[Interaction between PSF and cytokeratin 18 mediates PSF relocation to cell membrane and maintains chemosensitivity of myeloid leukemia].

OBJECTIVE: To identify the chaperone of polypyrimidine tractor-binding protein-associated splicing factor (PSF) in myeloid leukemia cells, and to explore the mechanism and redistributive pattern to cell surface of PSF in chemo-sensitive HL60 cells and resistant HL60/DOX cells. METHODS: The eukaryotic expression vector of PSF was transfected with liposomes transiently, then flow cytometry was used to detect the expression level of PSF on the cell surface 24 h, 48 h and 72 h after vector transfections. We constructed a chimeric expression vector, streptavidin binding peptide (SBP)-PSF, meanwhile this vector was transfected and made SBP-PSF fusion protein overexpress. In addition, we used streptavidin magnetic beads to precipitate the cellular chaperonin of PSF and then identified its chaperonin by mass spectrometry (MS). Lentiviral vectors containing cytokeratin18 (K18) interference sequences were transfected into 293T cells to prepare lentivirus. HL60 and HL60/DOX cells were infected with lentivirus to obtain stable interfering K18 cell lines. Next, flow cytometry was used to test the membrane relocation level of PSF. Together, these methods confirmed the similar or different mechanisms of the PSF redistributing to membrane synergistically mediated by K18 in HL60 and HL60/DOX cells. RESULTS: The expression of membrane relocated PSF was detected every day for three days (at the end of 24 h, 48 h and 72 h) after transient overexpression. The expressing rate of PSF on the cell surface was 22.4%±3.5%, 37.9%±6.0%, 58.3%±8.8%, respectively in sensitive HL60 cells, while that was 4.7%±0.5%, 3.9%±0.6%, 2.9%±0.6% , respectively in resistant HL60/DOX cells. The difference of expressing rate on each day was significant, P<0.01. We identified K18 detected by co-immunoprecipitation and mass spectrum assay which was the cellular chaperone of PSF. We found that K18 knockdown decreased the PSF expression level which redistributed on cell surface from 48.9%±5.4% to 6.2%±1.0% in sensitive HL60 cells, and from 9.11%±1.2% to 2.21%±0.51% in resistant HL60/DOX cells, respectively. CONCLUSION: K18 is the intracellular chaperonin of PSF. The interaction of PSF and K18 mediates its redistribution to cell membrane in sensitive cells. While in resistant cells, PSF failed to relocate at the cell surface and accumulated in cells, which mediated resistance to chemotherapeutics.

[Application and research advances of delayed sural neurotrophic vascular flap for diabetic foot ulcers].

Diabetic foot ulcer is one of the serious complications of diabetes. Diabetic wounds are of great difficulty to repair, causing a high amputation rate and a great burden to patients and their family members and society. Researches showed that the delayed sural neurotrophic vascular flap has a great effect in repairing diabetic foot ulcers. This article mainly reviewed the clinical status and research advances of the delayed sural neurotrophic vascular flap in repairing diabetic foot ulcers, intending to provide a reference for its application and research.

[Clinical repair strategy for ischial tuberosity pressure ulcers based on the sinus tract condition and range of skin and soft tissue defects].

Objective: To investigate the clinical repair strategy for ischial tuberosity pressure ulcers based on the sinus tract condition and range of skin and soft tissue defects. Methods: The study was a retrospective observational study. From July 2017 to March 2023, 21 patients with stage Ⅲ or Ⅳ ischial tuberosity pressure ulcers who met the inclusion criteria were admitted to the First Affiliated Hospital of Nanchang University, including 13 males and 8 females, aged 14-84 years. There were 31 ischial tuberosity pressure ulcers, with an area of 1.5 cm×1.0 cm-8.0 cm×6.0 cm. After en bloc resection and debridement, the range of skin and soft tissue defect was 6.0 cm×3.0 cm-15.0 cm×8.0 cm. According to the depth and size of sinus tract and range of skin and soft tissue defects on the wound after debridement, the wounds were repaired according to the following three conditions. (1) When there was no sinus tract or the sinus tract was superficial, with a skin and soft tissue defect range of 6.0 cm×3.0 cm-8.5 cm×6.5 cm, the wound was repaired by direct suture, Z-plasty, transfer of buttock local flap, or V-Y advancement of the posterior femoral cutaneous nerve nutrient vessel flap. (2) When the sinus tract was deep and small, with a skin and soft tissue defect range of 8.5 cm×4.5 cm-11.0 cm×6.5 cm, the wound was repaired by the transfer and filling of gracilis muscle flap followed by direct suture, or Z-plasty, or combined with transfer of inferior gluteal artery perforator flap. (3) When the sinus tract was deep and large, with a skin and soft tissue defect range of 7.5 cm×5.5 cm-15.0 cm×8.0 cm, the wound was repaired by the transfer and filling of gracilis muscle flap and gluteus maximus muscle flap transfer, followed by direct suture, Z-plasty, or combined with transfer of buttock local flap; and transfer and filling of biceps femoris long head muscle flap combined with rotary transfer of the posterior femoral cutaneous nerve nutrient vessel flap; and filling of the inferior gluteal artery perforator adipofascial flap transfer combined with V-Y advancement of the posterior femoral cutaneous nerve nutrient vessel flap. A total of 7 buttock local flaps with incision area of 8.0 cm×6.0 cm-19.0 cm×16.0 cm, 21 gracilis muscle flaps with incision area of 18.0 cm×3.0 cm-24.0 cm×5.0 cm, 9 inferior gluteal artery perforator flaps or inferior gluteal artery perforator adipofascial flaps with incision area of 8.5 cm×6.0 cm-13.0 cm×7.5 cm, 10 gluteal maximus muscle flaps with incision area of 8.0 cm×5.0 cm-13.0 cm×7.0 cm, 2 biceps femoris long head muscle flaps with incision area of 17.0 cm×3.0 cm and 20.0 cm×5.0 cm, and 5 posterior femoral cutaneous nerve nutrient vessel flaps with incision area of 12.0 cm×6.5 cm-21.0 cm×10.0 cm were used. The donor area wounds were directly sutured. The survival of muscle flap, adipofascial flap, and flap, and wound healing in the donor area were observed after operation. The recovery of pressure ulcer and recurrence of patients were followed up. Results: After surgery, all the buttock local flaps, gracilis muscle flaps, gluteus maximus muscle flaps, inferior gluteal artery perforator adipofascial flaps, and biceps femoris long head muscle flaps survived well. In one case, the distal part of one posterior femoral cutaneous nerve nutrient vessel flap was partially necrotic, and the wound was healed after dressing changes. In another patient, bruises developed in the distal end of inferior gluteal artery perforator flap. It was somewhat relieved after removal of some sutures, but a small part of the necrosis was still present, and the wound was healed after bedside debridement and suture. The other posterior femoral cutaneous nerve nutrient vessel flaps and inferior gluteal artery perforator flaps survived well. In one patient, the wound at the donor site caused incision dehiscence due to postoperative bleeding in the donor area. The wound was healed after debridement+Z-plasty+dressing change. The wounds in the rest donor areas of patients were healed well. After 3 to 15 months of follow-up, all the pressure ulcers of patients were repaired well without recurrence. Conclusions: After debridement of ischial tuberosity pressure ulcer, if there is no sinus tract formation or sinus surface is superficial, direct suture, Z-plasty, buttock local flap, or V-Y advancement repair of posterior femoral cutaneous nerve nutrient vessel flap can be selected according to the range of skin and soft tissue defects. If the sinus tract of the wound is deep, the proper tissue flap can be selected to fill the sinus tract according to the size of sinus tract and range of the skin and soft tissue defects, and then the wound can be closed with individualized flap to obtain good repair effect.

[Clinical effects of island posterior femoral composite tissue flaps in the repair of sinus cavity pressure ulcers in the areas of ischial tuberosity and greater trochanter].

Objective: To explore the clinical effects of island posterior femoral composite tissue flaps in the repair of sinus cavity pressure ulcers in the areas of ischial tuberosity and greater trochanter. Methods: The retrospective observational study was conducted. From December 2018 to December 2021, 23 patients with sinus cavity pressure ulcers in the areas of ischial tuberosity and greater trochanter who met the inclusion criteria were admitted to Ganzhou People's Hospital, including 16 males and 7 females, aged 45 to 86 years. The size of pressure ulcers in ischial tuberosity ranged from 1.5 cm×1.0 cm to 8.0 cm×5.0 cm, and the size of pressure ulcers in greater trochanter ranged from 4.0 cm×3.0 cm to 20.0 cm×10.0 cm before debridement. After treatment of underlying diseases, debridement and vacuum sealing drainage for 5 to 14 days were performed. All the wounds were repaired by island posterior femoral composite tissue flaps, with area of 4.5 cm×3.0 cm-24.0 cm×12.0 cm, pedicle width of 3-5 cm, pedicle length of 5-8 cm, and rotation radius of 30-40 cm. Most of the donor site wounds were sutured directly, and only 4 donor site wounds were repaired by intermediate thickness skin graft from the contralateral thigh. The survival of composite tissue flaps, wound healing of the donor and recipient sites and the complications were observed. The recurrence of pressure ulcers, and the appearance and texture of flaps were observed during follow-up. Results: A total of 32 wounds in 23 patients were repaired by island posterior femoral composite tissue flaps (including 3 fascio subcutaneous flaps, 24 fascial flaps+fascio subcutaneous flaps, 2 fascial flaps+fascial dermal flaps, 2 fascial flaps+fascio subcutaneous flaps+femoral biceps flaps, and one fascial flap+fascio subcutaneous flap+gracilis muscle flap). Among them, 31 composite tissue flaps survived well, and a small portion of necrosis occurred in one fascial flap+fascio subcutaneous flap post surgery. The survival rate of composite tissue flap post surgery was 96.9% (31/32). Twenty-nine wounds in the recipient sites were healed, and 2 wounds were torn at the flap pedicle due to improper postural changes, and healed one week after bedside debridement. One wound was partially necrotic due to the flap bruising, and healed 10 days after re-debridement. Thirty-one wounds in the donor sites (including 4 skin graft areas) were healed, and one wound in the donor site was torn due to improper handling at discharge, and healed 15 days after re-debridement and suture. The complication rate was 12.5% (4/32), mainly the incision dehiscence of the flap pedicle and the donor sites (3 wounds), followed by venous congestion at the distal end of flap (one wound). During the follow-up of 3 to 24 months, the pressure ulcers did not recur and the flaps had good appearance and soft texture. Conclusions: The island posterior femoral composite tissue flaps has good blood circulation, large rotation radius, and sufficient tissue volume. It has a high survival rate, good wound healing, low skin grafting rate in the donor site, few postoperative complications, and good long-term effect in the repair of sinus cavity pressure ulcers in the areas of ischial tuberosity and greater trochanter.

[An interlaboratory comparison study on the detection of RUNX1-RUNX1T1 fusion transcript levels and WT1 transcript levels].

Objective: To investigate the current status and real performance of the detection of RUNX1-RUNX1T1 fusion transcript levels and WT1 transcript levels in China through interlaboratory comparison. Methods: Peking University People's Hospital (PKUPH) prepared the samples for comparison. That is, the fresh RUNX1-RUNX1T1 positive (+) bone morrow nucleated cells were serially diluted with RUNX1-RUNX1T1 negative (-) nucleated cells from different patients. Totally 23 sets with 14 different samples per set were prepared. TRIzol reagent was added in each tube and thoroughly mixed with cells for homogenization. Each laboratory simultaneously tested RUNX1-RUNX1T1 and WT1 transcript levels of one set of samples by real-time quantitative PCR method. All transcript levels were reported as the percentage of RUNX1-RUNX1T1 or WT1 transcript copies/ABL copies. Spearman correlation coefficient between the reported transcript levels of each participated laboratory and those of PKUPH was calculated. Results: ①RUNX1-RUNX1T1 comparison: 9 samples were (+) and 5 were (-) , the false negative and positive rates of the 20 participated laboratories were 0 (0/180) and 5% (5/100) , respectively. The reported transcript levels of all 9 positive samples were different among laboratories. The median reported transcript levels of 9 positive samples were from 0.060% to 176.7%, which covered 3.5-log. The ratios of each sample's highest to the lowest reported transcript levels were from 5.5 to 12.3 (one result which obviously deviated from other laboratories' results was not included) , 85% (17/20) of the laboratories had correlation coefficient ≥0.98. ②WT1 comparison: The median reported transcript levels of all 14 samples were from 0.17% to 67.6%, which covered 2.6-log. The ratios of each sample's highest to the lowest reported transcript levels were from 5.3-13.7, 62% (13/21) of the laboratories had correlation coefficient ≥0.98. ③ The relative relationship of the reported RUNX1-RUNX1T1 transcript levels between the participants and PKUPH was not always consistent with that of WT1 transcript levels. Both RUNX1-RUNX1T1 and WT1 transcript levels from 2 and 7 laboratories were individually lower than and higher than those of PKUPH, whereas for the rest 11 laboratories, one transcript level was higher than and the other was lower than that of PKUPH. Conclusion: The reported RUNX1-RUNX1T1 and WT1 transcript levels were different among laboratories for the same sample. Most of the participated laboratories reported highly consistent result with that of PKUPH. The relationship between laboratories of the different transcript levels may not be the same.