Cancer is a complex disease that develops over time through accumulated mutations in DNA that transform normal cells into a cancerous state. To fully capture the complexity of the cancer genome, computational methods have been developed to summarize the mutational patterns of cancer, distinguish causal oncogenic mutations, and determine clinically useful mutational patterns. In this review, we survey different computational approaches with an emphasis on important clinical roles and provide insights into better integration of computational methods for clinical use.Since the rise of next-generation sequencing technologies, the catalogue of mutations in cancer has been continuously expanding. To address the complexity of the cancer-genomic landscape and extract meaningful insights, numerous computational approaches have been developed over the last two decades. In this review, we survey the current leading computational methods to derive intricate mutational patterns in the context of clinical relevance. We begin with mutation signatures, explaining first how mutation signatures were developed and then examining the utility of studies using mutation signatures to correlate environmental effects on the cancer genome. Next, we examine current clinical research that employs mutation signatures and discuss the potential use cases and challenges of mutation signatures in clinical decision-making. We then examine computational studies developing tools to investigate complex patterns of mutations beyond the context of mutational signatures. We survey methods to identify cancer-driver genes, from single-driver studies to pathway and network analyses. In addition, we review methods inferring complex combinations of mutations for clinical tasks and using mutations integrated with multi-omics data to better predict cancer phenotypes. We examine the use of these tools for either discovery or prediction, including prediction of tumor origin, treatment outcomes, prognosis, and cancer typing. We further discuss the main limitations preventing widespread clinical integration of computational tools for the diagnosis and treatment of cancer. We end by proposing solutions to address these challenges using recent advances in machine learning. Cancer has historically been studied using genetic techniques, with the goal to identify gene-driver mutations that confer selective advantage and drive cells into a cancerous state. Driver mutations are distinguished from passenger mutations, which accumulate in the genome due to the changes undergone in the cancer cell as it becomes cancerous ,2. TheseBox 1Targeted therapiesTherapies targeting a specific protein associated with a diseaseSynthetic lethalityA type of interaction wherein a single event is tolerable but co-occurrence of two or more events is lethal Driver mutationA mutation that provides a selective advantage to a cell and transforms a cell into a cancerous statePassenger mutationA mutation that is a result, but not a direct cause, of a cell becoming cancerousMutagenic processAnything that causes damage to DNA or induces mutations in DNA, such as UV light, radiation, or alkylating agentsNon-negative matrix factorization (in progress)Unsupervised mathematical method wherein a single large nonnegative matrix is decomposed into two or more smaller matrices COSMIChttps://cancer.sanger.ac.uk/cosmic, accessed on 12 March 2023Catalogue of Somatic Mutations in Cancer: Signature 1Mutation signature associated with ageSignature 2Mutation signature associated with the mutagenic effects of APOBEC activitySignature 4Mutation signature associated with tobacco smokeSignature 7Mutation signature associated with UV exposureSignature 10Mutation signature associated with POLE proofreading errorsSignature 16Mutation signature associated with alcohol consumptionSignature 18Mutation signature associated with the mutagenic effects of the MUTYH geneDDRDNA damage repair, a network of processes that repairs damaged DNAMMRMismatch repair, a DDR pathway involved in detecting and repairing DNA mismatches BERBase-excision repair, a pathway that repairs typically small-scale mutations by first removing only the base and leaving an abasic site, which is later removed and replaced with other nucleotidesNERNucleotide-excision repair, a pathway that repairs mutations by entirely removing mutated sections of DNAHRHomologous recombination, a pathway repairing double-strand DNA damage that uses another strand of DNA as a template for repairNHEJNon-homologous end joining, a pathway repairing double-strand DNA damage that involves attaching two strands of broken DNA together. Logistic regressionA regression model for supervised classification LASSO logistic regressionA regression model that uses L1 regularizationRandom rorestAn ensemble machine-learning model that combines decision trees produced by baggingICIImmune-checkpoint inhibitors, a class of cancer drugs that suppresses pro-tumor immune-system regulatory effectsSupervised learningMachine-learning strategies wherein the classes of outcomes are knownUnsupervised learningMachine-learning strategies wherein the task of the model is to cluster the data into previously unidentified classes or discover the underlying classesNeural networkA machine-learning model that connects the input data to a desired output classification, where nodes connected by edges apply non-linear transformations to the data passed through the networkDeep learningMachine-learning models that are composed of multiple layers of neural networks stacked over one another (giving rise to the term “deep”) OverfittingFitting a particular data point too well and therefore failing to predict on other dataUnderfittingNot fitting the data well enough and inferring simplified decision rules that may not be optimized for any datasetGraph convolutional networks Neural-network architectures that represent graph data for learning tasksOne of the earliest and most important computational tools to uncover patterns of mutations arising through different mutagenic processes  is mutatMutation signatures mathematically model certain types of mutations that cluster together based on co-occurrence in tumors ,25,26 FB. The orComputationally, the first mutation-signature methods relied on the mathematical principle of non-negative matrix factorization (NMF) , where aMany developments and refinements of the methods to generate mutation signatures have been suggested. Several rely on variations in NMF ,25,31,32Once derived, the mutation signatures are then associated with potential biological, environmental, or cancer-related phenomena, and mutations that occur in these signatures may be extracted to investigate potential clinical relevance B. The laConcurrent with the development of mutation signatures was the recognition that these signatures may potentially be used in a clinical context for prognoses and treatment outcomes ,24. WithDNA damage repair (DDR) is a complex network comprising multiple DNA-repair pathways, damage-tolerance processes, and cell-cycle checkpoints, with multiple interacting components assessing and maintaining genomic integrity ,56,57. IDisruption in DDR pathways induces genome-wide mutagenesis, and some DDR pathways are linked to responses to specific treatments, including chemoradiation and targeted therapies. Mutation signatures become useful in such cases, as they can examine patterns of DDR deficiencies throughout the genome.This concept has been most clearly shown in applications to HR-deficient cancers. Loss of HR results in increased sensitivity to inhibition of the BER gene PARP1. The absence of PARP allows for unrepaired single-strand breaks to accumulate, and these breaks collide with replication forks and induce cytotoxic double-strand breaks. When HR deficient, cells are unable to repair those breaks, leading to genomic instability and cell death ,63. TherOther treatments targeting HRD cancers are currently in clinical trials, where mutation signatures may become useful. These treatments target different proteins involved in the HR pathway, for example, ATR inhibitors . ATR inhMSH2, MSH6, PMS2, and MLH1) are clinically available for MMR , an, an76],  for MMR ,79, rese for MMR ,81, posi for MMR ,82,83 Fu for MMR .Other associations between cancer treatments and distinct DDR pathways include ERCC2 helicase in the NER pathway . MutatedOther than canonical DDR pathways, proofreading errors also induce distinct mutation signatures, potentially allowing for the development of similar methods to MMR and HR mutation-signature tools. For example, POLE proofreading errors are associated with Signature 10 , which cDue to their ability to elucidate associations between exogenous or endogenous mutagenesis and cancer, mutation signatures are useful for studying clinical phenomena when the underlying mechanisms and genetic markers are unknown. Therefore, these signatures may be useful for clinical development and discovery .Radiation therapy has long been recognized as a potential driver of new cancers ,100, butMutation signatures are being used to investigate the effects of other cancer treatments on the genome, allowing both a better understanding of the mechanism of the treatments and potential indications or contra-indications of the treatment. For example, using mutation signatures, 5-FU was found to induce numerous T > G substitutions throughout the genome, indicating a potential tumorigenic effect of this chemotherapy drug  89]. Fu. Fu89]. Mutation signatures have also driven discovery of clinically relevant environmental carcinogens through patterns of mutations in the genome. Aristolochic acid (AA) is a chemical found in plants used in herbal remedies. In different cancers, and in bladder cancers in particular, the presence of AA-associated signatures provided evidence that AA has a mutagenic effect on the genome, demonstrating the potential of mutation signatures as a screening tool  90,91,9,991,92. Another promising use of mutation signatures is as a biomarker for different cancer types or cell types. Mutation signatures were used to distinguish different cell types within esophageal adenocarcinoma, with the potential to directly target these different subtypes for different therapy treatments  97]. Re. Re97]. The clinical potential of mutation signatures in other contexts has been mentioned in multiple studies, for example, for predicting immunotherapy response ,86,87. IIn addition to mutation signatures, other methods have been developed to discover patterns of cancer mutations that drive cancer development and underlie clinical outcomes . The majMethods have also shifted from focusing on features of single genes to accounting for more complex patterns, such as gene networks and pathways  Figure C. These Other than discovery of driver mutations, methods have used pathway and network information to identify patterns of mutations to predict treatment outcomes, allowing for more biologically interpretable models  129,130130. An eMutations in a single gene or within a specific pathway may not be sufficient for characterizing cancer development or clinical outcomes. More complex patterns and interactions between mutations confer more information for clinical-prediction tasks. Methods to identify combinations of mutations were used to distinguish tumors from healthy tissues , to findSome methods have incorporated tumor mutations with other types of data to predict response to cancer therapies D 154]. . 154]. FDespite substantial efforts to identify clinically relevant cancer mutations and patterns, complex patterns beyond single-gene mutations have not been integrated into the clinic. There are several challenges for computational approaches that have prohibited the clinical success of data-driven mutational patterns, which are outlined below, along with potential ways forward to overcome these challenges .A major and fundamental challenge to overcome is the difficulty of recapitulating associations between mutational patterns and clinical features across multiple studies A 163,16. This isTools are also being developed to assist non-specialists with ML applications . One exaAnother important challenge to overcome in the path to clinical integration is the issue of biologically interpretable results B 113,13,188. An To address this complexity, many interpretation approaches have been proposed to provide explanations for the trained models’ predictions and the features driving the model to make a specific prediction . LIME isAnother form of explanatory methods is through biological-network explanations  210,211211. BiolAnother challenge for uncovering complex patterns of mutations is linked to the sparse nature of mutation data themselves C. MutatiAnother factor that can lead to sparsity is missing data. Missing data can result from experimental design or different types of human errors ,236. In Another challenge is linked to how mutations are accumulated in cancer evolution. As cancer develops, mutations arise in certain cell lineages, and tumor mutations are therefore clonal and not homogenous ,249,250.With the introduction of next-generation sequencing, numerous causal and actionable mutations have been identified and used clinically as biomarkers or for new targeted therapies. Due to the increasing realization of the vast complexity underlying tumorigenesis, future clinical breakthroughs are likely to increasingly rely on computational methods to identify these clinically actionable patterns of mutations. Mutation signatures allow for exploration of intricate patterns of mutations in cancer, effectively identifying mutational patterns to describe DDR pathways and environmental effects. However, mutation signatures require extensive sequencing of cancer genomes, limiting clinical applications beyond these purposes. Other methods have been developed to uncover complex patterns of mutations for clinical use. These include methods that identify drivers of cancer, methods that predict clinical outcomes by integrating mutations with biological pathways, and methods incorporating other types of omics. However, such methods have yet to be integrated into the clinic. The major challenges for clinical integration of computationally driven mutational patterns are lack of reproducibility, the difficulty of interpreting complex models, and issues associated with intrinsic attributes of cancer-mutational data, such as sparsity and clonality. State-of-the-art computational and machine learning can be adjusted to address these issues, improving the interpretation of complex models and enhancing reproducibility. With the consistent accumulation of cancer-genomic datasets and the complexity of cancer genomes, many of the next great clinical breakthroughs in cancer research will rely on computational tools to fully understand the complicated patterns of mutations that characterize cancer.


Chagas disease (ChD) represents a significant health burden in endemic regions of Latin America and is increasingly being recognized as a global health issue. The cardiac involvement in ChD, known as Chagas cardiomyopathy (ChCM), is the most severe manifestation and a leading cause of heart failure and mortality in affected individuals. Echocardiography, a non-invasive imaging modality, plays a crucial role in the diagnosis, monitoring, and risk stratification of ChCM.This consensus recommendation aims to provide guidance on the appropriate use of echocardiography in ChD. An international panel of experts, including cardiologists, infectious disease specialists, and echocardiography specialists, convened to review the available evidence and provide practical recommendations based on their collective expertise.The consensus addresses key aspects related to echocardiography in ChD, including its role in the initial evaluation, serial monitoring, and risk assessment of patients. It emphasizes the importance of standardized echocardiographic protocols, including the assessment of left ventricular function, chamber dimensions, wall motion abnormalities, valvular involvement, and the presence of ventricular aneurysm. Additionally, the consensus discusses the utility of advanced echocardiographic techniques, such as strain imaging and 3D echocardiography, in assessing myocardial mechanics and ventricular remodeling. Trypanosoma cruzi, with some of the most serious manifestations affecting the cardiovascular system. It is a chronic, stigmatizing condition, associated with poverty and affecting close to six million people globally  and B2 [asymptomatic global LV dysfunction] .Development of heart failure symptoms in a patient with structural cardiac changes identified by echocardiography is the hallmark of ‘Stage C’ illness. Finally, ‘Stage D’ chronic ChD patients are those at NYHA functional class IV with the clinical picture of decompensated, advanced heart failure generally associated with diffuse biventricular dysfunction and chambers dilatation 1862.18Detection of cardiac involvement in the early stages of disease is particularly challenging, but of importance in determining requirements for follow up and risk stratification. Patients with true stage A disease need much less regular follow up, while those with incipient disease need more regular monitoring. Decline in ventricular systolic function parallels the staging of cardiac impairment and serves as the strongest predictor of survival in chronic ChD 63. HowevAsymptomatic Chagas patients with normal ECG and chest radiography: A normal ECG has strong negative predictive value for cardiovascular events and the majority of patients with a normal ECG will not develop cardiomyopathy. Those who do so, will have a 10 to 30 year lag period before worsening. A baseline echocardiogram is theoretically recommended 3 however,For subsequent, follow-up echo there is also no urgent requirement when a baseline echo is normal, while ECG and X-rays remain normal and the patient remains asymptomatic.Asymptomatic patients with ECG abnormalities: baseline echocardiography is recommended and the frequency of follow-up echo depends on the clinical status of the patient. Although studies on the progression of the disease are limited, it seems to be slow in asymptomatic patients with a normal echo . Hence i362The purpose of echocardiographic follow-up is to identify patients with moderate or severe LV dysfunction, who are often still asymptomatic but who could benefit from early administration of guideline-directed medical therapy 626365. O6263If significant LV dysfunction is present, recommendations for follow-up echocardiographic evaluation are similar to those for other forms of dilated cardiomyopathy. An echocardiogram should be repeated if there has been a deterioration in clinical status. An echocardiogram is also advised 3–6 months after optimization of standard therapies for heart failure with reduced ejection fraction (HFrEF) to determine the need for additional novel pharmacological agents like Angiotensin Receptor and Neprilysin Inhibitor Sacubitril-Valsartan or Sodium-Glucose Transporter 2 inhibitors dapagliflozin and empagliflozin and/or implanted devices .Patients with symptomatic ChCM may present with diffuse hypokinesis. Nunes et al compared patients with idiopathic dilated cardiomyopathy versus patients with diffuse dilated ChCM, finding a worse prognosis in Chagas disease patients . SimilarCardiac magnetic resonance and radionuclide imaging provide complementary information to ECG and echocardiography in ChCM .123I-MIBG), SPECT, or PET with 18-Fluorodeoxyglucose, several findings and tissue characterization may be achieved, like microvascular disease, fibrosis-scars, edema, denervation [676869707172737475Using different cardiac magnetic resonance techniques and contrast images (late gadolinium enhancement), as well as radionuclide imaging with iodine-123-metaiodobenzylguanidine (Chagas disease is a serious public health concern in endemic countries and has become an emerging health problem in non-endemic areas due to migration and results in substantial morbidity and mortality. Echocardiography is a safe and cost-effective imaging tool to identify cardiac involvement in Chagas disease, offering the possibility of earlier diagnosis and prognostic information to improve management of patients. Thus, a comprehensive Doppler echocardiographic exam of patients with ECG abnormalities is fundamental, in order to detect ventricular abnormalities that may require treatment and offer prognostic information.Despite the advantages of echo, it remains out of reach of many patients in the resource limited settings where the disease is most prevalent. Ironically it is in these settings that potentially the greatest benefits could be achieved in better managing patients at the point of care.


Although late-stage clinical attrition has been long considered as the most significant issue facing the pharmaceutical industry, the probability of technical success in the clinic is largely related to decisions made years earlier in the preclinical stages of Research and Development (R&D); it is at these early stages that decisions are made regarding the molecular target, modality of intervention, drug design and clinical candidate selection. Accordingly, model-informed drug development approaches that have proven useful in the clinic  modeling, physiologically based pharmacokinetic (PBPK) modeling, pharmacokinetic-pharmacodynamic (PKPD) modeling) are increasingly leveraged to support decisions in the earlier preclinical stages of R&D. These advances, however, have not been well-represented in the literature. This topic illustrates efforts to apply modeling in target verification, lead compound optimization, clinical candidate selection, and human efficacious dose prediction, with an emphasis on how modeling and simulation is being used to advance hypothesis driven research and support decision making in preclinical research. As a collection, the papers included in this topic will allow researchers to better understand the impact and limitations that such modeling has in real-world drug research, and, in turn, facilitate insight and guidance for future research in quantitative pharmacological modeling and simulation.Presented as a high-level overview, authors from several pharmaceutical companies shared their collective experiences about how modeling and simulation approaches have been used to inform various decision points from discovery to first-in-human clinical trials . Target in vivo drug-target interactions and hence clinical outcomes.Beyond prospective predictions, retrospective analysis of existing clinical data through PBPK modeling can provide valuable information about target engagement required for efficacy at the site of action that may not be easily assessed using experimental methods . These sModeling preclinical data generated by novel tools can further help to better understand the system, facilitate applications of these tools in drug discovery, and provide the foundation for preclinical-to-clinical translation . ComputaIn conclusion, this topic highlights exciting new approaches to advance preclinical drug development and help reduce attrition along the drug development pipeline.


Background: Because of the aging population, the number of low falls in elderly people with pre-existing anticoagulation is rising, often leading to traumatic brain injury (TBI) with a social and economic burden. Hemostatic disorders and disbalances seem to play a pivotal role in bleeding progression. Interrelationships between anticoagulatoric medication, coagulopathy, and bleeding progression seem to be a promising aim of therapy. Methods: We conducted a selective search of the literature in databases like Medline (Pubmed), Cochrane Library and current European treatment recommendations using relevant terms or their combination. Results: Patients with isolated TBI are at risk for developing coagulopathy in the clinical course. Pre-injury intake of anticoagulants is leading to a significant increase in coagulopathy, so every third patient with TBI in this population suffers from coagulopathy, leading to hemorrhagic progression and delayed traumatic intracranial hemorrhage. In an assessment of coagulopathy, viscoelastic tests such as TEG or ROTEM seem to be more beneficial than conventional coagulation assays alone, especially because of their timely and more specific gain of information about coagulopathy. Furthermore, results of point-of-care diagnostic make rapid “goal-directed therapy” possible with promising results in subgroups of patients with TBI. Conclusions: The use of innovative technologies such as viscoelastic tests in the assessment of hemostatic disorders and implementation of treatment algorithms seem to be beneficial in patients with TBI, but further studies are needed to evaluate their impact on secondary brain injury and mortality. Traumatic brain injury (TBI) remains a significant health problem of our times with high socioeconomic burden ,2. The pRecent observational data from the pan-European CENTER-TBI study on patients with isolated TBI have confirmed laboratory coagulopathy in 16 percent of patients with no intake of antiplatelet drugs and/or anticoagulants before trauma versus 34 percent of patients with pre-injury antiplatelet and/or anticoagulant therapy upon hospital admission . In geneIn the first 24 h after admission to the hospital, we can observe a doubling in the prevalence of coagulopathy in patients with TBI , and a cThe occurrence of coagulopathy upon hospital admission has frequently been identified as a powerful predictor for prognosis after TBI . AggregaIn general, coagulopathy does not cause hemorrhage in the absenteeism of any (micro)vascular injury or failure, including blood-brain barrier (BBB) malfunction or breakdown. Experimental imaging studies have shown secondary bleeding from ruptured arterioles and venules localized to the periphery of existing hematomas and into congested areas of perihematoma tissues ,41. AlteDirect injury to the blood vessel or defragmentation from microvascular failure triggers the release of tissue factor (TF) which promotes the extrinsic coagulation pathway  with conHypoperfusion and shock play a pivotal role in the development of hemostatic failure ,31. ShocThe role of platelets in the context of TBI-associated coagulopathy remains poorly defined. It is understood that low platelet counts and platelet dysfunction contribute to hemostatic failure and disease progression ,58. WhilIn vivo and in vitro models are very useful in TBI research. Common in vivo models are weight drop, fluid percussion, controlled cortical impact injury, blast injury, penetrating ballistic-like brain injury and acceleration. In contrast to these, there are three usually used in vitro models called immortalized cell lines, dissociated primary cultures and organotypic cultures from different individuals . The mosIn contrast to in vivo models, in vitro models have better reproducibility but transfer from cell culture to clinical reality is difficult because of the complexity of TBI. Immortalized cell lines are useful for examining cellular proliferation, differentiation and molecular pathways in cells, and there are established cell lines for specific research fields . Dissoci® and TEG®) with platelet function assays and thrombin generation tests, may have advantages over conventional coagulation assays (CCAs) in the early detection of hemostatic abnormalities after TBI and in monitoring and guiding subsequent therapies [®) or thromboelastometry (ROTEM®), shear forces are measured optically or electromechanically between a wire and a cup in whole blood samples when pro-coagulatory agents are added. Depending on the clotting process, information about platelet function, fibrin polymerization and fibrinolysis is provided within minutes. While CCAs only reflect the initiation process of coagulation and only a small part of thrombin generation, “point of care” viscoelastic assays provide timely and more detailed information on the overall hemostatic potential [® Ecarin clotting time and Russel Viper venom/RVV clotting time) are sensitive to the detection of clinically-relevant NOAC plasma drug levels [Functional hemostatic assays, for example, viscoelastic tests  are the most transfused blood products, although evidence for transfusion threshold in patients with TBI is still missing . SeveralExperimental resuscitation with fresh frozen plasma concentrates (FFP) in different models of TBI and hemorrhagic shock was associated with reduced lesion size, downregulation of inflammatory genes, and improved protein expression profiles for blood–brain barrier (BBB) integrity ,82. The The pragmatic randomized controlled Clinical Randomisation of an Antifibrinolytic in Significant Haemorrhage (CRASH)-3 trial demonstrated, for the first time, an improvement in a pharmacological intervention for the least-specific TBI sub-groups . A stati® and TEG®) have successfully been integrated into diagnostic and therapeutic approaches in high-risk patients with active hemorrhage and furthermore in patients with TBI [Viscoelastic assays (ROTEMwith TBI ,66,72,91with TBI ,72,91,92with TBI . In the with TBI . Anotherwith TBI . This TBwith TBI .This review is intended for clinical practitioners and provides an overview of current knowledge of pathophysiology and therapeutic strategies in TBI, given therapeutic strategies are based on data or clinical experience of their authors. Nevertheless, treatment options mentioned in this review need further research, and big cohort studies are missing, especially for new reversal drugs. Potential side effects from thromboembolic events to death have been reported. In everyday clinical practice, this review can give some advice for therapeutic considerations, but individual risk-benefit analyses are needed for every patient. This review does not relate to patients with pre-existing hemostatic disorders or children suffering from TBI.The use of viscoelastic tests, in addition to conventional coagulation laboratory parameters, seems to be beneficial in patients with TBI and especially in patients with anticoagulatoric medication. Because of the aging population, more older patients with anticoagulatoric medication will suffer from TBI. Diagnostic and therapeutic approaches will be needed to improve survival and functional outcome. Point-of-Care diagnostic and Goal-directed-therapies seem to be promising fields of research. Further studies are needed to evaluate their impact on secondary brain injury and mortality and to define precise therapeutic thresholds.


The value, speed of completion and robustness of the evidence generated by TB treatment trials could be improved by implementing standards for best practice.A global panel of experts participated in a Delphi process, using a 7-point Likert scale to score and revise draft standards until consensus was reached.Eleven standards were defined: Standard 1, high quality data on TB regimens are essential to inform clinical and programmatic management; Standard 2, the research questions addressed by TB trials should be relevant to affected communities, who should be included in all trial stages; Standard 3, trials should make every effort to be as inclusive as possible; Standard 4, the most efficient trial designs should be considered to improve the evidence base as quickly and cost effectively as possible, without compromising quality; Standard 5, trial governance should be in line with accepted good clinical practice; Standard 6, trials should investigate and report strategies that promote optimal engagement in care; Standard 7, where possible, TB trials should include pharmacokinetic and pharmacodynamic components; Standard 8, outcomes should include frequency of disease recurrence and post-treatment sequelae; Standard 9, TB trials should aim to harmonise key outcomes and data structures across studies; Standard 10, TB trials should include biobanking; Standard 11, treatment trials should invest in capacity strengthening of local trial and TB programme staff.These standards should improve the efficiency and effectiveness of evidence generation, as well as the translation of research into policy and practice. Momentum towards global TB elimination has been slow, with a reversal of progress during the COVID-19 pandemic.Our aim is to present standards to complement the guidelines for good clinical practice (GCP) established by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH),We identified a global panel of experts representing clinical trialists, academics, civil society and implementers with TB programme knowledge and clinical trial experience. Special attention was given to ensure adequate regional representation and diversity. A total of 70 experts were invited and 40 responded within the specified time frame. A core group of eight people generated key domains for which potential standards could be developed and drafted initial statements that were then discussed and revised by the larger group. A 7-point Likert scale was used to assess agreement with initial draft statements  and subsequent revisions. Standards were accepted if 90% of respondents assigned a score of 5 or higher; standards with less than 80% agreement were dropped and those with 80–90% agreement were reviewed. Additional standards and domains could also be suggested, and similar statements were consolidated as appropriate. A total of 13 domains and 39 statements were developed, and 11 standards were retained after multiple rounds of feedback and consensus building. Panel members were then invited to comment on the ‘final’ standards and accompanying text that constituted the draft manuscript. The final version was approved by all contributors with >90% consensus achieved for all standards.High-quality data on TB regimens are essential to inform clinical practice and programmatic management.Recently updated WHO guidelines for the treatment of drug-resistant TB, including isoniazid-resistant and MDR/RR-TB, are based on low certainty of evidence.Although more investment in drug and regimen development is required, the TB drug development pipeline has improved considerably in recent years.The research questions addressed by TB treatment trials should be relevant to affected communities, who should be included in all trial stages from design to reporting.Community engagement is crucial for the success of clinical trials, especially in TB research, where obstacles such as stigma, limited healthcare access, social inequality and discrimination are common.23When communities are engaged early and thoroughly, they may ensure culturally appropriate study design, promote community sensitisation to current and future trials and facilitate acceptability, including enhanced trial recruitment and retention.27Effective community engagement requires investment, equity-based and pragmatic approaches tailored to community priorities, needs and capacities see . There m29TB treatment trials should make every effort to be as inclusive as possible to increase the generalisability of the findings.Mycobacterium tuberculosis strains can differ globally, with strain-specific differences in drug susceptibility or immune response,There is both a clinical and ethical imperative for an inclusive approach in clinical trials. Ultimately, clinicians will need to treat all people who present for TB care and will need knowledge about safety and efficacy in as broad a range of people as possible to be able to do so. TB disease manifestations and burden, as well as pharmacokinetics and pharmacodynamics may vary among individuals with comorbidities , people who use drugs, pregnant/breastfeeding people, children and those with extrapulmonary TB. Ethically there is a clear social justice requirement for broad-based access to clinical trials,The approach to inclusivity in trials does raise challenges, with broad inclusion likely to increase the complexity and potentially reduce the power to determine key outcomes due to increased heterogeneity. Furthermore, increasing inclusion will increase costs and can increase trial duration. Limited trial resources therefore favours the use of groups who are easier to recruit and retain, rather than a more representative range of participants. However, the resulting interventions may not be deliverable or beneficial to all groups within a population and further marginalise people with TB at highest risk of poor outcomes. Not only are such practices discriminatory,The most efficient trial designs should be considered to improve the evidence base as quickly and cost-effectively as possible, without compromising quality.It has been recognised that observational, non-randomised studies are useful for evidence generation,41In 2022, funding for TB research first exceeded USD1 billion, but this remains far below what is needed.Trial governance should be in line with accepted good clinical practice.TB clinical trials require governance that adheres to internationally recognised guidelines and principles.Trials should investigate and report strategies that promote optimal engagement in care.Non-adherence to TB treatment increases the risk of treatment failure, relapse and acquisition of drug resistance.Adherence is typically verified through treatment observation, but application of this observation can vary considerably. For example, DOT could refer to every dose being observed by a healthcare worker, trained lay provider, family member or via a plethora of digital technologies,Trials do not always define how adherence was encouraged and assured, even when there may be concerns about drug intolerability. Strategies to reduce participant drop-out are similarly diverse and variably described. Common approaches include providing food security and financial incentives for every completed trial visit,63Where possible TB trials should include pharmacokinetic and pharmacodynamic components to generate information about treatment failure and toxicity.The relationship between treatment response (pharmacodynamics) and drug exposure (pharmacokinetics) is important to inform optimal treatment strategies. In early drug development, the parameter that correlates best with treatment response  is identified, and exposure-response relationships for monotherapy are explored. In later-phase studies, pharmacokinetic data are important to understand factors that influence variability in exposures . As some drugs display a high inter-patient variability in drug exposure, pharmacokinetic data can be important in exploring reasons why some treatments are less well-tolerated or less effective in certain populations,Traditional pharmacokinetic assessments can be expensive and difficult to perform on many patients. Several new strategies can improve the feasibility and reduce the costs.Outcomes should include frequency of disease recurrence, as well as post-treatment sequelae.TB recurrence remains one of the most important endpoints for Phase III clinical trials for pulmonary TB. Inclusion of whole-genome sequencing can differentiate treatment relapse from re-infection and ideally should be routinely included in TB clinical trials.Post-TB treatment sequelae can be either TB disease-related or persisting adverse drug events. Commonly these include post-TB lung disease, mental health disorders, visual impairment, hearing impairment, renal impairment and neurological impairment. The prevalence of respiratory impairment post-cure is estimated at 33% for DS-TB and 59% for MDR/RR-TB.In addition to physical impacts, TB has multidimensional and complex adverse effects on people’s psychological, economic and social well-being. Treatment generally improves quality-of-life (QOL) measures, although physical health frequently recovers before mental well-being. Impairment in QOL often remains post-treatment completion, with decreased work capacity, stigma and psychological issues.TB treatment trials should aim to harmonise key outcomes and data structures across studies to facilitate robust analyses of pooled data.Pooling data from clinical trials allows us to test hypotheses that are not possible with individual trials alone. Pooled studies of TB treatment are commonly performed through individual patient data meta-analysis (IPD-MA), allowing for more detailed evaluation of treatment outcomes than meta-analysis of summary effect estimates.In a recent systematic review of 31 pulmonary TB treatment trials, most trials used a composite binary outcome that combined treatment failure, relapse, death and treatment changes as ‘unfavourable’ in their primary efficacy outcome.TB treatment trials should include biobanking to add value from complementary studies.Biobanking is the process of acquiring and storing biological specimens with matched clinical data and patient meta-data in an organised system, with information on the activities related to collection, preparation, preservation, analysis and distribution of the biological specimens obtained.One of the challenges of biobanks is the unspecified future use of specimens. Furthermore, trial funding is typically limited, with no capacity to support longer-term specimen storage, which differs from registry-linked, publicly funded centralised biobanks. However, trials that include complementary studies, which may be exploratory in nature, are often permitted to have biobanks linked into the trial. Specimens collected from clinical trial participants are highly valuable given randomised selection. It is important they are optimally utilised, including access to these samples by other investigators, to justify the generous donations made by participants and to recover the substantive costs associated with specimen collection and storage.Treatment trials should invest in training and capacity strengthening of local trial and TB program staff.Clinical trials should include capacity strengthening efforts beyond skills acquired through other forms of research or routine programme strengthening.Capacity strengthening should also aim to improve routine programme practices and enhance research capacity beyond immediate trial needs. There should be real efforts to identify, train and mentor investigators at sites with the long-term goal of enabling them to design and execute independent trials. Providing training opportunities to both trial-specific and TB programme staff may enhance cohesion and understanding for staff not directly involved in trial activities. Participating in and understanding the requirements of a rigorous trial deepens understanding of the evidence-base that informs treatment recommendations. Training with these broader benefits in mind could therefore improve patient care in the longer-term. Healthcare services may benefit through improved practices in standard procedures, infection control, laboratory services, documentation of care, communication about patient management and greater job satisfaction. Staff given opportunities to enhance their skills during a clinical trial may become future leaders or advocates who enable locally appropriate application of research findings, including driving health system and policy improvement. These investments should be relative to site capacity with due recognition of historical power and funding imbalances. Particularly at trial sites in low-resource settings, efforts should be prioritised to directly empower locally based researchers to have leading roles, offer quality career development opportunities and ensure effective information exchange so that site staff gain from being part of larger trial programmes.102Several areas of discussion within the expert group did not achieve consensus and require further consideration. For example, issues related to inclusion of disease manifestations with unique features (such as TB meningitis) may require specific evidence on optimal management, including drug penetration into sanctuary sites, appropriate markers of treatment success and consideration of targeted adjunctive treatment to minimise sequelae. Host-directed therapies could potentially contribute to improved clinical outcomes, shortened treatment duration and reduced post-treatment disability, and should be considered in future clinical trials. Consideration may be given to the role of surgery in addition to therapeutics in those with extensive cavitary disease. Evidence on relapse rates and post-TB sequelae with new regimens will also provide guidance for patient follow-up.Inadequate funding for TB trials hampers the pace of evidence generation and translation, and increases the global impact of TB on affected communities. The standards presented here aim to guide best practice in TB trial design and implementation to promote innovation and progress in support of the End TB Strategy. Incorporation of these standards may increase costs, but this should be offset by increased efficiency and utility. For example, the proposed standards should stimulate innovation, improve international collaboration and data sharing, as well as encourage greater involvement of affected communities. All these elements are essential to find solutions to reduce disease burden and achieve TB elimination.Click here for additional data file.


However, the mechanisms of the neuronal transdifferentiation and the induced neuronal subtypes are not yet well understood. This review analyzes the mechanisms of resident cellular transdifferentiation based on a review of the relevant recent literature, describes different molecular approaches to obtain different neuronal subtypes, discusses the current challenges and improvement methods, and provides new ideas for exploring therapeutic approaches for spinal cord injury.Spinal cord injury causes varying degrees of motor and sensory function loss. However, there are no effective treatments for spinal cord repair following an injury. Moreover, significant preclinical advances in bioengineering and regenerative medicine have not yet been translated into effective clinical therapies. The spinal cord’s poor regenerative capacity makes repairing damaged and lost neurons a critical treatment step. Reprogramming-based neuronal transdifferentiation has recently shown great potential in repair and plasticity, as it can convert mature somatic cells into functional neurons for spinal cord injury repair  Spinal cord injuries (SCI) can be traumatic or non-traumatic. Traumatic SCI represent the most common type in clinical practice and is the focus of this review. Unlike the other regions of the adult brain, the spinal cord cannot produce new neurons during adulthood . TraumatCongenital neurogenetic processes are temporally and spatially limited under pathological conditions and are usually insufficient for functional neural repair. Therefore, neural regeneration must be promoted using exogenous factors to replace damaged and lost neurons. Biomaterial-based approaches mainly scaffolds, fibers, conduits, hydrogels, and sheets, hold therapeutic potential to promote axonal regeneration and motor function recovery . Specifiin situ and is more suitable for in vivo tissue repair. This strategy inherits the unique age-related characteristics of transdifferentiated neurons and provides an advantage in mimicking the cellular processes that occur with aging  and small molecules, such as miR-9/9*-124, can induce neuronal transdifferentiation and are effective transdifferentiating effectors, inducing the conversion of fibroblasts to cultured neurons , were first reported to directly reprogram fibroblasts into functional neural precursor cells . Subsequ as Sox2 , Ascl1 -positive neuroblasts, which require further treatment with the histone deacetylase inhibitor VPA to differentiate into mature neurons  techniques , in vivoreaction  can helpreaction .Although the induction of transdifferentiation in the injured environment requires the involvement of endogenous factors (TF or miRNAs), ectopic expression of these factors is also necessary to unleash the full neurogenic potential of endogenous glial cells . NotablyIn most cases, miRNAs do not act alone, but in concert with different TFs or controller elements to create the distinct network required for transdifferentiation. For example, miR-124 alone is required in concert with TFs to induce neuronal transdifferentiation . We founin vivo, and the transdifferentiation efficiency reached 80%. Thus, overcoming this obstacle could effectively improve the outcomes of transdifferentiation. A key event in the downregulation of glial cell-to-neuron transformation induced by PTBP1 is the inhibition of gene regulatory loops. PTB (PTBP1) is an RNA-binding protein mainly involved in RNA metabolism and plays a key role in neurogenesis . InhibitIn another part of the loop, when PTBP1 levels are increased, the paralog nPTB is downregulated, and nPTB controls mRNA splicing in newborn neurons . Furtherin situ has ensued. For example, while NeuroD1 and shPTB can have a positive effect on disease function improvement, by lineage tracing YFP-expressing astrocytes, it was surprising to find that AAV-mediated NeuroD1 or shPTB did not provide evidence of astrocyte-to-neuron conversion. Moreover, it was found that the overall neuronal density remained unchanged. Thus, a possible causal mechanism for misdirecting the origin of the neuron is that the GFAP promoter cell specificity can be altered by either a trans or cis mechanism of downstream factors or expressed genes induced by NeuroD1 or shPTB. Specifically for PTBP1, the authors did not observe a significant reduction in endogenous PTBP1 expression when using CRISPR-CasRx knockdown in mouse cortex, nor did they observe glial cell transdifferentiating into neurons during lineage tracing; And when knockdown was performed using shRNA, although endogenous PTBP1 knockdown, no transdifferentiation of genealogically labeled astrocytes into neurons was observed   . Forskol (FICBY) . Hu W. e (FICBY) . Interes (FICBY) . Notably (FICBY) .In summary, using small molecules is a promising approach for facilitating the induction of cellular transdifferentiation into neurons. It is important to fully understand the molecular mechanisms regulated by each molecule and modulate the strength of signaling pathways to generate more precise and effective transdifferentiation strategies.in vitro and in vivo, showing that Brn2 and Myt1l may be specific factors for dopaminergic neuronal subtypes  induces ATN transformation in vivo. Upon analysis, a high proportion of ciNs with glutamatergic neuronal characteristics (72.8%) was observed, which occurred mainly in the cortex  by a variety of small molecule protocols eriments , only a e cortex . Interese cortex . This sue cortex . Howeverin vivo implantation capacity, and sensitivity to disease stimuli  and other factors . The proOverexpression of the TFs OCT4 and KLF4 induces astrocyte reprogramming and inhibits astrocyte migration. Thus, this may be a direct result of reprogramming the resulting NPC to differentiate into specific cell subtypes, likely an oligodendrocyte, thereby promoting myelin regeneration and subsequent recovery of the injured spinal cord . Anotherin vivo, providing a new potential method for clinical application. In addition, the comprehensive protocol of the transdifferentiation strategy combined with rehabilitation has achieved higher effectiveness and timeliness in treating SCI and is expected to achieve higher clinical feasibility in follow-up treatment  is still in the early stages of development and has some limitations. For example, neuronal transdifferentiation is relatively inefficient in human cells compared to mouse cells, and in vivo applications of transdifferentiation remain limited to animal experiments (However, eriments . In addiY-MF, W-CC, and W-JZ prepared the manuscript and contributed to writing the original—draft and visualization. Y-SY, YZ, X-LC, and M-QP drew the figures and tables and contributed to the writing—review and editing. SL and H-FH reviewed and finalized the manuscript and contributed to funding acquisition, validation, and resources. All authors had full access to every data in this review and took responsibility for the data’s integrity and the data analysis’s accuracy.


It is vital to ramp up crop production dramatically by 2050 due to the increasing global population and demand for food. However, with the climate change projections showing that droughts and heatwaves becoming common in much of the globe, there is a severe threat of a sharp decline in crop yields. Thus, developing crop varieties with inbuilt genetic tolerance to environmental stresses is urgently needed. Selective breeding based on genetic diversity is not keeping up with the growing demand for food and feed. However, the emergence of contemporary plant genetic engineering, genome-editing, and synthetic biology offer precise tools for developing crops that can sustain productivity under stress conditions. Here, we summarize the systems biology-level understanding of regulatory pathways involved in perception, signalling, and protective processes activated in response to unfavourable environmental conditions. The potential role of noncoding RNAs in the regulation of abiotic stress responses has also been highlighted. Further, examples of imparting abiotic stress tolerance by genetic engineering are discussed. Additionally, we provide perspectives on the rational design of abiotic stress tolerance through synthetic biology and list various bioparts that can be used to design synthetic gene circuits whose stress-protective functions can be switched on/off in response to environmental cues. Climate change is constantly altering the environment in which agricultural practices and crops evolved over the years . Plant dThe cascades of regulatory pathways are activated in plants during an abiotic stress response, enabling them to react and adapt to their environment efficiently . UnderstTraditional breeding strategies are constrained by limited genetic diversity with higher productivity under stress and the finite efficient selection methodology. Using traditional breeding, few varieties have been introduced with enhanced abiotic stress tolerance in field conditions . GeneticThe upstart field of synthetic biology (SynBio) can play a major role in overcoming these complex challenges , 13. PlaThe effective design of genetic circuits is a prerequisite for producing sentinel plants with desirable characteristics . Plant gThus, this chapter will discuss the potential applications of synthetic biology approaches for improving abiotic stress tolerance in crops. In particular, we will focus on the current understanding of the molecular mechanisms involved in the regulation of the major abiotic stresses, namely, heat, cold, drought, and salinity in plants, followed by summarizing the validated and predicted bioparts which can be further explored for improving abiotic stress tolerance in crops by adopting synthetic biology.A primary stress sensing/perceiving mechanism translates the abiotic stress stimuli into a biological signal. In plants, the identification of abiotic stress sensors has been a challenging task due to the redundant nature of multiple sensors and the criteria used to define primary sensors. Addressing these limitations, four principal characteristics for defining a stress sensor have been proposed , 24: (1)Plants get exposed to temperature changes that vary in range, intensity, and duration daily and seasonal. Temperature changes affect enzyme kinetics, membrane fluidity, and protein folding makes it challenging to distinguish temperature-induced physiological changes from the actual sensing mechanism . Plants 2+ dependent CG[T/G] cis-elements [elements . A mutatcis-regulatory sequences are predominantly found in the promoter region of genes. Their presence/absence/variation in position/sequence can affect the expression of the gene, resulting in induction, decrease, or even lack of expression. The most often utilised constitutively overexpressed promoters include the 35S promoter of the cauliflower mosaic virus (CaMV), and promoters are derived from plant actin and ubiquitin genes. The constitutive expression can have unforeseen effects on plant growth and development. It might result in overexpression of a specific transgene at the incorrect developmental stage or in tissues that are not ordinarily expressed. For example, constitutive overexpression of rice DREB1 (OsDREB1) in Arabidopsis exhibited resistance to salt, cold, and drought [AtDREB1A under the control of an Arabidopsis stress-inducible promoter, AtRD29A, resulted in increased abiotic stress tolerance in transgenic Arabidopsis and rice, addressing the problem of growth retardation [Furthermore,  drought , 389; hoardation , 391.de novo fabrication of new gene circuits and switches and restructuring signalling pathways [Synthetic biology uses fundamental engineering principles by employing naturally existing components for the rational design of new biological modules , 393. Supathways . This nepathways –397. Synpathways , 399.Recent advances in understanding systems biology of plant and application of bioinformatics tools have revealed a thorough understanding of regulatory components and processes operating at the cellular level. Such knowledge empowers the construction of synthetic modules using an infinite source of biological parts. Synthetic biology uses a top-down strategy to change what is already in nature. Synthetic biology diverges from the past biological approaches as it aims to build and develop something novel but still inspired by nature . This isE. coli lac repressor and the activation domain of Gal4. A tissue-specific promoter is used to drive the expression of the GR-LhG4 transcription factor that remains in the inactive state in the cytoplasm. The presence of DEX allows fusion protein to move into the nucleus, leading to transcription activation of the reporter gene controlled by a synthetic promoter pOp6 that includes 35S core promoter and six copies of the LhG4 binding site. This approach led to the generation of a suite of transgenic driver lines. Following crossing, these lines can induce tissue-specific expression of the reporter gene in the resulting progeny.The first application of the synthetic biology approach in plant systems includes the inventing of synthetic regulatory elements  and switches to modify of spatiotemporal gene expression and the engineering of signalling networks , 404. Th2 concentrations in the proximity of the RuBisCO enzyme enhances the efficiency of CO2 fixation and thus have a positive implication for plant productivity. The cyanobacteria and algae possess innovative carbon-concentrating mechanisms (CCMs) to enhance RuBisCO efficiency of CO2 fixation. Therefore, introducing these microautotrophic CCMs to enhance the photosynthetic ability of plants has shown to be an effective strategy [Synthetic biologists have also been working to enhance the photosynthetic rate in plants for maximizing plant productivity . Reenginstrategy , 409. Plstrategy . However2 from plants. To address this issue, enzymes that can convert glycolate to glycol-aldehyde have been engineered using synthetic biology approach. Under in vitro conditions, these engineered enzymes and other endogenous enzymes could recycle glycolate straight to RuBP  without releasing CO2 [In vivo, several studies have shown that tailored carbon-conserving Calvin cycles may synthesise acetyl-CoA directly from C3 sugars without releasing CO2 [Photorespiration is another natural process where RuBisCO binds oxygen resulting in the release of COsing CO2 . In vivosing CO2 . Anothersing CO2 . Hence, The creation of plant sentinel biosensors is also a key growing application of synthetic biology in the plant area . The desSimilarly, crops with efficient water utilisation capacity are being developed by controlling the production of ABA via an engineered PRY1 receptor with sensitivity to a fungicide Mandipropomid. The spray of this agrochemical on the plants under drought conditions activates ABA . These mArabidopsis, and tobacco, and such engineered devices are being progressively adapted for crops.In addition to providing proof of concept, the development of these synthetic techniques offers increasing prospects to reprogram plant development and metabolism and improve agricultural traits. Because these synthetic devices and platforms are plug-and-play, they have the potential to modulate gene expression patterns at numerous levels while not interfering with plant growth, development, or fitness , 403. EvCombining synthetic biology approaches with existing genetic engineering practices offers enticing opportunities for the rational development of abiotic stress-resistant crops. Orthogonality, or the capacity of genetic components and circuits to operating independently of one another and the host’s regulatory activities, is crucial in synthetic biology . OrthogoSynthetic promoters and TFs must be well designed and employed to control of gene expression using the endogenous plant cell transcriptional machinery , 420. SyArabidopsis plants [Transcription factors (TFs) play an essential role as regulators of stress-related gene expression Section . Each TFs plants . A systes plants . Thus, sArabidopsis overexpressing these synthetic regulators, Lee et al.[Arabidopsis lines. Similarly, after further refinement, this strategy could be utilised to maximize the epigenetic control of stress-responsive genes or master stress regulatory genes that are either less epigenetically regulated or regulated by several epigenetic regulators.Synthetic transcription regulators can also be constructed to use epigenetics to control gene expression at the transcriptional level. In transgenic ee et al. reportedArabidopsis to regulate endogenous and transgenes via posttranscriptional regulation [Another recently pursued technique is applying riboswitch, a stem-loop RNA structure with regulatory and ligand-binding domains, to modulate mRNA stability and translation in plants . A synthgulation . These rEngineering the crassulacean acid metabolism (CAM) pathway in plants to improve water-use efficiency and thrive in water-scarce conditions like semiarid deserts is another potential approach for imparting stress tolerance . DroughtA deep understanding of plant abiotic stress perception, signalling, and response processes is a prerequisite for crops that can maintain yield stability under stress conditions. In recent years, intensive “omics” based investigations have revealed activation of complex stress-responsive regulatory networks upon the perception of external stressors by plants. These interconnected networks involve various biological parts such as sensor proteins, enzymes, transcription factors, epigenetic, and posttranslational modifiers. While genetic engineering approaches involving the modulation of individual bio parts have yielded promising outcomes, the rational design of stress-responsive genetic circuits based on synthetic biology principles is urgently required.Various genes that can be selected as bioparts for the assembly of stress-protective genetic circuits have been identified. Genes that play significant roles in plant abiotic stress tolerance as functionally tested in transgenic plants are summarized in table, providing a valuable database of bioparts for the rational design of synthetic circuits. These include genes encoding transcription factors, chaperones, stress sensor protein kinases, enzymes that can scavenge reactive oxygen species, and enzymes that promote the accumulation of protective osmolytes.Besides protein-coding genes, several noncoding RNAs have emerged as potential bioparts to be deployed as tools for enhancing plant abiotic stress tolerance. Among noncoding RNAs, various miRNAs have been functionally validated for imparting abiotic stress tolerance. One of the main issues of concern is that while enhancing the ability of plants to tolerate stress conditions, the constitutive expression of protective genes can have detrimental effects on plant growth phenotype and yield. An on-demand protective functional module that gets activated in a spatially and temporally regulated manner can be fabricated using conditional or tissue-specific promoters is desirable to avoid undesirable consequences. Also, as mentioned, the use of transcription factors or other regulatory components upstream of the protective network enhances the risk of unwanted pleiotropic effects. Additional research is thus warranted to characterise the function of additional direct action candidate genes based on omics and comparative genomics approaches.Another consideration for designing abiotic stress-tolerant crop plants is the combinatorial action of various stressors in field situations. As discussed, these combined stresses such as heat and drought can have synergistic negative consequences for plant growth and yield. Further, there is a gap in our knowledge regarding stress-responsive regulatory circuits required to protect reproductive development in plants. It has become clear that exposure to environmental stressors such as heat and drought during the flowering phase of plant growth can lead to male sterility, failure of fertilization, and seed set. Thus, a priority is to design protective gene regulatory circuits that are temporally activated in the target reproductive tissues in responding to abiotic stresses. Thus, one of the primary foci for future research in abiotic stress tolerance is to achieve a comprehensive picture of the stress vulnerability of plant reproductive cells. The use of single-cell transcriptomics will enable uncovering of potentially significant cell-specific stress-responsive genes that may be yet undiscovered.Translation of synthetic biology approaches in plant systems depends upon efficient protocols for genetic transformation and control over the expression of inserted genes. Fortunately, for most of the major food crops such as wheat, corn, canola, soybean, and relatively facile methods of transgene addition are now available. The CRISPR technology for genome editing has already been implemented in major crop genera. The availability of diverse methodologies for genome manipulation and the engineering of synthetic circuits hold the potential for ushering in a new era for developing crop genotypes that can sustain yield stability in the face of multiple abiotic stress linked with climate challenges to agricultural productivity.


The emergence of antibiotic-resistant “superbugs” poses a serious threat to human health. Nanomaterials and cationic polymers have shown unprecedented advantages as effective antimicrobial therapies due to their flexibility and ability to interact with biological macromolecules. They can incorporate a variety of antimicrobial substances, achieving multifunctional effects without easily developing drug resistance. Herein, this article discusses recent advances in cationic polymers and nano-antibacterial materials, including material options, fabrication techniques, structural characteristics, and activity performance, with a focus on their fundamental active elements. Bacterial infection has always been a major threat to human life and a serious global challenge . The deaAt present, small molecular drugs and traditional antibiotics are often unable to solve the clinical dilemma caused by multi-drug-resistant bacteria infections, so it is urgent to research and develop new types of antimicrobial agents. With the rapid development of technology, researchers have put forward many valuable concepts to build antibacterial and antibiofilm platforms, including cationic polymers, chitosan-based polymers, antimicrobial peptides, and nanomaterials such as metal/metal oxide-based nanoparticles (NPs). Different from the traditional small molecular antimicrobial agents, cationic polymers and nano-antimicrobial agents have become new antibacterial materials with excellent development potential because of their long active time, high chemical stability, and rare drug resistance. These new antibacterial materials work can be briefly classified into the following aspects. 1) loading small molecular antimicrobial agents, antimicrobial peptides, and other bactericidal ingredients; 2) showing inherent bactericidal activity by inflicting chemical or physical damage on bacteria, such as silver nanoparticles; 3) being activated by light to produce photothermal/photoacoustic and photodynamic effects; 4) being stimulated by peroxide or photoacoustic to produce a large number of reactive oxygen species (ROS); 5) combining two or more bactericidal modes to synergistically optimize the therapeutic effect.In this review, we illustrate that how cationic polymers and nanomaterials could be used to combat bacterial infections. Firstly, the current main mechanisms of antibacterial activity of cationic agents and nanomaterials are comprehensively discussed. Secondly, the properties and design elements of cationic polymers and nanomaterials that exert good efficacy are reviewed, and the strategies of different antibacterial materials and the advantages and disadvantages of each antibacterial agent are presented . FinallyThe antibacterial mechanisms of cationic polymers and nano-antimicrobial agents are different from that of traditional antibiotics, so their damage to bacteria rarely leads to drug resistance. In this section, we briefly describe the antibacterial mechanisms of these two kinds of antimicrobial agents.Currently, the majority of cationic antimicrobial polymers developed are amphiphilic macromolecules with surface active properties. Surfactants’ adsorption capacity, high binding affinity to bacterial cell membranes, and appropriate lipophilicity allow them to efficiently damage the structure of membranes and then cause cell lysis . For GraIn particular, cationic antimicrobial polymers such as cationic antimicrobial peptides (AMPs) usually break bacterial cells through a series of steps, while some kinds of AMPs can enter the bacteria through cell penetration or endocytosis and interact with biological macromolecules to inhibit the growth of bacteria. AMPs can cause DNA damage, RNA synthesis inhibition, protein synthesis inhibition, protein folding inhibition, enzymatic activity inhibition, and cell wall synthesis inhibition . SeveralThe ultra-small size and unique chemical properties of metallic nanomaterials provide advantages for the presentation of antibacterial activity. Metal nanomaterials have a variety of antibacterial mechanisms. Teichoic acid and lipopolysaccharide on the bacterial surface make them electronegative, which makes positively charged nanomaterials adsorb on the bacterial surface through electrostatic interaction . As a re2− through electron transfer (type I). In type I reactions of PDT, unsaturated phospholipid molecules in bacterial cell membranes are predominantly hydrogenated. Further chemical reactions between molecules and oxygen result in lipid peroxides, which compromise the structural integrity of bacterial cell membranes and raise membrane permeability   light-induced 1O2 is more threatening than other ROS (·OH and O2−), making it more effective in antibacterial effect.Photodynamic therapy (PDT) is a clinical procedure that uses reactive oxygen species (ROS) generated by photosensitizers to destroy surrounding biomolecules and kill the pathogenic bacterium . The maneability . To creaType II) . TherefoUnder light excitation, photosensitizers can be used as photodynamic antibacterial materials to kill bacterial cells through light-dependent ROS production. In addition, some materials can decay back to the ground state by emitting fluorescence, or produce thermal energy by consuming energy through non-radiative relaxation pathways, namely the photothermal effect . These pIn addition to their direct use as antimicrobials, metallic nanomaterials have also been used as nanocarriers to transport existing antimicrobials. Antimicrobial agents can be loaded onto metal nanoparticles’ surfaces by covalent attachment or self-assembly . In summCationic agents are a group of therapeutic agents carrying positive charges which can be naturally derived or synthesized. The positively charged agents are shown to interact with negatively charged bacterial membranes and can be imported into bacterial cytoplasm . For man0 and copolymer PE31 that contained 31% ethyl methacrylate groups. Both polymers displayed good antibacterial activity against S. mutans and effectively killed the planktonic bacteria. At a high enough concentration , both polymers were able to significantly diminish biofilm biomass by 80% upon two-hour incubation.Cationic polymers are a group of supramolecular systems carrying a significant amount of positively charged sites on the surface and can therefore interact with the negatively charged bacterial cells. Their efficacy against planktonic bacterial species has been widely explored. In particular, gram-negative pathogens are known to be a major health burden, especially with the rapid emergence of antimicrobial-resistant strains. For example, many studies constructed amphiphilic polymer systems containing a significant portion of cationic residues and therefore exhibit significant electrostatic interaction with the anionic bacterial membrane. To mimic naturally occurring host defense peptide (HDP) that mediates selective membrane disruption of negatively charged bacterial membrane over zwitterionic mammalian cell membranes, a synthetic polymer with cationic charges is developed. However, many studies utilize AMP-mimicking polymers based on a global amphiphilic framework that cannot be fine-tuned for the selective killing of bacterial cells. To address this, in vivo toxicity and high biocompatibility. One such polymer is poly (2-oxazoline) (POX) which has been explored and shown to be non-toxic and biocompatible. in vitro and in vivo antibacterial efficacy against MRSA-biofilm infection.To ensure the polymeric mimics of HDPs can be used therapeutically, it is vital to develop such polymers with low Typical cationic polymers might induce broad non-specific toxicity due to electrostatic interactions of the positively charged polymers with biomacromolecules, membranes, and extracellular materials. Therefore, responsive charged polymers that can be rationally configured with “on-demand” antimicrobial and antibiofilm abilities are becoming a promising strategy. Chronic biofilm infections are often associated with inflammation and local bacterial fermentation that would induce an acidic microenvironment that can be exploited by pH-sensitive polymers. in vitro or in vivo toxicity against mammalian cells. Though demonstrating the potential to counter biofilm-associated biofilm infection, many polymeric micelles face the challenge of rapid host immune response and degradation that leads to low bioavailability and short blood circulation time. Similar strategies can be applied to construct on-demand micelles that target specific biofilm life cycles. in vitro and in vivo and could be applicable as an advanced drug delivery strategy against biofilm infections  have demonstrated promising in vivo models, showing great promise as a chitosan-based wound healing material in biofilm-related infections.A major drug administration hurdle in wound biofilm treatment is poor infection site adhesion and limited biofilm penetration. Therefore, polymers need to be designed to exhibit adhesive properties besides good antibiofilm efficacy. in vivo mouse models while having good biocompatibility in mammalian cell lines. Another study investigated mouthwashes incorporated with chitosan content in alleviating biofilm formations of oral microorganisms  using quaternary ammonium chitosan, N, N, N-trimethyl chitosan (TMC) along with liposome and doxycycline, namely TMC-Lip-DOX NPs . The NPsrganisms . ChitosaH. pylori. The pH-sensitive properties of the polymer allowed the postponed release of amoxicillin to enhance its local concentration at infection sites of GIT. The nanoparticle demonstrated effective inhibition of H. pylori growth while exhibiting minimal toxicity against mammalian cells.Although pH is commonly used for the targeted delivery of therapeutic agents, under acidic conditions, many polymers suffer from poor retention time due to degradation and inactivation. Chitosan-based materials with excellent biocompatibility, mucoadhesive properties, and feasibility for further chemical modifications make them ideal for oral drug delivery to GIT. In summary, owing to its advantageous properties like stretchability, biocompatibility, high density of functional groups, mucoadhesive, and degradability, chitosan and its derivatives are shown to be a promising antibiofilm strategy. Its wider clinical translation as combination therapy could provide further insights into its efficacy in the eradication of established biofilms and associated chronic infections.Antimicrobial peptides (AMPs) are small molecular proteins with antimicrobial activity in organisms in nature, which are an important line of defense against pathogens. The main pathways of their action are bacterial cell membrane destruction, and immune and inflammatory response regulation . RecentlTo reveal the bactericidal mechanism of AMPs, monitoring the dynamic interaction between AMPs and bacteria in real-time is necessary. Due to its high sensitivity and easy operation, fluorescence imaging technology has been widely used to visualize the dynamic interaction between molecules and organisms in the microscopic environment . Linkingin vivo and in vitro, and also has the function of promoting wound healing  in some liposomes and loaded it into a hydrogel of PEG and gold nanorods. The 860 nm laser stimulated the gold nanorods to lyse the liposomes through the photothermal effect, thereby releasing antimicrobial peptides and producing bactericidal activity against P. aeruginosa (gram-negative bacteria) and S. aureus (gram-positive bacteria) n wounds . Khan etfections . Comparefections . To prevacteria) .S. aureus and S. epidermidis which are often associated with infection of biological materials  or Bac2A (RLARIVVIRVAR-NH2) hydrogels on clinical medical devices directly in contact with blood to provide good bactericidal activity and prevent bacterial adhesion. The in vitro and in vivo evaluation of the anti-infective showed promising antibacterial effects.Infection of hospital medical equipment is common, so the demand for safe and non-toxic medical device coatings has increased significantly. HHC10 is an antimicrobial peptide screened by computer-aided drug design with good antibacterial activity . A hydroaterials . In addiemolysis . Liu et E. coli. The results showed that HHC10-NP could be bactericidal through the multi-mode mechanism, that was, it could directly interact with bacteria to dissolve its membrane and cause its death by inducing apoptosis pathways in host cells. In addition, HHC10-NP was significantly less cytotoxic to RAW264.6 than nude HHC10. Inspired by this result, they encapsulated HHC-8 (KIWWWWRKR) and MM-10 (MLLKKLLKKM) in polycaprolactone nanoparticles and investigated the effect of the nanoparticles on the activity of the AMPs. The results showed that the protection provided by AMP encapsulation increased the membrane permeability of antimicrobial peptides and enhanced the accumulation of antibiotics in mycobacterium, thus producing a synergistic effect . Compared with antimicrobial peptide magainin, CPP (R9)-magainin conjugate effectively crosses the lipid bilayer, increasing antibacterial activity by 2–4 times against gram-positive bacteria and 4–16 times against gram-negative bacteria . In addibacteria .2), a complete replacement library of 12 amino acid peptides was screened and optimized 12-Mer peptides were designed by substituting each amino acid with the most favorable substitution. It had excellent antibacterial activity against E. coli  and machine learning techniques. Antimicrobial peptides, including HHC10 and HHC36, with comparable or even higher activity than many traditional antibiotics, were obtained . They inin vitro drug release, antibacterial performance and cytotoxicity, and in vivo bone growth of an AMP loaded with a calcium phosphate-coated titanium (CaP-AMP) implant in a rabbit model. The results showed rapid release of antimicrobial peptides in the body for the first few hours, followed by slow release. It had good antibacterial activity against S. aureus and P. aeruginosa but had no effect on bone growth on the implant . + . + . These pong time . The cum323 days  to achie2+ and ROS have been suggested as possible antibacterial mechanisms for CuNPs. Cu2+ are also toxic to bacterial cells. Copper nanoparticles (CuNPs) presents a broad-spectrum of antibacterial activity. Over the past 2 years CuNPs has made great attention because of its remarkable antibacterial activity. Compared to AgNPs, CuNPs are more widely used in antimicrobial therapy because they are essential elements for living organisms and are more cost-effective. The set free of copper ions CuIn addition to the metal-based nanoparticles mentioned above, AuNPs are significantly more inert and biocompatible than AgNPs and CuNPs, making them potentially more effective therapeutic antibacterial agents. Although the majority of antibacterial metal or metal oxide-based nanoparticles cause cellular death primarily through the generation of ROS, the antimicrobial activity of AuNPs does not generate any ROS-related processes . Cui et MRSA biofilm under near-infrared (NIR) light irradiation. Besides that, these AuNPs showed no damage to the healthy tissues around the biofilm and they might be found potential application in healthcare as useful antibacterial agents.Apart from these two possible mechanisms, AuNPs, also known as “light-directed nano heaters,” can effectively kill bacteria by converting the absorbed light energy into heat energy through a non-radiative decay process . Xu et aS. aureus, including ZnO, CuO, TiO2, Al2O3, Mg2O, CeO2. Compared to other metal oxide-based nanoparticles, ZnO showed the most significant growth inhibition and had a special antibacterial activity under visible light. To further improve the antibacterial activity and biosafety of ZnONPs, surface modification is the most effective and commonly used method. In vitro antibacterial experiments have shown that the CS-ZnO nanocomposite exhibited better antibacterial activity against a variety of bacteria compared to flavone rutin. Implant related infections in surgical procedures are a very difficult problem in clinical practice. Loading zinc oxide nanomaterials as a coating on the surface of medical devices may help solve such infections. Wang et al. sputtered ZnONPs onto a titanium substrate to form a uniform nano film covering material for soft tissue implantation in mice  have gain more attentions than others due to their attractive properties such as good antibacterial activity, environmental friendliness, low cost, and high surface area and volume ratio . Jones e in mice . The matCompared to traditional antibiotics, metal/metal oxide-based nanoparticles undoubtedly have stronger and broader antibacterial activity due to their special antibacterial mechanisms and physicochemical properties. However, due to their small size, metal/metal oxide-based nanoparticles have poor stability and are prone to aggregation, while also having the disadvantage of strong toxicity. However, the surface of metal/metal oxide-based nanoparticles is easy to modify and functionalize, and using this advantage is expected to overcome the above problems.Polymeric nanomaterials, due to their advantages of low toxicity, good biocompatibility, and easy degradation, more and more researchers are interested in the construction of antibacterial agents based on polymer nanoparticles . PolymerS. aureus and MRSA infections, they have successfully created vancomycin-loaded pH-responsive chitosan nanoparticles that release their payload at an acidic pH. Two naturally occurring polymers are proteins and carbohydrates, and they have been widely used in the preparation of polymeric nanomaterials . NaturalBy imitating the chemical structure of AMPs, the synthesized polymeric nanoparticles can acquire intrinsic antimicrobial effects by adding hydrophobic and positively charged groups to the polymer chain. The cationic moiety enables electrostatic adsorption of the polymer with negatively charged bacterial surfaces, while the hydrophobic moiety increases the membrane penetration properties . Liu et With the rapid development of biological and nanoparticle technologies, more progress has been made in polymeric micelles (PMs) as carrier systems. Core-shell structure polymeric micelles could be prepared from various biodegradable and biocompatible polymers through a self-assembly process and they are usually used to improve the physicochemical and pharmacokinetic properties of drugs. Polymer micelles are usually formed by the self-assembly of individual polymer molecules, which are synthetic amphiphilic di- or tri-block copolymers consisting of hydrophilic and hydrophobic blocks. In summary, polymeric nanoparticles not only have good biocompatibility and structural stability, but also can load antibiotics, photosensitizers and other materials to further improve their antibacterial activity. In addition, it can also be applied to the surface of medical devices or mixed with some medical dressings to prevent infection and promote wound healing. This will make polymeric nanomaterials become a promising antimicrobial agent for clinical development.in vitro -Urirophages . It was rophages .In addition, because bacteria have difficulty surviving from physically destroyed cell structures, cationic liposomes can also destroy bacterial membranes and walls through electrostatic interactions, so cationic liposomes have specific antibacterial mechanisms and antibacterial activity, and this specific mechanism also effectively reduces the risk of enhanced bacterial resistance . CationiIt is generally accepted that nanoemulsions are a mixture of water, oil, surfactants, and co-surfactants. It has received a lot of attention within the field of food antimicrobials because of its high optical transparency, thermodynamic stability, targeting action, and biocompatibility as a drug carrier . EssentiSalmonella typhimurium. Other co-surfactants that played the same role are gelatin and soy lecithin, which Li et al. blended into thyme to prepare nanoemulsions that increased water vapor transmission and demonstrated broad-spectrum antibacterial activity  Biocompatibility and selectivity: These still are the most important challenge. Poor biocompatibility  and poor target selectivity are still prevalent in most of systems, and new strategies are needed to meet the stringent criteria in clinical settings.(2) Mechanistic understanding: Most work has focused on determining the final antimicrobial agents’ outcome, but more in-depth studies using biological antimicrobial mechanisms are of great interest to help advance our understanding of bacterial-antimicrobial agent interactions and rationalize the design of new and more effective antimicrobial agents.(3) Nanomaterial construction and antimicrobial efficiency: The antimicrobial performance of nanomaterials depends on the components and construction forms of the materials, and it is difficult to meet the practical application requirements with complex antimicrobial systems or lower antimicrobial effects.In vitro biological experiments provide information about the fast and efficient antimicrobial efficacy of nanomaterials; however, the results of in vitro experiments do not directly reflect the real effect of nanomaterials in vivo, such as metabolism, biodistribution, organ clearance efficiency, biodegradation, and immune responses at the organ and systemic levels, toxic response, inflammatory manifestation situation, etc.(4) Animal experimental models: (5) Clinical translational research: The design and development of a wide range of antimicrobial agents are most often aimed at achieving practical clinical applications, and clinical translational research on antimicrobial agents requires the concerted efforts of multidisciplinary experts.In this review, recent advances in the use of cationic polymers and nanomaterials for fighting bacteria and their biofilm infections are summarized. Although these nanomaterials have all made good research progress in the field of antimicrobial activity, they are still many challenges away from practical application.Despite the challenges, antimicrobial materials, including cationic polymers and nanomaterials, are emerging as new tools in modern medicine. Among them, polymeric nanoparticles have the characteristics of good biocompatibility and high structural stability. AMPs can be coated with polymeric nanoparticles to regulate the excessive electrostatic action of AMPs to reduce toxicity. In addition, ultra small metal nanoclusters can also be dispersed in polymer nanoparticles to improve their poor stability and easy aggregation. They can be made into medical device coatings or wound dressings, which may have great prospects in clinical anti-infective applications. Therefore, the development and application of novel antimicrobial materials are of great importance.