Microbial-transcriptome integrative analysis of heat stress effects on amino acid metabolism and lipid peroxidation in poultry jejunum.

Despite the significant threat of heat stress to livestock animals, only a few studies have considered the potential relationship between broiler chickens and their microbiota. Therefore, this study examined microbial modifications, transcriptional changes and host-microbiome interactions using a predicted metabolome data-based approach to understand the impact of heat stress on poultry. After the analysis, the host functional enrichment analysis revealed that pathways related to lipid and protein metabolism were elevated under heat stress conditions. In contrast, pathways related to the cell cycle were suppressed under normal environmental temperatures. In line with the transcriptome analysis, the microbial analysis results indicate that taxonomic changes affect lipid degradation. Heat stress engendered statistically significant difference in the abundance of 11 microorganisms, including Bacteroides and Peptostreptococcacea. Together, integrative approach analysis suggests that microbiota-induced metabolites affect host fatty acid peroxidation metabolism, which is correlated with the gene families of Acyl-CoA dehydrogenase long chain (ACADL), Acyl-CoA Oxidase (ACOX) and Acetyl-CoA Acyltransferase (ACAA). This integrated approach provides novel insights into heat stress problems and identifies potential biomarkers associated with heat stress.

Trends in Distributions of Hearing Threshold Levels by Ages: A Comparison of the ISO 7029 and Newly Available Country-Specific Data.

Hearing thresholds provide essential information and references about the human auditory system. This study aimed to identify changing trends in distributions of hearing threshold levels across ages by comparing the International Organization for Standardization (ISO) 7029 and newly available data after publishing ISO 7029. To compare ISO 7029 and newly available hearing threshold data after publishing ISO 7029, four country-specific datasets that presented average hearing threshold levels under conditions similar to ISO 7029 were utilized. For frequencies between 125 Hz and 8,000 Hz, the deviations of hearing threshold values by ages from the hearing threshold of the youngest age group for each data point were utilized. For frequencies from 9,000 Hz to 12,500 Hz, the median threshold information was utilized. Hearing threshold data reported after publishing ISO 7029 from the four countries were mostly similar to the ISO 7029 data but tended to deviate in some age groups and sexes. As national hearing threshold trends change, the following ISO 7029 revision suggests the need to integrate hearing threshold data from different countries.

Improving Accuracy and Reliability of Hearing Tests: An Exploration of International Standards.

This study explores the internal standards for hearing tests and benefits of implementing international standard protocols, including the International Organization for Standardization (ISO) and International Electrotechnical Commission (IEC), and discusses how ISO and IEC standards provide a framework for designing, calibrating, assessing hearing test instruments and methods, and exchanging and comparing data globally. ISO and IEC standards for hearing tests improve accuracy, reliability, and consistency of test results by applying standardized methods and environments. Moreover, they promote international harmonization and data interoperability, enabling information exchange and research collaboration. Those standards for hearing tests are beneficial but have challenges and limitations, such as variation in equipment and calibration, lag in updating standards, variation in implementation and compliance, and lack of coverage of clinical aspects, cultural diversity, and linguistic diversity. These affect the quality and interpretation of test results. Adapting ISO or IEC standards locally would improve their applicability and acceptability, while balancing customization and compatibility with global standards.

Triple ligation-based formation of a G-quadruplex for simultaneous detection of multiple miRNAs.

The simultaneous detection of multiple microRNAs (miRNA) is of great necessity but has not been extensively studied. This prompted our study, which involved the development of a triple ligation-based system for detecting three miRNAs at the same time. We designed a multi-ligation-padlock (MLP) probe that consists of three parts, each of which is complementary to two different miRNAs at the same time. In the presence of all three miRNAs, the probe becomes circularized, but in the absence of even one target, the probe remains linear. The first part of the MLP probe (MLP1) contains a T7 promoter part that can initiate RNA synthesis for any given target condition. However, it also includes a G-quadruplex complementary segment, which can only form a parallel RNA G-quadruplex through rolling circle transcription by the circularized template in the presence of all three targets. In this case, the application of our parallel G-quadruplex sensing fluorescent probe lutidine DESA (LutD) produces a strong signal. However, in the absence of any one of the targets, the RNA G-quadruplex cannot be formed and ultimately the LutD probe does not generate any signal. This difference in the signal intensity represents the presence or absence of all the target miRNAs. With our system, we were able to detect miRNA 21 at levels as low as 1.13 fM, miRNA 146a as low as 1.37 fM, and miRNA 25b as low as 1.51 fM within 45 minutes, confirming that our novel system can selectively and sensitively diagnose triple miRNAs.

Multiple gene detection using a selective fluorophore probe-RNA hybridization/graphene oxide quenching system.

Herein, we introduce a novel assay for multiple-gene recognition by ligation-double transcription mediated fluorometric profiling. We demonstrated the capability of the system to identify potential multi-gene classifiers for diagnostic use by employing a combination of a ligation-double transcription approach with a selective fluorophore probe-RNA hybridization/graphene oxide quenching system. The system is efficient and requires only 45 minutes for total experimentation and offers high sensitivity (369.6, 408, and 407.8 copies per mL for the O, E, and N genes of SARS-CoV-2, respectively) and specificity (selective to until two mismatches). Our system is expected to expedite the precise diagnosis of RNA-virus-related diseases with multiple gene classifiers. By focusing on distinct viral genes, our method allowed for the detection of various RNA viruses in a variety of sample pools.

Blocker-dUThiophene poly tailing-based method for assessing methyl transferase activity.

In this report, we present a method for the selective and sensitive detection of methyl transferase activity. The method uses a dsDNA probe that contains C3 spacers and is coupled with dUThioTP-TdT polymerase-based poly-tailing. The short dsDNA probe is designed with C3 spacers at both 3' ends to prevent any type of tailing reaction. However, the probe contains a methyl transferase recognition sequence that can methylate adenosines in the palindromic part of both strands. When a specific DpnI endonuclease is introduced, it selectively cleaves the dsDNA probe such that both strands are methylated, unblocking the probe into two separate dsDNA forms with exposed 3' OH groups. This makes the probe susceptible to tailing in the presence of a TdT tailing polymerase. The unblocked probe is then subjected to fluorescent dUThioTP-based tailing, which produces a strong fluorescent signal that indicates the presence of methyl transferase activity. In the absence of methyl transferase, the probe remains in the blocked state and does not undergo fluorescence. This method has a limit of detection of 0.049 U/mL with good selectivity and the potential for accurate MTase analysis.

Rolling circle transcription/G-quadruplex/QnMorpholine probe for highly selective and sensitive detection of alkaline phosphatase activity.

In this study, we combined a rolling circle transcription (RCT) system producing 22AG G-quadruplex RNA with a QnMorpholine (QNM) fluorescent probe for the selective and sensitive detection of alkaline phosphatase (ALP). ALP is involved in various biological functions, with monophosphate cleavage being one of its characteristic properties. Here, we developed a padlock RCT probing system in which a large amount of RCT 22AG RNA G-quadruplex was produced in the absence of ALP, providing a high fluorescence signal. In contrast, no RNA G-quadruplex was produced in the presence of ALP, with minimal fluorescence. This huge deviation in signal intensity allowed us to identify the presence or absence of ALP in a test sample. Under practical conditions, our system allowed the differentiation for ALP even when it was present at an extremely low concentration (0.0085 U/L), along with very high specificity. The simplicity and efficiency of this approach for ALP detection suggest its potential for use as a reliable diagnostic tool.

Consistency of Product Quality for SB5, an Adalimumab Biosimilar.

BACKGROUND: Biologics, regardless of whether they are biosimilars or reference products, are inherently variable due to their size, complexity, and the manufacturing process involved to produce them. Since a drift or evolution of quality attributes of a biologic may impact its clinical safety or efficacy, it is critical for the manufacturer to carefully control the manufacturing process and monitor the quality attributes of a biologic. OBJECTIVE: The aim of this study was to demonstrate that the quality profile of the SB5 drug product has been consistent over its production history from 2013 to 2022. SB5 is a biosimilar referencing adalimumab (Humira, trademark of AbbVie Biotechnology Ltd) and SB5 has been approved by 14 regulatory authorities including the European Commission in August 2017 (brand name Imraldi™) and the US Food and Drug Administration in July 2019 (brand name Hadlima™). METHODS: A total of 93 SB5 drug product batches manufactured between 2013 and 2022 were analyzed for a series of release parameters to evaluate the consistency in their critical quality attributes including purity, charge variants, and functional activities (TNF-α binding activity and TNF-α neutralizing potency). RESULTS: The purity, charge variants, and functional activities of all batches were consistent over time and within the stringent acceptance criteria defined by regulatory agencies to ensure the safety and efficacy of SB5. CONCLUSION: The data presented in this study provide evidence that the quality of SB5 has remained consistent and tightly controlled even through process changes such as manufacturing site transfers and change in formulation.

Multiple ligation-Assisted recombinase polymerase amplification for highly sensitive and selective colorimetric detection of SARS-CoV-2.

In this paper we present a new method for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), targeting a specific region "N gene." Under isothermal reaction conditions, we integrated ligation (Lig; high selectivity) and recombinase polymerase amplification (RPA; high sensitivity) processes, obtaining a robust method of detection. For point-of-care testing, we incorporated our laboratory-produced pyrophosphate ion (PPi)-sensing probe (PK-probe) for colorimetric analysis of the reaction. The total detection system was efficient and effective at diagnosing this RNA virus-mediated disease rapidly (30 min). In a full-genome SARS-CoV-2 study, our PK-probe/Lig-RPA system functioned with a limit of detection of 1160 copies/ml, with a single-mismatch level of selectively, and it was highly selective even in the presence of bacterial genomes commonly found in the human mouth and nose. This robust, straightforward, selective, efficient, and ultrasensitive colorimetric detection method, with potential for point-of-care analysis, should also be effective in detecting a diverse range of other RNA-based diseases.

Association of serum uric acid Levels with metabolic syndromes in Korean adolescents.

Introduction: The study findings investigated uric acid reference values and their association with a cluster of cardiometabolic risk factors among adolescents using the Korea National Health and Nutrition Examination Survey (KNHANES). Methods: A retrospective cross-sectional study was conducted using the KNHANES database from 2016 to 2018, involving a total of 2,462 participants aged between 10 and 18 years. Based on age- and sex-specific percentile curves for serum uric acid (SUA) levels from the KNHANES, we examined the correlation between cardiometabolic risk factors and serum uric acid levels. Results: The percentile values of SUA varied with sex and age. In male subjects, SUA levels tended to increase from 10 to 14 years of age and plateaued after 14 years of age. Moreover, the overall uric acid level in females was found to be lower than that in males; the levels tended to increase at approximately 10 to 12 years old but were relatively consistent according to age. Mean uric acid levels increased according to obesity status in both males and females. However, correlation analysis revealed that SUA levels were associated with several metabolic risks even after adjusting for obesity. The detailed metabolic syndrome (MetS) components that were observed to be associated with an increase in uric acid levels were different between males and females, but overall, high uric acid levels increased MetS risk. Additionally, a significant increase in MetS-related odds ratio (OR) for components, including waist circumference (WC), triglyceride (TG) levels, and low high-density lipoprotein cholesterol (HDL-c), was observed. However, differences between sexes were apparent, with a more pronounced increase in OR based on SUA levels in girls. Discussion: SUA levels were closely associated with MetS and its components, even in nonobese subjects. Therefore, high SUA levels in children and young adolescents should be closely monitored to prevent MetS.

Association between uric acid and height during growth hormone therapy in children with idiopathic short stature.

Background: Serum uric acid (UA) within appropriate levels is reported to be beneficial in patients with idiopathic short stature (ISS). This study aimed to evaluate the association between serum UA levels and height standard deviation scores (SDS) in patients with ISS during growth hormone (GH) therapy. Methods: A longitudinal study (LG Growth Study) of 182 children (mean age: 7.29±2.60 years) with ISS was performed. All participants were in the prepubertal stage and treated with GH, and the data within a treatment period of 30 months were analyzed. Results: In the adjusted Pearson's correlation, UA was significantly correlated with height SDS after controlling for sex, age, and body mass index (BMI) SDS (r=0.22, p=0.007). In the adjusted multiple regression analyses, the height SDS was significantly associated with UA after controlling for sex, age, and BMI SDS (ß=0.168, p=0.007). Within the 30-month treatment period, the UA levels significantly increased as the height SDS increased, and the mean UA levels at baseline and 30 months after treatment were 3.90±0.64 mg/dL and 4.71±0.77 mg/dL, respectively (p=0.007). Discussion: In conclusion, UA is related to height SDS, and GH treatment leads to a significant increase in UA without hyperuricemia. Elevated UA is considered a favorable outcome of GH therapy, and further studies are needed to determine its role as a monitoring tool.

Updated reference ranges for aminotransferase levels of Korean children and young adolescents based on the risk factors for metabolic syndrome.

We investigated the reference values of liver enzymes based on cardiometabolic risks among children and adolescents using the Korea National Health and Nutrition Examination Survey. A total of 8091 subjects aged 10-18 years were included from the data from 2007-2017. Overall, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and the AST/ALT ratio varied with sex and age. AST levels tended to decrease with age, but ALT levels had a U-shaped curve, which resulted in a gradual increase in the AST/ALT ratio after age 13. The prevalence of MetS was strongly associated with elevated AST or ALT and a decreased AST/ALT ratio. The prevalence ratios of the development of MetS were also elevated in groups with high levels of AST and ALT and a low AST/ALT ratio. Particularly in the combined ALT and AST/ALT analyses, borderline-high levels also showed a high prevalence ratio of MetS. Liver enzymes were also involved in the increase in the adjusted mean values for each risk factor for MetS. Here, we provided updated reference values for liver enzymes based on the analysis between population-based data and cardiometabolic risk factors; AST, ALT and the AST/ALT ratio might be useful in the early diagnosis and treatment of MetS.

22AG G-quadruplex RNA/QnMorpholine-mediated fluorimetric detection of miR-21.

Herein we report a simple ligation/transcription-mediated system, using a 22AG G-quadruplex RNA secondary structure and a fluorescence-inducing QnMorpholine probe, for the detection of miR-21. In the presence of the target miR-21, two oligonucleotide probes (promoter and reporter) were ligated, thereby transcribing the 22AG RNA sequence, a complement of the reporter probe. In contrast, in the absence of this target-induced ligation, the reporter complement could not be transcribed to produce the 22AG RNA sequence. Subsequent addition of the QnMorpholine probe resulted in binding with the 22AG G-quadruplex RNA, thereby generating high fluorescence; no fluorescence occurred in the absence of this secondary structure. Hence, the presence of miR-21 was evidenced by a target-induced high-intensity signal. This simple one-pot fluorimetric system, which could detect miR-21 of up to 3.08 femtomolar in less than 30 min, holds promise as a diagnostic tool for selective and sensitive miRNA detection.

Metabolic risk assessment in children and adolescents using the tri-ponderal mass index.

We assessed the risk of metabolic syndrome in children and adolescents who were classified using the tri-ponderal mass index (TMI) with data from the Korea National Health and Nutrition Examination Survey (KNHANES). Data from 10 to 18-year-old subjects that were overweight or obese (n = 1362) were extracted from the KNHANES 2007-2018. Weight classifications were determined by TMI and included overweight and Class I, Class II, and Class III obesity. The standard deviation scores (SDS) of weight, waist circumference, and body mass index (BMI) as well as cardiometabolic risk factors, including blood pressure, serum glucose levels, total cholesterol (T-C), triglycerides, HDL-c, and low-density lipoprotein cholesterol (LDL-c), worsened with the severity of obesity. Most risk factors showed a linear association with the severity increase, except for fasting glucose levels, T-C, and LDL-c. The prevalence of cardiometabolic risks also increased with the severity of obesity, which developed earlier in boys than in girls. The risk of metabolic syndrome significantly increased with the severity of obesity in both unadjusted and adjusted analyses. TMI reflected the severity of obesity and predicted the risk of metabolic syndrome and its components. Therefore, clinical applications of TMI could be a useful to identify the incidence of childhood obesity and metabolic syndromes.

Point-of-care COVID-19 testing: colorimetric diagnosis using rapid and ultra-sensitive ramified rolling circle amplification.

In this paper, we report a molecular diagnostic system-combining a colorimetric probe (RHthio-CuSO4) for pyrophosphate sensing and isothermal gene amplification (ramified rolling circle amplification)-that operates with high selectivity and sensitivity for clinical point-of-care diagnosis of SARS-CoV-2. During the polymerase phase of the DNA amplification process, pyrophosphate was released from the nucleotide triphosphate as a side product, which was then sensed by our RHthio-CuSO4 probe with a visible color change. This simple colorimetric diagnostic system allowed highly sensitive (1.13 copies/reaction) detection of clinical SARS-CoV-2 within 1 h, while also displaying high selectivity, as evidenced by its discrimination of two respiratory viral genomes (human rhino virus and respiratory syncytial virus) from that of SARS-CoV-2. All of the reactions in this system were performed at a single temperature, with positive identification being made by the naked eye, without requiring any instrumentation. The high sensitivity and selectivity, short detection time (1 h), simple treatment (one-pot reaction), isothermal amplification, and colorimetric detection together satisfy the requirements for clinical point-of-care detection of SARS-CoV-2. Therefore, we believe that this combination of a colorimetric probe and isothermal amplification will be useful for point-of-care testing to prevent the propagation of COVID-19.

Dual-site ligation-assisted loop-mediated isothermal amplification (dLig-LAMP) for colorimetric and point-of-care determination of real SARS-CoV-2.

A probing system has been developed based on dual-site ligation-assisted loop-mediated isothermal amplification (dLig-LAMP) for the selective colorimetric detection of SARS-CoV-2. This approach can induce false-positive and -negative detection in real clinical samples; dLig-LAMP operates with improved selectivity. Unlike RT-LAMP, the selectivity of dLig-LAMP is determined in both the ligation and primer binding steps, not in the reverse transcription step. With this selective system in hand, we developed a colorimetric signaling system for point-of-care detection. We also developed a colorimetric probe for sensing pyrophosphate, which arises as a side product during the LAMP DNA amplification. Thus, dLig-LAMP appears to be an alternative method for improving the selectivity problems associated with reverse transcription. In addition, combining dLig-LAMP with colorimetric pyrophosphate probing allows point-of-care detection of SARS-CoV-2 within 1 h with high selectivity.

Unnatural nucleotide-based rkDNA probe combined with graphene oxide for detection of alkaline phosphatase activity.

In this study we developed a fluorescent double-stranded DNA, incorporating an unnatural dUrk nucleotide, that we used as a probe for the detection of alkaline phosphatase (ALP) based on enzymatic cleavage of the non-fluorescent complementary strand. Primer extension performed using the unnatural nucleotide triphosphate dUrkTP and the natural deoxynucleotide triphosphates dATP, dCTP, and dGTP provided a simple fluorescent DNA strand that hybridized with the 5́-monophosphate non-fluorescent complementary strand. When applying the 5́-phosphate recognition and cleavage properties of lambda exonuclease (λ-exo), this probe could bind to graphene oxide (GO) and quench the fluorescence (in the absence of ALP) or not bind to GO and retain its fluorescence (in the presence of ALP). We obtained strongly fluorescent DNA strands through simple incorporation of multiple A sites in the complementary sequence, thereby increasing the number of dUrk residues during primer extension. This unnatural nucleotide-based rkDNA probing system exhibited high fluorescence differentiation for discriminating the status of ALP. This rkDNA-GO probing system appears to be a promising tool for monitoring the activity of disease-associated enzymes.

Structural insights into inhibition of PRRSV Nsp4 revealed by structure-based virtual screening, molecular dynamics, and MM-PBSA studies.

BACKGROUND: Porcine reproductive and respiratory syndrome respiratory sickness in weaned and growing pigs, as well as sow reproductive failure, and its infection is regarded as one of the most serious swine illnesses worldwide. Given the current lack of an effective treatment, in this study, we identified natural compounds capable of inhibiting non-structural protein 4 (Nsp4) of the virus, which is involved in their replication and pathogenesis. RESULTS: We screened natural compounds (n = 97,999) obtained from the ZINC database against Nsp4 and selected the top 10 compounds for analysing protein-ligand interactions and physicochemical properties. The five compounds demonstrating strong binding affinity were then subjected to molecular dynamics simulations (100 ns) and binding free energy calculations. Based on analysis, we identified four possible lead compounds that represent potentially effective drug-like inhibitors. CONCLUSIONS: These methods identified that these natural compounds are capable of inhibiting Nsp4 and possibly effective as antiviral therapeutics against PRRSV.

Comparison of the clinical characteristics and outcomes of pediatric patients with and without diabetic ketoacidosis at the time of type 1 diabetes diagnosis.

PURPOSE: We investigated the possible effects of diabetic ketoacidosis (DKA) at the initial diagnosis of type 1 diabetes mellitus (T1DM) on the clinical outcomes of pediatric patients. METHODS: Medical records of children and adolescents with newly diagnosed T1DM seen in the Ajou University Hospital from January 2008 to August 2020 were reviewed and analyzed. RESULTS: Among 129 diagnosed T1DM patients, 40.3% presented with DKA. Although demographic and basic characteristics did not differ between DKA and non-DKA patients, DKA patients needed a significantly higher insulin dosage than non-DKA patients for 2 years after diagnosis. However, control of glycated hemoglobin was not different between the DKA and non-DKA groups during the observation period. In the biochemical analysis, C-peptide, insulin-like growth factor-1, and insulin-like growth factor binding protein 3, high-density lipoprotein cholesterol, free T4, and T3 values were lower, but thyroid-stimulating hormone, initial serum glucose, uric acid, total cholesterol, triglyceride, and low-density lipoprotein cholesterol values were higher in DKA patients than non-DKA patients at the diagnosis of T1DM; however, these differences were temporarily present and disappeared with insulin treatment. Other clinical outcomes, such as height, thyroid function, and urine microalbumin level, did not vary significantly between the DKA and non-DKA groups during 5 years of follow-up. CONCLUSION: DKA at initial presentation reflects the severity of disease progression, and the deleterious effects of DKA seem to impact insulin secretion. Although no difference in long-term prognosis was found, early detection of T1DM should help to reduce DKA-related islet damage and the socioeconomic burden of T1DM.

Directly arylated oligonucleotides as fluorescent molecular rotors for probing DNA single-nucleotide polymorphisms.

We developed direct arylated oligonucleotide based molecular rotor (AOMR) to discriminate perfect matched DNA sequence from one base mismatched sequences. Quinolinium salts attached to vinyl aniline would be excellent fluorescent analogs with molecular rotor properties and are suitable for the detection of microenvironment change arising from dynamic motions with match-mismatch DNA base pairs. We applied direct N6 arylation of the adenosine located in natural oligonucleotide as a tool to incorporate the molecular rotor (Quinolinium salts attached vinyl aniline) and used it to discriminate perfect matched DNA sequence from one base mismatch sequences. The fluorescence and quantum yield of arylated oligonucleotide based molecular rotor (AOMR), particulary, RMAQn reveals 28.3 times higher discrimination factor with perfect matched sequence (RMAQn:T) (QY = 0.17) compare to single strand RMAQn (QY = 0.006) and one base mismatched sequence (RMAQn:G, RMAQn:A, and RMAQn:C) at λmax = 600 nm (orange emission), which would be useful for in vivo application. RMAQn:T duplex also showed high brightness (6068), 32.9 times higher than single strand RMAQn (192), as a result of restricted rotation of the Quinolinium salts attached vinyl aniline on adenosine moiety with perfect matched sequence compare to the mismatch sequences. Arylated oligonucleotide based molecular rotor (AOMR) proves to be an unprecedented sensitivity in detecting local dynamics of nucleic acids and also would be simple and cost-effective method to prepare SNP probe.