Mitophagy is triggered by cues that signal the mitochondrial damage such as disturbances in mitochondrial fission-fusion dynamics, mitochondrial membrane depolarisation, enhanced ROS production, mtDNA damage as well as developmental cues such as erythrocyte maturation, removal of paternal mitochondria, cardiomyocyte maturation and somatic cell reprogramming. As research on the mechanistic aspects of this complex pathway is progressing, emerging roles of new players such as the NIPSNAP proteins, Miro proteins and ER-Mitochondria contact sites (ERMES) are being explored. Although diverse aspects of this pathway are being investigated in depth, several outstanding questions such as distinct molecular players of basal mitophagy, selective dominance of a particular mitophagy adapter protein over the other in a given physiological condition, molecular mechanism of how specific disease mutations affect this pathway remain to be addressed. In this review, we aim to give an overview with special emphasis on molecular and signalling pathways of mitophagy and its dysregulation in neurodegenerative disorders.Macroautophagy (henceforth autophagy) an evolutionary conserved intracellular pathway, involves lysosomal degradation of damaged and superfluous cytosolic contents to maintain cellular homeostasis. While autophagy was initially perceived as a bulk degradation process, a surfeit of studies in the last 2 decades has revealed that it can also be selective in choosing intracellular constituents for degradation. In addition to the core autophagy machinery, these selective autophagy pathways comprise of distinct molecular players that are involved in the capture of specific cargoes. The diverse organelles that are degraded by selective autophagy pathways are endoplasmic reticulum (ERphagy), lysosomes (lysophagy), mitochondria (mitophagy), Golgi apparatus (Golgiphagy), peroxisomes (pexophagy) and nucleus (nucleophagy). Among these, the main focus of this review is on the selective autophagic pathway involved in mitochondrial turnover called mitophagy. The mitophagy pathway encompasses diverse mechanisms involving a complex interplay of a multitude of proteins that confers the selective recognition of damaged mitochondria and their targeting to degradation  Through extensive research in the past 2 decades, while intricate molecular details of this pathway have been unravelled, several unanswered questions particularly about the capture and delivery of damaged mitochondria to lysosomes remain to be elucidated. In this review, we are trying to focus on two major aspects regarding mitophagy pathway, firstly the mechanisms through which lesser explored molecular players aid in the completion of this pathway, and secondly, the molecular details of how dysregulation in this pathway contribute to pathological conditions such as neurodegenerative disorders.Mitochondria are highly dynamic intracellular organelles that maintain cellular homeostasis by coordinating a myriad of functions such as ATP generation, regulation of cell death, maintenance of calcium homeostasis, lipid and carbohydrate metabolism, and intracellular signalling . As thesThe morphological dynamics of the mitochondria are maintained by interconnected processes that control the fission-fusion dynamics enabling the organelle to respond to a plethora of cellular stresses by rapid changes in their structure and function. Anatomically, mitochondria form tubular structures that is divided into outer mitochondrial membrane (OMM), inner mitochondrial membrane (IMM), and intermembrane space . Each ofCaenorhabditis elegans model system suggest its role in the clearance of damaged mitochondria on treatment with drugs that affect the function of mitochondria . DRP1 present in the cytosol is recruited to the membrane of the mitochondria, by the receptors present on the outer membrane surface where it self-oligomerises into complexes that enhance its hydrolytic activity . DRP1 aschondria . It was chondria . This stThe fusion machinery is regulated by mitofusin proteins (MFN1 and MFN2) on the outer mitochondrial membrane and they also belong to a family of transmembrane GTPases. MFN1, owing to its higher GTPase activity regulates mitochondrial fusion and docking process much more efficiently than MFN2 . MFN2 hain vitro GTPase assay (m-chlorophenylhydrazone (CCCP) trigger actin filaments to rapidly associate with mitochondrial fragments in an Actin Related Protein 2/3 (Arp2/3) dependent manner (Filamentous actin (F-actin) plays a critical role in maintaining the morphological homeostasis of the organelle by aiding efficient docking of DRP1 protein onto the mitochondrial membrane at the time of fission . DRP1 prse assay  suggestit manner . Anothert manner . Also, it manner . These rt manner . Actin ct manner . Additiot manner .via its ubiquitin binding ability. This recruitment enhances the assembly of actin cages around the damaged mitochondria resulting in their “bite-sized” fragments. While these fragments are unable to fuse back to the neighbouring healthy mitochondria, they promote the assembly of the downstream autophagic machinery leading to its engulfment  proteins , a unique unconventional myosin, has binding sites for ubiquitin and different autophagy adaptor proteins . It is tgulfment . MYO6 thgulfment . Non-musgulfment  increasigulfment . NMIIB igulfment . Inhibitgulfment . Anotherproteins  or direcproteins . Loss orproteins .Saccharomyces pombe, depolymerisation of the microtubules resulted in enhanced fragmentation of the mitochondria  and its absence resulted in the fragmentation of the mitochondria preceded by the dissociation of the organelle from the track. This study also shows that the physical association of the microtubular track with the mitochondria impedes its scission by inhibiting the assembly of the fission-related protein Dnm1  onto the OMM (In vitro studies suggest a role of kinesin family member 1B (KIF1B) protein in mitochondrial transportation  in microtubules impact mitochondrial trafficking, positioning, and functionality inside a cell. A major chunk of the mitochondrial motility is coordinated by the microtubular tracks and hence any disturbances in its assembly are manifested as perturbations in fission-fusion dynamics . In Saccchondria . On the n cycles . The pron cycles . In fiss the OMM . Ubiquit the OMM . Mitocho the OMM . Release the OMM . Dynein  the OMM . Similar the OMM . Defects the OMM . The ret the OMM . Kinesin the OMM . In vitrortation  and kineortation . Trafficchondria . While Tchondria , TRAK1 bchondria . Over thchondria . Miro1/2chondria . Miro-mechondria .Mitophagy is triggered with the binding of PRKN to the OMM and subsequent phosphorylation by PINK1 leading to its enhanced activation, which in turn results in ubiquitination of mitochondrial proteins on OMM by PRKN. PINK1 has also been reported to phosphorylate Miro1/2 proteins which promotes PRKN-mediated ubiquitination of the Miro complex followed by its proteasomal degradation . PhosphoA recently reported process known as “Mitocytosis” characterised migrasome mediated removal of the damaged mitochondria. Authors have observed accumulation of damaged mitochondria inside migrasomes on inducing mitochondrial stress. According to the study, damaged mitochondria restricted their binding to dynein, hence inhibiting their retrograde movement and enhancing its peripheral localisation. Trafficking of damaged mitochondria towards the plasma membrane by kinesin family member 1B (KIF5B) and subsequent anchorage on the plasma membrane by MYO19 motor protein along with the fission protein complex orchestrated the efficient loading of damaged mitochondria into the migrasomes. As the cell underwent migration, migrasomes were shed off from the cell, thus also clearing the damaged mitochondria that hitchhiked in it .Mitophagy is a complex process wherein several protein complexes are recruited onto the damaged mitochondrion in a hierarchical manner targeting it for lysosomal degradation mediated by the autophagic machinery. Different mitochondrial stress signals such as hypoxia, membrane depolarisation, ROS accumulation, mtDNA damage, activate different pathways that flag the damaged mitochondria, eventually resulting in their elimination through autophagy pathway. Activation of these diverse pathways occurs as a result of PINK1-Parkin dependent ubiquitination of mitochondrial membrane proteins, ubiquitination independent receptor mediated pathways , or Syntvia their LC3 interacting regions (LIR motifs), bringing autophagic machinery to the flagged site. Mitophagy receptors like Atg32, BNIP3, BCL2L13, NIX, FKBP8, FUNDC1, cardiolipin , remain inactive under steady-state levels and are activated by different PTMs only on specific cues that trigger the mitochondrial damage. Mitophagy in the budding yeast Saccharomyces cerevisiae is induced when yeast cells are grown till their stationary phase, or grown on non-fermentable carbon sources, or subjected to nitrogen starvation , Nuclear Dot Protein 52 kDa (NDP52), Neighbor of BRCA1 gene 1 (NBR1), and Optineurin (OPTN) by Richard Youle’s group revealed insights into the contribution of each adaptor in the mitophagy pathway. Through this study, it was reported that PINK1 mediated phosphorylation of Ubiquitin at Ser65 results in the recruitment of autophagic adaptors, OPTN and NDP52, independent of Parkin , TBC1 domain family member 15 (TBC1D15) and TBC1 domain family member 17 (TBC1D17) functioning downstream of Parkin activation, work in concert with Fis1 and LC3 to recruit Rab7 onto the mitochondrial surface shaping mitophagosome during mitophagy . It was Unlike the canonical autophagy pathway, wherein several of the fusion machinery components such as SNAREs, Rabs and HOPS complex proteins are involved in the fusion of mature autophagosomes with the lysosomes , the fusPhosphorylation and dephosphorylation of key mitochondrial and mitophagy-related proteins constitute one of the most important PTMs in the mitophagy pathway. The initial phosphorylation of Parkin by PINK1 at Ser65 in its ubiquitin domain triggers a feedforward loop recruiting more Parkin from the cytosol onto the mitochondrial surface  as ment. PhosphoDrosophila models of PD respectively, led to alleviation of PD-like phenotypes in these organisms , a progressive neurodegenerative disease with characteristic extracellular amyloid-beta plaques and intracellular Tau containing neurofibrillary tangles, is the leading cause of dementia worldwide. The clinical etiology of AD has been linked to both genetic and sporadic factors, leading to progressive degeneration of neurons, loss of memory, and cognitive decline of the affected individual . MitochoDysregulation of mitophagy in AD. Impairment in the mitophagy pathway is a phenotype that has been closely associated with AD. A study by Fang et al. showed that levels of mitophagy related proteins such as Bcl2L13, PINK1, BNIP3L/NIX, p-ULK1 (Ser555), p-TBK1, FUNDC1, AMBRA1, and MUL1 is downregulated in AD patient brains and iPSC-derived cortical neuronal cultures generated from AD patients, indicative of defective mitophagy pathway . Howeverin vitro and in vivo AD models caused dysregulation in the mitophagy pathway by enhancing the recruitment of Parkin and Ubiquitin-C-terminal hydrolase L1 (UCHL-1) to the mitochondria. The authors also described that partially suppressing mitophagy in these models rescued the truncation induced neurotoxicity and restored mitochondrial density in the synapses  is one of the common neurodegenerative diseases characterized by progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc), leading to classic Parkinsonian motor symptoms such as tremor, bradykinesia, rigid muscles, involuntary movements, and postural instability . Lewy boDrosophila and rat models. In PINK1 or Parkin loss of function model in Drosophila, mitophagy pathway was impaired leading to accumulation of swollen and damaged mitochondria with disintegrated cristae, dopaminergic degeneration, locomotion defects, severe flight muscle degeneration, male sterility, and reduction in life span , impaired mitophagy by decreasing the recruitment of OPTN to damaged mitochondria  gene is known to cause autosomal dominant form of PD and accounts for most of familial PD cases . The patitophagy . Two inditophagy . Additiochondria . This wachondria . The impchondria .et. al. identified mutations in Miro1 that are linked to PD pathogenesis , a lysosomal enzyme that converts glucosylceramide to ceramide and glucose, is one of the most common genetic risk factors for PD pathogenesis, contributing to 7–20% of all PD cases  (As lysosomes are the end points for the degradation of damaged mitochondria PD cases . InteresPD cases . A recen444P/WT) . Neurons444P/WT) . Initiat444P/WT) .Collectively, mitochondrial dysfunction and impairment in the mitophagy pathway can be considered as one of the strong candidates contributing to PD pathogenesis, owing to its predominant occurrence in various PD models. Therefore, therapeutic interventions targeting mitochondrial health and mitophagy together with the existing PD medications could be developed as potential treatment strategies for alleviating PD pathogenesis.Since the discovery of the link between PD and the key players of mitophagy pathway - PINK1 and Parkin had been made, extensive research has gone into elucidating the pathway and identifying the effect of PD associated mutations on PINK1-Parkin pathway. Mechanistic details of mitophagy impairment in different PD models carrying specific mutations are emerging recently. While mutations in key genes like PINK1, Parkin, LRRK2, GBA are explored in more detail, there still remains the need for investigations in greater depth to identify the mechanisms in less explored PD associated genes. As PINK1 and Parkin KO mice fail to exhibit PD related phenotypes, the existence of compensatory mitophagy pathways that are PINK1-Parkin independent requires further exploration. Additionally, detailed research is also required in exploring the status of mitophagy pathway in sporadic PD. Understanding the molecular mechanisms of mitophagy impairment in these models will help to devise efficient therapeutic strategies to alleviate the disease.Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by the abnormal expansion of CAG repeats that encodes the polyQ tract at the N- terminus of the huntingtin protein. In the normal population, CAG repeats of huntingtin protein ranges from 6 to 35 units, whereas the affected individuals show CAG repeats more than 35 units which progressively triggers the disease process leading to characteristic disease symptoms such as chorea, dystonia, impaired gait and posture, cognitive decline and psychiatric disorders . Mutant et.al. described the accumulation of damaged mitochondria in Drosophila expressing mHTT (Httex1p Q93), suggesting an impairment in its clearance  to the mitochondria, which promotes excessive mitophagy leading to neuronal death (mt-Keima mouse model expressing human Huntingtin’s transgene (HTT) also showed marked reduction in mitophagy . A studychondria . In addichondria . A recenchondria . mHTT fuchondria . Contraral death . HoweverIn the recent years, studies probing the mechanism of mitophagy impairment in multiple HD model systems have been emerging. Independent studies showed that mHTT affected multiple steps in the mitophagy pathway such as initiation, cargo recognition, interaction with autophagosomes and transport of autophagosomes, thereby impeding the clearance of damaged mitochondria. The major studies have predominantly focused on PINK1-Parkin mitophagy although the status of Parkin independent mitophagy pathways in HD remains unexplored. Also, comprehensive mechanistic studies probing the effect of varying number of CAG repeats on mitophagy impairment is lacking. These detailed studies, if performed, can provide insights into the degree of mitophagy impairment caused due to the varying number of CAG repeats - an information which can then be used to develop effective and personalized therapeutic strategies to ameliorate the HD pathology.Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of motor neurons that arise from both brain and spinal cord, thereby causing muscle weakness, muscular atrophy, dysarthria and paralysis in affected individuals . ApproxiG93A mutation, while degenerated mitochondria accumulated in neuromuscular junctions (NMJs), a concomitant increase in the number of mitophagosomes was not observed, suggesting compromised mitophagy. Furthermore, levels of mitophagy proteins like Parkin, PINK1, BNIP3 and p62 was also reduced in SOD1G93A mice  of FIS1 (residues 101–152), an OMM protein a. mito-Rosella: A mitophagy reporter protein with pH-insensitive RFP variant, DsRed.T3 and a pH-sensitive GFP variant, pHluorin, tandemly tagged to MTS of citrate synthase at its N-terminus b. mito-mRFP-EGFP: A pH-sensitive reporter protein constructed by inserting MTS sequences of human cytochrome C oxidase subunit VIII, N-terminally in frame with mRFP-EGFP c. All the above-mentioned reporter proteins share similar principle for monitoring mitophagy flux. Upon induction of mitophagy, mitochondria expressing the tandem reporters are targeted to lysosomes, where the pH sensitive  fluorophores get quenched and pH insensitive  fluorophores show stable expression. This differential quenching property of these tandem mitophagy reporters help to visualize and quantitate the mitochondria targeted to lysosomes as a read out of mitophagy flux.1) Tandemly tagged pH sensitive reporters to analyze mitophagy flux. mt-mKeima: A pH sensitive mitophagy reporter protein developed by tagging tandem repeats of COX VIII MTS to mKeima. Keima is pH sensitive protein exhibiting dual excitation spectra and is stable at lysosomal pH. Upon mitophagy induction, the mitochondria expressing mt-mKeima targeted to lysosomes show an excitation peak at 550 nm whereas the mitochondria in cytosol shows an excitation peak at 438 nm. The dual excitation of the reporter helps to distinguish the mitochondria in lysosomes and cytosol, and hence can be used as a readout for mitophagy 2. MitoTimer: A fluorescent reporter protein constructed by tagging MTS of COX VIIIA to DsRed1-E5 (Timer). The fluorescence of DsRed1-E5 shifts from green to red, as the protein matures 3. mito-SRAI : A novel fluorescent mitophagy reporter protein constructed by fusing a tandem repeat of cytochrome C oxidase subunit VIII MTS to N-terminus of TOLLES-YPet (SRAI) fusion construct. TOLLES  is an acid-fast CFP that is stable at lysosomal pH whereas YPet is a YFP variant that is both acid and protease sensitive and hence gets degraded in lysosomes. Due to the differential stability of these fluorescent proteins in lysosomes, mito-SRAI can be used as a mitophagy reporter that can distinguish and quantitate the mitochondria in cytosol and lysosomes 4. Mitophagy pathway encompasses autophagy-mediated degradation of damaged mitochondria. Scientific contributions spanning over 2 decades have unraveled several molecular players governing mitophagy, which include mitochondrial fission-fusion dynamics, different post-translational modifications that fine-tune the process and recruitment of autophagy-related adaptor proteins on OMM. Mitochondrial membrane dynamics closely orchestrate the mitophagy pathway by either facilitating fission mediated separation of the damaged part or re-fusing the impaired mitochondria to the healthy pool to dilute out the impact of dysfunction. Mitochondrial damage initiates a series of events resulting in the localization of several proteins onto OMM. The recruitment of these proteins is very context-dependent and is also regulated by different post-translational modifications. There are only a few pieces of evidence wherein late-stage regulators such as SNARE and Rab proteins of the mitophagy pathway have been identified. Therefore, more studies characterising the functional players involved in mitophagosome expansion and fusion is required. Although well explored, the mitophagy pathway is surrounded with several controversies. Mitochondrial fission has been believed to precede mitophagy, but recent studies on MiD51, DRP1 and STX17 suggest otherwise. There are contrasting reports with respect to mitochondrial fission-fusion dynamics and mitophagy, and therefore careful spatio-temporal characterisation of proteins is required. As mitophagy is a predominant quality control pathway for maintaining mitochondrial homeostasis, any dysfunction in this highly orchestrated pathway leads to a myriad of disease conditions. Neurodegeneration is one such disease, and recent studies investigating the molecular details of mitophagy dysregulation in these disorders shed light into the mechanistic aspects of the disease pathology. However, extensive studies probing the mechanism of mitophagy dysregulation in neurodegenerative disorders is required to address the contradictory results and for better understanding of this pathway. The research outcome from such studies will help provide new insights for developing novel therapeutic strategies to ameliorate these debilitating diseases.


Mosaic chromosomal alterations (mCAs) are structural alterations that include deletions, duplications, or copy-neutral loss of heterozygosity. mCAs are reported to be associated with survival, age, cancer, and cardiovascular disease. Previous studies of mCAs in large population-based cohorts  have demonstrated a steady increase of mCAs as people age. The distribution of mCAs in centenarians and their offspring is not well characterized. We applied MOsaic CHromosomal Alteration (MoChA) caller on 2298 genome-wide genotype samples of 1582 centenarians, 443 centenarians’ offspring, and 273 unrelated controls from the New England Centenarian Study (NECS). Integrating Log R ratio and B-allele frequency (BAF) intensities with genotype phase information, MoChA employs a Hidden Markov Model to detect mCA-induced deviations in allelic balance at heterozygous sites consistent with genotype phase in the DNA microarray data. We analyzed mCAs spanning over 100 k base pairs, with an estimated cell fraction less than 50%, within samples with genome-wide BAF phase concordance across phased heterozygous sites less than 0.51, and with LOD score of more than 10 for the model based on BAF and genotype phase. Our analysis showed that somatic mCAs increase with older age up to approximately 102 years, but the prevalence of the subjects with mCAs tend to decrease after that age, thus suggesting that accumulation of mCAs is less prevalent in long-lived individuals. We also used Poisson regression to show that centenarians and their offspring tend to accumulate less mCA  compared to the controls.


To systematically review the evidence on the treatments of sleep disturbancesin individuals with acquired brain injury.PubMed, Embase, Web of Science, and PsycINFO were searched from inception toJanuary 2021.Eligibility criteria were (1) participants with mild to severe acquired braininjury from traumatic brain injury and stroke (⩾three months post-injury),(2) individuals aged 16 years and older, (3) participants with self-reportedsleep disturbances, (4) controlled group studies and single case studies, and (5) interventions aimed at treatment of sleepdisturbances. Two researchers independently identified relevant studies andassessed their study quality using the revised Cochrane assessment of biastool (RoB 2.0) and the risk-of-bias in N-of-1 trials (RoBiNT) scale.The search yielded 655 records; 11 studies met the inclusion criteria andwere included, with a total of 227 participants . Two studies includedpharmacological therapy, six studies examined the effects of cognitivebehavioral therapy and three studies investigated alternative interventionssuch as acupuncture.Although there was heterogeneity in the study quality of the includedstudies, their outcomes suggest that cognitive behavioral therapy isrecommended as treatment of choice for improving sleep in individuals withacquired brain injury, especially for patients with mild to severe traumaticbrain injury. Future research should examine the effects of cognitivebehavioral therapy in more high-quality randomized controlled designs. In the context of this review, acquired brain injury includes traumatic braininjury and stroke. It is estimated that fifty to seventy percent of individuals withtraumatic brain injury (50%) or stroke (67%) in the chronic phase of recovery sufferfrom sleep disturbances, which is much higher than the incidence in the generalpopulation.24 Up to a thirdof the persons with acquired brain injury experience insomnia, which is defined asan inability to sleep, particularly associated with problems of falling asleep andmaintaining sleep.57 Since manystudies did not diagnose sleep complaints as sleep disorders, this review focuses onsleep disturbances refering to sleep problems that occur at night and arecharacterized by the inability to initiate and maintain sleep. Several studies havereported a poorer sleep quality and a reduced sleep efficiency (ratio of time spentasleep compared with time spent in bed) due to more frequent awakenings atnight.810 Following acquired braininjury, sleep disturbances can exacerbate other injury-related symptoms (e.g.cognitive functioning and fatigue) and have a negative impact on recovery.12 The highprevalence of sleep disturbances, its negative outcomes and also the persistingnature of sleep disturbances stress the need for effective sleep interventions forindividuals with acquired brain injury.Sleep disturbances are commonly reported following acquired brain injury and have anegative impact on functioning. Although clear guidelines exist for treatment of sleep disturbances, littleresearch has been done on the effects of these treatments in people with acquiredbrain injury. As a result, sleep disturbances are often not part of standard carewithin the longer-term outpatient rehabilitation of patients with acquired braininjury.14Available treatment options for sleep disturbances in the general population could bedivided into pharmacological treatment, cognitive behavioral therapy (CBT) or other,alternative interventions (e.g. acupuncture). However, a general overview including both pharmacological andnon-pharmacological treatments for sleep disturbances following acquired braininjury is lacking. Our review aims to systematically review the evidence on thetreatments of sleep disturbances in individuals with acquired brain injury. Specificaims are to evaluate the effectiveness of pharmacological treatment, cognitivebehavioral therapy and other interventions in sleep disturbances and to discuss theimplications of the findings for clinical practice, based on the study quality ofthe included studies.Recently, a systematic review on non-pharmacological treatment for insomnia showedbeneficial effects of cognitive behavioral therapy in patients with acquired brain injury. to search, extract and evaluate the interventions. An electronic search wasconducted in the following international databases: Web of Science, PubMed, Embase,and PsycINFO with the latest search performed on January 11, 2021. The search wasnot limited by any restrictions regarding publication date or language. The searchstrategy was developed in collaboration with a clinical librarian and wasconstructed using the following themes: sleep disturbance, acquired brain injury andintervention. Identification of key search terms was followed, using synonyms of thesearch terms. Key search terms were combined using the Boolean “OR”; themes werecombined using the Boolean “AND”. The search strategy was adjusted accordingly tooptimize the specificity of the search. The search strategy of each database isincluded in Supplemental Appendix 1.A systematic review was performed to evaluate interventions for sleep disturbances inindividuals with acquired brain injury. The present systematic review followed thePreferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines (PRISMA)Studies were included based on the following criteria: (1) participants with mild tosevere acquired brain injury following a traumatic brain injury or a stroke (⩾threemonths post-injury); (2) participants aged 16 years and older; (3) participants withself-reported sleep disturbances; (4) (randomized) controlled group studies andsingle case experimental studies; and (5) interventions aimed at treatment of sleepdisturbances. Self-reported sleep disturbances encompass difficulties in initiatingand maintaining sleep. Single case studies are defined as experimental, controlledinterventions studied in a single case. Articles were excluded if they (1) werereview articles or meta-analyses and (2) gray literature.Two independent reviewers  determined eligibility criteria and systematicallyscreened the records by titles and abstracts with respect to inclusion and exclusioncriteria. Of the selected articles, full-texts were reviewed to verify that they metthe criteria. Lack of agreement between the reviewers on this was settled throughdiscussion.Data extraction was performed by two independent researchers . A dataextraction table was created, summarizing the most relevant data. The followinginformation was extracted for each intervention study: authors, year of publication,study type, study design, method, sample , intervention, primary outcome measure, findings, maintenance ofeffects, and study quality. A third researcher was consulted when consensus was notreached.Methodological quality of the included studies, including risk of bias, was assessedby two independent reviewers , using guidelines specified for each researchdesign. Randomized group studies were evaluated using the revised Cochraneassessment of bias tool (ROB 2.0 tool). The risk-of-bias in N-of-1 trials (RoBiNT)scale was used for the assessment of single case studies. The percentage ofagreement between reviewers was 87% in terms of rating the methodological quality.Consensus with scoring was reached through discussion. Any lack of consensus wasdiscussed with a third researcher.The electronic search (11th January 2021) yielded a total of 655 records . A total of 521 recordsremained after removal of duplicates. Then, the records were screened by title andabstract. Forty full-text records remained and were examined in detail. A total of11 studies met the inclusion criteria and were included .The 11 studies are summarized in 1724 and two studies examinedstroke patients.26 The included studies involved 227 participants . One of theincluded studies recruited only participants with mild traumatic brain injury. All studies included adults with mean ages ranging from 27 to 60 years.The severity of traumatic brain injury was not reported in four of the groupstudies.26A total of nine studies examined individuals with traumatic braininjury,26 Three types of objectivesleep assessments were used to gauge the effectiveness of the treatments forsleep disturbances. Actigraphy, a reliable and objective sleep measure, was themost common assessment of sleep quality.23 Actigraphy is acontinuous measurement of sleep rhythms and wake periods via a recording deviceon the wrist.Out of the 11 studies, 10 used subjective self-report sleep measurements as aprimary outcome, including questionnaires on sleep quality such as thePittsburgh Sleep Quality Index and questionnaires on the insomnia severity asmeasures by the Insomnia Severity Index. The most common was the self-reportedsleep diary with sleep quality as the primary outcome. The overall risk of bias is included in  and one study obtained a low risk of bias. The risk of bias in the studies aimed at behavioral treatment was ratedwith “some concerns” for all three studies.25 There was only a risk ofbias related to the measurement of outcome because patients were aware of thetreatment received. It is possible that this knowledge could have influencedtheir self-report on sleep quality following the intervention. However, thetreatment as usual groups were also receiving active treatments in whichpatients also worked on their personal goals which diminishes this risk of bias.This diminished the risk of bias. Evaluating the study quality of thealternative interventions, two studies obtained a low risk of bias21 and onestudy was rated with “some concerns”.The Cochrane assessment of bias tool (RoB 2.0) was used to assess the quality ofthe included randomized controlled trials. The overall risk of bias is included in 26 and one study was classified as “substantial risk of bias”. A major limitation included inadequate sampling of the targetbehavior.To rate the internal and external validity of the included single case studies,the Risk of Bias in N-of-1 trials (RoBiNT) was used.30 Pharmacological treatmentincluded melatonin and amitriptyline. Melatonin intake (2 mg daily), which couldbe also considered as supplement, approximately two hours prior to bedtime, hada positive effect on sleep quality, but not on sleep onset latency in patientswith traumatic brain injury. No follow-up assessment was conducted. This study obtained a low risk ofbias with the use of a randomized controlled crossover design as a majorstrength. Kemp et al. compared the effect of melatonin (5 mg) with amitriptyline (25 mg) onsleep disturbances in individuals with mild to severe traumatic brain injury.Either melatonin or amitriptyline intake did not show improvements on sleepquality, sleep duration, sleep latency, or daytime alertness compared tobaseline. No follow-up measurement was conducted. This study was rated with“some concerns”. There was a risk of bias in selection of the reported result asthere was no information on a statistical analysis protocol or trialregister.Two studies examined pharmacological treatment to improve sleep quality inpatients with acquired brain injury.26 reported significantimprovements in self-reported sleep quality. Two pilot randomized controlledtrials examined the effect of cognitive behavioral therapy compared to treatmentas usual on sleep quality in 15 stroke patients and in 24 individuals with mild to severe traumatic brain injury. In the first study, the cognitive behavioral therapy interventionconsisted of eight sessions and addressed both fatigue and sleep in oneintervention. Sleep interventions included stimulus control, bedtimerestriction, hypersomnia management, relaxation, and cognitive restructuring.Sleep quality significantly improved in the cognitive behavioral therapy groupcompared to the treatment as usual group and the results maintained atfour-month follow-up. Within the cognitive behavioral therapy group, four out ofeight patients showed a clinically and significant improvement on the PittsburghSleep Quality Index, which measures sleep quality. The second study foundsignificant improvements in sleep quality in the cognitive behavioral group ascompared to treatment as usual. Cognitive behavioral therapy included six modules: psychoeducation, sleephygiene, cognitive restructuring, sleep interventions , strategies for mental and physicalfatigue, and relapse prevention. Nguyen et al. conducted a follow-up measurementand results showed that effects were maintained at two-month follow-up. Bothpilot studies consist of a strong randomized controlled trial design, althoughconclusions are limited due to the small study sample. The study quality of bothpilot studies was rated with “some” concerns. The only concern was that patientswere aware of the treatment received and that this knowledge could haveinfluenced their self-report on sleep quality. Therefore, there was a risk ofbias in the measurement of outcome. Nevertheless, it is not very likely that theoutcome was influenced by knowledge of the intervention as participants will notknow the details and possible effects of the cognitive behavioral interventioncompared to treatment as usual.All six cognitive behavioral therapy studies Cognitive behavioral therapy consisted of psychoeducation, relaxationtraining, sleep restriction, cognitive therapy, information about theenvironment and sleep, and mindfulness meditation. The cognitive behavioraltherapy group showed significant improvements in self-reported sleep quality ascompared to an online education control group. Theadom et al. also used actigraphy, but they found no significant group differences onobjective measures of sleep. No follow-up measurement was conducted. Thecognitive behavioral intervention was administered online, which offers acheaper and more accessible option. This study was rated with “some concerns”. There was no appropriate analysis used to estimate the effect ofassignment to intervention. However, there was no potential for a substantialimpact on the result of the failure to analyze participants in the group towhich they were randomized.Another pilot randomized controlled trial examined the effect of cognitivebehavioral therapy on sleep quality in 24 individuals with mild or moderatetraumatic brain injury.25All three pilot randomized controlled studies showed that cognitive behavioraltherapy is an effective intervention for improvement of sleep quality inpatients with acquired brain injury.26 and polysomnography. Cognitive behavioral therapy for insomnia interventions consisted ofseven or eight sessions and included standard cognitive behavioral therapy forinsomnia techniques such as psychoeducation, sleep hygiene, stimulus control,sleep restriction, relaxation, and cognitive restructuring. Clinically andsignificant improvements were found in 14 out of 17 patients. Effects weremaintained at a follow-up assessment between two weeks and three months. Thestudies of Ouellet and Morin and Herron et al. obtained a very high risk of bias and one single casestudy was rated with a “substantial risk of bias”. One of the concerns was thattreatment adherence was not assessed in the single case studies.In addition, three single case studies with a total of 17 patients with acquiredbrain injury (12 following traumatic brain injury and 5 post-stroke) reportedpositive effects of cognitive behavioral therapy for insomnia on severalmeasures of sleep quality and sleep duration, using sleep diaryreportsThe cognitive behavioral interventions for treatment of sleep disturbancesconsisted of a combination of cognitive, behavioral, and educational components.Common and effective techniques were psychoeducation, cognitive restructuring,sleep hygiene, sleep interventions , strategies for mental and physicalfatigue, and relapse prevention. and in 60 veterans with mild traumatic brain injury. In the first study, participants in de acupuncture group receivedacupuncture at different points twice weekly for five weeks. Acupuncture wasinserted on points in Kidney 3, Heart 3, Bladder 60, Liver 3, Large intestine,Pericardium 7, Governor Vessel 20, ear points Shen Men, and Tranquilizer. Eachsession lasted 20 minutes. The treatment regime was known as “standard dose”acupuncture protocol. Participants in the control group used their prescribedsleep medication. The acupuncture group showed significant improvement in theperception of sleep quality, but not on actigraphy total sleep time, as comparedto controls. This study used a strong randomized controlled design and the risk ofbias was low. However, a limitation is the lack of an active control group andno follow-up assessment. The second study compared a 10-session acupuncturetreatment with a sham acupuncture treatment. Acupoints included standardized and individualized points. Dosage andtreatment regime is not reported. The sham acupuncture group received shamneedles without skin penetration. The acupuncture group showed largerimprovements in sleep quality and on actigraphy measures posttreatment, however,these results were not maintained at follow-up. The study quality was rated witha low risk of bias.There are also studies that investigated treatments for insomnia usingalternative approaches . Two controlled group studies examined the impact ofacupuncture on sleep in 24 patients with chronic traumatic brain injury This study is a randomized controlled trial with crossover design inwhich a three-day home-based warm footbath (41°C) intervention was compared tothree days of usual care. The risk of bias was low. Sleep onset latency andshorter wake time after sleep onset (WASO), as measured with actigraphy,improved following the warm footbath compared to usual care. Sleep efficiencyand total sleep time did not improve. Importantly, the positive effects of thewarm footbath intervention were very small: sleep onset latency was shortened by5.11 minutes. Furthermore, the effects diminish within days after patients stoptaking the warm footbath in the evening.Another alternative intervention comprised a home-based warm footbath aimed atimproving sleep quality in 23 patients with traumatic brain injury and insomnia.The present systematic review aimed to provide an overview of the research on thetreatments of sleep disturbances in individuals with acquired brain injury.Although it is clear that sleep disturbances following acquired brain injury arecommon and have a negative impact on daily functioning, the number ofwell-conducted studies regarding the treatment of sleep disturbances in acquiredbrain injury are limited.30 six studies examined theeffects of cognitive behavioral therapy26 and three studiesinvestigated alternative interventions21 such as acupuncture.Evidence for pharmacological therapy in patients with sleep disturbances due toacquired brain injury is scarce. Some positive effects of short-term melatoninwere found in one study, however long-term effects are unknown. Furthermore, one study comparedthe effect of amitriptyline with melatonin but found no improvement in sleepquality for either drug.To discuss the implications of the findings for clinical practice, we evaluatedthe results based on the study quality of the included studies. Studies wererated with a low risk of bias, with “some concerns,” or with a high risk ofbias. For the treatment of sleep disturbances following acquired brain injury,two studies included pharmacological therapy,26 Reliable change analysesshowed a clinically reliable improvement on sleep quality measures in 18 out of25 acquired brain injury patients (72%).26 It should be noted thatthe current cognitive behavioral studies were either pilot studies25 or singlecase experimental design studies26 Although cognitivebehavioral therapy has a potential in treating sleep disturbances followingacquired brain injury with effects lasting up to several months, future studiesare needed to examine the efficacy of cognitive behavioral therapy in wellperformed randomized controlled trial designs with larger samples and long-termfollow up assessments.Effects of cognitive behavioral therapy were positive in all six studies up to athree- to four-month follow-up assessment.21 however, these effects did not maintain at follow-up. Furthermore, Huang et al. recruited only participants with mild traumaticbrain injury. The results of this study should therefore be interpreted withcaution, as the findings cannot be generalized to the traumatic brain injurypopulation at large. A home-based warm footbath (41°C) shortened the sleep onsetlatency and resulted in a shorter wake time after sleep onset, but the effectswere very small and therefore not clinically relevant. Yet, it is a simple home-based intervention that, perhaps in combinationwith other interventions, could facilitate patients with acquired brain injuryand sleep problems to fall asleep in the evening.Other investigated interventions included acupuncture and a home-based warmfootbath. Two studies showed positive effects of acupuncture on self-reportedsleep quality,32 However, pharmacological agents can induce sedation,tolerance, and dependence. Since pharmacological interventions are still recommended for treatmentof sleep disturbances following acquired brain injury, regardless of theirside-effects, it is of paramount importance to evaluate and compare theeffectiveness of both pharmacological and non-pharmacological interventionsdirectly for treatment of sleep disturbances in acquired brain injurypatients.This review suggests that compared to pharmacological treatment, non-behavioralinterventions seem an effective treatment for sleep disturbances followingacquired brain injury. However, pharmacological agents are routinely prescribedby clinicians and especially administration of short-term medication is stillthe treatment of choice for sleep disturbances following traumatic brain injuryand stroke.35 Compared topharmacological treatments, cognitive behavioral therapy has the same beneficialeffects on short-term and better long-term effects than benzodiazepines orbenzodiazepine receptor agonists.37 Melatonin and acupunctureare not recommended due to low quality evidence and only short-termeffects.35 Although the evidence concerning treatment of insomniain the acquired brain injury population is not as well established, the resultsof this systematic review are in line with the clinical guidelines for sleepdisturbances in the general population.In the general population, cognitive behavioral therapy is recommended for thetreatment of sleep disturbances.Although cognitive behavioral therapy is recommended in the general populationfor treatment of sleep disturbances, there appears to be circumstances wherecognitive behavioral therapy is not feasible . Then, pharmacological therapies,acupuncture, or perhaps a simple home-based warm footbath may be moreappropriate alternatives. However, future trials should further investigate theefficacy of these interventions and especially in individuals with severe braininjuries to draw stronger conclusions.2128 By focusing on sleepdisturbances including problems with sleep onset and maintenance, we reached abroader group of patients. Individuals with sleep disturbances experiencedifficulties in everyday functioning and should be able to receive treatment,without meeting all diagnostic criteria for a formal sleep disorder.To our knowledge, this is the first systematic review providing a generaloverview including both pharmacological and non-pharmacological treatments forsleep disturbances following acquired brain injury. A strength is that weappraised the quality of the studies using the revised Cochrane assessment ofbias tool, a valid tool for rating the risk of bias. Taking study quality intoaccount, strengthens the value of clinical recommendations made. Moreover, thissystematic review focused on treatments for sleep disturbances followingacquired brain injury. Most studies require merely self-reported sleepdisturbances as an inclusion criterium and did not diagnose a sleep disorderaccording to international standards.39 Second, in order to maintain a certain qualityconcerning the study designs, this review only included controlled group andsingle case experimental studies, resulting in a relatively small number ofincluded studies. Despite the selection on designs, the quality of the studieswas mixed. As a result of the limited quantity and quality of the studies, ameta-analysis could not be performed. Because of the limited number of studiesand the variation in quality, the conclusions of this review may be consideredglobal. Third, in the context of this review, acquired brain injury includedtraumatic brain injury and stroke. Only two studies  focused on stroke patients.26 Nevertheless, cognitivebehavioral therapy has a promising potential in treating sleep disturbancesfollowing acquired brain injury. However, other interventions included onlyparticipants with traumatic brain injury which means that findings of theseinterventions are limited to the traumatic brain injury population. Finally, itshould be noted that only two studies examined pharmacological treatment toimprove sleep quality. In fact, none of the pharmacological studies investigatedthe effect of benzodiazepines on sleep quality. Clinical guidelines, however,still recommend benzodiazepines for treatment of sleep disturbances. One study examined the effect of benzodiazepines in persons with acquiredbrain injury from an inpatient rehabilitation ward. However, the time since injury of the included participants is unknownand therefore outside the scope of this review. Shan and Ashworth compared the effect of lorazepam (0.5–1.0 mg daily) with zopiclone(3.75–7.5 mg daily) on sleep duration in patients with acquired brain injury andinsomnia. Results showed no difference between the two interventions. This studywas rated with a high risk of bias as it did not have a placebo arm, did not usevalidated measures of sleep quality and did not include baseline measures. Amajor limitation included a high risk of bias in the outcome measurement, whichconsisted of recording the total sleep time as measured by the nursing staff.Based on the study design, no conclusions could be drawn regarding the effect oflorazepam or zopiclone on sleep quality in patients with acquired brain injury.The limited number of studies examining pharmacological treatment of sleepdisturbances following acquired brain injury, preclude us from drawingconfirmative conclusions.However, the present review also has a number of limitations. First, theobjective of this systematic review was to provide an overview including bothpharmacological and non-pharmacological interventions for treatment of sleepdisturbances following acquired brain injury (three months post-injury). As aconsequence, this review excluded studies with individuals in the (sub)acutephase of acquired brain injury (<three months post-injury). We are thereforeunable to determine the extent to which the findings generalize to samples ofindividuals with sleep problems and acquired brain injury in the acute phase ofinjury since the type of sleep disturbances can vary considerably per phase ofinjury.Although there was heterogeneity in the study quality of the included studies, theiroutcomes suggest that cognitive behavioral therapy is recommended as treatment ofchoice for sleep disturbances following acquired brain injury, with positiveshort-and long-term effects on sleep duration and sleep quality. Since otherinterventions included only participants with traumatic brain injury, findings ofthese interventions are therefore limited to the traumatic brain injury population.A cognitive behavioral intervention of eight sessions including standard techniquessuch as psychoeducation, sleep hygiene, cognitive restructuring, sleep interventions and relapseprevention, is recommended for individuals with mild to severe traumatic braininjury and stroke in the chronic phase of recovery (range 25–48 months post-injury).Furthermore, an online form of cognitive behavioral therapy seems to be an effectivemethod for patients with traumatic brain injury, which is increasingly available forpatients and a cost-effective intervention.With respect to future research in this area, more research of higher quality shouldfurther examine long-term effects of (cognitive) behavioral sleep therapies as wellas their specific predictive variables of treatment success.Cognitive behavioral therapy is recommended for treatment of sleepdisturbances in patients with acquired brain injurySix to eight sessions of cognitive behavioral therapy in patients withmild to severe traumatic brain injury improves sleep qualityClick here for additional data file.Supplemental material, sj-pdf-1-cre-10.1177_02692155211014827 for Treatments forsleep disturbances in individuals with acquired brain injury: A systematicreview by Louise Pilon, Nikita Frankenmolen and Dirk Bertens in ClinicalRehabilitation


In situ tumor ablation techniques, like radiotherapy, cryo- and heat-based thermal ablation are successfully applied in oncology for local destruction of tumor masses. Although diverse in technology and mechanism of inducing cell death, ablative techniques share one key feature: they generate tumor debris which remains in situ. This tumor debris functions as an unbiased source of tumor antigens available to the immune system and has led to the concept of in situ cancer vaccination. Most studies, however, report generally modest tumor-directed immune responses following local tumor ablation as stand-alone treatment. Tumors have evolved mechanisms to create an immunosuppressive tumor microenvironment (TME), parts of which may admix with the antigen depot. Provision of immune stimuli, as well as approaches that counteract the immunosuppressive TME, have shown to be key to boost ablation-induced anti-tumor immunity. Recent advances in protein engineering have yielded novel multifunctional antibody formats. These multifunctional antibodies can provide a combination of distinct effector functions or allow for delivery of immunomodulators specifically to the relevant locations, thereby mitigating potential toxic side effects. This review provides an update on immune activation strategies that have been tested to act in concert with tumor debris to achieve in situ cancer vaccination. We further provide a rationale for multifunctional antibody formats to be applied together with in situ ablation to boost anti-tumor immunity for local and systemic tumor control. Conventional vaccines comprise of antigen(s) often supplemented with immune adjuvants to support the induction of an effective immune response. Besides, adjuvants can function as a slow release system, ensuring prolonged and continuous presentation and stimulation of the immune system . The appin situ availability of ablated tumor material  following ablation  against inhibitory immune checkpoints, inhibition of immunosuppressive cells like regulatory T cells (Treg) or myeloid derived suppressor cells (MDSC) or by scavenging anti-inflammatory cytokines, such as transforming growth factor beta (TGFβ).Examples of immune activation strategies that are mostly applied together with tumor ablation include stimulation with pattern recognition receptor (PRR) agonists, adjuvants and agonistic antibodies. However, tumors have evolved mechanisms to create an immunosuppressive tumor microenvironment (TME), parts of which may admix with the antigen depot upon ablation. Development of a successful in situ tumor ablation techniques.Delivery of these immunomodulators to the relevant locations, i.e. the tumor and tumor dLNs, is often essential for enhancing anti-tumor specific immune responses following ablation. Targeting also mitigates potential toxic side-effects. Antibodies can exhibit tumor targeting abilities, either through their specificity for tumor antigens or ablation-associated factors, such as extracellular DNA and histones. Recent advances in antibody engineering enabled the creation of novel antibody formats with multiple functions, such as bispecific antibodies and protein-linked antibodies . Multifuin situ tumor ablation. Furthermore, we postulate new combination strategies involving multifunctional antibody formats to be applied together with in situ ablation to boost the anti-tumor immunity for local and systemic tumor control.Here, we review immune activation strategies and approaches that counteract the immunosuppressive TME that have been combined with via different approaches, e.g. ionizing radiation or extreme temperatures. Radiotherapy (RT) has been a strong pillar in cancer therapy and the majority of cancer patients undergo RT at one point during treatment , microwave ablation (MWA), laser ablation and thermal high-intensity focused ultrasound (HIFU) employ different sources of energy to heat the target region , 11. Celvia drainage or absorbed as part of the physiologic healing response  and cytokines  are important for initiation of antigen-specific T cells. Thus, proper DC maturation is essential for efficient immune response induction. The ability to load and mature DCs directly in situ by tumor ablation plus immune activation is thus an appealing strategy to develop a cancer vaccine.DCs, the most potent APCs of the immune system, have the unique ability to initiate and direct immune responses. DCs in the vicinity of or recruited to the local ablation site acquire and process tumor antigens, and subsequently present them to naïve T cells . Alternain situ tumor ablation.Adjuvants can boost the magnitude and duration of the adaptive immune response. One of the ways through which adjuvants act is by serving as, or inducing, DAMPs and/or pathogen-associated molecular patterns (PAMPs) that trigger PRRs on immune cells resulting in their activation. Alternatively, adjuvants can function as slow release system. Although numerous different adjuvants exist, we will focus on nucleic acid-sensing PRR agonists, as well as the potential of saponin-based adjuvants (SBAs) applied in combination with in vivo as evidenced by their capacity to upregulate co-stimulatory molecules and enhanced production of pro-inflammatory cytokines needed for DC-mediated T cell priming. The nucleic acid-sensing TLRs include TLR9, TLR3 and TLR7/8. CpG oligodeoxynucleotides (CpG) are short unmethylated single stranded-synthetic DNA molecules, which were one of the first adjuvants to be combined with in situ tumor ablation  is a ligand for TLR3 and has shown promising preclinical results in combination with a minimal 9 Gy single dose RT. The combination treatment greatly reduced tumor growth at primary and abscopal sites and enhanced survival in different mouse models –33. The TLR7/8 agonists gained fame through Imiquimod, an adjuvant formulation topically applied in the treatment of skin cancers . TopicalDNA normally resides in the nucleus and mitochondria; hence, its presence in the cytoplasm serves as a danger signal. This aberrant localization of DNA is sensed by the DNA binding enzyme cyclic GMP-AMP synthase (cGAS). Upon recognition, cGAS dimerizes and stimulates the production of cyclic-GMP-AMP (cGAMP) which can directly bind stimulator of interferon genes (STING) resulting in type I interferon (IFN) production . Cytopla+ T cells were observed  receptor family, such as CD40/CD40 ligand (CD40L) and CD27/CD70. CD40 engagement on DCs by CD40L expressed by CD4+ T helper cells or agonistic CD40 mAbs trigger DC activation to provide signals for the licensing and expansion of CD8+ cytotoxic T cells . Ligation of these receptors delivers co-stimulatory signals necessary for full T cell activation –65. RT i therapy –68. CD13by PD-L1 . Combiniby PD-L1 , 70, 71.To date, all agonistic antibodies investigated have shown promising results in combination with RT and the addition of adjuvants might further improve their function  fused to an Fc-tail clinical development and not yet applied in context with tumor ablation. We will here review antibody formats that could be beneficial in combination with tumor ablation to create an in situ cancer vaccine  presentation is of importance for an ion site , 125. Inion site . GM-CSF ion site . Severaleduction –130 and eduction , mediatehigh B78 melanoma cells. This response of RT and hu14.18-IL2 in melanoma could be augmented by addition of anti-CTLA-4 mAb antigen loading of DCs without prior knowledge of tumor antigens or epitopes as in conventional DC vaccination. The induction of an efficient immune responses following ablation, however, requires addition of immune stimuli to eradicate local tumors and metastases and to provide long-term protection. Numerous immune activating strategies have shown to be suitable to act in concert with ablation generated tumor debris to achieve in situ cancer vaccination. More detailed knowledge about how effective immune activation strategies can work in concert with tumor ablation, such as timing and dose, is required to guide rationale ablation combination strategies. Although in situ ablation plus immune activating strategies ensure that the immune system is well instructed and initiated, the immunosuppressive environment that immune cells encounter upon arrival at the TME is still a potential bottleneck. Therefore, additional removal of inhibitory influences provides the possibility to further boost anti-tumor immune responses and enhance in situ ablation efficacy.in situ cancer vaccine.Multifunctional antibodies stimulating immune activation as well as counteracting immunosuppression can simultaneously affect multiple key processes in the anti-tumor immune response. They hold great promise for targeted cancer treatment with limited systemic toxicities and deserve further exploration as potential strategy to achieve a successful RB, FS and GA wrote the manuscript. JF, MV and LC reviewed the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the foundation ‘Villa Joep’, a Radboud Institute for Molecular Life Sciences (RIMLS) PhD grant awarded to GA and a grant from the Dutch Cancer Foundation (KWF) awarded to GA (KUN2015-7604).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.


Currently, more potent next generation cereblon E3 ligase modulators (CELMoDs®) - iberdomide (IBER) and CC-92480 are in clinical development. With an expanding number of active agents/therapeutic modalities and a myriad of combinatorial possibilities, physicians and drug developers share an opportunity and challenge to combine and sequence therapies to maximize long-term patient benefit. Understanding drug mechanisms and their application in combination settings as well as the unique disease biology considerations from newly diagnosed (NDMM), relapsed/refractory (RRMM), and maintenance settings will be vital to guide the development of future MM therapies centered on a backbone of IMiD or CELMoD agents. Key aspects of drug activity are critical to consider while evaluating potential combinations: direct antitumor effects, indirect antitumor cytotoxicity, immune surveillance, and adverse side effects. In addition, the treatment journey from NDMM to early and late MM relapses are connected to genomic and immune changes associated with disease progression and acquisition of resistance mechanisms. Based on the types of combinations used and the goals of therapy, insights into mechanisms of drug activity and resistance may inform treatment decisions for patients with MM. Here we focus on the evolving understanding of the molecular mechanisms of CRBN-binding drugs and how they can be differentiated and suggest a strategic framework to optimize efficacy and safety of combinations using these agents.Multiple Myeloma (MM) is an incurable malignancy with current treatment choices primarily comprising combination regimens implemented with a risk-adapted approach. Cereblon (CRBN)-targeting immunomodulatory agents (IMiDs CRBN) E3 ligase complex, and trigger recruitment, polyubiquitination and subsequent degradation of substrate proteins. A simple framework (d~1.0–1.5 uM) . –1.5 uM)  and CC-9. –1.5 uMIn vitro studies with LEN and POM demonstrated broad co-stimulatory effects in primary human T and NK cells, including induction of proliferation, cytokine secretion and cytotoxicity [IMiD and CELMoD agents have shared as well as unique substrates; their downstream cellular effects may be differentiated by the presence, abundance of, and relative preference for these key substrate proteins of functional consequence . Among ttoxicity –19. AddePreclinical models showed enhanced immune co-stimulatory activities of IBER when compared to LEN and POM, including increased IL-2 secretion and granzyme-B degranulation in stimulated peripheral blood mononuclear cells (PBMCs) and enhanced immune-mediated killing of MM cells , 20. FurBiomarker analyses of tumor and immune samples from clinical trials in RRMM settings demonstrate mechanistic differences among IMiD and CELMoD therapies. Loss of CRBN expression was initially suggested as a mechanism of resistance to LEN , 13. MutImmunomodulatory data from patients treated with IMiD therapies showed an increase in bone marrow T lymphocytes , 32. Mor+ cells, leading to maturation arrest of myeloid precursors, and subsequent neutropenia [+ cells following IMiD exposure was found to be Ikaros-mediated and the subsequent maturation arrest at the promyelocyte stage shown to be reversible [IMiD therapies have both overlapping and distinct toxicities. Neutropenia and thrombocytopenia are common serious adverse events (SAE) in patients treated with IMiD compounds. Previous studies with LEN plus dexamethasone (LENdex) showed 30–40% grade 3/4 neutropenia in RRMM patients , 39. In tropenia , 43. Theversible . In addiversible , 43.To date, with limited clinical experience grade 3/4 neutropenia rates with IBER plus dexamethasone (IBERdex) are ~30% [The foundational role of IMiD agents in the treatment of MM evolved from several attractive features of these drugs making them ideal partners for combination therapy. Initial studies with LENdex showing activity in the RRMM setting allowed for the addition of several agents to this backbone: carfilzomib, DARA, elotuzumab  –47. SimiHow might we propose a rational combination approach for the CELMoD therapies in view of cell autonomous , immunomodulatory  and adverse effects of these agents? The design and selection of the new CELMoD agents relative to previous generation IMiDs has ensured superior pro-apoptotic and anti-proliferative activities while concurrently enhancing immunomodulation through augmentation of the NK cell compartment and T cell proliferation/activation. Additionally, with these more potent agents, attention has also been directed towards controlling dose-limiting cytopenias, which are known safety signals associated with IMiD agents . CELMoD Rational combination approaches for CELMoDs and approved agents should be based on the overlapping mechanisms of each contributing therapeutic modality. For instance, PIs are known as immunosuppressive agents that exert anticancer activity by proapoptotic effects. An ideal CELMoD therapy may add to the pro-apoptotic activity while counterbalancing the immunosuppressive nature of the PI . In FiguLastly, the hallmark traits of the CELMoD agents may also provide guidance for settings and patient subpopulations in which clinical benefit may be derived. For instance, a CELMoD with enhanced antitumor activity but no appreciable increase in safety signal may be well positioned in NDMM patients or as maintenance treatment. Alternatively, patients in the RRMM setting that may display poor prognostic features may benefit from an agent with dialed-up apoptotic activity . ClinicaInterrogating the molecular mechanisms of action of IMiD/CELMoD agents over the past several years has brought to light common and unique properties of these agents in preclinical and clinical settings. Further understanding of two key areas about the properties of these agents is critical to drive their future development. First, better understanding of the molecular mechanistic features of CELMoD compounds will help differentiate them clinically from IMiD agents and from one another. The basic framework described here provides a roadmap for guidance. Analysis of biomarkers from clinical studies will complement this effort. Second, and more importantly, learning from clinical studies to form an integrated view of the desirable “sweet spot” profile  for comb


Glass-Fiber-Reinforced Polymer (GFRP) laminates are widely used in the automotive and marine industries such as auto bodies and boat hulls. Decreasing the weight and improving the reparability of GFRP parts will cut down material usage, fuel consumption and repair costs. This study shows a bio-inspired helicoidal stacking configuration that significantly improves the impact performance and fiber damage resistance of GFRP laminates. For similar impact performance in terms of perforation energy, the helicoidal GFRP laminate is 20% lighter than the conventional quasi-isotropic GFRP laminate. Upon impact, delaminations and matrix splits link-up and grow extensively throughout the helicoidal laminate. This effectively reduces fiber damage and improves impact performance. Because helicoidal GFRP laminates are resistant to fiber damage and composite healing agents can effectively repair non-fiber damage, embedding healing agents into helicoidal GFRP results in lightweight, inexpensive and healable laminates. Glass-Fiber-Reinforced Polymer (GFRP) laminates have been widely adopted in the automotive and marine industries due to their low cost and good mechanical performance ,2,3. SinMany researchers have attempted to strengthen and toughen GFRP parts by adding micro/nano-reinforcements ,6,7,8 orDrawing inspiration from nature, it is proposed that the mechanical performance of GFRP laminates can be improved through bio-inspired structural design. Many crustaceans’ exoskeletons are composed of layers of aligned chitin fibers in a similar fashion to manmade fiber-reinforced polymer laminates . The weaIn the helicoidal structure, the orientation of the fibers in one layer is offset at a slight angle with respect to the fibers in the layer below, forming a spiraling helicoidal configuration. Inspired by these findings, several researchers have conducted studies that mimic the helicoidal structure using CFRP laminates ,24,25,26Several studies ,24,25,26This study investigates the impact performance of helicoidal GFRP laminates together with conventional quasi-isotropic GFRP laminate as reference. Helicoidal GFRP laminates with fewer plies were fabricated to determine the percentage weight reduction achieved while maintaining similar impact performance as the reference laminate. A lightweight and healable helicoidal GFRP laminate was produced from investigating the damage patterns to understand the cause of failure.2 squares. They were then laid manually to form 61-ply quasi-isotropic and helicoidal laminates, as well as lighter 49-ply and 46-ply helicoidal configurations, as shown in Quasi-isotropic and helicoidal laminates were fabricated from unidirectional G10000/6510 Glass fiber-epoxy prepregs. The mechanical properties of the material are shown in The specimens were cured in an autoclave. The layups and curing cycle are shown in Out-of-plane impact tests were conducted to evaluate the energy required to perforate the specimens. As shown in Additional light weight helicoidal laminates that match the perforation energy of QI61 were fabricated and cured using the same process as described in Dispersing healing agents within plies is commonly adopted for composite healing ,31,32,33The healable specimens were then subjected to a low energy impact. After impact, the damaged specimens were healed in a hot-press. The specimen was firstly heated to 200 °C over 10 min to soften the healing agent before a pressure of 2 bar was applied. The specimen was then heated to 220 °C over 20 min to allow the Nylon6 healing agent to melt and flow into the cracks. The specimen was then cooled to room temperature over 15 min.As shown in As shown in Although spiraling matrix split was deduced through theoretical study and experimental CT-scan by Suksangpanya et al.  and Liu As shown in The reduction in fiber damage in helicoidal laminates is a consequence of the extensive sub-critical spiraling matrix split. The highly diffused combination of matrix splits and delaminations in the spiraling matrix split reduces stresses along the fiber direction which minimizes fiber damage ,36,37,38Perforation of GFRP laminates is the result of an impactor breaking through the fibers along its path. Since helicoidal GFRP laminates are more resistant to fiber damage and the impact energy is dissipated through fiber damage as well as the massive spiraling matrix split, this is the reason why helicoidal GFRP laminate excels in impact performance.Several studies on composite intrinsic healing technology have shown that damaged epoxy resin can be healed effectively with thermoplastic healing agents ,31,32,33HL49 specimens with embedded Nylon6 healing agent following the fabrication process described in The result shows that by adding a small amount of healing agent between each ply, the GFRP helicoidal laminates can achieve good healing after low energy impact.This study shows that by stacking GFRP laminate helicoidally, the weight of the laminates can be reduced by 20% while maintaining the same perforation energy as the conventional quasi-isotropic GFRP laminate under impact. Since a large quantity of GFRP laminates are used in the automotive and marine industry as auto body parts and boat hulls, and since the lighter helicoidal GFRP laminates can meet the same safety requirement as the conventional GFRP laminate, adopting helicoidal GFRP laminates is beneficial for the industry and the environment as it saves material and cuts down fuel consumption while not introducing additional costs.During impact, a highly diffused spiraling matrix split develops in helicoidal GFRP laminates, reducing fiber damage. In contrast, damage is highly localized in conventional GFRP quasi-isotropic laminates, leading to localized delaminations and extensive fiber damage. Helicoidal GFRP laminates are more resistant to perforation because of the better resistance to fiber damage and the development of diffused spiraling matrix splits. Helicoidal GFRP laminates thus show reduced fiber damage under low energy impact. As such, healable helicoidal GFRP laminate can be produced by simply adding thermoplastic healing agents between plies.


Plants are faced with various biotic and abiotic stresses during their life cycle. To withstand these stresses, plants have evolved adaptive strategies including the production of a wide array of primary and secondary metabolites. Some of these metabolites can have direct defensive effects, while others act as chemical cues attracting beneficial (micro)organisms for protection. Similar to aboveground plant tissues, plant roots also appear to have evolved “a cry for help” response upon exposure to stress, leading to the recruitment of beneficial microorganisms to help minimize the damage caused by the stress. Furthermore, emerging evidence indicates that microbial recruitment to the plant roots is, at least in part, mediated by quantitative and/or qualitative changes in root exudate composition. Both volatile and water-soluble compounds have been implicated as important signals for the recruitment and activation of beneficial root-associated microbes. Here we provide an overview of our current understanding of belowground chemical communication, particularly how stressed plants shape its protective root microbiome. These studies have revealed new exciting insights into the chemical diversity of plant metabolites. Plants synthesize more than 200,000 primary and secondary metabolites, including volatile and soluble compounds . While perpenes) ,7. For eractions ,9,10.In their natural environment, plants are often exposed to a variety of biotic and abiotic stresses such as drought, salinity, nutrient limitations, pests and pathogens. To withstand these different stresses, plants have developed sophisticated adaptive mechanisms including the production of bioactive secondary metabolites. Some of these metabolites can have direct defensive effects while others can warn neighboring plants to mount their own defenses or lead to the recruitment of beneficial (micro)organisms that minimize the intensity of plant stresses both above-and belowground. It is well-known that, in response to attacks by aboveground herbivores, plants have evolved a ”cry-for-help” strategy where they recruit beneficial organisms to help to overcome the imposed stresses . For exaRecent studies have indicated that plants roots have also evolved a “cry-for-help” strategy to recruit beneficial soil (micro)organisms to minimize the damage caused by these stresses ,16,17,18Increasing our fundamental knowledge of belowground chemical interactions can provide a basis for developing new strategies for the sustainable crop production. This review provides an overview of belowground, stress-induced chemical communication. We give a summary of the various soluble and volatile metabolites released by plants roots exposed to biotic and abiotic stresses and discuss their role in assembly of a plant protective microbiome and stress alleviation. Furthermore, we discuss the challenges, opportunities and future directions in this emerging research field.Lupinus albus L.) to soil P deprivation induces the production of carboxylate [Plants release a significant fraction of their photosynthetically fixed carbon belowground in the form of root exudates , consistboxylate ,28. Orgaboxylate ,30,31.Glycine max (soybean) and Zea mays (maize) secrete citrate with a strong metal chelating capability, thereby reducing the uptake of harmful Al3+ by the plants [Exudation of low molecular-weight organic acids (LMWOA) into the rhizosphere has also been reported as a survival strategy of plants to improve nutrient acquisition in metal-contaminated soils. For example, in acidic soils with high aluminum (Al) concentrations, roots of e plants . Furthere plants . In addie plants . These ce plants .Arabidopsis thaliana grown in iron (Fe)-deficient soils (due to high pH) produce the coumarin scopoletin that reduces Fe3+ to Fe2+, thereby enhancing Fe-bioavailability to plants upon alkaline stress [Arabidopsis mutants lacking 2-oxoglutarate-dependent dioxygenase Feruloyl-CoA 69-hydroxylase1 (F6′H1), an enzyme involved in scopoletin biosynthesis, was significantly impaired in growth on synthetic agar media supplemented with low Fe [Hordeum vulgare) infected with the soil-borne pathogenic fungus Fusarium graminearum [t-cinnamic acid [Ocimum basilicum) upon infection by the oomycete pathogen Pythium ultimum [Another class of compounds secreted by plants in response to different stresses are the phenolics. For instance, roots of e stress ,35. Furth low Fe . Increasminearum . Among tmic acid . Similar ultimum .Fusarium verticillioides and the belowground herbivore Diabrotica balteata [an2 mutant) were more sensitive to drought. These results suggest that the terpenoids, apart from their role in direct defense against pests and pathogens, are associated with drought tolerance in maize plants. In another study, the semi-volatile diterpene rhizathalene was produced by Arabidopsis roots exposed to the root-feeding insect (Bradysia) [Z)-γ-bisabolene have been reported in root exudates of Arabidopsis [S)-limonene appears to be involved in the direct defense against plant pathogenic fungus Magnaporthe oryzae [Increased accumulation of the non-volatile terpenoids zealexins and kauralexins was found for roots of maize following infection of soil-borne fungus balteata . Interesradysia) . Furtherbidopsis , both wibidopsis . Similare oryzae .Collectively, these findings indicate that the same compound classes can be released by roots of a particular plant species exposed to different stresses, suggesting a general role of some exudate constituents in mediating plant resistance against both biotic and abiotic stresses. The schematic overview on the direct role of root exudates on plant resistance against biotic and abiotic stresses can be seen in Arabidopsis leaves by Pseudomonas syringae pv tomato (Pst) attracted the beneficial bacterium Bacillus subtilis FB17 to the roots, that in turn triggered systemic resistance against subsequent infections by Pst [Arabidopsis leaves by the downey mildew pathogen Hyaloperonospora arabidopsidis resulted in the enrichment of specific bacterial genera in the rhizosphere, in particular Microbacterium sp., Stenotrophomonas sp., and Xanthomonas sp. [Triticum turgidum L. var durum) naturally infected by the crown-rot pathogen Fusarium graminearum enriched for Stenotrophomonas rhizophila (SR80) in the rhizosphere and root endosphere [Root-associated microorganisms are essential for plant growth and health. Past and present plant microbiome studies have indicated that root microbiota are not merely passengers, but instead, they improve plant immune functions  and enhas by Pst . Similaronas sp. . When aponas sp. . In a redosphere . Upon redosphere .Arabidopsis roots upon Pst infection [Pst infection enhanced the expression of Aluminum-Activated Malate Transporter1 (MLT1) which became the key regulator for the recruitment of Bacillus subtilis FB17 on Arabidopsis roots following foliar Pst infection [Fusarium oxysporum f.sp. cucumerinum increased tryptophan but reduced raffinose exudation; these changes enhanced root colonization by the beneficial bacterium Bacillus amyloliquefaciens SQR9 [Trichoderma harzianum [Pseudomonas putida KT2240 [Colletotrichum graminicola [Spodoptera frugiperda [Evidence is mounting that the recruitment of beneficial microbiota by plants under siege is, at least in part, driven by changes in the exudate profiles. These studies include the early work on malic acid secreted by nfection . This wanfection . In anotens SQR9 . Furtherarzianum . Benzoxaa KT2240 . In the minicola . In a reugiperda .Trifolium pratense) grown in Fe-deficient soil accumulated phenolic compounds, which stimulated a siderophore-secreting Pseudomonas sp. When tested in plants, the siderophores produced by the bacterium were effective in solubilizing Fe, thereby improving its uptake by clover plants in Fe-deficient soils [Arabidopsis roots are known to produce the coumarin scopoletin under Fe-deficiency [Limonum sinense secreted several organic acids including 2-methyl butyric acid and palmitic acid with positive effects on the growth and chemotaxis of Bacillus flexus KLBPM 491, a beneficial bacterium naturally found on L. sinense. Re-introduction of this strain into soil significantly promoted growth of L. sinense seedlings under salinity stress [While the concept of root exudate-mediated “cry-for-help” is shown for plants under biotic stresses, several studies suggest that this concept may also apply to plants exposed to abiotic stresses. For instance, roots of red clover  into the rhizosphere. It is estimated that VOCs account for approximately 1% of the total secondary metabolites released by roots . Plant VThuja occidentalis damaged by larvae of the black vine weevil (Otiorhynchus sulcatus) when given a choice in a Y-tube olfactometer filled with sand [Diabrotica virgifera virgifera emit the sesquiterpene (E)-β-caryophyllene which attracts entomopathogenic nematodes that infest and kill the root-feeding larvae [Similar to soluble compounds, belowground plant VOCs can serve as direct and indirect plant defenses. For example, VOCs emitted from glucosinolate or cyanogenic glycoside conversion (such as cyanides and isothiocyanates) may serve as direct plant defenses as they are toxic to a wide range of belowground herbivories and pathogens ,65. Planith sand . In anotg larvae .13C-labelled leaf litter accounted for fractions in microbial biomass (located separately from the decomposition site), suggesting that VOCs can be carbon sources for soil microbes [Root VOCs can act as both signaling molecules and nutrient sources for soil microbes , and henmicrobes . In anotmicrobes . This fiFusarium culmorum, the root of sand sedge plant (Carex arenaria) emitted a specific blend of VOCs -limonene oxide) which clearly differed from the healthy plants. Interestingly, this specific blend of VOCs was able to attract certain beneficial bacteria within a synthetic bacterial community from a distance of approximately 12 cm. Furthermore, these recruited bacteria were able to inhibit the growth of F. culmorum [Evidence is available that under a biotic stress situation, root VOCs are involved in the recruitment of beneficial bacteria into the rhizosphere. This phenomenon had been clearly shown in a recent study by Schulz-Bohm et al.  who founculmorum .Fusarium oxysporum alters the root VOCs profile. The infected plant roots emitted VOCs such as benzonitrile, benzothiazole, dimethyl trisulfide, formic acid and a terpene-like compound, which are well-known for their antifungal activities. Interestingly, the infected and healthy plant roots did not show significant difference in the attraction of bacteria with biocontrol properties. However, these results were obtained only based on few selected bacteria; therefore, follow-up studies should be performed using total microbial communities to reveal whether the attraction of bacteria is significantly different between healthy and infected plant roots. Together, these findings suggest a novel strategy by which stressed plants can recruit and activate from distance soil microbes into the rhizosphere facilitating their adaptation. However, the role and mechanisms of root VOCs in the assemble and function of rhizosphere microbiome remain largely unexplored.Recently, a unique olfactometer-choice assay was applied to assess the migration of selected beneficial bacteria towards the roots of healthy and infected tomato plants in a soil system . The stuVicia faba L.) plants with symbiotic arbuscular mycorrhiza fungi (AMF) reduced the release of sesquiterpenes [E)-caryophyllene and (E)-β-farnesene, and aphid attractiveness to VOCs was negatively associated with the proportion of sesquiterpenes in the sample. Hence, the AMF have a key bottom-up role in insect host location by increasing the attractiveness of aphids to plant VOCs. Recent study revealed that the plant-beneficial Pseudomonas putida induced the production of indole and β-caryophyllene VOCs in maize plants and triggered ISR against the maize anthracnose fungus Colletotrichum graminicola [Interestingly, both plant-beneficial and plant-pathogenic microbes can modify the plant VOCs profile. For example, the pretreatment of faba bean (terpenes . The AMFminicola .S. lycopersicum L.) inoculated with plant growth-promoting rhizobacterium Bacillus amyloliquefaciens [Furthermore, microbe induced plant VOCs can interfere with plant-to-plant communication. Recently, the effect of volatiles on microbial communities and neighboring plants was investigated in tomato plants (efaciens . The stuConsidering the importance of root-emitted VOCs (next to the water-soluble exudates) on the assembly of protective-microbiome under stress conditions, future studies should elucidate more on the specificity or generality of root-emitted VOCs under different stresses, and investigate whether such volatile emissions can mediate the recruitment of beneficial microbes and activation of their beneficial traits.New developments in metabolomics offer a great potential to gain new insights into the mechanisms underlying stress-induced belowground chemical interactions as it allows for the discovery of novel compounds or combinations of compounds that directly or indirectly alleviate (a) biotic stresses. In general, detection of plant metabolites is extremely challenging, as there is no single-instrument platform available to effectively measure the overall coverage of plant metabolites and standards for many root exudates are currently lacking. To date, mass spectrometry-based metabolomic techniques (such as LC-MS and GC-MS) are the most sensitive for simultaneous analysis of a large number of soluble and volatile compounds . EspeciaMost studies, so far, are focused on plant metabolites induced by individual stress under controlled conditions, but in nature, plants are typically subjected to a multiple stress factors at the same time. To date, little is known about plant metabolic responses to multiple stresses that occur either simultaneously or sequentially. Although several stresses have similarities , each stress can lead to specific metabolic responses. Furthermore, the chemical responses to stress can depend on stress severity and duration . For exaWhile responses to stresses are studied predominantly with young plants very little is learned about how mature plants perceive stresses and recruit or activate beneficial members of the root microbiome through chemical cues. Root exudates have been shown to change significantly during plant development and having an impact on microbiome composition . In the The majority of studies on stress-induced plant metabolites are based on experiments with domesticated plants and hence, we know very little about the responses of the wild relatives in natural ecosystems. Both in agricultural and natural ecosystems, plants are subject to multiple biotic and abiotic stresses. Even if they are resilient to a single stress, plants are often compromised to tolerate multiple stresses occurring at the same time. It is plausible that plants under multiple stresses produce different chemical cues, resulting in the attraction of different microbes compared to plants under single stress stimuli. Interactive effects of biotic and abiotic stress responses are reflected in the plant’s hormonal signaling networks. Crosstalk in this signaling network regulates secondary metabolite biosynthesis and the metabolites produced by the plant may serve as cues for soil microbes that may mitigate the adverse effects.Apparently plant roots have evolved a “cry-for-help” strategy whereby they recruit beneficial soil microorganisms that can help to overcome stresses . HoweverSerratia plymuthica upregulated their terpene synthase gene and produced sodorifen together with as yet structurally unknown terpenes as a response to terpenes produced by the fungal root pathogen Fusarium culmorum [Arabidopsis seedlings to the Serratia produced sodorifen induced expression of two plant defense-related genes PDF1.2 and PR1, coinciding with reduced infections by the pathogen [Furthermore, several metabolites  are commonly produced by both plant roots and plant associated organisms. The biotic and abiotic stresses such as drought, salinity, nutrient limitations, and pathogens are stress factors not only perceived by plants but also plant microbiome. Hence it is intriguing to know who is the first to sense the stress; is it the plant or plant associated microbes? Previous study revealed that the rhizosphere bacteria of the species culmorum . Furtherpathogen . Hence, Climate change is expected to lead to increased frequency and severity of drought and rainfall events in the near future. These extreme events will have a strong impact on the belowground chemical communication, as water is the major medium for moving molecules into the soil space. Under such condition, volatile compounds can play even more important role in belowground chemical interactions as they can easily diffuse in the gas phase which is not possible for soluble compounds. In addition to abiotic stresses, plants are constantly facing attacks by pests and pathogens which are also expected to increase due to climate change. Plant-associated microbes can alleviate both biotic and abiotic stresses through mechanisms as diverse as induction of plant resistance, direct antagonism of pathogens, increased nutrient availability, or modulation of plants’ hormonal balance. The rhizosphere microbiome thus is a reservoir of efficient helpers that plants can specifically recruit to help them cope with one or multiple stresses. Therefore, deciphering belowground chemical communication can provide a fundamental knowledge for developing the multiple-stress resistant crops in the future.