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It had been observed that homozygous albumin knockout mice (Alb -/- ) exhibit low plasma free fatty acid (FFA) concentration and improved blood glucose regulation. However, it was not yet known to what extent heterozygous albumin knockout (Alb +/- ) mice would display a similar phenotype. Alb -/- , Alb +/- , and wild-type (WT) female mice were studied on a low-fat diet (LFD) or high-fat diet (HFD). On both diets, decreased plasma FFA concentration, and improved glucose tolerance test were observed in Alb -/- , but not in Alb +/- , compared to WT. Plasma adiponectin concentration showed greater elevation in Alb -/- than Alb +/- . Consistent with that, adiponectin gene expression was significantly higher in Alb -/- mice than in Alb +/- and WT mice. A dose-dependent response was observed for hepatic Acadl gene expression showing higher Acadl gene expression in Alb -/- mice than in Alb +/- and WT mice. In conclusion, although female Alb +/- mice exhibited some slight differences from WT mice (e.g., increased plasma adiponectin and hepatic Acadl gene expression), Alb +/- mice did not exhibit improved glucoregulation in comparison to WT mice, indicating that a minor suppression of albumin expression is not sufficient to improve glucoregulation. Furthermore, it is now clear that although the response of female mice to HFD might be unique from how males generally respond, still the complete albumin deficiency in Alb -/- mice and the associated FFA reduction is capable of improving glucoregulation in females on this diet. The present results have implications for the role of albumin and FFA in the regulation of metabolism., (© 2024. The Author(s).)

The legume albumin-1 gene family, arising after nodulation, encodes linear a- and b-chain peptides for nutrient storage and defense. Intriguingly, in one prominent legume, Clitoria ternatea, the b-chains are replaced by domains producing ultra-stable cyclic peptides called cyclotides. The mechanism of this gene hijacking is until now unknown. Cyclotides require recruitment of ligase-type asparaginyl endopeptidases (AEPs) for maturation (cyclization), necessitating co-evolution of two gene families. Here we compare a chromosome-level C. ternatea genome with grain legumes to reveal an 8 to 40-fold expansion of the albumin-1 gene family, enabling the additional loci to undergo diversification. Iterative rounds of albumin-1 duplication and diversification create four albumin-1 enriched genomic islands encoding cyclotides, where they are physically grouped by similar pI and net charge values. We identify an ancestral hydrolytic AEP that exhibits neofunctionalization and multiple duplication events to yield two ligase-type AEPs. We propose cyclotides arise by convergence in C. ternatea where their presence enhances defense from biotic attack, thus increasing fitness compared to lineages with linear b-chains and ultimately driving the replacement of b-chains with cyclotides., (© 2024. The Author(s).)

Liver-directed adeno-associated viral (AAV) vector-mediated homology-independent targeted integration (AAV-HITI) by CRISPR-Cas9 at the highly transcribed albumin locus is under investigation to provide sustained transgene expression following neonatal treatment. We show that targeting the 3' end of the albumin locus results in productive integration in about 15% of mouse hepatocytes achieving therapeutic levels of systemic proteins in two mouse models of inherited diseases. We demonstrate that full-length HITI donor DNA is preferentially integrated upon nuclease cleavage and that, despite partial AAV genome integrations in the target locus, no gross chromosomal rearrangements or insertions/deletions at off-target sites are found. In line with this, no evidence of hepatocellular carcinoma is observed within the 1-year follow-up. Finally, AAV-HITI is effective at vector doses considered safe if directly translated to humans providing therapeutic efficacy in the adult liver in addition to newborn. Overall, our data support the development of this liver-directed AAV-based knockin strategy., Competing Interests: Declaration of interests F.E., F.D.A., R.F., M.L.S., and A.A. are listed as inventors on the patent WO2023213831 “Homology independent targeted integration for gene editing” related to this work. A.A. is founder, shareholder, and consultant of InnovaVector s.r.l. and of AAVantgarde Bio s.r.l. R.F. is currently an employee of AAVantgarde Bio s.r.l. T.C., M.B., and G.A. are listed as inventors of CAST-Seq (EP3856928B1)., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Hatchability could be quite different among individuals of indigenous chicken breed which might be affected by the egg quality. In this study, hatchability was individually recorded among 800 forty-wk-old Huainan partridge chickens. The chickens were then divided into high and low hatchability groups (HH and LH group) with 50 birds in each group. Egg quality was further determined in the 2 groups. Eight birds from each group were selected for slaughtering and tissue, responsible for egg formation, collection for structure observation by staining and candidate gene expression by transcriptome analysis. The hatchability in HH was 100% and 61.18% in LH. The eggshell thickness and shell strength were significantly lower, while the albumen height and Haugh unit were significantly higher in HH group than those in LH group (P < 0.05). The magnum weight and index, and the expression of polypeptide N-acetylgalactosaminyltransferase 9 (GALNT9), which responsible for thick albumen synthesis, in HH group were also significantly higher than that of LH group (P < 0.05). Compared with the LH group, there were 702 differentially expressed genes (DEGs) in HH group, of which 402 were up-regulated and 300 were down-regulated. Candidate genes of calbindin 1 (CALB1) and solute carrier family 26 member 9 (SLC26A9), which regulate calcium signaling pathway so as to affect Ca 2+ transportation, exhibited significant high and low expression, respectively, in HH group compared to those in LH group (P < 0.05). Therefore, indigenous chicken with high expression of GALNT9 in magnum to form thick albumen to provide more protein for embryo, while high CALB1 and low expression of SLC26A9 to decrease Ca 2+ transportation so as to form a thinner eggshell and provide better gas exchange during embryo development., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Tissue-specific gene manipulations are widely used in genetically engineered mouse models. A single recombinase system, such as the one using Alb-Cre, has been commonly used for liver-specific genetic manipulations. However, most diseases are complex, involving multiple genetic changes and various cell types. A dual recombinase system is required for conditionally modifying different genes sequentially in the same cell or inducing genetic changes in different cell types within the same organism. A FlpO cDNA was inserted between the last exon and 3'-UTR of the mouse albumin gene in a bacterial artificial chromosome (BAC-Alb-FlpO). The founders were crossed with various reporter mice to examine the efficiency of recombination. Liver cancer tumorigenesis was investigated by crossing the FlpO mice with FSF-Kras G12D mice and p53 frt mice (KPF mice). BAC-Alb-FlpO mice exhibited highly efficient recombination capability in both hepatocytes and intrahepatic cholangiocytes. No recombination was observed in the duodenum and pancreatic cells. BAC-Alb-FlpO-mediated liver-specific expression of mutant Kras G12D and conditional deletion of p53 gene caused the development of liver cancer. Remarkably, liver cancer in these KPF mice manifested a distinctive mixed hepatocellular carcinoma and cholangiocarcinoma phenotype. A highly efficient and liver-specific BAC-Alb-FlpO mouse model was developed. In combination with other Cre lines, different genes can be manipulated sequentially in the same cell, or distinct genetic changes can be induced in different cell types of the same organism. NEW & NOTEWORTHY A liver-specific Alb-FlpO mouse line was generated. By coupling it with other existing CreERT or Cre lines, the dual recombinase approach can enable sequential gene modifications within the same cell or across various cell types in an organism for liver research through temporal and spatial gene manipulations.

The high potential of siRNAs to silence oncogenic drivers remains largely untapped due to the challenges of tumor cell delivery. Here, divalent lipid-conjugated siRNAs are optimized for in situ binding to albumin to improve pharmacokinetics and tumor delivery. Systematic variation of the siRNA conjugate structure reveals that the location of the linker branching site dictates tendency toward albumin association versus self-assembly, while the lipid hydrophobicity and reversibility of albumin binding also contribute to siRNA intracellular delivery. The lead structure increases tumor siRNA accumulation 12-fold in orthotopic triple negative breast cancer (TNBC) tumors over the parent siRNA. This structure achieves approximately 80% silencing of the anti-apoptotic oncogene MCL1 and yields better survival outcomes in three TNBC models than an MCL-1 small molecule inhibitor. These studies provide new structure-function insights on siRNA-lipid conjugate structures that are intravenously injected, associate in situ with serum albumin, and improve pharmacokinetics and tumor treatment efficacy., (© 2024. The Author(s).)

Background: Chronic kidney disease (CKD) is highly prevalent among Indigenous Australians, especially those in remote regions. The Tiwi population has been isolated from mainland Australia for millennia and exhibits unique genetic characteristics that distinguish them from other Indigenous and non-Indigenous populations. Notably, the rate of end-stage renal disease is up to 20 times greater in this population compared to non-Indigenous populations. Despite the identification of numerous genetic loci associated with kidney disease through GWAS, the Indigenous population such as Tiwi remains severely underrepresented and the increased prevalence of CKD in this population may be due to unique disease-causing alleles/genes., Methods: We used albumin-to-creatinine ratio (ACR) and estimated glomerular filtration rate (eGFR) to estimate the prevalence of kidney disease in the Tiwi population (N = 492) in comparison to the UK Biobank (UKBB) (N = 134,724) database. We then performed an exploratory factor analysis to identify correlations among 10 CKD-related phenotypes and identify new multi-phenotype factors. We subsequently conducted a genome-wide association study (GWAS) on all single and multiple phenotype factors using mixed linear regression models, adjusted for age, sex, population stratification, and genetic relatedness between individuals., Results: Based on ACR, 20.3% of the population was at severely increased risk of CKD progression and showed elevated levels of ACR compared to the UKBB population independent of HbA1c. A GWAS of ACR revealed novel association loci in the genes MEG3 (chr14:100812018:T:A), RAB36 (rs11704318), and TIAM2 (rs9689640). Additionally, multiple phenotypes GWAS of ACR, eGFR, urine albumin, and serum creatinine identified a novel variant that mapped to the gene MEIS2 (chr15:37218869:A:G). Most of the identified variants were found to be either absent or rare in the UKBB population., Conclusions: Our study highlights the Tiwi population's predisposition towards elevated ACR, and the collection of novel genetic variants associated with kidney function. These associations may prove valuable in the early diagnosis and treatment of renal disease in this underrepresented population. Additionally, further research is needed to comprehensively validate the functions of the identified variants/genes., (© 2024. The Author(s).)

This study was designed to perform an association analysis and identify SNP markers associated with production traits of Japanese quail using restriction-site-associated DNA sequencing. Weekly body weight data from 805 quail were collected from hatching to 16 weeks of age. A total number of 3990 eggs obtained from 399 female quail were used to assess egg quality traits. Egg-related traits were measured at the beginning of egg production (first stage) and at 12 weeks of age (second stage). Five eggs were analyzed at each stage. Traits, such as egg weight, egg length and short axes, eggshell strength and weight, egg equator thickness, yolk weight, diameter, and colour, albumen weight, age of first egg, total number of laid eggs, and egg production rate, were assessed. A total of 383 SNPs and 1151 associations as well as 734 SNPs and 1442 associations were identified in relation to quail production traits using general linear model (GLM) and mixed linear model (MLM) approaches, respectively. The GLM-identified SNPs were located on chromosomes 1-13, 15, 17-20, 24, 26-28, and Z, underlying phenotypic traits, except for egg and albumen weight at the first stage and yolk yellowness at the second stage. The MLM-identified SNPs were positioned on defined chromosomes associated with phenotypic traits except for the egg long axis at the second stage of egg production. Finally, 35 speculated genes were identified as candidate genes for the targeted traits based on their nearest positions. Our findings provide a deeper understanding and allow a more precise genetic improvement of production traits of Galliformes, particularly in Japanese quail., (© 2023. The Author(s).)

Hepatic lipid droplets (LDs) are implicated in ectopic lipid accumulation. The core of LDs, triacylglycerol (TAG), is synthesized from the esterification of fatty acids to a glycerol-3-phosphate (G-3-P) backbone. Albumin transports plasma free fatty acids, and previously albumin knockout (Alb -/- ) mice were shown to exhibit lower hepatic TAG levels than wildtype (WT). Exercise is a beneficial strategy to alter hepatic metabolism, but its impacts on reducing hepatic lipids are far from satisfactory. The aim of this study was to investigate the combined effect of albumin deficiency and acute exercise on hepatic LDs. Eight-week-old male Alb -/- and WT mice were divided into sedentary and exercise groups. Exercised mice performed a 30-min high-intensity exercise bout. Results showed that sedentary Alb -/- mice had smaller hepatic LDs (P < 0.0001), associated with mitochondria, while WT mice exhibited larger LDs, surrounded by glycogen granules. Following acute exercise, hepatic LDs in Alb -/- mice reduced by 40% in size, while in WT increased by 14% (P < 0.0001). The maintenance of WT hepatic LDs was associated with elevated G-3-P level (P < 0.05), potentially derived from glycogen (R = -0.32, %change in glycogen versus LD content, P < 0.05). The reduction in Alb -/- mice LDs after exercise was possibly due to their low glycogen level. In conclusion, Alb -/- mice exhibited an enhanced capacity for reducing hepatic LD size and content in response to exercise. These findings suggest that modulating albumin's functions combined with exercise could be a potential strategy to reduce ectopic lipid deposition in the liver., (© 2023. The Author(s).)

The fusion of human serum albumin (HSA) with human lactoferrin (hLF) (designated as hLF-HSA) has improved the pharmacokinetic properties and anti-proliferative activities of hLF against cancer cells. In this study, we evaluated the anti-migratory activities of hLF and hLF-HSA against the human lung adenocarcinoma PC-14 cell line using wound healing and Boyden chamber assays. Despite the unexpected hLF-induced migration, hLF-HSA clearly demonstrated the complete inhibition of PC-14 cell migration. To examine the mechanism underlying the enhanced PC-14 cell migration by hLF alone but suppressed migration by hLF-HSA, we focused on the matrix metalloproteinase (MMP) family of endopeptidases because MMPs are often reported to play important roles in facilitating the migration and metastasis of cancer cells. Furthermore, hLF is a transactivator of MMP1 transcription. As expected, treatment of cells with hLF and hLF-HSA led to the upregulation and downregulation of MMP1, respectively. In contrast, MMP9 expression levels, which are often associated with cancer migration, were unchanged in the presence of either protein. An MMP inhibitor attenuated hLF-induced migration of PC-14 cells. Therefore, specific enhancement and suppression of MMP1 expression by hLF and hLF-HSA have been implicated as causes of a marked increase and decrease in PC-14 cell migration, respectively. In conclusion, the fusion of HSA with hLF (hLF-HSA) promoted its anti-migratory effects against cancer cells. Therefore, hLF-HSA is a promising anti-cancer drug candidate based on its improved anti-migratory activity towards cancer cells., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Objective: Workers in secondary aluminum production plants are occupationally exposed to polycyclic aromatic hydrocarbons (PAHs). We aimed to monitor the concentrations of PAHs in air and in serum of workers at two secondary aluminum production plants. We also investigated the potential risk of lung cancer development among PAHs exposed workers with emphasis on the role of A1AT mutation and APEX1 gene polymorphisms., Methods: This study included 177 workers from administrative departments and production lines. Blood samples were obtained for estimation of benzo(a)pyrene diol epoxide albumin adduct (BPDE-Alb adduct), anti-Cyclin-B1 marker (CCNB1) and squamous cell carcinoma antigen (SCCAg). Genes' polymorphism for human apurinic/apyrimidinic endonuclease (APEX1) and alpha-1-anti-trypsin (A1AT) gene mutation were detected., Results: There was a significant increase in the level of BPDE-Alb adduct among exposed workers in comparison to non-exposed group. Moreover, 41.67% of exposed workers in El Tebbin had BPDE-Alb adduct level ≥ 15 ng/ml versus 29.6% of workers in Helwan factory. There was a significant increase in tumor markers (SCCAg and CCNB1) among workers whose BPDE-Alb adduct ≥ 15 ng/ml. There was a significant increase in the level of BPDE-Alb adducts in exposed workers carrying homozygous APEX1 genotype Glu/Glu. Furthermore, exposed workers with the Glu/Glu genotype had high tumor markers levels. There was a significant increase in levels of BPDE-Alb adducts in workers carrying A1AT mutant allele. Moreover, workers with mutant A1AT genotype had significantly high tumor markers (SCCAg and CCNB1) levels., Conclusion: Therefore, we conclude that aluminum workers may be at a potential risk of lung cancer development due to PAHs exposure. Although PAHs concentrations in air were within the permissible limits, yet evidence of DNA damage was present as expressed by high BPDE-albumin adduct level in exposed workers. Also, elevation of tumor markers (SCCAg and CCNB1) in exposed workers points to the importance of periodic biological monitoring of such workers to protect them from cancer risk., (© 2022. The Author(s).)

Diabetic nephropathy (DN) is a severe kidney-related complication of type 1 and type 2 diabetes and the most frequent cause of end-stage kidney disease. Extracellular vesicles (EVs) present in the urine mainly derive from the cells of the nephron, thus representing an interesting tool mirroring the kidney's physiological state. In search of the biomarkers of disease progression, we here assessed a panel of urinary EV miRNAs previously related to DN in type 2 diabetic patients stratified based on proteinuria levels. We found that during DN progression, miR145 and miR126 specifically increased in urinary EVs from diabetic patients together with albuminuria. In vitro, miRNA modulation was assessed in a model of TGF-β1-induced glomerular damage within a three-dimensional perfusion system, as well as in a model of tubular damage induced by albumin and glucose overload. Both renal tubular cells and podocytes undergoing epithelial to mesenchymal transition released EVs containing increased miR145 and miR126 levels. At the same time, miR126 levels were reduced in EVs released by glomerular endothelial cells. This work highlights a modulation of miR126 and miR145 during the progression of kidney damage in diabetes as biomarkers of epithelial to mesenchymal transition.

Hybrid sturgeon is the main species of sturgeon cultured in China, with the advantages of a fast growth rate, early sexual maturity, fertile offspring, and more stable genetic traits. In May 2021, a large number of deaths characterized by superficial hemorrhage and liver damage occurred in a sturgeon farm in Yichang, Hubei Province, which posed a significant risk to hybrid sturgeon captive breeding. We isolated a pathogenic bacterium named D-59 from the diseased sturgeon with apparent symptoms. The pathogen was identified as Staphylococcus sciuri using 16S rRNA gene phylogenetic analysis combined with biochemical identification. Regression experiments showed that D-59 exhibited clinical signs similar to those of diseased sturgeon in the farm after intraperitoneal injection into hybrid sturgeon. High-throughput sequencing of gut microbes in D-59-infected sturgeon showed that the number of gut microbial species decreased in infected sturgeon, the number of some intestinal commensal bacteria decreased, and the balance of the intestinal microorganisms was disrupted. Histopathological sections indicated many inflammatory cells, congestion, and even necrosis in the tissue of diseased sturgeon. Analysis of blood indexes revealed an increase in the proportion of mononuclear cells and a decrease in the proportion of lymphocytes in the peripheral blood of diseased sturgeon. Significantly elevated serum levels of aspartate aminotransferase and alanine aminotransferase, whereas alkaline phosphatase, total protein, albumin, and globulin were decreased in diseased sturgeon. Antimicrobial susceptibility tests demonstrated that D-59 is susceptible to florfenicol, enrofloxacin, and neomycin sulfate. This study aimed to highlight the dangers of Staphylococcus sciuri infection during hybrid sturgeon culture and to provide recommendations for diagnosis and treatment., Competing Interests: 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., (Copyright © 2022 Zhang, Xue, Xiao, Liu, Jiang, Meng, Fan, Liu and Zhou.)

Dapagliflozin, an inhibitor of sodium-glucose cotransporter 2 (SGLT2), is a new type of oral hypoglycemic drugs which can promote glucose excretion in the kidney. Studies have shown that dapagliflozin has renoprotective effect in the treatment of type 2 diabetes. However, the underlying mechanism remains unclear. Here, we combined integrated RNA sequencing and network pharmacology approach to investigate the molecular mechanism of dapagliflozin for diabetic nephropathy (DN). Dapagliflozin significantly relieved glucose intolerance, urinary albumin/creatinine ratio (UACR) and renal pathological injuries of db/db mice. The LncRNA and mRNA expression in kidney tissues from control group (CR), db/db group (DN) and dapagliflozin group (DG) were assessed by RNA sequencing. We identified 7 LncRNAs and 64 mRNAs common differentially expressed in CR vs DN and DN vs DG, which were used to construct co-expression network to reveal significantly correlated expression patterns in DN. In addition, network pharmacology was used to predict the therapeutic targets of dapagliflozin and we constructed component-target-pathway network according to the results of RNA sequencing and network pharmacology. We found that SMAD9, PPARG, CD36, CYP4A12A, CYP4A12B, CASP3, H2-DMB2, MAPK1, MAPK3, C3 and IL-10 might be the pivotal targets of dapagliflozin for treating DN and these genes were mainly enriched in pathways including TGF-β signaling pathway, PPAR signaling pathway, Chemokine signaling pathway, etc. Our results have important implication and provide novel insights into the protective mechanism of dapagliflozin for treating DN., Competing Interests: 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., (Copyright © 2022 Bai, Xie, Liu, Chen, Yu, Zhang, Luo, Chen, Zhao and Xiao.)

Methylmalonic acidemia (MMA) is an inborn error of metabolism mostly caused by mutations in the mitochondrial methylmalonyl-CoA mutase gene (MMUT). MMA patients suffer from frequent episodes of metabolic decompensation, which can be life threatening. To mimic both the dietary restrictions and metabolic decompensation seen in MMA patients, we developed a novel protein-controlled diet regimen in a Mmut deficient mouse model of MMA and demonstrated the therapeutic benefit of mLB-001, a nuclease-free, promoterless recombinant AAV GeneRideTM vector designed to insert the mouse Mmut into the endogenous albumin locus via homologous recombination. A single intravenous administration of mLB-001 to neonatal or adult MMA mice prevented body weight loss and mortality when challenged with a high protein diet. The edited hepatocytes expressed functional MMUT protein and expanded over time in the Mmut deficient mice, suggesting a selective growth advantage over the diseased cells. In mice with a humanized liver, treatment with a human homolog of mLB-001 resulted in site-specific genome editing and transgene expression in the transplanted human hepatocytes. Taken together, these findings support the development of hLB-001 that is currently in clinical trials in pediatric patients with severe forms of MMA., Competing Interests: The authors are/were employees of LogicBio Therapeutics. LogicBio has filed pending patent applications published as WO2020/032986 (pertaining to non-disruptive gene therapy for the treatment of MMA) and WO2020/214582 (pertaining to monitoring gene therapy). This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Methylmalonic acidemia (MMA) is a rare and severe inherited metabolic disease typically caused by mutations of the methylmalonyl-CoA mutase (MMUT) gene. Despite medical management, patients with MMA experience frequent episodes of metabolic instability, severe morbidity, and early mortality. In several preclinical studies, systemic gene therapy has demonstrated impressive improvement in biochemical and clinical phenotypes of MMA murine models. One approach uses a promoterless adeno-associated viral (AAV) vector that relies upon homologous recombination to achieve site-specific in vivo gene addition of MMUT into the last coding exon of albumin (Alb), generating a fused Alb-MMUT transcript after successful editing. We have previously demonstrated that nuclease-free AAV mediated Alb editing could effectively treat MMA mice in the neonatal period and noted that hepatocytes had a growth advantage after correction. Here, we use a transgenic knock-out mouse model of MMA that recapitulates severe clinical and biochemical symptoms to assess the benefits of Alb editing in juvenile animals. As was first noted in the neonatal gene therapy studies, we observe that gene edited hepatocytes in the MMA mice treated as juveniles exhibit a growth advantage, which allows them to repopulate the liver slowly but dramatically by 8-10 months post treatment, and subsequently manifest a biochemical and enzymatic response. In conclusion, our results suggest that the benefit of AAV mediated nuclease-free gene editing of the Alb locus to treat MMA could potentially be therapeutic for older patients., Competing Interests: Declaration of Competing Interest N.R. and J.L. are employees of LogicBio Therapeutics and receive salary support and stock options from LogicBio Therapeutics. S.G. and N.C. are former employees and shareholders of LogicBio Therapeutics., (Published by Elsevier Inc.)

We have previously reported that a major quantitative trait locus (QTL) responsible for susceptibility to salt-induced stroke in the stroke-prone spontaneously hypertensive rat (SHRSP) is located in a 3-Mbp region on chromosome 1 covered by SHRSP.SHR-(D1Rat23-D1Rat213)/Izm (termed Pr1.31), a congenic strain with segments from SHRSP/Izm introduced into the stroke-resistant SHR/Izm. Here, we attempted to narrow down the candidate region on chromosome 1 further through analyses of subcongenic strains constructed for the target region. Simultaneously, salt-induced kidney injury was evaluated through the measurement of urinary albumin and the gene expression of renal tubular injury markers (Kim-1 and Clu) to explore a possible mechanism leading to the onset of stroke. All subcongenic strains examined in this study showed lower susceptibility to salt-induced stroke than SHRSP. Interestingly, Pr1.31 had the lowest stroke susceptibility when compared with newly constructed subcongenic strains harboring fragments of the congenic sequence in Pr1.31. Although Kim-1 and Clu expression after 1 week of salt loading in Pr1.31 did not differ significantly from those in SHRSP, the urinary albumin level of Pr1.31 was significantly lower than those of the other subcongenic strains and that of SHRSP. The present results indicated that, although the congenic fragment in Pr1.31 harbored the gene(s) related to salt-induced organ damages, further genetic dissection of the candidate region was difficult due to multiple QTLs suggested in this region. Further analysis using Pr1.31 will unveil genetic and pathophysiological mechanisms underlying salt-induced end organ damages in SHRSP.

The study aims to explore the clinicopathological features and whether the nonsense mutations of CLCN5 gene have effect on the renal expression of CLC-5 protein and megalin/cubilin complex in children with Dent-1 disease. The clinicopathological features and genetic examination of three patients with Dent-1 disease were investigated. The expression of CLC-5 and megalin/cubilin complex in renal tissues was detected by using immunohistochemistry method. Urinary albumin, α1-microglobulin, β2-microglobulin, retinol binding protein, and calcium levels were measured by immunonephelometry. Urinary calcium and low molecular weight proteinuria (LMWP) were enhanced in three patients, and two presented with nephrotic range proteinuria. Focal glomerular obsolescence, minor tubulointerstitial injury, and focal calcification in corticomedullary junction were found in one patient. Nonsense mutations of CLCN5 gene from their mothers were identified in all three patients with Dent-1 disease; however, the expression of CLC-5 protein was not decreased in renal tubular cells. As the receptor complex of albumin and LMWP reabsorption, the expression of megalin/cubilin in the brush border of proximal tubules was decreased in Dent-1 patients. Even if the renal CLC-5 protein is expressed normally, the reduced expression of megalin/cubilin in the brush border of renal proximal tubules may be helpful to understand the physiopathology of Dent-1 disease with nonsense mutations of CLCN5 gene.

Objective: To investigate the utility of albumin RNAscope in situ hybridization in the diagnosis and differential diagnosis of hepatocellular carcinoma and its mimics. Methods: One hundred and fifty-two cases of hepatocellular carcinoma and its mimics and 33 cases of normal tissue were selected from the pathology database of the Department of Pathology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine from January 2013 to December 2019. Tissue microarrays were constructed and RNAscope in situ hybridization was performed to detect the expression of albumin mRNA. Results: No albumin mRNA expression was detected in normal tissues except for the liver. All hepatocellular carcinoma regardless of its degree of differentiation and primary or metastatic nature had detectable albumin mRNA, with strong and diffuse staining in 90.7% (49/54) of cases. While the positive rate of HepPar-1, Arg-1 or one of them by immunohistochemistry was 87.0% (47/54), 85.2% (46/54) and 92.6% (50/54) respectively. The positive rates of albumin mRNA in intrahepatic cholangiocarcinoma and biphenotypic hepatocellular carcinoma were 7/15 and 9/10, respectively. The former showed focal or heterogeneous staining, while the latter showed strong and diffuse staining. The positive rate of hepatoid adenocarcinoma was 8/19, and the albumin expression could be diffuse or focal. Sporadic cases of poorly differentiated gastric adenocarcinoma and metastatic colon adenocarcinoma showed focal staining of albumin mRNA. Conclusions: Detection of albumin mRNA by RNAscope in situ hybridization is of great value for the diagnosis and differential diagnosis of HCC, and the sensitivity may be improved by combining with HepPar-1 and Arg-1. It also offers different diagnostic clues according to different expression patterns.

Circadian rhythms are endogenously generated physiological and molecular rhythms with a cycle length of about 24 h. Bioluminescent reporters have been exceptionally useful for studying circadian rhythms in numerous species. Here, we report development of a reporter mouse generated by modification of a widely expressed and highly rhythmic gene encoding D-site albumin promoter binding protein ( Dbp ). In this line of mice, firefly luciferase is expressed from the Dbp locus in a Cre recombinase-dependent manner, allowing assessment of bioluminescence rhythms in specific cellular populations. A mouse line in which luciferase expression was Cre -independent was also generated. The Dbp reporter alleles do not alter Dbp gene expression rhythms in liver or circadian locomotor activity rhythms. In vivo and ex vivo studies show the utility of the reporter alleles for monitoring rhythmicity. Our studies reveal cell-type-specific characteristics of rhythms among neuronal populations within the suprachiasmatic nuclei ex vivo. In vivo studies show Dbp -driven bioluminescence rhythms in the liver of Albumin-Cre;Dbp KI/+ "liver reporter" mice. After a shift of the lighting schedule, locomotor activity achieved the proper phase relationship with the new lighting cycle more rapidly than hepatic bioluminescence did. As previously shown, restricting food access to the daytime altered the phase of hepatic rhythmicity. Our model allowed assessment of the rate of recovery from misalignment once animals were provided with food ad libitum. These studies confirm the previously demonstrated circadian misalignment following environmental perturbations and reveal the utility of this model for minimally invasive, longitudinal monitoring of rhythmicity from specific mouse tissues.

The native pumpkin 2S albumin, a multifunctional protein, possess a variety of potential biotechnologically exploitable properties. The present study reports the characterization of recombinant pumpkin 2S albumin (rP2SA) and unraveling of its potential DNA/RNA binding site. The purification and characterization of the rP2SA established that it retains the characteristic α-helical structure and exhibited comparable DNase, RNase, antifungal and anti-proliferative activities as native protein. In vitro studies revealed that rP2SA exhibits potent antiviral activity against chikungunya virus (CHIKV) at a non-toxic concentration with an IC 50 of 114.5 μg/mL. In silico studies and site-directed mutagenesis were employed to unravel the potential DNA/RNA binding site. A strong positive charge distribution due to presence of many arginine residues in proximity of helix 5 was identified as a potential site. The two of the arginine residues, conserved in some 2S albumins, were selected for the mutation studies. The mutated forms of recombinant protein (R84A and R91A) showed a drastic reduction in DNase and RNase activities suggesting their presence at binding site and involvement in the nuclease activity. A metal binding site was also identified adjacent to DNA/RNA binding site. The present study demonstrated the structural and functional integrity of the rP2SA and reports potential antiviral activity against CHIKV. Further, potential DNA/RNA binding site was unraveled through mutation studies and bioinformatics analysis., Competing Interests: Declaration of competing interest The authors have no conflict of interest to disclose., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Chicken eggs play an important role as food resources in the world. Although genetic effects on yolk and albumen contents have been reported, the number of chicken genotypes analyzed so far is still limited. To investigate the effect of genetic background on 10 egg traits, 19 yolk amino acid traits, and 19 albumen amino acid traits, we evaluated a total of 58 eggs from five genotypes: two Japanese indigenous breeds (Ukokkei and Nagoya) and three hybrids (Araucana cross, Kurohisui, and Boris Brown) under a floor rearing system. One-way ANOVA revealed significant effects of genotype on 10 egg traits, 8 yolk amino acids (Asp, Glu, Ser, Gly, Thr, Tyr, Cys, and Leu), and 11 albumen amino acids (Asp, Glu, Asn, Ser, Gln, His, Ala, Tyr, Trp, Phe, and Ile) contents. Moderate to strong positive phenotypic correlations among traits within each trait category (size and weight traits, yolk amino acid traits, and albumen amino acid traits), whereas there were basically no or weak correlations among the trait categories. However, a unique feature was found in the Araucana cross indicating moderate positive correlations of amino acids between yolk and albumen. These results suggest that genetic factors can modify not only the size and weight of the egg and eggshell color but also yolk and albumen free amino acids contents., Competing Interests: The authors have declared that no competing interests exist.

Deficiency of factor V is a congenital autosomal recessive coagulopathy associated with mutations in the F5 gene that results in mild-to-severe bleeding episodes. Factor V is a component of the prothrombinase complex responsible for accelerating conversion of prothrombin to thrombin. At the present time there are no therapeutic factor V concentrates available. This study was designed to lay the preliminary foundations for future cell-based therapy for patients with severe factor V deficiency. The study showed that hepatospheres, which produce coagulation factors VIII, IX, and V, synthetize and store intracellular glycogen and express albumin levels up to 8 times higher than those of undifferentiated cells. Factor IX and factor V gene expression increased significantly in hepatospheres as compared to undifferentiated cells, whereas factor VIII gene expression remained constant. The factor V protein was detected in the hepatospheres´ secretome. Considering the enormous potential of mesenchymal stem cells as therapeutic agents, this study proposes a highly reproducible method to induce differentiation of mesenchymal stem cells from human placenta to factor V-producing hepatospheres. This strategy constitutes a preliminary step towards a curative treatment of factor V deficiency through advanced therapies such as cell therapy., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Haemoglobin (Hb)-albumin (HSA) trimers were synthesized using five distinct Hb variants in which the structures were genetically and chemically tuned as an artificial O 2 carrier and used as a red blood cell (RBC) substitute. The trimers were found to have moderately low O 2 affinity ( p 50 = 23-34 Torr, 37 °C) and high co-operativity, yielding a maximum O 2 transport efficiency 1.8-fold higher than that of human RBCs.

The construction of an in vitro 3D cellular model to mimic the human liver is highly desired for drug discovery and clinical applications, such as patient-specific treatment and cell-based therapy in regenerative medicine. However, current bioprinting strategies are limited in their ability to generate multiple cell-laden microtissues with biomimetic structures. This study presents a method for producing hepatic-lobule-like microtissue spheroids using a bioprinting system incorporating a precursor cartridge and microfluidic emulsification system. The multiple cell-laden microtissue spheroids can be successfully generated at a speed of approximately 45 spheroids min -1 and with a uniform diameter. Hepatic and endothelial cells are patterned in a microtissue spheroid with the biomimetic structure of a liver lobule. The spheroids allow long-term culture with high cell viability, and the structural integrity is maintained longer than that of non-structured spheroids. Furthermore, structured spheroids show high MRP2, albumin, and CD31 expression levels. In addition, the in vivo study reveals that structured microtissue spheroids are stably engrafted. These results demonstrate that the method provides a valuable 3D structured microtissue spheroid model with lobule-like constructs and liver functions., (© 2021 The Authors. Advanced Materials published by Wiley-VCH GmbH.)

We performed a morphological study of the bone tissue after implantation of a cryogenically structured albumin sponge containing a bioregulator isolated from blood serum into an extensive experimental defect of the femur. By day 90, no complete reparation of the bone tissue was achieved in the control group (without implantation of 3D carrier), a loose spongy bone is formed at the site of the defect. After implantation of the 3D carrier without serum bioregulator, the defect was closed, but the formed bone was loose and contained no inflammation foci. After the defect was filed with the albumin sponge with the bioregulator, the repair pattern corresponded to the processes of epimorphic tissue regeneration. The results suggest that cryogenically structured protein material in combination with a serum bioregulator ensured complete restoration of the bone tissue.

Aims: To prolong the short lifespan of oxyntomodulin (OXM) for treating obesity and diabetes, we designed a novel fused OXM analog, containing an albumin-binding sequence, a protease cleavable tetrapeptide, and a mutated OXM., Main Methods: We screened two albumin-binding sequences (S3 and S6) to construct OXM derivatives, termed S3-2 (with two cysteines) and S6-0 (without cysteine). After peptides were synthesized, isothermal titration calorimetry (ITC) was applied to assess binding-affinity for HSA. Further in vivo acute efficacies evaluation and candidate selection were performed in diabetic db/db mice via oral glucose tolerance test (OGTT) and glucose-lowering duration test. Chronic efficacy test of selected candidate was also performed in diabetic mice., Results: Firstly, S3-2 and S6-0 with purity over 99% were prepared. ITC measurements demonstrated that S3-2 and S6-0 associate with HSA with high-affinity (K d  = 12.81 ± 1.11 nM and 26.98 ± 2.39 nM, respectively). Then hypoglycemic efficacies showed deoxidation S3-2 (S3-2re) showed longer hypoglycemic duration than the oxidation one (S3-2ox), and better blood glucose level (BGL) control effect than S6-0. OGTTs in diabetic mice revealed the glucose-lowering efficacies of S3-2re were similar to Liraglutide. The protracted antidiabetic effects of S3-2re were further confirmed by multiple OGTTs in db/db mice. Furthermore, twice weekly injection of S3-2re to db/db mice achieved beneficial effects on body weight gain, glucose tolerance, postprandial BGL and obesity. Moreover, S3-2 produces significantly protective effects on the impaired renal functions of the diabetic mice., Conclusion: S3-2re exhibits outstanding therapeutical potential as a candidate drug for treating the obesity and diabetes., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The aim of the study was to determine shell quality of eggs laid by some strains of native breed hens of different ages, with special consideration of their effect on lysozyme concentration and enzymatic activity. Evaluation was made of the eggshells from 6 breeds/strains of laying hens covered by the gene pool protection program in Poland: Greenleg Partridge (Z-11), Yellowleg Partridge (Ż-33), Rhode Island Red (R-11), Rhode Island White (A-33), Sussex (S-66), and Leghorn (H-22). Significant (P ≤ 0.01) differences were established for all the shell quality characteristics between hen strains. As the birds aged, shell weight and porosity increased, and shell compression strength decreased in all the experimental groups. Lysozyme content was lowest in white-shelled eggs (H-22) and highest in cream-colored and light brown eggs (Z-11, Ż-33, and R-11). Furthermore, age of hens had a greater effect on lysozyme concentration and activity in the eggs than on shell quality traits. Regardless of the layer genotype, eggs from older hens showed higher lysozyme concentration and enzymatic activity., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Albumin messenger RNA (mRNA) in situ hybridization is a sensitive and specific biomarker for hepatocellular carcinoma (HCC). Intrahepatic cholangiocarcinoma (ICC) shows variable sensitivity, whereas extrahepatic cholangiocarcinoma (ECC) and metastatic carcinoma are generally negative. We studied the clinical utility and limitations of albumin mRNA detection in a cohort of HCCs, ICCs, ECCs, bile duct adenomas, bile duct hamartomas, and metastatic carcinomas to the liver; and investigated the variability in sensitivity observed for this biomarker in ICCs. We identified 122 cases of hepatobiliary lesions and metastatic carcinomas. Albumin mRNA detection was performed using RNAscope run on formalin-fixed, paraffin-embedded tissue sections. ICCs were categorized according to the classification proposed by Hayashi and colleagues into the small duct, large duct, and indeterminate subtypes. Albumin mRNA was detected in all 17 HCCs and focally in 6/8 (75%) of bile duct adenomas. All 28 nonhepatic carcinomas, 13 bile duct hamartomas, and 9 ECCs were negative. Albumin mRNA was found in 38/47 (80.9%) of ICC with 35/37 (94.6%) in the small duct subtype, 2/3 (66.7%) in the indeterminate subtype, and 1/7 (14.3%) of the large duct subtype (P<0.003). Albumin mRNA detection is a sensitive and specific biomarker for HCCs. It is highly sensitive and moderately specific in the diagnosis of ICC with small gland morphology, but not ICCs with large duct morphology and in metastatic carcinoma. The variability in the sensitivity of albumin mRNA expression in ICCs may depend on the subtypes of ICC., Competing Interests: The authors declare no conflict of interest., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Intrahepatic cholangiocarcinomas (ICCs) show morphologic diversity, ranging from tumors composed of nonmucinous small ducts to mucin-producing large duct tumors to tumors with mixed hepatocellular carcinoma features. Diagnosing ICCs can be difficult, especially on biopsy, not only because of the morphologic diversity, but also because metastatic tumors are often in the differential diagnosis. Recently, branched DNA-based albumin RNA in situ hybridization (ISH) has been shown to be a potential sensitive and specific marker for ICC with 99% sensitivity. Using a different RNA ISH technology, we evaluated the expression of albumin RNA ISH in ICC. We performed RNA ISH for albumin using RNAscope on 43 ICCs in a triplicate tissue microarray. Albumin RNA ISH was positive in 18 of 43 (42%) ICCs. Five of the 6 (83%) combined hepatocellular carcinoma-CC were positive in the CC component. None of the tumors with mucin production were positive (0/9). In our cohort, albumin RNA ISH showed a sensitivity of 42% in ICCs, supporting the morphologic diversity of ICCs. Albumin RNA ISH does not appear to be a highly sensitive marker for ICC and hence cannot be used as a stand-alone marker for ICC., Competing Interests: The authors declare no conflict of interest., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Purpose: Complications following total knee arthroplasty (TKA) lead to patient morbidity and cost. While acute phase reactants, such as c-reactive protein (CRP) and fibrinogen, have been used to predict complications following TKA, the extent and duration of changes in albumin levels following TKA are unknown. It is hypothesized that like CRP and fibrinogen, albumin, and the fibrinogen/albumin ratio (FAR) represent useful measures of the acute phase response (APR) following TKA. The purpose of this study was to describe the longitudinal course of albumin and FAR in healthy patients following TKA, relative to established biomarkers, and examine if the variance in albumin or FAR correlates with patient comorbidities., Methods: This retrospective cohort study of patients undergoing TKA at a tertiary medical center. CRP, fibrinogen, and albumin values were collected pre- and post-operatively. An age-adjusted Charlson comorbidity index (CCI) was utilized as a measure of patient comorbidity status., Results: The median preoperative albumin value was 4.3 g/dL, which dropped to 3.6 g/dL on postoperative day 1 following TKA. The albumin value returned to 93% of the baseline by postoperative week 2. The course of albumin inversely mirrored the course of CRP (r = -0.41). Median preoperative FAR was 0.087 g/L, which rose to 0.130 g/L by postoperative week 2 and returned to baseline by postoperative week 6. While preoperative FAR strongly correlated with postoperative week 2 values (r = 0.74), there was a weak positive correlation between age-adjusted CCI and pre-operative FAR (r = 0.24) in patients undergoing primary TKA., Conclusion: Albumin levels follow a predictable postoperative decline that inversely correlates with CRP in healthy patients following TKA. Given the low cost and abundance of laboratories offering albumin levels, direct albumin levels and/or albumin ratios such as FAR may be underutilized biomarkers for monitoring the APR following TKA., Competing Interests: The authors have read the journal’s policy and have the following competing interests: EA’s spouse works as a paid employee of Exsomed. GGP is a paid consultant for DJO Global and is a board member of the American Association of Hip and Knee Surgeons. JGS is a member of OrthoPediatrics surgeon’s advisory board and education advisory board, receives research support from OrthoPediatrics and PXE International, and receives pharmaceuticals related to research but not related to this submission from IONIS. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare.

Activation of quiescent hepatic stellate cells (HSCs) to myofibroblasts plays a key role in liver fibrosis. We had previously shown that albumin and its derivative, R-III (a retinol-binding protein-albumin domain III fusion protein), inhibited HSC activation by sequestering retinoic acid (RA) and that R-III administration reduced carbon tetrachloride (CCl 4 )-induced liver fibrosis. In this study, we aimed to elucidate the mechanism of action of albumin downstream of RA sequestration. Nuclear factor-κB p65 was evenly distributed in the cytoplasm in activated mouse HSCs, whereas albumin expression or R-III treatment (albumin/R-III) caused the nuclear translocation of p65, probably via RA sequestration, resulting in a dramatic increase in interleukin-1beta (IL-1β) expression. Albumin/R-III in turn induced the phosphorylation of Smad3 at the linker region, inhibiting its nuclear import in an IL-1β-dependent manner. Consistent with the in vitro results, the level of IL-1β mRNA expression was higher in CCl 4 /R-III-treated livers than in CCl 4 -treated livers. These findings reveal that albumin/R-III inhibits the transforming growth factor-β-Smad3 signaling as well as the retinoic acid receptor-mediated pathway, which probably contributes to the inhibition of HSC activation, and suggest that R-III may be an anti-fibrotic drug candidate.

Cubilin (CUBN) and amnionless (AMN), expressed in kidney and intestine, form a multiligand receptor complex called CUBAM that plays a crucial role in albumin absorption. To date, the mechanism of albumin endocytosis mediated by CUBAM remains to be elucidated. Here, we describe a quantitative assay to evaluate albumin uptake by CUBAM using cells expressing full-length CUBN and elucidate the crucial roles of the C-terminal part of CUBN and the endocytosis signal motifs of AMN in albumin endocytosis. We also demonstrate that nuclear valosin-containing protein-like 2 (NVL2), an interacting protein of AMN, is involved in this process. Although NVL2 was mainly localized in the nucleolus in cells without AMN expression, it was translocated to the extranuclear compartment when coexpressed with AMN. NVL2 knockdown significantly impaired internalization of the CUBN-albumin complex in cultured cells, demonstrating an involvement of NVL2 in endocytic regulation. These findings uncover a link between membrane and nucleolar proteins that is involved in endocytic processes., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Cisplatin is one of the most potent chemotherapy drugs to treat cancers, but its clinical application remains limited due to severe nephrotoxicity. Several approaches have been developed to minimize such side effects, notably including chronotherapy, a well-known strategy based on the circadian clock. However, the component of the circadian clock machinery that particularly responses to the cisplatin stimulation remains unknown, including its functions in cisplatin-induced renal injury. In our present study, we demonstrated that Bmal1, as a key clock gene, was induced by the cisplatin stimulation in the mouse kidney and cultured human HK-2 renal cells. Gain- and loss-of-function studies indicated that Bmal1 facilitated cisplatin-induced renal injury both in vivo and in vitro, by aggravating the cell apoptotic process. More importantly, RNA-seq analysis revealed that Bmal1 triggered the expression of hallmark genes involved in renal hepatization, a critical event accompanied by the injury. At the molecular level, Bmal1 activated the transcription of hepatization-associated genes through direct recruitment to the E-box motifs of their promoters. Our findings suggest that Bmal1, a pivotal mediator induced renal injury in response to cisplatin treatment, and the therapeutic intervention targeting Bmal1 in the kidney may be a promising strategy to minimize the toxic side-effects of cisplatin in its clinical applications.

Bread wheat ( Triticum aestivum L.) is one of the most valuable cereal crops for human consumption. Its grain storage proteins define bread quality, though they may cause food intolerances or allergies in susceptible individuals. Herein, we discovered a diversity of grain proteins in three Ukrainian wheat cultivars: Sotnytsia, Panna (both modern selection), and Ukrainka (landrace). Firstly, proteins were isolated with a detergent-containing buffer that allowed extraction of various groups of storage proteins (glutenins, gliadins, globulins, and albumins); secondly, the proteome was profiled by the two-dimensional gel electrophoresis. Using multi-enzymatic digestion, we identified 49 differentially accumulated proteins. Parallel ultrahigh-performance liquid chromatography separation followed by direct mass spectrometry quantification complemented the results. Principal component analysis confirmed that differences among genotypes were a major source of variation. Non-gluten fraction better discriminated bread wheat cultivars. Various accumulation of clinically relevant plant proteins highlighted one of the modern genotypes as a promising donor for the breeding of hypoallergenic cereals.

Conjugation of antisense oligonucleotide (ASO) with a variety of distinct lipophilic moieties like fatty acids and cholesterol increases ASO accumulation and activity in multiple tissues. While lipid conjugation increases tissue exposure in mice and reduces excretion of ASO in urine, histological review of skeletal and cardiac muscle indicates that the increased tissue accumulation of lipid conjugated ASO is isolated to the interstitium. Administration of palmitic acid-conjugated ASO (Palm-ASO) in mice results in a rapid and substantial accumulation in the interstitium of muscle tissue followed by relatively rapid clearance and only slight increases in intracellular accumulation in myocytes. We propose a model whereby increased affinity for lipid particles, albumin, and other plasma proteins by lipid-conjugation facilitates ASO transport across endothelial barriers into tissue interstitium. However, this increased affinity for lipid particles and plasma proteins also facilitates the transport of ASO from the interstitium to the lymph and back into circulation. The cumulative effect is only a slight (∼2-fold) increase in tissue accumulation and similar increase in ASO activity. To support this proposal, we demonstrate that the activity of lipid conjugated ASO was reduced in two mouse models with defects in endothelial transport of macromolecules: caveolin-1 knockout (Cav1-/-) and FcRn knockout (FcRn-/-)., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The current treatment for liver failure is restricted to surgical liver transplantation, which is technically complicated, limited by the shortage of available organs and presents major risks to the patient. Bone marrow mesenchymal stem cells (BMSCs) represent promising sources of hepatocyte-like cells for cell transplantation treatment. However, a safe and efficient induction method for their differentiation remains to be defined. Here we further optimized an effective technique by combining high-dose treatment with hepatocyte growth factor (HGF) and ultrasound stimulation. The optimized ultrasound parameter (1.0 W/cm 2 intensity, 1 MHz frequency, 20% duty cycle, 100 Hz pulse repetition frequency, 60-s irradiation duration, triple times in three days) combined with different HGF doses (10, 20 and 50 ng/ml) was used to treat BMSCs. The results showed that the specific hepatic markers, including α-fetoprotein (αFP/AFP), cytokeratin 18 (CK18), albumin (ALB) and glycogen, were increased in a dose-dependent manner. Their concentration was then further increased when ultrasound irradiation was administered ( P < 0.05), as indicated by PCR, Western blot and immunofluorescence staining as well as a glycogen synthesis test. Furthermore, analysis of the hepatocyte-derived chemokines showed elevated stromal cell-derived factor 1alpha (SDF-1α) and C-X-C chemokine receptor type 4 (CXCR4) after HGF treatment. Again, concentrations of those chemokines were further increased by ultrasound radiation ( P < 0.05). The observed increased effect was sustained for 21 days. To summarize, we further defined the optimal combination of HGF and ultrasound treatment to increase the differentiation and chemotaxis of BMSCs in a safe, sustained and efficient manner. These findings provide a new perspective for stem cell orientation in the field of tissue engineering.

Introduction:  A phase 3b extension study evaluated the long-term safety and efficacy of a recombinant fusion protein-linking coagulation factor IX (FIX) with albumin (rIX-FP) for the routine prophylaxis and on-demand treatment of bleeding in pediatric hemophilia B patients., Methods:  Previously treated patients aged <12 years with moderate to severe hemophilia B enrolled in a 3-year extension study following a phase 3 pivotal study in which they received weekly rIX-FP prophylaxis. In the extension study, they could maintain or extend their prophylaxis interval to every 10 or 14 days if they were well controlled on the 7-day regimen., Results:  Compared with their initial regimen, by the end of the study, dosing intervals were the same, extended, and shortened in 16, 4, and 4 patients, respectively. Very low annualized spontaneous bleeding rates (AsBRs) were observed; median AsBR was 0.0 for the 7- and 10-day regimens, and 1.1 for the 14-day regimen. The 7- and 14-day regimens were comparable in preventing spontaneous bleeds; mean (95% confidence interval) difference in AsBR of -1.2 (-2.6 to 0.3) bleeding episodes/year/subject. Overall, 96% of bleeding episodes were successfully treated with one or two injections of rIX-FP. Patients on a 14-day regimen maintained a mean steady-state trough FIX level of >7.2 IU/dL. No patient developed an inhibitor., Conclusion:  This extension study demonstrated the long-term safety and efficacy of weekly rIX-FP in pediatric patients. Additionally, it showed that adequate bleed protection can be achieved with 10- or 14-day rIX-FP regimens in selected pediatric patients while maintaining safety., Competing Interests: G.K.: Research grant support from Alnylam, BPL, Bayer, Baxalta, CSL Behring, Opko biologics, Pfizer, Shire; consultant for Alnylam, Bayer, Opko biologics, Pfizer, Shire, Roche; honoraria/speakers bureau for Bayer, CSL, Pfizer, Roche. H.C.: Research support from CSL Behring, LFB, Novo Nordisk, Octapharma, Shire/Takeda, SOBI; honoraria from Bayer, LFB, Octapharma, Pfizer, Roche, SOBI; travel support from Bayer, LFB, Octapharma, Pfizer, Roche, Sobi; C.M.: Research support from Bayer, Biotest, CSL Behring, Shire/Takeda; honoraria from Bayer, Biotest, CSL Behring, Novo Nordisk, Pfizer, Roche, Takeda; travel support from Biotest, Bayer, CSL Behring, Novo Nordisk. S.H.: received honoraria for speaking from Bayer Healthcare, Baxalta Innovation, Biotest, CSL Behring, Novartis Pharma, Novo Nordisk, Octapharma, Pfizer; research grants from Bayer Healthcare, Baxalta, Biotest, CSL Behring, Novo Nordisk, Octapharma, Pfizer; T.L.: Research grant support and honoraria from Bayer, CSL Behring, Novo Nordisk, Octapharma, Pfizer, Roche, Shire, SOBI; honoraria from Uniqure; Y.L. and W.S. are employees of CSL Behring; E.S.: Research grant support from CSL Behring; honoraria for speaking and/or for consulting from Bayer, Bioverativ, CSL Behring, Grifols, Kedrion, Novo Nordisk, Octapharma, Pfizer, Roche, Shire/Takeda, SOBI, Spark, Uniqure., (Georg Thieme Verlag KG Stuttgart · New York.)

Background/aim: The outcomes of ramucirumab after lenvatinib failure for hepatocellular carcinoma (HCC) patients with alpha fetoprotein (AFP) levels of ≥400 ng/ml are unknown., Patients and Methods: Of 12 patients treated with ramucirumab after lenvatinib failure, 10 patients were enrolled in this retrospective study., Results: The disease control rate of 80% at 6 weeks and the median time to progression of 3.1 months were the same by both the Response Evaluation Criteria in Solid Tumors (RECIST) and the modified RECIST. AFP reduction was seen in 5 patients at 2 weeks and in 3 patients at 6 weeks. The incidence of grade 3 adverse events was low at 10%. The albumin-bilirubin scores within 6 weeks did not worsen., Conclusion: Ramucirumab might have potential therapeutic efficacy and safety in advanced HCC patients after lenvatinib failure. Further studies are needed to confirm the outcomes of ramucirumab after lenvatinib failure., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Purpose: Non-alcoholic fatty liver disease (NAFLD) improves after bariatric surgery. The aim of this study was to determine whether peripheral blood mononuclear cell albumin gene expression was related to NAFLD and whether albumin (ALB) and alpha fetoprotein (AFP) expression could be detected in whole blood and visceral adipose tissue., Methods: Using a retrospective case control study design, RNA isolated from peripheral blood mononuclear cells from patients prior to undergoing bariatric surgery was used for pooled microarray analysis. Quantitative polymerase chain reaction (QPCR) was used to analyze whole blood and visceral adipose tissue. Liver histology was obtained via intra-operative biopsy and clinical data extracted from the electronic health record., Results: The albumin (ALB) gene was the second most up-regulated found in microarray analysis of peripheral blood mononuclear cell RNA from patients with hepatic lobular inflammation versus normal liver histology. Transcript levels of ALB were significantly different across those with normal (n = 50), steatosis (n = 50), lobular inflammation (n = 50), and peri-sinusoidal fibrosis (n = 50) liver histologies, with lobular inflammation 3.9 times higher than those with normal histology (p < 0.017). Albumin expression levels decreased in 11/13 patients in paired samples obtained prior to and at 1 year after Roux-en-Y gastric bypass surgery. ALB expression could be detected in 23 visceral adipose tissue samples obtained intra-operatively and in 18/19 available paired whole blood samples. No significant correlation was found between ALB expression in visceral adipose tissue and whole blood RNA samples. Alpha fetoprotein expression as a marker of early hepatocytic differentiation was detected in 17/17 available VAT RNA samples, but in only 2/17 whole blood RNA samples., Conclusion: Albumin RNA expression from blood cells may serve as a biomarker of NAFLD. Albumin and alpha fetoprotein appear to be ubiquitously expressed in visceral adipose tissue in patients with extreme obesity.

Efficient enrichment and transmembrane transport of cytotoxic reagents are considered to be effective strategies to increase the efficiency and selectivity of antitumor drugs targeting solid tumors. In the present study, a recombinant protein ABD‑LDP‑Ec consisting of the albumin‑binding domain (ABD), the apoprotein (LDP) of lidamycin (LDM) and an EGFR‑targeting oligopeptide (Ec) was prepared by DNA recombination and bacterial fermentation, and was integrated with the enediyne chromophore (AE) of lidamycin to generate its enediyne‑integrated analogue ABD‑LDP‑Ec‑AE. ABD‑LDP‑Ec exhibited high binding capacity to both albumin and EGFR‑positive pancreatic cancer cells, and was internalized into the cytoplasm through receptor‑mediated endocytosis and albumin‑driven macropinocytosis of K‑ras mutant cells. In animal experiments, ABD‑LDP‑Ec demonstrated notable selective distribution in pancreatic carcinoma xenografts by passive targeting of albumin captured in the blood and was retained in the tumor for 48 h. ABD‑LDP‑Ec and ABD‑LDP‑Ec‑AE exhibited inhibitory activity in cell proliferation and AsPC‑1 xenograft growth, and ABD‑LDP‑Ec‑AE increased the tumor growth inhibition rate by 20% compared with natural LDM. The results indicated that the introduction of ABD‑based multi‑functional drug delivery may be an effective approach to improve the efficacy of antitumor drugs, especially for K‑ras mutant cancers.

Background: Euthyroid individuals with familial dysalbuminemic hyperthyroxinemia (FDH) have often falsely elevated serum free thyroxine (fT4) concentrations determined by different automated immunoassays. Methods: We measured serum fT4 using direct dialysis coupled with tandem mass spectrometry (fT4 DDMS) in individuals with the common albumin gene mutation ( ALB R218H) from 14 FDH families and compared them with results obtained by direct immunometric assay (fT4 DIMM) and free thyroxine index (fT4I). Results: While all 14 individuals with FDH had elevated total serum T4, the fT4 measured by DIMM was elevated in 12, by fT4I in 5, and by DDMS in 1. Conclusion: The latter method greatly reduced the discordance of fT4 results relative to thyrotropin in FDH.

Background: Durable transgene expression from plasmid DNAs is the key to gene therapy with non-viral vectors. A comparison of the durability of transgene expression from plasmid DNAs with the CpG-free and -containing backbones is important., Methods: We constructed plasmid DNAs with the CpG-containing backbone, various transcription regulatory sequences with and without CpG, and the gene encoding Gaussia princeps luciferase, which is apparently non-immunogenic. The tail vein hydrodynamics-based method was used for plasmid injection into mice, and the luciferase activity in serum was tracked for 28 days., Results: The plasmid DNAs containing the albumin promoter [with or without the cytomegalovirus (CMV) enhancer] and the elongation factor (EF)1α promoter plus the CMV enhancer exhibited long-term luciferase expression. The expression from the plasmid DNA containing the albumin promoter without the CMV enhancer was maintained for at least 24 weeks and was similar to that from the corresponding CpG-free plasmid DNA., Conclusions: The results obtained in the present study suggest that special sequences/systems are unnecessary for durable transgene expression from plasmid DNAs when the proper transcription regulatory sequences are used., (© 2019 John Wiley & Sons, Ltd.)

Aims: The histological distinction of intrahepatic cholangiocarcinoma (ICC) from metastatic adenocarcinoma remains a challenge. The primary goal was to evaluate the diagnostic value of morphology and albumin expression in the diagnosis of ICC., Methods: We evaluated morphological patterns in 120 ICCs and 677 non-hepatic adenocarcinomas and performed in situ hybridisation (ISH) stain for albumin in the former cohort (retrospective cohort). We also identified 119 samples from primary and metastatic lesions, the validation cohort, in which albumin ISH was performed as part of the diagnostic workup. Targeted sequencing was performed on selected cases. We also mined existing expression profiling data including cases from The Cancer Genome Atlas (TCGA) (41 760 unique samples)., Results: In the retrospective cohort, 45% of ICCs and <1% of non-hepatic adenocarcinomas showed a cholangiolar pattern; albumin ISH was positive in 93% of ICCs with significant intratumorous heterogeneity. In the validation cohort, 29% of ICCs showed a cholangiolar pattern and 88% expressed albumin, while all metastatic non-hepatic neoplasms were negative (n=37) (sensitivity 88% and specificity 100%). Targetable genetic alterations ( IDH mutations and FGFR2 fusions) were identified in 31% of ICCs (10 of 32). An analysis of the TCGA data validated the specificity of the albumin assay., Conclusions: The cholangiolar pattern and albumin RNA ISH distinguishes ICC from metastatic adenocarcinoma with high specificity. Given the high prevalence of targetable mutations in ICC, albumin RNA ISH is an essential component in the workup of tumours of uncertain origin. A specific diagnosis of ICC could trigger molecular testing and uncover targetable genetic alterations., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Hemophilia A, a bleeding disorder resulting from F8 mutations, can only be cured by gene therapy. A promising strategy is CRISPR-Cas9-mediated precise insertion of F8 in hepatocytes at highly expressed gene loci, such as albumin (Alb). Unfortunately, the precise in vivo integration efficiency of a long insert is very low (~ 0.1%)., Results: We report that the use of a double-cut donor leads to a 10- to 20-fold increase in liver editing efficiency, thereby completely reconstituting serum F8 activity in a mouse model of hemophilia A after hydrodynamic injection of Cas9-sgAlb and B domain-deleted (BDD) F8 donor plasmids. We find that the integration of a double-cut donor at the Alb locus in mouse liver is mainly through non-homologous end joining (NHEJ)-mediated knock-in. We then target BDDF8 to multiple sites on introns 11 and 13 and find that NHEJ-mediated insertion of BDDF8 restores hemostasis. Finally, using 3 AAV8 vectors to deliver genome editing components, including Cas9, sgRNA, and BDDF8 donor, we observe the same therapeutic effects. A follow-up of 100 mice over 1 year shows no adverse effects., Conclusions: These findings lay the foundation for curing hemophilia A by NHEJ knock-in of BDDF8 at Alb introns after AAV-mediated delivery of editing components.

Hemophilia A is a monogenic disease with a blood clotting factor VIII (FVIII) deficiency caused by mutation in the factor VIII (F8) gene. Current and emerging treatments such as FVIII protein injection and gene therapies via AAV-delivered F8 transgene in an episome are costly and nonpermanent. Here, we describe a CRISPR/Cas9-based in vivo genome editing method, combined with non-homologous end joining, enabling permanent chromosomal integration of a modified human B domain deleted-F8 (BDD-F8) at the albumin (Alb) locus in liver cells. To test the approach in mice, C57BL/6 mice received tail vein injections of two vectors, AAV8-SaCas9-gRNA, targeting Alb intron 13, and AAV8-BDD-F8. This resulted in BDD-F8 insertion at the Alb locus and FVIII protein expression in the liver of vector-, but not vehicle-, treated mice. Using this approach in hemophilic mice, BDD-F8 was expressed in liver cells as functional human FVIII, leading to increased plasma levels of FVIII and restoration of blood clotting properties in a dose-dependent manor for at least 7 months, with no detectable liver toxicity or meaningful off-target effects. Based on these findings, our BDD-F8 genome editing approach may offer an efficacious, long-term and safe treatment for patients with hemophilia A.

Background: Stem cell-derived pancreatic β-like cells hold great promise for treating diabetes. Gallbladder belongs to the extrahepatic bile duct system and possesses stem-like cells. These stem cells could be expanded in vitro and have the potential of differentiating into hepatocytes, cholangiocytes, or pancreatic cells. As the gallbladder is highly available, gallbladder stem cells provide a new cell source of pancreatic β-like cells. In this study, we aimed to investigate an approach for the generation of pancreatic β-like cells from gallbladder stem cells (GSCs) without genetic modification., Methods: A CK19Cre ERT ;Rosa26R-GFP mouse was used to isolate CK19 + cells, which represented EpCAM + stem cells in the gallbladder. They were cultured in the modified Kubota's medium for expansion and further analyzed. Then, we developed a strategy to screen a combination of small molecules that can generate insulin-secreting cells from gallbladder stem cells. These cells were identified with markers of pancreatic cells. Finally, they were seeded into the cellulosic sponge and transplanted to the diabetic mice for functional examination in vivo., Results: Gallbladder stem cells could be expanded for more than 15 passages. They expressed typical hepatic stem cell markers including CK19, EpCAM, Sox9, and albumin. By screening method, we found that adding Noggin, FR180204, and cyclopamine could efficiently induce gallbladder stem cells differentiating into insulin-secreting cells. These cells expressed Pdx1, Nkx6.1, and insulin but were negative for Gcg. After transplantation with the cellulosic sponge, they could ameliorate hyperglycemia in the diabetic mice., Conclusion: This study provides a new approach which can generate insulin-secreting cells from the gallbladder without genetic modification. This offers an option for β cell therapy in treating type 1 diabetes.

Objectives: Albumin messenger RNA (mRNA) expression is a marker of hepatocellular differentiation. Most published data are from review of tissue microarrays, and albumin in situ hybridization (ISH) expression across several tumor types is incompletely characterized., Methods: Sections from 221 tumors were evaluated for albumin mRNA. Immunohistochemistry was used to confirm diagnoses. Albumin ISH was performed according to manufacturer-provided instructions. Fifty-nine cases were evaluated with both commercial ISH assays., Results: Albumin mRNA was detected in all hepatocellular carcinomas (HCCs) and 81% of intrahepatic cholangiocarcinomas. Lung (20%), gallbladder (39%), hepatoid pancreatic (n = 1 of 1) adenocarcinoma, breast invasive ductal carcinoma (18%), yolk sac tumor (25%), and acinar cell carcinoma (29%) showed expression. Both assays were concordant in 93% of cases., Conclusions: Albumin ISH was expressed in all HCCs studied. It was also positive in intrahepatic cholangiocarcinoma and patchy positive in gallbladder adenocarcinoma and a subset of other neoplasms, which can be a potential pitfall., (© American Society for Clinical Pathology, 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Remodeling of the gene regulatory network in cells is believed to be a prerequisite for their lineage reprogramming. However, its key regulatory factors are not yet elucidated. In this article, we investigate the role of PIWI proteins and provide evidence that one of them, MIWI2, is elicited during transdifferentiation of fibroblasts into hepatocyte-like cells. In coincidence with the peak expression of MIWI2, we identified the appearance of a unique intermediate epigenetic state characterized by a specific Piwi-interacting RNA (piRNA) profile consisting of 219 novel sequences. Knockout of MIWI2 greatly improved the formation of the induced hepatocytes, whereas overexpression of exogenous MIWI2 completely abolished the stimulated effect. A bioinformatics analysis of piRNA interaction network, followed by experimental validation, revealed the Notch signaling pathway as one of the immediate effectors of MIWI2. Altogether, our results show for the first time that temporal expression of MIWI2 contributes negatively to cell plasticity not only in germline, but also in developed cells, such as mouse fibroblasts. Stem Cells 2019;37:803-812., (©2019 The Authors. Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press 2019.)