Emerging data has highlighted the role of the nutrient-sensing enzyme O-GlcNAc transferase (OGT) as an epigenetic modifier essential in coordinating cellular transcriptional programs and metabolic homeostasis. OGT utilizes the end-product of the hexosamine biosynthetic pathway to modify proteins with O-linked β-D-N-acetylglucosamine (O-GlcNAc). The levels of the modification are held in check by the O-GlcNAcase (OGA). Studies from model organisms and human disease underscore the conserved function these two enzymes of O-GlcNAc cycling play in transcriptional regulation, cellular plasticity and mitochondrial reprogramming. Here, we review these findings and present an integrated view of how O-GlcNAc cycling may contribute to cellular memory and transgenerational inheritance of responses to parental stress. We focus on a rare human genetic disorder where mutant forms of OGT are inherited or acquired de novo. Ongoing analysis of this disorder, OGT- X-linked intellectual disability (OGT-XLID), provides a window into how epigenetic factors linked to O-GlcNAc cycling may influence neurodevelopment.Cellular identity in multicellular organisms is maintained by characteristic transcriptional networks, nutrient consumption, energy production and metabolite utilization. Integrating these cell-specific programs are epigenetic modifiers, whose activity is often dependent on nutrients and their metabolites to function as substrates and co This adaptation requires changes in transcriptional programs that allow an appropriate gene regulatory network response. One way this is achieved is through epigenetic regulation: heritable modifications of DNA and histones that influence complex networks impacting transcription, DNA replication, and DNA repair . Importahylation . Here, wNeurodevelopment is one such process that is heavily coordinated by the crosstalk between the genome, environment and metabolic flux. Alterations during this process could lead to neurodevelopmental disorders, which constitute a broad range of disorders that originate during development of the central nervous system. As more and more sequences become available from neurodevelopmental disorder patients, it is becoming increasingly clear the essential role chromatin modifiers and epigenetic regulators play in these diseases. Of the hundreds of genes with mutations or copy number variations that are causative of neurodevelopmental disorders, chromatin regulation is the second most common association behind synaptic function . InteresO-GlcNAc transferase uses the end-product of the hexosamine biosynthetic pathway (HBP), UDP-GlcNAc, to add a single GlcNAc monosaccharide onto serines and threonines of target intracellular proteins. To form UDP-GlcNAc, the HBP incorporates intermediate metabolites derived from carbohydrates, amino acids, fat, and nucleotides  and activation by the trithorax group (trxG). In Drosophila, the gene encoding OGT was first described genetically by mutations that caused homeotic phenotypes including antenna-to-leg and wing-to-haltere transformations (super sex combs (sxc) for its loss of function phenotype. Like PcG genes Polycomb (Pc) and extra sex combs (esc), sxc mutations affect the development of the sex combs, a structure on the forelimbs of Drosophila males. Similar to other components of the PcG, sxc was required for the repression of Hox genes in tissues where they should not be expressed. Derepression of Hox genes could explain the homeotic transformations associated with sxc mutations. Epistasis experiments confirmed this, as sxc flies lacking the Hox gene Ubx had more normal wing development  demonstrated genetic interactions with sxc. Flies carrying sxc and ph mutations had enhanced sxc phenotypes, with greater numbers of sex combs, stronger developmental defects, and lethality even earlier in development  is not O-GlcNAc modified in Drosophila  and sin-3 (histone deacetylase), which is itself a predicted interactor with OGT-1  and continuing into adulthood. Without mutagenesis, gcy-5::gfp expression was limited to ASE neurons, but mutations in seven genes caused ectopic expression of the reporter. Three independent missense mutations were isolated in the ogt-1 gene. These three mutations were at highly conserved residues within the catalytic domains of OGT-1, in close proximity to sites previously reported to be necessary for enzymatic activity in human OGT (dot-1.1 (an H3K79 methyltransferase) and pmk-1/p38-alpha MAPK. In mammalian neuroblastoma cells, OGT and p38 MAPK have been shown to physically interact to drive O-GlcNAcylation of specific targets  analysis of histones isolated from mammalian cells   . In addi1 (MDC1) . The aut pathway .O-GlcNAc when cells are stressed by ROS , which cause damage, cellular stress, and DNA breaks. As such, DDR-related factors are prominent among proteins that accumulate d by ROS . Reciprod by ROS , pointinA damage . The eletic mice . Recentlem cells . Likewisuse ESCs . Previouuse ESCs . Through pathway   followed by guanines. This epigenetic mark is involved in a variety of functions in the mammalian genome, including X-chromosome inactivation, gene silencing, genomic stability, cellular identity, and genomic imprinting . Two modO-GlcNAcylation of TET1 was shown to regulate TET1 stability  maintenance also requires balanced t uptake .O-GlcNAcylation has been extensively studied in the brain due to its critical role during development described above, and in neurodegenerative diseases like Alzheimer’s -CREB signaling, and memory loss in Alzheimer’s disease. Alzheimer’s disease is associated with a decrease in O-GlcNAcylation, which has been shown to influence aggregating proteins such as tau  is a transcriptional co-activator that controls energy and nutrient homeostasis by coordinating gene expression. PGC-1α has been shown to form a complex with OGT and be O-GlcNAcylated at Ser333. Moreover, increased glucose levels lead to FOXO1 (Forkhead box other 1) O-GlcNAcylation via the PGC-1α/OGT complex, enhancing transcriptional activity  are described as nutritional sensors for lipid metabolism, glucose homeostasis and inflammation, and are posttranslationally modified by phosphorylation, acetylation, and O-GlcNAcylation  expression. SREBP-1c is a major player of gene expression in hepatic lipogenesis  downregulation of gluconeogenesis genes by initiating inhibitory phosphorylation of FOXO1, and (2) promotion of lipogenic pathways through activation of SHREBP-1c . O-GlcNAcylation . Increasogenesis . LXR hasnditions . The effnt cells . The LXRnt cells , providiO-GlcNAcylation. High glucose or OGT expression increased ChREBP O-GlcNAcylation, stabilizing the protein and increasing expression of its target genes. Oga overexpression in mouse livers markedly reduced ChREBP O-GlcNAcylation and decreased abundance of ChREBP targets  is a key factor of energy metabolism in the liver and is regulated by  targets . These rf ChREBP . Moreovef ChREBP . These dO-GlcNAc is able to alter patterns of gene expression in response to nutrient status and stress. Mutations in OGT may impair its targeting of specific pathways and disrupt their normal function in responding to the environment and coordinating development.Through regulation of transcription factors, Prenatal development is a particularly vulnerable time, when tight regulation of transcriptional networks is required to transform a fertilized egg into a multicellular organism requiring complex tissue development. In mammals, energy flow from the mother to the fetus is mediated by the placenta, which transfers macronutrients, gases, and metabolites into fetal circulation. Thus, the placenta transmits nutritional and stress information from the mother to the developing fetus. Glucose is the primary fuel for the fetus, which is provided by maternal transfer through the placenta. The fetal brain is a particularly nutritionally demanding tissue during fetal development. Brain regions are more sensitive to nutrient availability at particular gestational stages. Interestingly, a recent hypothesis to explain the male-biased presentation of neurodevelopmental diseases focuses on sex differences in the placenta in relaying signals to the developing brain regarding maternal perturbations .O-GlcNAcylation of placental proteins correlates with maternal glycemic index . Through a ChIP-seq experiment on placental tissue, the researchers identified a correlation between O-GlcNAc occupancy and Hsd17b3 expression and that O-GlcNAc is significantly reduced by exposure to early prenatal stress  termed OGT-CDG  and chromatin modifiers such as the Polycomb complexes (de novo mutations at OGT N567K have been shown to exhibit extreme skewing in X-inactivation (98%) although it is unclear which of the two X chromosome are inactivated (O-GlcNAc are affected and this reduction can be recapitulated in vitro and in other model systems (de novo mutation arising in female probands. OGT could contribute to DNA methylation/demethylation associated with the X-inactivation process. In addition, the role of OGT in Polycomb repression previously discussed raises the possibility that alterations in O-GlcNAc cycling could contribute to the skewing observed.The X chromosome has a unique pattern of inheritance compared to autosomal chromosomes. The X chromosome in males is inherited from the mother making recessive X-linked mutations predominant in males. The significance of the X-chromosomal location OGT has been previously discussed . In femaomplexes . Interesctivated . In thes systems . Given tOGT through activation of the transcription factor C/EBP-β (OGA leads to an elevation in OGT protein levels (O-GlcNAc cycling (O-GlcNAc regulates this process, suggesting a distinct feedback mechanism limiting the production of certain isoforms in response to O-GlcNAc elevation. In one study, an internal silencing site has been identified which appears to alter splicing of OGT mRNA, limiting its translation (O-GlcNAc levels (O-GlcNAc. However, lowering OGA levels can have a dramatic effect on neurodevelopment. We recently reported that mice in which Oga was deleted in the brain showed numerous phenotypes including microcephaly, enlarged ventricles, hypotonia, and developmental delay, strongly suggesting a possible link between OGT-XLID variants and perturbations of OGA levels (MGEA5/OGA as one of the genes closely associated with intelligence (In many instances, variants in OGT associated with XLID resulted in a reduction in levels of OGA , b. OGA  C/EBP-β , in addin levels . The gen cycling  (Hanover cycling . O-GlcNAnslation . A similc levels . Thus, hA levels . In addilligence . Studieslligence , althouglligence . So clealligence .O-GlcNAc transferase strongly associates with HCF-1 and heavily modifies it. In combination, OGT-HCF-1 forms interactions with numerous epigenetic complexes (omplexes . In addiomplexes , 2018. Somplexes , b. Mutaomplexes , suggestO-GlcNAc transferase interacts with numerous protein complexes associated with epigenetic regulation including the Polycomb repressive complexes, the pluripotency network associated with Oct4, Sin3A-HDAC complexes, and many others (O-GlcNAc modification leading to a more pleiotropic deregulation of development. Improper regulation of these epigenetic complexes is known to impact development. For example, mutations in PRC2 proteins EZH2, SUZ12, and EED can cause Weaver Syndrome, an autism spectrum disorder that presents with developmental delay (y others . The mutal delay . These oO-GlcNAc turnover in those complexes are under continuous development.At the present, it is difficult to examine these interactions quantitatively, but sensitive methods to examine both protein-protein interactions and O-GlcNAc levels due to compensatory changes in the O-GlcNAcase expression. Patients with OGT-XLID show numerous developmental and neurodevelopmental deficits resulting in a form of intellectual disability. This intellectual disability phenotype results from changes in neurodevelopment which strongly suggests that O-GlcNAc addition and removal may play a particularly important role in the functioning and development of the brain. This is perhaps not surprising given the importance of glucose and its metabolites in the physiology of the central nervous system. In addition, the complexity of the human brain originates from a finely tuned developmental process influenced by both genome and environment (in utero when glucose is provided primarily by maternal transfer through the placenta. This underscores the importance of maternal nutritional and environmental state and proper placental glucose metabolism for fetal brain development.The identification of OGT-XLID as a rare human disorder suggests that non-synonymous variants of OGT can be tolerated to a limited degree. These OGT variants are hypomorphic, leading to only modest changes in ironment . A partiDevelopment of the human cortex is incredibly sensitive, in part due to the requirement for two waves of rapid proliferation of progenitor cells . During O-GlcNAc pathway is integrated in human physiology. We have highlighted the role of O-GlcNAc cycling in numerous epigenetic complexes regulating development and differentiation. We have also examined the role of O-GlcNAc cycling in stem cell differentiation and the regulation of DNA damage response signaling. Finally, we have argued that compensatory mechanisms may be in place to limit the impact of the OGT mutations including X-inactivation of OGT, intron retention of OGT transcripts, OGA down regulation by Polycomb repression, intron retention, and transcription. These highly varied modes of regulation serve to buffer the effects of the OGT mutations, but may themselves have phenotypic consequences.Thus, the functional and morphological features of the human brain render it highly vulnerable to both genetically and environmentally induced alterations. In addition, there is an increasing awareness that chromatin regulation may be central to understanding neurodevelopmental disorders . The insDK, LA, and JH edited the manuscript. All authors contributed to the literature search, wrote specific sections of the manuscript, and agreed on the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: Acute pericarditis is a rapid inflammatory condition of the pericardium with both infectious and non-infectious etiology. Most acute pericarditis is self-limited, with a small portion evolving rapidly. The definitive diagnosis of acute pericarditis often requires detailed physical examination, ECG, echocardiography, blood analysis and chest X-ray. It's usually challenging to distinguish acute pericarditis from ST-elevated myocardial infarction (STEMI) due to the similar ECG characteristics (ST segment change). Here we present a case of purulent pericarditis probably caused by esophageal perforation.Case: A 52 year-old male presented with chest pain and dyspnea for 16 h. ST-segment elevation and positive cardiac markers lead to the initial diagnosis of ST-elevated myocardial infarction. Coronary angiography demonstrated normal coronary artery, while transthoracic echocardiography (TTE) showed massive pericardial effusion. Then, pericardiocentesis was performed with 250 ml of yellowish-green pus-like fluid extracted. A detailed history examination revealed a week history of possible esophageal perforation caused by a fishbone. And a further computed tomography (CT) demonstrated the presence of pneumomediastinum, and effusions in mediastinum, which lead to the diagnosis of purulent pericarditis. However, the patient's family refused further treatment and the patient died soon after discharge.Conclusion: The differential diagnosis of chest pain should include acute pericarditis, which can be equally critical and fatal. And it's important to note the peculiar characteristics of acute pericarditis, which include concave and diffused ST-segment elevation, PR segment depression, and the ratio of ST-segment elevation to T wave >0.24 in lead V6. Moreover, comprehensive medical history and physical examination are crucial to the differential diagnosis of chest pain patients. To quickly and effectively identify high-risk chest pain individuals is of great clinical importance and essential in emergency medical practice. From the results of a large multi-center study, chest pain patients accounted for about 5% of all emergency visits , and acuAcute pericarditis is an important cause of non-ischemic related chest pain with ST segment changes, which makes it challenging to be distinguished from AMI. There are various causes associated with it, both infectious  and non-infectious  . The defA 52 year-old male with continuous chest pain and dyspnea for 16 h was admitted to our hospital. Physical examination was unremarkable, and vital signs were normal. His initial troponin-T level was 20.9 ng/L , and the NT-proBNP level was 2,774 ng/L (0–227). Twelve-lead electrocardiogram (ECG) findings included ST-segment elevation at the inferior and lateral leads  .9/L) and elevated neutrophil count (88.2%). Considering the possibilities of an acute pericarditis, a pericardiocentesis was performed. And about 250 ml of yellowish-green pus-like fluid was extracted, with biochemical examination revealed elevated lactic dehydrogenase (TLDH)  and positive pus cells . Therefore, purulent pericarditis was suspected. Consequently, a furthered detailed history exam was conducted, which then, the patient further revealed a week history of sore throat with prior fever as well as dysphagia for 4 days that self-resolved. Furthermore, the patient complained that a “fishbone got stuck in his throat more than a week ago.” Due to the combined presentation of chest pain, dyspnea, history of esophageal injury, and purulent pericardiocentesis fluid, we suspected a pathogenesis of esophageal perforation resulting in mediastinal infection leading to purulent pericarditis.Due to the typical presentation of chest pain, positive biomarkers and ECG findings, the initial diagnosis was ST-Elevation myocardial infarction (STEMI). The patient was immediately transferred for emergency coronary angiography (CAG), which demonstrated normal patent left and right coronary arteries . Next, aMoreover, computed tomography (CT) further demonstrated the presence of pneumopericardium, effusions in mediastinum and pericardium . The heaAcute pericarditis is an important cause of non-ischemic related chest pain with ST segment changes. It is worthy to note that the characteristics of the chest pain is usually sharp, stabbing-like, and occasionally dull. Furthermore, the pain is often pleuritic and positional, and friction rub may occur in around 85% of the cases .Compared to STEMI, patients with acute pericarditis often presents with ST-segment elevation and PR-segment depression, and around 80% of these patients may also have elevated troponin. Nevertheless, the ECG characteristics of ST-segment elevation in acute pericarditis is concaved and diffused. Whereas, the changes are concaved and localized in AMI and limited to leads III, AVF and V12 in pulmonary embolism. One of the specific ECG features of pericarditis is that the ratio of ST-segment elevation to T wave is >0.24 in lead V6 (which was approximately 0.4 in this case)  is rare, and only few similar cases were ever published before . From thThe biggest limitation in this study is that the suspected esophageal perforation was not confirmed by endoscope, surgery or autopsy. However, due to the patient's history and existing evidence, the heart team highly supported this diagnosis. In this case, we highlighted the importance of ECG analysis and an accurate history in the early differential diagnosis of life-threatening pericarditis.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Although researchers have suggested that religiosity is a multidimensional construct, less is known about the long-term effects of religiosity profiles on Generation X (born between 1965 and 1980) adults’ psychological and familial wellbeing over the life courses. Thus, the goal of this study is (1) to identify unobserved profiles of young-adult Gen-Xers’ religiosity based on religious attendance, religious intensity, spirituality, and religious ideology, (2) to investigate demographic factors that predict membership in these religiosity latent classes, and (3) to examine how these profiles of religiosity predict Gen-Xers’ psychological wellbeing  and familial wellbeing  in early and middle adulthood. We selected 462 Gen-Xers from the Longitudinal Study of Generation in the 2005 (mean age = 30.25) and the 2016 waves (mean age = 41.25). In terms of data analysis, the three-step latent class analysis was conducted. We identified four religiosity profiles among young-adult Gen-Xers: strongly religious, weakly religious, literalists but not religious, religious but not literalists. Less educated single Gen-Xers were more likely to be in the strongly religious class, and less likely to be in other classes. Gen-Xers in the strongly religious class reported high scores of psychological and familial wellbeing than those in other religiosity classes. Given that the religiosity of the U.S. population has declined substantially over the past few decades, our findings indicate that religiosity is an important resource for Gen-X adults’ psychological and familial wellbeing.


Posterior fossa brain tumors (PFBT) are the most common solid tumor in children. Recent increases in survival rates are encouraging; however, survivors may experience a plethora of disease- and treatment-related complications that can persist into adulthood. Therapeutic exercise interventions have been shown to improve quality of survivorship in other pediatric cancer diagnoses. There is also evidence that goal-directed interventions are effective at improving motor activities, function, and self-care in children with complex health conditions. Yet, there is currently no evidence on the efficacy of goal-directed therapeutic exercise in pediatric PFBT survivors. The Physical ACTivity in Survivorship (PACTS) study aims to investigate the effects of a novel goal-directed therapeutic exercise program on cardiorespiratory fitness and physical activity-related goal attainment in pediatric survivors of PFBT.n = 48). Following baseline assessment, participants are randomized into either the intervention or usual care group. The intervention group will receive weekly individualized, goal-directed exercise therapy delivered face-to-face for 12 weeks, along with an accompanying home-based program (three sessions per week). Outcomes will be assessed at baseline, immediately post-intervention, and at 6- and 12-months post-intervention. The primary outcomes are cardiorespiratory fitness (Peak VO2) and physical activity-related goal attainment. Secondary outcomes are cardiorespiratory endurance, high-level mobility skills, functional muscle strength, habitual physical activity, gait, balance, quality of life, fatigue, participation, perceived movement skill competence and parameters of body composition.PFBT survivors, aged five to 17 years, who underwent surgery at least 12 months earlier and completed radiation therapy and/or chemotherapy at least 6 months prior will be recruited from the Queensland Children’s Hospital  (target PACTS is the first study to investigate the efficacy of goal-directed therapeutic exercise in children with PFBT and provide evidence needed to inform clinical practice recommendations for managing quality of survivorship in PFBT survivors.ACTRN12619000841178.The online version contains supplementary material available at 10.1186/s12887-021-02566-7. Posterior fossa brain tumors (PFBT) account for 25% of all pediatric cancer diagnoses and are the most common solid tumor in children , 2. ThisThe anatomical location of PFBTs, with or without administration of cranial radiation therapy, negatively impact the cerebellum and adjacent areas . ConsequPediatric PFBT survivors exhibit poor cardiorespiratory fitness, performing well below their age-matched peers and equivalent to children with congenital heart disease , 20. FolIntervention studies conducted in childhood cancer survivors indicate that therapeutic exercise is safe and feasible; however, studies conducted to date have focused predominantly on children with blood cancers  . To our Goal-directed therapy aims to promote functional performance and independence in everyday activities, where goals are set by the patients and their families , 38. An The Physical ACTivity in Survivorship (PACTS) study aims to evaluate the effects of a 12-week goal-directed therapeutic exercise program on cardiorespiratory fitness and physical activity-related goal attainment in PFBT survivors. We hypothesize that, compared with usual care, children who receive the intervention will achieve significantly greater gains in cardiorespiratory fitness and goal attainment, immediately post-intervention, with improvements maintained at 6- and 12-month follow-ups.Secondary aims are to evaluate the effects of the exercise program on cardiorespiratory endurance, high-level mobility skills, functional muscle strength, habitual physical activity, gait, balance, quality of life, fatigue, participation, perceived movement skill competence and parameters of body composition. We hypothesize that, compared with usual care, children who receive the intervention will achieve significantly greater gains in these secondary outcomes immediately post-intervention, with improvements maintained at 6- and 12-months follow-ups.n = 24) or usual care (n = 24). The study will be conducted in accordance with the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) guidelines [This study is a single-center randomized controlled trial. Participants will be randomized into the intervention arm . Using the randomization module in REDCap, participants will be randomized into the intervention or usual care group, stratified by treatment type . Within an exercise intervention, it is not possible to blind either therapist or participant; however, therapists will not take part in post-intervention outcome assessments. Trained assessors blinded to group allocation will collect outcome data.Participants randomized to the intervention group will receive weekly individualised, goal-directed exercise therapy delivered face-to-face for 12 weeks, along with an accompanying home-based program. Each face-to-face session will be delivered by a qualified physiotherapist or accredited exercise physiologist (referred to hereafter as therapist), for a 30–60-min duration. The intervention will adhere to accepted exercise prescription guidelines for children and adolescents, comprising physical exercise training (cardiorespiratory and strength-promoting activities) and goal-directed task specific practice , 44. At To supplement the face-to-face sessions, participants will complete a parent-led, home-based exercise program three times per week, up to 30 min of duration each time. The home-based program will replicate the essential elements of the supervised sessions in relation to activity type, intensity, and duration, and will also reflect the resources available at home. Prior to commencing face-to-face sessions, the therapist will complete a home visit to determine what space, equipment, and resources are available to complete the home-based exercise program safely and effectively. Parents will be given a detailed manual (including pictures and descriptions) for delivering the home-based component and will be encouraged to participate in the activities with their child. Peers and siblings may also be included to promote adherence to the home-based program. The home-based program will be monitored via heart rate monitors. The therapist will complete a follow-up home visit, between week four and week eight of the intervention, to provide an opportunity for participants and parents to ask questions about the home-based program, resolve any difficulties completing the program, and review the current activities.Due to the highly heterogenic nature of a goal-directed intervention, several strategies will be used to monitor compliance with the exercise program. First, the type, intensity and duration of each activity will be documented by the therapist using a standard form. The forms will prompt therapists to reflect on whether their session delivered the required dose of cardiorespiratory and goal-directed activities. Second, where possible, all sessions will be delivered by the same therapist to ensure consistency between sessions. Third, when consent is provided by parents or their guardian, intervention sessions will be video recorded and assessed by another member of the research team, with significant experience in pediatric exercise therapy. At the conclusion of the exercise intervention, parents will be asked to complete a questionnaire assessing their overall satisfaction with the program and whether the program met their expectations and needs.Participants randomized to the control group will receive care as usual. Usual care is highly variable for children who are PFBT survivors; however, it is anticipated that most participants will not receive direct therapy services for the duration of the study. Usual care may include any therapy  and visits to their treating medical practitioner.Upon meeting eligibility criteria and providing written consent, participants will complete a demographic and medical information questionnaire, provided to the parent up to 2 weeks prior to their scheduled baseline assessment. The following medical information will also be extracted from patient medical records by trained staff: definitive date of diagnosis, tumor type and grade, intraventricular shunt placement, extent of surgery, site and dose of radiation therapy, agent and cumulative dose of chemotherapy, hormone therapy, dose and frequency of Botulinum Toxin A (BoNTA), and physical impairment .At each assessment, the following anthropometric measures will be taken: height , weight , and waist circumference measured using a stretch resistant tape. Information about each participant’s usual care during the study period will be collected through a semi-structured parent interview.VO2) will be determined using the methods described by Day et al. [VO2) will be averaged over five second time windows. This measure has been shown to be reliable among children (ICC = 0.96) [A continuous ramped exercise test for determination of maximal aerobic capacity will be conducted on an electronically braked pediatric cycle ergometer . Following a 2-min warm-up at an output of 10 watts, power output will increase at a rate of 10 W per minute and cycling cadence will be maintained at 75 rpm (rpm) . The tesy et al. . Breath- = 0.96) .The Canadian Occupational Performance Measure (COPM) is a semi-structured interview used to identify participant issues of personal importance in the domains of self-care, productivity and leisure . Only goThe Modified Shuttle Test requires participants to walk/run back and forth along a 10-m course in time with a sound signal recording . Speed fHigh-Level Mobility Assessment Tool (HiMAT) is a 13-item tool assessing the performance of high-level mobility skills including walking, running, skipping, hopping, stairs, and bounding, among people who have sustained a brain injury . Raw scoFunctional muscle strength will be measured by identifying 30 s repetition maximum, preceded by a practice trial, for the following exercises: sit-to-stand, and half-kneel to stand, based on the protocol outlined in Verschuren et al. . These aDaily moderate-to-vigorous physical activity will be measured using the ActiGraph GT3X+ triaxial accelerometer . Monitors will be worn 24 h per day on their non-dominant wrist for seven consecutive days. Upon return of the accelerometer, the raw accelerometer data will be downloaded and processed into physical activity metrics using a random forest physical activity classification algorithm for children , 62. ThiThe GAITRite system  will be used to measure real-time temporal and spatial parameters of gait, including velocity, cadence, step length, stride length, heel-to-heel base of support, single leg support (left and right), double leg support, and toe in/out angles . ParticiBalance will be assessed using a PASCO force plate (PASCO PASport 22-axis force platform PS-2142). Participants will be assessed in three stances; bilateral, right unilateral and left unilateral, under two conditions; barefoot contact and a foam surface placed on the force plate. Displacement of center of pressure and mean sway velocity in mediolateral and anteroposterior directions will be measured , 67.A modified battery of the pediatric-modified Total Neuropathy Scale (peds-mTNS) will assess lower extremity neurological signs and symptoms, to evaluate their contribution to movement and balance findings. The peds-mTNS comprises three sets of questions on sensory symptoms, pain, motor function and autonomic function, as well as a five-part non-invasive neurological exam, including light touch sensation, vibration sensation, pin sensation, distal muscle strength and deep tendon reflexes . The pedThe Pediatric Quality of Life Inventory (PedsQL) Generic Core Scales 4.0 comprise 23 items measuring four dimensions of QoL; physical functioning, emotional functioning, social functioning and school functioning . Each itParents and children will also complete the PedsQL Brain Tumor Module . This diPediatric Quality of Life Inventory Multidimensional Fatigue Scale (PedsQL-MFS) will be completed by parents and children, including 18 items encompassing three subscales; general fatigue, sleep/rest fatigue, and cognitive fatigue. The format, instructions and Likert response scale are identical to the PedsQL Generic Core Scales, with higher scores indicating less fatigue. PedsQL-MFS has demonstrated internal consistency, reliability and validity among a diverse sample of pediatric cancer patients (aged 2–18) . ValiditThe Participation and Environment Measure for Children and Youth (PEM-CY) is a 20-item instrument completed by the parent and examines the frequency and level of participation across three settings; home, school and community . For eacThe Perceived Movement Skill Competence for Young Children is a pictorial tool that scores a child’s perceived movement competence for fundamental movement skills . It invo2) and body composition  data. Scans of the anterior-posterior spines L1-L4, left and right proximal femur will additionally be acquired. All scans will be performed as per standard protocols in lying supine and analyzed using the manufacturers pediatric software (EnCore version 14.1). Total body lean mass will be calculated as fat-free mass minus bone mineral content. DXA measured parameters of body composition and bone mineral density demonstrate a coefficient of variation of less than 2% [Total body scans using dual-energy X-ray absorptiometry  will acquire parameters of bone mineral density . This calculation assumes a two-tailed alpha level of 0.05, and standard deviations of 5.5 mL/kg/min and 1.5 points for peak VO2 [To detect the clinically important differences of 5.5 mL/kg/min for Peak VO2  and 2 poVO2  with 80%peak VO2 , respectAnalysis will follow standard principles for randomized controlled trials using two-group comparisons including all participants on an intention-to-treat basis. Between-group differences on the primary and secondary outcomes will be tested immediately post-intervention and at 6- and 12-month follow-up assessments using linear regression, with treatment group  as the main effect and baseline measures of the respective outcome, along with other potential confounders , as covariates.Ethical approval has been granted from the human research ethics committee of Children’s Health Queensland (HREC/19/QCHQ/50464) and the Queensland University of Technology (1900000696). The trial has been prospectively registered with the Australian New Zealand Clinical Trials Registry (ACTRN12619000841178). Before enrolling in the trial, full written and informed consent will be obtained from legal parents/guardians of all participants, verbal assent for children aged seven to 13 years, and verbal and written assent for children aged 14 years or older. Participant data will be managed on a secured electronic database (REDCap) and hardcopy forms stored securely at the research facility.A standardized protocol will be used to monitor adverse events that may occur during the face-to-face exercise sessions or home-based program. Adverse events commonly related to participating in physical activity may include, but are not limited to muscle soreness, falls, injury, exacerbation of existing symptoms, or any new or changed health problems since the previous contact. Adverse events will be graded using the Common Terminology Criteria for Adverse Events version 5.0 criteria . SeriousThis paper presents the rationale and design for a randomized controlled trial evaluating the efficacy of a 12-week goal-directed therapeutic exercise intervention in pediatric survivors of PFBTs. The increasing incidence and survival rates of PFBT indicate that there is a growing population of paediatric survivors of PFBT. However, there is a lack of research informing health care providers on effective interventions to manage the adverse effects of the disease and its treatment. To date, exercise interventions in childhood cancer survivors have implemented a structured exercise training approach, in group- and/or home-based settings. No studies have evaluated the efficacy of a goal-directed exercise intervention in any paediatric brain tumour survivor cohort, which may be associated with larger, more sustainable improvements physical functioning and habitual physical activity. Improvements in cardiorespiratory fitness and goal attainment may enhance the quality of survivorship and play a major role in mitigating the risk of disabling secondary health conditions later in life such as obesity, cardiovascular disease, osteoporosis, and metabolic disorders. The program of research will have strong potential for translation to clinical practice and pediatric rehabilitation services.Additional file 1. Standard Protocol Items: Recommendations for Intervention Trials (SPIRIT) Checklist”. The document contains a table listing the SPIRIT checklist criteria and the page number/s where each criterion is addressed in the manuscript.


The association between palliative care and life-sustaining treatment following emergency department (ED) resuscitation is unclear. This study aims to analyze the usage of palliative care and life-sustaining treatments among ED triage level I resuscitation patients based on a nationally representative sample of patients in Taiwan.A matched-pair retrospective cohort study was conducted to examine the association between palliative care and outcome variables using multivariate logistic regression and Kaplan–Meier survival analyses. Between 2009 and 2013, 336 ED triage level I resuscitation patients received palliative care services  under a universal health insurance scheme. Retrospective cohort matching was performed with those who received standard care at a ratio of 1:4 . Outcome variables included the number of visits to emergency and outpatient departments, hospitalization duration, total medical expenses, utilization of life-sustaining treatments, and duration of survival following ED triage level I resuscitation.The mean survival duration following level I resuscitation was less than 1 year. Palliative care was administered to 15% of the resuscitation cohort. The palliative care group received significantly less life-sustaining treatment than did the usual care group.Among patients who underwent level I resuscitation, palliative care was inversely correlated with the scope of life-sustaining treatments. Furthermore, triage level I resuscitation status may present a possible new field for starting palliative care intervention and reducing low-value care. Emergency department (ED) resuscitation refers to a critical time-sensitive state requiring immediate resuscitation , 2. The Early identification of the end-of-life state is key to avoiding low-value emergency care . MedicalIn January 2019, the Taiwan Ministry of Health and Welfare (MoHW) launched the Patient Autonomy Act, aimed at ensuring that all adults or surrogate(s) have access to counseling related to care options, including refusal of life-sustaining treatment for terminal diseases, irreversible comatose condition, vegetative state, advanced dementia, and other intractable illnesses or incurable diseases . MedicalThe National Health Insurance (NHI) program in Taiwan has provided hospice care since the first hospital-based hospice ward was opened in 1990, and palliative care since the Hospice Palliative Care Act was passed in 2000. The care settings include inpatient hospice wards and home and community hospice care. Palliative services were initially intended for patients with cancer or motor neuron diseases; however, it was expanded in 2009 to include eight categories of non-cancer patients . Since 2This nationwide retrospective cohort study was based on the National Health Insurance Research Database (NHIRD) of Taiwan. We obtained NHIRD data from a sample of roughly one million nationally representative claims for inpatient and ambulatory care between September 1, 2009, to December 31, 2013 , 27. TheIn accordance with regulations of the National Health Research Institutes, patient identification information was anonymized, such that informed consent was not required. This study was approved by the Institutional Review Board of Yang-Ming University (YM-107035E). No funding was received to support this research.Palliative care was extended to non-cancer patients in 2009; therefore, this study focused on the period between September 1, 2009, and December 31, 2013. Inclusion criteria included ED triage level I resuscitation during the study period. The palliative care sub-group included patients who received palliative care after post-triage level I resuscitation. Patients who received palliative care prior to the date of ED resuscitation were excluded. The date of the first palliative service was adopted as the index date. Patients in the palliative care subgroup were matched at a ratio of 1:4 with patients who received usual care in terms of event year, season, gender, age, and Charlson Comorbidity Index (CCI). Analysis was performed on the utilization of medical services from the index date until the patient died or the end of the study period. Cases were evaluated in terms of patient disposition, medical utilization, and medical outcomes.The selection of outcome and control variables was based on a conceptual framework describing interventions specific to life-sustaining treatment . The socBaseline patient characteristics, medical utilization, and outcomes were compared between (1) the palliative group, and (2) usual care group. Differences in outcome variables were examined across gender, age, survival status, occupation, income, and CCI. ICD-9 codes for principal diagnoses, including palliative services-related codes from inpatient and outpatient NHI claims in the year prior to death, were used to identify comorbidities. The Deyo-Charlson comorbidity index was calculated using ICD-9 codes and categorized as 0, 1, or ≧2 comorbid conditions. Survival duration was based on the interval between the date of ED resuscitation and the date of death or research termination. Continuous variables and categorical variables were compared between groups using t-tests and the Chi-squared test, respectively. The distribution of covariates between groups was also assessed using Chi-square tests and t-tests, where a p-value of < 0.05 indicated a difference of statistical significance. Data with a skewed continuous distribution was described using the median and 25th and 75th percentiles (interquartile range). Kaplan–Meier survival analysis was used to analyze between-group differences related to survival, medical expenses, and the usage of life-sustaining treatments, in conjunction with the log-rank test to determine statistical significance. To obtain the contribution of each predictor to the overall explanatory power of the model, we conducted further subgroup analysis for each stratum of gender, age group, income, living area, CCI and occupation of palliative service and life-sustaining treatment using the full model without stratifying Multivariate logistic regression was used with one dichotomous dependent variable; i.e., whether or not life-sustaining treatment was implemented. Predictor variables included gender, age, income, living area, and CCI. Adjusted odds ratios with 95% confidence intervals were derived from logistic regression analysis, with statistical significance set at a p-value < 0.05 following adjustment for risk factors. All analysis was performed using Statistical Analysis Software version 9.4 .n = 1344), resulting in a study cohort of 1680 resuscitation cases .Table p < .0001). The usual care group significantly exceeded the palliative group in terms of hemodialysis usage , cardiopulmonary resuscitation usage , and ventilator usage . Kaplan–Meier survival curves were significantly lower in the palliative group than in the usual care group   . Note thThis study compared patients who did or did not undergo palliative care after successful ED resuscitation. Note that this methodology could be confounded by the indication for palliative services. Since 2009, Taiwan’s NHI palliative care coverage expanded to eight non-cancer irreversible advanced stage organ-system dysfunctions . The NHIContemporary palliative care practice and research are disease-oriented (cancer and non-cancer disease) , 44 and This study has a number of limitations. First, the patients included in the palliative group were not selected at random. Nonetheless, claims data from the NHIRD is representative of critical care in Taiwan with respect to ED size, location, academic affiliation, and patient characteristics. Second, we were unable to determine the reasons underlying the assignment of participants to palliative care. Moreover, we were unable to measure the number of palliative care patients who received life-sustaining treatment or the corresponding timelines. Third, this study did not have access to data related to sociocultural factors, the nature of each case, personal medical experience, or family dynamics.The implementation of palliative care was correlated with fewer life-sustaining treatments following resuscitation. This study provides real-world data pertaining to post-resuscitation care, which could be of value to clinicians, patients, and their families undergoing with evidence-based counseling when facing life-sustaining treatment share decision making. We believe that ED triage level I resuscitation status is a potential starting point for early palliative case finding and end-of-life counseling.Additional file 1: Supplementary Table 1. Top 20 categories of single-level clinical classifications: Palliative and usual care groups


Introduction: The use of novel designer drugs has increased worldwide over the years. Etizolam is a designer benzodiazepine (BZD) that has raised concern because of its growing non-medical use, liability to tolerance and dependence, and related harms. Studies exploring the abuse liability and cognitive effects of etizolam outside the therapeutic doses are lacking.Aims: To explore the abuse liability of etizolam and the characteristics of patients affected by etizolam high-dose dependence in a nationwide tertiary referral addiction unit. To document the cognitive changes to etizolam high-dose use.Design and Methods: Sociodemographic and clinical data on subjects with etizolam high-dose use were retrospectively collected from a database of 1,293 patients consecutively admitted to the Addiction Medicine Unit, Verona University Hospital, Italy for detoxification from high-dose BZDs or Z-drugs dependence. Thorough neuropsychological testing explored the cognitive side effects of high-dose etizolam use.Results: We found eleven etizolam high-dose users, of which eight used etizolam only, and three used etizolam with other BZDs/zolpidem. All the patients were prescribed etizolam for medical reasons, i.e., anxiety and/or insomnia. Neuropsychological evaluation showed deficits of working memory, visuospatial memory and executive function in a 27-year-old woman who used etizolam 15 mg daily.Discussion: Our findings suggest that abuse and dependence liability of etizolam should be considered a public health and social problem. They offer preliminary evidence on the cognitive side effects of etizolam high-dose use.Conclusions: This report offers new information on the potential harms of etizolam in patients who are prescribed this drug for medical reasons. Benzodiazepines (BDZs) are gamma-amino-butyric acid type A (GABA-A) receptor positive allosteric modulators widely prescribed for anxiety, insomnia and other conditions .The increasing use of novel designer BZD derivatives has been recently reported in countries, where these chemical compounds do not have marketing authorization as medicinal products .Etizolam is a short-acting  thienodiazepine designer BZD with high affinity for the GABA-A receptor and anxiolytic and sedative properties . EtizolaA consistent increase in the non-medical use and the illicit drug market of etizolam has been reported since 2014, being this drug implicated in several deaths in Scotland, United Kingdom, and to a lesser extent in the United States and Sweden . A recenHigh-dose dependence of BZDs or related Z-drugs , i.e., daily intake ≥5 times the recommended maximum daily dosage  is an emThis report is aimed to (a) explore the liability of etizolam to abuse and the characteristics of patients affected by etizolam high-dose dependence in a nationwide tertiary referral addiction unit; (b) document the cognitive changes to etizolam in a high-dose user who underwent thorough neuropsychological evaluation.Subjects with high-dose etizolam were retrospectively collected from a database of 1,293 patients  aged >18 years and admitted (January 2003–December 2019) to the Addiction Medicine Unit, Verona University Hospital, Italy, a nationwide tertiary referral center for detoxification from high-dose BZD/Z-drug dependence with slow flumazenil infusion .High-dose BZD/Z-drug dependence was defined according to DSM-IV-TR criteria  with useWe collected socio-demographic and clinical variables of the patients. The dosage of BZD/Z-drugs was based on self-report. DDDE (mg) was calculated according to conversion tables . The diaNeuropsychological evaluation explored verbal memory, working memory, visuospatial memory, attention and executive function. Verbal memory was assessed by means of the Italian versions of the Verbal Paired Association (VPA)  and the All patients underwent a detoxification protocol with slow subcutaneous flumazenil infusion (40.5 μg/h for 24 h/day for 7 days) with a prophylactic antiepileptic treatment .The study was conducted according to the Declaration of Helsinki and approved by the ethics committee of the Verona University Hospital. All the patients gave written informed consent to the study.Among the patients admitted from January 2003 to December 2019, we found 11 patients  who used high-dose etizolam either as the only BZD (8 patients) or with other BZDs or Z-drugs . All the patients were prescribed etizolam for medical use  and obtained the drug through a prescription and a pharmacy. The number of patients was stable across years . Sociodemographic and clinical features of patients are reported in The remaining 1,282 patients used other BZD/Z-drugs high-doses .One patient  underwent neuropsychological evaluation that showed working memory, visuospatial memory, and executive function to be outside normal values . Our patients received on average 27.3 ± 29.3 mg etizolam daily, which is nearly ten times the maximum recommended daily dosage , with one patient taking 100 mg daily, i.e., >30 times the maximum daily dosage. These findings support the conclusion of the recent WHO-ECDD report that abuse/dependence liability of etizolam should be considered as a public health and social problem . They arEtizolam high-dose users represented 0.9% of our whole sample and did not increase during the nearly 20 years covered by our large database, suggesting some concern, but stable figures across time. The big size difference between etizolam and other BZDs/Z-drugs high-dose users hampered a reasonable statistical comparison. However, when compared to high-dose users of lormetazepam, i.e., the most common BZD of high-dose use in Italy , etizolaThe small number of etizolam high-dose users in our large sample is likely related to the number of prescriptions in the general population. Etizolam does not stand among the ten most prescribed BZD active principles in Italy, in that its defined daily dose (DDD) in 2018 was <0.5/1,000, while that of the most commonly ones ranged from 13.2 for lormetazepam to 0.7 for flurazepam . The absEtizolam negatively influenced most of the cognitive domains in the patient who underwent neuropsychological testing, in particular working memory, visuospatial memory, and executive function, some of them being <2 SDs worse than normal values. This finding, despite being preliminary since stemming from a single patient, extends the notion that high doses of BZDs have an impact on cognition, even in younger patients , 24. BZDThe strengths of this study are that it represents the first series of etizolam high-dose users, and offers new information on the harms of this BZD derivative from a nationwide referral center in one of the few countries where it is marketed. The limitations are the retrospective design, the absence of systematic quantitative BZD measures to verify self-reported data, and neuropsychological data from a single patient that suggest caution in generalizing our findings on cognitive side effects of etizolam.In conclusion, a small number of patients who use etizolam for therapeutic reasons appear to transition to high-dose use requiring specialist care. This report offers new information on the potential harms related to etizolam and extends them to patients who are prescribed this drug for medical reasons. Future studies should confirm our findings in larger populations and in other countries where etizolam is marketed.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics Committee of the Verona University Hospital. The patients/participants provided their written informed consent to participate in this study.ST, EM, AB, AF, RC, and FL designed the study and gathered the data. ST analyzed the data. ST, EM, AB, AF, and RC drafted the manuscript. FL and ST revised the manuscript. All authors approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer, AM, declared a past co-authorship with one of the authors, FL, to the handling Editor.


The new era of nanotechnology has produced advanced nanomaterials applicable to various fields of medicine, including diagnostic bio-imaging, chemotherapy, targeted drug delivery, and biosensors. Various materials are formed into nanoparticles, such as gold nanomaterials, carbon quantum dots, and liposomes. The nanomaterials have been functionalized and widely used because they are biocompatible and easy to design and prepare. This review mainly focuses on nanomaterials responsive to the external stimuli used in drug-delivery systems. To overcome the drawbacks of conventional therapeutics to a tumor, the dual- and multi-responsive behaviors of nanoparticles have been harnessed to improve efficiency from a drug delivery point of view. Issues and future research related to these nanomaterial-based stimuli sensitivities and the scope of stimuli-responsive systems for nanomedicine applications are discussed. Nanotechnology research has had a significant impact on the field of medicine. Particularly, nanomaterials, such as liposomes, dendrimers, polymers, metallic nanoparticles (NPs), and nanogels, are currently being developed and used in various biomedical applications. These nanomaterial systems play an important role not only in sensing, biomedical imaging, and diagnosis but also in drug delivery. These smart carrier systems have been widely developed because they are responsive to various exogenous  and endogenous  stimuli.A variety of nanoparticles are suitable for drug delivery; thus, the development of nanoparticle drug-delivery systems with stimuli-responsive properties has gained considerable attention ,2,3,4. TIn this review, endogenous and exogenous stimuli, along with dual- and multi-stimuli-responsive drug delivery, are discussed . This reThe redox potential of tumor cells is 100- to 1000-fold higher than that of other cells . GlutathThe development of drug-delivery systems based on GSH-responsive assemblies has gained increasing attention. GSH can destabilize disulfide linkages in nanoparticles in a redox-mediated manner. Block copolymers contain disulfide linkages between hydrophilic and hydrophobic blocks that are GSH-responsive. This enables the formation of micelles, which are also known as ‘shell-sheddable’ micelles. When these micelles interact with GSH, they become destabilized and release therapeutic materials ,15.High concentrations of reducing materials in intracellular compartments help maintain polymers with disulfide linkages; however, polymers containing hydrophobic blocks  aBy adding reducing agents, spherical micelles were reproduced, and it showed that these selenium-containing nanocapsules were responsive to oxidizing and reducing conditions.N-vinyl pyrrolidone) (PVPON), poly (methyl methacrylate) (PMAA), and a disulfide cross-linker that could deliver plasmid DNA and doxorubicin [Disulfide linkages have been frequently used to design nanocarriers. For example, redox-responsive capsules were prepared by combining poly  is gaining much attention as a therapeutic target due to its upregulation in the tumor microenvironment. For instance, Mahdi Ghavami et al. prepared the phospholipase sensitive liposome (PSL) as a drug-delivery system, in which liposome degradation is caused by a tumor cell-secreted phospholipase A2 (sPLA2). sPLA2 is responsible for triggering the release of active PNA (peptide nucleic acid) as an antisense agent from PSL. The results showed that >80% of phospholipase was utilized to activate the PNA release. Thus, phospholipase may act usefully as a triggered agent to release drugs from nano-delivery platforms [Enzyme-responsive systems offer unique features, such as biorecognition, process efficiency, sensitivity, selectivity, and catalytic efficacy. In these systems, the drug payload is released upon enzyme-assisted degradation of polymeric moieties. Typically, two classes of enzymes serve as triggers in enzyme-responsive drug delivery: Proteases (or peptidases) and phospholipases . Proteaslatforms .An enzyme-responsive nanomaterial adapting an HPMA triblock copolymer was developed . The polThere are recent developments in microRNA responsive systems that are employing nanomaterials to deliver microRNA (miRNA) specifically to the target. miRNA is defined as a class of small non-coding RNAs, including short nucleotide sequences, 20–24 mer nucleotides that can serve as key regulators of gene expression . PreviouThe efficiency of delivering miRNA to the target has been significantly improved by loading miRNA with nanoparticles (NPs) protecting the miRNA from the external microenvironment, thereby reducing degradation and enhancing circulation time as well ,42. NanoGold nanoparticles (AuNPs) have gained much attention as nucleic acid delivery nanocarriers by their biocompatibility, facile synthesis, and tunable size and shape ,44. The Hyaluronic acid (HA), which is a highly hydrophilic natural polysaccharide, has been used to link PEI and PEG to a controlled-release miRNA and/or DOX . Liang eExogenous stimuli-responsive systems have the potential to overcome interpatient variability. In these systems, drug release can be precisely controlled by external factors. Exogenous physical stimuli mainly include temperature, light, magnetic fields, and ultrasound, which can be applied directly to the tissue of interest to stimulate drug release ,51,52,53The main advantage of most external stimuli-responsive systems is that drugs can be controlled—releasing at the desired site, which minimizes off-target effects in the surrounding healthy tissue. However, some treatments that involve injecting material followed by activation with ultrasonic waves, advanced light sources, or strong magnetic fields may not always be practical or cost-effective. Another problem with exogenous stimuli is the depth of penetration needed to cause a drug-delivery system to release its payload . To addrTemperature- or thermo-responsive systems are one of the most widely explored exogenous stimuli-responsive systems for treatment and diagnostic applications ,56. The N-isopropyl acrylamide) (PNIPAM) conjugated carbon nanomaterials were prepared with drug molecules to treat tumors [There are two strategies for a thermo-responsive drug release. One strategy involves drug carriers that are designed for a burst drug release when exposed to high temperatures. For example, polycaprolactone (LCST) and the upper CST (UCST) . A decreSpecific wavelengths of light  can change the stability or structural degradation of responsive nanomaterials in light-responsive drug-delivery systems to release drugs at precise locations.Owing to a low penetration capability, visible and ultraviolet light are not suitable for in vivo therapeutic applications. However, a NIR-responsive system is a promising technique that uses light to control drug release because it displays better penetration and limited tissue damage . There aThe photothermal effect involves the conversion of light to heat by a photothermal agent contained in the nanocarrier. This heat stimulates the heat-sensitive material and disrupts the nanostructure, breaks the hydrophobic and hydrophilic linkages, or creates a phase transition that leads to rapid drug release at the tumor site. Recently, Li et al. produced multiple nanostructured lipid carriers encapsulated by liposomes that were loaded with the hydrophilic drug AMD3100 and the hydrophobic NIR photothermal agent IR780 . NIR ligβ-cyclodextrin cap [β-cyclodextrin cap, which led to drug release. In summary, TPA is a promising strategy for controlled drug delivery because a NIR laser has high spatial and temporal resolutions, deep tissue penetration, and low scattering losses. However, this technique requires a focal pulsed laser with high energy density to treat a small infection area. Therefore, this method is not ideal for in vivo experiments.TPA relies on the excitation by two absorbed photons of identical or different frequencies . For instrin cap . The TPAThe UCNP technique can convert NIR light to UV light to activate high energy light-sensitive materials . To illuw/w). The release rate of DOX was accelerated under NIR-II irradiation owing to the on/off switching stimulus results. This indicates that NIR-II-induced local hyperthermia can release DOX from InSe-DOX in a controlled manner and at a predetermined time. DOX release can reach a maximum under acidic conditions with irradiation. Thus, the pH-responsiveness, NIR-II sensitivity, and switchable release characteristics of the InSe nanoplatform make it advantageous as a targeted chemotherapy delivery method [Huang et al. reported that ultrathin InSe nanosheets were efficient NIR-II-responsive drug-release agents . PEGylaty method .Magnetic systems are commonly used for body imaging  and a controllable drug release through external stimuli . They arThe basic mechanism of magnetic stimuli-responsive drug-release systems involves their ability to generate heat via an alternate magnetic frequency. MNPs can function as transducers to convert hysteresis loss and Néel relaxation to heat under an alternating current magnetic field. Two treatment mechanisms based on magnetic-responsive systems have been reported. One mechanism involves magnetic field-induced hyperthermia , and the3O4) nanoparticles (SPIONs) for theranostic purposes [Hyperthermia-based magnetic systems have been explored for drug delivery and tumor inhibition applications due to their magnetic responses . Thirunapurposes . They lopurposes . The imppurposes .Ultrasound technology has been used in therapeutic applications, such as imaging-guided drug delivery, because it is safe, penetrates tissues, is non-invasive, and has better spatiotemporal control since it can precisely focus on a target area. The thermal and mechanical effects, as well as the radiation forces generated by ultrasound waves, are responsible for stimulating a carrier drug release .Paris et al. synthesized mesoporous silica nanoparticles (MSNPs) PEGylated by the thermo-responsive linker ) . When anElectric fields are among the various physical triggers that have helped increase the efficiency of drug therapy. High-intensity exogenous electric fields can directly influence the permeability of cellular membranes, be used as a stimulus for drug delivery, or serve as therapeutic tools for wound healing and the restoration of tissue integrity.The mechanism underlying this approach involves the reduction of the conducting polymer when a potential is applied. This causes the drug that is incorporated into the polymer to be discharged into the surrounding environment by electrostatic repulsion. The rate of drug release depends on the polymer’s morphology (including its density) or electrochemical properties or the characteristics of the solution surrounding the drug-delivery system . Recently, Neumann et al. reported a strategy for electro-responsive drug delivery . They utIn addition to the above single stimulus-responsive nanoparticles, combined triggers that use magnetic fields and pH, temperature and redox potential, temperature and enzyme presence, temperature and pH, and redox potential and pH combined with temperature and redox potential can be considered for smart-responsive drug-delivery systems ,79,80,81N-isopropyl acrylamide) (pNIPAAm) because its LCST is approximately 32 °C, and its gel structure collapses above the LCST; thus, at body temperature (37 °C), the hydrogel shrinks. Combinations of pNIPAAm with pH-responsive polymers, such as polyacrylic acid and polyacrylamide, create dual-responsiveness [Nanoparticles triggered by pH and temperature are the most widely investigated dual-sensitive nanosized smart-targeting nanotherapeutics. Various polymers that are both pH- and temperature-responsive have gained considerable interest in the last decade as carriers of chemotherapeutic drugs because tumor tissues have a lower pH and a higher temperature than healthy tissues. The most commonly studied thermo-responsive polymer is poly -sensitive nanosystems can be adopted for the transdermal drug-delivery applications since they can penetrate the deeper layers of skin tissues. Yamazaki et al. studied the effect of liposomal drug delivery on melanocytes via the transdermal route. Owing to the deep penetration, the modified liposomes delivered antioxidants or UV-protective agents to melanocytes residing in deep skin tissues .Chen et al. prepared dual-stimuli-responsive microcapsules with drug loading and controlled release behaviors . In theiTwo of the most interesting intrinsic triggers are pH and redox potential because certain dysfunctional conditions such as cancer can locally alter them . Cancer N-(2-hydroxypropyl) methacrylamide) copolymer-doxorubicin conjugate-based nanoparticles [Tang et al. fabricated dual-sensitive and biodegradable core-crosslinked HPMA  and hydrophobic tocopherol succinate (TOS) . A vesicLight is an external trigger with the energy to cause precise changes in the organization of the functional groups or dielectric constants of polymers sensitive to electromagnetic radiation.2 produced by ICG. The core expanded gradually, but the shell shrank as the oxidation process increased, and the size of the Pt-ICG NPs became larger after NIR irradiation. The oxidation of tellurium made the hydrophobic block of the polymer more hydrophilic, which facilitated the free diffusion of water in and out of the nanoparticle core. This chemical property and the structural changes enable increased CDDP release from the nanoparticles [Li et al. prepared controlled-release nanoparticles (CDDP—cisplatin) that can reduce water infiltration and enhance the nanocarrier’s stability and half-life in plasma . After iarticles .Redox potential and light dual-stimuli-responsive systems are advantageous in cancer therapy and targeting applications. NIR light, as a stimulating factor, has been widely studied by researchers. Photothermal therapy based on photothermal conversion agents that can convert NIR light to heat can inhibit tumor cells. It is worth mentioning that a photothermal treatment is based on external NIR light that can deeply penetrate human tissues without invasive damage.You et al. created ICG- and CDDP-loaded smart nanoparticles that were NIR- and redox potential-responsive  to target tumors . The pre3O4 particles, can be combined with pH- or redox potential-responsive materials to construct multi-stimuli-responsive delivery systems that can control the drug release.Magnetic properties and magnetic field intensity should be considered when designing magnetic field-responsive delivery systems. Magnetic nanoparticles, particularly Fe3O4 (PCI)-graphene oxide (GO) nanocomposites [Zhang et al. prepared a magnetic/pH dual-responsive nanocomposite via the amide condensation of PEGylated citric-coated Femposites . The DOXmposites . Therefo3O4 nanoparticles, hydrophobic drugs, and green fluorescent dyes were encapsulated in microcapsules (approximately 500 nm) targeted by a red fluorescent dye to construct a chitosan-based microcapsule with both redox potential and magnetic field-responsiveness. The results indicate that the loaded drug was released slowly in the presence of GSH and a magnetic field.Another study assembled folic acid onto chitosan . Oleic aSystems sensitive to more than two stimuli are becoming increasingly attractive. When systems with more than two sensitivities, such as pH, temperature, and redox potential, magnetic fields, and temperature, are combined in the same matrix, they are called multi-responsive systems ,97,98. TMulti-responsive nanocarriers that are sensitive to temperature and redox potential can effectively release their drug payload in the tumor microenvironment. Hegazy et al. reported magnetic mesoporous silica nanoparticles (MMSNPs) that have iron oxide nanoparticles as a core inside a mesoporous silica coating . They caThrough in vitro loading and the triggered release of DOX, some results indicate that MMSNPs-PNIPAAm NPs exhibit both a high drug-loading capacity (19.6%) and encapsulation efficiency (73.8%) and successfully encapsulate DOX based on an absorbance peak at 480 nm. In terms of a temperature-triggered DOX release, this result shows that thermo-sensitive nanocarriers retain their drug payload at room temperature and rapidly release it near a locally-heated tumor (40–42 °C) to counteract blood-passage time and washout from the tumor.Regarding redox potential stimuli-responsiveness, the addition of tris(2-carboxyethyl)phosphine  causes rapid aggregation into macroparticles in water. In the presence of TCEP, a drug release from MMSNPs-PNIPAAm/DOX NPs reached 45% compared to less than 20% without TCEP over 24 h. It was concluded that the drug release from the nanoparticles was responsive to a reducing environment owing to the cleavage of disulfide bonds. PNIPAAm/DOX NPs exposed to a magnetic field were able to release significantly more DOX than the NPs exposed to a temperature below the LCST (25 °C) at the same time. Other results indicated that both a magnetic field and a high temperature enhanced DOX release over time and reached 86% after 24 h, applying reducing potential and temperature together resulted in an 80% release. In summary, simultaneously applying thermal, redox potential, and magnetic-sensitive environments significantly improved the drug release from PNIPAAm/DOx NPs that reached 98% within 24 h . The amoVarious multifunctionalities, including pH, light, and enzyme stimuli-responsiveness, can be added to nano-drug-delivery systems to produce theranostics that can detect tumors and release drugs at the tumor site.Copper sulfide nanoparticles (CuS NPs), which are composed of NIR-resonant materials, can be used in theranostics . CuS is CuS produces heat upon exposure to NIR, which is used as hyperthermic phototherapy against cancerous cells. A drug release is controlled by the presence of HA and an enzyme that cleaves HA inside the liposomes. In vitro experiments showed that only small amounts of DOX are released in an acidic environment and under NIR irradiation. In the presence of HA, the DOX release increased 26.6% from a pH of 7.4 to a pH of 4.5 within 14 h, and the DOX release was approximately 39.7% under NIR irradiation. This indicates that the enzymatic degradation of the HA coating facilitated DOX diffusion from the hollow interior into the surrounding medium. Under NIR irradiation, the DOX release was further enhanced; this may be caused by the heat produced by hollow mesoporous CuS, which decreases the viscosity of the surrounding fluid and facilitates drug diffusion .3O4/SiO2 NPs) and conducted an in vitro experiment on A549 and Hela cells to probe the cytotoxicity. The results showed that the Fe3O4NP coated surface could minimize the oxidative stress and iron homeostasis alteration and thus reduce the overall toxicity. From their study, it can be observed that Fe3O4/SiO2 NPs are efficient platforms for preventing dissolution, hindering the toxic ions, and enhancing the stability of the particles in the reduction of cytotoxic effects [Nanomaterials are advantageous in numerous biomedical applications due to their potential benefits with high drug loading, surface chemistry, biodegradability, and drug release characteristics accompanied by stimuli-sensitives such as light, temperature, ultrasound, etc. However, some limitations include toxicity, biocompatibility with certain types of cell membranes, or the size/shape of nanocarriers that need to be solved to optimize the efficiency of drug release in chemotherapy applications for safety purposes. In order to overcome the toxicity or cytotoxicity of nanomaterials that are harmful to humans, some approaches are proposed to develop compatible nanomaterials such as by surface modification with different biodegradable molecules. For example, Malvindi et al.  prepared effects . Finally effects  compared2) groups. AuNPs with PEG-SH showed no cytotoxicity compared with PEG and PEG-NH2 nanoparticles.The results collected from testing analyzed nanoparticles in the range of 1–300 µM showed that the lowest cytotoxicity could be seen in spherical nanoparticles. Another work carried by Reznickova et al.  evaluate2 particles to protect cells under harsh oxidative conditions. Therefore, nanomaterials with biodegradability hold a promising future in nanomedicine for overcoming problems associated with biocompatibility properties.Biodegradability is another essential factor that needs to be considered because it affects the stability of drug-carrier particles in the biological medium. Some biodegradable nanomaterials such as PLGA micelles, PCL nanoparticles, chitosan nanoparticles, dendrimers, and liposomes with their specific composition and administration strategy are utilized to improve therapeutic effects and to decrease side-effects . For insRecently, wide-ranging applications of nanotechnology and nanomaterials have made profound impacts on the pharmaceutical industry and, consequently, give rise to nanomedicine. The primary features of stimuli-responsive nanoparticles applied in biomedical applications, especially drug delivery, have attracted the interest of many researchers owing to their high loading capacity, biocompatibility, and stimuli-responsive drug release characteristics. Such characteristics also provide a promising avenue to enhance the effectiveness of traditional drugs and promote the rapid development of disease diagnostics and treatments.Despite significant advancements being made with nanomaterials in the biomedical or pharmaceutical areas at large, or specifically regarding drug delivery, many limitations remain. Biocompatibility, biodegradability, lower toxicity, and safer elimination of smart nanomaterials are some of the obstacles that need to be considered before designing a nanomaterial-based drug-delivery system. Additionally, the size of the carrier is another important feature that requires consideration, and administrative issues involved in using polymeric-based smart drug-delivery systems are another major drawback in this field. To solve these problems, future investigations should explore and design drug-delivery systems that use novel polymeric materials that are biocompatible and non-toxic.From a long-term perspective, dual- and multi-stimuli-responsive nanomaterials  may pave the way for unique nanosystems that are beneficial in biomedical applications .


Mycobacterium tuberculosis (M. tuberculosis), is one of the leading causes of human deaths globally according to the WHO TB 2019 report. The continuous rise in multi- and extensive-drug resistance in M. tuberculosis broadens the challenges to control tuberculosis. The availability of a large number of completely sequenced genomes of M. tuberculosis has provided an opportunity to explore the pangenome of the species along with the pan-phylogeny and to identify potential novel drug targets leading to drug discovery. We attempt to calculate the pangenome of M. tuberculosis that comprises a total of 150 complete genomes and performed the phylo-genomic classification and analysis. Further, the conserved core genome (1251 proteins) is subjected to various sequential filters  resulted in identification of eight putative broad-spectrum drug targets. Upon molecular docking analyses of these targets with ligands available at the DrugBank database shortlisted a total of five promising ligands with projected inhibitory potential; namely, 2′deoxy-thymidine-5′-diphospho-alpha-d-glucose, uridine diphosphate glucose, 2′-deoxy-thymidine-beta-l-rhamnose, thymidine-5′-triphosphate, and citicoline. We are confident that with further lead optimization and experimental validation, these lead compounds may provide a sound basis to develop safe and effective drugs against tuberculosis disease in humans.Tuberculosis, caused by  Mycobacterium tuberculosis (M. tuberculosis).According to the 2019 global World Health Organization (WHO) report, tuberculosis (TB) is among the top ten causes of death and the leading cause of a single infectious agent (above HIV/AIDS) [M. tuberculosis establishes infection inside the human body by overcoming the immune responses directed by the host against the foreign pathogen. Once inside, the bacteria infiltrate the host macrophages and persist inside them for years thus causing a chronic infection that reflects the failure of host immunity [M. tuberculosis has been attributed to spontaneous mutations in the bacterial genome, followed by the emergence of these mutant strains at the expense of wild type strains [TB is majorly an airborne disease where immunity . The inc strains . This, iM. tuberculosis genome sequence in 1998 along with further developments in the genomics and associated disciplines has enabled us to understand the importance of many proteins encoded in its genome [The availability of the first s genome ,5. Especs genome . These gM. tuberculosis genome. Further, we explored the structural association of these potential targets with FDA-approved drugs using molecular docking to shortlist promising lead molecules to aid drug development against TB.Various protocols in bioinformatics have helped scientists to process biological data of pathogens in order to prioritize drug targets, while the parallel developments in the cheminformatics help researchers access ligand databases such as the DrugBank and perform virtual screening for in silico-aided drug discovery ,8. UtiliM. tuberculosis.Developing a new drug through conventional methods takes at least ten years of comprehensive research and huge funding; Nevertheless, the inclusion of computer-aided analyses at the initial stages can decrease the associated time and costs ,12. In sM. tuberculosis were downloaded from the NCBI. Their information such as the accession number, strain name, and genome statistics are provided in M. tuberculosis complete genomes revealed that there were 5009 gene families in total (pangenome), among them, 1251 proteins were common (core genome) in all the studied genomes. The ratio of the core and pangenome size was found to be 0.25, thus the core forms 25% of the pangenome. This signifies the low level of genetic diversity in the M. tuberculosis strains. This trend is also visible in the pan-core genome plot, which projects that the global gene repertoire of this species is difficult to alter considerably in the future  thus accounted for 30.5% of the average genome size. Similarly, the minimum number of protein-encoding genes (3622) was present in strain RGTB423 while the maximum number of protein-encoding genes (4599) was present in strain 2279.M. tuberculosis genomes is believed to be very high, with limited genetic variability between/across its various strains. Our genome-level analysis also indicates that the pangenome of this species is almost closed, with minimum variations. Multiple studies have corroborated this claim [The level of conservation in the is claim ,15,16. Tis claim . Due to M. tuberculosis for the identification of therapeutic targets. Targets found to be human homologs could adversely affect the host metabolism, therefore, all those proteins were excluded in the first step that were identified as human homologs. The Rv numbers (gene identifiers or virulent strain of Mycobacterium tuberculosis) and the gene annotation were also conducted on these core proteins; the core genomes-associated data is provided in M. tuberculosis pathogenicity  did not show similarity to any drug target in the DrugBank database thus it is potentially a novel drug target worthy of experimental testing. Virtual screening may be performed with ligands from other ligand databases in order to find potent inhibitors of this protein. Literature studies were conducted on the seven proteins from trcR has a crucial role in intracellular survival of M. tuberculosis and also in the regulation of Rv1057 expression, i.e., the β-propeller gene activated by the envelope stress [trcR and trcS two-component system genes are transcribed in broth-grown M. tuberculosis validated through reverse transcription PCR analysis. Moreover, through the SCOTS technique the expression of these genes are observed in liquid (broth) medium and after 18 h of M. tuberculosis growth in cultured human primary macrophages [M. tuberculosis [Among them, e stress . In anotrophages . Neverthrculosis . We beliM. tuberculosis through inhibitors is a good strategy, as has been recognized by earlier studies [The dTDP-4-dehydrorhamnose reductase, another protein classified as a drug target by our study, is already known to be a high-confidence drug target . This pr studies ,24.mtrA  with glucose-1-phosphate thymidylyltransferase  in that case. Nevertheless, our docking analyses proposed the 2′deoxy-thymidine-5′-diphospho-alpha-d-glucose compound as a high-ranked lead compound to develop a drug against tuberculosis.Interestingly, 2′deoxy-thymidine-5′-diphospho-alpha-l-rhamnose ligand exhibited docked energy of −9.1 kcal/mol with glucose-1-phosphate thymidylyltransferase . The core genome was then subjected to various sequential filters of subtractive genomics  to select potential drug targets. Finally, these proteins were subjected to molecular docking with ligands from the DrugBank database to identify potential lead compounds that could be useful to develop a drug against tuberculosis.A total of 150 complete genome sequences of M. tuberculosis strains and their associated proteomes were retrieved from the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/) available at the National Center for Biotechnology Information (NCBI). These strains had been earlier isolated from all the geographical regions of the World.The complete genome sequences off 150 M. tuberculosis [Pangenome analysis was conducted on the genomes using the Bacterial Pan Genome Analysis (BPGA) tool to obtain highly conserved proteins of rculosis . For thirculosis . BPGA ca−3) was used to compare the core genome with the human genome [To aid the preliminary identification of novel therapeutic targets in the core genome/proteome, we used an in-house pipeline VacSol for non-homology, essentiality, and virulence screening steps . To avoin genome . From thn genome . The DEGn genome ,46. The n genome . MvirDB M. tuberculosis were further filtered based on physicochemical parameters such as molecular weight, pI (isoelectric point), grand average of hydropathicity (GRAVY) value, and aliphatic index using the ProtParam tool [To identify potential drug targets, the non-human homolog, essential, and virulence-associated core proteins of ram tool . Low molram tool . A higheram tool . After pram tool .Finally, druggability assessment of the shortlisted proteins was performed using BLASTp against the DrugBank database at default settings . The druThe sequences of drug targets were checked for the availability of crystal structures, if any, from the RCSB Protein DataBank . Structu