Underground Pipeline Leakage Risk Assessment in an Urban City.

Underground pipeline safety is a concern among civilians in populated urban cities. Due to the potential for considerable damage from underground pipeline leakages, it is critical to identify potential risk areas. This study developed a simplified risk value using risk assessment software (ALOHA) and geography information systems (SuperGIS and Surfer) to produce potential risk maps for underground pipeline leakage in a major urban city. A risk assessment of areas affected by underground pipeline leakage was performed for vapor diffusion, thermal radiation from combustion, and overpressure from an explosion. The results are applicable to disaster management departments and agencies in highly populated cities.

Analyses of a whole-genome inter-clade recombination map of hepatitis delta virus suggest a host polymerase-driven and viral RNA structure-promoted template-switching mechanism for viral RNA recombination.

The genome of hepatitis delta virus (HDV) is a 1.7-kb single-stranded circular RNA that folds into an unbranched rod-like structure and has ribozyme activity. HDV redirects host RNA polymerase(s) (RNAP) to perform viral RNA-directed RNA transcription. RNA recombination is known to contribute to the genetic heterogeneity of HDV, but its molecular mechanism is poorly understood. Here, we established a whole-genome HDV-1/HDV-4 recombination map using two cloned sequences coexisting in cultured cells. Our functional analyses of the resulting chimeric delta antigens (the only viral-encoded protein) and recombinant genomes provide insights into how recombination promotes the genotypic and phenotypic diversity of HDV. Our examination of crossover distribution and subsequent mutagenesis analyses demonstrated that ribozyme activity on HDV genome, which is required for viral replication, also contributes to the generation of an inter-clade junction. These data provide circumstantial evidence supporting our contention that HDV RNA recombination occurs via a replication-dependent mechanism. Furthermore, we identify an intrinsic asymmetric bulge on the HDV genome, which appears to promote recombination events in the vicinity. We therefore propose a mammalian RNAP-driven and viral-RNA-structure-promoted template-switching mechanism for HDV genetic recombination. The present findings improve our understanding of the capacities of the host RNAP beyond typical DNA-directed transcription.

Effect of forced use of the lower extremity on gait performance and mobility of post-acute stroke patients.

[Purpose] The purpose of this study was to investigate the effects of a forced-use training program on gait, mobility and quality of life of post-acute stroke patients. [Subjects] Twenty-one individuals with unilateral stroke participated in this study. All participants had suffered from first-ever stroke with time since onset of at least 3 months. [Methods] A single-blinded, non-equivalent, pre-post controlled design with 1-month follow-up was adopted. Participants received either a forced-use or a conventional physical therapy program for 2 weeks. The main outcomes assessed were preferred and fastest walking velocities, spatial and temporal symmetry indexes of gait, the timed up and go test, the Rivermead Mobility Index, and the Stroke-Specific Quality of Life Scale (Taiwan version). [Results] Forced-use training induced greater improvements in gait and mobility than conventional physical therapy. In addition, compared to pre-training, patients in the conventional physical therapy group walked faster but more asymmetrically after training. However, neither program effectively improved in-hospital quality of life. [Conclusion] The forced-use approach can be successfully applied to the lower extremities of stroke patients to improve mobility, walking speeds and symmetry of gait.

Caregiver-mediated intervention can improve physical functional recovery of patients with chronic stroke: a randomized controlled trial.

Background and Purpose. Patients with chronic stroke may benefit from continuing rehabilitation training after hospital discharge. This study examined whether caregiver-mediated, home-based intervention (CHI) could improve physical functioning and social participation in these patients. Methods. A single-blind, randomized, controlled 12-week trial conducted with 51 patients from 3 hospitals in Taiwan who had chronic stroke (>6 months; Brunnstrom recovery stages III-V). Patients and their caregivers in the intervention arm (n = 25) were given weekly personalized CHI trainings designed by a physical therapist. Patients in the control arm (n = 26) received visits from the therapist without intervention. All were evaluated for physical recovery through the Stroke Impact Scale, Berg Balance Scale, 10-Meter Walk Test, 6-Minute Walk Test, and Barthel Index at baseline and endpoint. Caregivers were evaluated with the Caregiver Burden Scale. Results were analyzed through Mann-Whitney U test. Results. CHI significantly improved scores of the Stroke Impact Scale: strength (control vs intervention, respectively: 1.4 vs 15.5; P = .002), mobility (-0.5 vs 13.7; P < .001), composite physical (-0.7 vs 11.2; P < .001), and general recovery domain (0.2 vs 17.4; P < .001). CHI also significantly improved free-walking velocity (-1.4 vs 7.5 cm/s; P = .006), 6-minute walk distance (-10.5 vs 15.8 m; P = .003), Berg Balance Scale score (-0.8 vs 4.5; P = .006), and Barthel Index score (0.6 vs 7.2; P = .008). CHI did not significantly increase caregiver burden at endpoint. Conclusion. CHI can improve physical functional recovery and, possibly, social participation in patients with chronic stroke.

Structure of dihydromethanopterin reductase, a cubic protein cage for redox transfer.

Dihydromethanopterin reductase (Dmr) is a redox enzyme that plays a key role in generating tetrahydromethanopterin (H4MPT) for use in one-carbon metabolism by archaea and some bacteria. DmrB is a bacterial enzyme understood to reduce dihydromethanopterin (H2MPT) to H4MPT using flavins as the source of reducing equivalents, but the mechanistic details have not been elucidated previously. Here we report the crystal structure of DmrB from Burkholderia xenovorans at a resolution of 1.9 Å. Unexpectedly, the biological unit is a 24-mer composed of eight homotrimers located at the corners of a cubic cage-like structure. Within a homotrimer, each monomer-monomer interface exhibits an active site with two adjacently bound flavin mononucleotide (FMN) ligands, one deeply buried and tightly bound and one more peripheral, for a total of 48 ligands in the biological unit. Computational docking suggested that the peripheral site could bind either the observed FMN (the electron donor for the overall reaction) or the pterin, H2MPT (the electron acceptor for the overall reaction), in configurations ideal for electron transfer to and from the tightly bound FMN. On this basis, we propose that DmrB uses a ping-pong mechanism to transfer reducing equivalents from FMN to the pterin substrate. Sequence comparisons suggested that the catalytic mechanism is conserved among the bacterial homologs of DmrB and partially conserved in archaeal homologs, where an alternate electron donor is likely used. In addition to the mechanistic revelations, the structure of DmrB could help guide the development of anti-obesity drugs based on modification of the ecology of the human gut.

Nitrogen dilution effect on the flammability limits for hydrocarbons.

Theoretical models to predict the upper/lower flammability limits of hydrocarbons diluted with inert nitrogen gas are proposed in this study. It is found that there are linear relations between the reciprocal of the upper/lower flammability limits and the reciprocal of the molar fraction of hydrocarbon in the hydrocarbon/inert nitrogen mixture. Such linearity is examined by experimental data reported in the literature, which include the cases of methane, propane, ethylene and propylene. The R-squared values (R(2)) of the regression lines of the cases explored are all greater than 0.989 for upper flammability limit (UFL). The theoretical slope of the predictive line for lower flammability limit (LFL) is found to be very close to zero for all explored cases; and this result successfully explains the experimental fact that adding inert nitrogen to a flammable material has very limited effect on LFL. Because limit oxygen concentration (LOC) could be taken as the intersectional point of the UFL curve and LFL curve, a LOC-based method is proposed to predict the slope of the UFL curve when experimental data of UFL are not available. This LOC-based method predicts the UFL with average error ranging from 2.17% to 5.84% and maximum error ranging from 8.58% to 12.18% for the cases explored. The predictive models for inert gas of nitrogen are also extended to the case of inert gas other than nitrogen. Through the extended models, it was found that the inert ability of an inert gas depends on its mean molar heat capacity at the adiabatic flame temperature. Theoretical calculation shows that the inert abilities of carbon dioxide, steam, nitrogen and helium are in the following order: carbon dioxide>steam>nitrogen>helium; and this sequence conforms to the existing experimental data reported in the literature.

Carbon dioxide dilution effect on flammability limits for hydrocarbons.

Theoretical models to predict the upper/lower flammability limits of a mixture composed of hydrocarbon and inert carbon dioxide are proposed in this study. It is found theoretically that there are linear relations between the reciprocal of the upper/lower flammability limits and the reciprocal of the molar fraction of hydrocarbon in the hydrocarbon/inert gas mixture. These theoretical linear relations are examined by existing experimental results reported in the literature, which include the cases of methane, propane, ethylene, and propylene. The coefficients of determination (R(2)) of the regression lines are found to be larger than 0.959 for all aforementioned cases. Thus, the proposed models are highly supported by existing experimental results. A preliminary study also shows the conclusions in present work have the possibility to extend to non-hydrocarbon flammable materials or to inert gas other than carbon dioxide. It is coincident that the theoretical model for the lower flammability limit (LFL) in present work is the same as the empirical model conjectured by Kondo et al.

Detection of hepatitis delta virus recombinants in cultured cells co-transfected with cloned genotypes I and IIb DNA sequences.

It was reported previously that hepatitis delta virus (HDV), the only animal virus in which replication is performed by cellular RNA polymerase(s), undergoes RNA recombination. However, the previous RNA transfection system was somewhat limited in terms of practical application. Cultured cells were transfected with plasmids expressing replication-competent genotypes I and IIb HDV genomic RNAs to develop a better system for studying the fundamental aspects of HDV RNA recombination and HDV-related RNA species were examined using restriction fragment length polymorphisms and sequence analysis of cloned RT-PCR products. This novel experimental system generated efficiently recombinants between the two parental HDV sequences, but not between replication-defective HDV constructs. The genome organization of the HDV recombinants produced in this system resembled that observed previously in cultured cells co-transfected with genome I and IIb RNAs. These data indicate that replication-dependent HDV RNA recombination can be catalyzed by host RNA polymerases in cultured cells co-transfected with two cloned HDV sequences. This new DNA-based system is simpler than the previous RNA-based method of study, and generates a higher recombination frequency, facilitating study of HDV RNA recombination.

RNA recombination of hepatitis delta virus in natural mixed-genotype infection and transfected cultured cells.

Most RNA viruses encode their own RNA polymerases for genome replication, and increasing numbers of them appear to be capable of undergoing RNA recombination. Here, we provide the first report of intergenotypic recombination in hepatitis delta virus (HDV), the only animal RNA virus that requires host RNA polymerase(s) for viral replication. In vivo, we analyzed RNA species derived from the serum of a patient with mixed genotype I and genotype IIb HDV infection by using multiple restriction fragment length polymorphism (RFLP) assays and sequence analysis of cloned reverse transcription (RT)-PCR products. Six HDV recombinants were isolated from 101 tested clones, and HDV recombination in this patient was further confirmed by RT-PCR with genotype-specific primer pairs. Analysis of the recombination junctions suggested that the HDV genome rearrangement occurred through faithful homologous recombination. We then used an RNA cotransfection cell culture system to investigate HDV RNA recombination in vitro. We found that HDV recombinants could indeed be detected in the transfected cells; some of these possessed recombination junctions identical to those identified in vivo. Furthermore, using a PCR-independent RNase protection assay, we were able to readily identify the recombined HDV RNA species in cultured cells. Taken together, our results demonstrate that HDV RNA recombination occurs in both natural HDV infections and cultured cells, thereby presenting a novel mechanism for HDV evolution.

Initiation of RNA replication of cloned Taiwan-3 isolate of hepatitis delta virus genotype II in cultured cells.

Hepatitis delta virus (HDV) genotype II is the predominant genotype in Taiwan and is associated with less progressive disease than genotype I. Although the Taiwan-3 (T3) clone was the first genotype II HDV isolated in Taiwan, its replication in cultured cells has not previously been established. Here, we demonstrate that cloned T3 HDV is capable of replicating in cultured cells. Furthermore, we show that: (1). the replication level of T3 clones is 100-fold lower than that of a genotype I HDV prototype of Italian origin; (2). both forms of the genotype II T3 delta antigen are expressed; and (3). T3 HDV undergoes RNA editing during replication, with 4.8% of the T3 genomes showing evidence of editing. The low level of RNA replication may be related to the milder clinical outcomes of genotype II HDV infections.

Molecular cloning and expression of the hepatitis delta virus genotype IIb genome.

Analysis of hepatitis delta virus (HDV) genome sequences has revealed multiple genotypes with different geographical distributions and associated disease patterns. To date, replication-competent cDNA clones of HDV genotypes I, II, and III have been reported. HDV genotypes I, II, and IIb have been found in Taiwan. Although full-length sequences of genotype IIb have been published, its replication competence in cultured cells has yet to be reported. In order to examine this, we obtained a full-length cDNA clone, Taiwan-IIb-1, from a Taiwanese HDV genotype IIb isolate. Comparison of the complete nucleic acid sequence of Taiwan-IIb-1 with previously published genotype IIb isolates indicated that Taiwan-IIb-1 shares 98% identity with another Taiwanese isolate and 92% identity with a Japanese isolate. Transfection of Taiwan-IIb-1 into COS7 cells resulted in accumulation of the HDV genome and appearance of delta antigens, showing that cloned HDV genotype IIb can replicate in cultured cells.