Albumin Gene Research Articles

AAV-mediated genome editing is influenced by the formation of R-loops

Recombinant adeno-associated viral vectors (rAAV) hold an intrinsic ability to stimulate homologous recombination (AAV-HR) and are the most used in clinical settings for in vivo gene therapy. However, rAAVs also integrate throughout the genome. Here, we describe DNA-RNA immunoprecipitation sequencing (DRIP-seq) in murine HEPA1-6 hepatoma cells and whole murine liver to establish the similarities and differences in genomic R-loop formation in a transformed cell line and intact tissue. We show enhanced AAV-HR in mice upon genetic and pharmacological upregulation of R-loops. Selecting the highly expressed Albumin gene as a model locus for genome editing in both in vitro and in vivo experiments showed that the R-loop prone, 3’ end of Albumin was efficiently edited by AAV-HR, whereas the upstream R-loop-deficient region did not result in detectable vector integration. In addition, we found a positive correlation between previously reported off-target rAAV integration sites and R-loop enriched genomic regions. Thus, we conclude that high levels of R-loops, present in highly transcribed genes, may promote rAAV vector genome integration. These findings may shed light on potential mechanisms for improving the safety and efficacy of genome editing by modulating R-loops and may enhance our ability to predict regions most susceptible to off-target insertional mutagenesis by rAAV vectors.

Unraveling modulation effects on albumin synthesis and inflammation by Striatin, a bioactive protein fraction isolated from Channa striata: In silico proteomics and in vitro approaches

Hypoalbuminemia, associated with inflammation in severely ill patients, can emerge due to decreased albumin production. Transforming growth factor-beta (TGF-β) and nuclear factor-kappa B (NF-κB) are critical signaling pathways responsible for decreased albumin expression. This study explores the protein content and modulation effects of Striatin on albumin synthesis and inflammation, employing in silico proteomics and in vitro investigations. In the in silico proteomics realm, LC/MS-MS protein sequencing, 3D modeling, protein-protein docking simulations, 100 ns molecular dynamics (MD) simulations, and MM/PBSA binding free energy calculations were carried out. Complementing this, in vitro studies examined Albumin gene expression and extracellular secretion in HepG2 cells subjected to lipopolysaccharides-induced hypoalbuminemia. Furthermore, the study probed Striatin's influence on the NF-ᴋB expression, given albumin's role as a negative acute-phase protein. The results unveiled nucleoside diphosphate kinase (NdK) and parvalbumin (PV) as the prominent constituents within Striatin. Notably, NdK and PV exhibited the ability to disrupt hydrogen bonds with specific residues in both TGF-β and NF-κB complexes, thereby enhancing their flexibility, akin to the action of the FKBP12 complex (antagonist complex). In the in vitro experiments, Striatin demonstrated a dose and time-dependent inhibition of hypoalbuminemia, with peak efficacy observed at a concentration of 20 μg/mL. At this concentration, Striatin also suppressed NF-κB expression when co-incubated with lipopolysaccharides. While these findings suggest potential inhibitory effects of Striatin on TGF-β and NF-κB activities, they are preliminary and warrant further investigation. This study highlights Striatin's potential as a therapeutic agent for inflammation-related hypoalbuminemia, though additional research is needed to fully validate these results.

Inhibitors of LAMP used to detect Tenacibaculum sp. strain Pbs-1 associated with black-spot shell disease in Akoya pearl oysters, and additives to reduce the effect of the inhibitors

Black-spot shell disease is an unresolved disease that decreases pearl quality and threatens pearl oyster survival. In previous studies, the bacterium Tenacibaculum sp. strain Pbs-1 was isolated from diseased Akoya pearl oysters Pinctada fucata, and a rapid, specific, and sensitive loop-mediated isothermal amplification (LAMP) assay for detecting this pathogen was established. This technology has considerable potential for routine diagnosis of strain Pbs-1 in oyster hatcheries and/or pearl farms; therefore, it is vital to identify substances in environmental samples that might inhibit LAMP and to find additives that can reduce the inhibition. In this study, we investigated the effects of six chemicals or proteins, otherwise known as conventional PCR inhibitors, on LAMP, using the DNA of strain Pbs-1 as template: humic acid, urea, iron (III) chloride hexahydrate, melanin, myoglobin, and Ethylenediamine-N,N,N′,N′-tetraacetic acid, disodium salt, dihydrate (EDTA; pH 6.5). Next, to reduce the effects of identified inhibitors, we tested the addition of bovine serum albumin (BSA) or T4 gene 32 protein (gp32) to the LAMP assay. When 50 ng of DNA template was used, 4 ng/μL of humic acid, 0.05% melanin, and 10 mM of EDTA (pH 6.5) inhibited the LAMP reaction, whereas myoglobin, urea, and FeCl3 had no effect. When 50 pg of DNA template was used, 4 ng/μL of humic acid, 0.05% melanin, 4 μg/μL of myoglobin, 10 μg/μL of urea, and 10 mM of EDTA inhibited the LAMP reaction. Thus, it was shown that the gene-amplification inhibitory effect of melanin, humic acid, and urea could be reduced by adding BSA or gp32 to the LAMP reaction mixture. This technique could be applied as part of a protocol to prevent mass mortalities of pearl oysters; moreover, the results enhance our knowledge about substances that inhibit LAMP and methods to reduce the inhibition, which have rarely been reported.

Serum Afamin in Prediabetic Individuals in Association with Insulin Resistance and Glycemic Indices: A Case-Control Study

Background: Prediabetes is a metabolic disorder characterized by higher serum glucose levels than normal and below the diagnostic threshold for diabetes mellitus. Afamin is a glycoprotein mainly excreted from the liver and facilitates vitamin E transport. Afamin is one of the albumin gene family that includes albumin, α-Fetoprotein, and vitamin D-binding protein. Aim: This study aimed to evaluate serum afamin levels in prediabetic individuals and ascertain the association of serum afamin with insulin resistance and glycemic indices. Material and Methods: A case-control study was performed at Azadi Teaching Hospital, Kurdistan Region, Iraq, consisted of 88 participants, 44 prediabetic individuals and 44 apparently healthy individuals as a control group. Prediabetic individuals were obtained from relatives of patients with diabetes mellitus visiting Azadi Teaching Hospital, whereas, healthy individuals were chosen from the medical staff and college employees. The diagnosis of prediabetes was made according to the American Diabetes Association criteria. Serum afamin were assessed by Enzyme-Linked Immunosorbent Assay, while other biochemical parameters such as HbA1c, glucose and insulin were analyzed by Cobas 6000 (Roche, Hitachi/ Germany). Results: Mean level of serum afamin was higher in prediabetic individuals (115.31±62.74) compared to their level in the control group (91.57±43.46), with a statistically significant difference (p = 0.042). The mean age of prediabetics was (44.05±10.56) with males predominant (63.6%) and 70.5% of them were more than 40 years and 88.6% were obese. Conclusion: The study concluded the presence of higher mean serum afamin levels among prediabetic individuals compared to the healthy control.

Predicting prognosis in colorectal cancer patients with curative resection using albumin, lymphocyte count and RAS mutations

Colorectal cancer (CRC) poses a significant global health challenge, demanding reliable prognostic tools to guide treatment decisions. This study introduces a novel prognostic scoring system, the albumin-total lymphocyte count-RAS index (ALRI), integrating serum albumin, lymphocyte count, and RAS gene mutations. A cohort of 445 stage I–III CRC patients undergoing curative resection was analyzed, revealing ALRI's association with clinicopathological factors, including age, tumor location, and invasion depth. The ALRI demonstrated superior prognostic value, with a cutoff value of 2 distinguishing high and low-risk groups. The high-ALRI group exhibited elevated rates of recurrence. Univariate and multivariate analyses identified ALRI as an independent predictor for both 5 year recurrence-free survival (RFS) and overall survival (OS). Kaplan–Meier curves illustrated significant differences in RFS and OS between high and low-ALRI groups, emphasizing ALRI's potential as a prognostic marker. Importantly, ALRI outperformed existing nutritional indices, such as controlling nutritional status and neutrophil-to-lymphocyte ratio, in predicting overall survival. The study underscores the comprehensive insight provided by ALRI, combining inflammatory, nutritional, and genetic information for robust prognostication in CRC patients. This user-friendly tool demonstrates promise for preoperative prognosis and personalized treatment strategies, emphasizing the crucial role of inflammation and nutrition in CRC outcomes.

Abstract LB111: CORSA study - Telomere length measurement of 5371 Austrian colorectal cancer and adenoma patients and healthy controls: Relationships to tumor stage, course of disease and to clinical, life style and health parameters

Abstract CORSA study - Telomere length measurement of 5371 Austrian colorectal cancer and adenoma patients and healthy controls: relationships to tumor stage, course of disease and to clinical, life style and health parametersPetr Hanak1, Michal Kroupa1,3,4, Uttarilli Anusha1, Ludmila Vodickova1,3,4 Andrea Gsur2, Stefanie Brezina2, Pavel Vodicka1,3,4 1 Department of Molecular Biology of Cancer, Institute of Experimental Medicine of the Czech Academy ofSciences, Videnska 1083, 142 20 Prague, Czech Republic.2 Center for Cancer Research, Medical University of Vienna, Vienna, Austria;3 Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University, Prague, Czech Republic 4Faculty of Medicine and Biomedical center in Pilsen, Pilsen, Czech Republic Presenting author: Petr Hanák (e-mail: petr.hanak@iem.cas.cz)Telomeres are important parts of human chromosomes, playing a pivotal role in maintenance of genomic integrity. If their function is perturbed, it may trigger many pathologies, including colorectal cancer (CRC). In the present study, comparison of assessed relative telomere length (RTL) and tumor stage, progression of the disease and various health status and lifestyle parameters is investigated. Relative telomere length was measured in samples of DNA from peripheral blood lymphocytes of CRC patients by monochromatic multiplex qPCR. Repetitive telomere motif TTAGG was amplified from specific primer pair simultaneously with the reference albumin gene, the second one amplified from another set of primers specific to this gene. The qPCR reaction is based on intercalating dye Syto 9, which does not distinguish specific target. A separation of amplification of one target from the other one is based on division in time of both amplifications and on different levels of annealing temperatures of primer pairs. Relative telomere length was assessed in all patient samples. Obtained data correspond to our previously observed values. Here, we observe significant decreasing in RTL in the order healthy controls, adenoma and CRC patients (controls vs adenoma patients P<0.0001, controls vs CRC patients P=0.0001). Detailed statistical analyses and assignment to all relevant groups including health and lifestyle parameters is currently in progress. Acknowledgement & Funding: The study was funded by the Grant Agency of Czech Republic (21-27902S), National Institute for Cancer Research (Programme EXCELES, ID Project No. LX22NPO5102) - Funded by the European Union – Next Generation EU and Ministry of Health of the Czech Republic (NU22J-03-00028) Citation Format: Petr Hanák. CORSA study - Telomere length measurement of 5371 Austrian colorectal cancer and adenoma patients and healthy controls: Relationships to tumor stage, course of disease and to clinical, life style and health parameters [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB111.

SARS-COV-2 ORF9b Dysregulate Fibrinogen and Albumin Genes in a Liver Cell Line.

Individuals experiencing severe cases of Coronavirus Disease 2019 (COVID-19) exhibited elevated fibrinogen levels and decreased albumin levels, potentially linked to the presence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) proteins. Consequently, our study endeavors to examine the impact of SARS-CoV-2 ORF9b on the expression of fibrinogen and albumin genes within the Hep-G2 cell line. In this study, the Hep-G2 liver cell line was utilized alongside the plasmid pcDNA3.1 hyg+ containing ORF9b from the SARS-CoV-2 strain originating in Wuhan. Transfection procedures were executed, and the transfected cells were selected utilizing hygromycin B. Validation of ORF9b expression was conducted through SYBR green-based real-time PCR, and the expression of the Fibrinogen α (FGA), Fibrinogen β (FGB), Fibrinogen γ (FGG), and Albumin (ALB) genes was quantified using the same method. The real-time PCR analysis revealed a significant upregulation of fibrinogen genes-α (P=0.03), β (P=0.02), and γ (P=0.029) in Hep-G2 cells containing ORF9b compared to control cells. Furthermore, the findings indicated a markedly lower expression level of albumin in Hep-G2 cells harboring ORF9b compared to the control cells (P=0.028). The findings suggest that SARS-CoV-2 ORF9b could potentially influence the course of SARS-CoV-2 infection by triggering the expression of α, β, and γ fibrinogen gene chains while suppressing the albumin gene. Further investigations are warranted to validate these observations across various SARS-CoV-2 strains exhibiting differing levels of pathogenicity.

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.

Evaluation of Mir-1290 as a Possible Diagnostic Factor in the Serum of Oral Squamous Cell Carcinoma Patients with Qualitative Real-Time Polymerase Chain Reaction.

This study aimed to determine the incidence of microRNA (miRNA; miR-1290) in the serum of oral squamous cell carcinoma (OSCC) patients compared to a control group using the qualitative real-time polymerase chain reaction (PCR) method. Blood serum samples were obtained from patients diagnosed with OSCC and confirmed through biopsy. The samples were collected from patients referred to the Mashhad Dental Faculty and Ghaem Hospital. The OSCC group consisted of 17 patients, while the healthy group included 15 individuals. RNA was extracted from the patient samples, and samples with an A260/280 ratio between 1.8 and 2.0 (indicating acceptable RNA quality) were immediately converted into complementary DNA (cDNA) using albumin and cDNA reference genes. The SYBR green real-time reverse transcriptase PCR method was used to measure the presence of miR-1290 in the blood samples. A total of 32 patients were examined in this study, including 17 women (53.1%) and 15 men (46.9%). The mean age was 46.7 years in the healthy group and 54.6 years in the SCC group, indicating a significant difference (P<0.05). The expression level of the miR-1290 gene was higher in patients with SCC compared to the healthy group (P=0.000). While the expression level of miR-1290 was higher in grade 3 and advanced stage than in grades 2 and 1 and early stage, the differences were not statistically significant (P=0.173 and P=0.564 for grade and stage, respectively). The expression level of miR-1290 may increase in SCC patients compared to healthy individuals, making it a potential circulating biomarker. Further investigations for diagnostic utility would be warranted.

Abstract B114: Mutational "lineage traces" of an acinar cell-of-origin in human pancreatic ductal adenocarcinoma

Abstract A clinical challenge of pancreatic ductal adenocarcinoma (PDAC) is that most cases are detected late, after intractable metastasis is well underway. This also poses a challenge to our basic understanding of PDAC development, as the early steps of neoplasia are separated by a gulf of years or decades from accessible biospecimens and cell lines. Although mouse genetic models of PDAC have helped to fill in this gap, key questions remain incompletely answered. For example, what is the cell of origin of human PDAC – and does the answer to this question actually matter at a clinical level? In mouse, PDAC can be induced from both acinar and duct cells, with the former apparently more susceptible to transformation while the latter appears harder to transform, but capable of generating more aggressive tumors. Meanwhile, human PDAC has been divided into two major subtypes based on genomic data, classical and basal-like, the latter of which appears to be more aggressive. Is there a relationship between cell-of-origin, tumor subtype and clinical outcomes? This question requires the ability to directly infer cell-of-origin in human tumors, an approach to which we report here. Studies in human non-pancreatic cancers have identified somatic “passenger” (non-selected) mutations in several highly-expressed genes of the normal organ – for example, mutations in the ALB (albumin) gene in hepatocellular carcinoma, or SFTP genes (surfactant proteins) in lung adenocarcinoma. These might indicate a mutational process occurring in normal cells, prior to transformation, and constituting a “lineage mark” for the cell of origin – however, as these genes are also highly-transcribed in the cancer cells themselves, their mutations may arise after tumorigenesis is underway. This problem does not apply to acinar genes and PDAC: we and others have established that an initiating event in Kras-induced transformation of acinar cells is complete downregulation of acinar-specific gene products. Therefore, if a mechanism of transcription-induced mutation applies in the pancreas, we would expect it to leave a trace in acinar-derived PDAC. We have completed a large-scale analysis (n&amp;gt;2,000) of human whole genome sequencing data, across several carcinoma types including pancreas and liver, focused on unbiased detection of tissue-specific mutations similar to those previously found in the ALB and SFTP genes. Remarkably, we find robust enrichment of mutations in acinar-specific genes in PDAC, affecting approximately 25% of tumors. This constitutes the first in vivo genetic evidence for an acinar cell-of-origin in human pancreatic cancer, and opens the door to comparative genomic, transcriptomic and outcomes analyses of acinar-derived tumors. These are underway, and the results will be described in our presentation. Citation Format: Charles Murtaugh. Mutational "lineage traces" of an acinar cell-of-origin in human pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B114.

Exposure to smoky coal combustion emissions and leukocyte Alu retroelement copy number.

Household air pollution (HAP) from indoor combustion of solid fuel is a global health burden that has been linked to multiple diseases including lung cancer. In Xuanwei, China, lung cancer rate for non-smoking women is among the highest in the world and largely attributed to high levels of polycyclic aromatic hydrocarbons (PAHs) that are produced from combustion of smoky (bituminous) coal. Alu retroelements, repetitive mobile DNA sequences that can somatically multiply and promote genomic instability have been associated with risk of lung cancer and diesel engine exhaust exposure. We conducted analyses for 160 non-smoking women in an exposure assessment study in Xuanwei, China with a repeat sample from 49 subjects. Quantitative PCR was used to measure Alu repeat copy number relative to albumin gene copy number (Alu/ALB ratio). Associations between clusters derived from predicted levels of 43 HAP constituents, 5-methylchrysene (5-MC), a PAH previously associated with lung cancer in Xuanwei and was selected a priori for analysis, and Alu repeats were analyzed using generalized estimating equations. A cluster of 31 PAHs reflecting current exposure was associated with increased Alu copy number (β:0.03 per standard deviation change; 95% confidence interval (CI):0.01,0.04; P-value = 2E-04). One compound within this cluster, 5-MC, was also associated with increased Alu copy number (P-value = 0.02). Our findings suggest that exposure to PAHs due to indoor smoky coal combustion may contribute to genomic instability. Additionally, our study provides further support for 5-MC as a prominent carcinogenic component of smoky coal emissions. Further studies are needed to replicate our findings.

In silico identification of molecular mechanisms for stroke risk caused by heavy metals and their mixtures: Sponges and drugs involved

This study used various approaches and databases to evaluate the molecular processes and identify miRNA sponges and drugs associated with the pathophysiology of stroke caused by heavy metals and their combinations. We found that the genes ALB (albumin), IL1B (Interleukin-1β), F2 (coagulation factor II), APOA1 (apolipoprotein A1), IL6 (Interleukin 6), and NOS2 (nitric oxide synthase 2) were linked to the development of strokes by 18 chemicals and a combination of cadmium, copper, and lead. These results may point to the significance of detoxification and neuroinflammation in stroke as well as the potential for targeting these genes in future stroke therapies. ALB and IL1B were the most common and significant genes. The "selenium micronutrient network," "vitamin B12 metabolism," and "folate metabolism" were shown to be the most significant pathways connected to the risk of stroke brought on by combined heavy metals. The two main cellular elements that may increase the risk of stroke caused by heavy metals were discovered to be "blood microparticle" and "endoplasmic reticulum lumen." We also observed an important chromosome (chr7p15.3), two transcription factors (NFKB2 [nuclear factor kappa B subunit 2] and NR1I2 [nuclear receptor subfamily 1 group, member 2]), and four microRNAs (hsa-miR-26a-5p, hsa-miR-9–5p, hsa-miR-124–3p, and hsa-miR-155–5p) associated with stroke caused by combined heavy metals. Additionally, for these miRNAs, we created and examined in silico microRNA sponge sequences. Triflusal and andrographolide have been identified as potential treatments for heavy metal-induced stroke. Taken together, heavy metals may be a significant contributor to the pathophysiology of stroke, but further investigation into the precise molecular pathways implicated in stroke pathophysiology is required to corroborate these findings.

Alterations in the Plasma Protein Expression Pattern in Congenital Analbuminemia—A Systematic Review

Albumin is a highly abundant plasma protein with multiple functions, including the balance of fluid between body compartments and fatty acid trafficking. Humans with congenital analbuminemia (CAA) do not express albumin due to homozygosity for albumin gene mutation. Lessons about physiological control could be learned from CAA. Remarkably, these patients exhibit an apparently normal lifespan, without substantial impairments in physical functionality. There was speculation that tolerance to albumin deficiency would be characterized by significant upregulation of other plasma proteins to compensate for analbuminemia. It is unknown but possible that changes in plasma protein expression observed in CAA are required for the well-documented survival and general wellness. A systematic review of published case reports was performed to assess plasma protein pattern remodeling in CAA patients who were free of other illnesses that would confound interpretation. From a literature search in Pubmed, Scopus, and Purdue Libraries (updated October 2022), concentration of individual plasma proteins and protein classes were assessed. Total plasma protein concentration was below the reference range in the vast majority of CAA patients in the analysis, as upregulation of other proteins was not sufficient to prevent the decline of total plasma protein when albumin was absent. Nonetheless, an impressive level of evidence in the literature indicated upregulated plasma levels of multiple globulin classes and various specific proteins which may have metabolic functions in common with albumin. The potential role of this altered plasma protein expression pattern in CAA is discussed, and the findings may have implications for other populations with hypoalbuminemia.

Precise Authenticity of Quinoa, Coix Seed, Wild Rice and Chickpea Components Using Optimized TaqMan Real-Time PCR.

Functional food such as, quinoa, coix seed, wild rice and chickpea have experienced rapidly increasing demand globally and exhibit high economic values. Nevertheless, a method for rapid yet accurate detection of these source components is absent, making it difficult to identify commercially available food with labels indicating the presence of relevant components. In this study, we constructed a real-time quantitative polymerase chain reaction (qPCR) method for rapid detection of quinoa, coix seed, wild rice and chickpea in food to identify the authenticity of such food. Specific primers and probes were designed with 2S albumin genes of quinoa, SAD genes of coix seed, ITS genes of wild rice and CIA-2 genes of chickpea as the target genes. The qPCR method could specifically identify the four wild rice strains, yielding, LODs of 0.96, 1.14, 1.04 and 0.97 pg/µL quinoa, coix seed, wild rice and chickpea source components, respectively. Particularly, the method allowed the identification of the target component with content below 0.01%. A total of 24 commercially available food samples of different types were detected by using the method and the results indicate that the developed method is applicable to the detection of different food matrices, as well as authenticity verification in deeply processed food.

Importance of the filamin A-Sav1 interaction in organ size control: evidence from transgenic mice.

The nucleocytoplasmic translocation of yes-associated protein 1 (YAP1) controls the growth of animal tissues and organs. YAP1 binds to transcription factors in the nucleus to activate the transcription of proliferation and anti-apoptotic genes. The Hippo pathway prevents the nuclear translocation of YAP1 by phosphorylating YAP1, while mechanical forces promote it by opening the nuclear pore complex and stimulating other signaling pathways. Recently we found that Protein salvador homolog 1 (SAV1), a component of the Hippo pathway, interacts with filamin A (FLNA) in a force-dependent manner, raising a possibility that the Hippo pathway is regulated by mechanical force through the FLNA-SAV1 axis. To test this hypothesis, we generated conditional knock-in (KI) mice expressing non-Flna-binding mutant Sav1 in hepatocytes by crossing with mice carrying Cre recombinase driven by the serum albumin (alb) gene promoter. Unexpectedly, the insertion of the flox cassette skipped exon 2, resulting in a shorter Sav1 in all the transgenic mice. Since exon 2 encodes a fragment containing a Flna-binding domain, we analyzed both point mutant KI and exon 2-deleted mutant mice. Here we show that disruption of the Flna-Sav1 interaction in the mouse liver promotes apoptosis and suppresses tissue and organ growth without affecting the phosphorylation level of Yap1. These results provide evidence that the growth of animal tissues and organs is regulated by apoptosis downstream of the force-dependent FLNA-SAV1 interaction, at least in part.

Identification of cryptic forms of the hybridogenic complex of European water frogs (&lt;i&gt;Pelophylax esculentus complex&lt;/i&gt;) in the conditions of transformed biotopes of the south of the Central Russian Upland based on DNA markers

AIM: The aim of the study was to carry out species identification of the hybridogenic complex of European water frogs (Pelophylax esculentus complex) in the conditions of transformed biotopes of the south of the Central Russian Upland based on molecular genetic markers&#x0D; MATERIALS AND METHODS: The study included 36 local populations (770 individuals) living in conditions of transformed biotopes of the south of the Central Russian Upland. Identification of cryptic forms was carried out by a Multiplex PCR. Two molecular genetic markers were used for amplification: intron 1 of the SAI-1 DNA serum albumin gene to determine hybrids and cryptic forms, and a fragment of the first subunit of the cytochrome oxidase COI mtDNA gene to determine maternal lines.&#x0D; RESULTS: According to the data obtained, pure R-type population systems predominate (58.33%) in the study region. Mixed RE-type population systems were identified in 14 localities, REL-type in the region is extremely rare and is noted only in one locality. No pure L-type, E-type, or mixed LE-type population systems have been identified. The study revealed a statistically significant (p 0.001) predominance of haplotypes of the Western form (Pelophylax ridibundus).&#x0D; CONCLUSIONS: The data obtained indicate active adaptive changes in the population structure of European water frogs in the study area. Degradation of water bodies caused by abiotic and anthropogenic factors forces amphibians to migrate to neighboring reservoirs in which hybridization of representatives of this complex occurs. Introgressive and hybrid forms of the marsh frog, as well as hybrid edible individuals with greater ecological plasticity and tolerance to anthropogenic pressure, displace populations of Pelophylax lessonae. Based on the above, we consider it necessary to include the pond frog (P. lessonae) in the Red Book of the Belgorod region.

Familial Dysalbuminemic Hyperthyroxinemia Induced by Mutation R218H of Albumin

Familial Dysalbuminemic Hyperthyroxinemia (FDH) is thought of no clinical significance, but tends to to misdiagnosed and mistreated as Graves’ hyperthyroidism with long time. In a Chinese family to explore clinical characteristics, hereditary features and gene variation of FDH and its effect on pregnancy. Methods: Two propostitus (a father and his son) with hyperthyroidism were re-diagnosed as FDH due to the canonical profile of measured thyroid hormones. After screening thyroid function of the whole family, eight affected of FDH were identified among total thirteen lineal relatives. They were studied in terms of clinical history, biochemical and hormone examination, thyroid imaging and TSH secreting test by dexamethasone and bromocriptine. Three members accepted gene analysis with the next generation sequencing followed by Sanger method. Productive history was investigated in five spouse of the whole family. Results: In the two propostitus, FDH exhibited very high total thyroxine (T4) and mild high total triiodinethyronine (T3), and normal free T4 and T3 besides normal thyroid stimulating hormone (TSH). In addition, ultrasound discovered diffuse goiter and enhanced blood flow, and 99mTc uptake increased, while TSH receptor antibody and pituitary MRI scan remained normal. Other six family members had similar changes of the thyroid hormones and normal TSH which could be suppressed dramatically by dexamethasone and bromocriptine, then were suspected of FDH and thereafter confirmed with gene analysis. Among these eight FDH, four were misdiagnosed with hyperthyroidism and mistakenly received anti-thyroid medication at least one year. Gene analysis of three members revealed R218H variation of albumin gene. Conclusions: R218H associated FDH shows canonical changes of thyroid hormones with increased thyroid function, high penetrance and misdiagnose rate with hyperthyroidism, it exerts no effect on pregnancy.

FOXA1/2 depletion drives global reprogramming of differentiation state and metabolism in a human liver cell line and inhibits differentiation of human stem cell-derived hepatic progenitor cells.

FOXA factors are critical members of the developmental gene regulatory network (GRN) composed of master transcription factors (TF) which regulate murine cell fate and metabolism in the gut and liver. How FOXA factors dictate human liver cell fate, differentiation, and simultaneously regulate metabolic pathways is poorly understood. Here, we aimed to determine the role of FOXA2 (and FOXA1 which is believed to compensate for FOXA2) in controlling hepatic differentiation and cell metabolism in a human hepatic cell line (HepG2). siRNA mediated knockdown of FOXA1/2 in HepG2 cells significantly downregulated albumin (p < .05) and GRN TF gene expression (HNF4α, HEX, HNF1ß, TBX3) (p < .05) and significantly upregulated endoderm/gut/hepatic endoderm markers (goosecoid [GSC], FOXA3, and GATA4), gut TF (CDX2), pluripotent TF (NANOG), and neuroectodermal TF (PAX6) (p < .05), all consistent with partial/transient reprograming. shFOXA1/2 targeting resulted in similar findings and demonstrated evidence of reversibility of phenotype. RNA-seq followed by bioinformatic analysis of shFOXA1/2 knockdown HepG2 cells demonstrated 235 significant downregulated genes and 448 upregulated genes, including upregulation of markers for alternate germ layers lineages (cardiac, endothelial, muscle) and neurectoderm (eye, neural). We found widespread downregulation of glycolysis, citric acid cycle, mitochondrial genes, and alterations in lipid metabolism, pentose phosphate pathway, and ketogenesis. Functional metabolic analysis agreed with these findings, demonstrating significantly diminished glycolysis and mitochondrial respiration, with concomitant accumulation of lipid droplets. We hypothesized that FOXA1/2 inhibit the initiation of human liver differentiation in vitro. During human pluripotent stem cells (hPSC)-hepatic differentiation, siRNA knockdown demonstrated de-differentiation and unexpectedly, activation of pluripotency factors and neuroectoderm. shRNA knockdown demonstrated similar results and activation of SOX9 (hepatobiliary). These results demonstrate that FOXA1/2 controls hepatic and developmental GRN, and their knockdown leads to reprogramming of both differentiation and metabolism, with applications in studies of cancer, differentiation, and organogenesis.

Population Pharmacokinetic Analysis for Model-Based Therapeutic Drug Monitoring of Tacrolimus in Chinese Han Heart Transplant Patients.

Tacrolimus has become the first-line immunosuppressant for preventing rejection after heart transplantation. The present study aimed to investigate genetic variants and clinical factors affecting the variability of tacrolimus in Chinese Han heart transplant patients using a population pharmacokinetic approach. The retrospective study included 53 hospitalized patients with 547 tacrolimus concentrations for analysis. Nonlinear mixed-effects modeling was used to develop the population pharmacokinetics model for tacrolimus in patients with heart transplants, followed by Monte Carlo simulations to design initial dosing regimens. In our study, the mutation rate of CYP3A4*18B (C>T) was 27.36%. An oral one-compartment model with first-order absorption and elimination was used to describe the pharmacokinetics of tacrolimus in heart transplant patients. In the final model, the estimated apparent clearance (CL/F) and volume of distribution (V/F) were 532.5L/h [12.20% interindividual variability, IIV] and 16.87 L (23.16% IIV), respectively. Albumin, postoperative time, and rs2242480 (CYP3A4*18B) gene polymorphisms were the significant covariates affecting CL/F, and creatinine clearance had significant effects on the V/F. The population pharmacokinetic model of tacrolimus in heart transplant patients can better estimate the population and individual pharmacokinetic parameters of patients and can provide a reference for the design of individualized dosing regimens.

The environmental pollutant, polychlorinated biphenyl 126, alters liver function in a rodent model of alcohol-associated liver disease.

The prevalence of alcohol-associated liver disease (ALD), a subtype of fatty liver disease (FLD), continues to rise. ALD is a major cause of preventable death. Polychlorinated biphenyl (PCB) 126 is an environmentally relevant, dioxin-like pollutant whose negative metabolic effects have been well documented. In human and animal studies,PCBhas been associated withthe severity of nonalcoholic fatty liver disease (NAFLD). However, few studies have investigated whether exposures to environmental toxicants can worsen ALD. Thus, the objective of the current study was to develop an alcohol-plus-toxicant model to study how an environmental pollutant, PCB 126, impacts rodent ALD pathology. Briefly, male C57BL/6J mice were exposed to 0.2mg/kg PCB 126 or corn oil vehicle four days prior to ethanol feeding using the chronic-binge (10-plus-one) model. Concentrations of macromolecules, including hepatic lipids, carbohydrates, and protein (albumin) were impacted. Exposure to PCB 126 exacerbated hepatic steatosis and hepatomegaly in mice exposed to the chemical and fed an ethanol diet. Gene expression and the analysis of blood chemistry showed a potential net increase and retention of hepatic lipids and reductions in lipid oxidation and clearance capabilities. Depletion of glycogen and glucose was evident, which contributes to disease progression by generating systemic malnutrition. Granulocytic immune infiltrates were present but driven solely by ethanol feeding. Hepatic albumin gene expression and plasma levels were decreased by ~50% indicating a potential compromise of liver function. Finally, gene expression analyses indicated that the aryl hydrocarbon receptor and constitutive androstane receptor were activated by PCB 126 and ethanol, respectively. Various environmental toxicants are known to modify or enhance FLD in high-fat diet models. Findings from the present study suggest that they interact with other lifestyle factors such as alcohol consumption to reprogram intermediary metabolism resulting in exacerbated ethanol-associated systemic malnutrition in ALD.