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The retention of contaminants within low-conductivity regions such as clay lenses and aquitards can greatly affect groundwater remediation processes. The aim of this study was to experimentally investigate the effects of the geometry of low-conductivity zones, conductivity contrast, and flow regime on solute flushing. We conducted a series of flushing tests in cylindrical models containing a cylindrical low-conductivity zone (i.e., low-K zone) embedded in a highly conductive medium (i.e., high-K zone). Seven models comprising four high-conductivity-contrast (SL, SS, LL, and LS), one medium-contrast (LLM), one low-contrast (LLL), and one homogeneous (H) models were considered. Experiments were conducted at two flow rates (Q = 0.6 and 26 cm3/min) for each heterogeneous model (SL, SS, LL, LS, LLM, and LLL) to compare the flushing processes in different flow regimes. First, we verified the validity of our experiments by comparing the results of the H model from an analytical solution with our experiment. The results of the high-contrast models showed that for a diffusion-dominated regime (Q = 0.6 cm3/min), the pore volume injected (PVI) required to flush out solute mass was much smaller than that in an advection-dominated regime (Q = 26 cm3/min). To evaluate the pore volumes required to flush out solutes for the four high-contrast models, we introduced a parameter P0.01, which is defined as the PVI needed for the relative concentration to become 0.01 at the middle of the low-K zone. P0.01 decreases with increasing the specific surface area of the low-K zone for diffusion-dominated regimes, while it increases with increasing the length of the low-K zone for advection-dominated regimes. We also determined the importance of the effect of K contrast on solute retention by comparing the results of three different models of K contrast (LL, LLM, and LLL).

For slug-injection tracer tests, tracer concentrations below the detection limit of the measurement instrument can cause truncation of the observed data. This study investigated the truncation effect on the estimation error of parameters based on analytical solutions and the results of a laboratory-scale experiment. Spatial moment analysis was performed to estimate the measured total mass and transport parameters, including the pore velocity and the longitudinal and transverse dispersivities. Increasing the travel distance and detection limit caused the measured mass and dispersivities to be underestimated regardless of the dimensionality because hydrodynamic dispersion occurs with increasing travel distance, which smoothens the concentration front. The one- and two-dimensional cases showed that the truncation effect on the measured mass and longitudinal dispersivity depended on dimensionality. In contrast, the pore velocity showed no such dependence; the center of mass did not change as the unmeasured portion due to truncation was increased because the plume, which exhibited a Gaussian distribution, was truncated symmetrically. In the experiment, the measured mass and dispersivities likewise depended on the travel distance and detection limit, but there were large differences in the detection limit at which the dimensionless parameter reached a value of zero between the experimental results and analytical solution. This is because the initial plume in the experiment was of a finite size. Thus, experimental design factors such as the scale, device, and dimensionality should be considered to minimize the estimation error of transport parameters, excluding the pore velocity.

Simultaneous expression of multiplex guide RNAs (gRNAs) is valuable for knockout of multiple genes and also for effective disruption of a gene by introducing multiple deletions. We developed a method of Tetraplex-guide Tandem for construction of cosmids containing four and eight multiplex gRNA-expressing units in one step utilizing lambda in vitro packaging. Using this method, we produced an adenovirus vector (AdV) containing four multiplex-gRNA units for two double-nicking sets. Unexpectedly, the AdV could stably be amplified to the scale sufficient for animal experiments with no detectable lack of the multiplex units. When the AdV containing gRNAs targeting the H2-Aa gene and an AdV expressing Cas9 nickase were mixed and doubly infected to mouse embryonic fibroblast cells, deletions were observed in more than 80% of the target gene even using double-nicking strategy. Indels were also detected in about 20% of the target gene at two sites in newborn mouse liver cells by intravenous injection. Interestingly, when one double-nicking site was disrupted, the other was simultaneously disrupted, implying that two genes in the same cell may simultaneously be disrupted in the AdV system. The AdVs expressing four multiplex gRNAs could offer simultaneous knockout of four genes or two genes by double-nicking cleavages with low off-target effect.

In recent years, antimicrobial-resistant bacteria have been isolated from different wildlife species that have not been exposed to antibiotics. Antimicrobial resistance profile analysis of wildlife-derived Escherichia coli is one of the ways to obtain information about changes in wildlife habitats. The Amami rabbit (Pentalagus furnessi) is one of the Japanese endemic wildlife species that requires conservation due to its declining population. However, detailed information on its ecology, including habitat, is unclear. We conducted antimicrobial susceptibility testing (broth microdilution assay) of 12 antimicrobials on 135 E. coli strains isolated from the feces of free-living Amami rabbits, collected between 2017 and 2020. No antimicrobial-resistant E. coli were isolated from any sample, indicating that antimicrobial-resistant E. coli were not widely distributed in the Amami rabbit population. In addition, these results suggested that the habitat of Amami rabbits may not be contaminated with antimicrobials derived from humans, Japanese livestock, and wildlife that come in contact with the rabbits. This is the first study to examine the antimicrobial resistance of bacterial isolates from the Amami rabbits for a multi-year period. In order to observe changes in wildlife habitats on the Amami-Oshima Island, inhabited by many rare species including Amami rabbits, continued surveillance of antimicrobial-resistant E. coli in wildlife is necessary.

The complicated replication mechanisms of hepatitis B virus (HBV) have impeded HBV studies and anti-HBV therapy development as well. Herein we report efficient genome replication of HBV applying adenovirus vectors (AdVs) showing high transduction efficiency. Even in primary hepatocytes derived from humanized mice the transduction efficiencies using AdVs were 450-fold higher compared than those using plasmids. By using an expression unit consisting of the CMV promoter, 1.03-copy HBV genome and foreign poly(A) signal, we successfully generated an improved AdV (HBV103-AdV) that efficiently provided 58 times more pregenomic RNA than previously reported AdVs. The HBV103-AdV-mediated HBV replication was easily and precisely detected using quantitative real-time PCR in primary hepatocytes as well as in HepG2 cells. Notably, when the AdV containing replication-defective HBV genome of 1.14 copy was transduced, we observed that HBV DNA-containing circular molecules (pseudo-ccc DNA) were produced, which were probably generated through homologous recombination. However, the replication-defective HBV103-AdV hardly yielded the pseudo-ccc, probably because the repeated sequences are vey short. Additionally, the efficacies of entecavir and lamivudine were quantitatively evaluated using this system at only 4 days postinfection with HBV103-AdVs. Therefore, this system offers high production of HBV genome replication and thus could become used widely.

We propose and demonstrate micro-channel fabrication by femtosecond-laser processing to arrange neuronal cells on multi-electrode arrays (MEAs). When neuronal cells were seeded in the micro-channels on MEAs, they elongated their neurites along the channel and finally formed the network patterned on MEAs. The spontaneous electrical activity of the neuronal networks on MEAs was evaluated by using electrophysiological systems, indicating that patterned network was functionally activated along the channels. The present method is applicable to analyze network dynamics of living neuronal cells patterned artificially.

To quantitatively investigate the time course of changes in corneal irregular astigmatism and their relation to best spectacle-corrected visual acuity (BSCVA) in eyes with keratoconus. Subjects were 47 patients (57 eyes) with keratoconus who had undergone videokeratography examinations at least twice, with an interval of 1 year or longer between each examination. By means of Fourier series harmonic analysis, topography data were separated into spherical component, regular astigmatism, asymmetry, and higher order irregularity. In eyes that showed apparent progression on color-coded maps, the yearly rate of change was significantly positive for spherical component (P = 0.03) and higher order irregularity (P = 0.006). In eyes with apparent progression, the yearly change rate of BSCVA was significantly positive (P = 0.001), but not in the eyes without apparent progression (P = 0.1655). In eyes with progression on maps, the yearly change rate of BSCVA was significantly correlated with the change rate of higher order irregularity per year (r = 0.770, P = 0.0004). The results indicated that progression of keratoconus is associated with deterioration of BSCVA, which is in turn significantly correlated with increases in corneal higher order irregularity. Jpn J Ophthalmol 2007;51:265–269 @ Japanese Ophthalmological Society 2007

First-generation adenovirus vectors (FG AdVs) expressing short-hairpin RNA (shRNA) effectively downregulate the expressions of target genes. However, this vector, in fact, expresses not only the transgene product, but also virus-associated RNAs (VA RNAs) that disturb cellular RNAi machinery. We have established a production method for VA-deleted AdVs lacking expression of VA RNAs. Here, we showed that the highest shRNA activity was obtained when the shRNA was inserted not at the popularly used E1 site, but at the E4 site. We then compared the activities of shRNAs against hepatitis C virus (HCV) expressed from VA-deleted AdVs or conventional AdVs. The VA-deleted AdVs inhibited HCV production much more efficiently. Therefore, VA-deleted AdVs were more effective than the currently used AdVs for shRNA downregulation, probably because of the lack of competition between VA RNAs and the shRNAs. These VA-deleted AdVs might enable more effective gene therapies for chronic hepatitis C.

First-generation adenovirus vectors (FG AdVs) are widely used in basic studies and gene therapy. However, virus-associated (VA) RNAs that act as small-interference RNAs are indeed transcribed from the vector genome. These VA RNAs can trigger the innate immune response. Moreover, VA RNAs are processed to functional viral miRNAs and disturb the expressions of numerous cellular genes. Therefore, VA-deleted AdVs lacking VA RNA genes would be advantageous for basic studies, both in vitro and in vivo. Here, we describe an efficient method of producing VA-deleted AdVs. First, a VA RNA-substituted “pre-vector” lacking the original VA RNA genes but alternatively possessing an intact VA RNA region flanked by a pair of FRTs was constructed. VA-deleted AdVs were efficiently obtained by infecting 293hde12 cells, which highly express FLP, with the pre-vector. The resulting transduction titers of VA-deleted AdVs were sufficient for practical use. Therefore, VA-deleted AdVs may be substitute for current FG AdV.