes in patients with ischemic stroke and ICH have yielded mixed results; some have found comparable activity limitation and recovery30, whereas others have found greater recovery after ICH31.Post-stroke recovery has been widely studied, but the majority of research has focused only on ischemic stroke32. A recent longitudinal study of patients with ICH characterized the time course of recovery of motor and sensory impairment and ambulation in 11 patients up to six months post-stroke33. Sensory, truncal, and lower limb motor impairment reached a plateau after three months, whereas there was continued improvement of upper limb movement and ambulation to six months. The course of motor recovery has been shown to depend on the integrity of the corticospinal tract (CST), as measured by transcranial magnetic stimulation34 or diffusion tensor tractography32. In a study of 36 patients with putaminal hemorrhage, motor function recovered in all patients up to four months post-stroke, but patients who had preserved CST integrity at baseline had greater recovery of motor function32. More studies are needed to better elucidate the natural history of recovery after ICH, as has been done for ischemic stroke.The majority of recovery after ICH occurs early, within the first few months post-stroke36. Different types of rehabilitation have been tested in preclinical models, including environmental enrichment38, skilled reach training37, constraint-induced movement therapy40, and aerobic training41. Enriched rehabilitation (ER) combines environmental enrichment  and task-specific training (most commonly skilled reach training) and was first used as a successful rehabilitative intervention in preclinical models of ischemic stroke43. Rehabilitation has been shown to be associated with improved behavioral recovery and enhanced neuroplasticity, including dendritic reorganization44, astrocytic plasticity45, and synaptogenesis in ipsilesional motor cortex and striatum46.Rehabilitation has been shown to lead to functional improvements in rodent models using both collagenase-induced and whole blood-induced ICH36. ER also significantly reduced the amount of perihematomal neuronal death even after one week of treatment, suggesting a possible neuroprotective effect of rehabilitation. This study also examined whether rehabilitation influenced iron toxicity and inflammation post-ICH; rehabilitation did not have an effect on levels of iron-binding proteins (ferritin and transferrin) or number of inflammatory cells in perihematomal tissue. The results of a follow-up study also support a neuroprotective role of rehabilitation; ER beginning one week after ICH was demonstrated to augment the clearance of toxic blood components, Hgb and iron, and reduce oxidative stress at the hematoma/perihematomal interface35. The authors speculate that rehabilitation upregulates pathways for Hgb clearance  and downregulates anticlearance pathways  to expedite hematoma resolution and limit secondary injury. Further investigation is needed into a possible neuroprotective role of rehabilitation in preclinical models, as well as the extent to which secondary injury processes contribute to the functional deficits after ICH in humans, and the ability to reduce secondary injury with rehabilitation in humans.In a collagenase-induced rodent model, rats that participated in a two-week course of enriched rehabilitation beginning one week after ICH improved their skilled reaching and walking ability13. Interestingly, the behavioral improvements in the whole-blood model, unlike those in the collagenase model, were not accompanied by a decrease in lesion volume or increase in dendritic length13. The authors propose that the lack of delayed injury in the whole-blood model compared with the collagenase model may account for these differences. It is still unknown which of these models better exemplifies what occurs after ICH in humans.In a whole-blood injection model, ER beginning one week after ICH also led to significant improvements in reaching ability49.Taken together, evidence from preclinical studies shows a benefit of early rehabilitation after ICH, but the mechanisms underlying the behavioral gains are incompletely understood and vary according to model. Further refinement of neurorehabilitative interventions in preclinical models, which focuses on factors such as the timing, intensity, schedule, and total dose of therapy, is also needed to guide the development of optimal rehabilitation paradigms for patients50. Similar results were seen in ICH patients who received the same type and dose of inpatient rehabilitation and had greater recovery of neurological impairment and reduced activity limitation by time of discharge, relative to patients with ischemic stroke31.Rehabilitation may be more effective after hemorrhagic stroke than after ischemic stroke, when matched for baseline clinical severity. In a preclinical study in size- and location-matched ischemic or hemorrhagic stroke, rodents with ICH were shown to have greater recovery of skilled walking ability than those with ischemic stroke52 or excluded ICH patients altogether from study53. In animal models of ischemic stroke, there is an early ‘sensitive period’ after stroke, during which rehabilitation leads to larger behavioral improvements than when rehabilitation is delayed, even by one week54. However, owing to the paucity of clinical trials, evidence is lacking for a ‘sensitive period’ in humans for ischemic or hemorrhagic stroke.There have been very few rehabilitation studies undertaken in patients with ICH. Many of the larger neurorehabilitation trials have included both hemorrhagic and ischemic stroke patients55. Patients with moderate motor impairment were randomly assigned to either very early rehabilitation (within 48 hours) or standard care (rehabilitation starting after seven days). The very early rehabilitation group had shorter lengths of stay, improved quality of life, greater independence in activities of daily living, and lower six-month mortality when compared with the standard-care group. Because this study was conducted in China, where rehabilitation is delivered by family members and is less standardized, it is unclear whether the results are generalizable to the general ICH population. Furthermore, information on known predictors of outcome, such as hematoma volume, was not available, and the outcomes were self-reported and subject to responder bias. Nevertheless, it is a promising result in favor of very early rehabilitation, which has been a topic of recent debate56.The effects of early rehabilitation after ICH were recently investigated in a multicenter, prospective, randomized controlled study of 243 patients with ICH in China57, and multiple studies have shown that early therapy is safe and feasible59. However, the results of the recently published A Very Early Rehabilitation Trial (AVERT) indicate that very early, intensive mobilization may be detrimental after stroke52. In this single-blinded, randomized controlled trial of 2,104 patients with ischemic or hemorrhagic stroke, patients who received very early mobilization (< 24 hours post-stroke with more frequent sessions) were more likely to have an unfavorable outcome than patients who received usual care52. In a pre-specified subgroup analysis, this effect was stronger in patients with severe stroke and ICH. Furthermore, providing early rehabilitation for patients with ICH is particularly challenging because these patients are often admitted to intensive care units and require close neurological and hemodynamic monitoring. One must ensure that rehabilitation interventions do not cause detrimental fluctuations in blood pressure and intracranial pressure that can lead to hematoma expansion, which is associated with worsened functional outcomes after ICH60.Most clinical guidelines support early initiation of rehabilitation after strokeReducing impairment and disability after ICH requires a multifaceted approach with advances in the acute medical and surgical treatment and rehabilitation of patients with ICH. Rehabilitation leads to significant behavioral improvements in animal models of ICH and is associated with enhanced neuroplasticity and reduced neuronal degeneration, although the mechanisms contributing to behavioral recovery are not yet understood. Clinical rehabilitation studies in patients with ICH have been limited; more studies are needed to characterize the natural history of recovery after ICH and to examine the effect of rehabilitative interventions specifically in this patient population. Therapies to enhance neuroprotection and neuroplasticity are also being developed and may be used in conjunction with rehabilitation to promote recovery.


Hepatocellular carcinoma (HCC) remains one of the most common and lethal malignancies worldwide despite the development of various therapeutic strategies. A better understanding of the mechanisms responsible for HCC initiation and progression is essential for the development of more effective therapies. The cancer stem cell (CSC) model has provided new insights into the development and progression of HCC. CSCs are specialized tumor cells that are capable of self-renewal and have long-term repopulation potential. As they are important mediators of tumor proliferation, invasion, metastasis, therapy resistance, and cancer relapse, the selective targeting of this crucial population of cells has the potential to improve HCC patient outcomes and survival. In recent years, the role of epithelial-to-mesenchymal transition (EMT) in the advancement of HCC has gained increasing attention. This multi-step reprograming process resulting in a phenotype switch from an epithelial to a mesenchymal cellular state has been closely associated with the acquisition of stem cell-like attributes in tumors. Moreover, CSC mediates tumor metastasis by maintaining plasticity to transition between epithelial or mesenchymal states. Therefore, understanding the molecular mechanisms of the reprograming switches that determine the progression through EMT and generation of CSC is essential for developing clinically relevant drug targets. This review provides an overview of the proposed roles of CSC in HCC and discusses recent results supporting the emerging role of EMT in facilitating hepatic CSC plasticity. In particular, we discuss how these important new insights may facilitate rational development of combining CSC- and EMT-targeted therapies in the future. Hepatocellular carcinoma (HCC) is the most commonly diagnosed malignancy of the liver and is the third most frequent cause of cancer mortality worldwide –4. HCCs The precise molecular mechanisms of HCC pathogenesis are unclear. HCC features significant genetic, phenotypic, and functional heterogeneity, with the potential to confound the success of many therapies. A molecular basis of heterogeneity in HCC was evidenced by studies that found markedly different molecular profiles among cells from clinical specimens –15. HCC Clonal evolution or stochastic model suggests that serial acquisition of mutations generates tumor cell heterogeneity and contributes to cancer progression. With each new advantageous mutation, a clonal growth of novel cell populations completely or partially overgrows the old , 17. In The second model of cancer stem cell (CSC) or cancer-initiating cell (CIC) theory supports the presence of a specific subpopulation of cancer cells that possess tumorigenic potential and generates tumor cell heterogeneity . AccordiPhenotype plasticity model posits that irreversibly differentiated cells can be converted back to an undifferentiated state or stem cell-like state given the appropriate stimulus , 23. ThiAccording to the CSC hypothesis, tumors are organized into a hierarchy of heterogeneous cell populations, and only a small subset of cells within a tumor, termed CSCs or CICs, have the ability to sustain tumor formation and growth –29. The + cancer cells exhibited stem cell-like properties, including higher proliferative potential, greater colony-forming efficiency, self-renewal, and differentiating capacity when compared to CD133– counterparts. Additionally, CD133+ cells could initiate tumor growth in vivo, suggesting validation of the CD133 marker for enrichment of CSCs in HCC [+ and EpCAM+ CSC populations, CD90+ CSCs were able to promote motility in non-motile epithelial-like EpCAM+ CSCs [CSCs have proven to play a central role in the development, maintenance, metastasis, and recurrence of HCC –42. Inits in HCC . Besidess in HCC , 45–48. s in HCC . Howevers in HCC . A recenAlthough a number of molecules have been identified as markers for CSC, there is no general consensus on the best CSC markers for HCC. Moreover, there is high variability in marker expression across established cell lines and HCC tumors and their suitability for therapeutic targeting has not been extensively evaluated , 51. HenEMT is a complex molecular and cellular reprograming process by which epithelial cells lose their differentiated characteristics and acquire mesenchymal features, including motility, invasiveness, ability to escape immune cells, and a heightened resistance to apoptosis . EMT is highCD24low cell surface marker profile characteristic of breast CSCs. Notably, these cell populations also acquired self-renewal properties and enhanced tumor-initiating ability [Recently, EMT has emerged as an important regulator of cancer cells exhibiting stem cell-like properties . The bes ability , 66. Sim ability , 67. Fur ability , 66, 68.Transcription factors Snail and Slug have been established as crucial regulators of EMT during embryonic development, organ fibrosis, and cancer progression, as they are potent repressors of E-cadherin expression. Overexpression of Slug but not Snail has been reported in the HCC cell line, HepG2, to induce EMT and enhance CSC marker CD133 . The EMTThe connection between EMT and CSC indicates that the EMT process is doubly dangerous for the cancer patient and may explain how CSCs maintain their aggressiveness in the long period of time when invading and migrating to surrounding tissues . NotablyEMT may provide a means to integrate the canonical CSC model with the clonal evolution model. Given that EMT drives a dedifferentiation-like process whereby non-CSCs can be reprogramed to gain a CSC phenotype, it supports a bidirectional CSC model. Chaffer et al. proposed that some tumors adhere to phenotypic plasticity model wherein dynamic bidirectional conversions between the CSC and non-CSC compartments are common and essential components of tumorigenicity. A study on breast cancer suggests that aggressive CSCs can be created de novo within a tumor by spontaneous mechanisms that pertains to a switch in cell phenotype and is not driven by additional genetic mutations . Another+/ALDHhigh and CD133+/EpCAM+ separated from Huh7, HepG2, and Hep3B, well-differentiated human HCC cell lines were deemed to be epithelial by expression of E-cadherin and lack of Vimentin. In contrast, poorly differentiated cell lines such as SkHep1, HLE, and HLF and double negative subpopulation from well-differentiated cell lines were characterized as mesenchymal due to the higher expression of Vimentin, Zeb1, and Snail; and transforming growth factor (TGF)-β and hedgehog pathway activation [+ cells. Further examination of both primary tumors and HCC cell lines demonstrated that CD90+ subpopulation of cells were mesenchymal and overexpressed CD44, c-KIT, and TWIST1, whereas EPCAM+ subpopulation were epithelial and expressed alpha-fetoprotein (AFP) and Albumin [Recent data suggests that CSCs are indeed plastic and can be associated with both epithelial and mesenchymal states. Occurrence of two distinct populations of CSCs termed EMT-CSCs with epithelial signature and MET-CSCs with mesenchymal signature have been reported in breast cancer. These CSCs have the capacity to transition between these cellular states. It has been envisioned that EMT-CSCs are located at the invasive edge of the tumor and allow the tumor to expand into new territory. On the other hand, MET-CSCs are located in the tumor interior and facilitate tumor cell growth. Furthermore, when the invasive edge becomes the interior of the tumor, the two CSCs can change cellular states . It is ctivation . Another Albumin . The pla+ (K19+) expressing human HCC cells exhibited CSC-like properties together with elevated EMT marker expression. Activation of TGF-β and Smad signaling axis in K19+ cells has been implicated in mediating EMT [+ cells expressed high levels of mesenchymal markers N-cadherin and Vimentin and low levels of epithelial marker E-cadherin. Knockdown of CD44 reversed EMT by repression of ERK/Snail pathway and inhibited lung metastasis of HCC cells in the metastatic model of HCC established by tail vein injection of luciferase-labeled HCC cells in nude mice [Recent studies in HCC have demonstrated that the EMT phenotype and CSC biology are intricately linked. Thus, identifying common molecular pathways regulating CSC and EMT attributes may be critical for the development of efficacious treatment strategies for HCC patients. Table ting EMT . Similarude mice . A recenude mice . Anotherude mice . These sPluripotency factors, oncogenes, and viral infection that result in the generation of CSCs in HCC, also confer these cells with EMT features. For instance, ectopic expression of pluripotency associated transcription factors Oct4 and Nanog that are essential for maintenance of stem cell phenotype endowed HCC cell line MHCC97-L with both CSC and EMT traits through the activation of signal transducer and activator of transcription 3 (Stat3)/Snail signaling . FurtherSimilarly, Artemin, an estrogen-regulated growth factor which promotes resistance to antiestrogen therapies, has been shown to modulate EMT features of HCC cells with downregulation of E-cadherin and upregulation of N-cadherin, Vimentin, Snail, and E47. Artemin promotes the metastatic properties and tumor-initiating capacity of HCC by AKT modulation of factors involved in EMT and stemness . ChronicRecent studies of non-coding RNAs are shedding light on the regulation of EMT and CSC in cancers , 86. StuIn addition to intracellular factors influencing EMT and CSCs, recent studies have found that interaction among cancer cells and stromal cells within the tumor microenvironment can induce EMT by secretion of mediators such as growth factors, cytokines, and extracellular matrix proteins . Accordi2+ signaling has implicated the importance of oxidative stress in promoting EMT and CSC in HCC [Rapid growth of cancer cells often creates insufficient supply of oxygen and results in hypoxic microenvironment. A hypoxic microenvironment is a common feature of HCC and is associated with malignant invasion, metastasis, EMT, and CSC , 93. A pC in HCC . A recenC in HCC . These sThe transcription factor hypoxia-inducible factor-1α (HIF-1α), a key mediator of the cellular response to hypoxia, is overexpressed in HCC . A recen+ HCC cells were resistant to 5-Fluorouracil treatment by high expression of a drug transporter, multidrug resistance protein 5 (MRP5) [Recent evidences indicate that HCC patients manifesting both CSC and EMT phenotypes are unresponsive to standard chemotherapies and have low progression free survival . This ma5 (MRP5) . Sorafen5 (MRP5) –101. Fur5 (MRP5) . IdentifThe association of EMT and CSCs may also form the basis for identifying novel targeting agents to improve clinical outcomes in HCC patients Fig. . One potAs EMT signaling is involved in development and maintenance of hepatic CSCs, an improved understanding of EMT signaling networks and identifying key molecular players linking these cellular phenotypes may uncover new therapeutic targets. For instance, HGF/MET signaling axis is a potential target as HGF neutralizing antibody effectively reduced activation of HGF/MET signaling and concurrently blocked promotion of EMT and CSC phenotypes and cisplatin resistance . InhibitSignaling networks mediated by microRNAs and EMT-inducing transcription factors tie the EMT process to regulatory networks that maintain stemness can serve as possible targets. For instance, overexpression of miR-125b has been effective in targeting EMT and CSC in HCC by regulating transcription factor SMADs 2 and 4 . AlthougImmunotherapeutic approaches comprise an alternative attractive therapy option, although current studies in targeting CSC and EMT in HCC with immunotherapy are somewhat limited. A recent study showed that EMT signature from multiple cancer types was associated with enrichment of multiple druggable immune targets . NotablyThe notion that CSC and EMT phenotypes play important roles in HCC progression, metastatic competence, therapy resistance, and relapse is a rapidly evolving concept that is contributing to our understanding of HCC pathogenesis and development of effective treatment options for this cancer. Emerging research data indicate that the existence of EMT and CSCs may be related to a high risk for recurrence and poor prognosis for many tumor types. Further investigations into CSC and EMT biology will require additional technological advances for the visualization, isolation, and better characterization of HCC CSCs and EMT phenotype, with robust biomarkers, and elucidation of the signaling pathways that are altered in these tumor cells. Improving our understanding of these cellular states may help to categorize potential targets for novel therapies to preclude relapse. Further research, especially characterization of EMT and CSC phenotypes in HCC is needed for more conclusive information.As the molecular circuitries underlying EMT and stemness appear closely intertwined, it will be vital to further delineate key molecular players that link these two cellular states. Recent data suggests that CSCs are very plastic and can be associated with both epithelial and mesenchymal states. This plasticity of CSCs further suggests that targeting either state alone may not be sufficient since the CSCs in the alternative state can rapidly regenerate targeted cell populations. Hence, future investigations will be needed to simultaneously target CSCs existing in both epithelial and mesenchymal states to achieve complete tumor eradication. It remains to be seen where combination of conventional chemotherapy with agents that target CSC and EMT states may fundamentally enhance anticancer treatment in HCC.


Musculoskeletal tissues respond to optimal mechanical signals  through anabolic adaptations, while mechanical signals above and below optimal levels cause tissue catabolism. If an individual's physical behavior could be altered to generate optimal mechanical signaling to musculoskeletal tissues, then targeted strengthening and/or repair would be possible. We propose new bioinspired technologies to provide real-time biofeedback of relevant mechanical signals to guide training and rehabilitation. In this review we provide a description of how wearable devices may be used in conjunction with computational rigid-body and continuum models of musculoskeletal tissues to produce real-time estimates of localized tissue stresses and strains. It is proposed that these bioinspired technologies will facilitate a new approach to physical training that promotes tissue strengthening and/or repair through optimal tissue loading. Musculoskeletal diseases, such as osteoarthritis and tendinopathy, impose substantial burden on individuals and health care systems. As a community of scientists and clinicians, we have been largely ineffective in managing musculoskeletal diseases, as current prevalence, incidence, and socioeconomic burden are at alarming levels and projected to increase sharply in coming decades  the known mechanical stimuli for promoting positive tissue adaptation in musculoskeletal tissues, (2) how local tissue stresses and strains can be estimated using computational methods, (3) an approach to estimating musculoskeletal tissue stresses/strains in real-time, and (4) challenges and future directions for research in this area.Mechanobiology is the study of the effect of mechanical stimuli on tissue biology. It is well-established that mechanical loading plays an essential role in 1) musculoskeletal tissue development throughout human maturation Carter, , (2) mai musculosMusculoskeletal tissue state encompasses tissue morphology and function, both of which may be non-invasively assessed using medical imaging. Morphology, which encompasses all spatial descriptions of an object, can be measured using different medical imaging modalities, such as computed tomography (CT), magnetic resonance (MR), and ultrasound (US).Computed tomography is well-suited to the study of bone and provides high-resolution images that can be automatically- or semi-automatically segmented to render volumetric representations  are lower than those associated with degenerated cartilages  is a well-established computational method used in many branches of engineering. In a FEM model, the real system is discretized into a field of elements of known geometries and material properties, from which constitutive equations may be developed. The model system dynamics are then equilibrated by imposing a set of boundary conditions, e.g., musculoskeletal tissue loads informed by a neuromusculoskeletal model. Halloran et al.  applied Generating personalized FEM models of tissue requires both morphology and material properties. As previously described, different imaging modalities can be used to directly acquire tissue-specific morphology, but non-invasive methods to estimate material properties are scarce. Musculoskeletal tissues have a heterogeneous multiphasic structure, resulting in anisotropies and non-linear time-varying behavior , computational demand may be substantially reduced by spatially averaging many microstructural features, such as trabecular and cortical bone architecture. However, spatial averaging neglects analysis of tissue anisotropy and micro-architecture, which are known to influence tissue function . However, improvements in image processing software, such as automatic segmentation and statistical shape modeling  and registration of body-worn sensor positions to anatomic models. Wireless EMG systems have been used effectively in research and clinical settings for many years, and are now being integrated with inertial measurement units and other sensors as standalone devices or embedded into garments. Future research should aim to first establish if these wearable sensor systems can match the performance of traditional motion capture systems, and then minimize the number of sensors required to drive neuromusculoskeletal models.Very little is currently known about the ability of individuals to volitionally modulate musculoskeletal tissue stresses and strains in response to real-time feedback. To our knowledge, musculoskeletal tissue stresses and strains have never been estimated in real-time, let alone used to modify physical behavior. To date, only two research groups (Manal et al., We know from previous studies people can use visual biofeedback to manipulate external biomechanical variables, muscle excitations, and tibiofemoral contact forces (Manal et al., in vitro and ex vivo studies of tissue mechanobiology to personalized in silico real-time models of musculoskeletal tissue loading. Integrating and translating these bioinspired technologies to clinical settings will prove challenging and resource intensive. Skepticism from clinicians accustomed to generic recommendations based on linear statistics is anticipated and will need to be overcome by demonstrating the efficacy and clinical utility of the proposed new approach. However, there awaits a wide spectrum of important clinical conditions to which these bioinspired technologies could be applied with the goal of reducing the socio-economic burden of musculoskeletal diseases.By optimizing the mechanical environment it may be possible to regulate musculoskeletal tissue mechanobiology, potentially preventing disease, or restoring degraded tissue to health. Consequently, modeling and controlling physical behavior of individuals has enormous implications for development and management of chronic musculoskeletal diseases such as osteoarthritis or tendinopathies. We have presented a framework to move from CP, DL, and DS contributed to conceptualize, draft, critically revise, and approve the final version of the article. RB, JC, MZ, and TB contributed to conceptualize, critically revise, and approve the final version of the article.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.


However, no device delivers real-time histopathologic information on treatment response to help clinicians tailor individual therapeutic strategies. We assessed the potential of multimodal nonlinear optical microscopy to monitor therapeutic responses, including tumoral and stromal responses. We found that two-photon excited fluorescence imaging can, without labeling, identify colloid response, inflammatory cell infiltration, vascular proliferation, and tumor regression. It can also directly detect rare residual tumor cells, which may be helpful for distinguishing tumor shrinkage from tumor fragmentation. In addition, second harmonic generation imaging shows the ability to monitor three types of fibrotic responses: mature, intermediate, and immature. We also determined nonlinear spectra, collagen density, and collagen orientation indexes to quantitatively analyze the histopathologic changes induced by neoadjuvant therapy in rectal cancer. Our work demonstrates that nonlinear optical microscopy has the potential to become a label-free, real-time,  Neoadjuvant radiochemotherapy has become a standard of care for patients with locally advanced rectal cancer. This preoperative treatment is associated with improved local control, reduced postoperative local recurrence, and higher sphincter preservation rate in rectal cancer patients and can improve postoperative survival and prognosis –3. StudiHowever, treatment response varies among patients, ranging from a pathologic complete response to complete resistance. If a patient achieves a clinical complete response, surgery may be avoided entirely. However, most clinicians choose to perform resection in these patients due to the current limitations of medical imaging technology. Ideally, tumor response to neoadjuvant therapy should also be used to determine operation timing because, after neoadjuvant therapy, a tumor needs to progress from necrosis to apoptosis to complete fibrosis, but these pathologic changes cannot be assessed accurately in preoperative evaluation. Therefore, real-time monitoring of disease response to therapy requires standardization to design risk-adapted preoperative and postoperative treatments and ultimately optimize patient outcomes. However, assessment of neoadjuvant therapy response is currently suboptimal .Fortunately, biological exploration is benefiting from continuing development of nonlinear optical imaging technique. This imaging technique is capable of high-resolution, high-penetration-depth, three-dimensional imaging of biological tissues and has the ability to detect cellular and subcellular tissue microstructure by excitation of intrinsic fluorescent molecules. As a result, in this study, we monitored therapy response to preoperative radiochemotherapy for rectal cancer using multimodal nonlinear imaging combining the second harmonic generation (SHG) and two-photon excited fluorescence (TPEF) signals.The top row in Figure The bottom row in Figure The top row in Figure The bottom row in Figure To determine the origin of nonlinear optical signals in normal muscular tissues and pretherapy and posttherapy muscular tissues in rectal cancer, emission spectroscopic investigations were performed using the spectral imaging scan mode under the same conditions. A total of 15 samples including five normal tissues, five pretherapy tissues, and five posttherapy tissues were selected for measuring the nonlinear emission spectra. The fresh tissue section of each sample was excited at an 810-nm excitation wavelength, and the emission signals were collected between 377 and 716 nm using a spectral detector under the lambda mode setting. As displayed in Figure P < 0.001). These results indicate that the pretherapy and posttherapy muscular tissues may contain more collagen fibers because of the desmoplastic response caused by tumor invasion and the fibrotic response induced by neoadjuvant therapy, respectively. Therefore, collagen content may be treated as an optical biomarker to discriminate between normal and abnormal tissues.Collagen content was also calculated to quantify stromal collagen changes. Quantitative results shown in Figure Figure Colloid response, which is a type of stromal response and is often seen in posttreatment rectal tumors, is defined by predominant colloid changes with or without residual tumor cells and is an important predictor of survival . In our Previous studies have demonstrated that stromal fibrosis induced by preoperative adjuvant treatment is an important indicator of wound healing , 16 and Figure P < 0.001) in the collagen orientation index between the three types of stroma. Hence, collagen orientation index can be used as a diagnostic parameter to distinguish between the three kinds of fibrous stroma.Compared with the intermediate and immature stroma, the most obvious feature of collagen fibers in mature stroma is that they are fine and well-organized. Therefore, measurement of the orientation of collagen fibers may provide better understanding of patterns of fibrosis for pathologists. As previously reported, the collagen orientation index ranges from 0 (perfectly random) to 1  and is measured by ImageJ software , 21. In Neoadjuvant radiochemotherapy has been proven to be a useful tool for tumor reduction and to increase operability and decrease the rate of local recurrence in colorectal cancer patients , 23. HowTherapy response in rectal cancer patients treated with preoperative radiochemotherapy is divided into two main groups: tumor response and stromal response, as shown in Figure The scenario of tumor fragmentation indicates that the degree of tumor regression is not informative for depth of infiltration because residual tumor cells may still be scattered throughout the rectal wall or mesorectum, and a report has indicated that normal-appearing mucosa may harbor microscopic tumor cells up to 3 to 4 cm in any direction . HoweverOur experimental data imply that without the use of any exogenous contrast agents, TPEF microscopy can directly identify a single tumor cell because of its subcellular-level resolution. In addition, as a continuing development of nonlinear microscopy, it can also scan a large area of the samples . TherefoThere are many diverse elements in tumor stroma, and these have traditionally been classified into three main categories: fibrous tissues, blood vessels, and inflammatory cells. Accordingly, stromal changes induced by preoperative radiochemotherapy mainly include fibrotic reaction, inflammatory reaction, blood vessel hyperplasia, colloid response, and so on. Our study shows that TPEF microscopy is able to monitor inflammatory cell infiltrates, vascular proliferation, and colloid reaction without the need for fluorescent markers because there are several endogenous fluorophores within rectal tissues, including nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) in cells and elastin in the blood vessel wall. Using traditional techniques, it is difficult to detect sparse residual tumor cells in mucin lakes on the resection margin, especially in frozen sections , which mTumor invasion may activate fibroblasts and cause a desmoplastic reaction, whereas tumor regression in posttreatment rectal carcinomas mostly results in fibrotic changes replacing neoplastic glands –19. As pex vivo samples, it demonstrated great potential for future clinical applications when in vivo nonlinear optical endoscopy is realized, especially for presurgery evaluation after neoadjuvant radiotherapy. As a novel optical tool, the exploitation of a compact and flexible nonlinear optical endoscope for in vivo imaging is attracting attention and research, and previous publications have shown that in vivo images of unstained colon, kidney, and liver from an anesthetized rat can be obtained using a compact and flexible multiphoton microendoscope [To summarize, nonlinear optical microscopic imaging possesses many unique advantages, such as label-free imaging, single-cell detection, high-penetration-depth, and reduced photobleaching and phototoxicity in the out-of-focus regions. As a result, it has been widely used in life sciences . Althougndoscope , 33. Thuin vivo imaging technique, it will help clinicians to alter treatment doses if needed, determine the operation plan and operation timing, and avoid overtreatment or forgo futile treatment.In summary, it is presented that multimodal nonlinear optical microscopy can be used to monitor therapy response to neoadjuvant treatment. Our results demonstrate that a combination of SHG and TPEF imaging can identify pathologic alterations such as tumor response and stromal response in rectal cancer patients treated with preoperative radiochemotherapy. In the near future, with further improvements in these nonlinear methods, this ability may impact treatment options in rectal cancer patients who have received preoperative radiochemotherapy because the effects of therapy can be assessed before surgery. When nonlinear microscopy develops into a real-time, label-free, The nonlinear optical imaging system used in this study has been previously described in detail , 35. In This study was conducted with the approval of the institutional review board at the Fujian Medical University Union Hospital, and signed informed consent was obtained from each patient. Posttherapy tumors penetrating into the muscular layer or deeper were selected for this study, and normal tissues and pretherapy cancerous tissues were also collected for comparison. The region of our interest was muscular tissues because once tumor infiltrates into or beyond the muscularis propria layer, it is classified as an advanced cancer and has a much worse prognosis.Thirty-two patients with adenocarcinoma of rectum who received neoadjuvant therapy were recruited. Patient age ranged from 38 to 83 years (59 ± 11 years), and the male-to-female ratio was 1.7. Once fresh tissue samples were removed by surgeons, they were sent to the pathology laboratory immediately, where each specimen was serially sectioned at 10 μm by cryostat microtome. Then five serial tissue slices were chosen for research; the middle slice was stained with H&E to confirm experimental results, and the other sections were used for nonlinear optical imaging. In addition, to avoid dehydration or shrinkage during the imaging process, a small amount of phosphate-buffered saline was applied to the specimen.All of the H&E-stained slices were reviewed by a certified pathologist, and images were then taken using a standard bright field light microscope  with a CCD . The results from nonlinear optical imaging were then compared with the H&E images obtained from light microscopy (40×) for confirmation.P values of ≤0.05 were considered statistically significant.To quantitatively assess collagen changes in stroma during neoadjuvant therapy, collagen content and collagen orientation index were measured, where collagen content was defined as the ratio of SHG to all pixels in each image and collagen orientation index was described by an index ranging from 0 (perfectly random) to 1 . These values were expressed as means and standard deviations. Statistical calculations were performed using the IBM SPSS Statistics 21, and


Malurus melanocephalus), before vs. after naturally occurring fire disturbance in Northern Territory, Australia. We utilized a social network framework to evaluate changes in social dynamics pre- vs. post-fire. Our study covered the non-breeding season in two consecutive years in which fires occurred, and individuals whose habitat was affected and those that were not affected by fire. Individuals in habitat affected by fires had stronger social ties (i.e. higher weighted degree) after fires, while those that were in areas that were not affected by fire actually had lower weighted degree. We suggest that this change in social connections may be linked to habitat. Before fires, fairywrens used habitat that had similar grass cover to available habitat plots randomly generated within our study site. Fire caused a reduction in grass cover, and fairywrens responded by selecting habitat with higher grass cover relative to random plots. This study demonstrates how changes in habitat and/or resource availability caused by disturbance can lead to substantive changes in the social environment that individuals experience.Individual level response to natural and anthropogenic disturbance represents an increasingly important, but as yet little understood, component of animal behavior. Disturbance events often alter habitat, which in turn can modify behaviors of individuals in affected areas, including changes in habitat use and associated changes in social structure. To better understand these relationships, we investigated aspects of habitat selection and social connectivity of a small passerine bird, the red-backed fairywren ( Social structure shapes processes ranging from disease and parasite transmission ,2, to inSocial organization can vary in different ecological environments, as measured along a gradient or in discrete habitats e.g., ,16). Rel. Rel16])Tursiops aduncus) living in the same area altered their social organization in response to changes in food availability caused by the presence vs. absence of prawn trawlers )A more thorough understanding of individual social behaviors and responses to environmental stressors can be critical for conservation and management of social species , particuS1 TableObservations of red-backed fairywren groups of color-banded birds in 2013 and 2014 are provided in a.csv table. Spatial period denotes whether groups were observed pre- or post-fire, and location was used to determine if individuals were affected by fires.(CSV)Click here for additional data file.


Additionally, several usability features have been updated and improved. Together, these improvements make antiSMASH up-to-date with the latest developments in natural product research and will further facilitate computational genome mining for the discovery of novel bioactive molecules.Many antibiotics, chemotherapeutics, crop protection agents and food preservatives originate from molecules produced by bacteria, fungi or plants. In recent years, genome mining methodologies have been widely adopted to identify and characterize the biosynthetic gene clusters encoding the production of such compounds. Since 2011, the ‘antibiotics and secondary metabolite analysis shell—antiSMASH’ has assisted researchers in efficiently performing this, both as a web server and a standalone tool. Here, we present the thoroughly updated antiSMASH version 4, which adds several novel features, including prediction of gene cluster boundaries using the ClusterFinder method or the newly integrated CASSIS algorithm, improved substrate specificity prediction for non-ribosomal peptide synthetase adenylation domains based on the new SANDPUMA algorithm, improved predictions for terpene and ribosomally synthesized and post-translationally modified peptides cluster products, reporting of sequence similarity to proteins encoded in experimentally characterized gene clusters on a per-protein basis and a domain-level alignment tool for comparative analysis of  Natural products, also referred to as secondary or specialized metabolites, are the basis of many drugs and are also important molecules for agricultural and nutritional applications; moreover, they play key roles in scientific research as chemical probes to study many aspects of molecular and cellular biology. The observation that the genomes of many microorganisms contain multiple biosynthetic gene clusters (BGCs) that code for the production of such molecules has led to a paradigm shift in natural products research: within the last 10 years, genome mining has been established as an important technology complementing the bioassay- and chemistry-driven classical natural products discovery process –4), suchThe comprehensive open-source BGC mining platform antiSMASH –8 was fitrans-AT polyketide synthase (PKS) assembly lines and TTA codon annotation. Moreover, an improved user interface was introduced, along with several other usability and efficiency improvements. The public antiSMASH web server is freely accessible at http://antismash.secondarymetabolites.org.Here, we report version 4 of antiSMASH, which includes several major extensions, such as gene cluster boundary prediction for fungal BGCs, improved chemistry predictions for terpene, ribosomal peptide and non-ribosomal peptide BGCs, comparative alignment of Estimating the boundaries of BGCs is a continuing challenge for genome mining tools. Traditionally, antiSMASH has opted for a ‘greedy’ approach by design, in order to ensure a greater likelihood of including all pertinent biosynthetic genes. The rationale behind this was that expert users would be better at estimating cluster boundaries than automated algorithms would. However, for certain purposes, it is still highly beneficial for users to review a computer-assisted estimate of where a BGC may start and end. For this reason, antiSMASH has now added two methods to predict the boundaries of BGCs. For fungal genomes, the Cluster Assignment by Islands of Sites (CASSIS) algorithm  is used et al. Since then, several new algorithms have been published to predict A-domain specificity  substantially expanded the training sets for these algorithms, introduced an additional (phylogenetics-based) algorithm (PrediCAT), benchmarked all algorithms systematically and constructed an ensemble prediction method  that outperformed each method individually. To benefit from the latest insights in this field, we have now replaced the previous prediction algorithms with the SANDPUMA predictions; these provide not only the ensemble outputs, but also the individual outputs of the underlying SVM, ASM, PrediCAT and pHMM algorithms. Since the benchmark comparison had shown the Minowa method  adenylation (A) domains: the support-vector machine (SVM) and active-site motif (ASM) prediction methods from NRPSPredictor2  and the cificity –21. Morea method  to be thIn addition to the prediction of non-ribosomal peptide chemistry, antiSMASH now also provides chemical structure predictions for the products of bacterial terpene synthases . To thisRibosomally synthesized and Post-translationally modified Peptides (RiPPs) constitute a growing area of natural products research. antiSMASH supports researchers in predicting 15 distinct classes of RiPP BGCs. Previously, antiSMASH predicted only lanthipeptide precursors using a relatively limited pHMM-based approach. The current version of antiSMASH now provides a more sophisticated prediction and classification for class I lanthipeptides as well as lasso peptides, sactipeptides and thiopeptides. Given that RiPPs start as gene-encoded precursor peptides prior to post-translational modification, amino acid sequence prediction provides a wealth of information regarding the structure of the final product. However, the open-reading frames (ORFs) encoding these peptides are often overlooked by automated analysis and can be highly sequence variable, necessitating the need for current precursor identification methods.To assist in identifying the precursor peptide-encoding gene, antiSMASH now utilizes the algorithm from the genome-mining platform Rapid ORF Description and Evaluation Online (RODEO) , which uFor the RiPP classes analyzed by the RODEO algorithm, antiSMASH reports: (i) the respective class of RiPP , (ii) a predicted leader peptide cleavage site and (iii) any potential C-terminal proteolytic processing. Given the post-translational simplicity of lasso peptides, a molecular mass is also calculated, accounting for the number of disulfide bridges. For thiopeptides, the macrocycle size and potential amidation are predicted as well. Molecular weight predictions are not given for the other RiPP subclasses owing to their extensive and variable post-translational modifications.trans-AT PKS , which uses reference phylogenies of ketosynthase (KS) domains to assign KS domains from identified gene clusters into clades that correspond to a certain type of polyketide chemistry. Based on this classification, the encoded assembly line is then aligned to reference assembly lines from known BGCs in MIBiG  do not reveal major insights between the natural product structures and the genes for the corresponding multidomain proteins that encode their biosynthetic enzymes. In order to better address this issue, we have now included an assembly line alignment method for in MIBiG  based onin MIBiG ). The asin MIBiG . This anStreptomyces and related genera are important producers of clinically used antibiotics, such as tetracyclines or erythromycin, or drugs to treat parasitic worms such as avermectin. These bacteria have GC-contents of >70% and thus a skew toward higher GC triplets in their codon usage. While genes involved in primary metabolism almost exclusively use CTC codons to code for Leu, key genes in secondary metabolism and cell differentiation often contain TTA codons. As the expression of the TTA-codon specific Leu-tRNA-gene bldA is tightly controlled and the Leu-tRNA only accumulates in later stages of growth, this offers an additional level of regulation  and plant sequences (http://plantismash.secondarymetabolites.org/). The main antiSMASH website is now focused on bacterial and archaeal sequences. The metabolic modeling functionality along with an EC number prediction option that were introduced in antiSMASH version 3 were removed again, as they led to extremely long run times and high server load. An updated version with improved reaction rules for secondary metabolite biosynthetic pathways will be released as a separate, but still closely linked program.In order to simplify selecting the correct input settings, separate submission pages were created for fungal sequences (In addition to GenBank- and EMBL-formatted files, gene annotations can now also be added to FASTA sequences by also uploading a GFF3-formatted file. To assist job submission and retrieval from third-party tools running upstream or downstream analyses such as the CRISPR single guide RNA finding tool CRISPy-web  or the AWith the new features now introduced Table , the antWith regard to chemistry prediction of the products of NRPSs and PKSs, we have opted to be conservative for the moment. The recently introduced PRISM pipeline  does a gWe continue to strive for interoperability with other services. For example, antiSMASH predictions are also available through the Joint Genome Institute's IMG-ABC  as well http://antismash.secondarymetabolites.org/. This website is free and open to all users and there is no login requirement. Source code is available from https://bitbucket.org/antismash/antismash/.antiSMASH is available from Supplementary DataClick here for additional data file.


Neuroanatomical correlations of naming and lexical-semantic memory are not yetfully understood. The most influential approaches share the view that semanticrepresentations reflect the manner in which information has been acquiredthrough perception and action, and that each brain area processes differentmodalities of semantic representations. Despite these anatomical differences insemantic processing, generalization across different features that have similarsemantic significance is one of the main characteristics of human cognition.We evaluated the brain regions related to naming, and to the semanticgeneralization, of visually presented drawings of objects from the BostonNaming Test (BNT), which comprises different categories, such as animals,vegetables, tools, food, and furniture. In order to create a model of lesionmethod, a sample of 48 subjects presenting with a continuous decline both incognitive functions, including naming skills, and in grey matter density(GMD) was compared to normal young adults with normal aging, amnestic mildcognitive impairment (aMCI) and mild Alzheimer’s disease (AD). Semanticerrors on the BNT, as well as naming performance, were correlated with wholebrain GMD as measured by voxel-based morphometry (VBM).The areas most strongly related to naming and to semantic errors were themedial temporal structures, thalami, superior and inferior temporal gyri,especially their anterior parts, as well as prefrontal cortices .The possible role of each of these areas in the lexical-semantic networks wasdiscussed, along with their contribution to the models of semantic memoryorganization. Lexical-semantic memory refers to the storage of this knowledge inthe brain by means of patterns of neuronal activity interpreted as linguistic symbols ofconcrete and abstract concepts. The relationship between brain anatomy and the storageof these patterns of information is not yet well understood. Several hypotheses havebeen proposed to explain how lexical-semantic memory is processed and stored in thebrain, and these have been guided by two main general models: a parallel distributedrepresentation,2 comprising ahomogeneous network of equivalent neuronal units that process every aspect of semantics,and a center processing model, which assumes that all memory elements are encoded in adelimited area of the brain. Neither of these models in its pure form satisfactorilyexplains the phenomena, and so a combination of these two theories has beenproposed.3Language is one of the most important characteristics that allows us to codify, signify,and retain our experience of the world.5 It has also been suggested that thetemporal lobe object representation system may be organized hierarchically, withincreasing convergence and integration of information occurring along its posterior toanterior axis.6One of the main characteristics of human cognition is the capacity to generalize acrossconcepts that have similar semantic significance but not necessarily similar specific attributes. The most striking evidence of deterioration ofthis generalizing capacity, manifested initially by semantic naming errors production,is semantic dementia (SD), in which there is a degeneration of the anterior portions ofthe temporal lobes, and is more intense on the left side. These patients havedifficulties in naming everyday objects and knowing their properties, with impairment ofall kinds of concepts in the context of otherwise well-preserved cognition, includingepisodic memory. Other diseases associated with lesions in the anterior parts oftemporal lobe show the same pattern of loss of knowledge, particularly in Herpes simplexvirus encephalitis, stroke, and Alzheimer’s disease (AD). In this sense, as proposed byother authors, the temporal lobe, particularly its anterior part, may constitute aconvergence zone for information coming from brain regions responsible for processingdifferent aspects of knowledge.9 Problems with naming and word findingare even more common in mild cognitive impairment (MCI) and are most common inAlzheimer’s disease (AD).11 MCI is a clinical entity applied topatients with objective cognitive problems, most commonly in episodic memory, withoutsignificant impairment of activities of daily life.12Naming complaints are very common in mentally healthy elderly people. Individuals overthe age of seventy attain significantly lower scores on these naming tests compared toscores by young adults.13 regardless of category . In order to create a model of the lesionmethod, a sample of subjects presenting with continuous decline both in cognitivefunctions, including naming skills, and in grey matter density (GMD) were compared tonormal young adults with normal aging, amnestic mild cognitive impairment (aMCI), andmild AD. Semantic errors on the BNT were correlated with whole GMD as measured byvoxel-based morphometry (VBM). This correlation was also performed for BNT total score(correct responses). We hypothesised that temporal lobes, especially their anteriorparts, are related with semantic naming error production in this sample of subjects. Themajority of structural neuroimaging studies in patients with language problems haveemployed volumetric measurements on magnetic resonance imaging (MRI) data sets. Thiskind of approach has used the region-of-interest method, which depends on a priorichoices and can be applied to a selected set of brain structures only. A whole-brain VBMapproach on the other hand, has the advantage of not only evaluating the previouslyhypothesized brain structures, but also potentially revealing unexpected areas of graymatter density changes and their correlation with neuropsychological scores.Our aims were to evaluate the brain regions related to naming performance and tospontaneous semantic naming errors on the Boston Naming Test (BNT),13A total of 48 subjects older than 50 years  were studied. Routine laboratory examinations for dementiaassessment  and brain computed tomography were carried out in all patients.The local ethics committee approved this study. Diagnosis of aMCI in our clinic iscarried out by trained neurologists using a standardized mental state battery,including evaluation of episodic memory, orientation, language, attention, abstractthinking, calculation, and visual perception. The diagnostic process consists of adetailed interview with the patient and informant . Diagnosis of MCI was made according to the criteria of the InternationalWorking Group on Mild Cognitive Impairment:(i) the person is neither normal nor demented;(ii) there is evidence of cognitive deterioration shown by eitherobjectively measured decline over time and/or subjective report ofdecline by self and/or informant in conjunction with objective cognitivedeficits; and(iii) activities of daily living are preserved and complex instrumentalfunctions are either intact or minimally impaired.14 score of 0.5, with an obligatoryand exclusive memory score of 0.5. This classification was performed using asemi-structured interview.A diagnosis of aMCI was determined if the clinical history and cognitive performancepointed to an exclusive memory deficit and Clinical Dementia Rating (CDR)15 including only patients classified as CDR 1. Exclusioncriteria were history of other neurological or psychiatric diseases, head injurywith loss of consciousness, use of sedative drugs in the 24 hours preceding theneuropsychological assessment, drug or alcohol addiction, and prior chronic exposureto neurotoxic substances. The control group comprised subjects with CDR 0 and noprevious history of neurological or psychiatric disease, or memory complaints.For probable AD diagnosis, the criteria of the National Institute of Neurological andCommunicative Disorders and Stroke (NINCDS) and Alzheimer’s Disease and RelatedDisorders Association (ADRDA) were employed,13, for which subjects were asked to name the presentedpictures, was administered to all subjects. BNT total score was calculated byadding the number of correct spontaneous responses to the number of correctresponses after a semantic cue, which consisted of a short explanation about thepicture  or asuperordinate category . The semanticcue was only given if the patient had failed to recognize the picture (forexample: dog instead of tree) or if he/she said that they did not know what thepicture was.The sixty-item BNTSemantic errors registered when the spontaneous answer was semantically relatedto the target word. Two independent researchers performed this classification,and the discordances were solved by consensus.16(Brazilian version);17 Reyauditory verbal learning test18- episodic memory delayed recall (RAVLT-A7) and CAMCOG’ssubscale of similarities ;19 visual perception subtests of Luria’s NeuropsychologicalInvestigation ;20 the forward (FDS) and backward digit span (BDS)subtests of the WAIS-R21 andthe Cornell Scale for Depression in Dementia (CSDD).23 Dataanalysis was performed using Systat software 12.0. The Kruskall-Wallis andMann-Whitney tests were used for inter-group comparisons of demographic andcognitive scores. Statistical significance was considered when p<0.05.All subjects were submitted to tests of verbal fluency (VF) - animals category; Mini Mental StateExaminationwww.mricro.com) and set theorigin of the coordinate system at the anterior commissure.High-resolution MRI was performed using a 2.0 T scanner .T1- and T2-weighted images were acquired in axial, coronal and sagittal planeswith thin slices. In addition, volumetric (3D) T1 gradient echo (GRE) imageswere acquired in the sagittal plane with 1 mm-thick slices . Before pre-processing all scans were checked forscanner artefacts and gross anatomical abnormalities. MRIcro was used to convertthe original DICOM format to ANALYZE format (www.fil.ion.ucl.ac.uk) run on MATLAB 7.5 was used to performvoxel-based morphometry (VBM). For segmentation, the “New Segment” toolbox fromSPM8b was employed. The algorithm used for segmentation is based on “UnifiedSegmentation”,24 whichin turn is based on a mixture of Gaussian and combines image registration,tissue classification and bias correction within one generative model. In orderto obtain a more accurate inter-subject alignment the DARTEL  registration model waschosen. In addition, to preserve the volume of each tissue (modulation step) thewarped images were scaled using the Jacobian determinants. Finally, thenormalized, segmented, modulated (Jacobian-corrected) and warped images weresmoothed by convolving with an isotropic Gaussian kernel with full width at halfmaximum of 10 mm to reduce interindividual gyral variation.25 After this pre-processing, theresulting normalized, modulated and smoothed data was used for statisticstreatment.SPM8b  to identify brain regionswhose GMD values were significantly correlated with the absolute number ofsemantic errors and BNT total score. Age, education, total intracranial volumeand global cognition (as measured by MMSE) were also included in the analysis asdependent variables. Total intracranial volume was obtained by the sum ofvolumes of grey matter, white matter and cerebrospinal fluid. For multipleregression analysis, the three groups were considered together  to increase data variance and enhance the correlation betweencerebral region and psychological function, assuming that the denser the graymatter, the better the naming performance and vice versa.Multiple regression analysis using Non-Parametric Mapping (NPM) software. To control for FWE, permutationanalysis was also performed using NPM, with 4000 permutationsanalyzed.27As shown in Multiple regression analysis revealed significant correlations between GMD andBNT score, mostly in the thalamus: right lateral dorsal nucleus and left medialdorsal nucleus; bilateral hippocampus; bilateral parahippocampal gyrus; leftsuperior temporal gyrus; left inferior frontal gyrus; bilateral superior frontalgyrus; left middle frontal gyrus, and other areas shown in Our results support the hypothesis of a continuum in brain pathology and cognitivedecline among the three groups, particularly regarding their spontaneous answersduring BNT picture naming, which indicates that our lesion model was satisfactorilytested. Several brain regions were found to be negatively correlated with semanticerrors on the BNT , and positively correlated with BNT score. A discussion follows on thepossible role of each of these areas in the lexical-semantic networks and theircontribution to the models of semantic memory organization.28 It is wellestablished that episodic memory for events encoded during semantic categorizationis better remembered than when subjects do not associate the target event with aparticular previously learned characteristic, which indicates a close relationshipbetween semantic and episodic memories. It is also possible that, through repetitionand rehearsal, new information could be abstracted from its episodic context andrepresented as semantic memory.29In addition, it has been demonstrated that amnesic patients with lesions in themedial temporal lobes have impaired acquisition of new semantic memories.30 Our results concur with those of arecent VBM study in patients with early AD,31 which also found strong GMD correlations in the medialtemporal structures with naming performance, mainly with the most anterior part ofthe parahippocampus and other parts of the perirhinal cortex. As proposed by theseauthors, the primary role of this region is combining the different representationsof a given object, as part of a process of multimodal synthesis spread acrossdifferent cortical areas. Thus, lesion of these brain structures in early AD wouldisolate the hippocampus from the multisensory input of the neocortex, resulting inreduction of retrieval efficiency, rather than loss of representation.Medial temporal structures such as the hippocampus and parahippocampal gyrus have awell-known role in episodic memory processes. Recently, they have also beenassociated with lexical-semantic memory. In fact, episodic and semantic memories arehighly interactive.31 Researchershave proposed that the thalamus could modulate the mechanism for semantic objectrecall via synchronizing of electrical brain rhythms.32 Kraut et al. (2003)33 studied a word-word feature-binding task usingevent-related fMRI. They found two distinct loci of thalamic signal change, oneanterior in the dorsomedial nucleus, and the other posterior in the pulvinar. Basedon these findings and previous electrophysiological studies, the authors proposed aneural mechanism in which the dorsomedial nucleus is involved in the early search orobject generation and activates other prefrontal regions specifically involved intask-related working memory or language functions. Our findings support the ideathat the thalamus is directly involved in lexical-semantic memory activities,possibly with an integrative role, since its nuclei were correlated with both BNTtotal score and semantic errors.The role of the thalamus in lexical-semantic memory is less well understood than thatof other significant areas demonstrated in our study. Recent electrophysiologicaland functional neuroimaging studies have established the involvement of the thalamusin the process of feature binding, which results in the recall of the object insemantic memory.37 Grossmanet al. (2004)37 studied VBM andconfrontation naming in AD, frontotemporal dementia, and corticobasal degeneration,and found a left lateral temporal atrophy as a common source of impaired namingacross these patient groups. Another VBM study of semantic dementia38 showed that ATL activation peaksaligned closely with areas of strongest grey matter reduction, mostly with atrophyof the left anterior temporal lobe. We found correlations, particularly in theanterior parts of the STG, bilaterally but stronger on the left side, and in theanterior parts of the ITG, albeit weaker and less spread out than in the STG. Ourfindings support the notion that the anterior temporal lobe (ATL), predominantly itssuperior part, is strongly related to semantic generalization, since the subjectswere asked to name pictures of different categories and a close relationship wasobserved between semantic errors, regardless of their specific categories.The involvement of neocortical temporal regions in semantic memory is betterunderstood and has been extensively demonstrated.6 as evidenced by functional neuroimaging, transcranialmagnetic stimulation, and lesion studies.41 On functionalimaging studies, the LIPFC is more active when subjects make semantic judgmentsregarding words than when they make non-semantic judgments for the samewords,39 and also whenthey make semantic judgments for line drawings.42 The role of the LIPFC is crucial when the semantic tasksrequire cognitive control of semantic or lexical retrieval, particularly duringselection among competing alternatives. A study suggests that the LIPFC does notsupport retrieval of semantic knowledge per se.43 Rather, this retrieval is done entirely by the posteriorneocortex based upon cues presented through bottom-up processes, and the specificrole of the LIPFC would be to select those retrieved representations that aretask-relevant from among competing, irrelevant representations.The prefrontal cortex is also related to the lexical-semantic system, often in anasymmetrical manner, with the left side more involved than the right. The leftinferior prefrontal cortex (LIPFC) has been regarded as a “semantic working memorysystem” responsible for retrieving, maintaining, monitoring, and manipulatingsemantic representations stored elsewhere,44 In such cases,making a semantic error  might indicate difficulty in selecting the appropriate phonological responseto answer a particular semantic question. In fact, activation of the LIPFC has beenelicited by phonological tasks such as discrimination of visually and auditorilypresented words45 with thegreatest activation more posteriorly near Broca’s area.39 These and other studies46 have also suggested a domain-specificity of theanterior LIPFC (BA 45/47) for controlled semantics and of the posterior LIPFC (BA44/6) for controlled phonology. However, more recent studies48 have argued against domain-specificity and fordomain-preferentiality in LIPFC. Thus, it may be hypothesized that the LIPFC isactivated to the extent that lexical and semantic information must be rehearsed,temporarily stored, and selected in working memory to perform a particular task.Patients with left prefrontal lesions often have difficulty retrieving words inresponse to specific cues (e.g. words beginning with a specific letter or names ofobjects belonging to a specific semantic category), even when there is noaphasia.Our study has some limitations including the relatively small sample size and thefact that the BNT is not well balanced in terms of psycholinguistic variables.Despite this factor, the BNT is one of the most widely used naming tests in clinicalpractice, and it continues to be a well-accepted measure of naming impairment inbrain-damaged patients. Notwithstanding the limitations of this study, we foundevidence that several brain areas are related to the process of higher-ordersemantic generalization, particularly the thalamus, medial temporal lobe, prefrontalcortex (left more than right), and bilateral anterior temporal lobes (predominantlySTG and ITG).


The purpose of this study is to describe some of the latest advances in using hydrogels for cancer melanoma therapy. Hydrogel formulations of polymeric material from natural or synthetic sources combined with therapeutic agents have gained great attention in the recent years for treating various maladies. These formulations can be categorized according to the strategies that induce cancer cell death in melanoma. First of all, we should note that these formulations can only play a supporting role that releases bioactive agents against cancer cells rather than the main role. This strategy involves delivering the drug via transdermal pathways, resulting in the death of cancerous cells. Another strategy utilizes magnetic gel composites to combat melanoma via hyperthermia therapy. This review discusses both transdermal and hyperthermia therapies and the recent advances that have occurred in the field. According to the recent statistics from the Skin Cancer Foundation, one in six Americans will develop some form of skin cancer during their lifetime . These cIn the pathogenesis of melanoma, the melanocytic skin tumors include an extensive variety of benign and malignant skin lesions with different clinical, morphological, and genetic profiles . Major aAlthough the primary cutaneous melanoma can be managed by surgery, the advanced metastatic melanoma cannot be managed by surgery alone; therefore, it demands better therapeutic methods .α (INF-α), and granulocyte macrophage colony-stimulating factor (GM-CSF). Theses cytokines stimulate the patient's immune system to fight cancer. In general, this strategy was successful in fighting malignant cells. However, the main problem of conventional melanoma chemotherapy treatments is the powerful adverse effects, because the neoplastic agents do not distinguish cancer cells from healthy cells [In the conventional melanoma chemotherapy treatment, antineoplastic agents have been a principal tool for combating cancer , specifihy cells , 13. Forhy cells , 15. TheNew forms of treatments to attack cancer cells are required while simultaneously decreasing the side effects caused in healthy cells , 17. To Given the adverse side effects caused by conventional therapies in patients with melanoma, a new field, large carrier-based drug delivery systems, has emerged that fights the cancerous cells while subverting the side effects. These types of drug delivery systems possess drug carriers such as nanoparticles , dendrimHydrogels are three-dimensional, hydrophilic polymeric networks that are capable of absorbing large amounts of water, biological fluids, or molecules . AnotherA successful dermatological treatment depends on both the active compound and the physicochemical properties of the delivery system. Transdermal and topical delivery systems are biomaterials that carry a specific drug in contact with and through the skin . Transde in vitro and in vivo assays in melanoma.Traditionally, paclitaxel (PTX) was administered intravenously to treat skin cancer. However, since PTX could not differentiate between healthy cells and cancer cells, it has created many unwanted side effects, sometimes resulting in the patient's death. Therefore, to minimize the cytotoxicity and reduce side effects, targeted delivery of PTX to the cancerous cells, while not affecting the healthy cells, needs to be developed. Ta et al. have developed an approach where the researchers have encapsulated the drug PTX in hydrogels . However in vitro and in vivo assays in melanoma. Furthermore, it was demonstrated that 5-Fu-loaded hydrogel could act as a biodegradable drug depository capable of providing constant release of 5-Fu after intratumoral injection, thus increasing the chemotherapeutic effect of 5-Fu while decreasing its systemic toxicity. Working on the encapsulation of the bioactive agents in hydrogel scaffolds and subsequent release, Seo and coworkers obtained interesting results. Durable retention of 5-fluorouracil (5-Fu), an antineoplastic agent in a hydrogel based on polyethylene glycol, polycaprolactone, and poly-L-lactic acid copolymers (MCL), within a tumor was demonstrated. Please see β-cyclodextrin supported in-situ-forming hydrogels. The therapeutic effect of in vitro Cur on melanoma has been investigated. The solubility, stability under light, erosion, release in vitro, transdermal permeability efficiency, and cytotoxicity of Cur inclusion complexes have been improved by encapsulating Cur in hydrogel scaffolds. Therefore, Cur encapsulated in hydrogel is a promising formulation for melanoma treatment. Nevertheless, future studies on the developed formulation are necessary for validating the therapeutic effect in an in vivo setting [Since the beginning, classical pharmacology had to adapt to disadvantages of some antineoplastic agents such as low hydrophobicity. In this context, enormous efforts have been made to correct this problem with the usage of hydrogels. For instance, curcumin (Cur) is a hydrophobic polyphenol with antiproliferative and antiangiogenic activities. However, its weak water solubility and photochemical instability have been the major obstacles for its formulation. Cur has been encapsulated into the hydroxypropyl- setting . in vitro melanoma cell lines. Furthermore, the hydrogel and ibuprofen combination has been demonstrated to be capable of inhibiting cell migration [There is evidence that inflammation stimulates tumor invasion or cancer metastasis . Inflammigration . in vivo melanoma model in male mice. Moreover, this hydrogel has proven to be biodegradable and biocompatible [In recent years, studies have been carried out with the goal of achieving efficient release of doxorubicin (DOX) through a formulation based on hydrogel. Some research studies have investigated chemotherapy for melanoma in depth. These studies have utilized a natural protein from silk, sericin, with dextran to form hydrogels and later incorporate DOX to use a drug delivery system. The hydrogel loaded with DOX, subcutaneously injected, significantly suppressed tumor growth in thempatible . in situ contain both drugs due to the inclusion of the amphiphilic macromolecular cross-linker. The absorption procedure is illustrated in  In vivo assays (murine melanoma cancer) demonstrated the ability of the formulation as a depository to reduce the amount of drug being transferred to the bloodstream, consequently decreasing the toxic effects of the drug. The gel exhibited tumor inhibition efficacy. The efficiency of the system for combined DOX and PTX therapy has been confirmed by significant prolongation of the survival rate of the tumor bearing mice [Also, advances have been made in the combination therapy, where two or more drugs are used to combat cancer. This type of combinatorial system was developed based on an injectable hydrogel of glycol chitosan benzaldehyde capped poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (GC-OHC-PEO-PPO-PEO-CHO). This formulation has been prepared as a flexible vehicle for intratumor delivery of two anticancer drugs with different solubility characteristics, DOX and PTX. The GC-OHC-PEO-PPO-PEO-CHO injectable gel is able toing mice . In vitro antiproliferative activity of investigated complexes against melanoma cancer cell lines was tested. The results of the in vitro release study at different pH values confirmed that the synthesized complex is a highly effective pH-triggered drug delivery system for advanced anticancer therapy [Tomic's group synthesized and evaluated a series of dual-sensitive poly(2-hydroxypropyl acrylate/itaconic acid) (P(HPA/IA)) hydrogels as a potential highly effective antiproliferative drug delivery system. Ni(II) complex with oxaprozin, a hydrophobic antiproliferative agent, was synthesized and loaded into the P(HPA/IA) hydrogels. The studies showed that loaded antiproliferative agents did not annul pH and temperature sensitivity of the investigated hydrogels. therapy . in vitro after encapsulation into hydrogel based on chitosan [A few more alternatives of drug delivery systems have also been developed. A new injectable biodegradable scaffold for the delivery of living T-lymphocyte cultures was developed. Using combinations of sodium hydrogen carbonate and phosphate buffer as gelling agents, novel injectable chitosan-based biocompatible thermogels (CTGels) have been achieved. They have excellent mechanical characteristics and cytocompatibility. It was determined that T-cells remained cytotoxically functional to kill cancer cells of melanomachitosan . HoweverFinally, a new anticancer therapy strategy that utilizes enzymes to treat cancer was proposed by Matricardi et al. Authors of this study employed bovine serum amine oxidase (BSAO) to treat cancer. This enzyme converts polyamines that are overexpressed in malignant cells into hydrogen peroxide and aldehyde(s), thus inducing high cytotoxicity in cancer cells. In this regard, to improve delivery efficiency, they developed a formulation where BSAO is covalently immobilized into injectable nanohydrogels (NHs) based on cholesterol-graft-hyaluronic acid (HA-CH), a biocompatible conjugate that immediately leads to self-assembled structures in aqueous solutions . AgostinOwing to the molecular complexity of numerous cancers, combination therapy is becoming increasingly important for a better long-term prognosis and to reduce side effects. Combinatorial drug therapy for the treatment of a disease normally refers to either the simultaneous administration of two or more pharmacological drugs or the combination of different types of therapy, for instance, chemotherapy and radiotherapy . In contChemotherapy is most commonly used for the treatment of cancer patients but also develops undesirable side effects. In recent years, innovative treatment approaches such as hyperthermia with gold nanoparticles, magnetic nanoparticles, and the combination of hyperthermia and chemotherapy are being investigated for improving patient's quality of life . MagnetiLi and coworkers carried out studies in melanoma tumor-bearing mice; the result obtained demonstrated that an enhanced cancer chemoradiotherapy was achieved by a Pluronic F127-based thermosensitive hydrogel loading AuNPs and DOX (Au-DOX-Gel), in which DOX acted as a chemotherapeutic while polyethylene glycol (PEG) modified gold nanoparticles that (AuNPs) killed tumor cells by enhancing the radiation dose. On the other hand, studies carried out by Agostinelli et al. corroborated that the toxic enzymatic oxidation products generated by BSAO and polyamines can be used along with hyperthermia (42°C) or with other drugs such as lysosomotropic compounds in the treatment of solid tumors such as melanomas  .The strategies that have been developed in the last years and that are described in this article are summarized in  in vivo noninvasively is highly desired, mainly with the goal of avoiding or decreasing side effects.Developing a drug delivery system for effective cancer therapy where we can track the activityOne of the greatest advantages of developing a drug delivery system for a targeted therapy for melanoma skin cancer compared with other cancers is its easier accessibility. Perhaps for this reason there are several advances in this area (drug delivery system using melanoma cancer model). On the other hand, there are several investigations that have designed drug delivery systems with numerous antineoplastic agents that are used to treat different types of cancer, which included breast cancer, colon cancer, and ovarian cancer. The previously mentioned approaches could be tested in melanoma skin cancer as well.The usage of formulations based on hydrogels in melanoma skin cancer as a delivery system of antiproliferative agents has several advantages over other drug delivery systems and conventional therapies. The polymer engineer can design and synthesize hydrogel with molecular-scale control over structures such as cross-linking density and with tailored properties, such as biodegradation, degradation rate, pore size, mechanical strength, and chemical and biological response to stimuli such as pH, enzymes, and temperature. Another important benefit of hydrogel is the low cost compared with the other polymeric formulations such as nanoparticles, microparticles, and dendrimers. Therefore, the modifiable and highly tunable properties of hydrogel make it a preferred choice in melanoma cancer therapy. In the future, it is necessary to make changes such as concentration of antineoplastic agents applied and stage of cancer. These parameters were not discussed during this review because this is not the purpose of this work.