[Influence of work engagement and self-efficacy of nurses on clinical practice ability in burn intensive care unit].

Objective: To analyze the influence of work engagement and self-efficacy of nurses on clinical practice ability in burn intensive care unit (BICU), and to explore its potential pathways of action. Methods: A cross-sectional survey was conducted. From May to October 2020, a total of 30 hospitals with BICU in China were selected by stratified sampling method. Among BICU nurses who met the inclusion criteria, their clinical practice ability, work engagement, and self-efficacy were evaluated by self-evaluation scale of oriented problem-solving behavior in nursing practice (OPSN), Utrecht work engagement scale (UWES), and general self-efficacy scale (GSES), respectively. The total scale scores of each index and the average item scores were recorded. The self-designed general data questionnaire was used to investigate the nurses' gender, age, marital status, education background, working years, professional title, and the economic region of the hospital that they belonged to. The total scale scores of the above-mentioned three evaluation indexes were compared after the classification of nurses according to general data, and the data were statistically analyzed with independent sample t test or one-way analysis of variance. Pearson correlation analysis was used to analyze the correlation between the total scale scores of the three evaluation indexes. Based on the total scale scores of the above-mentioned three evaluation indexes, a structural equation model was established, the mediation analysis of the relationship among the three evaluation indexes and the pathway analysis of the structural model were conducted, and the Bootstrap method was used to verify the pathways of action. Results: A total of 401 questionnaires were distributed, and 337 valid questionnaires were returned, with a valid return rate of 84.04%. The total scale scores of clinical practice ability, work engagement, and self-efficacy of 337 nurses were 98.2±11.7, 67.7±18.6, and 26.6±5.6, respectively, and the average item scores were 3.9±0.5, 4.5±1.2, and 2.7±0.6, respectively. Among the 337 nurses, the majority were female, aged 40 or below, married, and had a bachelor's degree with work experience of ≤10 years; both nurses with professional nurse title and nurses from the Southeast region accounted for about 50%. There were statistically significant differences in the total scale score of clinical practice ability among nurses with different ages, education backgrounds, working years, and professional titles (with F values of 3.26, 4.36, 3.12, and 2.80, respectively, P<0.05). There was statistically significant difference in the total scale score of work engagement among nurses with different working years (F=4.50, P<0.05). There were statistically significant differences in the total scale score of self-efficacy among nurses with different ages, working years, and professional titles (with F values of 4.91, 4.50, and 2.91, respectively, P<0.05). The total scale score of nurses' work engagement was significantly positively correlated with the total scale score of clinical practice ability and the total scale score of self-efficacy (with r values of 0.30 and 0.51, respectively, P<0.05). The total scale score of nurses' self-efficacy was significantly positively correlated with the total scale score of clinical practice ability (r=0.37, P<0.05). The model had good adaptability, and the intermediary model was established. Nurses' work engagement had a significantly positive effect on both self-efficacy and clinical practice ability (with ß values of 0.54 and 0.16, respectively, P<0.05), and nurses' self-efficacy had a significantly positive effect on clinical practice ability (ß=0.29, P<0.05). Work engagement had a direct effect on self-efficacy and clinical practice ability, and self-efficacy had a direct effect on clinical practice ability and played a mediating role between work engagement and clinical practice ability. Bootstrap validation showed that self-efficacy played a significantly mediating role in the influence of work engagement on clinical practice ability (with effect size of 0.16, with 95% confidence interval of 0.08-0.24, P<0.05), accounting for half of the total effect of work engagement on clinical practice ability (with effect size of 0.32). Conclusions: BICU nurses have an above-average level of clinical practice ability, a medium level of self-efficacy, and a high level of work engagement. Work engagement and self-efficacy are positively correlated with clinical practice ability. Work engagement can directly affect clinical practice ability or indirectly affect clinical practice ability through the mediating role of self-efficacy.

[Clinicopathological analysis of pseudostratified ependymal tubules in ovarian mature teratoma].

Objective: To investigate the morphology and immunohistochemical (IHC) expression of pseudostratified ependymal tubules in ovarian mature teratoma (MT). Methods: Five cases of ovarian MT with pseudostratified ependymal tubules were collected from Shenzhen Hospital(Futian) of Guangzhou University of Chinese Medicine and the Eighth Affiliated Hospital of Sun Yat-sen University from March 2019 to March 2022. In addition, 15 cases of ovarian MT with monolayer ependymal epithelium from Shenzhen Hospital (Futian) of Guangzhou University of Chinese medicine and seven cases of immature teratoma (IMT) from Hainan Provincial People's Hospital from March 2019 to March 2022 were collected as control. The morphologic characteristics and immunophenotypes of pseudostratified ependymal tubules, monolayer ependymal epithelium, and primitive neural epithelial tubules were observed and compared by H&E stain and IHC expression pattern of genes related to the differentiation status of neuroepithelium, namely SALL4, Glypican3, nestin, SOX2, Foxj1, and Ki-67. Results: Mean age of the five patients of ovarian MT with pseudostratified ependymal tubules was 26 years (range from 19 to 31 years). Two tumors were located in the left ovary and three in the right. All five cases were excised, and clinical follow-up was available (mean follow-up 1.5 years; range 0.5 to 3 years). No recurrence was noted in any cases. The pseudostratified ependymal tubules of ovarian MT, which were lined with columnar or oval epithelia up to 4-6 layers, were morphologically similar to the primitive neuroepithelial tubules of IMT and different from monolayer ependymal epithelium of ovarian MT. By immunohistochemistry, SALL4 and Glypican3 were negative, Foxj1 was positive and Ki-67 index was lower in the pseudostratified ependymal tubules and the monolayer ependymal epithelium of ovarian MT. However, the primitive neuroepithelial tubules of IMT showed variably expression of SALL4 and Glypican3, were negative for Foxj1 and high Ki-67 index. All the above three groups expressed nestin and SOX2. Conclusions: The pseudostratified ependymal tubules of ovarian MT, which have morphological similarities to the primitive neuroepithelial tubules of IMT, are similar to the monolayer ependymal epithelia of the MT in immunophenotype. IHC assessment of Foxj1 and Ki-67 is helpful to differentiate the pseudostratified ependymal tubules of ovarian MT from the primitive neuroepithelial tubules of IMT.

[Indications, selection, and effect of flap application in repairing scar carcinoma in the lower leg and ankle].

Objective: To explore the indications, selection, and effect of flap application in repairing scar carcinoma in the lower leg and ankle. Methods: A retrospective cohort study was conducted. From June 2008 to December 2018, six male patients with scar carcinoma in the lower leg and ankle were treated in Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University, aged 48-64 years, with the area of lesion ranging from 3 cm×2 cm to 15 cm×6 cm. After extended resection, the defect area ranged from 8 cm×5 cm to 22 cm×9 cm, with tissue of tendon or bone exposed. Free anterolateral thigh perforator flap, latissimus dorsi myocutaneous flap, or pedicled sural neurovascular flap was selected to repair the wound according to the location of wound in the lower extremity, selection of operation position, the location of the anastomotic vessels in the recipient area, and whether there was good skin and soft tissue available in the lower leg. The size of flap was 11 cm×8 cm-26 cm×10 cm. The donor site of free flap or myocutaneous flap was closed directly by suturing in 5 cases, and the donor site of pedicled flap was repaired with full-thickness skin graft in 1 case. The blood supply and survival of flap, quality of skin graft survival, and complication were observed postoperatively. During the follow-up period, the recurrence and metastasis of scar carcinoma, and the appearance and function of donor and recipient sites were observed. Results: All the patients completed the operation successfully, all the transplanted flaps survived with good blood supply, and the skin graft in one donor site survived well. The wounds in the donor and recipient sites of all the patients healed well without infection, effusion, or dehiscence, etc. All the patients were followed up for 1-5 years. No local recurrence or distant metastasis of scar carcinoma was found. The quality of the transplanted flaps was good. The shape of the recipient area was quite good, and the function of the affected limb was fine. The appearance of the donor area was good without dysfunction. Conclusions: Flap transplantation is suitable for the patients with tendon and bone exposure after the excision of scar carcinoma in the lower leg and ankle. The flap can be selected according to the location of scar carcinoma, operation position, the location of anastomotic vessels in the recipient area, and whether there is good skin and soft tissue available in the lower leg. The free anterolateral thigh perforator flap or latissimus dorsi myocutaneous flap is an ideal choice for repair, which can be obtained in a large area, and the donor site can be directly sutured without affecting the function.

Two-Dimensional Dirac Semimetals without Inversion Symmetry.

Realizing stable two-dimensional (2D) Dirac points against spin-orbit coupling (SOC) has attracted much attention because it provides a platform to study the unique transport properties. In previous work, Young and Kane [Phys. Rev. Lett. 115, 126803 (2015)PRLTAO0031-900710.1103/PhysRevLett.115.126803 proposed stable 2D Dirac points with SOC, in which the Berry curvature and edge states vanish due to the coexistence of inversion and time-reversal symmetries. Herein, using the tight-binding model and k·p effective Hamiltonian, we present that 2D Dirac points can survive in the presence of SOC without inversion symmetry. Such 2D Dirac semimetals possess nonzero Berry curvature near the crossing nodes, and two edge states are terminated at one pair of Dirac points. In addition, according to symmetry arguments and high-throughput first-principles calculations, we identify a family of ideal 2D Dirac semimetals, which has nonzero Berry curvature in the vicinity of Dirac points and visible edge states, thus facilitating the experimental observations. Our work shows that 2D Dirac points can emerge without inversion symmetry, which not only enriches the classification of 2D topological semimetals but also provides a promising avenue to observe exotic transport phenomena beyond graphene, e.g., nonlinear Hall effect.

Mercury oxidation coupled to autotrophic denitrifying branched sulfur oxidation and sulfur disproportionation for simultaneous removal of Hg0 and NO.

Coupling elemental mercury (Hg0) oxidation, autotrophic denitrifying sulfur oxidation, and sulfur disproportionation offers technological potential for simultaneous Hg0 and nitric oxide (NO) removal. This study shed light on simultaneous demercuration and denitration of flue gas by a sulfur-oxidizing membrane biofilm reactor (MBfR). Removal efficiency of Hg0 and NO attained 92% and 83%, respectively in long-term operation. Taxonomic and metagenomic study revealed that a tremendous variety of Hg0-oxidizing bacteria (MOB) (Thiobacillus, Truepera, etc.), denitrifying/sulfur-oxidizing bacteria (DSOB) (Thioalkalivibrio, Thauera, etc.), sulfur-disproportionating bacteria (SDB) (Desulfobulbus, Desulfomicrobium, etc.), and multi-functional bacteria (Halothiobacillus, Thiobacillus, etc.) significantly increased in abundance during growth under feeding of Hg0 and NO in simulated flue gas. The comprehensive employment of sequential chemical extraction processes, inductive coupled mass spectrometry, X-ray diffraction, X-ray photoelectron spectroscopy, and scanning electron microscopy coupled to energy disperse spectroscopy confirmed that Hg0 was finally biologically oxidized to crystallized metacinnabar (ß-HgS) extracellular micromolecular particles. Our findings provided mechanistic insights that MOB, DSOB, and multi-functional bacteria synergistically bio-oxidized Hg0 as the initial electron donor to Hg2+ and denitrified NO as the terminal electron acceptor to N2. SDB disproportionated S0 branched from S2O32- into S2- and SO42-, and ß-HgS formation from Hg2+ and disproportionation-derived S2-, thermodynamically favored Hg0 bio-oxidation. This novel biotechnique can be a cost-effective and environmentally friendly alternative to flue gas Hg0 and NO treatment. KEY POINTS: • Combination of Hg0 bio-oxidation and autotrophic denitrifying sulfur oxidation achieved simultaneous Hg0 and NO removal. • Thiosulfate disproportionation reinforced Hg0 bio-oxidation for Hg0 removal. • Mercury-oxidizing bacteria, denitrifying/sulfur-oxidizing bacteria, and sulfur-disproportionating bacteria synergistically accomplished Hg0 and NO removal.

Bioconversion of Hg0 into HA-Hg for simultaneous removal of Hg0 and NO in a denitrifying membrane biofilm reactor.

Bacterial mercury oxidation coupled to denitrification offers great potential for simultaneous removal of elemental mercury (Hg0) and nitric oxide (NO) in a denitrifying membrane biofilm reactor (MBfR). Four potentially contributory mechanisms tested separately, namely, membrane gas separation, medium absorption, biosorption and biotransformation, which contributed 4.9%/7.2%, 8.1%/8.9%, 38.8%/9.5% and 48.2%/84.9% of overall Hg0/NO removal in MBfR. Herein, Hg0 bio-oxidation, oxidative Hg0 biosorption and denitrification played leading roles in simultaneous removal of Hg0 and NO. Living microbes performed simultaneous Hg0 bio-oxidation and denitrification, in which Hg0 as electron donor was biologically oxidized to oxidized mercury (Hg2+), while NO as the terminal electron acceptor was denitrified to N2. The Hg2+ further complexed with humic acids in extracellular polymeric substances via functional groups (-SH, -OH, -NH- and -COO-) and formed humic acids bound mercury (HA-Hg). Non-living microbial matrix performed oxidative Hg0 biosorption, in which Hg0 may be physically adsorbed by cellular matrix, then non-metabolically oxidized to Hg2+ via oxidative complexation with -SH in humic acids and finally cleavage of S-H bond and surface charge transfer led to formation of HA-Hg. Therefore, bioconversion of Hg0 to HA-Hg by Hg0 bio-oxidation and oxidative Hg0 biosorption coupled with NO denitrification to N2 dynamically cooperated to accomplish simultaneous removal of Hg0 and NO in MBfR.

Photocatalytic membrane combined with biodegradation for toluene oxidation.

Photocatalytic membrane coupled to biodegradation offers potential for degrading volatile organic compounds (VOCs) in photocatalytic membrane biofilm reactor. An intimately coupled photocatalysis and biodegradation reactor was operated in continuous operation for 500 days to treat simulated waste gas containing toluene. Toluene removal efficiency obtained 99%, with the elimination capacity of 550 g m-3·h-1. Membrane photocatalysis coupled to biodegradation was created to improve toluene removal from 11 to 20%. The dominant genera were Lysinibacillus, Hydrogenophaga, Pseudomonas at 30 d, Rudaea, Dongia, Litorilinea at 230 d xyl, Tod, Tcb, Bed, Tmo, Tbu, Tou, Dmp, Cat were functional genes of toluene metabolism, as shown by16S rDNA and metagenomic sequencing. Photocatalysis destroyed part of the toluene into biodegradable intermediates that were immediately mineralized by microorganisms in biofilm, some toluene was directly degraded by toluene degrading bacterial community into carbon dioxide and water. The novel hybrid photocatalytic membrane biofilm reactor is a cost-effective and robust alternative to VOCs treatment.

Simultaneous mercury oxidation and NO reduction in a membrane biofilm reactor.

This work demonstrates bacterial oxidation of mercury (Hg0) coupled to nitric oxide (NO) reduction in a denitrifying membrane biofilm reactor (MBfR). In 93 days' operation, Hg0 and NO removal efficiency attained 90.7% and 74.1%, respectively. Thauera, Pseudomonas, Paracoccus and Pannonibacter played dual roles as Hg0 oxidizers and denitrifiers simultaneously. Denitrifying bacteria and the potential mercury resistant bacteria dominated the bacterial community. Denitrification-related genes (norB, norC, norD, norE, norQ and norV) and enzymatic Hg0 oxidation-related genes (katG, katE) were responsible for bacterial oxidation of Hg0 and NO reduction, as shown by metagenomic sequencing. XPS, HPLC-ICP-MS and SEM-EDS indicated the formation of a stable mercuric species (Hg2+) reasulting from Hg0 oxidation in the biofilm. Bacterial oxidation of Hg0 was coupled to NO reduction in which Hg0 served as the initial electron donor while NO served as the terminal electron acceptor and thereby redox between Hg0 and NO was formed. MBfR was capable of both Hg0 bio-oxidation and denitrifying NO reduction. This research opens up new possibilities for application of MBfR to simultaneous flue gas demercuration and denitration.

[Spectomycin B1 induces VEGFR2 de-SUMO modification to inhibit angiogenesis in nasopharyngeal carcinoma].

Objective:To explore the new mechanism of spectomycin B1 in inhibiting angiogenesis of nasopharyngeal carcinoma and to provide a theoretical basis for targeted gene therapy of nasopharyngeal carcinoma. Method:Human nasopharyngeal carcinoma CNE1 cells were divided into two groups, the control group and spectomycin B1 group. Western blot was used to detect the expression levels of small ubiquitin-related modified protein（SUMO） 1 and vascular endothelial growth factor receptor 2（VEGFR2）. The angiogenesis assay was used to detect the angiogenic ability of CNE1 cells, and the apoptosis was detected by flow cytometry. The model of nasopharyngeal carcinoma-bearing mice was established, spectomycin B1 was administered, tumor volume and weight were measured, and protein expression of CD31 was detected by immunohistochemistry and microvessel density was compared. Result:Spectomycin B1 could reduce deSUMOylation of VEGFR2 protein by 4.05 times, significantly reduce the angiogenic ability of CNE1 cells, and increase the apoptosis rate by 20.68%. In the tumor-bearing mouse model, spectomycin B1 treatment could inhibit subcutaneous tumor growth rate and weight, and the blood vessel density decreased by 40.04%. Conclusion:Spectomycin B1 can inhibit neovascularization of nasopharyngeal carcinoma by inducing deSUMOylation of VEGFR2 protein.

[Application of Chinese version of ACE-â ¢ in type 2 diabetes mellitus patients with mild cognitive impairment].

Objective: To investigate the application and best cut-off value of Chinese version of Addenbrooke's cognitive examination-Ⅲ(ACE-Ⅲ) in type 2 diabetes mellitus (T2DM) patients with mild cognitive impairment. Methods: A total of 18 T2DM patients with normal cognitive function (NCI group) and 40 T2DM patients with mild cognitive impairment (MCI group) treated in outpatient clinic or ward of Department of Neurology and Endocrinology in Fujian Medical University Union Hospital between January 2015 and February 2016 were enrolled. Mini Mental State Scale (MMSE), Montreal cognitive assessment scale (MoCA), Activity of Daily Living Scale (ADL) and the Chinese version of ACE-Ⅲ were used to assess cognitive function of subjects and to assess the value of ACE-Ⅲ in the diagnosis of T2DM patients with mild cognitive impairment. Results: The Cronbach's alpha of the Chinese version of ACE-Ⅲ is 0.768. ACE-Ⅲ and MoCA were correlative (r=0.768, P<0.001). The area under the receiver operating characteristic (ROC) curve for ACE-Ⅲ was 0.906 (95%CI: 0.827-0.985). When the cut-off value for diagnosis was 87.5, the maximum Youden index was 0.769, with a sensitivity of 0.825 and a specificity of 0.944. Patients in MCI group got a lower score in the sub-items of attention/orientation, memory, verbal fluency, language and visual space of ACE-Ⅲ compared to those in NCI group, and the differences were statistically significant (t=5.336, P<0.001; t=5.530, P<0.001; t=4.556, P<0.001; t=5.301, P<0.001; t=2.821, P=0.008). Conclusion: The Chinese version of ACE-Ⅲ had good internal consistency reliability, and it could effectively detect impairment of general cognitive function and single cognitive domains in T2DM patients.

Nanoemulsion formulation of florfenicol improves bioavailability in pigs.

Nanotechnology applications in medicine have seen a tremendous growth in the past decade and are being employed to enhance the stability and bioavailability of lipophilic substances, such as florfenicol. This study aimed to examine the pharmacokinetic properties of the formulated oil-in-water florfenicol-loaded nanoemulsion (FF-NE). FF-NE and florfenicol control (Nuflor) were administered to the pigs at a dose of 20 mg/kg. Nanoemulsion formulation of florfenicol was highly influenced in vivo plasma profile. The in vivo absorption study in pigs indicated that Cmax (14.54 µg/mL) was significantly higher in FF-NE, 3.42 times higher than the marketed formulation. In comparison with the control group, the relative bioavailability of formulated nanoemulsion was up to 134.5%. Assessment of bioequivalence using log-transformed data showed that the 90% confidence intervals (90% CI) of Cmax and AUC0-∞ were 2.48-4.60 and 1.21-1.72, respectively.

Relationship between SPOP mutation and breast cancer in Chinese population.

SPOP protein has been found to have ubiquitin ligase activity. Mutations in SPOP gene have been recently reported in some cancers such as prostate, gastric, colorectal cancer. We investigated SPOP DNA mutation in tumor tissues collected from 70 Chinese female breast cancer patients in Southwestern China by DNA sequencing. The results did not show mutation in our tissue samples, indicating that a mutation in the SPOP gene may not be associated with breast cancer, particularly in Chinese women. This DNA mutation analysis or DNA genotyping may provide useful and important information for genetic counseling and personalized medical treatment for different types of cancers.

Astrocytic Cx 43 and Cx 40 in the mouse hippocampus during and after pilocarpine-induced status epilepticus.

Astrocytes have now been well accepted to play important roles in epileptogenesis by controlling gliotransmitter release and neuronal excitability, contributing to blood-brain barrier dysfunction and involving in brain inflammation. Recent studies indicate that abnormal expression of gap junction protein connexin (Cx) may also be a contributing factor for seizure generation. To further address this issue, we investigated the progressive changes of Cx 43 and Cx 40 in the mouse hippocampus at 4 h, 1 day, 1 week and 2 months during and after pilocarpine-induced status epilepticus (PISE). The co-localization of Cx 43 and Cx 40 with glial fibrillary acidic protein (GFAP) was also examined. We observed that Cx 43 and Cx 40 protein expression remained unaltered at 4 h during and at 1 day (acute stage) after PISE. However, their expression was significantly increased in CA1 and CA3 areas and in the dentate gyrus at 1 week (latent stage) and 2 months (chronic stage) after PISE. Double immunofluorescence labeling indicated the localization of Cx 43 and Cx 40 in astrocytes. Combined with progressive neuronal loss in the mouse hippocampus, our results suggest that the increase in gap junctions in the neuronoglial syncytium of reactive astrocytes may be implicated in synchronization of hippocampal hyperactivity leading to neuronal loss and epileptogenesis.

Residue depletion of colistin in swine after intramuscular administration.

A newly formulated colistin sulphate solution was prepared in a previous study as a potential agent for intramuscular injection and its effectiveness, toxicity and pharmacokinetics were investigated. In order to provide more information to establish scientific guidance for safe use of this preparation, its residue depletion in swine tissues following intramuscular administration was investigated in this experiment. Fifty healthy cross-bred piglets (13.3 +/- 0.9 kg) were used in this study. Five animals were kept as untreated controls and the other 45 animals were intramuscularly injected with the colistin preparation at a dose of 2.5 mg/kg of body weight. From the treated piglets, 5 animals were randomly selected and sacrificed at different withdrawal times. Liver, kidney and muscle tissues were sampled to examine the colistin residue levels by microbiological assay. The results showed that the colistin residue in liver and muscle decreased quickly and could not be detected at 1 day after the final dosing. However, the residue depletion in the kidneys was much slower than that in other tissues and even a small quantity of drug could be detected at 14 days after withdrawal. Using the method recommended by the Committee for Veterinary Medical Products (CVMP), a withdrawal time of 10 days was established for the safe use of the newly formulated colistin sulphate solution.

Vasoactive intestinal peptide in rats with focal cerebral ischemia enhances angiogenesis.

We studied the effect of vasoactive intestinal peptide (VIP) on angiogenesis in the ischemic boundary area after focal cerebral ischemia. Adult male Sprague-Dawley rats underwent middle cerebral artery occlusion for 2 h. A single dose of VIP was given via i.c.v. injection at the beginning of reperfusion. Immunohistochemistry and Western blotting were performed to assay angiogenesis and brain levels of vascular endothelial growth factor (VEGF) protein, respectively. In addition, the expression of VEGF and its receptors (flt-1 and flk-1), as well as endothelial proliferation, was measured using rat brain microvascular endothelial cells. Immunohistochemical analyses revealed significant (P<0.05) increases in the numbers of bromodeoxyuridine (BrdU) positive endothelial cells and microvessels at the boundary of the ischemic lesion in rats treated with VIP compared with rats treated with saline. Western blotting analysis showed that treatment with VIP significantly (P<0.05) raised VEGF levels in the ischemic hemisphere. In addition, treatment with VIP increased flt-1 and flk-1 immunoreactivity in endothelial cells. In vitro, incubation with VIP significantly (P<0.01) increased the proliferation of endothelial cells and induced the expression of VEGF, flt-1 and flk-1 in endothelial cells. The stimulatory effect of VIP on the proliferation of endothelial cells was significantly (P<0.01) inhibited by SU5416, a selective inhibitor of VEGF receptor tyrosine kinase. Our data suggest that treatment with VIP enhances angiogenesis in the ischemic brain, and this effect may be mediated by increases in levels of VEGF and its receptors.

The quantification of ADAMTS expression in an animal model of cerebral ischemia using real-time PCR.

BACKGROUND: ADAMTS1 and ADAMTS8 are proteases involved in extracellular matrix proteolysis and antiangiogenesis, but little is known about their expression and function in cerebral ischemia. We investigated the changes in ADAMTS1 and ADAMTS8 in a rat model of permanent middle cerebral artery occlusion (pMCAO). The expressions of glyseraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin, cyclophilin, and RPL13A were examined in order to validate the appropriate housekeeping genes for a long duration after inducing cerebral ischemia. METHODS: Male Sprague-Dawley rats were subjected to pMCAO, and ischemic penumbra was collected at 2, 24 h, 3, 7, and 21 days after inducing ischemia, ADAMTS1, ADAMTS8, and the four housekeeping genes were quantified using real-time polymerase chain reaction (PCR). RESULTS: The expression of beta-actin increased up to 21 days, and that of GAPDH decreased at 24 h after pMCAO, with no statistically significant changes in RPL13A and cyclophilin being detected. ADAMTS1 mRNA increased at 2 h after pMCAO, peaked at 24 h, and remained at a high level until 21 days. The expression of ADAMTS8 mRNA decreased at 2 and 24 h after pMCAO, followed by a slight increase at 3 days, and then decreased again at 7 days. CONCLUSION: The results suggest that RPL13A and cyclophilin are two appropriate housekeeping genes for the rat pMCAO model up to 21 days. ADAMTS1 mRNA levels increased, but ADAMTS8 decreased after pMCAO. Our data provide new insight into the mechanism of brain ischemia and self-repair after injury.

Influence of guanidine on proteinase K resistance in vitro and infectivity of scrapie prion protein PrP(Sc).

As the scrapie prion protein PrP(Sc) is rich in beta-sheets it aggregates into prion rods, which show infectivity and proteinase K (PK) resistance. Consequently, dissociation of prion rods and breakdown of beta-sheets in PrP(Sc) by denaturation results in loss of both infectivity and PK-sensitivity. In this study, the effects of guanidine (Gdn), which solubilizes and denatures proteins by breaking down their higher structure, on the solubility, the PK-resistance in vitro and the infectivity of PrP(Sc) of scrapie strain 263K was examined. The infectivity was assayed by intracerebral inoculation into hamsters. Brain tissues of scrapie-infected hamsters were used for preparation of homogenates and crude extracts of PrP(Sc). A treatment of PrP(Sc) with Gdn enhanced its PK-sensitivity in a dose-dependent manner. The PK-resistance in vitro of PrP(Sc) denatured with lower concentrations of Gdn (<2.5 mol/l) could partially resume by renaturation. Gdn markedly reduced or, at higher concentrations, even destroyed the infectivity of PrP(Sc). On the other hand, the infectivity of PrP(Sc) inactivated by denaturation could be partially restored by renaturation. These results confirmed our assumption that all the alternations in the PK-resistance and the infectivity of PrP(Sc) caused by Gdn resulted from changes in its higher structure. However, it should be emphasized that a complete loss of PK-resistance of PrP(Sc) may not necessarily mean its full non-infectivity.

Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: implications for gene therapy.

The development of stem-cell gene therapy is hindered by the absence of repopulation assays for primitive human hematopoietic cells. Current methods of gene transfer rely on in vitro colony-forming cell (CFC) and long-term culture-initiating cell (LTC-IC) assays, as well as inference from other mammalian species. We have identified a novel human hematopoietic cell, the SCID-repopulating cell (SRC), a cell more primitive than most LTC-ICs and CFCs. The SRC, exclusively present in the CD4+CD8- fraction, is capable of multilineage repopulation of the bone marrow of nonobese diabetic mice with severe combined immunodeficiency disease (NOD/SCID mice). SRCs were rarely transduced with retroviruses, distinguishing them from most CFCs and LTC-ICs. This observation is consistent with the low level of gene marking seen in human gene therapy trials. An SRC assay may aid in the characterization of hematopoiesis, as well as the improvement of transduction methods.

Colocalization of retrovirus and target cells on specific fibronectin fragments increases genetic transduction of mammalian cells.

Hematopoietic cells are important targets for genetic modification with retroviral vectors. Attempts at human gene therapy of stem cells have achieved limited success partly because of low gene transfer efficiency. Chymotryptic fragments of the extracellular matrix molecule fibronectin used during infection have been shown to increase transduction of human hematopoietic progenitor cells. Here, we demonstrate that this enhanced gene transfer into mammalian target cells is due to direct binding of retroviral particles to sequences within the fibronectin molecule. Transduction of mammalian cells, including murine long-term repopulating hematopoietic cells, is greatly enhanced when cells are adherent to chimeric fragments containing these retroviral binding sequences. In addition, colocalization of retrovirus and target cells on fibronectin peptides allows targeted transduction of specific cell types by exploiting unique ligand/receptor interactions.

Interleukin-11 stimulates multilineage progenitors, but not stem cells, in murine and human long-term marrow cultures.

Interleukin-11 (IL-11) is a bone marrow microenvironment-derived growth factor with pleiotropic effects on a variety of hematopoietic cells. To more accurately assess the effects of IL-11 on stem and progenitor compartments within the hematopoietic microenvironment (HM), we added recombinant human (rh) IL-11 to human and murine long-term bone marrow cultures (LTMC) and analyzed primitive (high proliferative potential-colony forming cells [HPP-CFC], long-term culture-initiating cells [LTC-IC], and long-term reconstituting stem cells) and progenitor (day 12 colony forming unit-spleen [CFU-S12], colony forming unit-megakaryocyte [CFU-Mk] and colony forming unit-granulocyte/macrophage [CFU-GM]) compartments throughout the duration of the cultures. rhIL-11 (100 ng/mL) added twice weekly resulted in significantly increased nonadherent (NA) cellularity, CFU-GM, and CFU-Mk production in human LTMC. Addition of rhIL-11 to murine LTMC was associated with a 5- to 40-fold increase in CFU-GM and a four- to 20-fold increase in day 12 CFU-S in NA cells. However, IL-11 had no significant effect on total HPP-CFC concentration and decreased the size of the more primitive stem/progenitor compartment as evidenced by both decreased LTC-IC frequency in human LTMC and decreased frequency of long-term reconstituting stem cells in murine LTMC. These data suggest that IL-11 may increase commitment of stem cells into a multipotential progenitor compartment.