A correlate of protection (CoP) is urgently needed to expedite development of additional COVID-19 vaccines to meet unprecedented global demand. To assess whether antibody titers may reasonably predict efficacy and serve as the basis of a CoP, we evaluated the relationship between efficacy and in vitro neutralizing and binding antibodies of 7 vaccines for which sufficient data have been generated. Once calibrated to titers of human convalescent sera reported in each study, a robust correlation was seen between neutralizing titer and efficacy (ρ = 0.79) and binding antibody titer and efficacy (ρ = 0.93), despite geographically diverse study populations subject to different forces of infection and circulating variants, and use of different endpoints, assays, convalescent sera panels and manufacturing platforms. Together with evidence from natural history studies and animal models, these results support the use of post-immunization antibody titers as the basis for establishing a correlate of protection for COVID-19 vaccines. Despite this, to meet unprecedented global demand, additional vaccines are needed even as placebo-controlled efficacy trials are becoming infeasible 22.1Inclusion criteria for immunogenicity and vaccine efficacy data are described in the Supplemental Appendix. At the time of analysis, seven vaccines met these criteria: Pfizer, Moderna, Gamaleya, AstraZeneca, Sinovac, Novavax, and Johnson & Johnson.2.2Vaccine efficacy (VE) was computed as one minus risk-ratio times 100, and the risk-ratio for each study was calculated as specified in the study protocol/primary publication. Correlation was the Spearman's rank correlation coefficient (ρ) between the readouts on the x- and y-axes; both x and y data were fit using a natural log transform. The dashed fit line was computed using locally estimated scatterplot smoothing (LOESS) regression . We applied a non-parametric Bayesian approach to evaluate the quality of VNA and binding antibodies as trial-level surrogate endpoints 3We first evaluated peak geometric mean titers (GMT) of VNA and binding antibodies 1–4 weeks following the recommended vaccination regimen as reported by each manufacturer but found low correlations with efficacy A, 1B, moUsing a non-parametric approach, we modeled the relationship of each of the candidate biomarkers with VE across the seven vaccines and attempted to predict the VE of each vaccine based on its biomarker response (Methods). For both the VNA ratios A and bin4In this study, a robust correlation was seen between antibody titers and efficacy across seven different vaccines, with higher titers correlating with higher vaccine efficacy, despite uncontrolled variables across the studies. First, these vaccines represent four vaccine manufacturing platforms with varying levels of engagement of humoral and cell-mediated immunity: mRNA, adenoviral vector, protein subunit, and inactivated virus vaccines. Second, our analysis relies upon calibration to HCS panels selected by each developer, which vary widely in sample size and representation of the range of disease severities. As both neutralizing and binding antibody responses to natural infection correlate significantly with severity of disease, the specific HCS panel selected may significantly impact ratios generated in our analysis Third, these clinical studies were conducted on geographically diverse study populations, which were subject to different forces of infection and circulating variants. Variability in the timing and location of the Phase III efficacy studies included in our analysis suggests that the efficacy point estimates will be differentially impacted by circulating variants of concern (VOCs) which are more likely to escape protection afforded by vaccination As additional data have become available, three issues challenge the interpretation and application of this model to further vaccine development. First, trials of several vaccines  have shown substantial efficacy following the first dose despite low neutralization titers, with many subjects with titers from the pre-dose two sample below the lower limit of quantification. This suggests either relatively low neutralization assay sensitivity or non-neutralizing antibodies and T cell responses may be functionally important, which is supported by the evidence that binding antibody titers appear robust at the same post-Dose 1 time points The results reported here support the use of post-immunization antibody levels as the basis for a CoP. We propose that the next steps toward achieving consensus on a CoP include the following: First, to establish comparability of antibody measurements among laboratories by (i) using the WHO International Standard (NIBSC 20/136) to express VNA titers in IU/ml and binding antibody titers in BAU/ml and (ii) establishing a relevant proficiency panel. Second, to agree on an assay, most likely a neutralization assay meeting performance-based criteria, to serve as the gold standard assay for CoP and perhaps to allow validation of secondary assays by strong correlations with the gold standard. Third, where possible, to calculate the protective threshold in each Phase III study based either on the distribution of antibody titers in random samples of vaccinees and controls A CoP agreed to by regulators will support the demonstration of efficacy of new vaccines using traditional criteria for establishing non-inferiority based on the proportion of vaccinees achieving the protective threshold and comparable GMTs, relative to an approved comparator vaccine. Since the relationship between antibody responses and efficacy was consistent across all manufacturing platforms evaluated to date, even though they may differ in their induction of other antibody functions or T cell responses, an argument can be made that any approved vaccine can serve as a comparator for a future candidate vaccine. Even if authorized for use based on an immunologic readout, full licensure should require confirmatory effectiveness evaluations to closely follow any conditional or accelerated approval process.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dr. Plotkin consults for Janssen and Moderna; Dr. Siber reports personal fees from Clover Biopharmaceuticals, other from COVAXX, personal fees from CanSino, personal fees from CureVac, personal fees from Valneva, personal fees and other from Affinivax, outside the submitted work; Dr. Gilbert reports grants and non-financial support from SanofiPasteur, outside the submitted work; Dr. Ambrosino reports personal fees from COVAXX, personal fees from Clover Biopharmaceuticals, outside the submitted work.


The lack of an appropriate platform for a better understanding of the molecular basis of hepatitis viruses and the absence of reliable models to identify novel therapeutic agents for a targeted treatment are the two major obstacles for launching efficient clinical protocols in different types of viral hepatitis. Viruses are obligate intracellular parasites, and the development of model systems for efficient viral replication is necessary for basic and applied studies. Viral hepatitis is a major health issue and a leading cause of morbidity and mortality. Despite the extensive efforts that have been made on fundamental and translational research, traditional models are not effective in representing this viral infection in a laboratory. In this review, we discuss in vitro cell-based models and in vivo animal models, with their strengths and weaknesses. In addition, the most important findings that have been retrieved from each model are described. Orthohepadnavirus and the family of Hepadnaviridae. HCV is a small virus (55–65 nm in size) with an RNA genome and is categorized into the genus of Hepacivirus and the family of Flaviviridae  and Huh7  are two human hepatoma cell lines which are widely used in antiviral studies, especially those regarding HBV. These cell lines support the virus replication when transfected with HBV ,49. SomeTo investigate various viruses’ interactions, Jian and collaborators developed a scalable and visualizable HAV/HCV coinfection model in Huh-7 cells. Their finding revealed that the simultaneous presence of HAV-HCV did not affect the viral RNA synthesis of both viruses. They suggested that indirect interactions may lead to the suppression and clearance of HCV in HAV/HCV coinfected patients . Sun andTherefore, to better mimic the viral life cycle and host-virus interactions, more representative and functional cell types are urgently needed. Pluripotent stem cells (PSCs) are a renewable source of cells and can be obtained via different protocols . Since tNevertheless, although great efforts are directed toward improving differentiation protocols to achieve better maturation, little progress has been achieved so far. Therefore, there is a need to provide a suitable niche that is more similar to the in vivo architecture, such as spheroids, organoids, bioprinted microtissues, and microfluidic chip devices .Cell polarity, micro-patterned complexity, and cellular interactions are absent in 2D culture systems. Because of the drawbacks of 2D model systems, researchers are looking for alternative three-dimensional (3D) models. The created 3D models, including spheroids, organoids with multicellular structures, and their specific extra-cellular matrix (ECM), were shown to exhibit higher tissue-specific microenvironmental complexity, more mature cells, and better physiological functionality compared to their simple 2D counterparts . Additio3D liver spheroids are multicellular aggregates that exhibit complex cellular communications and polarity in vitro and have characteristics that resemble complex native tissues. These 3D models have been developed through different approaches, such as scaffold-free and scaffold-based 3D cultures, and may be created from primary hepatocytes, established cell lines, and hPSCs-derived cells .Several studies have established liver spheroid models to study a hepatotropic virus’s life cycle in the liver tissue. Chong and colleagues generated primary human hepatocyte spheroids from uninfected liver resections. Spheroids were inoculated with an HCV-positive serum. The obtained data showed that spheroids had a differentiated phenotype and expressed putative HCV receptors. Furthermore, the HCV RNA was detected in the cells, as well as in the supernatant of the culture media . Aly andIn a study by Sainz and colleagues, Huh7 cells were cultured in a 3D rotating wall vessel to generate multilayered 3D liver aggregates. These 3D structures expressed high levels of Phase I and Phase II related xenobiotic drug metabolism genes, as well as hepatocyte-specific transcripts compared to the Huh7 monolayer culture. The data showed increased expression and organization of different proteins composing cell adhesion and tight junction proteins, polarity-related proteins, and HCV receptors. Thus, these 3D Huh7 aggregates could be a model to study the HCV infection effects on the function of liver cells . MurakamFu and colleagues dedifferentiated human primary hepatocytes into expandable liver progenitor-like cells (HepLPCs) using certain chemical agents. These HepLPCs could be redifferentiated into functional hepatocytes. The obtained data showed that redifferentiation in the spheroid culture conditions resulted in upregulation of the viral entry receptor, NTCP, compared to 2D culture. These liver spheroids have the potential to support the HBV infection and secreted newly produced virions. Furthermore, the researchers have shown that a reverse transcriptase inhibitor, entecavir, and the NTCP substrate, tauroursodeoxycholic acid, block HBV binding in liver spheroids and act as a suitable platform for the study of host interactions with HBV and antiviral drugs .HCC) development. Song and colleagues established HBV DNA-secreting HCC cell lines (AMC-H1 and AMC-H2) from infected patients. Then, they generated patient-derived 3D multicellular tumor spheroids consisting of HBV-infected HCC cells, fibroblasts, human umbilical vein endothelial cells (HUVECs), and human hepatic stellate cells to screen potentially effective treatment strategies for precision cancer medicine. The data demonstrated that these multicellular tumor spheroids exhibited a clear selective response to anticancer drugs as opposed to homogeneous HCC spheroids. The use of a coculture system in spheroid formation is one step forward to the native tissue environment  cyclohexane-1,3-dione (NTBC) that prevents the accumulation of toxic tyrosine catabolites, due to the lack of Fah . MoreoveThe establishment of TK-NOG severely immunodeficient mice expressing herpes simplex virus type 1 thymidine kinase has enabled the repopulation of human hepatocytes without the need for administration of exogenous drugs. A single ganciclovir injection can induce a liver injury , and thiSince the models described above are immunodeficient, neither pathogen- or drug-induced immune responses can be investigated. Thus, such models cannot be applied to study the immunopathology of hepatotropic viral infections.+ hematopoietic stem cells [–/–Il2rg–/–SirpaRag2NODAlb-uPAtg/tg mice with engrafted human hepatocytes and immune cells. Such a model can reproduce the full HBV cycle and produce HBV-specific antibodies, similar to humans. Thus, solving the immune system-related issues of humanized animal models would provide new insight into hepatotropic viral infections.To study these aspects, several approaches have been offered. One of them uses AFC8 chimeric immune mice. Compared to those mentioned above, AFC8 mice undergo the transplantation of both human hepatocytes and CD34em cells ,146. As em cells . Moreoveem cells . Yuan etem cells . They crAlthough the studies on the viral adaptation are limited and the efficacy of this approach is still unclear, it can be of interest to reveal the fundamentals of pathogen-host interactions and host-related features B.While infecting a nonspecific host, viruses usually stop their life cycle at certain stages. For instance, mouse cells can maintain HCV replication, but new virions are not released from them ,150. OneAdeno-associated viral systems have also been used for the delivery of hepatotropic viruses into murine hepatocytes. Hydrodynamic injection of viral vectors containing partial or full-length hepatitis viruses’ genomes is an efficient technique to deliver the genetic material in the liver of mice . This apSome studies have indicated that transfected models can induce innate  and adapGao and colleagues investigated the effects of interleukin-33 in an HBV mouse model. Interleukin-33 inhibited HBV via the ST2 receptor in the HBV mouse model. The results have shown that NK cell-derived antiviral cytokines may have a critical role in the early viral clearance during the HBV infection . One stuSeveral factors, including a plasmid backbone, mouse strain, age, and sex, can affect the level and duration of replication of the hepatitis viruses. The use of a lentiviral backbone instead of an AAV vector led to increased and prolonged HBV replication ,166. HBVsiRNA) and short hairpin RNA (shRNA) to minimize the HBV and HCV expression and liver failure. For instance, McCaffrey and colleagues cotransferred plasmids expressing shRNAs homologous to HBV mRNAs into mice with HBV to reduce the replicated viral genomes, viral RNAs, and secreted HBV surface antigen (HBsAg) [Transfected mouse models can be used for the evaluation of broad-spectrum antiviral compounds for liver disease. Several studies used small interfering RNA ( (HBsAg) . In anot (HBsAg) . Thus, s (HBsAg) .A promising novel approach is the use of the HBV-specific clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system for selective elimination of cccDNA and the viral protein. In hydrodynamic-based mouse models, using HBV-specific guide RNA (gRNAs) efficiently inhibits levels of HBV-expressing vectors and facilitates the clearance of the intrahepatic HBV without any toxicity ,174.Huang and colleagues used a transfected mouse model to evaluate the therapeutic effect of hepatitis delta antigens (HDAg) in the HDV infection. The results showed that the Tat-enhanced delivery of the C terminus of the HDAg-L (TAT-HA-HDAg L (198–210)) fusion protein suppressed the viral particles’ assembly and secretion of HDV and could be used as a new therapeutic compound against the HDV infection .The most significant benefit of hydrodynamic injection is the possibility to investigate the immune responses during the acute phases of infections which appear when viral sequences are not integrated into the host genome. Another privilege of this system is that the replication of HBeAg or HBsAg seroconversion proceeds without the manifestation of obvious signs and symptoms of liver diseases . Most trVarious model systems have been established for modeling individual aspects of the hepatotropic viruses’ life cycle or the complete infection cycle in vivo and in vitro. Since each system has its benefits and caveats, selecting the best model is a challenging task. Today, many biomedical studies depend on one of the two approaches, either 2D cell culture experiments or animal models. However, these systems have some drawbacks. Cells cultivated in a 2D culture have been shown to follow different adaptations in terms of gene expression patterns and cell physiology, as compared to those grown under 3D conditions. Studying the hepatotropic viruses in animal models is limited because most of them are species-specific. Moreover, conducting research with animal models in this field raises ethical concern. There are several limitations to the use of humanized liver in chimeric mice, including their immunodeficient background, pathogenesis, and immune response, which hamper their broad application . Thus, f


Neuronal Ceroid Lipofuscinosis Type 7, Ataxia-Telangiectasia, and Epilepsy of Infancy with Migrating Focal Seizures are presented. These case studies discuss individualized testing protocols, accounting for disease presentation, cognitive and motor function. We outline specific considerations for developing assessments for detecting changes in sensory processing in diverse patient groups and safety monitoring trials of early phase investigational intrathecal drug therapies. QST may complement information obtained from the standard neurologic examination, electrophysiologic studies, skin biopsies, and imaging. QST has limitations and challenges, especially in non-verbal subjects, as shown in the three cases discussed in this report. Future directions call for collaborative efforts to generate sensory datasets and share data registries in the pediatric neurology field.Several neurological disorders may be amenable to treatment with gene-targeting therapies such as antisense oligonucleotides (ASOs) or viral vector-based gene therapy. The US FDA has approved several of these treatments; many others are in clinical trials. Preclinical toxicity studies of ASO candidates have identified dose-dependent neurotoxicity patterns. These include degeneration of dorsal root ganglia, the cell bodies of peripheral sensory neurons. Quantitative sensory testing (QST) refers to a series of standardized mechanical and/or thermal measures that complement clinical neurologic examination in detecting sensory dysfunction. QST primarily relies on patient self-report or task performance . This brief report illustrates individualized pragmatic approaches to QST in non-verbal subjects receiving early phase investigational intrathecal drug therapies as a component of clinical trial safety protocols. Three children with neurodevelopmental disorders that include  Precision medicine is an emerging area for disease prevention and treatment strategies that takes into account individual variability in genes and environment. A growing number of neurological disorders may be amenable to precision medicine using treatment with gene-targeting therapies such as antisense oligonucleotides (ASOs) or viral vector-based gene therapy. The US Food and Drug Administration (FDA) and European Medicines Agencies (EMA) have approved several of these treatments for clinical applications; many others are in clinical trials.Recent advances in ASO drug development have resulted in a rapid transition from bench-to-bedside candidates with the FDA recently approving 12 ASO therapies between the years 2016 and 2020 alone . It is cClinicians are faced with challenges in identification and measurement of drug-related neurotoxicity in patients with neurological disorders using non-invasive bedside approaches. Quantitative sensory testing (QST) refers to a series of standardized measures that complement clinical neurologic examination in detecting sensory dysfunction –7. QST pMonitoring patient safety and drug toxicity during clinical trials is a critical component in drug development. Optimal safety biomarkers are quantitative and permit within-subject comparisons and group comparisons relative to age-specific norms. Developing broadly applicable safety biomarkers is challenging for patients with rare neurologic disorders. Sample sizes are small (or even N-of-1), and there is heterogeneity among different neurologic disorders or even participants with the same condition.Hyposensitivity (elevated sensory thresholds) occurs with sensory loss, due to injury or disease , 9 or teQST provides insight into large myelinated Aδ and small unmyelinated C fiber function . TypicalAdult QST protocols are standardized and studied extensively. A widely used protocol involves a battery of 13 non-invasive assessments, developed by the German Neuropathic Pain network (DFNS) . A DFNS-Standardized protocols permit comparison of one individual to normative values and patients with neuropathic conditions using multicenter databases , 19–23. Modifications to the DFNS protocol have been made for typically developing children as young as 6 years old. Reference values have been reported for age and gender , 22. OurFor term and preterm neonates, infants, and non-verbal children, surrogate approaches to sensory evaluation have recorded stimulus-evoked behaviors  –38. We rLimited information has been reported regarding QST protocol design in non-verbal children. From PubMed search, we could not identify publications describing protocols for sensory assessments during drug trials for children with communicative, cognitive, and motor constraints.individualized testing protocols, accounting for disease presentation, cognitive and motor function for three children receiving spinal administration of a personalized ASO in an N-of-1 protocol  for clinical administration , 41, 42.Lower extremities were tested because animal studies show more behavioral and histologic signs of toxicity around lumbar than thoracic or cervical spinal levels. Baseline assessments before treatment, and repeated tests pre- and post-drug administration were performed. The duration of treatments ranged from 12 to 36 months.The primary outcome measure was the lowest mechanical stimulus intensity required to evoke nocifensive behavior  or verbal and non-verbal cues (Case 2) when applied to the foot. In all cases caregivers were present during the assessments and they engaged with the physiologist in order to help relax the patient or providing feedback about responses as needed.All studies were conducted with local ethics approval and written informed parent consent.Patient 1 had typical neurodevelopment through infancy. By age 3, she began to develop a progressive decline of cognitive and motor function, blindness, seizures, and spasticity. At age 6, whole genome sequencing led to a diagnosis of neuronal ceroid lipofuscinosis Type 7 . She demonstrated frustration through making fussy vocalizations and becoming more active.Study optimization: We opted to stimulate the left side because the patient had a broader range of motion, making leg flexion withdrawal feasible. Musical distraction techniques (familiar movie soundtrack) and parent interaction (soothing singing) were used while performing the test to help induce relaxation and reduce spurious leg movement. The patient was assessed while awake for all study time-points.Interpretation:Patient 2 underwent newborn screening, which showed a risk for severe combined immunodeficiency. At 7-months, she was diagnosed with ataxia-telangiectasia. At evaluation, at age 2.8 years, she presented with delays in speech and adaptive skills, and mild ataxia. Testing with the use of Vineland Adaptive Behavior Scales (Vineland-III) showed moderately low scores (3rd percentile) at baseline indicating that the patient was behind peers in development. Peabody Developmental Motor Scale (PDMS-2) scores were low for locomotion (2nd percentile), stationary motor (9th percentile) and object manipulation (9th percentile) at baseline.Means of communication: Patient 2 used non-verbal communication to indicate “yes” and “no,” vocalized to identify objects, and signs and gestures to indicate distress and frustration.Study optimization: The challenges faced were in ensuring stimulus-response specificity and patient engagement. We opted to (1) perform multiple trains of stimuli by applying each stimulus three times in three groups and documenting a positive response when 2/3 groups elicited a response; (2) alternate stimulus application location across the feet, so stimuli were “unexpected” (increasing attention and preventing habituation); (3) utilize relaxation techniques (play or eating); (4) protocol adaptations accordingly with developmental gains Verbal and non-verbal cues were considered positive responses, e.g., immediate changes in attention, leg withdrawal. The patient was assessed while awake for all study time-points.Interpretation:Patient 3 developed unrelenting seizures from day 4 of life onward. At age 4-months, she was diagnosed with a potassium channel mutation causing epilepsy of infancy with migrating focal seizures. At evaluation, the patient was preverbal with significant developmental delay and presented multiple seizures a day. Vineland Adaptive Behavior Scales (Villand-III) scores were low (<1st percentile) in all domains and indicated profound developmental delays across all domains at baseline. Motor evaluation showed severe gross hypotonia thoughout with minimal to no head control appreciated in any positioning; she required assistance for all mobility.Means of communication: Patient 3 had difficulty engaging in fundamental skills for attending to her environment and eliciting parent support for her needs. Parents were able to report behavious that suggested patient was hungry  or unsettled .Study optimization: Being of toddler-age, the patient was often napping during the time window allocated for baseline data collection . We opted to take measurements when possible during both wakefulness and sleep states to ensure the measures were not floored.Interpretation: Sensory thresholds were stable over time but considerably lower during natural sleep compared to awake state (ke state . AlthougVarious types of assessments can be performed to understand neurophysiologic function including QST (measurement of sensory thresholds), electrophysiologic testing (nerve fiber excitability), and recording of cortical sensory-evoked potentials. Here we have outlined individualized pragmatic approaches to QST in non-verbal research-participants undergoing treatment with novel ASOs as a component of clinical trial safety protocols. We have attempted to identify the most reliable sensory testing parameters given the patient age, state, disease presentation and cognitive ability. There are several advantages to our approach for use in these challenging patient populations.One of the biggest challenges when working with pre/non-verbal populations lies in ensuring reliability of testing. The real-world data provided in this paper exemplifies how factors such as age and state can influence variability outcomes. We note that there is very little data available in this field, and as such, have largely drawn from our own experiences in this field. Where concerns over reliability of testing arise, it may be appropriate to complement sensory evaluation with more direct measures of nerve function. Alternative approaches include nerve conduction, electromyography, and cortical sensory-evoked potentials.Electrophysiologic studies including nerve conduction velocity (NCV) and electromyography (EMG) have well-established interpretations in evaluation of peripheral nerve functioning. Conduction velocity in the upper extremities and lower extremities  can be evaluated. Recording from one muscle supplied by the nerve distal to the site of stimulation is performed using surface electrodes, and/or a needle electrode. Slowing of nerve conduction velocity and reductions in amplitude usually indicate the presence of lesions affecting the axon of the peripheral nerve or loss of axons, respectively . ImportaCortical sensory-evoked potentials may provide additional objective information. Recording electrodes are placed over the scalp, and cutaneous stimuli are delivered. Cortical responses are analyzed in a similar approach to NCV/EMG. Laser, contact-heat, and intraepidermal electrical stimulation are the three main types of stimuli that can be used reliably. Thermal modalities primarily investigate small-diameter Að* fiber dysfunction (rather than C-fibers), and the central lesions involving the spinothalamic tract . IntraepChallenges faced when designing a sensory testing study in a neurologically challenged population is in adapting to disease progression. Many genetic neurologic disorders are associated with neuropathies that may progress over time. Here, we opt to utilize the flexion withdrawal reflex as an outcome measure because of its primitive nature. However, for some patients, spasticity may develop and make it difficult to identify what is evoked vs. spontaneous limb movement. In this scenario, we recommend applying multiple stimuli and testing on multiple occasions.We additionally provide a protocol that aims to generate objective and quantitative data. In pre- or non-verbal populations, experienced care givers may be used as a proxy for self-report although this approach is confounded by many challenges and biases. For example, patients may exhibit pain behaviors when they do not have pain and make it difficult to discern signs of pain, or observers may overestimate pain when unblinded to the application of a painful stimulus . HoweverWe show that our protocol can be used as a paragmatic approach to evaluate patient safety and drug toxicity during clinical trials were there is an potential risk for spinal toxicity. We additionally highlight the importance of interpreting the sensory testing results with other clinical examination assessments such as those performed by a neurologist and the physical therapist.We propose provisional recommendations for performing QST in children with neurologic disorders receiving early phase investigational intrathecal drug therapies based on our initial experience with N-of-1 trials.Protocol design must balance obtaining necessary information while limiting session duration and exposure to noxious stimuli; e.g., adults might complete a QST battery lasting 1-h, whereas 5-year-olds might only tolerate a mechanical detection paradigm lasting 15-min.Documentation of experimental variables, e.g., patient positioning, sleep state, and distraction technique .Documentation of degree of developmental disability across language, cognitive and motor domains (as appropriate).For neonates, young infants, or older children with developmental delay or neurologic regression who cannot provide self-report, mechanically evoked leg flexion withdrawal responses offer an indirect measure of sensory responsiveness. The outcome measure is the lowest mechanical stimulus intensity required to produce a response.Age-varying changes in mechanical threshold must be considered. The threshold required to evoke the reflex rapidly increases during the first 2 months of life and then changes very little up the first year , 36, 38.Patients with adequate understanding of the test but limited ability to verbalize the responses can be aided with alternative response cues, e.g., clicking a button or eye-blinking.Patients with movement disorders  presenting with stiff or restless legs make it difficult to evoke withdrawal responses. Optimizing positioning and distraction techniques facilitate relaxation and cooperation.Attention span and concentration affect the quality of the results by increasing data variability. Cooperation is enhanced in a quiet, calming environment and engaging through play or distraction .We recommend performing:A series of baseline evaluations on successive days that reflect changes in a patient's state and acknowledges that a degree of learned behavior will occur with longitudinal studies.Quality assurance tests where multiple assessors or study sites are involved.We have outlined specific considerations for developing individualized assessments for detecting changes in sensory processing in diverse patient groups and safety monitoring in N-of-1 trials of novel medications . QST ' legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.CB, LC, and CD conceptualized and wrote the manuscript. LC, CD, CB, and TY critically revised the manuscript. All authors contributed to the article and approved the submitted version.This work is supported by the Sara Page Mayo Endowment for Pediatric Pain Research and Treatment, and by the Cardiovascular & Critical Care Cluster of Clinical Research Excellence at Boston Children's Hospital.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Over the past 20 years, the field has been driven by the discovery of photoreceptor-based interaction systems, the engineering of light-actuatable binder proteins, and the development of photoactivatable compounds as dimerization inducers. This perspective is to categorize mechanisms and design approaches of these dimerization systems, compare their advantages and limitations, and bridge them to emerging applications. Our goal is to identify new opportunities in combinatorial protein design that can address current engineering challenges and expand  In chemogenetic and optogenetic approaches, PPIs are controlled by chemicals  and ligon (CID) ], respecon (CID) , the PPIon (CID) .opto-binders, which can target unmodified endogenous proteins. A first opto-binder was a split green fluorescent protein (GFP) antibody comprising two fragments genetically tagged with a photoswitchable heterodimerization system   . Presumade novo design, but some successes were recently achieved by screening combinatorial protein libraries , for example, variable flexible loops like antibody complementarity-determining regions or residues on a relatively rigid binding surface. To obtain a high percentage of protein sequences in the library that might be correctly translated and stably folded, the randomization is usually guided by amino acid distributions found in experimentally validated natural  or engiertoires  is ideal., phage , ribosom., phage , and mRN., phage . An impoThe first application of combinatorial binder libraries is to select dimerization binders against the light or dark form of a photoreceptor to create heterodimerization systems. This approach can circumvent unavoidable constraints of natural dimerization binders and provide the flexibility to select the protein size and binding properties. Typically, dimerization binders are obtained by screening a combinatorial library against the light or dark form of a photoreceptor as the positive selection and the other form as the negative selection . CandidaAvena Sativa phototropin 1 (AsLOV2)  (Acryochloris marina cyanobacteriochrome (CBCR) (9) with a humanized scaffold was screened by phage display against the DrBphP photosensory module. Column-based biopanning was designed for the selection of both the conformational specificity and dimerization reversibility. The library screening was demonstrated to be highly efficient; in vivo actuators constructed with selected dimerization binder clones showed drastically improved light-induction specificity and a significantly lower dark activity than a similar natural Rhodopseudomonas palustris BphP1 (RpBphP1) system and its derivative. Although the protein display screening is efficient, it is necessary to validate the expression of in vitro selected binders by cell-based assays  were screened by mRNA and ribosome display against a dark-form LOV2 domain of (AsLOV2)  and a li(DrBphP)  to obtai(DrBphP)  and a cGe (CBCR) . We recee (CBCR) . In this., yeast  or mamma., yeast ]. Based AsLOV2 domain was inserted into specific loops away from their target binding sites, so the light-triggered unfolding of the Jɑ helix in AsLOV2 allosterically regulates the binding site conformation. In the affibody-based opto-binders  the photo-uncaging of drugs, 2) the binding of drugs to endogenous targets, and 3) the drug-induced dimerization. Although such CID systems are yet to be reported, based on available methods for the CID engineering  and chemTo create such systems, a critical need is to design CID systems for any given drug. Recent advances were made by selecting dimerization binders from phage-displayed libraries of antigen-binding fragments (Fabs)  and nanoin vivo actuation needs. They can potentially generate functional data for large-scale protein sequences which are useful for developing deep learning methods (in vivo applications.The proposed combinatorial approaches will leverage ongoing efforts on designing photoswitchable dimerization systems for  methods  to impro methods ] can fac methods , electri methods , heat (C methods , and mec methods ] can als


Pseudomonas strains. We found that the effect of probiotic consortium inoculation was richness-dependent: consortia that contained more Pseudomonas strains reached higher densities in the tomato rhizosphere and had clearer beneficial effects on multiple plant growth characteristics. Crucially, these effects were best explained by changes in the resident community diversity, composition and increase in the relative abundance of initially rare taxa, instead of introduction of plant-beneficial traits into the existing community along with probiotic consortia. Together, our results suggest that beneficial effects of microbial introductions can be driven indirectly through effects on the diversity and composition of the resident plant rhizosphere microbiome.Plant growth depends on a range of functions provided by their associated rhizosphere microbiome, including nutrient mineralization, hormone co-regulation and pathogen suppression. Improving the ability of plant-associated microbiomes to deliver these functions is thus important for developing robust and sustainable crop production. However, it is yet unclear how beneficial effects of probiotic microbial inoculants can be optimized and how their effects are mediated. Here, we sought to enhance tomato plant growth by targeted introduction of probiotic bacterial consortia consisting of up to eight plant-associated  Many species mineralize nutrients, produce growth hormones or prevent diseases ,2. HowevOne way to increase microbiome-associated multifunctionality is the introduction of additional microbes into the soil . For a lIn addition to directly introducing functions to the rhizosphere, inoculated microbes could have indirect effects via alteration of the diversity, composition and functioning of the resident rhizosphere microbiome ,17. PrevPseudomonas spp. strains that all are well studied and have previously been shown to have beneficial effects on plants  [in vitro performance of each consortium regarding bacterial traits linked to plant-beneficial functions . The mean effects of individual plant-beneficial functions were further analysed by a weighted multifunctionality index [Pseudomonas strain present in consortia (strain identity effects). First, we measured the consortium effects on plant growth, nutrient assimilation and protection against the soil-borne pathogen Ralstonia solanacearum [Pseudomonas consortia, and assessed the probiotic inoculant colonization. Finally, structural equation modelling (SEM) was used to compare the contribution of direct and indirect effects of the inoculated consortia on plants. We hypothesized that introduction of Pseudomonas consortia could promote plant growth, and that this promotion could be magnified with increasing consortium richness. Mechanistically, such effects could be driven by improved Pseudomonas establishment and introduce novel functional traits into the microbiome directly, or indirectly via changes on the composition and functioning of the existing resident microbiome.In this study, we employed a biodiversity-ecosystem functioning framework  to assess S1–S3) . We firsty index , which snacearum . Second,. 2 (a)Pseudomonas spp. as model organisms: P. fluorescens 1m1–96, P. fluorescens mvp1–4, P. fluorescens Phl1c2, P. fluorescens Q2–87; P. kilonensis F113, P. protegens Pf-5, P. protegens CHA0 and P. brassicacearum Q8r1-96 . These strains are well-characterized model biocontrol strains and widely used in plant growth promotion and pathogen-suppression studies [phlD, which is involved in the production of antimicrobial polyketide 2,4-diacetylphloroglucinol (DAPG). This gene was used as a molecular marker to quantify the abundances of introduced Pseudomonas strains, as the background density of this gene was very low in the used soil . Ralstonia solanacearum QL-Rs1115 strain was used as a model soil-borne pathogen in in vitro and in vivo experiments. All strains were store at −80°C, and prior to all experiments, one single colony of each bacterial strain was selected and prepared by grown overnight in nutrient broth medium, washed three times in 0.85% NaCl buffer and adjusted to a density of 108 cells ml−1.We used eight strains of  studies ,26,29. T (b)Pseudomonas consortia in 48 treatments that contained 1, 2, 4 or 8 Pseudomonas spp. strains (four richness levels) following a substitutive design  [Pseudomonas species were mixed in equal amounts in all consortia to keep the total bacterial abundances the same between different richness levels and immediately used for subsequent experiments. The same substitutive experimental design was used in both the in vitro assays and in vivo greenhouse experiments.We assembled a total of 37 distinct able S2) ,30,31. I (c)in vitro functional traits of the assembled consortia were measured in previously published studies [The original data related to  studies ,15. In t (d)in vitro Pseudomonas traits were used to compute a multifunctionality index for each consortium using a weighted average standardized calculation method [All measured n method . Brieflyn method . To avoin method  to ident (e)Lycopersicon esculentum, cultivar ‘Jiangshu’) were germinated on water-agar plates for 3 days before sowing into seedling plates containing 60Co-sterilized seedling substrate . Germinated tomato plants were transplanted to seedling trays containing non-sterile agricultural soil at the three-leaf stage (12 days after sowing). Two tomato plants per cell (600 g of soil) were transplanted to total of eight separate cells resulting in sixteen seedlings per seedling tray (370 × 272 × 83 mm). Each tray was treated as one biological replicate resulting to a total of 104 trays. Half of these trays (n = 52) were used for the plant growth experiment, and the other half (n = 52) for the plant protection experiment; more detail included in the electronic supplementary material.We assessed the effect of consortium richness on microbiome diversity and function in two separate greenhouse experiments. In both experiments, we grew tomato plants in non-sterile agricultural soil collected from a tomato field in Qilin town of Nanjing, China, which has been used for tunnel greenhouse farming for over 15 years . Surface (f)in vitro Pseudomonas consortium multifunctionality index. For example, aboveground plant biomass and protection against pathogen were assigned to one cluster and each of them received a weigh of 0.5 .All measured plant traits  were used to calculate weighted average plant growth index . To avoi (g) (i)2O  and centrifuged  before the soil pellets were transferred into 2 ml tubes and stored at −80°C for subsequent experiments. We extracted DNA with the Power Soil DNA Isolation Kit . Briefly, DNA from 0.3 g of soil pellets per sample was extracted following the manufacturer's protocol. DNA fragment size was checked on 1% agarose gel, and DNA concentration and purity were determined with a NanoDrop 1000 spectrophotometer  prior to downstream analyses.Rhizosphere soil from the plant growth promotion experiment was collected by gently removing plants from the trays before shaking off excess soil and collecting the soil attached to the root system. Two randomly selected plants from different cells per seedling tray were collected and pooled together. Samples were then suspended into 30 ml of sterile H (ii)Pseudomonas consortia based on 16S rRNA and phlD gene copies per gram of soil, respectively; more detail included in the electronic supplementary material, methods.We used quantitative PCR (qPCR) to quantify the abundance of total bacteria associated with plant rhizosphere soil as well as the introduced  (iii)a and electronic supplementary material, figure S3 for a reproduction of figure 4c with DADA2 pipeline).We used the 563F (5′-AYT GGG YDT AAA GVG-3′) and 802R (5′-TAC NVG GGT ATC TAA TCC-3′)  primer pfigure 4 (iv)We characterized the biodiversity of resident bacterial communities using phylogenetic abundance evenness (PAE), an index accounting for both evenness and phylogenetic distribution  (more de (h)Pseudomonas consortia on the resident rhizosphere microbiome community composition, diversity and weighted mean of plant growth index, generalized linear models (GLMs) were used. Consortium richness was treated as a factor, phlD abundance in rhizosphere was log-transformed, and their effects on subsequently measured parameters were assessed with GLMs using a Gaussian distribution in two models with different factor directions following a subsequent ANOVA analysis in R. The effect of microbial inoculation on rhizosphere microbiome community composition was determined with a redundancy analysis (RDA) based on the taxa presence–absence OTU table or a principal coordinates analysis (PCoA) based on the weighted Bray–Curtis distance metric, consortium richness (factor with five levels) and phlD gene copy numbers in the rhizosphere, using the R function vegan: rda. Coordinates were used to draw two-dimensional graphical outputs, and the effect of consortium richness and establishment success on rhizosphere microbiome community composition was tested using the R function vegan: adonis. To explore the significance of Pseudomonas strain properties for the shift in resident microbiome, the effect of in vitro plant-beneficial functions measured at the consortium level were used to explain variation in the resident rhizosphere microbiome composition, which was presented by using canonical correlation analysis (CCA). Stepwise GLMs were used to test the effect of in vitro plant-beneficial functions, such as breadth of carbon catabolism, antibacterial activity, phytohormone production and nutrient availability, on resident microbiome composition. Finally, we used SEM to examine direct and indirect effects linking consortium inoculants with plant growth by accounting for multiple potentially correlated effect pathways [To analyse the effect of richness and abundance of probiotic pathways  (more de. 3 (a)Pseudomonas strains excelled at expressing a specific subset of plant-beneficial functions, including ability to consume different carbon resources, production of auxin, gibberellin, siderophores or antibacterial activity (a). For example, P. protegens Pf-5 produced the highest amount of siderophores but the lowest levels of auxin (a). By contrast, P. fluorescens 1M1-96 produced the highest levels of auxin but had a very weak antibacterial activity (a). These differences suggest that Pseudomonas strains were specialized in different plant-beneficial functions and could potentially show complementary effects when combined in multi-strain consortia. To test this, we used weighted means of all the individual plant-beneficial functions and combined them into predicted consortium multifunctionality index. It was found that consortium richness correlated positively with the consortium multifunctionality index , and increasing the consortium richness promoted the expression of all the individual plant-beneficial functions when measured directly in vitro . Some of the individual functions were further influenced by the presence of certain strains. For instance, consortia containing the P. protegens CHA0 and Pf-5 strains showed the highest levels of antibacterial activity . However, despite certain strong identity effects, consortium richness remained a major significant predictor even after accounting for the inclusion of each strain.Each of the studied activity a. For exof auxin a. By conactivity a. These  (b)in vitro measurement of plant-beneficial functions, each Pseudomonas strain had unique impacts on plant growth on their own. For example, P. kilonensis F113 inoculation led to a high aboveground plant biomass, P. protegens CHA0 inoculation led to a high concentration of potassium and phosphorous in plant tissues, while P. brassicacearum Q8r1-96 inoculation led to the highest protection against R. solanacearum pathogen (a). Single Pseudomonas strain inoculations affected only a limited number of plant growth characteristics , increasing consortium richness led to an improvement of multiple plant growth characteristics (b). All individual plant growth characteristics were positively correlated with consortium richness except for plant tissue nitrogen concentration (b). As a result, the weighted average plant growth index also correlated positively with the probiotic consortium richness (b). By contrast to the in vitro plant-beneficial functions expressed by consortia, the Pseudomonas strains had no significant identity effects on the plant growth phlD gene abundances were below 0.001% (relative to 16S rRNA gene abundances) in the non-inoculated control treatments , confirming that Pseudomonas strains containing this gene were rare in the non-sterile agricultural soil used in the pot experiments. By contrast, phlD gene abundances ranged between 0.01% and 0.25% of the total 16S rRNA gene abundances in samples with inoculated Pseudomonas strains. Specifically, we found that phlD gene abundances increased along with consortium richness gradient  and only mvp1–4 strain had a marginally significant positive identity effect on the relative phlD gene abundances .The background  (d)p = 0.0280; figure 3a), qualitatively similar results  were also obtained using weighted Bray–Curtis distance metric. Interestingly, a clear increase in resident microbiome diversity was observed along with consortium richness (figure 3b). Moreover, the effects of consortium richness on microbiome diversity remained significant even after accounting for Pseudomonas strain identity effects . One explanation for the increase in the resident community diversity is that Pseudomonas inoculants potentially indirectly favoured rare species by competing more intensively with relatively abundant taxa. In support for this, we found that rare taxa at phylum, family and OTU levels were more likely to increase in abundance along with the richness of the inoculated consortia . By contrast, the most abundant taxa were more likely to decrease in response to Pseudomonas inoculations . At a coarser taxonomic level, we observed that resident microbiome responses to Pseudomonas inoculants were conserved with some phyla. For instance, OTUs belonging to the phyla Candidate division WS6, Thermotogae, Spirochaetae, Gracilibacteria and Parcubacteria consistently increased along with the Pseudomonas consortium richness . Finally, we conducted covariance network analysis to assess whether the introduced Pseudomonas strains were embedded in the resident microbiome. Despite the limited taxonomic resolution of amplicon sequencing, we could identify two Pseudomonas sp. OTUs  putatively matching with two introduced Pseudomonas strains (CHA0 and mvp1–4 strains). These OTUs covaried positively with a range of other OTUs present in the resident rhizosphere microbiome, including Parcubacteria, Proteobacteria, Actinobacteria, Bacteroidetes and Saccharibacteria phyla .We found that irrespective of inoculant richness, all probiotic consortia had similar negative effects on the abundance of resident rhizosphere bacteria compared to a non-inoculated control treatment . All inoculated consortia changed the composition of the resident rhizosphere microbiome. However, this impact was magnified with increasing consortium richness ( (e)in vitro predicted poorly the individual plant growth characteristics . The only notable exception was a positive relationship between the consortium phosphate solubilization capacity and the plant shoot phosphorus content . Similarly, the multifunctionality index of probiotic consortia did not explain the positive effects of inoculants on the plant growth . By contrast, variation on the CCA2 was best explained by the niche breadth of the probiotic consortia . As access to resources is crucial for inoculant establishment, these data suggest that probiotic consortia could have shaped the resident microbiome through resource competition. By contrast, the antibacterial activity of Pseudomonas consortia was not retained in the final model .To gain additional insight into underlying mechanisms, we used CCA to describe the resident microbiome composition and assessed if . 4Pseudomonas strain identity effects were found, the improvement of plant growth was poorly explained by plant-beneficial functions provided by different consortia members. Instead, positive effects on the plant growth were best explained by consortium-mediated shifts in the resident microbiome, which were associated with phytohormone production and resource competition by the probiotic consortia. Together these findings suggest that probiotic bacteria can be used to steer the existing resident rhizosphere microbiome in order to improve plant growth.Here we used a biodiversity–ecosystem functioning framework to directly assess how the diversity of probiotic bacterial inoculants affect microbiome structure and plant growth. Specifically, we explored if the potential benefits were driven directly by introduced consortia via introduction of essential functions to the microbiome, or indirectly via changes in the resident bacterial community. It was found that increasing probiotic consortium richness increased inoculant colonization success, and was associated with improved plant growth, nutrient assimilation and protection from pathogen infection. Crucially, inoculants caused shifts in the resident microbiome and these effects were magnified with increasing probiotic consortium richness, leading to an increase in the abundance of rare taxa and overall microbiome biodiversity. While some significant Pseudomonas abundances in the rhizosphere. While certain Pseudomonas strains had strong identity effects, the effect of consortium richness remained significant even after their removal. Moreover, the densities of four- and eight-strain consortia were up to ten times higher than the best-performing single-strain inoculants, which indicates that richness effects were driven by emergent consortium level effects, making it ultimately difficult to disentangle the relative importance of density versus diversity effects. Such unexpectedly strong community performance is called transgressive overyielding [Pseudomonas strains and the resident microbiome or the plant. The fact that inoculation of probiotic bacteria resulted in reduction in the abundance of resident bacteria suggests that Pseudomonas bacteria probably promoted competition with the resident community. In support for this, the consortium resource niche breadth increased along with the richness gradient, which might have allowed strains to sequester nutrients more efficiently in the rhizosphere [Pseudomonas consortium richness.Multi-strain consortia could perform better together due to ecological complementarity between plant-beneficial functions they can provide at consortium level, or due to other emergent diversity effects arising in complex microbe–microbe–plant communities . While tyielding , and couzosphere . HoweverPseudomonas consortia reduced competitive exclusion of rare species by having disproportionally stronger competitive effect on the more abundant taxa. While additional work is required to unravel exact mechanisms, increase in the relative abundance of rare taxa was associated with decrease in the abundance of dominant taxa (c), indicative of highly asymmetric responses by different resident microbiome taxa. For example, four- and eight-strain probiotic consortia had proportionally large, positive effects on Parcubacteria [Pseudomonas bacteria may have functioned as a keystone group favouring the rare or dormant species and their associated functions in the soil [Interestingly, probiotic consortium richness and establishment success were positively associated with relatively larger and non-random changes in the diversity and composition of resident rhizosphere microbiome, with four- and eight-strain consortia showing the clearest effects. Specifically, diverse probiotic consortia were associated with more even resident bacterial communities and clearer increase in the abundance of rare bacterial taxa. One potential explanation for this could be inhibition of dominant organisms via production of secondary metabolites by the introduced consortia . Alternaant taxa c, indicabacteria , a phylubacteria . As thesthe soil . The rarthe soil .Pseudomonas phytohormone production also had a significant effect, which suggest potential complex microbiome–plant feedback mediated by hormonal signalling [Variovax bacterial strains in synthetic rhizosphere communities can restore the root growth in Arabidopsis thaliana via effects on bacterially produced ethylene and auxin in the rhizosphere [R. solanacearum [Pseudomonas strains, or how changes in the resident community diversity and composition were linked to expression of plant growth-promoting genes, metagenomics and transcriptomics approaches and directly tailored mechanistic experiments are needed in the future. Furthermore, metabolomic and plant transcriptomics studies would be important for inferring the chemical signalling between the plant and rhizosphere microbiome [In addition to resource catabolism, gnalling ,47. For zosphere . Finallynacearum . We founnacearum . However. 5We conclude that plant growth can be improved using species-rich inoculants that have indirect beneficial effects for the plant through compositional changes in the resident rhizosphere microbiome. We observed a positive response by rare taxa, highlighting the importance of rare biosphere for plant–microbe interactions . This caClick here for additional data file.


Environmental-friendly techniques based on plant stress memory, cross-stress tolerance, and seed priming help sustainable agriculture by mitigating negative effects of dehydration stress.The frequently uneven rainfall distribution caused by global warming will lead to more irregular and multiple abiotic stresses, such as heat stress, dehydration stress, cold stress or the combination of these stresses. Dehydration stress is one of the major environmental factors affecting the survival rate and productivity of plants. Hence, there is an urgent need to develop improved resilient varieties. Presently, technologies based on plant stress memory, cross-stress tolerance and priming of seeds represent fruitful and promising areas of future research and applied agricultural science. In this review, we will provide an overview of plant drought stress memory from physiological, biochemical, molecular and epigenetic perspectives. Drought priming-induced cross-stress tolerance to cold and heat stress will be discussed and the application of seed priming will be illustrated for different species. Dehydration stress is one of the major environmental factors affecting the survival rate and productivity of plants. Dehydration leads to low water availability and adversely affects food security  than the non-trained plants when exposed to a subsequent dehydration episode  found that a pre-treatment of drought acclimation cycles reduces leaf wilting, induces thicker cuticular layers and more open stomata under a subsequent drought stress, compared to a direct application of drought without pretreatment , superoxide dismutase (SOD) and catalase (CAT) decreasing the content of hydrogen peroxide (H2O2) and malondialdehyde (MDA)  showed that the wheat plants pre-exposed to a moderate water deficit had higher concentrations of ABA compared to non-primed plants, and the pre-exposure eventually resulted in higher grain yields , an essential phytohormone, increases in response to dehydration through a complex equilibrium of synthesis, degradation or conjugation Kim . A studyArrhenatherum elatius plants which had been pre-exposed twice to drought stress was higher than that of plants which only encountered a single drought stress  and a memory is maintained by feedback loops and cytosol partitioning  indicated that repeated stress conditions lead to the acquisition of a stable epigenetic memory on the level of DNA methylation ) changed during the transition from dehydration to rehydration in A. thaliana  onto turnip (B. rapa subsp. rapa) indicates that drought stress can be memorized and transmitted from turnip rootstock to rapeseed scion through the modification of histone H3K4me3 at the Δ1-pyrroline-5-carboxylate synthetase 1–2 (P5CS1-2) gene  plants  showed that pre-exposure to drought stress during stem elongation improved tolerance to high temperature occurring later during grain filling  by Zhang et al.  significantly improved germination as well as seedling growth under drought stress conditions  obtained better germination parameters, growth and higher yield under water shortage conditions  on wheat yield was evaluated in a field experiment  had longer roots and shoots under water deficit conditions  decreased, which indicates a gain of stress tolerance and also points to a stress memory  and recently genetic engineering  have limitations, such as the length of time, large man power, or the restrictions based on biosafety issues and environmental protection problems. Hence, alternative technologies emerged as promising solutions. As we describe in this review, methods involved in plant stress memory, cross-stress tolerance, and seed priming have become effective and favorable techniques for environmentally friendly and sustainable agriculture Fig. . CurrentDB and XL conceived the manuscript. XL and WQ wrote the manuscript. DB, XL and WQ revised the article.