In the US, medication assisted treatment, particularly with office-based buprenorphine, has been an important component of opioid dependence treatment among patients with iatrogenic addiction to opioid analgesics. The predictors of initiating buprenorphine for addiction among opioid analgesic patients have not been well-described.We conducted a time-to-event analysis using data from the North Carolina (NC) Prescription Drug Monitoring Program (PDMP). Our outcome of interest was time-to-initiation of sublingual buprenorphine. Our study population was a prospective cohort of all state residents receiving a full-agonist opioid analgesic between 2011 and 2015. Predictors of initiation of sublingual buprenorphine examined included: age, gender, cumulative pharmacies and prescribers utilized, cumulative opioid intensity (defined as cumulative opioid exposure divided by duration of opioid exposure), and benzodiazepine dispensing.Of 4.3 million patients receiving opioid analgesics in NC between 2011 and 2015 (accumulated 8.30 million person-years of follow-up), and a total of 28,904 patients initiated buprenorphine formulations intended for addiction treatment . In adjusted multivariate models, the utilization of 3 or more pharmacies  or 6 or more controlled substance prescribers  was associated with buprenorphine initiation. A dose-response relationship was observed for cumulative opioid intensity . Benzodiazepine dispensing was negatively associated with buprenorphine initiation .Opioid analgesic patients utilizing multiple prescribers or pharmacies are more likely to initiate sublingual buprenorphine. This finding suggests that patients with multiple healthcare interactions are more likely to be treated for high-risk opioid use, or may be more likely to be identified and treated for addiction. Future research should utilize prescription monitoring program data linked to electronic health records to include diagnosis information in analytic models. Iatrogenic addiction to opioid analgesics has been prominently discussed as a driver of the overdose epidemic in the United States (US). PatientsIn the ongoing opioid overdose epidemic in the United States, a leading demand-reduction paradigm suggests the need for abstinence from illicitly manufactured or diverted opioids. The medicalized treatment of opioid use disorder (OUD) is a major component of abstinence promotion. Medication assisted treatment (MAT) is a major OUD treatment modality, often characterized by the coupling of counseling and other mental health therapies and psychosocial supports with the use of an opioid agonist or antagonist. At present, there are three active pharmaceutical ingredients (APIs) used in MAT in the US: the antagonist naltrexone, and the agonists methadone and buprenorphine.Buprenorphine-based MAT has been widely embraced,, 13 in pCurrently, buprenorphine is widely available in outpatient settings and can be obtained for outpatient use from any physician who obtains a “buprenorphine waiver” and completes eight hours of training as required by DATA 2000. These trainings are administered annually nationwide by the Substance Abuse and Mental Health Services Administration (SAMHSA). DATA 2000 also established key regulations of buprenorphine MAT that make it unique among controlled substances (CS). Waivered physicians are limited in the maximum number of buprenorphine MAT patients they can serve at any one time. Newly-waived physicians also must complete a probationary period with even lower patient limits. In addition to maintenance dosing, waivered physicians may also use buprenorphine for shorter periods of opioid detoxification.Buprenorphine is the only form of MAT prescribed by outpatient community physicians and dispensed by community pharmacies. As the only form of addiction treatment that is routinely captured in Prescription Drug Monitoring Program (PDMP) data, buprenorphine dispensing outcomes have been examined by others who have used these data., 20 StatIn this study, we used PDMP data to identify the predictors of transitions from opioid analgesic use (ostensibly for pain management) to outpatient sublingual buprenorphine use. We hypothesized that increasing cumulative exposure to opioids, increasing numbers of physicians and pharmacies utilized, male gender, younger age and benzodiazepine dispensing would increase the likelihood of buprenorphine use after prescribed opioid analgesic exposure. We were also interested in determining if there were thresholds or breakpoints in cumulative prescribed opioid analgesic exposure that may predispose a patient to subsequent MAT.We conducted a retrospective cohort study of prospectively collected data among North Carolina (NC) residents, from January 1, 2011 to December 31, 2015, who received opioid analgesics (our exposure) to determine the predictors of progressing to outpatient sublingual buprenorphine use (our outcome), using time-to-event statistical models. Our sole data source was the state PDMP.Records of all controlled substance prescriptions dispensed and recorded in the Controlled Substances Reporting System  between January 1, 2011 and December 31, 2015 (60 months) were obtained from the NC Division of Mental Health, Developmental Disabilities, and Substance Abuse Services (DMHDDSAS). Cohort members were tracked longitudinally via unique identifiers created by the data vendor  using a proprietary method based on their names, dates of birth, and addresses. Unique prescriber and pharmacy identifiers were derived from Drug Enforcement Administration (DEA) license numbers.Relevant controlled substances dispensed were identified using National Drug Code (NDC) numbers. Product dosage and formulation details were abstracted using natural language processing and merged with drug metadata previously collected by our team: the active pharmaceutical ingredient (API), class , formulation , and dose., 22 OralWe constructed a cohort of prescription opioid analgesic (OA) recipients residing in North Carolina using the 2011–2015 PDMP data. To be eligible for cohort retention, NC residents had to receive at least one additional CS after their first dispensed OA, to reduce unobservable time bias. Cohort members were considered outcome-positive at the time of their first dispensed prescription for MAT-indicated formulations of buprenorphine , at which point follow-up stopped. Outcome-negative cohort members were censored at the time of their last filled CS.Additional covariates captured through the PDMP were cohort members’ age, sex, cumulative pharmacies and prescribers utilized, cumulative opioid intensity (defined as cumulative opioid exposure divided by duration of opioid exposure), and dispensing of benzodiazepines. Age was captured as an 11-level categorical variable, using ages 40–44 as the referent category, traditionally an age group with the highest overdose mortality. Sex and benzodiazepine exposure were modeled as binary, non-time-varying covariates. Cohort members with any history of benzodiazepine dispensing at cohort entry or during follow-up were considered exposed to benzodiazepines. Cumulative totals of pharmacies and prescribers used to-date by cohort members for any CS were captured as time-varying 3- and 6-level categorical variables, respectively, based on empirical examination of probability distributions.In order to assess whether total exposure to opioid analgesics may influence subsequent buprenorphine MAT, cumulative morphine milligram equivalents (MMEs) per prescription were calculated by multiplying the number of units dispensed by the days supply by a conversion factor., 23 CumuParameterization and the proportional hazards assumptions of the above measures were assessed empirically using frequency distributions and histograms, Kaplan-Meier survival plots, Nelson-Aalen cumulative hazard plots, and log of negative-log survival plots. We estimated hazard ratios using multivariate Cox proportional hazards regression, with robust standard errors to account for multiple observations per cohort member. Interactions with time-varying benzodiazepine exposure and cumulative MME exposure were also assessed, based on a directed acyclic graph (DAG) model of buprenorphine initiation and iatrogenic addiction . The 201To improve computational efficiency, regression models were estimated on a randomly sampled dataset containing 10% of all censored opioid analgesic patients and 100% of patients initiating buprenorphine. Thus, our regression models used all outcome-positive patients and a 10% random sample of outcome-negative patients, where the outcome was defined as initiating buprenorphine. We created sample weights based on the inverse probability of sampling , and used survey analysis methods to ensure that our point estimates and confidence intervals were statistically correct. This approach was validated against univariate models of the binary predictors that used the entire  dataset of 54,594,418 records. All population-based epidemiologic measures (such as the size of population studied and the incidence rate for initiating buprenorphine) were computed using the entire dataset.Analyses were conducted in a distributed server environment using Stata/MP 14.1 and Stata/IC 15.0 .This study was approved by the institutional review board of the University of North Carolina at Chapel Hill (IRB 17–0889).Trends of CS and OA dispensing in North Carolina are shown in Among the full agonist opioids , MMEs ofApproximately 4.3 million patients were included in the cohort of opioid analgesic recipients contributing 8.30 million person-years of follow-up during the 2011–2015 period, of which 28,904 initiated buprenorphine . Our sampled analysis dataset included a 10% sample of 432,594 censored patients and all patients who initiated buprenorphine .Gender was similar among those who initiated sublingual buprenorphine  compared to those who were censored . The mean age at cohort entry was lower among the buprenorphine initiators  than among censored patients . Controlled substance dispensing differed between the groups, insofar as outcome-positive sublingual buprenorphine initiators were exposed to considerably more opioid substances through prescriptions. Buprenorphine-initiating patients filled an average of 13.0 opioid analgesic prescriptions (median: 5), while their censored counterparts filled an average of 8.3 (median: 3). Additionally, buprenorphine initiators contributed an average 620 days of follow-up (SD: 475), 78 fewer days of follow-up compared to non-initiators . Furthermore, mean cumulative opioid analgesic exposure was more than twice as high among sublingual buprenorphine initiators: 24,252 MMEs  versus 10,353 MME (median: 600).In unadjusted Cox models of buprenorphine initiation, statistically significant associations were observed for all predictors . Male geth-and-higher-percentile of cumulative opioid exposure experienced a steadily increasing hazard of buprenorphine initiation up to 4 times higher in the highest decile than patients in the lowest decile . The associations observed in the multivariable adjusted model for these three measures are also graphically demonstrated in In calendar-time adjusted, multivariable models, these associations persisted. Younger male OA patients had higher hazard ratios of buprenorphine initiation, particularly ages 20–34 . The negative association between dispensed benzodiazepines and buprenorphine initiation increased in magnitude . The effects of total pharmacies and prescribers were somewhat attenuated. Patients using 3 or more pharmacies to fill controlled substances , and patients with 6 or more controlled substance prescribers utilized  remained very strongly associated with buprenorphine initiation. Patients in the 20As Prescription Drug Monitoring Programs have been widely promoted as an important supply-reduction tool, we also sought to assess their potential utility for demand-reduction: that is, by identifying patients filling prescriptions for opioid analgesics with a high likelihood of initiating MAT with buprenorphine. Buprenorphine initiation itself must be characterized as a key event in the health trajectory of a patient with OUD. Whether intended for MAT, detoxification, or tapering, the formulations of buprenorphine used as our outcome are all forms of demand-reduction that address the addictive potential of OAs while continuing to provide effective analgesia. Therefore, while buprenorphine initiation constitutes a potentially positive event in a patient’s history, it is also a possible marker of the need for improved pain and opioid management.It is generally accepted that outpatient exposure to opioid analgesics can lead to iatrogenic addiction, which in turn can be treated with MAT, including (but not limited to) sublingual buprenorphine. While the intermediate outcome of iatrogenic addiction itself is not readily observable in large population-based data sources, we were able to observe the subsequent transition to buprenorphine, which is a key factor in clinical resource allocation in the opioid crisis in the US. Not surprisingly, on an individual level, cumulative exposure to opioids was associated with increased likelihood of buprenorphine dispensing, ostensibly for MAT, with a near-monotonic dose-response relationship. What was surprising, however, was that the likelihood of receiving sublingual buprenorphine was elevated at doses well below the clinical threshold of 90 MME/day used in the Centers for Disease Control and Prevention (CDC) guideline for preventing overdose, with HR 3.75  for 51-to-113 cumulative daily MME. This obsOur finding that buprenorphine initiation was notably less likely among OA patients who were ever dispensed benzodiazepines was also unexpected. A previous study found that 1 in 8 buprenorphine MAT prescriptions were used concomitantly with benzodiazepines. Since thOA patients with increasing numbers of CS-prescribing physicians, as well as increasing numbers of pharmacies used, had a greatly increased rate of initiating buprenorphine, even after for controlling for the intensity of cumulative opioids. The number of providers-to-date in particular constitutes a proxy measure of the total volume of healthcare interactions patients have. It is possible that patients with a high volume of healthcare interactions are good candidates for outpatient MAT with buprenorphine, or initiate buprenorphine to address high-risk opioid use because of the coordination of multiple prescribers.The simplified directed acyclic graph (DAG) model of the PDMP data utilized  suggestsThough these proxies may have utility for purposes such as law enforcement, they do not reflect key risk and protective factors such as nonfatal overdose, inpatient opioid exposure, quality and integration of specialist care received (including pain management), and insurance status. The gap between data elements observed and unobserved in PDMP data can be bridged through the linkage of PDMP data with electronic health records, particularly ED data concerning nonfatal overdose, and treatment program quality assurance data. A more complete estimation of the quality of healthcare interactions would also allow population-level evaluation of medical home approaches to pain management. Linkage of this nature may allow clinicians and researchers to better address the biases present in this model of OA use and MAT. However, these linkages naturally raise many challenging data privacy questions.Our study was conducted using PDMP data from 2011 through 2015, and the results should be interpreted in light of the history of that period in the US. The “triple wave” framework of prescription, heroin, and fentanyl overdose epidemics is a useful model for considering this period. 2011 is The use of unlinked PDMP data for a survival analysis presents certain limitations related to the measurement of time-at-risk. After the dispensing of the first recorded OA, patients can only contribute time at-risk as long as they continue to fill CS prescriptions in North Carolina. Therefore, a patient would have to fill at least two prescriptions for a CS during this time period, the first of which was for an OA. Given that competing risks included death (including overdose), leaving NC, and entry into other forms of addiction treatment or MAT, we considered this conservative approach superior to assuming that recipients of a single OA were still alive at the end of the follow up period. Strong negative associations observed in older patients may be attributable to competing risks not detectable in the PDMP, including death, hospitalization or inpatient addiction treatment, underutilization of MAT in this population, or the use of transdermal buprenorphine as an alternative pain management approach. Many of these concerns can be directly addressed in future studies via PDMP linkage as described above.Days supply, which was used to calculate average MME, is a field that is created by the pharmacist at the point of dispensing and has not been validated. Our models necessarily assumed that opioid analgesics and buprenorphine were taken as prescribed; ingestion of diverted opioids  was not observable in the data source. The PDMP thus may undercount opioid analgesic exposure for those using other opioids, or over-represent exposure if the prescriptions were diverted to others. Use of diverted buprenorphine products is itself a complex phenomenon, often with motivations related to withdrawal self-management and self-detoxification, outside the scope of our analyses of a patient’s first dispensed buprenorphine prescription.Without mOur findings and conclusions must also be considered in light of the undertreatment of opioid use disorder during the study period. Older patients who received opioid analgesics, in particular, had very low hazard ratios of buprenorphine initiation. MAT remains underutilized relative to the total population of individuals with opioid use disorder.In prescription drug monitoring data, opioid analgesic patients utilizing multiple prescribers or pharmacies were more likely to initiate buprenorphine. Patients with multiple healthcare interactions may be more likely to have high-risk opioid use, including dependence, identified and treated on an outpatient basis. More intense cumulative opioid exposure also increased the rate of buprenorphine initiation. The dispensing of benzodiazepines to opioid analgesic patients may be an indicator of patient characteristics associated with low coordination among multiple providers. Increased specificity of these metrics and other reductions in bias will be possible through linkage of PDMP data with electronic health records.


While conventional cancer treatments, such as surgery, radiotherapy and chemotherapy, have been combined for decades in an effort to treat cancer patients, the emergence of novel fields of cancer research have led to a renewed interest in combining conventional treatments with more innovative approaches. The realisation that cancer progression is not exclusively due to changes in the cancer epithelial cells, but also involves changes in the tumour microenvironment, has opened new avenues for combination treatments. Here we discuss the use of combination therapies presented at the 55th Irish Association for Cancer Research (IACR) Annual Conference, highlighting examples of novel therapeutic strategies which, combined with conventional therapies, may greatly enhance not only the overall outcome for patients, but also the quality of life for cancer survivors. Among the novel treatment strategies, immune metabolism, epigenetic therapies and physical exercise are presented. In addition, novel technologies in the field of precision medicine, which will be useful to discover new therapeutics and to stratify patients for combination treatments, are also discussed. For many decades, cancer treatment was limited to only a few options for patients. These included surgery and radiation therapy for solid localized tumours, and chemotherapy for blood-related cancers and solid metastatic tumours. These therapies have been used as single treatments or in combination for a long time. Recently, with the advent of targeted therapies, a big emphasis has been put on the biological mechanisms underlying response/resistance to targeted agents. As a result, our understanding of the many pathways involved in cancer progression and the ways in which they can be targeted has improved dramatically, with combinatorial strategies involving multiple targeted therapies or “traditional” chemotherapeutics, such as the taxanes and platinum compounds, being found to have a synergistic effect . HoweverThe use of immunotherapy in the treatment of cancer has received considerable attention in recent years. Natural killer cells (NKs) are members of the innate lymphoid cell population and, as their name suggests, they have a role in eliminating cells that are known to be dangerous to the host organism, including cancer cells, viral-infected cells and foreign cells . Prof. DStudies by Prof. Finlay’s group have shown that the cellular fuels available to immune cells have a big impact on their function. They found that in cytokine-activated NK cells, robust induction of glycolysis and oxidative phosphorylation (OXPHOS) are essential for effective NK cell anti-cancer functions . Their gThe 5-azacytosine DNA methyltransferase inhibitors (DNMTis) have established a presence in the clinic for the treatment of myelodysplastic syndrome and acute myelogenous leukaemia. These agents act as S-phase specific inhibitors of the DNA methyltransferase enzymes and cause global decreases in DNA methylation . HaematoThe importance of exercise in the cancer journey was recently highlighted in a report by the Clinical Oncology Society of Australia, with the clear recommendation that exercise should be embedded as part of standard practice in cancer care . Pre-opeDr. Noel McCaffrey, medical director of ExWell (a community-based chronic rehabilitation exercise service) and his colleague Dr. Lisa Loughney (PhD.), along with local consultant surgeons , are conducting a programme of work investigating community-based (in a gym facility) exercise programmes in the pre-operative setting. Their first pilot study investigated the compliance, adherence and effectiveness of a community-based pre-operative exercise programme in people with a newly diagnosed prostate cancer and colorectal cancer (CRC) scheduled for surgery. Thirty-two surgical oncological (15 prostate cancer and 17 CRC participants) were recruited and assessed to measure health-related (HR) components of fitness  and HRQoL. An exercise programme was prescribed in the time available prior to surgery with repeat assessments pre-operatively. Exercise training was delivered over a median interquartile range (IQR) of 4 (3–4) weeks and 2 (1–3) weeks for the prostate cancer and CRC participants with >80% adherence. This pilot study showed that participants had acceptable compliance and adherence rates to the community-based pre-operative exercise programme and that it significantly increased lower body strength and HRQoL.A qualitative sub-study, as part of the above pilot study, investigated the effects of the pre-operative exercise programme on perceived wellbeing and HRQoL in the prostate cancer group. Following completion of the exercise programme (within 1 week before surgery), 11 participants took part in a semi-structured interview which covered four broad HRQoL domains, including physical, psychological, social and spiritual wellbeing. Findings showed that engagement in the pre-operative exercise programme provided participants with: (1) a teachable moment; (2) acted as a vehicle to recovery; (3) a sense of optimism and (4) social connectedness. This qualitative study showed that the exercise programme enhanced wellbeing and improved perceived HRQoL. Further research is required to explore this in a larger, adequately-powered sample.The research group have just completed a phase 1 study examining the feasibility and effectiveness of a community-based pre-operative exercise programme (initiated immediately after cancer diagnosis) in people with oesophageal and gastric cancer and scheduled for neo-adjuvant cancer treatment (NCT) followed by surgical resection. The rationale for this research study is based on previous research that showed NCT significantly reduces physical fitness prior to surgery ,34. Low In keeping with the theme of the importance of physical exercise in the cancer journey, Dr. Gillian Prue and her team from Queen’s University Belfast (QUB) adopt a precision oncology approach utilising exercise as an anti-cancer treatment. There is sufficient evidence demonstrating the favourable effects of exercise on symptom control and quality of life (QOL) in prostate cancer, such as in countering the effects of androgen deprivation therapy ,36,37. EMany exercise oncology trials adopt a generic, linear approach to training , but to maximise outcome, the ‘principles of training’ —traditionally used for athletes—should be applied to exercise prescriptions . The useDr. Gillian Prue and her team examine the survival advantage that can be achieved through targeted, tailored, exercise medicine. This is currently being achieved via a global research trial being led by Prof. Rob Newton and colleagues in Edith Cowan University, Perth, Australia. This Movember-funded study, which is part pf the Movember Global Action Plan (GAP4), is entitled INTERVAL–MCRPC , a multicentre, randomised, controlled, phase III study . The triIn addition, to provide evidence on the feasibility of exercise interventions in patients for whom high intensity exercise is not suitable, Dr. Prue’s team are currently running a parallel Northern Ireland-specific study based upon an exercise intervention  and CRC trial) developed and recently tested in CRC survivors by colleagues from Ulster University. EXACT-MCRPC (EXACT-metastatic castrate-resistant prostate cancer) offers the benefits of participating in a multicomponent physical activity programme to those men with MCRPC who are ineligible for the INTERVAL programme that contains high intensity exercise. The aim is to ensure that all men have the opportunity to capitalise on the benefits of increased physical activity by offering a lower intensity lifestyle physical activity intervention. This is the first of its kind in this advanced and unwell population. This feasibility study is providing preliminary evidence on the acceptability, feasibility and efficacy of moderate intensity physical activity among men with very advanced cancer, and setting the benchmark for all other cancer patients.Dr. Prue’s team is also planning to extend this research programme to pancreatic cancer. Unlike other gastrointestinal cancers (such as CRC), epidemiological evidence of the association between level of physical activity and pancreatic cancer risk and/or progression remains limited, but some evidence suggests greater volumes decrease risk ,43. GiveHuman biological processes are driven by a complex network of events leading to specific functional phenotypes. These include genomic, epigenomic, transcriptomic, epi-transcriptomic and proteomic networks that cooperate together to deliver a specific biological function. New technologies, as well as advances in analytical techniques, have revolutionized ‘omic’ science and allowed for a more in-depth and integrative understanding of biological processes that lead to various diseases including cancer . The EurDr. Charmsaz is currently focused on understanding epi-transcriptomic alterations in ER-positive endocrine-resistant breast cancer and is in the process of integrating these data with proteomics to identify novel therapeutic targets for treatment of aggressive breast cancer.In the past decades, we witnessed tremendous advances in cancer research which have opened new and exciting avenues for the future of cancer treatment and management. Combination therapies certainly played a huge part in this process. Each plenary session at the 2019 IACR meeting covered innovative approaches to cancer therapy which combine traditional therapies with novel ways of targeting cancer progression, summarised in The combination therapies presented at the 2019 IACR Conference highlighted the latest findings in the field of cancer therapeutics. These innovative approaches will offer additional therapeutic options to cancer patients. While more research is needed to refine our understanding of the molecular mechanisms underlying drug combinations, the development of ‘omics’ technologies will allow us to better predict the response to combination therapies, thus improving our ability to use them in the clinic.


In the pursuit to understand intestinal epithelial cell biology in health and disease, researchers have established various model systems, from whole animals  with experimentally induced disease to transformed human carcinomas. The obvious limitation to the ex vivo or in vitro cell systems was enriching, maintaining, and expanding differentiated intestinal epithelial cell types. The popular concession was human and rodent transformed cells of mainly undifferentiated cells, with a few select lines differentiating along the path to becoming goblet cells. Paneth cells, in particular, remained unculturable. The breakthrough came in the last decade with the report of conditions to grow mouse intestinal organoids. Organoids are 3-dimensional ex vivo “mini-organs” of the organ from which the stem cells were derived. Intestinal organoids contain fully differentiated epithelial cells in the same spatial organization as in the native epithelium. The cells are suitably polarized and produce and secrete mucus onto the apical surface. This review introduces intestinal organoids and provide some thoughts on strengths and weaknesses in the application of organoids to further advance our understanding of the intestinal epithelial–microbe relationship. Over thcrobiome . In thiscrobiome ,6. Entercrobiome , review crobiome , or focunfection . Here, wNot limited to the intestines, organoids are generally defined as three-dimensional assemblages of cells that grow from organ-specific progenitors or stem cells and resemble a miniaturized ex vivo organ from which they have been derived ,11. CurrBy including multiple cell types of the organ from which the stem cells were derived, organoids have a unique potential to model human disease. They are self-renewing and can be readily cryopreserved (which obviates the need for continued access to primary tissue), and they can be genetically and environmentally manipulated in highly controlled settings . Importa+ ISCs organize into crypt-like structures after receiving niche signals, most notably Wnt3 and Epidermal Growth Factor (EGF), from Paneth cells sandwiched between ISCs in the crypt [®) and a cocktail of the niche factors R-spondin, EGF, and Noggin is provided in culture media. Wnt is a critical factor for maintaining stemness and for driving the proliferation of TA cells. Wnt normally occurs in a spatial gradient, being most concentrated in the crypt base. In the absence of Wnt, near the top of the crypt, TA cells differentiate into epithelial subtypes such as enterocytes, goblet cells, enteroendocrine cells, tuft cells, and M cells [The human intestine is lined with a single-cell layer of columnar epithelia that forms an essential barrier to environmental challenges arising in the lumen. To maintain this barrier, especially in the context of pathogen- or stress-induced tissue damage, intestinal epithelial cells possess a high rate of self-renewal. This renewal process begins with intestinal stem cells (ISCs) residing in intestinal crypts that give rise to rapidly dividing transit-amplifying (TA) cells which form the rest of the crypt and flow up the villus where they differentiate into various epithelial cell subtypes and eventually die. There are two populations of ISC in the adult intestinal crypt: a quiescent ISC  and actively proliferative ISCs, which are characterized by expression of Lgr5 ,16. To ghe crypt ,18. For  M cells . Dependi M cells . NotablyBoth organoids and the human gut epithelium are comprised of multiple cell types; ISCs differentiate into these, including the abundant enterocytes, goblet cells, Paneth cells, enteroendocrine cells and Tuft cells. Differentiation of cells within the enteroids is hallmarked by products commonly associated with cells in vivo such as alkaline phosphatase and sucrase-isomaltase in enterocytes, mucins in goblet cells, and lysozyme in Paneth cells ,20. HumaNotwithstanding the evidence that cell phenotypes are programed into the local stem cell, enteroids and colonoids lack epithelial-adjacent tissues including enteric nerves, vasculature, gut-associated lymphoid tissues, luminal food products and interface with the microbiome, all of which may provide extrinsic signals to epithelial cells. In addition to food, presumably, the native intestinal epithelium is bathed in a filtrate of microbial products, which is not commonly emulated in standard organoid culture conditions. Yet it is unequivocal that microbial products impact epithelial cell physiology. One example of a microbial product that when added to organoid cultures clearly modified development is the TLR3 agonist poly I:C. Exogenous poly I:C increased enteroid surface area but reduced colonoid crypt budding associated with reduced numbers of lysozyme positive cells in both types of intestinal organoid . A diffeCells becoming polarized is critical to studying microbial infection of intestinal epithelial cells. The application of microbes or microbial products limited to the apical cell surface is challenging in organoid cultures since while the cells in organoids polarize, the apical cell surface faces a closed “lumen” (resulting in secreted products and dead cells accumulating in the lumen). A recent noteworthy adaptation that addresses access to the apical membrane was reported by Co et al. (2019) . Acting Organoids generated from fresh human tissue and therefore derived from ASC, possess cell types and important physiological properties of the organ from which they were obtained. On the other hand, there is great interest in the potential for using human embryonic and iPSCs to generate organoids in order to circumvent the scarce availability and accessibility to primary human tissue samples; however, there is compelling evidence that intestinal organoids derived from iPSCs are similar to fetal human intestine in terms of expressed progenitors and genes . Finkbeihttp://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/2006/ucm108576.htm). The availability of organoids provides a model system to study and test clinically relevant drugs. Certainly, using organoids for safety screening is likely to be more informative than previous approaches relying on 2-D monolayer cell lines that do not have the complex cell types and interactions present in vivo. Primary cell lines and tissue biopsies, although possibly preserving relevant cell signaling pathways and physiological properties, like cell lines are at risk of contamination by other cells, change ploidy, and mutate and/or change phenotype after being passaged over an extended period of time [Human diseases are often modeled in rodents. According to the U.S. Secretary of Health and Human Services, “Nine out of ten experimental drugs fail in clinical studies because we cannot accurately predict how they will behave in people based on laboratory and animal studies” (FDA (2006) FDA Issues Advice to Make Earliest Stages of Clinical Drug Development More Efficient.  of time . Organoi+ stem cells and engrafted in damaged colonic tissue in vivo, with engrafted mice ultimately achieving higher body weights than the controls [One such personalized, donor-specific application is using organoids as a cell source for tissue regeneration. Demonstrated in mice, colonoids have been grown from Lgr5controls . This ap2 levels and do not provide a suitable environment for studying anaerobic bacteria living in the gut. Organoids are grown in an extracellular matrix plug and can be used to establish an O2 gradient that will allow the bacteria to survive [2 availability, another refinement of monolayer cell culture approaches related to oxygen availability is to establish an air–liquid interface on the apical aspect of the cells. Using a porcine cell line, it was reported that growing cells with an air–liquid interface had a profound effect on cell physiology, principally leading to enhanced oxidative phosphorylation and suppressed glycolysis [Organoids also enable the study of the interaction between anaerobic bacteria and the human gut. Cell lines are grown in standard O survive . Yet desycolysis . This haAlong the evolutionary path of intestinal epithelial cell models, investigators have eagerly applied their model to better understand host interactions with pathogenic or commensal microbes. Consequently, much of our understanding of the epithelial cell response is drawn from experiments using transformed and undifferentiated cells and these were challenged with incompatibilities with the infectivity of organism, reducing confidence that outcomes can be applied to the living human. Notwithstanding the shortcoming discussed previously, organoids offer new opportunities for answers to questions about mechanisms of infection and host response. First, organoids can affirm (or dispute) our understanding of intestinal epithelial biology established from transformed cells. Second, using organoids allows specific examination of fully differentiated non-dividing versus dividing non-differentiated epithelial cell response to microbial challenges. Lastly, organoids permit an in-depth examination of unique (patient or engineered) genotype–phenotype associations in responses to microbes, which is highly relevant to disorders thought to arise from abnormal host–microbe interactions, such as the inflammatory bowel diseases. However, a challenge remains in accessing the apical side of the cells where microbes would typically encounter epithelial cells, which some investigators have overcome using microinjections and the “apical outside”, described earlier. Here, we review a few examples of research that highlight how using organoids is re-writing our understanding of microbe/intestinal epithelial cell interactions.Enteric viruses, like the human noro and rota viruses, are among the most significant causes of acute gastroenteritis; combined, they are responsible for approximately 70% of episodes . What isThe innate response mounted by the intestinal epithelium against viral infections is critical in controlling and possibly lowering the viral load during the initial infection. When exposed to HRV infection, enteroids mounted an innate immune response that mainly activated transcriptional pathways leading to production of type III interferons (IFNs). However, the induced IFNs were insufficient to curtail the HRV infection while adding type I IFNs to the system was successful in restricting viral infection . HoweverPrior to work conducted using organoids, our understanding was that human norovirus strains replicated in B cells, but since the virus can infect B cell-deficient patients, there must be another host cell type . ConsequSalmonella spp. are gram-negative enteric pathogens that annually cause millions of cases of gastroenteritis and typhoid fever worldwide. Salmonella spp. invades intestinal epithelial cells via bacterial-mediated endocytosis by preferentially binding to the highly specialized M cells on Peyer’s Patches. Studies of the pathogenesis of S. typhimurium have commonly been undertaken in mice; however, S. typhimurium dissemination occurs in mice with the infection becoming systematic, while in humans the disease typically remains localized in the intestines [Salmonella disrupts tight junctions, that epithelial cells activate NF-κB as an inflammatory response to infection, and that there is a significant decrease in the number of stem cell marker Lgr5+ cells in enteroids [testines . Studiesnteroids . Althougnteroids .Salmonella infection of mice has exposed another reciprocal relationship in which epithelial cells in infected mice make IL-23, which can act on IELs to secrete IL-22; IL-22 promotes anti-microbial peptide synthesis and secretion by epithelial cells, particularly Paneth cells. As an example of how pure epithelial cell cultures may not tell the entire story, including during infection, the intimate relationship between intestinal epithelial cells and intraepithelial lymphocytes (IELs) will be touched on here. In the healthy intestine, interleukin (IL)-7 made by epithelial cells acts on IELs , while ISalmonella, pathogenic strains of E. coli cause gastrointestinal disease world-wide and effective treatments and vaccines are highly sought after. When analyzing the pathogenesis of enteroaggregative Escherichia coli in enteroids, researchers observed unique patterns of adherence occurring in difference segments of the intestines taken from different donors [Like t donors . This maAs attractive as organoids are in recapitulating the native epithelium and potential applications, there are some noteworthy limitations that need to be considered when interpreting research outcomes. One concern about organoid development is reproducibility; it is not known whether all organoids grown under the same conditions will have the same phenotype . Unlike


In this article, Singh et al. have shown that feeding innate immune-deficient mice a diet enriched in soluble fibers  but not insoluble fibers could induce liver cancer  Singh e. Fiber-iT5KO) mice, by feeding the mice an inulin-containing diet (ICD). They observed that the sera of ~40% of mice without metabolic syndrome showed a yellow color, caused by elevated bilirubin levels during the experiments. Finally, based on their observations, the authors concluded that refined fermentable fibers can induce cholestasis followed by liver cancer in mice , which are important for human health (Holscher, Clostridium cluster XIVa, which performs a key enzymatic transformation of primary to secondary bile acids in the gut (Ridlon et al., Clostridium cluster XIVa and the secondary bile acids produced by them play important roles in promoting obesity-associated liver cancer (T5KO mice, and that oncogenic bacteria are transmissible to susceptible mice, as demonstrated by co-housing or cross-fostering. The article did not identify specific oncogenic bacteria. Overall, these studies on relationships between gut microbiota and liver cancer suggest that Clostridium cluster XIVa, Clostridium cluster XI, or other strains catalyzing the transformation from primary to secondary bile acids could be promoters of liver oncogenesis.The authors of this study showed that Clostridia species are enriched in the guts of mice with live cancer, including the r cancer . The accr cancer . Bile acr cancer . The curBJ wrote the commentary. RW, JZ, and YC helped to write.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.


Oncolytic viruses are under intense development and have earned their place among the novel class of cancer immunotherapeutics that are changing the face of cancer therapy. Their ability to specifically infect and efficiently kill tumor cells, while breaking immune tolerance and mediating immune responses directed against the tumor, make oncolytic viruses highly attractive candidates for immunotherapy. Increasing evidence indicates that a subclass of oncolytic viruses, which encodes for fusion proteins, could outperform non-fusogenic viruses, both in their direct oncolytic potential, as well as their immune-stimulatory properties. Tumor cell infection with these viruses leads to characteristic syncytia formation and cell death due to fusion, as infected cells become fused with neighboring cells, which promotes intratumoral spread of the infection and releases additional immunogenic signals. In this review, we discuss the potential of fusogenic oncolytic viruses as optimal candidates to enhance immunotherapy and initiate broad antitumor responses. We provide an overview of the cytopathic mechanism of syncytia formation through viral-mediated expression of fusion proteins, either endogenous or engineered, and their benefits for cancer therapy. Growing evidence indicates that fusogenicity could be an important feature to consider in the design of optimal oncolytic virus platforms for combinatorial oncolytic immunotherapy. Cancer immunotherapy represents a promising new aspect of cancer treatment that aims at activating the patient’s own immune system to eradicate the tumor. Ideally, an effective immunotherapy activates the cancer-immunity cycle that starts off with a first round of tumor cell killing, and the activation of the immune system to prime a broad antitumor immune response. This would then induce a second round of tumor cell killing, in which the endogenous immune cells of the patient are activated and directed against specific tumor antigens to eradicate the remaining tumor cells and metastases, as well as provide long-term protection to the patient against recurrence .This process, however, is best achieved by rationally combining different therapeutics ,3. WhileFusion is a common cellular process that enveloped viruses utilize to mediate the merging of the viral envelope with the host membrane during infection and internalization as a critical first step in their virus life cycle. Virus–cell fusion is achieved by one or more viral surface glycoproteins, denoted as fusogenic membrane glycoproteins (FMGs) or simply fusion proteins, which interact with receptors and coreceptors on target membranes, and induce distinct fusion processes according to their protein structure . In addiOncolytic viruses with fusogenic glycoproteins have some desirable advantages over their non-fusogenic counterparts, influencing both direct oncolytic and immune-stimulatory effects, which may translate to therapeutic benefits upon clinical translation. Tumor cells infected with these viruses form syncytia. As infected cells fuse with neighboring cells, intratumoral spread of the infection is facilitated . AnotherThe syncytia are viable for only a short time, before they undergo immunogenic cell death (ICD) . FusogenOver the last two decades, major breakthroughs were made in the understanding of the proteins and protein complexes that are responsible for mediating fusion between viral envelopes and their host cells. It is now clear that all characterized fusion proteins are similar, in that they mediate membrane fusion through irreversible conformational changes involving a trimer-of-hairpins motif, as well as through the induction of a common pathway of membrane dynamics to coerce membranes to join together via lipid junctions ,26. MembAlthough the predominant function of fusogenic membrane glycoproteins is to mediate the process of infection via fusion of the virus envelope with the target cell, a side product of this is the induction of fusion between infected and non-infected cells. Viral glycoproteins are shuttled to the membrane surface of infected cells, and interact with their receptors on neighboring cells. A sequence of conformational changes prompts the fusion of these cells, and over time, causes the formation of merged multinucleated giant cells . These sO-methyl)-fluoromethylketone (Z-VAD-FMK) [Apoptosis of syncytia was noted in cells infected with human immunodeficiency virus (HIV)-1 ,32. ApopVAD-FMK) . This hoVAD-FMK) . FurtherVAD-FMK) . +) cells upon fusion of dying cells with neighboring, “healthy” cells [An important feature of syncytial cell death is a significant bystander effect on neighboring, uninfected cells. “Contagious apoptosis” was displayed by HIV-infected cluster-of-differentiation-4 positive  and adjuvanticity  . AlthougImmunogenic cell death is associated with the release of danger signals such as heat-shock proteins, which stimulate the uptake of dead cell-associated antigens ,45. A reApart from dead tumor cells, the virus infection itself is detected by Toll-like receptors (TLRs), cytosolic DNA sensors, retinoic acid inducible gene (RIG)-like receptors or nucleotide-binding oligomerization domain (NOD)-like receptors, depending on the virus. This causes the release of intracellular danger signals, which alert the innate and adaptive immune system. This anti-viral response may also be able to activate an antitumor immunity relying on cross-presentation of tumor antigens from infected cells on antigen-presenting cells (APCs). Virus–cell fusion is sensed by the innate immune system, and activates a stimulator of interferon genes (STING)-dependent signaling pathway that leads to the production of type I interferon (IFN) and molecules encoded by IFN-stimulated genes (ISGs). A similar response occurred following cell–cell membrane fusion . For thePerhaps due to the evolutionary advantage associated with the ability to induce fusion, many different virus families adopted this mechanism of host cell entry and/or spread. Keeping in line with the focus of this review, we only discuss those fusogenic viruses that are oncolytic.Although all enveloped viruses enter the cytoplasm via membrane fusion, the mechanism can vary depending on the virus ,49. The Of the paramyxoviruses, the measles virus (MV) and Newcastle disease virus (NDV) were extensively applied as oncolytic virus therapeutics, and to a lesser extent, the mumps (MuV) and Sendai virus (SeV) also show promise. These viruses initiate infection via attachment to their cellular receptors, allowing fusion of the viral envelope to the host cell membrane. This process is coordinated by the activities of two discrete transmembrane glycoproteins: an attachment protein and a fusion (F) protein. The receptor for most paramyxoviruses is a molecule containing sialic acid residues, although it was demonstrated that MV has at least 3 different receptors: the complement regulatory molecule, CD46, the signaling lymphocyte-activation molecule , and the cell adhesion molecule, Nectin-4 . The vir0, which must be proteolytically cleaved to form F1 and F2 for fusion activity. These F1 and F2 polypeptides are disulfide-linked, and are derived from the carboxyl- and amino-terminal domains, respectively [Although the F protein directly mediates membrane fusion, paramyxoviruses require the co-expression of the attachment protein in order for fusion to occur . This evectively . The seqectively . As negative-strand RNA viruses, paramyxoviruses are particularly sensitive to the anti-viral actions of type I IFNs. Cancer cells acquire various mutations along the IFN signaling pathway during their malignant transformation, which help them to escape from host regulation ,57, and In addition to the potential tumor-targeting advantage of using sialic acid residues as receptors for paramyxovirus attachment, the sialidase (neuraminidase) activity of the HN protein of SeV, NDV, and other paramyxoviruses provides a unique potential advantage through the ability to remove sialic acid residues from the surface of tumor cells ,62,63. TDue to the many attractive properties of paramyxoviruses as oncolytic agents, several viruses from this family were investigated for their potential as cancer therapeutics. Sendai virus, although a highly transmissible respiratory virus in rodents, is considered apathogenic in humans. Preclinical studies using recombinant SeV (rSeV) demonstrate that the virus can spread extensively in tumor xenografts, leading to tumor growth inhibition for a wide variety of tumor entities, while leaving healthy surrounding cells unharmed ,69. ThesNDV is an avian virus that is extensively studied for its oncolytic potential. Depending on their virulence in their avian hosts, NDV strains are classified into three categories: velogenic (highly pathogenic), mesogenic (moderately pathogenic), and lentogenic (mildly pathogenic). This classification is based on the resultant mean death time in embryonated chicken eggs . The virAlthough a human pathogen, the measles virus (MV) was explored for its potential as an oncolytic virus for over a decade, and evidence for its anticancer effects, both preclinically and clinically, is growing ,88,89. PThe mumps virus (MuV), although explored to a much lesser extent than the other oncolytic paramyxoviruses in the preclinical setting, was applied in clinical investigations for various human cancers more than forty years ago in Japan with promising outcomes ,97,98. TOne of the greatest advantages of oncolytic viruses is the possibility to engineer individualized vectors depending on the patient’s needs. This may include introducing additional safety features, improving efficacy of tumor cell killing, or adding any other kind of therapeutic gene. Since the early 2000s, various groups demonstrated that oncolytic virotherapy could be improved by introducing fusogenic proteins see .+ cells when compared with non-syncytial cells [With these engineered viruses, the advantages of fusion for viral replication and spread become evident in direct comparison with their non-fusogenic counterparts. Since syncytia are viable for at least 24 h after formation, this form of cellular adaptation permits the intratumoral spread and release of viral progeny within the confines of the tumor. Extensive syncytium formation mediated by the reovirus FAST proteins triggers localized cell-to-cell transmission of the infection, followed by enhanced progeny virus release from apoptotic syncytia . Moreoveal cells . The fusal cells . Engineeal cells . In a sial cells ,103.GALV/CD) were tested in various cell lines in vitro and tumor lesions in vivo (see GALV retains its ability to cause tumor shrinkage in subcutaneous lesions of human squamous cell carcinomas (Fadu) or human fibrosarcomas (HT1080) in the flank of BALB/c homozygous nude (nu/nu) mice, whereas non-fusogenic OncoVEX does not [+ and CD8+ cells were also detected in tumors [The effects of fusogenic oncolytic viruses in vivo are important for predicting their potential value in clinical translation. A replication-competent adenovirus expressing GALV (ICOVIR16) was tested in different tumor models, and showed enhanced antitumor activity in subcutaneous SkMel-28 and NP18 lesions after both intratumoral and systemic administration compared to its non-fusogenic counterpart (ICOVIR15) . The antvivo see . A strikdoes not . VSVΔM51n tumors . Addition tumors . PreclinUntil now, viruses engineered to express heterologous fusion proteins were not yet applied to clinical studies; however, the application of naturally fusogenic oncolytic viruses, namely the paramyxoviruses, was extensively explored in clinical studies. Although we only provide a brief summary of clinical trials of oncolytic viruses that induce cell–cell fusion, a more comprehensive review can be obtained elsewhere ,115,116.Administration of oncolytic NDV vectors via various routes of administration up to relatively high doses resulted in mild-to-moderate adverse effects, such as mild conjunctivitis, laryngitis, and flu-like symptoms in clinical investigations . NDV str11 TCID50) [Along with NDV, MV was also investigated in numerous phase I and II clinical trials. These trials all involve attenuated MV-Edmonston, and either the unmodified Edmonston Zagreb (MV-Ez) strain or engineered vectors expressing the NIS or CEA reporter genes. The earliest reported clinical trial involving MV was a phase I investigation of MV-Ez injected intratumorally in patients with cutaneous T cell lymphoma . Impress TCID50) .As previously mentioned, mumps virus was utilized during the 1970s in several clinical trials in Japan, with both wild-type and attenuated vectors in patients with advanced malignancies ,97,129. Little or no toxicity at effective doses;Tumor-selective replication;Rapid spread throughout the tumor mass, resulting in efficient tumor debulking;The ability to induce potent adaptive antitumor immune responses.Extensive preclinical and clinical research in the field of oncolytic virotherapy, spanning the course of more than a century, recently culminated in groundbreaking advancements. The concept of using viruses as therapeutic agents transcended science fiction thriller plots to becoming a clinical reality in the management of cancer patients. Although oncolytic viruses are now generally accepted as promising cancer treatment agents, there remains a huge logistical challenge to make a comprehensive comparison of the myriad oncolytic virus vectors under development, in order to determine which are the most suitable for clinical translation. Furthermore, it is highly questionable that there will ever exist a “one-size-fits-all” virus that is effective in all cancer entities and patient settings. However, despite these points, both basic scientists and clinicians alike would agree that certain features are ubiquitous in the conceptualization of an ideal vector platform. The essential aspects include the following:We believe that the abundant research reviewed here would argue that oncolytic viruses that encode for a fusogenic protein, whether it be endogenously expressed or introduced via viral engineering, represent optimal OV platforms that fulfill all of the desirable requisites. Due to the widespread applicability of virus engineering and reverse genetics rescue systems, heterologous fusion proteins can be engineered into a variety of oncolytic platforms to improve not only the direct oncolytic effect, but also the immune-stimulatory effect. The ability of these viruses to spread directly from cell to cell through fusion minimizes the release of viral progeny into the surrounding healthy tissue and systemic circulation, which we believe to be a key benefit in reducing off-target effects and avoiding viremia. This strategy could also function to minimize the counterproductive effects of neutralizing antibodies, as the virions are essentially hidden from inactivation due to their intracellular spread. Furthermore, because a single fusogenic virion can potentially lead to the incorporation of hundreds of neighboring cells into the growing syncytium, the virus has the ability to efficiently destroy tumors without the need for high titers of virus production within the tumor. This concept is supported by in vitro data indicating that, despite the fact that adenovirus-mediated GALV expression has a substantially inhibitory effect on virus replication at low multiplicities of infection (MOIs), a similar level of cytotoxicity can still be achieved compared to that of the control Ad vector .Despite the accumulating data supporting the advantages of oncolytic viruses capable of inducing cell–cell fusion, it is expected that rationally designed combination therapies will offer the best chance of providing long-term survival benefits and harnessing the full potential of the host’s antitumor immune response. Particularly, immune checkpoint inhibitors revolutionized cancer therapy for certain tumor entities, but their efficacy depends on an immunologically active tumor microenvironment, and many solid tumors have proven difficult to treat by this approach . FurtherDespite the many potential benefits of fusogenic viruses as oncolytic agents, a valid safety concern should be considered, in that these viruses could potentially cause fusion of tumor cells with surrounding healthy cells, since the fusion process is not tumor specific. However, these viruses were applied in a multitude of clinical trials over several decades, and to our knowledge, there are no reports of fusion occurring within healthy tissue. Although it is not completely clear why this is the case, we believe that it is, at least in part, due to the capsule surrounding the tumor, which creates a physical barrier, preventing direct contact of the tumor cells with the surrounding tissue. Preclinical in vivo investigations on the tumor specificity of the measles virus was limited by the fact that it only enters human cells, meaning that, for the most part, xenograft mouse models are employed. These studies are naturally biased in the sense that virus replication is artificially restricted to the tumor, due to the lack of virus receptors on the mouse cells. This limitation was partially overcome with the generation of human CD46 transgenic mice ,133. In As we are currently witnessing major advancements in the field of viroimmunotherapeutics, and it is expected that increasing numbers of OV platforms will reach mainstream clinical application for cancer patients in the near future, this is indeed an exciting time to be working in the field of oncolytic virus development. Whether these vectors will be sufficiently effective in cancer destruction on their own, or whether they will be implemented as part of combination therapies, remains to be seen. Nevertheless, we predict that the development of fusogenic vectors for improved spread, potent immune stimulation, and enhanced safety will become a valuable strategy in the advancement of OV vectors for clinical translation.


Speech comprehension in noisy environments, such as a busy restaurant, becomes increasingly challenging as we get older. Consequently, many older adults shy away from these complex auditory situations, which can lead to social isolation . To dateTo demonstrate the importance of the interaction between attention and memory during auditory scene analysis and to assess age differences in this interaction, Alain et al.  used a dpresented simultaneously from three different locations. During the retention interval, participants were presented with a retro-cue indicating which item, from a three-item memory array, should be held in ASTM. Younger adults took advantage of this information, and they oriented their attention toward the cued item in ASTM. In young adults, retrospectively attending to an item in ASTM was associated with enhanced event-related potential amplitude and decreased alpha and low beta power. These neural modulations were attenuated in older adults. These results suggest that older adults may have difficulty in orienting attention to a specific item in ASTM. For instance, older adults may not have as much control over their attentional spotlight as younger adults. One possibility is that older adults are more reluctant than young adults to “leave out” information and by default, use a more effortful strategy of maintaining all, even task-irrelevant, items in ASTM. In other words, older adults may have held all three items in ASTM, despite being retro-cued to the one task-relevant item.Further research on the dynamic interactions between attention and memory is important for understanding how sound objects are represented, maintained, and accessed in the brain.In the Alain et al. study, the task was fairly challenging because three environmental sounds from three different locations were presented simultaneously. Hence, it remains unclear whether older adults would benefit from a retro-cue to orient their attention to short-term memory if fewer items were presented. Fewer auditory objects might ease the segregation and representation of the incoming auditory signal in ASTM. A better understanding of these mechanisms could help mitigate auditory-related communication problems encountered by aging individuals, and the social isolation that is often associated with them. The knowledge gained from studies on attention to auditory memory should foster the development of interventions using neurostimulation and/or behavioral techniques that could delay or prevent these age-related changes. In summary, Alain et al.’s findings underscore the importance of attention to memory in understanding age-related changes in processing real-world auditory environments and pave the way for new avenues of inquiry into aging and auditory cognition.


For a particular species, in general, only the reference genome is well established and annotated. Computational tools based on sequence alignment have been developed to investigate the gene models of individuals belonging to the same or closely related species. During this process, inconsistent alignment often obscures genome annotation lift over and leads to improper functional impact prediction for a genomic variant, especially in plant species. Here, we proposed the zebraic striped dynamic programming algorithm, which provides different weights to genetic features to refine genome annotation lift over. Testing of our zebraic striped dynamic programming algorithm on both plant and animal genomic data showed complementation to standard sequence approach for highly diverse individuals. Using the lift over genome annotation as anchors, a base-pair resolution genome-wide sequence alignment and variant calling pipeline for de novo assembly has been implemented in the GEAN software. GEAN could be used to compare haplotype diversity, refine the genetic variant functional annotation, annotate de novo assembly genome sequence, detect homologous syntenic blocks, improve the quantification of gene expression levels using RNA-seq data, and unify genomic variants for population genetic analysis. We expect that GEAN will be a standard tool for the coming of age of de novo assembly population genetics.With the broad application of high-throughput sequencing, more whole-genome resequencing data and  The deciversity , therebyiversity .de novo assembly genomes, the reference coordinates can be lifted with the genome sequence alignment-based variants calling.With the growing quantity of genomic information available, the ability to integrate information from different species and/or accessions becomes an interesting challenge. One of the crucial steps is to “lift over” the reference coordinates to another genome sequence . For whoAs previous studies point out, the same variants, especially INDELs and nearby SNPs, might be represented in multiple ways. The left alignment and genome-wide multiple-sequence alignment (MSA) have been proposed to solve this problem in population genetics studies . While tArabidopsis thaliana, Cardamine hirsuta  genome sequences. First, by using the A. thaliana and D. melanogaster genomes, we develop concepts that underlie the algorithms of GEAN as a tool to project a reference genome annotation to a new genome sequence assembly of the same species and solves inconsistent alignment problems. We then consider complex crop genomes and inter-species genomes and try to transform the genome annotation of the inbred maize line B73 to Mo17 and A. thaliana to C. hirsuta. We also show that the lift over genome annotation can help perform variant calling for the de novo assembly genome sequence and genome-wide MSA. As far as we know, GEAN is the first implementation that has been designed to handle the inconsistent alignment problem for gene structure annotation lift over.Here, we provide a semi-global sequence alignment algorithm to refine genome annotation lift over by giving distinct weights to different genetic regions. We named this weighted-sequence alignment approach as zebraic dynamic programming (ZDP). ZDP is accelerated by extending the striped Smith–Waterman (SSW) algorithm ; we name hirsuta , DrosophAn INDEL/SNP ratio of ∼25% has been observed in plants, animals, and humans . At leasA. thaliana accessions  between the number of realigned transcripts and identical by state (IBS) index for both species , GEAN complemented the annotation lift over with orthologue-based annotation , the resn method , and tran method . As illun method  for eachA. thaliana accessions ratio, coef = 5)$count). Sequence diversity  of these genes was significantly higher than that of the genome-wide background . Gene ontology (GO) analysis was conducted using agriGO  and performed structural variant calling with Assemblytics , intergenic region] directly and only uses an overlapping sliding window to perform MSA only for elements longer than sliding window size, which is a configurable parameter.The ORF state has been defined by updating the previous rules  as folloConsidering the presence of INDELs, SVs, and sequence fragment rearrangements, the coordinates of certain orthologous haplotype sequence fragments are different for each individual. Lift over is a way of mapping reference coordinates from one genome assembly to another.de novo assembly sequence, considering the similar range entries as input 1298], Guizhou Provincial Education Department [QJH-KY-Z [2018]420], Qiannan Science and Technology Bureau [QNKHXKJSNX(2018)5], Research and Innovation Team of Qiannan Normal University for Nationalities , and Scientific Research Project of Qiannan Normal University for Nationalities  grant to FW.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.


Walled-eyed monocular internuclear ophthalmoplegia (WEMINO) syndrome is a sub-variant of internuclear ophthalmoplegia (INO) and involves the same clinical findings with the addition of exotropia of the ipsilateral eye. Causes typically include multiple sclerosis (MS) and ischemia (hemorrhagic or embolic) but can be secondary to blunt trauma as seen in our presented case.A 27-year-old man presented with new-onset visual changes, diplopia, and strabismus following a motor vehicle collision. Physical exam showed left ocular exotropia and slight hypertropia on forward gaze with deficiency of left convergence and disconjugate eye movements on horizontal gaze with right nystagmus on rightward gaze. Imaging showed hyperintensities in the right middle cerebellar peduncle and left temporal-occipital white matter likely consistent with diffuse axonal injury but otherwise nonspecific. The patient was treated conservatively with left eyepatch and exhibited improvement of exotropia and diplopia at 1 week follow up.Common causes of WEMINO syndrome include MS and ischemia with no prior reports, to our knowledge, being secondary to the blunt trauma seen in our case. Patients with WEMINO present with the typical signs of failure of ipsilateral adductive movement during lateral along with ipsilateral exotropia. Management involves treating the underlying disorder, if possible, with conservative measures with traumatic origins.Until now, WEMINO syndrome secondary to trauma has not been previously documented. Our patient was effectively treated with conservative measures alone. Additionally, the left abducting eye will exhibit nystagmus. Those with WEMINO exhibit a similar failure of monocular abduction with contralateral gaze but also have exotropia of the affected eye with primary gaze. Therefore, a patient with right WEMINO would be unable to adduct the right eye with left lateral gaze as well as having rightward deviation when instructed to focus frontward. WEBINO is the bilateral sibling of WEMINO and exhibits the same pathology except with involvement of both eyes . The basis of all INO types involve damage to the Medial Longitudinal Fasciculus (MLF), a white matter tract that connects the paramedian pontine reticular formation  to the medial rectus nuclei of the contralateral eye. This tract serves to connect the lateral gaze initiation of the paramedian pontine reticular formation (PPRF) via cranial nerve (CN) VI of one eye, with medial adduction of the contralateral eye via CN III to allow for synchronous eye movement towards a horizontal direction. MLF lesions of any etiology result in dissociation of medial rectus activation with lateral rectus movement and present with the clinical findings described above.Wall-eyed monocular internuclear ophthalmoplegia, called WEMINO syndrome and its bilateral homologue WEBINO syndrome are both sub-variants of internuclear ophthalmoplegia (INO). INO presents clinically as a failure of monocular adduction during attempted lateral gaze with simultaneous lateral nystagmus of the contralateral abducting eye. When instructed to look to their left, those with a Causes of WEMINO and WEBINO are the same as those of general INO and include stroke, multiple sclerosis, tumors, infection, hydrocephalus, metabolic disturbance, toxins, degenerative disease, inflammatory disease, and trauma. However, by far the most common include multiple sclerosis and cerebrovascular accident (CVA) or stroke . Here, w2A 27-year old male with no significant past medical history presented to emergency department (ED) of our Level 1 Trauma Center with a 1-day history of new-onset visual changes, diplopia, and strabismus following a motor vehicle collision. He was an unrestrained front seat passenger of a vehicle traveling approximately 40 mph when it was involved in a side impact on his side. While he reported a head strike from the initial impact, he did not lose consciousness. He was able to ambulate after the incident though he did experience unsteady gait and stumbling. He also stated that while visual blurriness was not initially present after the accident, onset began several hours afterwards and was associated with his left eye being deviated outwards and unable to move medially. Additional complaints at presentation to the ED included visual blurriness, vertical diplopia, left forehead pain, mild photophobia, strabismus, and improved but still present unsteady gate. He denied any significant headache or any focal numbness or weakness of his face or limbs at intake. His only past medical history was significant only for a concussion from playing football in the remote past.Physical exam revealed a GCS of 15 and was significant for bilateral pupil exam normal, but left ocular extropia and slight hypertropia on forward gaze with deficiency of left convergence and disconjugate eye movements on horizontal gaze with nystagmus. Right ocular movement was not disturbed in any direction, but horizontal nystagmus appeared on rightward gaze. No ptosis was present in either eye. Visual acuity was grossly normal; visual fields showed no defects bilaterally; and normal finger-to-nose test bilaterally. Laboratory values were all within normal limits.A non-contrast computed tomography (CT) brain showed possible small right frontal hemorrhagic contusion and small subarachnoid hemorrhage. CT of the facial bones was negative for cranio-facial fractures. Subsequent magnetic resonance imaging (MRI) of the Brain , Fig. 3 Our traumatic brain injury neurology team was consulted and concluded that given the patient's presentation, post-traumatic DAI as the etiology. A follow up non-contrast CT brain was the next day showed resolution of the punctate right frontal lobe contusion and the SAH. They also ordered a CT Angiogram of brain to rule out a vascular etiology and to shed light on his symptomatology. The results showed no hemodynamically significant stenosis, no aneurysmal dilatation or dissection of the anterior and posterior cerebral circulation. The anterior and posterior communicating arteries were unremarkable, the arterial Circle of Willis complete and the brain vessels intact and patent.The ophthalmology evaluation revealed normal visual acuity bilaterally, normal visual fields bilaterally, and normal intraocular pressure. Slit lamp and dilated pupillary examinations were unremarkable. After group discussions with neuro-radiology, ophthalmology, and trauma neurology the patient was diagnosed the patient with Wall-Eyed Monocular Internuclear Ophthalmoplegia (WEMINO), a rare subtype of Internuclear Ophthalmoplegia (INO). Our patient was treated conservatively, with short-term application of left eye patch and discharged home. On follow-up visit in the trauma clinic 1 week later, it was found that his left ocular exotropia was less pronounced, and he stated that his diplopia was improving as well. After 12 week follow up, the patient had complete recovery of eye function with correction of the exotropia and was back to work without issues.33.1INO is an ocular movement disorder classified as a pre-nuclear cerebral disorder and known to be associated with damage to the medial longitudinal fasciculus (MLF). The MLF pathway carries inter-nuclear neurons to connect nuclei of the brain stem, including the nucleus of cranial nerve (CN) VI, also known as abducens nerve, in the pons to the contralateral sub-nucleus of CN III, also known as the oculomotor nerve, in the midbrain that innervates the medial rectus muscle. See . UltimatHistorically, specialists have separated INO into anterior (front) and posterior (back) varieties based on location of the lesion and clinical sequelae, but MRI scans have shown that this classification is unreliable. It is classically characterized by presence of impaired adduction of the ipsilateral eye with nystagmus of the contralateral abducting eye. The pathogenesis leading to the extropia manifestations of WEMINO syndrome is unclear with multiple potential origins suggested in previous studies. According to a literature review conducted by Wu et al., either direct oculomotor nucleus or pontine lesions can explain the exotropia during primary gaze. The former being responsible for medial rectus instability with the latter potentially leading to “overstimulation” of the ipsilateral abducens as proposed by Komiyama et al. ,4. Howev3.2The most frequent causes of WEMINO and WEBINO include cerebrovascular accident (CVA)/ stroke and demyelinating disorders such as Multiple Sclerosis (MS), with infection, tumors, vasculitis, and iatrogenic injury from neurosurgical procedures being less common . As WEMI3.3Treatment of WEMINO/WEBINO should be dictated by the discovered/presumed underlying etiology. In patients with MS, high-dose intravenous steroids may help alleviate symptoms by accelerating the resolution of the causative plaque. Dalfampridine or teriflunomide may be utilized as disease modifying therapy to improve axonal function in MS in the case that interferon and Glatiramer are not available. Infectious causes should be dealt with targeted pharmacotherapy and metabolic or toxic causes managed accordingly. INO due to trauma or demyelination usually resolves spontaneously over 3–6 months while INO secondary to cerebral infarction or tumor however is typically permanent. As shown in our presented case, treatment of WEMINO and WEBINO due to trauma is typically conservative, and aimed at symptom control using Fresnel prisms lenses or ipsilateral eyepatch as well as treatment of the underlying cause, if possible. Those patients suffering from intolerable diplopia and persistent visual derangement may benefit from botulinum toxin injections of opposing extraocular muscles  to minimize exotropia symptoms. Surgical correction is typically last-line therapy to utilize in persisting diplopia due to permanent causes, as well as in patients that suffer from persistent deficits ,6.4To our knowledge, we report the first case of WEMINO syndrome secondary to a blunt trauma origin. Our patient initially presented with left ocular exotropia and slight hypertropia on forward gaze with deficiency of left convergence and disconjugate eye movements on horizontal gaze with right ocular nystagmus on rightward gaze. MRI imagining was correlative with diffuse axonal injury. Our patient was treated conservatively with a left eye patch and showed complete clinical resolution at 12-week follow up.None.This report was conducted in compliance with ethical standards. Informed written consent has been obtained and all identifying information is omitted.Informed written consent has been obtained and all identifying information is omitted.Shaikh Hai, Adel Elkbuli, Dessy Boneva, Kyle Kinslow, Mark McKenney– Conception of study, acquisition of data, analysis and interpretation of data.Shaikh Hai, Adel Elkbuli, Dessy Boneva, Kyle Kinslow - Drafting the article.Shaikh Hai, Dessy Boneva– Management of case.Shaikh Hai, Adel Elkbuli, Kyle Kinslow, Dessy Boneva, Mark McKenney – Critical revision of article and final approval of the version to be submitted.This is a case report study.Dessy Boneva.Mark McKenney.Shaikh Hai.Not commissioned, externally peer-reviewed.None.


The purpose was to review the most recent literature on neuroimaging in the Kleine-Levin syndrome (KLS). We aimed to investigate if frontotemporal and thalamic dysfunction are key KLS signatures, and if recent research indicates other brain networks of interest that elucidate KLS symptomatology and aetiology.In a comprehensive literature search, we found 12 original articles published 2013–2018. Most studies report deviations related to cerebral perfusion, glucose metabolism, or blood-oxygen-level-dependent responses in frontotemporal areas and/or the thalamus. Studies also report dysfunction in the temporoparietal junction and the oculomotor network that also were related to clinical parameters. We discuss these findings based on recent research on thalamocortical networks and brain stem white matter tracts.The hypothesis of frontotemporal and thalamic involvement in KLS was confirmed, and additional findings in the temporoparietal junction and the oculomotor system suggest a broader network involvement, which can be investigated by future high-resolution and multimodal imaging. The Kleine-Levin syndrome (KLS) or periodic idiopathic hypersomnia is a puzzling disorder when it comes to both symptomatology and aetiology, and consequently also treatment. The cardinal KLS symptom is recurrent hypersomnia, with sleep episodes that can last as long as 2 weeks and recur several times a year. During sleep episodes, the patients are also troubled with one or more cognitive (such as language or memory impairment), psychiatric , or behavioural dysfunctions  . BetweenOver the years, several functional neuroimaging approaches have been applied to elucidate KLS aetiology and possibly assist in the diagnosis . One freBox 1 Neuroimaging methods and what they measureThe aim of the current review is to report the most recent literature on neuroimaging in KLS. We specifically aimed to investigate if frontotemporal and thalamic dysfunction are key KLS signatures, and if recent research indicates other brain networks of interest that could elucidate KLS symptomatology and aetiology., reporting findings in idiopathic hypersomnia. We also excluded clinically related articles only reporting structural MRI or CT without pathological findings.In order to obtain an update of recent literature on neuroimaging in KLS, we searched for articles in Web of Science and PubMed using the keywords (Kleine-Levin syndrome or KLS) and (PET or SPECT or MRI or fMRI or MRS or DTI or neuroimaging) during the period 2013–2018. We excluded review articles, articles without neuroimaging data, and articles that were not specifically reporting KLS findings, e.g.In the literature search, we found 12 original articles on neuroimaging in KLS published 2013–2018, whereof five articles were case reports ••, 10–18A large SPECT study by Kas et al. included 41 asymptomatic KLS patients, whereof 11 also were scanned during symptomatic periods, and 15 healthy controls , 19, theThe above studies investigated perfusion, glucose metabolism, BOLD responses and functional connectivity on a group-level comparing asymptomatic KLS patients with healthy controls or KLS patients in symptomatic vs. asymptomatic episodes. Results show statistically significant group- or state-dependent differences in frontotemporal and also parietal regions. However, what does these results on group level say about the status of individual patients? Can we use these neuroimaging methods for clinical patient assessment and diagnosis? A SPECT study by Vigren et al. reported temporal or frontotemporal hypoperfusion in 48% of 24 KLS patients investigated during their asymptomatic period or after remission . This hiIn summary, three different neuroimaging methods show functional abnormalities in frontotemporal, and sometimes also in parietal regions, that are persistent during asymptomatic episodes suggesting involvement of primarily language, but also executive and salience networks, in KLS. This conclusion is supported by the reported deficiencies in verbal memory, both episodic and working memory , 13 and Other key findings in the KLS neuroimaging literature is thalamic dysfunction in the form of increased BOLD responses , 16, hypAn extended fMRI study in 18 asymptomatic KLS patients could reproduce findings of increased BOLD responses in the left thalamus during a verbal working memory task . When maA combined fMRI and magnetic resonance spectroscopy (MRS) study in 14 asymptomatic KLS patients and 15 healthy controls reported inverse correlation between BOLD responses and N-acetylaspartate (NAA) concentration in the thalamus . NAA is Functional connectivity between the thalamus and the executive and salience networks, respectively, were increased in KLS patients when they performed an effortful working memory task . During In summary, thalamic malfunction continues to be a primary hypothesis in KLS aetiology as functional neuroimaging show involvement of the thalamus in the asymptomatic state with worsened dysfunction during symptomatic episodes., hypersexuality, binge eating, pathological gambling, and compulsive shopping, reviewed in [Abnormal neuroimaging patterns in KLS are also observed in the striatum, especially in the caudate nucleus and the putamen , 11, 12.iewed in . Howeveriewed in –30. In aiewed in  which coiewed in , 32. Thuiewed in .out of body experiences [.A significant finding in the study by Kas et al.  was hypoeriences –39.One resting-state fMRI study including 12 asymptomatic KLS patients and 14 healthy controls reports that KLS patients have reduced functional connectivity between the dorsal pons and the frontal eye field in Brodmann area (BA) 8 ••. Kas eKLS neuroimaging literature evidence broad involvement of thalamocortical networks, without findings of structural modifications in clinical neuroradiology. The thalamus is a primary subcortical hub having a crucial modulatory role in facilitating cortical arousal, information transmission and consciousness . There iInterestingly, areas with increased BOLD responses or increased functional connectivity in KLS patients , 16 invo, locus coeruleus, raphe nucleus, and the pedunculopontine nuclei, and the mesencephalon-tegmental area with projections to the thalamus, hypothalamus and the basal forebrain  of white matter tracts is an obvious alternative to the functional neuroimaging methods reviewed here. Further, multimodal functional imaging is probably necessary to inquire into the seeming uncoupling of cerebral blood flow and metabolism in KLS, as well as to delineate vascular, metabolic and neural contributions to KLS pathology.


Macrophages are often viewed through the lens of their core functions, but recent transcriptomic studies reveal them to be largely distinct across tissue types. While these differences appear to be shaped by their local environment, the key signals that drive these transcriptional differences remain unclear. Since Wnt signaling plays established roles in cell fate decisions, and tissue patterning during development and tissue repair after injury, we consider evidence that Wnt signals both target and are affected by macrophage functions. We propose that the Wnt gradients present in developing and adult tissues effectively shape macrophage fates and phenotypes. We also highlight evidence that macrophages, through an ability to dispatch Wnt signals, may couple tissue debridement and matrix remodeling with stem cell activation and tissue repair. Careful mutational analyses of the beta-subunit of the GM-CSF receptor (GM-CSF-R) revealed molecular strategies that could enhance monocyte/macrophage differentiation at the expense of granulocyte differentiation  was found to be a gene target and negative feedback regulator of Toll receptor signaling . SpecifiListeria infection  contamination of recombinant Wnt preparations or overlapping physiochemical properties of LPS and acylated Wnt proteins remains unclear. Thus, the use of recombinant Wnts to interrogate inflammatory pathways should be considered judiciously and verified using forced expression, knock-down or targeted gene loss approaches.Another complication relevant to the study of inflammatory Wnt signaling in macrophages is that β-cat can also be robustly upregulated by the Th2-cytokine, IL4 . This inrophages . WhetherArg1, Sti1, IL10, and decreased expression of IL1b, respectively . Two rectively . Anal cells .While evidence for Wnt-expression in macrophages relevant to tissue development, repair, and cancer progression appears clear, it should be noted that the absolute abundance of Wnt expression by macrophages is substantially less than other cell types within the same tissues, as evidenced by various single-cell RNA sequencing data sets that have become available  and by sGrowing knowledge at the intersection of macrophage biology and Wnt signaling reveals multiple roles for Wnt signals coming from and within the monocyte-macrophage lineage. Wnt signals received by macrophages are important for their various phagocytic roles, such as modulating the immune response in the setting of infection, tissue repair after injury, malignancy detection, and progression. Conversely, macrophages can be a source of Wnt signals critical for tissue morphogenesis and healing after injury. A key future direction will be to understand how tissue-specific macrophage identities are shaped by their local environment. Since Wnts have been classically shown to act locally, typically only a few cell diameters away from the source of Wnt secretion  evidenceAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.