OBJECTIVE To study the ameliorative effect and mechanism of Forsythia suspensa-Lonicera japonica herb pair on acute lung injury （ALI） based on serum exosomal microRNA （miRNA）. METHODS The rats were randomly divided into a blank group （normal saline）， model group （nomal saline）， and F. suspensa-L. japonica herb pair group （2.55 g/kg）， with 10 rats in each group. Except for the blank group， the other groups were used to establish an ALI model by intratracheal dripping of 5 mg/ mL lipopolysaccharides. After modeling， each group was given relevant medicine/normal saline intragastrically， once a day， for 3 consecutive days. After the last medication， the pathological status of lung tissue was observed； lung wet-to-dry weight ratio and leukocyte counts in bronchoalveolar lavage fluid （BALF） were determined. The levels of inflammatory factors [tumor necrosis factor-α（TNF-α）， interleukin-1β （IL-1β）， IL-10] in BALF were determined. Exosomes were isolated from rat serum， and high- throughput sequencing technology was employed to screen differentially expressed miRNA within the exosomes， followed by Kyoto Encyclopedia of Genes and Genomes （KEGG） enrichment analysis. Based on the screened differentially expressed miRNA and the enriched KEGG pathways， in vitro cellular experiments were conducted for validation. RESULTS The animal experimental results demonstrated that after intervention with the F. suspensa-L. japonica herb pair， the wet-to-dry weight ratio， the number of leukocytes in BALF， as well as the levels of TNF-α and IL-1β in BALF of ALI rats were all significantly reduced （P＜0.01）， while the level of IL-10 was significantly increased （P＜0.01）. The results of high-throughput sequencing experiments revealed that the F. suspensa-L. japonica herb pair could significantly up-regulate the expressions of miR-345-3p， miR-194-5p， miR-653-5p， and others in exosomes. Among them， the KEGG pathways involved in the target genes of differentially expressed miRNA included the hypoxia-inducible factor-1（HIF-1） signaling pathway， among others. The results of cellular E-mail：huang.haiying@126.com validation experiments showed that overexpressed miR-345-3p could significantly elevate the level of IL-10 in the cell supernatant （P＜0.01）， while significantly reducing the levels of TNF-α and IL-1β in the cell supernatant， as well as the mRNA and protein expression levels of protein kinase B1， phosphatidylinositol 3- kinase， and HIF-1α （P＜0.01）. CONCLUSIONS F. suspensa-L. japonica herb pair can alleviate inflammatory responses and thereby exert a therapeutic effect in improving ALI by up-regulating the expression of miR-345-3p in serum exosomes and inhibiting the activity of the HIF-1 signaling pathway.

Background::The goal of the assisted reproductive treatment is to transfer one euploid blastocyst and to help infertile women giving birth one healthy neonate. Some algorithms have been used to assess the ploidy status of embryos derived from couples with normal chromosome, who subjected to preimplantation genetic testing for aneuploidy (PGT-A) treatment. However, it is currently unknown whether artificial intelligence model can be used to assess the euploidy status of blastocyst derived from populations with chromosomal rearrangement.Methods::From February 2020 to May 2021, we collected the whole raw time-lapse videos at multiple focal planes from in vitro cultured embryos, the clinical information of couples, and the comprehensive chromosome screening results of those blastocysts that had received PGT treatment. Initially, we developed a novel deep learning model called the Attentive Multi-Focus Selection Network (AMSNet) to analyze time-lapse videos in real time and predict blastocyst formation. Building upon AMSNet, we integrated additional clinically predictive variables and created a second deep learning model, the Attentive Multi-Focus Video and Clinical Information Fusion Network (AMCFNet), to assess the euploidy status of embryos. The efficacy of the AMCFNet was further tested in embryos with parental chromosomal rearrangements. The receiver operating characteristic curve (ROC) was used to evaluate the superiority of the model. Results::A total of 4112 embryos with complete time-lapse videos were enrolled for the blastocyst formation prediction task, and 1422 qualified blastocysts received PGT-A ( n = 589) or PGT for chromosomal structural rearrangement (PGT-SR, n = 833) were enrolled for the euploidy assessment task in this study. The AMSNet model using seven focal raw time-lapse videos has the best real-time accuracy. The real-time accuracy for AMSNet to predict blastocyst formation reached above 70% on the day 2 of embryo culture, and then increased to 80% on the day 4 of embryo culture. Combing with 4 clinical features of couples, the AUC of AMCFNet with 7 focal points increased to 0.729 in blastocysts derived from couples with chromosomal rearrangement. Conclusion::Integrating seven focal raw time-lapse images of embryos and parental clinical information, AMCFNet model have the capability of assessing euploidy status in blastocysts derived from couples with chromosomal rearrangement.

Objective To explore the clinical application value of high frequency ultrasound combined with acoustic radiation force impulse in patients with diabetic sarcopenia(DSA).Methods A total of 196 patients with type 2 diabetes mellitus(T2DM)who were hospitalized in the Endocrinology Department of the First Affiliated Hospital of Longyan City,Fujian Medical University were enrolled in this study from January 2022 to February 2023.All the participants were divided into two groups:simple T2DM group(n=120)and combined DSA group(DSA,n=76)according to the limb skeletal muscle mass index(ASMI).During the same period,40 healthy individuals who underwent physical examination were selected as the normal control(NC)group.The general information and biochemical indicators were analyzed in each group.And the muscle thickness,pinnate angle,muscle bundle length and shear wave velocity of the medial head of the right gastrocnemius muscle were compared among the groups.Results The thickness,feathery angle,muscle bundle length,and shear wave velocity of the medial head muscle of the right gastrocnemius muscle were lower in DSA group than in NC and T2DM groups(P＜0.05).Pearson correlation analysis showed that ASMI was positively correlated with body weight,BMI,TC,albumin,total protein,muscle thickness,feather angle,muscle bundle length,and shear wave velocity(P＜0.05),while negatively correlated with age,FPG and HbA1c(P＜0.05).Binary logistic regression analysis showed that muscle thickness and shear wave velocity are influencing factors for DSA.The analysis of the receiver operating characteristic(ROC)curve of the subjects showed that the area under the ROC curve of muscle thickness and shear wave velocity was 0.826 and 0.877(P＜0.05).The diagnostic thresholds for DSA were 14.1 mm and 1.63 m/s,with sensitivity of 0.953 and 0.903,and specificity of 0.971 and 0.941.Conclusions Ultrasound measurement of the muscle thickness and shear wave velocity of the medial head of the right gastrocnemius muscle can be used to diagnose diabetic sarcopenia.

Pleomorphic adenoma (PA) is the most common benign tumour in the salivary gland and has high morphological complexity. However, the origin and intratumoral heterogeneity of PA are largely unknown. Here, we constructed a comprehensive atlas of PA at single-cell resolution and showed that PA exhibited five tumour subpopulations, three recapitulating the epithelial states of the normal parotid gland, and two PA-specific epithelial cell (PASE) populations unique to tumours. Then, six subgroups of PASE cells were identified, which varied in epithelium, bone, immune, metabolism, stemness and cell cycle signatures. Moreover, we revealed that CD36⁺ myoepithelial cells were the tumour-initiating cells (TICs) in PA, and were dominated by the PI3K-AKT pathway. Targeting the PI3K-AKT pathway significantly inhibited CD36⁺ myoepithelial cell-derived tumour spheres and the growth of PA organoids. Our results provide new insights into the diversity and origin of PA, offering an important clinical implication for targeting the PI3K-AKT signalling pathway in PA treatment.
Humans ; Adenoma, Pleomorphic/genetics* ; Phosphatidylinositol 3-Kinases ; Proto-Oncogene Proteins c-akt ; Transcriptome ; Myoepithelioma

Objective:To observe the clinical effect of four-point fixation in patients with posterior chamber intraocular lens dislocation.Methods:A retrospective case series study was adopted.Sixteen patients (16 eyes) with posterior chamber intraocular lens dislocation who underwent suture suspension techniques with four-point fixation in Changzhou No.2 people's Hospital from January 2015 to January 2018 were enrolled.Postoperative effects were observed during follow-up, ranging from 6 to 13 months.The preoperative and 6-month postoperative uncorrected visual acuity (UCVA), best corrected visual acuity (BCVA), corneal endothelium cell count and astigmatism were measured and the differences were compared, and the relationships between total astigmatism and corneal astigmatism or intraocular lens induced astigmatism were analyzed, and the postoperative position of intraocular lens and complications were observed.This study adhered to the Declaration of Helsinki.The study protocol was approved by an Ethics Committee of Changzhou No.2 People's Hospital (No.2015-C-012-01).Written informed consent was obtained from each patient before surgery.Results:The mean preoperative UCVA (LogMAR) and BCVA (LogMAR) were 1.09±0.24 and 0.48±0.20, respectively, which were significantly improved to 0.30±0.12 and 0.26±0.13 at 6 months after operation, respectively.And the differences were statistically significant ( t=11.782, 3.795; both at P<0.01).The preoperative and 6-month postoperative corneal endothelium cell count were (2 270±360)/mm 2 and (2 032±327)/mm 2, respectively, and the difference was not significant ( t=1.921, P=0.074).The 6-month postoperative mean total astigmatism, corneal astigmatism and intraocular lens induced astigmatism were (-1.47±0.82)D, (-1.34±0.61)D and (-0.22±0.35)D, respectively.There was a highly positive correlation between total astigmatism and corneal astigmatism ( r=0.885, P<0.05), but there was no significant correlation between total astigmatism and intraocular lens induced astigmatism ( r=-0.432, P=0.095).No dislocation, deviation or torsion of intraocular lens were observed during the follow-up.Varying degree of symptoms of iridocyclitis were observed during early stage after operation, which disappeared after treatment.There were two cases of high intraocular pressure, which were normal after treatment.No retinal detachment, choroidal detachment, expulsive suprachoroidal hemorrhage, endophthalmitis, corneal endothelial decompensation or other complications occurred during and after operation. Conclusions:There is a stable position of intraocular lens, good visual acuity and few complications after four-point fixation with suture and suspension, which is a feasible method to treat dislocated intraocular lens.

β-Thalassemia is a global health issue, caused by mutations in the HBB gene. Among these mutations, HBB -28 (A>G) mutations is one of the three most common mutations in China and Southeast Asia patients with β-thalassemia. Correcting this mutation in human embryos may prevent the disease being passed onto future generations and cure anemia. Here we report the first study using base editor (BE) system to correct disease mutant in human embryos. Firstly, we produced a 293T cell line with an exogenous HBB -28 (A>G) mutant fragment for gRNAs and targeting efficiency evaluation. Then we collected primary skin fibroblast cells from a β-thalassemia patient with HBB -28 (A>G) homozygous mutation. Data showed that base editor could precisely correct HBB -28 (A>G) mutation in the patient's primary cells. To model homozygous mutation disease embryos, we constructed nuclear transfer embryos by fusing the lymphocyte or skin fibroblast cells with enucleated in vitro matured (IVM) oocytes. Notably, the gene correction efficiency was over 23.0% in these embryos by base editor. Although these embryos were still mosaic, the percentage of repaired blastomeres was over 20.0%. In addition, we found that base editor variants, with narrowed deamination window, could promote G-to-A conversion at HBB -28 site precisely in human embryos. Collectively, this study demonstrated the feasibility of curing genetic disease in human somatic cells and embryos by base editor system.
APOBEC-1 Deaminase ; genetics ; metabolism ; Base Sequence ; Blastomeres ; cytology ; metabolism ; CRISPR-Cas Systems ; Embryo, Mammalian ; metabolism ; pathology ; Female ; Fibroblasts ; metabolism ; pathology ; Gene Editing ; methods ; Gene Expression ; HEK293 Cells ; Heterozygote ; Homozygote ; Humans ; Point Mutation ; Primary Cell Culture ; Promoter Regions, Genetic ; Sequence Analysis, DNA ; beta-Globins ; genetics ; metabolism ; beta-Thalassemia ; genetics ; metabolism ; pathology ; therapy

Genome editing tools such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) have been widely used to modify genes in model systems including animal zygotes and human cells, and hold tremendous promise for both basic research and clinical applications. To date, a serious knowledge gap remains in our understanding of DNA repair mechanisms in human early embryos, and in the efficiency and potential off-target effects of using technologies such as CRISPR/Cas9 in human pre-implantation embryos. In this report, we used tripronuclear (3PN) zygotes to further investigate CRISPR/Cas9-mediated gene editing in human cells. We found that CRISPR/Cas9 could effectively cleave the endogenous β-globin gene (HBB). However, the efficiency of homologous recombination directed repair (HDR) of HBB was low and the edited embryos were mosaic. Off-target cleavage was also apparent in these 3PN zygotes as revealed by the T7E1 assay and whole-exome sequencing. Furthermore, the endogenous delta-globin gene (HBD), which is homologous to HBB, competed with exogenous donor oligos to act as the repair template, leading to untoward mutations. Our data also indicated that repair of the HBB locus in these embryos occurred preferentially through the non-crossover HDR pathway. Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRSIPR/Cas9-mediated editing.
Blastocyst ; CRISPR-Cas Systems ; Hemoglobins, Abnormal ; genetics ; metabolism ; Humans ; Zygote

Objective To investigate the clinical use of array comparative genomic hybridization (aCGH) with fluorescence in situ hybridization (FISH) in preimplantion genetic diagnosis (PGD)for reciprocal and Robertsonian translocation carriers.Methods From Jan.2012 to Jun.2013,a total of 220 PGD cycles from 151 reciprocal translocation and 62 Robertsonian translocation carrier couples,including 33 cycles for reciprocal translocation carriers and 22 cycles for Robertsonian translocation carriers performed using array CGH,and 119 cycles for reciprocal translocation carriers and 46 cycles for Robertsonian translocation carriers performed using FISH were retrospectively studied.The rate of accurate diagnosis was compared between two methods.Results Normal and/or balance rates of the two translocated chromosomes detected by aCGH for both reciprocal and Robertsonian translocation carriers were 38.20％ (123/322) and 67.20％ (127/189),significantly higher than 15.39％ (195/1 267) and 30.75％ (202/657) by FISH (all P ＜0.05).Abnormal rates of the two translocated chromosomes detected by aCGH for both reciprocal and Robertsonian translocation carriers were 59.32％ (191/322) and 30.69％ (58/189),significantly lower than 83.03％ (1 052/1 267) and 67.43％ (443/657) by FISH (all P ＜ 0.05).And the rate of aneu ploidy in non-translocated chromosome from reciprocal translocation embryos was 20.19％ (65/322),which was significantly lower than 38.62％ (13/189) from Robertsonian translocation embryos (P ＜ 0.01).Conclusions Normal and/or balance rates of the two translocated chromosomes detected by array CGH were significantly higher than FISH.And the rate of aneuploidy in non-translocated chromosomes from reciprocal translocation embryos was significantly lower than that from Robertsonian translocation embryos.

Objective To investigate the characteristics and risk factors of the injured children Under the age of 14 in Dongguan area and provide the basis for making the specific protective measures.Methods A retrospective analysis of 1707 clinical cases of the injured children in the emergency department of our hospital from 2006 to 2010.Results The incidence rate of child injury in Dongguan area is high,accounting for 3.25％ of the emergency people at the same period.Fall-wound is at the first place of the causes of injury,accounting for 30.52％ ; traffic accident injury and bums are in the second place,accounting for 19.22％ and 11.42％ ; the incidence rate of 7-10 years-old child injury is the highest,38.25％ ; the incidence rate of boys injury is higher than girls; the incidence rate of the child injury of the children in these family such as their parents are separated,their mother are just senior high school graduates or the per capita monthly income for the whole family is less than RMB 3000 yuan is higher.There are 7 different degree of disability cases caused by injury and 10 death cases resulted from injury.Conclusions To carry out the pointed health education,average up the level of safety protection of parents and teachers,to strengthen the management of child injury surveillance and damage hazards,so as to minimize the occurrence of child injury.

OBJECTIVETo assess the value of array comparative genomic hybridization (array CGH) technique for preimplantation genetic diagnosis (PGD).

METHODSArray CGH was performed on three types of cells, which included 3-5 cells isolated from B2/C38/A1 embryonic stem cell lines, single cells isolated from two discarded normal fertilized embryos, and 10 blastocysts biopsied from 5 couples undergoing PGD for chromosomal translocations. For the 10 blastocysts, 8 were abnormal embryos, 1 appeared to be normal but showed arrested development, and 1 embryo was without any fluorescence signals. 24sure V3 or 24sure+ array chips were applied for CGH analysis. The results were analyzed with a BlueFuse Multi software.

RESULTS(1) The results of cells from B2/C3/A1 embryo stem cells by array CGH were consistent with karyotyping analysis. (2) For the 6 single cell samples from two discarded embryos, 2 blastomeres from one embryo were diagnosed as with aneuploidy and a normal karyotype, respectively. Two out of 4 blastomeres biopsied from another embryo were normal, whilst the remaining two were diagnosed with aneuploidies of -22 and +13. Repeated detection with 24sure+ array was consistent with the 24sure V3 result. (3) Ten cell masses from 10 embryos in PGD cycles were successfully analyzed with array CGH, among which four were confirmed with fluorescence in situ hybridization (FISH) on day 3. In two of them, array CGH confirmed FISH diagnosis. For the remaining two, additional aneuploidies for chromosomes not tested by FISH were discovered by array CGH. Another embryo diagnosed as no signal by FISH was found to have trisomy 13 by array CGH. The remaining 5 embryos also showed discordant results by FISH and array CGH. One embryo from a Robertsonian translocation carrier was found to have monosomy 13 by FISH but trisomy 14 and additional aneuploidies by both 24sure V3 and 24sure+ chips. One embryo with many fragments and arrested development by D5 showed discordant results by FISH and array CGH. However, the FISH and array CGH results for other two embryos from this reciprocal translocation carrier were consistent. Three embryos with inconsistent results by FISH and array CGH had a chromosomal translocation involving q11 region.

CONCLUSIONArray CGH is useful for PGD applications for its capability to detect structural chromosomal abnormalities through screening of aneuploidies. However, the 24sure V3 array may not suit detection of translocations with breakpoints close to the q11 region of chromosomes.

Cell Line ; Comparative Genomic Hybridization ; Embryonic Stem Cells ; metabolism ; Female ; Humans ; In Situ Hybridization, Fluorescence ; Male ; Pregnancy ; Preimplantation Diagnosis ; Reproducibility of Results ; Translocation, Genetic