Leveraging the 'trans-cleavage′ activity of Cas proteins, the CRISPR/Cas system has successfully transitioned from a gene-editing tool to a new-generation molecular diagnostic platform. This technology demonstrates notable advantages in point-of-care testing of biomarkers, including rapid detection, operational simplicity, and portability. However, it still faces critical challenges such as insufficient sensitivity and limited multiplex detection capability. Recent studies indicate that through innovative strategies including protein engineering, microfluidic integration, and artificial intelligence algorithm optimization, CRISPR/Cas-based diagnostic technology is advancing molecular diagnostics towards field-deployable and intelligent systems, thereby providing novel solutions for intelligent on-site biomarker screening.

Leveraging the 'trans-cleavage′ activity of Cas proteins, the CRISPR/Cas system has successfully transitioned from a gene-editing tool to a new-generation molecular diagnostic platform. This technology demonstrates notable advantages in point-of-care testing of biomarkers, including rapid detection, operational simplicity, and portability. However, it still faces critical challenges such as insufficient sensitivity and limited multiplex detection capability. Recent studies indicate that through innovative strategies including protein engineering, microfluidic integration, and artificial intelligence algorithm optimization, CRISPR/Cas-based diagnostic technology is advancing molecular diagnostics towards field-deployable and intelligent systems, thereby providing novel solutions for intelligent on-site biomarker screening.

  Objective  To understand the current status of surgical treatment for hemophilia osteoarthropathy (HO) in China.  Methods  Using an online questionnaire, select domestic hospitals that partici-pated in the compilation of the 'Guideline for perioperative management of hemophilia patients undergoing orthopaedic surgery in China ', in addition to members of the National Joint Surgery Group, and the Orthopedic Branch of the Chinese Medical Association for targeted investigation and analysis.  Results  A total of 17 domestic hospitals were included, all of which were general hospitals. Hospitals that started HO surgery treatment before 2000 accounted for 35.29%. A total of 3057 surgical cases of HO were reported by those hospitals. The most commonly performed surgical procedures were hip and knee joint replacement. The most commonly used coagulation factor replacement regimen was recombinant coagulation factor preparation. Ten hospitals reported finding patients with transfusion-related infectious diseases. Bleeding and hematoma formation were the most frequently reported surgical complications. Excessive length of hospital stay and high economic costs were the most frequently reported problems.  Conclusions  Surgical treatment for HO in 17 hospitals is mainly carried out in some large comprehensive medical centers in the eastern region. Compared with the patient base, the popularity and number of surgeries are still relatively insufficient. It is necessary to further standardize the treatment system by standardizing factor replacement and strengthening rehabilitation to improve surgical treatment outcomes.

BACKGROUND:General y considered bone marrow cells obtained by adherent method are mesenchymal stem cells, and they can differentiate into osteoblasts, adipocytes and chondrocytes.
OBJECTIVE:To explore the differentiation capacity of bone marrow adherent cells into osteoclasts.
METHODS:Primary mouse mesenchymal stem cells were obtained using adherent method, and bone marrow cells were obtained through adherence 1-5 days. Both of them were cultured with normal medium and inducing medium containing m-csf and RANKL. After 9 days, cells were stained by alkaline phosphatase and tartrate-resistant acid phosphatase. The passage 2 mesenchymal stem cells were divided into four groups, which were induced by mock, m-csf, RANKL and m-csf+RANKL, respectively. After 9 days of culture, the cells were stained by alkaline phosphatase and tartrate-resistant acid phosphatase.
RESULTS AND CONCLUSION:Primary culture of adhered cells was uniform. Alkaline phosphatase and tartrate-resistant acid phosphatase staining of primary and passaged bone marrow adherent cells induced with m-csf+RANKL were positive. This result showed that there were cells that could be induced into osteoclasts in the primary and passaged bone marrow adherent cells. Alkaline phosphatase and tartrate-resistant acid phosphatase staining showed differences of adherent cells at different times after the induction, indicating that adherent cells at different times had different differentiation capacity.

AIM: To obtain a high and stable expression analog of human basic fibroblast growth factor by genetic engineering. METHODS: The cysteins 78 and 96 of natural hbFGF polypeptide was substituted with serines by means of site-directed mutagenesis. Using pET- 3c as vector, the mutated polynucleotide was cloned and then transferred into BL21 (DE3)plysS. After induction by IPTG, the analog was obtained and analyzed by SDS - PAGE. RESULTS: After purification the form of soluble mutant increased remarkably but the forms of dimmer and higher multimer were reduced greatly to no more than 8% of the total recombinant protein. By MTT assay, the analog showed the same biological activity. This new analog represented a desirable complementation for native hbFGF to develop pharmaceutical drug in clinical use. CONCLUSION: Substitution of certain amino acids of polypeptide without altering native protein' s bioactivity to get the analog is an effective means to increase stability of foreign protein and its solubility in E. coli.

AIM: To study the expression of murine interleukin-12(mIL-12) gene in B16F10 melanoma cells. METHODS: mIL-12 gene was inserted into pcDNA3.1 eukaryotic expression vector by DNA recombination and then transfected into B16F10 melanoma cells by electroporation. The integration and expression of mIL-12 gene in B16F10 melanoma cells were detected by PCR,RT-PCR and Western blot after positive cell clones had been screened. RESULTS: mIL-12 gene was confirmed to have been transfected and expressed in B16F10 cells on three levels of DNA, mRNA and protein. CONCLUSION: mIL-12 gene can successfully be transfected and expressed in B16F10 melanoma cells in vitro , which lays a foundation for the further investigation of mIL-12 gene tumor vaccine.

AIM: To screen the cytotoxicity of degradable calcium metaphosphate (dCMP) compared with hydroxyapatite (HA). The proliferation and differentiation abilities of human marrow mesenchymal stem cells (MSC) were used to exhibit the cytotoxicity. METHODS: The cell morphology of MSC was analysed after direct contact with dCMP at different time points by scanning electron microscopy analysis. The degradation products of dCMP and HA were analysed with inductively coupled plasma torch and ion chromatography. The cytotoxic effect of degradation products of dCMP was evaluated by FACS, quantitative assay of ALP and ARS, respectively. RESULTS: dCMP enhanced the proliferation of MSC, but didn't interfere the osteogenic differentiation process of MSC and its mineralization. HA inhibited the proliferation of MSC and the mineralization of osteogenic differentiated MSC, while it did not interfere the osteogenic differentiation process of MSC. CONCLUSION: dCMP had a better cytocompatibility with MSC than HA, which might allow for its use as skeleton scaffolds.

To improve the expression level of non fusion hbFGF in E coli , the coding sequence of human bFGF gene, which had been cloned from primarily cultured human fibroblast, was mutated according to the principle of lowering the GC content and increasing the codon preference After being ligated into pET 3c and transformed into BL21(DE3), the recombinant induced by IPTG Expression level was up to 30% of the total bacterial protein The result indicated that optimizing of the TIR would promote the expression level of recombinant protein

AIM: To construct retroviral expression vector pLXmIL-12SN containing mouse IL-12 (mIL-12) gene and detect the gene expression in B16F10 cells. METHODS: Vector pGCp35-IRES-p40SN was digested to obtain p35-IRES-p40 (mIL-12 gene fragment) and retroviral vector pLXSN was digested by restriction enzyme, and then they were linked by ligase. The recombinant vector that mIL-12 gene fragment had correctly been inserted into pLXSN was identified by sequencing. The recombination vector with mIL-12 gene was transfected into B16F10 cell and was detected by RT-PCR. RESULTS: Expression vector pLXmIL-12SN was successfully constructed. mIL-12 gene was confirmed to express in B16F10 cells at the level of mRNA. CONCLUSION: Acquired vector pLXmIL-12SN and confirmed mIL-12 gene expression lays a foundation for mIL-12 gene therapy to eye diseases.