Donkeys originally evolved as a desert animal, and unlike the horse (which flees or runs away from danger), the donkey fights to avert danger. Hence, donkeys are more stoic and tend to express fear, pain, and discomfort in more subtle ways than horses. For owners and practitioners, it can prove to be challenging to identify donkeys in a state of pain or discomfort until the animal has reached an advanced degree of disease, at which point veterinary intervention may be too late. This study aims to identify signs of pain from both facial and body parameters in donkeys undergoing a surgical procedure. Scores were based on noted signs of discomfort/pain from the observed body language of the face, such as the eyes, ears, nose, nostrils, and muzzle, along with their overall body appearance. The study demonstrated that developing a scoring system donkey grimace scale proved to be accurate in identifying discomfort related to pain. However, the accuracy of the scale can be influenced by the observers’ gender, level of donkey knowledge, and experience.p < 0.001), affected observers’ ability to accurately score images. The study suggests that the most significant indicators of pain in donkeys are overall appearance and abnormal body stance provided their sensitivity, specificity and accuracy values of 63.18%, 62.07%, and 62.60%, respectively.The objectives of this study were to establish a donkey ethogram, followed by a donkey grimace scale to be applied to donkeys pre- and post-castration and to test if there was a notable difference in scores based on observer knowledge, gender, and experience, which could reveal possible discomfort/pain. Nine healthy male adult donkeys were surgically castrated. Fifty-four photos were selected from frontal, lateral, and body views taken pre- and post-castration. Observers ranging from minimal to extensive knowledge and levels of experience based on education and hours/month spent with donkeys scored six photos/donkey on a scale of 0–2 . Scores were based on body language and facial parameters: Ears down, ears back, eye white showing, glazed look, orbital tightening, eyes round shape, nostril tension, eyes narrow shape, muzzle tension, and abnormal stance and overall perception of the animal being in pain. Level of experience and knowledge, as well as gender significantly ( The misconception that donkeys do not feel pain to the degree that horses do, by suggesting donkeys have a higher level of pain tolerance, combined with a difficulty to identify indicators of pain in donkeys , has likAnimal pain has been described as “an aversive sensory experience that elicits protective motor actions, results in avoidance and may modify species-specific traits of behavior, including social behavior” . Since aAs donkeys become increasingly popular as companion animals along with the use of donkeys for draft animals and growing use for production , there is a growing need to understand more about their behavior related to their overall well-being from both an owner and veterinary perspective. Previous studies have yielded facial expression ethograms  and compNine adult male standard donkeys , ranging from 2 to 10 years old were photographed pre- and post-castration. All donkeys were clinically evaluated by the attending veterinarian  and his team. Any donkey not deemed healthy based on physiological parameters and overall appearance did not undergo castration. The evaluation of pain based on the evidence reported by Dyson et al. , who sugDonkeys were acclimated to the farm and arrived 10 days before surgery and housed in a group in paddocks with shelter, ad libitum hay, and free access to water. All donkeys were part of a private donkey rescue.A total of 270 photographs were initially taken for the present study. All photos were taken with an Apple mini iPad . The photos were identified depending on whether they were taken pre- or post-castration. Five photos were taken of each donkey from the following positions: One frontal photograph, one lateral photograph from the right side, one lateral photograph from the left side, one body photograph from the right side and one body photograph from the left. Each donkey was photographed 48 h, 24 h, 0 h before surgery and 8 h, 24 h and 48 h post-surgery on the farm. Fifty-four photos were randomly selected (27 pre- and 27 post-castration) to build the materials used for the surveys as suggested by Dalla Costa et al. , seekingDonkeys were anesthetized beginning in the morning by a standard xylazine/ketamine protocol : xylazinTwelve observers  with ages ranging from 20 to 70 years volunteered to participate in the donkey ethogram training and castration image survey. Those who volunteered had various levels of donkey experience and knowledge and included veterinarians, veterinary students, researchers, and donkey owners or were a combination of the aforementioned descriptors. All levels of knowledge and experience were equally represented in their frequency in the sample. All observers were then placed into categories based on the amount of time they spent with donkeys (interaction); minimal (<4 h/month), intermediate (4–6 h/month), and extensive (>6 h/month), and knowledge: Minimal , moderate , and extensive knowledge . Observer sample distributed across knowledge and experience levels as follows—33.3% of the observers presented minimal interaction with donkeys and minimal knowledge, 25% of the observers presented intermediate interaction with donkeys and moderate knowledge, and 41.7% of the observers presented extensive interaction with donkeys and extensive knowledge. The observers in the sample were chosen seeking an almost-equal representation of sexes, interaction and knowledge levels, to prevent the bias derived from the potential overrepresentation of some categories over the rest.An ethogram displaying key body language often used to evaluate pain in other animals was developed to train observers to evaluate this body language for signs of change Refer to . Each obThe survey included 54 photographs taken with an Apple mini iPad, six pictures per donkey: Lateral, frontal head, and lateral body view (one pre and post castration photograph/view). Each observer took the survey once per day on their own device , for three consecutive days to ensure intraobserver reliability. Photographs were presented in a digital form and survey to the observers in the same order on each consecutive day. Observers were blinded to the treatment of each donkey photograph, meaning the observer did not know whether each picture was pre-castration or post-castration. The database comprised of 18,072 scores.Observers were asked to score eleven parameters per photograph of each of the nine donkeys. The observer responded with a score ranging from 0 to 2  for each of the body language and facial parameters: Ears down, ears back, eye white showing, glazed look, orbital tightening, eyes round shape, nostril tension, eyes narrow shape, muzzle tension, and abnormal stance . The obsInterobserver reliability was assessed at two levels. First, the measurements of every observer were compared for days one to three, and second, measurements of scores across the total panel for the summation of all three days were compared. Intraobserver reliability was tested by comparing the responses of the same observer over three consecutive days scoring the survey. The intraclass correlation coefficient of the Reliability Analysis procedure of the Scale package in SPSS Statistics, Version 25.0, IBM Corp., Armonk, NY, USA, (2017) was used to assess inter (across observers) and intraobserver (for the observations of the same observer across the different days) reliability. Responses were then classified as successful or not, depending on whether the observers had identified signs of discomfort related to pain or not compared to pre-castration images ,21. Per p > 0.01), a nonparametric approach was considered. Frequency analysis and Pearson’s Chi-squared were performed to test for differences in the probability of success to identify pain signs  across factors: Castration status, observer gender, donkey knowledge, donkey interaction, body language. Frequency analysis and Pearson’s Chi-squared were performed using the Crosstab task from the descriptive statistics procedure in SPSS Statistics, Version 25.0, IBM Corp. (2017). Pearson’s chi-square test is used to determine whether there is a statistically significant difference between the expected frequencies and the observed frequencies in one or more categories of a contingency table [Before testing for the probability of success to identify signs of pain, Kolmogorov-Smirnov  and Levecy table , with eacy table ). As stacy table , when saOverall sensitivity, specificity, and accuracy were computed to determine the efficacy of the technique to report a comprehensive, accurate assessment of pain-related body language signs across body areas and on the whole, as a way to identify potential body language which should be considered better predictors for pain-related conditions versus those which may report misleading information. Sensitivity is the percentage of donkeys’ post castration being identified as in pain. Specificity is the percentage of donkeys pre castration being identified as not in pain. Accuracy is the likelihood of detecting either a true positive or true negative .The study was reviewed and approved by the University of California Davis’ Institutional Animal Care and Use Committee, but no protocol number was given, since only observations were being conducted by the research team. Castrations were scheduled and performed by a veterinary nongovernmental organization on-farm (in the field) independent of the study. No additional permission was needed following the recommendations of Royal Decree-Law 53/2013 and its credited entity, the Ethics Committee of Animal Experimentation from the University of Córdoba, given the application of the protocols present in this study followed the premises cited in the 5th section of its 2nd article, as the animals assessed were used for credited zootechnical use.All observers showed a Cronbach’s α parameter above 0.70 (average measures), suggesting that each observer reliably scored across all test days. Reliability coefficients  above 0.85 are generally regarded as high and those between 0.65 and 0.85 as moderate. The total panel across all three days reported a Cronbach’s α parameter above 0.90 (average measures), suggesting total panel reliably scored across days. It was likely for the panel to score the same for the same photo on the three repeated sessions. Additionally, interobserver reliability for the whole panel was tested, and the value of 0.97 (average measures) suggests agreement between observers was excellent. Hence, any of the members is valid, reliable, and provides consistent enough measures to be considered independently.p < 0.001) difference between female and male observers in their ability to correctly identify signs of pain after castration (2). Partial eta squared (ηp2) was 0.08, which meant that the effect of gender accounted for 8% of the differences in knowledge plus associated error variance. Sample size and possible factor combinations were not sufficient to justify the performance of univariate follow-up statistical analyses to determine whether significant differences could be ascribed to the interaction between knowledge and gender—however, these factors separately, as some of the categories, could be misrepresented. The study of the interaction between knowledge and gender may imply the multiplication of the number of possibilities within the interaction factor. Hence, instead of considering two variables (knowledge and gender) with two  and three levels , respectively, we would be considering one variable (knowledge level-gender) with six levels . Then, in the context of our sample and to maintain the balance in the representativity of each possibility, clustering both variables under an interaction term may reduce the frequency of cases across levels to two cases per level. This offers lower chances to compare than in a case in which the variables of knowledge level and gender are considered separately [There was a significant  were found for the distribution of the intensity of signs identified by the observers between pre castration and post castration images , therefore the subtle signs of pain may go unnoticed. Even when the disease or condition becomes chronic, the signs may remain unnoticed, due to the donkey having become habituated to the discomfort. This study began to define signs of discomfort related to pain in an acute phase versus chronic, in hopes of providing a tool to assist practitioners and owners in noticing these subtle behavioral changes and ultimately lead to providing treatment for pain ,34,35,36The ability to recognize signs of pain in this study was significantly dependent on gender , experieResults of the present study indicated that observers with more donkey experience and knowledge are less likely to miss indicators of pain . This is likely because by spending more time observing donkeys, one may become more skilled at noticing behavioral deviations from “normal”, and therefore, be more likely to pick up on subtle signs. These results suggest that practitioners and owners can become more successful at identifying when a donkey is painful by spending more time observing and becoming familiar with donkey behavior in general. Previous studies examining donkey behavior have confirmed that because donkeys demonstrate different clinical signs of pain/illness when compared to horses, previous knowledge and an understanding of the donkey can be crucial to correct examination, diagnosis and medical treatment of the donkey ,21,35,36Practitioners and owners should be aware of differences between donkey and horse medication dosages, and appropriate donkey dosages should be administered ,21,35,36The ability to recognize subtle body language signals may prove to be challenging to those who have limited experience working with donkeys. Observing the overall body posture and reactions of donkeys may improve our chances of recognizing pain in donkeys. This indicates the importance of observing more than just the face of the donkey to identify signs of pain. Facial indicators, such as ears, eyes, and nostrils, may be more easily influenced by external factors, such as other donkeys and the environment of the donkeys, thus it is important to look at the donkey as a whole for signs of behavior related to pain. Gender, experience or interaction with donkeys, and knowledge level of observers impacted the observer’s ability to correctly score a post castration donkey as painful. Female observers were more likely than male observers to provide a higher score for post castration pictures and were less likely to score a false-positive. By spending more time observing donkeys, one may become more skilled at noticing behavioral deviations from “normal”, and therefore, be more likely to pick up on subtle signs. Conclusively, identifying the appearance of a normal donkey may assist in decreasing the number of donkeys that go untreated, due to the subtleness of their behavioral pain indicators, as the biggest obstacle of treating pain in donkeys may be identifying the pain in the first place.


Subsequently, they were treated daily with either placebo or MZe786 for six days, when the heart tissue was harvested to assess cardiac mitochondrial activity. Here, we show that the loss of HO-1 disturbed cardiac mitochondrial respiration and reduced mitochondrial biogenesis. The overexpression of sFlt-1 resulted in the inhibition of the cardiac mitochondrial activity in Hmox1+/− mice. The present study demonstrates that the hydrogen sulfide (H2S) releasing molecule, MZe786, rescues mitochondrial activity by stimulating cardiac mitochondrial biogenesis and antioxidant defense in Hmox1−/− mice and in Hmox1+/− mice exposed to a high sFlt-1 environment.Hypertensive disorder in pregnancy is a major cause of maternal and perinatal mortality worldwide. Women who have had preeclampsia are at three to four times higher risk in later life of developing high blood pressure and heart disease. Soluble Flt-1 (sFlt-1) is elevated in preeclampsia and may remain high postpartum in women with a history of preeclampsia. Heme oxygenase-1 (Hmox1/HO-1) exerts protective effects against oxidative stimuli and is compromised in the placenta of pregnant women with preeclampsia. We hypothesized that sFlt-1 inhibits cardiac mitochondrial activity in HO-1 deficient mice. HO-1 haplo-insufficient mice (Hmox1 Preeclampsia is a pregnancy-related complication that affects more than 10 million women a year and is the leading cause of maternal and perinatal morbidity and mortality worldwide ,3. It isIn the last decade, the hypothesis that preeclampsia arises due to “increase in the level of endogenous soluble Flt-1 (sFlt-1) that may antagonize the beneficial effects of vascular endothelial growth factor (VEGF)”  has been2S) belongs to a family of labile biological mediators called gasotransmitters. It is synthesized by numerous mammalian tissues by three enzymes—cystathionine γ-lyase (CSE), cystathionine β-synthetase (CBS), and 3-mercaptopyruvate sulfurtransferase (3-MST) [2S inhibited oxidative stress through cysteine S-thiol to scavenge free radicals in the atherosclerosis mouse model [2S has also been shown to promote vasodilatation [2S and other gasotransmitters, such as HO-1 by-product, carbon monoxide (CO), was suggested to modulate vascular function. H2S may have a role in the control of CO bioavailability under specific pathophysiological conditions [2S donor increased HO-1 and its by-product CO [2S in volume overload-induced Chronic Heart Failure (CHF) in rats and demonstrated that H2S may play a protective role in volume overload-induced CHF by upregulating HO-1 [Hydrogen sulfide (H (3-MST) . Many re (3-MST) ,32. H2S se model  and actise model . H2S haslatation , exhibitlatation , protectlatation , and stilatation . Interplnditions . Human moduct CO . Anothering HO-1 .2S-releasing molecule, which protects the gastric mucosa from its anti-cyclooxygenase activity [2S release helps to rebalance the redox system as a result of increased H2S/gluthathione (GSH) formation, increased HO-1 promoter activity and reduced 8-isoprostane. MZe786 has also been shown to improve endothelial function in animal models of ischemia-reperfusion injury, possibly by modulating levels of glutathione and homosysteine [MZe786 is a novel synthesis Hactivity . H2S relsysteine ,44. +/− mice. We further tested whether a hydrogen sulfide-releasing molecule, MZe786, could rescue sFlt-1-induced mitochondrial dysfunction in the heart of Hmox1−/− mice. We have previously demonstrated using serum obtained from women with preeclampsia that increased levels of sFlt-1 leads to metabolic perturbations accountable for mitochondrial dysfunction in cultured endothelial cells. Furthermore, sFlt-1 exacerbated mitochondrial reactive oxygen species formation and mitochondrial membrane potential dissipation . As the Recombinant adenovirus-encoding mouse sFlt-1 (Ad-sFlt-1) under the control of the CMV promoter was a gift from Professor Richard Mulligan  and adenovirus containing the CMV promoter without an insert (Ad-CMV) was purchased from Vector Biolabs, Pennsylvania, USA and stored at −80 °C. All animal experiments were carried out using procedures approved by the Aston University Ethical Review Committee in compliance with the UK Home Office License Number 3003453 in accordance with the “Guidance on the operation of Animals” under the United Kingdom Animals (Scientific Procedures) Act 1986.2S-releasing dithio-thione moiety, synthesized by Professor Anna Sparatore. The drug was prepared in a drug carrier (0.5% carboxymethyl cellulose in PBS) fresh every day.MZe786  phenyl ester; chemical structure of MZe786, as shown +/−) mice were originally obtained from Professor Anupam Agarwal . Female wildtype (Hmox1+/+), Hmox1+/−, or knockout (Hmox1−/−) mice between the ages of twelve to twenty weeks were used. Mice lacking HO-1  received the drug carrier or 50 mg/kg of MZe786 treatment via gavage for six days before the heart was harvested for analysis. Animals were injected with 0.5 × 109 PFU/mL Ad-sFlt-1 or empty vector (Ad-CMV) via the tail vein. Dose titration studies were used to determine the non-toxic dose of Ad-sFlt-1 and 0.5 × 109 PFU increased sFlt-1 expression without inducing any toxic effect in mice. Mice were euthanized via cervical dislocation and the heart was harvested.Heterozygous Hmox1  and centrifuged at 10,000× g for 6 min at 4 °C. The pellet was re-suspended in 1.2 mL 15% Percoll in BSA-free MIM. In a separate tube, a gradient of Percoll was prepared by adding 1.2 mL 24% Percoll followed by 1.5 mL 40% Percoll (prepared in BSA-free MIM) added to the bottom of the tube. Next, the pellet was slowly layered on top of the 24% Percoll. A visible layer (mitochondria) was observed between the 24 and 40% Percoll layers after centrifuging the tube at 30,000× g for 8 min at 4 °C. The mitochondrial layer was collected using a 5 mL syringe needle and transferred to a new tube containing 2 mL BSA-free MIM and centrifuged at 16,000× g for 10 min at 4 °C. The pellet was transferred to a separate tube containing 5 mL BSA-free MIM and centrifuged at 7000× g for 10 min at 4 °C, after which the final pellet was collected and re-suspended in 50 μL BSA-free MIM. The total protein content was determined using the Bradford Assay Reagent .Mitochondria were isolated from mouse hearts by homogenization and density gradient centrifugation using a method adapted from Sakamuri et al. . Mice we2, 10 mM KH2PO4, 0.2% (w/v) fatty acid-free BSA, pH 7.4).Mitochondrial respiration was assessed using an XF24 Extracellular Flux Analyzer  using a method adapted from Boutagy et al. . In prepg for 10 min at 4 °C to allow mitochondria to attach to the plate bottom. Then, 450 μL of pre-warmed MAM-containing substrate was added to the respective wells. Finally, the microplates were incubated for 10 min at 37 °C in a non-CO2 incubator before the measurement of mitochondrial respiration was started. Once in the XF24 Analyzer, oxygen consumption rates were calculated by plotting oxygen concentration (pmol O2) vs. time (min). Mitochondrial oxygen consumption was measured after the sequential injections of ADP 1 mM, 2 μM oligomycin (ATP synthase inhibitor), FCCP 4 μM, and antimycin A 4 μM. Oxygen consumption rates measured after ADP injections are representative of the maximal coupled respiration, or state 3. To determine the oxygen consumption rate following the ATP/ADP ratio approaching equilibrium, we measured the rates of oxygen consumption after oligomycin injection (state 4o). For these parameters, states 3 and 4o were used to quantify the behavior of mitochondria by calculating the RCR, calculated by dividing the respiration determined in state 3 by that in state 4o [To effectively determine the mitochondrial activity in response to complex I and II substrates, MAM was supplemented with either: (a) 10 mM pyruvate/2 mM malate; (b) 10 mM succinate/2 μM rotenone; (c) 40 μM palmitoyl-L-carnitine/2 mM malate. MAM containing only substrate was loaded to blank wells A1, C3, B4, and D6. After plating the mitochondria, the microplate was centrifuged at 2000× ® 480 SYBR® Green I Master  and its specific primers. RT-PCR was performed using the following run conditions: Pre-incubation (1 cycle); amplification (45 cycles); melting curve (1 cycle); cooling (1 cycle). Relative gene expression was calculated using the 2−ΔΔCT method. A comparison of ND1 DNA expression, relative to HK2 DNA expression, provided a measure of the mtDNA copy number to nDNA copy number ratio (mtDNA/nDNA) [RNA was extracted from heart tissue using the RNeasy mini-kit  and quantified using the Nanodrop ND 1000 spectrophotometer . RNA was converted to cDNA using the EvoScript Universal cDNA Master  as indicated by the manufacturers’ guidelines. The relative expressions of mouse genes—PGC1-α, thioredoxin (Txn1), glutaredoxin (Glrx), hexokinase 2 (HK2), and ND1—were quantified by real-time PCR on a Lightcycler 480  using the LightCyclerNA/nDNA) .p < 0.05, p < 0.01, and p < 0.001 were considered statistically significant.Data were plotted as means ± SEM. Statistical analysis was performed using Kruskal–Wallis with Dunn’s post hoc test, using GraphPad Prism software . Values of −/− mice. Using a XF24 Seahorse Analyzer, we evaluated the capacity of mitochondria for substrate oxidation by calculating the mitochondrial respiratory control ratio (RCR). The RCR is defined as the respiration in state 3 divided by that in state 4o [HO-1 has been proposed to exert cytoprotective effects over oxidative stress-mediated hypertrophy, fibrosis, and metabolic dysregulation in heart tissue . From a state 4o . −/− mice displayed a significant reduction in the calculated RCR when compared to Hmox1+/+ (p = 0.003). The administration of MZe786 to Hmox1−/− mice showed a significant restoration in this parameter in comparison to Hmox1−/− cardiac mitochondria (p = 0.0159), as shown in −/− mice for the oxidation of palmitoyl-L-carnitine. Our results show a significant compromise in fatty acid oxidation in the absence of HO-1, as shown in +/+ (p = 0.004). Similar to pyruvate-dependent complex I activity, MZe786 was able to improve the oxidation of palmitoyl-L-carnitine in comparison to Hmox1−/− mice (p = 0.03). Correspondingly, we explored the ability of cardiac mitochondria for the oxidation of succinate (complex II substrate) in the presence of rotenone (complex I inhibitor). State 3 and state 4o respiration was used to calculate the RCR, as shown in −/− mitochondria are less able to respire using succinate as a substrate (p = 0.0034). Interestingly, MZe786 protected complex II-driven OXPHOS improved the calculated RCR when compared to Hmox1−/− hearts (p = 0.01), as shown in First, we measured the ability of cardiac mitochondria for the oxidation of pyruvate and palmitoyl-L-carnitine at the mitochondrial complex I in the presence of malate (complex II inhibitor) by calculating the RCR. Oxygen consumption rates representative of state 3 and state 4o respiration were used to calculate the RCR, as shown in Once we established that the loss of HO-1 leads to signs of mitochondrial dysfunction in the heart, we sought to evaluate the role of MZe786 in the restoration of the cardiac mitochondrial function. As previously reported, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α) plays a key role in cardiac metabolism by enhancing the mitochondrial biogenesis signal ,54 and pp = 0.0013). MZe786 significantly increased PGC1-α transcription when compared to wild type (p = 0.0014) and Hmox1−/− (p = 0.0006) hearts, as shown in p = 0.011), while MZe786 significantly enhanced the mtDNA/nDNA in Hmox1−/− when compared to both Hmox1+/+ and Hmox1−/− hearts (p < 0.0001), as shown in The loss of HO-1 significantly reduced the gene expression of PGC1-α in cardiac tissue in comparison to their wild type counterparts (−/− in comparison to Hmox1+/+ hearts (p = 0.12). The administration of MZe786 significantly stimulated Txn1 gene expression when compared to Hmox1+/+ (p = 0.02) and Hmox1−/− (p = 0.007) cardiac tissue, as shown in −/− hearts (p = 0.007), while MZe786 was able to restore its expression (p = 0.002). There was no significant difference between cardiac Glxr gene expression the in wild type compared to MZe786-treated Hmox1−/− mice (p = 0.14), as shown in We next focused on the ability of MZe786 to stimulate antioxidant gene expression in the absence of HO-1. Our results showed a slight reduction in thioredoxin (Txn1) gene expression in Hmox1+/−, single Hmox1 allele) has been associated with the dysregulation of angiogenic balance, leading to signs of fetal growth restriction and preeclampsia [+/− mice and evaluated the respiratory efficiency in mitochondria isolated from heart tissue using an XF24 Seahorse Analyzer. Haploinsufficiency of Hmox1 . In Ad-sFlt-1 mice treated with MZe786, we evidenced the significant restoration of the pyruvate-dependent RCR in comparison to the Ad-sFlt-1 injected mice (p = 0.0043). No significant difference was evidenced between the Ad-CMV and Ad-sFlt-1 + MZe786 treated mice (p = 0.9), as shown in p = 0.19). MZe786 enhanced the respiratory efficiency in cardiac mitochondria from Ad-sFlt-1 in comparison to Ad-sFlt-1 counterparts (p = 0.01). No difference was evidenced when comparing Ad-CMV with Ad-sFlt-1 + MZe786 treated mice (p = 0.9), as shown in p = 0.03). MZe786 improved the respiratory efficiency in Ad-sFlt-1 injected Hmox1+/− mice (p = 0.004) while no statistical difference was observed when compared to Ad-CMV (p = 0.9), as shown in +/−, suggesting that the fatty acid-driven mitochondrial function might play a key role in sustaining the energetic demands in Hmox1+/− hearts. Therefore, events dysregulating this metabolic pathway might lead to mitochondrial dysfunction in environments of reduced HO-1. To evaluate the capacity of Hmox1+/− mice in comparison to Ad-CMV (p = 0.0034). Mice injected with Ad-sFl-1 and administered with MZe786 were able to significantly increase PGC1-α transcription in comparison to those that were given Ad-sFlt-1 alone (p = 0.019). The expression of cardiac PGC1-α was not different when Ad-CMV and Ad-sFlt-1 + MZe786 mice were compared (p = 0.24), as shown in p < 0.0001), while MZe786 enhanced the cardiac mtDNA/nDNA in Ad-sFlt-1 + MZe786 treated mice (p < 0.0001). No statistical difference in the cardiac mitochondrial content was observed when Ad-CMV and Ad-sFlt-1 + MZe786 treated mice were compared (p = 0.43), as shown in Since MZe786 were shown to improve the mitochondrial biogenesis signal and antioxidant defense in Hmox1 knockout hearts, we explored whether MZe786 would exert similar effects in environments of high sFlt-1 and the partial loss of Hmox1. PGC1-α gene expression was significantly reduced in heart tissue from Ad-sFlt-1-injected Hmox1+/− mice showed the reduced transcription of antioxidant genes Txn1 and Glrx  when compared to their wild type counterparts. The administration of MZe786 significantly stimulated Txn1 and Glrx gene expression in the hearts from Ad-sFlt-1-injected Hmox1+/− mice . No statistical difference between Txn1 and Glrx gene expression was observed when compared to Ad-CMV , as shown in Finally, we observed that cardiac tissue from Ad-sFlt-1-injected Hmox1Several studies have demonstrated that the risk of developing cardiovascular disease is significantly higher in women with a history of preeclampsia ,58,59. ITwo decades ago, we demonstrated that HO-1 protein expression is reduced in placentas from pregnancies complicated with preeclampsia . More reThe myocardial tissue has the ability to metabolize different substrates such as fatty acids, amino acids, glucose, lactate, and ketone bodies, depends on the bioavailability of substrates in the cardiac environment . Typical−/− mice. In the case of pyruvate, it has been shown that its metabolism requires mitochondrial import mediated by the carrier-regulated process [In response to complex I substrates, pyruvate and palmitoyl-L-carnitine, we evidenced a reduced respiratory capacity in Hmox1 process . Likewis process . Interes process . Our res+/− mice further evidenced the role of sFlt-1 to impair the cardiac mitochondrial activity in a reduced HO-1 setting. In these experiments, non-pregnant females were exposed to high sFlt-1. As Akhter and colleagues previously reported, circulating levels of sFlt-1 remain higher in women post preeclampsia even one year after delivery (25). The administration of sFlt-1 to non-pregnant animals results in high blood pressure, proteinuria, and endotheliosis, characteristics of preeclampsia [The delivery of sFlt-1 to Hmox1clampsia . Therefo+/− mice. This was accompanied by reduced PGC1-α gene transcription and mitochondrial content in comparison to the control. These observed effects were consequently attributable to sFlt-1-induced damage to the mitochondria. We recently demonstrated that sFlt-1, dose-dependently disturbs the endothelial cellular bioenergetics and promotes mitochondrial specific reactive oxygen species production [The efficiency of the cardiac mitochondrial activity was significantly impaired in sFlt-1-treated Hmox1oduction . Likewisoduction . Based o2S, and carbon monoxide, the gaseous product of HO-1. H2S may have a role in increasing the expression of HO-1 and, subsequently, the CO level, therefore excreting protective effect. A proposed mechanism for this involves the Keap1/Nrf2 pathway. The persulfidation of Keap1 Cys151 by the reaction of H2S with oxidized Cys151 by H2S-derived HS· leads to the dissociation and nuclear translocation of Nrf2, ultimately resulting in an increased HO-1 protein level [2S, which is produced in endothelial cells and participates in the fine regulation of endothelial integrity and functions [2S bioavailability was suggested to be a novel indicator of endothelial dysfunction progression and is manifested in different forms in multiple pathologies, including preeclampsia [2S bioavailability, may benefit diseases with unmet medical needs.Hydrogen sulfide donors have been demonstrated to exert protective effects on the cardiac oxidative metabolism in different scenarios of cardiovascular disease ,74,75,76in level ,77,78. Eunctions ,80. DistThis study shows that the hydrogen sulfide releasing molecule, MZe786, protects cardiac mitochondrial efficiency through complexes I- and II-driven OXPHOS and improves antioxidant gene transcription in low HO-1 settings. As previously shown, exogenous hydrogen sulfide donors sustain the mitochondrial membrane potential, inhibit apoptosis, and suppress reactive oxygen species generation in a model of cardiac hypertrophy in mice . FurtherInterestingly, hydrogen sulfide-induced cardio-protection has been linked to enhanced antioxidant defenses via the upregulation of reduced glutathione levels  and Trx-+/+ mice. Similarly, the partial loss of Hmox1 reduced the mitochondrial capacity in comparison to the total loss of HO-1, even though the reduction in the respiratory efficiency was less pronounced. These observations suggest that HO-1 is necessary to support the cardiac mitochondrial function. Although, one allele provides protection, as demonstrated by the cardiac mitochondrial function, in high sFlt-1, this protection is abrogated. MZe786 also improved the respiratory capacity in these low HO-1 and high sFlt-1 environments, allowing for the protection of the mitochondrial activity in response to mitochondrial substrates, pyruvate, palmitoyl-L-carnitine, and succinate. Together, these observations demonstrate that MZe786 can provide beneficial effects on the cardiac metabolism, allowing for the restoration of the mitochondrial activity and providing antioxidant-enhanced protection.Our study shows that MZe786 improved mitochondrial capacity when the high sFlt-1 environment was accompanied by the partial or complete loss of HO-1 function in animal models. Although the total deficiency of Hmox1 resulted in a marked reduction in mitochondrial oxidative capacity, MZe786 improved the respiratory efficiency and restored antioxidant defenses to levels similar to the Hmox1Here, we provide first-time evidence demonstrating that the hydrogen sulfide-releasing molecule, MZe786, enhances cardiac mitochondrial activity and protects the antioxidant capacity in a reduced HO-1 and high sFlt-1 environment, mimicking the molecular impairments encountered in preeclampsia. Our results provide new insights into the molecular mechanisms associated with long-term cardiovascular disease observed in in women post preeclampsia and suggest MZe786 as an effective therapeutic molecule to relieve the sFlt-1-induced mitochondrial damage in the heart.Deficiency in HO-1 disturbed the mitochondrial activity and these effects were associated with reduced mitochondrial content and suppressed the antioxidant capacity. This suggests that the loss of HO-1 may impair the cardiac tissue ability to sustain metabolic demands through the mitochondria and to respond to oxidative stress-induced damage. In these settings, MZe786 was shown to protect the antioxidant capacity and mitochondrial function, suggesting that this molecule may be beneficial in protecting cardiac tissue against events leading to impairments in the antioxidant capacity of the heart. Consequently, the partial deficiency of HO-1 in a high sFlt-1 environment provided a more realistic setting, mimicking the preeclampsia-like molecular defects in the heart. Soluble Flt-1 impaired the mitochondrial activity and suppressed antioxidant gene transcription in the cardiac tissue. The administration of MZe786 protected the mitochondrial activity and upregulated the antioxidant genes. Our results suggest that MZe786 protects the heart from sFlt-1-induced mitochondrial damage in an environment of low HO-1 via the upregulation of key antioxidant genes and by exerting protective effects on the mitochondrial activity in the hearts.


In contrast, feed supplementation with 2% OMW, compared with the control diet, produced no noteworthy increase in BWG or the feed conversion rate (FCR). Antibiotic addition, on the other hand, increased serum cholesterol (p ≤ 0.05). After 42 days, neither OMW nor antibiotic addition affected organ mass. In contrast, antibiotic addition reduced the small intestine percentage, crypt depth and villus height (p ≤ 0.05). The Newcastle disease vaccine (NDV) antibody titer improved after feed supplementation with 1% OMW comparing with the control and antibiotic diet group. Furthermore, OMW supplementation decreased the heterophil-to-lymphocyte H/L ratio (p ≤ 0.05). The use of OMW led to a reduction in the malondialdehyde (MDA) content of the breast and liver and an increase in glutathione peroxidase. It helped to reduce glutathione, glutathione reductase, and glutathione S-transferase. In conclusion, the impact of OMW were dose-dependent, and the use of 1% OMW in broiler diets enhanced their growth and immunity. Nonetheless, supplementation with 2% OMW produced conflicting results.Oyster mushroom waste (OMW) is a by-product of the agriculture industry with valuable antimicrobial, antioxidant, antifungal, and prebiotic properties. This by-product might be a useful alternative to antibiotic growth stimulators in poultry nutrition. The purpose of this research was to test the impact of OMW on the immune responses and on the morphology of intestine of broiler chickens. Four dietary therapies with five replicas of 15 birds in each, totalling 300 day- Ross 308 broiler chickens, were utilized in this study. Control chickens were fed a mixed diet that included a maize-soybean meal complemented by 1 and 2% OMW in addition to the basal diet. Furthermore, Enramycin (125 g/kg) was added to the control diet as an antibiotic. Throughout this experiment, performance was studied as well as the immune response to the Newcastle Disease Virus (NDV) and intestinal morphological traits. A substantial surge was noted in body weight gain (BWG) and feed intake (FI) of chickens after the addition of 1% OMW ( Antibiotics have historically been used as promotors of growth in poultry feed and as treatment for contaminated chickens. For decades, conventional artificial nutritional additives, like antibiotic growth promoters, antioxidants, antifungal agents, and anti-parasitic agents are used in poultry feed . HoweverWorldwide, feed supplies for animal nutrition are in shortage, the problem get even difficult after COVID-19 due to many consequence negative effects on crop cultivation, harvest and production as well as trade. Hence, the use of agro-industry as feedstuffs in animal nutrition represents an important source of protein, vitamins, minerals and antioxidants and visible solution –5. This Pleurotus ostreatus stem waste is abundantly available due to rise in the mushroom's cultivation. The stems are treated as agricultural by-product, although they have medicinal and nutritive values. The popularity of mushrooms for human consumption has also lead to environmental pollution  stem waste as a phytogenic feed additive and substitute for antibiotics in broilers diets. In general, there have been insufficient studies in the examination of the health status and performance in broilers due to the efficiency of mushroom waste. For long term, antibiotics have been overused in the industry of poultry to raise production efficiency and health status has resulted in hazards effects on human health. This work investigates medicinal mushrooms usage in poultry feed dispose of antibiotics, without impacting optimal efficiency. Biologically active substances are found in many medicinal mushrooms like glycoproteins, polysaccharides, and other polymers that may function as preferable nutritional supplements in chickens as well as immunomodulating agents. The aim of the research was, therefore, to test the effectiveness of Pleurotus ostreatus oyster mushroom waste (OMW) in broiler chickens on growth output, serum cholesterol, immune status, and antibody response.ollution –9. Howevficiency . The weificiency . GastroiThe experimental protocol of the present work was approved by King Abdulaziz University, Jeddah, Saudi Arabia. The University recommend that animal protections, welfare, rights, and minimum stress levels, and avoided any suffering or harm to creatures according to Royal Decree number M59 in 14/9/1431H.An overall number of 300 unsexed 1-day old chicks were assigned to be fed four different experimental treatments randomly (Ross 308). Five duplicated pens with 15 birds in each pen with a ratio of 1:1 for each treatment were chosen. All birds in this study were raised on a commercial poultry farm and kept in-floor pens that have litters made of wood shreds. A totally randomized design was used in this experiment, following are the dietary treatments:  1% OMW cPleurotus osteratus) and mushroom growth plan rejections. Mushroom waste was prepared from the base of the stem of the mushroom. After harvesting, the mushrooms were weighed, placed in net bags, air/sun-dried, and eventually, the OMW was collected. After the sun drying, when a substantial volume of water was lost, OMW was further exposed to oven drying to eliminate water thoroughly. Upon drying in the oven, mushrooms were held at a consistent temperature of 60°C, and once the samples showed a steady weight, the process was stopped.Mushroom waste consisted mainly of oyster mushroom stem base , total triglycerides (TG), and high- and very-low-density lipoprotein cholesterol (HDL-C and VLDL-C). A spectrophotometer was used to measure serum lipids. Biochemical analysis of serum samples was performed using commercial diagnostic kits , and VLDL-C determination was carried out by dividing TC by 5.The H/L ratio was calculated from a blood sampling obtained on day 42. In this procedure, heparin-containing syringes were used to prevent the development of blood clots. Greenwald, May, and Giemsa dyes were used to stain blood smears . Under oBirds used in this experiment were vaccinated individually using an eyedropper with Newcastle Disease Virus (NDV) commercial vaccine at 6-day age, and in drinking water at the ages of 16 and 26 days. Also, three chickens from each treatment were bled at the age of 42 days to obtain serum for NDV antibody measurement. As defined , antibodThree chickens at age of 42 days, were selected from every pen, then were slaughtered according to the Islamic  method . Organs The measurement of villus height (micrometers) started from villus's edge until the villus-crypt junction, while depth of the crypt was measured starting at the base until the transition area amongst the crypt and villus. Tissues of the liver, breast, and thigh muscle were collected after slaughter by eliminating the connective tissue, skin, and fat. Specimens were packed in vacuum at −40°C to prepare them for processing. With regarding to measuring the antioxidant, tissues were defrosted than processed for five days at 4°C. Afterward, they were subjected to antioxidant activity and the oxidation of lipid in storage at 0 and 5 d. In this experiment, many antioxidants were measured (reduced glutathione (GSH), glutathione reductase (GR), glutathione peroxidase (GSH-Px), glutathione S-transferase (GST), and malondialdehyde (MDA).Using the approaches of , glutathp ≤ 0.05.One Way 2008 SAS statistical model was utilized in this study to analyse the variance in data appropriate for a completely randomized model were analyzed according to SAS Institute . Using tModel:p ≤ 0.05). However, supplementing broiler with 1% OMW resulted in greater BWG than the control and other treatments. In contrast, BW was not different when fed 2% OMW compared with the control. Supplementation with 1% OMW resulted in a noteworthy surge in FI (p ≤ 0.05) during overall times, whereas no effect of 2% OMW was observed. There was no effect of Enramycin on FI relative to the control (p ≤ 0.05). The improvement in FI seen in chickens fed the diet supplemented with 1% OMW could be the explanation for the significant increase observed in broiler BW. However, this increase may also be attributed to the presence of oligosaccharides in the cell walls of mushrooms  FCR comparing with control conditions. However, for the entire experimental period, 2% OMW did not affect this ratio (p ≤ 0.05). In comparison with the control, Enramycin was found to considerably increased LBW, ADFI, and ADG, and improved FCR. Antibiotics effectively boost growth efficiency by altering the gastrointestinal microbiota (in particular by destroying pathogens), thus reducing inflammation of the intestinal mucosa  stem waste supplementation (1 and 2%), TC decreased relative to the control. At both levels of OMW supplementation, TC was recorded to be (p < 0.05) which is lower than antibiotic-supplemented groups and the control groups. In this study, no substantial change was witnessed in low-density lipoprotein cholesterol (LDL-C) nor TG.The data in Moreover, no statistically significant differences were found for blood total protein, total albumin, globulin, or urea nitrogen concentrations at day 42 between treatment groups and control . UltimatAgaricus blazei) powder has been shown to reduce TC, yet it had no influence on serum TG in broilers (Pleurotus ostreatus), TC was reduced, yet other serum lipids were not affected  in OMW diets comparing to those fed with the antibiotic and control diets. The optimal responses for antibody titer were found in broiler fed 1% OMW (P < 0.05) in OMW and antibiotic groups illustrated greater (p < 0.05) serum immunoglobulin (IgG and IgM) levels than the control and group  in pullet-fed diets of oyster mushroom waste (p < 0.05) comparing to the antibiotic-fed and control diets in the pullet of experiment. Bai et al. . The physiological stress indicator is the ratio of heterophils to lymphocytes as the number of heterophils increases in moderately stressful settings (settings . Since tsettings  presentep ≤ 0.05) relative to control birds at 42 days of age, even though OMWs did not affect (p ≤ 0.05).p ≤ 0.05) decline these indices in the ileum. Given the variations in villus height and crypt depth of the jejunum, no significant effect of OMW was observed on the ratio of villus height to crypt depth, although this ratio was amplified in the ileum (p ≤ 0.05). The safety and health of the gut and improved absorption ability of the intestines were attributed and correlated to the higher villus height and lower crypt depth. In this analysis, the high fiber content of mushroom may have led to structural and body alternations in the small intestines of birds, but no impact the ratio of villus height to crypt depth was noticed. Gross and Siegel  displays antioxidant characters as a functional dietary supplement and food. However, Agaricus brasiliensis is considered a possible auxiliary to treat patients suffering from rheumatoid arthritis because of their ability to alleviate oxidative stress  or TNF-α-activated cells. Brugnari et al. (Pleurotus ostreatoroseus (Agaricomycetes) mushroom. Kumar et al. (Nowadays, the characteristics of antioxidant for various medicinal mushrooms are well understood. Few earlier researchers indicated that the antioxidant functions of the polysaccharides and oligosaccharides present in medicinal mushrooms . Butylatons role . A reseaons role  showed te stress . Muszynse stress  reportedi et al.  also notr et al.  found thData from the relevant literature showed inconsistent outcomes on the impact of natural dietary antioxidants and their antioxidant enzyme activity. , 56–88 PCollectively this research suggests that feeding oyster mushroom waste at a 1% level could have positive impacts on improving the performance, the humoral immune response to disease vaccines, and delayed poultry meat lipid oxidative rancidity and being more effective than 2% inclusion or antibiotic-supplemented-groups. Besides, the use of oyster mushroom waste in animal nutrition could decrease the environmental pollution. More studies are required to recognize the optimum inclusion level of oyster mushroom waste on performance enhancement and to detect the inner mechanisms of metabolic rate of lipid with oyster mushroom waste in broilers. Alternative feed resources and locally produced feeds may be useful tool for animal nutrition after COVID-19 outbreak due to expected decrease in feedstuffs for animal nutrition and increase the severity of competition between animal and human with absolute primacy given to fulfill human requirements.All datasets generated for this study are included in the article/supplementary material.The animal study was reviewed and approved by Animal Production Research Institute. Written informed consent was obtained from the owners for the participation of their animals in this study. King Abdulaziz University, Jeddah, Saudi Arabia, approved the experimental procedures, which protect animal welfare, rights, and stress levels, and avoided any suffering or harm to creatures according to Royal Decree number M59 in 14/9/1431H.KA set up the field work, design the experiments, and first draft of the manuscript. RH, MS, and MA contributed to the experimental setup and analyzing statistics, interpretation of results, and proofreading of the manuscript. All authors approved the final version.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.


The concept of reductive radical-polar crossover (RRPCO) reactions has recently emerged as a valuable and powerful tool to overcome limitations of both radical and traditional polar chemistry. The concept of reductive radical-polar crossover (RRPCO) reactions has recently emerged as a valuable and powerful tool to overcome limitations of both radical and traditional polar chemistry. Especially in case of additions to carbonyl compounds, the synergy of radical and polar pathways is of great advantage since it enables the use of traditional carbonyl electrophiles in radical reactions. The most recent and synthetically important transformations following this line are summarised in the first part of this review. The second part deals with transformations, in which the concept of RRPCO promotes the usage of alkyl halides as electrophiles in radical reactions. SCE),One of the most studied and fundamental transformations of organic chemistry is the addition of nucleophiles to carbonyls for the synthesis of alcohols. This transformation can be performed in a multitude of ways with high levels of diastereo- and enantioselectivity. While these reactions have been extensively developed in polar chemistry, radical pathways for this transformation remain underexplored. The development of such radical reactions would enable a wider variety of nucleophiles to be used, and hence generate valuable new product motifs. Furthermore, in combination with visible-light-mediated photochemistry, radical chemistry could be used to promote elusive transformations with high levels of functional group tolerance.pathways . In the via C–C-β-scission. Here, Glorius et al. showed that this obstacle could be overcome by adding alkyl radicals to protonated carbonyls to form alkoxy radical cations intermediates, which are less prone to C–C-β-scission and can be more easily reduced than alkoxy radicals  . In this2.2.12(dtbbpy)][PF6]; ppy: 2-phenylpyridine, dtbbpy: 4,4′-di-tert-butyl-2,2′-bipyridine) was used to generate trifluoromethyl radicals, which were added to substituted styrenes to form benzyl radicals. These stabilised benzyl radical intermediates were then reduced by the photocatalyst to form carbanions, which were trapped with carbon dioxide to deliver the carboxylic acid products. Evidence for the presence of an anionic species in this reaction was obtained by trapping with deuterium oxide instead of carbon dioxide. Furthermore, thermodynamic considerations of the two electron transfer steps by redox potential comparison also supported this mechanistic proposal. This challenging dicarbofunctionalization reaction highlighted the ability of this powerful synergy between radical and polar chemistry to generate valuable building blocks from simple reagents.Whilst the groups of Ryu and Sonoda were the first to describe the RRPCO concept in the 1990's,iv)-mediated ligand-to-metal charge transfer. Owing to the instability of such radicals, a C–C-β-scission generates a more stabilised alkyl radical. At this point, the photocatalyst-induced RRPCO takes place, which is followed by a nucleophilic addition of the generated anion to the pendant aldehyde moiety. Lactone formation is caused after ester hydrolysis, delivering the bridged product motifs. Once again, the interplay of radical and polar pathways results in the formation of complex products from simple starting materials in a single step.In 2018, the group of Zhiwei Zuo reported on a dual cerium- and photoredox-catalysed bridged lactone formation starting from cycloalkanols and Michael acceptors .9 At firvia two consecutive reductions of an imine intermediate by the excited state photocatalyst. This anion then adds to carbon dioxide to form the α-amino acid product. The existence of a carbanion intermediate was suggested by deuteration experiments and trapping with other electrophiles, such as carbon disulfide or isothiocyanates. The high regioselectivity for the α-carboxylation in this transformation is remarkable and is complementary to classical carboxylations of enamides that typically proceed in the β-position.In 2018, the group of Da-Gang Yu reported a highly chemo- and regioselective visible-light-mediated carboxylation of enamides and imines for the synthesis of α,α-disubstituted α-amino acids .10 An α-d7 did not cause any deuteration, contradicting a putative hydrogen atom transfer (HAT) step. In conclusion, this cross-electrophile coupling underlines the high potential of RRPCO, overcoming traditional philicity limitations.Later in 2018, the group of Yu also disclosed an elegant external-reductant-free cross-electrophile coupling between tetraalkylammonium salts and carbonyls .11 Mechavia 4CzIPN -1,3-dicyanobenzene)-mediated oxidation of benzyl carboxylates followed by rapid decarboxylation. The stabilised benzylic radical is converted to an anion through electron transfer from the reduced photocatalyst. After the RRPCO, nucleophilic addition to aliphatic aldehydes and ketones takes place, generating alcohols as products. Again, deuterium incorporation by addition of deuterium oxide signifies the intermediary formation of anions. The successful addition to aliphatic carbonyl compounds instead of aromatic ones reveals the improvement gained through RRPCO in comparison to radical chemistry alone, where to date only aromatic carbonyls could be used as acceptors.The group of König reported on a similar transformation .12 Here,via oxidation and subsequent deprotonation of a thiol HAT catalyst. The thiyl radical then abstracts a benzylic H atom to generate a benzylic radical and reform the thiol catalyst. As in the previous reaction, the stabilised benzylic radical is then converted to an anion by electron transfer from the reduced photocatalyst and subsequently adds into carbon dioxide to give the carboxylic acid products. The authors not only showed a broad substrate scope but further demonstrated the utility of their protocol by preparing four drug molecules in single steps. Interestingly, even if multiple benzylic C–H sites were present in the molecule, the monocarboxylated products were obtained selectively. This powerful C–H carboxylation clearly underlines the power of RRPCO to convert simple starting materials to high-value products.The group of König has since reported on an elegant carboxylation of benzylic C–H bonds by merging HAT catalysis with RRPCO .13 First2.2et al. employed the RRPCO concept during their studies of the synthesis of the paxilline indoloterpenoids -nodulisporic acid C and further developed an intermolecular version, which allowed a 14-step total synthesis of the terpenoid forskolin.15iii)-alkyl complex. This species transmetallates to a Cr(iii)-alkyl complex enabled by a Cr(ii) complex. This step is considered as the RRPCO step, since this step is only possible beginning from a chromium(ii) species, indicating that an alkyl radical is captured by the chromium(ii) complex to form the Cr(iii)-alkyl species. The in situ formed Cr(iii)-alkyl complex is able to add to carbonyls, in this case aliphatic and aromatic aldehydes, to afford alcohols as products.In 2018, Shenvi and co-workers reported on a branch-selective addition of olefins to aliphatic aldehydes by using a complex mixture consisting of a cobalt catalyst, chromium trichloride, phenylsilane as a superstoichiometric reductant and 1-fluoro-2,4,6-trimethylpyridinium tetrafluoroborate as a substoichiometric oxidant .16 The Cvia the RRPCO pathway (ii) species to the RAE. Following the principle of RRPCO, this radical is captured by the excess chromium(ii) salt to form a Cr(iii)-alkyl complex. This nucleophilic species is consequently able to add to aliphatic and aromatic aldehydes as electrophiles. The presence of trimethylsilyl (TMS) chloride in the reaction mixture lead to the isolation of TMS-protected alcohols. Baran and co-workers expanded the scope of the starting materials of traditional alkyl-Nozaki–Hiyama–Kishi reactions to RAE or carboxylic acids.In 2019, Baran and co-workers were able to facilitate a decarboxylative Nozaki–Hiyama–Kishi reaction  pathway .17 They 2.3ii) salts in enabling reactions following the RRPCO principle. Nevertheless, the requirement of stoichiometric chromium salts to obtain the desired reactivity is unfortunate, since chromium salts are widely considered to be physiologically hazardous and in case of chromium(ii) expensive and oxygen-sensitive. Furthermore, in enantioselective reactions, (over-)stoichiometric quantities of chiral ligands would be needed, limiting the practicability of such protocols.ii)- and Ir-photocatalytic ([Ir(dF(CF3)ppy)2(dtbbpy)][PF6]; dF(CF3)ppy: 2--5-trifluoromethylpyridine) system (iii)-allyl precursor in this reaction, which represents the first of example of a Nozaki–Hiyama–Kishi reaction based upon a C–H functionalization. Mechanistically, the reaction proceeds via oxidation of the allyl compound by the excited photocatalyst, which is followed by rapid proton-abstraction to give an allylic radical. This species undergoes the RRPCO through radical addition to the Cr-catalyst, forming a Cr(iii)-allyl complex. Nucleophilic addition to the aldehyde is facilitated by the Zimmerman-Traxler transition state, thereby providing high diastereomeric ratios of the branched anti-products. Finally, reduction of the chromium(iii) species by the reduced photocatalyst closes both catalytic cycles. This example constitutes a rare case of a highly diastereoselective radical reaction and underlines the great potential of RRPCO pathways.The protocols of Shenvi and Baran clearly show the power of chromium system .19 Both,In 2019, Kanai and co-workers then extended the dual chromium- and photocatalytic system through the addition of chiral ligands to foster an asymmetric allylation of aldehydes .20 The m3.The principle of RRPCO was also used to enable nucleophilic substitution reactions using alkyl halides as electrophiles. The Molander group was the first to apply this concept in the context of visible-light photoredox catalysis in 2018. In this work, they described the facile cyclopropanation of alkenes using iodomethyl silicates Aggarwal and co-workers also showed the utility of RRPCO processes in two transformations, a cyclopropanation  and a cy4.e.g. a chromium(ii) species. Importantly, these transformations allow complex molecules to be assembled in a fast step-economic fashion that would not be possible using either radical or polar chemistry alone. Furthermore, RRPCO enables highly challenging diastereo- or enantioselective radical transformations to be realized. To date, RRPCO reactions have been carried out using carbonyl compounds or alkyl halides/tosylates as electrophiles. Since this field of RRPCO is still in its infancy,Reductive radical-polar crossover reactions are initially radical reactions that are converted into traditional nucleophilic reactions by means of a single electron reduction. This reduction is either promoted by a photoredox catalyst or a redox active metal, There are no conflicts to declare.


Myopic macular degeneration (MMD) is a primary cause of blindness and visual impairment in many parts of the world. A review of clinical practice guidelines (CPGs) for intervention selection are required with the increasing demand for MMD management in clinical practice as well as in national health services. Therefore, we aim to systematically review CPGs for MMD and assist the recommendations development of the Package of Eye Care Interventions (PECI) program of the World Health Organization.A systematic review of CPGs published on MMD between 2010 and April 2020 was conducted. Guidelines were evaluated using the Appraisal of Guidelines for Research and Evaluation II (AGREE II) tool. Cochrane systematic reviews were also included when the evidence from included CPGs were inadequate or contradict.After applying exclusion criteria and conducting the quality appraisal, two CPGs were finally included. The average of the AGREE II ratings for the identified Guidelines were 56 and 63 respectively (7 for each item). To provide further information on interventions for MMD, one Cochrane review on MMD was additionally identified and included in the study. Intravitreal anti-vascular endothelial growth factor (anti-VEGF) drugs were recommended for patients with myopic choroidal neovascularization (mCNV) as first-line therapy to improve vision and reduce central macular thickness, and ranibizumab showed significant effectiveness compared to photodynamic therapy (PDT). PDT was recommended to be performed in those resistant to the treatment by one CPG but lacked of adequate description and support. Data extracted from the Cochrane systematic reviews indicated that anti-VEGF therapy for mCNV had significant effectiveness in improving visual acuity and reducing CMT compared to PDT with moderate to low certainty of evidence. Ranibizumab and bevacizumab were considered as equally effective with moderate certainty.The outcomes of this review suggest that high quality clinical practice guidelines for MMD management are limited. Intravitreal injection of anti-VEGF agents was recommended as an effective intervention to treat myopic CNV as the first-line treatment, while there was inadequate guidance for the application of PDT in myopic CNV management. The use of other interventions for MMD were not recommended at this time and additional evidence is called for. Myopic macular degeneration (MMD), also known as degenerative myopia or pathological myopia, is an important cause of blindness and visual impairment in many parts of the world, especially in areas with a high prevalence of myopia ,2. It isVarious treatment strategies for MMD have been explored in the last few decades, including macular surgical management,-14 laserGiven the increasing need for eye care, the World Health Organization (WHO) has highlighted the role of eye care in contributing to the Sustainable Development Goals (SDGs), and is developing an evidence-based Package of Eye Care Interventions (PECI). The methodology for the development of the PECI has been previously published. In briefThe systematic review of CPGs is following the procedures reported in the methodology paper previously . ExclusiA systematic literature search of academic databases  and guideline databases (as listed in Appendix S1 of the All the titles and abstracts of articles identified from the literature searches were screened independently by two authors (G.L and S.S). Abstrackr, a semi-automated online citation screening program, was utilized where possible . ConcernTwo authors (Y.C and X.H) independently evaluate the quality of the included CPGs using “Appraisal of Guidelines for Research and Evaluation” (AGREE II) . Items 4Following evaluation with the AGREE II tool, guidelines were excluded if the average rating of the two authors for items 4, 7, 8, 12 or 22 was less than 3, or the total rating for all 9 items was below 45. Final selection of a maximum of 5 CPGs according to the following criteria: 1) quality 2) publication time and 3) comprehensiveness  with the agreement of the whole study group.Information related to the recommendation, including type of recommendation, dosage, target population, the strength of recommendation and the quality of the evidence supporting the recommendation, were extracted by one author (Y.C or X.H) and re-reviewed by another author. Disagreements were discussed and resolved by the two researchers and a third author (S.K.). The process was repeated for all the Guidelines until agreement on the recommended eye care interventions was reached.With respect to the published protocol no changes were performed. The quality check and the methodological support for this study have been provided by WHO/CEV.When the evidence from included CPGs were inadequate or contradict, Cochrane Systematic Reviews (CSRs) were sought. The Information Specialist for CEV identified CSRs published in the last 10 years by searching the Cochrane library using the term “myopic macular degeneration. The following search limits were defined for the search strategy in the Cochrane library: 1) Content type: Cochrane Reviews, 2) Cochrane Library publication date: last 10 years, 3) Search word variations: Yes. The search was run on 23 July 2020 and retrieved 7 CSRs. The CEV Information Specialist pre-screened the results and forwarded PDFs of two CSRs that were potentially relevant for inclusion in the review.Two members of the group (Y.C and X.H) independently performed title and abstract screening. Systematic reviews were excluded if they were older than 10 years or the review was not an intervention or diagnostic test accuracy review that was specifically related to the target eye condition. After comparing the decisions of both researchers, only those CSRs where there was agreement between both authors (Y.C and X.H) were included.After combining all searches from academic and guideline databases and professional association webpages, 3778 CPGs were identified and underwent independent title and abstract screening. A total of 3575 CPGs met the exclusion criteria and 179 were duplicates, leaving a total of 34 CPGs that were considered potentially relevant and were retrieved to undergo full-text screening and quality appraisal. Of the identified 34 guidelines, we excluded 32 Guidelines for the following reasons: 29 because the content was not relevant to MMD after full-text screening , 3 becauAfter conducting quality appraisal on the remaining CPGs, and checking for quality, publication time and comprehensiveness, we finally included the following guidelines: “Intravitreal injection of anti-vascular endothelial growth factor agents for ocular vascular diseases; Clinical practice guideline”(2018)  developeThe included CPG developed by Nikkhah et al. recommended intravitreal anti-VEGF drugs for patients with myopic choroidal neovascularization (mCNV) to improve vision and to reduce central macular thickness. Intravitreal bevacizumab was recommended to be the first-line injection for these patients, and photodynamic therapy (PDT) performed in those resistant to the treatment. The evidence for the recommendations came from 1 meta-analysis, 1 RCT and 1 review, and the evidence level was graded as high. The CPG also recommended that highly myopic patients aged less than 50 years should have funduscopy to ensure early detection of MMD. For older patients with risk factors of CNV recurrence , periodic examinations are recommended, but the evidence level of this recommendation was low since it was only supported by case series. In the NICE CPG, intravitreal ranibizumab is recommended as an option for mCNV, and concluded that ranibizumab is a treatment with clinical effectiveness for visual impairment caused by mCNV, supported by 3 RCTs. But the long-term improvement (after 3 months) in BCVA brought by ranibizumab is uncertain. The recommendations and quality of evidence are summarized in To complement the evidence from the CPG, all Cochrane reviews on the topic area were also checked. In total one Cochrane review on MMD was identified, “Anti-vascular endothelial growth factor for choroidal neovascularisation in people with pathological myopia (Review)” (2016) . In the An overall view of the strength of recommendation and quality of evidence given in each Guideline is reported in The current study reviewed CPGs and Cochrane systemic reviews for MMD, with the aim of providing evidence on eye care interventions for MMD to aid in the development of the PECI. A total of 2 CPGs and 1 Cochrane systemic review were included after a standard screening process. Anti-VEGF therapy was recommended to improve the vision in patients with myopic CNV, among which ranibizumab showed greater effectiveness compared with PDT, while PDT was suggested to use in resistant cases with no solid evidence. Overall, current high-quality CPGs and meta-analysis can provide some guidance for the management of myopic CNV, but the types of interventions involved are limited.In recent years, anti-VEGF agents have been introduced and rapidly become an important therapy in the treatment of ocular vascular diseases, including age-related macular degeneration, diabetic macular edema, retinal vein occlusion and mCNV -24,66-70Among the available treatments for mCNV, PDT with verteporfin has a long history as an established and approved treatment and has been shown to achieve satisfying short-term outcome in improving vision of mCNV patients -19,77-81Despite the increasing demands for clinical management of MMD and other complications of pathological myopia, this study found few high-quality CPG. The CPG developed by Nikkhah et al provided clinical recommendations for 5 different ocular vascular diseases and evidence levels of each intervention, but presented a lack of details in terms of clinical effectiveness, cost-effectiveness, and technology of implementation. NICE, as a large government agency that develops guidelines, has only one comprehensive CPG for MMD. Though one Cochrane systemic review has been added to the current study, in total only recommendations on, and evidence for, two interventions were identified. The amount of benefit with surgical technology, including submacular surgery or macular translocation for mCNV, corticosteroid injection and combined therapy remains unclear. In an excluded CPG written by Cheung et al, a detailed recommendation of interventions were provided, that anti-VEGF therapy was considered as the first-line treatment with Class I recommendation, vPDT and intravitreal TA+ vPDT were Class IIa recommendation, macular surgery, laser photocoagulation and intravitreal TA + anti-VEGF therapy were Class IIb recommendation . Our maiThere are some inherent limitations in this study. Based on the criteria for identifying guidelines to inform the development of the PECI, only two guidelines were included. Though there have been enough evidence supporting ophthalmologists to use anti-VEGF as first-line therapy for mCNV, guidance is still insufficient for those who have contradictions, specific conditions or failure in taking anti-VEGF therapy. Second, MMD is most prevalent in countries where English is not the primary language. Given the language barrier in publications, some guidelines for MMD may have been omitted in the current analysis. Future study with larger searching database are required to provide better-quality data to help establish the best management strategy for MMD. Third, to assure the objectivity of the results, three papers providing assessment on more than one intervention for MMD were excluded due to possible conflicts of interest. The efficacy and utility of alternative treatments could not be assessed adequately.There are limited clinical practice guidelines for MMD management met the inclusion criteria of PECI development. Based on existing evidence, intravitreal injection of anti-VEGF agents is suggested to be an effective therapy for myopic CNV and should be the first-line treatment. The application of PDT for mCNV in clinical practice is still lack of adequate guidance. The use of other interventions for MMD is not recommended at this time and further solid evidence is called for.


Graph theoretical analysis allowed for the identification of acute and chronic network changes and revealed preserved small world topology before and after seizure onset. The most prominent acute change in network organization during seizures was the segregation of cortical regions from the remaining brain. Stronger connections between thalamic with limbic regions compared with preseizure state indicated network regularization during seizures. When comparing between strains, intrathalamic connections were prominent in NEC, on local level represented by higher thalamic strengths and hub scores. Subtle differences were observed for retrosplenial cortex (RS), forming more connections beyond cortex in epileptic rats, and showing a tendency to lateralization during seizures. A potential role of RS as hub between subcortical and cortical regions in epilepsy was supported by increased numbers of parvalbumin-positive (PV+) interneurons together with enhanced inhibitory synaptic activity and neuronal excitability in pyramidal neurons. By combining multimodal fMRI data, graph theoretical methods, and electrophysiological recordings, we identified the RS as promising target for modulation of seizure activity and/or comorbidities.Resting state-fMRI was performed to explore brain networks in Genetic Absence Epilepsy Rats from Strasbourg and in nonepileptic controls (NEC) during monitoring of the brain state by simultaneous optical Ca Absence epilepsy is a nonconvulsive form of epilepsy, accounting for 10–17% of all pediatric epilepsies . AlthougAbsence epilepsy is a network disease. The central element in the pathology and specifically in initiation, maintenance, and termination of the characteristic spike-and-wave discharges (SWDs) is the cortico-thalamo-cortical circuitry . CombineIn animal models of absence epilepsy, similar brain network components as in humans are involved . ActivatHere, we acquired rs-fMRI data from GAERS before and during seizures, and from NEC. GAERS  represenWe applied graph theoretical analysis of rs-fMRI data to characterize brain networks and to identify potential acute and chronic network changes in this absence epilepsy model.n = 25), and NEC (n = 20) from the breeding facility of the Institute of Physiology I, University of Münster . Adult GAERS exhibit frequently occurring absence seizures with a prevalence of 100% (Experiments were performed in line with ARRIVE guide lines with adult (>3 months), female GAERS . During the experiment, breathing rate, expiratory CO2, and rectal temperature were monitored and kept in physiological ranges. Rs-fMRI data were acquired at 9.4 T (Bruker Biospin MRI GmbH) using a gradientecho echo planar imaging sequence (GE-EPI) with TR 1 s, TE 18 ms, resolution 0.32 × 0.35 mm2, slice thickness 1.2 mm, 9 contiguous slices, and 30-min scan time per data set.For rs-fMRI and simultaneous Can = 13), one or two rs-fMRI measurements were performed under Neurolept anesthesia before seizures’ onset (preseizure state). SWD onset was identified in Ca2+-recordings (n = 7) underwent the same procedure and 30-min rs-fMRI data were acquired ~90 min after switching to Neurolept. Complete data sets (rs-fMRI and Ca2+-recordings) from three different groups were used for analysis: GAERS preseizure: number of animals n = 9, number of data sets N = 12; GAERS seizure: n = 8, N = 14; NEC: n = 7, N = 17 . The first 15 min of raw data were used. In case any shift in rat head position was detected, continuous 15-min epochs with stable translation (<0.2 mm) and rotation (<0.3°) parameters were selected from 30-min data sets. For data quality assurance, a cortical mask in slice 6 was used to estimate temporal SNR  in the NEC control group . At this density, all networks were fully connected. We determined clustering coefficient, shortest path length, degree, strength, and hub score for each node and data set. The degree specifies the number of connections associated with one node. The strength is the sum of link weights of a node. A high hub score index indicates the importance of brain regions with above average high number of connections (degree), a high level of betweeness centrality, and short average path length  edges in total  were transcardially perfused with 4% (w/v) phosphate-buffered paraformaldehyde (PFA). After removing from the skull, brains were fixed overnight in 4% PFA and later in 30% (w/v) sucrose for 48–72 h. Immunohistochemical staining was performed on free-floating 40-μm-thick coronal sections. First, sections were rinsed three times (10 min) in phosphate-buffered saline (PBS). Then, slices were incubated for 2 h in PBS supplemented with normal goat serum (10% [v/v]), Triton-X100 (0.3% [w/v]), and bovine serum albumin (3% [w/v]). Finally, sections were incubated overnight at 4 °C in primary antibodies: monoclonal mouse anti-parvalbumin  and monoclonal mouse anti-neuronal nuclear protein . After incubation with one of the primary antibodies, sections were washed three times for 10 min in PBS and then transferred to the secondary antibody solution  for 2 h. In the last step, sections were washed three times for 10 min in PBS and mounted with a mounting medium containing the nuclear marker 4′,6-diamidino-2-phenylindole  for confocal microscopy (Nikon eC1plus) equipped with ×10 objective (Leica). DAPI staining was used to define boundaries and layers of the granular RS; 40-μm-thick slices were scanned with 2-μm Z-stack steps and then combined together as one picture. Cells were counted in a 960 × 760 μm field of view in 3 slices from each animal with ImageJ .Female rats  and GAERS (n = 12) ranging in age from postnatal day P105–P120. After decapitation, the skull cap was surgically removed caudal to Bregma and a block of brain tissue containing the RS was submerged in ice-cold aerated (O2) saline containing (in mM): sucrose, 200; PIPES, 20; KCl, 2.5; NaH2PO4, 1.25; MgSO4, 10; CaCl2, 0.5; dextrose, 25; pH 7.35 was set with NaOH. Coronal slices (250–300 μm thick) were prepared with a vibratome. Slices were transferred to a holding chamber and kept submerged  in artificial cerebrospinal fluid (ACSF) containing (in mM): NaCl, 125; KCl, 2.5; NaH2PO4, 1.25; NaHCO3, 24; MgSO4, 2; CaCl2, 2; dextrose, 10; pH was adjusted to 7.35 by aerating the solution with carbogen (95% O2 and 5% CO2 gas mixture).Experiments were performed on NEC  connected to an upright microscope (ZEISS Axio Examiner.D1). All experiments were done in deep layer (Layer 5) of the cortex at 30–32 °C. Pyramidal neurons of Layer 5 were visually identified .VM = VP—10 mV; with VM = membrane potential and VP = pipette voltage).Membrane currents were measured with glass microelectrodes pulled from borosilicate glass capillaries  with a resistance of 2.5–3.5 MΩ. Patch electrodes were connected to a HEKA EPC-9 patch clamp amplifier (HEKA Elektronik) via a chlorinated silver wire. Access resistances were between 8 and 20 MΩ. Series resistance compensation of ≥50% was routinely applied. Experiments were controlled by the PatchMaster recording software (HEKA Elektronik) at a sampling rate of 10 kHz, with a low-pass filter of 2–3 kHz. All recordings were corrected offline for a liquid junction potential of 10 mV  were recorded from tissue slices of 3 GAERS and 4 NEC in the presence of GABA receptor blockers (10-μM CGP and 25-μM gabazine) at −60 mV. The micropipette was filled with a potassium-gluconate (K-gluconate)-based intracellular solution containing (in mM): K-gluconate, 88; K3-citrate, 20; NaCl, 10; HEPES, 10; MgCl2, 1; CaCl2, 0.5; BAPTA, 3; Mg-ATP, 3; Na2-GTP, 0.5. The pH of 7.25 was set with KOH. Osmolality of the internal solution was 295 mOsm/kg. Spontaneous inhibitory postsynaptic currents (sIPSCs) were measured from 3 GAERS and 3 NEC and were pharmacologically isolated using glutamatergic receptor blockers (20 μM AP-5 and 10 μM DNQX) at −60 mV. The internal solution contained a high molarity of Cl− (in mM): KCl, 110; NaCl, 10; EGTA, 11; HEPES, 10; Phosphocreatin, 15; Mg-ATP, 3; Na-GTP, 0.5; MgCl2, 1; CaCl2, 0.5. pH was adjusted to 7.25 with KOH. Osmolality was set at ~290 mOsm/kg. Spontaneous currents were recorded for 5 min . The frequency and the amplitude of the events were analyzed offline manually using Clampfit  software.Spontaneous synaptic events were measured in voltage clamp mode. During recordings slices were continuously oxygenated and perfused (~2 mL/min) with ACSF containing (in mM): NaCl, 125; KCl, 2.5; NaHm) were obtained by fitting negative voltage deflections (induced by hyperpolarizing current injections of −40 pA) to single or double exponentials. Input resistance (Rin) was calculated according to Ohm’s law: Rin = ΔV/I. The membrane capacitance was estimated using the following formula: Cm = τm/Rin. The anomalous rectification (or voltage sag amplitude) of the current injection at −240 pA was calculated as the differences between the maximal (at the beginning of the hyperpolarizing current injection) and steady state voltage deflection (at the end of the hyperpolarizing current injection). For depolarizing current steps, the number of action potentials (APs) was determined. For the spike in a train of APs, threshold and amplitude were determined. In addition, the number of rebound spikes, that is, the number APs following release from hyperpolarizing, was determined.Current clamp method was used to study the electrical properties of the pyramidal neurons. Recordings were done in brain slices of 6 GAERS and 6 NEC in ACSF (as described above). Micropipettes were filled with K-gluconate-based intracellular solution (as described above). The resting membrane potential (RMP) was measured in current clamp mode immediately after opening the membrane with zero current injection. Recordings were done at RMP and at −60 mV. To measure active and passive properties of the electrical membrane, 1000-ms hyperpolarizing (up to −240 pA) and depolarizing (up to 360 pA) current steps with 40-pA increments were injected as square-wave current pulses. Analysis was done offline using FitMaster software (HEKA Elektronik). Membrane time constants -corrected correlation matrices was performed. Network based statistics (NBS) were applied by correcting this t-test for multiple testing by 1000 times permutation  refer to numbers of neurons and for immunostaining—(n) to numbers of animals. Statistical differences between groups were established with repeated measures ANOVA, independent samples, two-tailed t-test with post hoc Tukey’s multiple comparisons test. Significance levels are indicated as P values. *, **, and *** indicate P < 0.05, P < 0.01, and P < 0.001, respectively.Statistical analysis was performed with IBM-SPSS (version 24). All results are presented as mean ± SEM. For patch clamp recordings, numbers given in text  after switching from Isoflurane to Neurolept. In seizure state, SWD epochs alternated with short epochs of rest  and second, after SWD onset (seizure state) A. Applyiz-values of the correlation coefficients between individual nodes . Global network parameters . Compared with NEC, shortest path length was low . Global clustering coefficient  intracortical connections compared with the preseizure state. NBS furthermore indicated that during seizures, particularly connections within and between basal ganglia and limbic regions (green) and within thalamic regions (blue) were significantly stronger. In the preseizure state  segregated from the remaining brain network A and shore state B, connecP < 0.004) intracortical connections . RS showed distinct connectivity patterns , we observed a tendency toward higher sEPSC frequencies in NEC  compared with GAERS   in comparison to NEC  A. While  = 0.07) B. sIPSCs = 0.07) C. The amn = 10; GAERS: −66.6 ± 0.9 mV, n = 11; F = 0.02, df = 18, P = 0.2) and right  hemispheres. When challenged with positive current steps, pyramidal neurons from NEC revealed higher firing rates in the left compared with right hemisphere (Rin). Rin was significantly higher in the right hemisphere of epileptic rats both at RMP  and at −60 mV . Except membrane time constant (τm), passive and active membrane properties, such as membrane capacitance (Cm), membrane time constant (τm), voltage sag amplitude, AP threshold and AP amplitude, and number of rebound spikes, were not statistically different between groups under both experimental conditions  and Interdisziplinäres Zentrum für Klinische Forschung Münster (core unit PIX).supplemetary_data_tgab023Click here for additional data file.


TEX19) is a specific human stem cell gene identified as cancer‐testis antigen (CTA), which emerged as a potential therapeutic drug target. TEX19.1, a mouse paralog of human TEX19, can interact with LINE‐1 retrotransposable element ORF1 protein (LIRE1) and subsequently restrict mobilization of LINE‐1 elements in the genome.Testis expressed 19  of Outcomes of this study revealed amino acids that potentially stabilize the predicted interaction interface between TEX19 and LIRE1. Of these SNPs, 37 were predicted to play a probably damaging role for the protein, three of them  located at the binding site of LIRE1 and could disturb this binding affinity.This information can be verified by further in vitro and in vivo experimentations and could be exploited for potential therapeutic targets. interaction pattern between TEX19 and LIRE1 proteins Formation of Van der Waals, electrostatic interactions and hydrogen bonding and formation of TEX19 and LIRE1complex The 3D structure of TEX19 protein was predicted by using the Iterative‐Threading ASSEmbly Refinement (I‐TASSER) server  software  for possible damaging effect on the protein. Sorting Tolerant From the Intolerant (SIFT) server was used for the prediction of the deleterious effect of mutations. For the prediction of disease‐related SNPs, we used Predictor of human Deleterious Single‐Nucleotide Polymorphisms (PhD‐SNP), and SNPs&GO servers. Project HOPE webserver was used to analyze the effect of single point mutation on protein structure.Different software were used for prediction of the effect of missense single nucleotide polymorphisms (SNPs) on the structure and function of the 2.5ConSerfserver. BioEdit version 2.7.5  and was then validated by PROSA statistic  analysis not only reflects the change of protein backbone versus simulation time but also indicates the divergence of the structure. The RMSD of the complex became stable at 15 ns. The RMSD value of modeled helicase was 0.45 nm Figure . The val3.4Table 3.5Residues of TEX19  showing hydrogen bonds with LIRE1 were among conserved sequences. Phe35 of TEX19 which have an interaction with LIRE1 predicted among highly conserved buried residues Figures  and 5.4In silico methods such as docking and molecular dynamic (MD) simulations are used to find the correct conformation of a ligand and its receptor and have been used previously in drug design (Bissaro et al., The authors have declared no conflict of interest.Conceptualization, O.R.A, F.A., and Y.H.; methodology, F.A., Y.H., H.A., and N.A.; software, Y.H., H.A., and N.A.; validation, F.A., Y.H., H.A., and N.A.; formal analysis, Y.H., H.E.A., and N.A.; investigation, Y.H., H.A., L.M., S.A., and N.A.; resources, F.A., Y.H., H.A., and N.A.; data curation, F.A., Y.H., H.A., L.M., and N.A.; writing—original draft preparation, O.M.A., F.A., and Y.H.; writing—review and editing, F.A., Y.H., H.A., S.A., L.M., N.A.,O.R.A., H.E.A., and O.M.A.; funding acquisition, F.A., N.A., S.A., and O.R.A.


Release of phosphocreatine kinase-myocardial band (73.86 ± 1.95 vs. 25.93 ± 0.66 IUL−1) and lactate dehydrogenase (299.01 ± 10.7 vs. 152.3 ± 16.7 IUL−1) was also decreased in the RIPC-treated group. These results demonstrate the cardioprotective effects of the hepatic remote preconditioning against the injury caused by I/R in the isolated perfused hearts.It has been convincingly demonstrated that remote ischemic preconditioning (RIPC) can make the myocardium resistant to the subsequent ischemia reperfusion injury (IRI), which causes severe damages by mainly generating cell death. However, the cardioprotective effects of the hepatic RIPC, which is the largest metabolic organ against I/R, have not been fully studied. The aim of our research is whether remote liver RIPC may provide cardioprotective effects against the I/R-induced injury. Here, we generated an I/R mice model in four groups to analyze the effect. The control group is the isolated hearts with 140-min perfusion. I/R group added ischemia in 30 min following 90-min reperfusion. The third group (sham) was subjected to the same procedure as the latter group. The animals in the fourth group selected as the treatment group, underwent a hepatic RIPC by three cycles of 5-min occlusion of the portal triad and then followed by induction of I/R in the isolated heart. The level of myocardial infarction and the preventive effects of RIPC were assessed by pathological characteristics, namely, infarct, enzyme releases, pressure, and cardiac mechanical activity. Subjected to I/R, the hepatic RIPC minimized the infarct size (17.7 ± 4.96 vs. 50.06 ± 5,  Acute myocardial ischemia accounts for the most common cause of hospitalization in Western countries. Myocardial infarction is recognized as a heart attack when myocardial ischemia exceeds a threshold, which causes irreversible cardiac cell damage or even death  . The FVB mice were kept in a room with controlled temperature and lighting . All animals had free access to chow and water. All protocols on animals were approved by the Ethics Committee of Beijing Anzhen Hospital , Capital Medical University. All experimental procedures were carried out following the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health .via a ventral penile vein. A small incision was made in the midline of the abdomen to expose the liver. The procedure of hepatic RIPC was performed by a three-cycle of 5 min before establishing the I/R in the isolated perfused heart. To prepare the isolated rat heart, the heart was excised and the aorta was cannulated for anterograde perfusion. The non-recirculating Krebs–Henseleit buffer solution was used at a flow rate of 10 ml/min. The buffer solution is saturated with the gas mixture of 95% O2 plus 5% CO2 (pH = 7.4) and kept at a temperature of 37°C. A pressure transducer  was used to detect the coronary perfusion pressure. All isolated hearts were subjected to 30-min ligation of the left anterior descending coronary artery, followed by 90 of reperfusion, except for the control group.The mice were anesthetized by 50 mg/kg thiopental sodium and then 250 IU heparin was injected A total of 24 mice were randomly divided into four experimental groups. In the control group, the Langendorff isolated heart perfusion was established in 150 min. However, in the I/R group, the isolated hearts were exposed to ischemia for 30 min and then reperfusion for 90 min. The procedure conducted on the third (sham-operated) group was the same procedure as the I/R group except that a sham operation on the liver. The animals in the fourth group underwent a remote liver IPC by three cycles of 5-min occlusion of the portal triad before establishing the I/R in the isolated perfused heart .The coronary effluent samples were collected respectively before ischemia and after 0, 5, 30, and 60 min of reperfusion. The collected samples were stored at −20°C until analyzing to measure the level of creatine kinase-myocardial band (CK-MB) and lactate dehydrogenase (LDH). Enzyme-linked immunosorbent assay kits were used for the analysis: serum CK-MB  and serum LDH .via an elastic water-filled balloon connected to a pressure transducer . The initial end-diastolic pressure was adjusted to 10 mmHg and after that, the pressure signals were recorded for analysis using Chart 5.0.1 Data Recording Software . The heart rate, LVDP, and the percentage of the rate pressure produce (RPP%) were calculated from basic measurements.Left ventricular-developed pressure (LVDP) was determined At the end of the procedure of each experiment group, the heart was collected from the perfusion apparatus, and then it was sectioned from the apex to the base into 2-mm-thick slices. The infarct size, the area at risk of ischemic injuries, and the intensity of the I/R-induced myocardial injury were quantified using standard methods.p-value of at least <0.05 was considered significant.Significant differences were analyzed by the one-way ANOVA to compare between the experimental groups. A p < 0.0001) declined in the I/R and sham-operated groups compared to the control group. However, the RPP% in the group exposed to the hepatic RIPC reached the same level as the control group. The effects of hepatic RIPC on the rate pressure of the isolated heart subjected to 30-min ischemia followed by 90-min reperfusion are shown in The levels of the heart rate and LVDP were immediately decreased upon the commencement of the ischemia and reached zero level during the ischemia in the groups exposed to the global ischemia. The values of the hemodynamic parameters obtained in the preconditioned and non-preconditioned hearts at different times of the preischemia and postischemia are shown in p < 0.001) reduction in infarct size to 15.60%. The representative images of the infarct size data are shown in The effect of hepatic RIPC on I/R-induced myocardial infarct size in the isolated perfused hearts is shown in p < 0.001) increased enzyme releases were observed in the isolated perfused hearts subjected to I/R. However, there was significant (p < 0.001) reductions in the release of lactate dehydrogenase from 299.01 ± 10.7 to 152.3 ± 16.7 IUL−1 and phosphocreatine kinase-MB from 73.86 ± 1.95 to 25.93 ± 0.66 IUL−1 in the preconditioned group . The current results suggest the cardioprotective effectiveness of liver RIPC against I/R-induced myocardial cell injury. Consistently, it has been reported that hepatic RIPC had significantly diminished the incidence of I/R-induced arrhythmias in isolated rat hearts , National Natural Science Foundation (81970525), Beijing Hospitals Authority Incubating Program (No. PZ2021007), and Beijing Hospitals Authority Youth Program (No. QML20200604). The sponsors had no role in the study design, data collection and analysis, decision to publish, or manuscript preparation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.