Androgen receptor (AR) is a key member of nuclear hormone receptors with the longest intrinsically disordered N-terminal domain (NTD) in its protein family. There are four mono-amino acid repeats  located within its NTD, of which two are polymorphic (polyQ1 and polyG). The length of both polymorphic repeats shows clinically important correlations with disease, especially with cancer and neurodegenerative diseases, as shorter and longer alleles exhibit significant differences in expression, activity and solubility. Importantly, AR has also been shown to undergo condensation in the nucleus by liquid-liquid phase separation, a process highly sensitive to protein solubility and concentration. Nonetheless, in prostate cancer cells, AR variants also partition into transcriptional condensates, which have been shown to alter the expression of target gene products. In this review, we summarize current knowledge on the link between AR repeat polymorphisms and cancer types, including mechanistic explanations and models comprising the relationship between condensate formation, polyQ1 length and transcriptional activity. Moreover, we outline the evolutionary paths of these recently evolved amino acid repeats across mammalian species, and discuss new research directions with potential breakthroughs and controversies in the literature. The protein family of nuclear hormone receptors (NHRs) includes several hormone-sensitive transcription factors (TFs), which were discovered and initially characterized as tissue-specific intracellular receptors whose functions are regulated by specific endocrine hormones , 2. SequMost NHRs have multiple different isoforms that are shorter than the canonical isoforms. A shorter isoform of AR primarily expressed in castration-resistant prostate cancer (CRPC), ARv7, indicates poorer prognosis in prostate cancer (PCa), as it lacks the LBD and remains overly active even in absence of the hormone, the mechanism of which is still not fully understood . Our undIn its inactive state, AR localizes in the cytoplasm sequestered by heat shock proteins (HSPs) . In a meIntriguingly, besides physiological cytoplasmic sequestering, HSPs can also affect AR signaling in PCa by triggering the degradation of the receptor, thereby resulting in lower transactivation , 29. EveIn this review, we initially focus on the structure-function relationship of AR’s polymorphic  and non-polymorphic  mono-amino acid repeat regions, the evolution and phylogenetic differences thereof, also highlighting current controversies in the literature. In the end, we list a few well-known and recently identified research gaps and propose future research directions with high potential for great breakthroughs.Polyglutamine repeat tracts are frequent in the proteome from yeast to humans and they are over-represented in the activation domain of TFs . In yeasin vitro NMR experiments. In this NMR study of the first 153 residues of the NTD, it was shown that the polyQ1 stretch has alpha-helical structure, while the rest of the sequence displayed no persistent secondary structure  has recently been reported to undergo reversible amyloid fiber formation under mild oxidative conditions . In a foIt is important to note that polyQ1 displays not only an increased aggregation propensity upon pathological expansion , it alsoIt has been well-established in the literature that Wnt signaling pathway is often misregulated in disease, especially in cancers like PCa, where it drives oncogenic proliferation , 80. EleN = 153) with normal androgen serum level, and found a significant correlation between the length of polyQ1 and defective sperm production. They found that longer polyQ1 repeats (≥28) increased the risk of impaired spermatogenesis four fold. However, later many more studies came to conflicting results, some showing correlation while others don’t  region is also located within the intrinsically disordered NTD of AR, and is encoded by three GGT triplets followed by one GGG and two more GGT triplets, and a variable number of GGC triplets. It has been shown that the length of this region has an effect on the translation of the protein itself , 125 andMoreover, it has been established that across certain races and ethnic groups the range of polyG/GGN repeat variation exhibits differences , and henIn light of the molecular details mentioned above, it is not surprising that the polymorphic length of GGN repeats, and consequently the polyG tract, is a risk factor in certain cancer types , 127 corN = 59), and patients with any of the two allele types have higher odds of developing malignancy, although the frequencies of the polymorphisms seem to be independent of each other. A large meta-analysis on the relationship between PCa and AR polymorphisms in the Caucasian population showed that short polyG of max. 16 repeats imposed the same amount of risk for PCa than a short polyQ with less than 22 repeats, while the combination of both short polyQ and polyG doubles the odds ratio of PCa risk (95% CI: 1.29–3.29) (N = 178) from around 33 months after PCa diagnosis; furthermore, long GGC repeats were associated with a worse prognosis and survival at all disease levels of stage and grade. In men from the Canary Islands (N = 72), an immunohistochemistry study showed that the polyG length was negatively correlated to prostate specific antigen (PSA) staining intensity, especially in samples with simultaneously shorter polyQ or from the more severe type of PCa with Gleason score of at least 7  . Edwards29–3.29)  found th least 7 .N = 226), longer GGC repeats (≥17 GGC) are more frequently associated with endometrial cancer (ECa) as compared to the control population (N = 207), shorter polyG was found to be more frequently associated with benign type of ECa with slower cancer progression and better outcomes (N = 257) than average polyQ (>22)  , 108.N = 557), and found that those with shorter than 23 GGN repeats (N = 44) had higher concentration of inhibin B, higher levels of progressive sperm and of correct morphology, and a higher fraction of Fas-positive sperm. Men with min. 24 GGN (N = 153) or min. 25 CAG (N = 118) both had higher estradiol levels, while those with 23 or fewer CAG had higher sperm DNA fragmentation (As AR has a key role in androgen insensitivity syndrome (AIS), a disease often leading to defects in virilization and infertility, investigating the role of mutations, including the polymorphic GGN/polyG alleles, are of high importance. Grigorova et al.  found thentation .GGN/polyG polymorphisms of AR may also correlate with certain measures of cognitive performance and risks of neurological conditions. For example, in a Chinese cohort of healthy individuals, a significant association was detected between polyG length and verbal memory of women . While iAndrogen receptor-NTD also contains a polyproline stretch (polyP at aa. 374-381) of eight amino acids, consecutively, which is conserved from human to rodents . SubstitStructurally, polyP sequences are known to fold into polyproline helices, and AlphaFold  does modIt is also noteworthy that huntingtin, the protein involved in Huntington’s disease, also has mono-amino acid repeats of both polyQ and polyP. For this protein, it was demonstrated that the polyP region chaperones the polyQ region, and without polyP the polyQ repeat is more prone to aggregation , 133–136The important role of the polyP region of AR is also highlighted by its sensitivity to mutations. The P380R substitution is cataloged in AIS , causingType 1 NHRs are a major and well-studied group of NHRs that bind bipartite hormone elements in homodimeric form . They evAcross mammalian species, AR’s DBD and LBD are fully conserved with only a single amino acid (glutamate) insertion in the sheep DBD, two mismatches in the DBD and one mismatch in the LBD of spotted hyena . These pGoing further in evolution toward vertebrates, there is a high degree of conservation of DBD, which can be explained by its function to bind to conserved DNA recognition elements . MutatioAndrogen receptor-NTD conservation across mammalian species is also relatively high with the exception of the polymorphic polyQ1 and polyG regions, however, non-mammalian vertebrates have significantly lower conservation of the NTD , 155, anMus musculus, Rattus norvegicus) and rabbits (Oryctolagus cuniculus) occupy a basal position, decisively distant from the primates’ clade, to which it is closely related in the nuclear phylogenies. Another notable exception is the bat’s AR, which appears to be closest to horses, and is positioned in a very nested position within the phylogeny. It is also worth mentioning that bat (Myotis lucifugus) appears to have the longest branch length among all the species considered, suggesting that its AR amino acid sequence has diverged the most compared to all the other orthologs considered, based on its position on In this article, we present a multiple sequence alignment  and corrWith this considered, one can appreciate that a significant part of these small changes among mammalian ARs occur around polymorphic regions. The polymorphic polyglutamine repeat (polyQ1) features in higher primates, being shorter or interrupted with other residues or even absent from mammals to frog and fish, which is also the case for other polyQ-containing and neurodegenerative disease related proteins . Two earLithobates catesbeianus) completely lacks the polyQ1 repeats (M. lucifugus), horse  and the Artiodactylian clade (Cetaceans and Ruminants), which show 4–10 polyQ1 repeats. In the primate clade, a range of repeat lengths is observed, from the 2 of the basal lemur (Eulemur fulvus collaris) to the 23 repeats in Homo sapiens, the latter representing the highest value observed in this set of sequences (O. cuniculus), which shows a long polyQ1 repeat (∼15) despite its early divergence in the phylogeny  . Interes repeats , highligequences . An intehylogeny .L. catesbeianus) lacks the polyQ2 repeat, while the ancestral state is maintained throughout most of the mammalian evolution (R = −0.16). Interestingly, carnivores and rats have the longest (min. 20 residues) polyQ2, but often interrupted by arginines or histidines , a basal state of 5–10 repeats for most of the mammalian species (R = 0.47), furthermore significant inverse correlation between polyG and polyQ2 length (Spearman’s R = −0.54) across mammalian species.The polyG region of AR is ∼86 amino acids N-terminally from AR-DBD, and it is longest in humans (∼23 residues), followed by apes (17–22 residues), Old World monkeys (15 residues), New World monkeys (8–14 residues) and other non-primate species (<10 residues) in order . Its evo species . The exc species . In mousIn summary, mono-amino acid repeats do not occur in non-mammalian species, not even the non-polymorphic polyP despite its high conservation of sequence (max. 2 substitutions) and length (eight amino acids) across mammals .N = 89) the polyG ranges between 14 and 22 repeats with 17–19 being the most common, while in bonobos (N = 54) only alleles with 18 and 19 repeats were found with 87 and 13% frequency (N = 10) polyG ranges between 21 and 24, with 21 being the most frequent allele, while polyQ length stays invariantly 4 + 5 (polyQ1 + polyQ2) (N = 47) polyG varies between 11 and 14, and similarly the fewest repeats , most frequent CAG allele had 10 repeats, both in males and females , followed by 10T (24.39 and 31.25%)–meaning one CAG was mutated to CAT–, then 13 repeats (7.32 and 9.09%) and finally 12 repeats  in order (N = 172), three polyQ1 alleles  and three polyQ2 alleles  were discovered with 11 being the most common in both of them  (N = 31) polyQ1 with 10 CAG was way more common (67.7%) than 11 CAG (32.3%), and 12 CAG was not represented at all despite the 18.6% expected occurrence (N = 31) had a tendency for shorter polyQ1 length with 10 or 11 CAG repeats , while none of the dogs with PCa had 12 CAG in polyQ1  . Interescurrence . Doberman polyQ1 . Ochiai rder N = , three prder N = , three pAlthough the relationship between fewer CAG repeats in polyQ1 and increased risk of PCa is seemingly established, it is still debated . For exaAnother point of debate is the negative correlation between the polymorphic CAG and GGC repeats, as various studies confirmed it , 115, whYet another disputed area is the transcription activity of AR, which is generally thought to be inversely correlated with the length of both CAG repeats and GGN repeats. Nonetheless, some early studies were unable to detect these differences with varying CAG repeats , 176. AlFurthermore, more research is clearly needed to clarify whether CAG repeats encoding for polyQ1 can influence cognitive function. An interesting study assessed cognitive impairment  by Mini-Mental State Examination (MMSE) in predominantly Caucasian elderly men, and found an association between longer CAG repeats and poorer performance . HoweverIn this review, we summarized efforts in determining the risk of length variations in the polymorphic regions of AR to certain types of cancer. These studies have mostly focused on a specific population in a single country, aside from a few exceptions, including the studies by Ackerman et al.  and KittStudies exploring the polymorphic nature of polyQ1 and polyG repeats in certain subpopulations, and the clinical associations thereof are dominant in the literature, nonetheless efforts should also focus on the exact mechanisms how these regions function. It is still not totally clear, how longer GGC repeats result in lower protein abundance, and whether shorter than average GGC repeat length could result in higher intracellular concentration. PolyG length certainly varies across species from short to long (see Section “Evolution and phylogenetics of the polymorphic repeat regions”), which tempts us to wonder if AR abundance is again higher in those animals in comparison to humans. Moreover, what is the interplay between polyG/GGC repeats and polyQ1/CAG repeats that makes them correlate throughout evolution? Does the polyG/GGC stretch affect the structure and function of AR on the protein level, or it only regulates the translation efficiency on transcript level? For example, the polyG region is adjacent to the binding segments of the ralaniten-like drug candidates , 63, 180A key missing area to further explore, is the modulatory role of polyQ1 and polyG and the effect of their length, respectively, as well as in certain combinations on biomolecular condensation, i.e., the formation of nuclear foci, by AR. It has been known for a long time that many TFs have a non-homogeneous distribution in the nucleus and form foci  at the DNA target site –186. LiqHOTAIR, PCAT1, HOXA11-AS-203, SOCS2-AS1, LBCS, GAS5 with a poorly understood mechanism . These RNAs comprise a huge part of the human transcriptome  and are echanism , 208. Inechanism . It is orecently , 210. Th1-AS-203 . In a foinvasion . The NTDRNAs often facilitate LLPS , which hHopefully, addressing these research gaps will enable potential breakthroughs in understanding these polymorphisms and their cross-talk, with implications in diagnostics of patients with AR alleles representing moderate to high risk to certain diseases and in developing therapeutics that are not affected by these polymorphisms or therapeutics that counterbalance the effect of overly long or short alleles.in vitro and in vivo studies are required to elucidate the contribution of the different domains as well as the two polymorphic regions to the condensate formation. Including co-factors and other crucial partners to these future studies could enable better understanding of the transition from physiological to pathological states, and explaining some of the controversies around these regions. Within NTD, elucidating the mechanism by which polyQ1 and polyG affects the functional repertoire of AR, would also enable the therapeutic targeting of these protein segments. Effect of polyQ1 length of AR on PCa and neurodegenerative disease like SBMA has been confirmed but there is a need for validation on the effect of polyG and polyP and their interplay before targeting. There are different possibilities to target repeat associated diseases for AR at DNA, RNA and protein level.Given the pace with which molecular and cell biology, genetics, diagnostics, and drug discovery develop, one can foresee a number of potential breakthroughs in the field focusing on better understanding and modulating of AR. It would be crucial to understand the molecular mechanism of the LLPS behavior of AR with regards to its activity. Further systematic On RNA level one way of targeting these polymorphic regions is by antisense oligonucleotides (ASOs) and stabilized miRNA analogs, which inhibit the translation of mRNA, this represents a fast-developing modality of drug design for repeat-associated diseases like Huntington’s disease –217, myoAt the DNA level, another approach could be a CRISPR/Cas9-based therapy for genetic engineering to restore the wild-type repeat number of the polymorphic regions, a method that is already developed for other repeat-associated diseases like Huntington’s disease –227, DucInhibiting intramolecular or intermolecular interactions of AR-NTD is yet another way of interfering with its pathogenic malfunctioning. However, this is particularly challenging due to the intrinsically disordered nature of the NTD. IDRs have been considered to be undruggable for a long while, although new success stories of upcoming molecules targeting IDRs have mostly dissolved this dogma –237, incRecent advances in understanding phase separation of AR provide opportunities to modulate condensates, thus targeting enhanceosomes and transcriptional condensates of AR variants may hold the future for drug discovery , 243–245Mono-amino acid repeats are present in many organisms including animals, plants, and fungi. For example, polyQ regions with increasing length affect solubility, stability, and abundance of proteins. Changes in hydrophobicity and secondary structure could result in oligomer formation, which potentially leads to condensation and aggregation.in vitro, and in cellulo aggregates in SBMA  with properties compatible with drug development. One can expect to see more studies directly addressing the influence of the adjacent polyG region with length polymorphism, or the effect of the partially interacting polyQ1 region on the drug binding properties of transactivation unit AR-Tau5 in the coming years. Alternative to protein-protein/DNA interaction inhibitors, targeted protein degradation inducing compounds offer a complementary approach to interfere with the overactive signaling or aggregating oligomers of a protein. To the best of our knowledge PROTACs against AR are all based on drugs interacting with LBD, however one could also envision degraders developed from ralaniten-like compounds. Moreover, therapeutic targeting of AR can also concentrate on the DNA and mRNA level by CRISPR/Cas9-based technologies and ASOs, which methods have a relatively lower entry barrier for drug development but well-known challenges in delivery and stability. However, as AR phase separates in the nucleus to form transcriptional condensates  it might be an important property for drugs to be able to partition into these condensates to exert their effects. Alternatively, condensate modulators can also be applied to hinder LLPS formation or dissolve condensates and thereby inhibit the constitutive transcriptional activation. As the number of investigational condensate modulators are rapidly growing, it is not far-fetched to expect these compounds to come of age in the near future.TL, AM, JA, and GR: conceptualization, investigation, and writing – original draft. GR and TL: visualization. TL: project administration. TL and PT: supervision. TL, AM, JA, GR, and PT: writing – review and editing. PT: funding acquisition. All authors contributed to the article and approved the submitted version.


This pilot study aimed to assess the safety and feasibility of an EMG-driven rehabilitation robot in patients with Post-Viral Fatigue (PVF) syndrome after COVID-19. The participants were randomly assigned to two groups (IG—intervention group and CG—control group) in an inpatient neurological rehabilitation unit. Both groups were assessed on admission and after six weeks of rehabilitation. Rehabilitation was carried out six days a week for six weeks. The patients in the IG performed additional training using an EMG rehabilitation robot. Muscle fatigue was assessed using an EMG rehabilitation robot; secondary outcomes were changes in hand grip strength, Fatigue Assessment Scale, and functional assessment scales . Both groups improved in terms of the majority of measured parameters comparing pre- and post-intervention results, except muscle fatigue. Muscle fatigue scores presented non-significant improvement in the IG and non-significant deterioration in the CG. Using an EMG rehabilitation robot in patients with PVF can be feasible and safe. To ascertain the effectiveness of such interventions, more studies are needed, particularly involving a larger sample and also assessing the participants’ cognitive performance. COVID-19 is a condition caused by a novel severe acute respiratory syndrome, which has been known for the last two years. A SARS-CoV-2 infection manifests itself through a wide spectrum of symptoms, from asymptomatic to life-threatening and possibly, fatal ,4,5,6. TPVF had previously been reported after the onset of other epidemics. In 2003, there was an outbreak of the SARS-CoV-1 virus, which caused the SARS epidemic. Tansey et al.  assessedSome COVID-19 patients subsequently develop PVF ,15. DuriThe symptoms that patients reported after COVID-19 were not limited to those typical of PVF. The most common Post-COVID complications were those related to patients’ physical and cognitive functioning. Very often, those symptoms were dominated by neuromuscular and immune exhaustion such as a lack of strength (as a polyneuropathy), disorders of neurological functions (such as cognitive disorders), aches and pains, problems with sleeping but also those with moving and receiving sensory stimuli, disorders of the immune system, and functions of the urogenital and digestive systems ,21.To cover the symptoms of patients after recovery from COVID-19, the synonymous terms “Long-COVID” and “Post-COVID Syndrome” are used. There is no clear, unanimous definition of Long-COVID . AccordiDue to a lack of unified definitions, PVF and Long-COVID pose a challenge to modern medicine. There is a growing need for research and the development of efficient therapeutic options for patients. Post-COVID rehabilitation is usually comprised of light aerobic and breathing exercises. Still, patients’ needs may vary from case to case. Notably, patients with myalgic encephalomyelitis or chronic fatigue syndrome may present adverse responses to exercise. These patients may thus need a different approach . The preA pilot prospective clinical study was conducted in the Neuro-rehabilitation Ward of the Provincial Hospital in Poznan, Poland, between January and November 2021.In our study, we included 30 adults admitted to the inpatient neurological rehabilitation unit. The sample size was decided based on previous pilot studies concerning biofeedback and/or robotic interventions ,27,28. EWe included participants, all of whom were transferred directly from Intensive Care Units (ICU) due to PVF after experiencing a severe course of COVID-19, according to the criteria presented by Carod-Artal . The excThe study participants were randomly assigned to two groups (IG—intervention group and CG—control group), thus constituting a 1:1 randomized study . The total stay of each patient in the Rehabilitation Unit was six weeks. Both groups were assessed twice: on admission and after six weeks.The main outcome measure was muscle fatigue during the two types of rehabilitation treatment. Additionally, the functional changes after rehabilitation were analyzed after two types of neuro-rehabilitation.Sociodemographic data were collected from all subjects; additionally, the following functional assessment tools were used: Functional Independence Measure (FIM), Barthel Index (BI), handgrip strength (HGS), Fatigue Assessment Scale (FAS), and muscle fatigue assessment model using the LUNA Rehabilitation Robot .The Functional Independence Measure (FIM) is used to assess the patient’s level of disability and a change in their status in response to rehabilitation or medical intervention. FIM is an 18-item instrument that comprises measures of independence for self-care, including transfers, locomotion, communication, sphincter control, and cognition . Each itThe Barthel Index is an ordinal scale used to measure performance in activities of daily living (ADL). BI measures the degree of assistance required by an individual on ten items of mobility and self-care activities: feeding, personal toileting, bathing, dressing and undressing, moving on and off a toilet, controlling the bladder, controlling bowel, moving from wheelchair to bed and returning, walking on a level surface and ascending and descending stairs. The Index is a three-item ordinal rating scale, and each item is rated in terms of whether the patient can perform the task independently-2 points, with some assistance-1 point, or is dependent on help 0 points. The maximum final score is 20 (assistance in ADL is not needed) .Handgrip strength was measured using a JAMAR Hand Dynamometer . The measurement was performed in a sitting position in an armless chair, with the forearm in a 90-degree flexed position. The average of three measurements (in kilograms) was recorded .Fatigue Assessment Scale (FAS) is a 10-item fatigue questionnaire to assess fatigue in the general population. Five questions reflect physical fatigue, and another five (questions 3 and 6–9)—mental fatigue. The total score ranges from 10 to 50. A total FAS score of <22 indicates no fatigue, and a score of ≥22 indicates the presence of fatigue .Muscle fatigue data were also retrieved from the EMG-rehabilitation robot regarding the work of the muscles of the two-headed arm by means of the robot’s muscle fatigue assessment model (isometric contraction tension). The test of EMG-based fatigue was performed on the Biceps Brachii muscle by surface electrodes. The electrodes were placed on the line between the medial acromion and the fossa cubit at 1/3 from the fossa cubit according to SENIAM . The samNeurological rehabilitation in both study groups was carried out six days a week for six weeks using neuromuscular re-education techniques (such as PNF—Proprioceptive Neuromuscular Facilitation and Bobath therapy), exercises for movement coordination and balance, with combined progressive endurance training. After the participants achieved the ability to maintain a standing position and conduct active and resistance exercises, progressive endurance training was performed from 35 percent of max HR (heart rate) to 70 percent of max HR  according to the equation max HR = 220 − age 30 min per day. PNF or Bobath therapy was conducted 45 min per day. The patients in the IG had this training for 75 min per day, and the CG had this form of therapy for 120 min a day.Each patient (in both IG and CG) worked individually with a psychologist and speech therapist five times a week for 30 min. Additionally, occupational therapy was performed daily for a minimum of 30 min.The patients in the IG, according to the intervention model of rehabilitation, performed additional training using an EMG rehabilitation robot—the EMG-rehabilitation robot . The EMG-rehabilitation robot enables the isokinetic-isotonic training of selected muscle groups using reactive electromyography (EMG-triggered), providing assisting robot movement, which is activated by the patient’s muscle bioelectric activity signal measured in real-time with surface electrodes. During exercises with the EMG robot, the participants conducted flexion and extension of the elbow and flexion, extension, and abduction in the glenohumeral joint. The muscles of the upper limb  were used to trigger the movement. Luna EMG is a rehabilitation robot specifically designed to support the physiotherapy of neurological patients with muscle weakness. It is an all-in-one platform for complex personalized therapy for patients suffering from neurological conditions. It tackles clinical issues such as muscle weakness, mobility disorders, gait problems, and range of motion restrictions, specifically by automating the process of personalized, motivating physiotherapy based on electromyography combined with force and position sensing. The robot was attached to the patient through extensions–interchangeable mechanical parts that are connected to the patient by straps or by the grip. The movements are controlled by a Windows application from a therapist panel, which provides a User Interface, patient management, reporting module, and Internet connectivity for the purpose of remote diagnostics and oversight.The device performs isokinetic, isotonic, and isometric exercises. The “EMG-triggered robotic movement” technology works actively with patients, even if no movement is observable. The EMG-rehabilitation robot detects the EMG activity of the muscle and provides appropriate assistance during the movement. If no movement or muscle activity is present, the device provides passive assistance.The EMG-rehabilitation robot follows a “hands-off” methodology to improve the independence of the patient, even when their mobility and muscle activity are very limited. In the wake of the COVID-19 pandemic, this approach seems to be beneficial since it limits direct contact between the patient and the therapist while maintaining proper rehabilitation measures.2 test was used to compare two groups of nominal variables. p < 0.05 was considered statistically significant.Statistical analysis was performed with the Statistica 13.3 software . The normality of the distribution was checked with the Shapiro-Wilk test. In the absence of a normal distribution and for ordinal variables, the Mann–Whitney test was used to compare the results in two groups. The Wilcoxon signed-rank test was used for intragroup comparisons. Data are presented as medians and range. The χA total of 30 participants (15 in IG and 15 in CG) were included  at baseline .Both the IG and CG improved in terms of the majority of measured parameters comparing the pre- and post-intervention results, except for muscle fatigue measured by EMG. Muscle fatigue scores presented non-significant improvement in IG and non-significant deterioration in CG. The results of the pre-post comparison are presented in The comparison of mean changes of measured parameters did not reveal any statistically significant differences between the study groups. The results are presented in The anonymized EMG charts from pre- and post-intervention measurements of all of the participants are included in the In this pilot study, the feasibility of the concept of using the EMG—rehabilitation robot in exercises for patients with PVF syndrome was verified positively. The study participants reported no adverse outcomes, and no dropouts from the study were observed. The lack of data from two participants resulted from transferring them to another facility. It must be noted that not only no adverse outcomes were reported but also improvement in both groups was observed. Based on that, conducting further research on this topic following the methodology presented in this study can be safe for patients and is not likely to negatively impact their outcomes of rehabilitation.The use of robots in rehabilitation started on a larger scale in 1990 , and thiAs Yong  stated, Our study demonstrated that implementing an EMG robot-rehabilitation protocol in PVF patients is feasible and safe. Similar results were obtained by different authors in studies using comparable devices. Kim et al.  conducteIt should be noted that our study has some limitations. First of all, the lack of significant difference in terms of improvement between the IG and CG could result from the too-small sample size. As the present study aimed to assess the feasibility and safety of the presented protocol, further research involving larger samples is needed to establish the actual influence of exercises on a rehabilitation robot on muscle fatigue and fatigue-related outcomes. A further limitation is that PVF is often accompanied by cognitive disorders, and exercises on the EMG—rehabilitation robot required notable focus and psychological effort to perform the exercises and measurements correctly. Further studies are needed to analyze if exercising on an EMG—rehabilitation robot affects cognitive abilities. The third limitation results from the fact that this study is, to the best of our knowledge, the first one assessing an EMG-driven device in the therapy of PVF. While it can be perceived as a strength of the study, it must be pointed out that there exists no literature with which to relate our results. While most of the studies assessing the effectiveness of such devices target stroke patients, due to the different nature and causes of PVF and stroke-related impairments, the effectiveness of such devices can differ between those groups. The different exercise regimens in the studied groups can also be viewed as a limitation.According to the results obtained in this feasibility study, using an EMG-robot in the rehabilitation of patients with PVF can be feasible and safe. To ascertain the effectiveness of such interventions in this group of patients, more studies are needed, particularly involving a larger sample and also assessing the participants’ cognitive performance.


Editorial on the Research TopicHealth systems, rehabilitation care and COVID-19: Challenges and opportunitiesCOVID-19 has considerably challenged health systems worldwide, especially acute and intensive care services, and has had a profound impact on the rehabilitation sector. COVID-19 has increased the need for rehabilitation services, either for those recovering from more severe cases potentially requiring hospitalization, prolonged intensive care, or experiencing Post Intensive Care Syndrome (PICS) , 3. On tLugo-Agudelo et al. while the second describes the impact and challenges in Bangladesh Uddin et al. A third paper presents a protocol for a study on rehabilitation and return-to-work of professionals who acquired COVID-19 in the workplace Muller et al. The fourth paper reports the results of an interdisciplinary workshop on the challenges of conducting rehabilitation research during the COVID-19 pandemic, and approaches to address the related complexities were proposed Noguchi et al.In this special collection, we are publishing four papers. Two papers are dealing with the impact of the COVID-19 pandemic on rehabilitation services: one tackles the countries' response to persons with disabilities (PWD) Lugo-Agudelo et al. They found information for 98 countries, with official documents only in 32 countries. The search focussed on all measures and was not specific to rehabilitation, though the authors noted that PWD may be an important rehabilitation user group. Indeed, the WHO, as well as the Centers for Disease Control and Prevention both declared PWD as more likely to become infected or develop serious illness  pandemic protocol adjustments to public health guidelines; (2) participant accessibility to ensure equity, safety, and feasibility; (3) adapted knowledge dissemination such as smaller networking events, and revisiting timeframes.Noguchi and a team of collaborators organized a two-hour online workshop attended by 25 rehabilitation researchers to discuss the challenges they had experienced and strategies employed to conduct rehabilitation research during the COVID-19 pandemic This collection contributes to the knowledge about the impact and challenges of COVID-19 on rehabilitation services in health systems across the world, non-pandemic related rehabilitation research and introduces an observational study that will examine the effect of a comprehensive rehabilitation programme for patients who got infected with the SARS-CoV-2 virus at work, also on the long-term.We are convinced that a “new” disease like COVID-19, for which evidence on many aspects is being continuously generated and compiled , require


Streptomyces tricolor HM10 was evaluated through an anemia-induced rat model to study its potential to accelerate recovery from anemia. Rats were subjected to an iron-deficient diet (IDD) for 42 days. Anemic rats (ARs) were then divided into six groups, and normal rats (NRs) fed a standard diet (SD) were used as a positive control group. For the recovery experiment, ARs were treated as a group I; fed an IDD (AR), group II; fed an SD (AR + SD), group III, and IV, fed an SD with an intraperitoneal injection of 1 μg Sid Kg−1 (AR + SD + Sid1) and 5 μg Sid Kg−1 (AR + SD + Sid5) twice per week. Group V and VI were fed an iron-enriched diet (IED) with an intraperitoneal injection of 1 μg Sid Kg−1 (AR + IED + Sid1) and 5 μg Sid Kg−1 (AR + IED + Sid5) twice per week, respectively. Weight gain, food intake, food efficiency ratio, organ weight, liver iron concentration (LIC) and plasma (PIC), and hematological parameters were investigated. The results showed that ~50–60 mg Sid L−1 medium could be producible, providing ~25–30 mg L−1 purified Sid under optimal conditions. Remarkably, the AR group fed an SD with 5 μg Sid Kg−1 showed the highest weight gain. The highest feed efficiency was observed in the AR + SD + Sid5 group, which did not significantly differ from the SD group. Liver, kidneys, and spleen weight indicated that diet and Sid concentration were related to weight recovery in a dose-dependent manner. Liver iron concentration (LIC) in the AR + IED + Sid1 and AR + IED + Sid5 groups was considerably higher than in the AR + SD + Sid1 AR + SD + Sid5 groups or the AR + SD group compared to the AR group. All hematological parameters in the treated groups were significantly closely attenuated to SD groups after 28 days, confirming the efficiency of the anemia recovery treatments. Significant increases were obtained in the AR + SD + Sid5 and AR + IED + Sid5 groups on day 14 and day 28 compared to the values for the AR + SD + Sid1 and AR + IED + Sid1 groups. The transferrin saturation % (TSAT) and ferritin concentration (FC) were significantly increased with time progression in the treated groups associatively with PIC. In comparison, the highest significant increases were noticed in ARs fed IEDs with 5 μg Kg−1 Sid on days 14 and 28. In conclusion, this study indicated that Sid derived from S. tricolor HM10 could be a practical and feasible iron-nutritive fortifier when treating iron-deficiency-induced anemia (IDA). Further investigation focusing on its mechanism and kinetics is needed.Iron-deficiency-induced anemia is associated with poor neurological development, including decreased learning ability, altered motor functions, and numerous pathologies. Siderophores are iron chelators with low molecular weight secreted by microorganisms. The proposed catechol-type pathway was identified based on whole-genome sequences and bioinformatics tools. The intended pathway consists of five genes involved in the biosynthesis process. Therefore, the isolated catechol-type siderophore (Sid) from  Iron, a trace metal, is crucial in maintaining normal human metabolism and is required by most organisms. It is used in numerous living processes, including oxygen transport, redox reactions, and nucleic acid synthesis ,2. Iron-Siderophores are low-molecular-weight compounds (200–2000 Da) secreted by microbes, fungi, and plants under limited iron conditions . Under i3+ complexes. Depending on membrane affinity and length, siderophores could anchor a particular gradient outside the cell membrane, thereby enhancing the ability to capture iron from seawater [Moreover, ferric iron considers the only state that siderophores could chelate. When ferric iron is reduced to ferrous iron, siderophores will release it. This is an important strategy for microbes to combat the iron stress environment ,19. The seawater ,21.Streptomyces reduced the viability of malignant melanoma cells significantly. At the same time, other siderophores  were used in cancer therapy as iron chelators [A siderophore is a biological molecule with many applications, and various bacteria can produce it. It can be utilized in multiple fields such as agricultural, medicinal, and environmental applications ,23,24. Ihelators ,28.O-trensox. Furthermore, siderophores can rescue non-transferrin-bound iron in blood serum [Klebsiella pneumoniae can act as antimalarial agents and can be used to treat malaria caused by Plasmodium falciparum [The iron-conveying abilities of siderophores are used to deliver drugs. Drug delivery was shown to occur in cells by forming conjugating among antimicrobials such as albomycins and siderophores . Sideropod serum . Sideroplciparum .Streptomyces, Gram-positive bacteria, are famous for producing various secondary metabolites. These secondary metabolites have significant biological and clinical usages. Secondary metabolite biosynthesis is usually controlled by complex regulatory networks that respond to various biotic and abiotic stresses in the surrounding environment. Streptomyces are considered to be one of the most common producers of secondary metabolites  [s, etc.) ,32,33,34Synechococcus sp. PCC7002 could be practical and feasible iron-nutritive fortifiers. However, no studies have clearly demonstrated the effectiveness of using siderophores to enhance iron absorption in human food models. In anemia conditions, a lack of sufficient healthy red blood cells to carry adequate oxygen to the body’s cells and low hemoglobin content causes fatigue and weakness. Iron-deficiency-induced anemia is the most common type of anemia worldwide, caused by a shortage of iron in the body. Bone marrow requires iron to produce hemoglobin. When iron levels are low, our bodies cannot produce enough hemoglobin for red blood cells. Therefore, the main objective of this study was to study the potential application of purified Sid from S. tricolor HM10 bacteria to accelerate recovery from iron-deficiency-induced anemia in a rat model.Siderophores could potentially become exciting molecules for combating IDA. However, despite the literature showing promising potentialities related to siderophores, particular attention needs to be paid to the applicability, dose, and mechanisms of siderophores. Moreover, to the best of the authors’ knowledge, the literature review mainly highlighted the purification and applications of siderophores, with only a few studies referring to in vivo applications ,36. The S. tricolor HM10 strain (accession: MN527236) was used for siderophore production, as previously established by Rehan et al. [2HPO4, 1.50; MgSO4.7H2O, 1.50; agar, 15.0 (g/L), pH 7.2) with a ratio of 1:9. In Sid production, a fresh culture of S. tricolor HM10 was cultivated on King’s B medium supplemented with FeCl3 (5 μM) for 72 h at 28 °C with continuous agitation (170 rpm min−1). Afterward, the culture broth was centrifugated at 10,000× g for 20 min−1. Sid concentration and type were determined in clear supernatant using Arnow’s Assay [n et al. , with min et al.  to King’’s Assay  for cate’s Assay  for hydr’s Assay , after t’s Assay . Before S. tricolor HM10 was sequenced using Oxford Nanopore, assembled, and annotated. The genome is available in the NCBI database with accession number JAJREA000000000. In terms of searching the entire genome, the catechol-type pathway putatively coding for siderophores close to petrobactin was identified using blast search and antiSMASH [The whole genome of ntiSMASH ,46.−1 DW). The radical scavenging activity was measured spectrophotometrically based on the bleaching of DPPH radicals in purple solution according to Yawadio Nsimba et al. [−1).The TPC-purified Sid was determined by using Folin–Ciocalteu reagent according to Yawadio Nsimba et al. , and thea et al. , and ABTa et al. . The finThe study protocol was approved by the Institutional Animal Ethics Committee (IAEC) of Qassim University, KSA (21-07-06 on 26 January 2022). It was regulated by the Purpose of the Control and the Supervision of Experiments on Animals (CPCSEA) Committee under the National Committee of Bioethics (NCBE), Implementing Regulations of the Law of Ethics of Research on Living Creatures. Fifty-six female Wistar rats (6–8 weeks) weighing 150–200 g were housed in polypropylene laboratory cages (four each) in a room at 24 ± 1 °C under inverted 12 h light–dark cycle conditions. Before the feeding experiment, the rats were allowed to adapt to the AIN-93G standard diet (SD) for one week, as Suehiro et al.  recommen−1. The animals were given unlimited access to deionized water. Anemic rats were separated into six groups during the anemia recovery period, each having eight rats. Anemic rats (ARs) in group I was fed an IDD for another four weeks. Group II (AR + SD) received the SD, group III (AR + SD + Sid1) received the SD and received purified Sid at 1 μg Kg−1 rat twice per week, and group IV (AR + SD + Sid5) received the SD and received pure Sid at 5 μg Kg−1 rat twice each week. The iron-enriched diet (IED) was created with double the iron content of the standard diet (SD) and administered to groups V and VI. Group V (AR + IED + Sid1) received pure Sid intraperitoneally twice a week at a dose of 1 μg Kg−1 rat. Group VI (AR + IED + Sid5) received pure Sid intraperitoneally twice a week at a dose of 5 μg Kg−1 rat. The animals were given deionized water ad libitum during iron-deficient anemia induction, while ordinary tap water was offered to drink throughout the recovery period. Rats were anesthetized and slaughtered, and their spleen and liver were taken and weighed before the liver was perfused with physiological saline to eliminate blood at the end of the experiment. The relative weight of organs was calculated using the following equation:−1 purple capped tubes incorporated with disodium salt of ethylenediaminetetraacetic acid (EDTA-2Na) from the tail vein of rats at the end of the 0, 2nd, and 4th week. The hemoglobin level (g dL−1) and hematocrit (%) were analyzed on the day of blood collection using CBC . The plasma iron (μg dL−1) was determined using a photometric–colorimetric test based on the chromazurol B (CAB) protocol according to Garčic [−1) was determined using an enzyme immunoassay test kit according to White et al. [−1) was determined using an in vitro quantitative kit . At the same time, serum transferrin iron saturation (TSAT %) values were calculated using the following equations:Five hundred microliters of blood were collected in 1.5 mg mLo Garčic , and fere et al. . The totThe statistical analysis was conducted using the SPSS program (ver. 23), applying the experimental design variance analysis using one-way ANOVA for growth parameters, food intake, feed efficiency, liver, kidney, and spleen weight, and iron concentration. In contrast, two-way ANOVA was applied for hematological parameters, iron concentration, transferrin saturation %, and ferritin concentration in plasma. The significance level was 0.05, and Tukey’s test was applied following Steel et al. .S. tricolor HM10 strain (Accession: MN527236) was used for Sid production, which was first detected on King’s B agar medium . Sid detection and type determination in clear supernatant was achieved according to Arnow’s method (step 4). Only 25–30 mg L−1 could be isolated and purified by applying the established protocol using acidified supernatant [The r medium , step 1.ernatant . To testS. tricolor HM10. The proposed pathway consisted of the gene that encodes diaminobutyrate-2-oxoglutarate transaminase family protein (gene A), iron transporter (gene B), the IucA/IucC family protein for siderophore biosynthesis (gene C), ferric iron reductase FhuF-like transporter (gene D), and the acetyltransferase (GNAT) domain (gene E). The proposed pathway consisted of five genes with predicted Enterochelin-like Sid production  are shown in −1 of Sid showed no significant differences. Consequently, ARs had significantly higher relative liver weights than AR groups fed an SD or IED and injected with 1 or 5 μg Kg−1 of Sid. Diet and Sid concentration were clearly related to the relative weight of the organ, and reductions in relative organ weights took place in a dose-dependent manner. The SD and AR groups demonstrated that an IDD affected the relative kidney weight. However, there was no significant difference in relative kidney weight between ARs fed an SD or IED with 1 and 5 μg Kg−1 of Sid groups and SD groups. The SD group had the highest relative spleen weight, which differed significantly from the AR group. ARs fed an SD or IED with 1 or 5 μg Kg−1 Sid did not display significantly different results from the SD and AR + SD groups. The LIC was significantly lower in the AR group than in the SD group. Among iron-deficient groups, the values observed in the AR + IED + Sid1 and AR + IED + Sid5 groups were considerably higher than those observed in the AR + SD + Sid1 and AR + SD + Sid5 groups or in the AR + SD group.The relative weights of the liver, kidneys, and spleen and liver iron concentration (mg g12 L−1), HGB (g dL−1), HCT (%), MCV (fL), MCH (pg), and MCHC (g dL−1), are determined through complete blood counts (CBCs) for blood samples taken at the start (day 0) and at the end of the recovery period (day 28) for different experimental groups are illustrated in The hematological parameters, such as RBC , respectively. Significant increases were seen during the recovery period when ARs were fed an SD or an SD or IED with 1 or 5 μg Kg−1 Sid. The PIC values in the AR + SD group were significantly lower than in the other four groups on days 14 and 28. Significant increases were observed in AR + SD + Sid5 and AR + IED + Sid5 groups on day 14 and day 28 compared to the values for the AR + SD + Sid1 and AR + IED + Sid1 groups. The TSAT levels in the SD and AR groups were 59.93% and (10.34–19.84%), respectively, at the beginning of the recovery period. No significant difference was recorded in the SD group until the end of the recovery period. On the contrary, the TSAT in the AR group was significantly decreased with time progression. At the beginning of the recovery period, ferritin concentration in the SD and AR blood serum was 71.92 and (12.48–19.56) ng mL−1  and activates catecholate siderophore production [Bacillus anthracis can produce two catechol-type siderophores (petrobactin and bacillibactin) under iron-limited conditions. Bacillibactin siderophores are highly sensitive to iron concentration, whereas petrobactin is less dependent on Fe [E. coli [Pseudomonas aeruginosa [The proposed catechol-type siderophore pathway was identified based on genome sequencing and bioinformatic tools, i.e., antiSMASH online software. The intended pathway contains five genes similar to the petrobactin biosynthetic gene cluster from oduction . The absoduction . Bacillunt on Fe . Catechont on Fe . Our stu[E. coli  and Pseuruginosa . Iron plruginosa . As is eruginosa ,62,63. Iruginosa ,5. Interruginosa ,65.−1 from Sid showed the highest weight gain. Although AR groups were fed an IED containing double iron content, the weight gain was not significantly changed. It might be because a diet containing high inorganic salt such as ferric citrate had side effects on the stomach and affected appetite and digestion [The rats’ final body weight, weight gain, food intake, and food efficiency ratio compared to the SD group during the recovery period were carefully investigated. During the recovery period, the AR group fed an SD and ARs fed an SD with different concentrations of injected Sid exhibited significant changes in weight gain. It could be noticed that Sid injection improved the weight gain in a dose-dependent manner in the presence of available iron in the diet. The increases in the weight gain might have been due to improved metabolism and increased fat and muscle mass, which was observed in the feed efficiency ratio . Remarkaigestion . Feed efigestion .−1 of Sid had significantly lower relative liver weights than in the AR group, reflecting the improvement in body weight. Indeed, the higher relative weight may explain the higher organ weight and lower body weight. Diet and Sid concentration were clearly related to weight recovery in a dose-dependent manner, similarly noted in [Consequently, the relative weights of the liver, kidneys, and spleen were linked to growth and body weight gain. Weight gained in the organs might have been due to improved metabolism and increased fat and muscle mass, as observed and indicated ,39. Consnoted in ,38. IDA noted in . Indeed,noted in ,38. This−1 resulted in more significant improvements in the hematological parameters than feeding an SD alone or even an IED with Sid injection. Accordingly, considerable increases in HGB, HCT%, MCV, MCH, and MCHC values promoted recovery from iron-deficiency-induced anemia. Iron is mainly absorbed in the duodenum; there are two types of iron in the diet: non-heme and heme iron, each with a different absorption mechanism. Non-heme iron is taken up into intestinal epithelial cells by the divalent metal transporter 1 (DMT1), though the iron in the intestinal tract would then need to be solubilized [Surprisingly, a noticeable improvement in the hematological parameters such as RBC, HGB, HCT, MCV, MCH, and MCHC was observed. Immediately after inducing anemia, the RBC, HGB, HCT, MCV, MCH, and MCHC were monitored every two weeks up to 4 weeks. Significant changes were found in all parameters in all experimental groups, confirming the efficiency of the anemia recovery treatments. After 2 weeks, the remarkable improvements in HGB, HCT%, MCV, and MCHC in treated AR were evident in the efficiency of Sid in the presence of iron in the diet to attenuate anemia. This may be because Sid could anchor a specific gradient outside the cell membrane, depending on membrane affinity and length, thereby enhancing the ability to capture iron from surrounding matrixes ,21. The ubilized . Howeverubilized . The solubilized . Sideropubilized . As Sid ubilized ,70. The ubilized .−1 Sid. Likewise, the TSAT was associated significantly with serum iron (SI), as evidently presented [−1 Sid on days 14 and 28. On the other hand, indigestible saccharides, such as water-soluble dietary fibers and oligosaccharides, are reportedly fermented in the large intestine by intestinal bacteria to produce short-chain fatty acids such as acetic acid, propionic acid, n-butyric acid, or organic acids such as lactic acid and succinic acid. These acids acidify the lumen, increase the number of minerals solubilized in the intestine, and promote iron absorption in the intestine [As previously hypothesized, Sid could anchor a specific gradient outside the cell membrane, depending on membrane affinity and length, thereby enhancing the ability to capture iron from surrounding matrixes ,21. Ironresented ,38. Signntestine ,75,76.Indeed, in small species, intraperitoneal injection is used instead of intravenous access, and it can be used to safely administer large volumes of fluid. A higher affinity between siderophores and iron than between transferrin and iron is recognized. The catecholate-type siderophore has stronger iron affinities than transferrin in comparison to other categories of siderophores. A siderophore–iron complex is expected to enter the cell through simple diffusion (according to the low iron concentration inside the cells regarding anemia induced by iron deficiency) via the endocytosis of the transferrin receptor (TfR1) Siderocalin/Lipocalin 2/Neutrophil Gelatinase Associated Lipocalin, which is an innate immune system protein with bacteriostatic activity, or ATP-binding cassette (ABC) transporters, the energy-dependent efflux transporters. Red pulp macrophages (RPMs) and Kupffer cells (KCs) express complete machinery for RBC clearance and heme iron recycling ,80,81,82Noticeably, at present, we did not delineate the detailed mechanism of these changes, and further experiments are needed to elucidate the exact underlying mechanisms. Combing our results with clearly reviewed studies, we suggest that the mechanism of action of Sid is to be effective in chelating iron in the entire intestine and binding to the cell membrane and will then find a way to transfer iron elements into the cell. However, to further confirm this hypothesis, we need to design a signaling experiment to elucidate iron pathways. In addition, the possibility of excess iron absorption needs to be examined using an iron-repleted diet and prolonged feeding conditions.S. tricolor HM10 was evaluated through an anemia-induced rat model to study its potential to accelerate recovery from IDA. This study revealed that Sid significantly improved weight gain and iron-deficiency-induced anemia was effective during recovery from anemia and could be a practical iron-nutritive fortifier. Combining our findings with previously mentioned studies, we suggest that Sid’s mechanism of action is effective in chelating iron in the entire intestine, binding to the cell membrane, and then finding a way to transfer iron elements into the cell. Noticeably, at present, we did not delineate the detailed mechanism of these changes, and the design of a signaling experiment to confirm this hypothesis is highly recommended. In addition, the possibility of excess iron absorption needs to be examined using an iron-repleted diet and prolonged feeding conditions.Isolated Sid from


Reactive oxygen species (ROS) play important roles in plant growth and defense. In this study, we determined that the cpr5 mutant exhibited excessive ROS accumulation. However, the mutation of respiratory burst oxidase homolog D (RBOHD), a reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase responsible for the production of ROS signaling in plant immunity, did not suppress excessive ROS levels in cpr5. Furthermore, the cpr5 mutant showed low levels of ascorbate peroxidase 1 (APX1), an important cytosolic ROS-scavenging enzyme. APX1 overexpression in the cpr5 background removed excessive ROS and restored the pleiotropic growth phenotype. Notably, APX1 overexpression did not reduce the resistance of cpr5 mutant to virulent strain Pseudomonas syringae pv. tomato (Pst) DC3000 and avirulent strain Pst DC3000 (avrRpt2). These results suggest that the removal of excessive ROS by APX1 overexpression restored the cpr5 growth phenotype while conserving pathogen resistance. Hence, our study provides a theoretical and empirical basis for utilizing CPR5 in the breeding of crops with disease resistance by effective oxidative stress management via APX1 expression.Breeding crops with enhanced immunity is an effective strategy to reduce yield loss caused by pathogens. The  The sistance . The CPRsistance , acting sistance ,3. The creenings ,5,6. Thenditions ,7,8. Phynditions . cpr5 haignaling ,11. Howe2O2) [cpr5 [cpr5 can be uncoupled by removing excessive ROS.Reactive oxygen species (ROS) are molecules containing active oxygen. They include superoxide, the hydroxyl radical, singlet oxygen, and hydrogen peroxide (H2O2) . ROS pla2O2) ,14. Howe2O2) . Many di2O2) ,17. It h2) [cpr5 ; howeverapx1 mutants exhibit enhanced sensitivity to various abiotic stressors such as high temperature, high light intensity, salt, drought, and heavy metals [2O2–APX1 reaction upon treatment with avirulent bacteria and lipopolysaccharide (LPS), an elicitor from the outer membrane of Gram-negative bacteria [ROS are generated during photosynthesis in chloroplasts, respiration in mitochondria, and photorespiration in peroxisomes . They ary metals ,23,24,25bacteria .delt9 (defective in LPS-triggered ROS production) mutation [CPR5 gene. delt9 and cpr5 showed reduced light signals in the luminol–H2O2–APX1 reaction but enhanced ROS levels according to colorimetric and fluorescent dye staining. The introduction of rbohD into the cpr5 mutants did not suppress ROS accumulation. In contrast, APX1 overexpression restored ROS levels in cpr5. We also determined that APX1 overexpression restored the pleiotropic growth phenotype but not pathogen resistance in the cpr5 mutants. Overall, our results suggest that cpr5-mediated disease resistance could be uncoupled from plant growth impairment by removing excessive ROS via APX1 overexpression.In this study, we established that the previously found mutation ,28 was aArabidopsis thaliana used in the present study was the Columbia-0 (Col-0) ecotype. Seeds of the cpr5 , rbohD (CS9555), and adr1-L2  T-DNA insertion mutant were obtained from the Non-Profit Arabidopsis Share Center (https://www.arashare.cn). APX::APX-GFP was used as previously described [The genetic background of the wild-type and mutant L2 salk_1422 T-DNAv/v) sodium hypochlorite (NaOCl) for 10 min, rinsed thrice with sterilized water, and sown on half-strength Murashige and Skoog (1/2 MS) agar containing 0.5% (w/v) sucrose. The seedlings were raised in a growth chamber  at 22 °C, 14 h light/10 h dark photoperiod, 75% RH, and 15,000 lx. For the mature plant assays, 7-day-old seedlings were transplanted in soil  and raised in a growth chamber  at 22 °C, 14 h light/10 h dark photoperiod, and 15,000 lx.The seeds were surface-sterilized by immersion in 10%  on the Illumina X-ten System . A genome-wide single-nucleotide polymorphisms (SNP) analysis detected the genomic region with relatively high SNP index and harboring a candidate SNP in CPR5.The 35S::CPR5-HA construct, cDNA sequences without CPR5 stop codons were amplified by PCR, cloned into pDONR/Zeo plasmids via BP (gateway) cloning, and subcloned into pGWB14 vectors by LR (gateway) reaction. The constructs were then electroporated into Agrobacterium tumefaciens GV3101. Transgenic plants were obtained using the Agrobacterium-mediated floral dipping transformation method [The primers used for gene cloning and plasmid construction are listed in n method .® qPCR Master Mix (Vazyme Biotech) was used for the qRT-PCR, and ACTIN7 was the reference gene. Relative gene expression levels were calculated by the 2−ΔΔCt method [Total foliar RNA was extracted with TRIzol reagent (Thermo Fisher Scientific). The cDNA was synthesized using 1 μg total RNA and a HiScript III 1st-Strand cDNA Synthesis Kit . ChamQTM SYBRt method . All priv/v) Triton X-100, and 1% (v/v) protease inhibitor cocktail . For CPR5 and RBOHD protein detection, the extraction buffer consisted of 50 mM Tris-HCl (pH 5.7), 2% (w/v) sodium dodecyl sulfate (SDS), 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1% (v/v) protease inhibitor. The tissues were ground, resuspended in the protein extraction buffer, and incubated on ice for 30 min. Cell debris was removed by centrifugation at 12,000× g and 4 °C for 15 min. The extracted proteins were separated by 8% SDS-PAGE (for CPR5 and RBOHD) and by 12% SDS-PAGE (for APX1) and transferred to polyvinylidene difluoride (PVDF) membranes at 80 V and 4 °C for 3 h. The membranes were blocked with 5% (w/v) nonfat milk and incubated with horseradish peroxidase (HRP)-conjugated α-HA , α-RBOHD , α-cAPX (Agrisera), and α-ACTIN  antibodies. Signals were detected with a SuperSignal West Femto Trial Kit (Thermo Fisher Scientific).Protein extraction and immunoblot analysis were performed as previously described . APX1 pr2O2.APX activity was measured as previously described . Briefly2) from 4-week-old plants were incubated overnight in water with light exposure in a 96-well plate. Then, 50 μL of a solution consisting of 200 mM luminol and 50 mg/mL LPS  was added to each well, and the chemiluminescence signals were recorded with a Photek HRPCS5 camera .ROS signals were measured by luminol-based chemiluminescence assay according to a previously described procedure . Leaf di2HPO4, and 0.05% (v/v) Tween 20 and incubated in the dark at 24 °C for 6–8 h until brown precipitate was observed in the leaves. The leaves were then decolorized with de-staining solution ( ethanol:acetic acid:glycerol = 3:1:1 (v/v/v)). Leaves that remained dark brown contained H2O2 and were viewed under a light microscope . The relative DAB staining intensity per unit leaf area was quantified, and the H2O2 content was determined using the ImageJ software .DAB staining was performed as described previously with some modifications . Excised2DCFDA staining assay was performed according to a previously reported method with slight modifications [2DCFDA  in 10 mM PBS buffer in the dark for 30 min. Images were captured under an Olympus FV3000 confocal laser scanning microscope  with a 488 nm filter. ROS signals were visualized in the range of 501–550 nm, and chlorophyll autofluorescence was detected in the range of 640–735 nm.The Hications . The exc2PO4 alkalized to pH 7.4 with KOH in the dark at 24 °C for 30 min. The leaf segments were then rinsed thrice in the dark with 10 mM KH2PO4 alkalized to pH 7.4 with KOH. The samples were then mounted on microscope slides with the stripped  leaf surfaces facing the coverslip. The mesophyll cell layers were immediately examined with an Olympus FV3000 confocal laser scanning microscope at 488/585–615 nm excitation/detection.Mitochondrial superoxide was imaged by MitoSOX Red staining . MitoSOX2O2 was measured by titanium sulfate assay as previously described with slight modifications [g and 4 °C for 15 min. Then 0.1 mL of 5% (w/v) Ti(SO4)2 and 0.2 mL NH4OH were added to 0.4 mL of the supernatant. The suspension was centrifuged at 5000× g for 15 min, and the precipitate was collected. Then 1 mL acetone was added to it, and the mixture was centrifuged at 5000× g for 15 min until a white precipitate formed. The precipitate was then dissolved in 1 mL of 2 M H2SO4, and absorbance was read at 415 nm. The output was converted to μmol H2O2/g leaf tissue.The Hications . BrieflyThe trichomes on living leaves were photographed with a Nikon Digital Sight DS-Fi2 camera mounted on a Nikon SMZ18 microscope.Pseudomonas syringae pv. tomato (Pst) DC3000 or Pst DC3000 (avrRpt2) at 1 × 105 CFU/mL. Then, 2 or 3 d after infiltration, three 1.5-cm2 leaf disks were ground in 500 mL of 10 mM MgCl2, and the suspension was serially diluted and plated on NYG medium consisting of 5 g/L peptone, 3 g/L yeast extract, 2% (w/v) glycerol, 1.5% (w/v) agar, and rifampicin. The colonies were enumerated 2 d after plating.Leaves of 4-week-old Arabidopsis plants were infiltrated with 2O2 reaction [defective in LPS-triggered ROS production (delt) were previously isolated from an ethyl methanesulfonate (EMS)-mutagenized Arabidopsis seedling population [delt9 mutant exhibited lower luminescence signals than the wild-type (Col-0) after the LPS treatment  35S promoter in the delt9 background (35S::CPR5-HA/delt9). We measured the CPR5-HA protein levels in the transgenic lines by immunoblotting with an α-HA antibody , a helper nucleotide-binding leucine-rich repeat protein (NLR), is upregulated in double mutants with loss-of-function of BRASSINOSTEROID INSENSITIVE 1 (BRI1)-ASSOCIATED RECEPTOR KINASE 1 (BAK1) and its closest paralog BAK1-LIKE 1 (BKK1) . The preus study , we exam in cpr5 , suggestcpr5 generated reduced luminol–H2O2–APX1 signals but elevated cytosolic H2O2 levels. Hence, cpr5 might have low endogenous APX1 levels. We tested this hypothesis by measuring APX1 protein abundance in the cpr5 mutants via immunoblotting with an α-cAPX antibody. Arabidopsis contains three cytosolic APX isoforms, including APX1, APX2, and APX6. APX1 exhibits the highest expression under normal growth conditions and accounts for ~50% of soluble APX activity [apx1 (named delt4) [delt4 mutant, suggesting that this α-cAPX antibody can be used to detect APX1 proteins   was usedproteins A. The APild-type A. We theild-type B. The inactivity . To dete mutants C. The pAactivity D and lum mutants E,F. Coll2O2; hence, we measure the H2O2 levels in pAPX1::APX1-GFP/cpr5 transgenic plants using the DAB staining method. The brown precipitate in the cpr5 mutants disappeared after APX1 was introduced into the cpr5 background  was significantly downregulated in pAPX1::APX1-GFP/cpr5 compared to that in cpr5 . The amount of Pst DC3000 (AvrRpt2) was significantly reduced in pAPX1::APX1-GFP/cpr5 transgenic plants compared with cpr5 mutants, suggesting that APX1 overexpression further enhanced cpr5 resistance to Pst DC3000 (AvrRpt2). Notably, APX1 overexpression in the wild-type also enhanced its resistance to Pst DC3000 (AvrRpt2)  G.Taken tcpr5 mutants restored growth impairment while maintaining enhanced disease resistance in Arabidopsis. Hence, we propose that the trade-off between growth and defense in cpr5 mutants can be mitigated by regulating cytosolic ROS levels  mutants presented with excessive ROS accumulation and premature leaf senescence [mod1 indicates that most mutants in which excessive ROS and cell death were suppressed were deficient in mitochondrial complex 1, suggesting that mitochondrial ROS are essential to trigger cell death [MOD1, encoding an enoyl-acyl carrier protein reductase that participates in fatty acid biosynthesis in chloroplasts [mod1 cell death, similar to its function for cpr5 cell death [cpr5 mutants contained higher AOX1a and AOX1d transcript levels. These enzymes prevent excessive ROS formation in the mitochondria [cpr5. Furthermore, chloroplastic ROS induced by MAPK cascade activation can induce cell death [mekk1 and mpk4 accumulated abundant ROS and presented with an extremely dwarfed and premature leaf senescence phenotype resembling that of cpr5 [cpr5. Nevertheless, cytosolic APX1 overexpression can remove excessive ROS in cpr5. This discovery is consistent with the previous notion that APX1 is the master regulator of ROS homeostasis in intracellular organelles [ROS are by-products of cellular metabolism. In both normal and stressed plants, ROS are produced in the apoplast, chloroplasts, mitochondria, and peroxisomes . The cpre stress . Howeverd plants . Arabidonescence . Suppresll death . MOD1, eroplasts , triggerroplasts . NADPH oll death . Comparechondria ,53. Thesll death . The MAP of cpr5 . It is, ganelles .cpr5 mutants showed increased Pst DC3000 resistance mainly due to high levels of salicylic acid (SA), an important phytohormone in plant pathogen resistance [nahG introduction in cpr5 restored its defenses to the wild-type levels, whereas the premature leaf senescence in cpr5 was not fully recovered [cpr5. It is generally accepted that SA and ROS are mutually regulated and form a self-amplifying loop under biotic stress [rbohD mutants [rbohD into cpr5 neither removed its excessive ROS nor restored premature leaf senescence. These results suggest that high SA levels might not be the main cause of excessive ROS accumulation in cpr5, which might originate from intracellular organelles. Thus, this provides a possibility to uncouple ROS-mediated growth retardation from SA-mediated defense. The sistance ,4,56. SAecovered , suggestc stress ,58. Howe mutants ,59,60. Ccpr5 mutants showed enhanced pathogen resistance but retarded growth. In the present study, we uncoupled plant growth from plant pathogen resistance by removing excessive ROS via APX1 overexpression.Plants have evolved complex mechanisms to balance growth and defense in the process of adapting to diverse biotic and abiotic stressors. The