AIM: To explore the diagnostic value of combining the corneal stress-strain index(SSI)with corneal biomechanical parameters for early keratoconus.METHODS:A retrospective study was conducted on 34 patients(53 eyes)with early keratoconus diagnosed and treated in our hospital from March 2022 to February 2024. Additionally, 112 normal volunteers(112 eyes)who underwent physical examinations in our hospital during the same period were selected as a healthy control group. The CorvisST equipment was utilized for measurement and recorded deformation with Scheimpflug camera to obtain 10 biomechanical parameters: first applanation time(A1T), first applanation length(A1L), velocity of initial applanation(Vin), second applanation time(A2T), second applanation length(A2L), velocity of outward applanation(Vout), highest concavity time(HCT), highest concavity depth of applanation(HCDA), highest concavity radius(HCR), and peak distance(PD), as well as stress-strain index(SSI), and the corneal biomechanical parameters of the two groups were compared. Furthermore, Logistic regression analysis was used to identify the risk factors for keratoconus, and ROC curves were plotted to analyze the biomechanical parameters of the cornea for early diagnosis of keratoconus.RESULTS:The SSI(0.77±0.17)in patients with keratoconus was lower than that in healthy controls(1.01±0.24; P<0.001). Patients with keratoconus had lower A1T, A1L, A2L, and HCR, and higher Vout, HCDA, and PD compared to healthy controls(all P<0.001). Logistic regression analysis showed that decreased SSI, A1T, A1L, A2L, and HCR, as well as increased Vout, HCDA, and PD, were risk factors for the development of keratoconus(P<0.001). ROC curve analysis showed that the AUC value for combined diagnosis of early keratoconus was 0.997, with a Youden's index of 0.954, sensitivity and specificity of 98.1% and 97.3%, respectively, and a 95% CI of 0.994-1.000.CONCLUSION:The combination of SSI and corneal biomechanical parameters holds diagnostic significance for early keratoconus, and the joint diagnostic value is even higher. It can be considered as a diagnostic or screening indicator for early keratoconus.

AIM: To analyze the influencing factors of postoperative dry eye complication after corneal transplantation and to build a nomogram prediction model.METHODS: Clinical data were collected on 117 patients who underwent corneal transplantation at our hospital from March 2021 to February 2023. They were divided into dry eye group(n=96)and non-dry eye group(n=21)according to whether there was a postoperative dry eye. The risk factors of postoperative complication of dry eye after corneal transplantation were analyzed, the nomogram prediction model for predicting postoperative complication of dry eye after corneal transplantation was constructed, and the internal validation of the model and the prediction efficacy were assessed by calibration curves and decision curves, respectively.RESULTS: Comorbid diabetes mellitus, comorbid sleep disorders, comorbid meibomian gland dysfunction, chronic eye drop abuse, chronic corneal contact lens wear, interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α)were the risk factors for the complication of dry eye after corneal transplantation(P<0.05). The nomogram model predicted a C-index of 0.890(95% CI 0.877-0.903). The nomogram model had a threshold >0.07, and the nomogram model provided higher net clinical benefit than the single index in all cases.CONCLUSION: The nomogram model built in this study based on the factors affecting the complication of dry eye after corneal transplantation has a good predictive value for the complication of dry eye after corneal transplantation.

Objective:To explore the efficacy of adjusting the dose of imatinib dose in the context of therapeutic drug monitoring (TDM) in patients with gastrointestinal stromal tumors (GISTs) who are receiving adjuvant therapy after complete resection of their tumors.Methods:This was a descriptive study. Inclusion criteria were (1) complete surgical resection with a pathological diagnosis of GIST, (2) postoperative adjuvant therapy with imatinib and dosage adjustment, (3) multiple TDM of imatinib, and (4) complete clinical, pathological, and follow-up data. The data of 70 patients with GISTs treated at Union Hospital, Tongji Medical College, Huazhong University of Science and Technology between January 2015 and December 2023 were collected retrospectively. The study cohort comprised 15 (21.4%) men and 55 (78.6%) women of median age 60 years (range: 25–82). Of the eligible patients, 49 (70.0%) were at high-risk, 14 (20.0%) at intermediate-risk, six (8.6%) at low-risk, and one (1.4%) at very low risk. Patients were followed up by the gastrointestinal stromal tumor clinic every 2–3 months and their plasma concentrations of imatinib were checked. The dose was adjusted to 300 mg/d or 200 mg/d depending on whether they had had ≥ grade III adverse reactions, and whether the first plasma concentration of imatinib was ≥ 1,500 μg/L or between the expected range of 760 μg/L–1,100 μg/L. Studied indicators included adverse reactions, quality of life before and after dose adjustment, and overall survival and recurrence-free survival (RFS) after dose adjustment.Results:Before dose adjustment, all 70 patients received 400 mg of imatinib daily, with initial TDM values of 1,900 ± 568 μg/L, for a median duration of 8.3 months. After dose adjustment, 60 patients received 300 mg daily, with a TDM of 1,216 ± 350 μg/L, whereas 10 received 200 mg daily, with a TDM of 1,023 ± 269 μg/L. The median duration of treatment after dose adjustment was 23.4 months. Compared with those whose dosages were not adjusted, the incidence of bone marrow suppression was significantly lower (74.3% [52/70] vs. 51.4% [36/70], χ 2=9.202, P=0.010); as were the incidences of edema (95.7% [67/70] vs. 50.0% [35/70], χ 2=40.526, P<0.001); skin reactions (70.0% [49/70] vs. 32.9% [23/70), χ 2=22.495, P<0.001); and gastrointestinal reactions (38.6% [27/70] vs. 10.0% [7/70], χ 2=15.899, P<0.001) in those whose dosages were adjusted. The average total scores for physical health before and after dose adjustment were 76 ± 5 and 88 ± 4, respectively; whereas the mental health scores were 75 ± 6 and 89 ± 4, respectively. The median follow-up period was 36 months (range 6–126). During the first 3 years of follow-up, five high-risk patients with non-gastric GISTs developed recurrences. The 3-year overall survival rate was 100%, and the 3-year RFS rate was 92.8%, high-risk patients having a 3-year RFS rate of 89.8%. Conclusion:The adverse reactions and quality of life of GIST patients with severe adverse reactions to adjuvant imatinib therapy after complete resection can be mitigated by appropriately reducing the dosage of imatinib under the guidance of TDM.

Objective:To explore the efficacy of adjusting the dose of imatinib dose in the context of therapeutic drug monitoring (TDM) in patients with gastrointestinal stromal tumors (GISTs) who are receiving adjuvant therapy after complete resection of their tumors.Methods:This was a descriptive study. Inclusion criteria were (1) complete surgical resection with a pathological diagnosis of GIST, (2) postoperative adjuvant therapy with imatinib and dosage adjustment, (3) multiple TDM of imatinib, and (4) complete clinical, pathological, and follow-up data. The data of 70 patients with GISTs treated at Union Hospital, Tongji Medical College, Huazhong University of Science and Technology between January 2015 and December 2023 were collected retrospectively. The study cohort comprised 15 (21.4%) men and 55 (78.6%) women of median age 60 years (range: 25–82). Of the eligible patients, 49 (70.0%) were at high-risk, 14 (20.0%) at intermediate-risk, six (8.6%) at low-risk, and one (1.4%) at very low risk. Patients were followed up by the gastrointestinal stromal tumor clinic every 2–3 months and their plasma concentrations of imatinib were checked. The dose was adjusted to 300 mg/d or 200 mg/d depending on whether they had had ≥ grade III adverse reactions, and whether the first plasma concentration of imatinib was ≥ 1,500 μg/L or between the expected range of 760 μg/L–1,100 μg/L. Studied indicators included adverse reactions, quality of life before and after dose adjustment, and overall survival and recurrence-free survival (RFS) after dose adjustment.Results:Before dose adjustment, all 70 patients received 400 mg of imatinib daily, with initial TDM values of 1,900 ± 568 μg/L, for a median duration of 8.3 months. After dose adjustment, 60 patients received 300 mg daily, with a TDM of 1,216 ± 350 μg/L, whereas 10 received 200 mg daily, with a TDM of 1,023 ± 269 μg/L. The median duration of treatment after dose adjustment was 23.4 months. Compared with those whose dosages were not adjusted, the incidence of bone marrow suppression was significantly lower (74.3% [52/70] vs. 51.4% [36/70], χ 2=9.202, P=0.010); as were the incidences of edema (95.7% [67/70] vs. 50.0% [35/70], χ 2=40.526, P<0.001); skin reactions (70.0% [49/70] vs. 32.9% [23/70), χ 2=22.495, P<0.001); and gastrointestinal reactions (38.6% [27/70] vs. 10.0% [7/70], χ 2=15.899, P<0.001) in those whose dosages were adjusted. The average total scores for physical health before and after dose adjustment were 76 ± 5 and 88 ± 4, respectively; whereas the mental health scores were 75 ± 6 and 89 ± 4, respectively. The median follow-up period was 36 months (range 6–126). During the first 3 years of follow-up, five high-risk patients with non-gastric GISTs developed recurrences. The 3-year overall survival rate was 100%, and the 3-year RFS rate was 92.8%, high-risk patients having a 3-year RFS rate of 89.8%. Conclusion:The adverse reactions and quality of life of GIST patients with severe adverse reactions to adjuvant imatinib therapy after complete resection can be mitigated by appropriately reducing the dosage of imatinib under the guidance of TDM.

During the gene editing process mediated by CRISPR/Cas9, precise genome editing and gene knock-in can be achieved by the homologous recombination of double-stranded DNA (dsDNA) donor template. However, the low-efficiency of homologous recombination in eukaryotic cells hampers the development and application of this gene editing strategy. Here, we developed a novel CRISPR/Cas9-hLacI donor adapting system (DAS) to enhance the dsDNA-templated gene editing, taking the advantage of the specific binding of the LacI repressor protein and the LacO operator sequence derived for the Escherichia coli lactose operon. The codon-humanized LacI gene was fused as an adaptor to the Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus lugdunensis Cas9 (SlugCas9-HF) genes, and the LacO operator sequence was used as the aptamer and linked to the dsDNA donor template by PCR. The Cas9 nuclease activity after the fusion and the homology-directed repair (HDR) efficiency of the LacO-linked dsDNA template were firstly examined using surrogate reporter assays with the corresponding reporter vectors. The CRISPR/Cas9-hLacI DASs mediated genome precise editing were further checked, and we achieved a high efficiency up to 30.5% of precise editing at the VEGFA locus in HEK293T cells by using the CRISPR/SlugCas9-hLacI DAS. In summary, we developed a novel CRISPR/Cas9-hLacI DAS for dsDNA-templated gene editing, which enriches the CRISPR/Cas9-derived gene editing techniques and provides a novel tool for animal molecular design breeding researches.
Humans ; Animals ; Gene Editing ; CRISPR-Cas Systems/genetics* ; HEK293 Cells ; Homologous Recombination ; DNA

Clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9), the third-generation genome editing tool, has been favored because of its high efficiency and clear system composition. In this technology, the introduced double-strand breaks (DSBs) are mainly repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathways. The high-fidelity HDR pathway is used for genome modification, which can introduce artificially controllable insertions, deletions, or substitutions carried by the donor templates. Although high-level knock-out can be easily achieved by NHEJ, accurate HDR-mediated knock-in remains a technical challenge. In most circumstances, although both alleles are broken by endonucleases, only one can be repaired by HDR, and the other one is usually recombined by NHEJ. For gene function studies or disease model establishment, biallelic editing to generate homozygous cell lines and homozygotes is needed to ensure consistent phenotypes. Thus, there is an urgent need for an efficient biallelic editing system. Here, we developed three pairs of integrated selection systems, where each of the two selection cassettes contained one drug-screening gene and one fluorescent marker. Flanked by homologous arms containing the mutated sequences, the selection cassettes were integrated into the target site, mediated by CRISPR/Cas9-induced HDR. Positively targeted cell clones were massively enriched by fluorescent microscopy after screening for drug resistance. We tested this novel method on the amyloid precursor protein (APP) and presenilin 1 (PSEN1) loci and demonstrated up to 82.0% biallelic editing efficiency after optimization. Our results indicate that this strategy can provide a new efficient approach for biallelic editing and lay a foundation for establishment of an easier and more efficient disease model.
Alleles ; CRISPR-Cas Systems ; DNA End-Joining Repair ; Gene Editing/methods* ; Recombinational DNA Repair

Objective:To summarize and analyze the clinical data of Chinese patients with colony-stimulating factor 1 receptor (CSF1R)-related leukoencephalopathy, and clarify the phenotypic and genetic characteristics of Chinese patients.Methods:Medical history of patients with CSF1R-related leukoencephalopathy diagnosed from April 1, 2018 to January 31, 2021 in the department of neurology of 22 hospitals in China was collected, and scores of Mini-Mental State Examination (MMSE), Montreal Cognitive Assessment Scale (MoCA), magnetic resonance severity scale were evaluated. Group comparison was performed between male and female patients.Results:A total of 62 patients were included, and the male-female ratio was 1∶1.95. The age of onset was (40.35±8.42) years. Cognitive impairment (82.3%, 51/62) and motor symptoms (77.4%,48/62) were the most common symptoms. The MMSE and MoCA scores were 18.79±7.16 and 13.96±7.23, respectively, and the scores of two scales in male patients (22.06±5.31 and 18.08±5.60) were significantly higher than those in females (15.53±7.41 , t=2.954, P=0.006; 10.15±6.26, t=3.328 , P=0.003). The most common radiographic feature was bilateral asymmetric white matter changes (100.0%), and the magnetic resonance imaging severity scale score was 27.42±11.40, while the white matter lesion score of females (22.94±8.39) was significantly higher than that of males (17.62±8.74 , t=-2.221, P<0.05). A total of 36 CSF1R gene mutations were found in this study, among which c.2381T>C/p.I794T was the hotspot mutation that carried by 17.9% (10/56) of the probands. Conclusions:The core phenotypic characteristics of CSF1R-related leukoencephalopathy in China are progressive motor and cognitive impairment, with bilateral asymmetrical white matter changes. In addition, there exist gender differences clinically, with severer cognitive impairment and imaging changes in female patients. Thirty-six CSF1R gene mutations were found in this study, and c.2381T>C/p. I794T was the hotspot mutation.

Objective To study the effect of rapid rehabilitation nursing measures in elderly patients with hip fracture and its effect on postoperative pain and quality of life.Methods A total of 82 elderly patients with hip fractures were divided into control group and observation group by random number table method,with 41 cases in each group.The patients in the control group were treated with routine nursing intervention,and the patients in the observation group underwent rapid rehabilitation nursing intervention based on conventional fracture care.The pain degree,hip function and prognostic quality of life of the two groups were compared.Results The VAS score of the observation group was lower than that of the control group at 1 week and 1 month after surgery (P ＜ 0.05).The Harris scores of the two groups were higher than that of the control group at 1,5 month after operation,and observation group was higher than the control group (P ＜ 0.05).At 5 months after operation,the scores of psychological status,physical function and daily social life of the observation group were higher than that of the control group (P ＜ 0.05).Conclusion Rapid rehabilitation nursing measures can reduce postoperative pain,accelerate the recovery of the hip function and improve the quality of life of elderly patients with hip fracture.So it is worthy of clinical application.

Objective To study the effect of rapid rehabilitation nursing measures in elderly patients with hip fracture and its effect on postoperative pain and quality of life.Methods A total of 82 elderly patients with hip fractures were divided into control group and observation group by random number table method,with 41 cases in each group.The patients in the control group were treated with routine nursing intervention,and the patients in the observation group underwent rapid rehabilitation nursing intervention based on conventional fracture care.The pain degree,hip function and prognostic quality of life of the two groups were compared.Results The VAS score of the observation group was lower than that of the control group at 1 week and 1 month after surgery (P ＜ 0.05).The Harris scores of the two groups were higher than that of the control group at 1,5 month after operation,and observation group was higher than the control group (P ＜ 0.05).At 5 months after operation,the scores of psychological status,physical function and daily social life of the observation group were higher than that of the control group (P ＜ 0.05).Conclusion Rapid rehabilitation nursing measures can reduce postoperative pain,accelerate the recovery of the hip function and improve the quality of life of elderly patients with hip fracture.So it is worthy of clinical application.

Objective:To explore the adjustment factors to the migration and functional activity on macrophages in the uterine of inflammatory mice induced by LPS.Methods:150 Kunming female mouse were divided into control group (group A),LPS model group (group B),MCP-1 blocking-up group (group C),the mice uterines were extracted separately at hour 1,3,6,12,24.The number of CD14+ macrophages and the expression of CD14 macrophages were detected by Immunohistochemistry,ELISA detects the expression of TNF-α and MCP-1.Results:①Compared with group A,the number of CD14+ macrophages and the expression of CD14 in endometrium,myometrium,perimrtrium of group B were highly significantly increased (P<0.01) at every time points,the endometrium,myometrium of group C were closely to normal level at 1,3,6 h;compared with group B,the number of CD14+ macrophages and the expression of CD14 were highly significantly decreased(P<0.01)at 1,3,6 h of perimrtrium of group C and every time points of endometrium,myometrium of group C.②Compared with group A,the content of TNF-α and MCP-1 were highly significantly increased (P<0.01) at every time points of group B and 12,24 h of group C;compared with group B,the content of TNF-α and MCP-1 of group C were highly significantly decreased(P<0.01) at every time points.Conclusion:The migration of macrophages and the expression of CD14 and TNF-α in the uterine of inflammatory mice induced by LPS were regulated by MCP-1.