Dissecting phospho-motif-dependent Shc1 interactome using long synthetic protein fragments.

Activated receptor tyrosine kinases (RTKs) rely on the assembly of signaling proteins into high-dimensional protein complexes for signal transduction. Shc1, a prototypical scaffold protein, plays a pivotal role in directing phosphotyrosine (pY)-dependent protein complex formation for numerous RTKs typically through its two pY-binding domains. The three conserved pY sites within its CH1 region (Shc1CH1) hold particular significance due to their substantial contribution to its functions. However, how Shc1 differentially utilizes these sites to precisely coordinate protein complex assembly remains unclear. Here, we employed multiple peptide ligation techniques to synthesize an array of long protein fragments (107 amino acids) covering a significant portion of the Shc1CH1 region with varying phosphorylation states at residues Y239, 240, 313, and S335. By combining these phospho-Shc1CH1 fragments with integrated proteomics sample preparation and quantitative proteomic analysis, we were able to comprehensively resolve the site-specific interactomes of Shc1 with single amino acid resolution. By applying this approach to different cancer cell lines, we demonstrated that these phospho-Shc1CH1 fragments can be effectively used as a diagnostic tool to assess cell type-specific RTK signaling networks. Collectively, these biochemical conclusions help to better understand the sophisticated organization of pY-dependent Shc1 adaptor protein complexes and their functional roles in cancer.

Prevalence of Anxiety and Depression among Healthcare Workers During the Omicron Pandemic and Its Impact on Gastrointestinal Symptoms.

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Case report: Severe cholestatic jaundice associated with hyperthyroidism treated with methimazole.

RATIONALE: We present a case of a 43-year-old female patient diagnosed with hyperthyroidism. This study aims to demonstrate the rare association between hyperthyroidism and severe cholestatic jaundice, and the effectiveness of methimazole treatment. PATIENT CONCERNS: The patient developed severe jaundice, a typically mild symptom in most hyperthyroidism cases. DIAGNOSIS: The severe jaundice was suspected to be a result of cholestasis induced by hyperthyroidism, with other potential causes such as drug-induced or autoimmune liver dysfunction being ruled out. OUTCOMES: The patient was effectively treated with methimazole. Outcomes: Treatment with methimazole alleviated the severe cholestatic jaundice and restored normal thyroid function. LESSONS: The specific mechanism of cholestasis as a secondary complication of hyperthyroidism remains unclear, and there are no specific biochemical markers for cholestasis caused by this hormonal disease. This case underscores the possibility of severe jaundice as a clinical manifestation of hyperthyroidism, and highlights antithyroid drug treatment as an effective strategy for managing severe cholestatic jaundice.

A robust and scalable active-matrix driven digital microfluidic platform based on printed-circuit board technology.

Two-dimensional digital microfluidic platforms, on which droplets are actuated by electrowetting on dielectrics, have merits such as dynamic reconfigurability and ease for automation. However, concerns for digital microfluidic platforms based on low-cost printed circuit boards, such as the scalability of the electrode array and the reliability of the device operation, should be addressed before high throughput and fully automatic applications can be realized. In this work we report the progress in addressing those issues by using active-matrix circuitry to automatically drive a large electrode array with enhanced device reliability. We describe the design and the fabrication of a robust and scalable active-matrix driven digital microfluidic platform based on printed-circuit board technology. Reliable actuation of aqueous and organic droplets is achieved using a free-standing double-layer hydrophobic membrane. To demonstrate the versatility of the digital microfluidic platform, a pentapeptide is synthesized on the device within 30 minutes. With these improvements, a fully automatic, scalable, robust, reusable, and low-cost digital microfluidic platform capable of parallel manipulation of a large number of droplets can find numerous applications in chemical engineering, bioengineering and biomedical engineering.

Fully Integrated and Multiplexed Sample Preparation Technology for Sensitive Interactome Profiling.

Affinity purification coupled to mass spectrometry (AP-MS) is a popular approach for deciphering the architecture of protein interaction networks. Protein lysates (100 µg) are typically required for multistep sample processing in large volumes, which often causes sample loss and reduces the MS analysis sensitivity. Herein, we reported a fully integrated spintip-based AP-MS technology, termed FISAP, for multiplexed and sensitive interactome profiling. The FISAP device can be easily employed for routine use by introducing AP beads into a C18 StageTip. Taking advantage of the switchable functionalization of the C18 matrix by sodium dodecyl sulfate, all the sample preparation steps encompassing peptide or antibody-based AP, reduction, alkylation, tryptic digestion, tandem mass tag (TMT) labeling, and desalting can be performed in a single tip with a benchtop centrifuge in 4 h. Using a biotinylated tyrosine phosphorylated (pTyr) peptide as an affinity ligand, we mapped the pTyr-dependent interactome of the pY191 motif on the immune receptor CD28 cytoplasmic domain. When processing 50 µg of protein lysates, FISAP showed a comparable interactome identification performance but better quantification performance and lower background interference compared to the traditional tube-based method. Furthermore, a cost-effective on-column TMT labeling protocol was established and integrated into the FISAP pipeline with increased sensitivity. Compared to the tube-based method, the usage of a synthetic peptide probe and a TMT reagent was both reduced by 20 times. As low as 1 µg of protein lysates could be applied for interactome profiling. Finally, we expanded the applicability of the FISAP technology to epitope tag-based AP-MS for profiling the ILK/PINCH/Parvin complex using 100 times less protein lysate than a previous report. Collectively, FISAP is an easy-to-use and sensitive technology for quantitatively profiling protein complexes when the starting material and affinity reagent are the limitation, especially for applications in biomedical research and chemical biology.

Comprehensive analysis of key lncRNAs in HCV-positive hepatocellular carcinoma.

INTRODUCTION: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Despite the therapeutic advances in HCC in the past few decades, the mortality rate of HCC is still high. Hepatitis C (HCV) infection is one of the major etiological risk factors of HCCs. However, the underlying mechanisms of HCV-induced hepatocarcinogenesis remain largely unclear. MATERIAL AND METHODS: Our study represented the comprehensive analysis of differentially expressed lncRNAs in HCV-positive HCC for the first time by analyzing the public dataset GSE17856. Co-expression network and gene ontology (GO) analysis revealed the functions of those differentially expressed lncRNAs. RESULTS: We identified 256 upregulated lncRNAs and 198 downregulated lncRNAs in HCV- positive HCC compared to the normal liver tissues. Co-expression network and GO analysis showed that these lncRNAs were involved in regulating metabolism, energy pathways, proliferation and the immune response. Seven lncRNAs (LOC341056, CCT6P1, PTTG3P, LOC643387, LOC100133920, C3P1 and C22orf45) were identified as key lncRNAs and co-expressed with more than 100 differentially expressed genes (DEGs) in HCV-related HCC. Kaplan-Meier analysis showed that higher expression levels of LOC643387, PTTG3P, LOC341056, CCT6P1 and lower expression levels of C3P1 and C22orf45 were associated with shorter survival time in the TCGA dataset. CONCLUSIONS: We believe that this study can provide novel potential therapeutic and prognostic biomarkers for HCV-positive HCC.

Photoaffinity-engineered protein scaffold for systematically exploring native phosphotyrosine signaling complexes in tumor samples.

Phosphotyrosine (pTyr)-regulated protein complexes play critical roles in cancer signaling. The systematic characterization of these protein complexes in tumor samples remains a challenge due to their limited access and the transient nature of pTyr-mediated interactions. We developed a hybrid chemical proteomics approach, termed Photo-pTyr-scaffold, by engineering Src homology 2 (SH2) domains, which specifically bind pTyr proteins, with both trifunctional chemical probes and genetic mutations to overcome these challenges. Dynamic SH2 domain-scaffolding protein complexes were efficiently cross-linked under mild UV light, captured by biotin tag, and identified by mass spectrometry. This approach was successfully used to profile native pTyr protein complexes from breast cancer tissue samples on a proteome scale with high selectivity, achieving about 100 times higher sensitivity for detecting pTyr signaling proteins than that afforded by traditional immunohistochemical methods. Among more than 1,000 identified pTyr proteins, receptor tyrosine kinase PDGFRB expressed on cancer-associated fibroblasts was validated as an important intercellular signaling regulator with poor expression correlation to ERBB2, and blockade of PDGFRB signaling could efficiently suppress tumor growth. The Photo-pTyr-scaffold approach may become a generic tool for readily profiling dynamic pTyr signaling complexes in clinically relevant samples.

[Epidemiological investigation on endemic fluorosis along the Yellow River alluvial plain of Shandong province].

OBJECTIVE: To investigate the current prevalent status of endemic fluorosis in the floodplain area of the lower Yellow River in Shandong province. METHODS: According to "The National Technical Scheme for Endemic Disease Control in 2008", 16 counties were chosen to carry out the epidemiological survey of endemic fluorosis. Three villages were chosen in each county, to determine the fluoride content of drinking water and to check the dental fluorosis of children aged 8 to 12 year old, the skeletal fluorosis of adults over 16 years of age. Both children and adults were tested for urine fluoride. The content of fluoride in drinking water and urine was determined by F-ion selective electrode while dental fluorosis of children aged 8 to 12 years old was diagnosed by Dean's method and adults skeletal fluorosis by the National Standard for "Diagnosis of endemic skeletal fluorosis" (WS 192-2008). RESULTS: The investigation was taken place in 26 'improved-water-quality' villages in 16 counties, among which 19 villages had water fluoride content ≤ 1.00 mg/L and accounted for 73.08% (19/26), 7 villages had water fluoride content > 1.00 mg/L and accounted for 26.92% (7/26), with the highest water fluoride content as 3.73 mg/L. In 22 'yet to improve-water quality' villages in 16 counties, 5 villages had water fluoride content ≤ 1.00 mg/L (accounted for 22.73%), 17 villages had water fluoride content > 1.00 mg/L (accounted for 77.27%), with the highest water fluoride content as 3.38 mg/L. The overall rate of dental fluorosis among children aged 8 to 12 years old was 52.18% (1042/1997), with the index of dental fluorosis as 1.17 and the rate of dental damage as 8.01% (160/1997). The urinary fluoride values above 1.40 mg/L were found in 65.00% (845/1300) of children aged 8 to 12 years old, with the highest urinary fluoride concentrations as 18.53 mg/L. The rate of skeletal fluorosis by clinic and X-rays in adults older than 16 years old were 4.35% (1121/25 781) and 11.36% (5/44), respectively. The urinary fluoride values above 1.60 mg/L were found as 63.92% (606/948) in adults older than 16 years old, with the highest urinary fluoride concentrations as 21.35 mg/L. CONCLUSION: The status of endemic fluorosis had not been effectively controlled and the situation for endemic fluorosis control was still critical in the floodplain area of the lower Yellow River in Shandong province, suggesting that the preventive approaches on endemic fluorosis control should be strengthened.