Basic reproduction number of African swine fever in wild boars (Sus scrofa) and its spatiotemporal heterogeneity in South Korea.

BACKGROUND: African swine fever (ASF) is a hemorrhagic fever occurring in wild boars (Sus scrofa) and domestic pigs. The epidemic situation of ASF in South Korean wild boars has increased the risk of ASF in domestic pig farms. Although basic reproduction number (R0) can be applied for control policies, it is challenging to estimate the R0 for ASF in wild boars due to surveillance bias, lack of wild boar population data, and the effect of ASF-positive wild boar carcass on disease dynamics. OBJECTIVES: This study was undertaken to estimate the R0 of ASF in wild boars in South Korea, and subsequently analyze the spatiotemporal heterogeneity. METHODS: We detected the local transmission clusters using the spatiotemporal clustering algorithm, which was modified to incorporate the effect of ASF-positive wild boar carcass. With the assumption of exponential growth, R0 was estimated for each cluster. The temporal change of the estimates and its association with the habitat suitability of wild boar were analyzed. RESULTS: Totally, 22 local transmission clusters were detected, showing seasonal patterns occurring in winter and spring. Mean value of R0 of each cluster was 1.54. The estimates showed a temporal increasing trend and positive association with habitat suitability of wild boar. CONCLUSIONS: The disease dynamics among wild boars seems to have worsened over time. Thus, in areas with a high elevation and suitable for wild boars, practical methods need to be contrived to ratify the control policies for wild boars.

Transplantation of hMSCs Genome Edited with LEF1 Improves Cardio-Protective Effects in Myocardial Infarction.

Stem cell-based therapy is one of the most attractive approaches to ischemic heart diseases, such as myocardial infarction (MI). We evaluated the cardio-protective effects of the human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) stably expressing lymphoid enhancer-binding factor 1 (LEF1; LEF1/hUCB-MSCs) in a rat model of MI. LEF1 overexpression in hUCB-MSCs promoted cell-proliferation and anti-apoptotic effects in hypoxic conditions. For the application of its therapeutic effects in vivo, the LEF1 gene was introduced into an adeno-associated virus integration site 1 (AAVS1) locus, known as a safe harbor site on chromosome 19 by CRISPR/Cas9-mediated gene integration in hUCB-MSCs. Transplantation of LEF1/hUCB-MSCs onto the infarction region in the rat model significantly improved overall survival. The cardio-protective effect of LEF1/hUCB-MSCs was proven by echocardiogram parameters, including greatly improved left-ventricle ejection fraction (EF) and fractional shortening (FS). Moreover, histology and immunohistochemistry successfully presented reduced MI region and fibrosis by LEF1/hUCB-MSCs. We found that these overall positive effects of LEF1/hUCB-MSCs are attributed by increased proliferation and survival of stem cells in oxidative stress conditions and by the secretion of various growth factors by LEF1. In conclusion, this study suggests that the stem cell-based therapy, conjugated with genome editing of transcription factor LEF1, which promotes cell survival, could be an effective therapeutic strategy for cardiovascular disease.

Acute Anemia Induces Erythropoiesis in Rat Organ Surface Primo-Vascular Tissue.

The primo-vascular system (PVS) is a newly identified vascular tissue composed of primo-nodes (PNs) and primo-vessels (PVs). Previously, we reported erythropoietic activity in the organ-surface PVS (osPVS) tissue of rats with heart failure. In this study, we further investigated whether acute anemia could induce erythropoiesis in the PVS of rats, based on the hypothesis that erythropoiesis in osPVS tissue is due to anemia accompanying heart failure. Acute anemia was induced by an intraperitoneal injection of phenylhydrazine (PHZ). Circulating red blood cells (RBCs) and hematocrit decreased by 31.6%, whereas reticulocytes and white blood cells increased at day 3 and day 6 after PHZ treatment. All these parameters recovered to control levels at day 10. At days 3 and 6, we observed an increase in the size of the PNs (P < 0.05), the number of the osPVS tissue samples per rat (P < 0.01), and the proportion of osPVS tissue samples with red chromophore (P < 0.001), which was from the RBCs in the PVS tissue. The number of RBCs, estimated from the PN sections stained with hematoxylin and eosin, increased at day 6 in the rats with anemia (P < 0.01). All these anemia-induced changes in the osPVS tissue recovered to the control levels by day 10. Taken together, the results showed that the morphological and cytological changes in the osPVS tissue appear to be related to the erythropoietic activity induced by acute anemia in rats. This study confirmed the previous findings that the osPVS can exert erythropoietic activity in disease states accompanied by anemia, such as heart failure.

Physicochemical factors that affect electroporation of lung cancer and normal cell lines.

Electroporation is used for cancer therapy to efficiently destroy cancer tissues by transferring anticancer drugs into cancer cells or by irreversible tumor ablation without resealing pores. There is growing interest in the electroporation method for the treatment of lung cancer, which has the highest mortality rate among cancers. Improving the cancer cell selectivity has the potential to expand its use. However, the factors that influence the cell selectivity of electroporation are debatable. We aimed to identify the important factors that influence the efficiency of electroporation in lung cells. The electropermeabilization of lung cancer cells (H460, A549, and HCC1588) and normal lung cells (MRC5, WI26 and L132) was evaluated by the transfer of fluorescence dyes. We found that membrane permeabilization increased as cell size, membrane stiffness, resting transmembrane potential, and lipid cholesterol ratio increased. Among them, lipid composition was found to be the most relevant factor in the electroporation of lung cells. Our results provide insight into the differences between lung cancer cells and normal lung cells and provide a basis for enhancing the sensitivity of lung cancers cells to electroporation.

Influence of water and food consumption on inadvertent antibiotics intake among general population.

Antibiotic entry into the water environment has been of growing concern. However, few investigations have been performed to examine the potential for indirect human exposure to environmental antibiotic residues. We evaluated the contribution of drinking water and major food consumption to inadvertent intake of antibiotic residues among general human population in Korea. We estimated daily human intake of six antibiotics, i.e., sulfamethazine (SMZ), sulfamethoxazole (SMX), sulfathiazole (STZ), trimethoprim (TMP), enrofloxacin (EFX), and roxithromycin (RTM), by measuring the concentrations of the antibiotics and their major metabolites in urine from general population in Korea (n=541). In addition, we measured antibiotics from source water of drinking water as well as in tap water samples, and surveyed water consumption rates among the study population. To assess the contribution of dietary factor, we also surveyed consumption pattern for several major foods which are suspected of antibiotics residue. SMZ, Sulfamethazine-N4-acetyl (SMZ-N4), TMP, EFX, ciprofloxacin (CFX), and RTM were detected up to 448, 6210, 11,900, 6970, 32,400, and 151pg/ml in the urine samples, respectively. Estimates of daily intake of major antibiotics did not appear to be related with consumption of drinking water although antibiotics were frequently detected in source waters (10-67ng/l). Consumption of several foods correlated significantly with urinary excretion of several antibiotics. Daily intake estimates of EFX and CFX were associated with consumption of beef, pork, and dairy products; those of SMZ and TMP associated with pork and dairy products; and that of TMP related with raw fish. Daily antibiotics intake estimates however did not exceed the acceptable daily intake levels.

DiI staining of fine branches of Bonghan ducts on surface of rat abdominal organs.

Novel thread-like structures and corpuscles, designated Bonghan ducts (BHDs) and corpuscles (BHCs), are known to form a system of networked channels. Here, we tested the effectiveness of a fluorescent carbocyanine dye, DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), in staining BHDs and BHCs. DiI solution was infused into a BHC on the surface of a rat abdominal organ at a steady rate and the resulting labeling of neighboring BHCs connected via BHDs was examined, as identified by the red fluorescence of DiI. BHDs diameters tapered away from BHCs and formed tree-like branches with fine arborizations embedded in the membranous tissues at their terminal parts. In the proximal parts, DiI fluorescence appeared as continuous lines within BHDs, but a large portion of BHDs remained unstained. In the distal parts of BHDs, discontinuous elongated DiI microparticles were identified along the sinuses within BHDs. The results showed that inner spaces within the BHDs allowed DiI to flow and that BHDs have tree-like branches and terminal arborizations. In conclusion, DiI can be used in visualizing BHDs fine structures.

Gateway RFP-fusion vectors for high throughput functional analysis of genes.

There is an increasing demand for high throughput (HTP) methods for gene analysis on a genome-wide scale. However, the current repertoire of HTP detection methodologies allows only a limited range of cellular phenotypes to be studied. We have constructed two HTP-optimized expression vectors generated from the red fluorescent reporter protein (RFP) gene. These vectors produce RFP-tagged target proteins in a multiple expression system using gateway cloning technology (GCT). The RFP tag was fused with the cloned genes, thereby allowing us localize the expressed proteins in mammalian cells. The effectiveness of the vectors was evaluated using an HTP-screening system. Sixty representative human C2 domains were tagged with RFP and overexpressed in HiB5 neuronal progenitor cells, and we studied in detail two C2 domains that promoted the neuronal differentiation of HiB5 cells. Our results show that the two vectors developed in this study are useful for functional gene analysis using an HTP-screening system on a genome-wide scale.

A helix-induced oligomeric transition of gaegurin 4, an antimicrobial peptide isolated from a Korean frog.

Gaegurin 4 (GGN4), a novel peptide isolated from the skin of a Korean frog, Rana rugosa, has broad spectrum antimicrobial activity. A number of amphipathic peptides closely related to GGN4 undergo a coil to helix transition with concomitant oligomerization in lipid membranes or membrane-mimicking environments. Despite intensive study of their secondary structures, the oligomeric states of the peptides before and after the transition are not well understood. To clarify the structural basis of its antibiotic action, we used analytical ultracentrifugation to define the aggregation state of GGN4 in water, ethyl alcohol, and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP). The maximum size of GGN4 in 15% HFIP corresponded to a decamer, whereas it was monomeric in buffer. The oligomeric transition is accompanied by a cooperative 9 nm blue-shift of maximum fluorescence emission and a large secondary structure change from an almost random coil to an alpha-helical structure. GGN4 induces pores in lipid membranes and, using electrophysiological methods, we estimated the diameter of the pores to be exceed 7.3 A, which suggests that the minimal oligomer structure responsible is a pentamer.

Effects of corticotropin-releasing factor on plasticity of optically recorded neuronal activity in the substantia gelatinosa of rat spinal cord slices.

We examined the effects of corticotropin-releasing factor (CRF) on plasticity of optically recorded neuronal activity in the substantia gelatinosa (lamina II) of 12-18-day-old rat spinal cord slices stained with a voltage-sensitive dye. Single-pulse test stimulation to the dorsal root that activated A and C fibres evoked prolonged (>100 ms) light-absorption change in the lamina II. This response represents the gross membrane potential change of all elements along the slice depth. After conditioning high-frequency stimulation of A-fibre-activating strength, test stimulus elicited less neuronal activity [-27+/-1% (7), (average+/-SE (n)), P<0.01 (*) at 45-60 min after conditioning]. When CRF (1 microM, 10 min) was applied during conditioning, the neuronal activity was facilitated rather than suppressed [+20+/-3% (5), P<0.05]. CRF alone exhibited insignificant effect [-5+/-1% (4), P=0.2]. In the presence of the inhibitory amino acid antagonists bicuculline (1 microM) and strychnine (0.3 microM) in the perfusate, in contrast, the conditioning facilitated it [+27+/-1% (12)*], and CRF treatment during conditioning inhibited the facilitation dose-dependently [0.1 microM: +18+/-2% (5)*, 1 microM: +13+/-1% (7)*]. Although interneuronal actions might contribute, these results suggest that CRF may have dual effects on excitatory synaptic transmission within the lamina II depending upon cellular conditions: a conversion from the induction of long-term depression to long-term potentiation (LTP), and inhibition of LTP induction. Since the LTP is thought to be responsible at least in part for the persistent pain, CRF could regulate the induction.