Reevaluating the splice-altering variant in TECTA as a cause of nonsyndromic hearing loss DFNA8/12 by functional analysis of RNA.

PURPOSE: The aim of this study was to determine the genetic cause of early onset autosomal dominant hearing loss segregating in five-generation kindred of Chinese descent and provide preimplantation genetic testing (PGT)for them. METHODS: Clinical examination, pedigree analysis and exome sequencing were carried out on the family. Minigene-based splicing analysis, in vivo RNA analysis and protein structure prediction by molecular modeling were conducted on the candidate variant. PGT for the causative variation and chromosome aneuploidis based on SNP analysis has been used for avoidance of hearing loss in this family. RESULTS: All the affected individuals presented with moderate down-sloping hearing loss and whole-exome sequencing identified a novel splice-site variant c.5383+6T>A in the tested subjects within the TECTA locus. Genotyping of all the 32 family members confirmed segregation of this variant and the hearing loss phenotype in the extended family. Functional analysis of RNA and molecular modeling indicates that c.5383+6T>A is a pathogenic splice-site variant and should be considered as genetic cause of the hearing loss. Furthermore, a successful singleton pregnancy with no variation in TECTA c.5383+6 was established and a healthy male child was born by PGT. CONCLUSION: We have identified a novel variant c.5383+6T>A in TECTA ZA-ZP inter-domain, which could be attributable to the early-onset autosomal dominant hearing loss. The implications of our study are valuable in elucidating the disrupted RNA splicing and uncovering the genetic cause of hearing loss with TECTA pathogenic variants, as well as providing reproductive approaches to healthy offspring.

Neuroplasticity of visual brain network induced by hypoxia.

The effects of hypoxia on brain function remain largely unknown. This study aimed to clarify this issue by visual-stimulated functional magnetic resonance imaging design. Twenty-three college students with a 30-d high-altitude exposure were tested before, 1 week and 3 months after returning to sea level. Brain functional magnetic resonance imaging and retinal electroretinogram were acquired. One week after returning to sea level, decreased blood oxygenation level dependent in the right lingual gyrus accompanied with increased blood oxygenation level dependent in the frontal cortex and insular cortex, and decreased amplitude of electroretinogram a-wave in right eye; moreover, the bilateral lingual gyri showed increased functional connectivity within the dorsal visual stream pathway, and the blood oxygenation level dependent signals in the right lingual gyrus showed positive correlation with right retinal electroretinogram a-wave. Three months after returning to sea level, the blood oxygenation level dependent signals recovered to normal level, while intensively increased blood oxygenation level dependent signals in a broad of brain regions and decreased retinal electroretinogram were also existed. In conclusion, hypoxic exposure has long-term effects on visual cortex, and the impaired retinal electroretinogram may contribute to it. The increased functional connectivity of dorsal stream may compensate for the decreased function of retinal photoreceptor cells to maintain normal visual function.

Fibroblast growth factor receptor 1/Klothoß agonist BFKB8488A improves lipids and liver health markers in patients with diabetes or NAFLD: A phase 1b randomized trial.

BACKGROUND AND AIMS: BFKB8488A is a bispecific antibody targeting fibroblast growth factor receptor 1c and Klothoß. This phase 1b study assessed safety, tolerability, pharmacokinetics, immunogenicity, and pharmacodynamics of BFKB8488A in patients with type 2 diabetes mellitus (T2DM) or NAFLD. APPROACH AND RESULTS: Patients were randomized to receive multiple doses of BFKB8488A at various dose levels and dosing intervals (weekly, every 2 weeks, or every 4 weeks) or placebo for 12 weeks. The primary outcome was the safety of BFKB8488A. Overall, 153 patients (T2DM: 91; NAFLD: 62) were enrolled and received at least one dose of treatment. Of these, 102 patients (62.7%) reported at least one adverse event (BFKB8488A: 83 [68.6%]; placebo: 19 [59.4%]). BFKB8488A exhibited nonlinear pharmacokinetics, with greater than dose-proportional increases in exposure. The treatment-emergent antidrug antibody incidence was 22.7%. Overall, trends in exposure-dependent increases in high-density lipoprotein (HDL) and decreases in triglyceride levels were observed. Decreases in alanine aminotransferase and aspartate aminotransferase were 0.7% and 9.2% for medium exposure and 7.3% and 11.2% for high-exposure tertiles, compared with increases of 7.5% and 17% in the placebo group, respectively, at Day 85. In patients with NAFLD, the mean decrease from baseline liver fat was 13.0%, 34.5%, and 49.0% in the low-, medium-, and high-exposure tertiles, respectively, compared with 0.1% with placebo at Day 85. CONCLUSIONS: BFKB8488A was adequately tolerated in patients with T2DM or NAFLD, leading to triglyceride reduction, HDL improvements, and trends in improvement in markers of liver health for both populations and marked liver fat reduction in patients with NAFLD. ( ClinicalTrials.gov : NCT03060538).

Next-generation sequencing analysis of the molecular spectrum of thalassemia in Southern Jiangxi, China.

BACKGROUND: Thalassemia is an extremely prevalent monogenic inherited blood disorder in southern China. It is important to comprehensively understand the molecular spectrum of thalassemia in an area with such a high prevalence of thalassemia before taking appropriate actions for the prevention and treatment of this disorder. Herein, we explored the clinical feasibility of using next-generation sequencing (NGS) for large-scale population screening to illustrate the prevalence and spectrum of thalassemia in Southern Jiangxi. METHODS: Blood samples collected from 136,312 residents of reproductive age in Southern Jiangxi were characterized for thalassemia by NGS. A retrospective analysis was then conducted on blood samples determined to be positive for thalassemia. RESULTS: In total, 19,827 (14.545%) subjects were diagnosed as thalassemia carriers, and the thalassemia prevalence rate significantly varied by geographical region (p < 0.001). A total of 40 α-thalassemia genotypes including 21 rare genotypes were identified, with -@-SEA/αα being the most prevalent genotype. 42 ß-thalassemia genotypes including 27 rare genotypes were identified, with the most common mutation IVS II-654 C > T accounting for 35.257% of these ß-thalassemia genotypes. Furthermore, 74 genotypes were identified among 608 individuals with combined α- and ß-thalassemia. Notably, most individuals with rare thalassemia mutations had mildly abnormal hematologic parameters including microcytic hypochromia. CONCLUSIONS: Our findings demonstrate the great heterogeneity and diverse spectrum of thalassemia in Southern Jiangxi, emphasizing the importance and necessity of persistent prevention and control of thalassemia in this region. Additionally, our findings further suggest that NGS can effectively identify rare mutations and reduce the misdiagnosis rate of thalassemia.

Evaluating the Clinical Utility of a Long-Read Sequencing-Based Approach in Prenatal Diagnosis of Thalassemia.

BACKGROUND: The aim is to evaluate the clinical utility of a long-read sequencing-based approach termed comprehensive analysis of thalassemia alleles (CATSA) in prenatal diagnosis of thalassemia. METHODS: A total of 278 fetuses from at-risk pregnancies identified in thalassemia carrier screening by PCR-based methods were recruited from 9 hospitals, and PCR-based methods were employed for prenatal diagnosis. CATSA was performed retrospectively and blindly for all 278 fetuses. RESULTS: Among the 278 fetuses, 263 (94.6%) had concordant results and 15 (5.4%) had discordant results between the 2 methods. Of the 15 fetuses, 4 had discordant thalassemia variants within the PCR detection range and 11 had additional variants identified by CATSA. Independent PCR and Sanger sequencing confirmed the CATSA results. In total, CATSA and PCR-based methods correctly detected 206 and 191 fetuses with variants, respectively. Thus, CATSA yielded a 7.9% (15 of 191) increment as compared with PCR-based methods. CATSA also corrected the predicted phenotype in 8 fetuses. Specifically, a PCR-based method showed one fetus had homozygous HBB c.52A > T variants, while CATSA determined the variant was heterozygous, which corrected the predicted phenotype from ß-thalassemia major to trait, potentially impacting the pregnancy outcome. CATSA additionally identified α-globin triplicates in 2 fetuses with the heterozygous HBB c.316-197C > T variant, which corrected the predicted phenotype from ß-thalassemia trait to intermedia and changed the disease prognosis. CONCLUSIONS: CATSA represents a more comprehensive and accurate approach that potentially enables more informed genetic counseling and improved clinical outcomes compared to PCR-based methods.

Hypoxic White Matter Injury and Recovery After Reoxygenation in Adult Mice: Magnetic Resonance Imaging Findings and Histological Studies.

Cognitive dysfunction and brain white matter (WM) injury have been found in adults exposed to hypoxia. However, the mechanisms underlying these impairments remain unclear, and moreover, it is also unclear whether these impairments are reversible after reoxygenation. In this study, adult male mice were exposed to hypoxia for 15 days at a simulated altitude of 4300 m and then reoxygenated for 2 months. Control mice were raised under normoxic conditions. Mice showed a significant decrease in arterial oxygen saturation (SaO2) and an increase in heart rate and breath rate after hypoxic exposure, and they displayed anxiety-like emotion and impaired cognitions. Hypoxic mice showed decreased brain WM fractional anisotropy (FA) and increased mean diffusion (MD) mainly in the corpus callosum and internal capsule. The reason for the adult brain WM injury was myelin rather than axon. Further, the myelin injury was due to the obstruction of oligodendrocyte precursor cells (OPCs) differentiation and eventually led to behavioral deficits. More importantly, the changes in physiological indicators, behavioral disorders, and WM injury caused by hypoxia can be recovered after reoxygenation. Taken together, our data indicate that adult brain WM injury caused by hypoxia is reversible after reoxygenation and enhancing OPCs differentiation may be a promising therapy for clinical hypoxic diseases associated with brain injury. Schematic diagram of brain WM and behavioral changes induced by hypoxia/reoxygenation in adult mice.

Left ventricular strain and myocardial work in short-term peritoneal dialysis patients.

BACKGROUND: Initiation of dialysis encompasses new cardiovascular challenges on patients with end-stage renal disease (ESRD). This study used two-dimensional speckle-tracking echocardiography (2D-STE) to investigate the change of left ventricular (LV) myocardial function undergoing peritoneal dialysis (PD) within 1-3 months. METHODS: A total of 56 patients with ESRD and 27 healthy controls were enrolled in this prospective study. Mean duration of PD was 44.41 ± 16.44 days. We evaluated LV myocardial function of patients with ESRD in baseline and within 1-3 months after PD by 2D-STE with global longitudinal strains (GLS) and myocardial work (MW). Based on the level of serum phosphate before PD, patients were divided into two groups: the group with normal serum phosphate or hyperphosphatemia. RESULTS: Compared with healthy controls, patients with ESRD had impaired GLS (p < .001) and increased global work index (GWI) (p = .034), global constructive work (GCW) (p < .001), global wasted work (GWW) (p < .001), and lower global work efficiency (GWE) (p = .002). After PD therapy, GWI (p = .001), GCW (p < .001), and GWW (p = .023) decreased and closed to healthy subjects (p > .05) and no significant improvement was observed in GLS (p = .387). GLS of basal segments worsened in the hyperphosphatemia group (p = .005) and GWW reduced remarkably in the group with normal serum phosphate after PD treatment (p = .008). The change of left ventricular internal diameter in diastole (LVIDd) was the only parameter influenced GWI in post-dialysis patients (ß = 0.324, p = .013). CONCLUSIONS: Short-term PD treatment improved LV MW in ESRD patients. They benefited more when receiving treatment before the increase of serum phosphorus.

[Application of single sperm sequencing for the preimplantation genetic testing of a Chinese family affected with Spinal muscular atrophy].

OBJECTIVE: To assess the value of single sperm sequencing in preimplantation genetic testing for monogenic disease (PGT-M). METHODS: A Chinese couple with two children whom had died of Spinal muscular atrophy (SMA) and attended the Jiangxi Provincial Maternal and Child Health Care Hospital in June 2020 was selected as the subject. Eleven single sperm samples were isolated by mechanical immobilization and subjected to whole genome amplification. Real-time PCR and Sanger sequencing were used to detect the SMN1 variants in the single sperm samples. Genomic DNA of the wife, her parents and the husband, as well as one single sperm sample harboring the SMN1 variant and two single sperm samples without the variant were used for the linkage analysis. Targeted capture and high-throughput sequencing were carried out to test 100 single nucleotide polymorphisms distributed within 2 Mb up- and downstream the variant site. The haplotypes linked with the SMN1 variants were determined by linkage analysis. Blastocyst embryos were harvested after fertilizing by intracytoplasmic sperm injection. Cells from the trophoblasts of each embryo were biopsied and subjected to whole genome amplification and targeted capture and high-throughput sequencing to determine their carrier status. Chromosomal aneuploidy of wild-type embryos was excluded. An euploid embryo of high quality was transferred. Amniotic fluid sample was taken at 18 weeks of gestation to confirm the status of the fetus. RESULTS: Genetic testing showed that the couple both had deletion of exons 7 ~ 8 of the SMN1 gene. The wife has inherited the deletion from her father, while the husband was de novo. The haplotypes of the husband were successfully constructed by single sperm sequencing. Preimplantation genetic testing has indicated that 5 embryos had harbored the heterozygous variant, 4 embryos were of the wild type, among which 3 were euploid. Prenatal diagnosis during the second trimester of pregnancy has confirmed that the fetus did not carry the deletion. CONCLUSION: By single sperm sequencing and PGT-M, the birth of further affected child has been successfully avoided.

Investigating teachers' participation patterns in online teacher professional development: what is the relationship between participation frequency and participation quality?

Online teacher professional development (OTPD) opportunities are made available to teachers and draw increasing research attention. As the key characteristics of teachers' participation in OTPD, the frequency and quality of participation are increasingly concerned. However, the relationship between teacher participation frequency and participation quality is still unclear. Addressing this problem not only helps reveal teachers' participation patterns in OTPD, but also provides support for promoting teachers' online professional learning and improving OTPD organization and management. To identify teachers' participation patterns and the relationship between participation frequency and participation quality in OTPD, this study analyzed 5,064 log records of 415 teachers using lag sequential analysis, t-test, and Chi-square test. The findings indicated that teachers preferred shallow participation behaviors, such as sharing resources and experience, and seldom carried out deep participation/engagement behaviors (e.g., proposing knowledge topics, establishing teaching and research practices). Teachers with higher participation frequency had lower participation quality in OTPD and tended to repeat shallow participation behaviors. Finally, the study proposed some suggestions for better supporting teachers' participation in online professional development, such as strengthening the links between information sharing activities, knowledge construction activities, and teaching and research practices.

Impaired left ventricular systolic synchrony in patients with lupus Nephritis: A speckle tracking echocardiography study.

OBJECTIVES: We aimed to explore the value of two-dimensional speckle tracking echocardiography measurements of the global longitudinal strain (GLS) and left ventricular mechanical dispersion (LVMD) in the assessment of early stage left ventricular systolic dysfunction and heterogeneity of myocardial contraction in patients with lupus nephritis (LN). METHODS: Patients with LN and extra-renal systemic lupus erythematosus (SLE) and healthy participants in the control group underwent echocardiography for the traditional measurement of the left ventricular systolic and diastolic function and speckle tracking measurements of the GLS and LVMD. GLS was defined as the average value of the peak strain during systole of the left ventricular 17 segments, and LVMD was defined as the standard deviation. The demographic characteristics including age, sex, and body mass index (BMI) of all the participants were collected. The clinical and laboratory characteristics of the patients with LN were collected. RESULTS: We included 41 healthy control, 37 patients with extra-renal SLE, and 73 patients with LN. There were statistically significant differences in the GLS and LVMD between the extra-renal SLE and LN groups (GLS -19.36% vs. -17.61%, p < 0.001; LVMD 35.62 ms vs 42.96 ms, p<0.001). There was a statistically significant difference in the LVMD between the extral-renal SLE and control groups (35.62ms vs 25.51ms, p<0.001), but not in GLS (-19.36% vs -19.52%, p > 0.05). Multiple regression analyses were conducted in a subset of patients, and 24-hour proteinuria was independently associated with LVMD (ß [SE], 0.793 [0.302], p < .05). CONCLUSIONS: Patients with LN have more severe myocardial involvement than patients with extra-renal SLE. The asynchrony in myocardial contraction represented by the LVMD can be recognized earlier than that of the overall contractile functional impairment represented by GLS. In patients with LN, the 24-hour proteinuria was associated with LVMD. This indicates that the heterogeneity in the contractile function may be associated with the severity of renal damage.

Color Discrimination Provides Insight into the Relationship between Personality Cognition and Brain Morphology in the Western Mosquitofish (Gambusia affinis).

Recent studies on the differences in cognitive ability between individuals focused on two aspects: one is whether the individual differences in cognitive ability are related to brain size, the other is whether they pertain to certain personality traits. To explore these two hypotheses, we tested the personality traits, cognitive abilities, and brain volumes of western mosquitofish (Gambusia affinis). First, a color preference test was conducted to select two unbiased colors for G. affinis for subsequent cognitive tests. The results showed that G. affinis had a great preference for red and green to yellow and blue; therefore, the red-green combination was selected for the study of cognitive abilities. Then, we explored the relationship among cognition, personality, and brain morphology through cognitive abilities tests, personality traits, and brain volume measurements. We found that there was a trade-off among cognition, personality, and brain morphology. For example, more active individuals found food faster, but had also poor memory; Those individuals with larger corpus cerebelli were bolder while they were less likely to find food; The individuals that found food faster were more active and had a smaller inferior lobe. The color preference test provides a reliable way for selecting unbiased colors for behavioral studies in G. affinis. Meanwhile, our study indicates that there exists a balance mechanism among cognition, personality, and brain morphology.

Accurate and fast reconstruction for bioluminescence tomography based on adaptive Newton hard thresholding pursuit algorithm.

As a promising noninvasive medical imaging technique, bioluminescence tomography (BLT) dynamically offers three-dimensional visualization of tumor distribution in living animals. However, due to the high ill-posedness caused by the strong scattering property of biological tissues and the limited boundary measurements with noise, BLT reconstruction still cannot meet actual preliminary clinical application requirements. In our research, to recover 3D tumor distribution quickly and precisely, an adaptive Newton hard thresholding pursuit (ANHTP) algorithm is proposed to improve the performance of BLT. The ANHTP algorithm fully combines the advantages of sparsity constrained optimization and convex optimization to guarantee global convergence. More precisely, an adaptive sparsity adjustment strategy was developed to obtain the support set of the inverse system matrix. Based on the strong Wolfe line search criterion, a modified damped Newton algorithm was constructed to obtain optimal source distribution information. A series of numerical simulations and phantom and in vivo experiments show that ANHTP has high reconstruction accuracy, fast reconstruction speed, and good robustness. Our proposed algorithm can further increase the practicality of BLT in biomedical applications.

Diagnosis of left anterior descending branch-right ventricular fistula with giant coronary artery aneurysm by contrast echocardiography: A case report.

BACKGROUND: Coronary fistulae are communications between a coronary artery and a heart chamber or vessel. The final diagnosis is usually made by coronary angiography or computed tomographic (CT) angiography. Here we report a case by employing contrast echocardiography in diagnosis of a giant coronary aneurysm with right ventricle (RV) fistula. CASE PRESENTATION: The patient, a 29-year-old woman, referred to our institution with a complaint of palpitation occasionally. Transthoracic echocardiogram showed a spherical, echogenic structure in the apex of RV. Proximal to the aneurysm, the left anterior descending branch (LAD) remained enlarged (8-9 mm) and showed a fistulous communication with the echogenic structure. A contrast echocardiography was performed, and 4-5 cardiac cycle after the left ventricle was enhanced, the echogenic structure started to become more prominent and several fistulae were seen between RV and the echogenic structure. Computed tomography (CT) angiography and coronary angiography confirmed the dilation (9 mm in diameter) of the LAD with an aneurysm at the distal segment of the LAD, with a small amount of iodinated contrast agent flowing into the subsequent region of the RV, thereby characterizing a LAD-to-RV fistula. CONCLUSION: The final diagnosis of fistula is usually made by coronary angiography or CT angiography. However, contrast echocardiography is also a well-established method for the demonstration of intracardiac shunting. In this case, the contrast echocardiography clearly revealed one of the fistulae between the aneurysm and RV.

In Vitro Inhibitory Effects of Maqian Essential Oil against Ectopic Endometrial Stromal Cells and LPS-Induced Endometrial Epithelial Cells.

Previous studies revealed that MQEO (Maqian fruits essential oil), which is extracted from the fruit of Maqian (Zanthoxylum myriacanthum var. Pubescens), had a good anti-inflammatory effect, but the effect on endometriosis in vitro remains unknown. In the present study, the inhibitory effects of MQEO against the EESCs (ectopic endometrial stromal cells) were investigated. Cells were treated with a concentration gradient (from 0.025 % to 0.15 %) of MQEO for 24 h and cell viability was detected by CCK-8. In addition, apoptotic rates were investigated using flow cytometry. The effect of MQEO on cell migration was determined by wound-healing and transwell assay. The expression of apoptosis-associated and cell adhesion-related proteins was assessed by western blotting. The transcriptional levels of IL-1, IL-6 and TNF-α were determined by Real-time qPCR. RNA-seq was used to identify the DEGs (differentially expressed genes) in MQEO-pretreated EESCs. We found that the MQEO condition dosage-dependently reduced the cell viability of EESCs. Based on flow cytometry results, the number of apoptotic cells increased significantly with dosage. The wound-healing and transwell results showed that MQEO group exhibited a significantly decreased cell motility and migration ability in comparison with the normal group. Western blotting results showed that MQEO down-regulated the expression of Bcl-2, ICAM-1 (intercellular adhesion molecule 1) and CD44, but up-regulated the cleaved caspase-3 expression in EESCs. What's more, MQEO also inhibited the LPS-induced inflammation in human EECs (endometrial epithelial cells). RNA-seq revealed that 221 DEGs were up-regulated genes and 284 DEGs were down-regulated in MQEO-pretreated EESCs. Our data uncovered the beneficial effects of MQEO in endometriosis and provided new insights into the mechanism of the effect of MQEO on EESCs, suggesting MQEO could be a promising new therapeutic agent for endometriosis.

A cationic cyclodextrin derivative-lipid hybrid nanoparticles for gene delivery effectively promotes stability and transfection efficiency.

Genetic medicines hold great promise for treatment of a number of diseases; however, the development of effective gene delivery carrier is still a challenge. The commonly used gene carrier liposomes and cationic polymers have limited their clinical application due to their respective disadvantages. Lipid-polymer hybrid nanoparticles (LHNPs) are novel drug delivery system that exhibit complementary characteristics of both polymeric nanoparticles and liposomes. In this account, we developed the α-cyclodextrin-conjugated generation-2 polyamidoamine dendrimers-lipids hybrid nanoparticles (CDG2-LHNPs) for gene delivery. The pDNA/CDG2-LHNPs was stable during 15 days of storage period both at 4 °C, 25 °C, and 37 °C, whereas the particle size of pDNA/CDG2 and pDNA/liposomes dramatically increased after storage at 4 °C for 8 h. CDG2-LHNPs showed significantly superior transfection efficiencies compared to either CDG2 or liposomes. The mechanism of high transfection efficiency of pDNA/CDG2-LHNPs was further explored using pharmacological inhibitors chlorpromazine, filipin, and cytochalasion D. The result demonstrated that cell uptake of pDNA/CDG2-LHNPs was mediated by clathrin-mediated endocytosis (CME), caveolae-mediated endocytosis (CvME), and macropinocytosis together. pDNA/CDG2-LHNPs were more likely be taken up by cells through CvME, which avoided lysosomal degradation to a large extent. Moreover, the liposome component of pDNA/CDG2-LHNPs increased its cell uptake efficiency, and the CDG2 polymer component increased its proton buffer capacity, so the hybrid nanoparticles taken up by CME could also successfully escape from the lysosome. CDG2-LHNPs with stability and high-transfection efficiency overcome the shortcomings of liposomes and polymers applied separately, and have great potential for gene drug delivery.

Wooden-Tip Electrospray Mass Spectrometry Characterization of Human Hemoglobin in Whole Blood Sample for Thalassemia Screening: A Pilot Study.

Traditional analytical methods for thalassemia screening are needed to process complicated and time-consuming sample pretreatment. In recent decades, ambient mass spectrometry (MS) approaches have been proven to be an effective analytical strategy for direct sample analysis. In this work, we applied ambient MS with wooden-tip electrospray ionization (WT-ESI) for the direct analysis of raw human blood samples that were pre-identified by gene detection. A total of 319 whole blood samples were investigated in this work, including 100 α-thalassemia carriers, 67 ß-thalassemia carriers, and 152 control healthy samples. Only one microliter of raw blood sample was directly loaded onto the surface of the wooden tip, and then five microliters of organic solvent and a high voltage of +3.0 kV were applied onto the wooden tip to generate spray ionization. Multiply charged ions of human hemoglobin (Hb) were directly observed by WT-ESI-MS from raw blood samples. The signal ratios of Hb chains were used to characterize two main types of thalassemia (α and ß types) and healthy control blood samples. Our results suggested that the ratios of charged ions to Hb chains being at +13 would be an indicator for ß-thalassemia screening.

Drug discovery for X-linked adrenoleukodystrophy: An unbiased screen for compounds that lower very long-chain fatty acids.

X-linked adrenoleukodystrophy (XALD) is a genetic neurologic disorder with multiple phenotypic presentations and limited therapeutic options. The childhood cerebral phenotype (CCALD), a fatal demyelinating disorder affecting about 35% of patients, and the adult-onset adrenomyeloneuropathy (AMN), a peripheral neuropathy affecting 40%-45% of patients, are both caused by mutations in the ABCD1 gene. Both phenotypes are characterized biochemically by elevated tissue and plasma levels of saturated very long-chain fatty acids (VLCFA), and an increase in plasma cerotic acid (C26:0), along with the clinical presentation, is diagnostic. Administration of oils containing monounsaturated fatty acids, for example, Lorenzo's oil, lowers patient VLCFA levels and reduced the frequency of development of CCALD in presymptomatic boys. However, this therapy is not currently available. Hematopoietic stem cell transplant and gene therapy remain viable therapies for boys with early progressive cerebral disease. We asked whether any existing approved drugs can lower VLCFA and thus open new therapeutic possibilities for XALD. Using SV40-transformed and telomerase-immortalized skin fibroblasts from an XALD patient, we conducted an unbiased screen of a library of approved drugs and natural products for their ability to decrease VLCFA, using measurement of C26:0 in lysophosphatidyl choline (C26-LPC) by tandem mass spectrometry as the readout. While several candidate drugs were initially identified, further testing in primary fibroblast cell lines from multiple CCALD and AMN patients narrowed the list to one drug, the anti-hypertensive drug irbesartan. In addition to lowering C26-LPC, levels of C26:0 and C28:0 in total fibroblast lipids were reduced. The effect of irbesartan was dose dependent between 2 and 10 µM. When male XALD mice received orally administered irbesartan at a dose of 10 mg/kg/day, there was no reduction in plasma C26-LPC. However, irbesartan failed to lower mouse fibroblast C26-LPC consistently. The results of these studies indicate a potential therapeutic benefit of irbesartan in XALD that should be validated by further study.

Prostaglandin E2 promotes pathological retinal neovascularisation via EP4R-EGFR-Gab1-AKT signaling pathway.

Proliferative retinopathies, such as proliferative diabetic retinopathy (PDR) and retinopathy of prematurity (ROP) are major causes of visual impairment and blindness in industrialized countries. Prostaglandin E2 (PGE2) is implicated in cellular proliferation and migration via E-prostanoid receptor (EP4R). The aim of this study was to investigate the role of PGE2/EP4R signaling in the promotion of retinal neovascularisation. In a streptozotocin (STZ)-induced diabetic model and an oxygen-induced retinopathy (OIR) model, rats received an intravitreal injection of PGE2, cay10598 (an EP4R agonist) or AH23848 (an EP4R antagonist). Optical coherence tomography, retinal histology and biochemical markers were assessed. Treatment with PGE2 or cay10598 accelerated pathological retinal angiogenesis in STZ and OIR-induced rat retina, which was ameliorated in rats pretreated with AH23848. Serum VEGF-A was upregulated in the PGE2-treated diabetic rats vs non-treated diabetic rats and significantly downregulated in AH23848-treated diabetic rats. PGE2 or cay10598 treatment also significantly accelerated endothelial tip-cell formation in new-born rat retina. In addition, AH23848 treatment attenuated PGE2-or cay10598-induced proliferation and migration by repressing the EGF receptor (EGFR)/Growth factor receptor bound protein 2-associated binder protein 1 (Gab1)/Akt/NF-κB/VEGF-A signaling network in human retinal microvascular endothelial cells (hRMECs). PGE2/EP4R signaling network is thus a potential therapeutic target for pathological intraocular angiogenesis.

Protective roles of the TIR/BB-loop mimetic AS-1 in alkali-induced corneal neovascularization by inhibiting ERK phosphorylation.

Hydrocinnamoyl-L-valylpyrrolidine (AS-1), a synthetic low-molecule mimetic of myeloid differentiation primary response gene 88 (MyD88), inhibits inflammation by disrupting the interaction between the interleukin-1 receptor (IL-1R) and MyD88. Here, we describe the effects of AS-1 on injury-induced increases in inflammation and neovascularization in mouse corneas. Mice were administered a subconjunctival injection of 8 µL AS-1 diluent before or after corneal alkali burn, followed by evaluation of corneal resurfacing and corneal neovascularization (CNV) by slit-lamp biomicroscopy and clinical assessment. Corneal inflammation was assessed by whole-mount CD45+ immunofluorescence staining, and corneal hemangiogenesis and lymphangiogenesis following injury were evaluated by immunostaining for the vascular markers isolectin B4 (IB4) and the lymphatic vascularized marker lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), respectively. Additionally, corneal tissues were collected to determine the expression of 35 cytokines, and we detected activation of IL-1RI, MyD88, and mitogen-activated protein kinase (MAPK). The results showed that alkali conditions increased the number of CD45+ cells and expression of vascular endothelial growth factor (VEGF)-A, VEGF-C, and LYVE1 in corneas, with these levels decreased in the AS-1-treated group. Moreover, AS-1 effectively prevented alkali-induced cytokine production, blocked interactions between IL-1RI and MyD88, and inhibited MAPK activation post-alkali burn. These results indicated that AS-1 prevented alkali-induced corneal hemangiogenesis and lymphangiogenesis by blocking IL-1RI-MyD88 interaction, as well as extracellular signal-regulated kinase phosphorylation, and could be efficacious for the prevention and treatment of corneal alkali burn.

Prolonged duration of repolarization and decreased conduction velocity in the atrial myocardium after hypothermic ischemia-reperfusion may be related to expressions of inward rectifier potassium channel 2.1 protein and connexin 40.

OBJECTIVES: The study aimed to determine the role of inward rectifier potassium channel 2.1 protein and connexin 40 expressions in regulating the duration of repolarization and conduction velocity of right atrial myocardium in rats following hypothermic ischemia-reperfusion. METHODS: The Langendorff isolated rat cardiac perfusion models were divided into control (C) and hypothermic ischemia-reperfusion groups, with 8 models in group C and 16 models in group ischemia-reperfusion. Depending on the incidence of atrial arrhythmia after reperfusion, the models in group ischemia-reperfusion were further divided into reperfusion non-atrial arrhythmia or reperfusion atrial arrhythmia subgroup. Right atrial monophasic action potential duration at 50% and 90% of repolarization after 30 minutes of continuous perfusion in group C and group ischemia-reperfusion (T0), 105 minutes of continuous perfusion in group C or after 15 minutes of reperfusion in group ischemia-reperfusion (T1) and 120 minutes of continuous perfusion in group C or 30 minutes of reperfusion in group ischemia-reperfusion (T2) were recorded. Right atrial conduction velocity and effective refractory period were recorded at T2. Then, the expressions of inward rectifier potassium channel 2.1 protein and connexin 40 in the right atrial myocardium were detected. RESULTS: Monophasic action potential duration at 50% and 90% were higher at T1 and T2 than those at T0 in subgroup reperfusion atrial arrhythmia (p < 0.05); monophasic action potential duration at 50% in subgroup reperfusion atrial arrhythmia were larger than group C and subgroup reperfusion non-atrial arrhythmia at T1 and T2 (p < 0.05); monophasic action potential duration at 90% in subgroup reperfusion atrial arrhythmia were larger than group C and subgroup reperfusion non-atrial arrhythmia at T1 and T2 (p < 0.05); effective refractory period in subgroup reperfusion atrial arrhythmia was greater than that in group C and subgroup reperfusion non-atrial arrhythmia, and the conduction velocity and the expressions of inward rectifier potassium channel 2.1 protein and connexin 40 were significantly lower than group C and subgroup reperfusion non-atrial arrhythmia (p < 0.05). CONCLUSIONS: The prolonged duration of repolarization and a decrease in conduction velocity of the atrial myocardium occur in rats after hypothermic ischemia-reperfusion. These observed effects may be related to the downregulated expressions of connexin 40 and inward rectifier potassium channel 2.1.