RNA-binding proteins (RBPs) are ubiquitous components within cells, fulfilling essential functions in a myriad of biological processes. These proteins interact with RNA molecules to regulate gene expression at various levels, including transcription, splicing, transport, localization, translation, and degradation. Understanding the intricate network of RBP-RNA interactions is crucial for deciphering the complex regulatory mechanisms that govern cellular function and organismal development. Ultravidet (UV) cross-linking and immunoprecipitation (CLIP) stands out as a powerful approach designed to map the precise locations where RBPs bind to RNA. By using UV light to create covalent bonds between proteins and RNA, followed by immunoprecipitation to isolate the protein-RNA complexes, researchers can identify the direct targets of specific RBPs. The advent of high-throughput sequencing technologies has revolutionized CLIP, enabling the identification of not only the types but also the exact sequences of RNA bound by RBPs on a genome-wide scale. The evolution of CLIP has led to the development of specialized variants, each with unique features that address specific challenges and expand the scope of what can be studied. High-throughput sequencing CLIP (HITS-CLIP) was one of the first advancements, significantly increasing the throughput and resolution of RNA-protein interaction mapping. Photoactivatable-ribonucleoside-enhanced CLIP (PAR-CLIP) introduced the use of photoactivatable ribonucleosides to enhance cross-linking efficiency and specificity, reducing background noise and improving the detection of low-abundance RNA-protein interactions. Individual-nucleotide resolution CLIP (iCLIP) further refined the technique, achieving unprecedented precision by resolving individual nucleotides involved in RBP binding, which is particularly valuable for studying the fine details of RNA structure and function. Despite the remarkable progress, there remains room for improvement in CLIP technology. Researchers continue to seek methods to increase sensitivity, reduce technical variability, and improve the reproducibility of results. Advances in sample preparation, data analysis algorithms, and computational tools are critical for addressing these challenges. Moreover, the application of CLIP to more diverse biological systems, including non-model organisms and clinical samples, requires the development of tailored protocols and the optimization of existing ones. Looking forward, the field of RNA biology is poised to benefit greatly from ongoing innovations in CLIP technology. The exploration of non-canonical RNA-protein interactions, such as those involving long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs), promises to reveal new layers of cellular regulation and may lead to the discovery of novel therapeutic targets. Furthermore, integrating CLIP data with other omics approaches, such as proteomics and metabolomics, will provide a more comprehensive understanding of the dynamic interplay between RNA and its binding partners within the cell. In conclusion, the continuous refinement and expansion of CLIP techniques have not only deepened our knowledge of RNA biology but have also opened up new avenues for investigating the molecular underpinnings of health and disease. As the technology matures, it is expected to play an increasingly pivotal role in both basic and applied research, contributing to the advancement of medical science and biotechnology.

Objectives: Hypoparathyroidism (HP) is a rare endocrine disorder caused by parathyroid hormone (PTH) defi ciency. The PTH is a candidate gene for familial isolated hypoparathyroidism (FIH). This study aimed to investigate the pathogenicity of two novel rare variants (RVs) ofPTH through in vitro functional study. Methods: Targeted next-generation sequencing was used to identify candidate gene mutations. Clinical data were retrospectively collected. Wild-type (WT) PTH was used as a template for site-directed mutagenesis to create mutant eukaryotic expression plasmids, which were transfected into cells. Treated with or without 4-phenylbu tyric acid (4-PBA), the levels of intact PTH (iPTH) and PTH (1-84) were measured by chemiluminescence, and protein expression was assessed using Western blotting. Results: Two patients carrying PTH mutations (c.154G > A: p.Val52Ile, c.270G > T: p.Leu90Phe) were identified.Patient 1, a 45-year-old male, presented with carpal and pedal numbness, muscle cramps, and low serum calcium (1.29 mmol/L). Patient 2, a 12-year-old female, had muscle twitches, convulsions, low calcium (1.50 mmol/L), and iPTH of 4 pg/mL. The iPTH or PTH (1-84) levels in the medium transfected with mutant Val52Ile and Leu90Phe PTH decreased by 31%–38%, and 51%–96% compared to WT (allP < 0.05), which were not rescued by 4-PBA. No significant changes in intracellular PTH expression were observed. Conclusions In this study, two novel RVs of PTH(Val52Ile and Leu90Phe) were identified that may impair hormone synthesis and secretion. Our study has broadened the mutation spectrum of the PTH and shed light on potential mechanisms underlying FIH.

Objectives: Hypoparathyroidism (HP) is a rare endocrine disorder caused by parathyroid hormone (PTH) defi ciency. The PTH is a candidate gene for familial isolated hypoparathyroidism (FIH). This study aimed to investigate the pathogenicity of two novel rare variants (RVs) ofPTH through in vitro functional study. Methods: Targeted next-generation sequencing was used to identify candidate gene mutations. Clinical data were retrospectively collected. Wild-type (WT) PTH was used as a template for site-directed mutagenesis to create mutant eukaryotic expression plasmids, which were transfected into cells. Treated with or without 4-phenylbu tyric acid (4-PBA), the levels of intact PTH (iPTH) and PTH (1-84) were measured by chemiluminescence, and protein expression was assessed using Western blotting. Results: Two patients carrying PTH mutations (c.154G > A: p.Val52Ile, c.270G > T: p.Leu90Phe) were identified.Patient 1, a 45-year-old male, presented with carpal and pedal numbness, muscle cramps, and low serum calcium (1.29 mmol/L). Patient 2, a 12-year-old female, had muscle twitches, convulsions, low calcium (1.50 mmol/L), and iPTH of 4 pg/mL. The iPTH or PTH (1-84) levels in the medium transfected with mutant Val52Ile and Leu90Phe PTH decreased by 31%–38%, and 51%–96% compared to WT (allP < 0.05), which were not rescued by 4-PBA. No significant changes in intracellular PTH expression were observed. Conclusions In this study, two novel RVs of PTH(Val52Ile and Leu90Phe) were identified that may impair hormone synthesis and secretion. Our study has broadened the mutation spectrum of the PTH and shed light on potential mechanisms underlying FIH.

Objectives: Hypoparathyroidism (HP) is a rare endocrine disorder caused by parathyroid hormone (PTH) defi ciency. The PTH is a candidate gene for familial isolated hypoparathyroidism (FIH). This study aimed to investigate the pathogenicity of two novel rare variants (RVs) ofPTH through in vitro functional study. Methods: Targeted next-generation sequencing was used to identify candidate gene mutations. Clinical data were retrospectively collected. Wild-type (WT) PTH was used as a template for site-directed mutagenesis to create mutant eukaryotic expression plasmids, which were transfected into cells. Treated with or without 4-phenylbu tyric acid (4-PBA), the levels of intact PTH (iPTH) and PTH (1-84) were measured by chemiluminescence, and protein expression was assessed using Western blotting. Results: Two patients carrying PTH mutations (c.154G > A: p.Val52Ile, c.270G > T: p.Leu90Phe) were identified.Patient 1, a 45-year-old male, presented with carpal and pedal numbness, muscle cramps, and low serum calcium (1.29 mmol/L). Patient 2, a 12-year-old female, had muscle twitches, convulsions, low calcium (1.50 mmol/L), and iPTH of 4 pg/mL. The iPTH or PTH (1-84) levels in the medium transfected with mutant Val52Ile and Leu90Phe PTH decreased by 31%–38%, and 51%–96% compared to WT (allP < 0.05), which were not rescued by 4-PBA. No significant changes in intracellular PTH expression were observed. Conclusions In this study, two novel RVs of PTH(Val52Ile and Leu90Phe) were identified that may impair hormone synthesis and secretion. Our study has broadened the mutation spectrum of the PTH and shed light on potential mechanisms underlying FIH.

[Objective] To study the relationship between the expression intensity of HLA-Ⅰ platelet antibodies in patients with platelet transfusion refractoriness (PTR) and platelet cross-matching, and to further evaluate other factors in order to provide relevant data support for improving platelet transfusion efficiency and optimizing platelet transfusion regimens. [Methods] Luminex single antigen flow cytometry was used to detect platelet specific antibodies in 35 patients with hematological disease. Subsequently, the Capture-P method was employed to perform 102 crossmatchings between plasma with HLA-Ⅰ antibodies and platelets with known HLA-Ⅰ genotypes. The cross-matching results were assessed and the clinical transfusion outcomes were tracked. [Results] The positive detection rate of HLA-Ⅰ and HPA antibodies in this study was 48.6% (17/35). The negative rate of cross-matching in 102 cases was 37.3% (38/102). Multiple factors affect platelet cross-matching, such as HLA-Ⅰ antibody expression level and antibody type, antigen expression level, cross-reactivity group and eplets. Among them, the expression level and antibody type of HLA-Ⅰ antibody are the main influencing factors. However, the effectiveness of clinical platelet transfusion is not completely determined by the compatibility of platelet cross-matching. [Conclusion] In addition to avoiding strong positive HLA-Ⅰ antibodies, clinical matching should also be vigilant against the serological cross-incompatibility caused by weak positive HLA-Ⅰ antibodies. It may be necessary to establish HLA-Ⅰ low expression antigen database as a better alternative platelet donor selection strategy, and gradually explore the effectiveness of ‘low expression mismatch’ strategy for clinical platelet transfusion.

Objectives: Hypoparathyroidism (HP) is a rare endocrine disorder caused by parathyroid hormone (PTH) defi ciency. The PTH is a candidate gene for familial isolated hypoparathyroidism (FIH). This study aimed to investigate the pathogenicity of two novel rare variants (RVs) ofPTH through in vitro functional study. Methods: Targeted next-generation sequencing was used to identify candidate gene mutations. Clinical data were retrospectively collected. Wild-type (WT) PTH was used as a template for site-directed mutagenesis to create mutant eukaryotic expression plasmids, which were transfected into cells. Treated with or without 4-phenylbu tyric acid (4-PBA), the levels of intact PTH (iPTH) and PTH (1-84) were measured by chemiluminescence, and protein expression was assessed using Western blotting. Results: Two patients carrying PTH mutations (c.154G > A: p.Val52Ile, c.270G > T: p.Leu90Phe) were identified.Patient 1, a 45-year-old male, presented with carpal and pedal numbness, muscle cramps, and low serum calcium (1.29 mmol/L). Patient 2, a 12-year-old female, had muscle twitches, convulsions, low calcium (1.50 mmol/L), and iPTH of 4 pg/mL. The iPTH or PTH (1-84) levels in the medium transfected with mutant Val52Ile and Leu90Phe PTH decreased by 31%–38%, and 51%–96% compared to WT (allP < 0.05), which were not rescued by 4-PBA. No significant changes in intracellular PTH expression were observed. Conclusions In this study, two novel RVs of PTH(Val52Ile and Leu90Phe) were identified that may impair hormone synthesis and secretion. Our study has broadened the mutation spectrum of the PTH and shed light on potential mechanisms underlying FIH.

Objectives: Hypoparathyroidism (HP) is a rare endocrine disorder caused by parathyroid hormone (PTH) defi ciency. The PTH is a candidate gene for familial isolated hypoparathyroidism (FIH). This study aimed to investigate the pathogenicity of two novel rare variants (RVs) ofPTH through in vitro functional study. Methods: Targeted next-generation sequencing was used to identify candidate gene mutations. Clinical data were retrospectively collected. Wild-type (WT) PTH was used as a template for site-directed mutagenesis to create mutant eukaryotic expression plasmids, which were transfected into cells. Treated with or without 4-phenylbu tyric acid (4-PBA), the levels of intact PTH (iPTH) and PTH (1-84) were measured by chemiluminescence, and protein expression was assessed using Western blotting. Results: Two patients carrying PTH mutations (c.154G > A: p.Val52Ile, c.270G > T: p.Leu90Phe) were identified.Patient 1, a 45-year-old male, presented with carpal and pedal numbness, muscle cramps, and low serum calcium (1.29 mmol/L). Patient 2, a 12-year-old female, had muscle twitches, convulsions, low calcium (1.50 mmol/L), and iPTH of 4 pg/mL. The iPTH or PTH (1-84) levels in the medium transfected with mutant Val52Ile and Leu90Phe PTH decreased by 31%–38%, and 51%–96% compared to WT (allP < 0.05), which were not rescued by 4-PBA. No significant changes in intracellular PTH expression were observed. Conclusions In this study, two novel RVs of PTH(Val52Ile and Leu90Phe) were identified that may impair hormone synthesis and secretion. Our study has broadened the mutation spectrum of the PTH and shed light on potential mechanisms underlying FIH.

Objective To explore clinical effect of closed reduction percutaneous elastic intramedullary nail assisted by arthrography in the treatment of radial neck fracture in children.Methods A retrospective analysis was performed on 23 chil-dren with radial neck fracture treated with arthrography assisted closed reduction and percutaneous elastic intramedullary nail internal fixation(arthrography with elastic nail group)from January 2019 to December 2022,including 12 males and 11 fe-males,aged from 2 to 12 years old with an average of(7.36±1.89)years old;According to Judet fracture types,14 children were type Ⅲ and 9 children were type Ⅳ.In addition,23 children with radial neck fracture were selected from January 2015 to December 2018 who were treated with closed reduction and percutaneous elastic intramedullary nail fixation(elastic nail group),including 11 males and 12 females,aged from 2 to 14 years old with an average of(7.50±1.91)years old;Judet classi-fication included 15 children were type Ⅲ and 8 children were type Ⅳ.Operative time and intraoperative fluoroscopy times were compared between two groups.Metaizeau evaluation criteria was used to evaluate fracture reduction,and Tibone-Stoltz evaluation criteria was used to evaluate functional recovery of elbow between two groups.Results Both groups were followed up for 12 to 24 months with an average of(16.56±6.34)months.Operative time and intraoperative fluoroscopy times of elastic nail group were(56.64±19.27)min and(21.13±7.87)times,while those of joint angiography with elastic nail group were(40.33±1 1.50)min and(12.10±3.52)times;there were difference between two groups(P＜0.05).According to Metaizeau evaluation,11 patients got excellent result,9 good and 3 fair in joint angiography with elastic nail group,while in elastic nail group,5 ex-cellent,13 good,4 acceptable,and 1 poor;the difference between two groups was statistically significant(P＜0.05).According to Tibone-Stoltz criteria,14 patients got excellent result,8 good,and 1 fair in joint arthrography with elastic nail group;while in elastic nail group,12 patients got excellent result,9 good,1 fair and 1 poor;there was no significant difference between two groups(P＞0.05).Conclusion Compared to percutaneous elastic intramedullary nail fixation,closed reduction assisted by arthrography has advantages of reduced operation time,decreased intraoperative fluoroscopy frequency,and improved fracture reduction.Arthrography enables clear visualization of the anatomical structures of radius,head,neck,bone,and cartilage in children,facilitating comprehensive display of fracture reduction and brachioradial joint alignment.This technique more pre-cisely guides the depth of elastic intramedullary nail implantation in radius neck,thereby enhancing surgical efficiency and success rate.

Objective To retrieve,evaluate and summarize the best evidence on the management of ocular complications in intensive care patients ventilated in prone position,and to provide references for clinical practice.Methods Evidence on management of ocular complications in intensive care patients ventilated in prone position was systemically retrieved in the guideline websites,professional association websites and databases,such as the BMJ Best Practice,UpToDate,Cochrane Library,Joanna Briggs Institute,PubMed,Web of Science,Science Direct,Embase,CNKI,Wanfang and other databases,including guidelines,clinical decisions,evidence summaries,expert consensuses,group standards,systematic reviews and scoping reviews,published from January 2013 to June 2023.The guidelines were individually evaluated by 4 researchers,and the remaining literature was individually evaluated by 2 researchers.The literature that met the criteria was extracted and graded.Results A total of 15 articles were involved,including 6 guidelines,2 clinical decisions,2 evidence summaries,3 systematic reviews,1 scoping review and 1 group standard.Finally,6 evidence topics and 16 pieces of best evidence were formed,including organization and training,risk identification,eye assessment,eye protection,position management and regular observation.Conclusion This study summarized the best evidence on the management of ocular complications in intensive care patients ventilated in prone position.In the application,the best evidence should be selected according to the clinical situation,so as to reduce the incidence of ocular complications and improve the quality of life of patients.

Objective:To explore the mechanism of peritoneal dialysis solution (PDS)-induced peritoneal microinflammation through activation of ceramide (CER) in peritoneal dialysis model mice.Methods:Thirty 5-week-old male C57BL/6 mice weighing about 22 g were used to set up peritoneal dialysis models, and then were randomly divided into 4 groups: sham operation group (1.5 ml sterilized water, n=7), high glucose-PDS group (1.5 ml 4.25% PDS, n=8), high glucose-PDS+ acid sphingomyelinase (ASMase) inhibitor desipramine (DES) group (1.5 ml sterilized water+10 mg/kg DES, n=8), high glucose-PDS+Src kinase inhibitor PP2 group (1.5 ml sterilized water +1 mg/kg PP2, n=7), with intraperitoneal injection once a day. After 28 days, the mice were sacrificed to retain peritoneal tissues. HE staining and Masson staining were used to observe the histological changes of peritoneum. Immunohistochemistry was used to detect the Toll-like receptor 4 (TLR4) and macrophages. High performance liquid chromatography, liquid chromatography/mass spectrometry and immunofluorescence were used to detect the expression of ASMase and CER. Real-time quantitative PCR was used to detect the mRNA levels of c-Src, p-Src, interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). Western blotting was used to detect the protein levels of c-Src, and p-Src. Enzyme-linked immunosorbent assay was used to detect the serum C reactive protein (CRP), IL-6 and TNF-α. Results:(1) High glucose-PDS led to peritoneal hyperplasia, collagen deposition and fibrosis in the peritoneal dialysis mice, indicating successful modeling. Compared with high glucose-PDS group, peritoneal hyperplasia, collagen deposition and fibrosis of mice treated with DES and PP2 were significantly improved (all P<0.05). (2) Compared with sham operation group, ASMase activation and CER level of peritoneal tissues were significantly higher in high glucose-PDS group, and DES could significantly inhibit activated ASMase and increased CER expression caused by high glucose-PDS (both P<0.05). PP2 had no significant effect on ASMase activation and CER level (both P>0.05). (3) Compared with sham operation group, there were more TLR4 and macrophage positive staining cells in peritoneal tissues in high glucose-PDS group, and the mRNA expression levels of IL-6 and TNF-α in peritoneal tissues and serum CRP, IL-6 and TNF-α were higher (all P<0.05). DES and PP2 could significantly inhibit the increased TLR4, macrophages and related inflammatory factors induced by high glucose-PDS (all P<0.05). (4) Compared with sham operation group, c-Src and p-Src mRNA and protein expression levels of peritoneal tissues in high glucose-PDS group were significantly higher (all P<0.05). PP2 significantly inhibited the increased p-Src mRNA and protein levels caused by high glucose-PDS (both P<0.05), but had no significant effect on the mRNA and protein expression levels of c-Src (both P>0.05). DES had no significant effect on the mRNA and protein expression levels of c-Src and p-Src (all P>0.05). Conclusions:High glucose-PDS may enhance the expression of CER through stimulating the activity of ASMase, phosphorylate Src, activate TLR4 and induce inflammatory damage of peritoneum in peritoneal dialysis model mice.