Intriguingly, IsiA can also function as an independent pigment protein complex in the thylakoid membrane and facilitate the dissipation of excess energy, providing photoprotection. The molecular basis of the IsiA-mediated excitation quenching mechanism remains poorly understood. In this study, we demonstrate that IsiA uses a novel cysteine-mediated process to quench excitation energy. The single cysteine in IsiA in the cyanobacterium Synechocystis sp. strain PCC 6803 was converted to a valine. Ultrafast fluorescence spectroscopic analysis showed that this single change abolishes the excitation energy quenching ability of IsiA, thus providing direct evidence of the crucial role of this cysteine residue in energy dissipation from excited chlorophylls. Under stress conditions, the mutant cells exhibited enhanced light sensitivity, indicating that the cysteine-mediated quenching process is critically important for photoprotection.Oxygenic photosynthetic organisms have evolved a multitude of mechanisms for protection against high-light stress. IsiA, a chlorophyll  This pi in IsiA . Since t in IsiA , 34. AltN” motif  located N” motif . An aligN” motif . InteresN” motif , a valinSynechocystis strain using a CRISPR/Cas12a (Cpf1) system  system . The resomplexes . In thisa Qy bands with an absorption maximum at 670.8 nm . Th. ThisiA  content . In addia contents, while the WT showed an ∼30% increase in phycobilin and an ∼50% increase in Chl a contents. On the other hand, under high-light and iron-depleted conditions, phycobilin and Chl a contents in both strains decreased pronouncedly. Most striking was the change in the Chl a content of the C260V mutant, an ∼55% reduction. The higher phycobilin-to-Chl a ratio in the C260V mutant under high-light and iron-depleted conditions  in 3 days than the WT strain  is facilitated by electron transfer between the excited BChl a and the thiyl radical at a cysteine residue  in IsiA with a valine. The essentially identical absorption spectra of C260V and WT IsiA (as well as WT PSI-IsiA and PSI-C260V IsiA) demonstrate that C260V IsiA maintains all of the Chl 673.8 nm  and D, i673.8 nm , 47. WhiSynechocystis sp. PCC 6803 (PDB accession number 6NWA) at a 3.5-Å resolution (a5 (505.q), Chl a6 (506.q), and Chl a14 (514.q). In particular, the edge-to-edge distances from Chl a5 and Chl a6 are 5.7 Å and 3.7 Å, respectively. These distances are short enough to facilitate electron transfer between these Chl a molecules and C260, the basis for the proposed Cys-mediated excitation quenching mechanism (Synechococcus sp. PCC 7942 (PDB accession number 6KIF) and Thermosynechococcus vulcanus (PDB accession number 6K33) also exhibit similar associations between these conserved chlorophylls and the unique Cys residue  sequence and the cysteine coding sequence, into the KpnI site on the editing vector. The resulting plasmid, pSL2854, was verified by sequencing and transferred to WT Synechocystis cells using the Escherichia coli strain containing the pRL443 and pRL623 plasmids in triparental conjugation  system reported previously . The ediA plasmid to generate the C260V-His strain was constructed by replacing the kanamycin resistance gene in the plasmid that was used to generate an IsiA-His strain  with a gSynechocystis cells were grown photoautotrophically in BG11 under 30 μmol photons m−2 s−1 of white light at 30°C. After 5 days, cells were harvested and washed three times with YBG11−Fe, a modified medium without any added iron (−2 s−1 (low light) or 800 μmol photons m−2 s−1 (high light). For iron-starved liquid cultures, BG11 was replaced with YBG11−Fe with or without the addition of the chelator deferoxamine (DFB) to a final concentration of 50 μM, depending on the experimental settings. The OD730 was continuously recorded every 10 min over the course of the growth experiments. After 3 days of growth, cells were harvested and counted. One milliliter of each culture was used to obtain the absorption spectra, and the rest of the cultures were divided based on the same cell number, resuspended in RB , and then stored at −80°C for future use.Wild-type and C260V ded iron , 56. Theg for 15 min and washed with RB once. The resulting pellet was resuspended in RB, solubilized with β-d-dodecyl maltoside (DDM) , and incubated on ice in the dark with gentle stirring for 30 min. The sample was then centrifuged at 30,000 × g for 30 min, and the resulting supernatant, the solubilized thylakoid membranes, was stored at −80°C for future use.The cells were thawed on ice prior to thylakoid membrane extraction. Cells were broken by bead beating as described previously , 58, wit−2 s−1 as mentioned above. The cells were harvested after 3 days and diluted to an OD730 of 0.01, and the cells were counted on an automated cell counter  with further manual curation to improve accuracy.Cell cultures were grown in MC-1000 multicultivators in BG11 and YBG11−Fe under 200 or 800 μmol photons m−1 cm−1 for P700 was used to estimate the photoactive PSI content.Cells were harvested after 3 days and adjusted to the same cell numbers. With the addition of 10 μM 3--1,1-dimethylurea (DCMU) and 20 μM dibromothymoquinone (DBMIB), which block linear and cyclic electron flow, respectively, the absorbance at 705 nm of P700 in each sample was recorded for 5 s under saturating light on a JTS-10 pump probe spectrophotometer (Bio-Logic Instruments). A molar extinction coefficient of 70 mMSolubilized thylakoid membranes of C260V and WT strains were prepared as mentioned above. The solubilized thylakoid membranes were fractionated by denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by protein immunoblotting as described previously , 60. Fraa concentration was estimated by a methanol extraction method (A620 − A730) − 0.0355 × (A678 − A730), where A620, A678, and A730 are the absorbances at 620, 678, and 730 nm, respectively, in a 1-cm path length . Time-resolved fluorescence (TRF) experiments were carried out using two different setups. For the recording of images of fluorescence profiles of PSI-IsiA supercomplexes at 77 K, a setup based on a Hamamatsu (Japan) universal streak camera described previously  was used


Multiple system atrophy (MSA) is a rapidly progressive, fatal neurodegenerative disease of uncertain aetiology that belongs to the family of α-synucleinopathies. It clinically presents with parkinsonism, cerebellar, autonomic, and motor impairment in variable combinations. Pathological hallmarks are fibrillary α-synuclein (αSyn)-rich glial cytoplasmic inclusions (GCIs) mainly involving oligodendroglia and to a lesser extent neurons, inducing a multisystem neurodegeneration, glial activation, and widespread demyelinization. The neuronal αSyn pathology of MSA has molecular properties different from Lewy bodies in Parkinson’s disease (PD), both of which could serve as a pool of αSyn (prion) seeds that could initiate and drive the pathogenesis of synucleinopathies. The molecular cascade leading to the “prion-like” transfer of “strains” of aggregated αSyn contributing to the progression of the disease is poorly understood, while some presented evidence that MSA is a prion disease. However, this hypothesis is difficult to reconcile with postmortem analysis of human brains and the fact that MSA-like pathology was induced by intracerebral inoculation of human MSA brain homogenates only in homozygous mutant 53T mice, without production of disease-specific GCIs, or with replication of MSA prions in primary astrocyte cultures from transgenic mice expressing human αSyn. Whereas recent intrastriatal injection of Lewy body-derived or synthetic human αSyn fibrils induced PD-like pathology including neuronal αSyn aggregates in macaques, no such transmission of αSyn pathology in non-human primates by MSA brain lysate has been reported until now. Given the similarities between αSyn and prions, there is a considerable debate whether they should be referred to as “prions”, “prion-like”, “prionoids”, or something else. Here, the findings supporting the proposed nature of αSyn as a prion and its self-propagation through seeding as well as the transmissibility of neurodegenerative disorders are discussed. The proof of disease causation rests on the concordance of scientific evidence, none of which has provided convincing evidence for the classification of MSA as a prion disease or its human transmission until now. Multiple system atrophy (MSA) is a fatal adult-onset neurodegenerative disorder of uncertain aetiology with a mean incidence of 0.6–0.7 cases per 100,000 person-years. It is clinically featured by various combinations of parkinsonism, cerebellar impairment, autonomic and motor dysfunction due to the degeneration of striatonigral, olivopontocerebellar, and autonomic nervous systems ,3,4 causC) into the pathologic isoform PrPSc rather than by maturation in a real viral genome [C into PrPSc [Sc aggregation also enable the self-templating of these pathologic proteins [In a series of articles, Prusiner et al. assessed whether αSyn aggregates may act as a prion disease ,46,48,49l genome ,55. In pto PrPSc ,57. The to PrPSc ,59. Manyproteins ,60. Simiproteins . Specifiproteins . In DLB,proteins . Proteinproteins . The difproteins  and haveproteins .C) mediates αSyn uptake, localization, and toxicity in vitro and in vivo confirmed previous results which showed that PrPC internalizes soluble misfolded αSyn, indicating its important role in its internalization required for the intercellular spread of αSyn [Sc) [Using this modern definition, αSyn aggregates can be classified as prions as they are able to self-multiply during disease, leading to cellular transmission and the spreading of protein aggregates ,66. Endo of αSyn . Cell in of αSyn . Aggrega of αSyn . This noSyn [Sc) . The abiSyn [Sc) . VariousSyn [Sc) ,83,84), Syn [Sc) ,86,87. ASyn [Sc) ,85,88. MSyn [Sc) ,89. The Syn [Sc) , whereasSyn [Sc) . Their rSyn [Sc) ,92.This mechanism would include the misfolding (from α-helix to β-sheet) of native proteins that aggregate into “seeds” that structurally have the capacity to corrupt proteins in their physiological conformation and induce their misfolding. Aggregated αSyn moreover can disrupt glial function, thus contributing to neurodegeneration through various pathways . This prIt has been suggested to change the definition of prions to “proteinaceous nucleating particle”, to avoid the infectious implication and to highlight the molecular action of the agents . The terThe theory that the prion-like mechanisms may underlie neurodegenerative disorders was supported by the demonstration of Lewy pathology after therapeutic transplantation in people with PD, which was discovered not only in the host neurons but also in grafted dopamine neurons about two decades after transplantation. It was hypothesized that prion-like transfer of αSyn might underlie the unexpected pathology in these patients ,99,100. The inoculation of wild-type (WT) mice with synthetic mouse αSyn pre-formed fibrils (PFFs) into striatum induced widespread αSyn pathology and dopaminergic neuronal loss in the substantia nigra compacta, whereas αSyn knockout mice inoculated with PFFs did not develop αSyn pathology . This suThe intrastriatal injection of PFF αSyn in rodent brains induced a PD-like propagation of Lewy body (LB) pathology together with significant nigrostriatal neurodegeneration. The injection of exogenous αSyn PFFs into the putamen of non-human primates (cynomolgus monkeys) resulted in a significant reduction in dopaminergic neurons in the ipsilateral substantia nigra (−29.3%), downregulation of the dopamine markers tyrosine hydroxylase and Nurr1, associated with LB-like intraneuronal αSyn-positive inclusions; all taken together indicative of early PD . PreviouαSyn has a prion-like property, the propensity to aggregate, that converts its physiological protein conformation into a pathogenic one, forming disease-causing fibrils. The aggregation of these fibrils and subsequent inclusion formations are suggested to interfere with vesicular trafficking and organelle functions in neurons . HoweverThe intracerebral injection of insoluble αSyn into WT mice induced prion-like propagation of phosphorylated αSyn pathology even one month after injection, while injection into αSyn-knockout mice failed to induce any pathology . AbundanHomozygous tg mice expressing human αSyn with A53T mutation, termed TgM83+/+ mice, spontaneously develop motor deficits at about one year of age, along with widespread αSyn pathology . InoculaC oligomers are developed based on the observation that only oligomers, not monomers, are infectious [The mechanisms of the seeding of αSyn pathology in the nervous system are defined by several factors that can differently influence the pathology among strains, thereby causing distinct disease entities ,144,145.fectious .There are several challenges to the hypothesis that MSA is a prion-disease. First, endogenous WT αSyn is insufficient to propagate αSyn pathology and mutated αSyn is needed as a template. Tg Nbm mice inoculated with the PFFs of brain homogenates from MSA brains developed “prions”, while the control sample did not. Transmission of αSyn “prions” to a second synucleinopathy model and their ability to propagate between two distinct mouse lines while retaining strain-specific properties were suggested to provide compelling evidence that MSA is a prion disease . AnotherFurthermore, GCIs, the morphological hallmarks of MSA, have never been identified in WT mouse brains inoculated with MSA-derived αSyn . In factC and αSyn. PrPC knockdown in neuroblastoma cells was shown to attenuate the uptake of recombinant αSyn oligomers with a similar effect observed when comparing αSyn uptake in mouse primary hippocampal neurons, prepared from WT or PrPC knockout mice. The latter developed lower levels of αSyn aggregates in the cortex, striatum, thalamus, and hippocampus, suggesting that PrPC may facilitate the uptake and aggregation of αSyn oligomers [C and αSyn oligomers and noted that PrPC neither binds to αSyn oligomers nor mediates their detrimental effects [C, and it is possible that future studies could demonstrate that both PrPC-dependent and -independent pathways could play a role in the pathogenesis of synucleinopathies [Sc was required to promote the efficient internalization and spreading of abnormal αSyn between cells. However, recent studies showed that PrPSc was able to efficiently propagate in the brain of animals even in the absence of αSyn, suggesting that this protein may not act as a key modulator of prion propagation. This suggests that αSyn may take part in this process of self-propagation but is not specifically required for sustaining prion conversion and propagation [Another problem is the relation between PrPligomers . They fuligomers , providiligomers . However effects . It coulopathies . Accordiopathies . On the pagation . Prion spagation . Moreovepagation . A reviepagation . Furtherpagation ,162. Up pagation  before apagation .In conclusion, one may postulate that even if the prion-like spreading of αSyn in experimental model systems may justify the view that the progression of neurodegeneration in MSA reflects the cell-to-cell spread of pathological αSyn, this is not sufficient to define MSA as a classical prion disease. After the injection of αSyn fibrils into the olfactory bulb of WT mice, despite the transneuronal spreading of αSyn aggregates to over 40 other brain regions, even at 18 months PI, there was no loss of mitral cells in the olfactory bulb. The lack of progression of αSyn pathology may be due to the compromise of the neuronal circuitry, and the activation of proteolytic mechanisms in resilient neurons may counterbalance the spread of pathogenic αSyn . This un


PSII) declined significantly in ‘P’ under salt stress. The photochemical quenching was reduced while nonphotochemical quenching rose in ‘P’ under salt stress. Interestingly, the regenerants  exhibited high apparent salt tolerance by maintaining quite stable Chl fluorescence parameters. Rising NaCl concentration led to a substantial increase in foliar proline, malondialdehyde and soluble carbohydrates accumulation in ‘P’. On the contrary, ‘R18’, ‘R19’, ‘R23’ and ‘R30’ exhibited a decline in soluble carbohydrates and a significant enhancement in starch under salinity conditions. The water status reflected by midday leaf water potential (ψl) and leaf osmotic potential (ψπ) was significantly affected in ‘P’ and was maintained a stable level in ‘R18’, ‘R19’, ‘R23’ and ‘R30’ under salt stress. The increase in foliar Na+ and Cl− content was more accentuated in parental plants than in regenerated plants. The leaf K+, Ca2+ and Mg2+ content reduction was more aggravated under salt stress in ‘P’. Under increased salt concentration, ‘R18’, ‘R19’, ‘R23’ and ‘R30’ associate lower foliar Na+ content with a higher plant tolerance index (PTI), thus maintaining a normal growth, while foliar Na+ accumulation was more pronounced in ‘P’, revealing their failure in maintaining normal growth under salinity stress. ‘R18’, ‘R19’, ‘R23’ and ‘R30’ showed an obvious salt tolerance by maintaining significantly high chlorophyll content. In ‘R18’, ‘R19’, ‘R23’ and ‘R30’, the enzyme scavenging machinery was more performant in the roots compared to the leaves. Salt stress led to a significant augmentation of catalase, ascorbate peroxidase and guaiacol peroxidase activities in the roots of ‘R18’, ‘R19’, ‘R23’ and ‘R30’. In contrast, enzyme activities were less enhanced in ‘P’, indicating lower efficiency to cope with oxidative stress than in ‘R18’, ‘R19’, ‘R23’ and ‘R30’. ACC deaminase activity was significantly higher in ‘R18’, ‘R19’, ‘R23’ and ‘R30’ than in ‘P’. The present study suggests that regenerated plants ‘R18’, ‘R19’, ‘R23’ and ‘R30’ showed an evident stability in tolerating salinity, which shows their potential to be adopted as interesting selected mutants, providing the desired salt tolerance trait in eggplant.Previously, an efficient regeneration protocol was established and applied to regenerate plants from calli lines that could grow on eggplant leaf explants after a stepwise in vitro selection for tolerance to salt stress. Plants were regenerated from calli lines that could tolerate up to 120 mM NaCl. For further in vitro and in vivo evaluation, four plants with a higher number of leaves and longer roots were selected from the 32 plants tested in vitro. The aim of this study was to confirm the stability of salt tolerance in the progeny of these four mutants . After three years of in vivo culture, we evaluated the impact of NaCl stress on agronomic, physiological and biochemical parameters compared to the parental control (‘P’). The regenerated and control plants were assessed under in vitro and in vivo conditions and were subjected to 0, 40, 80 and 160 mM of NaCl. Our results show significant variation in salinity tolerance among regenerated and control plants, indicating the superiority of four regenerants  when compared to the parental line (‘P’). In vitro germination kinetics and young seedling growth divided the lines into a sensitive and a tolerant group. ‘P’ tolerate only moderate salt stress, up to 40 mM NaCl, while the tolerance level of ‘R18’, ‘R19’, ‘R23’ and ‘R30’ was up to 80 mM NaCl. The quantum yield of PSII (Φ Soil salinity is a major abiotic factor, influencing over 1000 million ha of land in several parts of the world, and is one of the main current constraints in developing irrigated agriculture . An exceThe investigation of salt tolerance during different growth stages is important for elucidating saline limits at each developmental phase . Salt stSalts influence plant growth by increasing the osmotic pressure in the soil and interfering with plant nutrition . Carbohy2O2 produced in peroxisomes and chloroplasts diffuses to the cytosol and is converted to hydroxyl radicals by the Fenton reaction [2−), singlet oxygen (O2), hydrogen peroxide (H2O2) and hydroxyl radicals (OH−)) in plant cells under stress contributes to the damage of cellular components, including DNA, proteins and membrane lipids, and leads to the destruction of photosynthetic pigments [2O2, which is further scavenged by CAT and various peroxidases  to H2O.Abiotic stress contributes to the production of reactive oxygen species (ROS) in plants. ROS are continuously produced in chloroplasts, mitochondria and peroxisomes in higher plants . Howeverreaction . The ovepigments ,26. Abiopigments . Accordipigments , to scavpigments . The autpigments  showed t+ with K+, Ca2+ and Mg2+, and may reduce the availability of the beneficial nutrients [On the other hand, salinity tolerance usually correlates with high amounts of compatible solutes and efficient compartmentation, as shown in a large set of studies . Both sautrients .Salinity also affects photosynthesis mainly through a reduction in leaf area, chlorophyll content and stomatal conductance, and to a lesser extent through a decrease in photosystem II efficiency .A.Additionally, in susceptible tomato cultivars  chlorophPlants develop several strategies to overcome NaCl stress, including the plant growth-promoting bacteria (PGPB) living in the rhizosphere and rhizoplane of the plants ,38,39. TSolanum melongena L.) is one of the most popular and economically important vegetable crops worldwide [Eggplant  in vitro selection and screened for salt tolerance . These sThe present study aimed to identify possible stable salt-tolerant genotypes among the progeny of somaclonal variants and characterize them through agronomic, physiological and biochemical parameters.We analyzed the progeny of selected eggplant somaclonal variants, which had been obtained previously by a stepwise increasing salinity  in vitro selection and screened for salt tolerance. These somaclones were regenerated on the calli lines that could tolerate up to 120 mM NaCl. Out of thirty two plants tested in vitro, four plants with a higher number of leaves and root length were used for further in vitro and in vivo evaluation.Although it does not fully reproduce the field behaviour of plants, the germination kinetics under controlled saline conditions provide a trend about the differential behaviour of the studied cultivars in response to the applied stress.We studied the impact of increasing NaCl on germination kinetics as assessed by the final germination , mean daily germination  and mean germination time  of the parent and regenerant lines .In the control treatment, FG was high, reaching 84.3% in the parental control (‘P’) and 100% in all regenerants . A salt concentration of 40 mM significantly reduced FG in ‘P’ (63.5%) but had little effect on ‘R18’ (100%), ‘R19’ (100%), ‘R23’ (97%) and ‘R30’ (95%). When the salt concentration increased, the FG decreased significantly. The highest NaCl concentration (160 mM) significantly reduced FG in all lines evaluated. This reduction was clearly stronger in ‘P’ (91.7%) than in ‘R18’ (60%), ‘R19’ (57.1%), ‘R23’ (61.1%) and in ‘R30’ (64.5%) A.A salt concentration of 40 mM NaCl already reduced MDG in ‘P’ B. A highMGT is a measure of the rapidity of germination, with lower values indicating faster germination. The evaluation of the impact of salinity on the MGT showed a clear difference between the parental and regenerated lines. The first dissimilarity was observed in the control treatment, showing that germination was faster in ‘R18’, ‘R19’, ‘R23’ and ‘R30’ than in ‘P’. The higher salt concentration increased MGT significantly from 80 mM NaCl on. At 160 mM NaCl, MGT doubled in all lines compared to the controls C.The effect of increasing NaCl concentration on parent and regenerant growth parameters, as evaluated by plant height (PH), leaf number (LN), aerial fresh weight (FW), aerial dry weight (DW) and tissue water content (TWC) of eggplant seedlings after 45 days of growth, is shown in Increasing salinity level had no effect on LN in ‘R18’, ‘R19’, ‘R23’ and ‘R30’ but decreased the same parameter significantly in ‘P’ by 24% and 68% at 40 mM and 80 mM NaCl, respectively, compared to controls. However, PH was decreased for 80 mM NaCl in ‘P’, ‘R18’, ‘R19’, ‘R23’ and ‘R30’ with a reduction of 60%, 55%, 50%, 34.5% and 46%, respectively. For the parental control, LN declined from 40 mM NaCl and PH was reduced from 80 mM on . The FW of all lines decreased as the salinity increased . AlthougUnder salinity conditions, DW declined in all lines. The most important decline was found in ‘P’ by 78.3% for 80 mM NaCl . On the The impact of increasing NaCl levels on plant–water relations, as assessed by the midday leaf water potential (ψl) and leaf osmotic potential (ψπ) of parents and regenerants, is given in l in the parental control and attained −1.97 MPa after 30 days at the highest salt stress concentration (160 mM NaCl)  A. In conc stress A.π showed a similar trend to Ψl under salinity conditions. After 30 days of salt stress application, a salt concentration of 160 mM NaCl decreased Ψπ significantly in ‘P’ and attained −2.59 MPa. On the contrary ‘R18’, ‘R19’, ‘R23’ and ‘R30’ were able to maintain their Ψπ at a more stable level , aerial dry weight (DW) and tissue water content (TWC) of parents and regenerants, is shown in All salinity levels contributed to a significantly higher shoots biomass in the regenerated plants when compared to the control parental line . Indeed,The impact of salt stress on the yield parameters (fruit number per plant and mean fresh weight per fruit) of parents and regenerants subjected to an increasing salt stress level for five weeks and harvested 45 days after flowering is given in There was a significant difference in the number of fruits observed between the regenerants and the parental line. In the absence of salt stress, all regenerated plants produced more fruit than the parental line. Compared to the parental line, all plants produced from somaclonal lines also yielded more fruit at a salt concentration up to 80 mM NaCl, with the increase ranging from 225%  to 255% (‘R23’) . At 120 The effect of different salt concentrations  after 30 days under NaCl stress on the chlorophyll fluorescence parameters of parents and regenerants  is shown in v/Fm occurred in ‘P’ for the highest salt concentration (160 mM NaCl). However, ‘R18’, ‘R19’, ‘R23’ and ‘R30’ were hardly affected by salinity in all treatments . After 30 days of salt stress imposition, a significant reduction in Featments A.PSII was found only in ‘P’ after 30 days of salt stress imposition . In ‘P’, proline accumulation was hugely increased depending on the intensity of salt stress. The strongest increase in proline accumulation was found in ‘P’, at 80 mM NaCl A.The same trend observed for proline was also found in MDA under saline conditions. In ‘P’, MDA rose 7-fold in the 80 mM NaCl level compared to the control, whereas the MDA increase in ‘R18’, ‘R19’, ‘R23’ and ‘R30’ was, respectively, 2.2-fold, 2.1-fold, 1.6-fold and 2.4-fold for the highest salt level (80 mM NaCl) compared to the respective controls C.Increasing salt concentration contributed to a significant rise in proline content in the parental control and regenerated plant lines. However, proline’s increase was less accentuated in ‘R18’, ‘R19’, ‘R23’ and ‘R30’ than in ‘P’. A salinity level of 160 mM NaCl increased proline accumulation, respectively, by 89.1%, 59.01%, 55.7%, 55.5% and 51.6% in ‘P’, ‘R18’, ‘R19’, ‘R23’ and ‘R30’ relative to controls B. Although MDA content rose significantly in the parental control and regenerated plant lines, the accumulation was more accentuated in ‘P’ than in ‘R18’, ‘R19’, ‘R23’ and ‘R30’. The highest salt concentration (160 mM NaCl) caused MDA to increase, respectively, 8-fold, 3-fold, 2.4-fold, 2-fold and 2.8-fold in ‘P’, ‘R18’, ‘R19’, ‘R23’ and ‘R30’ compared to controls. Therefore, the lipid peroxidation level was higher in the parental control than in the regenerated plants under salinity conditions D. It is The impact of NaCl concentration on carbohydrates as assessed by glucose, fructose, sucrose, and starch levels in the leaves of parents and regenerants is given in The parental control line exhibited a significant elevation in foliar glucose, fructose and sucrose content under increasing salinity A–C. In cSalt stress augmentation generated a significant rise in starch content in ‘R18’, ‘R19’, ‘R23’ and ‘R30’; however, there was significantly less starch piling up in ‘P’ D.3−, Cl− and on Na/K and Na/Ca ratios in control parent and regenerant leaves is shown in The effect of NaCl salinity on the accumulation of K, Ca, Mg, Na, P, NO+ and Cl- accumulation in parent and all regenerants , ‘R19’ (23.7%), ‘R23’ (30%) and ‘R30’ (25.8%) was less accentuated than for ‘P’ (42.1%) when compared to their respective controls  decreased Mg content in ‘P’ and ‘R23’ . +/K+ and Na+/Ca2+ ratios rose significantly by increasing salinity for the parental control and regenerated plants, it was evident that these ratios were higher in ‘P’ than in ‘R18’, ‘R19’, ‘R23’ and ‘R30’, for all salinity treatments  was calculated based on the total fresh weight (FW) in salt-stressed plants per total FW in control plants.+ content. We obtained a significant negative correlation for parental control and all regenerated plants . While salt concentration rose, ‘R18’, ‘R19’, ‘R23’ and ‘R30’ maintained high PTI and low foliar Na+ content, thus contributing to normal growth  of parental control and somaclonal lines subjected to 40 mM, 80 mM and 160 mM NaCl was estimated and linked to the foliar Nal growth . In contf growth .The deaminase activity of the enzyme 1-aminociclopropane-1-carboxylase (ACC) is one of the key traits used by PGPB to decrease ethylene levels under salt stress. The ACC deaminase converts ACC into ammonia and α-ketobutyrate.Fifteen bacteria were isolated from the roots of ‘P’, ‘R18’, ‘R19’, ‘R23’ and ‘R30’ on enrichment media, of whic, four strains had the capacity of growing on DF minimal salt medium supplemented with 3 mM ACC as a nitrogen source, implying ACC deaminase activity. The ACC deaminase activity of these four isolates, named ‘ACC1’, ‘ACC2’, ‘ACC3’ and ‘ACC4’, was further quantified in terms of α-ketobutyrate production.The ACC deaminase activity of isolates was assessed by α-ketobutyrate production via the catalyzation of the deamination reaction of the sole nitrogen source, ACC in DF minimal salt broth media at 540 nm. The ACC deaminase activity of the tested isolates exhibited variation, ranging from 400 to 1600 nmol α-ketobutyrate per mg of cellular protein per hour A–D. −1 h−1), in 40 mM NaCl (ranging from 410 to 535 nmol α-ketobutyrate mg protein−1 h−1), in 80 mM NaCl (ranging from 850 to 920 nmol α-ketobutyrate mg protein−1 h−1) and in 160 mM NaCl (ranging from 1500 to 1600 nmol α-ketobutyrate mg protein−1 h−1)  A–D. The The quantification of IAA production by four ACC deaminase-producing isolates was achieved at 530 nm through supplementation of L-tryptophan to the growth media. The results are shown in The accumulation of IAA was significantly more enhanced in regenerants than in the parental line in all treatments for all isolates. The highest IAA production was reached for the regenerated plants by ‘ACC2’ for 80 mM NaCl (ranging from 36.5 µg/mL to 40.1 µg/mL) and 160 mM NaCl (ranging from 44.5 µg/mL to 46 µg/mL for 160 mM NaCl) and by ‘ACC3’ for 80 mM NaCl (ranging from 38.5 µg/mL to 40.5 µg/mL) and 160 mM NaCl (ranging from 43 µg/mL to 45.1 µg/mL for 160 mM NaCl) C,D.l, TWC, FW and DW had a negative correlation with PCA1. The loadings of ψl, MDA, starch and proline had a negative correlation with PCA2. For regenerated and parental lines, the scores of the PCA moved to lower FW, DW, TWC, chlorophyll, qp, Fv/Fm, PSII and ψl and higher MDA, proline, starch, Na/K ratio in leaves, APX-L, CAT-L, POD-L, POD-R, APX-R and CAT-R values under increasing NaCl concentration. In parental line P, control and salt-stressed plants were highly separated along PCA1 in comparison to somaclones ‘R18’, ‘R19’, ‘R23’ and ‘R30’ (A scores scatter plot of the first two PCAs (explaining 63.5% of the variation) shows a clear distinction between the regenerated plants and parent after 30 DSS . The loand ‘R30’ . ControlSalinity is still considered the most important abiotic stress threatening agriculture . SomacloThe germination of seeds is the most vulnerable stage of development; therefore, the germinating seeds of most plant species do not tolerate salt. This has already been demonstrated by numerous vegetables such as lettuce  and beanIt should be noted that in our experiment, the germination of the control seeds decreased significantly at 80 mM NaCl. The same finding was reported for eggplant, in which a strong inhibition of germination at 100 mM NaCl was observed . MoreoveSimilar results, describing a drastic decrease in seed germination, were found in tomato at 100 mM NaCl and in red beet, bell pepper, melon and broccoli at 150 mM NaCl  and in PThe salt sensitivity of eggplant during germination was confirmed at further growth stages. The decrease in growth parameters  was more pronounced for ‘P’ compared to ‘R18’, ‘R19’, ‘R23’ and ‘R30’ . Indeed,2 uptake, contributing to the dysfunction of the photosynthetic apparatus and the decrease in growth [2 partial pressure . Moreoviewed by ,72,73,74To verify the impact of increasing salinity on the photochemistry of PSII, we performed Chl a fluorescence measurement in salt-tolerant eggplant mutants selected in vitro. Our study shows that increasing salinity has no significant effect on Fv/Fm in all regenerants after 30 days of salt stress. Similar findings have been reported for salt-tolerant tomato  and wheaThe study of ΦPSII reflects PSII activity and adaptation . It is cSimilar to ΦPSII, increasing salt stress also contributed to lower qp in ‘P’, ‘R18’, ‘R19’, ‘R23’ and ‘R30’. However, the qp decrease was less pronounced in regenerants than in the parental control. As stated by , qp refl2 under abiotic stress. Stomata closure contributes to a limitation of transpiration rates [As highlighted by , photochon rates  and a reon rates  with incon rates . During on rates ,85,86,87Photosystem II is very vulnerable to salt stress. The absence of a significant effect of salinity on ΦPSII in all regenerated plants can be explained by the presence of an effective electron sink. Interestingly, the plants manage to maintain an effective conversion system of excitation energy via the downregulation of PSII RCs under salt condition . PhotoreThe decline in the non-photochemical energy loss is likely the main mechanism used to cope with photodamage . The parIt is clear that the increase in the NPQ of ‘P’ contributed greatly to counteract the decrease in Fv/Fm. The increased NPQ dissipates some of the abundant excitation energy at the expense of photochemical utilization ,92, leadUnder salinity conditions, foliar water potential and osmotic potential/EC changes are interrelated. It is obvious that the capacity of plants in maintaining a stable water potential difference between leaves and nutrient solution is of immense importance in order to impede desiccation. Salt stress decreased leaf turgor and atmospheric vapor pressure .In , it was l and ψπ at relatively stable levels under increasing salinity levels, while the parental control failed to do so. It is evident that the decrease in ψl in ‘P’ occurred simultaneously with the reduction in ψπ, thus keeping the same turgor status as the control. The aggravated decline in foliar ψπ in parental controls might be explained by a substantial accumulation of Na+ and Cl−  represents an efficient stress indicator. All regenerated plants succeeded in maintaining higher and quite stable TWC compared to the parental control regardless of the salt concentration. Our findings indicate the ability of ‘R18’, ‘R19’, ‘R23’ and ‘R30’ in maintaining enhanced turgor and cell expansion using a performant plasmolytic process in order to overcome salt stress ,98. To cProline catabolism is upregulated under increasing salinity concentrations by impeding its oxidation, which contributes to its substantial accumulation. Our results show that proline rose under salinity conditions in both parental control and regenerated plants. However, proline accumulation increase was more pronounced in ‘P’ than in ‘R18’, ‘R19’, ‘R23’ and ‘R30’. In the current work, it is noteworthy that the enhancement of proline accumulation seems to be induced by the metabolic damage generated by salt stress rather than appearing as a tolerance feature ,104.Saline conditions contribute to oxidative stress . A high The increase in carbohydrates as a key factor involved in osmotic adjustment was stated previously by many authors ,112. TheOur results indicate that regenerated plants exhibited the ability to overcome a long and increasing salt stress, while they were successful in maintaining better growth by maintaining a higher chlorophyll content under salt stress conditions. In contrast, a high salinity level led to a chlorophyll content reduction in the parental control line, which contributed to an early leaf senescence and plant withering. As emphasized by , salinitThis is in line with earlier reports ,120,121 + and Ca2+ contents is crucially needed for preserving the integrity and functioning of cell membranes. The rise of Na+ and Cl− content is considered as an osmoregulation process adopted by plants under salinity condition, while a decline in K+ and Ca2+ occurred in stressed plants [As stated by , the avad plants .+, Ca2+, Mg2+ and NO3− in the parental control and in all regenerated plants. However, all the regenerated plants exhibited reduced leaf concentration of Na+ and Cl− and succeeded in keeping a more enhanced accumulation of K+, Ca2+ and Mg2+ compared to the parental control. This is consistent with earlier reports obtained by [In the present work, rising salinity level engendered an increase in foliar Na and Cl concentration associated with a reduction in Kained by ,18 for sIt is worth noting that the parental control line showed an accentuated reduction in K content. K deficiency is mainly due to the strong competition between the absorbed NaCl and specifically K uptake ,66. Ther+ and Cl− uptake and enhancing the absorption of other crucially required ions, such as K+ [To accomplish performant growth via normal cellular functioning, a reduced cytosol Na/K ratio is of immense importance in all plants. The imposition of salt stress and its continuity contributes to the inhibition of the K-specific transporters by Na, while competing with K uptake. Therefore, an excessive toxic concentration of Na is combined with a K deficiency ,126. Othch as K+ . - from the cytosol through vacuole compartmentalization [Moreover, to overcome the substantial amount of foliar Na, many glycophytes are provided with the mechanism of excluding Na and Cllization ,128. The high salt tolerance in the regenerants is closely related to a lower amount of accumulated salt ions in leaves. This might be explained by a more efficient mechanism of salt exclusion in ‘R18’, ‘R19’, ‘R23’ and ‘R30’ leaves (more tolerant) compared to ‘P’ (susceptible) .A substantial salt ion accumulation contributes to the alteration of osmoregulation, the disturbance of membrane potential equilibrium and the loss of turgor . Consequ+ concentration was evident. This result agrees with earlier findings. The authors of [+ tissue tolerance, such as the high accumulation of osmoregulants and intracellular compartmentation. The same hypothesis was adopted by [A positive correlation between PTI and leaf Nathors of , workingthors of , workingopted by  as an alUnder increasing salinity, the antioxidative machinery is upregulated in the regenerated plants. The effective protection developed by ‘R18’, ‘R19’, ‘R23’ and ‘R30’ against ROS in eggplant may be due to an efficient antioxidative system. These results are in line with many earlier reports ,133. 2O2 in peroxisomes is scavenged by CAT, and H2O is transformed into water and molecular O2. The enhanced activities of CAT and APX effectively decreased the rising amount of H2O2, thus leading to the amelioration of the cell membrane integrity and stability. In the parental control and the regenerated plants, CAT activity rose proportionally with the salt stress level. In ‘R18’, ‘R19’, ‘R23’ and ‘R30’, the most elevated salt concentration (160 mM NaCl) generated the highest CAT activity, thus leading to improved protection against ROS in all the regenerated plants. This result agrees with previous reports, stating that many salt-tolerant species exhibited higher CAT activity, such as maize [As emphasized by ,135, theas maize , sesame as maize , potato as maize , melon [as maize  and wheaas maize . Severalas maize ,137,141.2O2 in higher plants [The susceptibility of the parental control could be due to the suppression of APX activity induced by rising salinity concentrations. This result is in concordance with previous reports dealing with Triticum aestivum , potato r plants ,143. 2O2 in eggplant under salinity conditions. POD, which acts at both the intracellular and extracellular level, is an effective enzyme involved in the elimination of H2O2 [Our findings show that POD enzyme activities in parental control and regenerated plants were more enhanced with increasing salinity levels and were more elevated in ‘R18’, ‘R19’, ‘R23’ and ‘R30’  than in ‘P’ . It is noteworthy that several previous studies stated that POD enzymes are associated with salt tolerance in melon , soybean of H2O2 . 2O2 in eggplant. The salt tolerance response of the regenerated plants could be engendered by its enhanced capacity in downregulating ROS production or by its improved ability to overcome ROS compared to the parental control. Our findings indicate that antioxidant enzyme machinery may have a strong contribution in scavenging HPopulus euphratica, [Plantago. This is in line with earlier results reported by  working hratica,  working hratica,  working ACC deaminase activity exhibited by PGPR has been proved to play a key role in improving growth and stress tolerance in plants under stressed conditions ,150. ACCUnder salt stress, PGPB contribute in enhancing IAA additional production, which may be useful in stimulating root growth, counteracting the inhibition impact of salinity on root and shoot growth and ameliorating plant physiological features . In our study, all regenerants exhibited higher IAA accumulation in all isolates and specifically in ‘ACC2’ and ‘ACC3’ compared to the parental line in response to increasing salinity, which might be explained by their superior capacity to tolerate salt stress. This is consistent with previous work reported by ,157,158.+ was one of the criteria explaining the majority of dissimilarities among parental and regenerants lines, as well as the characteristics related to the antioxidant machinery, proline, PSII, MDA and the leaf water potential.In addition, the PCA analysis showed that NaIn this study, the progeny of somaclonal variants showed significant dissimilarities for several characters when compared to the parental line, and a higher level of salt tolerance. The statistical analysis of the whole set of data included in the current study confirmed the change in genetic stability. Regenerants with consistent performance at advanced generations are assumed to bear gene or chromosome variations, which seem to be stable and heritable. The success in applying somaclonal variation for salt tolerance depends on the rate and type of somaclonal variation obtained. Therefore, the low phenotypic variation among regenerants could be due to low physiological disturbance during tissue culture. Moreover, the identification of regenerants with putatively increased salt tolerance showed the positive impact of somaclonal variation in improving crop species for desired characters, besides the utility of culture-induced variation in genetic upgradation.In this study, the stability of the salt tolerance of four previously obtained somaclonal variants was evaluated by characterizing the agronomic, physiological and biochemical parameters of their progeny. Seedlings from the parent plants and the regenerated plants were evaluated for salt tolerance under in vitro and realistic greenhouse conditions. Under the imposed salt stress, the results are consistent for germination kinetics, seedling growth and morphological and physiological responses, indicating that salt tolerance could be confirmed at both the germination and young plant stages. Metabolic homeostasis was less disturbed in the salt-tolerant plants, as indicated by the lower amount of MDA and loss of water content, despite the accumulation of proline and soluble carbohydrates. The salt tolerance of the regenerants was demonstrated by a stable primary photosynthetic apparatus and an effective antioxidant enzyme system. Two potential strains, ACC2 and ACC3, were found to possess other growth-promoting properties, such as the production of IAA. The efficiency of these strains in reducing salt stress and promoting plant growth was evident. The evaluation of the physiological and biochemical performance of the progeny of the in vitro regenerated plants demonstrates the great potential of callus culture for the development of variable genotypes and the selection of desirable traits in eggplant, such as genetically stable salt tolerance. From a more fundamental perspective, research on the level of field tolerance to other abiotic stresses of these genotypes selected for salt tolerance could be of interest. In previous work, an efficient regeneration protocol was developed to regenerate plants on salt-stress-tolerant calli lines derived from eggplant (‘Bonica F1’) leaf explants after a stepwise in vitro selection on media with increasing salinity  . Plants We performed two experiments: the first was an in vitro (germination and seedling growth) experiment (Exp 1), and the second was an in vivo (greenhouse) experiment (Exp 2).w/v) sucrose in 0.7 L glass vessels. The pH was adjusted to 5.8 with 1 N NaOH before autoclaving. NaCl was added to the medium at concentrations of 0 (control), 40, 80 and 160 mM.Thiram-pretreated seeds of ‘Bonica’  were rinsed with 70% alcohol for some seconds and washed with distilled water. Then, they were surface sterilized for 20 min in a 5% HazTab solution  and 0.02% Dreft , followed by a solution of mercuric chloride (0.5%) for 10 min. After three rinses with sterile distilled water, the seeds were germinated on agar-solidified (0.8%) MS  medium w–2 s–1.Four seeds per vessel were used for every treatment and cultivar, in five replicates. The cultures were maintained in a growth chamber at 28 ± 2 °C, and a 16 h photoperiod regime was provided by cool-white fluorescent lamps with a photon flux density of 36 µmol mEach 24 h for one week, we investigated the germination (radicle to emergence) and the time to germinate. We determined the final germination percentage, the mean daily germination (MDG) and the mean germination time (MGT). The mean daily germination (MDG) and the mean germination time (MGT) were calculated according to . After exposition to salt stress for 45 days (45 DSS), we randomly selected one seedling of each treatment. We determined seedling length, number of leaves, aerial fresh (FW) and dry weight (DW). The samples were then oven-dried at 70 °C for 24 h to determine the dry weight (DW). The tissue water content (TWC) was calculated using the formula: TWC = (FW − DW/FW).A minimum of two seedlings per vessel were pooled, ground in liquid nitrogen and stored at −80 °C until metabolite analysis.−2 s−1, a constant temperature of 25 °C and 70% relative humidity (RH). After 25 days, seedlings showing their second true leaf were transferred to 2-litre plastic pots filled with peat and kept in a heated greenhouse  at the Department of Plant Physiology and Biotechnology, National Institute of Agricultural Research, Tunisia. For the next 36 days, each seedling was irrigated with 250 mL of Hoagland solution [Germination took place in trays filled with peat and moistened with distilled water. The trays were placed in a controlled growth chamber at a photon flux density of 150 µmol msolution  at full midday) of the youngest fully expanded leaves. We determined the leaf osmotic potential (ψπ) according to [After being exposed to salt stress for 30 days (30 DSS), we randomly selected eight plants of each treatment. We determined shoot and leaf fresh weight (FW). The samples were then oven-dried at 70 °C for 48 h to determine the dry weight (DW). The tissue water content (TWC) was calculated using the formula: TWC = (FW − DW/FW). The Scholander pressure chamber  was used to determine the leaf water potential . After 30 min of dark adaptation, Fv/Fm was estimated as (Fm − F0)/Fm, where Fm (induced by a short pulse (0.6 s) of saturating light (3450 µmol m–2 s–1)) and F0 were the maximum and minimum fluorescence, respectively [–2 s–1) and saturating pulses every 25 s, the maximum (Fm’) and the constant-state (Fs) fluorescence signals were measured in the light-adapted leaves. Then, the actinic light was stopped and the far-red pulse was applied to obtain the minimal fluorescence after the PSI excitation (F0’). ΦPSII was calculated as (Fm’ − Fs)/Fm’ and qp was estimated as (Fm’ − Fs)/(Fm’ − F0’) [PSII × PAR × 0.84 × 0.5, where the absorbed photon energy (PAR) was assumed to be equally distributed between PSI and PSII, and 0.84 was the assumed light absorptance of the leaf. Measurements were applied on the youngest, fully developed leaf after 5, 10, 15, 20, and 30 d of salt stress (DSS) in four replicates.Chl ectively . After 4’ − F0’) . NPQ, wh’ − F0’) . The eleFor all plants and for all salt concentrations, we determined leaf chlorophyll content by means of a chlorophyll meter . We performed three SPAD measurements from three fully expanded leaves from the lower, middle and upper canopy. Thus, chlorophyll content of each plant was assayed as an average of nine SPAD readings per plant. Chlorophyll data were assessed on two dates: five and ten weeks after beginning salt stress imposition.After 30 days of salt stress, we reaped fully developed upper leaves (2 leaves/replicate in a bulked sample) between 12 h and 14 h from four plants, in each treatment (1 plant/block) and for each variety. We ground harvested leaf material in liquid nitrogen and stored it at −80 °C until analysis.g for 5 min [Soluble sugars were extracted by 80% ethanol at 70 °C for 10 min and further at 45 °C for 3 h, followed by centrifugation at 5000× or 5 min . HPLC wa+.The rest of the ethanol insoluble material was washed two times with ethanol 80% and the pellet residue was treated with HCl 1 M for 2 h at 95 °C for starch hydrolysis. Starch extraction and quantification were achieved according to  and basew/v) sulfosalicylic acid. The determination of proline was carried out using a calibration curve and expressed as µg proline g−1 FW [We quantified proline according to . In shore g−1 FW .Lipid peroxidation was investigated through the determination of malondialdehyde (MDA) ,165. In w/v) polyvinylpolypyrrolidone (PVPP). The whole extraction procedure was carried out at 4 °C. A centrifugation of the homogenates was performed at 4 °C for 15 min at 130,009× g, and enzyme activity was measured using the supernatants. Protein was quantified as described by [For protein and enzyme extractions, 0.5 g of leaf and root samples were homogenized with 50 mM potassium phosphate buffer (pH 7.8) containing 1 mM EDTA-2Na and 7%  at 240 nm. This activity was measured in a reaction mixture containing 1900 µL of potassium phosphate buffer , 100 µL sample and 1000 µL H2O2 (30 mM). CAT activity was expressed as µmol H2O2 decomposed min−1 mg−1 proteins.According to , catalas2O2 started the reaction and ascorbate oxidation was determined at 290 nm for 1 min. Activity was quantified using the extinction coefficient, e = 2.8 mM−1 cm−1. Each sample was measured in three repetitions. Results are expressed as µmol oxidized ascorbate min−1 mg−1 proteins.Ascorbate peroxidase (APX) activity (EC 1.11.1.11) was determined according to . The rea2O2. The absorbance elevation generated by the oxidation of guaiacol to tetra guaiacol was recorded for 3 min at 436 nm. POD activity was estimated from the extinction coefficient, ε = 25.5 mM−1 cm−1. Results are expressed as µmol oxidized guaiacol min−1 mg−1 proteins.Guaiacol peroxidase (POD) activity (EC 1.11.1.7) was quantified according to . The reaLeaf and root samples were harvested from four plants in every treatment (1 plant/block) and for every cultivar, washed, oven-dried at 70 °C for 48 h and finally grounded. The determination of P, K, Ca, Mg, S and Na contents was performed by ICP-OES after dry-ashing at 550 °C. A potentiometric analysis using an ion-selective electrode  for chlorides was achieved.The root samples of Bonica and Black Beauty were collected from 6 pots for each cultivar in every treatment during May and June 2021 in a heated glasshouse  at the Department of Plant physiology and Biotechnology, National Institute of Agronomic Research, Tunisia. Four plants/cultivar/treatment were collected from each pot and brought to the laboratory in closed plastic bags for further analysis. The roots were separated from each plant, crushed and mixed together to form one composite pool of root sample.The isolation of bacteria and the qualitative determination of ACC deaminase activity were achieved according to . Briefly−1 h−1.The quantitative determination of ACC deaminase activity was performed according to . This me3 + 70% perchloric acid). Development of red color (which indicates the formation of indolic compounds) with addition of Salkowski reagent and cell free culture supernatant (4:1) was measured by UV–vis spectrophotometer at 530 nm [−1.According to  the bactt 530 nm . The conp = 0.05). We performed the principal component analysis (PCA) on starch, chlorophyll, proline, MDA, PSII, qp, Fv/Fm, ψl FW, DW, TWC, Na/K ratio in leaves, POD, APX and CAT activity in leaves/roots of parental control and regenerated plants grown for 30 days under salt stress. PCAs with eigenvalues > 1, thus explaining more than a single parameter alone, were examined. For these principal components, a varimax rotation was applied to the resulting factor loading. According to [A completely random design was used in conducting all analyses. The detection of significant dissimilarities between treatments or varieties was completed using SPSS Statistics 21 after subjecting all collected data to a one-way analysis of variance (ANOVA). The comparison of means was performed using the Tukey’s multiple range test (rding to , this ro


Shifting LED wavelengths from 595 nm to 633 nm and from 595 nm to 613 nm resulted in a biomass yield decrease of ~50% and ~80%, respectively. When 630-nm light is blocked, lettuce displayed expanded plant structures and the absence of purple pigmentation. This report presents a new and feasible approach to plant photobiology studies, by removing certain wavelengths to assess and investigate wavelength effect on plant growth and photosynthesis. Findings indicate that amber light is superior to red light for promoting photosynthetic activity and plant productivity, and this could set precedence for future work aimed at maximizing plant productivity in controlled environment agriculture.Red and blue light are the principal wavelengths responsible for driving photosynthetic activity, yet amber light (595 nm) has the highest quantum efficiency and amber-rich high pressure sodium lamps result in superior or comparable plant performance. On this basis, we investigated how lettuce plant growth and photosynthetic activity were influenced by broad and narrow light spectra in the 590–630 nm range, by creating amber and red light-emitting diode (LED) spectra that are not commercially available. Four different light spectra were outfitted from existing LEDs using shortpass and notch filters: a double peak spectrum  that excluded 630-nm light, 595-nm, 613-nm, and 633-nm light emitting at an irradiance level of 50 W·m Plant lighting experiments show that red (600–700 nm) light plays a critical role in photosynthetic activity and plant development within the photosynthetically active radiation (PAR) spectrum 400–700 nm) ,2. Pione00–700 nmSince the first data using 660-nm LEDs for plant lighting were reported by Bula et al. ,8,9. DeeOcimum basilicum) and lettuce , was observed when grown under high proportions of amber light . Relative humidity and temperature of the LI-COR environment was controlled at 50% and 23 °C, the same as in the germination growth chamber. The CO2 concentration and flowrate in the Whole Plant Arabidopsis Chamber were set to 400 µL·L−1 and 400 µL·min−1, respectively. Whole plant photosynthetic rate measurements were collected on three independent biological replicates for each lighting condition. The measurement of photosynthetic rates lasted 30 min to one hour in duration with 4 s per data acquisition period for each replicate. The photosynthetic rate stabilized after 20 min of light exposure. Plant leaf area was measured using ImageJ 1.48v software . Imagery acquired with ImageJ software was used to determine the whole plant photosynthetic rate on a per unit leaf area basis.Whole plant photosynthetic rate analyses of two-week old lettuce plants were carried out using a LI-6400 Portable Photosynthesis System  equipped with a Whole Plant Arabidopsis Chamber , in which different light treatments were provided by the external LED light sources , as described above. The irradiance level was set to 50 W·m−2 for two weeks: an amber LED assembly, an amber assembly with the notch filter, a red LED assembly, and a red LED assembly with the shortpass filter. Peak wavelengths, spectral compositions, and light intensities  [2 levels, and photoperiod) in the chamber with the LED treatments were the same as the germination chamber. A timer connected to the DC power supply controlled the 16-h photoperiod.After emergence of the fourth true leaf (2 weeks after germination in the germination chamber), lettuce plants were immediately transplanted to a growth chamber with the LED lighting system. Plants were cultivated in 13-L hydroponic tanks  under the four following different LED light treatments at an irradiance level of 50 W·mux (YPF) ) of the After growing the plants for two weeks under the different LED light treatments, they were harvested and sampled for biomass yield and morphological analyses, including shoot fresh mass, dry mass, and leaf area. Biomass yield (fresh and dry mass) was determined with a balance . To determine dry mass, plant samples were dried at a temperature of 75 °C for no less than 72 h. All biomass yield and growth parameters, including shoot fresh mass, shoot dry mass, and leaf area, were measured and reported under each light treatment.p < 0.05).Statistical analyses were performed using JMP 10 software . Tukey–Kramer’s HSD was used for multiple comparisons among spectral treatment means obtained from significant one-way analysis of variance (ANOVA) tests  and 244.5 ± 4.21 µmol·m−2·s−1 (YPFD).Shortpass and notch filters incorporated into the experimental LED lighting system resulted in single and double peak LED spectra  that madTibbitts . As we sLactuca sativa cv. Breen) were treated with 595 + 655-nm, 595-nm, 633-, or 613-nm LED light spectra for two weeks. Lettuce plants exhibited the largest size when grown under the 595 + 655-nm light treatment, followed by the 595-, 633-, and 613-nm light treatments  [L. sativa var. capitata) plants [L. sativa, cv. Grand Rapids) under HPS lamps is reportedly caused by a high proportion of amber light [−2·s−1 [−2·s−1) [−2·s−1 (243−267 µmol·m−2·s−1). Therefore, when comparing data from these studies and the current investigation, data suggest that, within the amber wavelength range, plants respond differently to irradiance levels of amber light. Low irradiance levels of amber light result in higher plant productivity, and these data support those reported by Gajc-Wolska, Kowalczyk, Metera, Mazur, Bujalski and Hemka [Higher shoot fresh mass, shoot dry mass, and leaf areas (approximately 50–80% higher) were observed in lettuce plants grown under the 595- and 595 + 655-nm light treatments when compared to the 613-nm and 633-nm light treatments, all with similar irradiance levels. For the amber wavelengths (595- and 595 + 655-nm), similar plant productivity is reported using HPS lamps with and without sunlight for greenhouse grown tomato (uck’ F1)  and lett) plants . Howeverer light . If we c [−2·s−1 . In cont[−2·s−1) ,19. The nd Hemka . In contnd Hemka ,21.−2·s−1, whereas Cope, Snowden and Bugbee [−2·s−1 and 100–300 µmol·m−2·s−1, respectively.High photosynthetic activity was measured in lettuce under 613- and 633-nm light treatments, yet lettuce leaf elongation and poor leaf development were observed. This suggests that 613- and 633-nm LED light affected plant morphology in a way that is similar to that of deep-red light ,33. Furtd Bugbee  and Johkd Bugbee  reportedAnother reason for low correlation between photosynthetic activity and plant development with 613- and 633-nm light treatments could be interactive effects between plant architecture and photosynthetic activity. Because of instrument and chamber size limitations, two-week old plants were used for whole plant photosynthetic rate analyses. As plants grow, different photosynthetic rates are induced, and plant architecture is impacted. When plants are cultivated in a bigger environment, this results in different light interception efficiencies for leaves grown under different light treatments over the plant’s lifespan . We obseA low correlation between photosynthetic activity and plant development has been reported with narrow-spectrum amber light ,37,38. NProtea cynaroides) [Euphorbia pulcherrima) [Cucumis sativus cv. Jinchun No. 4) [Blocking 630-nm light resulted in different leaf coloration and plant architecture when compared to the 595- and 595 + 655-nm light treatments. Sole or supplemental 650–660 nm light enhancing fresh/dry mass gain and leaf expansion has been observed in lettuce plants ,46,47. Taroides) , poinsetherrima) , and cucPurple coloration was observed in lettuce leaves grown under the 595-nm light treatment, but not when grown under 595 + 655-nm light, where in 630-nm light was blocked via the notch filter. Red and purple coloration in fruit and leaves is typically due to anthocyanin, a polyphenolic pigment . AnthocyIn summary, we investigated the influence of wavelength on lettuce plant growth and photosynthetic performance by filtering amber and orange/red light, yielding different spectra with the same irradiance level. Findings suggest that it is inappropriate to simply predict plant growth and development using photosynthetic data, and that the impact of wavelength on plant photomorphogenesis should be considered. This study further highlights the necessity of high controllability on amber and red light, as they may have positive or negative effects on photosynthetic activity, plant growth and development, and pigment accumulation. These data, coupled with previous studies, provide compelling evidence that amber light is superior to red light for promoting photosynthetic activity and plant productivity. To this end, our data add to our understanding of the impact of 590–630 nm light on both photosynthetic activity and plant development.


Phagocytes, which include neutrophils, monocytes, macrophages, and dendritic cells, protect the body by removing foreign particles, bacteria, and dead or dying cells. Phagocytic integrins are greatly involved in the recognition of and adhesion to specific antigens on cells and pathogens during phagocytosis as well as the recruitment of immune cells. β2 integrins, including αLβ2, αMβ2, αXβ2, and αDβ2, are the major integrins presented on the phagocyte surface. The activation of β2 integrins is essential to the recruitment and phagocytic function of these phagocytes and is critical for the regulation of inflammation and immune defense. However, aberrant activation of β2 integrins aggravates auto-immune diseases, such as psoriasis, arthritis, and multiple sclerosis, and facilitates tumor metastasis, making them double-edged swords as candidates for therapeutic intervention. Therefore, precise regulation of phagocyte activities by targeting β2 integrins should promote their host defense functions with minimal side effects on other cells. Here, we reviewed advances in the regulatory mechanisms underlying β2 integrin inside-out signaling, as well as the roles of β2 integrin activation in phagocyte functions. Phagocytosis is the mechanism by which microorganisms are engulfed and killed, and it is the main process by which immune cells disassemble pathogens to present antigens. This is important for the innate immune response and initiating adaptive immune responses. Phagocytosis is a special form of cell endocytosis, whereby cells ingest solid particles through vesicles, including microbial pathogens –3. WhileIntegrins are essential cell-surface adhesion molecules that are widely expressed on cell membranes. As cell adhesion receptors, integrins transduce intracellular and bidirectional intercellular signals , 9, and β2 integrins play a major role in regulating phagocyte adhesion and migration to inflamed organs and other immunological processes, such as phagocytosis , 13 Ta. The actIntegrin αLβ2 is critical for the adhesion of blood phagocytes (such as neutrophils and monocytes) to the vascular endothelium –35, as wβ2 integrins are important for the fusion of human  but not D of ~26 µM  or ~460 nM  , and a flexible cytoplasmic tail –59 Fig, 61–66: ulation) . These r2+ treatment, the αX K313I, F315E, and I317H mutations exhibited increased monoclonal antibody 24 (mAb24) binding, which indicates headpiece opening, but unchanged KIM127 antibody binding, which indicates extension. A previous electron microscopy study showed that mAb24 exclusively binds to extended but not bent αXβ2 integrins  conformation was described for β2 integrins , 71. By ntegrins . This cacis and auto-inhibit neutrophil adhesion and aggregation  and recruit talin-1 to the plasma membrane to interact with the β2 cytoplasmic tail  is a FERM  domain with four subdomains: F0, F1, F2, and F3. Structural studies revealed that the F3 subdomain binds to the cytoplasmic tail of β2 integrins, leading to integrin conformational change, the critical final step of integrin activation –90. Thervia a short amino acid sequence present in its 28 amino acid tail -dependent phagocytosis exhibit distinct characteristic. The activation of αMβ2 prior to challenge with particles is required for αMβ2-mediated phagocytosis. The engulfment process in αMβ2-dependent phagocytosis is initiated by surface-tethering of particles, that then induces an invagination in the phagocyte plasma membrane into which the particle sinks, drawn by F-actin cytoskeletal forces . ObviousAs mentioned above, talin-1 and kindlin-3 bind to the integrin β cytoplasmic tail, which activates integrins . Talin-1via its interaction with VASP.RIAM has been shown to play an important role in complement-dependent phagocytosis . SuppresIntegrins are well-established mediators of cell adhesion and migration, yet underlying mechanisms and signaling pathways continue to be revealed . Furthervia Rho GTPases to modulate actin cytoskeleton dynamics. During integrin-dependent uptake, signaling involves either RhoA (for αMβ2-mediated phagocytosis) or Rac1/Cdc42 activity. This suggests that the particle engulfment in integrin-dependent phagocytosis may share similar actin-regulating pathways with general Fc-receptor-dependent phagocytosis modes.Critical gaps remain in our knowledge of phagocytic integrin signaling. Several alternative mechanisms regulate talin-1 recruitment, but their contributions and significance are obscure. The Rap1-talin-1 interaction is evolutionarily conserved and may contribute to short-term adhesions , whereasStudies on β2 integrins indicate that integrin-mediated phagocytosis is an extension capacity of integrin-mediated cell adhesion. Besides β2 integrins, other integrins may also be involved in phagocytosis, including those in non-leukocytes. Integrins bind to ECM components, such as fibrinogen , fibronectin , vitronectin , or collagen . However, it is not clear which integrins are involved in phagocytosis. Those integrins known to induce actin remodeling might support particle uptake but need to be further evaluated. As far as we know, integrins αVβ3 and αVβ5 are involved in apoptotic-cell (AC) uptake . RGD ar. The remAn improved understanding of phagocytosis is important since it is involved in bacterial clearance, antigen presentation, inflammation resolution, and progression of chronic inflammatory or auto-immune diseases. β2 integrins are clearly important in phagocytosis, although their general role is just emerging. Investigating the detailed molecular mechanism of integrin functions in the complex phagocytotic process is a fascinating challenge. β2 integrins are a valuable clinical target . HoweverHS and KZ contributed equally to this work. HS prepared figures. HS and KZ drafted the manuscript. HS, KZ, LH, and ZF edited and revised the manuscript. ZF approved the final version of the manuscript. All authors contributed to the article and approved the submitted version.This research was supported by funding from the National Institutes of Health, USA  and a startup fund from UConn Health.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: Persisters are important reasons for persistent infections, and they can lead to antibiotic treatment failure in patients and consequently chronic infection. Staphylococcus aureus small colony variants (SCVs) have been shown to be related to persistent infection. Mutations in the genes of the heme biosynthesis pathway lead to the formation of SCVs. However, the relationship between heme production genes and persister has not been tested.Methods:HemA and hemB were knocked out by allelic replacement from S. aureus strain USA500 separately, and then, the heme deficiency was complemented by overexpression of related genes and the addition of hemin. The stress-related persister assay was conducted. RNA-sequencing was performed to find genes and pathways involved in heme-related persister formation, and relative genes and operons were further knocked out and overexpressed to confirm their role in each process.Results: We found that heme biosynthesis deficiency can lead to decreased persister. After complementing the corresponding genes or hemin, the persister levels could be restored. RNA-seq on knockout strains showed that various metabolic pathways were influenced, such as energy metabolism, amino acid metabolism, carbohydrate metabolism, and membrane transport. Overexpression of epiF and operon asp23 could restore USA500∆hemA persister formation under acid stress. Knocking out operon arc in USA500∆hemA could further reduce USA500∆hemA persister formation under acid and oxidative stress.Conclusion: Heme synthesis has a role in S. aureus persister formation. Staphylococcus aureus in 1944  and inserted into pCM29  were counted for the USA500, USA500∆hemA, USA500∆hemB, USA500∆hemA::pRB473-hemAX, and USA500∆hemB::pRB473-hemB strains at 0, 3, 6, 10, 16, 24, 36, and 48 h by plating 10 μl aliquots of 10-fold serial dilutions on TSA in triplicate. For the hemin complementation experiment, all strains were grown to the stationary phase, then diluted to 1:1,000 in 5 ml of TSB medium, and incubated at 37°C and 220 rpm. Hemin was added at a concentration of 5 μg/ml from the beginning of growth. All experiments were repeated three times.All strains were grown to the stationary phase, then diluted to 1:1,000 in 5 ml of TSB medium, and incubated at 37°C and 220 rpm. Among them, USA500∆hemA, USA500∆hemB, USA500∆hemA::pRB473-hemAX, and USA500∆hemB::pRB473-hemB mutants were cultured for 28 h, while USA500 was cultured for 24 h so that all of them reached an equivalent stationary phase. The final bacterial concentration was adjusted to the same level (approximately 1 × 109 CFU/ml) to minimize the quorum sensing influence on the persister assay. After centrifugation, 1 × 109 CFU of the stationary phase bacteria were resuspended in 1 ml of TSB and incubated under different stress conditions, including heat , acid , oxidative stress , and ciprofloxacin . Aliquots were withdrawn from each treatment at different time points, and live bacteria were counted as described above. For the hemin complementation experiment, hemin was added at a concentration of 5 μg/ml from the beginning of growth before all kinds of treatment. The bacterial load detection limit was 101 CFU. All the experiments were replicated at least three times. Because USA500∆hemA and its derivatives were very sensitive to all stresses, to compare their differences, the heat and oxidative stress conditions were changed to 55°C and 10 mM H2O2, respectively.All strains were diluted to 1:100 in the stationary phase in a fresh TSB. Because of their slight growth deficiency, the USA500∆The previously constructed transposon mutant library was further screened for mutants vulnerable to levofloxacin (12.5 μg/ml) under the same conditions . The trahemA insertion mutants were treated with levofloxacin (12.5 μg/ml) in TSB medium for 3–6 days at 37°C. The replica was transferred to Tryptone Soy Agar (TSA) plates and compared with the wild-type strain.To confirm their decreased survival phenotype, hemA and hemB, as described previously . Total RNA was isolated from these five strains using the RNeasy mini kit  according to the manufacturer’s protocol. For library construction, rRNA was depleted from the total RNA extracted, followed by RNA fragmentation. After first- and second-strand cDNA synthesis, end repair, adding poly(A) tails, adapter ligation, PCR, and product purification, the library was successfully constructed. For quality analysis, SOAP  and reverse transcribed using the PrimeScript RT reagent kit . Real-time PCR was performed using SYBR Premix Ex Taq II  with the primers listed in −∆∆Ct method . For in vitro experiments, data from three independent experiments were pooled. p < 0.05 was considered statistically significant. The PossionDis algorithm was used to screen for differentially expressed genes using the RNA-seq data.Statistical analysis was performed using the one-way ANOVA with Dunnett’s hemA  and 3.8 log (p < 0.0001) after 1 h, respectively  and 1.2 log (p < 0.0001) lower than those in the wild-type strain after 1 h, respectively, and both mutants were eliminated after 2 h  to the medium could also rescue persister levels in both ∆hemA and ∆hemB strains to almost those in the wild-type strain under heat, inorganic acid stress, and oxidative conditions  and 1.40-log(p < 0.01) lower than the wild-type strain and ∆hemA::pRB473-hemAX and ∆hemB::pRB473-hemB were 0.74-log (p < 0.01) and 0.37-log lower (p > 0.05). After 7 days, persister levels of ∆hemA and ∆hemB were 1.83-log (p < 0.001) and 1.18-log (p < 0.01) lower than the wild-type strain and ∆hemA::pRB473-hemAX and ∆hemB::pRB473-hemB were 1.11-log (p < 0.01) and 0.47-log (p > 0.05) lower. These results showed that persister level of ∆hemA and ∆hemB mutants was reduced under ciprofloxacin stress and complementing the respective genes may not always restore the phenotype.When the cells were treated with ciprofloxacin at the concentration of 400 mg/ml , after 5S. aureus, and the lack of heme biosynthesis could result in severe deficiency in persister formation under various stress conditions.The data indicate that heme was involved in persister formation in hemA and ∆hemB strains to investigate the downstream genes and pathways after the heme biosynthesis pathway was disrupted, which could provide a clue for the deficiency of persister formation. A total of 64 genes were downregulated, and 23 genes were upregulated in both ∆hemA and ∆hemB mutants , nucleotide metabolism , carbohydrate metabolism (acsA1), transporters , lipid metabolism (lip), carotenoid biosynthesis (crtM), and environmental information processing . Some of these differentially expressed genes were clustered in the same operons , butanoate metabolism , arginine biosynthesis , transporters (USA300HOU_0762 and USA300HOU_0897), and a multidrug efflux pump (USA300HOU_2333 and USA300HOU_2334).The upregulated genes were associated with energy metabolism , USA300HOU1605–09, and asp23  operons and epiF (involved in Quorum sensing), and constructed two double-knockout strains, including ∆arc (encoding arginine/ornithine antiporter) and ∆alsSD (involved in Butanoate metabolism) in USA500∆hemA, to further investigate the mechanism of heme effects on persister formation.Since some operons were entirely upregulated or downregulated, they were more likely to be consequent downstream genes or complementary genes of heme deficiency. We therefore constructed strains overexpressing four downregulated genes or operons in USA500∆hemA mutant, with 0.56 and 0.93 log higher survival than that in the ∆hemA control after 2 h and 3 h treatment, respectively (p < 0.05). However, the double ∆hemA∆arc mutants had 1.16log fewer CFU than that in the USA500∆hemA mutant after 2 h treatment . The double USA500∆hemA∆arc mutant had 5.35 log fewer CFU than those in the control after 3 h treatment  showed defective persistence under various conditions, such as acid, antibiotics, and heat stress . This work was also supported by Research grants from the Shanghai Science and Technology Committee  and Key Discipline Construction Plan from Shanghai Municipal Health Commission (GWV-10.1-XK01).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Avaliar a utilidade do pico de fluxo da tosse para predizer o desfecho daextubação em pacientes que obtiveram sucesso no teste derespiração espontânea.®,Lilacs, Ibecs, Cinahl, SciELO, Cochrane, Scopus, Web of Science e literaturacinzenta. Utilizaram-se os critérios Quality Assessment of DiagnosticAccuracy Studies para avaliar a qualidade da metodologia e o risco deviés dos estudos. A heterogeneidade estatística darazão de verossimilhança (LR) e razão de chancediagnóstica (RCD) do diagnóstico foram avaliadas comutilização de gráficos em floresta, teste Q de Cochrane um gráfico crosshair summary Receiver Operating Characteristic,utilizando um modelo com múltiplos pontos de corte.A busca cobriu as bases de dados científicos MEDLINEInicialmente obteve-se, nas bases de dados, um total de 3.522referências; dentre estas, selecionaram-se para análisequalitativa 12 estudos que incluíram 1.757 participantes. Muitosestudos apresentavam um risco de viés incerto em termos daseleção de pacientes e do fluxo e tempo. Dentre os 12 estudosincluídos, sete tinham alto risco e cinco risco incerto para o itempadrão de referência. O desempenho diagnóstico do picode fluxo da tosse para o resultado da extubação foi baixo amoderado quando se consideram os resultados de todos os estudosincluídos, com +LR de 1,360 , -LR de 0,218 e razão de chance diagnóstica de 6,450. Uma análise de subgrupos que incluiu somenteestudos com valores de corte entre 55 e 65 L/minuto demonstrou desempenholigeiramente melhor, porém ainda moderado.A avaliação do pico de fluxo da tosse, considerando valor decorte entre 55 e 65 L/minuto, pode ser útil como medida complementarantes da extubação. São necessários estudos commelhor delineamento para elucidar o melhor método e equipamento pararegistrar o pico de fluxo da tosse, assim como o melhor ponto de corte. Increases in MV duration are associated withincreased mortality and complications such as ventilator-associated pneumonia,ventilator-associated lung injury, atelectasis and pneumothorax, amongothers.,6, 7 It can be carried out using various techniques, such as lowventilatory pressure support, continuous positive airway pressure (CPAP), automatictube compensation, or total removal of the mechanical ventilatory support byconnecting a “T”-shaped piece in the endotracheal tube to an enriched oxygensource. A trial isconsidered successful when the patient tolerates 30 minutes or more of eithertechnique., 9 Although SBT has been proven tohave high accuracy in predicting the weaning outcome, 12.4% to 21% of subjects whosucceed in this test require reintubation within 48 to 72 hours. is recommended in the current weaning guidelines to assess the patient’sability to breathe spontaneously.. Changes were made in the initial protocol toinclude the possibility of further subgroup analyses. This addendum to the protocolaimed to enable the analysis of subgroups, including studies with similarcharacteristics, such as the use of rescue therapy, the cough stimulation method,equipment used, cutoff values, etc. However, subgroup analyses were only carried outin the presence of a sufficient number of studies with homogeneous characteristics.This systematic review was reported according to the Preferred Reporting Items forSystematic Reviews and Meta-Analyses (PRISMA) checklist.http://clinicaltrials.gov) for unpublished and ongoingstudies.The searches of the databases were carried out on April 3rd, 2020 guided by twoexperienced librarians and two researchers. They covered the following databasesand portals: MEDLINE® via PubMed®; BVS Portal, includingLiteratura Latino-Americana e do Caribe em Ciências da Saúde(Lilacs) and Índice Bibliográfico Español en Ciencias de la Salud(Ibecs) scientific databases; Cummulative Index to Nursing and Allied HealthLiterature(Cinahl); Scientific Electronic Library Online (SciELO); Cochrane;Scopus; Web of Science; Embase®; and gray literature. Additionally, wesearched a clinical trials registry  and (cough or “peak expiratory flow rate”)and (“airway extubation” or weaning or “ventilator weaning” or extubation). Forthe Embase® database, the search strategy was  and coughing or “peak expiratory flow”) andweaning or “ventilator weaning” or “extubation failure.”This research was designed to obtain the highest possible sensitivity, while itsspecificity was ensured by manual reviews of the retrieved results. FG and NAindependently examined the titles and abstracts resulting from the electronicsearch to exclude obviously irrelevant articles. After this stage, the fulltexts of the other studies were evaluated. The two reviewers discussed the textsto reach a consensus when there was a disagreement.The inclusion criteria were as follows: (1) type of study: prospective orretrospective peer-reviewed studies in English, Portuguese or Spanish; (2)population: subjects older than 18 years under MV for more than 24 hours whowere successful in the SBT and considered able to be extubated; (3) indextest/assessment: measurement of cough peak flow prior to the extubation process;and (4) predefined results: the expected outcome of the cough peak flowassessment’s ability to predict extubation success or failure. The followingwere excluded: abstracts, letters, editorials, expert opinions, reviews and casereports; studies with tracheotomized subjects; and studies with subjectsextubated for clinical comfort.Two reviewers extracted the data independently using a predefined data extractionform. The data extracted included the first author’s name, year of publication,study design, cough assessment method (voluntary or involuntary), instrumentused for measurement, use of rescue therapy , sample size(n), extubation failure or success (n), sensitivity and specificity, cutoff,area under the Receiver Operating Characteristic (ROC) curve (AUC), positive andnegative predictive value, relative risk, odds ratio and positive and negativelikelihood ratios. Articles by the same author were carefully examined to avoidduplication of the included studies, and any disagreement was resolved byconsensus. was used to assess themethodological quality and risk of study bias. This tool is structured in fourdomains that present the main sources of bias, including patient selection,index testing, reference standard and flow and time. Each domain is assessed forbias risk and, except for the flow and time domains, for test applicability.The Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) and thepackages diagmeta andmada, asrecommended.Statistical analysis was performed by ASF using R Project version3.6.2Confidence intervals (95%CIs) were calculated using Wilson’s method. Thestatistical heterogeneity of likelihood and DORs were evaluated using forestplots and Cochran’s Q statistic, in which each study was weighted by the use ofan inverse variance model; significant heterogeneity was detected when p <0.10 due to the number of studies included. To quantify the extent of heterogeneity, we usedHiggins’s I2 statistic to measure the percentage of variability amongsummary indices that was caused by heterogeneity rather than chance .Positive and negative likelihood ratios  and the diagnostic odds ratio  were calculated for eachstudy. Pooling of the indices was performed with the bivariate model ofZwindermann & Bossuyt. A smoothed curve was thenfitted across the studies to represent the relationship between the truepositive and false positive (1-specificity) fractions of each study, from whichthe area under the ROC (AUROC) curve was calculated. To investigate whethervariations in the diagnostic threshold affected the shape of the sROC curve, thethreshold effect was tested using the regression equation log (DOR) = a + b·S,where S is a measure of the diagnostic threshold (null hypothesis: b = 0). Asubgroup analysis was conducted with studies that reported a cutoff value in therange of 55 to 65L/min. Publication bias was assessed by funnel plots for theDOR using Deeks’ funnel plot asymmetry test. Significant asymmetry (p < 0.10)indicates the presence of publication bias.The paired sensitivity and specificity values of each study are presented on acrosshair summary ROC (sROC) plot using the multiple cutoff model.We initially identified 3,522 references in the databases, and after the removalof 818 duplicates, we obtained 2,704 studies. Among these, we discarded 2,654articles after reading the titles and abstracts. We examined the full texts of50 articles and excluded 38 that did not meet the inclusion criteria. Finally,12 articles were selected for inclusion in this review .. Ten (71%) results from ninestudies used cutoff values in the range of 56 to 62.4L/minute. The total numberof successful cases was 1,677 (84%), with an average cough peak flow value of83.6 ± 17.9L/minute (range 63.6 to 125.8L/minute). The total number offailures was 321 (16%), with an average cough peak flow value of 55.5 ±11.1L/minute (range 36.3 to 75.8L/minute). The details of all included studiesare shown in tables 1 and 2.Most studies presented a low to unclear risk of bias in the overall analysis,except for the item “reference standard.” Among the 12 included studies, sevenpresented a “high risk” and five an “unclear risk” due to a lack of objectiveclinical criteria for reintubation and the nonexclusion of subjects who werereintubated for laryngospasm. The use of NIV as rescue therapy was observed infour studies and was classified as “unclear risk” for bias in the item “flow andtime”. Three studies including reintubated subjects were also assigned as having“unclear risk” in the item “flow and time.” Three studies reported that thesubjects were ready to wean but did not describe specific criteria, so weassigned them to the “unclear risk” classification in the item “patientselection”  and 3.2 = 4.5%), -LR  or DOR .Regarding the confusion matrix analysis, 10 (71%) results reported sensitivityand specificity values alongside the area under the ROC curve. Four (29%)results reported positive and negative predictive values. Five (36%) resultsreported positive likelihood values, and four (29%) reported negative likelihoodvalues. Five (36%) studies also reported the relative risk, whereas only three(21%) reported the odds ratio. The pooled summary probabilities  and 5 shThe sROC curve yielded a maximum sensitivity of 0.767 (95%CI 0.353 - 0.967) and aspecificity of 0.536 (95%CI 0.158 - 0.882) and an area under the curve of 0.696, consistent with the moderate diagnostic accuracy of the cough peak flowfor extubation (2 = 0%), -LR, or DOR . Due to the limited number of studies, nosubgroup analysis was conducted for the sROC curve. Likewise, we observed noevidence of a threshold effect .Six (43%) studies reported a cutoff value in the range of 55 to 65L/minute. Thepooled summary probabilities showed that the diagnostic performance of the coughpeak flow for extubation was slightly higher than the overall quantitativesynthesis, with a +LR of 1.390 (95%CI 1.270 - 1.540), -LR of 0.176 (95%CI 0.109- 0.267), and DOR of 8.400 (95%CI 4.740 - 13.600). No evidence of heterogeneitywas observed for +LR , comparedvoluntary and involuntary cough assessments ,concluding that voluntary coughing is not invasive and has a greater capacity topredict extubation failure than involuntary coughing in cooperativesubjects. As the coughreflex response may be directly proportional to the stimulation, it is not knownwhether a stronger stimulus, such as mechanical stimulation with a catheter orinstilling a higher saline volume, would produce better results. Nevertheless, theinstillation of saline solution could be uncomfortable and could lead to transitorydesaturation. Thus, voluntary measures would be more suitable for cooperativeindividuals, whereas involuntary stimulation could be reserved for noncooperativesubjects. Another alternative is using voluntary testing first in cooperativesubjects. If the cough peak flow is below the success threshold, an effectiveinvoluntary stimulation would be applied to discard false-negative results due tothe low motivation of the subjects.The evaluation of cough peak flow is an objective method to predict successfulextubation, which can be carried out voluntarily or involuntarily. Most of thestudies in this review evaluated voluntary coughing, which depends on the patient’smotivation, effective coordination and the preservation of respiratory neuromuscularactivity. Two studies evaluated the subjects in an involuntary way - i.e., onetriggered by the cough reflex.versus AUC = 0.83 for the ventilator, p =0.26). Similarly,Gobert et al. used a flow meter built into a ventilator, showing good predictivecapacity (AUC = 0.61) and highlighting the advantage of not requiring additionalcosts to purchase a device or having to disconnect the patient. Although a measurement using themechanical ventilator is more practical, some aspects must be taken into account,such as how much the ventilator circuit resistance reduces the cough peak flow(perhaps determining a lower cutoff), what frequency response and sampling frequencyare needed in the ventilator acquisition system and what the most appropriateventilatory parameters are necessary to perform the measurement. Other technicalaspects can also affect the reliability of the cough peak flow measurements,regardless of whether they are made with a ventilator or with an external device.The European Respiratory Society (ERS) carried out a complete—although not specifically designedfor the target population of our study—update on respiratory muscle tests since itslast recommendations in 2002, advising on the importance of the standardization ofpeak expiratory flow measurements. Aspects such as the number of maneuvers, patientposition and possible discrepancies between different measurement instruments werepointed to as important contributors to different results across the studies. Theauthors of the articles selected for this review used different measurementinstruments, and generally, they did not describe how the maneuvers were performed.The position of the subjects, level of consciousness, endotracheal tube size, numberof measurements and the interval(s) between them, acceptability criteria for themeasurements, and how the researchers performed the verbal stimulation are examplesof data not reported in most studies.Most of the studies used an external spirometer to record the cough peak flow.However, two studies used mechanical ventilator displays. Bai et al. concluded thatboth methods had good predictive accuracy for reintubation  also received an “unclear” or “highrisk” rating in the “index test” criterion.The inclusion of subjects who had already been extubated was scored as “unclear risk”in the “flow and time” criterion,, which might be confounders for determining the best cutoff. Age andsex are directly related to the predicted peak expiratory flow. Therefore, it islikely that women and older people may have a cough peak flow that is lower butwithin a normal range. Moreover, Despite the methodological limitations, different measurement instruments and ways inwhich the cough peak flow was measured, we observed that nine of the 12 studies inthis review had cough peak flow cutoff values between 56 and 62.4L/minute,presenting high sensitivity and specificity to predict success in plannedextubation.When we analyzed the subgroup of studies with cutoff values between 55 and65L/minute, the diagnostic performance of the cough peak flow was slightly higherthan the overall quantitative synthesis, reinforcing the current assumption that thebest cutoff is approximately 60L/minute. Their resultsshowed that for subjects with a cough peak flow ≤ 70L/minute, NIV reducedreintubation compared to the control group . Subjects with cough peak flow >70L/minute did not benefit from NIV, which strengthens the hypothesis that NIV asrescue therapy may have been a source of bias in another three cough peak flowstudies included in this review.,35, 39The rationale of cough peak flow as a predictor of extubation outcome in subjects whosucceed in an SBT is based on secretion retention during the postextubation perioddue to cough ineffectiveness. Therefore, measures should be taken to optimize airwayclearance and to prevent reintubation in subjects who present with a low cough peakflow. Some evidence reinforces this premise, such as a study from Duan et al., whodivided their sample of 356 individuals who succeeded in an SBT into subjectseligible for treatment with NIV or conventional oxygen therapy (controlgroup). and the moderatediagnostic performance found in the statistical analysis.As limitations of this review, we found that the included studies used differentmethods, rescue therapies, extubation failure criteria, populations, and devices toperform the measurements. These differences, the lack of relevant information, andsome other methodological limitations of the included studies make it difficult todevise recommendations for recording the cough peak flow and the best cutoff pointassociated with the extubation outcome. Moreover, along with a possible presence ofpublication bias, all of these abovementioned issues may have contributed to theasymmetry observed in the funnel plot analysis (46) and The cough peak flow recording is promising for improving approaches to subjectsduring the process of mechanical ventilation withdrawal. The studies included inthis review make it very clear that reduced cough peak flow values are associatedwith extubation failure. The cutoff of approximately 60L/minute seems to have thebest accuracy. However, recommendations about how to perform the measurement arenecessary so that well-designed studies using standardized protocols can in thefuture determine the best cutoff associated with the extubation outcome.