Endogenous Reference Research Articles

β-Hydroxy β-Methyl Butyric Acid (HMB) and Its Potential Doping Relevance: A Pilot Study on Its Urinary Excretion Profile.

β-hydroxy β-methyl butyric acid (HMB), either as the free acid or in the form of its calcium salt, is a component of several dietary supplements marketed to enhance sports performance, due to its role in protein synthesis. In this pilot study, we investigated the urinary excretion profile of HMB, an endogenous metabolite of the essential amino acid leucine. The endogenous reference ranges of HMB, in human urine samples, were monitored by collecting samples from 20 volunteers. Data obtained were compared with those from controlled excretion studies, following the intake of a 3-g oral dose of HMB. Urine samples were analyzed through a targeted procedure employing a conventional GC-MS system operating in SIM mode. Our results show that the excreted urinary concentration of HMB significantly exceeds the range of endogenous concentrations for at least 24 h, making possible to detect its exogenous origin.

Circulating miR-323-3p as a novel potential plasma biomarker for multimorbidity burden and cognitive decline in middle-aged and older adults: results from the National Institute for Longevity Sciences–Longitudinal Study of Aging in Japan

BackgroundSeveral circulating microRNAs (miRNAs) in the blood are indicators of chronic diseases, but their association with multimorbidity burden is unknown. The aim of this study was to assess the association of plasma levels of circulating miR-323-3p and miR-135-3p with age, cognitive performance, and number of comorbidities in middle-aged and older individuals. MethodsData from 295 community dwellers (≥40 years) who participated in the second wave (2000‒2002) of the National Institute for Longevity Sciences-Longitudinal Study of Aging in Japan were analyzed. miRNAs were isolated from the plasma, and miR-323-3p and miR-135-3p levels were measured using quantitative reverse-transcription polymerase chain reaction and normalized, with miR-16 as an endogenous reference gene. Cognitive performance was assessed using the Information and Similarities subtests and the Digit Symbol Substitution Test of the Wechsler Adult Intelligence Scale-Revised Short Form (WAIS-R-SF). Correlation tests and multivariate general linear regression analyses were performed to examine the relationship among circulating miRNA levels, burden of multimorbidity, and cognitive performance. ResultsThe mean age of the participants was 59.1 ± 10.3 years. Pearson's correlation analysis revealed that the miR-323-3p level positively correlated with age and number of comorbidities but negatively correlated with WAIS-R-SF subtest performance. In men, miR-323-3p level negatively correlated with the performance of all three WAIS-R-SF subtests. The general linear regression analysis showed that the miR-323-3p level increased in participants with four comorbidities compared with those with one comorbidity. ConclusionCirculating miR-323-3p level is associated with the burden of multimorbidity and decreased cognitive performance in middle-aged and older adults.

COMPARISON OF YIELD AND QUALITY OF CIRCULATING CELL-FREE DNA FROM K2EDTA AND K3EDTA COLLECTIONS IN HEALTHY SUBJECTS

Background: Circulating cell-free DNA (cfDNA) is a biomarker for various clinical applications, including detecting and monitoring cancer. However, blood collection tubes can affect the yield and quality of cfDNA. Since specific cfDNA collection tubes are costly, K2EDTA and K3EDTA anticoagulant tubes are alternatives in routine clinical laboratories. Objectives: This study aimed to compare the efficiency of cfDNA extraction from plasma collected in K2EDTA and K3EDTA tubes and evaluate implementation for molecular diagnostics. Methods: Blood samples from 38 healthy subjects were collected in K2EDTA and K3EDTA tubes that were processed within 2 hours. The extracted cfDNA was measured and performed using SYBR Green-based qPCR for three endogenous reference genes (GAPDH, HPRT1, TFRC). The cfDNA yield and the amplification efficiency of these genes were compared between K2EDTA and K3EDTA tubes using the Mann-Whitney U test. Results: There were no significant differences in cfDNA concentration between K2EDTA and K3EDTA tubes (p=0.051). However, qPCR analysis revealed significantly higher copy numbers of TFRC and HPRT1 in K2EDTA tubes than in K3EDTA tubes (p<0.05). No significant difference was found for GAPDH. Conclusion: The results indicate that K2EDTA and K3EDTA tubes are an alternative option for cfDNA analysis if samples are processed quickly after a blood draw, which offers flexibility and cost savings in resource-limited areas.

Suppression of CNS APOE4 Expression by miRNAs Delivered by the S2 AAVrh.10 Capsid-modified AAV Vector.

The homozygous APOE4 genotype is the major risk factor for the development of early Alzheimer's disease. Genome engineering studies in mouse models of human APOE4-dependent pathology have established that reduction of APOE4 expression can rescue the phenotype. We hypothesized that APOE4 could be suppressed in the CNS of APOE4 homozygotes using adeno-associated virus (AAV) expression of microRNAs (miRNA) designed to hybridize to APOE mRNA. We screened 9 different miRNAs targeting APOE following transfection in HEK293T and Huh7 cells. Optimal APOE suppression was obtained with mir2A (targeting coding region nt330-351) and mirN4 (3' untranslated region nt1142-1162). miRNA expression cassettes were designed with two copies of each of these two miRNAs co-expressed with a mCherry transgene. To optimize delivery of these miRNAs, an engineered AAVrh.10 variant was identified from a screen of multiple peptide insertions into capsid loop IV and substitutions in loop VIII. This led to identifying the AAV.S2 capsid with enhanced transduction of both neurons and glia and enhanced distribution in the brain. The engineered capsid was used to deliver the APOE miRNA suppression cassette to the hippocampus of TRE4 mice (human APOE4 knock-in replacement of the murine apoE locus). Two weeks after intra-hippocampus administration, regional expression of miRNA at the injection site was quantified at the mRNA level relative to an endogenous reference. The AAV.S2 capsid provided 2.31 ± 0.37-fold higher expression of miRNA over that provided by AAVrh.10 (p<0.05). In the targeted region, a single intra-hippocampus AAV.S2 administration suppressed hippocampal APOE4 mRNA levels by 76.5 ± 3.9% compared to 41.3 ± 3.3% with the same cassette delivered by the wildtype AAVrh.10 capsid (p<0.0001). We conclude that an expression cassette with two different miRNAs targeting APOE4 delivered by the AAV.S2 capsid will generate highly significant suppression of APOE4 in the CNS.

Improving the Determination of Carbon Isotope Ratios of Endogenous Steroids Found in Human Serum.

The determination of serum concentrations of testosterone (T) and 4-androstenedione (A4) was implemented into the steroidal module of the Athlete Biological Passport in 2023. Monitoring T, A4, and the ratio of T/A4 in a longitudinal manner enables the detection of the misuse of low-dose T administrations especially in female athletes, whereas urinary markers of the steroid profile may not be influenced significantly. In contrast to the urinary steroid profile, knowledge on confounding factors regarding serum concentrations of T and A4 is yet comparably scarce, and corroborating exogenous sources of the target analytes by isotope ratio mass spectrometry (IRMS) is desirable. In a recent study, it was demonstrated that carbon isotope ratios (CIRs) of serum steroids can be determined if analyte concentrations permit. The therein-employed method utilized 2D-GC/IRMS, and only a limited number of potential endogenous reference compounds were available. The here-presented approach uses complementary analyte purification strategies, addressing previous limitations. A high-performance liquid chromatography cleanup was developed and fully validated for serum steroids in order to enable all doping control laboratories to potentially implement this method alongside existing protocols for urinary steroids. Besides the already-investigated endogenous steroids cholesterol, dehydroepiandrosterone sulfate, androsterone sulfate, and epiandrosterone sulfate, two additional steroids were included in the test menu, namely, pregnenolone sulfate and 5-androstene-3β,17β-diol sulfate. Serum steroid concentrations down to 25 ng/mL were found to allow robust CIR determinations, and a reference population encompassing 124 male and female athlete samples was investigated to enable the calculation of population-based thresholds for all relevant steroid combinations.

Two-Dimensional Liquid Chromatography Purified GC/C/IRMS Doping Control Method: Analysis of Endogenous and Exogenous Sources in Urine Samples from Asian Subjects Administered a Low Dose of AICAR

AICAR (5-amino-4-imidazolecarboxyamide ribonucleoside), as a metabolic modulator, is classified in the S4 category by the World Anti-Doping Agency (WADA). Carbon Isotope Ratio Mass Spectrometry (CIR) is the mainstream method for distinguishing the endogenous and exogenous sources of AICAR in urine due to the significant individual difference in the concentration. The purpose of this study is to establish a gas chromatography combustion Isotope Ratio Mass Spectrometry (GC/C/IRMS) method for AICAR based on efficient two-dimensional liquid chromatography (2D-HPLC) separation. MethodIn this study, an automated 2D-HPLC separation technique was used to separate and purify AICAR and endogenous reference substances in urine samples. Then, AICAR was derivatized with 3-TMS as the main derivative product, while the endogenous reference compounds remained in their original form. Subsequently, the developed GC/C/IRMS method was utilized for the detection of the target and reference substances. Followed, we evaluated the applicability of this method using urine samples from two Asian males administered a low dose of AICAR (3 grams). ResultsThe advantages of this study include: 1) reduced sample pretreatment time: the established 2D-HPLC separation method can separate the target and endogenous reference substances in one step; 2) low interference: the isotope chromatograms have low background interference, and the separation of endogenous reference substances is purer; 3) more accurate result calculations: this method only requires derivatization and result correction for AICAR, with the endogenous reference substances measured in their original form, reducing biases from corrections of multiple substances. The detection method performed well, with a concentriton limit of 2500 ng/mL, meeting the needs of routine detection concentrations. The CIR results from volunteer samples indicated that samples collected within 16 hours post-administration exceeded the threshold set in the literature. ConclusionThis study successfully established a 2D-HPLC-GC/IRMS method that integrates CIR as the most stable indicator for distinguishing the internal and external sources of AICAR. After administering a low dose of AICAR to the Asian population, exogenous drug characteristics were manifested within 16 hours. This observation, when compared to the 40-hour detection window cited in the literature, suggests that the length of the detection window is positively correlated with the dosage of the test drug.

A portable multifunctional biosensor based on a newly screened endogenous reference gene for pork adulteration detection

The adulteration of animal-derived food is a widespread problem with meat adulteration emerging as a global concern. To address the need for on-site rapid detection and visual analysis, a portable multifunction biosensor (PMB) was developed. This device integrates Recombinase Polymerase Amplification (RPA), CRISPR/Cas12a assay, and a custom-designed 3D-printed multifunction device, specifically for the visual and rapid detection of pork in food. Initially, a screening model was constructed using Perl bioinformatics based on chromosomal gene information, and the LOC110262058 gene was selected as a reliable endogenous reference gene for pigs. Subsequently, a one-tube RPA-CRISPR/Cas12a system was established to simplify the procedures, converting pork-specific signals into fluorescence while preventing cross-contamination. In addition, an independently designed 3D-printed multifunction device was developed to support the RPA-CRISPR/Cas12a system for one-tube detection, incorporating functions such as heating, centrifugation, and imaging. The reaction conditions of the PMB were optimized to reduce the reaction time to 20–30 min with a sensitivity of 0.01 g/100 g. The analysis of actual samples indicated that the PMB has high selectivity and practical effectiveness for pork detection. The proposed biosensor offers several detection advantages, including output, ease of use, stability, and independence from complex equipment, making it a powerful method for rapid pork detection in food.

Ces44T as an endogenous reference gene in real-time quantitative PCR detection of tiger nut (Cyperus esculentus) ingredients in food

Tiger nut (Cyperus esculentus) is a highly adaptable and nutritious plant that can be used to produce health-enhancing food products, edible oil, and functional ingredients. To ensure transparency, reliability, and accurate labeling throughout the food supply chain, accurate methods for identifying food ingredients are indispensable. In this study, the Ces44T gene was identified and validated as the endogenous reference gene for tiger nut. A real-time quantitative PCR assay targeting the Ces44T gene was developed, demonstrating exceptional specificity in distinguishing tiger nut from other plant species, and exhibiting remarkable stability across different tiger nut cultivars. Standard curves were established to validate the reliability and repeatability of the assay, displaying good linearity (R2 > 0.99) and PCR efficiency (90–110 %). The assay was estimated to have a limit of detection (LOD) of 0.0033 ng of tiger nut genomic DNA per reaction and a limit of quantification (LOQ) of 0.013 ng. We applied this assay to both laboratory-prepared samples and commercial food products, confirming its excellent specificity and robust performance. In conclusion, the developed real-time quantitative PCR assay provides an accurate means for the identification and quantification of tiger nut ingredients in various samples.

Reference Genes for Expression Analyses by qRT-PCR in Enterobacter cancerogenus.

The Enterobacter cancerogenus strain EcHa1 was isolated from the dead larvae of Helicoverpa armigera, and has the potential for biocontrol of some Lepidoptera insects. In order to screen insecticidal-related genes by qRT-PCR, stable endogenous reference genes used for normalizing qRT-PCR data were selected and evaluated from 13 housekeeping genes (HKGs). The expression levels of the HKGs were determined using qRT-PCR under different experimental conditions, including two culture temperatures and three bacterial OD values. Five stability analysis methods (Ct, BestKeeper, NormFinder, geNorm, and RefFinder) were used to comprehensively rank the candidate genes. The results showed that the optimal reference genes varied under different experimental conditions. The combination of gyrA and gyrB was recommended as the best reference gene combination at 28 °C, while gyrA and rpoB was the best combination at 37 °C. When the OD values were 0.5, 1.0 and 2.0, the recommended reference gene combinations were ftsZ and gyrA, rpoB and gyrB, and gyrA and pyk, respectively. The most suitable reference genes were gyrA and gyrB under all experimental conditions. Using gyrA and gyrB as the reference genes for qRT-PCR, EcHa1 was found to invade all tissues of the H. armigera larvae, and expressed a candidate pathogenic factor Hcp at high levels in gut, Malpighian tubules, and epidermis tissues. This study not only establishes an accurate and reliable normalization for qRT-PCR in entomopathogenic bacteria but also lays a solid foundation for further study of functional genes in E. cancerogenus.

Addressing the unsolved challenges in microRNA-based biomarker development: Suitable endogenous reference microRNAs for SARS-CoV-2 infection severity

Circulating cell-free microRNAs (miRNAs) are promising biomarkers for medical decision-making. Suitable endogenous controls are essential to ensure reproducibility. We aimed to identify and validate endogenous reference miRNAs for qPCR data normalization in samples from SARS-CoV-2-infected hospitalized patients. We used plasma samples (n = 170) from COVID-19 patients collected at hospital admission (COVID-Ponent project, www.clinicaltrials.gov/NCT04824677). First, 179 miRNAs were profiled using RT–qPCR. After stability assessment, candidates were validated using the same methodology. miRNA stability was analyzed using the geNorm, NormFinder and BestKeeper algorithms. Stability was further evaluated using an RNA-seq dataset derived from COVID-19 hospitalized patients, along with plasma samples from patients with critical COVID-19 profiled using RT-qPCR. In the screening phase, after strict control of expression levels, stability assessment selected eleven candidates (miR-17-5p, miR-20a-5p, miR-30e-5p, miR-106a-5p, miR-151a-5p, miR-185-5p, miR-191-5p, miR-423-3p, miR-425-5p, miR-484 and miR-625-5p). In the validation phase, all algorithms identified miR-106a-5p and miR-484 as top endogenous controls. No association was observed between these miRNAs and clinical or sociodemographic characteristics. Both miRNAs were stably detected and showed low variability in the additional analyses. In conclusion, a 2-miRNA panel composed of miR-106a-5p and miR-484 constitutes a first-line normalizer for miRNA-based biomarker development using qPCR in hospitalized patients infected with SARS-CoV-2.

GMOIT: a tool for effective screening of genetically modified crops

BackgroundAdvancement in agricultural biotechnology has resulted in increasing numbers of commercial varieties of genetically modified (GM) crops worldwide. Though several databases on GM crops are available, these databases generally focus on collecting and providing information on transgenic crops rather than on screening strategies. To overcome this, we constructed a novel tool named, Genetically Modified Organisms Identification Tool (GMOIT), designed to integrate basic and genetic information on genetic modification events and detection methods.ResultsAt present, data for each element from 118 independent genetic modification events in soybean, maize, canola, and rice were included in the database. Particularly, GMOIT allows users to customize assay ranges and thus obtain the corresponding optimized screening strategies using common elements or specific locations as the detection targets with high flexibility. Using the 118 genetic modification events currently included in GMOIT as the range and algorithm selection results, a “6 + 4” protocol (six exogenous elements and four endogenous reference genes as the detection targets) covering 108 events for the four crops was established. Plasmids pGMOIT-1 and pGMOIT-2 were constructed as positive controls or calibrators in qualitative and quantitative transgene detection.ConclusionsOur study provides a simple, practical tool for selecting, detecting, and screening strategies for a sustainable and efficient application of genetic modification.

In silico approach of 2,5-Diketopiperazines from marine organisms to neurodegenerative diseases

In this paper we present an in silico studies of new biological active of 2,5-Diketopiperazines candidates, from Marine Organisms to neurodegenerative diseases particular for the neurodegenerative central nervous system to Alzheimer's, Huntington and Parkinson’s diseases. A total of 35 DKPs were studied, by structural similarity analysis obtained MAO-A/B, β/γ-Secretase, COX-1/2 enzymes targets obtained, to continue molecular docking studies compared to endogenous substrates and reference inhibitors, finding a multitarget potential to increase dopamine levels and decrease β-amyloid and PGE2 levels, which makes them excellent molecules for studies against neurodegenerative diseases. DKP4, DKP23 and DKP25 as inhibitors of the 6 enzymes and DKP15, DKP19, DKP21, DKP26 and DKP33 for β-Secretease specifically, the rest with multitarget potential, denoting that the DKP ring serves as a base to generate multitarget or unitarget compounds through modifications in substituents. Finally, DKPs present low bioaccumulation in the body, no toxicity, high feasibility of crossing hematoencephalic membrane and activity on the CNS, which makes them an interesting set of molecules for the search for alternatives against neurodegenerative diseases.

MR Spectroscopy Assessment of Daily Variations of GABA Levels within the Parietal Lobe and Anterior Cingulate Gyrus Regions of Healthy Young Adults.

The changes that occur in the gamma-aminobutyric acid (GABA) levels within specific brain regions throughout the day are less clear. To evaluate the daily fluctuations of GABA levels within the parietal lobe (PL) and anterior cingulate gyrus (ACC) regions and explore their association with melatonin (MT) levels, heart rate (HR), and blood pressure. Prospective. 26 healthy young adults (15 males and 11 females aged 22-27 years). 3.0T, T1-weighted imaging, Mescher-Garwood point resolved spectroscopy (MEGA-PRESS) sequence. The acquired GABA signal contained the overlapping signals of macromolecules and homocarnosine, hence expressed as GABA+. The creatine (Cr) signal was applied as an endogenous reference. The GABA+, GABA+/Cr were measured at six different time points (1:00, 5:00, 9:00, 13:00, 17:00, and 21:00 hours) using MEGA-PRESS. The blood pressure, HR and sputum MT levels, were also acquired. The one-way repeated-measures analysis of variance (ANOVA) was used to evaluate the GABA, blood pressure, HR, and MT levels throughout the day. A general linear model was used to find the correlation between GABA and blood pressure, HR, and MT. P < 0.05 was statistically significant. Significant variations in GABA+/Cr and GABA+ levels were observed throughout the day within the PL region. The lowest levels were recorded at 9:00 hour (GABA+/Cr: 0.100 ± 0.003，GABA+:1.877 ± 0.051 i.u) and the highest levels were recorded at 21:00 hour (GABA+/Cr: 0.115 ± 0.003, GABA+:2.122 ± 0.052 i.u). The MT levels were positively correlated with GABA+/Cr (r = 0.301) and GABA+ (r = 0.312) within the ACC region. GABA+/Cr and GABA+ in ACC are positively correlated with MT. GABA levels in the PL have diurnal differences. These findings may indicate that the body's GABA level change in response to the light-dark cycle. 1 TECHNICAL EFFICACY: Stage 2.

Two-dimensional high performance liquid chromatography purification of underivatized urinary prednisone and prednisolone for compound-specific stable carbon isotope analysis.

The gas chromatography-combustion isotope ratio mass spectrometry (GC/C/IRMS) confirmation procedure for prednisone (PS) and prednisolone (PSL) is still a great challenge for the doping control laboratory due to the many structurally similar steroids present in urinary matrices. This study aims to establish an innovative online two-dimensional high performance liquid chromatography (2D-HPLC) purification method for measuring the carbon isotope ratios (CIRs) and achieving the identification of the synthetic forms of these two endogenous anabolic androgenic steroids (EAASs). Initially, the one-dimensional chromatographic column was used to separate and purify endogenous reference compounds (ERCs), and the co-elution fluids containing PS and PSL were switched to a two-dimensional chromatographic column for further purification through an online transfer system. Then the purified compounds were analyzed using GC/C/IRMS after sample pretreatments. The results showed that the minimum detection concentration of PS and PSL reached 30 ng mL-1, and no isotope fractionation occurred during the entire collection and preparation process. This method has been validated with the WADA technical document and showed good sensitivity and selectivity, demonstrating its practical applicability for urine samples in doping control laboratories.

Surgical resection of lung cancer inhibits mRNA expression of GOT2 gene encoding kynurenine aminotransferase in leukocytes.

Lung cancer is the most common malignant tumour. More than 80% of all diagnosed cases are non-small cell carcinoma which can be effectively treated by radical resection. Despite significant progress in the field of diagnostic and therapeutic methods, the results of lung cancer treatment are still unsatisfactory. Lung cancer is detected relatively late, which leads to an unfavourable prognosis. Kynurenine aminotransferases are an important element of the kynurenine pathway of tryptophan metabolism, which has recently aroused great interest from the aspect of possible use as a target point of personalized therapies in malignant tumours.The aim of the study was to analyze the expression of the selected gene of kynurenine aminotransferases GOT 2 at the mRNA level in peripheral blood leukocytes of patients with lung cancer. The mRNA expression of the GOT 2 gene was tested on blood samples from 50 patients treated surgically for non-small cell lung cancer.The control group consisted of 15 healthy individuals.The determination of mRNA expression of the GOT 2 gene was performed using the real-time PCR method.The GAPDH gene was used as the endogenous reference level. The mRNA expression of the GOT2 gene on the 6th day after surgery was statistically significantly lower than before surgery (p = 0,05). In the study group, the average LogRQ mRNA expression of the GOT2 gene before the procedure was 0.192082±0.292174 in woman. This was statistically significantly higher than in men whose average LogRQ mRNA expression of the GOT2 gene before the procedure was 0.004210±0.235065 (p=0.0183). Surgical resection of lung cancer results in inhibition of GOT2 mRNA expression in leukocytes. Further studies are expected to show whether it may be used as a target point for personalized therapies in lung cancer.

Evaluating the stability of host-reference gene expression and simultaneously quantifying parasite burden and host immune responses in murine malaria

The efficacy of pre-erythrocytic stage malaria antigens or vaccine platforms is routinely assessed in murine models challenged with Plasmodium sporozoites. Relative liver-stage parasite burden is quantified using reverse transcription quantitative PCR (RTqPCR), which relies on constitutively expressed endogenous control reference genes. However, the stability of host-reference gene expression for RTqPCR analysis following Plasmodium challenge and immunization has not been systematically evaluated. Herein, we evaluated the stability of expression of twelve common RTqPCR reference genes in a murine model of Plasmodium yoelii sporozoite challenge and DNA-adenovirus IV 'Prime-Target' immunization. Significant changes in expression for six of twelve reference genes were shown by one-way ANOVA, when comparing gene expression levels among challenge, immunized, and naïve mice groups. These changes were attributed to parasite challenge or immunization when comparing group means using post-hoc Bonferroni corrected multiple comparison testing. Succinate dehydrogenase (SDHA) and TATA-binding protein (TBP) were identified as stable host-reference genes suitable for relative RTqPCR data normalisation, using the RefFinder package. We defined a robust threshold of 'partial-protection’ with these genes and developed a strategy to simultaneously quantify matched host parasite burden and cytokine responses following immunisation or challenge. This is the first report systematically identifying reliable host reference genes for RTqPCR analysis following Plasmodium sporozoite challenge. A robust RTqPCR protocol incorporating reliable reference genes which enables simultaneous analysis of host whole-liver cytokine responses and parasite burden will significantly standardise and enhance results between international malaria vaccine efficacy studies.

Determination of Appropriate Endogenous Reference Genes for RT-qPCR Analysis in Syrian (Golden) Hamsters and Mongolian Gerbils

Purpose: The use of hamsters and gerbils has increased significantly in a variety of fields, including biological rhythms, reproductive biology, immunology, oncology, and many others. Material and Methods: The most stable genes in Syrian hamsters (Mesocricetus auratus) and Mongolian gerbils (Meriones unguiculatus) were assessed using 32 reference genes for normalization in RT-qPCR analysis. Adrenal, cerebral cortex, heart, hypothalamus, kidney, liver, lung and testis tissues were used to extract and purify RNAs. GeNorm was used to determine the gene expression stabilities of 14 candidate endogenous genes from each tissue that was compatible for both animals. Results: Under our experimental conditions, we discovered that two endogenous genes are adequate for each tissue to perform RT-qPCR normalization. There were differences in the most stable genes between species and tissues. Conclusion: We suggest that combinations of endogenous genes ought to be carefully chosen under various experimental circumstances.

The Identification of the Banana Endogenous Reference Gene MaSPS1 and the Construction of Qualitative and Quantitative PCR Detection Methods.

Endogenous reference genes play a crucial role in the qualitative and quantitative PCR detection of genetically modified crops. Currently, there are no systematic studies on the banana endogenous reference gene. In this study, the MaSPS1 gene was identified as a candidate gene through bioinformatics analysis. The conservation of this gene in different genotypes of banana was tested using PCR, and its specificity in various crops and fruits was also examined. Southern blot analysis showed that there is only one copy of MaSPS1 in banana. The limit of detection (LOD) test showed that the LOD of the conventional PCR method is approximately 20 copies. The real-time fluorescence quantitative PCR (qPCR) method also exhibited high specificity, with a LOD of approximately 10 copies. The standard curve of the qPCR method met the quantitative requirements, with a limit of quantification (LOQ) of 1.14 × 10-2 ng-about 20 copies. Also, the qPCR method demonstrated good repeatability and stability. Hence, the above results indicate that the detection method established in this study has strong specificity, a low detection limit, and good stability. It provides a reliable qualitative and quantitative detection system for banana.

Reference dependence and endogenous anchors

AbstractIn a complete market, we find optimal portfolios for an investor whose satisfaction stems from both a payoff's intrinsic utility and its comparison with an endogenous reference as modeled by Kőszegi and Rabin. In the regular regime, arising when reference dependence is low, the marginal utility of the optimal payoff is proportional to a twist of the pricing kernel. High reference dependence leads to the anchors regime, whereby investors reduce disappointment by concentrating significant probability in one or few fixed outcomes, or “anchors.” Multiple equilibria arise because anchors may not be unique. If stocks follow geometric Brownian motion, the model implies that investors with longer horizons choose larger stocks holdings.

Assessment of neurotransmitter imbalances within the anterior cingulate cortex in women with primary dysmenorrhea: An initial proton magnetic resonance spectroscopy study

PurposeThe neural pathophysiology underlying primary dysmenorrhea (PDM), which leads to poor mode and changes in central pain modulatory systems, remains largely unknown. The objective of this study was to investigate the changes in glutamate/glutamine (Glx) and gamma-aminobutyric acid (GABA+) levels within anterior cingulate cortex (ACC), and their associations with clinical indicators in PDM women. MethodsUsing 3 T proton magnetic resonance spectroscopy (1H-MRS), we acquired and compared ACC-Glx and ACC-GABA+ levels in PDMs (N = 41) and age- and education-matched healthy controls (HCs) (N = 39) during both the menstrual and periovulatory phases, and between menstrual and periovulatory phases within each group. Total creatine (Cr referencing) level was used as an endogenous reference. The correlations of ACC-neurotransmitter levels with clinical characteristics and the correlations of ACC-Glx with ACC-GABA+ levels in the two groups were analyzed. ResultsCompared to HCs or the periovulatory phase, PDMs exhibited significantly increased ACC-Glx levels (p < 0.05) during the menstrual phase. Positive correlations between GABA+ and Glx levels (r = 0.385, p = 0.025) were found in PDMs during the menstrual phase. ACC-GABA+ levels were associated with self-rating distress scale (SDS) scores (GABA+/Cr: r = 0.369, p = 0.045) and pain catastrophizing scale (PCS) scores (GABA+/Cr: r = 0.373, p = 0.042) in PDM group in only the menstrual phase. ConclusionOur study represents the first report of ACC-GABA+/Glx imbalances in PDMs during the menstrual phase, which may underlie the mechanisms mediating depression and painful catastrophic symptoms.