How acidic amino acid residues facilitate DNA target site selection.

Despite the negative charge of the DNA backbone, acidic residues (Asp/Glu) commonly participate in the base readout, with a strong preference for cytosine. In fact, in the solved DNA/protein structures, cytosine is recognized almost exclusively by Asp/Glu through a direct hydrogen bond, while at the same time, adenine, regardless of its amino group, shows no propensity for Asp/Glu. Here, we analyzed the contribution of Asp/Glu to sequence-specific DNA binding using classical and ab initio simulations of selected transcription factors and found that it is governed by a fine balance between the repulsion from backbone phosphates and attractive interactions with cytosine. Specifically, Asp/Glu lower the affinity for noncytosine sites and thus act as negative selectors preventing off-target binding. At cytosine-containing sites, the favorable contribution does not merely rely on the formation of a single H-bond but usually requires the presence of positive potential generated by multiple cytosines, consistently with the observed excess of cytosine in the target sites. Finally, we show that the preference of Asp/Glu for cytosine over adenine is a result of the repulsion from the adenine imidazole ring and a tendency of purine-purine dinucleotides to adopt the BII conformation.

Heterologous Expression and Purification of a CRISPR-Cas9-Based Adenine Base Editor.

CRISPR-cas9-guided adenine base editors (ABEs) site-specifically convert the A-T base pair to G-C base pair in genomic DNA. The intracellular delivery of ABE proteins preassembled with guide RNAs (gRNAs) has shown greatly reduced off-target effects compared with that of plasmids or viral vectors containing ABE and gRNA-encoding sequences. For efficient gene editing by the ribonucleoprotein delivery method, the ABE-gRNA complexes need to be prepared in high purity and quantity. Here we describe the expression and purification procedure of ABEmax, one of high-efficiency ABE versions.

Therapeutic adenine base editing of human hematopoietic stem cells.

In ß-thalassemia, either γ-globin induction to form fetal hemoglobin (α2γ2) or ß-globin repair to restore adult hemoglobin (α2ß2) could be therapeutic. ABE8e, a recently evolved adenine base editor variant, can achieve efficient adenine conversion, yet its application in patient-derived hematopoietic stem cells needs further exploration. Here, we purified ABE8e for ribonucleoprotein electroporation of ß-thalassemia patient CD34+ hematopoietic stem and progenitor cells to introduce nucleotide substitutions that upregulate γ-globin expression in the BCL11A enhancer or in the HBG promoter. We observed highly efficient on-target adenine base edits at these two regulatory regions, resulting in robust γ-globin induction. Moreover, we developed ABE8e-SpRY, a near-PAMless ABE variant, and successfully applied ABE8e-SpRY RNP to directly correct HbE and IVS II-654 mutations in patient-derived CD34+ HSPCs. Finally, durable therapeutic editing was produced in self-renewing repopulating human HSCs as assayed in primary and secondary recipients. Together, these results support the potential of ABE-mediated base editing in HSCs to treat inherited monogenic blood disorders.

Preservation of red blood cell antigenicity in a new storage solution in vitro.

INTRODUCTION: Red blood cell (RBC) storage solution is used for suspending and preserving RBCs for later use in in vitro immunohematology testing. Proper RBC preservation is crucial for obtaining accurate results in RBC phenotyping and pretransfusion antibody screening tests. Haemolysis or RBC antigen degradation during storage can result in inaccurate RBC phenotyping, thereby decreasing the sensitivity of pretransfusion antibody screening and identification assays. The conventional RBC storage solutions usually contain adenosine, adenine, and antibiotics. We designed an RBC storage solution and determined whether it could preserve RBC integrity for 70 days. MATERIALS AND METHODS: The new storage solution has a different formula from that of the conventional solution-in particular, it is strengthened with polyethylene glycol (PEG). The extent of haemolysis and hemagglutination reactivity of the RBC antigen systems, Rh, Duffy, Kidd, Lewis, MNS, P1, and the rare antigen Mia (which has a low prevalence antigen in most parts of the world but a higher prevalence in Taiwan), in the new RBC storage solution was compared with that of the conventionally preserved RBC storage solution. RESULTS: The RBCs preserved in the new solution for 70 days retained a similar haemolysis grade as those preserved in the control solution for 28 days. Although both solutions largely preserved RBC antigenicity, the decline in RBC hemagglutination scores in new solution often occurred later than that in the control solution in most antigen phenotyping assays, especially labile antigens such as D, P1, and M. CONCLUSION: The new solution reduces haemolysis more effectively and preserves antigenicity throughout the 70-day storage period. Moreover, Mia antigen is more stable in the experimental group.

Systematic Design of Adenosine Analogs as Inhibitors of a Clostridioides difficile-Specific DNA Adenine Methyltransferase Required for Normal Sporulation and Persistence.

Antivirulence agents targeting endospore-transmitted Clostridioides difficile infections are urgently needed. C. difficile-specific DNA adenine methyltransferase (CamA) is required for efficient sporulation and affects persistence in the colon. The active site of CamA is conserved and closely resembles those of hundreds of related S-adenosyl-l-methionine (SAM)-dependent methyltransferases, which makes the design of selective inhibitors more challenging. We explored the solvent-exposed edge of the SAM adenosine moiety and systematically designed 42 analogs of adenosine carrying substituents at the C6-amino group (N6) of adenosine. We compare the inhibitory properties and binding affinity of these diverse compounds and present the crystal structures of CamA in complex with 14 of them in the presence of substrate DNA. The most potent of these inhibitors, compound 39 (IC50 ∼ 0.4 µM and KD ∼ 0.2 µM), is selective for CamA against closely related bacterial and mammalian DNA and RNA adenine methyltransferases, protein lysine and arginine methyltransferases, and human adenosine receptors.

Intermediate products of purine metabolism in an experimental model of pancreatic necrosis.

BACKGROUND AND AIM: Determine the level of purines in the blood plasma of experimental animals at three stages of induced pancreatic necrosis. Find out the potential of purines as predictors of the severity of pancreatitis. METHODS: The experiment was carried out on white outbred rabbits. The pancreatic necrosis was modeled by introducing self-bile into the pancreatic parenchyma. The pancreas of rabbits, after isolation, was subjected to microscopic description. Blood was also taken from rabbits to determine the plasma levels of adenine, guanine, hypoxanthine, xanthine, and uric acid. RESULTS: 12 hours after the administration of self-bile, the level of xanthine significantly increases and the concentration of uric acid in the blood plasma increases by 3 times. 24 hours after the introduction of self-bile, there is a slight decrease in the level of adenine, xanthine and uric acid, and the indicators of purine metabolism remain elevated. 48 hours after the introduction of self-bile, the levels of guanine, hypoxanthine and xanthine are reduced. CONCLUSIONS: The concentration indices of absolute and relative intermediate products of purine metabolism were increased at the initial stage of pancreatic necrosis. The activity of enzymes and metabolites of purine metabolism involved in the formation of reactive oxygen species and free radicals increased. The hypothesis that intermediate products of purine metabolism can be predictors of pancreatic necrosis was confirmed.

Adenine base editing efficiently restores the function of Fanconi anemia hematopoietic stem and progenitor cells.

Fanconi Anemia (FA) is a debilitating genetic disorder with a wide range of severe symptoms including bone marrow failure and predisposition to cancer. CRISPR-Cas genome editing manipulates genotypes by harnessing DNA repair and has been proposed as a potential cure for FA. But FA is caused by deficiencies in DNA repair itself, preventing the use of editing strategies such as homology directed repair. Recently developed base editing (BE) systems do not rely on double stranded DNA breaks and might be used to target mutations in FA genes, but this remains to be tested. Here we develop a proof of concept therapeutic base editing strategy to address two of the most prevalent FANCA mutations in patient hematopoietic stem and progenitor cells. We find that optimizing adenine base editor construct, vector type, guide RNA format, and delivery conditions leads to very effective genetic modification in multiple FA patient backgrounds. Optimized base editing restored FANCA expression, molecular function of the FA pathway, and phenotypic resistance to crosslinking agents. ABE8e mediated editing in primary hematopoietic stem and progenitor cells from FA patients was both genotypically effective and restored FA pathway function, indicating the potential of base editing strategies for future clinical application in FA.

Therapeutic Ultrasound Halts Progression of Chronic Kidney Disease In Vivo via the Regulation of Markers Associated with Renal Epithelial-Mesenchymal Transition and Senescence.

Low-intensity pulsed ultrasound (LIPUS), a therapeutic type of ultrasound, is known to enhance bone fracture repair processes and help some tissues to heal. Here, we investigated the therapeutic potential of LIPUS for the treatment of chronic kidney disease (CKD) in two CKD mouse models. CKD mice were induced using both unilateral renal ischemia/reperfusion injury (IRI) with nephrectomy and adenine administration. The left kidneys of the CKD mice were treated using LIPUS with the parameters of 3 MHz, 100 mW/cm2, and 20 min/day, based on the preliminary experiments. The mice were euthanized 14 days after IRI or 28 days after the end of adenine administration. LIPUS treatment effectively alleviated the decreases in the body weight and albumin/globulin ratio and the increases in the serum renal functional markers, fibroblast growth factor-23, renal pathological changes, and renal fibrosis in the CKD mice. The parameters for epithelial-mesenchymal transition (EMT), senescence-related signal induction, and the inhibition of α-Klotho and endogenous antioxidant enzyme protein expression in the kidneys of the CKD mice were also significantly alleviated by LIPUS. These results suggest that LIPUS treatment reduces CKD progression through the inhibition of EMT and senescence-related signals. The application of LIPUS may be an alternative non-invasive therapeutic intervention for CKD therapy.

Uremic Myopathy and Mitochondrial Dysfunction in Kidney Disease.

Alterations in muscle structure and function in chronic kidney disease (CKD) patients are associated with poor outcomes. As key organelles in muscle cell homeostasis, mitochondrial metabolism has been studied in the context of muscle dysfunction in CKD. We conducted a study to determine the contribution of oxidative metabolism, glycolysis and fatty acid oxidation to the muscle metabolism in CKD. Mice developed CKD by exposure to adenine in the diet. Muscle of CKD mice showed significant weight loss compared to non-CKD mice, but only extensor digitorum longus (EDL) muscle showed a decreased number of fibers. There was no difference in the proportion of the various muscle fibers in CKD and non-CKD mice. Muscle of CKD mice had decreased expression of proteins associated with oxidative phosphorylation but increased expression of enzymes and transporters associated with glycolysis. In cell culture, myotubes exposed to uremic serum demonstrated decreased oxygen consumption rates (OCR) when glucose was used as substrate, conserved OCR when fatty acids were used and increased lactate production. In conclusion, mice with adenine-induced CKD developed sarcopenia and with increased glycolytic metabolism but without gross changes in fiber structure. In vitro models of uremic myopathy suggest fatty acid utilization is preserved compared to decreased glucose utilization.

The gut microbe Bacteroides fragilis ameliorates renal fibrosis in mice.

Renal fibrosis is an inevitable outcome of various manifestations of progressive chronic kidney diseases (CKD). The need for efficacious treatment regimen against renal fibrosis can therefore not be overemphasized. Here we show a novel protective role of Bacteroides fragilis (B. fragilis) in renal fibrosis in mice. We demonstrate decreased abundance of B. fragilis in the feces of CKD patients and unilateral ureteral obstruction (UUO) mice. Oral administration of live B. fragilis attenuates renal fibrosis in UUO and adenine mice models. Increased lipopolysaccharide (LPS) levels are decreased after B. fragilis administration. Results of metabolomics and proteomics studies show decreased level of 1,5-anhydroglucitol (1,5-AG), a substrate of SGLT2, which increases after B. fragilis administration via enhancement of renal SGLT2 expression. 1,5-AG is an agonist of TGR5 that attenuates renal fibrosis by inhibiting oxidative stress and inflammation. Madecassoside, a natural product found via in vitro screening promotes B. fragilis growth and remarkably ameliorates renal fibrosis. Our findings reveal the ameliorative role of B. fragilis in renal fibrosis via decreasing LPS and increasing 1,5-AG levels.

Kif26b contributes to the progression of interstitial fibrosis via migration and myofibroblast differentiation in renal fibroblast.

Kinesin family member 26b (Kif26b) is essential for kidney development, and its deletion in mice leads to kidney agenesis. However, the roles of this gene in adult settings remain elusive. Thus, this study aims to investigate the role of Kif26b in the progression of renal fibrosis. A renal fibrosis model with adenine administration using Kif26b heterozygous mice and wild-type mice was established. Renal fibrosis and the underlying mechanism were investigated. The underlying pathways and functions of Kif26b were evaluated in an in vitro model using primary renal fibroblasts. Kif26b heterozygous mice were protected from renal fibrosis with adenine administration. Renal expressions of connective tissue growth factor (CTGF) and myofibroblast accumulation were reduced in Kif26b heterozygous mice. The expression of nonmuscle myosin heavy chain II (NMHCII), which binds to the C-terminus of Kif26b protein, was also suppressed in Kif26b heterozygous mice. The in vitro study revealed reduced expressions of CTGF, α-smooth muscle actin, and myosin heavy chain 9 (Myh9) via transfection with siRNAs targeting Kif26b in renal fibroblasts (RFB). RFBs, which were transfected by the expression vector of Kif26b, demonstrated higher expressions of these genes than non-transfected cells. Finally, Kif26b suppression and NMHCII blockage led to reduced abilities of migration and collagen gel contraction in renal fibroblasts. Taken together, Kif26b contributes to the progression of interstitial fibrosis via migration and myofibroblast differentiation through Myh9 in the renal fibrosis model. Blockage of this pathway at appropriate timing might be a therapeutic approach for renal fibrosis.

Alterations in Primary Carbon Metabolism in Cucumber Infected with Pseudomonas syringae pv lachrymans: Local and Systemic Responses.

The reconfiguration of the primary metabolism is essential in plant-pathogen interactions. We compared the local metabolic responses of cucumber leaves inoculated with Pseudomonas syringae pv lachrymans (Psl) with those in non-inoculated systemic leaves, by examining the changes in the nicotinamide adenine dinucleotides pools, the concentration of soluble carbohydrates and activities/gene expression of carbohydrate metabolism-related enzymes, the expression of photosynthesis-related genes, and the tricarboxylic acid cycle-linked metabolite contents and enzyme activities. In the infected leaves, Psl induced a metabolic signature with an altered [NAD(P)H]/[NAD(P)+] ratio; decreased glucose and sucrose contents, along with a changed invertase gene expression; and increased glucose turnover and accumulation of raffinose, trehalose, and myo-inositol. The accumulation of oxaloacetic and malic acids, enhanced activities, and gene expression of fumarase and l-malate dehydrogenase, as well as the increased respiration rate in the infected leaves, indicated that Psl induced the tricarboxylic acid cycle. The changes in gene expression of ribulose-l,5-bis-phosphate carboxylase/oxygenase large unit, phosphoenolpyruvate carboxylase and chloroplast glyceraldehyde-3-phosphate dehydrogenase were compatible with a net photosynthesis decline described earlier. Psl triggered metabolic changes common to the infected and non-infected leaves, the dynamics of which differed quantitatively (e.g., malic acid content and metabolism, glucose-6-phosphate accumulation, and glucose-6-phosphate dehydrogenase activity) and those specifically related to the local or systemic response (e.g., changes in the sugar content and turnover). Therefore, metabolic changes in the systemic leaves may be part of the global effects of local infection on the whole-plant metabolism and also represent a specific acclimation response contributing to balancing growth and defense.

Molecular Recognition of FDA-Approved Small Molecule Protein Kinase Drugs in Protein Kinases.

Protein kinases are key enzymes that catalyze the covalent phosphorylation of substrates via the transfer of the γ-phosphate of ATP, playing a crucial role in cellular proliferation, differentiation, and various cell regulatory processes. Due to their pivotal cellular role, the aberrant function of kinases has been associated with cancers and many other diseases. Consequently, competitive inhibition of the ATP binding site of protein kinases has emerged as an effective means of curing these diseases. Decades of intense development of protein kinase inhibitors (PKIs) resulted in 71 FDA-approved PKI drugs that target dozens of protein kinases for the treatment of various diseases. How do FDA-approved protein kinase inhibitor PKI drugs compete with ATP in their own binding pocket? This is the central question we attempt to address in this work. Based on modes of non-bonded interactions and their calculated interaction strengths by means of the advanced double hybrid DFT method B2PLYP, the molecular recognition of PKI drugs in the ATP-binding pockets was systematically analyzed. It was found that (1) all the FDA-approved PKI drugs studied here form one or more hydrogen bond(s) with the backbone amide N, O atoms in the hinge region of the ATP binding site, mimicking the adenine base; (2) all the FDA-approved PKI drugs feature two or more aromatic rings. The latter reach far and deep into the hydrophobic regions I and II, forming multiple CH-π interactions with aliphatic residues L(3), V(11), A(15), V(36), G(51), L(77) and π-π stacking interactions with aromatic residues F(47) and F(82), but ATP itself does not utilize these regions extensively; (3) all FDA-approved PKI drugs studied here have one thing in common, i.e., they frequently formed non-bonded interactions with a total of 12 residues L(3),V(11), A(15), K(17), E(24),V(36),T(45), F(47), G(51), L(77), D(81) and F(82) in the ATP binding. Many of those 12 commonly involved residues are highly conserved residues with important structural and catalytic functional roles. K(17) and E(24) are the two highly conserved residues crucial for the catalytic function of kinases. D(81) and F(82) belong to the DFG motif; T(45) was dubbed the gate keeper residue. F(47) is located on the hinge region and G(51) sits on the linker that connects the hinge to the αD-helix. It is this targeting of highly conserved residues in protein kinases that led to promiscuous PKI drugs that lack selectivity. Although the formation of hydrogen bond(s) with the backbone of the hinge gives PKI drugs the added binding affinity and the much-needed directionality, selectivity is sacrificed. That is why so many FDA-approved PKI drugs are known to have multiple targets. Moreover, off-target-mediated toxicity caused by a lack of selectivity was one of the major challenges facing the PKI drug discovery community. This work suggests a road map for future PKI drug design, i.e., targeting non-conserved residues in the ATP binding pocket to gain better selectivity so as to avoid off-target-mediated toxicity.

Swim Training Affects on Muscle Lactate Metabolism, Nicotinamide Adenine Dinucleotides Concentration, and the Activity of NADH Shuttle Enzymes in a Mouse Model of Amyotrophic Lateral Sclerosis.

In this study, we aim to verify whether swim training can improve lactate metabolism, NAD+ and NADH levels, as well as modify the activity of glycolytic and NADH shuttle enzymes and monocarboxylate transporters (MCTs) in skeletal muscle of amyotrophic lateral sclerosis (ALS) mice. ALS mice (SOD1G93A) (n = 7 per group) were analyzed before the onset of ALS, at first disease symptoms (trained and untrained), and the last stage of disease (trained and untrained), and then compared with a wild-type (WT) group of mice. The blood lactate and the skeletal muscle concentration of lactate, NAD+ and NADH, MCT1 and MCT4 protein levels, as well as lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) activities in skeletal muscle were determined by fluorometric, Western blotting, liquid chromatography-MS3 spectrometry, and spectrometric methods. In the untrained terminal ALS group, there were decreased blood lactate levels (p < 0.001) and increased skeletal muscle lactate levels (p < 0.05) as compared with a WT group of mice. The amount of nicotinamide adenine dinucleotides in the ALS groups were also significantly reduced as well as LDH activity and the level of MCT1. Swim training increased lactate levels in the blood (p < 0.05 vs. ALS TERMINAL untrained). In addition, cytosolic MDH activity and the cMDH/LDH 2.1 ratio were significantly higher in trained vs. untrained mice (p < 0.05). The data indicate significant dysfunction of lactate metabolism in ALS mice, associated with a reduction in muscle anaerobic metabolism and NADH transporting enzymes, as well as swim-induced compensation of energy demands in the ALS mice.

Extracellular DNA concentrations in various aetiologies of acute kidney injury.

Extracellular DNA (ecDNA) in plasma is a non-specific biomarker of tissue damage. Urinary ecDNA, especially of mitochondrial origin, is a potential non-invasive biomarker of kidney damage. Despite prominent tissue damage, ecDNA has not yet been comprehensively analysed in acute kidney injury (AKI). We analysed different fractions of ecDNA, i.e. total, nuclear and mitochondrial, in plasma and urine of children, and different animal models of AKI. We also analysed the activity of the deoxyribonuclease (DNase), which is contributes to the degradation of ecDNA. Patients with AKI had higher total and nuclear ecDNA in both, plasma and urine (sixfold and 12-fold in plasma, and 800-fold in urine, respectively), with no difference in mitochondrial ecDNA. This was mainly found for patients with AKI due to tubulointerstitial nephritis and atypical haemolytic uremic syndrome. Increased plasma ecDNA was also found in animal models of AKI, including adenine nephropathy (fivefold), haemolytic uremic syndrome (fourfold), and ischemia-reperfusion injury (1.5-fold). Total urinary ecDNA was higher in adenine nephropathy and ischemia-reperfusion injury (1300-fold and twofold, respectively). DNase activity in urine was significantly lower in all animal models of AKI in comparison to controls. In conclusion, plasma total and nuclear ecDNA and urinary total ecDNA is increased in patients and animals with particular entities of AKI, suggesting a mechanism-dependent release of ecDNA during AKI. Further studies should focus on the dynamics of ecDNA and its potential role in the pathogenesis of AKI.

Lactobacillus acidophilus KBL409 Reduces Kidney Fibrosis via Immune Modulatory Effects in Mice with Chronic Kidney Disease.

SCOPE: Intestinal dysbiosis has been reported to play an important role in the pathogenesis of various diseases, including chronic kidney disease (CKD). Here, to evaluate whether probiotic supplements can have protective effects against kidney injury in an animal model of CKD is aimed. METHODS AND RESULTS: An animal model of CKD is established by feeding C57BL/6 mice a diet containing 0.2% adenine. These model mice are administered Lactobacillus acidophilus KBL409 daily for 4 weeks. Features of adenine-induce CKD (Ade-CKD) mice, such as prominent kidney fibrosis and higher levels of serum creatinine and albuminuria are improved by administration of KBL409. Ade-CKD mice also exhibit a disrupted intestinal barrier and elevate levels of TNF-α, IL-6, and 8-hydroxy-2'-deoxyguanosine. These changes are attenuated by KBL409. Administration of KBL409 significantly reduces macrophage infiltration and promotes a switch to the M2 macrophage phenotype and increasing regulatory T cells. Notably, the NLRP3 inflammasome pathway is activated in the kidneys of Ade-CKD and decreases by KBL409. In primary kidney tubular epithelial cells treated with p-cresyl sulfate, short-chain fatty acids significantly increase M2 macrophage polarization factors and decrease profibrotic markers. CONCLUSIONS: These results demonstrate that supplementation with the probiotic KBL409 has beneficial immunomodulating effects and protects against kidney injury.

Mechanisms of the ethanol extract of Gelidium amansii for slow aging in high-fat male Drosophila by metabolomic analysis.

Gelidium amansii (GA) is a kind of red alga homologous to medicine and food and is distributed all over the world. Studies on GA are mainly focused on its polysaccharides, with little research on the ethanol extract. The ethanol extract of Gelidium amansii (GAE) was subjected to a reverse-phase column to obtain 7 components. Among them, 100% methanol solution (GAM), enriched with phytene-1,2-diol, exhibited the strongest DPPH free radical scavenging activity (IC50 = 0.17 mg mL-1). Subsequently, high-fat male flies (HMFs) were used as a model to explore the antioxidant and anti-aging effects of GAM in vivo. Studies showed that GAM can effectively prolong the lifespan of HMFs. When GAM concentrations were 0.2 and 1.0 mg mL-1, the average lifespan of HMFs was increased by 28.7 and 40.7%, respectively, while the longest lifespan of HMFs was increased by 20.55% and 32.88%, respectively. Further research revealed that GAM can significantly downregulate the levels of malondialdehyde (MDA) and protein carbonyl (PCO), and can significantly upregulate the levels of catalase (CAT) and total superoxide dismutase (T-SOD). In addition, by analyzing differential metabolites, we found that GAM relieves aging caused by oxidative stress by regulating amino acid, lipid, sugar, and energy metabolism. The GAM group significantly regulated the levels of adenine, cholic acid, glutamate, L-proline, niacin, and stachyose which tend to recover to the levels of the normal diet male fly (NMF) group. In general, our research provides ideas for the high-value utilization of GA and provides a lead compound for the research and development of anti-aging food or medicine.

Iron Supplementation Improves Skeletal Muscle Contractile Properties in Mice with CKD.

Background: Patients with chronic kidney disease (CKD) frequently have compromised physical performance, which increases their mortality; however, their skeletal muscle dysfunction has not been characterized at the single-fiber and molecular levels. Notably, interventions to mitigate CKD myopathy are scarce. Methods: The effect of CKD in the absence and presence of iron supplementation on the contractile function of individual skeletal muscle fibers from the soleus and extensor digitorum longus muscles was evaluated in 16-week-old mice. CKD was induced by the adenine diet, and iron supplementation was by weekly iron dextran injections. Results: Maximally activated and fatigued fiber force production was decreased 24%-52% in untreated CKD, independent of size, by reducing strongly bound myosin/actin cross-bridges and/or decreasing myofilament stiffness in myosin heavy chain (MHC) I, IIA, and IIB fibers. Additionally, myosin/actin interactions in untreated CKD were slower for MHC I and IIA fibers and unchanged or faster in MHC IIB fibers. Iron supplementation improved anemia and did not change overall muscle mass in CKD mice. Iron supplementation ameliorated CKD-induced myopathy by increasing strongly bound cross-bridges, leading to improved specific tension, and/or returning the rate of myosin/actin interactions toward or equivalent to control values in MHC IIA and IIB fibers. Conclusions: Skeletal muscle force production was significantly reduced in untreated CKD, independent of fiber size, indicating that compromised physical function in patients is not solely due to muscle mass loss. Iron supplementation improved multiple aspects of CKD-induced myopathy, suggesting that timely correction of iron imbalance may aid in ameliorating contractile deficits in CKD patients.

In vivo adenine base editing reverts C282Y and improves iron metabolism in hemochromatosis mice.

Hemochromatosis is one of the most common inherited metabolic diseases among white populations and predominantly originates from a homozygous C282Y mutation in the HFE gene. The G > A transition at position c.845 of the gene causes misfolding of the HFE protein, ultimately resulting in its absence at the cell membrane. Consequently, the lack of interaction with the transferrin receptors 1 and 2 leads to systemic iron overload. We screened potential gRNAs in a highly precise cell culture assay and applied an AAV8 split-vector expressing the adenine base editor ABE7.10 and our candidate gRNA in 129-Hfetm.1.1Nca mice. Here we show that a single injection of our therapeutic vector leads to a gene correction rate of >10% and improved iron metabolism in the liver. Our study presents a proof-of-concept for a targeted gene correction therapy for one of the most frequent hereditary diseases affecting humans.

[Development of an APRT-deficient CHO cell line and its ability of expressing recombinant protein].

Chinese hamster ovary (CHO) cells are the preferred host cells for the production of complex recombinant therapeutic proteins. Adenine phosphoribosyltransferase (APRT) is a key enzyme in the purine biosynthesis step that catalyzes the condensation of adenine with phosphoribosylate to form adenosine phosphate AMP. In this study, the gene editing technique was used to knock out the aprt gene in CHO cells. Subsequently, the biological properties of APRT-KO CHO cell lines were investigated. A control vector expressed an enhanced green fluorescent protein (EGFP) and an attenuation vector (containing an aprt-attenuated expression cassette and EGFP) were constructed and transfected into APRT-deficient and wild-type CHO cells, respectively. The stable transfected cell pools were subcultured for 60 generations and the mean fluorescence intensity of EGFP in the recombinant CHO cells was detected by flow cytometry to analyze the EGFP expression stability. PCR amplification and sequencing showed that the aprt gene in CHO cell was successfully knocked out. The obtained APRT-deficient CHO cell line had no significant difference from the wild-type CHO cells in terms of cell morphology, growth, proliferation, and doubling time. The transient expression results indicated that compared with the wild-type CHO cells, the expression of EGFP in the APRT-deficient CHO cells transfected with the control vector and the attenuation vector increased by 42%±6% and 56%±9%, respectively. Especially, the EGFP expression levels in APRT-deficient cells transfected with the attenuation vector were significantly higher than those in wild-type CHO cells (P < 0.05). The findings suggest that the APRT-deficient CHO cell line can significantly improve the long-term expression stability of recombinant proteins. This may provide an effective cell engineering strategy for establishing an efficient and stable CHO cell expression system.