Particulate Air Pollutants, Brain Structure, and Neurocognitive Disorders in Older Women.

Introduction: An increasing number of studies have suggested that exposure to particulate matter (PM) may represent a novel - and potentially amendable - environmental determinant of brain aging. The current longitudinal environmental epidemiological study addressed some important knowledge gaps in this emerging field, which combines the study of air pollution and neuroepidemiology. The investigators hypothesized that long-term PM exposure adversely influences global brain volume and brain regions (e.g., frontal lobe or hippocampus) that are critical to memory and complex cognitive processing or that are affected by neuropathological changes in dementia. It was also hypothesized that long-term PM exposure results in neurovascular damage and may increase the risk of mild cognitive impairment (MCI) and -dementia. Methods: The investigators selected a well-characterized and geographically diverse population of older women (N = 7,479; average age = 71.0 ± 3.8 years at baseline) in the Women's Health Initiative (WHI) Memory Study (WHIMS) cohort (1996-2007), which included a subcohort (n = 1,403) enrolled in the WHIMS-Magnetic Resonance Imaging (WHIMS-MRI) study (2005-2006). Residence-specific yearly exposures to PM ≤ 2.5 µm in aerodynamic diameter (PM2.5) were estimated using a Bayesian maximum entropy spatiotemporal model of annual monitoring data (1999-2007) recorded in the U.S. Environmental Protection Agency (U.S. EPA) Air Quality System (AQS). Annual exposures (1996-2005) to diesel PM (DPM) were assigned to each residential census tract in a nationwide spatiotemporal mapping, based on a generalized additive model (GAM), to conduct census tract-specific temporal interpolation of DPM on-road estimates given by the U.S. EPA National-Scale Air Toxics Assessment Program. Multiple linear regression and multicovariate-adjusted Cox models were used to examine the associations, with statistical adjustment for multiple potential confounders. Results: The investigators found that participants had smaller brain volumes, especially in the normal-appearing white matter (WM), if they lived in locations with higher levels of cumulative exposure (1999-2006) to PM 2.5 before the brain MRI scans were performed. The associations were not explained by sociodemographic factors, socioeconomic status, lifestyle factors, or other clinical characteristics. Analyses showed that the adverse effect on brain structure in the participants was driven primarily by the smaller WM volumes associated with cumulative PM2.5 exposures, which were present in the WM divisions of the association brain area (frontal, parietal, and temporal lobes) and corpus callosum. Increased DPM exposures were associated with larger ventricular volume, suggesting an overall atrophic effect on the aging brains. The participants tended to have smaller gray matter (GM) volumes if they lived in areas with the highest (i.e., fourth quartile) estimated cumulative DPM exposure in the 10 years before the brain MRI scans, compared with women in the first to third quartiles. This observed association was present in the total brain GM and in the association brain cortices. The associations with normal-appearing WM varied by DPM exposure range. For women with estimated cumulative exposure below that of the fourth quartile, increased DPM estimates were associated with smaller WM volumes. However, for women with increased cumulative DPM exposures estimates in the fourth quartile, WM volumes were larger. This pattern of association was found consistently in the association brain area; no measurable difference was found in the volume of the corpus callosum. These observed adverse effects of cumulative exposure to PM2.5 (linking exposure with smaller WM volumes) and to DPM (linking exposure in the highest quartile with smaller GM volumes) were not significantly modified by existing cardiovascular diseases, diabetes mellitus, obesity, or measured white blood cell (WBC) count. MRI measurements of the structural brain showed no differences in small-vessel ischemic diseases (SVID) in participants with varying levels of cumulative exposure to PM2.5 (1999-2006) or DPM (1996-2005), and no associations between PM exposures and SVID volumes were noted for total brain, association brain area, GM, or WM. For neurocognitive outcomes followed until 2007, the investigators found no evidence for increased risk of MCI/dementia associated with long-term PM exposures. Although exploratory secondary analyses showed different patterns of associations linking PM exposures separately with MCI and dementia, none of the -results was statistically significant. A similar lack of associations between PM exposures and MCI/dementia was found across the subgroups, with no strong indications for effect modification by cardiovascular diseases, diabetes mellitus, obesity, or WBC count. Conclusions: The investigators concluded that their study findings support the hypothesized brain-structure neurotoxicity associated with PM exposures, a result that is in line with emerging neurotoxicological data. However, the investigators found no evidence of increased risk of MCI/dementia associated with long-term PM exposures.To better test the neurovascular effect hypothesis in PM-associated neurotoxic effects on the aging brain, the investigators recommend that future studies pay greater attention to selecting optimal populations with repeated measurements of cerebrovascular damage and address the possibility of selection biases accordingly. To further investigate the long-term consequence of brain-structure neurotoxicity on pathological brain aging, future researchers should take the pathobiologically heterogeneous neurocognitive outcomes into account and design adequately powered prospective cohort studies with improved exposure estimation and valid outcome ascertainment to assess whether PM-associated neurotoxicity increases the risks of pathological brain aging, including MCI and dementia.

Identification of Synthetic and Natural Host Defense Peptides with Leishmanicidal Activity.

Leishmaniaparasites are a major public health problem worldwide. Effective treatment of leishmaniasis is hampered by the high incidence of adverse effects to traditional drug therapy and the emergence of resistance to current therapeutics. A vaccine is currently not available. Host defense peptides have been investigated as novel therapeutic agents against a wide range of pathogens. Here we demonstrate that the antimicrobial peptide LL-37 and the three synthetic peptides E6, L-1018, and RI-1018 exhibit leishmanicidal activity against promastigotes and intramacrophage amastigotes ofLeishmania donovaniandLeishmania major We also report that theLeishmaniaprotease/virulence factor GP63 confers protection toLeishmaniafrom the cytolytic properties of alll-form peptides (E6, L-1018, and LL-37) but not thed-form peptide RI-1018. The results suggest that RI-1018, E6, and LL-37 are promising peptides to develop further into components for antileishmanial therapy.

[Hyperactivation of PI3K/AKT/mTOR signal pathway impairs TNF-α-induced autophagy in mesenchymal stem cells from patients with ankylosing spondylitis].

OBJECTIVE: To investigate the changes in autophagy of mesenchymal stem cells (MSCs) from patients with ankylosing spondylitis and explore the mechanism for decreased autophagy in ASMSCs. METHODS: MSCs collected from 14 patients with AS (ASMSCs) and from 15 healthy donors (HDMSCs) were cultured in the absence or presence of 25 ng/mL TNF-α for 6 h. Autophagy of the cells was determined by immunofluorescence staining of GFP-LC3B, and the results were confirmed by detecting the protein expressions of autophagy markers LC3 II/LC3 I and P62. The mRNA expressions of the related genes were detected using qRT-PCR, and the protein expressions of the autophagy markers and signaling pathway-related molecules were determined with Western blotting. TG100713 was used to block the PI3K/AKT/mTOR signal pathway, and its effect on autophagy of ASMSCs was evaluated. RESULTS: ASMSCs showed significantly weaker GFP-LC3B puncta staining and lower protein expression levels of LC3 II/LC3 I but higher levels of P62 protein (P < 0.05), indicating a decreased autophagy capacity as compared with HDMSCs. TNF-α-induced ASMSCs showed significantly higher protein expressions of p-PI3K/ PI3K, p-AKT/AKT and p-mTOR/mTOR than HDMSCs (P < 0.05), suggesting hyperactivation of the PI3K/AKT/mTOR signaling pathway in ASMSCs. Blocking PI3K/AKT/mTOR signaling with TG100713 eliminated the difference in TNF-α-induced autophagy between HDMSCs and ASMSCs. CONCLUSION: In patients with AS, hyperactivation of the PI3K/AKT/mTOR signaling pathway results in decreased autophagy of the MSCs and potentially contributes to chronic inflammation.

Fever as a predictor of adverse outcomes in COVID-19.

BACKGROUND/INTRODUCTION: There are little data on outcomes of COVID-19 patients with the presence of fever compared to the presence of symptoms. AIM: We examined the associations between symptomology, presence of fever and outcomes of a COVID-19 cohort. DESIGN AND METHODS: Between 23 January and 30 April 2020, 554 COVID-19 patients were admitted to a tertiary hospital in Singapore. They were allocated into four groups based on symptomology and fever-Group 1: asymptomatic and afebrile, Group 2: symptomatic but afebrile, Group 3: febrile but asymptomatic and Group 4: symptomatic and febrile. The primary outcomes were intensive care unit (ICU) admissions and mortality. The composite end-point included ICU admissions, mortality or any COVID-19 related end-organ involvement. RESULTS: There were differences in ferritin (P=0.003), C-reactive protein (CRP) levels (P<0.001) and lymphopenia (P=0.033) across all groups, with the most favourable biochemical profile in Group 1, and the least in Group 4. Symptomatic groups (Groups 2 and 4) had higher ICU admissions (1.9% and 6.0%, respectively, P=0.003) than asymptomatic groups (Groups 1 and 3). Composite end-point was highest in Group 4 (24.0%), followed by Group 3 (8.6%), Group 2 (4.8%) and Group 1 (2.4%) (P<0.001). The presence of fever (OR 4.096, 95% CI 1.737-9.656, P=0.001) was associated with the composite end-point after adjusting for age, pulse rate, comorbidities, lymphocyte, ferritin and CRP. Presence of symptoms was not associated with the composite end-point. DISCUSSION/CONCLUSION: In this COVID-19 cohort, presence of fever was a predictor of adverse outcomes. This has implications on the management of febrile but asymptomatic COVID-19 patients.

A clinical analysis of diabetic patients with hand ulcer in a diabetic foot centre.

AIMS: The aim of the study was to explore the prevalence and clinical characteristics of hand ulcer in hospitalized patients with diabetes. METHODS: We analysed 17 subjects with hand ulcer among diabetic inpatients, who were admitted to the Diabetic Foot Care Center, Department of Endocrinology and Metabolism at the West China Hospital of Sichuan University from April 2003 to December 2008. RESULTS: The prevalence of diabetic hand ulcer among hospitalized patients (0.37%) was significantly lower than that of diabetic foot ulcers (9.7%, P = 0.000). The mean age was 62.1 +/- 9.4 years. The average known durations of diabetes and glycated haemoglobin (HbA(1c)) were 5.3 +/- 4.9 years and 10.9 +/- 2.4%, respectively. All patients lived in the subtropical zone. Fifteen patients (88.2%) were diagnosed with diabetic peripheral neuropathy. Ten patients had hand infection. After therapy, the ulcers healed in 13 patients (76.5%) and none of them experienced amputation. The average hospital stay for patients with local infection was characteristically longer than that for patients without infection (P = 0.012). The prognosis of the hand ulcer was poorer in the patients who had diabetes for > 3 years compared with those who had diabetes for < 3 years (P = 0.009). CONCLUSIONS: Diabetic hand ulcer is a relatively rare complication of diabetes in South-West China. Long duration of diabetes, poorly controlled blood glucose, minor trauma and delayed treatment are the risk factors. Diabetic peripheral neuropathy may play an important role in the pathogenesis of hand ulcer. Early control of blood glucose with insulin and early anti-microbial therapy with appropriate antibiotics are crucial. Debridement and drainage are necessary for hand abscesses.

Pediatric Atypical Teratoid/Rhabdoid Tumors of the Brain: Identification of Metabolic Subgroups Using In Vivo 1H-MR Spectroscopy.

BACKGROUND AND PURPOSE: Atypical teratoid/rhabdoid tumors are rare, aggressive central nervous system tumors that are predominantly encountered in very young children. Our aim was to determine whether in vivo metabolic profiles correlate with molecular features of central nervous system pediatric atypical teratoid/rhabdoid tumors. MATERIALS AND METHODS: Twenty confirmed patients with atypical teratoid/rhabdoid tumors who underwent MR spectroscopy were included in this study. In vivo metabolite levels of atypical teratoid/rhabdoid tumors were compared with molecular subtypes assessed by achaete-scute homolog 1 expression. Additionally, brain-specific creatine kinase levels were determined in tissue samples. RESULTS: In vivo creatine concentrations were higher in tumors that demonstrated achaete-scute homolog 1 expression compared with those without achaete-scute homolog 1 expression (3.42 ± 1.1 versus 1.8 ± 0.8 IU, P < .01). Additionally, levels of myo-inositol (mI) (9.0 ± 1.5 versus 4.7 ± 3.6 IU, P < .05) were significantly different, whereas lipids approached significance (44 ± 20 versus 80 ± 30 IU, P = .07) in these 2 cohorts. Higher brain-specific creatine kinase levels were observed in the cohort with achaete-scute homolog 1 expression (P < .05). Pearson correlation analysis showed a significant positive correlation of brain-specific creatine kinase with absolute creatine (P < .05) and myo-inositol (P < .05) concentrations. CONCLUSIONS: In vivo MR spectroscopy may predict key molecular features of atypical teratoid/rhabdoid tumors at initial diagnosis, leading to timely patient risk stratification and accelerating the development of targeted therapies.

Signal Hyperintensity on Unenhanced T1-Weighted Brain and Cervical Spinal Cord MR Images after Multiple Doses of Linear Gadolinium-Based Contrast Agent.

BACKGROUND AND PURPOSE: The clinical implications of gadolinium deposition in the CNS are not fully understood, and it is still not known whether gadolinium tends to be retained more in the brain compared with the spinal cord. In this study, we assessed the effects of linear gadolinium-based contrast agents on the T1 signal intensity of 3 cerebral areas (dentate nucleus, globus pallidus, and the less studied substantia nigra) and the cervical spinal cord in a population of patients with MS. MATERIALS AND METHODS: A single-center population of 100 patients with MS was analyzed. Patients underwent 2-16 contrast-enhanced MRIs. Fifty patients received ≤5 linear gadolinium injections, and 50 patients had ≥6 injections: Fifty-two patients had both Gd-DTPA and gadobenate dimeglumine injections, and 48 patients received only gadobenate dimeglumine. A quantitative analysis of signal intensity changes was independently performed by 2 readers on the first and last MR imaging scan. The globus pallidus-to-thalamus, substantia nigra-to-midbrain, dentate nucleus-to-middle cerebellar peduncle, and the cervical spinal cord-to-pons signal intensity ratios were calculated. RESULTS: An increase of globus pallidus-to-thalamus (mean, +0.0251 ± 0.0432; P < .001), dentate nucleus-to-middle cerebellar peduncle (mean, +0.0266 ± 0.0841; P = .002), and substantia nigra-to-midbrain (mean, +0.0262 ± 0.0673; P < .001) signal intensity ratios after multiple administrations of linear gadolinium-based contrast agents was observed. These changes were significantly higher in patients who received ≥6 injections (P < .001) and positively correlated with the number of injections and the accumulated dose of contrast. No significant changes were detected in the spinal cord (mean, +0.0008 ± 0.0089; P = .400). CONCLUSIONS: Patients with MS receiving ≥6 linear gadolinium-based contrast agent injections showed a significant increase in the signal intensity of the globus pallidus, dentate nucleus, and substantia nigra; no detectable changes were observed in the cervical spinal cord.

Digital image analysis agrees with visual estimates of adult bone marrow trephine biopsy cellularity.

INTRODUCTION: Evaluation of cellularity is an essential component of bone marrow trephine biopsy examination. The standard practice is to report the results as visual estimates (VE). Digital image analysis (DIA) offers the promise of more objective measurements of cellularity. METHODS: Adult bone marrow trephine biopsy sections were assessed for cellularity by VE. Sections were scanned using an Aperio AT2 Scanscope and analyzed using a Cytonuclear (version 1.4) algorithm on halo software. Intraclass correlation (ICC) was used to assess relatedness between VE and DIA, and between MRI and DIA for a separate subset of patients. Trephine biopsy sections from a subset of patients with bone marrow biopsies uninvolved by malignancy were assessed for age-related changes. RESULTS: Interobserver VE agreement was good to excellent. The ICC value was 0.81 for VE and DIA, and 0.50 for MRI and DIA. Linearity studies showed no statistically significant trend for age-related changes in cellularity in our cohort (r = -.29, P = .06). CONCLUSIONS: Agreement was good between VE and DIA. It may be possible to use DIA or VE to measure cellularity in the appropriate clinical scenario. The limited sample size precludes similar determinations for MRI calculations. Further studies examining healthy donors are necessary before making definitive conclusions regarding age and cellularity.

Trends in the use of pulmonary artery catheterization in the aneurysmal subarachnoid hemorrhage population.

Use of the pulmonary artery catheter (PAC) has been controversial since the late 1980s. Multi-center observational and randomized controlled trials (RCTs) have concluded that PACs fail to decrease mortality. Subsequently, studies have looked for a decline in PAC use that corresponds to the literature and have indeed found that it exists. However, none to date have looked primarily at trends in the aneurysmal subarachnoid hemorrhage (aSAH) population. This study uses the Nationwide Inpatient Sample (NIS) from 2000-2010 to identify trends in PAC use among patients with aSAH. Trend analysis was assessed using a multivariable regression model with a calculation of slope of PAC frequency over time for pre-2005 and post-2005. Trends in mortality and routine discharge were also assessed for the same time period. 363,096 SAH patients were extrapolated using survey weights, of whom 6,988 had a PAC. Over time, PAC use declined, with a significant downward shift in the year 2005. Analyses also showed a decrease in mortality over the same time period. Our results show that PAC use among patients with aSAH decreased from 2000 to 2010. Similar to other studies, the decline appears to be temporally related to RCTs that showed a lack of benefit from PAC. Studies such as these have the potential to influence clinical practice through illumination of shifting opinions and approaches.

Study of immortalization and malignant transformation of human embryonic esophageal epithelial cells induced by HPV18 E6E7.

In order to study the effect of viruses and tumor promoters on the tumorigenicity of the esophagus, human embryonic esophageal epithelial cells were infected with human papilloma virus HPV18 E6E7-AAV in synergy with 12-O-tetradecanoylphorbol 13-acetate (TPA) to observe their malignant transformation. The cultured esophageal epithelial cells incubated with HPV18 E6E7-AAV were divided into two groups: the SHEEC1 group was exposed to TPA (5 ng/ml) for 4 weeks at the 5th passage of the cells; the SHEE group served as the control and was cultured in the same medium without TPA. The morphological phenotype, the DNA content during the cell cycle and the chromosomes were analyzed. The tumorigenicity was assessed by colony formation after cultivation in soft agar and transplanting the cells into nude mice. HPV18 E6E7 DNA was assayed by fluorescent in situ hybridization (FISH) and the polymerase chain reaction (PCR). The SHEE group, at its 20th passage, grew as a monolayer with the cells showing anchorage dependence and contact inhibition. The chromosome analysis showed diploidy, and soft-agar cultivation and injection into nude mice showed the cells to be non-tumorigenic. They were therefore immortalized cells. In contrast, the SHEEC1 group (TPA group) showed increased DNA synthesis and a proliferative index that was higher (45%) than that of the SHEE group (34%). The number of large colonies of dense multilayer cells (positively transformed foci) in soft agar was high in SHEEC1 group (4.0%) but low in the SHEE group (0.1%). Tumors resulting from transplantation were observed in all six nude mice injected subcutaneously with cells of the SHEEC1 group but no tumor developed in mice receiving cells of the SHEE group. In both groups of cells, HPV18 E6E7 DNA was positively detected by FISH and PCR. The malignant transformation of human embryonic epithelial cells was induced in vitro by HPV18 E6E7 in synergy with TPA. This is a good evidence for the close relationship between HPV and the etiology and pathogenicity of esophageal carcinoma. It is also a reliable model for studying the cellular and molecular mechanisms of carcinogenesis of esophageal carcinoma.

Diffusion tensor imaging correlates with the clinical assessment of disease severity in cervical spondylotic myelopathy and predicts outcome following surgery.

BACKGROUND AND PURPOSE: CSM is a common neurologic disease that results in progressive disability and eventual paralysis without appropriate treatment. Imaging plays a significant role in the evaluation of CSM and has evolved with recent technical advances. We sought to systematically explore the relationship between clinical disease severity and DTI in CSM, and to investigate the potential use of DTI in surgical decision-making models. MATERIALS AND METHODS: MR imaging studies and clinical assessments were prospectively collected on 30 patients with CSM. Spearman correlations were used to investigate associations between clinical disease severity and FA at the time of diagnosis. Clinical assessment was performed using mJOA, Nurick, Short Form-36, and NDI scores. Fifteen patients with CSM subsequently underwent decompressive surgery; Spearman correlation and logistic regression were applied to this cohort to study the relationship between baseline DTI measurements and postoperative outcome. Conventional imaging (spinal cord T2 signal intensity and degree of stenosis) was evaluated for comparison with DTI. RESULTS: At diagnosis, FA demonstrated a strong correlation with baseline mJOA (r = 0.62, P < .01) and Nurick (r = -0.46, P = .01) scores. After surgery, recovery of function demonstrated by improvement in NDI score was associated with higher FA values on preoperative DTI (r = -0.61, P = .04). Severely affected patients with CSM with disproportionately high FA tended to achieve greater mJOA scores after surgery compared with subjects with lower FA (P = .08). T2 signal intensity was associated with functional status at baseline but did not predict postoperative outcome; degree of stenosis lacked any significant correlation with clinical parameters. CONCLUSIONS: DTI may be a useful diagnostic tool for assessing disease severity in CSM. The predictive value of DTI regarding postoperative outcome may improve surgical decision-making and facilitate health care outcomes research.

[Synergistic effects of human papillomavirus and phorbol ester in cell transformation.].

BACKGROUND: To investigate the molecular mechanism of the synergistic effects of human papillomavirus (HPV) and phorbal esters (TPA) in cell transformation. METHODS: The expression of oncogenes and anti-oncogenes in 293 cell line treated with HPV and TPA was studied by Southern Blot and RNA dot blot. RESULTS: It was found that the synergistic effect induced the amplification of c-myc (4-8 times), increased expressing level of c-erbB-2 (32-64 times) and decreased expressing level of p16(1/4-1/8). CONCLUSION: The above results show that the synergistic effect has an important role in development of carcinoma.

[Immortalization of human fetal esophageal epithelial cells induced by E6 and E7 genes of human papilloma virus 18].

OBJECTIVE: For studying the relationship between HPV and esophageal carcinoma, an immortalized human fetal esophageal epithelial cell line (SHEE) was established. METHODS: The human fetal esophageal tissues were cultured and infected with HPV 18 E6E7 AAV. It was examined by light- and electron-microscope for morphological changes, by PCR and FISH for detection of HPV E6E7 and by soft agar culture and nude mice inoculatior for dectecting tumor transformation. RESULTS: The cell line has become immortal and has propagated cntinuously for more than 50 passages. After a long-term culture, the phenotype keeps the characteristics of primary epithelial cells. They showed as monolayer growth and anchorage dependent growth without forming colonies in softagar. They were nontumorigenic in nude mice. SHEE cells contained tonofilaments in its cytoplasm by electron microscopic examination and showed cytokeratin positive in immunohistochemical procedure. So it shows that the cells are squamous epithelium in origin. The cell line contained the HPV 18 E6 and E7 genes by FISH and PCR assay. CONCLUSION: Establishment of the esophageal epithelial cell line SHEE successfully immortalized with HPV 18, E6E7, supports that the HPV18 may be related to the etiology of esophageal carcinoma. It will facilitate further research on etiology and pathogenesis of esophageal carcinoma.

[A review on the application of myoblast on gene therapy and tissue engineering].

OBJECTIVE: Because of its special biological characteristics, myoblast might play a role in gene delivery and cell-to-biomaterial interactions. In this paper, the biological features of myoblast and its application on gene therapy and tissue engineering was discussed. METHODS: Documents about proliferation and differentiation of myoblast were reviewed in details. The prospects of its application on gene therapy and tissue engineering were also presented. RESULTS: Myoblast was important in muscle regeneration. The activation of myoblast to proliferate and differentiate was the very beginning of regeneration after injury. The cultured myoblast had high potential to proliferate, it was ready to fuse with each other and to form myotube (the special behavior of myoblast differentiation). Myoblast transplantation had been studied as a possible treatment for inherited myopathies, such as Duchenne muscular dystrophy. The transplanted myoblast could fuse with host myofibers, so the delivered target gene integrated into host. Several myoblast-mediated gene delivery system had been established, including the gene delivery of human factor IX (hFIX), erythropoietin (EPO) and clony stimulating factor-1 (CSF-1). Results from animal experiments demonstrated that myoblast-mediated gene delivery could be used as gene therapy for some inherited diseases. And recently, some authors have shown great interest in the interaction between myoblast and type I collagen gels. It was found that myoblast could keep on proliferating and differentiating in collagen gels and could form discoid, tubular materials. CONCLUSION: Myoblast has great importance in gene therapy and tissue engineering. It is suggested that more efforts should be made in this field.

[Biological characteristics of continuously subcultured human embryonic skeletal myoblasts].

OBJECTIVE: To investigate the biological characteristics of continuously subcultured human embryonic skeletal myoblasts, and choose the optimal seeding cells for muscle tissue engineering. METHODS: Human embryonic skeletal myoblasts were subcultured in vitro. The growth curve, rate of myotube formation(RMF) were used to evaluate the proliferative and differentiation ability of myoblasts, and to investigate the influence of fibroblasts contamination on myoblasts. RESULTS: The beginning 6 passages of myoblasts showed strong proliferative and differentiation ability. From the 8th to 20th passage, the rate of fibroblasts contamination was increased, it mainly showed the growth characteristics of fibroblasts with increased proliferation and low differentiation. After subcultured to the 20th passage, the degeneration of myoblasts was obvious. CONCLUSION: The myoblasts within 6 passages should be used as the seeding cells of muscle tissue engineering because of strong proliferative ability and high rate of myotube formation.

[The proliferation and differentiation of primary human embryonic skeletal myoblasts].

OBJECTIVE: To observe the proliferation and differentiation properties of primary human embryonic skeletal myoblasts cultured in vitro. METHODS: The skeletal muscle samples were obtained from 20 to 25-week abortion fetus, the family history of inherited myopathies of parental generation was negative. With a modified method of Blau, the muscle sample was digested with trypsin and collagenase. The isolated cell suspension was a mixture of myoblasts and fibroblasts, the latter was removed by repeated attachment to culture dishes. The morphological, immunohistochemical observation, the proliferation and differentiation of primary myoblasts were studied. RESULTS: The isolated myoblasts were spherical in cell suspension and spindle-like after attached to culture dishes. The myosin specialized immunohistochemical staining was strongly positive. A large quantity of skeletal muscle specialized creatine kinase (CK-MM) was synthesized in cultured myoblasts. Additionally, while the cell density of myoblasts increased, the monocyte myoblasts would fused to form multinucleated myotube. All those indicated that the cultured cells were myoblasts. Primary myoblasts proliferated quickly, the doubling time, measured in growth curve, was 4.8 days. CONCLUSION: A large number of myoblasts can be available with digestion and repeated attachment method. The cultured cells can be proved as myoblasts by morphological and immunohistochemical detection. The cultured myoblasts have good ability of proliferation and differentiation.

Association of human immunodeficiency virus type 1 Vif with RNA and its role in reverse transcription.

The vif gene of human immunodeficiency virus type 1 (HIV-1) is essential for viral replication, although the functional target of Vif remains elusive. HIV-1 vif mutant virions derived from nonpermissive H9 cells displayed no significant differences in the amount, ratio, or integrity of their protein composition relative to an isogenic wild-type virion. The amounts of the virion-associated viral genomic RNA and tRNA(3)(Lys) were additionally present at normal levels in vif mutant virions. We demonstrate that Vif associates with RNA in vitro as well as with viral genomic RNA in virus-infected cells. A functionally conserved lentivirus Vif motif was found in the double-stranded RNA binding domain of Xenopus laevis, Xlrbpa. The natural intravirion reverse transcriptase products were markedly reduced in vif mutant virions. Moreover, purified vif mutant genomic RNA-primer tRNA complexes displayed severe defects in the initiation of reverse transcription with recombinant reverse transcriptase. These data point to a novel role for Vif in the regulation of efficient reverse transcription through modulation of the virion nucleic acid components.

Roles of Pr55(gag) and NCp7 in tRNA(3)(Lys) genomic placement and the initiation step of reverse transcription in human immunodeficiency virus type 1.

To study in vivo tRNA(3)(Lys) genomic placement and the initiation step of reverse transcription in human immunodeficiency virus type 1, total viral RNA isolated from either wild-type or protease-negative (PR(-)) virus was used as the source of primer tRNA(3)(Lys)/genomic RNA templates in an in vitro reverse transcription assay. At low dCTP concentrations, both the rate and extent of the first nucleotide incorporated into tRNA(3)(Lys), dCTP, were lower with PR(-) than with wild-type total viral RNA. Transient in vitro exposure of either type of primer/template RNA to NCp7 increased PR(-) dCTP incorporation to wild-type levels but did not change the level of wild-type dCTP incorporation. Exposure of either primer/template to Pr55(gag) had no effect on initiation. These results indicate that while Pr55(gag) is sufficient for tRNA(3)(Lys) placement onto the genome, exposure of this complex to mature NCp7 is required for optimum tRNA(3)(Lys) placement and initiation of reverse transcription.

The role of Pr55(gag) in the annealing of tRNA3Lys to human immunodeficiency virus type 1 genomic RNA.

During human immunodeficiency virus type 1 (HIV-1) assembly, the primer tRNA for the reverse transcriptase-catalyzed synthesis of minus-strand strong-stop cDNA, tRNA3Lys, is selectively packaged into the virus and annealed onto the primer binding site on the RNA genome. Annealing of tRNA3Lys in HIV-1 is independent of polyprotein processing and is facilitated in vitro by p7 nucleocapsid (NCp7). We have previously shown that mutations in clusters of basic amino acids flanking the first Cys-His box in NC sequence inhibit annealing of tRNA3Lys in vivo by 70 to 80%. In this report, we have investigated whether these NC mutations act through Pr55(gag) or Pr160(gag-pol). In vivo placement of tRNA3Lys is measured with total viral RNA as the source of primer tRNA-template in an in vitro reverse transcription assay. Cotransfection of COS cells with a plasmid coding for either mutant Pr55(gag) or mutant Pr160(gag-pol), and with a plasmid containing HIV-1 proviral DNA, shows that only the NC mutations in Pr55(gag) inhibit tRNA3Lys placement. The NC mutations in Pr55(gag) reduce viral infectivity by 95% and are trans-dominant-negative, i.e., they inhibit genomic placement of tRNA3Lys even in the presence of wild-type Pr55(gag). This dominant phenotype may indicate that the mutant Pr55(gag) is disrupting an ordered Pr55(gag) structure responsible for the annealing of tRNA3Lys to genomic RNA.