POU6F1 promotes ferroptosis by increasing lncRNA-CASC2 transcription to regulate SOCS2/SLC7A11 signaling in gastric cancer.

OBJECTIVE: This study investigated the effect and mechanism of POU6F1 and lncRNA-CASC2 on ferroptosis of gastric cancer (GC) cells. METHODS: GC cells treated with erastin and RSL3 were detected for ferroptosis, reactive oxygen species (ROS) level, and cell viability. The expression levels of POU6F1, lncRNA-CASC2, SOCS2, and ferroptosis-related molecules (GPX4 and SLC7A11) were also measured. The regulations among POU6F1, lncRNA-CASC2, FMR1, SOCS2, and SLC7A11 were determined. Subcutaneous tumor models were established, in which the expressions of Ki-67, SOCS2, and GPX4 were detected by immunohistochemistry. RESULTS: GC patients with decreased expressions of POU6F1 and lncRNA-CASC2 had lower survival rate. Overexpression of POU6F1 or lncRNA-CASC2 decreased cell proliferation and GSH levels in GC cells, in addition to increasing total iron, Fe2+, MDA, and ROS levels. POU6F1 directly binds to the lncRNA-CASC2 promoter to promote its transcription. LncRNA-CASC2 can target FMR1 and increase SOCS2 mRNA stability to promote SLC7A11 ubiquitination degradation and activate ferroptosis signaling. Knockdown of SOCS2 inhibited the ferroptosis sensitivity of GC cells and reversed the effects of POU6F1 and lncRNA-CASC2 overexpression on ferroptosis in GC cells. CONCLUSION: Transcription factor POU6F1 binds directly to the lncRNA-CASC2 promoter to promote its expression, while upregulated lncRNA-CASC2 increases SOCS2 stability and expression by targeting FMR1, thereby inhibiting SLC7A11 signaling to promote ferroptosis in GC cells and inhibit GC progression.

Correlation between serum vitamin D level and uterine volume in girls with idiopathic central precocious puberty.

OBJECTIVES: Investigate serum vitamin D (vit D) levels' relation to uterine volume in idiopathic central precocious puberty (ICPP) girls and compare findings with normal peers. METHODS: Analyzed 278 ICPP cases from January 2017 to September 2022 alongside 239 normally developing girls. Collected clinical data and lab markers and performed subgroup analysis based on vit D levels. Correlation and regression analyses were conducted. RESULTS: The ICPP group exhibited elevated uterine volume and lower serum vit D compared to controls (p<0.05). A weak negative correlation was noted between vit D and uterine volume in ICPP (r=-0.193, p=0.004), and no such correlation in controls (r=-0.073, p=0.319). The ICPP vit D deficiency subgroup displayed higher uterine volume than the insufficiency and sufficiency subgroups (p<0.05). Uterine volume in the insufficiency subgroup exceeded the sufficiency subgroup (p<0.05). After adjusting for confounders, lower vit D is linked to increased ICPP uterine volume (non-standardized regression coefficient ß=-25.55, 95 % CI= -46.23, -4.87, p=0.016). A Limited correlation between vit D and uterine volume was seen in girls with normal pubertal timing. CONCLUSIONS: We demonstrated a correlation between vit D and uterine volume in ICPP girls, absent in normal peers. ICPP girls often exhibit lower vit D levels and increased uterine volume. Further research is vital for understanding vit D's role in ICPP pathogenesis and guiding prevention and treatment strategies.

18 F-FDG PET/CT Versus 18 F-FAPI PET/CT in a Case of Recurrent Malignant Phyllodes Breast Tumor.

ABSTRACT: A 52-year-old woman with medical history of surgery for left malignant phyllodes breast tumor found a mass on the left chest 3 months ago. A suspicion of recurrent malignant phyllodes breast tumor was made. The patient was enrolled in the clinical trial of 18 F-FAPI PET/CT in recurrent sarcoma (no. NCT05485792). 18 F-FAPI PET/CT and 18 F-FDG PET/CT were performed, and the images demonstrated intense uptake in a huge mass in the left anterior chest wall. Then the patient underwent extended resection of left chest wall tumor. The tumor proved to be recurrent malignant phyllodes breast tumor pathologically.

Head-to-head comparison of 18F-FAPI and 18F-FDG PET/CT in staging and therapeutic management of hepatocellular carcinoma.

BACKGROUND: Fluorine 18 (18F) fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) has limitations in staging hepatocellular carcinoma (HCC). The recently introduced 18F-labeled fibroblast-activation protein inhibitor (FAPI) has shown promising prospects in detection of HCC lesions. This study aimed to investigate the initial staging and restaging performance of 18F-FAPI PET/CT compared to 18F-FDG PET/CT in HCC. METHODS: This prospective study enrolled histologically confirmed HCC patients from March 2021 to September 2022. All patients were examined with 18F-FDG PET/CT and 18F-FAPI PET/CT within 1 week. The maximum standard uptake value (SUVmax), tumor-to-background ratio (TBR), and diagnostic accuracy were compared between the two modalities. RESULTS: A total of 67 patients (57 men; median age, 57 [range, 32-83] years old) were included. 18F-FAPI PET showed higher SUVmax and TBR values than 18F-FDG PET in the intrahepatic lesions (SUVmax: 6.7 vs. 4.3, P < 0.0001; TBR: 3.9 vs. 1.7, P < 0.0001). In diagnostic performance, 18F-FAPI PET/CT had higher detection rate than 18F-FDG PET/CT in intrahepatic lesions [92.2% (238/258) vs 41.1% (106/258), P < 0.0001] and lymph node metastases [97.9% (126/129) vs 89.1% (115/129), P = 0.01], comparable in distant metastases [63.6% (42/66) vs 69.7% (46/66), P > 0.05]. 18F-FAPI PET/CT detected primary tumors in 16 patients with negative 18F-FDG, upgraded T-stages in 12 patients and identified 4 true positive findings for local recurrence than 18F-FDG PET, leading to planning therapy changes in 47.8% (32/67) of patients. CONCLUSIONS: 18F-FAPI PET/CT identified more primary lesions, lymph node metastases than 18F-FDG PET/CT in HCC, which is helpful to improve the clinical management of HCC patients. TRIAL REGISTRATION: Clinical Trials, NCT05485792 . Registered 1 August 2022, Retrospectively registered.

Increased 18F-FAPI Uptake in Radiation-Induced Liver Injury.

ABSTRACT: A 51-year-old woman with breast cancer underwent a complete surgical resection and chemoradiotherapy approximately 3 months ago. Follow-up abdominal ultrasound detected a new lesion with decreased echogenicity in the hepatic segment IV/VIII. 18F-FDG PET/CT showed the hepatic lesion without abnormal uptake. The patient was subsequently enrolled in a clinical trial of 18F-FAPI PET/CT to assess the hepatic lesion. An intense 18F-FAPI activity was identified in the hepatic lesion. Finally, pathological analysis combined with imaging follow-up confirmed the diagnosis of radiation-induced liver injury.

Oriented Crystallization of Hydroxyapatite in Self-Assembled Peptide Fibrils as a Bonelike Material.

Controlling oriented crystallization is key to producing bonelike composite materials with a well-organized structure. However, producing this type of composite material using synthetic biopolymers as scaffolds is challenging. Inspired by the molecular structure of collagen-I, a collagenlike peptide─(Pro-Hyp-Gly)10 (POG10)─was designed to produce self-assembled fibrils that resemble the structure of collagen-I fibrils. In addition, the oriented mineralization of HAP crystals is formed in the fibrils that reproduces a bonelike material similar to collagen-I fibril mineralization. Unlike collagen-I fibrils, POG10 fibrils do not contain gap spaces. The molecular simulation results indicate that in addition to space confinement, the molecular field generated by POG10 can also confine the orientation of HAP, enriching our understanding of physical confinement and shedding light on the design of synthetic biopolymer scaffolds for bonelike material fabrication.

[18F]FAPI PET/CT in the evaluation of focal liver lesions with [18F]FDG non-avidity.

PURPOSE: This prospective study was aimed to investigate the potential utility of [18F]fibroblast activation protein inhibitor (FAPI) PET/CT for evaluating focal liver lesions (FLLs) with [18F]FDG non-avidity. METHODS: From January 2021 to March 2022, this prospective study included 80 FLLs that were not avid on [18F]FDG PET/CT from 37 patients, then underwent [18F]FAPI PET/CT. All patients with FLL(s) with biopsy-proof or follow-up confirmation were categorized into four subgroups (20 hepatocellular carcinomas [HCCs]/5 non-HCC malignancies/4 inflammatory FLLs/8 benign noninflammatory FLLs). The diagnostic value of [18F]FAPI for detecting liver malignancy was determined by visual evaluation. Differences in the maximum standardized uptake value (SUVmax) and lesion-to-background ratio (LBR) obtained from [18F]FAPI PET/CT among the four subgroups were analyzed by semiquantitative analysis. RESULTS: Among the thirty-seven enrolled participants (34 males; median age 57 years, range 48-67 years), on visual evaluation, the sensitivity, specificity, and accuracy of [18F]FAPI PET for detecting liver malignancy in the patient-based analysis were 96.0% (24/25), 58.3% (7/12), and 83.8% (31/37), respectively. On semiquantitative analysis, the SUVmax and LBR of [18F]FAPI PET in liver malignancy (33 HCC lesions; 19 non-HCC malignant lesions) were significantly higher than those in 11 benign noninflammatory FLLs [HCC: SUVmax: 6.4 vs. 4.5, P = 0.017; LBR: 5.1 vs. 1.5, P = 0.003; non-HCC: SUVmax: 5.5 vs. 4.5, P = 0.008; LBR: 4.4 vs. 1.5, P = 0.042]. Notably, there was no significant difference in the SUVmax of [18F]FAPI PET between 33 HCC lesions and 17 inflammatory FLLs (6.4 vs. 8.2, P = 0.37), but the LBR of [18F]FAPI PET in HCC were significantly lower than that in inflammatory FLLs (5.1 vs. 9.1, P = 0.003). CONCLUSIONS: [18F]FAPI PET/CT shows high sensitivity in detecting HCC and non-HCC malignancy with [18F]FDG non-avidity. [18F]FAPI might be a promising radiopharmaceutical for the differential diagnosis of benign noninflammatory FLLs and liver malignancy with [18F]FDG non-avidity.

68 Ga-FAPI Versus 18 F-FDG PET/CT in Recurrent Undifferentiated Pleomorphic Sarcoma of Colon Mesentery.

ABSTRACT: A 56-year-old woman presented with primary undifferentiated pleomorphic sarcoma of colon mesentery underwent complete surgical resection approximately 7 months ago. Abdominal contrast-enhanced CT showed a new mass in the descending colon, suggesting a high probability of tumor recurrence. Under a clinical trial, the patient underwent 68 Ga-FAPI and 18 F-FDG PET/CT to detect whether there are additional recurrent lesions. Compared with 18 F-FDG PET/CT, the recurrent undifferentiated pleomorphic sarcoma of colon mesentery, peritoneum, pelvic lymph node, and lung metastases showed higher uptake in 68 Ga-FAPI PET/CT. This case showed that 68 Ga-FAPI might be a promising radiopharmaceutical in the evaluation of undifferentiated pleomorphic sarcoma.

Decabromodiphenyl ether induces male reproductive toxicity by activating mitochondrial apoptotic pathway through glycolipid metabolism dysbiosis.

Decabromodiphenyl ether (BDE-209), an extensively used flame retardant, exists widely in the environment. Although male reproductive toxicity induced by BDE-209 has been reported, its mechanisms remain unclear. To explore the role of glycolipid metabolism in male reproductive toxicity and the potential mechanisms, forty male SD rats were divided into four groups and given gavage with BDE-209 at 0, 5, 50, and 500 mg/kg/d for 28 days. In vitro, the spermatogenic cell lines GC-2spd cells were divided into four groups: the control group, 32 µg/mL BDE-209 group, 32 µg/mL BDE-209 + 0.4 µM Fatostatin (the inhibitor of SREBP-1) group, and 0.4 µM Fatostatin group. Our results showed that BDE-209 decreased sperm quality and quantity, which was correlated with glycolipid metabolism dysbiosis of testis. The levels of glucose, triglyceride, and total cholesterol were negatively correlated with sperm concentration, and triglyceride and total cholesterol levels were negatively correlated with sperm motility, while positively correlated with the sperm malformation rate. Moreover, BDE-209 exposure activated the glycolipid metabolism pathways (PPARγ/RXRα/SCAP/SREBP-1) and mitochondrial apoptotic pathway, thereby inducing the apoptosis of spermatogenic cells. In vitro, BDE-209 caused triglyceride and total cholesterol disorder and apoptosis of GC-2spd cells, the lipid metabolism pathways inhibitor fatostain downregulated the elevation of triglyceride and total cholesterol concentrations, and suppressed apoptosis and the activation of the mitochondrial apoptotic pathway in GC-2spd cells caused by BDE-209. Our results indicated that BDE-209 induced male reproductive toxicity by causing glycolipid metabolism dysbiosis of testis resulting in activating of the mitochondrial apoptotic pathway in spermatogenic cells. The study provides new insight into the mechanisms of male reproductive toxicity caused by BDE-209.

In Situ Exosomal MicroRNA Determination by Target-Triggered SERS and Fe3O4@TiO2-Based Exosome Accumulation.

Exosomal microRNAs (miRNAs) have been proved to be important biomarkers for the early diagnosis of cancers. However, the accurate quantification of exosomal miRNAs is hampered either by laborious exosome isolation and lysis or by RNA extraction and the amplification process. Here, we reported an in situ platform for direct exosomal miRNAs from serum samples. First, locked nucleic acid (LNA)-modified Au@DTNB (DTNB is the Raman reporter molecule 5,5'-dithiobis-(2-nitrobenzoic acid)) was synthesized as surface-enhanced Raman scattering (SERS) tags to enter into exosomes and assemble with target miRNAs to induce hot-spot SERS signals. Second, Fe3O4@TiO2 nanoparticles were added to enrich the exosomes through affinity interaction of the TiO2 shell for further SERS detection. Based on the platform, target miRNAs can be directly qualified in situ with a detection limit of 0.21 fM, which is better or comparable with quantitative reverse transcription polymerase chain reaction (qRT-PCR) and other in situ methods reported before. Moreover, neither capture antibody nor ultracentrifugation pretreatment was needed in the whole detection procedure. Using exosomal miRNA-10b as a proof of concept, pancreatic ductal adenocarcinoma (PDAC) patients can be recognized from normal controls (NCs) with an accuracy of 99.6%. The simple and sensitive in situ exosomal miRNA detection assay can be seen as a noninvasive liquid biopsy assay for clinical cancer diagnostic adaption.

Resveratrol inhibits bile acid-induced gastric intestinal metaplasia via the PI3K/AKT/p-FoxO4 signalling pathway.

Gastric intestinal metaplasia (GIM) is the essential pre-malignancy of gastric cancer. Chronic inflammation and bile acid reflux are major contributing factors. As an intestinal development transcription factor, caudal-related homeobox 2 (CDX2) is key in GIM. Resveratrol has potential chemopreventive and anti-tumour effects. The aim of the study is to probe the effect of resveratrol in bile acid-induced GIM. We demonstrated that resveratrol could reduce CDX2 expression in a time- and dose-dependent manner in gastric cell lines. A Cignal Finder 45-Pathway Reporter Array and TranSignal Protein/DNA Array Kit verified that resveratrol could increase Forkhead box O4 (FoxO4) activity and that Chenodeoxycholic acid (CDCA) could reduce FoxO4 activity. Furthermore, bioinformatics analysis showed that FoxO4 could bind to the CDX2 promoter, and these conjectures were supported by chromatin-immunoprecipitation (ChIP) assays. Resveratrol can activate FoxO4 and decrease CDX2 expression by increasing phospho-FoxO4 nucleus trans-location. Resveratrol could increase FoxO4 phosphorylation through the PI3K/AKT pathway. Ectopic FoxO4 expression can up-regulate FoxO4 phosphorylation and suppress CDCA-induced GIM marker expression. Finally, we found a reverse correlation between p-FoxO4 and CDX2 in tissue arrays. This study validates that resveratrol could reduce bile acid-induced GIM through the PI3K/AKT/p-FoxO4 signalling pathway and has a potential reversing effect on GIM, especially that caused by bile acid reflux.

The role of 99mTc-MIBI SPECT/CT in patients with secondary hyperparathyroidism: comparison with 99mTc-MIBI planar scintigraphy and ultrasonography.

BACKGROUND: This study aimed to compare the sensitivity of 99mTc-MIBI SPECT/CT, 99mTc-MIBI planar scintigraphy and ultrasonography (US) in patients with secondary hyperparathyroidism (SHPT), and to explore the factors that affect the sensitivity of 99mTc-MIBI SPECT/CT. METHODS: In this retrospective study, forty-six patients with SHPT who underwent 99mTc-MIBI planar scintigraphy, 99mTc-MIBI SPECT/CT and US were enrolled. They underwent surgery within 1 month. We compared the sensitivity of the different imaging methods based on the lesions according to the pathological results. The parathyroid lesions on 99mTc-MIBI SPECT/CT images were divided into missed diagnosis group (MDG) and non-missed diagnosis group (NMDG). We compared the lesion to background ratio (LBR), maximum diameter, volume, the mean CT Hounsfield unit values (CTmean) and location of lesions between MDG and NMDG. RESULTS: The sensitivity of 99mTc-MIBI SPECT/CT, 99mTc-MIBI planar scintigraphy and US were 70.30% versus 48.48% versus 61.82%, respectively. The sensitivity of 99mTc-MIBI SPECT/CT combined US was 79.39%, which was higher than 99mTc-MIBI SPECT/CT with significant difference (P = 0.000). On 99mTc-MIBI SPECT/CT images, the LBR, maximum diameter and volume of lesions in MDG was smaller than those in NMDG with significant difference (P < 0.001). The average LBR, maximum diameter and volume of lesions in MDG and NMDG were 3.42 ± 1.28, 9.32 ± 2.69 mm, 208.51 ± 163.22 mm3 versus 6.75 ± 5.08, 15.03 ± 4.94 mm and 863.85 ± 1216.0 mm3, respectively. CONCLUSIONS: 99mTc-MIBI SPECT/CT exhibited the highest sensitivity among the three methods. When 99mTc-MIBI SPECT/CT combined with US, the sensitivity can be further improved. Lesions with lower MIBI uptake and smaller lesions on 99mTc-MIBI SPECT/CT images were easily missed.

DKK1 is epigenetically downregulated by promoter methylation and inhibits bile acid-induced gastric intestinal metaplasia.

Dickkopf-related protein 1 (DKK1) is essential to gastric cancer as an inhibitor of Wnt signaling. Gastric intestinal metaplasia (GIM) is an important precancerous lesion of gastric cancer that can be activated by bile acid reflux and chronic inflammation. However, the exact mechanism of DKK1 in bile acid-induced GIM has not been completely elucidated. We aimed to explore the epigenetic alterations and biological functions of DKK1 in the development of GIM. In the present study, bile acid was found to induce the expression of intestinal markers in gastric epithelial cells, whereas DKK1 was downregulated in response to bile acid stimulation. The mRNA and protein expression levels of DKK1 were decreased in GIM tissues as evidenced by qRT-PCR and immunohistochemical staining. Surprisingly, the methylation of the DKK1 promoter increased in GIM tissues, and we discovered 28 differential methylation sites of the DKK1 promoter in GIM tissues. Bile acid was able to induce the partial methylation of the DKK1 promoter, while 5-aza could increase DKK1 expression as well as decrease intestinal markers expression in gastric epithelial cells. In conclusion, the promoter methylation and downregulation of DKK1 might play important roles in the development of GIM, especially bile acid-induced GIM.

Measurements of nonvolatile size distribution and its link to traffic soot in urban Shanghai.

Measurements of particle size distribution and size-resolved particle volatility were conducted using a volatility tandem differential mobility analyzers (V-TDMA) in the urban area of Shanghai during wintertime in January 2014. The nonvolatile mode particles with VSF exceeding 0.85 were always externally mixed with more-volatile mode particles. The average VSF ranged from 0.58 to 0.65 for 100-400nm particles, increasing with the increase of particle size. On average, the nonvolatile mode contributed 15-20% of number fraction for 50-400nm particles. Due to their hydrophobic nature, the nonvolatile particles were not easily removed by wet deposition. The concentrations of the nonvolatile mode particles and NOx were well correlated, indicating that the nonvolatile mode particles were mostly attributed to be fresh traffic soot. The diurnal variations in ensemble VSF and number fraction of nonvolatile mode particles exhibited two peaks in clean days, corresponding to morning and evening rush hours. The VSF distributions of 50nm particles were similar during a transition between haze to clean periods whereas in the accumulation mode range, the number fraction of more-volatile mode and the amount of volatile materials in the more-volatile mode particles during haze periods are considerably larger than those in clean periods, indicating different contribution from transported sources.

A high-throughput, homogeneous, fluorescence polarization assay for inhibitors of hedgehog protein autoprocessing.

Hedgehog (Hh) signaling plays an important role in embryonic patterning and adult stem cell renewal but has recently been found also to be involved in certain stem cell cancers. One of the first steps in Hh signaling is the autoprocessing of Hh protein, in which the C-terminal domain (Hh-C) catalyzes a cholesterol-dependent autocleavage reaction that leads to the production of the cholesterol ester of the N-terminal Hh domain (Hh-N), thereby yielding a signaling molecule that activates the Hh pathway by binding to the Patched receptor. This article describes an in vitro, homogeneous assay system that measures changes in fluorescence polarization that accompany the cholesterol-dependent autocleavage of Hh protein. The assay system makes use of a modified Hh protein in which Hh-N, which is not essential for autocleavage, is replaced by a 25-residue peptide containing a tetracysteine motif, complexed with a bisarsenical fluorophore. The assay is quite robust and easily adapted to high-throughput screening in 384-well plates with Z' factors above 0.8. It has been used to screen the National Institutes of Health Clinical Collection, which has led to the identification of 2 compounds that inhibit the cholesterol-dependent autocleavage of Hh protein at micromolar concentrations.

An in vitro screening system for protein splicing inhibitors based on green fluorescent protein as an indicator.

This paper describes an in vitro fluorometric assay system for protein splicing based on the RecA intein of Mycobacterium tuberculosis and a modified green fluorescent protein (GFP). The assay takes advantage of the fact that polypeptides inserted adjacent to residue 129 of GFP cause the protein to form inclusion bodies when expressed in Escherichia coli and to be incapable of fluorophore formation. However, when the inserted polypeptide is an intein, the renatured fusion protein can undergo protein splicing and chromophore formation. Comparison of chromophore formation by renatured GFP-intein fusion and renatured GFP showed that under optimal conditions (pH 6.5 and 20 degrees C) protein splicing is significantly slower than GFP chromophore formation. Taking advantage of the reversible inhibition of protein splicing by zinc ion, a fluorometric protein splicing assay was developed in which the denatured fusion protein of GFP and the RecA intein was purified on a metal ion affinity column and renatured in the presence of 2 mM ZnCl2. When diluted into appropriate buffers, protein splicing could be initiated by the addition of a molar excess of EDTA and followed fluorometrically. This assay should be valuable as a high-throughput screening system for protein splicing inhibitors as potential antimycobacterial agents and as tools for studying the mechanism of protein splicing.

In vitro splicing of erythropoietin by the Mycobacterium tuberculosis RecA intein without substituting amino acids at the splice junctions.

Protein splicing is a self-catalyzed process involving the excision of an intervening polypeptide sequence, the intein, and joining of the flanking polypeptide sequences, the extein, by a peptide bond. We have studied the in vitro splicing of erythropoietin (EPO) using a truncated form of the Mycobacterium tuberculosis RecA mini-intein in which the homing endonuclease domain was replaced with a hexahistidine sequence (His-tag). The intein was inserted adjacent to cysteine residues to assure that the spliced product had the natural amino acid sequence. When expressed in Escherichia coli, intein-containing EPO was found entirely as inclusion bodies but could be refolded in soluble form in the presence of 0.5 M arginine. Protein splicing of the refolded protein could be induced with a reducing agent such as DTT or tris(2-carboxyethyl)phosphine and led to the formation of EPO and mini-intein along with some cleavage products. Protein splicing mediated by the RecA intein requires the presence of a cysteine residue adjacent to the intein insertion site. We compared the efficiencies of protein splicing adjacent to three of the four cysteine residues of EPO (Cys29, Cys33 and Cys161) and found that insertion of intein adjacent to Cys29 allowed far more efficient protein splicing than insertion adjacent to Cys33 or Cys161. For ease of purification, our experiments involved a His-tagged EPO fusion protein and a His-tagged intein and the spliced products (25 kDa EPO and 24 kDa mini-intein) were identified by Western blotting using anti-EPO and anti-His-tag antibodies and by mass spectroscopy. The optimal splicing yield at Cys29 (40%) occurred at pH 7.0 after refolding at 4 degrees C and splicing for 18 h at 25 degrees C in the presence of 1 mM DTT.