In vitro, BJF significantly improved cigarette smoke extract-induced mitochondrial functional impairment in L6 skeletal muscle cells through effects on mitochondrial membrane potential, mitochondrial permeability transition pores, adenosine triphosphate production, and mitochondrial respiration. In addition, BJF led to upregulated expression of mitochondrial biogenesis markers, including AMPK-α, PGC-1α, and TFAM and downregulation of mitophagy markers, including LC3B, ULK1, PINK1, and Parkin, with increased expression of downstream markers of the AMPK pathway, including mTOR, PPARγ, and SIRT1. In conclusion, BJF significantly improved skeletal muscle and mitochondrial function in COPD rats and L6 cells by promoting mitochondrial biogenesis and suppressing mitophagy via the AMPK pathway. This study suggests that BJF may have therapeutic potential for prophylaxis and treatment of skeletal muscle dysfunction in patients with COPD.Skeletal muscle dysfunction, a striking systemic comorbidity of chronic obstructive pulmonary disease (COPD), is associated with declines in activities of daily living, reductions in health status and prognosis, and increases in mortality. Bufei Jianpi formula (BJF), a traditional Chinese herbal formulation, has been shown to improve skeletal muscle tension and tolerance via inhibition of cellular apoptosis in COPD rat models. This study aimed to investigate the mechanisms by which BJF regulates the adenosine monophosphate-activated protein kinase (AMPK) pathway to improve mitochondrial function and to suppress mitophagy in skeletal muscle cells. Our study showed that BJF repaired lung function and ameliorated pathological impairment in rat lung and skeletal muscle tissues. BJF also improved mitochondrial function and reduced mitophagy via the AMPK signaling pathway in rat skeletal muscle tissue.  Chronic obstructive pulmonary disease (COPD), a common and preventable disease, is characterized by persistent airflow limitations caused by chronic inflammatory responses to noxious particles and gases . In ChinDevelopment of SMD is likely multifactorial, with inflammation and mitochondrial dysfunction playing predominant roles. Other pathophysiological factors, including oxidative stress, imbalance of protein synthesis/degradation, hypoxia, and cellular apoptosis have also been postulated to contribute to the muscle wasting experienced by COPD patients . Recent Decreased mitochondrial biogenesis and enhanced mitophagy play particularly important roles in mitochondrial impairment. Peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α has been shown to be a master regulator of mitochondrial biogenesis . PGC-1α Mitophagy, on the other hand, is closely associated with the AMPK pathway. AMPK directly facilitates phosphorylation of UNC-51-like kinase (ULK1), which stimulates the mitophagic activity of ULK1 . The PTETraditional Chinese medicine (TCM) has been successfully used to treat COPD for thousands of years. Lung-spleen qi deficiency syndrome is believed to be the main underlying pathologic mechanism for the SMD observed in patients with COPD . Bufei Jin vivo COPD rat model and an in vitro cigarette smoke extract (CSE)-induced skeletal muscle cell line to identify potential underlying mechanisms and to provide a basis for further research.In this study, we aimed to explore the mechanism by which BJF affects mitochondrial regulation via the AMPK pathway in an Klebsiella pneumoniae (strain: 46114) was obtained from the National Center for Medical Culture Collections . HongqiQu® filter cigarettes  were purchased from Henan Tobacco Industry . Aminophylline (APL) tablets were obtained from Shandong Xinhua Pharmaceutical Co., LTD. (lot H37020630) . 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) was purchased from Selleck Chemicals (lot 2627-69-2) . Dorsomorphin dihydrochloride (BML-275 dihydrochloride) was purchased from MedChemExpress (lot 1219168-18-9) .Thirty-six male and 36 female Sprague-Dawley (SD) rats with an average body weight of 200 ± 20 g were purchased from the Experimental Animal Center of Henan Province . The rats were housed in specific pathogen-free cages under standard temperature (25 ± 2°C) and light conditions, with a 12-h light/dark cycle, and free access to food and water. All animal studies were conducted in accordance with the Institute’s Guide for the Care and Use of Laboratory Animals and were approved by the Experimental Animal Care and Ethics Committee of the First Affiliated Hospital of the Henan University of Chinese Medicine.BJF, consisting of 12 herbs , was preSixty Sprague-Dawley rats were randomized into control and Bufei Jianpi groups. Rats in the Bufei Jianpi group were administered BJF (4.5 1g/kg/d) intragastrically 10 times once per day for 7 days. Rats in control group were administrated distilled water . On day 7, all rats were narcotized with 4% chloral hydrate and blood samples were collected, centrifugated at 3,500 rpm for 15 min, inactivated at 56°C, and stored in a −80°C freezer for further study.Klebsiella pneumonia suspension  was slowly dropped into the nasal cavities of rats every 5 days for the first 8 weeks. The successful generation of a COPD rat model was evaluated based on symptoms, lung function, and pulmonary pathology , 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR), Bufei Jianpi Formula + 5-Aminoimidazole-4-carboxamide ribonucleotide (BJF + AICAR), and Aminophylline (APL), with 12 animals in each group. The detailed procedures for creation of the COPD rat model have been described in a previous report ; howeverathology .D represents dose, I represents body shape index, and W represents body weight.Starting from week 9, rats in the control and model groups were intragastrically administered normal saline . BJF (4.51 g/kg/d) was administered to rats in the BJF and BJF + AICAR groups, while APL (2.7 mg/kg/d) was administered to rats in the APL group twice per day for 12 weeks. AICAR (25 mg/kg/d) was intraperitoneally administered to rats in the AICAR and BJF + AICAR groups once a day during week 20. The equivalent dosages of BJF and APL were calculated using the equation:All rats underwent necropsy after intraperitoneal injection of 10% chloral hydrate at 100 mg/kg of body weight at the end of week 20, and lung and quadriceps muscle tissues were collected for further analysis.Lung function was measured by a whole-body plethysmography system  at weeks 0, 4, 8, 12, 16, and 20. Tidal volume (TV), peak expiratory flow (PEF), and 50% tidal volume expiratory flow (EF50) were evaluated.After being fixed in 10% formalin for 72 h, left lung and quadriceps muscle tissues were embedded with paraffin, cut into 4-μm thick sections, and stained with hematoxylin and eosin (HE) for light microscopy.After being cut into sections with 1-mm thicknesses, fresh quadriceps muscle tissue was fixed in 2.5% glutaraldehyde for 2 h followed by 1% osmium tetroxide. Sections were then stained with uranyl acetate, dehydrated in a methanol series and propylene oxide, and embedded with Epon-812. Ultrathin sections were obtained, and ultrastructural changes and mitophagy of skeletal muscle cells were examined using a JEM-1400 transmission electron microscope .Mitochondrial characteristics, including mitochondrial membrane potentials (MMP), ATP production, and opening of mitochondrial permeability transition pores (MPTPs) were measured with a JC-1 kit , an ATP assay kit , and an MPTP assay , respectively, according to the manufacturer’s instruction and as previously described .2-humidified atmosphere at 37°C. When cells had grown by 70–90%, they were plated on dishes at a density of 1 × 105 cells/mL and incubated in 5% CO2-95% air for 24 h. On the next day, cells were treated with 10% cigarette smoke extract (CSE), 10% CSE + AICAR (2.5 ng/mL), or 10% CSE +10% BJF-containing serum in DMEM and harvested 24 h later.Rat L6 myoblasts  were cultured in Dulbecco’s modified Eagle’s medium (DMEM)  supplemented with 10% fetal bovine serum (FBS) in a 5% COCSE was prepared using a modified version of Sheridan’s method . BrieflyMitochondrial respiration was measured with a Seahorse XF Cell Mito Stress Test Kit  on a Seahorse XF24 Extracellular Flux Analyzer  according to the manufacturer’s instruction. L6 cells were seeded  onto Seahorse assay plates in DMEM media and treated with 10% CSE for 24 h. During the experiment, cells were switched to an XF assay medium (HCO3-free modified DMEM) supplemented with 10 mM of L-glutamine, 10 mM of glucose, and 10 mM of pyruvate, while a pH of 7.4 was maintained at 37 °C. After baseline measurement of oxygen combustion rate (OCR), a mitochondrial respiration test was performed by sequential additions of 1.0 μM oligomycin, 2.0 μM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone, and 0.5 μM rotenone/antimycin A. Several mitochondrial parameters were determined, including basal respiration, maximal respiratory capacity, proton leak, and non-mitochondrial respiration. The results were expressed as OCRs (pmol/min/lg protein).5 cells per well and treated with 10% CSE, 10% CSE + AICAR, or 10% CSE +10% BJF-containing serum for 24 h. Before the experiment, cells were treated with carbonyl cyanide m-chlorophenyl hydrazone (CCCP)  for 1 h as a positive control. Changes in mitochondrial membrane potentials of L6 cells in different groups were then assessed using the JC-1 mitochondrial membrane potential detection kit according to the manufacturer's instruction. Red fluorescence represented JC-1 aggregates, whereas green fluorescence represented the monomeric form of JC-1. Fluorescence intensity was analyzed by a Varioskan® Flash Microplate Reader . MMPs in each group were calculated as the ratio of red/green fluorescence. In addition, cells were visualized using confocal laser scanning microscopy at a 514-nm excitation and a 529-nm emission for green and a 585-nm excitation and a 590-nm emission for red.For measurement of mitochondrial membrane potentials, L6 cells were seeded in 6-well plates with a density of 1 ×105 cells per well for 24 h. After treatment with study drugs, cells were lyzed using a cell lysis reagent, and the ATP content in cell lysates was measured using an ATP assay kit  following the manufacturer’s protocol. The total and mitochondrial protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit .For measurement of ATP levels, L6 cells were seeded in 6-well plates with a density of 1 ×10GAPDH) was used as an internal standard.Expression levels of mRNA in quadriceps muscle tissuewere analyzed by semiquantitative polymerase chain reactions (qPCR). Total RNA was extracted from the quadriceps muscle using a total RNA isolation kit  according to the manufacturer's protocol. Real-time (RT)-qPCR was performed using a reverse transcriptase kit  and a SYBR Green Master Mix . To normalize the amount of total RNA in each reaction, a reference gene , anti-sirtuin 1 (SIRT1), anti-peroxisome proliferator-activated receptor (PPAR)γ , or anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH)  at 4°C overnight. After incubation with the HRP-coupled secondary antibody  at room temperature for 2 h, signals were detected using a super-enhanced chemiluminescence plus reagent and scanned and quantified by a ChemiDoc MP imaging system .Total proteins from cells or tissue samples were lyzed in a lysis buffer and quantified using a BCA assay. Ten-cell lysates were loaded into each well in 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to a polyvinylidene difluoride (PVDF) membrane. Membranes were blocked in 5% nonfat milk in tris-buffered saline and polysorbate 20 (TBST) for 2 h at room temperature and incubated with the indicated primary antibodies, including anti-AMPK-α, anti-P-values of < 0.05.Data are expressed as mean ± SD. Statistical differences between the groups were identified by one-way analysis of variance (ANOVA). Differences were considered to be significant at Epimedium A (0.6869 mg/g), Icariin (1.1195 mg/g), Nobiletin (0.0951 mg/g), BaohuosidⅠ (0.0806 mg/g) were identified and their structures had also been shown.Fingerprint of BJF The retention time values of the identified compounds were compared with that of the reference substances. A representative chromatogram of BJF was shown in To assess potential effects on skeletal and lung tissues, BJF was orally administered to COPD rats for 12 weeks using APL as a positive control. We found that BJF attenuated declines in body weight and respiratory function in COPD rats. As shown in As shown in We also found that BJF obviously reduced lung injury identified on histopathological examination. As shown in BJF also obviously improved morphological and ultrastructural changes in skeletal muscle of COPD rats. As shown in To confirm the effect of BJF on mitochondrial function, we measured the levels of mitochondrial functional indicators. As shown in BJF Promoted Expression of Mitochondrial Biogenesis Factors via Regulation of the AMPK Signaling Pathway in COPD RatsAMPK-α, PGC-α, and TFAM were markedly decreased in model rats. While treatment with BJF and APL significantly promoted expression of AMPK-α, PGC-α, and TFAM mRNA, AICAR upregulated levels of AMPK-α and PGC-α but not TFAM. As shown in p-AMPK-α, PGC-α, and TFAM were lowered in model rats compared with control rats. BJF, AICAR, and APL treatment resulted in dramatic increases in AMPK-α, p-AMPK-α, PGC-α, and TFAM protein expression.To explore the mitochondrial biogenesis-related mechanisms of BJF, we measured mRNA and protein levels of AMPK-α, PGC-α, and TFAM in skeletal muscle tissues of COPD rats. As shown in BJF Downregulated Expression of Mitophagic Factors via Regulation of the AMPK Signaling Pathway in COPD RatsLC3B, ULK1, PINK1, and Parkin mRNA levels were significantly upregulated in COPD rats in comparison to control rats. While treatment with BJF, AICAR, and APL downregulated expression of LC3B, ULK1, PINK1, and Parkin mRNA, AICAR upregulated levels of all the above-mentioned indicators. Additionally, protein expression of LC3B, ULK1, PINK1, and Parkin were markedly decreased in COPD rats compared with control rats. While BJF and APL treatment upregulated protein expression of LC3B, ULK1, PINK1, and Parkin, AICAR showed no effect on the level of LC3B . Our study measured the respiratory characteristics of mitochondria under cellular baseline and stress conditions. there is evidence indicated that mitochondrial aerobic respiration was inhibited in L6 skeletal muscle cells exposed to high-dose fructose, with significantly reduced oxygen consumption rate, basal and maximal respiration, and ATP synthesis . NeverthDifferent factors related to mitochondrial dysfunction, including abnormalities in mitochondrial biogenesis and mitophagy, may contribute to the loss of skeletal muscle strength that leads to functional declines in patients with COPD . AMPK, ap-ULK have also been observed in COPD patients  and the Scientific Research Fund for doctors of Henan University of Chinese Medicine (RSBSJJ2018-03).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.


HIBCH, NM_014362.3) gene mutation can cause HIBCH deficiency, leading to Leigh/Leigh-like disease. To date, few case series have investigated the relationship between metabolites and clinical phenotypes or the effects of treatment, although 34 patients with HIBCH mutations from 27 families have been reported. The purpose of this study was to analyze the phenotypic spectrum, follow-up results, metabolites, and genotypes of patients with HIBCH deficiency presenting with Leigh/Leigh-like syndrome and explore specific metabolites related to disease diagnosis and prognosis through retrospective and longitudinal studies. Applying next-generation sequencing, we identified eight patients with HIBCH mutations from our cohort of 181 cases of genetically diagnosed Leigh/Leigh-like syndrome. Six novel HIBCH mutations were identified: c.977T>G [p.Leu326Arg], c.1036G>T [p.Val346Phe], c.750+1G>A, c.810-2A>C, c.469C>T [p.Arg157*], and c.236delC [p.Pro79Leufs*5]. The Newcastle Pediatric Mitochondrial Disease Scale (NPMDS) was employed to assess disease progression and clinical outcomes. The non-invasive approach of metabolite analysis showed that levels of some were associated with clinical phenotype severity. Five (5/7) patients presented with elevated C4-OH in dried blood spots, and the level was probably correlated with the NPMDS scores during the peak disease phase. 2,3-Dihydroxy-2-methylbutyrate in urine was elevated in six (6/7) patients and elevated S-(2-caboxypropyl)cysteamine in urine was found in three patients (3/3). The median age at initial presentation was 13 months (8–18 months), and the median follow-up was 2.3 years (range 1.3–7.2 years). We summarized and compared with all reported patients with HIBCH mutations. The most prominent clinical manifestations were developmental regression/delay, hypotonia, encephalopathy, and feeding difficulties. We administered drug and dietary treatment. During follow-up, five patients responded positively to treatment with a significant decrease in NPMDS scores. Our research is the largest case series of patients with HIBCH mutations.3-Hydroxyisobutyryl-CoA hydrolase ( Leigh syndrome (LS), as the most common presentation of mitochondrial disorders in children, is a devastating neurodegenerative disease accompanied by symmetrical lesions in basal ganglia, brainstem, and with genetic and clinical heterogeneity. It sometimes presents in patients with normal laboratory findings, atypical neuroimaging or symptoms, but highly suggestive of LS, which is known as Leigh-like syndrome . It is rHIBCH , encodes the 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) protein. It is mainly responsible for converting 3-hydroxyisobutryl-CoA to 3-hydroxyisobutyric acid in valine catabolism, as well as 3-hydroxypropanoyl-CoA to 3-hydroxypropionic acid, a hypothetical minor pathway for propionic acid catabolism, which feeds into the valine catabolic pathway  in 2007  combined with metabolic examination and who presented with Leigh/Leigh-like Syndrome.Herein, we analyzed the genetic, metabolic, and clinical features and follow-up findings of 8 Chinese patients with HIBCH mutations who were from our large cohort of case series with 181 cases of genetically diagnosed Leigh/Leigh-like syndrome were recruited to participate in this study from October 2012 to December 2019.Eight patients with identified HIBCH mutations and test the parental origin of available family members.Due to different times of recruitment, we applied different NGS approaches: targeted panel sequencing (Patients 1 and 2)  and wholBriefly, genomic DNA was extracted from peripheral blood using TIANamp Blood DNA Kit . Then 200 ng high-quality genomic DNA were utilized for library preparation. Genomic DNA was enzymatic sheared, end repaired, phosphorylation of the 5′ prime ends, a-tailing of the 3′ends and ligated to sequencing adapters, then PCR amplified following standard library preparation SOP, by using KAPA Hyper Plus Kits . The post-PCR libraries were captured by SureSelectXT Human All Exon V6  or clinical exome analysis (6110 genes) , respectively. The final enriched libraries were sequenced with 2 × 150 bp on Illumina sequencers . The average sequencing depth for each sample was 149.3X (Patient 1), 174.9X (Patient 2), 148.7X (Patients 3 and 4), 133.9X (Patient 5), 130.5X (Patient 6), 132.02X (Patient 7), and 130.2X (Patient 8). Sequence data were aligned to the human genome reference (UCSC Genome Browser build hg19) using Burrows-Wheeler Aligner (BWA) . VariantHIBCH gene was firstly identified by WES as the loss of heterogeneity in the Patient 8. Droplet-based digital PCR (ddPCR) was performed in the patient and his father to verify this deletion. Using QX200™ Droplet digital™ PCR systems, we performed the processes of sample dispersion, amplification and Quantification in turn. The number of droplets was obtained in the droplet reader based on the fluorescence amplitude  were analyzed by electrospray tandem mass spectrometry (LC-MS/MS) for C4-OH . Samples were prepared as follows: a 3-mm diameter disc was punched in a microtiter plate, 100 μl internal standard solution was added, and the plate was covered. After gentle shaking at 45°C for 45 min, the solvent after elution was transferred into a new microtiter plate and analyzed immediately. An LCMS-8040  in positive mode was utilized for acylcarnitines and amino acids analysis. For MS/MS data acquisition, the peak including C4-OH (248.2>85) was observed with multiple reaction monitoring.Seven patients’ urine metabolites, including 2,3-dihydroxy-2-methylbutyrate (23DH2MB), were analyzed by standard gas chromatography-mass spectrometry (GC-MS). Briefly, 100 μl urine was incubated with 40 units urease at 37°C for 15 min. After adding an internal standard (20 μg of n-heptadecanoic acid), the sample was centrifuged to deproteinize by adding 1,000 μl of ethanol, then the supernatant was evaporated. The residue was completely dried under a nitrogen stream for 3 min and derivatized with 100 μl of N, O-bis(trimetnylsilily) trifluorcetamide and 10 μl of trimethylchlorosilane at 90°C for 40 min . A GCMS-Through retrospective re-analysis, quantitative urine screening for S-(2-caboxypropyl) cysteamine (SCPCM) was measured by LCMS/MS in urine of cases. This project was not routinely performed in the metabolic laboratory. The process included sample and label pretreatment. The SCPCM concentration in urine of 140 control children was determined by the detection and verification of an SCPCM standard, and the reference value was obtained using the SCPCM standard concentration curve.At the first visit, clinical data were collected retrospectively, consisting of demographic features, early developmental milestones before first symptoms, age at presentation, initial present, precipitating causes, metabolite findings, and radiographic data.During the follow-up period, all eight patients were assessed every 6 months to 1 year at our neurology clinic. Each follow-up evaluated motor, cognition and linguistics functions; muscle strength and tone; feeding situation; growth index; recurrence; metabolite analyses; and response to treatment.Follow-up MRI results were classified into four levels: progression, stable, evolution, and regression . The NewHIBCH mutations.We reviewed the data of 34 published patients, of which 4 cases had insufficient clinical data . The datHIBCH gene mutations and other gene mutations associated with Leigh/Leigh-like syndrome.We grouped confirmed genes of 181 patients with Leigh/Leigh-like syndrome according to pathomechanism including OXPHOS complexes , mitochondrial DNA-related protein synthesis, mitochondrial homeostasis, substrate, mitochondrial cofactors, and inhibitors. The age of onset, predisposing factors, initial symptoms, main symptoms, brain imaging, laboratory examinations, and clinical outcomes of 173 patients were investigated to analyze similarities and differences between subjects with HIBCH gene with compound heterozygous states (HIBCH gene in Patient 5 homozygous for the c.1027C>G (p.His343Asp) HIBCH variant. Our identified 6 novel mutation and 31 other reported mutation sites are summarized in Ten mutations, including six novel mutations, were identified by NGS of the s states , and accs states . No othes states . Two novs states . The forThere were 5 (5/8) patients with DBS showing elevated C4-OH during either the peak or recovery phase. Six (6/7) had increased 23DH2MB levels in urine , includiAdditionally, we found that the NPMDS scores during the peak phase may be correlated with the value of C4-OH and SCPCM. We also observed that patients with lower C4-OH during the peak phase were more likely to have a positive response to treatment.In this case series, eight patients  were from seven non-consanguineous Chinese families, and Patients three and four were siblings. The clinical features are summarized in The median age of diagnosis was 2.0 years (range 1.0–5.7 years). During the disease course, the frequently prominent clinical features were hypotonia (8/8), developmental delay (8/8), developmental regression (7/8), encephalopathy (6/8), feeding difficulties (5/8), dystonia (4/8), ataxia (4/8), metabolic acidosis (4/8), strabismus (4/8), nystagmus (3/8), and seizures (3/8). Video electroencephalogram (VEEG) in two cases (Patients 3 and 4) with tonic seizures showed bilateral central and apical areas with sharp and slow wave emission that was more remarkable on the right side. The VEEG of Patient 8 with focal seizures presented as continuous release of slow waves and multiple spikes in the right frontal and temporal regions. Two cases (Patients 3 and 8) developed status epilepticus during acute encephalopathy. Three  developed respiratory failure during acute encephalopathy, and all required intubation and ventilatory support for ∼1 week, of which one case (Patient 3) had a failed extubation and underwent a second intubation for ventilator-assisted breathing. Two cases (Patients 2 and 8) had two episodes of acute encephalopathy both induced by infection in 1 year. During acute encephalopathy period, two cases (Patient 2 and 4) were both associated with thyroid dysfunction  and one case (Patient 6) with hepatic dysfunction , and about half a year after the peak period, these indicators gradually returned to normal.Once the patients diagnosed, we fully informed the parents about treatments and recommended pharmacologic therapy and adopting a valine-restricted diet. The drugs consisted of antioxidants and OXPHOS complex cofactors including L-carnitine (50 mg/kg/day), coenzyme Q10 (10 mg/kg/day), thiamine (10 mg/kg/day), and riboflavin (10 mg/kg/day), additionally including some symptomatic drugs, such as levetiracetam, or baclofen. For the dietary treatment, we worked with nutritionists to develop a plan to limit valine intake with protein intake of 1.0–1.5 g/kg/d, recommended energy intake of 80 kcal/kg/d, and no restriction on carbohydrates. It was recommended to use maple glycosuria formula milk powder and supplement leucine and isoleucine.. The treated patients presented with significant decreased scores.Two cases (Patients 1 and 5) only used drug treatments, 3  initiated drug and diet therapy, and 3 cases  abandoned therapy. The NPMDS scores in the peak phase and at the last follow-up (recovery phase) were shown in The median duration between initial symptom onset and the last follow-up was 2.3 years (range 1.3–7.2 years). NPMDS scores during the peak phase and at the last evaluation, as well as MRI findings were showed in Patient 1 exhibited developmental regression following pneumonia at age 14 months but subsequently recovered. Unfortunately, at 4 years 5 months, she experienced acute encephalopathy due to a febrile infection, which again led to developmental regression, and she lost the skills of sitting and speaking. She received drug therapy thereafter and slowly recovered and gradually improved. The patient is currently 8 years 4 months old, with a normal growth index. She is independent and attends grade 3 at a normal elementary school but sometimes has trouble concentrating and wandering. She has some movement intolerance and walks with a slightly abnormal right lower limb posture. Despite having several fevers and other infections since initiating treatment, none have worsened her condition. Her last MRI scan showed significant improvement .For Patient 5, roseola infantum at age 13 months caused her condition to deteriorate, presenting with no head control, no language, and unable to sit unassisted. Following drug therapy, she gradually recovered. Now she is 2 years 4 months old, can understand basic sentences, walk a dozen steps unsupported, and speak 2 and 3 words.Patient 7 has received drug and diet treatments for 6 months and has improved. The therapy included drugs of L-carnitine 500 mg/day, coenzyme Q10 100 mg/day, thiamine 100 mg/day, riboflavin 100 mg/kg/day, and limit protein intake 12 g/d, no restriction on carbohydrates. At present she is 2 years 7 months, can walk without assistance but occasionally falls, can understand complex sentences, and pronounce monosyllables.Patient 6 have received the standard therapy of drug and diet plus baclofen (1 mg/kg/day) since the onset of encephalopathy for nearly 1 year. Despite four bouts of pneumonia during this time, his condition did not deteriorate. He is 3 years and 2 months old, with dystonia as the main manifestation. He can walk for a few steps with support, understand general language, and pronounce sequential words.Patient 8 was initiated on drug therapy plus levetiracetam (30 mg/kg/day) since onset at age 11 months with partial responses. However, 10 months later he experienced a second encephalopathy episode followed by frequent seizures and status epilepticus. Since being placed on a valine-restricted diet, he has shown a dramatic response. Although he has been seizure-free for 5 months, there has been no improvement in development. He is 2 years 4 months old, with no head control, language, or eye contact.The conditions of all three cases without long-term treatment  deteriorated. They had poor response to treatment in the early stage, and all eventually stopped therapy. At the last follow-up, Patient 2 was 4 years old, with height 90 cm (<3rd percentile) and weight 8.5 kg (<3rd percentile). She has poor head control with no eye contact or language. Patient 3 is currently 8 years old. He has no head control and is unable to sit unsupported or actively grasp objects. He knows simple words but is nonverbal. Patient 4 is presently 6 years 4 months old; his clinical course and physical examination are similar to that of his older brother (Patient 3).Six (6/8) patients underwent follow-up neuroimaging . Brain MThe patient's general condition, clinical, imaging, and metabolic characteristics were summarized in rized the clinical, genetic, metabolic and imaging characteristics of the 173 cases. Due to baseline heterogeneity and large differences in sample size, we only performed statistical descriptions of the data  and urine 23DH2MB (85.7 vs. 5.8%) were significant in the HIBCH mutation group. Among the 173 patients group, 10 cases showed elevated 23DH2MB levels, including 8 with ECHS1 mutation, 1 with SURF1 mutation, and 1 with MT-ND6 mutation, and the latter 2 patients showed slightly elevated 23DH2MB levels and normal C4-OH levels. This implied that elevated levels of C4-OH combined with 23DH2MB in patients with clinically diagnosed Leigh/Leigh-like Syndrome may serve as specific metabolites for HIBCH mutation patients.There were 43 other mutant genes identified in 173 cases with Leigh/Leigh-like syndrome . We summthe data . The symHIBCH mutations associated with Leigh/Leigh-like syndrome and reported their follow-up data providing additional natural history information. We also investigated the relationships between metabolite levels, disease severity, and clinical outcomes.We analyzed the clinical, metabolic, and genetic features of 8 patients with HIBCH mutations, 4 of which have insufficient clinical data. Twenty-six (26/32) had initial symptoms within 2 years of age, including seven cases who died within an onset age of 1 year old  patients described elsewhere, most of had poor responses to treatment and worse prognoses , is the first-line auxiliary diagnostic approach. However, the targeted NGS testing is costly, and it can not identify possible pathogenic variants in deep intron regions and larger deletions/duplications. If the metabolites and clinical phenotypes are well defined and consistent, it may be recommended to directly perform the less costly approach of Sanger sequencing. Unfortunately, we did not perform enzymatic detection of HIBCH. We should combine clinical, metabolic and genetic analysis to diagnose the disease. Early diagnosis and treatment may delay disease progression of the disease. The main limitations of our study were the small sample size and the absence of formal clinical trials.HIBCH gene variants in 8 patients with Leigh/Leigh-like syndrome, including 6 novel variants. The phenotype of Leigh/Leigh-like syndrome caused by HIBCH mutations was not significantly different from other gene mutations, except for more comment presentations with hypotonia, dystonia, and acute encephalopathy. The genotype-phenotype correlation requires further research. However, measurements of metabolites including C4-OH, 23DH2MB and SCPCM were relatively specific and also associated with disease severity, therapeutic effects, and possibly prognosis. The non-invasive tools of NGS and metabolite analyses should be considered first-line auxiliary diagnostic approaches. Patients with Leigh/Leigh-like syndrome with HIBCH mutations may have a positive prognosis, especially with early diagnosis and timely therapy.In conclusion, we identified 10 rare


Treatment with OBE inhibited the house dust mite (HDM)-induced increased phosphorylation of EGFR, ERK1/2 and AKT which resulted in the inhibition of HDM-induced increase in airway cellular influx, perivascular and peribronchial inflammation, goblet cell hyper/metaplasia, and also inhibited ex vivo eosinophil chemotaxis. Moreover, treatment with a combination of a low dose OBE and low dose dexamethasone resulted in a significant inhibition of the HDM-induced cellular influx, perivascular and peribronchial inflammation, goblet cell hyper/metaplasia, and increased the pERK1/2 levels, whereas neither treatment, when given alone, had any discernible effects. This study therefore shows that inhibition of the EGFR/ERK1/2/AKT-dependent signaling pathway is one of the key mechanisms by which OBE can mediate its anti-inflammatory effects in diseases such as asthma. Importantly, this study also demonstrates that combining OBE with steroids results in significantly enhanced anti-inflammatory effects. This action may have important potential implications for future asthma therapy.The treatment of allergic diseases, such as asthma, with both conventional and novel therapies presents a challenge both in terms of optimal effect and cost. On the other hand, traditional therapies utilizing natural products such as onion have been in use for centuries with demonstrated efficacy and safety but without much knowledge of their mechanims of action. In this study, we investigated if the anti-inflammatory effects of onion bulb extract (OBE) are mediated  Natural products have been the cornerstone of therapeutic agents for millennia and more recently an important source of therapeutic drugs with unique structural diversity and pharmacological actions . Many th. While the mechanisms of asthma still remain unclear, it is well recognized that chronic airway inflammatory disease is central to its pathogenesis and is mediated by inflammatory cells such as mast cells and eosinophils and is driven by specific Th2 and Th17 lymphocytes, cytokines, and chemokines  and long-acting beta-agonists (LABA) is a main treatment advocated by most asthma guidelines , a signiNovel monoclonal antibody-based therapy, in inflammatory disease management, has made an impact on disease control. For example, the anti-IgE antibody, omalizumab, has been used as a steroid sparing drug in patients with severe asthma, but unfortunately its use is limited  due to aAllium cepa L. (Family Amaryllidaceae) is one of the most commonly consumed vegetables and has also been used for medicinal purposes for numerous ailments such as ulcer wounds, scars, dysentery, inflammation, hypertension and also in respiratory conditions such as cough, asthma, and bronchitis   . In this closely , are necThe EGFR/ERK1/2/AKT signaling pathway has been recently shown to be an important signaling pathway, in both clincial and preclinical studies. However, whether the EGFR/ERK1/2/AKT signaling pathway is a target for OBE in a clincially relevant animal model of allergic asthma, is not known. Also whether OBE synergizes with steroids has not been studied previously. In this study, using a clincially relevant allergen (HDM), we investigated 1) whether the EGFR/ERK1/2/AKT signaling pathway is modulated by OBE, and 2) if OBE synergizes with dexamethasone to result in a greater anti-inflammatory action.Allium cepa, and a voucher specimen, number KOE-010, was deposited at the herbarium of Kuwait University (KTUH), College of Science, Kuwait.Fresh red onion bulbs were bought from the local market. The plant was identified as vacuo till dryness to obtain brownish syrupy residue. This extraction process was repeated as needed. The treatment stock solution was prepared using the residue and PBS as a vehicle.About 20 kg of fresh red onion was peeled, coarsely cut, and percolated three times, each using 10 L of dichloromethane. The dichloromethane layers of the percolates were separated from the aqueous layer, dried over anhydrous sodium sulphate, and then evaporated in –mass spectrometer Double Focusing Sector system (GC–MS DFS) fitted with a DB-5MS capillary column with 0.25 μm film thickness, 30 m length and 0.25 mm inner diameter, using helium as a carrier gas with a flow rate of 0.8 ml/min. The operating conditions were as follows: splitless injector with port temperature 250°C, detector temperature 280°C, and program temperature from 50 to 250°C at the rate of 6°C/min with 10 min hold time, and from 250 to 300°C at the rate of 10°C/min with 6 min hold time. The MS conditions were as follows: electron impact ionization mode, ionization energy 70 eV, ion source temperature 175°C, scan range m/z 40–900 Da. The qualitative identification of the compounds was based on computer matching with NIST MS Search 2.0 library and by comparison with data in the literature (Dichloromethane extract of onion bulbs (1 μl sample size) was analyzed on a Thermo high resolution gas chromatographyterature .ad libitum.Male BALB/c mice  were used in this study. All studies involving animals were in accordance with the ARRIVE guidelines for reporting experiments involving animals. All experimental protocols were approved by the Animal Welfare and Use of Laboratory Animals Committee in the Health Sciences Center, Kuwait University and were carried out in accordance with the EU Directive 2010/63/EU for animal experiments and the National Institutes of Health guide for the care and use of Laboratory animals . Animals were maintained under temperature-controlled conditions with an artificial 12 h light/dark cycle and were allowed standard chow and water Seven treatment groups  were established to determine whether OBE if given prophylactically inhibits the HDM-induced asthma phenotype. All mice were immunized once by intraperitoneal (i.p.) injection of 40 µg HDM in 0.2 ml of alu-Gel-S  on day 0. Mice were subsequently challenged for 3 days, days 14, 17, and 18 with HDM or PBS in the case of the control group. Mice in groups A (n = 11) and B (n = 13) were pretreated intraperitoneally with 0.2 ml of the drug vehicle, 1 h before each intranasal challenge with PBS and HDM, respectively. In the same manner, groups C (n = 10), D (n = 9), E (n = 10), and F (n = 15) were pretreated with the same volume of OBE at 10, 30, 60, and 100 mg/kg, respectively, and group G (n = 11) with dexamethasone (3 mg/kg), 1 h before each intranasal challenge with HDM  was performed to obtain BAL fluid, and then the lungs were excised for preparation for histology, Western Blot (WB), and immunofluorescence (IF) studies. In another separate group of animals, airway responsiveness was measured. For the histology/WB/IF studies, the OBE dose of 60 mg/kg was selected to represent the prophylactic approach as it gave an optimum effect.Six treatment groups  were established to determine whether combining OBE and dexamethasone would results in synergistic anti-inflammatory actions. Mice in groups A (n = 9) and B (n = 11) were pretreated intraperitoneally with 0.2 ml of the drug vehicle 1 h before each intranasal challenge with PBS and HDM, respectively. In the same manner, groups C (n = 11), D (n = 7), E (n = 11), and F (n = 9) were pretreated with the same volume of OBE at 30 mg/kg (OBE 30), OBE at 30 mg/kg in combination with dexamethasone (DEX) at 0.5 mg/kg (OBE 30 + DEX 0.5), 0.5 mg/kg (DEX 0.5 mg) and 3 mg/kg (DEX 3 mg), respectively, 1 h before each intranasal challenge with HDM. Treatment with OBE and/or dexamethasone was also repeated on days 14, 17, and 18  after sacrificing the animals with an over dose of halothane. The BAL cells were then counted using a particle-size counter , and cytosmears were prepared for differential count. The cells were stained with Diff-Quik, and a differential count of 200 cells was performed using standard morphologic criteria. Results are expressed as total cell count/ml and as total macrophages, lymphocytes, neutrophils, and eosinophils/ml in BAL fluid. For histological assessment, segments of the lung tissue were removed and fixed in 10% buffered formalin, embedded in paraffin wax and sectioned into 5-µm-thick slices. The sections were processed and stained separately with H&E stain and periodic acid–Schiff (PAS) according to standard methods as previously described . SectionRL) (cm H2O per ml/s).Airway responsiveness was measured 24 h after last HDM or PBS challenge, using a separate set of animals using the Buxco FinePointe series RC site  as described previously . BrieflyLung tissues were processed as described above. Immunofluorescence was performed as previously described . In shor3VO4, 50 mM NAF, 1 mM PMSF, 20 µM phenyl arsine, 10 mM sodium molybdate, 10 µg ml–1 leupeptin and 8 µg ml−1 aprotinin. Using a homogenizer, the tissues were homogenized for 10 s, three times. Samples were allowed to lyse completely by incubation on ice for 30 min. The lysates were then centrifuged at 13,000 rpm for 10 min at 4°C and the supernatants collected, and protein concentrations were estimated by Bio-Rad Bradford Protein Assay . Aliquots containing equal amounts of protein were subjected to SDS-PAGE and transferred electrophoretically onto nitrocellulose membrane . The membranes were blocked with 5% BSA and then incubated with ERK1/2 (137F5) , pERK1/2 (Thr202/Tyr204)  and β-actin antibody   at 4°C overnight. Membranes were incubated with appropriate secondary antibodies conjugated to horseradish peroxidase  to detect phosphorylated form of ERK1/2 (42/44 kDa), or total form of actin (45 kDa). The immunoreactive bands were detected with Super Signal Chemiluminescent Substrate  utilizing a Kodak autoradiography film (Care stream Biomax Xarfil 1660760). Images were then analyzed and quantified and the data were normalized to β-actin levels. The experiment was run twice with lung samples from three different mice in each treatment group (pooled) in each run.Appropriate lobes from the dissected lungs of the mice were snap-frozen in liquid nitrogen and stored at −80°C. Following that, the tissue samples were defrosted in ice then transferred to lysis buffer (pH 7.6) containing 50 mM Tris-base, 5 mM EGTA, 150 mM NaCl, 1% Triton 100, 2 mM Na™ Mouse Cytokine Array Kit . The procedure was done in line with the manufacturer’s protocols and as recently described  blood by erythrocyte sedimentation, followed by percoll gradient centrifugation as reported recently . Eosinop5) were then placed in the upper wells, and 500 µl of BAL fluid derived from mice challenged with PBS (vehicle) or with HDM, pretreated ex-vivo with either vehicle or OBE , were placed in the lower wells (37 °C/5%CO2) and eosinophils allowed to migrate for 1 h. The transmigrated eosinophils were determined by counting under the microscope by using a hemocytometer.Peripheral blood derived eosinophils were used for chemotaxis assay utilizing a Boyden chamber as previously described . PurifieL values were computed and were used as an index of airway responsiveness. For the histopathological assessment, a semi-quantitative 5-level lung pathology score was used to grade the degree of inflammation in each microscopic field at 20×. All data were initially assessed for normality. One-way analysis of variance (ANOVA) test followed by Bonferroni post hoc was used to compare differences between individual groups for both total and differential cell count as well as histopathological data and the immunofluorescence data for both the prophylactic and the synergistic studies. A two-way repeated measure analysis of variance followed by a Bonferroni post hoc test was used for the airway responsiveness data. The mean difference was considered as significant at a probability level of less than 0.05. All analyses were performed using GraphPad Prism.All numerical values were expressed as means ± SEM. Total cell counts represent the number of cells/ml of BAL fluid. Differential cell counts represent the absolute number of each cell type/ml of BAL fluid. Absolute RThe extraction process was repeated four times to afford 7.5 g (0.038% yield) of brownish syrupy residue (Allium cepa bulb (OBE) was analyzed to identify different compounds and confirm the identity of the plant. The identified compounds and their mass spectral data are listed in via a direct comparison and matching with the stored spectra in the GC–MS system.Dichloromethane extract of vs 11.0 ± 2.0 × 105 cells/ml) as well as in lymphocytes (0.03 ± 0.01 vs 1.9 ± 0.6 × 105 cells/ml) and eosinophils (0.04 ± 0.02 vs 4.9 ± 0.6 × 105 cells/ml),  developed a significant increase in total cell count (1.3 ± 0.3 vs 1.0 ± 0.07) and increase in bronchial mucus production and goblet cell hyper/metaplasia  demonstrating a marked degree in airway remodeling , achieving almost normal histological appearance that was very similar to the dexamethasone treatment group  showed normal histology  on the airway expression levels of various pro-inflammatory cytokines was determined. HDM challenge significantly enhanced the expression of the following interleukins (IL) by: IL-3 (147.4%), IL-4 (104.6%), IL-5 (5400%), IL-10 (152.0%), and tumor necrosis factor (TNF-α)   to methacholine in the HDM-challenged mice as compared to the PBS-treated control mice at a dose of 25 mg/ml (5.2 ± 0.2 vs 3.9 ± 0.2 cm H2O per ml/s) and 50 mg/ml (7.9 ± 0.5 vs 4.6 ± 0.3 cm H2O per ml/s)  . Our data show that there was a significant increase in airway responsiveness 24 h after the last intranasal HDM challenge as demonstrated by a significant increase in lung resistance  or dexamethasone (0.5 mg/kg) alone resulted in modest but significant reduction in the HDM-induced perivascular and peribronchial inflammation  or the low dose dexamethasone (0.5 mg/kg) did not result in marked inhibition of the HDM-induced phosphorylation of ERK1/2  at sub-maximal doses resulted in an enhanced anti-inflammatory effect at the cellular, histopathological, and molecular levels.The major finding of this study is that OBE produces anti-inflammatory actions in an established asthma model, partly, The use of natural products as therapeutic agents has been on the rise, not only due to their demonstrated efficacy  but possibly due to their perceived, and in many instances, real lack of adverse effects, even when consumed in large quantities . Based oOur data show that OBE dose-dependently decreased the total and differential cell influx into the airways, at both 60 and 100 mg/kg dose, confirming data from recent studies reporting its inhibitory effect on BAL fluid cellularity . In linevia inhibition of EGFR-dependent signaling perturbation. However, it is also likely that in addition to the EGFR pathway, OBE may inhibit other pro-inflammatory signaling pathways.The EGF/EGFR is a critical signaling pathway in the pathogenesis of asthma, and both EGF and EGFR levels have been shown to be consistently increased in both human asthma and in animal models of asthma . Howevervia inhibition of the EGFR/ERK1/2 dependent signaling , Kuwait University. GC-MS analyses were done at Research Sector Project Unit (RSPU), Faculty of Science, Kuwait University, supported by Grant numbers GS01/03.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.


Fragaria × ananassa ‘Camarosa’) fruit to different numbers and timing of MeJA applications, we performed three differential preharvest treatments  consisted of successive field applications of 250 μmol L–1 MeJA at flowering (M3), large green (M2 and M3), and ripe fruit stages . Then, we analyzed their effects on fruit quality parameters  along with anthocyanin and proanthocyanidin (PA) accumulation; the antioxidant-related enzymatic activity of catalase (CAT), guaiacol peroxidase (POX), and ascorbate peroxidase (APX); the total flavonoid and phenolic contents, antioxidant capacity, and ascorbic acid content (AAC) during post-harvest storage . We also evaluated the effect on lignin, total carbon and nitrogen (%C and N), lipid peroxidation, and C and N isotopes signatures on fruits. Remarkably, the results indicated that MeJA treatment increases anthocyanin and PA contents as well as CAT activity in post-harvest storage, depending on the number of preharvest MeJA applications. Also, M3 fruit showed a higher AAC compared to control at 48 and 72 h. Noticeably, the anthocyanin content and CAT activity were more elevated in M3 treatment comparing with control at all post-harvest times. In turn, APX activity was found higher on all MeJA-treated fruit independent of the number of applications. Unlike, MeJA applications did not generate variations on fruit firmness and weight, lignin contents,% C and N, and in lipid peroxidation and water/nitrogen use efficiency according to C and N isotope discrimination. Finally, we concluded that an increasing number of MeJA applications (M3 treatment) improve anthocyanin, PA, AAC, and CAT activity that could play an essential role against reactive oxygen species, which cause stress that affects fruits during post-harvest storage.Preharvest applications of methyl jasmonate (MeJA) have been shown to improve post-harvest fruit quality in strawberry fruit. However, the effectiveness of consecutive field applications at different phenological stages on the reinforcement of the antioxidant capacity remains to be analyzed. To determine the best antioxidant response of strawberry ( Fragaria × ananassa Duch.), a Rosaceae family member, is one of the most popular fruits grown worldwide due to its organoleptic attributes and abundance in nutrients, vitamins, and minerals , during an in vitro fruit ripening MeJA assay induces an increase in the red coloration of fruit skin along with anthocyanin levels (–1 MeJA in the Chilean strawberry fruit [Fragaria chiloensis (L.) Mill.] increase antioxidant capacity during post-harvest. Moreover, the antioxidant enzymatic related activity also has been associated with MeJA application. In Arabidopsis thaliana, the total activities of catalase (CAT), peroxidase (POX), superoxide dismutase (SOD), and glutathione reductase (GR) increased considerably in response to MeJA (The jasmonic acid (JA) and its endogenous plant hormone derived methyl jasmonate (MeJA) have been implicated in several physiological processes, mainly modulating plant defense responses, including antioxidant capacity against pathogens and abiotic stresses  althoughn levels . Other r to MeJA . In this–1 MeJA during strawberry fruit development and ripening. Then, we analyzed their effects mainly on fruit quality parameters, anthocyanins and proanthocyanidins (PAs) accumulation, antioxidant enzymatic, and non-enzymatic activity during four post-harvest storage times. This investigation aimed to determine the best combination between the MeJA application frequency and phenological stage of applications to enhance the antioxidant capacity of strawberry (F. × ananassa ‘Camarosa’) fruit. We found that three MeJA applications from flowering to ripe fruit stages improve the antioxidant response of the fruit during post-harvest storage.In this study, we performed three differential preharvest treatments consisted of successive field applications of 250 μmol LF. × ananassa ‘Camarosa’ distributed in three random plots were used per treatment. Flowers were marked in each plot (n = 180) to analyze the same biological material at each developmental stage planned in the experiment. Methyl jasmonate (MeJA) treatments consisted of different sequential applications as sprays on the plant performed through strawberry fruit development. In this sense, M3 treatment consisted in three different applications at flowering, after 24 days at the large green, and after 7 days at 100% red receptacle fruit stages; M2 treatment consisted in two applications at the large green, and after 7 days at 100% red receptacle; and M1 treatment consisted in one application at 100% red stage  at pH 4.3, and 0.05% (v/v) Tween-20 as surfactant. Distilled water plus 0.05% (v/v) Tween-20 was used as a control (C). The MeJA concentration was chosen as the minimum effective concentration according to previous field experiments on strawberry . Climatic conditions of this location are presented in ed stage . Each aprawberry , 2017. HFour times point evaluations were made at 0, 24, 48, and 72 h during post-harvest storage to check the long-term effect of different preharvest MeJA applications. One hundred eight fruits from each preharvest MeJA treatment were selected for the experiment based on uniform size, shape, and absence of surface damage. For each post-harvest time point, 27 fruits per treatment were separated into three groups of nine fruit each and packaged in transparent perforated plastic containers  and maintained at room temperature (25°C) and 40% relative humidity.n = 352) and expressed as grams per fruit (g⋅Fruit–1). Seventeen fruits from each treatment were weighed at each time point (three replicates of nine fruit each) during the post-harvest experiment, and weight loss was expressed as a percentage. Afterward, strawberry skin color from each treatment and post-harvest time point (three replicates of six fruit each) was measured using a colorimeter  and expressed according to the CIELAB scale where L∗, a∗, and b∗ values indicate lightness (dark to light), redness [green (−) to red (+)] and yellowness [blue (−) to yellow (+)], respectively. The dimensions of color Chroma [C = (a*2 + b*2)1/2] and Hue angle [h° = arctan (b∗/a∗)] were calculated from numerical values of a∗ and b∗ .Fruit firmness was measured at opposite sides of 18 fruits from each treatment and post-harvest time point (three replicates of six fruit each) using a texture analyzer  fitted with a flat 3 mm TA-39 probe suitable for firmness measurement in berries and small fruits. Each fruit was penetrated 3 mm at 1 mm sTM HOMOGENIZER 125, Kinematica, Lucerne, Switzerland) with a 12 mm rotor, and filtered. For SSC, 150 μL of each sample was measured using a digital refractometer , recorded as Brix degrees (°Bx) and expressed as g of sucrose per 100 g of fresh weight (FW). For TA, aliquots of 2.5 mL of each sample were diluted in distilled water  and titrating using semi-automatic titrator  with 0.025N NaOH to pH 8.2. Results were calculated as citric acid equivalents (CAE) per 100 g of FW, according to Soluble solids content (SSC) and titratable acidity (TA) analyses were conducted according to 13C and δ15N, respectively, in ‰) were determined using a combustion and gas preparation module , attached to an Isotope Ratio Mass Spectrometer . Combustion and reduction columns were operated at 1000 and 600°C, respectively, and ultra-grade high purity helium was used as the carrier gas. Combusted gas passed through a GC column operated at 100°C. An ultra-grade reference gas  was injected before each analysis for CO2 and N2 peak drift correction. Two laboratory standards  were run every 10 analytical samples to ensure analytical quality. These standards were previously calibrated against international reference materials . The long-term standard deviation of repeated dual-mode δ15N and δ13C measurements of the laboratory standards were ± 0.2 and ± 0.4‰, for δ13C and δ15N, respectively.Lyophilized leaf and fruit tissues were oven-dried to a constant weight, milled, and homogenized in a Spex ball micro mill. An aliquot of the milled samples (six fruits or leaves per treatment) was then weighted with a precision of ± 0.001 mg and encapsulated in tin capsules. Total carbon and nitrogen content (in% of dry weight) and carbon and nitrogen isotope composition (δ–1). The results were expressed as micromoles of MDA per g of FW.The level of lipid peroxidation in cell membranes was measured in terms of malondialdehyde (MDA), using 2-thiobarbituric acid-reactive substances (TBARS) method described by Pooled fruit receptacle (2 g) of each treatment were homogenized in 40 mL of acetone/water/acetic acid  solution . The res3, 100 μL of 1M CH3CO2K, and 2.8 mL of distilled water. After incubation at room temperature for 30 min, the OD of the reaction mixture was measured at 415 nm in a UV/Vis spectrophotometer , and the results were expressed as mg of quercetin equivalents (QE) per 100 g of FW.Total flavonoid content (TFC) was determined, according to Total phenolic content (TPC) was determined by The antioxidant capacity was measured by oxygen-radical absorbing capacity (ORAC) method and assayed for each AWA extract from each treatment and post-harvest time point (three replicates of three fruit each) as described by –1cm–1) reported by Total anthocyanin content (AC) was quantified by the pH differential method , with so–1) of catechin as standard. Results from each treatment and post-harvest time point (three replicates of three fruit each) were expressed as μg of catechin equivalent per g of FW.Total proanthocyanidin content (PA) was quantified by 4-dimethylaminocinnamaldehyde (DMAC) colorimetric method , with so–1) of hydrolytic lignin (Sigma-Aldrich) as standard. Results from each treatment and 0, 24, and 48 h post-harvest time point (three replicates of three fruit each) were expressed as μg of lignin per g of FW.Total lignin content was quantified, according to L-ascorbic acid (Sigma–Aldrich). Measurements were performed four times, and results were expressed as mg of ascorbic acid per 100 g of FW.Ascorbic acid content (AAC) was quantified using the 2,6-dichloroindophenol titrimetric method, according to AOAC method 967.21 . Fruit eThe determination of enzyme activities was performed through spectrophotometric activity assays at 25°C. Fruit receptacle (1 g) from each treatment at each post-harvest time point (three replicates of three fruit each) were ground with liquid nitrogen and homogenized in 5 mL of extraction buffer containing 100 mM potassium phosphate buffer (pH 7.8), 1 mM EDTA (pH 7.0) and 5% (w/w) polyvinylpolypyrrolidone (PVPP). Then, the homogenate was centrifuged for 15 min at 10,000 rpm at 4°C. The supernatant was taken as a crude enzyme extract and was used for estimated enzymatic activities, as described below.2O2, ε = 36 mM–1 cm–1) breakdown at 240 nm, as previously described (2O2. Results were expressed as mmoles of H2O2 decomposed per g of FW.The CAT activity was determined following the hydrogen peroxide (Hescribed . The rea–1 cm–1)  . The ass2O2-dependent oxidation of ascorbate by measure the rate of dehydroascorbate (DHA) formation at 290 nm (ε = 2.8 mM–1 cm–1) (L-ascorbic acid (Sigma-Aldrich), 20 μL 10 mM EDTA (pH 7.0), and 20 mL of crude fruit protein extract in a 300 μL-volume. The reaction started by adding 1.2 μL 88 mM H2O2. Results were expressed as mmoles of DHA per g of FW.The APX activity was determined following the H–1 cm–1)  with a fpost hoc test was used to evaluate the significance between treatments at each post-harvest time point . Values of p ≤ 0.05 were considered statistically significant. Principal component analysis (PCA) was used for all variables to discriminate between treatments and post-harvest times points, using the R software with the ggplot2 package (The experiment was performed using a randomized complete block design. All the measurements were conducted at triplicate and expressed as mean ± standard deviation (SD) (tables) or ± standard error of the mean (SEM) (figures). Data were analyzed by analysis of variance (ANOVA) using Infostat software (version 2016) . Tukey p package .F. × ananassa ‘Camarosa’) firmness (p ≤ 0.05).Different field MeJA applications seem to have little or no impact on fruit firmness and weight attributes. Regarding that, no differences between treatments on strawberry  were observed between control (28.92 ± 1.83) and M2 (30.97 ± 2.38) treatments at 0 h and a constant decrease from 0 to 72 h during post-harvest storage in M2 and M3 treatments. We observed an increase in redness (a∗ index) in M2 (30.74 ± 2.19) and M3 (30.23 ± 2.43) treatments at 0 h compared with control (27.59 ± 2.74), although this index decrease through storage time in those treatments. At 24 h of storage, results showed significant differences between control (28.60 ± 1.96) and M3 treatment (26.69 ± 1.69) in luminosity, and between control (24.43 ± 2.88) and M3 treatment (26.58 ± 2.39) in redness. No significant differences were observed at 48 and 72 h of post-harvest storage in any of the color parameters analyzed. The results on fruit skin luminosity and redness indicate that MeJA treatments could increase luminosity (whiter) and redness in fruits at 0 h, although this effect is loss from 24 to 72 h post-harvest storage , 24 (M3), and 48 h (M2) compared with control, which means that MeJA treatments impact this important parameter in strawberry flavor by increasing SSC and reducing TA values . In turn storage .Additionally, with the aim to verify if MeJA alters essential constituents of plants we measured the carbon (C) and nitrogen (N) contents (as% of dry weight) and estimated the C:N ratio in leaf  and frui13C and δ15N) in leaf and fruit at harvest and in lipid peroxidation during post-harvest fruit storage as measurements indicative of stress in different MeJA-treated samples. No differences were found in δ13C and δ15N stable isotope composition, used as a proxy for water use efficiency (WUE) and differential nutrient source/allocation, respectively, both in fruits or leaves  and from 0 to 48 h (M2) of post-harvest storage  and remained higher at 72 h (p = 0.0092) with an increase of 63.8 and 53.3%, respectively. However, no significant difference was found for M1 and M2 treatments concerning control at 72 h. Moreover, AAC values of M2 and M3 treatments remain stable during storage time points (The AAC is shown in e points .p < 0.0001). Higher values were found on M3 treatment fruits at 0, 24, 48, and 72 h compared to control . Also, CAT activity increased two-fold during the post-harvest storage period between 0 and 72 h for M3 treatment. Along with it, M2 treatment remained approximately 4-fold higher than control at all time points during the post-harvest storage. Nevertheless, no differences were found between M1 and control fruits. Related to POX activity , guaiacol peroxidase (POX), and ascorbate peroxidase (APX) is shown in activity , a similactivity , even thFinally, the complete dataset was analyzed by PCA  to identF. × ananassa ‘Camarosa’) fruit quality parameters during post-harvest storage, and it can be useful to understand the mechanism involved in hormonal field application and its effect on the storability of soft fruits.In the present research, we report a significant impact of different preharvest MeJA treatments on different fruit quality parameters such as weight loss, soluble solids content/titratable acidity (SSC/TA) ratio, skin color, total anthocyanin (AC), total proanthocyanidin (PA) and ascorbic acid contents (AAC) accumulation, and antioxidant enzymatic activities of catalase (CAT) and ascorbate peroxidase (APX) during post-harvest storage . It is k–1 MeJA) (–1 MeJA) (–1 MeJA) (–1 MeJA in large green and 100% red receptacle (M2 treatment) and in 100% red receptacle (M1 treatment) stages of strawberry (F. × ananassa ‘Camarosa’) fruit showed an effect on this attribute during post-harvest storage  , apricot–1 MeJA) , and str–1 MeJA) . In the  storage  and coul∗ and a∗ values in MeJA-treated fruits (M2 and M3) than control at 0 and 24 h, indicating a brighter red color in ‘Camarosa’ fruits at the beginning of the post-harvest storage as a result of the preharvest treatment , preharvest application of MeJA also increased skin red color during fruit development  (F. chiloensis)  , and Chiloensis) . All the–1) applied at flowering (–1 MeJA), our results point out that at least two successive applications of 250 μmol L–1 MeJA since flowering (M3) or early fruit developmental (M2) stages are required on commercial strawberry to increase that parameter. Along with this, raspberry fruits treated with foliage-berry spray of 100 μmol L–1 MeJA at the early light pink stage with two consecutive sprayings, had higher SSC and lower TA and therefore a higher ratio of SSC/TA than control fruits or those treated with less MeJA concentrations (–1)  . The rolons (–1) . HoweverIn relation to metabolic changes suggested by changes in N (%) and C:N ratio , the MeJ13C and δ15N isotopes has been reported for the support that the application of MeJA could induce a more significant water use efficiency (WUE) and a differential allocation of nutrients, respectively, in fruits and leaves of different species as a defense mechanism to face both in biotic and abiotic stresses  did not show a relation with those previously reported effects on the antioxidant capacity, total flavonoids content or total polyphenol content values up to 72 h of post-harvest storage. Similar results have been reported in post-harvest MeJA-treated strawberry  since no differences in total phenolic content and antioxidant capacity was observed up to 5 days after treatment  biosynthesis (FaMYB10) and structural  genes related to anthocyanin biosynthesis pathway of strawberry (F. × ananassa ‘Aromas’)  fruit treated with MeJA vapor at post-harvest, the anthocyanin content increased at 5 and 7 days post-harvest, being pelargonidin-3-glucoside, cyanidin-3-glucoside and pelargonidin-3-rutinoside the main augmented anthocyanins (–1) raised anthocyanins such as cyanidin 3-glucoside and cyanidin 3-rutinoside, respectively , and APX, along with non-enzymatic antioxidants such as phenolic compounds, flavonoids, ascorbic acid among others, which are produced as secondary metabolites exerting various protective roles . So, theananassa . Phytohoananassa . In the reatment . Further systems , becauseynthesis , and conAromas’) . In the Aromas’)  probablyocyanins . In raspectively . Besidesectively . Consideectively , our resF. × ananassa ‘Selva’ and ‘Queen Elisa’ cultivars) (de novo biosynthesis of ascorbic acid (F. × ananassa ‘Camarosa’) fruits during post-harvest, being the M3 treatment that reached the highest levels of CAT activity at all post-harvest times (–1 MeJA on strawberry fruits (F. × ananassa ‘Sabrosa’) notified an increased CAT and POX activities (–1 MeJA (–1) increase CAT, POX, and APX activities during lemon fruit development (2O2) in the strawberry plants, as has been early shown in several plant species  . In straltivars) , blueberltivars) , and loqltivars) . Experimbic acid . Besidesst times . Similarst times . Previoutivities . Additio(–1 MeJA . In turnelopment . In geneelopment . Probabl species . This in species .F. × ananassa ‘Camarosa’) fruit stages  can markedly improve fruit quality and reinforce the antioxidant capacity that suggests a better status of the fruit to deal with oxidative stress during post-harvest. It is important to note that the results presented in the current research are related to the Camarosa cultivar, and these could be different using other varieties. All strawberry cultivars present fruits with a different composition of antioxidants (Finally, as the PCA analysis shows differentiation between control and MeJA-treated fruits  and accooxidants , b that oxidants . By any The datasets generated for this study are available on request to the corresponding author.CF designed the research, supervised experiments, and acquired the funding. PZ, YC, OA-S, LF, and FA performed the experiments. PZ, OA-S, LF, FA, and CF analyzed the data. PZ, LF, and CF wrote the manuscript.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.


Our results revealed, for the first time, that short-term inhibition of Rho-associated protein kinases (ROCK) for 4 h prior to in vitro maturation (IVM) in a biphasic IVM approach improved oocyte nuclear maturation, producing more MII oocyte, through modulating the expression of cytokinesis- and antiapoptosis-related mRNA transcripts. This positive result suggests ROCK inhibitor as a potential candidate molecule to exploit in the control of oocyte meiotic maturation.n = 375) in a prematuration medium supplemented with ROCK inhibitor (RI) for 2 h, 4 h, 6 h, and 24 h before submission to normal in vitro maturation to complete 28 h. The control was cultured for 28 h in the absence of RI. In the first phase of experiment two, we cultured COCs (n = 480) in the presence or absence (control) of RI for 2 h, 4 h, 6 h, and 24 h, and conducted real-time relative quantitative PCR (qPCR) on selected mRNA transcripts. The same was done in the second phase, but qPCR was done after completion of normal IVM. Assessment of nuclear maturation showed that pre-IVM for 4 h yielded an increase in MII oocyte . As expected, the same group showed the highest degree (2) of cumulus expansion. In experiment 2, qPCR results showed significantly higher expression of ACTB and BCL2 in the RI group treated for 4 h when compared with the other groups. However, their relative quantification after biphasic IVM did not reveal any significant difference, except for the positive response of BCL2 and BAX/BCL2 ratio after 4 and 6 h biphasic IVM. In conclusion, RI prevents premature oocyte maturation and gave a significantly positive outcome during the 4 h treatment. This finding is a paradigm for future investigation on dromedary camel biphasic IVM and for improving the outcome of IVM in this species.This is the first report on a biphasic in vitro maturation (IVM) approach with a meiotic inhibitor to improve dromedary camel IVM. Spontaneous meiotic resumption poses a major setback for in vitro matured oocytes. The overall objective of this study was to improve in vitro maturation of dromedary camel oocytes using ROCK inhibitor (Y-27632) in a biphasic IVM to prevent spontaneous meiotic resumption. In the first experiment, we cultured immature cumulus–oocyte complexes (COCs,  Camelus dromedarius), with its natural ability to produce quality meat, milk, and fiber under very hot and hostile climatic conditions [Reproductive success in livestock ensures the continuation, survival, and conservation of species, and, by extension, food security. To maximize productivity and to address several reproductive challenges, assisted reproductive technologies (ARTs) have come to the rescue ,2,3. Thenditions , is reprnditions ,6. Few inditions .Studies on camel oocytes are still unfolding. So far, several attempts to improve camel oocyte maturation have been made ,9,10. OnThe biphasic IVM approach involves a temporary delay in meiosis resumption (holding the oocyte at the GV stage in vitro) in a prematuration phase using meiotic inhibitors before submission to normal in vitro maturation ,18. It iSeveral approaches have been examined to delay oocyte meiotic resumption. These include modulation of cAMP signaling  ,19,20,21Rho-associated protein kinases (ROCK) maintain embryonic stem cells properties, promoting their recovery and their post-thaw proliferation ,27. ReceThe role of ROCK in dromedary camel oocyte maturation and the potential effect of its inhibition to prevent spontaneous meiotic maturation in camel IVM have not been previously investigated. The aim of the present study, therefore, is to improve the in vitro maturation of dromedary camel cumulus–oocyte complexes using ROCK inhibitor (Y-27632) in biphasic IVM to prevent spontaneous meiotic resumption.Unless otherwise stated, reagents were obtained from Sigma-Aldrich Corp. . Media preparation and the experimental procedure were performed as described by ,32,33 unn = 320) were collected from a local slaughterhouse in Riyadh and transported within 3 h after slaughter to the laboratory in thermos flasks containing prewarmed saline 0.9% (w/v) maintained at 33–36 °C. Fluid from antral follicles (2–8 mm) was aspirated using an 18-gauge needle connected to a 10 mL syringe to obtain the cumulus–oocyte complexes (COCs). The aspirate was placed in a 60 mm sterile IVF dish. Then, COCs wash medium composed of tissue culture medium 199 (TCM-199) supplemented with 2 mM NaHCO3, 0.1% bovine serum albumin (BSA), 10% (v/v) fetal bovine serum (FBS), 5 μg/mL gentamycin sulfate, and penicillin/streptomycin was added and left to settle for 30 s. COCs with intact, uniform cytoplasm, and compact cumulus were selected under a stereomicroscope and randomly distributed for the experiments.Camel ovaries (Phase 1: Pre-IVM incubation period with RI for different durations .v/v) sterile camel follicular fluid, 10% fetal bovine serum (FBS), 10 μg/mL follicle stimulating hormone (FSH), 10 μg/mL luteinizing hormone (LH), 1 μg/mL 17β-estradiol, 20 ng/mL epidermal growth factor (EGF), 1 μL/mL insulin-transferrin-selenium (ITS), 0.3 μM cysteamine, 0.15 mg/mL L-glutamine, 1 μL/mL gentamycin sulfate and Sodium pyruvate. Then, COCs were incubated in a humidified chamber at 38 °C for different time periods . The control group was incubated without RI. Each group contained 15 COCs that were randomly selected. The experiment was replicated five times. The total number of COCs used was 375 in a completely randomized design.Selected COCs were transferred into new 60 mm dishes containing wash medium (mentioned above) for further washing. Washing was done three times. The COCs were further examined, selected and transferred to the pre-IVM culture medium that was supplemented with 10 µM Y-27632 (RI) to block meiotic resumption ,34. TogePhase 2: Normal IVMAfter pre-IVM for the respective treatments, all COCs were transferred into normal IVM medium (without RI supplement) in separate wells to complete the 28 h maturation period—the optimum time for dromedary camel oocyte maturation in vitro . TreatmeThe degree of cumulus expansion was assessed at the end of IVM culture as described previously . BrieflyTo assess the oocytes for the completion of meiosis I (extrusion of first polar body), COCs were denuded (stripped of cumulus cells) chemically by repeated pipetting in TCM-199 supplemented with 1 mg/mL hyaluronidase. This was done separately for each group. Denuded oocytes were washed three times in wash medium (TCM-199 supplemented with 10% FBS and penicillin/streptomycin). Thereafter, cumulus free oocytes were stained with 5 μg/mL bisbenzimide (Hoechst 33342) for 5 min to visualize the nuclear materials and polar body extrusions using an inverted microscope equipped with epifluorescence . Oocytes were classified as matured , immature , and degenerated .Oocyte morphometrics, including ooplasm diameter, zona pellucida thickness, oocyte diameter, and perivitelline space, were measured in three different areas of the oocyte using ImageJ 1.50i software  . FinallyEffect of biphasic IVM with RI on expression of cytokinesis- and apoptosis-related mRNA transcripts of dromedary camel COCs.BCL2, BAX, and CTSB) and cytokinesis-related genes , COCs for each group were collected in two phases:To determine the effect of RI on the expression of mRNA transcript of selected apoptotic-  in 1.5 μL sterile tubes and snap frozen at −80 °C until RNA extraction was done. The same was done for other groups and their respective controls.Phase 2: COCs were collected after biphasic IVM and randomly distributed into four groups (one control and three treatments). After pre-IVM for 2 h, 4 h, and 6 h, COCs were transferred into normal IVM medium to complete 28 h incubation. The control groups were matured in normal IVM medium without RI. For all groups, COCs were collected after 28 h of incubation. A total of 240 COCs was used, with 20 COCs per group. The experiment was replicated at least three times.® Green Premix (Applied Biosystems). Three replicates for each sample and primer were run. The fold change and relative quantification of cytokinesis or morphology-related  and apoptotic-related  transcripts were normalized to the house-keeping gene GAPDH and calculated with the 2−ΔΔCt method described by [Camelus dromedaries) GenBank sequences. Details of the primer set used are presented in Total RNA was isolated from the COCs collected for each group using the innuPREP RNA Mini kit  following the manufacturer’s guide. The concentration and quality of the total RNA was estimated using a NanoDrop™ 2000 spectrophotometer . High-capacity cDNA Reverse Transcription Kit  was used to synthesize cDNA (20 µL reaction volume) from the total RNA samples according to the manufacturer’s instructions. Briefly, RT master mix sufficient for 10 samples was prepared on ice. The components included 10 μL (100 ng) RNA, 2.0 μL of 10 × RT buffer, 0.8 µL of 25× dNTP mix (100 mM), 2.0 µL of 10× RT random primers, 4.2 µL of nuclease-free water, and 1.0 µL MultiScribe™ Reverse Transcriptase. RT reactions were run in four steps at 25 °C for 10 min, 37 °C for 120 min, then 85 °C for 5 min, and finally at 4 °C until the plate was removed. The cDNA samples were then stored at −20 °C until further reaction. Relative quantification of mRNA transcripts was performed using real-time PCR (Applied Biosystems). The reactions contained 1 μL (100 ng) cDNA, 1 µM forward primer, 1 µM reverse primer, 7 μL nuclease-free water, and 10 μL SYBRribed by  in relatt-test was used when the means of two groups were compared. When more than two conditions were being compared, data were analyzed using one-way ANOVA. When a significant difference was identified (p < 0.05), individual treatment differences were compared using Tukey-HSD test. All statistical analyses were carried out using R software (version 3.6.2) (R Core Team) with R Studio editor using the Agricolae package. Microsoft Excel was used to generate graphs. Pearson’s linear correlation coefficients were calculated to determine the correlation (r) between the means of oocyte maturation indices, morphometric parameters, and mRNA transcript expression, where r values > ±0.7 were considered strong positive/negative linear relationships, r > ±0.5 were considered moderate positive/negative linear relationships, and r <  ±0.5 were considered weak positive/negative linear relationships [COCs were randomly assigned to the experimental groups, and the experiments were repeated at least five times. An unpaired ionships .RI showed no impact on the cumulus morphology when COCs were cultured for 2, 4, 6 h when compared with control group . For cumThe first experiment determined the effect of biphasic IVM  on dromedary camel COC maturation. The COC maturation parameters examined were first polar body extrusion (meiosis I completion), cumulus morphology and expansion, and oocyte morphometry. The results shown in p < 0.05) was observed in all groups for all the parameters measured. The 6P group showed the smallest oocyte diameter (138.82 ± 1.11 µm), zona pellucida thickness (9.14 ± 0.25 µm), and ooplasm diameter (103.93 ± 0.7 µm). The control group had the thickest zona pellucida (12.97 ± 0.5 µm). The perivitelline space (PS) length was smaller in the control group (2.56 ± 0.41 µm) and the 4P group (3.19 ± 0.30 µm), while large PS length was observed in both the 2P (6.01 ± 0.30 µm) and 6P (6.55 ± 0.7 µm) groups. The ooplasm diameter in the 2P, 4P, and control group were not significantly different.To explore the effect of biphasic IVM with ROCK (pre-IVM with ROCK inhibitor for varying duration) on oocyte morphometry, COCs were denuded after IVM and observation and image capturing were achieved using an inverted microscope. The captured images were used to obtain morphometric parameters including oocyte diameter (OCD), zona pellucida thickness (ZP), length of perivitelline space (PS), and ooplasm diameter (OPD) see . As presACTB to be the only cytokinesis gene with markedly reduced expression level. A simultaneous reduction of both of proapoptotic transcripts  and antiapoptotic BCL2 was surprising. After inhibiting ROCK for 4 h (4P), we found a positive effect on the expression of cytokinesis related genes (except for TUBA1 which was significantly reduced) and anti-apoptotic BCL in 4P (BAX/BCL2 was significantly reduced compare to the 4C control group (BCL2 was reduced in the 6 h (6P) group (BCL2) transcripts were significantly lowered, while proapoptotic (BAX) transcripts had significant increase  group C. When wincrease D.ACTB, TUBA1, and CFL1) and apoptosis-related  genes. There was no significant difference (p > 0.05) in the relative quantification of ACTB, CFL1, BCL2, and CTSB amongst treatments (TUBA1 and BAX (p < 0.05) compared to the other groups.Next, we examined the effect of biphasic IVM (pre-IVM with RI for 2 h (2P), 4 h (4P), and 6 h (6P) followed by normal IVM (as illustrated in eatments . BiphasiACTB to be positively correlated with oocyte maturation (r = 0.65), cumulus expansion (r = 0.77), oocyte diameter (r = 0.68), and ooplasm diameter (r = 0.65). Similarly, for CFL1, there was positive correlation with oocyte maturation (r = 0.84), cumulus expansion (r = 0.52). TUBA1 showed inverse correlation with cumulus expansion (r = −0.65) and strong positive correlation with oocyte degeneration (r = 0.77). BAX and BAX/BCL2 ratio both showed negative correlation with polar body extrusion  and cumulus expansion (r = −0.58), with a positive correlation with oocyte degeneration . The relationship between BCL2 and oocyte diameter and ooplasm diameter was positive . For interrelations of mRNA transcript expression, ACTB directly correlated with BCL2 (r = 0.72) which showed direct correlation with TUB (r = 0.61). TUB showed direct correlation with BAX (r = 0.96) and inverse correlation with CFLN (r = 0.55). On the other hand, CTSB showed no relation with the studied genes, while it showed positive correlation with cumulus expansion (r = 0.58) and ooplasm diameter (r = 0.65), with negative correlation with oocyte degeneration (r = 0.57), which indicates a tendency for COCs to undergo apoptosis by the end of IVM stages.As shown in Since the recognition of in vitro maturation as a simple, promising, noninvasive, and cheaper procedure for embryo production in ARTs, extensive studies have been conducted on different species. Among livestock, cattle and pigs have both received the most attention in this area . In the Few studies have investigated the role of ROCK signaling in the maturation medium of oocytes in some animal species. Our findings identified ROCK as a key player in dromedary camel oocyte meiotic maturation as in the pigs ,40, cattOur studies showed the importance of ROCK in cytokinesis and regulation of dromedary camel oocyte nuclear maturation. We observed that long-term inhibition by ROCK (6 and 24 h RI biphasic IVM) resulted in lower incidence of polar body development and incomplete meiosis I. This indicates a gradual failure in the migration of the spindle to the cortex as a result of prolonged inhibition. As reported earlier, activation of ROCK causes increased cell migration, while the inhibition of ROCK signaling inhibits cell migration . Our resIt is well known that optimal cumulus cell expansion is essential for normal oocyte maturation, oocyte cytoplasmic maturation in particular ,46. ImpaACTB on polar body extrusion, oocyte and ooplasm diameter, and cumulus expansion showed the central role of β-actin in oocyte meiosis, spindle rotation, and cellular morphology. Moreover, TUBA1 increased with oocyte diameter, and CFLN increased with oocyte maturation and cumulus expansion, which supports the necessity of cytokinesis-related transcripts in the final stages of COC maturation. The resulting alterations and abnormalities in oocyte diameter and PB extrusion by RI are due to the direct effect of ROCK on tubulin and other cytoskeleton proteins’ organization [Our results revealed significant differences in oocyte morphometry for the various treatment groups, and these values were comparable to our previous findings . In the nization ,54,55,56nization ; therefoACTB, TUBA1, and CFL1 would show the highest transcript levels in the 4 h RI biphasic IVM group. Interestingly, the expression of ACTB and BCL2 were distinctly higher when we examined them immediately after the 4 h pre-IVM period . Considering the result of nuclear maturation, it would be expected that M period . This unACTB (beta-actin) from the actin family, whereas there are other members of the actin family (such as F- and G-actin) that could exert similar or greater effects on oocyte meiosis if ROCK is inhibited. Some reports considered the contribution of F-actin to be greater [Reports on ROCK for porcine , bovine  greater . In the  greater .BAX/BCL2 reflects the balance between apoptotic and antiapoptotic signals and determines the cell’s susceptibility to apoptosis and the fate of cell survival [BAX/BCL2 could also possibly explain the higher rate of nuclear maturation and fully expanded cumulus in the 4 h RI group. A higher level of BCL2 is able to repress apoptosis, while overexpression of BAX accelerates cell death by apoptosis. The high level of BAX  to 42%, but when there is special combination of RI with cysteamine and leukemia inhibitory factor, the maturation rate was improved to 67% , owing tOur results demonstrated, for the first time, that ROCK is involved in dromedary camel oocyte maturation, and that the inhibition of ROCK activity has profound effects on oocyte meiotic progression and polar body extrusion. Our findings further suggest that short-term inhibition of ROCK for 4 h pre-IVM in a biphasic IVM approach improves oocyte nuclear maturation, producing more MII oocytes, possibly as a result of modulating the activities of several downstream molecules that regulate the expression of cytokinesis and inhibit apoptosis. This positive result suggests ROCK inhibitor as a potential candidate molecule to exploit in the control of oocyte meiotic maturation.


Brain monitors which track quantitative electroencephalogram (EEG) signatures to monitor sedation levels are drug and patient specific. There is a need for robust sedation level monitoring systems to accurately track sedation levels across all drug classes, sex and age groups. Forty-four quantitative features estimated from a pooled dataset of 204 EEG recordings from 66 healthy adult volunteers who received either propofol, dexmedetomidine, or sevoflurane  were used in a machine learning based automated system to estimate the depth of sedation. Model training and evaluation were performed using leave-one-out cross validation methodology. We trained four machine learning models to predict sedation levels and evaluated the influence of remifentanil, age, and sex on the prediction performance. The area under the receiver-operator characteristic curve (AUC) was used to assess the performance of the prediction model. The ensemble tree with bagging outperformed other machine learning models and predicted sedation levels with an AUC = 0.88 (0.81–0.90). There were significant differences in the prediction probability of the automated systems when trained and tested across different age groups and sex. The performance of the EEG based sedation level prediction system is drug, sex, and age specific. Nonlinear machine-learning models using quantitative EEG features can accurately predict sedation levels. The results obtained in this study may provide a useful reference for developing next generation EEG based sedation level prediction systems using advanced machine learning algorithms.Clinical trial registration: NCT 02043938 and NCT 03143972. Optimal sedation level management is critical for a healthy outcome of patients undergoing surgical procedures/ in intensive care units which otherwise can lead to unwanted neurological and cardiovascular complications –4. In reTo overcome the limitation of drug specificity, in our preliminary work , we deveThe current study received ethical approval from the “The Independent Ethics Committee” (Medisch Ethische Toetsings Commissie) of the Foundation ‘Evaluation of Ethics in Biomedical Research’ (Stichting BEBO), Assen, The Netherlands.A detailed description of the experimental protocol and EEG recordings have been described in full in the original studies , 16. HowREMI) of 2 versus 4 ng/ml that was maintained throughout the study duration. Exclusion criteria were weight less than 70% or more than 130% of ideal body weight, pregnancy, diseases involving the neurological, cardiovascular, pulmonary, gastric, and endocrinological system, and recent use of psycho-active medication or intake of more than 20 g of alcohol daily.Thirty six age and gender stratified healthy volunteers (American Society of Anesthesiologists Class I) were included in this study (Table PROP) and remifentanil (CeREMI) are calculated using the pharmacokinetic-dynamic (PKPD) model of respectively Schnider et al.  versus sedated [MOAA/S 1 and 0], discarding the remaining MOAA/S scores.Time domain – (1) Nonlinear energy operator, (2) Activity (1st Hjorth parameter), (3) Mobility (2nd Hjorth parameter), (4) Complexity (3rd Hjorth parameter) [rameter) , (5) Roorameter) , (12) Burameter) ;Frequency domain – (13) Pδ=mean power in delta band (0.5–4 Hz), (14) Pθ=mean power in theta band (4–8 Hz), (15) Pα=mean power in alpha band (8–12 Hz), (16) Pσ=mean power in spindle band (12–16 Hz), (17) Pβ=power in beta band (16–25 Hz), (18) PT=total spectral power (0.5–25 Hz), (19–23) Pδ/PT, Pθ/PT, Pα/PT, Pσ/PT, Pβ/PT, (24–27) Pδ/Pθ,Pα/Pθ, Pσ/Pθ, Pβ/Pθ, (28–30) Pα/Pθ, Pσ/Pθ, Pβ/Pθ, (31–34) mean, standard deviation, skewness and kurtosis of frequency modulation (FM) [ Hz), 19– Pδ/PT, PEntropy domain – (37) Singular value decomposition entropy [ entropy , (38) sp entropy , (39) st entropy , (40) sa entropy , (41) Re entropy , (42) Sh entropy , (43) pe entropy , (44) fr entropy .Fig. We extracted these features separately for each bipolar frontal montage channel and then obtained a median across channels to combine the channel information. These features were then used to train the machine learning algorithm to obtain the probability of the sedated state for each 4 s EEG epoch.We used the area under the receiver operator characteristic curve (AUC) to evaluate the model performance. In addition, we also report sensitivity, specificity, F1-score for the best performing machine learning model.N-1 folds. In each iteration, we used N-1 EEG recordings for training the machine learning model and the left-out unseen recording for testing, resulting in a total of N iterations. In each fold, features in the training data were Z-score standardized (by subtracting the mean and dividing by the standard deviation) and the testing data features were normalized with respect to the Z-score normalization factor of the training data before using them for classification. We performed grid search to identify the optimal hyper-parameters of these models  , supportFirst, we performed binary classification to differentiate between awake and sedated state using pooled dataset during propofol, sevoflurane and dexmedetomidine infusion. Then we added remifentanil data to this pooled dataset to evaluate the robustness and stability of the machine learning models. By this way we identified the machine learning model that is invariant after the addition of new drug (remifentanil in this case).For significance analysis, we used Analysis of Variance (ANOVA) with the Tukey Honest Significant difference test. All tests were two-sided with alpha = 0.05. All of the coding and analysis was performed using the MATLAB 2018a scripting language . All experiments were performed on a local computer with windows 10 platform, Intel Xeon 4116 processor and 32GB RAM. The overall time spent to extract these features from a 4 s epoch was 0.5 s and prediction of a sedation probability using a trained model was 0.05 s.All results are reported as mean  unless stated otherwise. 95% confidence interval was obtained using bootstrapping with 1000 bootstraps.Figure The performance of different machine learning models to predict sedation levels using the proposed architecture is summarized in Table Pβ, Pβ/PT, standard deviation of FM, SVDE, and FD. After the inclusion of remifentanil 12 features had weights above 0.3: NE, mobility, complexity, BSR, Pα, Pσ, Pα/Pθ,standard deviation of FM, kurtosis of FM, SVDE, SE, and FD. This suggests that with the addition of remifentanil, the properties of EEG change and the ET-B algorithm requires more features to achieve comparable prediction performance.Fig. To evaluate the effect of age on the performance of the ET-B model, we divided the dataset into three sub groups: group1–18 to 35 years, group 2–35 to 50 years and group 3–50 to 70 years. We then performed three different training testing combinations: (i) train on group 1 test on groups 2 and 3, (ii) train on group 2 test on groups 1 and 3 and (iii) train on group 3 test on groups 1 and 2. Table To evaluate the influence of sex, we performed cross training and testing i.e., we trained the ET-B model on male and tested it on female and vice-versa. When trained and tested within the same sex the prediction performance of the ensemble model was similar: AUC = 0.88 (0.82–0.92) and 0.90 (0.85–0.94) for male and female, respectively. However, the overall performance dropped by 9% (0.79 (0.75–0.85)) and 8% (0.82 (0.77–0.88)) for male and female, respectively during cross training and testing.In recent years, there is a growing interest in developing EEG-based level of sedation monitors. However, among several unresolved important questions, it was not clear why these monitors failed to perform across different anesthetic drugs and patient groups. In this study, we compared the performance of four machine learning models trained on a large dataset of 204 EEG recordings. Using a large set of 44 QEEG features, the ensemble tree with bagging (ET-B) machine learning model achieved the best prediction performance of AUC > 0.85 to discriminate between awake and sedated states. There are four major contributions of this study: (i) we developed a technique for a drug-independent nonlinear machine learning based sedation level prediction system, (ii) we showed that individual features and/ or features derived from spectral domain are not sufficient for real-time sedation level prediction at population level, (iii) we demonstrated how addition of remifentanil affects the prediction performance of different features, and (iv) we demonstrated the importance of the inclusion of all age groups and sex to develop a robust patient-independent sedation level monitoring system.The EEG is the only technique available to accurately monitor sedation levels in real-time. One of the main issues in developing EEG based sedation level monitors is the “feature engineering”: which features should be used to accurately predict sedation states? Current EEG based sedation level monitors either use a single feature or few expert defined spectral features to predict sedation levels , 8. AddiIt should be noted that we only performed binary classification to discriminate between two extreme levels of sedation: awake and sedated. If the model is not robust in this scenario, it will not be efficient to discriminate multiple levels of sedation. However, we have already developed a method to estimate continuous level of sedation from binary classification via sigmoid transformation in our previous work . Except There are several limitations in this study. First, despite using advanced nonlinear machine learning algorithms, we did not achieve perfect discrimination between awake and sedated states (AUC = 1.0). Inclusion of additional data and/or QEEG features could help improve the performance. Second, we only used four anesthetic drugs in this study. Validation on another external dataset with combination of multiple drugs is required to explore the robustness of the proposed system. Third, we did not include pediatric (< 18 years) and data from elderly cohorts (>70 years) in this analysis due to the nature of the clinical trial. Fourth, we only used data from healthy volunteers which may not reflect the influence of disease severity/routine medications on the EEG.Despite the above mentioned limitations, the findings in this study suggests that by pooling data from different drugs, age and sex groups, it is possible to develop a robust realtime sedation level prediction system using advanced nonlinear machine learning algorithms. Features derived from traditional spectrogram alone are not sufficient to accurately predict levels of sedation. It is hoped that findings in this study would help understand the mechanism of anesthetics/sedatives on EEG and help in developing improved and robust sedation level monitoring systems.