Prostate cancer is the most common cancer in men, with over 52,000 new cases diagnosed every year. Diagnostics and early treatment are potentially hindered by variations in screening protocols, still largely reliant on serum levels of acid phosphatase and prostate-specific antigen, with tumour diagnosis and grading relying on histopathological examination. Current treatment interventions vary in terms of efficacy, cost and severity of side effects, and relapse can be aggressive and resistant to the current standard of care. For these reasons, the scientific community is looking for new chemotherapeutic agents. This review reports compounds and extracts derived from marine organisms as a potential source of new drugs against prostate cancer. Whilst there are several marine-derived compounds against other cancers, such as multiple myeloma, leukemia, breast and lung cancer, already available in the market, the presently collated findings show how the marine environment can be considered to hold potential as a new drug source for prostate cancer, as well. This review presents information on compounds presently in clinical trials, as well as new compounds/extracts that may enter trials in the future. We summarise information regarding mechanisms of action and active concentrations. Prostate cancer is the second leading cause of cancer-related death in males . On a glIn the US, prostate cancer currently accounts for 14% of newly diagnosed cancer cases, with an estimated 5.7% of cancer deaths in 2022 and a calculated 5-year survival rate for the period 2012–2018 of 96.8% . In termWhilst tumours from distant organs can spread to the prostate, over 95% of prostate cancers develop locally as primary adenocarcinomas, with approximately 4% attributed to transitional cell carcinomas of the epithelium of the urethra or ducts [Prostate cancer screening largely relies on measuring the serum levels of acid phosphatase and prostate specific antigen (PSA), even though the predictive value of this test has been questioned . DiagnosRegarding treatment, a prostate cancer diagnosis often results in one or more of the following courses of action: watchful waiting/surveillance, hormonal treatment, surgical removal of the primary tumour, curative or palliative brachytherapy, cryotherapy, or curative or palliative chemotherapy. Overall, tailored treatment plans are carefully evaluated and largely driven by tumour grade and stage, circulating PSA levels and calculated risks of recurrence . Stage IWhilst prostate cancers are generally seen as initially slow-growing and relatively benign tumours, treatment interventions vary in terms of their efficacy, cost and severity of side effects. Relapse, on the other hand, can be aggressive and resistant to the current standard of care. As such, the discovery of new chemotherapeutic agents as single-line treatment or in combination with radiotherapy, with both greater efficacy and preferably more favourable off-target side effect profiles, are desirable. Examples are recent studies focusing on T-type calcium channels as emerging promising therapeutic targets  and on tAround 70% of the planet’s surface is covered by water , and marA series of primary and secondary metabolites of marine organisms with potential applications for the prevention and treatment of different cancers have been reported to date ,25,26,27Using the search terms “prostate cancer” and “marine compounds” in PubMed, an increase in the number of publications on this topic in recent years is apparent . PreviouMarine microorganisms have attracted much attention in recent years because not only do they allow drug discovery without impacting marine ecosystems, but cultivation volumes are also readily scalable in the majority of cases. In addition, mass cultivation can be supported by photobioreactors, where parameters such as light, temperature, nutrients and degree of bubbling can be modified and controlled in order to induce the production of the metabolites of interest. Bioactivities of microorganism extracts/compounds against prostate cancer cells have been reported for marine fungi, bacteria, cyanobacteria and microalgae .Fusarium found on Little San Salvador Island, Bahamas. Because N-methylation can enhance both potency and selectivity for peptides, Liu et al. [Sansalvamide A is a cyclic depsipeptide, originally isolated from a marine fungus of the genus u et al.  synthesiIsaria felina KMM 4639 and subsequent fractionation of ethyl acetate extracts. They were, notably, isariketide A and isariketide B (with a hydroxyl group replacing the acetyl group in isariketide A), plus a third compound (compound 3), a derivative of isariketide A with an additional acetyl group [Aspergillus sulphureus, gave rise to an additional compound, Oxirapentyn L. The cytotoxic effects of isariketide A, isariketide B, compound 3 and Oxirapentyn L were assessed on different prostate cancer cell lines , as well as murine non-malignant splenocytes and erythrocytes. Isariketide A and compound 3 were found to be highly cytotoxic to cancer cells, whereas isariketide A showed no toxicity in normal cells until used at concentrations ≥ 100 μM. In addition, cytotoxicity was positively associated with the degree of acetyltation. Preliminary analyses of the mechanism of action of isariketide A and compound 3 suggested induction of apoptosis at 5 μM concentrations (48 h treatment). Interestingly, isariketide A and compound 3 demonstrated cytotoxicity in both hormone therapy-resistant cell lines 22Rv1 and PC3, as well as hormone-sensitive LNCaP cells, therefore presenting as promising drug substrates for drug-resistant prostate cancer therapy.A number of derivatives were isolated after cultivation of the fungus yl group . Co-cultMicrosporum sp. strain MFS-YL was collected from the surface of the marine red alga Lomentaria catenata, found in the Republic of Korea, and cultured for 30 days. The fermentation broth was extracted with ethyl acetate, fractionated and the pure compound physcion successfully isolated, as confirmed by nuclear magnetic resonance (NMR) and mass spectral (MS) analyses [The fungus analyses . PhyscioSZ-685C, a marine anthraquinone isolated from the fungi of mangroves from the South China Sea , demonstPlanctomycetes phyla, one of the less studied bacterial phyla for drug discovery, have been previously shown by in silico genomic analyses to hold potential for producing many secondary metabolites. This was exploited by Calisto et al., (2019) , who culStreptomyces sp. Mei37, isolated from the muddy sediment of Jade Bay on the southern German North Sea coast, and performed solid phase and ethyl acetate extractions. They identified five isoquinolinequinones, the known 3-methyl-7-(methylamino)-5,8-isoquinolinedione and four new derivatives, mansouramycin A-D, and tested them on various cancer cell lines, including DU-145 prostate cancer cells. Mansouramycin C was the most active, with IC50 values ranging from 0.24 to 1.11 µM. More recently, a secondary metabolite, named Lu01-M, was isolated from the marine actinomycetes Streptomyces sp., collected from marine sediments at 400 m depth in Taiwan. This compound was tested at different concentrations  for 24–72 h on various prostate cancer cell lines, including PC3, DU145 and LNCaP cells [Hawas et al.  culturedaP cells . FindingChromobacterium sp. [Chromopeptide A is a depsipeptide isolated from the marine sediment-derived bacterium rium sp. . This corium sp. , known trium sp. .Phyllospongia lamellosa were evaluated for possible extracellular L-asparaginase production [Escherichia coli (E. coli) (Elspar® and its pegylated form Oncaspar®) and L-asparaginase from the bacterium Erwinia chrysanthemi (Erwinase®) [Bacillus safensis MK541039 was evaluated in PC3 cells for 24 h, and a concentration-dependent decrease in cell viability, with an IC50 of 38.75 IU/mL, has been reported [The bacterial isolates 12 Y, 12 OR and 12 W from the marine sponge oduction . L-asparoduction . Differewinase®) . Howeverwinase®) ,53. PartLyngbya majuscule, collected from Papua New Guinea, produces aurilides B and C. Both compounds were tested on various cancer cell lines, and aurilide B exhibited high cytotoxicity (with GI50 less than 10 nM) against prostate cancer cells [L. majuscula from the western lagoon of Pulau Hantu Besar, Singapore [Nostoc sp. GSV 224 [The marine cyanobacterium er cells . Cyclodeingapore . This co GSV 224 . Its ana GSV 224 . Whilst Finally, other anti-prostate activities from marine microorganisms were observed for compounds isolated from microalgae . In partChemical structures available in the public database PubChem  of compoSimilarly to marine microorganisms, invertebrates are known to produce a series of cytotoxic compounds to defend themselves from predators . In the Jaspis johnstoni, were assessed in the human prostate cancer cell lines PC3, LNCaP and TSU-Pr1 [Xestospongia exigua. These compounds were shown to play a role in the inhibition of invasion of basement membrane gels by PC3 cells [In 1995, the antitumour effects of jasplakinolide, a cyclodipsipeptide produced by the marine sponge  TSU-Pr1 . Exposur TSU-Pr1 . In 2001C3 cells . In partGeodia japonica, was toxic to LNCaP cells (IC50 of 120µg/mL) and induced moderate oxidative stress, with the activation of NAPDH oxidase [Dysidea sp. At concentrations of 5 µg/mL, sintokamide A inhibited transactivation of the N-terminus of the androgen receptor (AR) in LNCaP cells [Negombata magnifica. Both of these compounds were found to have anti-migratory activity against highly metastatic PC3M cells in a wound-healing assay and potent anti-invasive activity against PC3M cells in a matrigel assay at concentrations ranging from 50 nM to 1 μM [In 2006, Liu et al. demonstrated that stellettin A , an isom oxidase . In 2008aP cells . In the  to 1 μM . Latrunc to 1 μM .Hemimycale arabica. Phenylmethylene hydantoin showed potent anti-invasive and cytotoxic effects against PC3M cells, with an EC50 of 150 µM [Eurypon laughlini, demonstrated growth suppression of human prostate cell lines LNCap, PC3MM2, PC3 and DU145, with IC50 of 0.8 µM, 4.7 µM, 1.4 µM and 0.85 µM, respectively [Psammoclema sp. led to the isolation of four new trihydroxysterols, named trihydroxysterol 1, 2, 3 and 4, related to aragusterol G. These compounds inhibited the growth of DU145 cells after 72 h [In 2009, (Z)-5-(4-hydroxybenzylidene)-hydantoin (phenylmethylene hydantoin) was isolated from the Red Sea sponge f 150 µM ,76,77,78ectively . Also inter 72 h . In 2009ter 72 h  synthesiter 72 h . FBA-TPQter 72 h .Chondrosia corticate. Halichondramide showed anti-metastatic activity correlated with the down-regulation of matrix metalloproteases (MMPs) and the modulation of the expression of various biomarkers associated with metastasis, including phosphatase of regenerating liver-3 (PRL-3), proteins p85 and p110, N-cadherin, E-cadherin and matrix metallopeptidase-2 and -9 , at both the transcriptional and translational levels. In addition, the compound showed potent growth inhibitory activity against PC3 cells, with an IC50 value of 0.81 µM [Hippospongia metachromia. This compound promoted hypoxia-inducible factor-1 (HIF-1) and G1 phase cycle arrest in LNCaP prostate cancer cells [In 2013, a trisoxazole-containing macrolide, named halichondramide, was isolated from the sponge  0.81 µM . Halicho 0.81 µM . In 2013er cells ,85,86.Pipestela candelabra sponge, belonging to three structural classes, including milnamide, hemiasterlin and geodiamolide. This led to the identification of three new members of the milnamide family: milnamide E, milnamide F and milnamide G, as well as a new member of the hemiasterlin family, hemiasterlin D [Hyrtios sp. Along with the known metabolites fascaplysin and reticulatate, these compounds showed cytotoxic activity against LNCaP cells, with IC50 values ranging from 0.54 to 44.9 μM [In 2014, 20 secondary metabolites were isolated from the terlin D . These cterlin D . In 2014 44.9 μM .Rhizochalina incrustata [In 2015, the anticancer activity of furospinosulin-1, a furanosesterpene isolated from an Indonesian marine sponge, was further assessed. It showed antiproliferative activity in DU145 cells in vitro, at 1–100 nM concentrations . In hypocrustata , was asscrustata . Rhizochcrustata , and it crustata . Regardicrustata ,95. In Pcrustata .Cliona viridis showed significant cytotoxic activity against PC3 cells, with an IC50 of 150 µg/mL. In particular, this extract decreased PC3 cell proliferation in a dose-dependent manner [Sponia sp. were evaluated in DU145 cells in 2-dimensional (2D) cell cultures [In 2018, the dichloromethane extract of the marine sponge t manner . In 2019cultures ,98. Two Haliclona gracilis. After 24 h treatment, five gracilosulfate compounds, gracilosulfate A, B, D, F, and G, inhibited the expression of PSA in 22Rv1 human hormone-independent prostate cancer cells [Spongiella sp. Antiproliferative activity against PCa cells was mediated by inhibition of androgen receptor (AR) signaling [More recently, seven new steroids belonging to the group of gracilosulfates were isolated from the sponge er cells . The cyter cells  highlighignaling . The AR-ignaling . This neignaling .Aplysia oculifera. These two compounds exhibited cytotoxic activity against a panel of cell lines, including PC3 cells, with IC50 values ranging from 2–8 μM [To our knowledge, to date, only three compounds have been isolated from mollusks with proven activities against prostate cancer cell lines: kahalalide F, oculiferane and epi-obtusane. As reported below, kahalalide F is an antitumor drug under clinical investigations. It is a dipsipeptide, isolated from mollusks, that induces oncosis in human prostate cancer cells . Kahalalm 2–8 μM .Cladiella pachyclados [Pseudopterogorgia elisabethae. Anti-neoplastic activities in PC3 cells were demonstrated both by reduction in cell viability and altering the concentration of intracellular reactive oxygen species through cytotoxic and apoptotic effects [Anthopleura anjunae were shown in DU145 cells [Leptogorgia sp. Moderate cytotoxic properties were reported after 72 h, with selectivity against human drug-resistant prostate cancer cells 22Rv1, compared with the non-cancerous prostate cells PNT2 [In 2010, two diterpenes, pachycladin A and pachycladin D, were isolated from the coral hyclados . Both cohyclados . Recentl effects . In 201845 cells . IC50 va45 cells . The maills PNT2 .100) from Pacific cod. TFD100 inhibited in vitro adhesion of androgen-independent PC3 cells to endothelial cells, angiogenesis and gal3-mediated T-cell apoptosis, and it also prevented PC3-induced metastasis in mice [5 from the starfish Culcita novaeguineae. These compounds were able to exhibit moderate cytotoxicity against LNCaP cells, with IC50 concentrations of 48.59 ± 2.30 mM, 39.6 ± 82.65 mM, 31.80 ± 1.59 mM and 57.0 ± 81.81 mM, respectively, assessed by determining the amount of sulforhodamine B (SRB) [In 2013, Guha et al.  purified in mice . In 2015 in mice  isolated B (SRB) . In a re B (SRB) .Chemical structures available in the public database PubChem  of compoThere are no currently available marine drugs on the market for prostate cancer specifically, but there are a number of compounds reported in pre-clinical and early phase I/II clinical trials. A keyword search on both the ClinicalTrials.gov database, as well as the Cochrane Library, with the terms ‘prostate cancer’, ‘marine*’, ‘marine extract’ and ‘marine compound’ revealed several distinct compounds of interest as potential therapeutic candidates, in addition to a number of dietary supplementation studies making use of marine omega-3 fatty acids . Of particular note are the pre-clinical applications of fucoidan and synthetic hemiasterlin, as well as the phase I/II studies on marine extracts in conjugate vaccines,  ,132,133.In pre-clinical trials, the inhibitory effects of a synthetic analogue of the tripeptide hemiasterlin, a marine sponge product, were assessed in different in vitro and in vivo mouse tumour models . PrelimiTwo vaccine conjugate studies have reported the use of hemocyanin, an extract from the snail-like marine mollusk keyhole limpet. Keyhole limpet hemocyanin (KLH) has been shown to possess immune system-fortifying properties in various animals and human studies, and it has been evaluated in conjunction with MUC-2, a protein present in many cancers, including prostate cancer, and brought into solution with the immunological saponin adjuvant QS21, a tree bark extract (NCT number: NCT00698711 ), as welDolabella Auricularia [Elysia rufescens and from the algae Bryopsis pennata, which the mollusk feeds on , in phase II trials for prostate cancer, in conjunction with patent (US20040067895A1), reporting its application against prostate cancer, in particular, androgen-independent prostate cancer [Dolabella auricularia, is the precursor compound of the reported auristatins, known to inhibit microtubules and possess apoptotic effects. Dolostatin 10 has been reported to be in phase II clinical trials in patients with hormone-refractory metastatic prostate adenocarcinoma. In addition, there is evidence for the assessment of the efficacy and safety of fucoidan in a phase I/II clinical trial study in Japan, although no linked reports of results could be located as yet (UMIN ID: UMIN000005432), with a different group of researchers reporting a role for fucoidan in raising natural killer (NK) cells in a mixed male population of tumour patients [DSTP3086S is an antibody-drug conjugate (ADC)-targeting six-transmembrane epithelial antigens of the prostate 1 (STEAP1) conjugated to monomethylauristatin E (MMAE) in phase I trials in patients with metastatic castration-resistant prostate cancer . MMAE isicularia . Tisotumicularia . MMAE, licularia . PSMA-ADicularia . Kahalale cancer . Dolastae cancer , originapatients . Finallypatients .Even if there are no approved marine-derived drugs against prostate cancer available yet, there are several compounds/extracts that have shown promising activities, and a number have progressed to clinical trials. The majority of those in clinical trials are antibodies directed against specific cancer cell markers conjugated with MMAE.Streptomyces sp. Mei37 (active concentrations 0.089–1.11 μM); Lu01-M from marine actinomycetes Streptomyces sp. ; and chromopeptide A from the marine bacterium Chromobacterium sp. (IC50 1.75–2.43 μM) (Eurypon laughlini (IC50 0.8–4.7 µM); helichondramide from the marine sponge Chondrosia corticate (IC50 0.81 μM); Kahalalide F from a mollusk (IC50 0.07–0.28 μM); and the synthetic-derived makalunamide analog FBA-TPQ (IC50 0.26–0.79 μM) -5,8-isoquinolinedione, isolated from the marine bacterium 2.43 μM) . The mos0.79 μM) .100, which prevented PC3 metastasis in mice; and synthetic hemiasterlin and fucoidan, currently reported in clinical trials against prostate cancer.The major part of the studies reported in this review made use of the prostate cancer cell lines PC3, LNCaP and DU145 and compared their susceptibility to the ‘normal’ human neonatal foreskin fibroblast cell line NFF. Cytotoxicity was typically evaluated by MTT assay and induction of apoptosis assessed by Annexin V assay, with treatment periods ranging from 48–72 h. A few of the compounds were also assessed in in vivo models, such as rhizochalinin, which was administered in PC3 and 22Rv1 human tumor xenograft models at a dose of 1.8 mg/kg/day and found to inhibit tumor growth; the extract TFDMicroorganisms are often considered more promising than macroorganisms because they can be cultivated in large volumes and quantities in eco-friendly and eco-sustainable ways and generally require shorter periods of cultivation. Furthermore, it is possible to use a One Strain Many Compounds-OSMAC approach, based on the modulation of growth parameters  to improve the production of target molecules. Last, but not least, -omics studies can be a promising approach to identify the metabolic pathways responsible for the synthesis of bioactive compounds and to identify the genes responsible for their synthesis. The identified genes could be used for genetic engineering and heterologous expression experiments in order to increase the production and purity of compounds of interest.Overall, the data reported in the current review show that marine organisms give rise to an array of chemical structures (from peptides to complex polyketides) with a vast range of different cellular targets. However, the molecular mechanisms activated can be variable, ranging from induction of apoptosis with associated down-regulation of anti-apoptotic proteins, such as Ras, Bcl-xL and Bcl-2, and up-regulation of pro-apoptotic proteins, such as caspase-3, cleaved PARP and Bax, to the arrest of cell cycle at G2/M (summarised in


Chronic heart failure (CHF) has become an increasing concern with the aging of the population. This study aims to evaluate the effectiveness and safety of Qili Qiangxin capsules (QLQX) for CHF.A systematic review and meta-analysis on clinical studies was conducted. The mechanisms of preclinical studies were summarized.We searched six electronic databases by 20 July 2022, and finally, 7 preclinical experiments (PEs) and 24 randomized controlled trials were included. The risk of bias was accessed by the SYRCLE and RoB 2.0 tool, respectively. PEs indicated that QLQX suppresses myocardial apoptosis, inhibits renin-angiotensin-aldosterone system activation, improves water retention, and enhances cardiocyte remodeling. In clinical studies, compared with routine treatment, QLQX could improve the indicators: clinical efficacy rate , left ventricular end-diastolic dimension , left ventricular ejection fraction , 6-minute walk distance , brain natriuretic peptide , N-terminal pro-brain natriuretic peptide , and adverse events .In summary, QLQX exerts a potential mechanism of utility on myocardial apoptosis and cardiac function and has noteworthy clinical adjuvant efficacy and safety in patients with CHF.https://www.crd.york.ac.uk/prospero/. Chronic heart failure (CHF) is a syndrome of venous blood stasis and arterial blood supply insufficiency due to impaired cardiac function and failure of pumping ventricular blood completely . The incOxidative stress and myocardial apoptosis are associated with the development and progression of CHF, particularly, the induction of HO-1 in CHF has been validated as an important cardioprotective adaptation against pathological left ventricular remodeling –6. QLQX The inclusion of intervention in the study, QLQX, is based on the Chinese New Drug Certificate , and the patent “A pharmaceutical composition and preparation method for the treatment of chronic heart failure” (patent number: ZL02146573.8). The quality control, prescription composition and chemical composition involved in the included studies are shown in This study was pre-registered  and conducted following the PRISMA statement . The cheInclusion criteria met the following requirements: (1) study type: preclinical experiments (PEs) or randomized controlled trials (RCTs); (2) cellular or animal models preclinically compatible with CHF, and patients clinically diagnosed with CHF; (3) QLQX alone or combined with RT as the intervention, and in preclinical experiments, the mechanism of QLQX was explored; (4) control group received RT or placebo; (5) there was no restriction on the outcome indicators of PEs, and the outcome indicators of RCTs were included as follows: clinical efficacy rate, cardiac function, 6-min walk distance (6-MWD), brain natriuretic peptide (BNP), N-terminal pro-brain natriuretic peptide (NT-proBNP), and adverse events. The exclusion criteria were as follows: (1) studies that included non-CHF patients; (2) repetitive studies, review, protocol, comment, case report, etc.; (3) PE or RCT included other Chinese medicine or related interventions in addition to QLQX; (4) data of studies was not available even though contacted with original authors.According to the Systematic Review Centre for Laboratory animal Experimentation (SYRCLE) , a risk The following data was extracted: (1) basic information of the included PEs  and RCTs ; (2) all outcome measures of experiments; (3) outcome measures including clinical efficacy rate, cardiac function, 6-MWD, BNP, NT-proBNP, and adverse events. Under the supervision of a researcher (HC), two researchers (HSL and JW) extracted the data of the PEs, and the other two researchers (CL and HZ) extracted the data of the RCTs.p-value < 0.05 indicated statistically significant; (5) Q statistics and I2 were used to evaluate the heterogeneity of each pooled analysis, and the p-value in Q statistics < 0.05 or I2 > 50% meant high heterogeneity; (6) the sensitivity analysis was carried out when any meta-analysis presented high heterogeneity; (7) as for the difference of interventions or classification of outcome measures, subgroup analyses were inclined to carry on.The Stata 17.0 software  was applied to statistical analysis: (1) random-effect model was employed, as different RT medicine or dosage may affect the therapeutic effect; (2) Cohen’s d and 95% CI were used for continuous variables; (3) relative risk (Relative Risk) and 95% CI were used for categorical variables; (4) A This study used GRADE  , a transA total of 2,235 studies were retrieved through the initial search, and 1,482 studies were obtained after removing duplicate studies. By screening the titles or abstracts, 177 studies were obtained, followed by the full-text accessment, and 7 PEs and 24 RCTs were included. The flow chart of the study screening is shown in Seven PEs –29 and 2Seven PEs –29 were 2+ influx, finally enhancing myocardial contractile function  = 5.43, P = 0.00, I2 = 0]. Egger test results showed that there was no published bias . The L’Abbe and funnel plots are respectively presented in Fourteen RCTs , 51, 53 p < 0.05; Q (13) = 46.66, p = 0.00, I2 = 71.31%, GRADE: moderate], and LVEDD  compared to the control group. However, there was no significant difference between QLQX plus RT and RT on decreasing LVESD in patients with CHF . Heterogeneity analysis indicated that differences in drug dosage and sample size might cause high heterogeneity.Fourteen studies reporting the results of LVEF, 7 studies reporting LVEDD and three studies , 44, 53 p < 0.05, GRADE: low]; but there was a higher heterogeneity . The heterogeneity analysis indicated that different durations led to high heterogeneity.Six studies , 46, 53 Q (7) = 152.55, p = 0.01, I2 = 98.16%] and were excluded from the pooled analysis. The forest plot in p < 0.05, Q (5) = 12.29, p = 0.00, I2 = 59.27%, GRADE: low]. Six studies  = 168.27, p = 0.00, I2 = 98.06%, GRADE: low] as shown in Eight studies , 47, 49  studies , 51, 53 p < 0.05, Q (6) = 12.01, p = 0.02, I2 = 51.05%, GRADE: low].Seven studies , 51, 53 From the clinical aspect of traditional Chinese medicine, QLQX could be used for the type of heart-Yang deficiency caused by Qi inadequacy and blood stasis in CHF . This st2– to H2O2, then catalase or glutathione peroxidase in the cytoplasm and mitochondria converts H2O2 to H2O and O2 (Based on our preclinical findings and previous studies, PI3K/AKT may be a noteworthy signaling pathway regulated by QLQX during the treatment of CHF. QLQX could activate this signaling pathway, up-regulating GSK3β, increasing Bcl-2 and decreasing Bax , 56. TheO and O2 . BesidesO and O2 . AnotherO and O2  indicateO and O2 , 60, altO and O2 . TherefoThere are some limitations in this study. First, even though one high-quality RCT  is incluQLQX has adjuvant efficacy and safety in patients with CHF and improves clinical efficacy rates, LVEF at moderate levels of evidence. QLQX could attenuate CHF by inhibiting apoptosis, suppressing RAAS activation, improving water retention, and enhancing cardiocyte remodeling.The original contributions presented in this study are included in the article/QH and HC conceived and designed the study. HC revised the manuscript. JM and HDL supervised the study and provided some comments. XX and JG performed the data analysis and wrote the manuscript. BS, YW, WY, and YJ assessed the literature. HSL, JW, CL, and HZ extracted the data. All authors contributed to the article and approved the submitted version.


Recent studies have reported that steamed and freeze-dried mature silkworms, also known as HongJam, have various health-promoting effects.The goal of this study was to elucidate changes in the various health-promoting effects of HongJam, after its digestion with a food-grade protease.Drosophila fed with food-grade alkaline protease-digested HongJam compared to those fed with non-digested HongJam. The differences in mitochondrial functions, chemical susceptibilities, and activations of signal transduction pathways between Drosophila supplemented with various feed were examined to elucidate the molecular and biochemical basis of healthspan-promotion and locomotor-improvement effects of protease-digested HongJam.We examined whether healthspan-promotion and rotenone-induced loss of motor-control prevention effects were enhanced in Drosophila model. By conducting a series of studies to reveal the molecular and biochemical basis for healthspan-promoting effects, we found that GS and GSD similarly enhanced mitochondrial activity, but GSD activated autophagy signaling more than GS. In addition, GSD feed (GSDf)-, GSD supernatant feed (GSDsupf)-, and GSD precipitate feed (GSDprecf)-reared Drosophila were also found to have increased resistance to an autophagy inhibitor compared to that of normal feed- or GS feed-reared Drosophila. Furthermore, we found that the rotenone-induced loss of motor control prevention effect was superior for GSDsup compared to GS, GSD, or GSDprec. This result may have occurred because GSDsup has more phenolic compounds and antioxidant activities than other samples.We first found that the healthspan-promotion effect of HongJam digested with a food-grade protease was different depending on the silkworm variety used for its production. Digestion with food-grade protease into White-Jade HongJam (WJ) as prepared from the White-Jade silkworm variety that spins white cocoons did not enhance its functionality. However, compared to Golden-Silk HongJam (GS), a food-grade protease-digested Golden-Silk HongJam (GSD) produced from the Golden-Silk silkworm variety that spins yellow cocoons, it further promoted the healthspan in a GSDsup contained more digested small peptides and free phytochemicals than other samples due to the digestion of proteins with a food-grade protease. Thus, GSDsup leads to further healthspan-promoting and locomotor-improvement effects than GS, GSD, or GSDprec. Bombycis Corpus cum Batryticatus, and silk moth extracts have been used as traditional Oriental medicines for treating various diseases, such as diabetes, hypertension, fever, stroke, and cerebral infarct  is a progressive degenerative disorder of the central nervous systems caused by genetic defects and/or environmental risk factors in humans. Behavioral symptoms of PD are including slowness of movement, tremor, rigidity, and difficulty with walking. The pathological hallmark of PD is the death of dopaminergic neurons in human brains . Among vosophila , 27. Theosophila , 29. In  control , 13, 15.While we analyzed the nutritional compositions and health enhancement effects between HongJam produced with various pulverization methods, we found that the health enhancement effects of HongJam depended on the sizes of particles and recovery rates , 30, 31.® alkaline protease (FP® AP) was efficiently used to hydrolyze the silk fiber Fibroin. Thus, we investigated whether digesting HongJam with FP® AP can enhance healthspan promotion and rotenone-induced loss of motor control prevention effects of HongJam.HongJam consists of approximately 70% crude proteins, 15% crude fatty acids, 3% crude ash, and 2% phytochemicals and vitamins , 30, 32.Bombyx mori were raised on mulberry leaves at the campus of the National Institute of Agricultural Science (NIAS), Wanju-gun, Jeolla-buk do, South Korea. HongJam, also known as steamed and freeze-dried mature silkworms, was produced as previously published . After dH2O was added to the mixtures to make 1 L of solution, an enzyme digestion was performed in a rotary stirrer at 55°C for 24 h. The FP® AP was deactivated by boiling at 90°C for 10 min. WJD and GSD were harvested and separated with supernatants and precipitates by centrifuging at 13,000 rpm for 10 min. The supernatants and precipitates were freeze-dried, and the weight ratio of dried supernatants to precipitates was 7:3.A previously published protocol was used . In brieTo determine the biophysical characteristics of samples, size exclusion chromatography was performed using an AKTA fast protein liquid chromatography system  equipped with a Superose™ 6 10/300 column  equilibrated with 0.1 M phosphate buffer . A gel filtration standard markers kit  was used to calibrate a Superose™ 6 10/300 column. The void volume (Vo) of the Superose™ 6 10/300 column was 7.5 ml. The ratios of elution volume/Vo of 200, 150, 66, 29, and 12.4 kDa protein standards were 1.988, 2.095, 2.174, 2.399, and 2.537, respectively. Radio immuno-precipitation assay (RIPA) buffer  containing Halt™ protease inhibitor cocktail  was used to extract proteins from samples. 0.3 ml (5.0 mg/ml) of samples were injected into AKTA FPLC and then separated for 70 min with a flow rate of 1.0 ml/min. UV absorbance changes at 215, 254, and 280 nm were monitored to reveal amounts of digested peptides, other bio-molecules with ring structures, and proteins, respectively, in samples.Drosophila melanogaster obtained from the Bloomington Drosophila Stock Center  was used to test whether the lifespan and healthspan of Drosophila supplemented with WJ, WJD, GS, or GSD were altered. Drosophila was raised with normal feed [Nf ] or HongJam feeds containing 6.24 g of WJ, WJD, GS, or GSD in Nf. The life expectancy and healthspans were investigated as previously published  or GSDprec feed (GSDprecf). The Drosophila were raised with Nf, GS feed (GSf), GSD feed (GSDf), GSDsupf, or GSDprecf, and the numbers of live flies and active flies were counted as previously published (To examine the effects of GSD supernatant (GSDsup) and GSD precipitate (GSDpepc) on ublished , 13, 24.Drosophila reared with Nf, GSf, or GSDf were measured as previously published , and then the debris was removed by three layers of medical gauze . Filtered lysates were centrifuged at 150× g for 5 min at 4°C and the supernatants were collected and then centrifuged at 900× g for 10 min at 4°C to collect the mitochondrial pellets. The pellets were resolved with 200 μl of lysis buffer and then divided into two tubes: one tube containing 155 μl for MitoCom I∼III and the other tube containing 45 μl of a mitochondrial sample with 5 μl of 10 mM n-D-β-D maltoside for MitoCom IV. All activities of MitoCom I∼IV were normalized by using the activities of Drosophila reared with Nf.The activities of mitochondrial complexes (MitoCom) I∼IV in 5-, 10-, 15-, or 20-day-old ublished , 12. TenDrosophila reared with diverse feeds were investigated to elucidate the signaling altered by GS or GSD. Dithiothreitol , fipronil , H2O2 , LiCl , and 3-methyladenine  were used to modulate various signal transduction pathways. Drosophila were reared with various feeds containing GS, GSD, GSDsup, or GSDprep, and then one hundred adults  were exposed to various signal transduction modulators mixed with 1.5% agar with 0.5 M sucrose and incubated at 28.0 ± 1.0°C. The numbers of live flies were counted every 3 days, and then those live flies were transferred to new tubes containing fresh feed with modulators.After treatment with various signal transduction pathway modulators, the changes in the survival rates of –ΔΔCT method was used to quantify the relative expression levels of the genes.The expression changes of 36 genes related to the unfolded protein response (UPR), autophagy, and the target of rapamycin (Tor)/AKT/phosphatidylinositol 3-kinase (PI3K) were investigated by performing real-time quantitative PCR (RT–qPCR). In addition, the expression changes of six gustatory receptors (Gr), Gr64af, were investigated. The heads and bodies of 7- or 15-day-old adult flies were collected to extract the total RNA using TRIzol reagents according to the manufacturer’s protocol. After the quality of the total RNA was determined using the A260/A280 and 28S rRNA/18S rRNA ratios, DNase I  was used to remove genomic DNA contaminants, and then cDNA was synthesized using Superscript IV (Thermo Fisher Scientific). The DNA sequences of the oligomers and PCR conditions are listed in 1 (Drosophila.The STRING database (DB)1  was usedDrosophila model, Nf-, GSf-, GSDf-, GSDsupf-, or GSDprecf-reared Drosophila were treated with 0.2 M rotenone in 0.1% dimethyl sulfoxide (DMSO), 1.5% agar, and 0.5 M sucrose (Dae-Jung). One hundred age-matched adult Drosophila  were collected and then exposed to 0.2 M rotenone. For the controls, Nf-, GSf-, GSDf-, GSDsupf-, or GSDprecf-reared Drosophila were exposed to only 0.1% DMSO in 1.5% agar and 0.5 M sucrose. The number of live Drosophila was counted every 3 days. Kaplan-Meier survival analyses were performed as described below.To investigate the effects of GS, GSD, GSDsup, or GSDprec on the onset and progression of rotenone-induced loss of motor control in a To quantify the amounts of flavonoids or polyphenolic compounds in various samples, the samples were mixed with 80% methanol  and then shaken for 90 min at 150 rpm using a rotary shaker . Mixed solutions were centrifuged to obtain their supernatants. Following filtration with a syringe filter  to remove small debris, the amounts of total phenolic compounds and flavonoids were measured as previously published , 14, 36.The antioxidant activities of GS, GSD, GSDsup, and GSDprep were examined by performing a 1,1-di-phenyl-2-picryl-hydrazyl (DPPH) assay and a ferric-reducing ability of plasma (FRAP) assay as previously published , 14, 36.post-hoc analysis were performed using Microsoft Excel for Windows 10  as previously published  and Tukey’s honestly significant difference (HSD) ublished , 12. AllDrosophila fed with different feeds or treated with various chemicals. In addition, Cox proportional hazard regression analyses and the log-rank tests were performed to obtain hazard ratios (HRs) and 95% confidential intervals (CIs), and p-values, respectively. Kaplan-Meier survival estimations, the log-rank tests, and Cox proportional hazard regression analyses were performed using the R program  as previously published , 25.47 days (53.8% up), 44.4% to 23.91 days (44.4% up), or 24.99 days (50.9% up), respectively , 18.2 days (98.2% up), 15.6 days (69.6% up), or 13.5 days (46.7% up), respectively  were upregulated, and 2 DEGs in Tor signaling (Pi3K59F and Akt1), UPR signaling (Xbp1 and PEK) and Grs (Gr64d and Gr64f) were downregulated , one DEG in Tor signaling (foxo), all six DEGs in UPR signaling , and two out of three Grs (Gr64d and Gr64f) were downregulated , one out of two Tor signaling (foxo), one out of three DEGs in UPR signaling (Xbp1), and one out of five DEGs in Grs (Gr64d) were downregulated , two DEGs in Tor signaling (foxo and S6k), all four UPR signaling , and four out of five DEGs in Grs  were upregulated. Except for the Grs, the DEGs showed very strong intra- and inter-signaling interactions , one DEG in Tor signaling (S6k), and all four Grs  were upregulated, while all four DEGs in UPR signaling  were downregulated , one DEG in Tor signaling (S6k), all four DEGs in UPR signaling , and one out of four DEGs (Gr64a) in Grs were upregulated , one DEG in Tor signaling (S6k), two out of six DEGs in UPR signaling (Atf6 and PEK), and one out of three DEGs in Grs (Gr64c) were upregulated , two DEGs in Tor signaling (Tor and foxo), three out of six DEGs in UPR signaling , and one out of four DEGs in Grs (Gr64d) were downregulated , one out of three DEGs in Tor signaling (S6k), three out of four DEGs in UPR signaling , and all three DEGs in Grs  were upregulated  was significantly reduced only when exposed to 100 mM DTT. The HRs of GSDf-reared Drosophila exposed to 50 mM  or 100 mM DTT  were significantly reduced. However, GSDsupf-reared Drosophila exposed to 50 mM  and 100 mM DTT  showed significantly increased HRs. In addition, GSDprecf-reared Drosophila exposed to 100 mM DTT only showed a significantly increased HR .After the osophila . When GSDrosophila reared with various feeds showed altered HRs when exposed to 10 nM or 50 nM of the GABAAR blocker fipronil  or 50 nM fipronil  were significantly reduced. Although the HRs of GSDf-reared Drosophila exposed to fipronil did not show any difference from those of Nf-reared Drosophila, GSDsupf-reared Drosophila exposed to 10 nm  or 50 nm fipronil  showed significantly reduced HRs. By contrast, GSDprecf-reared Drosophila showed a significantly increased HR when exposed to 50 nm fipronil .fipronil . The HRs2O2, the HRs of 0.3%  or 1.5% H2O2 -exposed GSf-reared Drosophila were significantly increased. Similarly, the HRs of 0.3%  or 1.5% H2O2  were significantly increased in GSDf-reared Drosophila. However, GSDsupf-reared Drosophila only showed a significantly increased HR when exposed to 0.3% H2O2 .For the oxidative stressor Hp < 0.005) of GSDprecf-reared Drosophila was significantly reduced. By contrast, when exposed to the autophagy inhibitor 20 mM 3-MA, the HRs of GSDf- , GSDsupf- , and GSDprecf  were significantly reduced.When exposed to the autophagy inducer 50 mM LiCl, the HR , the HRs of GSDf- (0.48), GSDsupf- (0.34), and GSDprecf-reared Drosophila (0.53) were significantly reduced , the average lifespans of GSf-, GSDf-, GSDsupf-, and GSDprecf-reared Drosophila were 22.69 days (18.5% up), 23.34 days (21.9% up), 25.75 days (34.5% up), and 23.89 days (24.8% up), respectively , GSDf- (0.53), GSDsupf- (0.41), and GSDprecf-reared Drosophila (0.69) were significantly reduced , the average lifespans of GSf-, GSDf-, GSDsupf-, or GSDprecf-reared Drosophila exposed to 0.2 M rotenone were 13.4 days (15.7% up), 13.98 days (20.7% up), 15.46 days (33.5% up), and 13.04 days (12.6% up), respectively , GSDsup extract (150.9 ± 1.30%), and GSDprec extract (131.3 ± 1.23%) compared with GS extract (100 ± 2.54%) (More than 1,900 species of edible insects worldwide are consumed as food and feed that supplies protein and lipids –42. CompThe advantage of silkworms compared to other edible insects is that their body wall is very thin and soft, so they can be consumed whole by steaming and freeze-drying . In prevDrosophila was compared in this study (Drosophila (Drosophila to the autophagy inhibitor 3-MA (Interestingly, we found that the effect of food-grade protease treatment on HongJam was different depending on the silkworm variety when comparing the healthspan-promoting effect. There was no difference in the healthspan-promoting effect between WJ and WJD made with the white-colored cocoon silkworm variety, while a significant healthspan-promoting effect was present in GSD compared to GS made with the yellow-colored cocoon silkworm variety . In a pris study . Howeveris study . As expeosophila , 4. Thistor 3-MA .Another important finding from this study was that GSDsup had an excellent inhibitory effect on the onset of rotenone-induced loss of motor control compared to GS, GSD, or GSDprec . The reaInterestingly, recent studies have reported that phytochemicals in food inhibit the onset of PD by activating autophagy signaling , 45–47. Drosophila than other samples. Nevertheless, further clinical and preclinical researches are needed to confirm the prevention or treatment of PD through activation of autophagy signaling by phytochemicals or other small molecular weight molecules in GSDsup.In summary, the reason GSDsup was more effective in healthspan promotion and prevention of the onset of rotenone-induced loss of motor control compared to other samples is that GSDsup may contain more free phytochemicals and small molecular weight peptides that enhance autophagy signaling and mitochondrial function of The original contributions presented in the study are included in the article/LXM, S-KK, Y-YJ, and PN performed experiments, analyzed data, and made 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.


Acinetobacter baumannii (AB) ventilator-associated pneumonia (VAP) represents a clinical challenge in the Intensive Care Unit (ICU) due to the negligible lung diffusion of this molecule and the low-grade evidence on efficacy of its nebulization. (2) Methods: We conducted a prospective observational study on 134 ICU patients with COS-AB VAP to describe the ‘real life’ clinical use of high-dose (5 MIU q8) aerosolized colistin, using a vibrating mesh nebulizer. Lung pharmacokinetics and microbiome features were investigated. (3) Results: Patients were enrolled during the COVID-19 pandemic with the ICU presenting a SAPS II of 42 [32–57]. At VAP diagnosis, the median PaO2/FiO2 was 120 [100–164], 40.3% were in septic shock, and 24.6% had secondary bacteremia. The twenty-eight day mortality was 50.7% with 60.4% and 40.3% rates of clinical cure and microbiological eradication, respectively. We did not observe any drug-related adverse events. Epithelial lining fluid colistin concentrations were far above the CRAB minimal-inhibitory concentration and the duration of nebulized therapy was an independent predictor of microbiological eradication : 1.069 (1.003–1.138), p = 0.039). (4) Conclusions: High-dose and prolonged colistin nebulization, using a vibrating mesh, was a safe adjunctive therapeutic strategy for COS-AB VAP. Its right place and efficacy in this setting warrant investigation in interventional studies.(1) Background: Colistin-only susceptible (COS)  Ventilator-associated pneumonia (VAP) caused by extensively drug-resistant (XDR) Gram-negative bacteria represent a clinical challenge for Intensive Care Unit (ICU) physicians, especially when old and potentially toxic drugs remain the only therapeutic armamentarium ,2,3,4.Despite the recent introduction of new antibiotics against difficult-to-treat bacteria, colistin is still used in the critically ill setting, especially as part of combination therapies . This drAcinetobacter baumannii (AB) VAP. ELF pharmacokinetics and pulmonary microbiome features are also described in a subset of patients.In light of that, the aim of this study is to describe the ‘real life’ clinical use of high-dose (5 MIU q8) nebulized CMS, administered by means of a vibrating mesh nebulizer, in a large cohort of hypoxemic critically ill patients with colistin-only susceptible (COS) This observational study prospectively included hospitalized patients across the two COVID-19 ICUs (75 beds) of the Fondazione Policlinico Universitario “A. Gemelli IRCCS” , between 1 March 2020 and 31 December 2021. Patients were eligible for inclusion if they were treated with high-dose nebulized colistin, for at least 48 h, due to a COS-AB VAP.®, Sanofi, Paris, France) dissolved in 6 mL of saline solution by nebulization over 30 min using a vibrating-mesh nebulizer  every 8 h. Solutions for nebulization were freshly prepared. During aerosol delivery, all patients were sedated and received assisted/controlled mechanical ventilation with a tidal volume of 6–8 mL/kg of predicted body weight and respiratory rates of 12–15 cycles/min. The humidifier was removed and the nebulizer was inserted near the Y-piece connector on the inspiratory armPatients received 5 MIU of CMS . A written informed consent or proxy consent was waived, due to the observational nature of the study, according to committee recommendations. All data were anonymous and identified with an admission code number.4 CFU/mL. VAP was defined as bacteremic when the microbiological diagnosis coincided with the same isolation in at least one blood culture in the absence of other specified sources of bacteremia  days, according to clinical status.During the study period, 1386 patients were admitted to the ICU, and >60% received invasive mechanical ventilation (IMV). One hundred thirty-four developed COS-AB VAP and were treated with nebulized high-dose colistin for a median duration of 10 days ,12,13. IDuring 1376 days of high-dose colistin nebulization, we did not observe any drug-related adverse events, and not even aerosolization was interrupted due to bronchospasm, Y-piece obstruction, mucus plugs, or respiratory deterioration. In the absence of a control arm, we could only document observational, crude outcome measures results: 81 (60.4%) healed from VAP, and 54 (40.3%) underwent microbiological eradication, with a 28-day and 90-day mortality rate of 50.7% and 58.2%, respectively. The durations of the MV and ICU stay after VAP diagnosis were 12 [7.5–19] days and 15 [9–27.75] days, respectively.2/FiO2, absence of septic shock, and longer durations of nebulized and intravenous colistin therapy. Multivariable logistic regression confirmed only SAPS II (OR (95%CI) 0.963 (0.940–0.986), p = 0.002), PaO2/FiO2 (OR (95%CI) 1.008 (1.001–1.015), p = 0.034), and duration of colistin nebulization (OR (95%CI) 1.069 (1.003–1.138), p = 0.039) as independent predictors of microbiological eradication , compared with a marked reduction in Proteobacteria, especially Acinetobacter, Bacteroidetes, and Actinobacteria.Lung microbiome analysis of surveillance BAL, at the phylum and genus level, was performed in a patient with microbiological eradication after 12 days of nebulized therapy . It showAcinetobacter baumannii (We investigated colistin and colistimethate (CMS) ELF concentration in seven patients after 72 h of nebulized therapy. One hour after nebulization, the median colistin and CMS concentrations were 121.7 [40.1–143.1] mcg/mL and 1445.3 [236.2–1918.2] mcg/mL, respectively. Twelve hours after nebulization, the median colistin and CMS concentrations were 122.6 [43.3–130] mcg/mL and 522.3 [222.3–636.5] mcg/mL, respectively. Colistimethate underwent extensive pulmonary hydrolysis to colistin, whose ELF concentration was far from the median MIC (1 mcg/mL) of isolated aumannii A,B.In a cohort of 134 patients with COS-AB VAP, treated with inhaled colistin at a dose of 5 MIU q8, delivered through a vibrating mesh nebulizer, we obtained a clinical cure of 60.4%, without observing any pulmonary adverse events. Longer therapy duration was associated with higher rates of microbiological eradication, observing very high colistin ELF concentrations and deep changes in lung microbiome communities.Current guidelines recommend avoiding nebulized antibiotics, including colistin, either as adjunctive or substitution therapy, for the treatment of lower-respiratory-tract infections, especially in patients with severe hypoxemia . Such a Pseudomonas aeruginosa, compared with 122 controls treated with intravenous antibiotics. However, up to now, only small case series, mainly focusing on pharmacokinetics, confirmed the feasibility of the above therapeutic approach [There is pre-clinical evidence to support vibrating-mesh nebulizers, over jet and ultrasonic ones. Although slightly larger, vibrating-mesh aerosol particles remain below 5 microns, reaching the distal lung, with substantially reduced nebulization time and residual volume ,21. Alveapproach ,24.In our cohort of patients, we did not observe any side-effects related to CMS aerosolization. Similarly, Benitez-Cano et al., in 27 patients undergoing high-dose nebulization (9–15 MIU/day), did not report any episode of bronchospasm, although the majority of patients were already receiving bronchodilators , and a mFrom a pharmacokinetic standpoint, CMS nebulization may overcome its negligible ELF penetration after intravenous administration . AlthougGiven the abovementioned heterogeneity in current published investigations, it is clear why we do not have robust evidence on the efficacy of nebulized CMS as a therapeutic strategy for difficult-to-treat VAP. Interestingly, in a recent observational study on 326 patients with COS Gram-negative VAP, the use of aerosolized CMS, although at a dosage of 2 MIU q 8 h through a jet nebulizer, was associated with a significantly lower DAY-14 clinical failure rate, without affecting mortality .Further, we do not have data on the optimal duration of nebulized therapy, although it is reasonable that longer courses (8–14 days) may be required to definitely eradicate the infection, as recently shown in a well-conducted meta-analysis of 11 randomized trials and 1210 patients . Thus, iThis paper has some limitations. First, its design is purely observational without a control arm, so we cannot draw any conclusion on the real clinical efficacy of inhaled CMS, in comparison with other combination strategies including new molecules. Second, we investigated the colistin pulmonary pharmacokinetic and lung microbiome in an exemplifying subset of patients. Finally, part of the patients also received combination intravenous treatment, whose contribution to the clinical observed results is difficult to ascertain.However, this the largest study where the clinical use of high-dose inhaled CMS through a vibrating-mesh nebulizer has been investigated, along with the evaluation of ELF colistin pharmacokinetics and the lung microbiome in a selected subgroup of patients.High-dose and prolonged colistin nebulization using a vibrating mesh was a safe adjunctive therapeutic strategy for COS-AB VAP. Its right place and efficacy in this setting warrant investigation in future interventional studies.


OsDHAR1) under the control of a stress-inducible sweet potato promoter (SWPA2). OsDHAR1-expressing transgenic plants exhibited improved environmental adaptability compared to wild-type plants, owing to enhanced ascorbate levels, redox homeostasis, photosynthetic ability, and membrane stability through cross-activation of ascorbate-glutathione cycle enzymes under paddy-field conditions, which enhanced various agronomic traits, including root development, panicle number, spikelet number per panicle, and total grain yield. dhar2-knockdown plants were susceptible to salt stress, and owing to poor seed maturation, exhibited reduced biomass (root growth) and grain yield under paddy field conditions. Microarray revealed that transgenic plants highly expressed genes associated with cell growth, plant growth, leaf senescence, root development, ROS and heavy metal detoxification systems, lipid metabolism, isoflavone and ascorbate recycling, and photosynthesis. We identified the genetic source of functional genomics-based molecular breeding in crop plants and provided new insights into the physiological processes underlying environmental adaptability, which will enable improvement of stress tolerance and crop species productivity in response to climate change.Abiotic stress induces reactive oxygen species (ROS) generation in plants, and high ROS levels can cause partial or severe oxidative damage to cellular components that regulate the redox status. Here, we developed salt-tolerant transgenic rice plants that overexpressed the dehydroascorbate reductase gene ( During this process, AsA is oxidized to monodehydroascorbate (MDHA) and further oxidized to dehydroascorbate (DHA) through spontaneous oxidation. AsA is subsequently regenerated from DHA and MDHA by the action of MDHA reductase (MDHAR) and DHA reductase (DHAR), respectively [Arabidopsis thaliana [DHAR expression has been reported to enhance tolerance toward abiotic stress. For example, the overexpression of wheat [Arabidopsis [DHAR genes has been shown to improve tolerance toward ozone, chilling, drought, and salt in transgenic tobacco, and the overexpression of the rice DHAR has been shown to increase the tolerance of transgenic Arabidopsis [Escherichia coli [DHAR is an important factor in the detoxification of abiotic stress-induced reactive oxygen species (ROS).In the AsA-GSH system, AsA peroxidase (APX) catalyzes the reduction of Hectively ,8,9. DHAectively . Therefoectively , rice [1thaliana , and swethaliana ; high DHof wheat , human [of wheat , and Arabidopsis ,18 DHAR bidopsis  and Eschhia coli  to salt DHAR-mediated abiotic stress response of plants grown under controlled conditions, the findings of these studies are unlikely to correspond to the stress responses of plants under natural conditions for several reasons [DHAR in mediating the environmental adaptability and productivity of rice grown under natural paddy fields has not been examined.Although several studies have examined the  reasons . First,  reasons ,22. Inde reasons ,22. HoweOsDHAR1-mediated AsA recycling and ROS-induced oxidative stress that results from environmental stress in paddy fields. Recently, we identified the AsA-regeneration mechanism  by determining the protein structure of thermostable OsDHAR1 [OsDHAR1 overexpression would increase the environmental adaptability and productivity of rice plants grown in paddy fields in response to multiple environmental stresses. It is important to analyze the function of stress-inducible genes, not only for further understanding of the mechanisms underlying stress tolerance and responses of higher plants, but also for improving the stress tolerance of crops by gene manipulation.Given the complexity associated with studying environmental stress responses in plants, in the present study, we aimed to elucidate the relationship between  OsDHAR1 . The mec OsDHAR1 . We postOsDHAR1-overexpressing plants under conditions of environmental stress, and to identify the target genes of stress-related transcription factors and potential cis-acting DNA elements by combining the expression data. The profiles of plant responses to environmental stresses are expected to enable the identification of regulators that will be useful in biotechnological approaches aimed at improving stress tolerance, in addition to providing new insights into the physiological processes involved in the mechanisms underlying environmental adaptability.Here, we used gene expression profiling and genetic network to analyze the expression patterns in Oryza sativa L. japonica cytosolic dehydroascorbate reductase (OsDHAR1) (accession no. AY074786) was cloned from rice leaves using reverse-transcription polymerase chain reaction (RT-PCR). The primers, OsDHAR-FC and OsDHAR-RC were used for PCR  were selected for subsequent experiments. For genotyping, both transgenic and wild-type (WT) plants were cultivated in natural paddy fields located at the Gunwi campus  of Kyungpook National University, Korea, from June to October. Seeds were germinated at 32 °C in a controlled growth chamber, transplanted into soil pots (12.5 cm diameter), and maintained at 28–32 °C under a 16-h light/8-h dark cycle. For the salt stress assay, 4-week-old seedlings were treated with 100 mM NaCl for 25 days. Leaves were harvested when seedlings were exposed to 100 mM NaCl for 4 days or grown for ∼4 weeks after transplantation in paddy fields. The harvested leaves were immediately soaked in liquid nitrogen and stored at −70 °C until subsequent use. In addition, a T-DNA insertion mutant line (3A-03259), a putative dehydroascorbate reductase gene  knockdown mutant, was provided by the Salk Institute Genomic Analysis Laboratory . Homozygous lines were identified as described for the DHAR1-overexpressing transgenic plants.The Ilmi variety of OsDHAR1 transgene consisted of two steps. First, genomic DNA was digested with restriction enzymes and ligated to an adaptor, and second, the fragments were subjected to PCR amplification using two primer sets that are specific to the OsDHAR1 transgene and adaptor regions. In the present study, genomic DNA (500 ng) was digested with 2 U of MspI and HincII and ligated using 5 U of T4 DNA ligase (Takara Bio) in 20-micoliter reactions; the reaction mixtures contained T4 DNA ligase buffer and 50 pmol of adaptors and were incubated for 1 h at 37 °C. The first PCR was conducted in 20-microliter reactions that contained 0.5 pmol of each primer  (Determination of the copy number and location of the  or RB2)  and 1 μL or RB2) .v/v) m-phosphoric acid. The homogenate was centrifuged at 12,000× g for 20 min and the clear supernatant was collected. The tAsA content was determined in a reaction mixture containing 100 μL crude extract, 500 μL 150 mM KH2PO4 buffer , and 100 μL 10 mM dithiothreitol, which reduced DHA to AsA. The reaction mixture was incubated at 25 °C for 10 min and supplemented with 100 μL of 0.5% (w/v) N-ethylmaleimide (NEM) to remove excess dithiothreitol. The AsA content was assayed by replacing dithiothreitol and NEM with 200 μL of deionized H2O as described previously. Color was developed in both reaction mixtures by adding 400 μL of 10% (w/v) trichloroacetic acid, 400 μL of 44% (v/v) o-phosphoric acid, 400 μL of α,α-dipyridyl prepared in 70% (v/v) ethanol, and 200 μL of 30% FeCl3. The reaction mixture was incubated at 40 °C for 1 h and the absorbance was quantified at 525 nm. The DHA concentration was estimated by subtracting the AsA content from the tAsA content.The total AsA (tAsA), AsA, and DHA contents were spectrophotometrically determined as described previously . Briefly2, 1 mM EDTA, 1 mM PMSF, and protease inhibitor cocktail ) with a mortar and pestle. The homogenate was centrifuged at 15,000× g for 20 min at 4 °C and the clear supernatant was used for enzymatic assays. To prepare crude protein extracts for APX and GSH reductase (GR) assays, leaf samples (0.2 g) were extracted in 1 mL of extraction buffer (mM potassium phosphate (pH 7.0), 7 mM β-ME, and EDTA-free protease inhibitor cocktail ). In addition, crude protein extract for the APX assay was prepared in the presence of 2 mM AsA. All enzyme assays were performed by monitoring the corresponding reaction mixtures at 25 °C using a UV-1800 UV-VIS spectrophotometer . MDHAR activity was assayed in 1 mL reaction mixture containing 50 mM potassium phosphate (pH 7.2), 0.2 mM NADH, 2 mM AsA, 1 U AsA oxidase, and crude extract. The absorbance was measured at 340 nm, the activity was calculated using an absorbance coefficient of 6.2 mM−1cm−1; and one unit of MDHAR activity was defined as the amount of enzyme that oxidized 1 nmol of NADH per min at 25 °C [−1cm−1 [2O2, and crude extract . Enzyme activity was recorded as the decrease in absorbance at 290 nm for 1 min, and the amount of oxidized AsA was calculated using an extinction coefficient of 2.8 mM−1 cm−1 [To prepare the crude protein extracts for the MDHAR and DHAR assays, leaf samples (0.2 g fresh weight) were homogenized at 4 °C in 1 mL of extraction buffer (50 mM Tris-HCl (pH 7.5), 3 mM MgClat 25 °C . DHAR ac [−1cm−1 , and one [−1cm−1 . APX actM−1 cm−1 . All enzg for 20 min at 4 °C and the clear supernatant was collected. Intracellular hydroperoxide levels were determined based on ferrous ion oxidation in the presence of xylenol orange, a ferric ion indicator. Briefly, crude extract (50 μL) was added to 950 μL of FOX reagent . The mixture was incubated at 25 °C for 30 min and then centrifuged to remove the flocculated material before measuring the absorbance at 560 nm [g for 10 min, and the absorbance of the sample was measured at 535 nm and compared with that of a control sample. Malondialdehyde concentration in the sample was calculated using an extinction coefficient of 1.56 × 105 M−1 cm−1 [−1)-HCl (pH 3.8), incubated for 8 h at 28 °C, and then cleared in boiling ethanol (96%) for 10 min before photographing [Leaf samples (0.2 g fresh weight) were homogenized at 4 °C in 1 mL extraction buffer (20 mM sodium phosphate (pH 7.2), 1 mM PMSF, and EDTA-free protease inhibitor cocktail (Roche)), using a mortar and pestle. The homogenate was centrifuged at 15,000× t 560 nm . Lipid pt 560 nm . BrieflyM−1 cm−1 . For 3,3Fv/Fm), which represents the maximum yield of the photochemical reaction of photosystem II at room temperature, using a Handy Fluor Cam FC 1000-H  [Fv/Fm values were measured as described previously [Five leaves were collected from the TR and WT rice plants grown for 4 weeks in the natural paddy field, after which ten leaf discs were cut (approximately 1 cm). The leaf discs were then incubated in water supplemented with 10 μM methyl viologen (MV) in the dark at 25 °C for 12 h to allow the MV to diffuse into the leaf discs. After pre-incubation, leaf discs were placed in a growth chamber (16 h light: 8 h dark) at 25 °C for 120 h. The conductivity of the suspension was measured using a conductance meter  before and after autoclaving at 121 °C for 15 min to release all electrolytes. Relative ion leakage was expressed as a percentage of total conductivity . MV-treaepublic) . Fv/Fm veviously .g for 10 min at 4 °C to remove any insoluble material. The supernatant containing the RNA was collected, mixed with 0.2 mL of chloroform, and centrifuged at 12,000× g for 15 min at 4 °C. The upper phase containing the RNA, was transferred to a new tube, and RNA was precipitated by mixing the solution with isopropyl alcohol (0.5 mL) and recovered by centrifuging the sample at 12,000× g for 10 min at 4 °C. The RNA pellet was washed briefly with 1 mL of 75% ethanol by centrifuging at 7500× g for 5 min at 4 °C. Finally, the RNA pellet was dissolved in DNase-free water and RNA quality and quantity were assessed using an Agilent Bioanalyzer 2100 . Gene expression was analyzed using GeneChip Rice Genome Arrays , which (each) comprised over 54,000 probe sets, and 11 pairs of oligonucleotide probes were synthesized in situ on the arrays. Biotinylated cRNA was prepared from 100 ng of total RNA, according to the standard Affymetrix protocol. Following fragmentation, 15 µg of RNA was hybridized for 16 h at 45 °C using a GeneChip Rice Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450 and scanned using the Affymetrix GeneChip Scanner 3000 7G. The data were analyzed using robust multi-array analysis—with default Affymetrix settings—together with the global scaling normalization method. The trimmed mean target intensity of each array was arbitrarily set at 100, and both the normalized and log-transformed intensity values were analyzed using GeneSpring GX 13.0 (Agilent Technologies). The fold-change filters used defined the up- and downregulated genes as those that are expressed at levels that were at least 200% less than 50% of the expression of the corresponding genes in the controls. Hierarchical clustering was performed using GeneSpring GX 13.0 (Agilent Technologies), with the clustering algorithm being the Euclidean distance and average linkage. The genetic network of the differentially expressed genes was framed using String  and ClueGo .Leaf samples (pooled from five plants per family) were harvested from WT and TR1 plants that had been grown for ∼30 days under natural paddy field conditions. TR1 plants were used as representative transgenic lines. Total RNA was extracted from rice leaves using the TRIzol reagent (Sigma-Aldrich) according to the manufacturer’s instructions. Following homogenization, 1 mL of the solution was transferred to a 1.5 mL Eppendorf tube and centrifuged at 12,000× 3 or T4 homozygous lines and their respective controls (WT plants) were transplanted into natural paddy fields located at the Kyungpook National University campus in 2020 (T3) and 2021 (T4). A completely randomized block design was employed with two replicates, and each plot comprised 12 plants, with two rows for each line, 0.2 m between plants, and 0.3 m between plots. When the plants reached maturity and the grains ripened, yield components were scored by digging up 8 rice plants per plot to a radius of 15 cm. After washing the plant roots, total plant weight (g), stem weight (g), and root weight were measured and number of panicles per hill and number of spikelets per panicle were counted. The filling rate (%) of the rice plants was calculated relative to 100% of WT rice plants, which were defined had expressed the degree of the filling rate as panicles and spikelets under field conditions. To measure the total grain weight (g), and 1000-grain weight (g), whole seeds per plant were collected and weighed, and then 1000 seeds were counted and weighed additionally. Unfilled and filled grains were separated, randomly counted, and weighed. The following agronomic traits were scored: total plant weight (g), culm weight (g), root weight (g), number of panicles per hill, number of spikelets per panicle, filling rate (%), total grain weight (g), and 1000-grain weight (g).To examine the yield parameters of the transgenic rice plants, three independent TAll experiments were performed with at least three independent repetitions, and the results are expressed as mean ± standard deviation (SD). All biochemical results of the transgenic plants were calculated relative to those of WT plants grown under normal conditions, which were defined as 100%.OsDHAR1-overexpressing plants, we produced 20 lines (T0 generation) of transgenic plants carrying the SWPA2::OsDHAR1 transgene construct. Fifteen seeds (T1 seeds) per primary transgenic line were cultivated in a paddy field and their progenies (T2 seeds) were harvested. To assess the stress tolerance of SWPA2::OsDHAR1 transgene copy was inserted into the rice genome  were identified B. In eace genome . In addiOsDHAR1 overexpression increases salt tolerance, the phenotypes and gene expression of the transgenic plants were examined under salt stress. There was no difference in the phenotypes of the transgenic and WT plants grown under normal conditions  and further analyzed the response of the mutant to salt stress. As a result, the dhar2-knockdown mutants showed hypersensitivity to salt stress  and WT plants was compared. Although salt stress increased the AsA levels in both transgenic and WT plants, the AsA levels of the transgenic plants were higher than those of the WT plants. Meanwhile, the DHA level of the transgenic plants was distinctly decreased compared to that of the WT plants, which subsequently resulted in an increased AsA/DHA ratio. Indeed, under conditions of salt stress, the AsA/DHA ratio of the transgenic plants was ∼1.67-fold higher than that of the WT plants, whereas, only a slight difference was observed under normal conditions  plants and WT plants into paddy fields using a completely randomized design. The plants were grown to the seed maturation stage, and agronomic parameters were scored from the vegetative to the reproductive stage. Transgenic plants performed better than the WT plants during both the vegetative and post-flowering harvest stages . These results indicated that OsDHAR1 overexpression significantly enhanced the rice grain yield and total biomass in response to various environmental stresses under paddy-field conditions.We postulated that t stages . These ot stages C. The rit stages A. On thec traits . More spT plants . Among tOsDHAR1-expressing transgenic plants exhibited increased grain yield and biomass under paddy-field conditions, we performed gene expression profiling to identify the mechanisms underlying OsDHAR1-mediated environmental tolerance. TR1 plants were used because in this line, the SWPA2::OsDHAR1 transgene was inserted into an intergenic region. We found that ∼404 genes were upregulated, and ∼525 genes were downregulated , plant secretory proteins, rapid alkalization factor 2, tubulin (OsTUB6), actin depolymerizing factor (OsADF3), annexin p35, photosystems, phytochrome-interacting protein, molybdopterin biosynthesis CNX2 protein, sodium/hydrogen exchanger protein, abiotic stress responsive protein (OsASR2), heavy metal transport/detoxification protein, heat- and acid-stable phosphoprotein, drought-induced protein, ABA/WDS induced protein, multidrug-resistance associated protein, jasmonate ZIM-domain protein (OsJAZ2), methyl chloride transferase which is harmless to ozone layer 1 (OsHOL1), aquaporin, glutaredoxins (OsGRX4 and OsGRX29), peroxidase (Prx59), MDHAR (OsMDHAR1), AsA peroxidases (OsAPX1 and OsAPX8), GR1, and AsA biosynthesis   . The higogenase) . OsMYB42/85, OsMYB45, Spl7, OsNAC103, OsbZIP49, OsWRKY1, OsWRKY10, OsWRKY69, and CBF-B), nuclear Y/CCAAT-box binding factor A (OsHAP2E), zinc fingers , TonB box, APETALA2/ethylene-responsive protein 129, ethylene responsive element binding factor (OsERF3), symbiosis-related disease resistance protein, multi-antimicrobial extrusion protein, apoptosis regulator Bcl-2 protein (OsBAG1), ATPase, physical impedance induced protein, alcohol dehydrogenase, lactate dehydrogenase, cadmium tolerant 1, flavonoid 3,5-hydroxylase 2, AsA peroxidase (OsAPX7), glutaredoxins (OsGRX2 and OsGRX16), ferredoxin III, GSH transferases , auxin transport protein (REH1), and auxin-up RNA26  involved in the AsA-GSH cycle , Na+ transporter (HKT1), Na+/K+ antiporter (HKT2), Na+ ATPase (ENA), and vacuolar pyrophosphatase (+-PPasevacuolar H) has been shown to improve salt tolerance in a variety of transgenic crop plants by increasing biomass (shoot and root) production, germination rate, and proline content by enhancing ion homeostasis [OsDHAR1 plays an important role in salt tolerance by improving the AsA pool and redox and ion homeostasis, indicating that DHAR expression in WT plants is insufficient to overcome salt stress.In the present study, SH cycle . The phyeostasis . Taken t2O2, which enhances redox homeostasis and proteostasis by minimizing oxidative damage through the buffering of stress-induced ROS [The transgenic plants exhibited a better phenotype than the WT plants under salt stress , which wuced ROS . During dhar2-knockdown transgenic lines, dhar2-1 and dhar2-2, which exhibited lower DHAR activity, were sensitive to salt stress  enhances the growth and survival of Chlamydomonas reinhardtii through the improvement of redox state, photosynthesis efficiency, GSH and AsA pools, and GR and APX activities, which subsequently improve resistance to ROS-induced oxidative stress [Arabidopsis, and lettuce [APX, GST, SOD, MDHAR, and CAT, increases salt tolerance in transgenic tobacco and rice by augmenting photosynthetic efficiency and plant growth [Arabidopsis DHAR-knockout mutants  that exhibited decreased tAsA and increased DHA were more sensitive to bright light and oxidative stress than the WT plants. The mutants possessed lower levels of reduced AsA, MDHAR, and DHAR activities and higher levels of hydroperoxide, which indicates that AsA can limit ROS scavenging under abiotic stress [CrDHAR1-knockdown microRNA lines with reduced DHAR expression and AsA recycling ability were sensitive to light and oxidative stress [In contrast, the t stress . Accordi lettuce , and thet growth . In contc stress ,47. CrDHEchinochloa crus-galli L. depends on higher activities of guaiacol peroxidase, APX, and GR than WT plants when exposed to 200 mM NaCl, whereas salt-sensitive plants with reduced enzyme activities exhibited slower growth rates, higher lipid peroxidation rates, and higher levels of ROS in their leaves [Vicia faba seedlings, exogenous supplementation of AsA was reported to alleviate oxidative stress [OsDHAR1 can be used to facilitate the development of improved rice cultivars with enhanced AsA content.The salt-tolerant barnyard grass r leaves . Moreovee stress ,49, whice stress . Taken tOsDHAR1, which are a downstream regulatory gene, are higher in the three independent homologous TR rice lines than in WT rice plants in paddy fields at transcriptional and translational levels, as a result, OsDHAR1 overexpressing TR plants have been shown to affect environmental adaptability and productivity in rice grown in natural paddy fields, which are frequently exposed to abiotic and biotic stressors , compared to WT plants . Indeed, AsA content is correlated with seed development and seedling growth [In the present study, field-grown transgenic rice plants that overexpressed T plants . The rooT plants C. In parT plants C. In conc traits . SpecifiT plants . Among tg growth . AsA is g growth ,54,55. TOsLSK1 (large spike S-domain receptor-like kinase 1), OsLRK1 (leucine-rich repeat receptor-like kinase 1), OsEBS1 (enhancing biomass and spikelet number), AtSUC2 , and CaPLA1 (Capsicum annuum phospholipiase A1) have been reported to possess more tillers, larger spikelet hulls, longer panicles, more branches per panicle, heavier grains, and greater cellular proliferation than WT plants, thereby improving total grain yield [OsAGSW1 (ABC1-like kinase related to grain size and weight) has been shown to significantly increase grain size, grain weight, grain filling rate, and 1000-grain weight by regulating the number of external parenchyma cells and the development of vascular bundles, which results in wider and longer spikelet hulls [OsNRT2.3b (nitrate transporter), OsSPL14 , OsAMT (ammonia transporter), and OsPTR9 (peptide transporter/nitrate transporter 1 homologue) in transgenic rice enhances pH homeostasis by increasing the uptake of ions , improving grain yield, and improving nitrogen use efficiency [OsCKX2, which encodes cytokinin oxidase 2, increases tiller number and rice yield, whereas constitutive overexpression reduces tiller number and growth in a gene dose-dependent manner [OsDHAR1 do not. In addition, our findings also suggest that OsDHAR1 affects functional tolerance to environmental stresses and consequently leads to improvement in growth, seed maturation (shape and size), and productivity of rice plants grown in paddy fields. In previous studies, field-grown transgenic rice plants that overexpress cell rescue-related genes have exhibited improved growth, development, and yield. More specifically, plants that overexpressed in yield ,57,58,59et hulls . In addificiency ,62,63,64t manner . Taken tOsDHAR1 mediates the environmental adaptability of transgenic rice plants grown under paddy field conditions, despite the underlying mechanism being complex. In the OsDHAR1-expressing TR1 plants, genes involved in a wide range of functional categories, including cell cycle, transcription factors, amino acid (serine and threonine) metabolism, glycosphingolipid biosynthesis, photosynthesis process, secretory proteins, cell rescue systems , antioxidants (isoflavone and AsA biosynthesis), and metal detoxification, were upregulated. The elevated genes were networked to the gene ontology corresponding to the regulation of signal transduction, glycosphingolipid biosynthesis, hydrolase activity, protein complex subunit organization, membrane transporter activity, chlorophyll envelope, response to ROS, water-soluble vitamin biosynthesis, redox homeostasis, and isoprenoid biosynthesis -expressing transgenic rice plants exhibited significantly improved plant biomass, seed weight, and grain yield via regulation of auxin transport. However, the knockdown of BG1 decreased auxin sensitivity and grain size [On the other hand, the expression of genes involved in nitrogen metabolism, amino acid  biosynthesis, apoptotic process, cell adhesion, flavonoid biosynthesis, and auxin-metabolic pathway were downregulated in TR1 plants. The downregulated genes were networked to the gene ontology corresponding to the MAPK signaling pathway, (secondary active) membrane transporter activity, methyltransferase activity, aromatic amino acid family biosynthetic process, and phenylalanine ammonia-lyase activity . Interesain size .O. sativa L. japonica). In OsDHAR1-expressing TR1 plants promotes root growth and development, nutrient uptake, ion homeostasis, senescence regulation, pathogen resistance, photosynthesis optimization, redox homeostasis, proteostasis, and lipid metabolism, which together accelerate agronomic performance and productivity by regulating auxin metabolism.The total genetic network indicated that the differentially expressed genes formed a complex network and provided important information about the mechanisms underlying environmental adaptability . AlthougOsDHAR1 with respect to the alleviation of abiotic stress, especially under paddy field conditions. We found that OsDHAR1-expressing transgenic plants were more tolerant to environmental stress, including high salinity, and we attributed the OsDHAR1-mediated environmental adaptability to increased ROS-neutralizing ability. The observed ROS-neutralizing ability was achieved by enhancing the AsA pool, redox and ion homeostasis, proteostasis, lipid-based membrane stability, photosynthesis capacity, and auxin transport through mutual organic networks between the genes, which improved various agronomic traits, including plant growth (root development), biomass, panicle and tiller number, spikelet per panicle, seed maturation, and grain yield. Furthermore, our findings regarding the molecular, genetic, and phenotypic bases for the responses of rice to abiotic and biotic stresses will facilitate the application of molecular breeding, genetic engineering, and other integrated approaches for developing stress-tolerant crop cultivars.The present study characterized the functions of


In eukaryotic cells, the genome is organized in the form of chromatin composed of DNA and histones that organize and regulate gene expression. The dysregulation of chromatin remodeling, including the aberrant incorporation of histone variants and their consequent post-translational modifications, is prevalent across cancers. Additionally, nuclear envelope proteins are often deregulated in cancers, which impacts the 3D organization of the genome. Altered nuclear morphology, genome organization, and gene expression are defining features of cancers. With advances in single-cell sequencing, imaging technologies, and high-end data mining approaches, we are now at the forefront of designing appropriate small molecules to selectively inhibit the growth and proliferation of cancer cells in a genome- and epigenome-specific manner. Here, we review recent advances and the emerging significance of aberrations in nuclear envelope proteins, histone variants, and oncohistones in deregulating chromatin organization and gene expression in oncogenesis. Each chromosome occupies a unique sub-volume in the interphase nucleus, referred to as a chromosome territory . A chromThe double-membraned nucleus functions as the primary protector of the genome. In metazoans, the nucleus not only harbors the genome but also works in tandem with the differentially-compacted chromatin to regulate its tissue-specific spatial and functional organization. The nuclear envelope comprises the nuclear lamina that maintains nuclear integrity and regulates gene expression and is interspersed with Nuclear Pore Complexes (NPCs), whose primary function is to regulate nuclear transport . Though In metazoans, type V intermediate filament proteins, the lamins, maintain the structural and functional integrity of the nucleus . The nucChromatin in eukaryotes is organized as DNA wrapped around histone octamers, forming nucleosomes. Further, the linker histone H1 is incorporated with the nucleosomes constituting the fundamental units of the chromatin fiber—the chromatosomes . HistoneCancer cells exhibit a remarkable interplay between aberrant genome organization and deregulated transcription. The cancer genome harbors mutations in both coding and non-coding regions, selectively providing tumorigenic cells with a proliferative advantage to outcompete normal cells . For insvia the interaction with the SUN and KASH domain proteins  and NPCs. The INM is lined on the inner side by a protein meshwork referred to as the nuclear lamina, which is composed of type V intermediate filament proteins—the A- and B-type lamins. The A-type lamins, lamin A/C, are produced as two somatic isoforms of prelamin A by alternative mRNA splicing at exon 10 of the LMNA gene . Lamin Aproteins . These lAberrations in nuclear morphology, such as invaginations, blebs, and micronuclei, serve as histological markers for grading tumor progression and often correlate with carcinogenesis. For instance, Haematoxylin-Eosin (HE) staining of papillary thyroid carcinoma cells and colon adenocarcinoma revealed enlarged nuclei with irregular morphology compared to normal cells with smaller and spherical nuclei . FurtherThe stability and integrity of LINC complex proteins and nuclear lamins are crucial for maintaining chromatin organization and genome stability, aberrations of which are associated with various cancers . For insRemarkably, nuclear morphology plays a vital role in modulating cell fate in the continuum of cancer progression . In part−/− mice, cells derived from EMD-null mice showed a compensatory increase in lamin A expression  pathway proteins such as Rad51 in U2OS cells . Corresper cells . FurtherNuclear pore complexes (NPCs) are ∼120 nm-wide structures in the nuclear envelope, which mediate selective transport in and out of the nucleus. In vertebrates, NPCs comprise nucleoporins (Nups), a group of ∼30 proteins, to form a ∼125MDa protein complex . In addivia a similar mechanism  have been identified and characterized across a wide range of cancer types using high-density single nucleotide polymorphism (SNP) arrays . DetermiIt is well established that CIN and aneuploidy involving whole chromosomal and focal amplifications and deletions in the genome are defining features of cancer initiation and progression . InteresInterestingly, the LMNB1 gene shows a significant number of deletions across cancers. It is unique that LMNB1 and LMNB2 genes showed only deep deletions in ovarian cancers . In ovarWe examined the status of mutations in genes that encode the nuclear envelope proteins across cancers using cBioPortal. This analysis revealed recurrent mutations in the Nesprin-1 gene—SYNE1 . MarkedlThe genome is a highly dynamic collection of genes, their regulators, and massive stretches of DNA whose function is yet to be discovered. Maintenance of genome organization involves the concerted function of numerous proteins required for the regulation of chromatin organization and gene expression. The aberrant function of chromatin organizers is associated with cancers . Here, wCCCTC-binding factor (CTCF) is a conserved zinc-finger protein that functions as a chromatin organizer and transcription factor. In association with cohesin, CTCF regulates the organization of gene loci and alternative splicing primed by its sequence-specific binding to CTCF sites. In addition, CTCF functions as an insulator to restrict the expansion of repressive marks . The humAberrant expression or occupancy of CTCF is associated with breast, lung, endometrial, gastrointestinal, prostate, and skin cancers . Of noteAnalyzing patient data sets from TCGA reveals frequent loss of the CTCF gene in breast and prostate cancer patients, correlating with hypermethylation of CpG islands and hypomethylation of other parts of the genome. CTCF depletion in a prostate cancer cell line, HPECE6/E7, shows hypermethylation of CTCF binding sites, further downregulating respective gene expression . This inCTCF functions as a double-edged sword, acting both as an oncogene as well as a tumor suppressor in a cancer subtype-specific manner. Ovarian cancers exemplify the oncogenic potential of CTCF, where metastatic lesions display elevated CTCF expression. Further, the depletion of CTCF in ovarian cancer cell lines (SKOV3 and A2780) decreased cell migration by consistently downregulating three metastasis-associated genes, including CTBP1, SRC, and SERPINE . In contin vivo remains an area of active study. Moreover, it is intriguing that environmental factors, such as diet and social interaction, also impinge on CTCF function and, therefore, chromatin organization and function  proteins, SMC2 and SMC4, but differ in their non-SMC components. Interestingly, decreased condensin expression triggers CIN, consequently driving colorectal cancer progression . In addil ploidy . This agl ploidy . HoweverApart from chromatin compaction, condensins also play moonlighting roles that include facilitating enhancer RNA transcription and enhancer-promoter looping in condensin-bound ERα (Estrogen Receptor α)-sensitive enhancers in breast cancers by recruiting p300 and RIP140 . ImmunopNon-canonical histone variants occasionally replace canonical histones in the genome, often serving two main purposes. First, histone variants are dynamically incorporated throughout the interphase with the regular nucleosomal turnover of canonical histones to sustain nucleosomal stability. Secondly, additional regulatory domains, interactors, and PTMs in non-canonical histones offer supplementary mechanisms for the control of epigenetic regulation. Since cancers are characterized by large-scale remodeling of their epigenetic landscape, canonical histones in cancers are occasionally interchanged with histone variants . StructuThe histone variant H3.3 functions as a space-filling histone when canonical H3 is evicted from the nucleosome, thus maintaining nucleosomal stability . The incin vitro and in vivo studies reveal that both the oncohistones, H3.3K27M and H3.3G34W/L/V/R, reduce H3K27me3 levels, resulting in the aberrant expression of Polycomb-group (PcG)-mediated heterochromatinized genes, though the results are more promising in in vitro systems  . H3G34V/ systems . H3.3K27 systems . Likewis systems . A possi systems . Intrigu systems . It rema systems . The ratAll histones exist as multiple variants that modulate gene expression, barring histone H4, which has only one variant. Histone H2A and H2B cumulatively have 15 non-canonical histone variants, out of which 11 are H2A variants—H2A.X, H2A.Z.1, H2A.Z.2.1, H2A.Z.2.2, H2A.Bbd, H2A.J, H2A.B, TH2A, H2A.P, macroH2A.1.1, macroH2A.2, and macroH2A1.2, and four are H2B variants—H2BE, H2B.S.M, TH2B, and H2B.W , which aBoth H2A.Z and H3.3 maintain an open conformation of chromatin in nucleosome-depleted regions of the promoter for transcription factors to interact with gene promoters resulting in their transcriptional upregulation . ConsistH2A.Bbd, a member of the short H2A family, is a testis and brain-specific histone variant overexpressed predominantly in Diffuse Large B-cell Lymphomas (DLBCLs) . InteresEssentially, tail-domain mutants are well-characterized, but not limited to, H3.3. The same is true for globular domain mutations, which are better documented for histone H2 . MutatioApart from the role of histones in regulating transcription, histones are essential for modulating DNA damage response, genome organization, and chromosome maintenance. CENP-A, a centromere-specific H3 variant, is necessary and sufficient to ensure the structural and functional organization of the centromere. Heterochromatinization at the centromere is achieved by recruiting RNAi-based DICER machinery and SUV methyltransferases . MoreoveThe two fundamental functions of CENP-A include 1) centromere formation and maintenance and 2) nucleation of checkpoint assembly proteins involved in chromosomal segregation. The organization of the centromere is dynamic during the various cell cycle stages, contributing to chromatin reorganization. During the early G1 phase, CENP-A molecules form a rosette-like structure nucleated by HJURP, facilitating a 3D ring-like organization during the G1 phase . During INK4A, a cell cycle blocker (Cellular senescence is a state of dormancy where the cell ceases to divide. Senescence involves shortened telomeres, increased DNA damage, stalled replication, nuclear deformities, mitochondrial dysfunction, and aberrant genome organization . After a blocker .Remarkably, extensive topological changes in chromatin compartmentalization accompany senescence, obfuscating the spatial separation between the A and B compartments. Microscopy and polymer modeling of chromatin reveals that the spatial organization of chromatin compartments is drastically altered in tumor cells. Following this finding, an additional intermediate Compartment I has been proposed that interacts with both A and B compartments in normal tissue and inclines toward the B compartment in cancerous tissue . Cells uThe induction of senescence in cancer cells serves as a traditional therapeutic approach by targeting p53, mTOR, PI3K, and BCL-2 family proteins using senolytic agents . HoweverThe LADs are parts of chromatin domains that are largely in a state of repression. Surprisingly, the simultaneous loss of all lamin forms in mouse embryonic stem cells did not change the overall TAD structure but reorganized the inter- and intra-TAD interactions, further altering transcriptional output . It is iOvarian cancers harbor homozygous deletion in the LMNB1 gene, while the loss of lamin A/C leads to poor prognosis and enhanced metastatic potential of cells . In the Additionally, TCGA data retrieved from GEPIA2 shows that most of the cancers show upregulated levels of lamin B1. It is known that lamin B1 overexpression sequesters 53BP1, a crucial mediator of the NHEJ pathway . It remaLamin A regulates gene expression by interacting with various chromatin modifiers, the deregulation of which promotes cancers. For instance, lamin A directly interacts with and prevents the proteasomal degradation of SUV39H1, the writer of the H3K9me3 inactive mark , the dysIn cancers, chromatin organizers harbor mutations in various domains, resulting in their deregulated activity and altered binding to chromatin or lamins. For instance, sarcomas harbor high-frequency H3.3G34R and H3.3K36M mutations that directly prevent the binding of H3K36me2/3 writer NSD1/2, thus reducing PRC2-H3K36me2 interaction and increasing H3K27me3 levels . The intThe nuclear lamina is primarily associated with inactive histone marks at the nuclear periphery. However, lamins also modulate active euchromatin . How theIn summary, it is beyond any doubt that genetic mutations, aberrations in chromatin organization, incorporation of oncohistones, deregulated transcription, and defects in nuclear envelope organization and function collectively deregulate the cellular and molecular processes of cancers. Novel therapeutic targets will be identified by leveraging high-resolution single-cell approaches, such as sc-ChIP-seq, sc-ATAC-seq, and sc-Hi-C, with high-content imaging, including high-resolution FISH. Furthermore, molecular and biochemical assays remain the mainstay for the elucidation of molecular mechanisms. Collectively, these lines of evidence reveal that aberrant genome architecture serves as a precursor and promoter of cancer initiation and progression.