Throughout human life, bone is constantly in a delicate dynamic equilibrium of synthesis and resorption, hosting finely-tuned bone mineral metabolic processes for bone homeostasis by collaboration or symphony among several cell types including osteoclasts (OCs), osteoblasts (OBs), osteocytes (OYs), vascular endothelial cells (ECs) and their precursors. Beyond these connections, a substantial level of communication seems to occur between bone and other tissues, and together, they form an organic unit linked to human health and disease. However, the current hypothesis, which includes growth factors, hormones and specific protein secretion, incompletely explains the close connections among bone cells or between bone and other tissues. Extracellular vesicles (EVs) are widely-distributed membrane structures consisting of lipid bilayers, membrane proteins and intravesicular cargo (including proteins and nucleic acids), ranging from 30 nm to 1000 nm in diameter, and their characters have been highly conserved throughout evolution. EVs have targeting abilities and the potential to transmit multidimensional, abundant and complicated information, as powerful and substantial “dogrobbers” mediating intercellular communications. As research has progressed, EVs have gradually become thought of as “dogrobbers” in bone tissue—the “eternal battle field” —in a delicate dynamic balance of destruction and reconstruction. In the current review, we give a brief description of the major constituent cells in bone tissues and explore the progress of current research on bone-derived EVs. In addition, this review also discusses in depth not only potential directions for future research to breakthrough in this area but also problems existing in current research that need to be solved for a better understanding of bone tissues. Bone is a very well-adapted tissue in a delicate dynamic balance, hosting finely-tuned bone mineral metabolic processes for the homeostasis of both bone itself and other organs . EmerginIn the bone remodelling compartment (BRC), remodelling takes place throughout the whole life of organisms, including mammals, to maintain bone homeostasis via the replacement of senescent bone and repair of micro-damage . The norHowever, identification of the specific mechanisms and agents that are responsible for directly triggering and coordinating the processes of remodelling remains difficult. Considering the elaborateness of the regulation of bone remodelling, some communication methods among bone cells are needed to carry abundant information and help coordinate the regulatory processes.Extracellular vesicles (EVs) have complex membrane structures and range from 30 nm to 1000 nm in diameter –17. AlmoEVs are composed of lipid bilayers, membrane proteins and intravesicular cargo (including proteins and nucleic acids) , 24. TheClassification of EVs is based on their size and mechanisms of biogenesis . EVs canLeading researchers in the EV research area, such as Clotilde Thery, suggested that although exosomes and microvesicles have different mechanisms of biogenesis, the present isolation technology is not capable of separating the sub-populations of EVs . The terThe continuous evolution of EV research may be essential for understanding the underlying mechanisms of communications not only among bone cells but also between bone and other body systems. Finally, our understanding of physiology and pathology may be dramatically enhanced. In this review, we introduce the major constituent cells in bone tissues, present the current research progress on EVs derived from these kinds of bone cells, and then point out the problems existing in current research with possible future research directions to achieve a breakthrough in this area.Bone is constantly in a delicate dynamic equilibrium of both synthesis and resorption throughout the lives of animals including humans to withstand the demands of their activities. In the process of bone modelling and remodelling, bone-building cells (OBs) are responsible for osteogenesis (or bone synthesis), while bone-resorbing cells (OCs) are responsible for osteolysis (or bone resorption) . In addiOBs make up approximately 5% of the total resident cells in bones . OBs areTheoretically speaking, OBs can be classified as preosteoblasts, mature OBs, and bone-lining cells . This clOCs are derived from haematopoietic stem cells (HSCs) . In the According to current research, the most important signalling pathway regulating the differentiation of OCs is the receptor activator of nuclear factor kappa-B ligand (RANKL) signalling pathway. After receptor activator of nuclear factor kappa-B (RANK) is bound by RANKL, tumour necrosis factor receptor-associated factor 6 (TRAF6) is recruited to activate nuclear factor kappa-B (NF-κB) and the downstream cascades , 45. TheFurthermore, cross-talk between signalling pathways is also important in osteoclastogenesis, such as partial calcium-signalling pathway activation by RANKL , 51 and For a long time, PreOCs were considered to be simply precursors of OCs . HoweverSome signalling pathways of preOCs have been discussed above. In addition, macrophage colony-stimulating factor (M-CSF) plays an important role in triggering the proliferation and differentiation of preOCs via activation of MAPK-Erk and phosphoinositide 3-kinase (PI3K)/protein kinase B  signalling . HoweverHomo sapiens, are star-shaped OB-derived cells embedded in mineralized bone matrix that reside in the lacunae as an interconnected network and are bathed in the canalicular fluid [OYs, which are terminally-differentiated from OBs, are the most abundant sub-population of bone cells at over 90% –60. OYs,ar fluid –63. The ar fluid . Furtherar fluid . As reseThere are two fundamental and well-recognized concepts in the research area of bone biology –69: (1) The dendritic processes of OYs, which extend through the canaliculi, are essential for forming a highly-connected network not only between OYs but also between OYs and their neighbours, including OBs, OCs and cells in the blood supply in bone , 71. OYsSeveral signalling pathways are involved in OY-predominated regulation and control of OBs and OCs. These pathways are the major sources of sclerostin, the primary candidate for regulating OBs , 76, 77,Sclerostin has been reported to inhibit the Wingless-related integration site (Wnt) pathway and then keep the status ‘inhibition of OB differentiation’ Fig. . HoweverThus, OYs have the potential to be an orchestrator of mineral metabolism and bone remodelling , 88 and In addition, recent evidence implied that OYs regulate not only bone but also systemic processes of multiple organs. Sato et al. reported that ablation of OYs in mice (OY-less mice) leads to both severe lymphopenia and the loss of white adipose tissue . In a seIn the beginning, the endothelium was once considered to be just a ‘cellophane wrapper’ providing selective permeability to electrolytes and water, without any further function. As research progressed, more and more evidence has led to a more in-depth understanding of the multitudinous functions of the endothelium , 94. TheThe coupling of angiogenesis and osteogenesis plays an essential role in maintaining appropriate bone homeostasis , 98. TheSpecifically, the H-type vessel may be the key to coupling, while the L-type vessel  is not associated with promoting osteogenesis and coupling , 102. InIn mouse models, there is a very interesting phenomenon in which the total number of ECs in bone is not significantly different after maturation but the ratio of H-type:L-type ECs decreases with age after adolescence, and this phenomenon implies that the relationship between the transformation from H-type ECs to L-type ECs and the bone mass loss during ageing is caused by a loss of osteogenic ability . FurtherCurrent research shows that there are two main communication approaches: a direct action and a remote action. At present, a direct action approach has only been found between ECs and BMSCs. EC-derived PDGF-BB recruits PDGF-receptor β (Rβ)–expressing BMSCs into a pericyte fate . RecruitIn the bone marrow, BMSCs  are mononuclear stem cells that are osteogenic precursors and can differentiate into OBs and other kinds of skeletal cells , 109. ThHowever, after years of research, some problems have received increasing attention. The in vivo BMSCs have been reported to be very different from ex vivo expanded ‘BMSCs’ , 112, anNevertheless, optimistically speaking, there have been many achievements. In the field of application, SSCs  are very good seed cells for tissue repair and one of the representative cells used to observe the response of bone cells after repeated stimulation. The gaps in in vivo BMSC knowledge are tremendous opportunities for future research to better understand the fascinating mysteries of biology.Proteins and nucleic acids, contained within OB-derived EVs (OB-EVs), play important roles in intercellular communication in bone tissue . OB-EVs OB-EVs contain RANKL, which can be transferred to precursors of OCs. Via receptor–ligand interaction, OB-EVs can stimulate RANKL–RANK signalling to promote osteoclast formation . InhibitIn addition to RANKL, galectin-3 has also been found to be important in OB-EVs. Weilner et al. reported that EVs from elderly people can inhibit the osteogenic ability of BMSCs unlike those from younger people . InteresRecently, Ge et al. reported the results of a proteomics study on EVs derived from MC3T3 cells (mouse OBs) and showed that many proteins, which were highly enriched in EVs derived from MC3T3 cells, are enriched in osteogenesis-related pathways, including eukaryotic initiation factor 2 (EIF2) signalling, integrin signalling and mammalian target of rapamycin (mTOR) signalling . EIF2 siOB-EVs also contained microRNAs (miRNAs). Cui et al. found that several miRNAs in OB-EVs were increased during mineralization . ThroughProteins and nucleic acids in EVs derived from OCs (OC-EVs) may also play an important role in bone mineral metabolism. Huynh et al. found that OC-EVs, which contain RANK on the surface, are paracrine regulators of osteoclastogenesis . RANK-coIncreased levels of serum miR-214-3p contained within EVs were found in both elderly women with fractures and ovariectomized (OVX) mice . Li et aHuynh et al. found RANK in OC-EVs but not in EVs derived from preOCs (preOC-EVs) . More inAs briefly described in a previous section, Sato and his colleagues found that EVs derived from OYs (OY-EVs) may transfer their components, including miRNAs, to recipient cells via circulation in the blood . This fi2+-dependent contractions [Morrell et al. found that mechanical stimulation upregulates the production of EVs containing bone regulatory proteins, coupled with Caractions . miR-218ractions . Interesractions . These rmiR-31 is highly expressed in EC-derived EVs (EC-EVs) from elderly and osteoporotic patients . miR-31 EVs derived from BMSCs (BMSC-EVs) are also important participants in the remodelling of bone tissue via direct regulation of the proliferation and activity of OBs . Davis eHowever, most recent studies related to BMSC-EVs have concentrated on therapeutic use under a variety of conditions, including anti-inflammation, tissue repair and tissue protection. Another focus of BMSC-EV research is the relationship between BMSC-EVs and tumours. Nevertheless, the in-depth study of BMSC-EVs in bone cell communications under physiological and pathogenic conditions is still relatively insufficient. Certainly, BMSC-EVs have a powerful potential as therapeutic drugs for regenerative medicine or targets of tumour therapy, but revealing the specific details of BMSC-EVs in the communication of other bone tissue cells is important.Recently, the so-called ex vivo expanded ‘BMSCs’ have been identified as SSCs , 112. OpWith the development of research, EVs have gradually come to be thought of as correspondents or “dogrobbers” in bone tissue, the “eternal battle field” in a delicate dynamic balance of destruction and reconstruction. An in-depth understanding of the EVs in bone helps to understand both how bone maintains a delicate homeostatic balance and the pathogenesis of disease.Furthermore, there is another mystery—how bone communicates with other systems. The conventional hypothesis is that growth factors, hormones and specific proteins such as OCN participate in the communication between bone and other systems –4, 7–9. Due to the limitations of current research techniques, the functional similarities and differences among sub-populations of EVs cannot be explained clearly. Moreover, many studies do not make clear distinctions. We have used the term ‘EVs’ rather than the terms of their sub-populations in this review. However, the sub-populations of EVs is a topic attracting considerable interest, and increasing attention has been paid to it , 150. InOBs and OCs can exist within the periosteum and the bone marrow. There are obvious differences between these two sites. OBs/OCs in the periosteum have more possibilities to communicate with tendons, muscles and nerves. In addition, OCs in the periosteum are derived from circulating monocytes , while OOYs, the most abundant sub-population of bone cells, are the least-studied cell type. Current research suggests that sclerostin is a very important molecule. Micro-damage causes sclerostin shut-down and triggers bone remodelling. However, it is not clear whether sclerostin is present in a free state or in EVs. We suspect that sclerostin exists in EVs and that the EVs are present in canalicular fluid. Moreover, micro-damage may block the mobility of canalicular fluid and then cause a shut-down of EV-carried sclerostin. Follow-up studies should first confirm what is affected by the micro-damage—the OYs, the mobility of canalicular fluid, or both.There are many studies on EC-derived EVs. However, current studies have not focused on H-type ECs. Not only are there few studies on H-type ECs, but there is also almost no research on EVs derived from H-type ECs. Future research in this direction is likely to be one of the most important advances. Beyond that topic, there are large gaps with enormous research potential regarding the true nature of BMSCs, as mentioned in the previous section.EVs have gradually become a focus of the new generation of treatment and diagnosis. With all kinds of new technologies , we can Bone is a very well-adapted tissue in a delicate dynamic balance, hosting finely-tuned bone mineral metabolic processes for bone homeostasis by collaboration or symphony among several cell types including OCs, OBs, OYs, ECs and their precursors. EVs are widely-distributed vesicles, ranging from 30 nm to 1000 nm in diameter, with targeting abilities and potential to transmit multidimensional, abundant and complicated information. In this review, we introduce the major constituent cells of bone tissues and explore the progress of current research into bone-derived EVs and then point out the problems existing in current research with possible future research directions to break through in this area.


Job insecurity is a modifiable risk factor for poor health outcomes, and exposure to job insecurity varies by population groups. This study assessed if job insecurity exposure varied by migrant status and if the differences varied by gender, age, educational attainment, and occupational skill level. Data were from wave 14 of the Household Income and Labour Dynamics in Australia Survey. The outcome was job insecurity. Exposure was migrant status defined by (1) the country of birth (COB), (2) the dominant language of the COB, and (3) the number of years since arrival in Australia. Data were analysed using linear regression, adjusting for gender, age, educational attainment, and occupational skill level. These covariates were also analysed as effect modifiers for the migrant status–job insecurity relationships. Migrant workers, especially those from non-English speaking countries (non-ESC-born), experienced higher job insecurity than Australia-born workers; however, these disparities disappeared after 11+ years post-arrival. The migrant status–job insecurity relationships were modified by educational attainment. Unexpectedly, the disparities in job insecurity between non-ESC-born migrants and Australia-born workers increased with increasing educational attainment, and for those most highly educated, the disparities persisted beyond 11 years post-arrival. Our findings suggested that continuing language skill support and discrimination prevention could facilitate migrant integration into the Australian labour market. Psychosocial job stressors are recognized as modifiable risk factors for poor health outcomes ,2. Job iExposure to job insecurity varies by population groups. Workers with lower socioeconomic status are generally more likely to be exposed to job insecurity . For exaMigrant workers are a heterogeneous group. Besides the country of birth, the language proficiency of the host country (English in this study) and the number of years since arrival in the host country may have important influences on migrants’ job stressor exposures. Research suggests that language barriers reduce migrants’ opportunities to obtain quality employment  and, comMoreover, the interaction of migrant status with gender, age, and socioeconomic status may create more complex patterns of inequalities in job stressor exposures  due to sThe number of migrants in Australia is increasing, and nearly 60% are employed . DespiteThe data were procured from the Household Income and Labour Dynamics in Australia Survey (HILDA). This study was approved by the Deakin University Human Research Ethics Committee (No. 2017-226).HILDA is a nationally representative sample of Australian households, whose data collection began in 2001 and was conducted annually via a stratified three-stage clustered design . In AustThe respondents included in our analysis were aged 15 to 64 years, were employed, and responded to all the items on job insecurity in wave 14. In this wave, 15,231 observations were aged 15–64 years, and of these, 10,575 were employed. Among these respondents, 9043 answered all three items of the job insecurity scale and formed the analytic sample of this study.Migrant status was measured in three ways. The first was based on country of birth (COB) only; a binary variable coded as Australia-born versus overseas-born. The second was based on COB and the dominant language of the COB, a three-category variable coded as (1) Australia-born, (2) born in the main-English speaking country (main-ESC-born), and (3) born in the non-English speaking country (non-ESC-born) was generated as a crude proxy for measuring English language proficiency. The third was based on both COB and years since arrival in Australia. A four-category variable including (1) Australia-born, and overseas-born workers of those who (2) arrived ≤5 years ago, (3) arrived 6–10 years ago, and (4) arrived ≥11 years ago. This was generated to provide a crude measure of increasing acculturation over time.We formulated job insecurity using a previously developed scale in HILDA . Job insCovariates included gender , age , educational attainment , and occupational skill level. Based on the Australia and New Zealand Standard Classification of Occupation , occupatFirstly, we used Chi-squared tests to compare the prevalence of socio-demographic characteristics and occupational skill level by migrant status. Then, descriptive analysis was performed to calculate the mean and standard error of job insecurity by migrant status. Because the HILDA Survey involves a complex design and has unequal probabilities of selection and non-response, Summerfield et al.  suggesteSubsequently, linear regression analysis was conducted to examine the relationship between migrant status and job insecurity (as continuous outcomes). Then, gender, age, educational attainment, and occupational skill level were included in the linear regression one by one, and then simultaneously, to assess the potential for confounding ,18. HoweAll analyses were conducted using Stata 15.1  .p < 0.001) and workers in high-skill level jobs  compared to main-ESC-born workers. Further, Australia-born workers included a significantly lower proportion of workers with a postgraduate degree (12%) than overseas-born workers , which may be explained by Australia’s preference for skilled migrants. In contrast, Australia-born workers included a significantly higher proportion of workers between 15 to 24 years of age (21%) than overseas-born workers .n = 1184) were due to participants not responding to the self-completion questionnaire (SCQ) of the HILDA survey, which is where the job insecurity questions were asked. Other reasons included not being asked (n = 286), multiple responses to the SCQ (n = 12), and refused/not stated (n = 50). Among the 50 who refused to answer the items, 9 were missing all three items, 2 were missing two items, and 39 were missing one item. Based on these reasons, a total of 1479 (1184 + 286 + 9) were missing all three items of the job insecurity scale, plus a small number (n = 12) were excluded due to multiple responses , leaving 41 respondents that were missing one or two items of the job insecurity scale. Compared to the observations that were included in our analysis, these exclusions were more likely to be male , younger , lower educated , lower in skill level , and non-ESC-born migrants .There were 1532 respondents (14% of the employed sample) who were missing one, two, or all three items in the job insecurity scale; these were excluded from our analysis. According to the reasons for missing data provided by HILDA, the vast majority . There was no evidence of effect modification of the migrant status–job insecurity relationship by gender , age , or occupational skill level . The main effects and interaction effects of migrant status and educational attainment are shown in When effect modifiers were tested, the only significant interaction was found between educational attainment and migrant status based on COB (LR test results: COB × education: p = 0.02), while job insecurity of those with a postgraduate degree was significantly lower , than the Australia-born workers with high school or lower education levels. Within the migrant worker group , similar job insecurity levels were reported regardless of their educational level. The only exception occurred when migrant status was measured in terms of overseas-born, in which migrants with diploma degrees had significantly higher job insecurity  than migrants with high school or lower education levels.For completeness, we also made within-group comparisons. In Australia-born workers, the job insecurity of those with diploma degrees was significantly higher (β = 0.25, Consistent with our broad hypothesis, migrant workers, especially non-ESC-born and recently arrived migrants, experienced higher job insecurity than Australia-born workers. These disparities gradually decreased as years since arrival in Australia increased, becoming similar to Australia-born workers after 11+ years post-arrival, with a notable exception regarding highly educated migrants, for whom disparities persisted. Importantly, the migrant status–job insecurity relationships in our study were not confounded by gender, age, educational attainment, or occupational skill level. However, migrant status–job insecurity relationships were modified by educational attainment, and the disparities in job insecurity between non-ESC-born migrants and Australia-born workers increased with increasing educational attainment, contrary to expectation.There are a limited number of previous studies directly comparing job insecurity between migrant workers and native workers, with most of them reporting that migrant workers experienced higher job insecurity ,33,35,37With respect to research question one, our finding that the disparities in job insecurity were more significant among non-ESC-born migrants and decreased with increasing years post-arrival in Australia may indicate an acculturation effect. This may be explained by new migrants, especially those with low English proficiency, being more likely to find temporary or lower quality jobs , or haviMoreover, we found that higher educated migrant workers had similar job insecurity levels to lower educated migrants, which was inconsistent with previous working population-level results suggesting that higher levels of education are associated with greater job security ,17. ThisSome limitations should be considered in the interpretation of our findings. First, we used a cross-sectional design, so we could not formally conclude that migrant status caused more adverse job stressor exposures. However, migrant status was a stable characteristic and preceded job stressor exposures, with little possibility of reverse causation. Another possible limitation is the healthy worker effect, wherein migrants who dropped out the workforce due to poor working conditions may have been missed due to the cross-sectional design, thereby resulting in underestimation of the job stressor exposures of migrant workers. Second, we measured only subjective job insecurity, which may have varied by individual or population groups. For example, Erlinghagen  reportedThis study has particular strengths. It presents a thorough comparison of job insecurity of migrants and non-migrants using a national population-representative sample. We also used a predictively validated measure of job insecurity , and theIn conclusion, non-ESC-born workers have significantly higher levels of job insecurity than Australia-born workers, especially for those with high educational attainment. Although the disparity in job insecurity between Australia-born workers and migrant workers decreases with the increasing number of years post-arrival in Australia, the difference is still significant among highly educated migrants even 11+ years post-arrival. Considering the associations between job insecurity and a wide range of adverse physical and mental health outcomes, the persisting disparities in job insecurity among highly educated migrant workers deserves further attention, and future research on its causes and its effect on migrants’ health and well-being is warranted. Our results also suggest that policies and practices to improve migrants’ English language ability and to prevent discrimination could reduce disparities in job insecurity and facilitate the integration of migrants into the Australian labour market.


It has been reported that donor age affects patient outcomes after liver transplantation, and that telomere length is associated with age. However, to our knowledge, the impact of donor age and donor liver telomere length in liver transplantation has not been well investigated. This study aimed to clarify the influence of the length of telomere and G-tail from donor livers on the outcomes of living donors and recipients after living donor liver transplantation. The length of telomere and G-tail derived from blood samples and liver tissues of 55 living donors, measured using the hybridization protection assay. The length of telomeres from blood samples was inversely correlated with ages, whereas G-tail length from blood samples and telomere and G-tail lengths from liver tissues were not correlated with ages. Age, telomere, and G-tail length from blood did not affect postoperative liver failure and early liver regeneration of donors. On the other hand, the longer the liver telomere, the poorer the liver regeneration tended to be, especially with significant difference in donor who underwent right hemihepatectomy. We found that the survival rate of recipients who received liver graft with longer telomeres was inferior to that of those who received liver graft with shorter ones. An elderly donor, longer liver telomere, and higher Model for End-Stage Liver Disease score were identified as independent risk factors for recipient survival after transplantation. In conclusion, telomere shortening in healthy liver does not correlate with age, whereas longer liver telomeres negatively influence donor liver regeneration and recipient survival after living donor liver transplantation. These results can direct future studies and investigations on telomere shortening in the clinical and experimental transplant setting. Liver transplantation (LT) is a standard treatment for end-stage liver disease and liver malignancies. In a globally aging society, a declining pool for living donor liver transplantation (LDLT) and cadaver LT has become a critical issue. The possibility and safety of donations from marginal donors should be considered, particularly those of elderly and obese donors. It remains controversial whether donor age impairs recipient outcomes after LDLT . HoweverFor a long time, the liver was recognized as an organ that could regenerate; yet, the mechanism of liver regeneration is remains unclear. Eukaryotic organisms senesce as they get older, and organ function and regeneration ability decline. It has been reported that liver regeneration in elderly people and rats after hepatectomy slows down . The res5'-TTAGGG-3' at the ends of chromosomes, appear to be deeply involved in tissue regeneration, lifespan, and cell division [Telomeres, double-stranded DNA containing repeat sequences of division . It has division . Accordidivision . HoweverWe investigated the influence of telomere and G-tail length from donor blood and liver tissues on donor liver regeneration and recipient outcome after LDLT.Overall, 223 patients underwent LDLTs at Hiroshima University Hospital between 1991 and 2015. Blood and liver samples from 55 donors were collected at LDLT between 2010 and 2015. Written informed consent was obtained from all participants before surgery, in accordance with the Declaration of Helsinki. This study was approved by the Hiroshima University Institutional Review Board (HiM129-28). The procedure to protect the identity of the patients was subject to approval by the institutional review committee and met the guidelines of the responsible governmental authority.LDLTs were performed at Hiroshima University Hospital, following the Japanese Liver Transplant Society guidelines. Donors were healthy adults who voluntarily applied to donate their liver. The size of the graft had to be more than 0.8% of the recipient's body weight . Our donPerioperative volumetric measurements of hepatic morphology were performed as previously described , 9. ReseDonor outcomes included morbidity, post-hepatectomy-liver-failure (PHLF), and ERI. Recipient outcomes included morbidity and survival. The definition of PHLF was based on the International-Study-Group-of-Liver-Surgery (ISGLS), included the following: decrease in liver synthesis excretion, detoxification function, or symptoms of elevation of high bilirubin value; and international normalized ratio of prothrombin (PT-INR) value on day 5 (PT-INR-5), or later compared with the previous one if there was a high preoperative value, or a normal value that required “artificial supplements from the outside to maintain” , 12. TheQuantification of relative telomere length (RTL) and relative G-tail length (RGL), double-stranded and single-stranded, was performed using the telomere hybridization protection assay (HPA) method, as described previously , 14. TheAll statistical analyses were conducted using JMP 13 . The univariate analysis for continuous variables with normal distribution was compared using the Student's t-test. Continuous variables without a normal distribution were compared with Mann-Whitney's U test. A descriptive comparison was performed with the chi-squared test. Correlations between the presence of PHLF and continuous variables were expressed using Pearson's correlation coefficient. The area under the curve was calculated using the receiver operating characteristic (ROC) curve for the sensitivity and specificity of the value of the limit of telomere length. The difference between the two sides was considered statistically significant when the p-value was 0.05 or less. All relevant data are shown within Supporting Information files .Clinical characteristics of donors and recipients are shown in Next, we investigated the correlation between B-RTL and L-RTL, as well as that between B-RGL and L-RGL. L-RTL was not associated with B-RTL , and B-RWe investigated the correlation between telomere and G-tail length from blood and liver tissues and donor age. As shown in We reviewed preoperative characteristics of LDLT donors. Among 55 donors, 5 donors had PHLF, according to the ISGLS criteria. We were unable to identify any significant preoperative factors which affected PHLF . NietherWe assessed the factors which may affect postoperative liver regeneration. To investigate the differences between the regeneration rates after left and right hemihepatectomy, we examined ERI separately for left and right hemihepatectomy. Donor age was not correlated with ERI in any donor, regardless of the procedure they underwent . ERI wasWe reviewed the postoperative outcomes of recipients after LDLT . While tThe impact of donor age on LT has been analyzed in several studies , 15. SevLiver regeneration rate after hepatectomy has been shown to be inversely correlated with age , 23, 24.5'-TTAGGG-3' at the ends of chromosomes. Those repeat sequences (TTAGGG) are single strands of approximately 50 to 300 bases at the furthest 3' ends (G-overhangs), called G-tails. These G-tails are normally protected by forming a loop, except when telomerase, a telomere extension enzyme, interacts with the telomere, for instance, during DNA replication [Telomeres appear to be deeply involved in tissue regeneration, lifespan, and cell division . They arlication . Telomerlication .In the present study, B-RTL was inversely correlated with age, consistent with previous studies. However, B-RGL, L-RTL, and L-RGL were not correlated with donor age, and L-RTL was not correlated with B-RTL. Telomere shortening in liver tissues during aging has been reported in some studies –32: for This is the first study investigating the length of liver telomere/G-tail and liver regeneration after hepatectomy in LDLT. Several reports have investigated telomere length and telomerase activity in patients with liver disease. Aikata et al. reported that telomere repeats were shorter in the liver of patients with chronic diseases than in normal age-matched livers , and HarCellular senescence is considered to be a stress-response limiting the proliferation of damaged cells and leading to permanent cell-cycle arrest . The telTo our knowledge, this is the first study investigating the association between telomere length of donor tissues and recipient outcome in LDLT. In organ transplantation, it remains unclear whether donor tissue telomere length is associated with recipient survival. A previous study showed that longer donor telomere length is associated with improved recipient survival among hematopoietic cell transplant recipients with aplastic anemia . In contThis study has several limitations. First, although this is the first study to investigate specifically on telomere and G-tail length from healthy donor liver tissue, we had a relatively small cohort with a limited follow-up period, and the age of donors included was limited to less than 65 years only. Second, a cell cluster of whole liver tissue used in our study included not only hepatocytes, but also other cells including cholangiocytes, and sinusoidal cells. Third, we did not assess telomerase activity or the telomere/G-tail in transplant recipients. Fourth, we assessed telomere length by HPA assay, as a flow-based assay. Telomere length is measured as fluorescence intensity relative to internal control, not as an absolute length .Telomere shortening in healthy liver tissue was not correlated with age, whereas longer liver telomeres negatively impact donor liver regeneration and recipient survival after LDLT. These results can direct future studies and investigations on telomere shortening in the clinical and experimental transplant setting.S1 Table(XLSX)Click here for additional data file.


In this study, polarization-sensitive optical coherence tomography (PS-OCT) with local phase retardation imaging is used for the first time to visualize depth-resolved  birefringence of healthy human oral mucosa in vivo. Depth-resolved birefringence is shown to reveal the expected local collagen organization. To demonstrate proof-of-principle, 3D image stacks were acquired at labial and lingual locations of the oral mucosa, chosen as those most commonly affected by cancerous alterations. To enable an intuitive evaluation of the birefringence images suitable for clinical application, color depth-encoded en-face projections were generated. Compared to en-face views of intensity or conventional cumulative phase retardation, we show that this novel approach offers improved visualization of the mucosal connective tissue layer in general, and reveals the collagen fiber architecture in particular. This study provides the basis for future prospective pathological and comparative in vivo studies non-invasively assessing stromal changes in conspicuous and cancerous oral lesions at different stages.Stromal collagen organization has been identified as a potential prognostic indicator in a variety of cancers and other diseases accompanied by fibrosis. Changes in the connective tissue are increasingly considered for grading dysplasia and progress of oral squamous cell carcinoma, investigated mainly  ProgrPSR-POL) –9. WherePSR-POL) –8 and onPSR-POL) ,11. PSR-in vivo due to the limited field-of-view (FOV) and high magnification. As an interesting alternative, we propose polarization-sensitive optical coherence tomography (PS-OCT) for in vivo imaging of collagen fiber organization in the human oral mucosa, employing the intrinsic birefringence of collagen as the contrast mechanism, and combined with reconstructing the local, depth-resolved tissue birefringence. Even though optical coherence tomography (OCT) has inferior spatial resolution  than SHG microscopy , it is attractive due to its large imaging depth of ~1 mm in soft tissue, wide FOV (from a few to tens of square millimeters), fast acquisition rates and workflows, with the potential for real-time imaging under in vivo conditions, suitable for clinical translation [in vivo oral dysplasia and malignancies [PSR-POL is limited to histology and SHG microscopy is challenging nslation . Conventgnancies –16, but en-face parametric maps of birefringence averaged over appreciable fractions of a millimeter, with encouraging results [By extending OCT to measuring the polarization state of the light returning from the sample, additional tissue-specific contrast, such as birefringence of aligned fibrous structures, is obtained ,18. In m results ,27. The  results ,29. Sinc results –34 or th results –40. Focu results ,41, skin results ,36 and b results  imaging.in vivo with images of depth-resolved birefringence, reconstructed from measurements with PS-OCT. Moreover, for the first time, healthy oral collagen structures are visualized using a novel en-face color-based depth-encoded projection of depth-resolved birefringence obtained using PS-OCT. The current realization has an FOV of 2.1 mm × 2.1 mm, images collagen at depths up to 400 µm, and conveys all essential information conveniently in a single image. Our method and results suggest the way forward for further studies assessing malignant stromal alterations in oral cancer.To the best of our knowledge, for the first time, we visualize non-invasively the stromal collagen organization in healthy oral soft tissue The PS-OCT system setup was based on a wavelength-swept light source  centered at λ = 1310 nm with Δλ = 110 nm and a modified fiber-coupled Mach-Zehnder interferometer  was placed in the sample arm to generate two orthogonal linear polarization states with different delays (passive polarization multiplexing) –47. AfteFor the interpretation of the subsequent imaging results of non-birefringent and birefringent oral structures, the histological and anatomical background is briefly given below. In general, the human oral mucosa consists of stratified squamous epithelium (EP) and an underlying connective tissue distinguished into lamina propria (LP) and submucosa. In turn, the lamina propria is divided into two layers: the superficial papillary layer (PL), associated with an irregular convoluted interface consisting of finger-like projections of connective tissue extending into the lower aspect of the epithelium; and the deeper reticular layer (RL) of grid-like tight connective tissue. Whereas the collagen fibers of the PL are thin and loosely arranged, the RL contains closely packed and net-like arranged bundles of collagen fibers which tend to lie in a plane parallel to the surface.For this proof-of-principle study, representative mucosal structures of the readily accessible anterior oral cavity of one volunteer  were imaged by PS-OCT in contact mode with a standard scanner head in combination with a thick transparent optical quartz glass window. Multiple measurement points of the healthy non-altered oral mucosa were defined with advice from an experienced oral medicine specialist  the intensity signal, (e) the depth-resolved birefringence and (f) the cumulative phase retardation, visualizing single projection views within the RL at a depth of 265 µm below the surface , muscle structure can be identified by the strong birefringence signal at greater depth ∆n (∆n) for the depth range of 214-367 µm below the surface. AVG intensity gave better contrast by inspection than MIP intensity. The depth range representing the RL is manually determined from the averaged birefringence and intensity signals of the detected 1000 × 1000 A-scans versus depth.We anticipate that it may be beneficial in future clinical applications to visualize the collagen organization in a single data set  via the gence ∆n , MIP ∆n Δn en-face slices is color-encoded to highlight the variation of collagen fiber alignment versus depth, as shown in Δn, for the depth range containing the RL with tight collagen fiber content, was generated using the ‘Time Series Color Coder’ macro for Fiji software with a specifically designed isoluminant look-up table [en-face projection, which offers considerably enhanced contrast over the simple averaged (AVG) intensity signal. Additionally, the visibility of the grid-like collagen fiber network is greatly enhanced by means of simple MIP Δn.The 3D stack of up table  and is pThe labial and lingual oral mucosa offer the highest level of collagen fiber organization in the healthy oral cavity and are, besides the floor of the mouth, the most susceptible areas to carcinogenic alterations. Therefore, these readily accessible regions were used in this proof-of-principle study of depth-resolved birefringence imaging of healthy human oral mucosa.en-face images showing the transition from epithelium (EP) to connective tissue layer  by means of the intensity signal and the depth-resolved birefringence, as well as the cumulative phase retardation  birefringence Malignant alterations are frequently observed at rapidly changing transitions between different epithelial types. Therefore, the dorsal side of the volunteer’s tongue was imaged en-face projections , 8(c)In contrast, the collagen content is increased towards the ventral tongue but shows a high variability in fiber orientation, especially at larger depths. The parts with no birefringence and less backscattering intensity , 9(c)For completeness, the ventral side of the tongue was imaged en-face parametric images. In particular, our results demonstrate the great potential of depth-resolved birefringence for visualization of collagen fiber organization within the upper connective tissue layer. In detail, the healthy reticular layer (RL) we investigated, with measured thickness in the range 100-200 µm, is well suited to be represented by a single 2D MIP image. The conditions for imaging aligned collagen fibers within the RL of the oral lamina propria by PS-OCT are optimal because the fiber orientation is mainly perpendicular to the incident sample beam, particularly when using a glass plate in contact mode, as in the present study. Further, we have shown this configuration leads to strong backscattering signals, with good SNR, relatively large phase retardation and strong local birefringence contrast. Even though our OCT system, with spatial resolution of 12-14 µm, cannot directly resolve the fibrillar collagen  within En-face projections of the 3D PS-OCT data set provide new insights into the collagen fiber organization of the oral mucosa, especially in comparison to common PS-OCT cross-sectional (B-scan) imaging and ex vivo PSR-POL, which is conventionally based on cross-sectioning in depth. For histological biopsy examination of thin tissue slices by means of PSR-POL, it is challenging to assess the collagen fiber organization as cross-sections typically cut across the collagen fiber plane. Thus, depth-resolved birefringence presented as en-face images could provide an alternative non-invasive means to access information on the connective tissue of the oral mucosa during prospective preoperative examinations. At the very least, PS-OCT could serve as an adjunct to routine histopathology for studying stromal changes at the invading front of the tumor island, which, in turn, could help to develop a grading system for tumor behavior/progression in vivo. For in vivo imaging, a hand-held device will be necessary, with rigid or fiber-based endoscopic head [pic head ,51,52, cpic head  revealinin vivo. For this, the depth range used for the color-encoded MIP must be chosen appropriately depending on the tumor dimensions and the nature of its transition to the adjacent healthy RL.With regard to oral squamous cell carcinoma in particular, previous studies using polarized light microscopy have shown that distinct deposits of collagen with high birefringence, mainly around the tumor islands , are present in well-differentiated lesions –8,54. Thin vivo, with the main observation of high birefringence within the lamina propria caused by a high level of collagen organization. Looking forwards, color depth-encoded en-face projections may offer a more intuitive way of viewing OCT data, with the aim to make clinical assessment of the scanned oral area more efficient. The results of this study motivate future pathological and comparative studies assessing cancer-induced stromal changes in the human oral connective tissue non-invasively by PS-OCT.In summary, PS-OCT with depth-resolved birefringence based on a differential Mueller matrix formalism ,36 was u


Family caregiving of an older adult has become an essential element of the U.S. health care system, with 83 percent of long-term care provided to older adults coming from family members or other unpaid helpers. With the amount and type of care provided by families expected to increase, caregiving demands should be coupled with community and health care systems-based supports. While scientific research has demonstrated the value of providing education, skills training and support to family caregivers, health care and social service providers do not systematically include these interventions in their services. Thus, for the vast majority of family caregivers, caregiving support services remain extremely fragmented, if not elusive. This symposium provides four examples of how health care systems that frequently see patients with dementia and community-based organizations who provide ongoing supportive services to family caregivers, have adapted evidence-based caregiver interventions into branded service programs. Dr. Jinmyoung Cho will present racial/ethnic comparisons on the impact of community-based implementation of a caregiver education program, REACH-TX. Dr. Leah Hanson will introduce the implementation of Mindfulness-Based Dementia-Care (MBDC) within a health care system. Dr. Christine Jensen will address how caregivers can benefit from evidence-driven programs in health care settings. Lastly, Dr. David Coon will present two different approaches to translation of evidence-based programs through community-based organizations, with CarePRO embedded after completion of a clinical trial and EPIC embedded from the program’s initial pilot phase. The discussant, Dr. Alan Stevens, will highlight the needs of caregivers and support services recognized by all key stakeholders.


Rheumatoid arthritis (RA) is a type of inflammatory arthritis that affects ~1% of the general population. Although arthritis is the cardinal symptom, many extra-articular manifestations can occur. Lung involvement and particularly interstitial lung disease (ILD) is among the most common. Although ILD can occur as part of the natural history of RA (RA-ILD), pulmonary fibrosis has been also linked with methotrexate (MTX); a condition also known as MTX-pneumonitis (M-pneu). This review aims to discuss epidemiological, diagnostic, imaging and histopathological features, risk factors, and treatment options in RA-ILD and M-pneu. M-pneu, usually has an acute/subacute course characterized by cough, dyspnea and fever. Several risk factors, including genetic and environmental factors have been suggested, but none have been validated. The diagnosis is based on clinical and radiologic findings which are mostly consistent with non-specific interstitial pneumonia (NSIP), more so than bronchiolitis obliterans organizing pneumonia (BOOP). Histological findings include interstitial infiltrates by lymphocytes, histiocytes, and eosinophils with or without non-caseating granulomas. Treatment requires immediate cessation of MTX and commencement of glucocorticoids. RA-ILD shares the same symptomatology with M-pneu. However, it usually has a more chronic course. RA-ILD occurs in about 3–5% of RA patients, although this percentage is significantly increased when radiologic criteria are used. Usual interstitial pneumonia (UIP) and NSIP are the most common radiologic patterns. Several risk factors have been identified for RA-ILD including smoking, male gender, and positivity for anti-citrullinated peptide antibodies and rheumatoid factor. Diagnosis is based on clinical and radiologic findings while pulmonary function tests may demonstrate a restrictive pattern. Although no clear guidelines exist for RA-ILD treatment, glucocorticoids and conventional disease modifying antirheumatic drugs (DMARDs) like MTX or leflunomide, as well as treatment with biologic DMARDs can be effective. There is limited evidence that rituximab, abatacept, and tocilizumab are better options compared to TNF-inhibitors. Rheumatoid arthritis (RA) is the most common inflammatory arthritis with a worldwide prevalence of about 1% and a female predominance of about 3:1 . In clinA focused literature review including the keywords “methotrexate,” “pneumonitis,” “interstitial lung disease,” and “rheumatoid arthritis” was performed. In addition, articles from the personal archives of the authors or references from key papers were included if deemed relevant by the authors.The frequency of M-pneu has been reported to range between 0.3 and 11.6% . M-pneu Symptomatology mainly pertains to dry cough and dyspnea observed in more than 80% of the patients. Fever also occurs in more than 60% of them , 11, 12.Pathogenic mechanisms underlying M-pneu are unclear. It is considered by many investigators to be a hypersensitivity reaction, while interleukin-8 has been implicated in the pathogenesis . It shouSeveral risk factors have been identified , but it Pneumocystis jirovecii pneumonia (PJP), viral and atypical pneumonias, and ILD due to RA (RA-ILD), is difficult to be made  and bronchoalveolar lavage (BAL) might prove to be helpful as well. However, the differential diagnosis, which includes infections, like  be made .Performance of PFTs routinely for diagnostic or prognostic purposes is still under debate . Althougper se. Besides, none of these patients developed clinically significant lung disease in these studies  . In pedi studies .+ T cells in BAL is suggestive of M-pneu  might also be a useful diagnostic adjunct. In a study evaluating 44 patients with drug-induced lung injury, 75% underwent TBLB , more so than bronchiolitis obliterans organizing pneumonia (BOOP) : on chesThe most common histopathological pattern observed includes interstitial infiltrates by lymphocytes, histiocytes, and eosinophils with or without granulomas . GranuloIn suspected M-pneu MTX should be discontinued immediately. Often, treatment with steroids is required . Other iThe prognosis of M-pneu is generally good and most patients recover fully , howeverRA is not merely a disease of the joints. It is a true systemic inflammatory disease with effects on many organs and organ systems. A variety of pulmonary manifestations can be seen in RA including pulmonary nodules, pleural effusions, bronchiectasis, and, most importantly, ILD .ILD is a frequently under-recognized complication of RA. The estimated prevalence is heavily dependent on the ascertainment method used. Bongartz et al. reported a lifetime risk of 7.7%, a 9-fold increase over the general population . StudiesIncreasing evidence supports a primary role for the lung in initiating RA pathogenesis and RA-ILD may occur prior to the onset of the joint disease –72. KnowThe clinical findings in RA-ILD are similar to those previously described for M-pneu with dyspnoea and non-productive cough with or without fever predominating . Most tyThe diagnosis of RA-ILD can generally be made by a combination of clinical features as described above and congruent findings on chest imaging. It is important to remember that RA patients are, at least equally, and in often cases more likely, to develop other causes of dyspnea and cough than the general population. For example, the risk of infection, including atypical infections, pulmonary emboli, and lung cancer, are all increased in RA patients –82. PFTsBronchoscopy and BAL may be performed to rule out other diagnoses. BAL is frequently abnormal in RA-ILD, but the findings are non-specific and rarely diagnostically useful. In rare cases open lung biopsy may be needed to confirm a diagnosis, in general when an alternative diagnosis is suspected.Apart from treatment-related complications, the thoracic manifestations of RA are plentiful  and inclFindings on BAL are generally abnormal but non-specific in RA-ILD. Common findings include some form of neutrophil or lymphocytic predominant leucocytosis, or alterations in T-lymphocyte ratios , 84–86. Glucocorticoids remain an important part of the acute management of RA-ILD. The optimum longer-term management of RA-ILD is uncertain, however, given the known factors predictive of RA-ILD described above it is logical that good RA disease control should be the cornerstone of any strategy . This isPreliminary evidence of a particular role for Rituximab (RTX) is beginning to emerge , 91, 92.Other agents are currently under investigation in the treatment of RA-ILD: The anti-fibrotic tyrosine kinase inhibitor nintedanib has been shown to be effective in an animal model of RA-ILD; the same agent has demonstrable efficacy in RCTs in IPF and, recently, also in systemic sclerosis –95. AnotILD in general has a poor prognosis, however, this is even more true of RA-ILD, which has an ominous prognosis with a Hazard Ratio (HR) for death of 2.86 . OverallMethotrexate pneumonitis usually presents acutely but its incidence has been decreasing over time. Suspension of MTX and administration of glucocorticoid pulse therapy are usually required. In the long term, MTX therapy may associate with a lower incidence of RA-ILD, thus questioning the fear of progressive pulmonary fibrosis associated with this agent. Regarding bDMARDs, ATC, TCZ, or RTX appear more promising than TNF-i in patients requiring more intense immunosuppression although the evidence base for this remains weak.Future studies should aim at determining the exact prevalence of RA-ILD in early stage RA patients and will certainly rely on PFTs and imaging with CT at baseline and during the disease course to help identify patients at high risk for progression.GF, EN, and RC wrote the first draft of the manuscript. PK edited and revised the manuscript and drafted the figures. JL edited the manuscript and contributed figures. All authors revised the manuscript critically and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Several novel spin-selected dual-wavelength metalenses have been proposed and investigated based on the plasmonic metasurface consisting of two kinds of rotary rectangle gap nanoantennas (RGN), which are designed based on merging two or four polarity-inverse lenses corresponding to different wavelengths (765 and 1300 nm). The spin-selected dual-wavelength metalenses with two similar and two different vertical and lateral focal points have also been proposed respectively, which can focus two wavelengths with inverse spin states to arbitrary special positions. The three-dimensional metalens with four focal points have also been proposed, which can focus four beams with inverse spin states and different wavelengths to preset positions. Moreover, a spin-dependent achromatic metalens has also been proposed, which can focus left circularly polarized (LCP) incidence with different wavelengths to the same position. Our work opens up new avenues toward establishing novel spin-selected and wavelength-selected metadevices, and is significant for the development of spin-controlled photonics and particles manipulation. In addition, it provides a new idea for solving the problem of data transmission from optical fiber communication to visible light communication. With the developments in nanofabrication technology, metasurfaces consisting of nanoantennas have attracted great research interests, which can tailor the amplitudes, phase, and polarization states of the incident electromagnetic waves ,2,3,4. RFurthermore, two or more wavelength multiplexing metasurfaces have gained considerable attentions in recent years ,27,28,29In this paper, we propose several spin-related dual-wavelength metalenses, which are constructed by two kinds of rotary rectangle gap nanoantennas (RGN) corresponding to different wavelengths. To meet near-infrared optical fiber communication and future visible light communication, 765 nm and 1300 nm are selected as target wavelengths. Three kinds of spin-selected dual-wavelength metalenses are designed, in which different spin photons with different wavelengths can be focused to same or different positions respectively. A three-dimensional spin-selected dual-wavelength metalens is designed, four beams with two spin states and two wavelengths can be focused to the preset positions. One spin-dependent achromatic metalens is also designed, in which the LCP incident light with different wavelength can be focused to the same positions. These spin-selected and wavelength-selected metalenses are meaningful for the development of particles manipulation and detection techniques.As shown in Based on the idea of multiplexing, a dual-wavelength metalens can be achieved through arranging the two types of RGNs properly. To realize the wavelength-selective focusing, research on the resonance of the RGNs is crucial. Here, the finite element method is used to perform the numerical simulations. We set the height of the gold and glass substrate as 100 and 200 nm, respectively. Periodic boundary conditions are set along the x- and y-directions, and perfectly matched layers are given in the z-direction. x-axis can be expressed as follows:f is the focal length, λ is the incident wavelength, “+” corresponds to LCP incidence, and “−” corresponds to RCP incidence. In fact, the dual-wavelength metalens can be realized by alternately arranging two kinds of rotating RGNs A and B along x axis. The spin-selected metalens can be made up by two metalenses with inverse polarity, and the phase shift possess opposite signs for two polarity-inverse metalenses. Therefore, the spin-selected and wavelength-selected metalens can be made up by two polarity-inverse metalenses, which correspond to different incident wavelengths. For designing an ordinary planar metalens focusing at the X–Z plane, according to the Fermat’s principle, the phase distributions of the metalens along x-axis can be expressed as follows:We have designed a spin-selected dual-wavelength metalens with the same focusing length, and it can focus the LCP light and RCP light with different wavelengths of 765 nm and 1300 nm simultaneously. The metalens with a focal length of 8 μm can be realized through alternately arranging two kinds of RGNs. The phase distributions of the metalens along x-axis can be expressed as follows:x-axis can be expressed as follows:We have also designed two spin-selected dual-wavelength metalenses with the focus displacements along vertical and lateral directions, respectively. The phase distributions of the metalens with two longitudinal focuses along The spin-selected dual-wavelength metalens with the focus displacements along lateral directions can be designed. For the designed spin-selected dual-wavelength metalens with two lateral focuses along x-directions, f = 4 μm. The designed 3D spin-selected dual-wavelength metalens is shown in In order to testify the superiority of our designs, we try to design a 3D spin-selected dual-wavelength metalens, which can focus four incident beams with LCP and RCP in 765 and 1300 nm simultaneously. Through setting the focal positions as We finally designed an achromatic metalens for focusing LCP lights under 765 nm and 1300 nm simultaneously. Under the LCP incidence with the wavelength of 765 nm, the focusing phenomenon is shown in In conclusion, we have demonstrated several spin-selected dual-wavelength metalenses, which can be used to modulate the focusing performances of different incident wavelengths with inverse spin states. Through spatial interleaving two polarity-inverse metalenses that are designed for two different wavelengths, we can achieve spin-selected metalens manipulating at two wavelengths simultaneously. We respectively designed three spin-selected dual-wavelength metalenses, which can focus two wavelengths with inverse spin states on arbitrary positions. We designed a 3D spin-selected dual-wavelength metalens, which can focus four incident spin states at two wavelengths to preset positions simultaneously, which can be served as a linear/circular polarization and wavelength analyzer. We have also designed an achromatic metalens under LCP incidence for the wavelengths of 765 nm and 1300 nm. The research of spin-selected and dual- wavelength metalens paves the way to future applications in advanced imaging, color display technologies, and spin-based photonics devices.


Prehospital (ambulance) care can reduce morbidity and mortality from trauma. Yet, there is a dearth of effective evidence-based interventions and implementation strategies. Emergency Medical Services Traumatic Shock Care (EMS-TruShoC) is a novel bundle of five core evidence-based trauma care interventions. High-Efficiency EMS Training (HEET) is an innovative training and sensitization program conducted during clinical shifts in ambulances. We assess the feasibility of implementing EMS-TruShoC using the HEET strategy, and feasibility of assessing implementation and clinical outcomes. Findings will inform a main trial.We conducted a single-site, prospective cohort, multi-methods pilot implementation study in Western Cape EMS system of South Africa. Of the 120 providers at the study site, 12 were trainers and the remaining were eligible learners. Feasibility of implementation was guided by the RE-AIM  framework. Feasibility of assessing clinical outcomes was assessed using shock indices and clinical quality of care scores, collected via abstraction of patients’ prehospital trauma charts. Thresholds for progression to a main trial were developed a priori.The average of all implementation indices was 83% (standard deviation = 10.3). Reach of the HEET program was high, with 84% learners completing at least 75% of training modules. Comparing the proportion of learners attaining perfect scores in post- versus pre-implementation assessments, there was an 8-fold (52% vs. 6%) improvement in knowledge, 3-fold (39% vs. 12%) improvement in skills, and 2-fold (42% vs. 21%) increase in self-efficacy. Clinical outcomes data were successfully calculated—there were clinically significant improvements in shock indices and quality of prehospital trauma care in the post- versus pre-implementation phases. Adoption of HEET was good, evidenced by 83% of facilitator participation in trainings, and 100% of surveyed stakeholders indicating good programmatic fit for their organization. Stakeholders responded that HEET was a sustainable educational solution that aligned well with their organization. Implementation fidelity was very high; 90% of the HEET intervention and 77% of the implementation strategy were delivered as originally planned. Participants provided very positive feedback, and explained that on-the-job timing enhanced their participation. Maintenance was not relevant to assess in this pilot study.We successfully implemented the EMS-TruShoC educational intervention using the HEET training strategy in a single-site pilot study conducted in a low-resource international setting. All clinical outcomes were successfully calculated. Overall, this pilot study suggests high feasibility of our future, planned experimental trial. Trauma is a leading global cause of mortality in persons between 5 and 44 years of age . Each yeHigh-quality prehospital  care is a critical component of trauma care. Prehospital care can avert 54% of all mortality from emergency conditions, including trauma, in low-and-middle income countries –8.In low-and-middle income countries, limited resources and strained clinical services often mean traditional educational models  are difficult to implement and poorly attended, and result in variable outcomes –11. ContTM, a globally acclaimed training program with 46% mortality reduction in perinatal asphyxia of neonates in resource-limited settings [In 2016, we developed a novel educational intervention of bundled trauma care , which is implemented using a novel training strategy  based on adult-learner principles. In general, bundling of intervention components, usually 3 to 5 items, helps increase clinical uptake . The EMS2 with over 6 million people in 2011, has over 1 million persons estimated to live in dense, informal settlements, where gang warfare and interpersonal violence are major contributors to the burden of trauma [South Africa was the initial test site for EMS-TruShoC and HEET. South Africa, a middle-income country with high income inequality, has an exceptionally high prevalence of inter-personal violence resulting in 7 times the global mean trauma mortality rate, and loss of 1 million disability adjusted life years (DALYs) in 2000 . The Wesf trauma , 19.The Western Cape Government Department of Health operates a public emergency medical services (EMS) system that provides prehospital (ambulance) services for the Western Cape . WesternThe overarching purpose of this pilot study is to evaluate the feasibility of implementation and the feasibility of assessing outcomes. If the pilot study satisfies predetermined criteria for success, we will subsequently conduct an experimental trial to gain robust evidence regarding implementation of HEET and clinical effectiveness of EMS-TruShoC.The primary objective is to assess the feasibility of implementing EMS-TruShoC using the HEET strategy. The secondary objective is to evaluate feasibility of assessing implementation and clinical effectiveness outcomes.This is a single-site, prospective cohort, pilot study using a multi-method outcomes assessment. The delivery and feasibility assessment of HEET implementation was guided by an implementation science framework, RE-AIM  , 22. CliThe logic model underpinning this implementation science project is congruent with PRECEDE-PROCEED, a widely used model in public health for bringing change in behavior. The model suggests that behavior change is influenced by both individual and environmental factors . We posiThe pilot was conducted at one busy ambulance base, called “Northern Division,” within the Cape Town metropolitan area. The Northern Division base operates 13–15 ambulances daily, each staffed by a pair of providers, in 12-h shifts beginning at 07:00 and 19:00. In 2017, providers at the Northern Division transported about 7000 trauma patients, with 39% due to gunshot wounds and 43% from assaults and transport related injuries . TraumatNorthern Division has about 120 providers at the ranks of basic, intermediate, and advanced life support . All received international standard training in trauma care in their foundational education. Staff attrition is about 5% per year, and providers generally feel burned out and frustrated by the strained trauma care system . BurnoutEMS-TruShoC is a bundle of 5 core, evidence-based, trauma interventions . The EMS-TruShoC core bundle includes the following: (i) on-scene hemorrhage control, (ii) scene time less than 10 min, (iii) large bore intravenous catheter insertion, (iv) high-flow oxygen administration, and (v) transport to a trauma center . Each coHEET consists of structured trainings, divided into 8 sessions or modules, which were delivered by peer trainers  in 15-min sessions occurring every other week, during shifts, and in the back of ambulances. A small subset of all available ambulances were reserved for training, and training time was protected from being dispatched into service. Using peer facilitators in a 2:1 learner-to-facilitator ratio, HEET promotes learning intimacy and relationship building. Furthermore, training is conducted within the patient compartment of the ambulance so as to better approximate clinical context and environment while minimizing operational impact. Content is intentionally repeated to encourage retention. For example, the components of the core bundle are defined in each training session. Training sessions have a deliberate focus on the three domains of learning: knowledge transfer, skills practice, and self-efficacy enhancement. Assessments similarly focus on all three domains. Training modules use common clinical case scenarios to promote relevance and enhance adult-learning. In this pilot, HEET facilitators were ILS or ALS providers who elected to participate, and were specially trained in a 2-day program. A Western Cape EMS-appointed HEET Steering Committee oversaw the program. The Committee was comprised of six senior educators and two quality improvement managers—each with over 15 years of experience in Western Cape EMS. A subset of this committee with most familiarity of Northern Division (the “HEET Team”) conducted the pilot implementation.Feasibility of implementation was the primary outcome, and feasibility of assessing implementation and clinical outcomes data were secondary outcome. We assessed implementation outcomes using the RE-AIM framework , 22, 28 We planned to enroll all EMS providers working clinical shifts during the pilot study since the future study will pragmatically enroll all providers at participating ambulance bases; hence, no sample size estimates were calculated for this pilot study. The HEET Team solicited 12 volunteer facilitators through word of mouth to all ALS and ILS providers. Participation requirement of facilitators was simply a keen interest and being available at work during planned implementation months. Benefits of facilitation, emphasized by the HEET Team, included gaining educational credits, satisfying maintenance of certification requirements, and relationship-building with learners. Although facilitators volunteered, all remaining BLS and ILS staff on duty at the Northern Division were required to participate as learners since this was an on-the-job activity.(i)Learner assessments—assessed knowledge, attitudes, and skills using verbal case vignettes, observation, and written questionnaires, respectively; collected pre- and post-training(ii)Training rosters—collected names, training dates/times; completed by shift managers after each training session(iii)Training evaluations—provided anonymous feedback on quality of content, satisfaction, relevance, and length of training; confidentially completed by learners and facilitators after each training session(iv)Exit survey—assessed overall program experience, satisfaction, problems, willingness to adopt; completed by learners, facilitators, and base managers upon completion of program(v)EMS clinical charts—retrospectively abstracted by a research associate using a validated chart abstraction tool; charts were completed by Northern Division providers during routine trauma careFacilitators were trained by the HEET Team in September, 2017. Implementation was conducted at the Northern Division from October to December, 2017. There were 5 sources for quantitative data:Investigators  and trained research associates collected qualitative data upon program completion. We conducted interviews with learners, facilitators, base managers, and the HEET Steering Committee regarding their experiences with the program. Interviews were one-on-one, private, and semi-structured. We took detailed notes during interviews, then transcribed notes into a Microsoft Word  document. All data collectors were blinded to clinical outcome.Overall criteria for evaluating success (feasibility) of the pilot study were defined as follows: (i) continue to main study without modifications, i.e., feasible as-is; (ii) continue with modifications and/or close monitoring; (iii) stop—main study not feasible .Reach, Efficacy, Adoption, and Implementation Fidelity. Maintenance, defined as the existence of the institutionalized program after 6 months, was not applicable to this pilot study (as trainings lasted 10 weeks), and therefore not assessed. Each RE-AIM dimension contained one or more indices  was assessed by appraising the completeness of qualitative and quantitative data within the RE-AIM framework. The presence of data in most (defined as ≥ 80%) indices would favorably suggest progression to main study without any modifications, whereas presence of data in some (40–79%) indices would suggest modest modifications needed, while the presence of data in only a few (< 40%) indices would suggest very poor feasibility of implementation outcomes data collection in the future trial.Qualitative data were thematically analyzed, by reviewing interview notes, to provide participant and stakeholder experiences. This was not a formal mixed-method study as there was no a priori plan to integrate nor co-analyze quantitative with qualitative data. Considering the pilot nature of this study, a formal deductive and thematic analysis, using independent reviewers and consensus discussion, was not conducted.Feasibility of calculating clinical outcomes in the future trial was assessed in our pilot study by the success of calculating the average change in patients’ shock index, and separately, the average change in providers’ quality of traumatic shock care score. Shock index is heart rate divided by systolic blood pressure  . ProvideThe primary feasibility outcome dictated the overall feasibility of the pilot study, while the secondary feasibility outcomes helped qualify whether modifications would be needed for the future main trial.In total, 121 Northern Division providers participated: 12 were trained as facilitators , and 109 providers  were on duty during the implementation period and eligible to participate as learners  learners completed at least 6 of 8 (75%) training modules. During interviews, learners explained that training during work time facilitated attendance, and was a major factor for high participation rates Table . Learner% learnerknowledge post-training  versus pre-training (4 out of 68 = 6%). There was a 3-fold improvement in the proportion of learners achieving perfect performance of traumatic shock skills post-training  compared with pre-training . There was a 2-fold improvement in proportion with high self-efficacy in recognizing and managing traumatic shock, assessed by the written questionnaire in the post-training  versus pre-training  phase. Participants mentioned that the peer-led format, the case-based discussion, and hands-on nature of trainings enhanced knowledge and skills transfer  met study criteria and were fully abstracted into the study database. Of those cases abstracted, 501 68%) were excluded for multiple reasons Fig. , with 248% were e7% met stTen of 12 (83%) facilitators actively participated. Two ALS facilitators could not participate in trainings as one went on extended sick leave, and the other was promoted to management. Overall, the participating facilitators were very representative  compared with the larger pool of eligible facilitators. Qualitative data indicate that, similar to learners, facilitators felt the on-the-job timing, the relative simplicity of training, and enjoyable format, facilitated their high rates of participation. Survey data from key stakeholders’ (managers and HEET Steering Committee) found that an overwhelming majority (92%) support the adoption and incorporation of this program into Western Cape EMS , and the intervention was adapted by excluding non-core (airway and breathing management) content to shorten modules 3 and 4. Both adaptations allowed a catch-up phase, and from module 5 onwards, administrators and participants were accustomed with the training regimen, allowing the program to run and conclude on schedule. Overall, 90% of the intervention  was delivered as intended—about 10% of non-core (“airway” and “breathing”) content was removed from training materials to accelerate the overall program. Overall, 77% of the implementation strategy was delivered as originally planned, calculated by averaging the following three implementation metrics . The major contributory factor for high participation was the scheduling of training sessions during work hours, and the inclusion of HEET as part of change of shift procedures. Coupled with the enjoyable training format, learners and facilitators were able to frequently participate. Further, training times were kept short via tight monitoring by EMS shift managers (who released ambulances into service as soon as possible), and by facilitators who were trained to be sensitive to training time.Second, we found that specific clinical data were missing with high frequency. In our future study, our primary aim is the change in shock index scores . We noteThird, collecting clinical effectiveness data disproportionately consumed the largest proportion of research effort. We required multiple steps to collect clinical data, starting with a query of the EMS database, followed by manual screening and exclusion procedure, and ending with abstraction into our study database. We excluded 71% of 739 cases originally abstracted  stemmed from slow engagement by shift managers at the base. Shift managers are responsible for ensuring vehicles are staffed and leave the ambulance base on schedule, including other logistic responsibilities. In this pilot, while shift managers were informed about the implementation, they were not formally included in the planning and logistics until implementation. As a result, feedback suggests that while they were supportive of the program, they were disconnected from the details and not adequately invested, resulting in poor oversight and ownership of HEET. The future trial will be modified to formally include managers early and consistently in the implementation process, as recommended in “designing for dissemination” and stakeholder engagement practices .The RE-AIM framework was successfully applied to this work. RE-AIM is a well-known public health framework useful to guide both program and delivery strategy planning and evaluation of implementation outcomes , 22. RE-Our experience from this pilot study demonstrate three important concepts: first, that EMS-TruShoC can be practically implemented with HEET, and evaluated, in a busy EMS base in Africa; second, that relevant clinical outcomes data  EMS-TruShoC are highly feasible to collect; and third, that there is a modest clinical outcomes benefit that warrants further assessment. We received generally positive and enthusiastic feedback from participants and stakeholders about the intervention and implementation strategy. Our pilot experience holds exciting promise for future implementation and research efforts.Study strengths included the multi-method  approach, the systematic assessment of important implementation issues using the RE-AIM framework, and inclusion of both learner and clinical patient outcomes. There are a few limitations of this pilot study. First, the study was conducted in only one site and we did not formally assess organizational context at the Northern Division base—while the future study will be conducted in the same EMS organization but with multiple sites, defining and understanding local context may be important for replication. Second, we did not assess cost-effectiveness—while we have a gross estimation of the resources and inputs, a formal micro-costing analysis may help predict costs of the future trial . Third, In this single-site pilot study, we successfully implemented the EMS-TruShoC educational intervention using the HEET training strategy, in a low-resource international setting. We successfully collected and analyzed quantitative and qualitative data on implementation, educational, and clinical effectiveness that will help enhance the conduct of our future trial. Overall, this pilot study suggests excellent feasibility and value of our future, planned experimental trial.


Non-coding RNAs (ncRNAs) have been implicated in most cellular functions. The disruption of their function through somatic mutations, genomic imprinting, transcriptional and post-transcriptional regulation, plays an ever-increasing role in cancer development. ncRNAs, including notorious microRNAs, have been thus proposed to function as tumor suppressors or oncogenes, often in a context-dependent fashion. In parallel, ncRNAs with altered expression in cancer have been reported to exert a key role in determining drug sensitivity or restoring drug responsiveness in resistant cells. Acquisition of resistance to anti-cancer drugs is a major hindrance to effective chemotherapy and is one of the most important causes of relapse and mortality in cancer patients. For these reasons, non-coding RNAs have become recent focuses as prognostic agents and modifiers of chemo-sensitivity. This review starts with a brief outline of the role of most studied non-coding RNAs in cancer and then highlights the modulation of cancer drug resistance via known ncRNAs based mechanisms. We identified from literature 388 ncRNA-drugs interactions and analyzed them using an unsupervised approach. Essentially, we performed a network analysis of the non-coding RNAs with direct relations with cancer drugs. Within such a machine-learning framework we detected the most representative ncRNAs-drug associations and groups. We finally discussed the higher integration of the drug-ncRNA clusters with the goal of disentangling effectors from downstream effects and further clarify the involvement of ncRNAs in the cellular mechanisms underlying resistance to cancer treatments. We thus built a network of non-coding RNAs starting from a human-curated selection of papers and applied an ad-hoc data mining approach to dissect the network and identify the most important ncRNA/cancer drugs interactions and cliques. We obtained a fully connected network of 388 drug/ncRNA interactions (edges) and 5 unconnected pairs  for a number of conscious or unconscious reasons, scientists often end up chasing the most “popular” ncRNAs among others of “lesser pedigree.” Thus there is potentially a positive bias toward well-known ncRNAs in the overall scheme, and therefore in the final network. For this reason, we decided to keep all associations and, although the “degree” (number of associated drugs for an ncRNA) is important, we tried to avoid biased selections and included in our review all ncRNAs/pairs.Here we commence with describing the ncRNAs that are most prominent in relation to chemoresistance, as detailed in Figure .miR-21 has the highest scores  as it was associated with several drugs. The MIR21 gene is located at17q23.2, a region frequently amplified in several tumors  play an important role in tumorigenesis and are potential biomarkers for cancer diagnosis, treatment and prognosis in clinical settings  mRNA is a target of miR-141, that is involved in drug-induced apoptosis, conferring resistance to docetaxel-sensitive BC cells  is deleted or down-regulated in some cancer types   . In assou (DCIS) . In prosu (DCIS) . Furtheru (DCIS) . It alsou (DCIS) .let-7 family members are down-regulated in lung  and in cancer, eckpoint . miR-224eckpoint , 237. Ineckpoint . miR-224eckpoint .miR-29 family members are miRNAs that can play different roles in cancer  protein. It is used to treat solid tumors, as non small cell lung cancer (NSCLC). It acts by inhibiting the anti-apoptotic Ras signaling cascade . Recent Cisplatin is a platinum compound classified as alkylating like agent that interferes with DNA replication and is used to treat several solid malignancies . The effSubnetwork 3 incorporates several non-coding RNAs related with paclitaxel. This antineoplastic drug is a taxol derivative that blocks cell cycle progression by targeting beta-tubulin. Paclitaxel causes inhibition of mitosis and triggers the apoptotic process or the reversion of cell cycle. Paclitaxel is used to treat a number of solid cancers that include lung, ovarian, breast and pancreatic tumors . A numbein vitro and in vivo models of HCC  is a first-line treatment of CRC, hile for CRC in advanced stages is common to use oxaliplatin in combination with 5-FU (FOLFOX) . Severalreatment . Furtherreatment . Tan et reatment . Moreovereatment .in vitro models have pinpointed its role in chemoresistance: miR-302a exerts its function through inhibition of IGF1R and of downstream Akt signaling: events associated with enhanced 5-FU-induced cell death in colon cancer cells (5-Fluorouracil (5-FU) is a widely used therapeutic agent for treating a range of cancers, including advanced CRC , liver aer cells . The up-er cells , 383 ander cells . miR-96 er cells . miR-23aer cells , while mer cells . Like leer cells . miR-34aer cells , 388. Ther cells ; this loer cells . miR-204er cells  and has er cells .Most of non-coding RNAs disregulations related to doxorubicin, methotrexate and etoposide play a role in chemoresistance exacerbation or inhibition. They are involved in several pathways that regulate cell growth, autophagy, apoptosis and cell proliferation –401 or mTamoxifen, a selective modulator of estrogen receptor, is an effective first-line endocrine therapy that significantly improved relapse overall and relapse-free survival for many ER+ and endocrine-responsive patients. However, a significant proportion of the advanced ER+ BC patients do not respond . RecurreImatinib (IM) is a 2-phenyl-amino-pyrimidine, an ATP-competitive tyrosine kinase inhibitor (TKI) and one of the most potent inhibitors of ABL1. Imatinib was approved for clinical treatment of CML but the problem of drug resistance encouraged the development of new TKI generations . VariousFew ncRNA and drug combinations are not connected to the main network . One of these drugs is dactilosib, an imidazoquinoline derivative under phase II trial that works as dual inhibitor of PI3K and mTOR. It might improve conventional drug treatments and overcome some intrinsic adverse reactions of rapamycin and its derivates . Deng etin vitro screen of several miRNAs and drugs in BC, miR-126 augmented the potency of CDK4/6 or PIK3CA inhibitors in MCF7  and MDA-MB-453 (HER2+) cell lines (In several human cancers miR-144 and miR-451 were identified as tumor suppressor ncRNAs . In termll lines .SV contributed to design and critical revision. FC, LM, and FB performed collection and curation of data. FC assembled data and wrote the article. LM and FB contributed to network analysis. CA contributed to network analysis and writing. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In dogs, melanomas are relatively common tumors and the most common form of oral malignancy. Biological behavior is highly variable, usually aggressive, and frequently metastatic, with reported survival times of three months for oral or mucosal melanomas in advanced disease stages. Classical clinical management remains challenging; thus, novel and more efficacious treatment strategies are needed. Evidence-based medicine supports the role of the immune system to treat neoplastic diseases. Besides, immunotherapy offers the possibility of a precise medicinal approach to treat cancer. In recent years, multiple immunotherapeutic strategies have been developed, and are now recognized as a pillar of treatment. In addition, dogs represent a good model for translational medicine purposes. This review will cover the most relevant immunotherapeutic strategies for the treatment of canine malignant melanoma, divided among five different categories, namely, monoclonal antibodies, nonspecific immunotherapy activated by bacteria, vaccines, gene therapy, and lymphokine-activated killer cell therapy. Melanoma is a relatively common cancer of dogs arising from melanocytes. It accounts for 3% of all neoplasms and up to 7% of all malignant tumors, and it is the most common oral malignancy (56%). Primary melanomas can also occur in the nail bed, footpad, eye, gastrointestinal tract, and mucocutaneous junction ,2. The bIt is increasingly recognized that the immune system plays a critical role in the development and progression of cancer . The treThe aim of this review is to summarize the current and most relevant immunotherapeutic strategies for the treatment of CMM, divided among five different categories: monoclonal antibodies, nonspecific immunotherapy activated by bacteria, vaccines, gene therapy, and lymphokine-activated killer cell therapy. Studies performing at least two approaches fell in one category, chosen at the discretion of the author. This review focuses on the most relevant English-written, peer-reviewed papers of preclinical experiments and clinical data collected from client-owned dogs with spontaneously arising tumors. The search was based on PubMed, Web of Science, and Scopus using the Boolean chains “dog” OR “dogs” OR “canine” AND “melanoma” AND “immunotherapy” OR “vaccine”. The search was performed and revised within the time frame from October the 1st to December the 15th of 2018 and retrieved a total of 241 papers. Irrelevant, duplicated, or beyond-the-scope papers were excluded. 63 papers were included for the review.It is now recognized that established tumors have numerous mechanisms for suppressing the antitumor immune response, including production of inhibitory cytokines, recruitment of immunosuppressive immune cells, and upregulation of coinhibitory receptors known as immune checkpoints. The primary effector cells of the adaptive immune response against cancer are the T lymphocytes. Importantly, T-cell priming/activation is tightly regulated by costimulatory or coinhibitory signals known as immune checkpoints  that proHowever, blocking regulatory checkpoint molecules can also result in aberrant immune activation leading to undesirable off-target inflammation and autoimmunity. Despite the impressive benefits of the immune-checkpoint blockade, its use can be hampered by the occurrence of serious adverse events, which can affect multiple organs of the body, including skin, the gastrointestinal tract, the kidneys, both peripheral and central nervous systems, liver, lymph nodes, eyes, pancreas, and the endocrine system. They can range from mild to severe and can even be fatal . In dogs, a recent pilot clinical study provided the first evidence of clinical benefit in blocking the PD-1/PD-L1 pathway . PD-1 suHuman tumors originating from neuroectodermal cells, such as malignant melanoma, express high levels of disialogangliosides GD2 and GD3, making these antigens ideal targets for mAbs. In a study, the expression of disialogangliosides GD2 and GD3 on canine oral malignant melanomas and their in vitro ability to mediate antibody-dependent cellular cytotoxicity (ADCC) using murine anti-GD2 and -GD3 mAbs was assessed. The data from this study indicated that disialogangliosides GD2 and GD3 were expressed in fresh canine melanoma cells. In addition, mAbs reacted with these antigens and could target and trigger tumor killing by multiple canine effector populations. An additional effect when combined with interleukin (IL)-2 was also observed . In anotCorynebacterium parvum to treat dogs with OMM. A longer survival time was observed in dogs with stage II and III diseases, suggesting that C. parvum, when combined with surgery, may have antitumor activity in the canine melanoma model [Signature molecules released by bacteria termed pathogen-associated molecular patterns (PAMPs) activate innate immune responses (nonspecific) through the activation of so-called pattern recognition receptors (PRRs) and downstream cellular pro-inflammatory signaling . Importama model .A randomized, double-blinded clinical trial evaluated the efficacy of a liposome-encapsulated lipophilic derivative of muramyl dipeptide (L-MTP-PE) when used in a surgical adjuvant setting and administered alone or in combination with recombinant canine granulocyte macrophage colony-stimulating factor (GM-CSF) to treat 98 dogs with spontaneous OMM. In rodents, dogs, and humans, it was proven that L-MTP-PE, when administered in vivo, could activate monocytes and macrophages, resulting in antitumor activity. In this study, the L-MTP-PE administered alone and in combination had minimal antitumor activity. The study could not demonstrate any therapeutic advantage of GM-CSF over L-MTP-PE alone. However, there was suggestive evidence that L-MTP-PE resulted in the prolongation of survival in dogs with early (stage I) OMM . Oncolytic virotherapy is an emerging approach to treat cancer. However, oncolytic virotherapy in veterinary medicine is still in its very early stages. Antitumor mechanisms of oncolytic viruses are not fully elucidated, but they can selectively infect, replicate, and lyse tumor cells, and promote immune antitumor responses by different mechanisms ,20. DiffThe discovery of tumor-associated antigens (TAAs) has allowed for the development of techniques to specifically target neoplasms immunologically . TherapeWhole-cell tumor vaccines are another approach. Various protocols can be used to prepare whole tumor cells for patient vaccination . Whole-cGene therapy relies on the delivery of foreign DNA into cells also known as transfection. The transfer of DNA can be accomplished by non-viral vectors utilizing liposome delivery or DNA protein complexes, and by viral vectors. Viral vectors are genetically modified viruses, which are still able to transfer their genetic material to a host-cell . There aCytokines are able to promote adaptive as well as innate immune responses . IntratuSuperantigens are bacterial proteins that, due to their unique structure, are capable of activating a large number of T-cells . HoweverThe induction of apoptosis has been shown to enhance immune responses against tumor antigens. There is evidence that various pathways that promote intrinsic apoptosis are commonly inactivated in canine melanoma, and have been evaluated for OMM. Intratumoral administration of the human Fas-ligand gene was evaluated in a phase I clinical trial in four dogs with stage III disease. In three dogs, a 12.5–58% reduction of tumor burden was reported, and no adverse effects were observed in all dogs .Suicide gene therapy, also called gene-directed enzyme prodrug therapy, relies on the capacity of the gene product to convert a non-toxic prodrug into a toxic compound . This apAnother immunogene therapy approach is the adjuvant use of a xenogeneic antigen DNA vaccination encoded by a bacterial plasmid. Xenoantigens reported to treat canine OMM are the chondroitin sulfate proteoglycan-4 (CSPG4) and the human tyrosinase  genes. CTM to treat CMM cannot be recommended with confidence. The same approach with xenogeneic murine instead of the human tyrosinase gene has been studied in dogs with digit melanoma. In this study, the authors concluded that the vaccine was safe and appeared effective as an adjunctive treatment. An MST of 476 days was reported [In February 2010, OnceptTM was the first cancer vaccine to receive full approval from the US Department of Agriculture. On the other hand, the company withdrew the application for the European Medicine Agency in 2014 . Howeverreported . Cluster of differentiation 40 (CD40) is a co-stimulatory molecule  found on the surface of B-cells and antigen-presenting cells (APCs). When it binds to CD40 ligand (CD40L) expressed on T-cells, it activates the CD40 bearing cells and thus enhances humoral as well as cell-mediated immunity . CD40 al+ T-cells and a decreased ratio of CD4+ to CD8+ T-cells [T lymphocytes are important for anti-tumor immune responses. Therefore, enhancement of T lymphocytes may have utility in slowing, or halting, the progression of malignant tumors . Adoptiv T-cells . Althoug T-cells .Immunotherapy encompasses a wide range of different treatment modalities  and has