[Analysis on antimicrobial sensitivity of Neisseria meningitidis in China from 2005 to 2019].

Objective: To analyze the antimicrobial resistance characteristics of 538 Neisseria meningitidis isolated from 2005 to 2019 in China. Method: Total of 538 Neisseria meningitidis strains collected from 30 provinces in China from 2005 to 2019. Antimicrobial susceptibility test were performed based on the standards of clinical and laboratory standardization association (CLSI) including 11 recommended antibiotics. Gradient diffusion method was used to detect the antibiotic sensitivity of Neisseria meningitidis. Results: All 538 strains were sensitive to azithromycin, meropenem, chloramphenicol, rifampicin and ceftriaxone. As to other six antibiotics, the antibiotics sensitivity rates were cefotaxime (97.4%, 524 strains), ampicillin (87.7%, 472 strains), penicillin (84.8%, 456 strains), minocycline (95.2%, 512 strains), ciprofloxacin (24.9%, 134 strains) and trimethoprim/sulfamethoxazole (11.2%, 60 strains) respectively. Conclusions: Neisseria meningitidis isolated from 2005-2019 in China were all sensitive to azithromycin, meropenem, chloramphenicol, rifampicin and ceftriaxone. It should highlight Neisseria meningitidis resistant to cefotaxime, ampicillin and penicillin. Ciprofloxacin and sulfamethoxazole are not recommended as the priority choice for clinical treatment and prophylactic medication.

[Molecular characteristics of serogroup B neisseria meningitidis, China].

Objective: To investigate the molecular characteristics of serogroup B neisseria meningitidis in China. Methods: Total of 485 (100 strains isolated from cerebrospinal fluid or blood samples of encephalomyelitis cases, and 385 strains isolated from nasopharynx of healthy carriers) Meningococcal serogroup B (MenB) strains, isolated from 29 provinces of China between 1968 and 2016, were analyzed by multilocus sequence typing (MLST) and PorA typing methods. Further, the genetic diversity of three MenB vaccine proteins, FHbp, NadA and NHBA, were analyzed. Results: The 485 study strains belonged to 270 sequence types (STs), 107 of which (representing 211 strains) could be grouped into ten clonal complexes (CC). CC4821 has been the predominant lineage in China since 2005 (28.7%, n=139). The most common PorA types of MenB strains from invasive meningococcal cases were P1.5-2,2-2 (10.0%, n=10), P1.5-1,2-2 (9.0%, n=9) and P1.5-1,10-4 (9.0%, n=9). Four hundred and twenty one strains had intact fhbp gene; variant 1, 2 and 3 accounted for 12.8% (54 strains), 85.0% (358 strains) and 2.2% (9 strains) respevtively. Ten out of 432 strains (2.3%) contained complete nadA gene. All the 172 strains for which the nhba gene was sequenced had intact gene sequence which corresponded to 68 peptide types. Conclusion: CC4821 was the predominant CC of MenB strains in China; the vaccine proteins were diverse about the sequences. The vaccine proteins should be carefully selected when developing MenB vaccines in China.

A novel regulatory function for miR-29a in keloid fibrogenesis.

BACKGROUND: A growing body of evidence has shown that microRNA-29 (miR-29) plays a central role in the progression of fibrosis. However, the mechanisms underlying the role of miR-29 in keloid fibrogenesis remain unknown. AIM: To investigate the roles of miR-29 in dermal fibroblasts in the pathogenesis of keloids. METHODS: Primary fibroblasts from 9 patients with keloid and 6 healthy controls (HCs) were cultured and pretreated with transforming growth factor (TGF)-ß1. Next, fibroblasts were transfected with precursor miRNA and anti-miR-29a miRNA. TGF-ß1-associated miR-29 alterations were investigated by quantitative real-time PCR. Collagen I and collagen III protein levels were analysed by western blotting. RESULTS: miR-29a, miR-29b and miR-29c levels were significantly lower in keloid compared with healthy fibroblasts (P < 0.05), and in particular, miR-29a was especially markedly reduced (P < 0.001). Type I and type III collagen mRNA and protein levels were decreased in keloid fibroblasts transfected with pre-miR-29a (P < 0.05), whereas knockdown with anti-miR-29a increased type I and type III collagen mRNA and protein expression (P < 0.05) in the fibroblasts. Interestingly, pretreatment of fibroblasts with TGF-ß1 significantly decreased miR-29a (P < 0.05), whereas miR-29b and miR-29c were reduced to a lesser extent, which was not significant. CONCLUSIONS: These findings show that miR-29a exerts as a novel regulator in the fibrogenesis of keloid, suggesting that miR-29a might be a novel marker for keloid.

Altered circulating endothelial progenitor cells in patients with keloid.

Evidence has suggested that vascular endothelial growth factor (VEGF), a crucial growth factor in regulating endothelial progenitor cells (EPCs), plays a central role in keloid formation. However, the levels of circulating EPCs in patients with keloid have not yet been explored. The aim of this study was to determine the number of circulating EPCs in patients with keloid. Circulating EPCs (defined as CD45- CD34+CD133+VEGFR2+cells) and VEGF levels from 39 patients with keloid and 22 healthy controls (HCs) were assessed by flow cytometry and ELISA, respectively. EPCs were detectable in the peripheral blood of patients with keloid. The number of circulating EPCs and the levels of plasma VEGF were significantly higher in patients with keloid than in HCs. However, no correlation was found between the number of circulating EPCs and the serum VEGF levels. This study provides the first evidence that EPCs are increased in the peripheral blood of patients with keloid. Understanding the roles of EPCs in keloid fromation may lead to the development of novel therapeutic strategies for keloid.

Experimental study of survival of pedicled perforator flap with flow-through and flow-end blood supply.

BACKGROUND: Flap viability after transfer depends on blood flow from the arterial blood supply below the fascia. This study evaluated survival of a pedicle flap with a perforator lateral branch and flow-through blood supply, compared with that of a flap with a flow-end blood supply and perforator terminal branch. METHODS: Forty Sprague-Dawley rats, 20 in each group, were assigned to transfer of a superficial epigastric artery pedicle island flap with a flow-through or flow-end configuration of blood supply. Laser Doppler imaging was used to evaluate flap perfusion 2 h, 3 days and 5 days after surgery. The rats were killed on day 5, and lead oxide-gelatine-enhanced flap angiography and histology with haematoxylin and eosin staining was performed. Dorsal midline tissue was excised for quantification of vascular endothelial growth factor by western blot assay. RESULTS: On day 5 after surgery, the flow-through group exhibited a significantly greater mean(s.d.) flap survival area (97·8(3·5) versus 80·8(10·2) per cent; P = 0·003), microvascular density (303(19) versus 207(41) per mm(2) ; P < 0·001) and perfusion (8·64(0·14) versus 5·95(0·14) perfusion units; P < 0·001) than the flow-end group. The flow-through group exhibited more angiosomes connected by dilated vascular anastomoses between the skin and subcutaneous fasciae. CONCLUSION: The flow-through blood supply improved pedicle perforator flap survival. Surgical relevance Perforator flap failure is mainly the result of impaired blood supply, as a flow-end blood configuration is nourished only by the perforator terminal branch of the artery. This work showed that the flow-through blood supply nourished by the perforator lateral branch improved flap survival, with dilatation of collateral vascular anastomoses and increased neoangiogenesis. The use of a flow-through configuration improves perforator flap survival and could therefore minimize morbidity resulting from flap necrosis.

Role of the NO/cGMP pathway in postoperative vasodilation in perforator flaps.

BACKGROUND: The nitric oxide (NO)/cyclic guanylyl monophosphate (cGMP) pathway is one of the most important regulators of tissue perfusion. Here, we sought to elucidate the protective effects of the NO/cGMP pathway on the microcirculation of axial pattern skin flaps. MATERIAL AND METHODS: Overall 40 rats were divided into four groups (n = 10 each): group A, sildenafil was administered orally at 10 mg/kg daily; group B, sildenafil citrate (10 mg/kg, oral) and nitro-amino-methyl-L-arginine (L-NAME, intraperitoneal injection), a nitric oxide synthase inhibitor, were administered daily; group C, L-NAME was administered alone; and group D, no drugs were administered. After surgery, the surviving flap area was calculated as a percentage of total flap dimensions using the paper template technique. Angiography and imaging were performed to compare the macrovascular changes of the choke zones in the flaps. Histological examinations were performed to compare the differences in microvascular changes between the two choke zones. RESULTS: A significant improvement of flap survival area and a significant dilation of vessels in both choke zones were found after administration of sildenafil. We also found that the postoperative vasodilation of choke vessels could be altered by inhibition of NO synthase (NOS). Moreover, the vasodilatory effect prolonged by the phosphodiesterase 5 inhibitor sildenafil was attenuated after administration of L-NAME. L-NAME significantly reversed the protection afforded by sildenafil. CONCLUSIONS: Targeting the NO/cGMP pathway can dilate vessels along the axis of the flap, including the choke vessels, thus augmenting flap viability. Therefore, targeting of this pathway may have therapeutic applications.

[Effect of accurately localized mini anterolateral thigh perforator flap in repairing medium-sized skin and soft tissue defects in fingers].

Objective: To explore the effect of accurately localized mini anterolateral thigh perforator flap in repairing medium-sized skin and soft tissue defects in fingers. Methods: The study was a retrospective observational study. From December 2019 to September 2022, 15 patients with medium-sized skin and soft tissue defects who met the inclusion criteria in fingers were admitted to the Second Affiliated Hospital of Wenzhou Medical University, including 12 males and 3 females, aged 23 to 62 years. After debridement, the wounds were all accompanied by exposed tendons, bones, vessels and nerves, with an area from 4.0 cm×3.0 cm to 8.0 cm×3.5 cm. Computed tomography angiography and color Doppler ultrasonography examinations were performed on both lower limbs of the patient before surgery to accurately locate the anterolateral thigh perforators. When the flap with area from 6.0 cm×3.0 cm to 11.0 cm×4.0 cm was harvested, the flap was thinned. The artery and vein perforators of the flap were anastomosed respectively with the digital artery and dorsal metacarpal vein. If there was avulsion injury, infection, or burn in the recipient area, the main arterial and veinous vessels carried by the skin flap was anastomosed with the radial artery and accompanying vein. The lateral thigh cutaneous nerve carried by the flap was anastomosed with the stump of the digital nerve. The types of perforators of the lateral thigh artery were observed during operation and compared with the location of the vessels before operation. After operation, the survival and adverse complication of the flap were closely observed. During follow-up, the skin flap color, texture, and shape were observed; the wound healing in donor area was observed. At the last follow-up, the two-point discriminative distance of the affected finger pulp was measured, and the function of the affected finger was evaluated using the trial standard for the evaluation of functions of upper limbs of Hand Surgery Society of Chinese Medical Association, and the interphalangeal joint movement of the affected finger was observed; the patients' complaints about the adverse effects of flap resection on lower limbs were recorded. Results: During the operation, it was observed that the perforators of the flaps in 11 patients were the descending branch of the lateral circumflex thigh artery, in two patients, the perforators of skin flaps were the oblique branch of the lateral thigh artery, and the perforators in another two patients were the transverse branch of the lateral circumflex thigh artery, which were consistent with the preoperative vascular localization. After operation, all flaps survived without vascular crisis and infection. The patients were followed up for 6-12 months, the flaps had excellent color, texture, and appearance; only linear scars remained on the donor wound. At the last follow-up, the two-point discrimination distance in the finger pulp was 7-11 mm; the affected finger function was rated as excellent in 6 cases, good in 6 cases, and fair in 3 cases; the flexion and extension function of the finger was not affected; two patients complained of numbness in the lateral thigh after excision of the skin flap, and the other 13 patients had no complain of adverse complaints. Conclusions: The perforating branch in lateral thigh region can be accurately located by computed tomography angiography and color Doppler ultrasonography, accurate positioning of perforators before operation can reduce the damage to the donor area during the incision of the flap, the appearance and function of the affected finger can be restored to the maximum extent by thinning the transplanted flap and rebuilding the finger sensation. Therefore, it is an effective and reliable way to repair the medium-sized skin and soft tissue defects of fingers with the mini thigh anterolateral perforator flap.

Vitamin D: a novel therapeutic approach for keloid, an in vitro analysis.

BACKGROUND: Vitamin D and its metabolites play an important role in calcium homeostasis, bone remodelling, hormone secretion, cell proliferation and differentiation. Recent studies also suggest a beneficial role of vitamin D in slowing the progression of tissue fibrosis. However, their effects on dermal fibrosis and keloids are unknown. Objectives To investigate the effect of 1,25-dihydroxyvitamin D3 (1,25D) in the pathogenesis of tissue fibrosis by keloid fibroblasts (KFs). METHODS: KFs were cultured and exposed to different concentrations of 1,25D in the presence or absence of transforming growth factor (TGF)-ß1. KF phenotypes and protein production were analysed by real-time reverse transcriptase-polymerase chain reaction, Western blot, immunofluorescence and multiplex enzyme-linked immunosorbent assay techniques. Collagen synthesis was evaluated by measuring (3) H-proline incorporation. The effect of 1,25D on cell proliferation and viability was evaluated by Formazan assay, proliferating cell nuclear antigen expression and the colorimetric conversion of 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide. RESULTS: We confirmed the presence of vitamin D receptors (VDRs) in cultured keloid fibroblasts. Fibroblasts transfected with a vitamin D response element reporter construct and exposed to the active vitamin D metabolite 1,25D showed increased promoter activity indicating VDR functionality in these cells. Incubation of KFs with 1,25D suppressed TGF-ß1-induced collagen type I, fibronectin and α-smooth muscle actin expression. 1,25D also modulated plasminogen activator inhibitor-1 and matrix metalloproteinase-9 expression induced by TGF-ß1. Interestingly, 1,25D induced hepatocyte growth factor mRNA expression and protein secretion in keloid fibroblasts. CONCLUSIONS: This study highlights key mechanistic pathways through which vitamin D decreases fibrosis, and provides a rationale for studies to test vitamin D supplementation as a preventive and/or early treatment strategy for keloid and related fibrotic disorders.

Role of caveolin-1 in the pathogenesis of tissue fibrosis by keloid-derived fibroblasts in vitro.

BACKGROUND: Recent studies have suggested that caveolin-1 (cav-1) plays an important role in the regulation of transforming growth factor (TGF)-ß1 signalling and participates in the pathogenesis of tissue fibrosis. However, its effects on dermal fibrosis keloids are unknown. OBJECTIVES: To investigate the effect of cav-1 in the pathogenesis of tissue fibrosis by keloid fibroblasts. METHODS: Keloid fibroblasts were cultured and exposed to different concentrations of cav-1 cell-permeable peptides (cav-1p) in the presence of TGF-ß1. Keloid fibroblast phenotypes and protein production were analysed by real-time reverse transcriptase-polymerase chain reaction, Western blot, and multiplex enzyme-linked immunosorbent assay techniques. The effect of cav-1p on cell viability was evaluated by MTT assay. RESULTS: Cav-1 was markedly decreased in the keloid-derived fibroblasts. Moreover, cav-1p significantly reduced TGF-ß receptor type I levels and Smad2/3 phosphorylation in response to added TGF-ß1. Additionally, TGF-ß1 decreased cav-1 expression in human skin fibroblasts. Cav-1 was able to suppress TGF-ß1-induced extracellular matrix production in cultured keloid fibroblasts through regulation of the mitogen-activated protein kinase pathway. CONCLUSIONS: Cav-1 appears to participate in the pathogenesis of tissue fibrosis in keloid. Restoration of cav-1 function by treatment with a cell-permeable peptide corresponding to the cav-1 scaffolding domain may be a novel therapeutic approach in keloid.

CSN6 promotes malignant progression of oral squamous cell carcinoma by down-regulating TIMP-2.

OBJECTIVE: This study was designed to investigate the expression characteristics of CSN6 in oral squamous cell carcinoma (OSCC), and to further explore the mechanism of how it promotes the malignant progression of this cancer. PATIENTS AND METHODS: The expressions of CSN6 and TIMP-2 in tumor tissue samples and adjacent normal ones collected from 36 OSCC patients were detected via quantitative Real Time-Polymerase Chain Reaction (qRT-PCR), and the interplay between their expression levels and the clinical indicators or prognosis of OSCC patients was analyzed as well. Meanwhile, the expressions of CSN6 and TIMP-2 in OSCC cell lines were further verified via qRT-PCR. In addition, CSN6 overexpression and knockdown models were constructed using lentivirus in OSCC cell lines, CAL-27, and Tca8113. At the same time, transwell and cell wound healing assays were conducted to uncover the impact of CSN6 on the function of OSCC cells. Finally, the potential mechanism was explored using Luciferase reporter gene and recovery experiments. RESULTS: In this work, qRT-PCR results revealed that the level of CSN6 in tumor tissues of OSCC patients was remarkably higher than that in adjacent normal ones. Compared with patients with low expression of CSN6, those with high expression CSN6 had a higher incidence of lymph node or distant metastasis and a lower overall survival rate. In vitro experiments revealed that silencing CSN6 remarkably attenuated the invasive, as well as migration capacities of OSCC cells while overexpression of CSN6 conversely enhanced those. Subsequently, in OSCC cell lines and tissues, TIMP-2 expression was remarkably reduced, which was negatively correlated with CSN6 level. Bioinformatics and Luciferase reporter genes demonstrated that CSN6 can target the corresponding sites of TIMP-2 promoter. In addition, cell recovery experiments suggested the existence of a mutual regulation between CSN6 and TIMP-2, which may synergistically modulate the malignant progression of OSCC. CONCLUSIONS: The above results indicated that CSN6 was remarkably upregulated both in OSCC tissues and cell lines, which is remarkably relevant to the incidence of lymph node or distant metastasis and poor prognosis of OSCC patients. Additionally, we verified that CSN6 may promote OSCC malignant progression by regulating TIMP-2.

LncRNA LncOGD-1006 alleviates OGD-induced ischemic brain injury regulating apoptosis through miR-184-5p/CAAP1 axis.

OBJECTIVE: This study aimed to explore the effect of long non-coding RNA (LncRNA) LncOGD-1006 to ischemic stroke and the possible mechanism. MATERIALS AND METHODS: The primary brain microvascular endothelial cells (bEnd.3) of oxygen-glucose deprivation (OGD) was used as a mimic of ischemic stroke in vitro. RESULTS: The results showed that LncOGD-1006 was upregulated in bEnd.3 after OGD-induced. CONCLUSIONS: LncOGD-1006 might act as a ceRNA to inhibit apoptosis in bEnd.3 cells by targeting miR-184-5p/CAAP1 pathway.

Troglitazone suppresses transforming growth factor-beta1-induced collagen type I expression in keloid fibroblasts.

BACKGROUND: Peroxisome proliferator-activated receptor (PPAR)-gamma agonists are increasingly used in patients with diabetes and some studies have suggested a beneficial effect on organ fibrosis. However their effects on dermal fibrosis in keloids are unknown. OBJECTIVE: To investigate the effect of the PPAR-gamma agonist troglitazone on transforming growth factor (TGF)-beta1-induced collagen type I expression in keloid fibroblasts. METHODS: Keloid fibroblasts were cultured and exposed to different concentrations of troglitazone in the presence of TGF-beta1. The mRNA expression of PPAR-gamma was determined by semiquantitative reverse transcriptase-polymerase chain reaction. The protein of PPAR-gamma, Smad2, Smad3, phoshpo-Smad2/3 and collagen type I was determined by Western blotting and collagen synthesis was evaluated by measuring (3)H-proline incorporation. The effect of troglitazone on cell viability was evaluated by the colorimetric conversion of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide. RESULTS: PPAR-gamma was expressed at a moderate level in keloid fibroblasts. Troglitazone depressed TGF-beta1-stimulated collagen type I expression and collagen synthesis in keloid fibroblasts in a concentration-dependent manner. Moreover, troglitazone inhibited expression and phosphorylation of TGF-beta1-induced Smad2/3. Cell viability was unaffected. These inhibitory effects of troglitazone were reversed by the PPAR-gamma-specific antagonist GW9662. CONCLUSIONS: Our data suggest that PPAR-gamma is present in keloid fibroblasts and PPAR-gamma activation inhibits TGF-beta1-induced collagen type I expression at least in part by decreasing collagen synthesis. PPAR-gamma may be a promising therapeutic target for keloids.

Introgression of a disrupted cadherin gene enables susceptible Helicoverpa armigera to obtain resistance to Bacillus thuringiensis toxin Cry1Ac.

A disrupted allele (r1) of a cadherin gene (Ha_BtR) is genetically associated with incompletely recessive resistance to Bacillus thuringiensis toxin Cry1Ac in a Cry1Ac-selected strain (GYBT) of Helicoverpa armigera. The r1 allele of Ha_BtR was introgressed into a susceptible SCD strain by crossing the GYBT strain to the SCD strain, followed by repeated backcrossing to the SCD strain and molecular marker assisted family selection. The introgressed strain (designated as SCD-r1, carrying homozygous r1 allele) obtained 438-fold resistance to Cry1Ac, >41-fold resistance to Cry1Aa and 31-fold resistance Cry1Ab compared with the SCD strain; however, there was no significant difference in susceptibility to Cry2Aa between the integrated and parent strains. It confirms that the loss of function mutation of Ha_BtR alone can confer medium to high levels of resistance to the three Cry1A toxins in H. armigera. Reciprocal crosses between the SCD and SCD-r1 strains showed that resistance to Cry1Ac in the SCD-r1 strain was completely recessive. Life tables of the SCD and SCD-r1 strains on artificial diet in the laboratory were constructed, and results showed that the net replacement rate (R0) did not differ between the strains. The toxicity of two chemical insecticides, fenvalerate and monocrotophos, against the SCD-r1 strain was not significantly different from that to the SCD strain. However, larval development time of the SCD-r1 strain was significantly longer than that of the SCD strain, indicating a fitness cost of slower larval growth is associated with Ha_BtR disruption in H. armigera.

Influences of miR-320a on proliferation and apoptosis of fibroblast-like synoviocytes in rheumatoid arthritis through targeting MAPK-ERK1/2.

OBJECTIVE: To study the expression of micro ribonucleic acid (miR)-320a in synovial tissues of patients with rheumatoid arthritis (RA) and explore the influences of miR-320a on the proliferation and apoptosis of fibroblast-like synoviocytes (FLSs) in RA and its mechanism. PATIENTS AND METHODS: The expression level of miR-320a in synovial tissues of 40 healthy people and 32 RA patients was detected via reverse transcription-polymerase chain reaction (RT-PCR). The FLSs were isolated from RA patients, cultured in vitro and divided into Control group and miR-320a mimic group. The proliferation and apoptosis of FLSs in each group were observed. Finally, the expression level of mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK) 1/2 in each group was detected via Western blotting. RESULTS: The expression level of miR-320a in synovial tissues of RA patients was significantly lower than that in healthy people (p < 0.05). After miR-320a mimic was transfected into FLSs cultured in vitro, EdU staining and flow cytometry analysis were performed. The results revealed that the proportion of EdU-positive cells significantly declined in miR-320a mimic group, the proportion of cells in G0/G1 phase was increased, while the cells in G2/M and S phases were significantly decreased (p < 0.05). Above data indicated that the cell proliferation ability was significantly inhibited. In addition, the results of flow cytometry also showed that the apoptosis rate of FLSs in miR-320a mimic group was significantly higher than that in Control group (p < 0.05). The results of Western blotting manifested that the Bcl-2 associated X protein (Bax)/Bcl-2 ratio in miR-320a mimic group was also obviously increased (p < 0.05). According to further studies, the phosphorylation level of ERK1/2 in miR-320a mimic group was remarkably inhibited (p < 0.05). CONCLUSIONS: The expression level of miR-320a significantly declined in synovial tissues of RA patients. MiR-320a attenuated proliferation and promoted apoptosis of FLSs through inhibiting the activation of the MAPK-ERK1/2 signaling pathway.

MicroRNA-22 targets FMNL2 to inhibit melanoma progression via the regulation of the Wnt/ß-catenin signaling pathway and epithelial-mesenchymal transition.

OBJECTIVE: Melanoma is regarded as one common malignancy in skin cancers, and there is growing evidence that microRNAs (miRNAs) play a vital role in the oncogenesis of tumors. This study aimed to investigate the roles and mechanism of miR-22 in melanoma. PATIENTS AND METHODS: Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was utilized to detect the expressions of miR-22 and mRNA. The functions of miR-22 in melanoma cell proliferation, migration and invasion were investigated with functional assays, including MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and transwell assay. Western blots were utilized to examine the protein expressions. Luciferase reporter analysis was conducted to confirm the interactions between formin-like 2 (FMNL2) and miR-22 in melanoma cells. FMNL2 expression levels in melanoma tissues were investigated by immunohistochemistry (IHC) assays. RESULTS: The qRT-PCR analysis demonstrated significant decreased miR-22 expressions in melanoma tissues. Decreased miR-22 in melanoma tissues were correlated with adverse clinicopathologic features and poor prognosis. Functional assays indicated that upregulation inhibited melanoma cell proliferation, invasion and migration capacities. Luciferase reporter assays showed that FMNL2 was targeted by miR-22 in melanoma cells. Western blots indicated that miR-22 exerted anti-tumor functions by regulating the Wnt/ß-catenin and epithelial-mesenchymal transition (EMT). CONCLUSIONS: Our findings showed that miR-22 served as a tumor suppressor in melanoma progression, implying that miR-22 may function as a novel therapeutic target and prognostic biomarker for melanoma treatments.

Differential phosphorylation of azidothymidine, dideoxycytidine, and dideoxyinosine in resting and activated peripheral blood mononuclear cells.

The antiviral activity of azidothymidine (AZT), dideoxycytidine (ddC), and dideoxyinosine (ddI) against HIV-1 was comparatively evaluated in PHA-stimulated PBM. The mean drug concentration which yielded 50% p24 Gag negative cultures were substantially different: 0.06, 0.2, and 6 microM for AZT, ddC, and ddI, respectively. We found that AZT was preferentially phosphorylated to its triphosphate (TP) form in PHA-PBM rather than unstimulated, resting PBM (R-PBM), producing 10- to 17-fold higher ratios of AZTTP/dTTP in PHA-PBM than in R-PBM. The phosphorylation of ddC and ddI to their TP forms was, however, much less efficient in PHA-PBM, resulting in approximately 5-fold and approximately 15-fold lower ratios of ddCTP/dCTP and ddATP/dATP, respectively, in PHA-PBM than in R-PBM. The comparative order of PHA-induced increase in cellular enzyme activities examined was: thymidine kinase > uridine kinase > deoxycytidine kinase > adenosine kinase > 5'-nucleotidase. We conclude that AZT, ddC, and ddI exert disproportionate antiviral effects depending on the activation state of the target cells, i.e., ddI and ddC exert antiviral activity more favorably in resting cells than in activated cells, while AZT preferentially protects activated cells against HIV infection. Considering that HIV-1 proviral DNA synthesis in resting lymphocytes is reportedly initiated at levels comparable with those of activated lymphocytes, the current data should have practical relevance in the design of anti-HIV chemotherapy, particularly combination chemotherapy.

[Effects of pretreatment with dimethyloxalylglycine on the survival of multi-territory perforator flap in rat and related mechanism].

OBJECTIVE: To observe the effects of pretreatment with dimethyloxalylglycine (DMOG) on the survival of multi-territory perforator flap and the vessels of choke zone (CZ) 2 in rat, and to explore related mechanism. METHODS: Sixty adult SD rats were divided into group DMOG and normal saline group (NS) according to the random number table, with 30 rats in each group. Perforator flap with three angiosomes was made on the right dorsal side of rat, including deep iliac circumflex artery perforator, intercostal artery perforator, thoracodorsal artery perforator, as well as CZ 1 and CZ 2. Rats in group DMOG were intraperitoneally injected with 2 mL NS containing DMOG (40 mg/kg) 2 days before operation, 2 hours before operation, and 2 days after operation. Rats in group NS were intraperitoneally injected with equivalent volume of NS at the same time point. On post operation day (POD) 7, gross observation was conducted, and the survival rate of flap was calculated. On POD 7, the vascularity in CZ 2 and potential zone of flap was observed using angiography. On POD 7, new vessel in CZ 2 of flap was observed with HE staining, and the microvessel density (MVD) was calculated. On POD 7, the expression of vascular endothelial growth factor (VEGF) in CZ 2 of flap was detected by immunohistochemistry and Western blotting (respectively denoted as integral absorbance values and ratio of gray value), and blood flow volume of vessel in CZ 2 of flap was examined by laser Doppler perfusion imager. The sample number of each index was 6 in each group. Data were processed with t test. RESULTS: (1) On POD 7, rats in two groups all survived, and the flaps were not infected. In group DMOG, the necrotic area of flaps of rats with dark yellow crust and soft texture was observed approximately at the distal end of skin entry point of thoracodorsal artery perforator. In group NS, the necrotic area of flaps of rats with brownish black crust and hard texture was observed approximately at the distal end of CZ 2. The survival rate of flap of rats in group DMOG was (88±3) %, which was significantly higher than that in group NS [(82±3) %, t=3.38, P<0.01]. (2) On POD 7, there were clear vascular structure and many new vessels in CZ 2 of flaps of rats in group DMOG, with intact vascular structure in potential zone. On POD 7, there were unclear vascular structure and few new vessels in CZ 2 of flaps of rats in group NS, with disorder vascular structure in potential zone. (3) On POD 7, MVD in CZ 2 of flaps in rats of group DMOG was (29.2±2.2)/mm(2,) which was significantly higher than that of group NS [(20.3±3.6)/mm(2,) t=5.10, P<0.01]. (4) On POD 7, the expressions of VEGF in CZ 2 of flaps in rats of group DMOG detected by immunohistochemistry and Western blotting were 5 060±432 and 0.48±0.04 respectively, which were significantly higher than those of group NS (2 811±382 and 0.26±0.06, with t values respectively 9.54 and 5.67, P values below 0.01). (5) On POD 7, blood flow volume of vessel in CZ 2 of flaps in rats of group DMOG was (58±4) perfusion units (PU), which was significantly more than that of group NS [(46±4) PU, t=5.20, P<0.01]. CONCLUSIONS: DMOG can increase the survival rate of multi-territory perforator flap through promoting angiogenesis in CZ 2 of flap on the back of rat and improving blood supply of flap.

Altered activity of midbrain dopamine neurons following 7-day withdrawal from chronic cocaine abuse is normalized by D2 receptor stimulation during the early withdrawal phase.

Using in vivo single-unit recording in rats, we compared the effects of continuous cocaine infusion via minipump or single daily injections (both 40 mg/kg/d x 14 days, S.C.) on the activity of putative dopamine (DA) neurons in the substantia nigra pars compacta (SNC) and ventral tegmental area (VTA). On days 1-5 after cocaine withdrawal, animals were further treated with single daily injections of DA agonists. On withdrawal day 7 continuous cocaine caused a reduction in spontaneously active neurons in the SNC and reduced bursting in the VTA. In contrast, intermittent cocaine resulted in an increase in the number of active neurons in the VTA. These changes were all reversed by apomorphine or quinpirole given during the first 5 withdrawal days. The D1 antagonist SCH 39166 did not antagonize the effects of apomorphine in either region. The role of D2 receptors in modulating baseline DA activity during intermediate cocaine withdrawal is discussed.

Alterations in baseline activity and quinpirole sensitivity in putative dopamine neurons in the substantia nigra and ventral tegmental area after withdrawal from cocaine pretreatment.

Using in vivo single-unit recording, we compared in rats the-effects of continuous infusion and once-a-day injections of cocaine on the activity of single putative dopamine neurons in the substantia nigra and ventral tegmental area. After a 7-day withdrawal, we determined: (1) the number of spontaneously active neurons and their bursting patterns and (2) sensitivity of these neurons to intravenous quinpirole. In the substantia nigra, continuous cocaine infusion reduced the number of neurons without affecting the bursting patterns; daily injections were without effects. In the ventral tegmental area, continuous infusion reduced the bursting activity without affecting the number of neurons, whereas injections increased number of neurons without changes in the bursting pattern. Acute sulpiride normalized all the changes in both cell body areas. The quinpirole sensitivity was selectively increased in the nigral neurons following withdrawal from continuous infusion. Possible role of D2/autoreceptor mechanisms in these changes is discussed.

Selective depletion of DNA precursors: an evolving strategy for potentiation of dideoxynucleoside activity against human immunodeficiency virus.

Human immunodeficiency virus type 1 (HIV-1) is wholly dependent on its host cell for a variety of essential metabolites. Among the latter are the deoxynucleoside-5'-triphosphates (dNTPs) required for reverse transcription of the single-stranded RNA viral genome into double-stranded viral DNA. Since viral DNA synthesis has an absolute requirement for all four dNTPs, restriction of a single one of these is sufficient to inhibit HIV-1 replication. To date, this therapeutic strategy has been most successful when depletion of the individual dNTP is coupled with exposure to its corresponding chain-terminating dideoxynucleoside (ddN). While several examples of such combined therapy have been defined and studied in vitro, that which has been investigated most extensively at both the laboratory and the clinical level is ddATP exposure combined with dATP depletion [with dATP restriction being induced by the ribonucleotide reductase inhibitor hydroxyurea (HU) and ddATP generated from its prodrug 2',3'-dideoxyinosine (ddI)]. Several long-term clinical trials of the hydroxyurea/2',3'-dideoxyinosine combination have been completed, with plasma viral RNA being reduced to undetectable levels in a substantial fraction (one-third to one-half) of the patients treated. The major advantages of this and analogous combinations discussed in this review are their low cost relative to other current multiple drug protocols and their potential for retention of activity against drug-resistant HIV mutants.