Conclusion: We demonstrated that CEST MRI can be used for non-invasive, label-free detection of mannitol accumulation in the brain following BBBO treatment. This method may be useful as a rapid imaging tool to optimize the dosing of mannitol-based OBBBO and improve its safety and efficacy.Purpose: Mannitol is a hyperosmolar agent for reducing intracranial pressure and inducing osmotic blood–brain barrier opening (OBBBO). There is a great clinical need for a non-invasive method to optimize the safety of mannitol dosing. The aim of this study was to develop a label-free Chemical Exchange Saturation Transfer (CEST)-based MRI approach for detecting intracranial accumulation of mannitol following OBBBO. Methods: In vitro MRI was conducted to measure the CEST properties of D-mannitol of different concentrations and pH. In vivo MRI and MRS measurements were conducted on Sprague-Dawley rats using a Biospec 11.7T horizontal MRI scanner. Rats were catheterized at the internal carotid artery (ICA) and randomly grouped to receive either 1 mL or 3 mL D-mannitol. CEST MR images were acquired before and at 20 min after the infusion. Results: In vitro MRI showed that mannitol has a strong, broad CEST contrast at around 0.8 ppm with a mM CEST MRI detectability. In vivo studies showed that CEST MRI could effectively detect mannitol in the brain. The low dose mannitol treatment led to OBBBO but no significant mannitol accumulation, whereas the high dose regimen resulted in both OBBBO and mannitol accumulation. The CEST MRI findings were consistent with  The blood-brain barrier (BBB) poses a major challenge in drug delivery into the central nervous system. For example, the fact that less than 2% of FDA approved anticancer drugs can pass the BBB is thought to be a major factor in the in the low success rate in brain cancer clinical trials ,2. Thus,While numerous new strategies to deliver drugs beyond the BBB have been explored to date, such as microbubble-based high-intensity focused ultrasound (HIFU) and convection-enhanced delivery (CED), or receptor-mediated transcytosis (RMT), mannitol-based OBBBO is still considered an effective and safe approach that is continuously used in the clinic . However1H magnetic resonance spectroscopy (MRS) and magnetic resonance spectroscopic imaging (MRSI) for detecting mannitol [A critical hindrance to the widespread use of IA-injection of mannitol in the brain is the potential of mannitol deposition following OBBBO. It is well known that the accumulation of mannitol in the brain interstitium can cause a “rebound” phenomenon, i.e., a secondary reversal of the osmotic gradient and an increase in brain water content, followed by increased ICP ,23. Thismannitol ,29,30. HThis study aimed to develop an MRI approach for detecting mannitol accumulated in the brain following OBBBO. The mannitol molecule contains six hydroxyl(-OH) protons, which can be directly detected by Chemical Exchange Saturation Transfer (CEST) without the need for chemical labeling . CEST MRUnless otherwise noted, all chemicals were purchased from Sigma Aldrich . The D-mannitol (25% solution) used in animal studies was purchased from Hospira Inc. .n = 3 each) to receive either 1 mL or 3 mL D-mannitol at a 0.6 mL/min rate. The corresponding infusion time and total doses were 1.7 min (4 mL/kg or 1 g/kg) and 5 min (12 mL/kg or 3 g/kg), respectively.All animal protocols were approved by our Institutional Animal Care and Use Committee. Sprague-Dawley rats  were purchased from Charles River . Catheterization was performed as previously described ,50. In b1H-NMR measurements of mannitol solutions (100 mM in D2O or a mixture of 90% H2O and 10% D2O) were performed on a Bruker Avance III 500 MHz spectrometer  at room temperature (pH = 6.5). The 1H NMR spectra were acquired using a standard one-dimensional pulse sequence (zg30) with 128 scans. A total of 65,500 data points for each scan were acquired with a spectral width of 20.6 ppm and an acquisition time of 3.18 s. The data were recorded and processed with TopSpin 4.0.1.The 1 and T2 relaxation times of D-mannitol in PBS solution of different concentrations (5 to 100 mM) and pH (5.5 to 8.0) using a 11.7T Bruker Avance vertical bore MRI system equipped with a 15 mm sawtooth RF coil . CEST MRI was conducted using a modified rapid acquisition with relaxation enhancement (RARE) sequence as described previously [0 image was acquired using B1 = 0 µT at −50 ppm. Unless otherwise noted, the parameters of the saturation RF pulses were continuous wave (CW), Tsat = 3000 ms, B1 = 1.8 μT (150 Hz). Other imaging parameters were: TR/TE = 6.0 s/5 ms, RARE factor = 16, slice thickness = 2 mm, matrix size = 64 × 64, FOV = 15 × 15 mm2, spatial resolution= 0.23 × 0.23 mm2, number of averages (NA) = 2, total acquisition time = 16 min 48 s. The B0 inhomogeneity was measured using the Water Saturation Shift Reference (WASSR) method [sat = 500 ms, B1 = 0.5 μT (21.3 Hz) and the saturation frequency swept from −1 ppm to 1 ppm (step size = 0.1 ppm). T1 and T2 maps were acquired as described previously [2 preparation module , respectively. During MRI scans, the temperature of the samples was maintained at 37 °C.In vitro MRI was conducted to measure the CEST properties and Teviously ,51. Z-sp) method  using theviously  using a 1 = 1.8 µT, Tsat = 3 s, TR/TE = 5000/5 ms, RARE factor = 23, matrix size = 64 × 64 , FOV = 30 × 30 mm2, spatial resolution = 0.47 × 0.47 mm2, slice thickness = 0.6 mm, number of averages NA = 2, acquisition time = 8 m 40 s). Z-spectral acquisitions were acquired by sweeping saturation offsets  from −5 to +5 ppm (step=0.2 ppm) before and at 20 min after the infusion of mannitol at the prescribed volumes. The time interval of 20 min was chosen because the half-life of mannitol in rats was estimated to be 17–25 min using the reported human values of 70–100 min [0 inhomogeneity.All in vivo studies were conducted on a Biospec 11.7T horizontal MRI scanner  equipped with a rat brain surface array RF coil (receiver) and a 72 mm volume coil . All CEST MR images were acquired using the same RARE-CEST sequence  sequence .T1H MRS spectra (voxel size = 4 × 6 × 3 mm3) were acquired in the ipsilateral and contralateral hemispheres using a stimulated echo acquisition mode (STEAM) sequence  with outer volume suppression (OVS) and the variable power RF pulses and optimized relaxation delays (VAPOR) water suppression scheme according to previously published procedure [Following the post-mannitol CEST acquisition, single-voxel localized rocedure . A field0 correction was conducted first. Then a region of interest (ROI) mask was placed over each sample, and the mean value of water signal intensity (SΔω) at each saturation offset relative to the water protons (±Δω) was calculated. The normalized SΔω/S0, where S0 is the water signal intensity without saturation, was plotted as a function of Δω, which is traditionally called a Z-spectrum. To quantify the CEST effect, the magnetization transfer ratio asymmetry (MTRasym) was calculated as MTRasym = (S−∆ω − S+∆ω)/S0. As the peak position of MTRasym plots is dependent of the concentration of mannitol due to interference of the direct saturation, we used numerically integrated area under the curve (AUC) [asym plots between 0.2 and 2 ppm as an approximation of the signal integral. Because AUC values are also dependent on the frequency range, we normalized the AUC values by the spectral width (ppm) that used in the calculation. The resulting AUC values have a unit of % per ppm (%/ppm).All MRI data processing was performed using custom-written scripts in MATLAB . For CEST MRI data, pixel-wise Bve (AUC)  of MTRas1 changes, the T1 compensated inverse Z-analysis (AREX), defined by (S0/S+∆ω − S0/S−∆ω)/T1, were also calculated using the acquired T1 times [asym.To accurately quantify the CEST effect in the presence of substantial TT1 times ,60,61. O1A , the offsets of amide, amine, and magnetization transfer contrast (MTC) pools, and the T2 times of all the exchangeable protons (except the NOE pool) were fixed, with all other parameters set to fitting parameters. 2 relaxation time of the NOE pool, and concentrations of amide and NOE from the first step ROI fitting were fixed in the second step pixel-by-pixel fitting.To quantify the concentration of mannitol in different tissues, we also fitted the Z-spectra using 6-pool Bloch equations as described previously ,63,64. T1H-MRS was processed according to a previously published procedure [N-acetyl-aspartate (NAA) resonance was set at 2.02 ppm. After local baseline flattening around the integration region using a second-order polynomial baseline correction, the peaks at 3.03 ppm and 3.5–4 ppm  and mannitol, respectively), were quantified using their integrated peak areas.In vivo rocedure . As a reData were expressed as mean ± SD. The comparison of two groups was conducted using a two-tailed Student’s t-test.2O/D2O and D2O. Mannitol contains six exchangeable protons . Four distinct NMR signals of non-exchangeable protons with distinct scalar coupling patterns were observed in both D2O and H2O NMR spectra, at approximately 3.65, 3.73, 3.77, and 3.84 ppm, in good agreement with previous reports [asym peak between 0.2 and 2 ppm. It is be (in the intermediate to fast exchange regime), leading to a distinct signal broadening especially at positive frequency and.We first acquired and compared the NMR spectra of D-mannitol  in Hs red in a and eigs red in a. As sho reports . Figure asym curve is around 0.4 ppm at 1 mM and shifts to higher offsets at higher concentrations, i.e., 0.6 ppm at 40 mM. Quantified by the AUC (0.2–2 ppm) of the MTRasym plots, the mannitol CEST contrast is linearly correlated with concentration (2 = 0.9927). Fitting the Z-spectra to the Bloch equations allows more accurate estimation of the mannitol concentration for the faster exchange rates (2 = 0.9976). Based on the curves in 1 values (ex) of mannitol as a function of pH  × 2π (rad) ≈ 2500 rad/s).Subsequently, we characterized the CEST contrast of mannitol as a function of concentration and pH. As shown in 1 values , we estion of pH f, which sat and B1, which were also studied , respectively  resulted in marked changes in CEST contrast, indicating the accumulation of mannitol at the injection site, which was confirmed by asym analysis (p = 0.0433) and 20.0 ± 2.4%/ppm and 18.7 ± 2.6%/ppm in the contralateral hemisphere (p = 0.3787), respectively. In contrast, no significant change in CEST contrast was observed in the rats infused with 1 mL of 25% mannitol solution , while there was no significant difference the in low dose group (p = 0.3536).1 changes, we also attempted to quantify the CEST contrast enhancement by mannitol using the AREX approach. asym) plots in the ipsilateral and contralateral hemispheres in a rat receiving high dose mannitol. Comparing the ∆MTRasym (0.8 ppm) maps  was higher than that by the AREX method , attributed to T1 “contamination” in the MTRasym method.To accurately quantify the CEST effect in the presence of substantial Tpm) maps c and ∆ARpm) maps d revealsp = 0.0237), while there was no significant difference the in low dose group (p = 0.4297).The comparison of the mean ∆AREX values between the low and high dose groups (n = 3 each) is shown in 1 and T2 data (n = 3) was estimated to be 23.8 ± 17.8 mM, which is in good agreement with that measured by the MRS method (24.1 ± 4.9 mM), whereas the average concentration of mannitol was estimated to be 4.5 ± 3.7 mM in the low dose group (n = 3).Finally, we also attempted to quantify the CEST effect using Bloch fitting of the acquired CEST Z-spectral data together with T T2 data  to compuin vitro and exploited it for non-invasive and label-free detection of mannitol accumulated in the brain following intra-arterial infusion for OBBBO. While mannitol has been used as a hyperosmotic agent for various medical applications, including OBBBO, it can cause severe adverse events in some patients , including electrolyte abnormalities, acidosis, hypotension, acute renal failure congestive, and heart failure with pulmonary edema [1 and T2 times). These findings are consistent with previously reported mannitol accumulation in the acute cerebral ischemia [In the present study, we systematically characterized the inherent CEST MRI contrast of mannitol ry edema ,72,73,74ry edema ,25,26. Rry edema . It was ry edema . Cathetery edema ,17,18. Iischemia  or brainischemia ,29. In c1H-MRS/CSI method. Compared to 1H MRS, CEST MRI has relatively higher sensitivity, higher spatial resolution, and shorter acquisition time; hence it is more suitable for monitoring mannitol distribution in vivo in a clinical setting. Spin-lock (CESL) MRI has also been used to detect D-mannitol [ρ. Being used as an intravascular agent in those studies, no quantitative detection of tissue mannitol was performed. Here, we applied CEST MRI for detecting mannitol accumulated in the brain. Our study is the first attempt to perform quantitative analyses of tissue-accumulated mannitol using label-free CEST MRI directly by its hydroxyl protons. Further technical optimization is warranted to improve the accuracy of quantification. For example, we observed relatively large variations using a 6-pool Bloch fitting, likely due to low SNR of pixel-wise CEST data. We then demonstrated that AREX  was helpful in reducing the influence of T1 and T2. Still, future studies are needed to convert the measured AREX values into mannitol concentrations and validate such quantitation. Another technical hurdle is the potentially low sensitivity of CEST MRI detection at low field strengths, such as 3T. For example, the simulation  can be used to reduce the potential influence of tissue T1 changes [1 changes, the degree of ∆AREX increase was much smaller than that of ∆MTRasym, indicative of an overestimation of mannitol accumulation when the non-T1-compensated MTRasym was used. However, both methods show consistent spatial distribution of mannitol (1 changes. Moreover, we utilized Bloch equation fitting to extract mannitol concentration in the tissue. This method takes all factors, such as T1, T2, tissue water content, and other exchangeable protons, into consideration and hence potentially provides more accurate quantification of mannitol concentration. Indeed, the computed mannitol concentrations were in good agreement with MRS measurement. Tissue pH might also affect the accuracy of quantification. While both intracellular and extracellular pH values in the healthy brain are constant [1H-MRS are not prone to these factors, combining CEST and MRS can achieve both quantification accuracy and high spatial/temporal resolution for detecting mannitol.It should be noted that accurate determination of the concentration of mannitol using CEST MRI is not always straightforward because the CEST MRI signal is affected by a wide variety of factors. For example, T changes ,60,61. Omannitol . The AREconstant , patholoconstant . Hence, We characterized the CEST MRI contrast of mannitol and demonstrated that CEST MRI could be used for non-invasive, label-free detection of mannitol accumulated in the brain parenchyma following OBBBO. Thus, the present CEST MRI method provides a tool for monitoring the brain uptake of mannitol and adjusting the dose in mannitol-based treatments to obtain optimized efficacy and safety. Furthermore, there is traction to move guidance of IA infusions to cerebral vasculature from the current X-ray routine toward real-time MRI guidance. In this new environment, the reported MRI method has potential for improving the safety profile of OBBBO by continuously monitoring mannitol accumulation in the brain. It could be combined with previously described, drug-detecting CEST techniques to confirm adequate drug delivery after OBBBO.


Chlorella pyrenoidosa and functional characterization of transcription factors in Yarrowia lipolytica. We found that early nitrogen deprivation dramatically reduced fatty acid desaturation without increasing lipid content. The contents of palmitic acid (16:0) and oleic acid (18:1) dramatically increased to 2.14 and 2.87 times that of nitrogen repletion on the second day, respectively. Lipidomic analysis showed the transfer of polyunsaturated fatty acids from phospholipids and glycolipids to triacylglycerols, and an increase in lipid species with 16:0 or 18:1 under nitrogen deprivation conditions. Upregulated stearoyl-ACP desaturase and oleyl-ACP thioesterase promoted the synthesis of 18:1, but restricted acetyl-CoA supply revealed that it was the intensive lipid turnover instead of an attenuated Kennedy pathway that played an important role in the variation in fatty acid composition under early nitrogen deprivation. Finally, two differentially expressed SQUAMOSA promoter-binding proteins (SBPs) were heterologously expressed in Y. lipolytica, demonstrating their role in promoting the accumulation of total fatty acid and the reduction in fatty acid desaturation. These results revealed the crucial role of lipid turnover and SBPs in determining fatty acid desaturation under early nitrogen deprivation, opening new avenues for the metabolic engineering of fatty acid desaturation in microalgae.Nitrogen deprivation induces variations in fatty acid desaturation in microalgae, which determines the performance of biodiesel and the nutritional value of bioproducts. However, the detailed scenario and the underlying regulatory mechanism remain unclear. In this study, we attempt to outline these scenario and mechanisms by performing biochemical, lipidomic, and transcriptomic analyses in  Microalfixation . As the fixation , researcfixation . Nitrogefixation . HoweverScenedesmus sp., TAG dramatically increased with the decrease in monogalactosyldiacylglycerol (MGDG), phosphatidylglycerol (PG), diacylglycerol-N,N,N-trimethylhomoserine (DGTs), and phosphatidylethanolamine (PE), while digalactosyldiacylglycerol (DGDG) and lyso-DGTS first increased and then decreased , DGDG (18:3/16:4), SQDG (18:3/16:0) and highly unsaturated phosphatidylcholine (PC), DGTs, and PG mainly contributed  is accompanied by reduced phospholipids and glycolipids after nitrogen starvation. In ecreased . With reabundans . After 4ecreased . Polar ltributed . HoweverAuxenochlorella pyrenoidosa  pathway was first examined. Indeed, the increased 18:1 content was closely related to upregulated Δ9 fatty acid desaturase in nitrogen-depleted enoidosa . Similarabundans . In addiabundans , fatty aabundans , lysophoabundans , and diaabundans  also contabolism , whose ryl donor . The mutnd C18:3 . TherefoNannochloropsis oceanica  with 600 ml working volume of modified BG-11 medium at 25 ± 1°C and 6,000 lx. BG-11 medium consisted of (per liter) 1.5 g NaNO3, 0.04 g K2HPO4·3H2O, 0.075 g MgSO4·7H2O, 0.036 g CaCl2·2H2O, 0.006 g citric acid, 0.006 g ferric ammonium citrate, 0.001 g EDTA·2Na, 0.02 g Na2CO3, and 1 ml micronutrient solution. The micronutrient solution consisted of (per liter) 2.86 g H3BO3, 1.81 g MnCl2·4H2O, 0.222 g ZnSO4·7H2O, 0.39 g NaMoO4·2H2O, 0.079 g CuSO4·5H2O, and 0.0494 g Co(NO3)2·6H2O. Nitrogen repletion (1.5 g L−1 nitrate) was set as control, while the initial nitrate concentration was 0 g L−1 for nitrogen deprivation.C. pyrenoidosa cells with modifications to our previous method  was suspended in 4 ml of 6 M HCl and 6 ml of distilled water and boiled for 30 min until the carbohydrate was hydrolyzed into monosaccharide, which was detected by an iodine solution. The samples were then determined using the 3.5-dinitrosalicylic acid colorimetric method, according to the manufacturer’s instructions .The colorimetric approach was used to determine the protein concentration. Freeze-dried algae power (0.02 g) was suspended in 1 ml of buffer containing 2% (w/v) sodium dodecyl sulfate (SDS), 10% (v/v) glycerol, 50 mM Tris–HCl (pH 8), and 1 mM dithiothreitol (DTT). The samples were homogenized and centrifuged at 12,000 rpm for 20 min. The BCA kit  was used to determine the protein level of the supernatant fraction.−1, maintained for 4 min, finally reached 300°C with a temperature gradient of 30°C min−1, and maintained for 2 min. Helium as carrier gas was set to a column flowrate of 1 ml min−1. The mass spectrometry was performed in positive electron ionization mode at 70 eV, obtaining full scan spectra with an m/z range of 45−550. The FAME components were identified by matching their electron ionization mass spectra with those from the NIST 17 MS library. For quantification, nonadecanoic acid was added as an internal standard.After acidic transesterification of lipid for 2 h , the comC. pyrenoidosa cells under nitrogen repletion and deprivation on days 2, 6, and 12. Isopropanol:methanol  was used to redissolve lipids to a final concentration of 1 mg ml−1. Lipid substances were separated by a Vanquish UHPLC system equipped with an ACQUITY UPLC BEH C18 column , and mass spectrometry was performed on a Thermo Scientific Q Exactive hybrid quadrupole-Orbitrap mass spectrometer with an electrospray ionization source, using a previously reported method  was used for statistical analyses and lipidomic data mining (www.lipidontology.com) was used for ontology enrichment analyses , the data were extracted as raw files. LipidSearch 4.1  was used to perform identification, alignment, and quantification. By comparing the fragmentation ions, retention time, and high-resolution mass using the LipidSearch Database, the lipids were identified, which revealed the fatty acyl groups (FAs) on lipids. The sample names  and response areas were exported for further analyses. LINT-web  adjustment. Differentially expressed genes (DEGs) were identified according to |log2FC| ≥ 1 and p-adjust < 0.05. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/) was used to annotate and analyze the metabolic pathways.Total RNA was isolated from microalgae cells with Plant RNA Purification Reagent  according to the manufacturer’s instructions after 2 days of cultivation. The quality of the RNA was then evaluated using a 2100 Bioanalyzer  and quantified by the NanoDrop ND-2000 spectrophotometer . RNA sequencing and data analysis were performed by Majorbio (Shanghai Majorbio BioPharm Technology). Relevant RNA sequencing raw data were deposited in the GenBank repository under accession number PRJNA855982. The −ΔΔCT method (The FastKing RT Kit (With gDNase)  was used to synthesize cDNA. All PCR primers used in this study are listed in T method .Yarrowia lipolytica CLIB122 (Δura3 Δhis Δleu2) and plasmid pINA1313 were kindly provided by Professor Hairong Cheng (Shanghai Jiao Tong University). SBP1 and SBP2 were PCR amplified using the primers homo-SBP1-F/R and homo-SBP2-F/R listed in Y. lipolytica CLIB122 according to previously published methods . Statistical analysis was performed using GraphPad Prism 9 . The C. pyrenoidosa stopped growing on the second day, and the dry cell weight largely remained stable, eventually reaching 0.27 g L−1. This was only 23.48% of that under nitrogen repletion -based lipidomics. Principal component analysis (PCA) was employed to perform dimensionality reduction analyses on the dataset. The first two components explained 77.5% of the variance , and lysophosphatidylglycerol (LPG). The PC species, namely, 18:1/18:2_16:0 (prokaryotic pathway), 18:1/18:2/18:3_18:0, and 18:1_18:1/18:2/18:3 (eukaryotic pathway), increased significantly, while other species decreased on the second day , could remove C18:1 and C18:3, respectively, from MGDG . Under nCollectively, these results illustrate changes in lipid species with different fatty acid chain lengths and desaturation, supporting the crucial role of lipid turnover in TAG accumulation and variation of fatty acid desaturation.Most transcriptomic analyses in nitrogen-deprived microalgae focus on triacylglycerol synthesis and regard fatty acid desaturase as the main contributor to fatty acid desaturation . In thisTo validate the expression level, 13 differentially expressed genes were tested by RT-qPCR, of which 12 genes (92.86%) were differentially expressed consistently with transcriptome sequencing results , nitrate reductase (NR), and glutamine synthetase (GS), were upregulated , the key enzyme in the starch synthesis pathway, were upregulated, while α-amylase (α-AMY) and β-amylase (β-AMY) related to its hydrolysis were significantly downregulated Figure 4Most genes involved in fatty acid synthesis, including the key enzyme acetyl-CoA carboxylase (ACC), had no significant change, suggesting constant activity of the fatty acid synthesis pathway Figure 4Many genes involved in glycolysis were significantly downregulated. Of these, pyruvate kinase (PK), the rate-limiting enzyme for glycolysis, was also downregulated. Glycolysis converts sugar to pyruvate, which subsequently converts to acetyl-CoA as the substrate of fatty acid biosynthesis . AttenuaAlthough the glycerol kinase (GK), lysophospholipids acid acyltransferase (LPAAT), and phosphatidic acid phosphatase (PP) were all downregulated, key enzymes in triacylglycerol synthesis, diacylglycerol acyltransferase (DGAT), and major lipid droplet protein (MLDP) were upregulated Figure 4C. pyrenoidosa, including 16 SBPs, 14 MYBs, 9 ZFs, 8 RWP-RKs, 3 bZIPs, 2 AP2s, and 8 other transcription factors  can be found below: BioProject number PRJNA855982.RW and XM conceived the study. RW performed the experiments and data analyses. RW wrote the manuscript with contributions from XM who supervised the project. Funding acquisition, XM. All authors contributed to the article and approved the submitted version.This research was sponsored by the National Key R&D Program of China (2021YFA0909500). It was also supported by the Natural Science Foundation of Shanghai (No. 20ZR1426600).We thank Hairong Cheng (Shanghai Jiao Tong University) for providing yeast strain and plasmid and Dr. Feng Lei and Xin Li  for their excellent technical help.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.


Salmonella, polyamine levels and Mg2+ levels are coordinately regulated and that this regulation is critical for viability under both low and high concentrations of polyamines. Upon Mg2+ starvation, polyamine synthesis is induced, as is the production of the high-affinity Mg2+ transporters MgtA and MgtB. Either polyamine synthesis or Mg2+ transport is required to maintain viability. Mutants lacking the polyamine exporter PaeA, the expression of which is induced by PhoPQ in response to low Mg2+, lose viability in the stationary phase. This lethality is suppressed by blocking either polyamine synthesis or Mg2+ transport, suggesting that once Mg2+ levels are reestablished, the excess polyamines must be excreted. Thus, it is the relative levels of both Mg2+ and polyamines that are regulated to maintain viability. Indeed, sensitivity to high concentrations of polyamines is proportional to the Mg2+ levels in the medium. These results are recapitulated during infection. Polyamine synthesis mutants are attenuated in a mouse model of systemic infection, as are strains lacking the MgtB Mg2+ transporter. The loss of MgtB in the synthesis mutant background confers a synthetic phenotype, confirming that Mg2+ and polyamines are required for the same process(es). Mutants lacking PaeA are also attenuated, but deleting paeA has no phenotype in a polyamine synthesis mutant background. These data support the idea that the cell coordinately controls both the polyamine and Mg2+ concentrations to maintain overall cation homeostasis, which is critical for survival in the macrophage phagosome.Polyamines are organic cations that are important in all domains of life. Here, we show that in  Salmonella enterica serovars infect over 1 million people per year in the United States  is expressed under most of the conditions that were tested, but is also significantly induced in macrophages. We used low pH (pH 5.6) and Mg2+ starvation to mimic the conditions that Salmonella encounters in macrophages to see whether PhoPQ, which controls the expression of numerous genes in the phagosome, has any role in the regulation of paeA expression.We previously showed that PaeA is responsible for cadaverine and putrescine efflux from the cell under certain conditions . To bettmic data  suggestspaeA-lac transcriptional fusion (2+ replete (10 mM Mg2+) or Mg2+ deficient (10 μM Mg2+). As shown in paeA expression was slightly induced in Mg2+-limited conditions, and this induction was dependent on PhoPQ. Expression was further induced in the phoQ24 constitutive mutant background, independent of Mg2+ availability. These results show that paeA expression is controlled by the PhoPQ two-component regulatory system in response to host-like conditions. When the same in-locus paeA-lac fusion strain was used to infect BALB/c mice, the expression of paeA in bacteria isolated from infected mouse spleens was increased by 20-fold, compared to bacteria grown in LB medium  and the OD600 over a period of 48 h. As shown in paeA had no effect on the initial growth under Mg2+-sufficient or Mg2+-deficient conditions, although the maximum number of CFU was reduced under Mg2+-limited conditions. However, in the stationary phase, the ΔpaeA strain lost viability under Mg2+-deficient conditions, with the CFU being reduced by ~3 logs at 48 h. The OD600 was unaffected by the deletion of paeA, showing that the cells were not lysing under these conditions (The nditions .10.1128/mbio.02698-22.1FIG S12+ starvation conditions. (A) OD600 measurements for 600 is presented as the mean ± SD, n = 3. Unpaired t test  versus the parent strain that received the same treatment. (B) Survival of strains containing pWKS30-paeA or a vector plasmid after growth in Mg2+-replete or Mg2+-deficient conditions. CFUs were determined at 7.5 and 24 h. CFUs are presented as the mean ± SD, n = 3. Unpaired t test  versus Δdeg at the same Mg2+ concentration. (C and D) The wild-type, Δ7 (ΔcadB ΔpotE ΔSTM14_3648 ΔspeDE ΔpatA ΔpatD ΔcsiD-R ΔpaeA ΔmgtJI), and indicated Δ6 strains when grown in (C) neutral N-minimal medium (pH 7.4) or (D) acidic N-minimal medium (pH 5.5) with or without 10 mM MgCl2. CFUs are presented as the mean ± SD, n = 6. Unpaired t test  versus the Δ7 strain that received the same treatment or (lines) versus the wild-type that received the same treatment. Strains used: 14028, JS2430, JS2464, JS2465, JS2560, JS2561, JS2566, JS2567, JS2568, JS2569, JS2570, JS2571, JS2572, JS2573, JS2574, JS2575, JS2576, and JS2577. Download FIG S1, TIF file, 2.9 MB.PaeA is required for survival under MgCopyright © 2022 Iwadate et al.2022Iwadate et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the paeA phenotype. To test this hypothesis, we created a degradation deficient strain (Δdeg) that lacks all of the genes involved in cadaverine and putrescine degradation  into the ΔpaeA strain complemented the survival defect under Mg2+-deficient conditions (2+ starvation.PaeA is a putative cadaverine and putrescine efflux pump . Thereforadation . As shownditions . These r2+ starvation should depend on cellular polyamine synthesis. We constructed polyamine synthesis-deficient strains (Δsynth) that lacked all of the known genes for polyamine synthesis  as well as strains that have only one of the pathways intact (the Δ6 strains). We determined the viability of each strain under Mg2+-replete and Mg2+-deficient conditions. As shown in paeA+) strain maintained viability upon Mg2+ starvation, whereas the Δ7 strain and the other Δ6 strains that lack paeA lost viability. These results show that PaeA is the most important polyamine clearance system for survival under these Mg2+-deficient conditions.There are seven known or putative polyamine clearance pathways : the cad2+ but also acidic. Therefore, we investigated whether PaeA is important for survival in pH 5.5 media with or without Mg2+. As shown in paeA+) strain but also the Δ6 (cadB+) strain maintained viability in Mg2+-deficient conditions. First, these results show that the PaeA-dependent efflux of cadaverine and/or putrescine is important for survival under Mg2+-limited conditions in both acidic and neutral environments. Second, these results are consistent with the CadAB system being induced in low pH, leading to the production of cadaverine from lysine by CadA and export by the lysine:cadaverine antiporter CadB as a mechanism to maintain the cytoplasmic pH  versus the parent vector strain that received the same treatment. Strains used: JS2452, JS2584, JS2585, JS2586, JS2587, JS2588, JS2589, JS2590, JS2591, and JS2592. Download FIG S2, TIF file, 2.2 MB.Putrescine and spermidine synthesis is critical for growth and survival during MgCopyright © 2022 Iwadate et al.2022Iwadate et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Salmonella has three Mg transporters: CorA, MgtA, and MgtB. Under Mg2+-replete conditions, Mg2+ is transported primarily through CorA. However, CorA is known to be inactivated once cytoplasmic Mg2+ levels drop  complemented the survival defect under Mg2+ deficient conditions in the ΔmgtA ΔmgtB Δsynth strain .els drop . Under th strain . These r2+ starvation  or in medium with 0.01 mM of added MgCl2, both the wild-type and ΔpaeA strains showed higher tolerances to cadaverine, putrescine, and spermidine than did cells grown with 0.1 mM or higher concentrations of added MgCl2. Sensitivity to cadaverine and putrescine was greatly enhanced in the absence of PaeA, whereas sensitivity to spermidine was unchanged, as we previously reported  under Mg2+-limited conditions at 7.5 h. In contrast, the strain that synthesized only cadaverine showed similar CFU to those of the ΔmgtA ΔmgtB Δsynth strain. These results suggest that putrescine and spermidine are the most important polyamines for survival under Mg2+ starvation conditions.To clarify which polyamine is most important for growth and survival under Mgermidine . As showSalmonella has three independent putrescine synthesis pathways: SpeAB, SpeC, and SpeF . The initial intracellular concentration of Mg2+ in the wild-type cells decreased from approximately 11 mM to approximately 5 mM over 9 h, whereas the total polyamine concentration dropped from approximately 27 mM to 10 mM at 18 h. Note that the cells continued to divide, explaining some of the decrease in cellular concentrations. As expected, the loss of PaeA resulted in higher intracellular levels of polyamines, compared to the wild-type, over the course of the experiment, peaking at 3 h after the media shift, whereas the Mg2+ concentrations were similar to those of the wild-type. Note that the paeA mutant was likely starting to die after 12 h  in BALB/c mice. As shown in paeA mutant was approximately 2.3-fold attenuated, relative to the isogenic wild-type strain, and this difference was statistically significant. BALB/c mice are susceptible to Salmonella infections due to a mutation in Nramp1 (Slc11a1) , only putrescine (PUT+), or putrescine and spermidine (PUT+SPD+), with each competing against the isogenic wild-type strain. As shown in + and PUT+SPD+ strains competed evenly with the wild-type strain. The CAD+ strain showed decreased competitive fitness, which is consistent with published results  , a divalSlc11a1) . Recentllability , to test results , but thi2+ transport. As shown in mgtA ΔmgtB strain was dramatically attenuated, relative to the wild-type, and this finding is consistent with previous reports  concentration could trigger a lethal event from which the cell cannot recover. More work is required to understand the lethality caused by either low or high polyamine and Mg2+ concentrations.While the absence of polyamines leads to immediate lethality under low Mgry phase . Our pre2+ conditions and in the animal, despite their difference in charge. The cell has transporters for these compounds, and the addition of either suppresses lethality under low Mg2+ conditions, even in a strain incapable of converting putrescine to spermidine or modifying spermidine  is specifically coordinated and is required for enzymatic function. Likewise, there might be functions that specifically require one of the polyamines. However, polyamines seem to primarily play the roles of simple cations that are capable of replacing Mg2+ (hydrated) in fundamental cell processes. It is likely that this general concept holds true in all cells.In summary, we show that the cell coordinately regulates polyamine and MgSalmonella strains used are derivatives of Salmonella enterica serovar Typhimurium strain 14028. Deletions with concomitant insertions of antibiotic resistance cassettes were constructed using λ Red-mediated recombination as previously described  paeA promoter only, and (iii) a region that includes the msrA promoter, ORF, and paeA promoter  msrpromoter . The pri10.1128/mbio.02698-22.6TABLE S3Table S3, DOCX file, 0.04 MB.Primer list. Download Copyright © 2022 Iwadate et al.2022Iwadate et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 4)2SO4, 0.5 mM K2SO4, 1 mM KH2PO4, 0.1 M Tris, and 0.1% casamino acids) was supplemented with glycerol and MgCl2 as indicated  medium containing 1% NaCl. Strains containing the temperature-sensitive plasmids pCP20 and pKD46 were grown at 30°C. Antibiotics were used as follows: ampicillin, 50 μg/mL; chloramphenicol, 10 μg/mL; kanamycin, 50 μg/mL; tetracycline, 10 μg/mL; and apramycin, 50 μg/mL. The N-minimal medium  × 106/(OD600 × mL of cell suspension) and are presented as the mean ± standard deviation with n = 3.β-galactosidase assays were performed using a microtiter plate assay as previously described . Bacteri4 cells of the in-locus paeA-lac fusion strain that were grown in LB medium for 16 h. After 4 days of infection, the mice were sacrificed, and their spleens were homogenized. Bacterial cells were extracted from the splenic tissue using a protocol based on (2O. 40 U of DNase I were added and incubated for 20 min at room temperature (RT) with periodic mixing. The samples were centrifuged for 2 min, and the supernatants were discarded. The pellets were washed twice with sterile Z-buffer (in vitro in LB medium (16 h of growth) was measured using the same assay and was calculated per CFU of bacteria in the sample.BALB/c mice were infected intraperitoneally with 10based on . The splZ-buffer  and were2, were diluted 200-fold into the same medium and grown for 4 h. The cells were then washed with 0.85% NaCl three times, diluted to an OD600 value of either 0.005 or 0.05 into 10 mL of N-minimal medium (pH 7.4 or 5.5) with 15 mM glycerol, with or without 10 mM MgCl2, in a 125 mL baffled flask, and incubated at 37°C. Before and after incubation, serial dilutions of the cultures were plated on LB agar plates supplemented with 0.1% glucose and were incubated overnight at 37°C to determine CFU. For the growth curves, the OD600 of 250 μL of culture was measured in a BioTek ELx808 Absorbance Reader at the indicated time points. When necessary, the cultures were diluted in the original growth medium to accurately measure the OD600 value.Overnight cultures, grown in N-minimal medium (pH 7.4) supplemented with 15 mM glycerol and 10 mM MgCl2+ stress, we started cultures using a 10-fold higher cell density than those used in the experiments described above. First, we confirmed that the increased inoculum did not affect the phenotypes of the various mutant strains under Mg2+ starvation conditions (g) at the indicated time points and washed twice with 0.85% NaCl. The supernatant was removed. The cell pellet was frozen at −80°C and then suspended in 1 mL of methanol and disrupted via sonication. The samples were cleared via centrifugation, and the concentration of polyamines in the solvent was determined via liquid chromatography-mass spectrometry (LC-MS) at the Metabolomics Center, Roy J. Carver Biotechnology Center, University of Illinois at Urbana-Champaign. The concentration is reported as nmoles per (mL × OD600) and is converted to an intracellular concentration assuming 7.97 × 108 CFU per 1 ml of OD600 = 1 culture  and 2.3 × 10−15 L per cell  versus the paeA+ wild-type background + Mg2+ or  versus the paeA+ wild-type background minus Mg2+. Strains used: 14028, JS2430, JS2560, JS2561, JS2562, JS2598, JS2563, and JS2600. Download FIG S3, TIF file, 2.1 MB.The survival phenotypes of various mutants under MgCopyright © 2022 Iwadate et al.2022Iwadate et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the g) and washed twice with 0.85% NaCl. The cell pellets were dried at room temperature and were sent to the University of Georgia Center for Applied Isotope Studies for the determination of the total Mg2+ content. The concentration is reported as μg per g of dry weight and is converted to an intracellular concentration assuming 2.8 × 10−13 g per cell  with the indicated amount of MgCl2, and incubated for 4 h at 37°C. Then, the cells were washed three times, diluted to an OD600 value of 0.05 in 1 mL of 0.85% NaCl with HEPES buffer (pH 8.5) with the indicated amount of cadaverine, putrescine, or spermidine, and incubated at 37°C in 13 × 100 mm test tubes. At 0 and 24 h, serial dilutions of the cultures were plated on LB agar plates supplemented with 0.1% glucose, and they were incubated overnight at 37°C to determine the CFU.Polyamine stocks were prepared at 50 mM in 0.85% NaCl with HEPES buffer (pH 8.5). Cultures that were grown overnight in N-minimal medium (pH 7.4) supplemented with 15 mM glycerol and 10 mM MgClSalmonella strains were grown overnight in LB medium, mixed 1:1, and diluted to a target inoculum of approximately 1,000 CFU in 200 μL sterile PBS. For the Δsynth background strains, we used an inoculum of approximately 10,000 CFU. The mice were infected via the intraperitoneal route. Each inoculum was plated on LB medium to measure the total inoculum and was replica plated to the appropriate selective medium in order to calculate the input ratio for each strain. After 4 days of infection for the BALB/cAnNHsd mice or 5 days of infection for the C3H/HeNHsd mice, the animals were sacrificed via CO2 asphyxiation, and their spleens were removed and homogenized. Serial dilutions of the spleen homogenates were plated on LB medium and were replica plated to the appropriate selective medium in order to calculate the output ratio for each competition. The competitive index (CI) was calculated as (percent strain A recovered/percent strain B recovered)/(percent strain A inoculated/percent strain B inoculated). The statistical comparisons of individual competitions were done using Student’s t tests.All of the animal work was reviewed and approved by the University of Illinois IACUC and was performed under the protocol number 21197. The competition experiments were performed using 5 to 6-week-old mice. The BALB/cAnNHsd and C3H/HeNHsd mice were purchased from Envigo. The


Chronic glomerulonephritis (CGN) is one of the major causes of end-stage kidney disease. Zhen-wu-tang (ZWT), as a famous Chinese herbal prescription, is widely used in China for CGN therapy in clinic. However, the mechanism of ZWT in CGN has not been fully understood. The present study explored the therapeutic effect and the underlying mechanism of ZWT on mitochondrial function in cationic bovine serum albumin (C-BSA)-induced CGN model rats and tumor necrosis factor (TNF-α)-damaged mouse podocytes. The renal functions were measured by serum creatinine (Scr) and blood urea nitrogen (BUN). Renal pathological changes and ultrastructure of kidney tissues were evaluated by periodic acid-Schiff (PAS) staining and transmission electron microscopy. The levels of antioxidases, including mitochondrial catalase (CAT), superoxide dismutase 2 (SOD2), and peroxiredoxin 3 (PRDX3), in CGN rats were examined by real-time PCR. The mitochondrial functions of podocytes were measured by ATP concentration, mitochondrial membrane potential (MMP), and mitochondrial ROS (mtROS). For mitophagy level detection, the expressions of mitophagy-related proteins, including LC3, p62, heat shock protein 60 (HSP60), and translocase of outer mitochondrial membrane 20 (TOMM20), were measured by Western blot, as the colocation of LC3 and mitochondrial marker COX IV were evaluated by immunofluorescence. Our results manifested that ZWT ameliorated CGN model rats by a remarkable decrease in Scr and BUN, inhibition of mesangial matrix proliferation, protection against foot processes fusion, and basement membrane thickening. More importantly, ZWT protected against mitochondrial dysfunction by increasing the expressions of CAT, SOD2, and PRDX3 in CGN model rats, increased ATP content and MMP in podocytes, and decreased excessive mtROS. Furthermore, ZWT induced mitophagy in CGN through increasing the expression of LC3, and decreasing p62, HSP60, TOMM20, and ZWT also enhanced the colocation of LC3 to the mitochondria. We found that ZWT inhibited the PI3K/AKT/mTOR pathway, which could be disturbed by PI3K inhibitor LY294002 and agonist insulin-like growth factor 1. Moreover, ZWT reversed the inhibition of the AMPK pathway in CGN. Overall, ZWT ameliorated renal mitochondrial dysfunction probably by inducing mitophagy via the PI3K/AKT/mTOR and AMPK pathways. In 2017, 1.2 million people died from chronic kidney disease (CKD), and the global all-age mortality rate of CKD increased by 41.5% within the last 28 years . ChronicTreatise on Febrile Diseases, is composed of five traditional Chinese medicines including Aconitum carmichaelii Debeaux, Poria cocos (Schw.) Wolf, Atractylodes macrocephala Koidz, Paeonia lactiflora Pall, and Zingiber officinale Roscoe (Zhen-wu-tang (ZWT), recorded in the e Roscoe . Notablye Roscoe . HoweverMitochondrial damage and dysfunction participate in the pathogenesis of kidney diseases, which not only damage the renal cells but also affect the infiltrating inflammatory cells in the kidneys . Under tRecently, growing evidence has demonstrated that the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway plays an indispensable role in renal diseases to regulate autophagy . PI3K isAconitum carmichaelii Debeaux (Fuzi), Poria cocos (Schw.) Wolf (Fu ling), Atractylodes macrocephala Koidz (Bai zhu), Paeonia lactiflora Pall (Bai shao), and Zingiber officinale Roscoe (Sheng jiang) was purchased from Guangzhou Caizhilin Pharmaceutical Co., Ltd. , which constitutes ZWT in the ratio of 3:3:3:2:3. According to clinical usage, the daily intragastric doses were 4.2 kg/kg, 8.4 g/kg, 16.8 g/kg for low, middle, and high doses of ZWT , respectively. The water extract of ZWT was prepared as described previously , and blood urea nitrogen (BUN) were measured according to the instruction of the manufacturer .Kidney tissues of each group were fixed in 4% paraformaldehyde for 48 h, embedded in paraffin, and then were cut into 5-μm-thick sections. Afterward, sections were deparaffinized by xylene, hydrated by gradient ethanol, and then stained with periodic acid-Schiff (PAS). The sections were observed under a light microscopy .3 were fixed in 2.5% glutaraldehyde for 24 h and 1% leleonic acid for 2 h. Next, the samples were dehydrated with a series of ethanol and acetone, immersed with acetone and propylene oxide. Each sample was embedded and then sliced with ultramicrotome. Subsequently, the sections were dyed with uranium acetate and lead citrate, and the ultrastructure of the podocytes and the number of mitochondrial autophagosomes in the CGN model rats were observed under a Hitachi transmission electron microscope (TEM) . The foot process width and glomerular basement membrane thickness were calculated by using the ImageJ 1.48 software.Fresh kidney tissues in 1 mm−ΔΔCt method. All the primers were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd. The primer sequences used were as follows: CAT, 5′-CTGACTGACGCGATTGCCTA-3′ and 3′-GTGGTCAGGACATCGGGTTT-5′; SOD2, 5′-CACCGAGGAGAAGTACCACG-3′ and 3′-TGGGTTCTCCACCACCCTTA-5′; PRDX3, 5′-AGTGTGGAAGAACCACTCCG-3′ and 5′-TGGCTTGATCGTAGGGGACT-3′; GAPDH, 5′-ACAGCAACAGGGTGGTGGAC-3′ and 3′-TTTGAGGGTGCAGCGAACTT-5′.The total RNA was isolated from renal cortex homogenate using RNAiso Plus  under the instructions of the manufacturer, and then, cDNA was synthesized by Prime Script RT Reagent Kit with gDNA Eraser . Quantitative analysis of mitochondrial catalase (CAT), superoxide dismutase 2 (SOD2), and peroxiredoxin 3 (PRDX3) mRNAs were measured using the CFX96 Bio-Rad real-time PCR instrument  with TB Green Premix Ex Taq Kit . The mRNA expression levels were normalized to GAPDH through the 22. Proliferated podocytes were transferred to 37°C in IFN-γ-free medium for 14 days. The subsequent experiment was conducted with well-differentiated podocytes. ZWT-containing serum and normal serum from healthy male SD rats were prepared as in a previous study  at 33°C and 5% COus study . The podus study  Control us study  TNF-α grus study  2.5% ZWTus study  5% ZWT gus study  10% ZWT g, 4°C for 5 min. The ATP standard substance was diluted into 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 μM. Next, 100 μl of ATP working solution was added into a 96-well plate, and then 20 μl of podocyte lysis or standard substance was added, respectively. After incubation in the dark for 5 min, the luminescence intensity of each sample was measured by a Tecan M1000 Pro plate reader . The ATP levels were calculated and expressed as μM in reference to the corresponding standard curves.Intracellular ATP content was detected using an ATP assay kit . First, podocytes treated above were lysed, and the supernatant was obtained after being centrifuged at 12,000 × Mitochondrial membrane potential (MMP) was assessed using a JC-1 staining kit  in accordance with the instructions of the manufacturer. Briefly, podocytes were washed with phosphate buffer saline (PBS) three times and incubated with JC-1 dyeing solution for 20 min at 37°C in the dark. Subsequently, the cells were rinsed with PBS thrice and then observed by a confocal laser microscope  at ×400 magnification. The green fluorescence (Ex/Em = 514/529 nm) was used for monomer detection and the red fluorescence (Ex/Em = 585/590 nm) for aggregates. Five randomly chosen fields were photographed and calculated with the ratio of aggregates/monomers for fluorescence intensity analysis by using ImageJ 1.48 software.The mitoSOX red fluorophore dye  was used for the determination of mtROS accumulation. Upon treatment with TNF-α or ZWT-containing serum, podocytes were ringed with Hank’s balanced salt solution (HBSS) thrice and then incubated with 2.5 μM mitoSOX working solution at 37°C for 10 min with light protection. After being washed with HBSS three times, podocytes were observed by a confocal laser microscope  at ×400 magnification. Five randomly chosen fields were photographed, and the fluorescence intensity of mitoSOX was analyzed by using the ImageJ 1.48 software.Renal frozen sections, 4-μm thick, and cultured podocytes were fixed with paraformaldehyde at 4°C for 10 min, and then washed with PBS three times. Then samples were permeabilized with 0.5% Triton X-100 for 10 min, and blocked with normal goat serum at 37°C for 30 min. Next, all samples were incubated with LC3B  together with COX IV (Abcam) at 4°C overnight. After being washed with PBS, the sections or cells were incubated with goat-anti rabbit AF555 and goat-anti mouse AF488 antibodies purchased from Cell Signaling Technology  for 30 min at 37°C in the dark. Samples were added with DAPI (5 μg/ml) for 5 min at room temperature. The sections and cells were washed with PBS three times and then observed under a confocal laser microscope  at ×400 magnification. Five randomly chosen fields were photographed and calculated with the area ratio of colocalized LC3 to COX IV in glomerulus analyzed by using the ImageJ 1.48 software.The protein extracts of renal cortex and podocytes were prepared in RIPA lysis buffer containing phosphatase inhibitors and protease . Equal amounts of 30 μg of proteins were separated on 10% or 15% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) and transferred to polyvinylidene difluoride membranes . After that, the membranes were blocked with 5% bovine serum albumin (BSA) for 2 h at room temperature. Subsequently, membranes were incubated with corresponding primary antibodies at 4°C overnight as follows: LC3B (ab192890), p62 (ab56416) obtained from Abcam , HSP60 (#12165S), TOMM20 (#42406S), PI3K (#4257S), p-AKT (#4060S), AKT (#4691S), p-AMPKα (#2535S), AMPKα (#2532), p-mTOR (#2971S) and mTOR (#2983S) purchased from Cell Signaling Technology , GAPDH (D2817) from Santa Cruz . After being washed thrice with Tris-buffered saline Tween-20 (TBST), the membranes were incubated with secondary antibody conjugated with horseradish peroxidase purchased from Jackson Immuno Research  at room temperature for 1 h. Finally, these membranes were washed again with TBST and detected using an enhanced chemiluminescence kit . Quantification of the protein bands were analyzed using Image J 1.48 software.p < 0.05, the differences were defined as statistically significant.All data were analyzed using SPSS 20.0  and expressed as mean ± standard deviation (SD). The differences between groups were analyzed by one-way ANOVA. When For quality assessment, the main chemical components of ZWT were identified with UPLC-Q-TOF-MS. As shown in Serum creatinine (Scr) and blood urea nitrogen (BUN) are important indictors of renal function. As expected, in comparison with the model group, the levels of Scr and BUN were significantly decreased after ZWT treatment . Next, tMitochondrial dysfunction is involved in the pathogenesis of kidney diseases . As antiMitophagy is an important quality control mechanism for cell homeostasis by eliminating damaged mitochondria. To evaluate the regulatory effects of ZWT on mitophagy in CGN model rats, the expressions of autophagy-related proteins LC3 and p62, and mitochondrial proteins TOMM20 and HSP60, were detected. As shown in in vitro, mouse podocytes were simulated with TNF-α and treated with different proportions of ZWT-containing serum. Mitochondrial membrane potential (MMP) was detected by JC-1 probe. The findings showed that the ratio of aggregates/monomers increased under ZWT treatment, suggesting that the MMP was returned to normal /AKT/mTOR pathway has inhibitory roles on the autophagic pathway. Recently, some studies suggested that the PI3K/AKT/mTOR pathway is also involved in mitophagy . FurtherPI3K inhibitor LY294002 and agonist insulin-like growth factor-1 (IGF-1) were used alone or with ZWT to intervene in podocytes in this study. As shown in In the current study, we investigated the protective effects and regulatory mechanisms of ZWT in CGN. We found that ZWT protected kidney injury in CGN model rats. Furthermore, ZWT ameliorated mitochondrial dysfunction in renal tissue of CGN model rats and damaged podocytes induced by TNF-α. The mechanisms of ZWT above is related to inducing mitophagy through the PI3K/AKT/mTOR and AMPK pathways.Mitochondria are classically described as energy-producing organelles through generation of adenosine triphosphate (ATP). Mitochondria also plays a critical role in oxidative phosphorylation, fatty acid oxidation, and amino acid catabolism . MitochoAtractylodes macrocephala Koidz, atractylenolide III ameliorated cerebral ischemic injury by inhibiting mitochondrial fission dependent on the JAK2/STAT3/Drp1 pathway /AKT/mammalian target of rapamycin (mTOR) signaling pathway plays an important role in the regulation of cell survival, growth, and proliferation. mTORC1 is considered a negative regulator of autophagy because suppression of mTORC1 activates autophagy . DownregThe AMP-activated protein kinase (AMPK) is a master regulator of metabolism, which is regulated by a wide array of metabolic stresses. Recent studies revealed that AMPK are involved in various aspects of mitochondrial homeostasis, such as mitophagy . In the In conclusion, our present study provided evidence that ZWT protects against kidney injury and podocyte injury in CGN model rats by ameliorating mitochondrial function. Furthermore, the above protective mechanisms of ZWT are irrelevant to the induction mitophagy via the PI3K/AKT/mTOR and AMPK pathways.


T/T  were significantly higher in PCa patients when compared to control group and considerably increased the risk of disease by 2.29, 1.44, and 2.13 folds respectively. Interestingly, the results demonstrated that PCa patients with the dominant model (T/G + G/G vs. T/T) of VDBP had significantly lower serum levels of vitamin D and higher serum levels of total and free PSA in comparison to the controls. Furthermore, when compared to controls, PCa patients with the dominant model T allele (T/G + G/G vs. TT) of VDBP had significantly higher vitamin D, total PSA, and free PSA concentrations. Serum levels of 25(OH)-vitamin D and rs7041 T/G polymorphism of the VDBP gene could be potential risk factors for PCa.Prostate cancer (PCa) pathology has been linked to vitamin D, vitamin D receptors (VDRs), and vitamin D binding proteins (VDBPs). We sought to investigate the association between VDR rs2228570 and rs1544410 as well as VDBP rs7041 polymorphisms and serum 25-hydroxyvitamin D (25(OH)-vitamin D) levels in PCa patients. Blood samples were collected from 111 PCa patients and 150 age-matched healthy volunteers. The VDR rs2228570 T/C, rs1544410 G/A, and VDBP rs7041 T/G genotypes were determined using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). 25(OH)-vitamin D and PSA  serum levels were measured. The frequencies of VDBP genotypes T/G vs. T/T (56.5% vs. 44.5%,  Prostate cancer (PCa) is one of the main common causes of cancer-associated deaths in men worldwide . AlthougIn this case-control study, 111 PCa patients with a mean age of 67.7 ± 11.2 years old and 150 healthy controls (66.2 ± 11.3 years old) from the Kermanshah University of Medical Sciences clinic were enrolled. A full biography  was taken from each participant and all were physically examined by a urologist and data regarding age, history of smoking, height, weight, and pretreatment testosterone levels were collected. The clinical characteristics of medical records were documented, including Gleason score, family history, and type of treatment. The levels of prostate specific antigen (PSA) in all participants were measured in order to diagnose the condition. Histopathology confirmation was used to select patients with PCa. Control subjects with PSA levels >2.5 ng/ml and the ratio of free PSA to total PSA ≤0.1 or positive digital rectal examination (DRE) were excluded. Patients having any sort of cancer or a family history of PCa were not allowed to participate in the study. The ethnic background of patients and controls was western Iranian Kurdish.This research was approved by the ethical committee of Kermanshah University of Medical Sciences. We followed the guidelines approved by the Helsinki Research Ethics Committee of the Iranian Authority Ministry of Health. Written-signed consent was obtained from all included individuals.5 ml of peripheral blood samples were collected from all participants and genomic DNA was isolated from blood leukocytes using the phenol-chloroform method. The extracted DNA was kept at −20°C until it could be examined , 30.The VDR genotypes were determined by PCR-RFLP with forward and reverse primers 5′- AGCTGGCCCTGGCACTGACTCTGCTCT -3′and 5′- ATGGAAACACCTTGCTTCTTCTCCCTC -3′with minor modification . In brieThe forward; 5′-CAACCAAGACTACAAGTACCGCGTCAGTGA-3′, and reverse; 5′-AACCAGCGGAAGAGGTCAAGGG-3′, primers were used to determine VDR rs1544410 G/A SNP . ThermocThe forward 5′-AAATAATGAGCAAATGAAAGAAGAC-3′ and reverse 5′-CAATAACAGGAAAGAAATGAGTAGA -3′ primers were used to identify VDBP rs7041 T/G polymorphism . The theTotal PSA, free PSA, and vitamin D concentrations in serum were evaluated using standard ELISA procedures , and the fPSA/tPSA ratio was calculated.χ2 test was used to test the difference in the distribution of the haplotypes in patients and controls. Statistical significance was assumed at p ≤ 0.05. The genotypes and allele frequencies of VDR rs2228570 and rs1544410 and VDBP rs7041 in PCa patients were compared to the control group using the Chi-square (χ2) test in three different genotype models of co-dominant, the dominant/recessive, and the heterozygous. The Odds ratios (OR) and 95% confidence intervals (CI) were obtained by SPSS logistic regression . The t-test, ANOVA, and nonparametric independent sample Mann–Whitney analysis were used to compare the quantitative data.We calculated the allelic frequencies by the gene counting method. Pearson’s 2), p = 0.015]. In addition, the frequency of smoker PCa patients was significantly higher than smoker healthy subjects (58 (52.3%) vs. 41 (27.3%), p < 0.001). PCa patients have significantly lower serum levels of 25(OH)-vitamin D  and FPSA/TPSA ratio  and higher serum levels of free PSA  and total PSA  compared to control group. The odd ratio  and frequencies of genotypes of VDR rs2228570 and rs1544410 and VDBP rs7041 are shown in p = 0.015] and increased the risk of disease by 2.2 folds [2.29 ]. The distribution of T/G + G/G genotypes according to the dominant genetic model of VDBP rs7041 SNP  was significantly higher in PCa patients and increased the risk of disease by 2.13 times [2.13 ]. The influence of the dominant model of VDBP rs7041 SNP (T/G + G/G vs. T/T) genotypes on 25(OH)-vitamin D, total PSA, free PSA, concentration, and free PSA/total PSA ratio between PCa patients and control group is presented in p < 0.001) and T/G + G/G: 73.58 vs. 0.89 ng/L (p < 0.001)] free PSA  compared to control subjects. Interestingly in presence of both T/T and T/G + G/G genotypes, the free PSA/total PSA ratio was considerably higher in the control group when compared to PCa patients . The serum level of 25(OH)-vitamin D (ng/ml) was significantly higher in control subjects with T/G + G/G genotypes than in PCa patients . The PSA serum level  was highly linked with the grade of disease, according to the results shown in p < 0.001). However, depending on the Gleason Score, serum levels of 25(OH)-vitamin D were similar between low and high grades of illness. We found a negative correlation between 25(OH)-vitamin D and aggressive PCa.s -vitamin D and the risk of PCa. Our findings were consistent with Ahonen et al., Atoum et al., and Deschasaux et al., who reported an inverse association between serum levels of 25(OH)-vitamin D and PCa risk among the population from Jordan, Finland, and France respectively –36. Vitap = 0.015] which was associated to increase the risk of disease by 2.2 folds [2.29 ]. Moreover, we report an association between T/T and T/G + G/G genotypes in patients and the concentration of total PSA when compared to control subjects  free PSA . Finally, we reported that in presence of both T/T and T/G + G/G genotypes, the free PSA/total PSA ratio was considerably higher in the control group when compared to PCa patients .In conclusion, the frequencies of T/G genotypes of VDBP rs7041 SNP were significantly higher in PCa patients compared to control subjects [T/G vs. T/T, (61 (56.5%) vs. 61 (44.5%), Finally, from a clinical point of view, the presence of mutations in the VDBP gene in PCa patients, and decrease in the serum level of vitamin D as well as the increase in tPSA and fPSA levels, may increase the risk of PCa onset, therefore, determining the genotypes of these genes in different populations and linking them with the onset of PCa parallel to measuring serum PSA and vitamin D levels. Enriching the diet with vitamin D supplements may reduce the risk of PCa in susceptible individuals.


We evaluated the visual outcomes and complications of “endothelium-out” and “endothelium-in” Descemet membrane endothelial keratoplasty (DMEK) graft insertion techniques.ClinicalTrials.gov. Study designs included clinical trials, comparative observational studies, and large case series (≥25 eyes). PRISMA guidelines were used for abstracting data and synthesis. Random-effects models were employed for meta-analyses.Electronic searches were conducted in CENTRAL, Cochrane databases, PubMed, EMBASE, “endothelium-in” studies  compared to “endothelium-out” studies  at 6 months (p = 0.018). Graft re-bubbling rates were higher in the “endothelium-out” studies  compared to “endothelium-in” studies , although statistical significance was not reached (p = 0.440). Primary graft failure rates were comparable between the two groups (p = 0.552). Quality of evidence was considered low and significant heterogeneity existed amongst the studies.21,323 eyes (95 studies) were included. Eighty-six studies reported on “endothelium-out” techniques; eight studies reported on “endothelium-in” techniques. One study compared “endothelium-out” to “endothelium-in” techniques. Eighteen “endothelium-out” studies reported that 42.5–85% of eyes achieved best-corrected visual acuity (BCVA) ≥20/25 at 6 months; pooled proportion of eyes achieving BCVA ≥20/25 at 6 months was 58.7% . Three “endothelium-in” studies reported that 44.7–87.5% of eyes achieved BCVA of ≥20/25 at 6 months; pooled proportion of eyes achieving BCVA ≥20/25 at 6 months was 62.4% (95% CI 33.9–86.9%). Pooled mean endothelial cell loss was lower in the Reported rates of endothelial cell loss were lower in “endothelium-in” DMEK studies at 6 months compared to “endothelium-out” studies. Outcomes of “endothelium-in” techniques were otherwise comparable to those reported in “endothelium-out” studies. Given the technical challenges encountered in “endothelium-out” procedures, surgeons may consider “endothelium-in” techniques designed for easier intra-operative DMEK graft unfolding. “Endothelium-in” studies evaluating outcomes at longer time points are required before conclusive comparisons between the two techniques can be drawn. Loss of vision from diseases of the corneal endothelium is the predominant indication for corneal transplantations , 2. OverCurrently, there are two predominant techniques of EK performed worldwide: Descemet’s stripping automated endothelial keratoplasty (DSAEK) and Descemet membrane endothelial keratoplasty (DMEK) , 4, 11. Descemet membrane endothelial keratoplasty is the more recent advancement in EK surgery . In DMEKCurrent methods of DMEK graft transfer into the anterior chamber involve inserting the graft through a small clear corneal wound. Different surgical instruments have been described for the insertion of DMEK grafts. Examples of such instruments include glass injectors , 28 and “Endothelium-in” DMEK graft insertion techniques have been described more recently –37 Figu. In thesThis review aims to evaluate the published literature reporting the visual outcomes and complications of both “endothelium-out” and “endothelium-in” graft insertion techniques for DMEK.1. A study protocol for this systematic review is available in This review was submitted to PROSPERO International prospective register of systematic reviews (reference ID: 160657)We included publications in which the visual outcomes and complications of DMEK performed for the treatment of endothelial dysfunction were reported.Study designs included controlled clinical trials, prospective or retrospective comparative observational studies, and large case series (≥25 eyes). Small case series (<25 eyes), letters, reviews, published abstracts, and laboratory-based studies were excluded.Studies reporting only surgical outcomes of DMEK performed for graft failure (including repeat DMEK surgery) or specific high-risk disease groups  were also excluded. To avoid duplicate reporting of similar study populations, where the same group of investigators published several studies, earlier smaller studies were excluded if more recent larger studies reporting the same outcome measures were available.Information sources included all applicable electronic databases, all relevant articles in the reference list of any relevant articles, and all relevant articles which cite any relevant articles.2, PubMed, EMBASE, ClinicalTrials.gov.3 No date or language restrictions were set in our electronic searches. Key search terms were the MeSH headings Descemet’s membrane endothelial keratoplasty, Descemet membrane endothelial keratoplasty, and DMEK. The last electronic database search was performed on 30 June 2021. The search strategies for the relevant databases can be found in Electronic literature searches were conducted in the following databases: CENTRAL, Cochrane Library databasesCitations and abstracts obtained from electronic searches were examined. Replicated studies and evidently irrelevant studies were removed. Full text prints of relevant studies were retrieved; they were assessed against our inclusion criteria for this review.Only data from eyes that had received DMEK surgeries were included. Where studies reported on the outcomes of eyes that had undergone surgeries other than DMEK, these eyes were excluded from the review. The following details of each study were extracted for this review: study participants’ characteristics, study design, DMEK graft insertion techniques, and surgical outcome measures.The study design of each article was assessed and rated according to its level of evidence. A rating scale adapted from the Oxford Centre for Evidence-Based Medicine was used  Table 1Table 1.Cochrane Handbook for Systematic Reviews of Intervention  selection bias – allocation concealment, (c) performance/detection bias – masking of outcome examiners and participants , (d) attrition bias incomplete outcome data, and (e) reporting bias – selective outcome reporting. Each study was graded as “low risk” of bias, “high risk” of bias, or “unclear risk.” Any differences between the authors were resolved by discussion.Studies meeting the inclusion criteria were also assessed for risk of bias using Chapter 8 of the rvention . The folData on the following surgical outcome measures were obtained: visual outcomes, endothelial cell loss, and complications including graft detachment/re-bubbling, graft rejection, and graft failure. For direct comparison of visual outcomes, measures of visual acuities in Snellen were converted to the respective logarithm of the minimum angle of resolution (LogMAR) equivalents. The proportion of eyes that achieved a best-corrected visual acuity (BCVA) of 20/25 or better at a specific time points were also evaluated.All outcome measures  were discrete data, except mean endothelial cell loss where outcome measures were continuous data. Outcomes for eyes rather than individuals were used as the unit of analysis. Studies where both eyes received the same treatment were included.All relevant data were extracted from the published studies. These included the details of studies and their quantitative results, without having to request these data from the original investigators.® Statistical Software version 20.014; MedCalc Software Ltd., Ostend, Belgium; 2021).4Data analyses were performed according to Chapter 9 of the Cochrane Handbook for Systematic Reviews of Interventions . As publ2 test and I2 statistic to quantify heterogeneities across reports. Significant heterogeneity was defined as an I2 statistic of ≥50% and a Chi2 test p-value of <0.1. If all the effects of an outcome measure were in a similar direction, then we considered data-pooling to be acceptable even in the existence of heterogeneities.We identified dissimilarities between published studies which are expected to introduce heterogeneities. As some degree of heterogeneity would always exist due to the diversities in methodologies of studies, where appropriate, we employed the ChiElectronic searches generated a total of 1,603 citations. Publications not relevant to the review were removed. After removal of duplicated publications, abstracts of 579 records were screened and a further 463 records were removed. Full text copies of 116 articles were obtained and reviewed. We included 95 studies in this review; 21 studies that failed to meet the inclusion criteria were excluded. The PRISMA flow diagram is illustrated in Studies included in this review are summarized in Using the Oxford Centre for Evidence-Based Medicine rating  of the “The visual outcomes and complications reported in studies included are summarized in The reported mean length of follow-up of all studies ranged from 0 to 60 months (mean 12.8 ± 12.2 months).“Endothelium-out” studies: Thirty-four of the 87 studies (39.1%) reported the mean BCVA at 6 months after DMEK surgery; BCVA ranged from 0.0 to 0.49 LogMAR.Fifteen studies (17.2%) reported that 42.5–85% of eyes achieved a BCVA of 20/25 or better at 6 months. The random pooled proportion of eyes achieving BCVA of 20/25 or better at 6 months was 58.7% (95% CI 49.4–67.7%) (15 studies).“Endothelium-in” studies: Two of the nine studies (22.2%) reported the mean BCVA at 6 months after DMEK surgery; BCVA ranged from 0.09 to 0.10 LogMAR. Three studies (33.3%) reported that 44.7–87.5% of eyes achieved a BCVA of 20/25 or better at 6 months. The random pooled proportion of eyes achieving BCVA of 20/25 or better at 6 months was 62.4% (95% CI 33.9–86.9%) (3 studies).“Endothelium-out” studies: 67/87 (77.0%) studies reported data on percentage endothelial cell loss following DMEK surgery at various time points. The mean endothelial cell loss ranged from 19 to 53%. One study  at various time points. The reported mean endothelial cell loss range from 26.6 to 56.0%. The random pooled mean endothelial cell loss was 28.1 ± 1.3% at 6 months (7 studies) and 29.6 ± 1.2% at 12 months (1 studies).“endothelium-out” to “endothelium-in” techniques, pooled mean endothelial cell loss was lower in the “endothelium-in” group, compared to “endothelium-out” group at 6 months (p = 0.018). However, this was not statistically computable at 12 months as there was only 1 study for the “endothelium in” group.Comparing outcomes of “Endothelium-out” studies: Re-bubbling rates to treat DMEK graft detachments were reported in 77/87 (88.5%) studies and ranged from 0 to 82%. Fifty-eight (66.7%) studies reported primary graft failure rates which ranged from 0 to 21.0%. Thirty-five (40.2%) studies reported secondary graft failure rates which ranged from 0 to 7.0% at 15.3 ± 13.9 months. Fifty (57.5%) studies reported graft rejection rates; rates ranged from 0 to 7.0%.“Endothelium-in” studies: Re-bubbling rates to treat DMEK graft detachments were reported in all nine studies and ranged from 4.7 to 45.7%. Six of the nine studies (66.7%) reported primary graft failure rates which ranged from 0 to 3.0%. Three of the nine studies (33.3%) reported on secondary graft failures rates which ranged from 0 to 6.5%.“endothelium-out” and “endothelium-in” techniques were 26.2% (95% CI 21.9–30.9%) (74 studies) and 16.5% (95% CI 8.5–26.4%) (6 studies), respectively. Comparing outcomes of “endothelium-out” to “endothelium-in” techniques, graft re-bubbling rates were not statistically significant in the “endothelium-out” group (p = 0.440). The random pooled primary graft failure rates for “endothelium-out” and “endothelium-in” techniques were 2.9% (95% CI 2.03–4.02%) (58 studies) and 1.5% (95% CI 0.6–2.7%) (5 studies), respectively. Comparing outcomes of “endothelium-out” to “endothelium-in” techniques, there was no significant difference in primary graft failure rates between the two groups (p = 0.552).The random pooled graft re-bubbling rates for Although DMEK offers the advantages of faster visual rehabilitation, better visual and refractive outcomes –25, and In current clinical practice, the vast majority of DMEK surgeries performed are “endothelium-out” techniques. This was reflected in this systematic review. Of the 21,323 included eyes that underwent DMEK, 19,945 (93.5%) received their grafts through various “endothelium-out” insertion techniques. In these techniques, the DMEK graft is loaded into an injector and inserted into the anterior chamber as a scroll, with the endothelium on its outer surface. Injectors used included modified intraocular lens cartridges, implantable contact lens cartridges, intravenous tubing, or glass injectors . Direct Moreover, in “endothelium-out” techniques, there is often no control of the scrolled graft during insertion. Despite the use of intraoperative imaging , orientaThe unfolding of a scrolled DMEK graft and its central positioning on the recipient’s posterior stromal surface can also be problematic and time-consuming. To unfold the DMEK scroll after insertion into the anterior chamber, numerous approaches such as using air bubbles or jets of balanced salt solution in the presence of a shallow anterior chamber and the stroking of the corneal surface have been described . To overThe concept of “endothelium-in” DMEK insertion techniques have been recently introduced , 44, 45.This review included a total of 95 studies . Eighty-“endothelium-out” techniques were 26.2% (95% CI 21.9–30.9%) (74 studies). The random pooled primary graft failure rates for “endothelium-out” techniques was 2.9% (95% CI 2.03–4.02%) (58 studies).Evaluating the outcomes of “endothelium-out” techniques, the mean BCVA at 6 months after DMEK surgery ranged from 0.0 to 0.49 LogMAR (34 studies); 42.5–85% of eyes (15 studies) achieved a best-corrected visual acuity (BCVA) of 20/25 or better at 6 months. The mean endothelial cell loss ranged from 19 to 53%. The random pooled mean endothelial cell loss was 36.3 ± 6.4% at 6 months (27 studies) and 38.7 ± 7.2% at 12 months (12 studies). Rates of re-bubbling for graft detachments, primary graft failure rates, secondary graft failure rates, and graft rejection rates ranged from 0 to 82%, 0 to 21.0%, 0 to 7.0%, and 0 to 7.0%, respectively. The random pooled graft re-bubbling rates for “endothelium-in” techniques was 16.5% (95% CI 8.5–26.4%) (6 studies). The random pooled primary graft failure rates for “endothelium-in” techniques was 1.5% (95% CI 0.6–2.7%) (six studies).Of the eight “endothelium-in” studies reporting visual acuity data, the mean BCVA at 6 months after DMEK surgery ranged from 0.09 to 0.10 LogMAR (2 studies); 44.7–87.5% of eyes (3 studies) achieved a best-corrected visual acuity (BCVA) of 20/25 or better at 6 months. The mean endothelial cell loss ranged from 26.6 to 56.0% (7 studies). The random pooled mean endothelial cell loss was 28.1 ± 1.3% at 6 months (7 studies) and 29.6 ± 1.2% at 12 months (1 study). Graft detachment re-bubbling rates and primary graft failure rates ranged from 4.7 to 45.7% (nine studies) and 0 to 3.0% (six studies), respectively. Only one study reported on secondary graft failure rates in which there were none. None of the studies reporting on “endothelium-in” techniques reported the graft rejection rates. The random pooled graft re-bubbling rates for “endothelium-out” to “endothelium-in” techniques, pooled mean endothelial cell loss was lower in the “endothelium-in” studies compared to “endothelium-out” studies at 6 months (p = 0.018); this was not statistically computable at 12 months as there was only 1 study for “endothelium in” group. Although re-bubbling rates for graft detachments were higher in the “endothelium-out” studies compared to “endothelium-in” studies, statistical significance was not achieved (p = 0.440). There was no significant difference in primary graft failure rates between the two groups (p = 0.552).Comparing outcomes of This review has several limitations. The quality of available evidence was considered low (grade III and IV) with a significant number of studies judged as having high risks of bias . SignifiThe rates of endothelial cell loss were reported to be significantly lower in “endothelium-in” DMEK surgeries at 6 months following surgery compared to “endothelium-out” surgeries. Despite the above-mentioned limitations, visual outcomes and rates of complications of “endothelium-in” techniques from the small number of studies were noted to be comparable to those reported in “endothelium-out” studies. Given the intra-operative challenges following graft insertion encountered using “endothelium-out” techniques, surgeons may consider “endothelium-in” techniques designed for easier intra-operative DMEK graft unfolding after graft insertion. However, further well-conducted, adequately powered, randomized controlled trials and studies with longer duration of follow-up are needed before conclusive comparisons between the two techniques can be made.The original contributions presented in this study are included in the article/HO and JM: conceptualization and supervision. HO and HH: data curation. HO, HH, MA, and JM: formal analysis, investigation, methodology, writing draft, and review and editing. All authors approved the manuscript.JM holds a patent on the EndoGlide and receive royalties. The remaining 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.