Development and Clinical Validation of a Hook Effect-Based Lateral Flow Immunoassay Sensor for Cerebrospinal Fluid Leak Detection.

BACKGROUND AND OBJECTIVES: Rapid detection of cerebrospinal fluid (CSF) leaks is vital for patient recovery after spinal surgery. However, distinguishing CSF-specific transferrin (TF) from serum TF using lateral flow immunoassays (LFI) is challenging due to their structural similarities. This study aims to develop a novel point-of-care diagnostic assay for precise CSF leak detection by quantifying total TF in both CSF and serum. METHODS: Capitalizing on the substantial 100-fold difference in TF concentrations between CSF and serum, we designed a diagnostic platform based on the well-known "hook effect" resulting from excessive analyte presence. Clinical samples from 37 patients were meticulously tested using the novel LFI sensor, alongside immunofixation as a reference standard. RESULTS: The hook effect-based LFI sensor exhibited outstanding performance, successfully discriminating positive clinical CSF samples from negative ones with remarkable statistical significance (positive vs negative t-test; P = 1.36E-05). This novel sensor achieved an impressive 100% sensitivity and 100% specificity in CSF leak detection, demonstrating its robust diagnostic capabilities. CONCLUSION: In conclusion, our study introduces a rapid, highly specific, and sensitive point-of-care test for CSF leak detection, harnessing the distinctive TF concentration profile in CSF compared with serum. This novel hook effect-based LFI sensor holds great promise for improving patient outcomes in the context of spinal surgery and postsurgical recovery. Its ease of use and reliability make it a valuable tool in clinical practice, ensuring timely and accurate CSF leak detection to enhance patient care.

Attaching protein-adsorbing silica particles to the surface of cotton substrates for bioaerosol capture including SARS-CoV-2.

The novel coronavirus pandemic (COVID-19) has necessitated a global increase in the use of face masks to limit the airborne spread of the virus. The global demand for personal protective equipment has at times led to shortages of face masks for the public, therefore makeshift masks have become commonplace. The severe acute respiratory syndrome caused by coronavirus-2 (SARS-CoV-2) has a spherical particle size of ~97 nm. However, the airborne transmission of this virus requires the expulsion of droplets, typically ~0.6-500 µm in diameter (by coughing, sneezing, breathing, and talking). In this paper, we propose a face covering that has been designed to effectively capture SARS-CoV-2 whilst providing uncompromised comfort and breathability for the wearer. Herein, we describe a material approach that uses amorphous silica microspheres attached to cotton fibres to capture bioaerosols, including SARS CoV-2. This has been demonstrated for the capture of aerosolised proteins (cytochrome c, myoglobin, ubiquitin, bovine serum albumin) and aerosolised inactivated SARS CoV-2, showing average filtration efficiencies of ~93% with minimal impact on breathability.

Association between urinary iodine concentration and the risk of papillary thyroid cancer by sex and age: a case-control study.

Previous studies on dietary iodine intake and the risk of papillary thyroid cancer (PTC) have demonstrated inconsistent results. We aimed to evaluate the association between urinary iodine concentration (UIC), a surrogate biomarker for dietary iodine intake, and the risk of thyroid cancer stratified by sex and age in an iodine-sufficient area. A hospital-based case-control study was conducted in Seoul, South Korea. A total of 492 cases of newly diagnosed PTC and 595 controls were included. Compared with the lowest quartile of creatine-adjusted UIC (< 159.3 µg/gCr), the highest quartile (≥ 1037.3 µg/gCr) showed an increased risk of PTC (odds ratio [OR] 1.49, 95% confidence interval [CI] 1.04-2.13), especially in those who were < 45 years old (ptrend = 0.01) compared with those who were ≥ 45 years old (ptrend = 0.48). For those who were < 45 years old, a positive association between creatinine-adjusted UIC and the risk of PTC was observed in both men (q4 vs. q1, OR 4.27, 95% CI 1.14-18.08) and women (OR 1.97, 95% CI 1.04-3.78). For those who were ≥ 45 years old, no association was found in any sex. Creatinine-adjusted UIC was positively associated with the risk of PTC, especially in those who were younger than 45 years for both men and women.

Plasmon color-preserved gold nanoparticle clusters for high sensitivity detection of SARS-CoV-2 based on lateral flow immunoassay.

Lateral flow immunoassays (LFI) have shown great promise for point-of-care (POC) sensing applications, however, its clinical translation is often hindered by insufficient sensitivity for early detection of diseases, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This is mainly due to weak absorption signals of single gold nanoparticles (AuNPs). Here, we developed AuNP clusters that maintain the red color of isolated individual AuNPs, but increase the colorimetric readout to improve the detection sensitivity. The plasmon color-preserved (PLASCOP) AuNP clusters is simply made by mixing streptavidin-coated AuNP core with satellite AuNPs coated with biotinylated antibodies. The biotinylated antibody-streptavidin linker forms a gap size over 15 nm to avoid plasmon coupling between AuNPs, thus maintaining the plasmonic color while increasing the overall light absorption. LFI sensing using PLASCOP AuNP clusters composed of 40 nm AuNPs showed a high detection sensitivity for SARS-CoV-2 nucleocapsid proteins with a limit of detection (LOD) of 0.038 ng mL-1, which was 23.8- and 5.9-times lower value than that of single 15 nm and 40 nm AuNP conjugates, respectively. The PLASCOP AuNP clusters-based LFI sensing also shows good specificity for SARS-CoV-2 nucleocapsid proteins from other influenza and coronaviruses. In a clinical feasibility test, we demonstrated that SARS-CoV-2 particles spiked in human saliva could be detected with an LOD of 54 TCID50 mL-1. The developed PLASCOP AuNP clusters are promising colorimetric sensing reporters that present improved sensitivity in LFI sensing for broad POC sensing applications beyond SARS-CoV-2 detection.

Advanced trap lateral flow immunoassay sensor for the detection of cortisol in human bodily fluids.

Paper-based biosensors based on lateral flow immunoassay (LFI) are promising candidates for POC diagnosis because of their ease of use and rapid target detection. However, the low sensitivity of LFI limits its application, and signal amplification has been used in numerous studies to increase its sensitivity. We developed an advanced trap LFI (α-trapLFI), a simple-to-use sensor, with an additional step for signal amplification. Here, signal amplification is automatically implemented following delayed release of enhancement solution induced by water-soluble polyvinyl alcohol tape. As the polyvinyl alcohol tape is exposed to water, its polymer structure is perturbed (within 5 min), allowing ions to pass through. This new sensor was designed to have a short time delay between the flow of solutions used for the immunoassay and signal amplification. The α-trapLFI was subsequently used to detect cortisol with high sensitivity (9.1 pg∙mL-1) over a broad detection range (0.01-1000 ng∙mL-1) in bodily fluids. Furthermore, an excellent correlation was obtained by analyzing 20 human real saliva samples using this sensor and a conventional ELISA (R2 = 0.90). The new sensor will be helpful in detecting various small molecules for simple, rapid, and portable POC diagnosis of stress disorders.

A rapid assay provides on-site quantification of tetrahydrocannabinol in oral fluid.

Tetrahydrocannabinol (THC), the primary psychoactive ingredient of cannabis, impairs cognitive and motor function in a concentration-dependent fashion. Drug testing is commonly performed for employment and law enforcement purposes; however, available tests produce low-sensitive binary results (lateral flow assays) or have long turnaround (gas chromatography­mass spectrometry). To enable on-site THC quantification in minutes, we developed a rapid assay for oral THC analysis called EPOCH (express probe for on-site cannabis inhalation). EPOCH features distinctive sensor design such as a radial membrane and transmission optics, all contained in a compact cartridge. This integrated approach permitted assay completion within 5 min with a detection limit of 0.17 ng/ml THC, which is below the regulatory guideline (1 ng/ml). As a proof of concept for field testing, we applied EPOCH to assess oral fluid samples from cannabis users (n = 43) and controls (n = 43). EPOCH detected oral THC in all specimens from cannabis smokers (median concentration, 478 ng/ml) and THC-infused food consumers. Longitudinal monitoring showed a fast drop in THC concentrations within the first 6 hours of cannabis smoking (half-life, 1.4 hours).

COVID-19 vaccine safety monitoring in the Republic of Korea: February 26, 2021 to April 30, 2021.

OBJECTIVE: On February 26, 2021, coronavirus disease 2019 (COVID-19) vaccination was started for high-priority groups based on the recommendation of the Advisory Committee on Immunization Practices with 2 available COVID-19 vaccines (AstraZeneca and Pfizer-BioNTech) in Korea. This report provides a summary of adverse events following COVID-19 vaccination as of April 30, 2021. METHODS: Adverse events following immunization are notifiable by medical doctors to the Korea Immunization Management System (KIMS) under the national surveillance system. We analyzed all adverse events reports following COVID-19 vaccination to the KIMS from February 26 to April 30, 2021. RESULTS: In total, 16,196 adverse events following 3,586,814 administered doses of COVID-19 vaccines were reported in approximately 2 months (February 26 to April 30, 2021). Of these, 15,658 (96.7%) were non-serious adverse events, and 538 (3.3%) were serious adverse events, including 73 (0.5%) deaths. The majority of adverse events (n=13,063, 80.7%) were observed in women, and the most frequently reported adverse events were myalgia (52.2%), fever (44.9%), and headache (34.9%). Of the 73 deaths following the COVID-19 vaccination, none were related to the vaccines. CONCLUSION: By April 30, 3.6 million doses of the COVID 19 vaccine had been given in Korea, and the overwhelming majority of reports were for non-serious events. The Korea Disease Control and Prevention Agency continues to monitor the safety of COVID-19 vaccination.

Impact of Media Coverage on Influenza Vaccine Coverage in Elderly Individuals from 2020 to 2021 in the Republic of Korea.

Increased awareness of adverse events following immunization (AEFI) can disrupt vaccination programs. In South Korea, a report of alleged influenza vaccine-related deaths attracted significant media attention in 2020. We retrieved the vaccination coverage and AEFI data to determine their association with media coverage. Between 2015 and 2019, the vaccination coverage rate ranged between 80.5% and 83.3%; however, the vaccination coverage rate declined significantly from 2020 to 2021 to 73.6% (p < 0.0001). During the 43rd week of 2020, following a large amount of media coverage on vaccine safety issues, the number of cases with AEFI reached 60. Between 2015 and 2020, the mortality rate ratios for influenza vaccines and non-vaccines ranged between 0.1296 (95% confidence interval (CI), 0.1262-0.1331, p < 0.0001) and 0.1608 (95% CI, 0.1572-0.1644, p < 0.0001). Vaccine safety surveillance should be continued in conjunction with investigation and transparent risk communication to maintain public trust in vaccines and vaccinations.

Rapid and Simple Detection of Ochratoxin A using Fluorescence Resonance Energy Transfer on Lateral Flow Immunoassay (FRET-LFI).

The detection of mycotoxins is crucial because of their toxicity in plants, animals, and humans. It is very important to determine whether food products are contaminated with mycotoxins such as ochratoxin A (OTA), as mycotoxins can survive heat treatments and hydrolysis. In this study, we designed a fluorescence resonance energy transfer (FRET)-based system that exploits antibody-antigen binding to detect mycotoxins more rapidly and easily than other currently available methods. In addition, we were able to effectively counteract the matrix effect in the sample by using a nitrocellulose membrane that enabled fluorescence measurement in coffee samples. The developed FRET on lateral flow immunoassay (FRET-LFI) system was used to detect OTA at a limit of detection (LOD) of 0.64 ng∙mL-1, and the test can be completed in only 30 min. Moreover, OTA in coffee samples was successfully detected at a LOD of 0.88 ng∙mL-1, overcoming the matrix effect, owing to the chromatographic properties of the capillary force of the membrane. We believe that the developed system can be used as a powerful tool for the sensitive diagnosis of harmful substances such as mycotoxins and pesticides for environmental and food quality control monitoring.

Urinary iodine concentration and thyroid hormones: Korea National Health and Nutrition Examination Survey 2013-2015.

PURPOSE: An epidemiologic study that evaluated population's iodine nutrition status and its relationship with thyroid hormones is lacking in iodine-sufficient area. This nationwide study aimed to evaluate the iodine nutrition status in Korea and relationship between urine iodine concentration (UIC) and thyroid hormones. METHODS: A total of 8318 subjects of the Korea National Health and Nutrition Examination Survey VI (2013-2015) with UIC and thyroid hormone evaluation were included. Median UIC level and estimated 24-h iodine intake were calculated. The prevalence of iodine deficiency or excess was obtained using estimated average requirement or above the tolerable upper intake level cut-point method by estimated iodine intake. We analyzed UIC with regard to age, sex, social economic status, and geographic characteristics. RESULTS: The median UIC in general population and estimated iodine intake in adult population were 293.9 µg/L (above requirement according to World Health Organization classification) and 249.3µg/day, respectively. The prevalence of iodine deficiency and excess was 14.0 and 13.4%. The median UIC was higher among SAC [511 (299.9-948.5)] and lower among seventies [251.2 (98.9-761.6)] compared to other age groups. The median UIC increased with household income level (p for trend < 0.001). The subjects living in rural and inland region had lowest UIC among the enrolled subjects. The subgroups with higher median UIC were associated with higher mean TSH levels. CONCLUSIONS: This first nationwide study in Korea demonstrated that the median of UIC and estimated iodine intake lie at nearly 300 µg/L and 250 µg/day, respectively, which shows an overall excellent iodine nutrition.

Non-immune-related hypothyroidism and its relationship with excess iodine.

PURPOSE: In iodine-sufficient areas, autoimmune hypothyroidism has been regarded as the major subtype of hypothyroidism. Non-immune-related hypothyroidism has received little attention because it is considered to be rare. The aim of this study was to evaluate the prevalence of non-immune-related hypothyroidism in Korea and to identify its associating factors. METHODS: A total of 6434 participants in the Korea National Health and Nutrition Examination Survey VI (2013-2015) without known thyroid disease who were examined for thyroid stimulating hormone, free thyroxine, TPO Ab, and urine iodine concentration (UIC) were enrolled. The weighted proportions, demographic variables, and severity of immune-related and non-immune-related hypothyroidism were compared. To assess the effect of iodine on hypothyroidism in TPO Ab positive or negative populations, the weighted prevalence of hypothyroidism was assessed in each population according to UIC or estimated iodine intake subgroups. RESULTS: The prevalence of undetected hypothyroidism in Korea was 3.8% (n = 233). Of these 233 cases, 171 (71.8%) were non-immune-related. In the TPO Ab negative population, the prevalence of hypothyroidism was increased significantly in the subgroup with UIC between 250 and 749 µg/L (HR 2.12 [1.17, 3.83]) and ≥ 750 µg/L (HR 3.42 [1.93, 6.04]) or the subgroups with estimated iodine intake ≥ 750 µg/day (HR 2.81 [1.64, 4.80]). CONCLUSIONS: This nationwide study demonstrated that most cases of hypothyroidism in iodine-sufficient areas are non-immune-related and are associated with excess iodine above a certain level. More attention to this unrecognized but widespread potential health risk is needed.

High sensitive and broad-range detection of cortisol in human saliva using a trap lateral flow immunoassay (trapLFI) sensor.

Cortisol, a steroid hormone, is a main biomarker of psychological stress. Early diagnosis and proper treatment of such stress is crucial to prevent the excessive secretion of cortisol. However, cortisol has a low molecular weight and cannot provide sufficient recognition sites for sandwich immunoreaction; it has previously been measured using a competitive immunoassay instead of a general sandwich immunoassay. The disadvantage of this approach is that quantitative measurements are limited because of the narrow measurable range that is key for biosensors. To overcome this limitation, we propose a new detection platform that enables small molecules such as cortisol to be quantified with high detection sensitivity. A trap lateral flow immunoassay (trapLFI) sensor has deletion and detection zones instead of the test and control zones in general lateral flow immunoassay (LFI) sensors. The conjugates used to minimize possible detection targets at low concentration are gold nanoparticles that include an antibody against cortisol and an enzyme for signal generation. Target-bound conjugates are captured in the detection zone, whereas conjugates not binding with targets are trapped in the deletion zone. Using this platform, enzyme-catalyzed color signals increase in the detection zone and decrease in the deletion zone with the concentration of cortisol. The ratio of signal from deletion zone and detection zone supplied a wide analytical range (0.01-100 ng mL-1) with high detection sensitivity (9.9 pg mL-1). Analysis of 15 human saliva samples showed a good correlation with conventional ELISA results (R2 = 0.9432).

Age- and gender-specific reference intervals of TSH and free T4 in an iodine-replete area: Data from Korean National Health and Nutrition Examination Survey IV (2013-2015).

BACKGROUND: Establishment of the reference interval of thyroid-stimulating hormone (TSH) is critical in the diagnosis of thyroid dysfunction and is affected by age, gender, iodine nutrition, and ethnicity. The aim of this study was to determine the reference intervals of TSH and free thyroxin (FT4) from a large, nationwide data of Korea where iodine intake is more than adequate. METHODS: We analyzed data from the Korea National Health and Nutrition Examination Survey VI that measured serum TSH, FT4, and thyroid peroxidase antibody from 7,061 individuals (urinary iodine measurement in 6,565). Age- and gender-specific reference intervals were established from 95% confidence limits from the 2.5 to 97.5 percentile of TSH (log-transformed) and FT4 in reference populations. RESULTS: The geometric mean of TSH was 2.16 ± 0.01 mIU/L, with the lowest value found in the middle aged group (2.04 ± 0.02 mIU/L) and higher values noted in age groups of 10-19 and over 70 years (2.38 ± 0.02 and 2.32 ± 0.07 mIU/L, respectively). The association of TSH and age was U-shaped. The overall reference interval of TSH was 0.59-7.03 mIU/L. Mean FT4 was 1.25 ± 0.003 ng/dL (16.09 ± 0.039 pmol/L), and it showed a small but continuous decrease after 20 years of age (P < 0.001). There was a significant positive correlation between TSH and urine iodine concentration (r = 0.154, P < 0.001). CONCLUSIONS: The reference interval of TSH in Korea, where iodine intake is above the requirement, was 0.59-7.03 mIU/L and showed U-shaped change with age, which was a similar pattern to iodine intake. The reference interval of FT4 was 0.92-1.60 ng/dL. The geometric mean and upper limit of TSH were higher than those of Western populations, reflecting the paramount importance of iodine intake on thyroid function.

Pediatric sialolithiasis is not related to oral or oropharyngeal infection: A population-based case control study using the Korean National Health Insurance Database.

OBJECTIVES: Poor oral hygiene is one of the risk factors for sialolithiasis particularly in adults; however the etiology of sialolithiasis in pediatric patients remains largely unknown. The purpose of this study is to identify the association between sialolithiasis and the oral/oropharyngeal infections in the pediatric population, as surrogate indicators for oral hygiene and retrograde infections to the affected salivary gland. METHODS: This was a population based case-control study using the Korean National Health Insurance Database. We identified 10,095 pediatric patients, diagnosed with sialolithiasis, as cases (study period 2011-2015) and 50,475 age/gender/residence matched subjects without sialolithiasis, but with unrelated diseases (e.g., simple trauma in extremities) were set as controls. Logistic regression analyses were conducted to evaluate the association of sialolithiasis with oral or oropharyngeal infections. RESULTS: The morbidity rate of sialadenitis was much higher in the cases than the controls (32.92% vs 0.72%, p < 0.0001). By contrast, the prevalence of oral or oropharyngeal infections (stomatitis, gingivitis, periodontitis, and pharyngo-tonsillitis) was significantly lower in pediatric sialolithiasis patients in all age (0-18) groups. The adjusted odds ratios of the multivariate analyses also confirmed significantly less prevalence of the oral and oropharyngeal infections in pediatric sialolithiasis patients. CONCLUSION: Oral or oropharyngeal infections were inversely associated with pediatric sialolithiasis, suggesting that pediatric sialolithiasis may result from the intrinsic factors of the salivary gland itself, not from oral or oropharyngeal infections.

Coffee Intake and Liver Enzyme Association in Korean Immigrants and Japanese: A Comprehensive Cross-Sectional Study.

OBJECTIVES: Significant inverse association between coffee intake and the levels of liver enzymes has been reported. We demonstrated higher prevalence of metabolic syndrome in Korean immigrants (KIs) than in indigenous Japanese (IJs). The aim of this study was to investigate whether the association between coffee intake and liver enzyme levels was different between the 2 ethnic groups. METHODS: This study is a cross-sectional study including a total of 966 subjects comprising KIs and IJs. The association between the quintiles of coffee intake and dichotomous values of liver enzymes was evaluated by logistic regression analysis in KIs, IJs, a high-risk group (current smokers or alcohol drinkers ≥45 g/day), and a low-risk group (non-smokers and alcohol drinkers <45 g/day). RESULTS: In KIs, a significant inverse association between coffee intake and serum aspartate aminotransferase (AST) levels was observed. In the IJs, a significant inverse association between coffee intake and serum alanine aminotransferase levels was observed. In the high-risk group, a significant inverse association between coffee intake and serum AST and gamma-glutamyltransferase levels was observed. CONCLUSION: No difference was observed between KIs and IJs regarding the association between coffee and liver enzymes. Coffee might inhibit hepatic damage by alcohol drinking and smoking.

Resurgence of measles in a country of elimination: interim assessment and current control measures in the Republic of Korea in early 2014.

Since the beginning of 2014, the Republic of Korea has experienced a resurgence of measles cases. Among the 220 cases confirmed as measles during epidemiological weeks 1-20 (December 29, 2013 to May 17, 2014), 10 imported cases were identified. The predominant genotype was B3, which reflects the circulating measles virus in adjacent countries. Even with the verification of measles elimination in March 2014 by the World Health Organization, recent importation has been related to international travel. Targeted control measures have been implemented in addition to proper isolation and patient care. A vigilant surveillance system and high levels of vaccine coverage should be maintained to sustain the measles elimination status.

SRSF2 promotes splicing and transcription of exon 11 included isoform in Ron proto-oncogene.

The product of proto-oncogene Ron is a human receptor for the macrophage-stimulating protein (MSP). Upon activation, Ron is able to induce cell dissociation, migration and matrix invasion. Exon 11 skipping of Ron pre-mRNA produces Ron△165 protein that is constitutively active even in the absence of its ligand. Here we show that knockdown of SRSF2 promotes the decrease of exon 11 inclusion, whereas overexpression of SRSF2 promotes exon 11 inclusion. We demonstrate that SRSF2 promotes exon 11 inclusion through splicing and transcription procedure. We also present evidence that reduced expression of SRSF2 induces a decrease in the splicing of both introns 10 and 11; by contrast, overexpression of SRSF2 induces an increase in the splicing of introns 10 and 11. Through mutation analysis, we show that SRSF2 functionally targets and physically interacts with CGAG sequence on exon 11. In addition, we reveal that the weak strength of splice sites of exon 11 is not required for the function of SRSF2 on the splicing of Ron exon 11. Our results indicate that SRSF2 promotes exon 11 inclusion of Ron proto-oncogene through targeting exon 11. Our study provides a novel mechanism by which Ron is expressed.

3' Splice site sequences of spinal muscular atrophy related SMN2 pre-mRNA include enhancers for nearby exons.

Spinal muscular atrophy (SMA) is a human genetic disease which occurs because of the deletion or mutation of SMN1 gene. SMN1 gene encodes the SMN protein which plays a key role in spliceosome assembly. Although human patients contain SMN2, a duplicate of SMN1, splicing of SMN2 produces predominantly exon 7 skipped isoform. In order to understand the functions of splice site sequences on exon 7 and 8, we analyzed the effects of conserved splice site sequences on exon 7 skipping of SMN2 and SMN1 pre-mRNA. We show here that conserved 5' splice site sequence of exon 7 promoted splicing of nearby exons and subsequently reduced splicing of distant exons. However, to our surprise, conserved 3' splice site sequence of exon 7 and 8 did not promote splicing of nearby exons. By contrast, the mutation inhibited splicing of nearby exons and subsequently promoted splicing of distant exons. Our study shows that 3' splice sites of exon 7 and 8 contain enhancer for their splice site selection, in addition to providing cleavage sites.

PSF contacts exon 7 of SMN2 pre-mRNA to promote exon 7 inclusion.

Spinal muscular atrophy (SMA) is an autosomal recessive genetic disease and a leading cause of infant mortality. Deletions or mutations of SMN1 cause SMA, a gene that encodes a SMN protein. SMN is important for the assembly of Sm proteins onto UsnRNA to UsnRNP. SMN has also been suggested to direct axonal transport of ß-actin mRNA in neurons. Humans contain a second SMN gene called SMN2 thus SMA patients produce some SMN but not with sufficient levels. The majority of SMN2 mRNA does not include exon 7. Here we show that increased expression of PSF promotes inclusion of exon 7 in the SMN2 whereas reduced expression of PSF promotes exon 7 skipping. In addition, we present evidence showing that PSF interacts with the GAAGGA enhancer in exon 7. We also demonstrate that a mutation in this enhancer abolishes the effects of PSF on exon 7 splicing. Furthermore we show that the RNA target sequences of PSF and tra2ß in exon 7 are partially overlapped. These results lead us to conclude that PSF interacts with an enhancer in exon 7 to promote exon 7 splicing of SMN2 pre-mRNA.

Exon 9 skipping of apoptotic caspase-2 pre-mRNA is promoted by SRSF3 through interaction with exon 8.

Alternative splicing plays an important role in gene expression by producing different proteins from a gene. Caspase-2 pre-mRNA produces anti-apoptotic Casp-2S and pro-apoptotic Casp-2L proteins through exon 9 inclusion or skipping. However, the molecular mechanisms of exon 9 splicing are not well understood. Here we show that knockdown of SRSF3 (also known as SRp20) with siRNA induced significant increase of endogenous exon 9 inclusion. In addition, overexpression of SRSF3 promoted exon 9 skipping. Thus we conclude that SRSF3 promotes exon 9 skipping. In order to understand the functional target of SRSF3 on caspase-2 pre-mRNA, we performed substitution and deletion mutagenesis on the potential SRSF3 binding sites that were predicted from previous reports. We demonstrate that substitution mutagenesis of the potential SRSF3 binding site on exon 8 severely disrupted the effects of SRSF3 on exon 9 skipping. Furthermore, with the approach of RNA pulldown and immunoblotting analysis we show that SRSF3 interacts with the potential SRSF3 binding RNA sequence on exon 8 but not with the mutant RNA sequence. In addition, we show that a deletion of 26nt RNA from 5' end of exon 8, a 33nt RNA from 3' end of exon 10 and a 2225nt RNA from intron 9 did not compromise the function of SRSF3 on exon 9 splicing. Therefore we conclude that SRSF3 promotes exon 9 skipping of caspase-2 pre-mRNA by interacting with exon 8. Our results reveal a novel mechanism of caspase-2 pre-mRNA splicing.