Effect of acupuncture on atrial fibrillation stratified by CHA2DS2-VASc score-a nationwide cohort investigation.

OBJECTIVE: This research aimed to make statements regarding the reduction in atrial fibrillation (AF) risk due to acupuncture, stratified by CHA2DS2-VASc score. METHODS: The Kaplan-Meier method was performed to calculate cumulative incidence of outcomes for each group, and the log-rank test were performed to compare differences between groups. Incidences and hazard ratios (HRs) were estimated by univariate Cox proportional hazards models, and adjusted HRs (aHRs) were estimated by multivariate Cox proportional hazards models including demographic covariates and comorbid status. RESULTS: In CHA2DS2-VASc scores of 0-1, 2-3, 4-5 and >5, cases with acupuncture were all associated with decreased incidence of AF (aHR 0.46 with 95% CI 0.42-0.51, P < 0.001 in the CHA2DS2-VASc scores of 0-1; aHR 0.53 with 95% CI 0.50-0.57, P < 0.001 in the CHA2DS2-VASc scores of 2-3; aHR 0.56 with 95% CI 0.52-0.61, P < 0.001 in the CHA2DS2-VASc scores of 4-5; and aHR 0.64 with 95% CI 0.55-0.74, P < 0.001 in the CHA2DS2-VASc scores of >5). CONCLUSION: Protective effect of acupuncture on AF was observed in this study, and the effect was more obvious for those with fewer comorbidities.

Effect of air pollution on gout development: a nationwide population-based observational study.

OBJECTIVE: To investigate the effect of air pollution on gout development. METHODS: A total of 170318 participants were enrolled. These pollutants were considered: carbon monoxide (CO), fine particulate matter 2.5 (PM2.5), total hydrocarbons (THC) and methane (CH4). The yearly average concentrations were calculated from 2000 to 2011. Univariate and multivariate analyses by Cox proportional hazard regression models were adopted to estimate hazard ratios for gout in the Q2-Q4 concentrations of air pollutants compared with the Q1 concentration. RESULTS: In THC, relative to the Q1 concentration, the risk of gout was higher in participants exposed to the Q2-Q4 concentrations [adjusted hazard ratio (aHR), 1.10 with 95% confidence interval (CI), 1.01-1.19 in the Q2 concentration of THC; aHR, 4.20 with 95% CI, 3.93-4.49 in the Q3 concentration of THC; aHR, 5.65 with 95% CI, 5.29-6.04 in the Q4 concentration of THC]. In regard to CH4, when the Q1 concentration was defined as the reference, the risks of gout were increased for participants exposed to the Q2, Q3 and Q4 concentrations (aHR, 1.16 with 95% CI, 1.06-1.26 in the Q2 concentration of CH4; aHR, 2.37 with 95% CI, 2.20-2.55 in the Q3 concentration of CH4; aHR, 8.73 with 95% CI, 8.16-9.34 in the Q4 concentration of CH4). CONCLUSIONS: Association between air pollution and risk of gout was noted.

[Epidemic characteristics and dynamic changes of spatio-temporal distribution of hemorrhagic fever with renal syndrome in Guangzhou, 2010-2019].

Objective: To analyze the epidemic characteristics and spatio-temporal distribution of hemorrhagic fever with renal syndrome (HFRS) in Guangzhou from 2010 to 2019 and provide a basis for prevention and control strategies. Methods: The data of HFRS was from National Disease Reporting Information System and the epidemic investigation. A descriptive analysis was used. OpenGeoDa 1.2.0 software was used for global spatial autocorrelation and local spatial autocorrelation analysis. SatScan 9.6 software was used for detecting the hot spot area in time and space. ArcGIS 10.2 software was used for map visualization. Results: 1 298 cases of HFRS were reported, and three patients died in Guangzhou in 2010-2019. The annual incidence rate was 0.99/100 000. The proportion of 21-50 years old cases accounted for 70.88% and the male to female ratio was 2.98∶1. Most patients were house workers or unemployed, accounting for 31.28%, followed by business servants (accounting for 17.33%). The incidence peak in spring and winter accounted for 33.74% and 26.35% of the year. All districts reported cases in recent ten years. A total of 407 cases had been reported in Haizhu district, accounting for 31.36% of the total number of cases in the whole city. The annual incidence rate was 2.52/100 000. The number of reported cases and the annual incidence rate were the highest in Guangzhou. The clustered area showed that there was spatio-temporal clustering in Guangzhou. The aggregation area was mainly concentrated in the urban villages adjacent to Wan-mu orchard and the Haizhu Lake Wetland Park in Haizhu district (logarithmic likelihood ratio was 44.08, P<0.001). Conclusions: The prevalence and concentration of HFRS in winter and spring Guangzhou city from 2010 to 2019, showed a high incidence. Young and middle-aged men engaged in domestic and unemployed, and commercial services appeared the main risk groups. The urban-rural junction with many immigrants and low health environment, streets adjacent to Wan-mu orchard, and the Haizhu Lake Wetland Park in Haizhu district were the important regions for preventing and controlling HFRS. The government should formulate prevention and control measures to curb the rise and spread of the HFRS epidemic.

Maximizing the security of chaotic optical communications.

The practical application of chaotic optical communications has been limited by two aspects: the difficulty in concealing the time delay - a critical security parameter in feedback chaotic systems, and the difficulty of significantly enlarging the key space without complicating the implementation. Here we propose an architecture to break the above limits. By introducing a frequency-dependent group delay module with frequency tuning resolution of 1 MHz into the chaotic feedback loop, we demonstrate excellent time delay concealment effect, and an additional huge key space of 1048 can be achieved at the same time. The effectiveness is proved by both numerical simulation and experiment. Besides, the proposed scheme is compatible with the existing commercial optical communication systems, thus pave the way for high-speed secure optical communications.

Comparing maternal and perinatal outcomes in pregnancies complicated by preeclampsia superimposed chronic hypertension and preeclampsia alone.

AIM: The study was to determine whether preeclampsia with superimposed chronic hypertension results in worse maternal and perinatal outcomes than preeclampsia alone. MATERIALS AND METHODS: A retrospective study involving 850 pregnant women was conducted and divided into two groups: preeclampsia superimposed on chronic hypertension (group A, n = 84) and preeclampsia alone (group B, n = 766). The maternal and fetal outcomes of all subjects were collected and analyzed. RESULTS: There were no significant differences between the two groups in baseline information. However, the systolic and diastolic blood pressures in group A were significantly higher than those in group B (p < 0.05). The average interval between the onset of preeclampsia and the termination of pregnancy was significantly longer in group A as compared to group B. The incidence of serious maternal complications showed no differences between the two groups (p > 0.05). It showed a higher rate of neonatal respiratory distress syndrome and intracranial hemorrhage in group A than in group B (p < 0.05). CONCLUSIONS: Women in group A had higher risks of maternal and perinatal outcomes as compared to women in group B.

Increased titer and reduced lactate accumulation in recombinant retrovirus production through the down-regulation of HIF1 and PDK.

Many mammalian cell lines used in the manufacturing of biopharmaceuticals exhibit high glycolytic flux predominantly channeled to the production of lactate. The accumulation of lactate in culture reduces cell viability and may also decrease product quality. In this work, we engineered a HEK 293 derived cell line producing a recombinant gene therapy retroviral vector, by down-regulating hypoxia inducible factor 1 (HIF1) and pyruvate dehydrogenase kinase (PDK). Specific productivity of infectious viral titers could be increased more than 20-fold for single gene knock-down (HIF1 or PDK) and more than 30-fold under combined down-regulation. Lactate production was reduced up to 4-fold. However, the reduction in lactate production, alone, was not sufficient to enhance the titer: high-titer clones also showed significant enrollment of metabolic routes not related to lactate production. Transcriptome analysis indicated activation of biological amines metabolism, detoxification routes, including glutathione metabolism, pentose phosphate pathway, glycogen biosynthesis and amino acid catabolism. The latter were validated by enzyme activity assays and metabolite profiling, respectively. High-titer clones also presented substantially increased transcript levels of the viral genes expression cassettes. The results herein presented demonstrate the impact of HIF1 and PDK down-regulation on the production performance of a mammalian cell line, reporting one of the highest fold-increase in specific productivity of infectious virus titers achieved by metabolic engineering. They additionally highlight the contribution of secondary pathways, beyond those related to lactate production, that can be also explored to pursue improved metabolic status favoring a high-producing phenotype.

Metabolic pathways recruited in the production of a recombinant enveloped virus: mining targets for process and cell engineering.

Biopharmaceuticals derived from enveloped virus comprise an expanding market of vaccines, oncolytic vectors and gene therapy products. Thus, increased attention is given to the development of robust high-titer cell hosts for their manufacture. However, the knowledge on the physiological constraints modulating virus production is still scarce and the use of integrated strategies to improve hosts productivity and upstream bioprocess an under-explored territory. In this work, we conducted a functional genomics study, including the transcriptional profiling and central carbon metabolism analysis, following the metabolic changes in the transition 'parental-to-producer' of two human cell lines producing recombinant retrovirus. Results were gathered into three comprehensive metabolic maps, providing a broad and integrated overview of gene expression changes for both cell lines. Eight pathways were identified to be recruited in the virus production state: amino acid catabolism, carbohydrate catabolism and integration of the energy metabolism, nucleotide metabolism, glutathione metabolism, pentose phosphate pathway, polyamines biosynthesis and lipid metabolism. Their ability to modulate viral titers was experimentally challenged, leading to improved specific productivities of recombinant retrovirus up to 6-fold. Within recruited pathways in the virus production state, we sought for metabolic engineering gene targets in the low producing phenotypes. A mining strategy was used alternative to the traditional approach 'high vs. low producer' clonal comparison. Instead, 'high vs. low producer' from different genetic backgrounds (i.e. cell origins) were compared. Several genes were identified as limiting in the low-production phenotype, including two enzymes from cholesterol biosynthesis, two enzymes from glutathione biosynthesis and the regulatory machinery of polyamines biosynthesis. This is thus a frontier work, bridging fundamentals to technological research and contributing to enlarge our understanding of enveloped virus production dynamics in mammalian cell hosts.

Three-dimensional multipotent progenitor cell aggregates for expansion, osteogenic differentiation and 'in vivo' tracing with AAV vector serotype 6.

Multipotent adult progenitor cells (MAPCs) are bone marrow-derived stem cells with a high growth rate suitable for therapeutical applications as three-dimensional (3D) aggregates. Combined applications of osteogenically differentiated MAPC (OD-MAPC) aggregates and adeno-associated viral vectors (AAV) in bone bioengineering are still deferred until information with regard to expansion technologies, osteogenic potential, and AAV cytotoxicity and transduction efficiency is better understood. In this study, we tested whether self-complementary AAV (scAAV) can potentially be used as a gene delivery system in an OD-MAPC-based 'in vivo' bone formation model in the craniofacial region. Both expansion of rat MAPC (rMAPC) and osteogenic differentiation with dexamethasone were also tested in 3D aggregate culture systems 'in vitro' and 'vivo'. rMAPCs grew as undifferentiated aggregates for 4 days, with a population doubling time of 37 h. After expansion, constant levels of Oct4 transcripts, and Oct4 and CD31 surface markers were observed, which constitute a hallmark of undifferentiated stage of rMAPCs. Dexamethasone effectively mediated rMAPC osteogenic differentiation by inducing the formation of a mineralized collagen type I network, and facilitated the activation of the wnt/ß-catenin, a crucial pathway in skeletal development. To investigate the genetic modification of rMAPCs grown as 3D aggregates before implantation, scAAV serotypes 2, 3 and 6 were evaluated. scAAV6 packaged with the enhanced green fluorescent protein expression cassette efficiently mediated long-term transduction (10 days) 'in vitro' and 'vivo'. The reporter transduction event allowed the tracing of OD-rMAPC (induced by dexamethasone) aggregates following OD-rMAPC transfer into a macro-porous hydroxyapatite scaffold implanted in a rat calvaria model. Furthermore, the scAAV6-transduced OD-rMAPCs generated a bone-like matrix with a collagenous matrix rich in bone-specific proteins (osteocalcin and osteopontin) in the scaffold macro-pores 10 days post-implantation. Newly formed bone was also observed in the interface between native bone and scaffold. The collective work supports future bone tissue engineering applications of 3D MAPC cultures for expansion, bone formation and the ability to alter genetically these cells using scAAV vectors.

Scalable expansion of multipotent adult progenitor cells as three-dimensional cell aggregates.

Many applications of stem cell technologies require a large quantity of cells for which scalable processes of cell expansion and differentiation are essential. Multipotent adult progenitor cells (MAPCs) are adult stem cells isolated from the bone marrow with extensive self-renewal and broad differentiation capabilities. MAPCs are typically cultured surface adherent (2D) and at low cell density, making the large surface required for cell expansion a hindrance for many applications. This study demonstrates that MAPCs can be cultivated as aggregates in an undifferentiated state for at least 16 days, as levels of a number of transcripts, including Oct4, remained similar, Oct4 protein was unchanged, and differentiation to neural progenitor, endothelial cell and hepatocyte like cells was retained. Cultivation of these aggregates in stirred bioreactor lead to a 70-fold expansion in 6 days with final cell densities of close to 106/mL. Importantly, the MAPC aggregates recovered from stirred bioreactors could be differentiated to hepatocyte-like cells that expressed albumin, alpha-1-antitrypsin (AAT), and tyrosine amino transferase (TAT) transcripts and also secreted albumin and urea. This method of scalable expansion combined with differentiation of MAPCs can potentially be used for generating large numbers of MAPC and MAPC-derived differentiated cells.

Expansion and hepatic differentiation of rat multipotent adult progenitor cells in microcarrier suspension culture.

Many potential applications of stem cells require large quantities of cells, especially those involving large organs such as the liver. For such applications, a scalable reactor system is desirable to ensure a reliable supply of sufficient quantities of differentiation competent or differentiated cells. We employed a microcarrier culture system for the expansion of undifferentiated rat multipotent adult progenitor cells (rMAPC) as well as for directed differentiation of these cells to hepatocyte-like cells. During the 4-day expansion culture, cell concentration increased by 85-fold while expression level of pluripotency markers were maintained, as well as the MAPC differentiation potential. Directed differentiation into hepatocyte-like cells on the microcarriers themselves gave comparable results as observed with cells cultured in static cultures. The cells expressed several mature hepatocyte-lineage genes and asialoglycoprotein receptor-1 (ASGPR-1) surface protein, and secreted albumin and urea. Microcarrier culture thus offers the potential of large-scale expansion and differentiation of stem cells in a more controlled bioreactor environment.

Molecular orientation of evaporated pentacene films on gold: alignment effect of self-assembled monolayer.

Pentacene films deposited on self-assembled monolayers (SAMs) bearing different terminal functional groups have been studied by reflection-absorption IR, grazing angle XRD, NEXAFS, AFM, and SEM analyses. A film with pentacene molecules nearly perpendicularly oriented was observed on Au surfaces covered with an SAM of alkanethiol derivative of X-(CH2)(n)-SH, with X = -CH(3), -COOH, -OH, -CN, -NH(2), C(60), or an aromatic thiol p-terphenylmethanethiol. On the other hand, a film with the pentacene molecular plane nearly parallel to the substrate surface was found on bare Au surface. A similar molecular orientation was found in thinner ( approximately 5 nm) and thicker (100 nm) deposited films. Films deposited on different surfaces exhibit distinct morphologies: with apparently smaller and rod-shaped grains on clean bare Au surface but larger and islandlike crystals on SAM-modified surfaces. X-ray photoemission electron microscopy (X-PEEM) was used to analyze the orientation of pentacene molecules deposited on a SAM-patterned Au surface. With the micro-NEXAFS spectra and PEEM image analysis, the microarea-selective orientation control on Au was characterized. The ability to control the packing orientation in organic molecular crystals is of great interest in fabricating organic field effect transistors because of the anisotropic nature of charge transport in organic semiconducting materials.

Long-term enhancement of cytochrome P450 2B1/2 expression in rat hepatocyte spheroids through adenovirus-mediated gene transfer.

Tissue-like structures of cells organized in vitro have a great potential for a number of clinical and biomedical applications. Cell functions may be modulated with gene delivery, improving the characteristics of these structures. Hepatocytes that self-assemble into spheroids can be transduced through adenovirus-mediated gene transfer. An adenoviral vector (AdGFP) was employed to deliver a gene encoding for green fluorescent protein (GFP) in rat hepatocyte spheroids. GFP fluorescence was detected for at least one month. Furthermore, the rat cytochrome P450 2B1 gene (CYP2B1) was transferred through infection with a recombinant adenovirus (AdCYP2B1) in hepatocyte spheroids cultured in suspension. The CYP2B1/2 mRNA and apoprotein levels were continuously higher for over 23 days compared to phenobarbital-induced and control cultures. P450-catalyzed pentoxyresorufin-O-dealkylation activity was also high in the AdCYP2B1-infected spheroids. In these spheroid cultures, albumin and urea levels were similar to those in uninfected spheroid cultures, indicating that expression of the CYP2B1 transgene did not impair these liver-specific functions. Hepatocyte spheroids transduced by recombinant adenoviral vectors can be efficiently used for drug metabolism studies, in implantation, and in bioartificial liver devices.

Simultaneous analysis of spatio-temporal gene expression for cephamycin biosynthesis in Streptomyces clavuligerus.

Linkage between structural and regulatory genes implies that a direct correlation should exist between the spatio-temporal distribution of their expression. Green fluorescent protein (GFP) and cyan fluorescent protein (CFP) were used as reporters to analyze simultaneously expression of lysine-epsilon-aminotransferase (LAT) and its corresponding genetic regulator, CcaR. The isogenic strain containing lat::gfp and ccaR::cfp in the chromosome produced cephamycin C at levels similar to wild type Streptomyces clavuligerus. Confocal laser scanning microscopy revealed that expression of both LAT and CcaR in liquid culture was temporally dynamic and spatially heterogeneous in S. clavuligerus mycelia. During the early culture stage only a part of the mycelia began to express LAT and CcaR at low levels. As the culture aged, expression levels and the population of mycelia expressing LAT and CcaR increased and were followed late in the growth cycle by a reduction of the mycelia population expressing LAT and CcaR. The approach provides a precise simultaneous temporal-spatial expression profile and corroborates the regulatory linkage between ccaR and lat in S. clavuligerus.

Proteomic investigation of metabolic shift in mammalian cell culture.

Mammalian cells, under typical cultivation conditions, produce large quantities of lactate and ammonia that affect cell growth adversely and result in low cell concentration. Controlled nutrient feeding to maintain low concentrations of glucose and glutamine reduces metabolite production drastically, altering the metabolism of the cells. This metabolic shift results in higher cell concentration in continuous cultures and does not affect the specific productivity of the cells. We have taken a proteomics approach to investigate the differential protein expression with metabolic shift. Using two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS), we have found at least eight differentially expressed spots; two proteins were down-regulated, and the others were up-regulated with metabolic shift. These included metabolic enzymes, the brain form of phosphoglycerate mutase, which was down-regulated, and the precursor of the 23 kDa subunit of NADH-ubiquinone oxidoreductase, which was up-regulated. Another enzyme, the L1 isozyme of ubiquitin carboxyl-terminal hydrolase, which is involved in protein turnover and degradation, was also up-regulated in the metabolically altered cells. The remaining down-regulated spot had been identified as two isoforms of cytoplasmic actins, while three of the up-regulated spots were viral GAG polyproteins from various murine viruses. An unidentified protein was also up-regulated in the cells with altered metabolic state. This study shows the potential of using a proteomics approach in deciphering the intracellular changes in cells with physiological changes such as metabolism shift. The new insight into cell metabolism afforded by this analysis will greatly facilitate process optimization of continuous cell cultures.

Probing enhanced cytochrome P450 2B1/2 activity in rat hepatocyte spheroids through confocal laser scanning microscopy.

Cytochrome P450 (CYP450) enzymes are essential for xenobiotic metabolism. Although CYP450s are found in many tissues, CYP2B1/2 are primarily expressed in the rat liver. The constitutive expression in vivo of CYP2B1/2 is low but it is induced in the presence of various drugs such as phenobarbital (PB). In this study, CYP2B1/2 activity in cultured hepatocytes was assessed in situ with the introduction of a fluorogenic substrate, pentoxyresorufin. The product of 7-pentoxyresorufin-O-dealkylation (PROD), which is catalyzed specifically by CYP2B1/2, was detected using confocal laser scanning microscopy (CLSM). Primary hepatocytes cultured as monolayers on collagen-coated surfaces exhibited background PROD activity and minimal PB inducibility after 4 days in culture. In contrast, rat hepatocytes organized in compacted aggregates, or spheroids, exhibited higher levels of PROD activity and retained their ability for PB induction. The results from the CLSM analysis were verified by RT-PCR and Western immunoblotting analysis. Furthermore, CLSM in conjunction with image processing techniques and three-dimensional reconstruction revealed the localization of enhanced PROD activity in the center of spheroids. The results support the use of CLSM as a powerful tool for investigating CYP2B1/2 activity in cultured rat hepatocytes.

IS1550 from Mycoplasma fermentans is transposable in Escherichia coli.

An insertion sequence (IS)-like element (ISMil) was previously isolated from the incognitus strain of Mycoplasma fermentans. With polymerase chain reaction primers corresponding to the left and right terminal inverted repeats of ISMil, a 1.4-kb DNA fragment was amplified from the genome of the M64 strain of M. fermentans. This DNA fragment has structural characteristics similar to those of ISMil and is designated IS1550. One copy of IS1550 encoded two considerable overlapping open reading frames (ORFs), ORF1 and ORF2. A putative translation frame-shift signal AAAAAAG (A6G) was located near the 3'-end of ORF1. This signal might cause a -1 frame-shift to form a fused product of ORF1 and ORF2 with 444 amino acids, which has a significant similarity to the putative transposase of the IS3 family. This copy of IS1550 was shown to be transposable in Escherichia coli ISM612. Its transposition caused a 1465-bp deletion immediately adjacent to the 3'-end of the element and the creation of a pair of 3-bp direct repeats flanking the element at the new insertion site. On the basis of these results, IS1550 was considered a typical transposable element.

Analysis of temporal and spatial expression of the CcaR regulatory element in the cephamycin C biosynthetic pathway using green fluorescent protein.

The DNA-binding capability of a key secondary metabolite regulatory element (CcaR) in the Streptomyces clavuligerus cephamycin C pathway was investigated by gel mobility retardation and DNase I footprinting analysis. These results revealed that CcaR specifically binds to the promoter region of the lysine-epsilon-aminotransferase gene (lat). Green fluorescent protein (GFP) was subsequently used as a reporter to analyse in vivo expression of CcaR. The corresponding isogenic strain containing ccaR:gfp in the chromosome produced cephamycin C at levels similar to those of wild-type S. clavuligerus. Confocal laser scanning microscopy revealed that expression of CcaR in liquid culture was temporally dynamic and spatially heterogeneous in S. clavuligerus mycelia. The highly fluorescent seed culture mycelia quickly lost fluorescence upon inoculation into fresh culture medium. The characteristic green colour reappeared in a small portion of mycelia during mid-exponential growth phase. As the culture aged, the population expressing CcaR expanded, and the expression level increased. This was followed by a reduction in the CcaR-expressing population towards the end of the culture period. During peak expression, CcaR was distributed uniformly in mycelia, but became localized distal to the chromosome when the culture entered stationary phase. In solid phase analysis, abundant CcaR expression was evident in the substrate mycelia, but was completely absent in aerial hyphae. These results show regulatory linkage between ccaR and lat, whose expression profile showed a similar spatial decoupling between morphogenesis and antibiotic production. In addition, visualizing CcaR within S. clavuligerus mycelia demonstrates a distinct pattern of localization over the course of physiological differentiation.

The role of actin filaments and microtubules in hepatocyte spheroid self-assembly.

Cultured rat hepatocytes self-assemble into three-dimensional structures or spheroids that exhibit ultrastructural characteristics of native hepatic tissue and enhanced liver-specific functions. The spheroid formation process involves cell translocation and changes in cell shape, indicative of the reorganization of the cytoskeletal elements. To elucidate the function of the cytoskeleton, hepatocytes undergoing spheroid formation were treated with drugs that disrupt the different cytoskeletal components. Cytochalasin D, which targets the actin filaments, caused inhibition of spheroid formation. The role of microtubules in this process was assessed by incubating the cells with taxol or nocodazole. Perturbation of microtubules had minimal effects on spheroid assembly. Scanning electron micrographs showed no morphological differences between spheroids formed in control cultures and those formed in the presence of taxol or nocodazole. In addition, the effects of those agents on hepatocyte functions were investigated. Albumin secretion and cytochrome P450 2B1/2 activities of hepatocytes were comparable in spheroids formed in the presence of taxol or nocodazole to those formed in control cultures. The levels of these liver-specific activities were lower in cytochalasin D--treated cultures where only dispersed cells or cell clumps were found but spheroids had not found. Thus, hepatocytes require an intact actin network to self-assemble efficiently into functional tissue-like structures. Perturbation of the microtubule lattice does not impair the formation process. Events that transpire during hepatocyte spheroid self-assembly exhibit striking similarities to processes commonly observed in tissue morphogenesis. The results provide insight into the mechanisms that cells employ to organize into tissues and can contribute to our understanding of how to control the cellular assembly in tissue engineering and clinical applications.

Effects of homology length in the repeat region on minus-strand DNA transfer and retroviral replication.

Homology between the two repeat (R) regions in the retroviral genome mediates minus-strand DNA transfer during reverse transcription. We sought to define the effects of R homology lengths on minus-strand DNA transfer. We generated five murine leukemia virus (MLV)-based vectors that contained identical sequences but different lengths of the 3' R (3, 6, 12, 24 and 69 nucleotides [nt]); 69 nt is the full-length MLV R. After one round of replication, viral titers from the vector with a full-length downstream R were compared with viral titers generated from the other four vectors with reduced R lengths. Viral titers generated from vectors with R lengths reduced to one-third (24 nt) or one-sixth (12 nt) that of the wild type were not significantly affected; however, viral titers generated from vectors with only 3- or 6-nt homology in the R region were significantly lower. Because expression and packaging of the RNA were similar among all the vectors, the differences in the viral titers most likely reflected the impact of the homology lengths on the efficiency of minus-strand DNA transfer. The molecular nature of minus-strand DNA transfer was characterized in 63 proviruses. Precise R-to-R transfer was observed in most proviruses generated from vectors with 12-, 24-, or 69-nt homology in R, whereas aberrant transfers were predominantly used to generate proviruses from vectors with 3- or 6-nt homology. Reverse transcription using RNA transcribed from an upstream promoter, termed read-in RNA transcripts, resulted in most of the aberrant transfers. These data demonstrate that minus-strand DNA transfer is homology driven and a minimum homology length is required for accurate and efficient minus-strand DNA transfer.