A bisulfite-assisted and ligation-based qPCR amplification technology for locus-specific pseudouridine detection at base resolution.

Over 150 types of chemical modifications have been identified in RNA to date, with pseudouridine (Ψ) being one of the most prevalent modifications in RNA. Ψ plays vital roles in various biological processes, and precise, base-resolution detection methods are fundamental for deep analysis of its distribution and function. In this study, we introduced a novel base-resolution Ψ detection method named pseU-TRACE. pseU-TRACE relied on the fact that RNA containing Ψ underwent a base deletion after treatment of bisulfite (BS) during reverse transcription, which enabled efficient ligation of two probes complementary to the cDNA sequence on either side of the Ψ site and successful amplification in subsequent real-time quantitative PCR (qPCR), thereby achieving selective and accurate Ψ detection. Our method accurately and sensitively detected several known Ψ sites in 28S, 18S, 5.8S, and even mRNA. Moreover, pseU-TRACE could be employed to measure the Ψ fraction in RNA and explore the Ψ metabolism of different pseudouridine synthases (PUSs), providing valuable insights into the function of Ψ. Overall, pseU-TRACE represents a reliable, time-efficient and sensitive Ψ detection method.

Steric hindrance of N6-methyl in m6A and its application for specific loci detection.

We found that the N6 methyl group of N6-methyladenine is able to hinder the methylation of N6-methyladenine at the N1 position by DMS. Based on this, we have devised a novel method for detecting N6-methyladenine, which was successfully applied to identify specific m6A loci in 28S rRNA.

Identifying spatiotemporal trends of SARS-CoV-2 RNA in wastewater: from the perspective of upstream and downstream wastewater-based epidemiology (WBE).

Recently, many efforts have been made to address the rapid spread of newly identified COVID-19 virus variants. Wastewater-based epidemiology (WBE) is considered a potential early warning tool for identifying the rapid spread of this virus. This study investigated the occurrence of SARS-CoV-2 in eight wastewater treatment plants (WWTPs) and their sewerage systems which serve most of the population in Taoyuan City, Taiwan. Across the entire study period, the wastewater viral concentrations were correlated with the number of COVID-19 cases in each WWTP (Spearman's r = 0.23-0.76). In addition, it is confirmed that several treatment technologies could effectively eliminate the virus RNA from WWTP influent (> 90%). On the other hand, further results revealed that an inverse distance weighted (IDW) interpolation and hotspot model combined with the geographic information system (GIS) method could be applied to analyze the spatiotemporal variations of SARS-CoV-2 in wastewater from the sewer system. In addition, socio-economic factors, namely, population density, land use, and income tax were successfully identified as the potential drivers which substantially affected the onset of the COVID-19 outbreak in Taiwan. Finally, the data obtained from this study can provide a powerful tool in public health decision-making not only in response to the current epidemic situation but also to other epidemic issues in the future.

Development and Validation of a Breastfeeding Health Literacy Scale (BFHLS) for Taiwanese Pregnant Women.

BACKGROUND: Breastfeeding is the best source of nutrition for infants. Health literacy is a critical factor affecting breastfeeding rates. RESEARCH AIMS: The aim of this research was to develop and test the Breastfeeding Health Literacy Scale to determine its validity and reliability. METHODS: This study featured a cross-sectional telephone survey design. Researchers reviewed the literature and used expert opinions to develop the content-validated 30-item Breastfeeding Health Literacy Scale covering five dimensions. We examined internal consistency, exploratory factor analysis, and confirmatory factor analysis to assess reliability and construct validity. A Taiwanese government organization provided potential participants' contact information. After mailing an invitation letter, researchers phoned all participants to invite participation, obtain oral consent and complete a Breastfeeding Health Literacy Scale and collect demographic data. RESULTS: Participants (N = 300) had a mean age of 31.8 (SD = 4.66) years. The item-level content validity index was 0.67 to 1.00 and scale-content validity index was 0.94. After performing exploratory factor analysis, three factors were extracted. Examining content factor analysis for the three factors resulted in χ2/df = 2.05; p < .001; goodness of fit index = 0.90; Comparative Fit Index (CFI) = 0.96; and Root Mean Square Error of Approximation (RMSEA) = 0.06. Cronbach's alphas on the total scale and the subscales ranged from 0.87 to 0.94. Women with multigravida, breastfeeding information from physicians and nurses, and previous breastfeeding experience had better breastfeeding health literacy. CONCLUSIONS: Psychometric analysis demonstrated that the newly developed 20-item Breastfeeding Health Literacy Scale is a valid self-assessment instrument. Improving breastfeeding health literacy during pregnancy could help enable breastfeeding success.

A review on medical waste treatment in COVID-19 pandemics: Technologies, managements and future strategies.

Since the outbreak of COVID-19 few years ago, the increasing of the number of medical waste has become a huge issue because of their harmful impact to environment. A major concern associated to the limitation of technologies for dealing with medical waste, especially conventional technologies, are overcapacities since pandemic occurs. Moreover, the outbreak of new viruses from post COVID-19 should become a serious attention to be prevented not only environmental issues but also the spreading of viruses to new pandemic near the future. The high possibility of an outbreak of new viruses and mutation near the future should be prevented based on the experience associated with the SARS-CoV-2 virus in the last 3 yr. This review presented information and strategies for handling medical waste during the outbreak of COVID-19 and post-COVID-19, and also information on the current issues related to technologies, such as incineration, pyrolysis/gasification, autoclaves and microwave treatment for the dealing with high numbers of medical waste in COVID-19 to prevent the transmission of SARS-CoV-2 virus, their advantages and disadvantages. Plasma technology can be considered to be implemented as an alternative technology to deal with medical waste since incinerator is usually over capacities during the pandemic situation. Proper treatment of specific medical waste in pandemics, namely face masks, vaccine vials, syringes, and dead bodies, are necessary because those medical wastes are mediums for transmission of the SARS-CoV-2 virus. Furthermore, emission controls from incinerator and plasma are necessary to be implemented to reduce the high concentration of CO2, NOx, and VOCs during the treatment. Finally, future strategies of medical waste treatment in the perspective of potential outbreak pandemic from new mutation viruses are discussed in this review paper.Implications: Journal of the air and waste management association may consider our review paper to be published. In this review, we give important information related to the technologies, managements and strategies for handling the medical waste and control the transmission of SARS-CoV-2 virus, starting from proper technology to control the high number of medical waste, their pollutants and many strategies for controlling the spreading of SARS-CoV-2 virus. Moreover, this review also describes some strategies associated with control the transmission not only the SARS-CoV-2 virus but also the outbreak of new viruses near the future.

m6A modification negatively regulates translation by switching mRNA from polysome to P-body via IGF2BP3.

In the cytoplasm, mRNAs are dynamically partitioned into translating and non-translating pools, but the mechanism for this regulation has largely remained elusive. Here, we report that m6A regulates mRNA partitioning between polysome and P-body where a pool of non-translating mRNAs resides. By quantifying the m6A level of polysomal and cytoplasmic mRNAs with m6A-LAIC-seq and m6A-LC-MS/MS in HeLa cells, we observed that polysome-associated mRNAs are hypo-m6A-methylated, whereas those enriched in P-body are hyper-m6A-methylated. Downregulation of the m6A writer METTL14 enhances translation by switching originally hyper-m6A-modified mRNAs from P-body to polysome. Conversely, by proteomic analysis, we identify a specific m6A reader IGF2BP3 enriched in P-body, and via knockdown and molecular tethering assays, we demonstrate that IGF2BP3 is both necessary and sufficient to switch target mRNAs from polysome to P-body. These findings suggest a model for the dynamic regulation of mRNA partitioning between the translating and non-translating pools in an m6A-dependent manner.

Antibody-Free Fluorine-Assisted Metabolic Sequencing of RNA N4-Acetylcytidine.

N4-Acetylcytidine (ac4C) has been found to affect a variety of cellular and biological processes. For a mechanistic understanding of the roles of ac4C in biology and disease, we present an antibody-free, fluorine-assisted metabolic sequencing method to detect RNA ac4C, called "FAM-seq". We successfully applied FAM-seq to profile ac4C landscapes in human 293T, HeLa, and MDA cell lines in parallel with the reported acRIP-seq method. By comparison with the classic ac4C antibody sequencing method, we found that FAM-seq is a convenient and reliable method for transcriptome-wide mapping of ac4C. Because this method holds promise for detecting nascent RNA ac4C modifications, we further investigated the role of ac4C in regulating chemotherapy drug resistance in chronic myeloid leukemia. The results indicated that drug development or combination therapy could be enhanced by appreciating the key role of ac4C modification in cancer therapy.

Nanomolar range of FAM237B can activate receptor GPR83.

Our recent study confirmed that the mature neuropeptide FAM237A, also known as neurosecretory protein GL (NPGL), is an efficient agonist for GPR83. The paralog FAM237B was previously reported as a weak agonist for GPR83. In the present study, we prepared mature human FAM237B via an intein-fusion approach and demonstrated that it could cause a significant activation effect at the nanomolar range (1‒10 nM) in a NanoBiT-based ß-arrestin recruitment assay. Thus, FAM237B appears to be another endogenous agonist for GPR83 and future in vivo studies will be required to confirm this.

The ghrelin receptor GHSR has two efficient agonists in the lobe-finned fish Latimeria chalumnae.

The peptide hormone ghrelin (an agonist) and LEAP2 (an antagonist) play important functions in energy metabolism via their receptor GHSR, an A-class G protein-coupled receptor. Ghrelin, LEAP2, and GHSR are widely present from fishes to mammals. However, our recent study suggested that fish GHSRs have different binding properties to ghrelin: a GHSR from the lobe-finned fish Latimeria chalumnae (coelacanth) is efficiently activated by ghrelin, but GHSRs from the ray-finned fish Danio rerio (zebrafish) and Larimichthys crocea (large yellow croaker) have lost binding to ghrelin. Do fish GHSRs use another peptide as their agonist? In the present study we tested to two fish motilins from D. rerio and L. chalumnae because motilin is distantly related to ghrelin. In ligand binding and activation assays, the fish GHSRs from D. rerio and L. crocea displayed no detectable or very low binding to all tested motilins; however, the fish GHSR from L. chalumnae bound to its motilin with high affinity and was efficiently activated by it. Therefore, it seemed that motilin is not a ligand for GHSR in the ray-finned fish D. rerio and L. crocea, but is an efficient agonist for GHSR in the lobe-finned fish L. chalumnae, one of the closest fish relatives of tetrapods. The results of present study suggested that GHSR might have two efficient agonists, ghrelin and motilin, in ancient fishes; however, this feature might be only preserved in some extant fishes with ancient evolutionary origins.

Disease Diagnosis Based on Nucleic Acid Modifications.

Nucleic acid modifications include a wide range of epigenetic and epitranscriptomic factors and impact a wide range of nucleic acids due to their profound influence on biological inheritance, growth, and metabolism. The recently developed methods of mapping and characterizing these modifications have promoted their discovery as well as large-scale studies in eukaryotes, especially in humans. Because of these pioneering strategies, nucleic acid modifications have been shown to have a great impact on human disorders such as cancer. Therefore, whether nucleic acid modifications could become a new type of biomarker remains an open question. In this review, we briefly look back at classical nucleic acid modifications and then focus on the progress made in investigating these modifications as diagnostic biomarkers in clinical therapy and present our perspective on their development prospects.

Circular RNA hsa_circ_0067842 facilitates tumor metastasis and immune escape in breast cancer through HuR/CMTM6/PD-L1 axis.

BACKGROUND: Circular RNAs (circRNAs) have been shown to play diverse biological functions in the progression of multiple diseases. However, the impacts of circRNAs on breast cancer (BC) progression remains unclear. Therefore, the objective of this paper is to investigate the role and mechanisms of a functional circRNA in BC metastasis and immune escape. METHODS: This study used a circRNA microarray and identified a novel circRNA hsa_circ_0067842. The validation and characteristics of hsa_circ_0067842 were investigated using qRT-PCR, sanger sequencing, RNase R treatment, actinomycin D treatment and fluorescence in situ hybridization (FISH). Gain- and loss-of-function assays were performed to evaluate the biological function of hsa_circ_0067842 in BC progression and immune escape. Mechanistically, the interaction between hsa_circ_0067842 and HuR was explored by RNA pull down, mass spectrometry (MS), subcellular component protein extraction and immunofluorescence (IF). The regulatory mechanisms of hsa_circ_0067842/HuR/CMTM6/PD-L1 axis were investigated by qRT-PCR, western blot, FISH, immunoprecipitation and rescue assays. RESULTS: The expression of hsa_circ_0067842 was upregulated in BC tissues and cells, which was found to be significantly associated with poor prognosis, regardless of other clinical covariates. Function assays showed that hsa_circ_0067842 promoted the migration and invasion capacities of BC cells. Moreover, co-culture experiment with peripheral blood mononuclear cells (PBMCs) showed that hsa_circ_0067842 played a role in the immune escape of BC cells. Mechanistically, our study showed that hsa_circ_0067842 interacted with HuR, affecting its nuclear translocation, thus enhancing the stability of CMTM6. CMTM6 not only enhances the migration and invasion ability of BC cells, but also affects the ubiquitination of PD-L1 and inhibits its degradation. CONCLUSION: Collectively, our results demonstrated that hsa_circ_0067842 promoted BC progression through the HuR/CMTM6/PD-L1 axis, providing new insight and a potential target for BC prognosis and therapy.

[Laboratory testing strategies for human immunodeficiency virus (HIV) in blood donors].

Objective To propose the blood detection strategies for human immunodeficiency virus (HIV) among blood donors, and provide reference for the detection, early diagnosis and transmission blocking of HIV. Methods A total of 117 987 blood samples from blood donors were screened using the third- and fourth-generation ELISA HIV detection reagents. Western blot analysis was used to verify the reactive results of the third-generation reagent alone, or both the third-generation and fourth-generation reagents. HIV nucleic acid test was carried out for those with negative test results of the third- and fourth-generation reagents. For those with positive results of the fourth-generation reagent only, nucleic acid test followed by a confirmatory test by Western blot analysis was carried out. Results 117 987 blood samples from blood donors were tested by different reagents. Among them, 55 were tested positive by both the third- and fourth-generation HIV detection reagents at the same time, accounting for 0.047% and 54 cases were confirmed HIV-positive by Western blot analysis, and 1 case was indeterminate, then turned positive during follow-up testing. 26 cases were positive by the third-generation reagent test alone, among which 24 cases were negative and 2 were indeterminate by Western blot analysis. The band types were p24 and gp160 respectively detected by Western blot analysis, and were confirmed to be HIV negative in follow-up testing. 31 cases were positive by the fourth-generation HIV reagent alone, among which 29 were negative by nucleic acid test, and 2 were positive according to the nucleic acid test.Western blot analysis was used to verify that the two cases were negative. However, after 2~4 weeks, the results turned positive when the blood sample was retested by Western blot analysis during the follow-up of these two cases. All the specimens that were tested negative by both the third- and fourth-generation HIV reagents were validated negative by HIV nucleic acid test. Conclusion A combined strategy with both third- and fourth-generation HIV detection reagents can play a complementary role in blood screening among blood donors. The application of complementary tests, such as nucleic acid test and Western blot analysis, can further improve the safety of blood supply, thus contributing to the early diagnosis, prevention, transmission and treatment of blood donors potentially infected by HIV.

A reference interval study of ferritin and transferrin levels in donors from two blood centers.

Blood donors not only save the lives of patients but also play an important role in the development of medical and health services. Therefore, it is particularly important to pay attention to the blood health of blood donors who are at a high risk of iron deficiency. Detection of serum ferritin and transferrin is an important basis for the diagnosis of iron deficiency anemia. However, to the best of our knowledge, the levels of serum ferritin and transferrin, and the influencing factors, such as age and type of donation, in blood donors have not been clarified. In the present study, the serum ferritin and transferrin levels of donors from two blood centers were investigated. Demographic data were collected from the donors, and their serum ferritin and transferrin levels were tested. A total of 1,817 donors were enrolled and were eligible for evaluation. Reference intervals (RIs) for ferritin and transferrin were obtained from blood donors, and it was revealed that the ferritin and transferrin levels of blood donors were associated with age. Furthermore, serum transferrin levels were associated with the type of donation; the serum transferrin RI level was significantly higher in platelet-only donors compared with in whole blood donors. It was also demonstrated that ferritin levels were negatively associated with transferrin levels. The present study identified RIs for ferritin and transferrin levels in blood donors, and indicated that age and type of donation were important factors affecting ferritin and transferrin levels in blood donors. These findings may prove useful for blood donation recruitment and screening strategies in China, and could promote the health of blood donors.

Pregnancy incidence and associated risk factors of dichorionic triamniotic triplet under assisted reproduction: A large sample of clinical data analysis.

Background: Dichorionic triamniotic (DCTA) triplet pregnancies are rare in spontaneous pregnancy. The aim was to characterize the incidence and risk factors of DCTA triplet pregnancies after assisted reproductive technology (ART). Methods: A retrospective analysis of 10,289 patients, including 3,429 fresh embryo transfer (ET) cycle and 6,860 frozen ET cycle, was performed from January 2015 to June 2020. The effect of different ART parameters on the incidence of DCTA triplet pregnancies was evaluated by multivariate logistic regression analyses. Results: Among all clinical pregnancies after ART, the incidence of DCTA was 1.24%. 1.22% occurred in the fresh ET cycle, while 1.25% occurred in the frozen ET cycle. The number of ET and cycle type has no effect on the occurrence of DCTA triplet pregnancies (p = 0.987; p = 0.056, respectively). There were significant differences in DCTA triplet pregnancies rate among receiving intracytoplasmic sperm injection (ICSI) and receiving in vitro fertilization (IVF) [1.92% vs. 1.02%, p < 0.001, OR = 0.461, 95% confidence interval (CI) 0.315-0.673], blastocyst transfer (BT) versus cleavage-ET (1.66% vs. 0.57%, P < 0.001, OR = 0.329, 95% CI 0.315-0.673), and maternal age ≥ 35 years versus maternal age < 35 years (1.00% vs. 1.30%, P = 0.040, OR = 1.773, 95% CI 1.025-3.066). Based on the regression analysis of cycle type, DCTA triplet pregnancies rate was higher in maternal age < 35 years than in maternal age ≥ 35 years (1.35% vs. 0.97%, P < 0.001, OR = 5.266, 95% CI 2.184-12.701), BT versus cleavage-ET (1.47% vs. 0.94%; P = 0.006, OR = 0.346, 95% CI 0.163-0.735), and receiving ICSI was higher than receiving IVF (3.82% vs. 0.78%, p < 0.001, OR = 0.085, 95% CI 0.039-0.189) in fresh ET cycle. However, DCTA triplet pregnancies rate did not show difference in maternal age, insemination methods, and number of ET, and only BT was found to be associated with a higher DCTA triplet pregnancies rate in the frozen ET cycle (1.73% vs. 0.30%, p < 0.001, OR = 0.179, 95% CI 0.083-0.389). Conclusion: The prevalence of DCTA triplet pregnancies has increased after ART. Maternal age < 35 years, BT, and receiving ICSI are risk factors for DCTA triplet pregnancies, also in fresh ET cycle. However, in frozen ET cycle, BT is an independent risk factor for increased DCTA triplet pregnancies rate.

NSUN2-mediated M5c methylation of IRF3 mRNA negatively regulates type I interferon responses during various viral infections.

5-Methylcytosine (m5C) is a widespread post-transcriptional RNA modification and is reported to be involved in manifold cellular responses and biological processes through regulating RNA metabolism. However, its regulatory role in antiviral innate immunity has not yet been elucidated. Here, we report that NSUN2, a typical m5C methyltransferase, negatively regulates type I interferon responses during various viral infections, including SARS-CoV-2. NSUN2 specifically mediates m5C methylation of IRF3 mRNA and accelerates its degradation, resulting in low levels of IRF3 and downstream IFN-ß production. Knockout or knockdown of NSUN2 enhanced type I interferon and downstream ISGs during various viral infection in vitro. And in vivo, the antiviral innate response is more dramatically enhanced in Nsun2+/- mice than in Nsun2+/+ mice. The highly m5C methylated cytosines in IRF3 mRNA were identified, and their mutation enhanced cellular IRF3 mRNA levels. Moreover, infection with Sendai virus (SeV), vesicular stomatitis virus (VSV), herpes simplex virus 1 (HSV-1), or Zika virus (ZIKV) resulted in a reduction of endogenous NSUN2 levels. Especially, SARS-CoV-2 infection (WT strain and BA.1 omicron variant) also decreased endogenous levels of NSUN2 in COVID-19 patients and K18-hACE2 KI mice, further increasing type I interferon and downstream ISGs. Together, our findings reveal that NSUN2 serves as a negative regulator of interferon response by accelerating the fast turnover of IRF3 mRNA, while endogenous NSUN2 levels decrease during SARS-CoV-2 and various viral infections to boost antiviral responses for effective elimination of viruses.

Pneumocystis jirovecii diagnosed by next-generation sequencing of bronchoscopic alveolar lavage fluid: A case report and review of literature.

BACKGROUND: The advent of molecular targeted agents and immune checkpoint inhibitors has greatly improved the treatment of advanced renal cell carcinoma (RCC), thus significantly improving patient survival. The incidence of rare drug-related adverse events has gained increased attention. CASE SUMMARY: We report a patient with advanced RCC treated with multiple lines of molecular targeted agents and immune checkpoint inhibitors, who developed a pulmonary infection after treatment with everolimus in combination with lenvatinib. Determining the pathogenic organism was difficult, but it was eventually identified as Pneumocystis jirovecii by next-generation sequencing (NGS) of bronchoscopic alveolar lavage fluid (BALF) and successfully treated with trimethoprim-sulfamethoxazole. CONCLUSION: Rare pulmonary infections caused by molecular targeted agents are not uncommon in clinical practice, but their diagnosis is difficult. Evaluating BALF with NGS is a good method for rapid diagnosis of such infections.

FAM237A, rather than peptide PEN and proCCK56-63, binds to and activates the orphan receptor GPR83.

G protein-coupled receptor 83 (GPR83) is primarily expressed in the brain and is implicated in the regulation of energy metabolism and some anxiety-related behaviours. Recently, the PCSK1N/proSAAS-derived peptide PEN, the procholecystokinin-derived peptide proCCK56-63, and family with sequence similarity 237 member A (FAM237A) were all reported as efficient agonists of GPR83. However, these results have not yet been reproduced by other laboratories and thus GPR83 is still officially an orphan receptor. The peptide PEN and proCCK56-63 share sequence similarity; however, they are completely different from FAM237A. To identify its actual ligand(s), in the present study we developed NanoLuc Binary Technology (NanoBiT)-based ligand-binding assay, fluorescent ligand-based visualization, and NanoBiT-based ß-arrestin recruitment assay for human GPR83. Using these assays, we demonstrated that mature human FAM237A could bind to GPR83 with nanomolar range affinity, and could activate this receptor and induce its internalization with nanomolar range efficiency in transfected human embryonic kidney 293T cells. However, we did not detect any interaction of PEN and proCCK56-63 with GPR83 using these assays. Thus, our results confirmed that FAM237A is an efficient agonist of GPR83, but did not support PEN and proCCK56-63 as ligands of this receptor. Clarification of their pairing paves the way for further functional studies of the brain-specific receptor GPR83 and the so far rarely studied neuropeptide FAM237A in the future.

Tongxinluo enhances the effect of atorvastatin on the treatment of atherosclerosis with chronic obstructive pulmonary disease by maintaining the pulmonary microvascular barrier.

Atherosclerosis (AS) is a common comorbidity of chronic obstructive pulmonary disease (COPD), and systemic inflammation is an important mechanism of COPD with AS. Tongxinluo (TXL) improves the function of vascular endothelial cells. We aimed to prove that impairment of pulmonary microvascular barrier function is involved in COPD-mediated aggravation of AS and investigate whether TXL enhances the effect of Ato (atorvastatin) on COPD with AS by protecting pulmonary microvascular endothelial barrier function. In vivo, a COPD with atherosclerotic apolipoprotein E knockout (AS ApoE-/-) mouse model was established by cigarette smoke combined with a high-fat diet. The animals were administered TXL, Ato, and TXL + Ato once a day for 20 weeks. Lung function, lung microvascular permeability, lung inflammation, systemic inflammation, serum lipid levels, atheromatous plaque formation, and endothelial damage biomarkers were measured. In vitro, human pulmonary microvascular endothelial cells (HPMECs) were pretreated with TXL and incubated with cigarette smoke extract to establish the model. The permeability of the endothelial monolayer, inflammatory cytokines, endothelial damage biomarkers, and tight junction (Tj) proteins were determined. Cigarette smoking significantly exacerbated the high-fat diet-induced pulmonary function decline, pulmonary microvascular endothelial barrier dysfunction, inflammation, and atherosclerotic plaques. These changes were reversed by TXL-Ato; the combination was more effective than Ato alone. Furthermore, TXL protected the HPMEC barrier and inhibited inflammation in HPMECs. COPD aggravates AS, possibly through the destruction of pulmonary microvascular barrier function; thus, lung inflammation triggers systemic inflammation. In treating COPD with AS, TXL enhances the antiatherosclerotic effect of Ato, protecting the pulmonary microvascular barrier.

LEAP2 is a more conserved ligand than ghrelin for fish GHSRs.

Recently, liver-expressed antimicrobial peptide 2 (LEAP2) was identified as an endogenous antagonist and an inverse agonist of the ghrelin receptor GHSR. However, its functions in lower vertebrates are not well understood. Our recent study demonstrated that both LEAP2 and ghrelin are functional towards a fish GHSR from Latimeria chalumnae, an extant coelacanth believed to be one of the closest ancestors of tetrapods. However, amino acid sequence alignment identified that the 6.58 position (Ballesteros-Weinstein numbering system) of most fish GHSRs are not occupied by an aromatic Phe residue, which is absolutely conserved in all known GHSRs from amphibians to mammals, and is responsible for human GHSR binding to its agonist, ghrelin. To test whether these unusual fish receptors are functional, we studied the ligand binding properties of three representative fish GHSRs, two from Danio rerio (zebrafish) and one from Larimichthys crocea (large yellow croaker). After overexpression in human embryonic kidney 293T cells, the three fish GHSRs retained normal binding to all tested LEAP2s, except for a second LEAP2 from L. crocea. However, they displayed almost no binding to all chemically synthesized n-octanoylated ghrelins, despite these ghrelins all retaining normal function towards human and coelacanth GHSRs. Thus, it seems that LEAP2 is a more conserved ligand than ghrelin towards fish GHSRs. Our results not only provided new insights into the interaction mechanism of GHSRs with LEAP2s and ghrelins, but also shed new light on the functions of LEAP2 and ghrelin in different fish species.