[Effect of autologous platelet-rich plasma perfusion on cytokines in uterine drainage fluid of patients with intrauterine adhesions following hysteroscopic adhesiolysis].

Objective: To investigate the effect of autologous platelet-rich plasma (PRP) perfusion on the levels of cytokines in uterine drainage fluid in patients with moderate to severe intrauterine adhesions (IUA) following hysteroscopic adhesiolysis. Methods: Thirty patients with moderate to severe IUA who underwent hysteroscopic adhesiolysis at Beijing Obstetrics and Gynecology Hospital, Capital Medical University from November 2020 to March 2021 were randomly divided into two groups: the PRP group (15 patients with placement of intrauterine-suitable balloons and PRP infusion) and the control group (15 patients with placement of intrauterine-suitable balloons only). For all patients, the channel switch was opened 48 hours after the surgery. The drainage fluid of the uterine cavity was collected using syringes through the proximal end of the drainage channel switch at 24 hours after the surgery and through the drainage channel directly at 48, 72, 96, and 120 hours after the surgery, and the levels of related cytokines including platelet-derived growth factor BB (PDGF-BB), vascular endothelial growth factor A (VEGF-A), insulin-like growth factor 1 (IGF-1) and transforming growth factor-ß1 (TGF-ß1) in the drainage fluid of the uterine cavity were evaluated, respectively. Results: (1) The changes in volumes of uterine cavity drainage fluid: the total drainage fluid volumes of the PRP group and the control group in 120 hours after the surgery were (21.8±2.9) and (22.7±2.7) ml, respectively, and there was no statistically significant difference between the two groups (t=-0.847, P>0.05). No significant differences were found in the volumes of drainage fluid between the two groups at 72, 96, and 120 hours after the surgery (all P>0.05). (2) Variation in cytokine levels in the uterine cavity drainage fluid: ① PDGF-BB: median PDGF-BB levels at 24 and 48 hours after the surgery in the PRP group (6.6 and 9.6 µg/L, respectively) were significantly higher than those in the control group (4.7 and 2.7 µg/L, respectively; all P<0.05). There were no significant differences in PDGF-BB levels between the two groups at 72, 96, and 120 hours after the surgery (all P>0.05). ② VEGF-A: median VEGF-A levels at 24 and 48 hours after the surgery in the PRP group (3.5 and 2.8 µg/L, respectively) were significantly higher than those in the control group (1.6 and 1.2 µg/L, respectively; all P<0.05). There were no significant differences in VEGF-A levels between the two groups at 72, 96, and 120 hours after the surgery (all P>0.05). ③ IGF-1: median IGF-1 level at 48 hours after the surgery in the PRP group was significantly higher than that in the control group (39.5 vs 8.6 µg/L, P<0.05). No significant differences were found in IGF-1 levels at 24, 72, 96, and 120 hours after the surgery between the two groups (all P>0.05). ④ TGF-ß1: There were no significant differences in TGF-ß1 levles between the two groups at 24, 48, 72, 96, and 120 hours after the surgery (all P>0.05). Conclusions: PRP perfusion following hysteroscopic adhesiolysis may increase the levels of PDGF-BB, VEGF-A, and IGF-1 in the uterine cavity drainage fluid, which plays a beneficial role in improving wound microvascular formation, reducing adhesion reformation, and promoting endometrial regeneration and repair.

Renal tumours in a Tsc2(+/-) mouse model do not show feedback inhibition of Akt and are effectively prevented by rapamycin.

Tuberous sclerosis (TSC) is an inherited syndrome in which tumours in multiple organs are characterised by activation of mammalian target of rapamycin complex 1 (mTORC1). Previous work suggests that mTORC1 activation is associated with feedback inhibition of Akt, a substrate of mTORC2. This could limit TSC-associated tumour growth but lead to paradoxical promotion of tumour cell survival upon treatment with mTOR inhibitors. However, Akt/mTOR signalling has not been fully investigated in TSC-associated tumours and it has been uncertain whether mTOR inhibition can prevent TSC-associated renal tumourigenesis. In this study, we investigated Akt/mTOR signalling in renal tumours using a Tsc2(+/-) mouse model and tested whether mTOR inhibition could prevent renal tumourigenesis. We found that all renal lesions including cysts, adenomas and carcinomas exhibited activation of both Akt and mTORC1 as evidenced by increased protein expression and phosphorylation of Akt and mTOR and their downstream targets. Protein kinase Cα was also highly expressed and phosphorylated in these lesions, consistent with activation of mTORC2. Surprisingly, IRS proteins were highly expressed, in contrast to a striking decrease seen in cultured Tsc2(-/-) mouse embryonic fibroblasts, suggesting one mechanism through which loss of feedback inhibition of Akt may occur in mTORC1 hyperactivated Tsc-associated tumours. Long-term treatment with rapamycin reduced both Akt and mTORC1 activity in normal kidney tissues and blocked the development of all types of renal lesions. In conclusion, in contrast to previous studies, we found that Akt signalling is not inhibited in Tsc-associated renal lesions and that by partially inhibiting the Akt/mTOR pathway, rapamycin is highly effective in preventing Tsc-associated tumours.

mTORC1 drives HIF-1α and VEGF-A signalling via multiple mechanisms involving 4E-BP1, S6K1 and STAT3.

Recent clinical trials using rapalogues in tuberous sclerosis complex show regression in volume of typically vascularised tumours including angiomyolipomas and subependymal giant cell astrocytomas. By blocking mechanistic/mammalian target of rapamycin complex 1 (mTORC1) signalling, rapalogue efficacy is likely to occur, in part, through suppression of hypoxia-inducible factors (HIFs) and vascular endothelial growth factors (VEGFs). We show that rapamycin reduces HIF-1α protein levels, and to a lesser extent VEGF-A levels, in renal cystadenoma cells in a Tsc2+/- mouse model. We established that mTORC1 drives HIF-1α protein accumulation through enhanced transcription of HIF-1α mRNA, a process that is blocked by either inhibition or knockdown of signal transducer and activation of transcription 3 (STAT3). Furthermore, we demonstrated that STAT3 is directly phosphorylated by mTORC1 on Ser727 during hypoxia, promoting HIF-1α mRNA transcription. mTORC1 also regulates HIF-1α synthesis on a translational level via co-operative regulation of both initiation factor 4E-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase-1 (S6K1), whereas HIF-1α degradation remains unaffected. We therefore proposed that mTORC1 drives HIF-1α synthesis in a multifaceted manner through 4E-BP1/eIF4E, S6K1 and STAT3. Interestingly, we observed a disconnect between HIF-1α protein levels and VEGF-A expression. Although both S6K1 and 4E-BP1 regulate HIF-1α translation, VEGF-A is primarily under the control of 4E-BP1/eIF4E. S6K1 inhibition reduces HIF-1α but not VEGF-A expression, suggesting that mTORC1 mediates VEGF-A expression via both HIF-1α-dependent and -independent mechanisms. Our work has important implications for the treatment of vascularised tumours, where mTORC1 acts as a central mediator of STAT3, HIF-1α, VEGF-A and angiogenesis via multiple signalling mechanisms.

Detection of copy number changes at the NF1 locus with improved high-resolution array CGH.

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disease caused by various types of mutations in the NF1 gene. We have previously developed a locus-specific DNA microarray for detection of copy number changes at the NF1 locus by comparative genomic hybridization (CGH) analysis. The original array contains 183 probes pooled from 444 polymerase chain reaction (PCR) products. In the current work, we have used 493 probes derived from single PCR products (200--998 bp in size) to construct a higher resolution array with a smaller average probe size for molecular diagnosis of NF1. This has improved the average resolution from 12.6 kb in the previous array to 4.5 kb in the current version. The performance of the newly constructed microarray was validated with 14 well-characterized NF1 mutations for CGH analysis. These mutations represent deletions from approximately 7 kb to over 2 Mb in size. Using this array, we examined a total of 55 NF1 patients for copy number changes at the NF1 locus, detecting deletions in four of them. These results demonstrate that a locus-specific microarray constructed from single PCR products can efficiently detect copy number changes at the NF1 locus, providing a simple method for the molecular diagnosis of NF1.

A structurally defined mini-chromosome vector for the mouse germ line.

Yeast artificial mini-chromosomes have helped to define the features of chromosome architecture important for accurate segregation and replication and have been used to identify genes important for chromosome stability and as large-fragment cloning vectors. Artificial chromosomes have been developed in human cells but they do not have defined, experimentally predictable structures. Fragments of human chromosomes have also been introduced into mice and in one case passed through the germ line. In these experiments, however, the structure and sequence organization of the fragments was not defined. Structurally defined mammalian mini-chromosome vectors should allow large tracts of DNA to be introduced into the vertebrate germ line for biotechnological purposes and for investigations of features of chromosome structure that influence gene expression. Here, we have determined the structure and sequence organization of an engineered mammalian mini-chromosome, ST1, and shown that it is stably maintained in vertebrate somatic cells and that it can be transmitted through the mouse germ line.

Molecular genetics of neurofibromatosis type 1 (NF1).

Neurofibromatosis type 1 (NF1), also called von Recklinghausen disease or peripheral neurofibromatosis, is a common autosomal dominant disorder characterised by multiple neurofibromas, café au lait spots, and Lisch nodules of the iris, with a variable clinical expression. The gene responsible for this condition, NF1, has been isolated by positional cloning. It spans over 350 kb of genomic DNA in chromosomal region 17q11.2 and encodes an mRNA of 11-13 kb containing at least 59 exons. NF1 is widely expressed in a variety of human and rat tissues. Four alternatively spliced NF1 transcripts have been identified. Three of these transcript isoforms (each with an extra exon: 9br, 23a, and 48a, respectively) show differential expression to some extent in various tissues, while the fourth isoform (2.9 kb in length) remains to be examined. The protein encoded by NF1, neurofibromin, has a domain homologous to the GTPase activating protein (GAP) family, and downregulates ras activity. The identification of somatic mutations in NF1 from tumour tissues strongly supports the speculation that NF1 is a member of the tumour suppressor gene family. Although the search for mutations in the gene has proved difficult, germline mutation analysis has shown that around 82% of all the fully characterised NF1 specific mutations so far predict severe truncation of neurofibromin. Further extensive studies are required to elucidate the gene function and the mutation spectrum. This should then facilitate the molecular diagnosis and the development of new therapy for the disease.

Two single base polymorphisms in introns 41 and 16 of the NF1 gene.

We have characterized two intragenic polymorphisms in the neurofibromatosis type 1 (NF1) gene by direct sequencing of PCR products. The variants for these polymorphisms were initially detected on Hydrolink gels. One of the polymorphisms involves a G to A transition in intron 41 at the 28th base upstream of exon 42 with an observed 'G'/'A' heterozygosity of 0.42. The other polymorphism is a T to C transition in intron 16 at the 16th base upstream of exon 17 with an observed 'T'/'C' heterozygosity of 0.09. In combination with other documented polymorphisms in the NF1 gene, these variants should assist in genetic analysis of NF1 families.

Neurofibromatosis type 1 (NF1): the search for mutations by PCR-heteroduplex analysis on Hydrolink gels.

Despite the extensive search for disease causing mutations in exons 28-36 of the neurofibromatosis type 1 (NF1) gene, the NF1 specific mutations so far documented account for only a small proportion of all NF1 cases. In this study, we have used 8 sets of new primers to amplify sequences throughout the NF1 gene, including 10 different exons and their flanking intron sequences. The derived PCR products from 150 independent NF1 patients and 50 normal controls were examined by heteroduplex analysis on Hydrolink gels. Three novel mutations were identified and characterised. Two of these mutations include the same 3-bp deletion (AAT) within exon 17 with a silent codon change from ACA (threonine) to ACG (threonine) and a loss of the codon ATG (methionine). The third mutation is a 10-bp deletion (TTCTCTTGGA) within exon 44 resulting in the formation of an inappropriate stop codon. These results should be useful for the further elucidation of the molecular basis of NF1.

A de novo nonsense mutation in exon 28 of the neurofibromatosis type 1 (NF1) gene.

We have screened a total of 105 unrelated patients with neurofibromatosis type 1 (NF1) for mutations in exon 28 of the NF1 gene using heteroduplex analysis and single strand conformation polymorphism analysis. One novel mutation has been identified and characterised. This mutation involves a 13-bp deletion (AAACTGGCTGAGC or AACTGGCTGAGCA) from base position 5077 (or 5078) to 5089 (or 5090) of the cDNA coding sequence. This alteration leads to a reading frame shift with a premature amber termination signal (TAG) at codon 1694. In addition, there is a change from lysine to threonine at codon 1693. The truncated gene product is estimated to be 1125 amino acid residues shorter than the predicted normal protein (2818 amino acids).

DNase I-hypersensitive sites and transcription factor-binding motifs within the mouse E beta meiotic recombination hot spot.

The second intron of the E beta gene in the mouse major histocompatibility complex is the site of a meiotic recombination hot spot. We detected two DNase I-hypersensitive sites in this intron in meiotic cells isolated from mouse testes. One site appears to be constitutive and is found in other tissues regardless of whether or not they express the E beta gene. Near this hypersensitive site are potential binding motifs for H2TF1/KBF1, NF kappa B, and octamer transcription factors. Gel retardation studies with mouse lymphoma cell nuclear extracts confirmed that each of these motifs is capable of binding protein. The binding of transcription factors may contribute to the enhancement of recombination potential by altering chromatin structure and increasing the accessibility of the DNA to the recombination machinery.