Albumin Gene Research Articles

Evolutionary Transformations of Albumin Using the Example of Model Species of Jawless Agnatha and Bony Jawed Fish (Review)

Issues of the organization of albumin (a protein belonging to the superfamily of albuminoids) are considered using the example of model representatives of lower aquatic vertebrates: jawless Agnatha (Cyclostomata) and jawed bony lobe-finned (Sarcopterygii) and ray-finned (Actinopterygii: Chondrostei, Holostei, Teleostei) fish. A comparative analysis is carried out of the total length and the length of coding and noncoding sequences of the albumin gene; the number of exons, the length of introns, and the presence of repeated DNA elements in them; and the presence/absence of paralogous genes, their chromosomal affiliation, and the composition of syntenic groups. A comparative analysis of the length of the amino acid sequence, the number of domains, and S–S bonds in the albumin structure is also carried out. The issues of the emergence of the ancestral gene for albuminoids, the “expansion” and “narrowing” of the albuminoid repertoire in different groups of vertebrates, the appearance of a cluster of paralogous genes within a single chromosome in the Sarcopterygii branch and paralogous genes on different chromosomes in the Actinopterygii branch in the representatives of cartilaginous ganoids and teleost fish, the elongation of the gene and protein chain of albumin in the lamprey line, and losses of the albumin gene in the largest groups of teleost fish (Ostariophysi and Acanthopterygii) are discussed. The listed scenarios are considered in light of their consistency with the events of whole genome, local, and segment duplications. The problem of compensating for the functions of the “lost” albumin gene due to multiple osmotically active plasma proteins is discussed using the example of the group of teleost fish.

RNAi and CRISPR-Cas silencing E3-RING ubiquitin ligase AIP2 enhances soybean seed protein content.

The majority of plant protein in the world's food supply is derived from soybean (Glycine max). Soybean is a key protein source for global animal feed and is incorporated into plant-based foods for people, including meat alternatives. Soybean protein content is genetically variable and is usually inversely related to seed oil content. ABI3-interacting protein 2 (AIP2) is an E3-RING ubiquitin ligase that targets the seed-specific transcription factor ABI3. Silencing both soybean AIP2 genes (AIP2a and AIP2b) by RNAi enhanced seed protein content by up to seven percentage points, with no significant decrease in seed oil content. The protein content enhancement did not alter the composition of the seed storage proteins. Inactivation of either AIP2a or AIP2b by a CRISPR-Cas9-mediated mutation increased seed protein content, and this effect was greater when both genes were inactivated. Transactivation assays in transfected soybean hypocotyl protoplasts indicated that ABI3 changes the expression of glycinin, conglycinin, 2S albumin, and oleosin genes, indicating that AIP2 depletion increased seed protein content by regulating activity of the ABI3 transcription factor protein. These results provide an example of a gene-editing prototype directed to improve global food security and protein availability in soybean that may also be applicable to other protein-source crops.

Characterization of 2S albumin allergenic proteins for anaphylaxis in common buckwheat

2S albumin (2SA) is responsible for anaphylaxis following consumption of buckwheat in allergic individuals. To reduce allergen incidents, characterization of 2SA polypeptides is prerequisite, thus was analyzed in this study. Of the five 2S albumin genes (g03, g11, g13, g14, and g28), g03 was seemingly non-functional. The g14 content was 3- and 40-fold higher than that of g11/g28 and g13, respectively. The g11/g28 were more processed to a ∼8 kDa band from a 16 kDa band than g14 in seeds, agreeing with that g11/g28 have high similarity with Fag e 8kD. Meanwhile, anti-g13 produced only a single ∼10 kDa band. Modification of g13 and domain exchange between g13 and g14 suggested that the hydrophobicity of the first domain and the nature of some amino acids in g13 contributed, at least in part, to the lower apparent molecular weight of g13 than expected. Thus, g13 might be an unexplored and noteworthy allergen.

Abstract 2255: Alu retroelement copy number alterations and non-Hodgkin lymphoma

Abstract Background: Alu retrotransposons are repetitive DNA elements that make up nearly 10% of the human genome and reflect genomic instability. Genomic instability plays an important role in the pathogenesis of non-Hodgkin lymphoma (NHL). However, the relationship between Alu retrotransposons and future risk of NHL is unclear. Methods: We conducted a nested case-control study of NHL risk and Alu retroelement copy number within the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) study, a prospective cohort with prediagnostic blood samples from 29,133 male Finnish smokers. We measured Alu copy number in 107 incident NHL cases and 1:1 matched controls (± 5 years age). DNA samples extracted from blood were assayed in triplicate using a monochrome multiplex quantitative polymerase chain reaction. We used the albumin gene for the single copy gene to control for genome content in samples. The odds ratios (OR) and 95% confidence intervals (CI) for all NHL were estimated using conditional logistic regression models, adjusting for age at randomization, number of cigarettes smoked per day and smoking duration. Polytomous logistic regression models were fit to compute ORs with Alu copy number for NHL subtypes. To determine if the association might be driven in part by elevated Alu copy number among undiagnosed cases at the time of blood sample collection, we excluded cases diagnosed within the first two years of follow-up. Results: Higher Alu copy number (using the median as the cut-point) was associated with an increased risk of NHL, both overall [OR, 95% CI=3.80 (1.76-8.17)] as well as for the diffuse large B-cell lymphoma [OR, 95% CI=4.83 (1.50-15.55)] and chronic lymphocytic leukemia/small lymphocytic lymphoma (SLL) [OR, 95% CI=2.66 (1.10-6.40)] subtypes. These associations were similar after excluding individuals diagnosed with NHL within two years after blood draw. Conclusions: These findings, which require replication, suggest that higher Alu copy number in prediagnostic leukocytes may play a role in the pathogenesis of NHL. Citation Format: Wei Hu. Alu retroelement copy number alterations and non-Hodgkin lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2255.

Purslane (Portulaca oleracea L.) as a novel green-bioreactor for expression of human serum albumin (HSA) gene.

Transgenic plants showed high potential to become a valuable and safe source of bio-compounds that can be used as therapeutics without any require for pooled human blood products. Human serum albumin (HSA) is one of the best-selling pharmaceuticals in the world because it is utilized for treating several acute illnesses, including hypovolemia, burns, and hemorrhage. This work was aimed to investigate the production of recombinant HSA (rHSA) protein in a plant-based expression platform. For this, we used in-planta and tissue culture-based Agrobacterium-mediated transformation (TCBAT) procedures to insert HSA gene into purslane (Portulaca oleracea L.) genome. The purslane seeds and leaves were infected with A. tumefaciens strain LBA4404 containing the HSA gene on pBI121 plasmid, and then regenerated into transgenic plant on MS medium. The qRT-PCR, southern hybridization, western blotting, and ELISA analysis were accomplished to corroborate the insertion and expression of HSA gene in transgenic plantlets. The molecular asses indicated that HSA gene was successfully transferred and expressed in purslane plants using in-planta and TCBAT methods. The first attempt to express rHSA in purslane resulted in a low-level accumulation of the protein in the transgenic plant shoots. Therefore, we used a synthetic 5'UTR (synJ) to enhance HSA transcript stability and translation efficiency. The results suggested that the synJ caused pronounced enhancement of rHSA expression rate. The highest amount of rHSA protein was recorded in transgenic purslane generated by TCBAT method (33.92 ± 4.31µg/g FW).

Amelioration of aflatoxin acute hepatitis rat model by bone marrow mesenchymal stem cells and their hepatogenic differentiation.

Background and Aim:Bone marrow-derived mesenchymal stem cells (BM-MSCs) transplantation and their hepatogenic differentiated cells (HDCs) can be applied for liver injury repair by tissue grafting. Regenerative potentiality in liver cirrhosis models was widely investigated; however, immunomodulation and anti-inflammation in acute hepatitis remain unexplored. This study aimed to explore the immunomodulatory and evaluate twice intravenous (IV) or intrahepatic (IH) administration of either BM-MSCs or middle-stage HDCs on aflatoxin (AF) acute hepatitis rat model.Materials and Methods:BM-MSCs viability, phenotypes, and proliferation were evaluated. Hepatogenic differentiation, albumin, and mmmmmmmm-fetoprotein gene expression were assessed. AF acute hepatitis was induced in rats using AFB1 supplementation. The transplantation of BM-MSCs or their HDCs was done either by IV or IH route. Hepatic ultrasound was performed after 3-weeks of therapy. Cytokines profile (tumor necrosis factor-α [TNF-α], interleukin [IL]-4, and IL-10) was assessed. Hepatic bio-indices, serum, and hepatic antioxidant activity were evaluated, besides examining liver histological sections.Results:Acute AFB1 showed a significant increase in TNF-α (p<0.01), liver enzyme activities (p<0.05), as well as decrease in IL-4, IL-10, and antioxidant enzyme activities (p<0.05). Cytokines profile was ameliorated in groups treated with IV and IH BM-MCs, showed a negative correlation between IL-4 and TNF-α (p<0.05), and a positive correlation between IL-10 upregulation and TNF-α (p<0.01). In IV HDCs treated group, positive correlations between IL-4 and IL-10 downregulation and TNF-α were observed. However, in IH HDCs group, a significant positive correlation between IL-4 and IL-10 upregulation and TNF-α, were recorded (p<0.05). In addition, IV BM-MSCs and IH HDCs treatments significantly increased antioxidant enzymes activity (p<0.05). IV and IH BM-MSCs significantly ameliorated liver transaminase levels, whereas IH HDCs significantly ameliorated alanine aminotransferase activity and nitric oxide concentration (p<0.05).Conclusion:The administration routes of BM-MSCs did not demonstrate any significant difference; however, the IH route of HDCs showed significant amelioration from the IV route. On the other hand, it showed noticeable anti-inflammatory and immunomodulatory improvements in aflatoxicosis rats. Therefore, it can be concluded that acute hepatitis can be treated by a noninvasive IV route without the expense of hepatogenic differentiation. Further research using clinical trials that address several problems regarding engraftment and potentiation are needed to determine the optimal manipulation strategy as well as to achieve better long term effects.

Hepatocyte Differentiation from iPSCs or MSCs in Decellularized Liver Scaffold: Cell–ECM Adhesion, Spatial Distribution, and Hepatocyte Maturation Profile

ABSTRACT Mesenchymal stem cells (MSC) and induced pluripotent stem cells (iPSC) have been reported to be able to differentiate to hepatocyte in vitro with varying degree of hepatocyte maturation. A simple method to decellularize liver scaffold has been established by the Department of Histology, Faculty of Medicine, Universitas Indonesia, in SCTE IMERI lab.15 This study aims to evaluate hepatocyte differentiation from iPSCs compared to MSCs derived in our decellularized liver scaffold. The research stages started with iPSC culture, decellularization, seeding cell culture into the scaffold, and differentiation into hepatocytes for 21 days. Hepatocyte differentiation from iPSCs and MSCs in the scaffolds was characterized using hematoxylin–eosin, Masson Trichrome, and immunohistochemistry staining to determine the fraction of the differentiation area. RNA samples were isolated on days 7 and 21. Expression of albumin, CYP450, and CK-19 genes were analyzed using the qRT-PCR method. Electron microscopy images were obtained by SEM. Immunofluorescence examination was done using HNF4-α and CEBPA markers. The results of this study in hepatocyte-differentiated iPSCs compared with hepatocyte-differentiated MSCs in decellularized liver scaffold showed lower adhesion capacity, single-cell-formation and adhered less abundant, decreased trends of albumin, and lower CYP450 expression. Several factors contribute to this result: lower initial seeding number, which causes only a few iPSCs to attach to certain parts of decellularized liver scaffold, and manual syringe injection for recellularization, which abruptly and unevenly creates pattern of single-cell-formation by hepatocyte-differentiated iPSC in the scaffold. Hepatocyte-differentiated MSCs have the advantage of higher adhesion capacity to collagen fiber decellularized liver scaffold. This leads to positive result: increase trends of albumin and higher CYP450 expression. Hepatocyte maturation is shown by diminishing CK-19, which is more prominent in hepatocyte-differentiated iPSCs in decellularized liver scaffold. Confirmation of mature hepatocyte-differentiated iPSCs in decellularized liver scaffold maturation is positive for HNF4-a and CEBPA. The conclusion of this study is hepatocyte-differentiated iPSCs in decellularized liver scaffold is mature with lower cell–ECM adhesion, spatial cell distribution, albumin, and CYP450 expression than hepatocyte-differentiated MSCs in decellularized liver scaffold.

New insight into the roles of lipid transfer protein and seed storage albumin gene families involved in oil and protein accumulation in rapeseed (Brassica napus L.)

In rapeseed ( Brassica napus L.), lipid transfer protein (LTP) and seed storage albumin (SSA) gene families are involved in lipid and protein metabolism. Understanding the mechanism of oil biosynthesis has great value to increase oil production. The LTPs and SSA gene families were identified of Brassicaceae. The LTPs were divided into six groups according to their sequences, and LTPs of all five species in Brassicaceae also had the same pattern. The genes at a higher expression level were evenly distributed across five groups except group ii. The gene groups in these two gene families showed different expression patterns, with most of the LTP genes expressed at a higher level at 25 days after flowering (DAF), but SSA genes were highly expressed at 40 DAF stage. Gene Ontology (GO) enrichment analysis of the regulatory genes for LTPs also was performed, including response to biotic stimulus (GO:0009607), generation of precursor metabolites and energy (GO:0006091), electron transport chain (GO:0022900), oxidative phosphorylation (GO:0006119), adenosine triphosphate metabolic process (GO:0046034), and phosphorylation (GO:0016310). These results may facilitate further research to understand the expression patterns and regulatory mechanism of LTPs and SSAs.

A hierarchical regulatory network ensures stable albumin transcription under various pathophysiological conditions.

It remains unknown how patients with liver failure maintain essential albumin levels. Here, we delineate a hierarchical transcription regulatory network that ensures albumin expression under different disease conditions. We examined albumin levels in liver tissues and serum in 157 patients, including 84 with HCC, 38 decompensated cirrhosis, and 35 acute liver failure. Even in patients with liver failure, the average serum albumin concentrations were 30.55 g/L. In healthy subjects and patients with chronic liver diseases, albumin was expressed in hepatocytes. In patients with massive hepatocyte loss, albumin was expressed in liver progenitor cells (LPCs). The albumin gene (ALB) core promoter possesses a TATA box and nucleosome-free area, which allows constitutive RNA polymerase II binding and transcription initiation. Chromatin immunoprecipitation assays revealed that hepatocyte nuclear factor 4 alpha (HNF4α), CCAAT/enhancer-binding protein alpha (C/EBPα), and forkhead box A2 (FOXA2) bound to the ALB enhancer. Knockdown of either of these factors reduced albumin expression in hepatocytes. FOXA2 acts as a pioneer factor to support HNF4α and C/EBPα. In hepatocytes lacking HNF4α and C/EBPα expression, FOXA2 synergized with retinoic acid receptor (RAR) to maintain albumin transcription. RAR nuclear translocation was induced by retinoic acids released by activated HSCs. In patients with massive hepatocyte loss, LPCs expressed HNF4α and FOXA2. RNA sequencing and quantitative PCR analyses revealed that lack of HNF4α and C/EBPα in hepatocytes increased hedgehog ligand biosynthesis. Hedgehog up-regulates FOXA2 expression through glioblastoma family zinc finger 2 binding to the FOXA2 promoter in both hepatocytes and LPCs. A hierarchical regulatory network formed by master and pioneer transcription factors ensures essential albumin expression in various pathophysiological conditions.

Variations in the Human Serum Albumin Gene: Molecular and Functional Aspects

The human albumin gene, the most abundant serum protein, is located in the long arm of chromosome 4, near the centromere, position 4q11–3. It is divided by 14 intervening introns into 15 exons, the last of which is untranslated. To date, 74 nucleotide substitutions (mainly missense) have been reported, determining the circulating variants of albumin or pre-albumin. In a heterozygous state, this condition is known as alloalbuminaemia or bisalbuminaemia (OMIM # 103600). The genetic variants are not associated with disease, neither in the heterozygous nor in the homozygous form. Only the variants resulting in familial dysalbuminaemic hyperthyroxinaemia and hypertriiodothyroninaemia are of clinical relevance because affected individuals are at risk of inappropriate treatment or may have adverse drug effects. In 28 other cases, the pathogenic variants (mainly affecting splicing, nonsense, and deletions), mostly in the homozygous form, cause a premature stop in the synthesis of the protein and lead to the condition known as congenital analbuminaemia. In this review, we will summarize the current knowledge of genetic and molecular aspects, functional consequences and potential therapeutic uses of the variants. We will also discuss the molecular defects resulting in congenital analbuminaemia, as well as the biochemical and clinical features of this rare condition

Development and performance evaluation of whole-genome sequencing with paired-end and mate-pair strategies in molecular characterization of GM crops: One GM rice 114-7-2 line as an example

Basic data for the safety assessment of transgenic line involves the molecular characterization of the integration site of exogenous DNA, flanking sequences, copy number, and unintended plasmid backbone residues. However, performing a full molecular characterization remains challenging, especially for GMOs that possess complex exogenous DNA integrations. We established two whole-genome sequencing strategies: paired-end and mate-pair, to characterize the exogenous DNA integration of a human serum albumin gene into rice line 114-7-2, and evaluated the performance of these two strategies in the molecular characterization of transgenic line. The results showed the existence of two exogenous DNA insertion loci (Chr 01 and Chr 04) and their corresponding flanking sequences, five copies of the exogenous rHSA gene, and the presence of unintended residual plasmid backbone sequences. However, the WGS-MP strategy demonstrated higher efficiency, lower cost, and lower background noise compared with the WGS-PE analysis, especially for identification of the exogenous DNA integration site.

Biological activity and subchronic toxicity study of Allanblackia floribunda Oliv. on experimental rats

Allanblackia floribunda Oliv. (Elephant rice) belongs to the family Clusiaceae. It is confined to tropical Africa where it is used traditionally for the treatment of malaria, asthma and inflammatory diseases. This study evaluated the biological activity of methanol extract of A. floribunda's stem bark and leaf in addition to the seed oil as well as the subchronic toxicity of the stem bark extract. Standards procedures were used to screen the biological activity of A. floribunda. The subchronic toxicity profile was investigated by administering orally 100, 300 and 600 mg/kg b. wt of the stem bark extract to male Albino rats for 28 days consecutively. After the treatment period, changes in the various biochemical parameters and alteration in the expression of antioxidants, cytokine, Kidney injury molecule-1 (KIM-1) and albumin genes of the sensitive organs were used to assess the safety level of the stem bark extract. The stem bark extract had more phytochemicals and better antioxidant activities compared with the leaf extract and oil. Administration of the stem bark extract did not result in treatment related changes in markers for hepatic and renal injury (aspartate transaminase, alanine transaminase, total protein, albumin, globulin, creatinine and urea). The extract resulted in an increase in hepatic malondialdehyde. Also, hepatic and renal glutathione levels increased significantly. The blood composition was normal and the genes coding for albumin in the liver together with catalase in the liver and kidney were up-regulated. Tumor necrosis factor – alpha (TNFα) gene expression was down-regulated in the liver and up-regulated in the kidney while KIM-1 gene was down-regulated in the treated animals compared with the control. This study confirmed that A. floribunda's stem bark extract is richer in bioactive compounds than the leaf extract and seed oil. Furthermore, it is relatively safe to animals when used in moderate dose.

Deploying new generation sequencing for the study of flesh color depletion in Atlantic Salmon (Salmo salar)

BackgroundThe flesh pigmentation of farmed Atlantic salmon is formed by accumulation of carotenoids derived from commercial diets. In the salmon gastrointestinal system, the hindgut is considered critical in the processes of carotenoids uptake and metabolism. In Tasmania, flesh color depletion can noticeably affect farmed Atlantic salmon at different levels of severity following extremely hot summers. In this study, RNA sequencing (RNA-Seq) was performed to investigate the reduction in flesh pigmentation. Library preparation is a key step that significantly impacts the effectiveness of RNA sequencing (RNA-Seq) experiments. Besides the commonly used whole transcript RNA-Seq method, the 3’ mRNA-Seq method is being applied widely, owing to its reduced cost, enabling more repeats to be sequenced at the expense of lower resolution. Therefore, the output of the Illumina TruSeq kit (whole transcript RNA-Seq) and the Lexogen QuantSeq kit (3’ mRNA-Seq) was analyzed to identify genes in the Atlantic salmon hindgut that are differentially expressed (DEGs) between two flesh color phenotypes.ResultsIn both methods, DEGs between the two color phenotypes were associated with metal ion transport, oxidation-reduction processes, and immune responses. We also found DEGs related to lipid metabolism in the QuantSeq method. In the TruSeq method, a missense mutation was detected in DEGs in different flesh color traits. The number of DEGs found in the TruSeq libraries was much higher than the QuantSeq; however, the trend of DEGs in both library methods was similar and validated by qPCR.ConclusionsFlesh coloration in Atlantic salmon is related to lipid metabolism in which apolipoproteins, serum albumin and fatty acid-binding protein genes are hypothesized to be linked to the absorption, transport and deposition of carotenoids. Our findings suggest that Grp could inhibit the feeding behavior of low color-banded fish, resulting in the dietary carotenoid shortage. Several SNPs in genes involving in carotenoid-binding cholesterol and oxidative stress were detected in both flesh color phenotypes. Regarding the choice of the library preparation method, the selection criteria depend on the research design and purpose. The 3’ mRNA-Seq method is ideal for targeted identification of highly expressed genes, while the whole RNA-Seq method is recommended for identification of unknown genes, enabling the identification of splice variants and trait-associated SNPs, as we have found for duox2 and duoxa1.

Bioengineered 3D electrospun nanofibrous scaffold with human liver cells to study alcoholic liver disease in vitro.

Alcohol injury induces hepatic fibrosis which gradually progresses to cirrhosis, sometimes may lead to liver cancer. Animal models are less efficient in mimicking responses of human liver cells, whereas in vitro models discussed so far are majorly based on rodent cells. In this work, a coculture of primary human hepatocytes (PHHs) with LX-2 cells was established on the unmodified (C:F_0:0), collagen-I modified (C:F_1:0), fibronectin modified (C:F_0:1) and 3:1 collagen-I to fibronectin modified (C:F_3:1) 3D electrospun fibrous scaffolds. The effect of alcohol injury was evaluated on this cell-scaffold model at 0-40μl/ml alcohol concentrations over 14days of culture period by using the gold standard sandwich culture as the control. Among all the culture groups, C:F_3:1 scaffold was able to maintain translational and transcriptional properties of human liver cells at all concentrations of alcohol treatment. The study reveals that, PHHs on C:F_3:1 were able to maintain ~4-fold and ~1.6-fold higher secretion of albumin than the gold standard sandwich culture on Day 3 and Day 7, respectively. When treated with alcohol, at concentrations of 20 and 40μl/ml, albumin secretion was also observed to be higher (~2-fold) when compared to the gold standard sandwich culture. Again as expected, in C:F_3:1 culture group on 40μl/ml alcohol treatment, albumin gene expression decreased by ~2-fold due to alcohol toxicity, whereas CYP2C9, CYP3A4, CYP2E1 and CYP1A2 gene expressions upregulated by ~3.5, ~~4, ~5 and ~15-fold, respectively in response to the alcohol injury. LX-2 cells also acquire more quiescent phenotype on C:F_3:1 scaffolds when compared to the gold standard sandwich culture upon alcohol treatment. Thus, C:F_3:1 scaffold with human liver cells was established as the potential platform to scan alcohol toxicity at varied alcohol concentrations. Thus, it can pave a promising path not only to support functional healthy human liver cells for liver tissue engineering but also to examine potential drugs to study the progression or inhibition of alcoholic liver fibrosis in vitro.

Alginate-chitosan core-shell microcapsule cultures of hepatic cells in a small scale stirred bioreactor: impact of shear forces and microcapsule core composition

A small scale stirred bioreactor was designed and the effect of different agitation rates (30, 60 and 100 rpm) was investigated on HepG2 cells cultured in alginate-chitosan (AC) core-shell microcapsule in terms of the cell proliferation and liver-specific function. The microencapsulated hepatic cells could proliferate well when they were cultured for 10 days at 30 rpm while the cell-laden microcapsules showed no cell proliferation at 100 rpm in the bioreactor system. Albumin production rate, as an important liver function, increased also 1.8- and 1.5- fold under stirring rate of 30 rpm compared to the static culture and 60 rpm of agitation, respectively. Moreover, In comparison with the static culture, about 1.5-fold increment in urea production was observed at 30 rpm. Similarly, the highest expressions of albumin and P450 genes were found at 30 rpm stirring rate, which were 4.9- and 19.2-fold of the static culture. Addition of collagen to the microcapsule core composition (ACol/C) could improve the cell proliferation and functionality at 60 rpm in comparison with the cell-laden microcapsules without collagen. The study demonstrated the hepatic cell-laden ACol/C microcapsule hydrogel cultured in the small scale stirred bioreactor at low mixing rate has a great potential for mass production of the hepatic cells while maintaining liver-specific functions.

Integrative Analysis of Transcriptomic and Epigenomic Dynamics of Induced Pluripotent Stem Cell Derived Hepatic Organoids Using Single Cell RNA-Seq and ATAC-Seq

Previously, we developed a novel method by which to generate functionally mature human hepatic organoids derived from pluripotent stem cells (PSCs), and the functionality and maturity of the organoids were validated by various experiments including bulk RNA-seq. In this study, to identify the heterogeneity and dynamics of transcriptomes and epigenomes of the organoids at the single-cell level, we applied single-cell RNA-seq (scRNA-seq) and single-cell ATAC-seq (scATAC-seq) to analyze liver organoids treated with two medium conditions—hepatic medium (HM, expandable condition) and differentiation medium (DM, more matured condition). The cells were separated into 13 distinct clusters corresponding to stages ranging from early to late by both scRNA-seq and scATAC-seq, and DM liver organoids were more similar in more differentiated liver cells compared to those in HM. Mitochondrial gene expression was increased in differentiated hepatocytes, with ATAC-seq peaks enriched near the mitochondrial control region. Many transcription factors (TFs)—both activators and repressors—were revealed throughout the differentiation of liver organoids. Different patterns of expression of alpha-fetoprotein (AFP) and albumin (ALB) were observed between liver organoids and adult liver tissues, and several open chromatin regions (OCRs) near AFP and ALB genes were found to be differentially regulated between the liver organoids and tissues. HNF4A, YY1 (Yin Yang 1) and CTCF binding sites near AFP and ALB genes were differentially occupied between embryo and adult liver tissues. Our integrative analysis of transcriptomic and epigenetic dynamics of the liver organoids can contribute to the understanding of transcriptional regulatory networks during liver development and to the further development of mature in vitro human liver models.

Circulating tumor cells detected with a microcavity array predict clinical outcome in hepatocellular carcinoma.

The present study aimed to establish a novel isolation strategy for circulating tumor cells (CTCs) using a microcavity array (MCA) system and to evaluate the clinical significance of CTCs in hepatocellular carcinoma (HCC). We examined recovery rates of HCC cell lines spiked into whole blood in MCA assay. Circulating tumor cells were isolated from peripheral blood samples (3 mL) of 7 healthy donors (HD), 14 patients with liver cirrhosis (LC), and 31 patients with HCC using the MCA system. Additionally, we investigated the mRNA expression of liver‐specific genes in isolated CTCs using qPCR. The recovery rates were 65.1% (HepG2), 76.7% (HuH7), and 99.0% (PLC/PRF/5). In HD and patients with LC and HCC, the CTC positivity rate (CTCs ≥10) and average CTC number were as follows: HD 0% and 0.1, LC 14.3% and 5.3, HCC 54.8% and 47.6, respectively. The CTC positivity rate in HCC was significantly higher than that in LC (p < 0.05). The number of CTCs was significantly higher in metastatic HCC (102.2 ± 160.6) than in localized HCC (8.2 ± 7.7) (p < 0.05). The expression of AFP, glypican‐3, EpCAM, and albumin (ALB) genes was detected in isolated CTCs. The positive CTCs (CTCs ≥10) significantly reduced the cumulative survival in patients with HCC (p = 0.025), especially in localized patients with HCC (p = 0.046). The newly developed MCA system has the potential to isolate CTCs from HCC with high sensitivity, and mRNA expression could be measured from CTCs. Identification of positive CTCs can help predict clinical outcome of patients with HCC. Thus, analysis of CTCs in patients with HCC may provide important information as a novel biomarker in disease progression.

Albumin protects the liver from tumor necrosis factor α-induced immunopathology.

Besides its oncotic power, albumin exerts pleiotropic actions, including binding, transport, and detoxification of endogenous and exogenous molecules, antioxidant activity, and modulation of immune and inflammatory responses. In particular, recent studies have demonstrated that albumin reduces leukocyte cytokine production. Here, we investigated whether albumin also has the ability to protect tissues from the damaging actions of these inflammatory mediators. We circumscribed our investigation to tumor necrosis factor (TNF) α, which exemplifies the connection between immunity and tissue injury. In vivo experiments in analbuminemic mice showed that these mice exhibit a more pronounced response to a model of TNFα-mediated liver injury induced by the administration of lipopolysaccharide (LPS) and D-galactosamine (D-gal). A tissue protective action against LPS/D-gal liver injury was also observed during the administration of human albumin to humanized mice expressing the human genes for albumin and neonatal Fc receptor (hAlb+/+ /hFcRn+/+ ) with preestablished carbon tetrachloride (CCl4 )-induced early cirrhosis. The cytoprotective actions of albumin against TNFα-induced injury were confirmed ex vivo, in precision-cut liver slices, and in vitro, in primary hepatocytes in culture. Albumin protective actions were independent of its scavenging properties and were reproduced by recombinant human albumin expressed in Oryza sativa. Albumin cytoprotection against TNFα injury was related to inhibition of lysosomal cathepsin B leakage accompanied by reductions in mitochondrial cytochrome c release and caspase-3 activity. These data provide evidence that in addition to reducing cytokines, the albumin molecule also has the ability to protect tissues against inflammatory injury.

Species composition of green frogs (Pelophylax Esculentus Complex) of the Belgorod agglomeration based on DNA markers

On the basis of molecular genetic analysis of the intron-1 of the nuclear serum albumin gene (SAI-1) were identified 177 individuals of Pelophylax esculentus complex of 9 localities Belgorod. Two types of population systems R and RE were identified. Pure populations of L-type, E-type and LE-type as well as P. lessonae individuals were not identified.

A novel nonsense variation in the albumin gene (c.1309 A>T) causing analbuminaemia

Congenital analbuminaemia (OMIM # 616,000) is a rare autosomal recessive disorder, characterized by the near-complete absence, or very low levels, of serum albumin. The clinical diagnosis is usuall...