Hijacking of N-fixing legume albumin-1 genes enables the cyclization and stabilization of defense peptides.

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.

Metabolic responses to albumin deficiency differ distinctly between partial and full ablation of albumin expression in mice.

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.

Safe and effective liver-directed AAV-mediated homology-independent targeted integration in mouse models of inherited diseases.

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.

High albumen height by expression of GALNT9 and thin eggshell by decreased Ca2+ transportation caused high hatchability in Huainan partridge chicken.

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 Ca2+ 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 Ca2+ transportation so as to form a thinner eggshell and provide better gas exchange during embryo development.

Albumin promoter-driven FlpO expression induces efficient genetic recombination in mouse liver.

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-KrasG12D mice and p53frt 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 KrasG12D 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.

Structural optimization of siRNA conjugates for albumin binding achieves effective MCL1-directed cancer therapy.

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.

Novel genetic markers for chronic kidney disease in a geographically isolated population of Indigenous Australians: Individual and multiple phenotype genome-wide association study.

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.

Association analysis of production traits of Japanese quail (Coturnix japonica) using restriction-site associated DNA sequencing.

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.

Combining albumin deficiency and acute exercise reduces hepatic lipid droplet size in mice.

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.

Albumin fusion with human lactoferrin shows enhanced inhibition of cancer cell migration.

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.

Lung cancer risk in workers occupationally exposed to polycyclic aromatic hydrocarbons with emphasis on the role of DNA repair gene.

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.

An integrated RNA sequencing and network pharmacology approach reveals the molecular mechanism of dapagliflozin in the treatment of diabetic nephropathy.

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-ß 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.

Upregulation of miR145 and miR126 in EVs from Renal Cells Undergoing EMT and Urine of Diabetic Nephropathy Patients.

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-ß1-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.

Staphylococcus sciuri causes disease and pathological changes in hybrid sturgeon acipenser baerii × acipenser schrencki.

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.

Novel AAV-mediated genome editing therapy improves health and survival in a mouse model of methylmalonic acidemia.

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.

Growth advantage of corrected hepatocytes in a juvenile model of methylmalonic acidemia following liver directed adeno-associated viral mediated nuclease-free genome editing.

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.

[Value of albumin RNAscope in situ hybridization in diagnosis and differential diagnosis of hepatocellular carcinoma].

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.

A 3-Mbp fragment on rat chromosome 1 affects susceptibility both to stroke and kidney injury under salt loading in the stroke-prone spontaneously hypertensive rat: a genetic approach using multiple congenic strains.

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.

Renal Expression of CLC-5 and Megalin/Cubilin in Dent-1 Disease With Nonsense Mutations of CLCN5 Gene.

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, ß2-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.

Cell-Type-Specific Circadian Bioluminescence Rhythms in Dbp Reporter Mice.

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;DbpKI/+ "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.