Hordeum vulgare L.) is one of the most important cereal crops worldwide. In the Qinghai-Tibet Plateau, six-rowed hulless (or naked) barley, called “qingke” in Chinese or “nas” in Tibetan, is produced mainly in Tibet. The complexity of the environment in the Qinghai-Tibet Plateau has provided unique opportunities for research on the breeding and adaptability of qingke barley. However, the genetic architecture of many important agronomic traits for qingke barley remains elusive. Heading date (HD), plant height (PH), and spike length (SL) are three prominent agronomic traits in barley. Here, we used genome-wide association (GWAS) mapping and GWAS with eigenvector decomposition (EigenGWAS) to detect quantitative trait loci (QTL) and selective signatures for HD, PH, and SL in a collection of 308 qingke barley accessions. The accessions were genotyped using a newly-developed, proprietary genotyping-by-sequencing (tGBS) technology, that yielded 14,970 high quality single nucleotide polymorphisms (SNPs). We found that the number of SNPs was higher in the varieties than in the landraces, which suggested that Tibetan varieties and varieties in the Tibetan area may have originated from different landraces in different areas. We have identified 62 QTLs associated with three important traits, and the observed phenotypic variation is well-explained by the identified QTLs. We mapped 114 known genes that include, but are not limited to, vernalization, and photoperiod genes. We found that 83.87% of the identified QTLs are located in the non-coding regulatory regions of annotated barley genes. Forty-eight of the QTLs are first reported here, 28 QTLs have pleotropic effects, and three QTL are located in the regions of the well-characterized genes HvVRN1, HvVRN3, and PpD-H2. EigenGWAS analysis revealed that multiple heading-date-related loci bear signatures of selection. Our results confirm that the barley panel used in this study is highly diverse, and showed a great promise for identifying the genetic basis of adaptive traits. This study should increase our understanding of complex traits in qingke barley, and should facilitate genome-assisted breeding for qingke barley improvement.Barley ( Hordeum vulgare L.) was domesticated in Israel and Jordan in the southern part of the Fertile Crescent approximately 10,000 years ago . Barley is mainly used for food, fodder, alcoholic beverage ingredient, and is generally considered to be a healthful food , which inhibits flowering  in Arabidopsis is similar to HvVRN3 in that it induces the expression of HvVRN1 during periods of long daylight, promoting flowering  gene family via homology-based cloning  and Photoperiod-H2 (Ppd-H2) genes on chromosomes 2H and 1H, respectively   approach  on chromosome 7H. Recently, EigenGWAS, which combines the statistical framework of GWAS with eigenvector decomposition, is a novel approach for identifying regions of the genome under selection in any genetic data where the underlying population structure is unknown. EigenGWAS has been applied to studies in evolution, ecology, breeding, and human genetics (https://github.com/gc5k/GEAR/wiki/EigenGWAS).As the growth range of barley increased, it adapted to a wide spectrum of agricultural conditions. Studying selection signals in the barley genome is important to help us understand how this genome reacted to the various agricultural conditions experienced during domestication  in 308 qingke barley accessions to (1) understand the genetic diversity in the landraces and the modern varieties and the changes in population structure that occurred going from the landraces to the breeding varieties, (2) identify genetic loci associated with HD, PH, and SL by GWAS, and (3) identify loci that underwent selection for environmental adaptation using EigenGWAS. The findings of this study could facilitate a better understanding of the genetic mechanisms underlying the establishment of adaptive traits and genome-assisted selection in qingke barley breeding.Z = 50; Hemming et al., A total of 308 qingke barley accessions were used in this study; 206 qingke landraces, 72 qingke varieties, and 30 varieties . All the 308 accessions were planted in Tibet at three locations; Lhasa  in April 2018, Namling  on May 2017, and Nyingchi  in October 2017 with three replicates each. We used a randomized design to construct the field experiment. At each location, 30 seeds of each accession were planted in a plot with two rows of 150 cm long and 30 cm between rows. HD was measured as the number of days when the head first emerged from the flag leaf sheath on the main shoot in a plot  of target traits were calculated by AOV functionality in QTL IciMapping v.4.1  with non-linear regression as implemented in the R package  across the barley genome was calculated with TASSEL v5.2. The population structure of the 308 accessions was evaluated using principle component analysis (PCA) and a phylogenetic tree. Pairwise distances were estimated between genotyped individuals using an unbiased model of substitution frequencies. Distance estimates were then used to construct a phylogenetic tree using the Neighbor-Joining-like algorithm described by Saitou and Nei (The 308 accessions were genotyped using a newly developed genotyping-by-sequencing technology (tGBS) that eases the process of sorting high-quality GBS sequencing libraries and results in more accurate SNP calling  = 4.18, was also calculated. To balance the false positives and false negatives, a –log10(P) threshold of 4.00 was used for the GLM and 3.00 was used for the MLM. To determine whether the uncovered genetic architecture was appropriate, the identified QTL was used to predict the performance of the corresponding trait. The most significant SNP in each QTL region was fitted in the linear model with the original trait performance as the dependent variable. The adjusted coefficient of determination (R2) from the linear model was then calculated. The performance of QTL in different locations were estimated by iae = ia - a, where a was the averaged effect of QTL across locations, ai was the additive effect of QTL for each location, e is the number of locations, and aei was the additive by environment effect of QTL in each location  and a mixed linear model (MLM) as implemented in TASSEL v5.2 software . Gene annotation information was acquired by BARLEX: The Barley Genome Explorer , was used to separate loci under selection by treating top 10 eigenvectors  as phenotypes. We adjusted the p-value using a genomic control factor, denoted as PGC, to exclude the effect of the genetic drift  threshold of 4.00 was applied for EigenGWAS analyses in all 10 eigenvectors.EigenGWAS is a regression approach based on principal component analysis  across trials, with the exception of the replicates in PH_NM and SL_NM  in the variety subpopulation was higher than in the landrace subpopulation, particularly on chromosomes 2H, 4H, 5H, and 7H  family protein. A QTL at 24,897,375 on chromosome 5H significantly associated with PH_NM was found to be located 799 bp downstream of the gene HORVU5Hr1G009980 that encodes a tetratricopeptide repeat (TPR)-like superfamily protein. For SL_NM and SL_NC, a stable QTL at 593,522,597 bp on chromosome 2H is located 554 bp downstream of HORVU2Hr1G081800, which encodes a WPP domain interacting protein 2  near the vernalization gene HvVRN3, and a significant SNP  near the photoperiod gene PpD-H2.The annotation conducted on the 62 significant QTLs identified by GWAS  showed trotein 2 . In addiR2= 69.20%), PH_NM (R2= 64.07%), and SL_NC (R2= 42.37%)  . For SL_ 42.37%) . Looking 42.37%)  suggestsTo the best of our knowledge, few genetic studies have investigated the complex agronomic traits in Tibetan qingke barley . As a result, 48 of 62 identified QTLs were first reported in this study , the SNPs near HvVRN1, HvVRN3, and PpD-H2 were significant in this study. It suggests that HvVRN1, HvVRN3, and Ppd-H2 play an important role in the qingke barley population, and should be prioritized when attempting to improve HD and plant growth in qingke barley cultivars in the qingke barley-growing regions of the Qinghai-Tibet Plateau. Previous studies have shown that QTLs for PH and SL are located on different chromosomes depending on the different environments and treatments  were found in the landrace subpopulation than in the variety subpopulation. That is to say, the number of SNPs was higher in the varieties than in the landraces, indicating that the varieties grown in Tibet and the varieties from around the Tibetan area may be derived from the different landraces grown in the different regions. A GWAS identified 62 QTLs that are associated with HD, PH, and SL, and 114 known genes were mapped which include, but are not limited to, genes involved in vernalization and photoperiod. Of the 62 QTLs, 48 are first reported here as qingke specific QTLs, 52 (83.87%) were found to be in intergenic regions, 28 had pleotropic effects, and three QTL were in the regions of the well-characterized genes https://www.ncbi.nlm.nih.gov/sra/PRJNA606408.The datasets generated for this study can be found in the This study was designed by HL and DD. The experiment was performed under the support of GG, JZ, and TN. The evaluation of traits was conducted by NL, KD, PC, LG, and WC. Data were analyzed by ZL, NL, and HL. The manuscript was drafted by ZL, NL, and HL, and revised by GG and JW. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In early 2020, over 80,000 cases of coronavirus disease (COVID-19) were confirmed in China. Public prevention and control measures, along with efforts from all sectors of society, were undertaken to control and eliminate disease transmission.This paper describes Chinese citizens’ response to the epidemic, the preventive measures they implemented to avoid being infected, and the public strategies that were carried out by the government, health workers, etc. We also discuss the efficacy of these measures in controlling the epidemic in China.Information on the responses and behaviors of Chinese citizens were collected through a cross-sectional, internet-based survey using Dingxiang Doctor’s public account on WeChat. Information on public strategies implemented by all sectors of society to control the epidemic and data on new COVID-19 cases were collected from the internet, mainly from government websites. Standard descriptive statistics and multivariate logistic regression analyses were conducted to analyze the data.P<.001). With a series of strict public control measures, like nationwide health education campaigns, holiday extensions, the Examine and Approve Policy on the resumption of work, close management of working and living quarters, a health QR (Quick Response) code system, community screening, and social distancing policies, the number of new cases have decreased dramatically since February 12, 2020.A total of 10,304 participants responded to the survey, with 10,198 valid responses; 74.1% (n=7557) were female and 25.9% (n=2641) were male. Overall, 98.2%  of participants paid high or very high attention to the epidemic, with WeChat being their main information source . Over half the participants  were confident that the epidemic could be curbed in China; 92.4% (n=9427) opened windows for ventilation more frequently than usual; 97.9% (n=9986) used masks in public; 95.7% (n=9759) avoided large crowds and stayed at home as much as possible; and 97.9% (n=9988) washed their hands more often than usual. Women were more likely to practice these behaviors than men (The methods employed by Chinese citizens and authorities have effectively curtailed the spread of COVID-19, demonstrating that this pandemic can be brought under control as long as the right measures are taken. In December 2019, an outbreak of pneumonia associated with the novel coronavirus disease (COVID-19) was reported in Wuhan, Hubei Province, China . In justCOVID-19 is a highly contagious disease. It mainly spreads from person to person through respiratory droplets, similar to the common cold and influenza viruses . It can also be transmitted through contact with the secretions of infected individuals. The role of fecal–oral transmission is yet to be determined for COVID-19, but it was found to occur during the severe acute respiratory syndrome (SARS) outbreak . COVID-1Under such a situation, it is important to provide the public with adequate information on risks and precautions, like the proper use of masks, frequent handwashing, and the avoidance of large gatherings, to control the outbreak. From January 20, 2020, when person-to-person transmission was confirmed and made public in China by Dr Zhong Nanshan , a renowDuring the early part of the outbreak, genetic analyses conducted in China revealed that the virus was similar to, but distinct from, severe acute respiratory syndrome coronavirus (SARS-CoV). China’s emergency management of SARS was heavily criticized in 2003 ,13, and This paper aimed to describe Chinese citizens’ responses to the epidemic, the preventive measures they implemented to avoid being infected, and public strategies carried out by the government, health workers, etc. We also discuss the efficacy of these measures in controlling the epidemic in China.Data collection for our study comprised two parts. First, information on personal responses and preventive measures to avoid being infected with COVID-19 were collected through a cross-sectional, internet-based survey. The questionnaire collected sociodemographic data ; sources used to obtain information on COVID-19; the level of attention paid to the epidemic; respondents’ confidence in the curbing of the outbreak in China; places that participants had visited during the COVID-19 epidemic period prior to the survey; and preventive measures taken to avoid infection. A 5-point Likert scale  was used to evaluate the participants’ confidence in curbing the outbreak by China and their level of attention to the COVID-19 epidemic. The questions about preventive measures were as follows:What did you do to protect your families and friends?Did you open windows for ventilation more frequently than usual?Did you wear a mask in public?Did you avoid large crowds and stay at home as much as possible?Did you wash your hands more often than usual?Under the following circumstance, do you wash your hands after touching public goods, toilet use, returning home, coughing/sneezing, or before eating?Do you wash your hands with soap and running water in most cases?Do you cover your coughs/sneezes with tissue or your bent elbow in most cases?Questions about COVID-19 were based on the latest official report from the WHO, the Chinese Center for Disease Control and Prevention, and scientific literature. The original questionnaire was developed in Chinese by research group members from the Zhejiang Provincial Center for Disease Control and Prevention and refined over two rounds of Delphi method collaboration. A pilot study was conducted to verify its reliability.Second, information on public strategies carried out by government, health workers, companies, etc, to protect citizens from being infected were sourced from official government websites, national and local health commission websites, national and local centers for disease control and prevention websites, and official papers -26. DataParticipants were recruited via Dingxiang Doctor, a WeChat public account with 35 million users in China, used to disseminate health knowledge to the general population. A message stating “COVID-19, have you done enough to prevent it?” was created on the site, with a link to the questionnaire. Users who viewed the message could share it through the internet, allowing for news and information to spread quickly to many people. The study period was from January 31 to February 2, 2020.The study was approved by the Ethics Committee at Zhejiang Provincial Center for Disease Control and Prevention. Informed consent was obtained from all participants before their information was collected.P values <.05 were considered statistically significant (two-sided). Epidemic trends were depicted visually and main public strategies used in China in response to COVID-19 were chronologically described.Data were exported from Dingxiang Doctor to Microsoft Excel and analyzed using SPSS, version 19.0 (IBM Corporation). Standard descriptive statistics were used to summarize the data. Multivariate logistic regression analyses were conducted to explore differences in the practice of preventive behaviors between men and women. A total of 58,000 Dingxiang Doctor users visited the study page, 17.8%  of whom completed the questionnaire; 106 participants outside of China were excluded and 10,198 responses were included in the study. Among the participants, 74.1% (n=7557) were female and 25.9% (n=2641) were male; 47.4 (n=4770) were single, divorced, or widowed; 52.6% (n=5364) were married. The proportion of participants in the age groups <30 years, 30-49 years, and ≥50 years were 55.4% (n=5653), 39.8% (n=4059), and 4.8% (n=486), respectively. Most participants had an undergraduate degree , while participants with primary or lower, secondary, and postgraduate education comprised 5.1% (n=524), 10.8% (n=1105), and 13.6% (n=1390) of the sample, respectively. A total of 40.1% (n=4089) participants worked in the business and service industry, 17.7% (n=1809) worked in government institutions, 18.1% (n=1844) were students, 6.5% (n=666) were health workers, 7.3% (n=745) were housewives or househusbands, and 10.3% (n=1045) indicated “other” occupational statuses .Using a 5-point Likert scale, most participants indicated that they paid high attention to the epidemic. As seen in Most participants followed the recommendations of the public health authority. P<.05).Gender differences existed in the practice of all preventive behaviors, with women being more compliant with all hygiene measures than men. Except for differences in “washing hands after coughing/sneezing or before eating” and “told friends and family to avoid large gatherings,” all differences were statistically significant  participants were strongly confident, and 28.1% (n=2864) were confident. A total of 30.1% (n=3073) participants were neutral, 10.0% (n=1022) lacked confidence, and 2.2% (n=225) strongly lacked confidence .Since January 20, 2020, a series of strategies were implemented to curb the spread of the virus to the wider community. This is the first large-scale, nationwide study to report on the comprehensive preventive measures taken by Chinese citizens and strategies implemented by the government, health workers, factories, etc, to combat COVID-19 at a national level. We found that Chinese citizens responded quite well to the COVID-19 epidemic, by strictly following the recommendations of health authorities. The frequency of taking preventive measures by participants was high, with 92.4% of participants opening windows for ventilation more often than usual, 99.4% of participants using masks in public, 95.7% of participants avoiding large crowds and staying at home as much as possible, and 97.9% of participants washing hands more often than usual. However, responses varied among participants, and women showed higher compliance with all hygiene measures than men—a phenomenon that was also found in other studies -30. FurtCompared with measures taken to prevent the transmission of SARS in 2003 , we founWeChat, the largest standalone social media mobile app in China, was found to be one of the main sources of information on the COVID-19 epidemic for over 90% participants. In China, over 95% of adults own a mobile phone and over 1 billion access WeChat at least once a day. The app has become an integral part of Chinese daily life, with citizens using it to send messages, share updates, and access the latest news from the government, celebrities, enterprises, and so on . Our stuOur findings showed that over 80% participants had tried to influence their families and friends by telling them to avoid large gatherings and stay at home as much as possible, sharing epidemic information with them, and telling them to use masks when necessary. This finding supports previous findings that participants who lived with their families were more likely to use a higher number of precautionary measures than those who did not live with their families during the SARS outbreak in 2003 .Innovative measures combined with traditional strategies have facilitated China’s response to COVID-19. Traditional strategies, including active contact tracing, isolation and quarantine, a series of social distancing policies, and community containment were reinforced at a national level. China’s emergency management of SARS was heavily criticized in 2003 ,13; howeThis study has some limitations. First, selection bias might exist. Intrinsic limits, such as partial coverage of the population and missing responses from interviewees, are also present in our study, like other internet-based surveys ,35. PartOur study provided a broad description of preventive measures taken by Chinese citizens to avoid being infected with the novel coronavirus and the main strategies implemented by the government, health workers, factories, companies, and media to protect citizens. Chinese citizens followed the hygiene recommendations of health authorities very well; however, further education on practicing respiratory hygiene is still needed in China. In addition to traditional response strategies, an innovative health QR code system based on big data and mobile internet technologies has helped to prevent the spread of COVID-19 and facilitate the resumption of work and production. Online education has enhanced the capacity of health workers on the prevention, diagnosis, and treatment of COVID-19 in a safe and efficient way. With daily decreases in the number of new cases, we conclude that Chinese measures to curb the spread of COVID-19 has been effective. Our findings suggest that an epidemic of COVID-19 can be brought under control as long as the right measures are taken.


Older people are considered the most vulnerable population in the current COVID-19 (coronavirus disease-2019) pandemic. While the negative impacts on health in the present are important, negative impacts on future health should also be considered. During the COVID-19 pandemic, older people have had more restrictions placed on them. This includes restrictions on going out, meeting other people, and participating in activities, all of which may negatively affect their social relationships.The Japan Gerontological Evaluation Study (JAGES) has collaborated with more than 40 municipalities throughout Japan and has promoted evidence-based, gerontological research, targeting approximately 300 000 adults aged 65 years and older. To mitigate post-COVID-19 negative health impacts among older people, this article presents the results of JAGES, and discusses the importance of social relationships on the health of older people.1 older people who were in contact with others “from once a month to less than once a week” demonstrated a 1.40 times increase in the risk of functional disability, a 1.39 times increase in the risk of dementia, and a 1.15 times increase in the risk of premature death, when compared with individuals who reported they were in contact with others “frequently, every day (more than 9 per week).” Moreover, older people who were in contact with others “less than once a month” demonstrated a 1.37 times increase in the risk of functional disability, a 1.45 times increase in the risk of dementia, and a 1.34 times increase in the risk of premature death, when compared with individuals who were in contact with others “frequently, every day.” Similar results were also seen in a study by Aida et al.2 Therefore, to reduce the negative impact of COVID-19 on health among older people, contact with others at least once a week should be needed.First, since the COVID-19 pandemic may increase social isolation among older people, opportunities for contact with others should be maintained, with special attention paid to frequency of contact . Accordi3 compared with older males who ate with others, older males who ate alone and lived alone were 2.36 times more likely to demonstrate the onset of depression, while those who ate alone, but lived with others were 1.03 times more likely to develop depression. Similarly, compared with older females who ate with others, those who lived alone and ate alone were 1.31 times more likely to develop depression, while those who ate alone, but lived with others demonstrated a 1.21 times higher risk for the onset of depression.3 These results indicate that eating alone may increase the risk of depression, especially when combined with living alone, in older males. During the COVID-19 pandemic, older people living alone should receive special consideration, as they may face difficulties in going to restaurants or having lunches/dinners with others. Thus, the creation of opportunities to eat and communicate with others through virtual lunches and dinners should be considered.Second, while eating a healthy, balanced diet may be challenging during the COVID-19 pandemic, not only nutrition but eating status among older people should also be considered. According to Tani et al,5 and cognitive impairment,6 increased instrumental activities of daily living scores,7 decreased incidence of functional disability,811 and death.11 For example, Kanamori et al,10 found that older people who participated in one group demonstrated a 0.83 times lower incidence of disability, while those who participated in two groups demonstrated a 0.72 times, and those who participated in three or more different types of groups demonstrated a 0.57 times lower risk of disability than those who did not participate in any group. Similarly, Ide et al8 reported that the number of groups an older adult participated in was associated with lowered risk of functional decline in both rural and urban areas (0.76-0.92 times lower risks than those who did not participate in any group). Furthermore, compared with the active participant group , the sedentary group  exhibited a 1.65 times increase in risk, while the exercise-alone group  demonstrated a 1.29 times increased risk for incidence of functional disability.9 In other words, while regular exercise may reduce the incidence of functional disability, participation in a sports organization increases the preventive effects. Taken together, the above research suggests that participation in social activities is beneficial for health, and that if older people lose such opportunities for extended periods of time, their health may be negatively affected. Thus, the maintenance of social participation among older people during the pandemic is a critical health issue.Third, while social gatherings increase the risk of spreading the coronavirus, the benefits of social participation should also be taken into account. Using data from JAGES, various studies have presented the relationships between increased social participation and health, such as a decreased risk of dementia,The COVID-19 pandemic has restricted people’s lives, and its impact on health may be prolonged. This article has discussed the impact of social relationships on health among older people, based on cohort studies of JAGES, from a “super-aging” society. To ameliorate negative health impacts among older people post-COVID-19, these individuals should be encouraged to contact others, eat with others, and maintain organized social participation by practicing physical distancing. During mandated isolation, a minimum of weekly contact with others is recommended. Even non-face-to-face methods of contact could be beneficial, such as telephone, text, and/or video chat. Additionally, while eating with others and maintaining social participation in organized groups in-person may be difficult during a pandemic, participation could still occur via the internet. However, since internet use may be challenging for some older people, the construction of technological support networks may be needed. Avoiding pandemics such as COVID-19 may be impossible, but social relationships may help minimize the associated health risks in older individuals.


Prediction of patients at risk for mortality can help triage patients and assist in resource allocation.Develop and evaluate a machine learning-based algorithm which accurately predicts mortality in COVID-19, pneumonia, and mechanically ventilated patients.Retrospective study of 53,001 total ICU patients, including 9166 patients with pneumonia and 25,895 mechanically ventilated patients, performed on the MIMIC dataset. An additional retrospective analysis was performed on a community hospital dataset containing 114 patients positive for SARS-COV-2 by PCR test. The outcome of interest was in-hospital patient mortality.When trained and tested on the MIMIC dataset, the XGBoost predictor obtained area under the receiver operating characteristic (AUROC) values of 0.82, 0.81, 0.77, and 0.75 for mortality prediction on mechanically ventilated patients at 12-, 24-, 48-, and 72- hour windows, respectively, and AUROCs of 0.87, 0.78, 0.77, and 0.734 for mortality prediction on pneumonia patients at 12-, 24-, 48-, and 72- hour windows, respectively. The predictor outperformed the qSOFA, MEWS and CURB-65 risk scores at all prediction windows. When tested on the community hospital dataset, the predictor obtained AUROCs of 0.91, 0.90, 0.86, and 0.87 for mortality prediction on COVID-19 patients at 12-, 24-, 48-, and 72- hour windows, respectively, outperforming the qSOFA, MEWS and CURB-65 risk scores at all prediction windows.This machine learning-based algorithm is a useful predictive tool for anticipating patient mortality at clinically useful timepoints, and is capable of accurate mortality prediction for mechanically ventilated patients as well as those diagnosed with pneumonia and COVID-19. •Mortality predictions have not previously been evaluated for COVID-19 patients.•Machine learning may be a useful predictive tool for anticipating patient mortality.•Prediction can be estimated at clinically useful windows up to 72 h in advance. The out2Of particular interest during the COVID-19 pandemic is mortality prediction of COVID-19 patients, as well as those who have developed respiratory complications such as pneumonia and conditions requiring mechanical ventilation. Some prior studies predicting mortality in the mechanically vented subpopulation have used a logistic regression model. When applied on day 21  or 14 2,27 of me33.1Patient records were collected from the Medical Information Mart for Intensive Care (MIMIC) dataset, an openly available dataset developed by the MIT Lab for Computational Physiology, comprising de-identified health data associated with ~60,000 intensive care unit admissions . It inclPatient records of COVID-19 polymerase chain reaction (PCR) positive patients were collected from a community hospital and formatted in the same manner as the MIMIC dataset. A total of 114 patient encounters were collected between 12 March and 12 April 2020. Data collection was passive with no impact on patient safety. Dascena establishes de-identification by removing all protected health information (PHI) identifiers and by jittering all timestamps (including date of birth (DOB)) randomly either forwards or backwards in time. Studies performed on de-identified patient data constitute non-human subjects research, and thus this study has been determined by the Pearl Institutional Review Board to be Exempt according to FDA 21 CFR 56.104 and 45CFR46.104(b) (4): (4) Secondary Research Uses of Data or Specimens under study number 20-DASC-119.3.1.1For the MIMIC and community hospital datasets, we included only records for patients aged 18 years or older. We excluded patient records for which there were no raw data or no discharge or death dates. We then filtered for length of stay (LOS) for the different look aheads of 12, 24, 48, and 72 h. Data were discretized into 1 h intervals, beginning at the time of the first recorded patient measurement and hourly measurements were required for each input variable. Measurements were averaged to produce a single value in cases when multiple observations of the same patient measurement were taken within a given hour. This ensures that the measurement rate was the same across patients and across time. Missing values were imputed by carrying forward the most recent past measurement in cases where no measurement of a clinical variable was available for a given hour. For some patients with infrequent measurements of one or more vital signs, this simple imputation resulted in many consecutive hours with identical values.Our publication on the use of gradient boosted trees for sepsis detection and prediction describes the data processing in detail . Predict3.2The outcome of interest was in-hospital patient mortality, determined retrospectively for each patient. In the MIMIC dataset, we used the expire_flag field to identify the last stays of those patients. Similarly, the community hospital dataset contains a deceased flag that is either true or false to determine mortality.3.2.1The classifier was created using the XGBoost method for fitting “boosted” decision trees. We applied the XGBoost package for Python32 to the patient age and vital sign measurements and their temporal changes, where temporal changes included hourly differences between each measurement beginning 3 h before prediction time. Gradient boosting, which XGBoost implements, is an ensemble learning technique that combines results from multiple decision trees to create prediction scores. Each tree splits the patient population into smaller and smaller groups, successively. Each branch splits the patients who enter it into two groups, based on whether their value of some covariate is above or below some threshold—for instance, a branch might divide patients according to whether their temperature is above or below 100 °F. After some number of branches, the tree ends in a set of “leaves.” Each patient is in exactly one leaf, according to the values of his or her measurements. Each “leaf” of the tree is predicted to have the same risk of mortality. The covariate involved in each split and the threshold value are selected by an algorithm designed to trade off fit to the training data and accuracy on out-of-sample data by using cross-validation to avoid “over-fitting.” We restricted tree depth to a maximum of six branching levels, set the learning rate parameter of XGBoost to 0.1, and restricted the tree ensembles to 1000 trees to limit the computational burden.Hyperparameter optimization was performed using cross-validated grid search. We included a hyperparameter for the early stopping of the iterative tree-addition procedure to prevent overfit of the model on the training data and optimized across this hyperparameter using fivefold cross-validation. Due to computational and time constraints, hyperparameter optimization was performed across a sparse parameter grid, where the candidate hyperparameter values were chosen to span large ranges of viable parameter space. Cross-validated grid search was conducted to determine the optimal combination of candidate hyperparameters. While XGBoost has a large number of trainable parameters, computational and time constraints limited the set of parameters to be tuned to just those parameters with the largest impact on performance on the training data and most relevant to the prediction task.To validate the boosted tree predictor when training and testing was performed on data from the same institution, we used fivefold cross-validation. For each model, four-fifths of the patients were randomly selected to train the model and the remaining one-fifth were used as a hold-out set to test the predictions. To account for the random selection of the training set, reported performance metrics are the average performance of the five separately trained models arising from fivefold cross-validation, each of which was trained on four-fifths of the data and tested on the remaining fifth. For AUROC, we also reported the standard deviation of the five AUROC values obtained from cross-validation.For patients who died, we modeled mortality 12, 24, 48, and 72 h before death to evaluate the performance with a variety of lead times. For mechanically ventilated encounters, the time point was the start of ventilation for positive and negative class. Predictors were trained independently for each distinct lookahead time. In 12, 24, 48 and 72 h long lookahead predictions following a 3-h window of measurements, patients must have data for, respectively, 15, 27, 51 or 75 respective hours preceding the time of in-hospital mortality or the time of discharge. Accordingly, we selected patients with the appropriate stays for the training and testing of each lookahead.3.3To calculate the AUROC for rule-based predictors of mortality, we calculated quick Sepsis Related Organ Failure Assessment (qSOFA), Modified Early Warning Score (MEWS) and CURB-65 scores for patients in the MIMIC database. qSOFA has also been used to predict poor outcomes in pneumonia patients, including the need for mechanical ventilation, and has been shown to either match or outperform other outcome predictors such as SOFA, CRB, CRB-65 and the pneumonia severity index (PSI) ,31. Amon4XGBoost model training and testing was performed on the MIMIC dataset. Patient demographic information for all ICU encounters as well as each subpopulation are presented in The XGBoost ML algorithm predicted mortality in all ICU patients as well as mechanically ventilated and pneumonia patients more accurately than qSOFA, MEWS and CURB-65 at all prediction windows  . When trDetailed performance metrics for the XGBoost predictor on pneumonia and mechanically ventilated patients are presented in These results suggest that the XGBoost predictor is capable of predicting mortality in pneumonia, mechanically ventilated, and COVID-19 patients and outperforms the qSOFA, MEWS and CURB-65 mortality risk scores.5Accurate mortality prediction can assist with the allocation of limited hospital resources and optimize patient management. Additionally, advanced mortality prediction can facilitate decision making with family and caregivers. The commonly used MEWS , the APAStudies of mortality prediction on pneumonia and mechanically ventilated patients are particularly relevant for COVID-19 related lung complications. We have demonstrated that machine learning algorithms are useful predictive tools for anticipating patient mortality at clinically useful windows of 12, 24, 48, and 72 h in advance and have validated mortality prediction accuracy for COVID-19, pneumonia, mechanically ventilated, and all ICU patients , demonstA meta-analysis of studies focusing on predicting mortality in pneumonia patients showed that of the three commonly used prognostic scores which predicted mortality, the Pneumonia Severity Index (PSI) had the highest AUROC of 0.81. However, this index was used for predicting 30-day mortality specifically among patients with community acquired pneumonia . When trWhen trained and tested on the community hospital dataset, the XGBoost predictor obtained AUROCs of 0.91, 0.90, 0.86, and 0.87 for mortality prediction on COVID-19 PCR positive patients at 12-, 24-, 48-, and 72- hour windows, respectively . The algThere are several limitations to our study. The ML algorithm developed on the MIMIC dataset used only data from the ICU. Therefore, further research is required to evaluate performance of the algorithm in other patient care settings. Further, because the algorithm only utilized laboratory data and vital signs as inputs, it did not account for actions undertaken by the care team. These actions could signify aggressive treatment or withdrawal of treatment and could cause changes to algorithm inputs, potentially leading to variations in the algorithm's prediction score. On one hand, incorporating care team actions into algorithm inputs could be useful feedback to the care team in the sense that it may aid them in determining whether a given intervention was harmful or beneficial. On the other hand, accounting for actions undertaken by the care team may complicate the interpretation of what it means to “anticipate” mortality, given that the current state of knowledge of the care team is unknown. Finally, because this is a retrospective study, we cannot determine the performance of the mortality prediction algorithm in a prospective clinical setting. Prospective validation is required to determine how clinicians may respond to risk predictions as well as whether predictions can affect patient outcomes or resource allocation.6The ML algorithm presented in this study is a useful predictive tool for anticipating patient mortality at clinically useful windows up to 72 h in advance, and capable of accurate mortality prediction for COVID-19, pneumonia, and mechanically ventilated patients.Patients and the public were not involved in the design and conduct of the study, choice of outcome measures, or recruitment to the study due to the nature of data collection.Transparency declaration: RD affirms that the manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as originally planned  have been explained.N/A.Data has been deidentified and, as such, does not constitute human subjects research.All authors who have affiliations listed with Dascena  are employees or contractors of Dascena.ClinicalTrials.gov under study number NCT04358510.This study has been registered on Not commissioned, externally peer reviewed.The Guarantor is the one or more people who accept full responsibility for the work and/or the conduct of the study, had access to the data, and controlled the decision to publishLogan Ryan: Methodology, Investigation. Carson Lam: Conceptualization, Methodology, Formal analysis, Writing - original draft, Supervision. Samson Mataraso: Conceptualization, Methodology, Formal analysis, Writing - original draft, Supervision. Angier Allen: Investigation, Writing - original draft. Abigail Green-Saxena: Writing - original draft. Emily Pellegrini: Writing - original draft. Jana Hoffman: Conceptualization, Methodology, Formal analysis, Writing--Original Draft, Supervision. Christopher Barton: Supervision. Ritankar Das: had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis, Conceptualization, Methodology, Formal analysis, Writing - original draft, Supervision.


Lung adenocarcinoma is the most common histology in non-small cell lung cancer that has a large variety of histological and also pathological presentations. However, mediastinal lymph node involvement remains one of the most important prognosticators in these patients, and treatment can vary from upfront surgery to induction therapy or definitive radio-chemotherapy. One of the most intriguing issues regards the identification of the most appropriate treatment for a patient and one of the parameters that may indicate upfront surgical resection is the presence of single mediastinal station involvement. Moreover, another interesting argument regards the adjuvant therapy indication for these patients. For these reasons, we investigated the survival outcome of patients who underwent surgical resection for lung adenocarcinoma and single mediastinal station involvement, with the aim to investigate the prognostic factors in this class of patients.p = 0.003), and skip metastases (p = 0.005) were related to overall survival (OS). Multivariable analysis confirmed, as independent prognostic factors, age <66 years and NR with a three-year OS (3YOS) of 78.7% in NR < 10% vs. 46.6% in NR > 10%. In skip metastases, NR  and pT stage  were confirmed as independent prognostic factors. AD did not influence the OS of patients with singular positive lymph nodes (p = 0.41), while in patients with multiple lymph nodes and AD, a significantly better 3YOS was demonstrated, i.e., 49.1% vs. 30% (p = 0.004). In patients with N2 + N1 involvement, age (p = 0.002) and AD (p = 0.022) were favorable prognostic factors. Conclusions: Adenocarcinoma patients with single N2 station involvement had a favorable outcome in the case of skip metastases and low NR. Adjuvant therapy improves survival with multiple nodal involvement, while its role in single node involvement should be clarified.Background: Prognostic factors in patients with single mediastinal station (sN2) involvement continues to be a debated issue. Methods: Data on 213 adenocarcinoma patients with sN2 involvement and who had undergone complete anatomical lung resection and lymphadenectomy, were retrospectively reviewed. Clinical and pathological characteristics together with adjuvant therapy (AD) and node (N) status classifications (number of resected nodes (#RN), number of metastatic nodes (#MN), and node ratio (#MN/#RN = NR) were analyzed. Results: Univariable analysis confirmed that age (0.009), #MN (0.009), NR (0.003), #N1 involved stations ( The optimal therapeutic approach in patients with mediastinal lymph node involvement in non-small cell lung cancer is a very debated issue, with different possible strategies varying from upfront surgery to definitive radio- and chemotherapy to induction therapy plus surgery with different schedules ,2.For this reason, multidisciplinary assessment and identification of favorable prognostic factors is fundamental to the identification of the best therapeutic approach. For a better stratification of the node (N) parameter, the 8th edition of the tumor node metastases staging system (TNM) has proposed a sub-classification substantially based on the number of involved stations , with thIndeed, one of the clinical factors that could be an indicator for surgery is the presence of single N2 station involvement, i.e., a patient group presenting a quite good prognosis as compared with multiple N2 stations ,2,4. HowMoreover, considering other studies, the prognosis of patients with skip metastases remains controversial, and despite the presence of a slight survival advantage as compared with other types of N2 involvement, there is still no definitive conclusion on this issue ,5.Starting from this evidence, different studies have analyzed the validity of the proposal, and some studies have confirmed overlapping curves ,7, and pIndeed, in skip metastases adenocarcinoma, the prognosis seems to be better as compared with multiple hilar stations or single N2 station + hilar involvement, while in squamous cell carcinoma, the survival rate is poor in skip metastases as compared with multiple hilar stations involvement . SimilarIn consideration of these results, histology and the type of nodal involvement seem to be important factors in patients with single N2 station involvement, and their categorization may be useful for a better prognosis stratification.The aims of this study are to analyze the survival outcome, the prognostic role of lymph node involvement type, and the prognostic role of the adjuvant therapy in patients affected by lung adenocarcinoma with single N2 station involvement, who have undergone surgical treatment.Data on 4863 patients affected by NSCLC, who underwent surgical treatment from January 2002 to December 2014 among 7 institutions were collected and retrospectively analyzed. Patients without N2 involvement, N3 or distant metastases, incomplete resections, or those who had undergone neoadjuvant therapy as well as patients with non-adenocarcinoma histology were excluded from the analysis .Finally, the analysis was conducted on 213 adenocarcinoma patients with pathological single-station N2 involvement. According to the 8th TNM proposal, patients without N1 involvement were categorized as N2a1, while patients with single N2 + N1 involvement were categorized as N2a2. Although different centers were involved in this multicenter collaboration, the same policies in terms of preoperative workout were applied, including a whole-body computed tomography with contrast, 18-fluorodeoxyglucose positron emission tomography—CT (18F-FDG PET-CT) (when adopted and available in the involved center), and brain magnetic resonance in the case of suspected brain metastases. Lymph nodes larger than 1 cm on computed tomography or showing increased FDG uptake independently of the size were defined as metastatic nodes. As necessary, if pre-operative radiological evaluation was not exhaustive, in the case of N1 involvement or central tumors >3 cm, endobronchial ultrasound-guided transbronchial needle aspiration cytology or mediastinoscopy were performed for clinical staging.Surgery was performed by certified thoracic surgeons and consisted of major anatomical lung resection  associated with lymphadenectomy  according to the IASLC lymph node map and ESTS guidelines ,10. In pSampling: The removal of one or more lymph nodes guided by preoperative or intraoperative findings which are thought to be representative,Radical nodal dissection: All the mediastinal tissue containing the lymph nodes is dissected and removed systematically within anatomical landmarks.Lymphadenectomy was limited in the case of hemodynamic instability or in the case of sticky lymph nodes non dissociable from the mediastinal structures as infective disease results .In the case of lymph node fragmentation, lymph node parts were added to the corresponding node station for the histological analysis and counted as single node fragments. Pathological reports were reviewed and classified according to the 8th TNM edition .Oncologists and radiotherapists indicated and administered adjuvant therapy (AD) in accordance with pathological stage, patients’ clinical conditions and clinical guidelines were validated during the study period . In geneAlthough some inter-institutional variation could have been present, follow-up was comprised of clinical visits, blood tests, and radiological examinations (computed tomography and positron emission tomography when available and indicated) every 3 to 6 months after surgery for the first 2 years postoperatively, and then every 6 months for 5 years.Clinical and pathological characteristics  were analyzed together with adjuvant therapy and different N status classifications (number of resected nodes (#RN), number of metastatic nodes [#MN, node ratio (#MN/#RN), number of resected stations, number of positive hilar stations, and presence of skip metastases).Descriptive analyses, including clinical and demographic characteristics of the patients, were analyzed through the median and the range for continuous variables and the absolute value and relative frequencies for categorical variables.p ≤ 0.05 level. The hazard ratio (HR) and the 95% confidence interval (95% CI) were estimated using the Cox univariate model. A multivariate Cox proportional hazard model was developed using stepwise regression (forward selection) to compare the prognostic power of different factors. The enter limit and removal limit were p = 0.10 and p = 0.15, respectively. The assessment of interactions between significant investigation variables was taken into account when developing the multivariate model. Statistical evaluations were performed using SPSS Survival curves were calculated by the Kaplan–Meier product-limit method from the date of surgery until relapse or death. The log-rank test was used to assess differences between subgroups. Significance was defined at the The final analysis was conducted on 213 patients, who met the inclusion criteria. Clinical and pathological characteristics are reported in Median follow-up was 33 (range 1–152) months. Median three-year disease-free survival (3Y-DFS) and OS (3YOS) results were 35 (CI 95% 27–42) and 46 (CI 95% 36–56) months, respectively. Skip metastases were present in 89 patients and adjuvant therapy was performed in the majority of patients .p = 0.003), and presence of skip metastases (p = 0.005) were favorable prognostic factors for OS , low number of positive lymph nodes (0.009), low lymph node ratio (0.003), low number of N1 involved stations (s for OS .p = 0.003) and NR  as independent favorable prognostic factors. In particular, an optimal cut-off for NR was identified, and in patients with node ratio <10%, the 3YOS result was 78.7% vs. 44.6% (p = 0.001)  . In skip= 0.001) B.p = 0.001).Considering the number of involved lymph nodes, in patients with single lymph node involvement, the administration of AD did not improve survival A, while p = 0.024) was a significant prognostic factor, while AD improved DFS in patients with multiple lymph node involvement (p = 0.02).Regarding DFS, only tumor grading (p = 0.003)  A. Pathol = 0.050 .p = 0.41), while, patients, in the case of multiple lymph node involvement who underwent AD, had a significantly better 3YOS, i.e., 49.1% vs. 30% (p = 0.004).Regarding adjuvant therapy, in the case of singular lymph node involvement, the difference in OS was not significant (p = 0.012).In these patients, the administration of adjuvant therapies resulted in a significant improvement in terms of DFS  and the administration of adjuvant therapy, with a 3YOS of 54.4% vs. 26.7% in patients with or without adjuvant therapy  as the only independent prognostic factor. Considering the DFS, in the group of N2a2, the administration of adjuvant therapy result was an independent prognostic factor, with a 3YDFS of 53.4% vs. 24.2% .In patients with concomitant hilar involvement, a significant difference in terms of long-term survival was observed when stratifying by age , which may be a sign of an appropriate lymphadenectomy. Moreover, previous studies have reported a prognostic value especially in early stages and with a relatively small number of resected nodes, which may be the signal of a limited and partial lymph node assessment, suggesting that a high number of harvested nodes may improve staging accuracy ,14.In line with other studies, we reported the effectiveness of the NR in patients with lymph node involvement ,16 and sThe efficacy of the NR was confirmed in the subgroup of patients with skip metastases, permitting a further stratification for these patients; in this subset of patients, a significant survival benefit may be achieved by the administration of adjuvant therapy.The prognostic role of the number of metastatic nodes remains to be an intriguing issue, and when the entire population was considered, we observed a correlation with survival. Conversely, in accordance with the results of Katsumata et al. , the preIndeed, based on the anatomical lymphatic drainage, direct mediastinal involvement seems to be related to a direct connection to mediastinal nodes, without interception of hilar nodes. Although a codified route is present for the lymphatic drainage, crossing the hilar, and then the mediastinal lymph nodes, a direct connection to the mediastinal nodes is not common, occurring in only about 25% of anatomic cases . Such diTherefore, it may be correct to talk about first lymph node involvement rather than skip metastases ,19 as roThis concept was also reported in an analysis by Park et al. , which dIn this study, we reported a 3YOS in skip metastases with singular nodal involvement of 77.1% when adjuvant therapy was administered and of 72.4% without AD involvement, similar to patients with N1 involvement reported in literature [The prognostic role of the number of involved lymph nodes continues to be an intriguing issue with only recent studies that have analyzed its potential role showing promising results ,16,21. IHowever, one of the most important limitations of considering metastatic node numbers is the preoperative assessment difficulty. For this reason, parameters such as the NR may be considered especially for the pathological evaluation, trying to combine these factors with the optimal specific anatomical landmark.In this study, we confirmed its validity in patients with single-station involvement, showing a survival stratification comparable to the proposed classification based on the concomitant hilar involvement.Another interesting point regards the possibility of stratifying patients for adjuvant treatments. Indeed, adjuvant therapy is a fundamental treatment in patients with N2 involvement ,2,17, wiHowever, few data are present regarding its potential benefits according to the number of involved stations or number of lymph nodes, which may better define an indication for adjuvant therapy. Legras et al.  reportedWe reported a survival benefit in terms of overall survival in the overall population, and also considering N2a1 and N2a2 subgroups, also confirming the importance of this approach for patients in the favorable group (skip metastases). In particular, in skip metastases, we found that AD was a predictor for DFS but not for OS, and interestingly, we noticed that it might influence the prognosis based on the type of nodal involvement.Indeed, in these patients, in the case of singular lymph node involvement, AD did not ensure any survival advantage, while considering patients with multiple positive nodes, AD ensured a survival benefit of about the 25% at three years.Different studies have reported the survival advantages of patients with skip metastases or single-station involvement ,7,22,23,These results also seem to be confirmed in our study considering the number of positive lymph nodes, suggesting that this parameter may also be useful for indicating adjuvant therapy to reduce the risk of treatment administration without clear survival benefit. To our knowledge, this is the first study reporting this kind of analysis; further studies with larger populations are needed to confirm our findings. However, our results are based on platinum-based chemotherapy, while it would be interesting to evaluate the role of tyrosin kinase inhibitors or immunotherapy in the AD setting, performing a tailored therapy in this class of patients.Finally, age was an independent prognostic factor for survival in these patients, which is also in agreement with other studies that have analyzed this patient group ,24, confHowever, patient selection is a fundamental part of the surgical approach in the case of mediastinal involvement, and the inclusion of these parameters (age as well as performance status or Karnosky index) during multidisciplinary meeting ,12,25 maThis study presents some (unavoidable) limitations that are common in similar studies on this topic. Firstly, due to its retrospective nature, it is difficult to obtain information regarding clinical course, including patients, who underwent upfront surgery for limited N2 disease, adjuvant therapy, or patients with unexpected N2 involvement. However, surgery indication and AD administration were in accordance with the study period and were performed based on different TNM classifications (6th and 7th) that presented differences, especially regarding the T3 and the T4 categories. However, in this study, we considered patients without pleural effusion that were recategorized as M+ patients, and therefore AD was homogenously indicated in patients with N2 involvement irrespective of tumor dimension or infiltration. Another limitation was regarding the histological sub-classification for adenocarcinoma patients, which was not collected. Although we studied quite a homogeneous population of patients with single-station involvement, we believe it is the principal factor affecting the prognosis in these patients; it would be interesting to evaluate if different adenocarcinoma patterns also influence the prognosis in these patients. In addition, an appropriate analysis of patterns is also necessary to review pathological specimens for accurate adenocarcinoma classification; this could be the objective of specific studies.Our study confirms that lymph node factors such as the number of involved nodes, skip metastases, and in particular, the lymph node ratio are good prognosticators in patients with single-station mediastinal involvement. Adjuvant therapy continues to have a fundamental role in the case of multiple lymph node involvement, while its role in single metastatic nodes needs to be clarified.


Passeriformes, including European blackbirds (Turdus merula). Strikingly, no in vivo avian model exists so far to study this arbovirus. The domestic canary (Serinus canaria) is a passerine, which is considered as a highly susceptible model of infection by WNV. Here, we experimentally challenged domestic canaries with two different doses of USUV. All inoculated birds presented detectable amounts of viral RNA in the blood and RNA shedding via feathers and droppings during the early stages of the infection, as determined by RT-qPCR. Mortality occurred in both infected groups  and was not necessarily correlated to a pure neurological disease. Subsequent analyses of samples from dead birds showed histopathological changes and virus tropism mimicking those reported in naturally infected birds. A robust seroconversion followed the infection in almost all the surviving canaries. Altogether, these results demonstrate that domestic canaries constitute an interesting experimental model for the study of USUV pathogenesis and transmission.Usutu virus (USUV) is a neurotropic flavivirus closely related to West Nile virus (WNV). Its enzootic cycle mainly involves mosquitoes and birds. Human infection can occur with occasional, but sometimes severe, neurological complications. Since its emergence and spread in Europe over the last two decades, USUV has been linked to significant avian outbreaks, especially among  Flaviviridae, genus Flavivirus [Turdus merula) [Passer domesticus) [Strix nebulosa) [Melanitta nigra) [Usutu virus (USUV) is a mosquito-borne virus classified together with the West Nile virus (WNV) in the Japanese encephalitis virus (JEV) serogroup, of the family avivirus . It has avivirus ,3,4,5. Uavivirus ,9,10,11.avivirus . At leasavivirus ,14,15,16avivirus , includi merula) ,19,20,21esticus) ,23, greyebulosa) , and comAnser anser f. domestica) [Gallus gallus domesticus) [Passeriformes are suspected to be relevant hosts for the amplification of USUV [Serinus canaria) belongs to the same order (Passeriformes) as the European blackbird, which is highly susceptible to USUV infection. This species is accustomed to captivity and is more convenient to use in the laboratory than wild-caught European blackbirds [Experimental infections of avian species with USUV are scarce and restricted so far to the domestic goose (mestica)  and domeesticus) , which w of USUV , as in t of USUV . The domackbirds . Furtherackbirds . Epidemiackbirds ,29,30. HGenBank: KX555624) was used in this experiment. It was isolated from a pool of organs including the brain, spleen, kidney, and heart of a blackbird found dead during an episode of anomalous mortality, which occurred in 2010 in the province of Pesaro Urbino .Usutu virus strain UR-10-Tm belonging to the European lineage 2 (50) technique. The virus was amplified in African Green Monkey Vero cells  and titrated by the 50% tissue culture infective dose . The birds were transported to the biosafety level 2 (BSL-2) experimental animal facility of the Department of Pathology, Faculty of Veterinary Medicine, Liège, Belgium, where they were marked by a unique colored and numerated leg band and housed in randomly-composed groups of five per cage with water and grains supplied ad libitum. One week later, all birds were blood-sampled and tested for the presence of USUV and WNV antibodies prior to the experimental infection (see section: Detection of antibodies to USUV). The animal care and procedures performed in this experiment were approved and supervised by the Committee for Ethics in Animal Experimentation of the University of Liege, Belgium .n = 5), group A , and group B , then anesthetized via isoflurane inhalation. After weighting, groups A and B were inoculated using the intraperitoneal route with either a high dose  or a low dose  of USUV, respectively, dispersed in 100 µL of cell culture medium  supplemented with 1% penicillin/streptomycin). The control group was injected with an equivalent volume of the virus-free medium. After infection, each group was maintained in a separate wire cage with a removable floor that was cleaned daily.Birds were assigned to three groups: control . A 100 µL blood sample was collected from the jugular vein of each bird at 1, 3, 9 and 15 dpi to assess the course of RNAemia and antibody response. The blood was then added to phosphate-buffered saline (PBS) in a ratio of 1:5 and allowed to clot at 4 °C. All the birds were weighed and immature feathers were collected according to the same sampling schedule to reduce stress and repetitive anesthesia. Droppings were daily collected from the cages during the first week of infection and stored at −80 °C until use. Birds that succumbed to the infection were necropsied and 50 ± 1 mg of the brain, eye tissues, lung, liver, kidney, and intestines were harvested for PCR analysis. Other portions of these organs, as well as the heart and spleen, were fixed in 4% formalin for histological and immunohistological examinations. Approximately 10 ± 1 mg of immature feathers were, also, collected from each of these birds.Tissue samples preserved in formalin were embedded in paraffin wax, sectioned and then stained with hematoxylin and eosin. Slides were also processed for immunohistochemistry (IHC) as described in  using a RNA was extracted from 125 µl of diluted serum and the viral genome load was measured by RT-qPCR, as described in . TissuesSerum samples were screened for antibodies using a competitive ELISA kit  following the manufacturer’s instructions. This kit is able to detect immunoglobulins M and G directed against the envelope protein of WNV, which contains an epitope common to viruses from the JEV serocomplex, including USUV ,33. BlooSurvival curves were plotted and compared using the Log-Rank and the Gehan-Breslow Wilcoxon tests .p-values < 0.05 were considered statistically significant.Levels of RNAemia and virus shedding via droppings and feathers were checked for normality using Shapiro–Wilk and Kolmogorov–Smirnov statistics. The logarithmic transformation was performed to normalize the distribution of the data revealed as non-parametric. Data were then analyzed using ANOVA implemented in Rstudio. 2 = 2.322, P = 0.3131, and the Gehan-Breslow Wilcoxon tests χ2 = 2.305, P = 0.3158. For surviving canaries, no loss in body weight was observed (data not shown). However, a loss in the initial body mass ranging from 15.9% to 19.6% was recorded in the dead birds. No weight loss or fatality was detected in the control group.All the infected birds but one showed inactivity and fluffed feathers between days 5 and 9 pi. Two out of five birds from group A and one from group B succumbed without showing specific signs prior to death. The survival curves  did not At necropsy, dead canaries had splenomegaly and pallor of the liver. Histopathological investigations revealed severe satellitosis, neuronal necrosis, apoptosis, and neuronophagia in the brain of the canary 4 from group A a. The saAll three lethally infected canaries exhibited USUV antigen immunolabeled cardiomyocytes a. In theP < 0.005) and did not statistically differ between the infected groups (P = 0.56). No detectable RNAemia was found in the control group on days 1, 3, 9, and 15 pi.All birds, except controls, became infected with USUV, based on viral RNA detection by RT-qPCR in the serum as early as 1 dpi . Very hiP = 0.53 and P = 0.614 respectively). The sham-inoculated group did not shed viral RNA via the feathers or droppings during the experiment.In the virus-inoculated canaries, virus shedding was shown to occur from 2 to 5 dpi through the droppings in group A and from the first day to day 4 pi in group B, reaching a maximum at 1 or 2 dpi according to the group a. The deAll samples collected from the dead canaries at necropsy were USUV-positive by RT-qPCR, with high RNA amounts in their blood and tissues, as presented in The absence of a previous USUV (and WNV) infection was ensured by negative ELISA results on blood samples collected before starting the experiment. Serum samples from all the surviving canaries 15 days post-infection, with one exception, showed a positive reaction in the ELISA . Similar3.4 and 105.9 PFUs [3 TCID50 and 106 TCID50 challenge doses may have complied with the amount of USUV inoculated in the birds during a mosquito bite. However, in this experiment, morbidity and mortality rates did not statistically differ in a dose-dependent manner. In the three dead canaries, higher levels of RNAemia compared to the surviving ones were recorded during the infection, which might explain the fatal outcome in these birds, regardless of the infective dose. This result is similar to that described in the study of VanDalen et al. (2013), in which higher viremia was detected in American robins (Turdus migratorius) lethally infected with WNV, although inoculated with the lowest dose [In the present study, we questioned the susceptibility of domestic canaries to USUV. To our knowledge, this is the first report of experimental infection with this virus in a passerine. After their injection with two different doses of USUV, three out of the ten infected birds succumbed to infection, in contrast to a high mortality rate (5/5) reported after 5 days of their challenge with as few as 10 PFU of WNV . This su5.9 PFUs . Thus, test dose . In addiest dose  and VanDest dose . For virest dose . We did Cloacal and/or oropharyngeal shedding of USUV was previously described following natural  or experUSUV pathogenesis in the lethally infected canaries entailed early onset of viremia, followed by a rapid viral invasion of all systems, as the virus was detected by RT-qPCR in all organs examined, including the brain, heart, liver, spleen, skin, and kidney. This systematic infection is similar to that reported in naturally infected birds . Gross lThe dissemination of USUV to the eye was here shown by RT-qPCR. This is consistent with the detection of abundant USUV antigens within the retina previously reported in experimentally infected goose embryos . The necTwo weeks after the experimental infection, almost all the surviving canaries showed a humoral response and specific neutralizing antibodies against USUV, which further demonstrates their susceptibility to USUV infection. Nevertheless, a single specimen from the group infected with the lower dose of USUV presented a doubtful serological response using the ELISA examination and an undetectable neutralizing antibodies titer by our technique. This finding could be due to a certain heterogeneity in the genetic background of the outbred canaries used in this experiment, which mimics the very specific host-pathogen interaction that has been described in nature (, SupplemPasseriformes when naturally infected with USUV. Further studies in canaries using other USUV strains circulating in Europe might contribute to a larger understanding of USUV pathogenesis. In addition, alternative transmission routes of USUV can be assessed using this avian model.In summary, we established that canaries are susceptible to USUV infection and can shed viral RNA via droppings and feathers. Further, we showed that USUV-associated mortality was not necessarily correlated to a pure neurological disease. These findings match those observed in European blackbirds and other


Tree nuts show nutritional properties and human health benefits. However, they contain allergenic proteins, which make them harmful to the sensitised population. The presence of tree nuts on food labelling is mandatory and, consequently, the development of suitable analytical methodologies to detect nuts in processed foods is advisable. Real-Time PCR allowed a specific and accurate amplification of allergen sequences. Some food processing methods could induce structural and/or conformational changes in proteins by altering their allergenic capacity, as well as produce the fragmentation and/or degradation of genomic DNA. In this work, we analysed by means of Real-Time PCR, the influence of pressure and thermal processing through Instant Controlled Pressure Drop (DIC) on the detectability of hazelnut, pistachio and cashew allergens. The detection of targets in hazelnut, pistachio and cashew  is affected by the treatment to different extents depending on the tree nut. Results are compared to those previously obtained by our group in the analysis of different treatments on the amplificability of the same targets. Reduction in amplificability is similar to that reported for some autoclave conditions. Our assays might allow for the detection of up to 1000 mg/kg of hazelnut, pistachio and cashew flours after being submitted to DIC treatment in food matrices. Food allergy is a worldwide health concern that affects around 2% of the population, reaching 8% in children . The mosTo date, there is no treatment for food allergies, so patients must avoid any contact and consumption of allergenic ingredients. Thus, available sensitive and specific detection methods of hidden allergens in foods are essential in order to protect sensitized individuals and comply with current regulations. Enzyme-Linked Immunosorbent Assay (ELISA) is the most-used methodology for direct detection of proteins from specific foods, as well as other immunoassays, such as lateral flow. Many studies have demonstrated that certain thermal treatments might be useful to diminish the allergenic potential of nuts, influencing allergen structure ,8. The sFoods, including tree nuts, are frequently submitted to thermal and non-thermal processing in the food industry, to improve their organoleptic properties or ensure their safety . RoastinBefore this study, the development and validation of Real-Time PCR methods for pistachio, cashew and hazelnut were performed and published ,21,22. IPistachia vera Kerman) and hazelnut  were obtained from the Germoplasm Bank of Institut de Recerca i Tecnologia Agroalimentaries , whereas cashew nuts  were provided by Productos Manzanares S.L. . Three nut seeds have been used in this study: hazelnut, pistachio, and cashew. Pistachio  treatment, performed at the La Rochelle University (LaSiE). DIC treatment was carried out following a factorial experimental design previously described . In thisUntreated and DIC treated nut seeds were ground with a kitchen robot  and defatted with n-hexane (34 mL/g) for 4 h, followed by air-drying. Flours were passed through a 1 mm mill and stored at 4 °C until use.Triticum spelta L.) flour, in a final weight of 25 g. The mixture containing 10% of each nut  was prepared by adding 2.5 g of the nut flour to 22.5 g of spelt wheat flour, and followed by 10-fold dilutions, homogenizing with the kitchen robot. Moreover, for hazelnut, binary mixtures of DIC treated flours were also created from peanut defatted flour (Arachis hypogea L. from Productos Manzanares S.L.), in order to mimic the conditions described in our previous paper, used as a reference for this nut . I. IR2  and efficiency (91.52%) (N = 8), regarding efficiency and R2, as well as the practical LOD and limit of quantification (LOQ), both 10 mg of pistachio/kg of the mixture  A. The pe mixture B.Regarding the effect of DIC treatment at the particular conditions of 7 bar 120 s on Pis v 1 target, for the same spiked level  of the binary mixtures, Ct is significantly delayed compared to the untreated control, and linear regression was not obtained. In the case of 100,000 mg/kg-containing binary mixtures, the detection was delayed more than 10 cycles . Pis v 1This result is similar to the effect produced on the detectability of Pis v 1 when pistachio nuts were submitted to heat and pressure in an autoclave, which also combines heat and pressure. These treatments were carried out previously by our group  and a diRecently, Vicente and collaborators  studied R2 and efficiency of reaction within the minimal standards for Real-Time PCR publications . At 138 °C for 15 min in an autoclave, the detection and quantification were possible up to 1000 mg/kg, maintaining a linear correlation of the curve (from 105 to 1000 mg/kg) similar to DIC 7 bar for 120 s treatment. Ct means showed no significant differences between both treatments , pistachio (Pis v 1) and cashew (Ana o 1). This effect was less intense in the case of the cashew allergen. According to the available data regarding the effect of DIC processing on the IgG/IgE binding capacity of nuts, this system would allow us to detect traces of DIC 7 b 120 s-processed hazelnut and pistachio, and even quantify treated cashew, if the quantity of the allergenic ingredient was around 100,000–10,000 mg/kg in a food mixture.


Alcohol use is associated with an automatic tendency to approach alcohol, and the retraining of this tendency (cognitive bias modification [CBM]) shows therapeutic promise in clinical settings. To improve access to training and to enhance participant engagement, a mobile version of alcohol avoidance training was developed.The aims of this pilot study were to assess (1) adherence to a mobile health  app; (2) changes in weekly alcohol use from before to after training; and (3) user experience with regard to the mHealth app.A self-selected nonclinical sample of 1082 participants, who were experiencing problems associated with alcohol, signed up to use the alcohol avoidance training app Breindebaas for 3 weeks with at least two training sessions per week. In each training session, 100 pictures  were presented consecutively in a random order at the center of a touchscreen. Alcoholic beverages were swiped upward (away from the body), whereas nonalcoholic beverages were swiped downward (toward the body). During approach responses, the picture size increased to mimic an approach movement, and conversely, during avoidance responses, the picture size decreased to mimic avoidance. At baseline, we assessed sociodemographic characteristics, alcohol consumption, alcohol-related problems, use of other substances, self-efficacy, and craving. After 3 weeks, 37.89% (410/1082) of the participants (posttest responders) completed an online questionnaire evaluating adherence, alcohol consumption, and user satisfaction. Three months later, 19.03% (206/1082) of the participants (follow-up responders) filled in a follow-up questionnaire examining adherence and alcohol consumption.P<.001; n=410) and another reduction of 6.2 units at 3 months for follow-up responders . Posttest responders provided positive feedback regarding the fast-working, simple, and user-friendly design of the app. Almost half of the posttest responders reported gaining more control over their alcohol use. The repetitious and nonpersonalized nature of the intervention was suggested as a point for improvement.The 410 posttest responders were older, were more commonly female, and had a higher education as compared with posttest dropouts. Among those who completed the study, 79.0% (324/410) were considered adherent as they completed four or more sessions, whereas 58.0% (238/410) performed the advised six or more training sessions. The study identified a significant reduction in alcohol consumption of 7.8 units per week after 3 weeks (95% CI 6.2-9.4, This is one of the first studies to employ alcohol avoidance training in a mobile app for problem drinkers. Preliminary findings suggest that a mobile CBM app fulfils a need for problem drinkers and may contribute to a reduction in alcohol use. Replicating these findings in a controlled study is warranted. Problematic alcohol use is one of the most prevalent health problems in modern life. It has several negative personal, social, and economic consequences -4. When P=.05 and 10%, P=.04, respectively) in the CBM condition as compared with a placebo condition. This effect was found to be mediated by a change in approach tendencies in the latter study ) to postassessment (mean 25.5 [SD 20.4]).The average alcohol consumption of the posttest responders (n=410) decreased significantly by 7.8 units per week : male gender, unemployment, high level of baseline craving, high baseline AUDIT score, high level of self-reported concentration during sessions, and high adherence. When these factors were entered in a multivariate regression model, only gender, adherence, and craving remained significant predictors of a change in alcohol consumption.P<.001) and from postassessment to follow-up .The subsample of 206 participants that completed the follow-up assessment displayed a reduction in alcohol use over time. Their mean weekly alcohol consumption decreased from 31.6 (SD 23.2) units at baseline to 24.4 (SD 19.2) units at 3 weeks and to 18.2 (SD 17.3) units at follow-up 3 months later, resulting in a total decrease of 13.4 units a week. Pairwise comparisons in mixed-model analysis demonstrated the reductions for this subsample as significant both from baseline to postassessment  and from postassessment to follow-up .A mixed model for repeated measurements, in which all 410 participants were taken into account, produced similar results . Pairwis, with many participants also reporting being more aware of their alcohol use . However, 47.3% (194/410) of participants reported that they gained nothing from using the app.When posttest responders (n=410) were asked about what they gained from using the app over a 3-week period, almost half of the participants stated having the feeling of more control over their drinking Posttest responders had an overall CSQ score of 20.9 (SD 4.4) with an average score of 2.6 on a scale from 1-4 (item variances: 0.5), indicating moderate satisfaction. Participants were particularly positive about the simple, fast-working, user-friendly design of the app. Criticism and subsequent suggestions about the app mostly targeted elements concerning monotony and lack of personalization. Participants deemed app use as boring and monotonous owing to the repetition of the task and pictures. They suggested introducing motivational elements, such as levels or game options, as well as a shorter interval between swiping movements and subsequent pictures. Thus, the introduction of a greater variation in pictures and the possibility of choosing pictures was suggested.Of the 410 posttest responders, 318 (77.6%) had never sought help or used an intervention to reduce alcohol use previously. Additionally, 46 (11.2%) participants reported receiving extra help in reducing alcohol use during the Breindebaas training period in the form of a self-help program, help from a general practitioner, help from a professional in (addiction) care, or peer support. Of these 46 participants, 20 had never sought help before.Of the 206 participants that completed the follow-up questionnaire, 85 continued to use the app. The main reasons behind this decision were that using the app helped them to be more conscious of their alcohol use (n=51) and it assisted in maintaining their reduced drinking habit (n=15). Of the 121 participants who stopped using the app, the main reasons were doubts regarding the app’s effectiveness (n=40) and simply forgetting to use the app (n=33).To our knowledge, this study is the first to evaluate a mobile version of AAT training in a sample of problem drinkers among the general population. Given the debate on the effectiveness of CBM ,41, it iThe baseline characteristics, adherence to the intervention, change in alcohol consumption, and user experiences were studied. Participants in this study were comparable to groups analyzed in previous research via Web-based self-help interventions regarding the level of problematic alcohol use -44 and nThe intervention adherence among posttest responders was high. Most of the posttest responders  used the app four or more times, doing better than an online CBM trial by Wiers et al on alcohol, where 43.3% (136/314) of the participants completed the prescribed four sessions . The facIn this study, we observed a significant reduction in alcohol use among posttest responders immediately after using the mobile intervention and 3 months later. Although a reduction of 13 units per week is substantial for such a brief intervention, the results need to be seen in the context of a pilot study without a control condition for comparing the findings, especially given earlier observed main effects across CBM and control conditions . FurtherParticipants were mostly positive about the Breindebaas app. The simple, fast-working, user-friendly design resulted in participants reporting more awareness and control over their alcohol use. Nevertheless, a considerable portion of participants also reported gaining nothing from using the app. Reportedly, this was because of the repetitious and monotonous characteristics of the AAT and its lack of personalization. Regarding the lack of personalization, participants mainly indicated that some of the pictures contained beverages  that were not appealing to them at all. Wiers et al already indicated that personalizing alcohol-related stimuli is a potential way forward . StudiesA number of limitations should be addressed. First, as this study was set up as a pilot study with the aim to assess feasibility and adherence, no control group was allocated. Consequently, the change in self-reported alcohol consumption found in this study may well be the result of a placebo effect of the app, a nonspecific effect of engaging in any intervention, or even an effect of participating in a study. As already mentioned, just reporting alcohol use alone can have an effect on the reduction of drinking . NonetheIn summary, several suggestions from users and researchers provide the following insights for the future development of the Breindebaas app: (1) using personalized stimuli in the app; (2) adding more information about the working mechanism and effects of CBM, which can increase motivation; (3) including bias measurement in the app, so participant progress in bias scores can be tracked; and (4) adding motivational/gamification elements  to improve user adherence to the app. The addition of gamification elements was mentioned by users of the Breindebaas app and has shown promising results in other forms of cognitive training ,58. In a


Ovarian cancer (OC) is one of the deadliest cancers among women contributing to high risk of mortality, mainly owing to delayed detection. There is no specific biomarker for its detection in early stages. However, recent findings show that over-expression of specificity protein 1 (Sp1) is involved in many OC cases. The ubiquitous transcription of Sp1 apparently mediates the maintenance of normal and cancerous biological processes such as cell growth, differentiation, angiogenesis, apoptosis, cellular reprogramming and tumorigenesis. Sp1 exerts its effects on cellular genes containing putative GC–rich Sp1–binding site in their promoters. A better understanding of the mechanisms underlying Sp1 transcription factor (TF) regulation and functions in OC tumorigenesis could help identify novel prognostic markers, to target cancer stem cells (CSCs) by following cellular reprogramming and enable the development of novel therapies for future generations. In this review, we address the structure, function, and biology of Sp1 in normal and cancer cells, underpinning the involvement of Sp1 in OC tumorigenesis. In addition, we have highlighted the influence of Sp1 TF in cellular reprogramming of iPSCs and how it plays a role in controlling CSCs. This review highlights the drugs targeting Sp1 and their action on cancer cells. In conclusion, we predict that research in this direction will be highly beneficial for OC treatment, and chemotherapeutic drugs targeting Sp1 will emerge as a promising therapy for OC. Reportsfor SV40 .It is important to understand how a complex factor such as Sp1 is involved in basal transcriptional regulation in various genes. The encoded protein is involved in many cellular processes, including cell differentiation, cell growth, apoptosis, immune responses, response to DNA damage, and chromatin remodeling. Post-translational modifications such as phosphorylation, acetylation, glycosylation, and proteolytic processing significantly affect the activity of this protein, which can be an activator or a repressor . When SPSp1 gene is supported by 512 sequences from 447 cDNA clones [The gene encoding Sp1 is located in the q arm of the 12th chromosome in humans . AccordiA clones . In humaA clones ,20. Sp1 A clones . Sp1 hasA clones . The DNAA clones . The twoA clones . The C dA clones . The N-tA clones . The co-The uniqueness of the Sp1 TF is that it not only initiates transcription but also regulates the activation or repression processes. Growing evidence suggests that the transcriptional activity and stability of Sp1 is influenced by its post-translational modifications (PTMs). Sp1 undergoes acetylation, sumoylation, ubiquitylation, and glycosylation after translational ,27. AcetSp1 has a key role in regulating cyclins, CDKs, and CDKIs, which are critical components of cell cycle machinery . In the Tumorigenesis can be defined as an uncontrolled cell cycle progression. Defects in the Sp1 transcriptional activities act as a cause for tumorigenesis in many types of cancers, such as ovarian, breast, and gastric cancer. The transcription activity of Sp1 in cancer cells is enhanced by various oncogenes like Ras, Src, and Raf, especially through the p42/p44 mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway . Sp1 expIn OC, the most frequently altered pathways include JNK1  pathways, MAPK signaling, and the PI3K/AKT pathway . Rhox5 hAngiogenesis is the process of formation of new blood vessels within the cells, and it is an important prognostic factor for the pathophysiological conditions seen in OC cells. VEGF is one of the genes normally linked with most of the angiogenic processes in cancer cells. A few studies have proven that Sp1 promotes angiogenesis in OC via induction of VEGF expression by directly binding to its promoter site ,53,54. Isurvivin gene belongs to the inhibitor of apoptosis protein (IAP) family, which is a key agent for the anti-apoptosis process. The promoter region of the surviving gene has GC–rich sites, which are known to be the binding site for Sp1  has a very poor prognosis because of delayed diagnosis in most of the patients and resistance to some cytotoxic drugs. A major obstacle that jeopardizes OC chemotherapeutic treatment is multidrug resistance (MDR) . A remarptomyces . The intptomyces ,85. Besiptomyces . In a fuptomyces . Anotherptomyces . Similarptomyces . Anotherptomyces . Betulinptomyces ,90. Currptomyces . In a reptomyces . Similarptomyces .Drugs designed by applying the mechanism-based criteria, such as targeting important pro-oncogenes, their associated pathways, and their initiators, are approved for clinical use fairly easily. Various potential strategies are available for blocking or inhibition of Sp1. These include activation of ROS, proteasome, caspases, the cannabinoid receptors, and targeting Sp1 binding GC–rich regions. Recently, it has been reported that targeting Sp1 expression at the G2/M phase of cell cycle by radiotherapy helps reduce tumor development . InteracStem cells and Cancer stem cells appear to have similar regulatory signals in their microenvironments that contribute to their reprogramming and proliferative potential. But the mechanisms involved in reprogramming continue to remain enigmatic. Recently, switch genes have been identified, that convert glioblastomas from stem-like cells to differentiated forms. Sp1 transcription factors, were recognized as central regulators of the switch genes, displaying their potential role in cellular reprogramming . Sp1 is Xenopus laevis [Cellular reprogramming is a process where any dysfunctions in the cells can be retrieved by erasing any kind of epigenetic alterations in the somatic cells using Induced Pluripotent Stem Cells (iPSCs). This process of cell reprogramming or repairing was first anticipated by Dr. John Gurdon during an experiment on the cloning process of somatic cells in the s laevis . The effs laevis . Recentls laevis . Recentls laevis . The durs laevis . Thus, rs laevis . During s laevis . It has s laevis . Thus, tTill now the only valid and trust-worthy tool to detect cancer is the cytogenetic assay, where the chromosomal aberrations in the peripheral lymphocytes of the patients ,117. DesIn conclusion, we suggest that novel medicinal plant-based compounds must be developed for suppressing the oncogenic functions of Sp1 by targeting its specific sites in OC. A higher number of clinical or phase trials must be carried out to ascertain the effect of OC therapy routines on Sp1 signaling and to develop strategies for modifying the Sp1–targeted survival response. Finally, the biggest challenge is to deliver adequately dosed interventions, taking into account the many sources of interference for a specific, tissue-targeted or cell-targeted effect in OC.