Phytotoxicity of metals significantly contributes to the major loss in agricultural productivity. Among all the metals, copper (Cu) is one of essential metals, where it exhibits toxicity only at its supra-optimal level. Elevated Cu levels affect plants developmental processes from initiation of seed germination to the senescence, photosynthetic functions, growth and productivity. The use of plant growth regulators/phytohormones and other signaling molecules is one of major approaches for reversing Cu-toxicity in plants. Nitric oxide (NO) is a versatile and bioactive gaseous signaling molecule, involved in major physiological and molecular processes in plants. NO modulates responses of plants grown under optimal conditions or to multiple stress factors including elevated Cu levels. The available literature in this context is centered mainly on the role of NO in combating Cu stress with partial discussion on underlying mechanisms. Considering the recent reports, this paper: (a) overviews Cu uptake and transport; (b) highlights the major aspects of Cu-toxicity on germination, photosynthesis, growth, phenotypic changes and nutrient-use-efficiency; (c) updates on NO as a major signaling molecule; and (d) critically appraises the Cu-significance and mechanisms underlying NO-mediated alleviation of Cu-phytotoxicity. The outcome of the discussion may provide important clues for future research on NO-mediated mitigation of Cu-phytotoxicity. PGRs such as auxins (AUXs), cytokinins (CKs), gibberellins (GAs), brassinosteroids (BRs), ethylene (ET), jasmonic acids (JA), polyamines (PA), salicylic acid (SA), nitric oxide (NO), and strigolactones modulate physiological/biochemical and genetic processes and improve plant tolerance to major abiotic stresses including metals/metalloids , b.Earlier known as a vital signaling and effector molecule in animals, NO is known to occur also in plants, and act therein as a short-lived multifunctional gaseous signaling molecule . NO contGiven above, this paper is aimed to: (i) overview the aspects of the uptake, transport, and role of Cu in photosynthesis and nutrient-use-efficiency; (ii) highlight major aspects of Cu-phytotoxicity; (iii) discuss NO as a signaling molecule; and (iv) critically appraise the Cu-significance and mechanisms underlying NO-mediated alleviation Cu-toxicity in plants. Important clues for future research in this direction with the outcome of the facts are also discussed herein.2+ and Cu+ ions. Cu2+ frequently prefers coordination with oxygen in aspartic and glutamic acid and with nitrogen in histidine side chains. On the other, Cu+ has a higher affinity with the sulfur in methionine or cysteine. The list of major Cu-containing proteins in plants includes plastocyanin, cytochrome-c oxidase (COX), ethylene receptors, Cu/Zn–superoxide dismutase (Cu/Zn SOD), tyrosinase, plantacyanin, phenol oxidase, laccase, ascorbate, and amine oxidase . Composed of 12-14 sub-units, plant COX is the terminal enzymatic complex IV of the mitochondrial respiratory chain  redox repair , SPL7 modulates the Cu-uptake and homeostasis under Cu-deficiency (Copper (Cu) is an essential trace element of most living organisms on the earth including plants, where >30 types of proteins are known to possess Cu as their structural constituent . As an e oxidase . Cu main oxidase . It is lr plants . Cu alsory chain . Anotherry chain . Anotherry chain , and arery chain . Cu-depery chain . Cu-depell death . Polyphell death . AOs alsx repair . Polyphex repair . In addix repair . Cu-homeficiency . Thus, tOn the contrary, the condition of both Cu-deficiency and -elevation can bring severe consequences in plants . Plants 2+ ions from the rhizosphere, where the binding of Cu with various ligands facilitates the process (COPT1-6) involved in the uptake of Cu and secretion of metal ions  encoding COPT transporters. COPT1, one of the most characterized members of the Cu-transporter family has been reported to permit the entry of Cu into the cells from outside to the cytoplasm  and functional complementation in yeast . All theytoplasm . In addi  plants, Impacts of Cu on photosynthesis are well documented. The photosynthetic apparatus is susceptible to heavy metal toxicity, which in turn directly or indirectly significantly impact photosynthetic functions. Excess Cu has been reported to decrease the level of photosynthetic pigments such as chlorophyll . Cu conc+ . In Avicpparatus . In a reSurplus level of Cu restricts plant growth and development . The imp+ and Cl– accumulated in plant organs may compete with mineral nutrients and also disturb their uptake, translocation, and assimilation , The accumulation of ions such as Namilation . Higher milation . In sandmilation . In both, and Co . Elevate, and Co . In Suaee shoots . In a reT. aestivum L. under salt stress (S-nitrosylation that leads to the formation of S-nitrosothiols (SNOs). The reaction of NO with ROS such as superoxide anions also leads to the protein tyrosine nitration and yields nitrite (ONOO-). In fact, SNOs are the key signaling molecules largely involved in response to several stresses in plant biology (S-nitrosoglutathione (GSNO) is the most important among SNOs. This metabolite is considered as a major reservoir of NO. As a NO-reservoir, GSNO can be transported to other cells/tissues which confer NO as a long distance mobile signaling molecule. GSNO can also be converted into oxidized glutathione (GSSG) and NH3 by GSNO reductase (GSNOR) (S-transnitrosylation reactions (2-FA) have shown the importance of NO in cell signaling processes  . In addieactions . NO-deperocesses . NO2-FA stresses . A NO-mestresses  and in tstresses .Medicago truncatula; whereas, a decreased growth was observed with higher concentration of NO (2.5 mM) , a NO-donor  . The auta growth . DiffereNO-donor . SNP wasZea mays . HoweverZea mays . Comparece seeds . NO can ce seeds , induce pirillum .Solanum melongena seedlings was argued as a result of excessive quenching energy and an increase in quantum yield of PSII  and increased the content of PSII oxygen-evolving complex , alternative oxidase (AOX1a) and glutathione peroxidase (GPX)  . It has se (GPX) .Panax ginseng modulated the activity of H2O2-metabolizing enzymes including catalase (CAT), peroxidase, and ascorbate peroxidase (APX), and thereby increased the detoxification of H2O2 in roots (Triticum aestivum with NO was reported to reverse the inhibitory effect of Cu stress by increasing the activity of superoxide dismutase (SOD) and CAT, and reducing the lipoxygenase activity and membrane lipid peroxidation  alone or in combination with GSH (200 μM) minimized Cu-impacts by reducing Cu-uptake and eased the Cu-induced oxidative damage by amending GSH production. Not only GSH content was increased but also increased the content of ascorbate (AsA), and the ratios of GSH/GSSG and AsA/DHA, which in turn strengthened antioxidant defense system improved Cu-tolerance. Moreover the contribution of different metal-chelating ligands, such as metallothioneins (MTs) and phytochelatins (PCs) are crucial players and plays pivotal role in conferring resistance to heavy metal tolerance in plants dealing with high concentrations of various metal inclusion (Lemna esculentum (Solanum lycopersicum and this NO-induced expression of MTs-related genes were reversed by NO scavenger , NOS inhibitor [N-nitro-l-arginine methyl ester (L-NAME)] and NR inhibitor (tungstate), which confirmed the involvement of MTs in NO-mediated tolerance to Cu toxicity . These witnessed phenotypic changes in Cu-effected roots are possibly due to AUX action, because in roots, the pattern of AUX distribution is vital for healthy root development. In addition to this Arabidopsis, and reduced transport of AUX and its response by altering the PIN1 levels. Therefore, the supply of optimal level of NO might be a responsible for maintaining the AUX concentration and its distribution when plants countered the heavy metal stress (In naked barley (without hull), Cu-tolerance involved nitrate reductase-mediated NO-production . Furthertivities . Yuan etl stress . In Arabl stress . In anotThis review appraised the literature available on Cu-induced toxicity and its NO-mediated amelioration and underlying mechanisms in plants. It is clear that NO is a diffusible gaseous molecule and plays a key role in performing a number of biological functions in plants. NO acts as a signaling molecule in inducing the antioxidant system during oxidative stress. There is a remarkable progress in our understanding on the biological role of NO in plants particularly in case of salt stress. However, least information is available on the response of NO on Cu stress. Insights are required into the signaling pathways and the direct targets of NO particularly in Cu-exposed plants. Examination of the Cu-induced modulation of the NO biosynthetic pathways and its involvement in the physiological roles of NO in plants would be imperative in this regard. Some of the biosynthetic pathways of NO in plants are well known but how these pathways are interconnected and what is the mode of action in each tissue and organ are required to be elucidated.AM and NK conceived the idea. BR, ZS, and AM prepared the first draft. NA, AM, and NK corrected and improved the manuscript. All authors approved the final version for its publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The majority of mediastinal tumours develop asymptomatically and are often detected incidentally on a chest X-ray performed for another reason. Mediastinal tumours, although mostly asymptomatic, may cause non-specific symptoms associated with advanced tumour growth.We present a case of a 30-year-old woman who presented with exhaustion and lower back pain accompanied by severe headaches with symptoms of visual disturbances, followed by the typical Horner syndrome. Computed tomography revealed a tumour measuring 12 × 11 × 10 cm in the right cavity with features suggestive of teratoma. The patient underwent mediastinal tumour resection and thymectomy. The pathomorphological examination confirmed the primary diagnosis of mediastinal teratoma, but rare somatic type malignancy was detected. Therefore, the patient was referred for further oncological treatment.Mediastinal teratoma is an uncommon finding and usually asymptomatic. Despite its slow growth, it can grow enough to compress adjacent structures, causing symptoms similar to those presented in our patient.Radiologic imaging proves diagnostic in most cases. Despite the somatic type malignancy, surgical excision of the tumour using the en-bloc technique seems to be a sufficient option for the patient, and further oncological treatment is not always obligatory. The mos2A 30-year-old female patient with an active and healthy lifestyle was feeling fine until two months when exhaustion and lower back pain began. The symptoms were initially associated with intense work effort. A week later, she developed severe headaches with symptoms of visual disturbances, photophobia, orbital pain, and a general decline in immunity. Soon, the symptoms were accompanied by the loss of skin sensation in the upper part of the forehead, an uneven pupil diameter, and a dropping right eyelid, which prompted the patient to consult a physician. At the time of medical evaluation, a chest X-ray revealed an opacity in the right lung field extending from the mediastinum, obliterating the hilar and right lower lung shadow . A CT sc3Teratoma is a rare type of germ cell tumour containing tissues derived from one or more germ layers i.e., ectoderm, mesoderm, or endoderm. The vast majority of cases are benign, and in very few cases they are prone to malignancy and metastasis. Mediastinal teratoma is an uncommon finding with an incidence of 10–15% of all mediastinal tumours, and most often present in young adults with a mean age of 20–40 years old . As we rWe also want to draw the reader’s attention to the presence of typical Horner syndrome in our patient. It is of greatest importance to exclude myasthenia or paraneoplastic syndrome at an early stage of the diagnostic process. Laboratory tests that include ACHR, CRP, AFP, β-hCG, and other common tumour markers seem to be crucial . The rea4In conclusion, we reported a case of the mature mediastinal teratoma with somatic type malignancy including neuroblastoma and intestinal type of adenocarcinoma successfully treated with complete surgical resection. Despite the malignant component there was no need for further oncological treatment. As it was in our patient, radiologic imaging can with a high accuracy prove diagnostic. Therefore, especially in the era of COVID-19, chest X-rays should be still the basic and frequently performed screening study that may reveal chest tumours at a very early stage.The authors report no declaration of interest.None.This is a case report study. Informed patient written consent has been obtained and all identifying information was omitted.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.MR: Conceptualization; Supervision; Validation; Roles/Writing - original draft; Writing - review & editing; KK: Roles/Writing - original draft; Writing - review & editing; MB: Data curation; Investigation; Writing - review & editing; MJ: Supervision; Validation; Writing - review & editing; RP: Data curation; Investigation; Methodology; Supervision; Validation.Not applicable.Roman Przybylski.Not commissioned, externally peer-reviewed.


Several studies have evaluated surgical resection of pulmonary metastases as a standard treatment option for colorectal cancer (CRC) patients with resectable pulmonary metastases. However, the role of peri-operative chemotherapy after complete resection of pulmonary metastases from CRC patients is still controversial. This systematic review and meta-analysis is aimed to investigate the clinical efficacy of peri-operative chemotherapy after resection of CRC pulmonary metastases.PubMed, the Cochrane Library databases, and Embase were searched for studies evaluating the effect of peri-operative chemotherapy on the survival of patients with CRC after pulmonary metastasectomy. The hazard ratio (HR) was used for analyzing overall survival (OS) and progression-free survival (PFS)/recurrence-free survival (RFS)/disease-free survival (DFS).p < 0.05) and PFS/RFS/DFS  in patients who received pulmonary metastasectomy. Multivariate analysis also validated this result . There was a significant benefit in peri-operative group on OS and PFS/RFS/DFS in studies with R0 resection of pulmonary metastases  and metachronous pulmonary metastases .Eight studies were included in the final analysis. The outcome showed that peri-operative chemotherapy had a significant favourable effect on OS  contains supplementary material, which is available to authorized users. CRC is the third most common cancer in the world. It was estimated that more than one million cases and 600 thousands deaths occurred in 2012  were included.Studies that did not examine long-term follow-up were excluded.The quality of the selected studies was evaluated using the Newcastle–Ottawa Scale (NOS).The studies were independently examined by two investigators. First author, year of publication, study type, study population characteristics, number of subjects, length of follow-up, and outcomes were recorded. Publication bias was investigated by funnel plots.2 was used to evaluate heterogeneity across studies. A p value of < 0.05 was considered statistically significant.Stata software was used to evaluate the overall effect of peri-operative chemotherapy on survival. The results were obtained as the hazard ratio of PFS/RFS/DFS and OS. ITo date, there have been no reports of randomized trials that compared surgery to neo-adjuvant or adjuvant chemotherapy combined with surgery.Figure A total of 1936 patients in eight studies were evaluated in this meta-analysis, of whom 926 patients received surgery alone and 1010 patients received peri-operative chemotherapy and surgery . The outcome of the random effect model of the meta-analysis revealed that treatment with peri-operative chemotherapy significantly prolonged OS in comparison to treatment with only pulmonary metastasectomy   . Independent prognostic factors were verified using multivariate analyses in four studies. Peri-operative chemotherapy strongly affected PFS/RFS/DFS and OS by multivariate analysis compared to surgery alone     Tables . Three o) Tables .The need for peri-operative chemotherapy after pulmonary metastasectomy from CRC patients is still a matter of debate due to the lack of evidence. In this study, we evaluated eight retrospective studies concerning the efficacy of chemotherapy in these patients. The HR estimates suggested that patients benefit from peri-operative chemotherapy after resection of pulmonary metastases. For PFS/RFS/DFS, the peri-operative treatment group also improved survival. Multivariate meta-analyses were conducted to adjust for other variables that could have influenced survival, such as age, disease stage, CEA level, and tumor location. Our analyses demonstrated that peri-operative chemotherapy was an independent and favorable prognostic factor for OS and PFS/RFS/DFS, which is consistent with previous findings , intravenous 5-FU plus irinotecan (FOLFIRI), and molecular targeted agents. Recently, these new chemotherapeutic regimens have significantly improved the prognosis of patients with CRC Below is the link to the electronic supplementary material.


Securing clean indoor air laws is a major tobacco control accomplishment of the past 15 years. The public quickly adopted and supported such policies both in public and private spaces. Clean indoor air is now threatened by the emergence of e-cigarettes. E-cigarette aerosol contains nicotine, heavy metals, and carcinogens, and the long-term effect of secondhand exposure is unknown. Surveillance is necessary to track voluntary rules on smoking and vaping in the home.The Minnesota Adult Tobacco Survey (MATS) is a series of cross-sectional, random-digit–dial telephone surveys on smoking, vaping, and other tobacco-related behaviors, attitudes, and beliefs among Minnesota adults. MATS measured voluntary smoke-free rules in the home in 2014  and measured both smoke-free and vape-free home rules in 2018 .The prevalence of smoke-free home rules among Minnesota adults in 2018 was 91.5% , up slightly from 89.3%  in 2014. In comparison, 84.0%  reported vape-free home rules. Although 70.0%  of smokers in 2018 reported smoke-free home rules, only 23.3%  of e-cigarette users reported vape-free home rules. Living with children younger than 18 years significantly increased the odds of having smoke-free and vape-free home rules.Although widespread adoption of voluntary smoke-free and vape-free home rules demonstrates a positive change in social norms, most e-cigarette users allow vaping in their homes, including those who live with children younger than 18. Tracking voluntary smoke-free and vape-free home rules and efforts to encourage them are important to improve the public’s health. Adults are increasingly making their homes smoke-free and protecting children and other nonsmokers from dangerous secondhand smoke. E-cigarettes pose a new threat to clean indoor air, but few studies have examined whether homes are vape-free.This study provides the first statewide estimate of vape-free rules and shows that most adults who use e-cigarettes, including those who live with children, allow vaping in their homes and are potentially exposing others to secondhand aerosol.Clean indoor air efforts should promote smoke-free and vape-free homes.Securing clean indoor air laws is a major tobacco control accomplishment of the past 15 years. Still, secondhand tobacco smoke poses a serious health threat to nonsmokers  and encoEmerging in 2007, e-cigarettes have been marketed as a healthier alternative to combustible cigarettes, and some advertisements promoted their use where smoking is not allowed . E-cigarSeveral state and local governments have added e-cigarettes to their smoke-free laws. However, misleading industry marketing, acceptance of e-cigarettes by some public health advocates as a harm-reduction strategy for smokers, and slow regulation by the US Food and Drug Administration have contributed to the public’s perception that e-cigarette aerosol is low risk or even harmless . Adults www.clearwaymn.org/MATS. MATS was approved by the Minnesota Department of Health’s institutional review board.Data came from the 2014 and 2018 administrations of MATS. MATS is a series of cross-sectional, random-digit–dial landline and cell phone surveys of civilian, noninstitutionalized adults aged 18 or older living in Minnesota. MATS data were collected in 1999, 2003, 2007, 2010, 2014, and 2018. The MATS 2014 final sample included 9,304 respondents; the response rate was 25.2% for the landline survey and 18.2% for the cell phone survey. In 2018, MATS included 6,055 participants and yielded American Association for Public Opinion Research (AAPOR) response rates of 17.5% for the landline survey and 13.4% for the cell phone survey. The same screening, sampling, and refusal conversion protocols were used in both survey years. Weighting was applied to create unbiased population estimates based on the probability of selection resulting from the sampling plan. Weights were calibrated based on sex, race/ethnicity, location, and education totals from the US Census Bureau’s American Community Survey ?” Vape-free rules were measured similarly: “Which statement best describes the rules about using e-cigarettes or vaping devices inside your home (excluding porches and garages)?” Responses included not allowed anywhere, allowed in some places or at some times, or allowed anywhere. Only respondents who indicated that smoking or vaping was not allowed anywhere were considered to have rules against that activity.Covariates were age (4 categories), sex , race/ethnicity , education (4 categories), annual household income (4 categories), marital status (married or not), current smoker of cigarettes, cigars, or pipe (yes or no), current e-cigarette user (yes or no), and lives with a child younger than 18 years (yes or no). Respondents who reported currently smoking cigarettes, cigars, or pipe every day or some days and met minimum lifetime-use thresholds  were categorized as current smokers. Similarly, respondents who reported currently using e-cigarettes every day or some days were categorized as current e-cigarette users; however, no minimum lifetime use was required.2 to assess the change from 2014 to 2018 in the percentage of Minnesota adults with smoke-free rules and assess the bivariate association between smoke-free and vape-free rules and respondent characteristics. We used logistic regression to assess the unique association between respondent characteristics and smoke-free rules and vape-free rules in 2018. We entered the entire set of potential predictors simultaneously, testing for main effects only. Ordinal variables were dummy coded. We used odds ratios (ORs) and 95% CIs to estimate the likelihood of adults reporting smoke-free or vape-free rules.We performed all analyses using SPSS Statistics version 24 (IBM Corporation) for complex samples. We used pairwise deletion to maximize available data. We used the Pearson χ12 = 21.8; P < .001). Current smokers were less likely than nonsmokers to report having smoke-free rules . The percentage of smokers with smoke-free rules in 2018 was not significantly different from the percentage observed in 2014 .The prevalence of smoke-free home rules among Minnesota adults in 2018 was 91.5% , up slightly from 89.3%  in 2014  reported vape-free home rules in 2018. Having vape-free rules was significantly associated with all respondent characteristics assessed . The preAssociation of respondent characteristics with smoke-free rules in the multivariate analysis. When we controlled for other variables in the model, respondent sex, annual household income, and e-cigarette use status were no longer associated with smoke-free home rules  have rules against vaping in the home, but as expected, this percentage was lower than the percentage of adults that have smoke-free home rules (91.5%). Although the prevalence of vape-free home rules is moderately high overall, our findings underscore the importance of targeting messages to segments of the population that do not have vape-free home rules. The discrepancy between smoke-free and vape-free rules may be due to the belief by many that electronic cigarettes are less harmful than conventional cigarettes . This beDisparities in vape-free rules show marked similarity to historical disparities in smoke-free rules ,7,15. Ouclean air rules in the home — no smoking or vaping in the home —especially in homes where children live. Smoke-free home rules have played a unique role in discouraging smoking  by reduOur study has limitations. MATS did not assess the perceived harmfulness of secondhand aerosol or whether others in the respondent’s household vape, so we could not include these variables — which are likely to be associated with vape-free rules — in our analysis. Our study relies on self-report, and self-reported data are subject to some degree of social desirability and recall biases. Our data are from a state with strong antismoking norms and policies, so the findings may not generalize nationally or to other states.Although widespread adoption of voluntary smoke-free and vape-free home rules demonstrates positive social norm change, most e-cigarette users in our study allowed vaping in their homes, including those who live with children younger than 18 years. Tracking voluntary smoke-free and vape-free home rules is an important component of tobacco control and demonstrates where resources should be directed to improve the public’s health.


Bos taurus ssp. taurus x B. t. ssp. indicus) calves were compared with the performance of traditional PPD-tuberculin in both the skin test and in vitro interferon-gamma release assay (IGRA). The IFN-γ production from whole blood cells stimulated with both PPDs increased significantly from the 0 week baseline levels, while DST induced no measurable IFN-γ production in BCG-vaccinated calves. None of the 15 BCG-vaccinated calves were reactive with the DST skin test . In contrast, 10 of 15 BCG-vaccinated calves were classified as reactors with the PPD-based single intradermal test (SIT) . Taken together, the results provide strong evidence that the DST is highly specific and enables DIVA capability in both skin and IGRA assay format, thereby enabling the implementation of BCG vaccine-based bTB control, particularly in settings where test and slaughter remain unfeasible.In most low- and middle-income countries (LMICs), bovine tuberculosis (bTB) remains endemic due to the absence of control programs. This is because successful bTB control and eradication programs have relied on test-and-slaughter strategies that are socioeconomically unfeasible in LMICs. While Bacillus Calmette–Guérin (BCG) vaccine-induced protection for cattle has long been documented in experimental and field trials, its use in control programs has been precluded by the inability to differentiate BCG-vaccinated from naturally infected animals using the OIE-prescribed purified protein derivative (PPD)-based tuberculin skin tests. In the current study, the diagnostic specificity and capability for differentiating infected from vaccinated animals (DIVA) of a novel defined antigen skin test (DST) in BCG-vaccinated ( Mycobacterium tuberculosis complex (MTBC) in cattle. The disease poses a significant threat to public health and limits livestock productivity, resulting in an estimated economic burden of $3 billion globally, annually (Bovine tuberculosis (bTB) is a chronic, granulomatous, inflammatory disease that is predominantly caused by members of the annually . While bannually –4. Furthannually –7. Thus,M. bovis that was initially isolated from the udder of a tuberculous cow  at the Pasteur Institute and since has been used as a vaccine against TB in humans. First evaluation of BCG against bTB in cattle occurred 10 years prior, in 1912, and following promising initial reports, the vaccine has been extensively evaluated in both experimental and field trials. Encouragingly, numerous reports have demonstrated BCG-induced protection in field trials conducted in many different countries and settings  is a live attenuated strain of lous cow , 9. ThisM. bovis strain, including those antigens that are broadly cross-reactive with the BCG vaccine (M. avium subsp. avium strain) is used to improve specificity, although this is known to compromise sensitivity  is the international standard and is based on the use of PPD bovine (PPD-B)-tuberculin that represents culture media sterile filtrates containing a large number of antigens from a defined  vaccine . Upon insitivity . Also, ain vitro .Efforts in recent years have been focused on developing BCG-compatible diagnostic tests with the ability to differentiate infected from vaccinated animals (DIVA). Genomic and transcriptomic analyses have identified ESAT-6, CFP10, and Rv3615c as promising antigens for differential diagnosis of bTB , 15. Recin vitro assays in the face of BCG vaccination, suggesting that application of DST provides an opportunity to consider BCG vaccine-based bTB control programs in endemic settings, especially where test and slaughter are unfeasible.Hence, the current investigation was designed to assess the utility of DST as a DIVA skin test in the face of BCG vaccination. The results show that DST possesses high specificity both as a skin test reagent and in M. bovis viz, ESAT-6 , CFP-10 , and Rv3615c . A total of 13 peptides representing these three antigens were commercially synthesized at >98% purity by GenScript USA, Inc. and USV Private Limited, India (in vivo safety of DST was confirmed in B. taurus ssp. indicus under Good Laboratory Practice (GLP) conditions in India with repeat and overdosing experiments (unpublished).Bovine tuberculin (PPD-B) and avian tuberculin (PPD-A) were purchased from Prionics, Thermo Fisher, Schlieren, Switzerland. The defined antigen skin test (DST) is a peptide-based cocktail representing three antigens from B. taurus ssp. taurus X B. taurus ssp. indicus) were recruited from bTB-free farms near Chennai, India. The population of crossbred cattle in India increased by 26.9% in 2019 as compared to the previous census  at TANUVAS and Committee for the Purpose of Control and Supervision of Experimental Animals . The trial timeline is shown in The trial was conducted in India under field  conditions. In order to assess the skin test performance of DST, 3- to 6-month-old crossbred calves (s census . They ar6 CFU) of BCG Danish delivered in 0.5 ml, at ~3 to 6 months of age. This dose was decided based on previous studies that have shown relatively low doses of 104-106 CFU administered subcutaneously to be efficacious in inducing protective immunity   were injected in a 0.1-ml volume as recommended by the manufacturer and the OIE. The DST injection is composed of 10 μg of each of the 13 peptides . The dose was based on prior dose titration experiments performed in cross-bred calves (indicus) . The samindicus) .TM kit was used for the IFN-γ ELISA. The tuberculin antigens (purchased from Thermo Fisher), PPD-B and PPD-A, were used for in vitro stimulation of whole blood at a final concentration of 300 and 250 IU/ml, respectively, as per the kit instructions (antigens provided with the BOVIGAMTM kit were not used in the assays). The DST was used at 10 μg/ml in in vitro assays. Blood was collected for IGRA just prior to BCG vaccination at week 0, and just prior to skin test at weeks 4, 6, and 16. Whole blood was stimulated overnight  with antigens (PPDs and DST) in vitro. BOVIGAMTM enzyme-linked immunosorbent assay-based kits  were used to determine IFN-γ concentrations in whole blood culture supernatants. Results for antigen-stimulated cultures were expressed as background-corrected optical density at 450 nm .Bovine tuberculin (PPD-B) and avian tuberculin (PPD-A) antigens were purchased from Thermo Fisher Scientific. The BOVIGAMP = 0.05) was used for comparing delta IFN-γ OD response between pre- and post-vaccination time points. The IGRAs were performed at four time points during the duration of the trial, the area under the curves  . Confidee curves  were detin vitro assays, measurement of antigen-induced IFN-γ responses was performed using whole blood collected at weeks 0, 4, 6, and 16 post-vaccination from all BCG vaccinates and controls. Responses to avian and bovine PPD (PPD-A and PPD-B) were measured to determine vaccine-induced IFN-γ response To determine the diagnostic performance of DST in In stark contrast with the results of the PPDs, whole blood stimulated with DST did not result in any appreciable IFN-γ responses in either BCG vaccinated or unvaccinated control calves at any of the observed time points, suggesting that the DST is highly specific and exhibits no cross-reactivity in BCG vaccinated calves .We next tested specificity of DST in skin tests in BCG-vaccinated calves. Skin test responses to DST, PPD-A, and PPD-B were recorded 6 weeks post-BCG vaccination, the time point with the highest cell-mediated responses . The DSTBCG is the most widely used of all vaccines today (and the only vaccine available against human TB). While BCG is licensed for use in wildlife , it is nM. bovis isolate was made to undergo ~230 serial passages that resulted in its loss of virulence in various animal models and consideration for use as a vaccine in both cattle and humans  and demonstrated its considerable sensitivity in reliably detecting controls . Howevercontrols .Mycobacterium avium ssp. paratuberculosis in these geographies , it would be expected to provide superior specificity over PPD-B alone as observed in our current studies. It is also important to note that given the cross-reactivity between PPD-B and PPD-A, there are serious implications for the sensitivity of CCT in settings such as in India where there is likely a high burden of environmental mycobacteria. In such conditions of high background environmental mycobacterial exposure that drives PPD-A reactivity, CCT would result in a high proportion of false-negative results whereas SIT, performed with PPD-B alone, would result in a high proportion of false positives  in India to PPD-A and PPD-B at the start of the study  suggest pre-sensitization from high levels of exposure to environmental mycobacteria and pathogenic mycobacteria, including graphies , 28. Whie to DST  29). Al. AlMycob derived . Contrarositives . Hence, Another interesting observation is that animal IDs 69 and 80 remained relatively unresponsive to BCG immunization based on the trajectories of their immune responses as measured by IGRA over time . While tIn summary, this study confirms that DST allows not only discrimination between infected animals and those environmentally sensitized but also its utility as a much-needed DIVA test reagent in a challenging geographical context. Thereby, the DST has the potential to overcome a major hurdle for the implementation of BCG cattle vaccination programs as an intervention strategy for disease control.All datasets generated for this study are included in the article/The animal study was reviewed and approved by Institutional Animal Ethics Committee (IAEC) at TANUVAS and Committee for the Purpose of Control and Supervision of Experimental Animals .SSr, DG, KK, MVe, DB, MVo, and VK conceptualized and designed the study. SSr, SSu, SSh, MJ, and SK performed the experiments. KR, VM, SSe, and MVe carried out the field work. SSr analyzed the data. SSr drafted the paper. SSr, DB, MVo, and VK contributed to writing. All authors reviewed the manuscript.Animal and Plant Health Agency (APHA), the employer for MVo, holds three patents  for the use of Rv3615c in diagnostic tests for bTB. In addition, APHA (MVo) and Penn State (SSr and VK) are in the process of intellectual property protection filings for the DST. The remaining 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.


Objective: To compare the survival outcomes brought by different radiation dose schedules to bone lesions and different chemotherapy regimens in bone metastatic nasopharyngeal carcinoma (NPC).Background: The current treatment strategy for bone metastatic NPC patients was empirically given and poorly studied before. It is of necessity to optimize the treatment for bone metastasis to enhance the therapeutic effect and increase the proportion of long-term survived patients.Methods: Three hundred patients who received chemoradiotherapy from 2002 to 2018 were involved in the study. Demographics, laboratory results, and detailed treatment plans were recorded. Radiotherapy plans were classified into three categories based on the intensity, and the survival analysis was performed using log-rank test. Multivariable analysis was made by the Cox proportional regression model.Results: Patients who received 60–75 Gy/30–35 fractions of radiation to the metastatic bones had significantly longer bone relapse-free survival (BRFS) , overall survival (OS) , and progression-free survival (PFS) . The administration of paclitaxel, cisplatin and 5-fluorouracil regimen was also associated with better BRFS , PFS , and OS with borderline significance . In multivariable analysis, the post-treatment EBV DNA level and radical radiation dose were proved as independent prognostic factors for both BRFS and OS.Conclusions: Radiotherapy to metastatic bones with palliative dose prescription should not be considered in bone metastatic NPC patients. TPF chemotherapy regimen might help to improve the survivals in NPC patients but failed to be an independent protective factor. Nasopharyngeal carcinoma (NPC), which stems from the epithelium of nasopharynx, is a special subtype of head and neck cancers. Characterized by the poor differentiated nature, NPC is highly sensitive to chemoradiotherapy and excellent locoregional control rates can often be achieved in non-metastatic NPC. Nonetheless, distant metastasis is the major threat and cause of death faced by all NPC patients. It's reported that nearly 10% newly-diagnosed patients presented with synchronous distant metastasis  and apprP = 0.001), and similar results were also observed in Shen et al.'s (Radiotherapy (RT) to metastatic bones is widely applied to relieve pain, prevent skeleton-related events (SRE) and improve quality of life among various cancer types. Accumulated evidences have pointed out the survival benefit brought by local radiotherapy to BM plus chemotherapy in NPC patients. In Liang et al.'s study , a signiet al.'s  and He eet al.'s . Althouget al.'s . From siHerein, we investigated the real-world therapeutic strategy for bone metastatic NPC patients who received chemoradiotherapy, and a retrospective cohort study was performed with an attempt to find out the optimal chemoradiation plan and yield insight into future studies to establish specific guidelines.Patients treated in Sun Yat-sen University Cancer Center from January 2002 to December 2018 were consecutively evaluated for their eligibility. The diagnosis of BM was determined by at least one of the following examinations including computed tomography (CT) with contrast, magnetic resonance imaging (MRI) with contrast, positron emission tomography-computed tomography (PET/CT) and histologically proven metastatic lesion. The inclusion criteria were: (1) Patients were previously or concurrently diagnosed as NPC with pathological evidence. (2) Patients who had secondary BM received radical radiotherapy to the nasopharynx as an initial treatment. (3) Radiotherapy to the BM was performed. (4) Karnofsky performance status (KPS) ≥70. Patients were excluded if any of the following condition was met: (1) Radiotherapy was stopped halfway for any reason. (2) Coexistence of a second malignancy. (3) Incomplete medical records. (4) Patients who showed no evidence of progression were lost to follow up within 3 months after the BM-directed treatment. This study was approved by the Sun Yat-sen University Cancer Center Clinical Research Ethics Committee.All patients received multi-modality treatment including radiotherapy and chemotherapy. Chemotherapy was administered every 3 weeks for at least 4–6 cycles before the radiotherapy. Chemotherapy regimens included TP, paclitaxel plus cisplatin; PF, cisplatin plus 5-fluorouracil; GP, gemcitabine plus cisplatin; and TPF, paclitaxel plus cisplatin and 5-fluorouracil. Carboplatin and nedaplatin were also applicated as substitutes for cisplatin. If several regimens were applied, the regimen with which patients achieved major response was recorded. The adopted radiotherapy techniques for BM ranged from 2-dimentional (2D-RT) or 3-dimentional radiotherapy (3D-RT), intensity modulated radiotherapy (IMRT), volumetric modulated arc therapy (VMAT) to tomotherapy (TOMO). The dose-fractionation patterns were heterogeneous among patients from single fractionation schedule to radical dose regimen. In addition, all initially diagnosed patients received radiotherapy to the nasopharynx and neck. The prescribed dose to the gross tumor volume was 66–70 Gy and 60 Gy to the clinical target volume. The zoledronic acid was given to some patients with a dosage of 4 mg every 3–4 weeks. Epidermal growth factor receptor (EGFR) inhibitors such as cetuximab and nimotuzumab, immune checkpoint inhibitors including antibodies of CTLA-4, programmed cell death receptor (PD-1) and its ligand (PD-L1) and anti-angiogenic agents like endostar and apatinib were also considered as supplement to promote the therapeutic effect.in-situ bone relapse-free survival (BRFS), which referred to the interval from the diagnosis of BM to the in-situ relapse of the radiated bones. The second endpoints measured overall survival (OS) and progression-free survival (PFS), which, respectively referred to the interval from the diagnosis of BM to death caused by any reason or any tumor progression. Patients were censored at the date of last follow-up.Demographics, laboratory results and detailed treatment plans of all patients were recorded. If multiple BM relapses occurred, the information collected and evaluation was subjected to the first episode. The pre-treatment levels of ALP, lactic dehydrogenase (LDH), and EBV DNA were measured at the time when the BM was detected. The plasma EBV DNA test was not performed in patients treated before 2007. The osteolytic lesion was defined when the BM appeared as disrupted trabecular structure or area of faint density, while the osteogenic lesion was distinguished by sclerotic change or enhanced density. The follow-up information of all patients was collected. The primary endpoint was P-value of <0.05 was considered statistically significant.Continuous variables were dichotomized to categorical variables before analysis. The cut-off value of LDH was categorized using the upper normal limit 250 U/L), and the cut-off values of ALP and EBV DNA were, respectively determined as 110 U/L and 4,000 copies/ml based on previous literatures 50 U/L, a, 17. Thein-situ with or without invasions to other bones or organs.Clinical data of all patients who were diagnosed with bone metastatic nasopharyngeal carcinoma in Sun Yat-sen University Cancer Center from January 2002 to December 2018 were evaluated. After the inclusion and exclusion procedure, 300 patients were eligible for analysis . The basP = 0.003), OS , and PFS  (P = 0.004). Concerning the effect of fractionated dose on survival, it was found that a fractionated dose of ≤ 2 Gy helped to extend the BRFS , but brought no benefit to the OS and PFS.All patients accepted multimodality systemic treatment and radiotherapy was prescribed to the BM. The efficacy of different radiotherapy plans and chemotherapy plans were compared in = 0.041) . In addiin-situ relapse . But in the meantime, radical radiotherapy failed to bring BRFS benefits to patients with osteolytic lesions or with soft tissue involvement  and the absence/presence of soft tissue involvement. For patients with osteogenic BM, radical radiation plan significantly reduced the occurrence of olvement .Most of patients (91.7%) received platinum-based chemotherapy. The number and proportion of patients receiving TP, PF, GP, and TPF were 78 (25.4%), 75 (24.4%), 44 (14.3%), and 68 (22.1%), respectively. EGFR inhibitors such as cetuximab and nimotuzumab were administered to 59 (19.7%) patients, immune checkpoint inhibitors including CTLA-4, PD-1, and PD-L1 antibodies were given to 24 (8.0%) patients and anti-angiogenic agents to five (1.7%) patients. Furthermore, zoledronic acid was administered to 193 (64.3%) patients.P = 0.007) and PFS  benefits were achieved with the administration of TPF regimen compared to other chemotherapy plans (P = 0.058). As it was found that TPF was mostly administered to newly-diagnosed patients , we further explored the efficacy of TPF in newly-diagnosed cohort, and patients using the TPF regimen tended to exhibit better OS, PFS in contrast to other chemotherapy regimens (P = 0.009), but didn't show superiority in terms of PFS and BRFS . With regard to the treatment strategy, the administration of TPF regimen failed to be a prognostic variant, but radiotherapy to BM with radical dose was proved as an independent protective factor for both BRFS  and OS .Univariable analysis was performed first to screen out potential prognostic factors for BRFS and OS, and the multivariable analysis was made thereafter . CompareAs good results have been achieved in the treatment of locoregionally advanced nasopharyngeal carcinoma, more attention should be paid to the management of metastatic NPC. The present study is the first one to explore the combinatorial treatment strategy for bone metastatic patients, and found that the intensity of dose prescription to the BM was strongly correlated to the BRFS, OS, and PFS. Meanwhile, the survival results in terms of BRFS, OS and PFS favored the administration of TPF regimen. Patients benefited most from the combination of radical radiation dose and TPF chemotherapy. Furthermore, radical radiation dose to the BM was proved to be an independent protective factor.in-situ recurrence, the radical dose prescription significantly improved the OS and PFS. In summary, patients' predicted life expectancy and treatment goals should be considered when giving local radiotherapy. For patients with a chance of long-term survival, radical radiotherapy to the BM was preferred.The appropriate dose prescription and fractionation to the BM has been discussed in many cancers. Although multiple studies demonstrated the equivalent effect of short fractionation schedules (including single fraction) on pain palliation, the situation might be different in treating NPC patients. As our results showed that 170 (56.7%) patients had asymptomatic BM, relieving the pain was not the major concern for giving radiotherapy. Besides, many patients presented with oligometastasis, and longer survival durations were often observed among them, thus the short fractionation schedules, which sometimes required the need for retreatment , were leWith respect to the optimum chemotherapy regimens for metastatic NPC, it had been inconclusive and the PF regimen was the most empirically-used regimen for over decades . Zhang eThe study had several limitations. Primarily, because of the retrospective nature, selection bias may exist as clinicians were more inclined to give aggressive combinatorial treatment to patients with less metastatic burdens and satisfactory performance status. Secondly, given the scarcity of bone metastatic patients receiving local radiotherapy, we had to include patients with metastasis to other organs to guarantee the sample size for analysis, which might be a confounding variable while calculating PFS and OS. Furthermore, as the data was the representative of treating experience from single institute where the short-course hypofractionated (including single fraction) radiotherapy plans were seldomly used, the true efficacy of them in NPC patients remained unknown. Therefore, future studies which focus on the multi-modality treatment for bone metastatic NPC patients are in urgent need.In conclusion, this study showed that the intensity of radiation dose to the BM was strongly associated with the BRFS, OS, and PFS, and it remained independently protective in multivariable analysis. The administration of TPF regimen might help to improve OS, PFS and BRFS compared with other platinum-based regimens, and patients benefited most from the combination of radical local radiation and TPF chemotherapy.The datasets generated for this study are available on request to the corresponding author.This study was approved by the Sun Yat-sen University Cancer Center Clinical Research Ethics Committee.H-QM and L-QT: study concepts. X-YL, J-JY, and G-DJ: study design. X-SS, S-LL, and S-SG: data acquisition. L-TL: quality control of data and algorithms. X-YL, X-SS, and G-DJ: data analysis and interpretation. X-SS and X-YL: statistical analysis. X-YL, RS, and D-HL: manuscript preparation. X-SS, S-LL, and H-YM: manuscript editing. Q-YC and LG: manuscript review.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. The reviewer XL declared a shared affiliation, with no collaboration, with the authors to the handling editor at time of review.


Mycobacterium tuberculosis challenges the goal of elimination of tuberculosis in the near future. For the last 50 years, management of patients with tuberculosis has followed a standardized management approach. This standardization neglects the variation in human susceptibility to infection, immune response, the pharmacokinetics of drugs, and the individual duration of treatment needed to achieve relapse-free cure. Here we propose a package of precision medicine-guided therapies that has the prospect to drive clinical management decisions, based on both host immunity and M. tuberculosis strains genetics. Recently, important scientific discoveries and technological advances have been achieved that provide a perspective for individualized rather than standardized management of patients with tuberculosis. For the individual selection of best medicines and host-directed therapies, personalized drug dosing, and treatment durations, physicians treating patients with tuberculosis will be able to rely on these advances in systems biology and to apply them at the bedside.Tuberculosis is a bacterial infectious disease that is mainly transmitted from human to human via infectious aerosols. Currently, tuberculosis is the leading cause of death by an infectious disease world-wide. In the past decade, the number of patients affected by tuberculosis has increased by ~20 percent and the emergence of drug-resistant strains of  Tuberculosis is the leading cause of death attributed to a single microbial pathogen world-wide . The WorMycobacterium tuberculosis. Treatment was empiric or adopted to results of phenotypic drug-susceptibility testing that takes several weeks to months to become available. Patient-tailoring was limited to adjusting dosing to body weight, mostly in pediatric patients  nexiagnosis ; (II) hoiagnosis –9; or otiagnosis , (III) iiagnosis , and  and small insertions/deletion (indels). Acquisition of resistance genes via plasmids or horizontal gene transfer does not occur  in the future (M. tuberculosis complex strains has become possible by advances of next generation sequencing (NGS) techniques. NGS allows for rapid sequencing of full genomes or targeted sequencing of resistance genes (amplicon sequencing) even directly from patient specimens, i.e., sputum  and ReSeqTB (platform.reseqtb.org) are currently collecting genome sequencing data of global strain collection in combination with phenotypic DST data to further improve genotypic resistance predictions. Here, sequencing approaches represent an effective alternative to rapidly generate comprehensive resistance profiles of multidrug-resistant (MDR) strains allowing the design of a successful MDR-TB therapy for around 90% of the MDR-TB patients -12, interferon (IFN)-γ or tumor necrosis factor (TNF) pathways, which result in decreased intracellular killing of rculosis , 23. Howar space , 24–26. ar space , but theIn the pre-antibiotic era, treatment for tuberculosis was limited to host-directed therapy aiming at improving the nutritional and immune status of patients, e.g., with cod liver oil and sunlight , resultiEndotype characterization studies likely will need to occur at disease diagnosis and again weeks into therapy. Tuberculosis is the archetypical chronic infection, hence host immunity after months of chronic antigenic stimulation is unlikely to resemble host immunity at the beginning of infection. Upon initial antigenic stimulation, modulated by inositol triphosphate receptors, there is an influx of calcium that helps stimulate glycolysis and activates nuclear factor of activated T-cells (NFAT) to translocate into the nucleus  as an exHost genetic studies have thus far largely focused on susceptibility to active tuberculosis , 53, to With regard to anti-tuberculous treatment, genetic studies have focused on drug exposure and toxicity (“pharmacogenetics”). The strongest association established is between N-acetyltransferase (NAT)2-acetylator status and isoniazid drug concentrations, microbiological failure, relapse , and hepIn the most extreme disease phenotypes, targeted HDT may be most relevant. A Leukotriene A4 hydrolase (LTA4H) promotor genotype, important for the balance between pro- and anti-inflammatory eicosanoids , predictM. tuberculosis growth and central nervous system inflammation, was associated with higher mortality. Using a genome-wide association approach, so-called quantitative trait loci (QTLs) that associate with cerebrospinal fluid tryptophan levels were identified, and those same QTLs predicted survival in 285 other patients (https://grantome.com/grant/NIH/R01-AI145781-01). Other “omics” data that could be used in such an integrated approach are transcriptional data or proteomics.Genetic studies can be strengthened by an integrated or “cross-omics” approach. Among patients with tuberculous meningitis, using unbiased metabolomics, it was found that higher cerebrospinal fluid tryptophan, which can affect patients . The relM. tuberculosis genotype  is the concept of individualizing drug dosing by measuring the drug concentration in a patient's serum/plasma and adjusting the dose accordingly.Consequently, TDM is indicated in settings with a risk of low or high tuberculosis drug exposures: presence of altered drug absorption, distribution, metabolism, or excretion , comorbidities that may affect exposure to anti-tuberculosis drugs , drug-dLow anti-tuberculosis drug concentrations can lead to inefficient mycobacterial killing, treatment failure, relapse and selection of drug resistance , whereasThe TDM process comprises (1) obtaining blood samples, (2) measuring drug concentrations, and (3) interpreting the results.The optimal sampling time points and the number of samples depend on the assessed drug. Limited sampling strategies (LSS) predict the most informative sampling time points based on population pharmacokinetic data, i.e., “average” exposures from tuberculosis patients. With the help of LSS, sampling can often be reduced to three or less time points , 70.Analysis of the serum/plasma samples is ideally performed with high performance liquid chromatography mass spectrometry (HPLC-MS). HPLC-MS is highly sensitive and specific and allows simultaneous analysis of several tuberculosis drugs in one run of the assay .M. tuberculosis strain, as reflected in the minimal concentration that inhibits growth of M. tuberculosis . Dependent on the drug's mode of action, targets are defined as area under the curve (AUC)/MIC, maximal concentration (Cmax)/MIC, or time above MIC (T>MIC). Unfortunately, specific targets are not yet available for anti-tuberculosis drugs and MIC is seldomly determined. Alternatively, drug concentrations are compared to population pharmacokinetic data  with TDM data is a very promising concept to provide tailored high-dosage therapies in cases of low level bacillary drug resistance , 72. Morin-vitro models that help to substantiate target values  or the line-probe assays  are commercially available as rapid diagnostic methods that permit drug-resistance prediction for important first- and second-line drugs , 77. As Immunological assays could improve outcomes in populations in whom diagnosis is very difficult, such as children in whom reports of the T-cell activation marker (TAM-TB) test suggest a sensitivity and specificity of ~83 and 97%, respectively . ComplexIn addition, certain computer-aided diagnostic (CAD) tools may be able to identify tuberculosis patients with high accuracy (specificity of 98% and of sensitivity 90%) from digital chest-X-ray images .Mycobacterial culture is the most relevant measure of treatment response, but mycobacterial growth is slow and, as treatment progresses, the time to a positive result increases . Here, rM. tuberculosis-specific blood T cells may be able to inform about treatment efficacy as shown in adults and in children . The remaining 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.


Objective: To evaluate the current literature on the accuracy of fluoro-2-deoxy-D-glucose positron emission tomography-computed tomography (FDG PET-CT) for lymph node (LN) staging in urothelial carcinoma (UC), as robust evidence on the value of this technology in UC is still lacking.Methods: The Medical Literature Analysis and Retrieval System Online (MEDLINE)/PubMed, Cochrane Library, and Scopus databases were searched for eligible studies. We included all original studies evaluating FDG PET-CT in bladder or upper tract UC. The search results were restricted to the English language, and included prospective and retrospective studies without time restriction. We included only studies reporting the sensitivity and specificity of FDG PET-CT in detecting UC LN metastases.Results: We identified 23 articles meeting our inclusion criteria. In the preoperative setting, the sensitivity of FDG PET-CT for detecting LN metastases in patients with bladder cancer was widely variable ranging from 23% to 89%; the specificity ranged from 81% to 100%; and the overall accuracy ranged from 65% to 89%. During bladder cancer monitoring the sensitivity for detecting LN metastases ranged from 75% to 92% and the specificity ranged from 60% to 92%. The sensitivity for LN staging in upper tract UC ranged between 82% and 95%, with a specificity of 84–91%.Conclusion: Despite the inconsistencies in sensitivity between the reports, FDG PET-CT seems to have a high specificity for LN staging in patients with UC. Future prospective, well-designed studies are necessary to evaluate the role of FDG PET-CT in UC management.Abbreviations: FDG: fluoro-2-deoxy-D-glucose; LN: lymph node; PET: positron emission tomography; PRISMA: Preferred Reporting Items for Systematic Reviews and Meta-analyses; PSMA: prostate-specific membrane antigen; (N)(P)PV: (negative) (positive) predictive value; QUADAS-2: Quality Assessment of Diagnostic Accuracy Studies-2; SUVmax: maximum standard uptake value; (UT)UC: (upper urinary tract) urothelial carcinoma Urothelial carcinoma (UC) arises from the urothelium of the urinary tract potentially affecting the urinary bladder in 90–95% of cases and, less commonly, the upper urinary tract in 5–10%  . In the D-glucose (FDG) has been used to improve the staging and monitoring of patients with different malignancies, e.g. lung, breast, colorectal, as well as UC [In the last decade, positron emission tomography (PET) with the glucose analogue fluoro-2-deoxy-ll as UC . PET meall as UC . Metastall as UC . FDG PETll as UC . HoweverThis systematic review aimed to evaluate the diagnostic accuracy of the FDG PET-CT technique in the staging and monitoring of UC. We performed this review according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) statement . A comprElectronic databases were searched, including the Medical Literature Analysis and Retrieval System Online (MEDLINE)/PubMed, and Scopus. The databases of systematic reviews, e.g. the Cochrane libraries and Center for Reviews and Dissemination, were screened for eligible primary studies in April 2020, and saved for further screening and in-depth reading. The following keywords were used: ‘transitional cell carcinoma’, ‘urothelial cancer’, ‘urologic neoplasms’, ‘upper tract’, ‘upper tract urothelial cancer’, ‘bladder cancer’, OR ‘urinary tract cancer’, ‘urothelial cancer’, ‘FDG’ OR ‘Fluorodeoxyglucose’, ‘FDG-PET’, ‘PET-CT’.The flow of the information through the different stages of a systematic review  is shown in the PRISMA flow chart .Figure 1Data from each study were extracted by two independent reviewers. Data were collected from the included studies into data extraction tables, which were designed for this review and contained: author name, year of publication, study design, sample size, characteristics of the imaging technique, performance measures , and any relevant comment of the included study.The risk of bias was evaluated according to the Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2) tool. This tool is based on four domains: patient selection, index test, reference standard, and the timing of reference test. Also, the QUADAS-2 tool assessed applicability concerns of   on FDG-PET scans in 131 patients with bladder cancer. The SUVmax >2 FDG-PET scans had a sensitivity and specificity of 79% and 66%, respectively. In contrast, a SUVmax of >4 FDG-PET had a sensitivity and specificity of 61% and 84%, respectively, which may help in identifying patients with a poor prognosis .Tet al. [et al. [et al. [The performance of FDG PET-CT in the evaluation of UTUC has been examined in three retrospective studies ,21,22. T [et al.  evaluate [et al. ,21,22).TThe introduction of any technique to clinical practice requires proving the diagnostic accuracy with a low risk of harm. Non-invasive diagnostic techniques such as CT or MRI were found to be inadequate in the diagnosis and staging of UC . New tecet al. [Regarding the LN staging of UC, the variability in the sensitivity measures was more evident than in the monitoring phase. The highest sensitivity was estimated at 95%, while the lowest sensitivity was 7–23% . On the et al. , where 2Our present result is consistent with what has been found with the use of PET-CT with radiolabelled prostate-specific membrane antigen (PSMA) for diagnostic assessment of patients with prostate cancer . PSMA PEThe ability to exclude false positives in the staging is an advantage of this technique. While in staging tumours, specificity is more important than sensitivity because false positives can be subjected to unnecessary invasive investigations such as biopsies. Nevertheless, missing cancer cases due to low sensitivity may result in delayed treatment and associated adverse effects. Thus, several sequential diagnostic modalities are recommended to improve the net sensitivity of staging LN of UC. These findings demonstrate that studies assessing the role of PET-CT in LN monitoring revealed comparatively high sensitivity and specificity values of PET-CT in surveillance and proved its capability to impact individual treatment plans and change the clinical decision.et al. [18F-FDG is caused by inflammatory states and not by cancer.Generally, FDG PET-CT has higher diagnostic values in cancer monitoring than in cancer staging . This caet al. , who fouet al. . FurtherThe approach utilised suffers from the limitation that the included studies are mostly retrospective. Other limitations include heterogeneous patient populations, non-standardisation of techniques, variable follow-up durations, and different confirmatory tests. Furthermore, most of the included studies lacked statistical power due to small samples, which are far lower than the sample size recommended for diagnostic studies. There is a need for further well-designed studies to accurately determine the diagnostic accuracy of FDG PET-CT in patients with UC.The use of FDG PET-CT appears to have a high sensitivity for both UTUC and bladder UC in the detection of LN metastasis. Despite the high specificity of FDG PET-CT, the detection of LNs is still in the range of conventional images. Albeit the role of PET-CT could be considered as promising, more research is required to establish it as the imaging modality of choice, or at least to recommend its routine use in a selected group of patients.


Skull-base chordomas (SBC) are rare tumours with unfavourable outcomes, even when undergoing advanced treatments such as carbon-ion radiotherapy (CIRT). By retrospectively analysing imaging , treatment (dose maps) and clinical information available before treatment, the potential use of radiomics and dosiomics for risk modelling targeting SBC treated with CIRT was explored. Despite the small sample size, dosiomic features appear to be promising factors related to local control in SBC, with worse outcomes being associated to higher dose heterogeneity. Risk models exploiting all sources of information showed slightly inferior but good performance, suggesting that multi-parametric approaches are worth being pursued for patient risk stratification. This study is put forward as groundwork for radiomic analyses targeting SBC in CIRT.Skull-base chordoma (SBC) can be treated with carbon ion radiotherapy (CIRT) to improve local control (LC). The study aimed to explore the role of multi-parametric radiomic, dosiomic and clinical features as prognostic factors for LC in SBC patients undergoing CIRT. Before CIRT, 57 patients underwent MR and CT imaging, from which tumour contours and dose maps were obtained. MRI and CT-based radiomic, and dosiomic features were selected and fed to two survival models, singularly or by combining them with clinical factors. Adverse LC was given by in-field recurrence or tumour progression. The dataset was split in development and test sets and the models’ performance evaluated using the concordance index (C-index). Patients were then assigned a low- or high-risk score. Survival curves were estimated, and risk groups compared through log-rank tests (after Bonferroni correction α = 0.0083). The best performing models were built on features describing tumour shape and dosiomic heterogeneity , followed by combined (0.73/0.30 and 0.75/0.27) and CT-based models (0.77/0.24 and 0.64/0.28). Dosiomic and combined models could consistently stratify patients in two significantly different groups. Dosiomic and multi-parametric radiomic features showed to be promising prognostic factors for LC in SBC treated with CIRT. Particle therapy makes use of charged particles such as protons or carbon ions and is increasingly being adopted worldwide, with over 50 facilities built in the last ten years . AlthougSkull-base chordoma (SBC) is a rare but aggressive tumour, locally invasive and highly recurrent . Given tRadiomics refers to the automatic extraction of quantitative imaging features, which can be divided in shape, first order, textural and filter-based features, according to the specific characteristic described, to develop predictive models . The genDespite its potential clinical usefulness, the radiomic paradigm has been applied to few studies targeting SBC, mostly focused on diagnostic tasks . PromisiThe aim of this study was to explore radiomic approaches for predicting local control in SBC treated with CIRT. Towards this goal, radiomic and dosiomic features were extracted from routinely acquired pre-treatment imaging and dose maps, which were selected, combined, enriched with clinical information, and fed to time-to-event models. Such framework is, therefore, put forward as the groundwork towards the identification of the most promising radiomic (and dosiomic) workflow for deriving prognostic factors of SBC response to CIRT.Imaging , treatment (dose maps) and clinical data was retrospectively collected for 57 SBC patients treated with CIRT. Local control (LC), i.e., favourable outcome, was found in 70% of the patients after a median follow-up time of 35.2 months (range: 2.9–66.07 months). Clinical features  were recn = 45) within a cross-validation procedure and further tested on the remaining 20% (n = 12) of the data, to evaluate the models on totally unseen samples. Data was split randomly but ensuring that the proportion of samples associated to adverse and favourable LC was equal .Survival models were developed exploiting 80% of the dataset . Performance was assessed in terms of the concordance index (C-index) computed from a stratified five-fold cross-validation (CV) routine over the training set  for model development and over the hold-out test set (test C-index).Selected T1w-MRI and T2w-MRI features  belongedSelected CT features  describeSelected dosiomic features  mostly dThe signatures  associatOverall, r-Cox performed better than s-SVM , being tFollowing single modality analyses, imaging, dose and clinical information with the best validation C-indices were combined to evaluate the performance of a multi-parametric scenario. In the best-performing cases of comboAll models, the correlation-based feature selection method favoured clinical and dosiomic features, whereas the PCA-based method retained features from all modalities . TexturaRegarding models’ performance, the best r-Cox performed slightly better than the best s-SVM (0.75/0.28 vs. 0.73/0.30), but they did not outperform the best single-modality dosiomic models.The survival curves  of high-In this study, survival models were investigated to stratify SBC patients treated with CIRT according to the risk of an adverse local control. Information available before treatment was exploited, including radiomic features extracted from T1w-MRI, T2w-MRI and CT, dosiomic features and clinical factors recorded according to the clinical practice.By exploring various feature selection routines, it was possible to investigate different feature signatures while mitigating the mismatch between number of features and sample size.To further account for the problem of limited sample size, s-SVM was the chosen machine learning model, as it is relatively robust to overfitting , and it Additionally, the limited test set available for the current study hinders the evaluation of the test C-index alone, which often reached its maximum value. However, this over-optimism is expected to fade once the hold-out test data is expanded.p-values obtained on the hold-out test set. Such behaviour may be explained by different factors. Firstly, MR-radiomic features were extracted from gross tumour volumes (GTV) that had been delineated on a fused MR-CT and rigidly registered to MRIs, as no direct manual contour was available for each MR modality. As such, the contour may not exactly match the MR-visible tumour due to registration errors. Moreover, MRI were retrospectively collected and, even if most of the acquisition parameters were matched, some of them  varied more if compared to acquisition and computation parameters of CT and dose maps, respectively. This and the lack of test-retest data may cause non-reproducible features to be fed to the models [The MRI features that led to the best MRI-based models described shape, histogram and textural properties , but were models ,32 whiche models . Nonethee models , from whe models ,34,35. Ie models . In the Although CT offers a lower soft tissue contrast than MRI, the selected CT features  showed cDosiomic features turned out to be the most promising signatures , as showModels based on clinical features did not outperform dosiomic models but showed comparable results to radiomic models. Clinical variables were limited to those available for most of the patients but other factors, missing from the current evaluation , may be beneficial and their impact of LC in SBC treated with CIRT should be investigated . ClinicaWhen combining all sources of information, features leading to the best models within each group  were merged, selected, and evaluated (comboAll). The best validation C-indices slightly lowered with respect to those from dosiomic models  but improved with respect to those from radiomic and clinical models. In the best comboAll cases, the selected clinical features were anatomical location, optic pathway involvement, and/or gender. This agrees with recent studies  that invThe clinical usefulness of dosiomic and comboAll models is supported by the separation of survival curves obtained for low- and high-risk groups. Only the dosiomic s-SVM model could significantly separate low- and high-risk patients in both re-training and hold-out test sets. However, the significant results observed in the re-training set, although over-optimistic, suggest that statistical significance may be found in larger hold-out test sets when using s-SVM models. As only a single stratification cut-off  was evaluated and these results should be considered cautiously , this anMoreover, given the relatively small sample size and the mono-centric and retrospective study design, the reported results need to be validated by broader analyses, considering additional data either coming from the same or an external institution . In the Other than technical aspects, clinical and biological validations need to be addressed before radiomic and dosiomic features can be employed as biomarkers for SBC . This isPatients affected by SBC and treated with CIRT between 2013 and 2016 at the National Center of Oncological Hadrontherapy  were retrospectively selected. Inclusion criteria were: (i) prescribed biological dose of 70.4 Gy(RBE) delivered in 16 fractions, (ii) the availability of clinical follow-up at least at three months, and (iii) availability of pre-treatment T1-weighted and T2-weighted  MRI, planning CT and planned biological dose maps. Plans were optimized with a commercial treatment planning system  using a pencil beam algorithm for physical dose calculation and the local effect model (α/β = 2 Gy) for computing the 3D relative biological effectiveness (RBE) . PatientThe LC was chosen as the clinical endpoint and was calculated from the last day of therapy to the date of event or censoring. Recurrence or disease progression in the target volume (adverse LC) was clinically assessed on radiological imaging at follow-up, and was considered an event for survival analysis, whereas progression-free evaluation (favourable LC) referred to censored data. Clinical features consisted of age at the time of treatment, gender, GTV, anatomical location , biopsy-Within the clinical planning procedure, gross tumour volumes (GTVs) were manually delineated for treatment planning on the planning CT, with the support of a fused MRI and following institutional guidelines. Since manual MRI contours were not available and T1w- and a T2w-MRI were acquired on the same day of the planning CT, GTV contours were conveyed to both T1w- and T2w-MRI through a rigid registration with CT imaging . Before n = 14) were computed from the GTVs segmented on the CT, whereas first-order (n = 18) and textural features (n = 75) were computed for every modality separately . Textural features described spatial intensity patterns from gray level co-occurrence (GLCM), gray level run length (GLRLM), gray level size zone (GLSZM), gray level dependence (GLDM), and neighbouring gray tone difference (NGTDM) matrices. For all the modalities, features were extracted from the GTV. Details on the feature extraction routines employed are provided in the Features were extracted using the open-source software pyradiomics (v2.2.0) , which cDifferent methods for dimensionality reduction were tested to mitigate the unbalance between number of features and sample size  and to iA machine learning survival model based on linear survival support vector machines (s-SVM)  was adopn = 45) were assigned to the development set, to evaluate the model building procedure, and 20% (n = 12) to the hold-out test set, to evaluate the models on totally unseen data , various follow-up time durations were present. Folds were created 10 times, each time with a different data split (repeated stratified five-fold CV). During the development phase, feature selection was performed, as reported in During models’ development, a five-fold cross-validation routine was defined so that, in each fold  was evaluated separately. Then, the single-modality signatures that were associated to the best validation C-index, within each modality, were retained; they were combined into a multi-parametric feature set, to which clinical features were added (comboAll); and the development procedure was repeated .Subsequently, the single-modality and comboAll models with the highest validation C-index were tested on the hold-out dataset. Since the cross-validated development phase did not provide a unique model as output, models were re-trained on the whole development set and tested on the hold-out dataset. This allowed evaluating the procedure on totally unseen data as, in the development stage, data used to evaluate the models in the validation folds had been previously used to select features and to optimize models’ hyper-parameters. In this phase, models were evaluated in terms of C-index computed over the hold-out testset (test C-index). n = 6) was applied to each model, thus leading to a corrected α = 0.0083.As for evaluating the clinical applicability of the proposed procedure, the stratification cut-off was set to be the median value of the model’s output in the re-training set, and it was applied to both re-training and test data. The estimated survival curves .Radiomic and dosiomic analyses predicting the risk of adverse LC in SBC treated with CIRT were implemented for the first time, integrating MRI, CT, dose maps, and clinical features. Dosiomic and combined features showed promising results in terms of performance and generalization abilities, but a thorough validation is needed before these models can be applied in the clinical practice. Nevertheless, the reported findings support further investigations on radiomic and dosiomic approaches which may improve the understanding of how CIRT treatment affects LC in SBC.


RNA binding proteins (RBPs) play a vital role in post-transcriptional processes in all eukaryotes, such as splicing regulation, mRNA transport, and modulation of mRNA translation and decay. The identification of RBP binding sites is a crucial step in understanding the biological mechanism of post-transcriptional gene regulation. However, the determination of RBP binding sites on a large scale is a challenging task due to high cost of biochemical assays. Quite a number of studies have exploited machine learning methods to predict binding sites. Especially, deep learning is increasingly used in the bioinformatics field by virtue of its ability to learn generalized representations from DNA and protein sequences.In this paper, we implemented a novel deep neural network model, DeepRKE, which combines primary RNA sequence and secondary structure information to effectively predict RBP binding sites. Specifically, we used word embedding algorithm to extract features of RNA sequences and secondary structures, i.e., distributed representation of k-mers sequence rather than traditional one-hot encoding. The distributed representations are taken as input of convolutional neural networks (CNN) and bidirectional long-term short-term memory networks (BiLSTM) to identify RBP binding sites. Our results show that deepRKE outperforms existing counterpart methods on two large-scale benchmark datasets.https://github.com/youzhiliu/DeepRKE/.Our extensive experimental results show that DeepRKE is an efficacious tool for predicting RBP binding sites. The distributed representations of RNA sequences and secondary structures can effectively detect the latent relationship and similarity between k-mers, and thus improve the predictive performance. The source code of DeepRKE is available at The online version contains supplementary material available at (doi:10.1186/s12864-020-07239-w). RNA-binding proteins (RBPs) have important functions in many biological processes, e.g. gene regulation, RNA editing, mRNA processing and other biological processes , 2. It iWith the increasing volume of verified RBP binding sites, quite a few studies focused on developing computational prediction models based on the known RBP binding sites. As is shown in Table L is transformed into a matrix of size 4*L. DeepBind [Recently, deep learning  has achiDeepBind  is the fDeepBind  to prediDeepBind  and iDeeDeepBind  also adoDeepBind  exploitsDeepBind  uses k-mDeepBind  to prediDeepBind , BioVec DeepBind , seq2vecDeepBind , and GenDeepBind  heuristiDeepBind  to obtaiDeepBind  applies In this paper, we present a novel deep neural network, DeepRKE, which consists of CNN and bidirectional LSTM, to infer latent RBP binding sites. We used the word embedding model to build distributed representations of RNA sequences and secondary structures, and input them into a deep neural network to predict RBP binding sites. The skip-gram algorithm  uses theIn this part, First we evaluated DeepRKE with other five state-of-the-art methods on two large-scale benchmark datasets RBP-24  and RBP-We implemented the learning framework of DeepRKE to infer RNA-proteins binding site on RNAs, as shown in Fig. We evaluated DeepRKE with other multiple existing methods on two large-scale benchmark datasets, including RBP-24  and RBP-First, we conducted the performance evaluation on the RBP-24 dataset with GraphProt, deepnet-rbp, DeepBind and iDeepV that can handle variable sequences. Table On RBP-31 dataset, we also compared DeepRKE to iDeepS and Oli’s method . The AUCNote that the current state-of-the-art method iDeepS gets an average AUC of 0.86, which is still slightly worse than DeepRKE. In fact, our method performs better than iDeepS on quite a few sets of RBPs. For example, DeepRKE increases the AUC by 8.2%, 5.1%, 5%, 11%, 7.5% on the Ago/EIF, Aog2-MNase, Ago2-1, IGFBP1-3 and MOV10, respectively. Moreover, we compared DeepRKE with GraphProt, which is a structure profile-based method and demonstrates better performance than RNAcontext . GraphPrIn summary, DeepRKE achieves significant performance on both RBP-24 and RBP-31 datasets. DeepRKE not only outperforms the current methods for predicting RBP binding sites, but also successfully handles input sequences with variable length, which fails to be addressed by iDeepV and iDeepS.The results in Table As shown in Table Rather than traditional one-hot encoding, DeepRKE adopts distributed representations of RNA sequence to capture the high-order dependencies among nucleotides, leading to dimension-reduced feature vectors. To justify the advantage of distributed representation in identifying binding site of RNA-binding proteins, we removed the secondary structural profiles (as it can not be represented by one-hot encoding) and took one-hot encoding of RNA sequence as the input of deep learning framework. For convenience, we referred to this model as DeepRKE- -, indicating that both RNA secondary structure and distributed representations of RNA sequence are excluded when constructing the input.FigureIn our proposed learning framework, BiLSTM is used to identify long-term dependent information of the extracted sequence and secondary structure feature . For furIn this paper, we demonstrated that distributed representation can significantly improve the predictive ability in modeling protein-RNA binding. In fact, we can also obtain the distributed representations by using UTR sequences, and thus our method is not limited to the application of RBP-24 and RBP-31 benchmark datasets.With the incorporation of biLTSM layer and secondary structure information, our method outperforms all counterpart methods in predicting protein-RNA binding sites. However, we can make advantage of RNA tertiary structure or region type to further improve the performance, similar to deepen-rbp , iDeep . CompareNote that only a small fraction of RNA sequences have been detected by CLIP-seq assays, the vast majority of sequences are not found so far. Therefore, the number of negative samples is much higher than that of positive samples in the real world. However, we built balanced training sets in this study, for that imbalanced datasets often lead to biased machine learning models with preference on overwhelming class (e.g. most of sequences are classified to negative samples), which makes trained models useless. More verified RBP binding sites are expected to improve the predictive power of computational models.Although DeepRKE achieves state-of-the-art performance, it can not identify the binding motifs directly from learned convolve filters. We plan to extend DeepRKE to identify the binding motifs in our future work. For example, we can use the DeepRKE model to assign binding potential scores to all sequences of interest, and then select high-confidence candidates to extract the binding motifs.In this paper, we present a novel deep neural network model, DeepRKE, to predict RBP binding sites. DeepRKE combines the primary RNA sequence and secondary structure into a unified learning framework. The novelty of DeepRKE lies in that we use the skip-gram model in word2vec to learn the distributed representation of RNA sequences and RNA structure. Also, we introduced the BiLSTM layer into the deep learning model to capture the high-order interdependence of sequences and secondary structures. We evaluated DeepRKE on two RBP binding benchmark sets derived from the CLIP-seq, and the results demonstrated that DeepRKE achieves better AUCs than other competitive methods. Our results suggest that distributed representation of k-mers sequence helps to improve the prediction performance for identifying the binding sites of RNA-binding protein. The BiLSTM layer also contributes significantly to the enhancement of predictive ability.RBP-24 dataset is used by GraphProt  as the thttps://github.com/mstrazar/ionmf. In this dataset, the CLIP-seq data consists of 19 proteins with 31 experiments, and their annotations are based on human assembly hg19. As described in iONMF, each nucleotide within clusters of interaction sites derived from CLIP-seq is considered as binding sites. To reduce redundancy, the positive binding sites with the highest cDNA count and without consecutive sites on genome are further randomly selected. Finally, among those sites with less than 15 nucleotides apart, one site with the highest cDNA counts was selected as the positive sample. The negative sites were sampled from genes that were not identified as an interacting partner in any of 31 experiments. As a result, 4,000 cross-linked sites are used for training, 1,000 samples for validation, and other 1,000 samples for independent test.RBP-31 dataset includes training and test samples with fixed-length RNA sequences of 101 nucleotides collected in iONMF , which cTwo large-scale datasets derived from human CLIP-seq: RBP-24 and RBP-31 are used as the benchmark datasets. The detail of the datasets are as below: In addition, as DeepRKE requires RNA secondary structural sequence as input, we fed RNA sequence into RNAShapes  to obtaiWord2Vec is a model proposed for learning semantic knowledge from a large number of textual corpora in an unsupervised manner, which is widely used in natural language processing. We extend the usage of word2vec to obtain the distribution representation of k-mer sequences. Based on the theory of distribution hypothesis, distributed representation is a strategy to obtain the semantic representation of words by using the symbiotic matrix. Word embedding is a multi-dimensional vector of real value which is mapped from the vocabulary words or phrases. It can capture the potential relationship between context and the target word. Generally, Word2Vec provides two architectural options: CBOW and skip-gram. CBOW can predict the current word based on the surrounding context, while the skip-gram uses the current word to predict the surrounding context. In this paper, we use skip-gram to learn the distributed representation of k-mers.s1,s2,s3.....sk, skip-gram learns the word representations using the co-occurrence information of words within a context window. It maximizes the following objective function: Given word sequence m is the context window size, and the conditional probability p is defined as follows: where V is the size of vocabulary, s and vs, respectively.where logp(S0∣Si) is approximated using negative sampling: Because of the computational infeasibility, the σ=1/(1+exp(−x)).where 3=64 (sequences) and 53=125 (structures) with 3-mers.Recently, BioVec , seq2vecConvolutional neural network: In our method, we employ three CNNs as feature extractors [tractors , which ttractors . FinallyLong Short Term Memory networks: In order to further detect long-term interdependence of sequence and secondary structure, we introduced a bidirectional long and short-term memory network (BiLSTM) [(BiLSTM)  into ourx}h}C}o}As LSTM iterates over each element of input, it firstly determines what information the forgetting gate layer should exclude based on previous input. The input gate layer is then used to determine what information should be stored for the next layer and update the current state value. Finally, the output gate layer determines which parts of the state value should be output. Taking a sequence {σ is the Logistic Sigmoid function and tanh is the tanh function to force the values to be between -1 and 1. Wf,Wi,Wo,Uf,Ui and Uo are the weights and bf,bi,bc and bo are the bias.where ⊙ denotes element-wise multiplication, the In DeepRKE, a bidirectional LSTM (BiLSTM) is used to scan from left to right and from right to left, concatenating the output in each direction as a final output for subsequent classification. The number of neurons in this layer was set to 32, thereby the output size is 64.Additional file 1 The file includes the performance measures of competitive methods on the RBP-24 dataset. The AUC values of using DeepRKE- and without RNA secondary structure sequence, as well as the AUC values using DeepRKE- without RNA secondary structure and distributed representation of sequences.Additional file 2 The file includes the performance measures of competitive methods on the RBP-31 dataset. The AUCs of using DeepRKE- and without RNA secondary structure sequence, as well as the AUC values of using DeepRKE- without RNA secondary structure and distributed representation of sequences.


Sidekick (Sdk) gene, first described in Drosophila, encodes the single-pass transmembrane protein, Sdk, which is one of the largest among IgSF membrane proteins. Sdk first appeared in multicellular animals during the Precambrian age and later evolved to Sdk1 and Sdk2 in vertebrates by gene duplication. In flies, a single Sdk is involved in positioning photoreceptor neurons and their axons in the visual system and is responsible for dynamically rearranging cell shapes by strictly populating tricellular adherens junctions in epithelia. In vertebrates, Sdk1 and Sdk2 are expressed by unique sets of cell types and distinctively participate in the formation and/or maintenance of neural circuits in the retina, indicating that they are determinants of synaptic specificity. These functions are mediated by specific homophilic binding of their ectodomains and by intracellular association with PDZ scaffold proteins. Recent human genetic studies as well as animal experiments implicate that Sdk genes may influence various neurodevelopmental and psychiatric disorders, such as autism spectrum disorders, attention-deficit hyperactivity disorder, addiction, and depression. The gigantic Sdk1 gene is susceptible to erratic gene rearrangements or mutations in both somatic and germ-line cells, potentially contributing to neurological disorders and some types of cancers. This review summarizes what is known about the structure and roles of Sdks.Many of the immunoglobulin superfamily (IgSF) molecules play pivotal roles in cell communication. The  The immunoglobulin superfamily (IgSF) is a large group of cell surface or secreted proteins, characterized by the occurrence of a variable number of cognate 70–110 amino acid immunoglobulin (Ig)-like domains, originally noticed in antibodies . Most meSidekick (Sdk) gene was initially identified in a mutant screen of Drosophila melanogaster for defects in eye development. An Sdk-null mutant was identified by its rough-eye phenotype, and the gene was suggested to play a role in controlling proper photoreceptor development in the fly eye , was initially identified in a screen for molecular subset markers of retinal ganglion cells (RGCs) in the developing chick retina, and its close homolog, Sidekick-2 (Sdk2), was subsequently identified  Sdk, were identified. Mouse Sdks were also cloned using a differential gene expression analysis of HIV-infected versus non-infected kidney cells  domains, a transmembrane domain, and a ∼200-amino acid cytoplasmic domain . The FNITGFSSFV and -AGFSSFV, respectively . The mechanism and contribution of the two Sdks in their body plan is an interesting conjecture.It appears that most, if not all, animal phyla have Sdk or Sdk-like molecules . All verectively . Interesectively . Since ctebrates . LampreyC. elegans) possess one Sdk with -GFSSFV. Each of the non-bilaterians (cnidarians and one placozoa) also has a protein homologous to Sdk. These non-bilaterian Sdk-like proteins have a domain architecture identical to Sdk: 6 Ig and 13 FNIII domains, as well as one transmembrane and cytoplasmic domain. Their cytoplasmic domain is ∼400 amino acids, which is longer than that of vertebrate Sdks, and most strikingly, lacks -GFSSFV. Among cnidarians, Sdk-like proteins in corals and sea anemones end with -SFV, a canonical PDZ-binding motif. However, this -SFV is not present in Sdk-like proteins in Hydra and Trichoplax. These non-bilaterian animals are a group of the most primitive multicellular animals which appeared in the Precambrian age , Echinodermata , and Nematoda  for anchoring to PDZ domain proteins, indicating that it determines the localization of Sdk proteins. It is indeed required for synaptic localization in the retina  and cytons e.g., . MAGI prns e.g., . An intrns e.g., . In addin of Sdk . Polychan of Sdk . It is in of Sdk .Drosophila visual system consist of many ommatidia and transmit visual information to the underlying optic lobes via four neuropils:the lamina, medulla, lobula, and lobular plate. Each ommatidium contains eight photoreceptors (R1–R8) which project to either the lamina or medulla , although Drosophila Sdk is a homophilic adhesion molecule  to ensure epithelial integrity, dynamics, and function  Figures. In a Dr at tAJs . In an el mutant , other menitalia . Detaileenitalia  and dynaenitalia . Polychaenitalia . Sdk canenitalia . Super-renitalia , indicatenitalia , suggestSdk1 and Sdk2 are expressed by non-overlapping subsets of retinal neurons  form synapses on over 40 types of RGC dendrites. This results in the assembly of a synaptic neuropil, consisting of multiple sublaminae . FunctioSdk2 is expressed by restricted subsets of retinal neurons, including non-canonical glutamatergic interneurons called Vesicular glutamate transporter-3 (VGlut3)-positive amacrine cells (VG3-ACs), and an RGC type called W3B  are repaired by non-homologous end joining. Neurons often contain somatic genomic variations caused by this process. Sdk1 has been identified using an unbiased, high-throughput method, to map genomic regions harboring frequent DSBs in neural stem/progenitor cells -1-positive patients. odocytes . Sdk1, bhropathy , 2007. Stermined .SDK1 gene  and its transcript have been previously observed (SDK1:AMACR fusion and prostate cancer progression remains to be clarified.Finally, in some prostate cancer patients, gene fusions of observed . A causaSDK genes call for further analysis on their pleiotropic roles. Sdk is an evolutionarily conserved protein which first appeared in the Precambrian age and later duplicated to generate Sdk1 and Sdk2 when vertebrates emerged and evolved. The function of Sdk in primitive multicellular animals is totally unknown. Sdk proteins are concentrated at cell-cell junctions, including at tAJs in Drosophila, and at chemical synapses in vertebrates. Inspired by localization of Sdk at tAJs, more studies on vertebrates are required to reveal the precise localization of Sdk proteins at various cell-cell contacts, including synaptic sites, to understand detailed functions of Sdks in diverse neural circuits. Nonetheless, animals without Sdk genes are still viable (Sdk to remain in a variety of living and behaving animals.Sdks are unusually large membrane proteins that have been refractory to structural and biochemical studies. They are often overlooked in molecular screening and systems biology, where the 5’-end of long transcripts is underrepresented. However, recent reports on human l viable . It is pMY wrote the text and created the figures and table.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.