Development of saxitoxin biosynthesis gene sxtB-targeted qPCR assay for the quantification of toxic dinoflagellates Alexandrium catenella (group I) and A. pacificum (group IV) occurring in the Korean coast.

Toxic dinoflagellate Alexandrium can produce saxitoxins (STXs) and cause paralytic shellfish poisoning (PSP), and thus they are monitored for environmental safety management. Microscopic discrimination of dinoflagellates is difficult to distinguish between toxic and non-toxic species due to their similar morphology. Meanwhile, an alternative quantitative PCR (qPCR) assay is sensitive, rapid, and cost-effective for harmful species monitoring. Herein, we developed a novel qPCR assay to detect the STXs biosynthesis gene sxtB of Alexandrium catenella and A. pacificum, the leading cause of PSP outbreaks in Asian coasts and worldwide. The newly designed sxtB TaqMan probes target the species without any positive signal in other relative dinoflagellates. Deming regression analysis revealed that the sxtB copy number of A. catenella and A. pacificum was 3.6 and 4.1 copies per cell, respectively. During the blooming periods (April 13th-14th, 2020), only A. catenella cells were detected through the qPCR assay, ranging from 5.0 × 10 to 2.5 × 104 eq cells L-1. In addition, sxtB qPCR quantified more accurately compared to large subunit (LSU) rRNA targeting qPCR assay that overestimate cell density. Besides, the sensitivity of sxtB was higher compared to the microscope when the species were rarely present (5.0 × 102 cells L-1). These suggest that the sxtB qPCR assay can be applied to toxic Alexandrium monitoring in the Korean coast, even in the early stage of bloomings.

The Effects of COVID-19 Pandemic on the Recovery of Hip Fracture Patients.

Purpose: To figure out how complete control of family visits to prevent infection of coronavirus disease 2019 (COVID-19) affected the activity recovery of hip fracture patients admitted to nursing hospitals. Materials and Methods: Eighty-one patients with hip surgery in the two years prior to COVID-19 pandemic were classified as Group A, and 103 patients in the next two years were designated as Group B. The subjects' walking ability was evaluated by using the modified Koval index (MKI). In order to analyze the impact of the family visit control to the subjects, each group was classified into two different groups: (1) inpatients group who admitted to nursing hospitals and (2) home-treated patients. Additionally, statistical elements were processed in consideration of other factors that may affect the results of the experiment. Results: The MKI evaluated at 6 months postoperative was 3.31±1.79 in Group A and 2.77±1.91 in Group B, and it was meaningfully low after the pandemic (P=0.04). There was significantly low among both of Group A 2.74±1.76 and Group B 1.93±1.81 after the pandemic (P=0.03) among those treated at the nursing hospital. The rate of deterioration of the MKI was 35 (43.2%) in Group A and 57 (55.3%) in Group B, which increased by 12.1% after the pandemic. Conclusion: The pandemic had a negative effect on the recovery of postoperative activities of elderly hip fracture patients who admitted to nursing hospitals when family access was completely restricted to prevent infection.

Direct Anterior Approach for Total Hip Arthroplasty in the Elderly with Femoral Neck Fractures: Comparison with Conventional Posterolateral Approach.

BACKGROUND: The aim of this study was to determine if it was feasible and safe to perform total hip arthroplasty (THA) using the direct anterior approach (DAA) when compared with the conventional posterolateral approach (PA) in patients with femoral neck fractures. The time required to start walking was investigated to identify advantages of the muscle-sparing approach. Safety of the approach was judged based on the incidence and nature of all complications. METHODS: We retrospectively reviewed 67 THA cases due to femoral neck fractures from October 2015 to January 2019. The PA was used in 31 cases, and the DAA was used in 36 cases. The average operative time and amount of bleeding were evaluated. Cup inclination, anteversion, and leg length discrepancy (LLD) were also measured on radiographs. The time to start walking and complications (e.g., intraoperative fracture, infection, and dislocation) were recorded. RESULTS: The mean operative time was 84.35 ± 13.95 minutes in PA group and 99.22 ± 20.33 minutes in DAA group (p = 0.010). But after experiencing 20 cases using the DAA, there was no statistically significant difference in the operative time between the groups. The mean volume of bleeding was 428.73 ± 207.26 mL in the PA group and 482.47 ± 150.14 mL in the DAA group. There was no difference in the acetabular cup position between two groups. Ambulation was started at 3.94 days after surgery on average in the PA group and 3.14 days in the DAA group, showing a statistically significant difference. Intraoperative fracture and infection were not observed in either group. The incidence of LLD was 1 in each group. The dislocation rate was 3.2% (1 case) in the PA group and 5.5% (2 cases) in the DAA group. CONCLUSIONS: Although the DAA for THA was similar to the PA in terms of operative time, volume of bleeding, and complications, the DAA showed a great advantage in early rehabilitation as a muscle-sparing procedure in the elderly with femoral neck fractures.

Origins and characteristics of dissolved organic matter fueling harmful dinoflagellate blooms revealed by δ13C and D/L-Amino acid compositions.

We measured the concentrations of dissolved inorganic and organic nutrients, dissolved organic carbon (DOC), total hydrolyzable amino acids (THAA), fluorescent dissolved organic matter (FDOM), phytoplankton pigments, and δ13C-DOC during the summer of 2019 in the harmful dinoflagellate bloom regions of the southern coast of Korea. In the harmful dinoflagellate bloom region, the concentrations of inorganic nitrogen were depleted, inhibiting the growth of diatoms, while the concentrations of dissolved organic components (nutrients, DOC, FDOM, and amino acids) which fuel dinoflagellates were unusually high. Thus, we attempted to investigate the origins and characteristics of DOM which fuels the harmful dinoflagellate blooms. The δ13C-DOC values (- 22.2‰ to - 18.2‰) indicate that the elevated DOC concentrations result from in-situ biological production rather than terrestrial inputs. The enantiomeric (D/L) ratios of THAA indicate that dissolved organic nitrogen was more labile in the early stage of harmful dinoflagellate bloom and became more refractory in the final stage. Our results suggest that the marine production of bioavailable DOM plays an important role in initiating and sustaining harmful dinoflagellate blooms.

Gyrodiniellum shiwhaense n. gen., n. sp., a new planktonic heterotrophic dinoflagellate from the coastal waters of western Korea: morphology and ribosomal DNA gene sequence.

The heterotrophic dinoflagellate Gyrodiniellum shiwhaense n. gen., n. sp. is described from live cells and from cells prepared for light, scanning electron, and transmission electron microscopy. Also, sequences of the small subunit (SSU) and large subunit (LSU) of rDNA have been analyzed. The episome is conical, while the hyposome is ellipsoid. Cells are covered with polygonal amphiesmal vesicles arranged in 16 horizontal rows. Unlike other Gyrodinium-like dinoflagellates, the apical end of the cell shows a loop-shaped row of five elongate amphiesmal vesicles. The cingulum is displaced by 0.3-0.5 × cell length. Cells that were feeding on the dinoflagellate Amphidinium carterae Hulburt were 9.1-21.6 µm long and 6.6-15.7 µm wide. Cells of G. shiwhaense contain nematocysts, trichocysts, a peduncle, and pusule systems, but they lack chloroplasts. The SSU rDNA sequence is >3% different from that of the six most closely related species: Warnowia sp. (FJ947040), Lepidodinium viride Watanabe, Suda, Inouye, Sawaguchi & Chihara, Gymnodinium aureolum (Hulburt) Hansen, Gymnodinium catenatum Graham, Nematodinium sp. (FJ947039), and Gymnodinium sp. MUCC284 (AF022196), while the LSU rDNA is 11-12% different from that of Warnowia sp., G. aureolum, and Nematodinium sp. (FJ947041). The phylogenetic trees show that the species belongs in the Gymnodinium sensu stricto clade. However, in contrast to Gymnodinium spp., cells lack nuclear envelope chambers and a nuclear fibrous connective. Unlike Polykrikos spp., cells of which possess a taeniocyst-nematocyst complex, G. shiwhaense has nematocysts but lacks taeniocysts. It differs from Paragymnodinium shiwhaense Kang, Jeong, Moestrup & Shin by possessing nematocysts with stylets and filaments. Gyrodiniellum shiwhaense n. gen., n. sp. furthermore lacks ocelloids, in contrast to Warnowia spp., Nematodinium spp., and Proterythropsis spp. Based on morphological and molecular data, we suggest that the taxon represents a new species within a new genus.

Clinical efficacy of high-flow nasal oxygen in patients undergoing ERCP under sedation.

Hypoxemia can occur during endoscopic retrograde cholangiography (ERCP) and it is difficult to achieve adequate ventilation with the prone position. High-flow nasal oxygen (HFNO) has been recommended to be more effectively help ventilation than conventional low flow oxygen. The aim of this study was to evaluate the effect of HFNO during sedated ERCP and to identify predictors of desaturation during ERCP. The investigated variables were age, gender, American Society of Anesthesiologists classes (ASA), duration of exam, and sedative used for midazolam or/and propofol of 262 patients with sedated ERCP. The differences between categorical and continuous variables were analyzed using the Student's t test and the chi-square test. Desaturation (SpO2 ≤ 90%) occurred in 9(3.4%) patients among 262 patients during sedated ERCP. The variables found to predict desaturation were older age (p < 0.01), higher sedation dose for midazolam or propofol (p < 0.01), and use of midazolam (p < 0.01). Desaturation rate was lower during sedated ERCP with HFNO compared to the preliminary study with conventional low flow nasal oxygen. Patients with older age, higher sedation dose, or the use of midazolam might require close monitoring for desaturation and hypoventilation by nursing staff. The study shows the use of high-flow nasal oxygen reduces the incidence of desaturation during ERCP.

Alexandrium catenella (Group I) and A. pacificum (Group IV) cyst germination, distribution, and toxicity in Jinhae-Masan Bay, Korea.

To better understand the outbreaks of paralytic shellfish poisoning and bloom dynamics caused by Alexandrium species in Jinhae-Masan Bay, Korea, the germination and distributions of ellipsoidal Alexandrium cysts were investigated, and paralytic shellfish toxins (PSTs) profiles and contents were determined using strains established from germling cells. The phylogeny and morphological observations revealed that the germinated vegetative cells from ellipsoidal cysts collected from the surface sediments in Jinhae-Masan Bay belong to Alexandrium catenella (Group I) and A. pacificum (Group IV) nested within A. tamarense species complex. Cyst germinations of A. catenella (Group I) were observed at only 10 °C, whereas cysts of A. pacificum (Group IV) could germinate at temperature ranges of 10 to 25 °C. Maximum germination success (85%) for isolated cysts occurred at 15 °C, and the germling cells were A. pacificum (Group IV). The results indicate that the variation in water temperature in Jinhae-Masan Bay can control the seasonal variations in germination of cysts of A. catenella (Group I) and A. pacificum (Group IV). The germination rates of ellipsoidal Alexandrium cysts were different among sampling sites in Jinhae-Masan Bay, probably because of differences in distribution and abundance of A. catenella (Group I) and A. pacificum (Group IV) in the sediments. The ellipsoidal Alexandrium cyst concentrations were much higher in February than in August, however the distributions were similar. Gonyautoxins 3 and 4 (GTX-3 and GTX-4) contributed a large proportion (>90%) of the toxins produced by strains A. catenella (Group I) and A. pacificum (Group IV) established from germling cells, and the total cellular contents were higher in A. catenella (Group I) than in A. pacificum (Group IV).

Conditions of nutrients and dissolved organic matter for the outbreaks of Paralytic Shellfish Poisoning (PSP) in Jinhae Bay, Korea.

We measured the concentrations of nutrients, fluorescent dissolved organic matter (FDOM), and photosynthetic pigments in seawater during the springs of 2018 and 2019 in Jinhae Bay, Korea. The samplings were carried out during the severe and weak outbreaks of paralytic shellfish poisoning (PSP) in April 2018 and March 2019, respectively. The additional sampling campaigns were carried out before and after the PSP outbreak for the comparison. During the severe PSP outbreak, lower salinities, higher organic and total nutrients, and higher humic-like FDOM were observed. Although the environmental condition of April 2018 is favorable for the growth of dinoflagellates, the lowest peridinin (dinoflagellate index) and highest fucoxanthin (diatom index) concentrations were observed amongst all sampling periods. Thus, our results suggest that PSP could be more effectively produced by dinoflagellates in the course of the ecological shift by interspecific competition under the environmental condition favorable for dinoflagellates.

Tracing the sources of nutrients fueling dinoflagellate red tides occurring along the coast of Korea using radium isotopes.

It is a well held concept that the magnitude of red-tide occurrence is dependent on the amount of nutrient supply if the conditions are same for temperature, salinity, light, interspecific competition, etc. However, nutrient sources fueling dinoflagellate red-tides are difficult to identify since red tides usually occur under very low inorganic-nutrient conditions. In this study, we used short-lived Ra isotopes (223Ra and 224Ra) to trace the nutrient sources fueling initiation and spread of Cochlodinium polykrikoides blooms along the coast of Korea during the summers of 2014, 2016, and 2017. Horizontal and vertical distributions of nutrient concentrations correlated well with 224Ra activities in nutrient-source waters. The offshore red-tide areas showed high 224Ra activities and low-inorganic and high-organic nutrient concentrations, which are favorable for blooming C. polykrikoides in competition with diatoms. Based on Ra isotopes, the nutrients fueling red-tide initiation (southern coast of Korea) are found to be transported horizontally from inner-shore waters. However, the nutrients in the spread region (eastern coast of Korea), approximately 200 km from the initiation region, are supplied continuously from the subsurface layer by vertical mixing or upwelling. Our study highlights that short-lived Ra isotopes are excellent tracers of nutrients fueling harmful algal blooms in coastal waters.

Thermally Responsive Torsional and Tensile Fiber Actuator Based on Graphene Oxide.

Graphene-based actuators are of practical interest because of their relatively low cost compared with other nanocarbon materials, such as carbon nanotubes. We demonstrate the simple fabrication of graphene oxide (GO)-based fibers with an infiltrated nylon-6,6 polymer by wet spinning. These fibers could be twisted to form torsional actuators and further coiled to form tensile actuators. By controlling the relative twisting and coiling direction of the GO/nylon fiber, we were able to realize reversible contraction or elongation actuation with strokes as high as -80 and 75%, respectively, when the samples were heated to 200 °C. The tensile actuation showed a remarkably little hysteresis. Moreover, this GO/nylon actuator could lift loads over 100 times heavier than itself and generate a stable actuation at high temperatures over the melting point of the polymer. This novel kind of GO-based actuator, which has a multidirectional actuation, has potential for a wide range of applications such as artificial muscles, robotics, and temperature sensing.

Development of real-time RT-PCR for detecting viable Cochlodinium polykrikoides (Dinophyceae) cysts in sediment.

Morphological observations have confirmed that cysts are produced by dinoflagellates. However, finding a seed bed or unknown cysts in field samples by microscopy is extremely time consuming. Real-time PCR has been used to facilitate the detection of dinoflagellate cysts in sediment. However, DNA from dead vegetative cells remaining on the surface sediment may persist for a long period of time, which can cause false positive DNA detection. In this study, a non-quantitative RNA targeted probe using real-time RT-PCR was developed for detection of viable cysts in sediment. Large-subunit rRNA was used to develop a species-specific RNA targeted probe for the ichthyotoxic dinoflagellate Cochlodinium polykrikoides. The sediment samples were sieved and incubated at 30°C for 3h prior to RNA extraction to remove RNA from dead cells remaining in the sediment. Nested-PCR was conducted to maximize assay sensitivity. A field survey to determine the distribution of cysts at 155 sampling stations in the western and southern part of the Korean peninsula showed that C. polykrikoides cysts were detected at five sampling stations.

Enhanced contractility of vascular smooth muscle after brief exposure to arsenate.

Epidemiological studies indicate that arsenic exposure induces hypertension. We hypothesized that arsenate exposure modulates the contractility of vascular smooth muscle through the stress response. Intraperitoneal injection of sodium arsenate (15mg/kg) 16h before increased not only the blood pressure of rats but also the pressor response to preganglionic nerve stimulation (2 and 16Hz) or to bolus injection of vasopressin or phenylephrine in pithed rats as compared with the control rats. Exposure of rat aortic rings to 4mM sodium arsenate for 60min enhanced the contractile responses to KCl or phenylephrine as well as the HSP 70 expression 8h later, but did not affect the relaxation responses to acetylcholine, histamine, or sodium nitroprusside. These results suggest that brief exposure to arsenate is associated with enhanced contractility of vascular smooth muscle through the stress response.

Endothelial dysfunction after exposure to cobalt chloride enhanced vascular contractility.

Brief exposure to cobalt chloride augmented vascular contractility. We hypothesized that endothelial dysfunction plays a role in the augmentation of aortic contractility, after brief exposure to cobalt chloride. Rat aortic ring preparations were mounted in organ baths, exposed to cobalt chloride (0.3-300µmol/L) for 30min, and then subjected to contractile agents or relaxants 1 and 5h after the end of exposure. Presence of cobalt chloride did not affect the contractile response to phenylephrine. Brief exposure to cobalt chloride, however, even at 5h after the end of exposure, not only augmented contractile responses to KCl or phenylephrine but also attenuated the relaxant response to acetylcholine. The mechanical denudation of endothelium or inhibition of endothelial nitric oxide synthase with 100µmol/L N(ω)-nitro-l-arginine methyl ester abolished the augmentation of contractile responses. Pre-treatment with 150units/mL of superoxide dismutase also abrogated the augmented contractile responses. Brief exposure to cobalt chloride did not affect the contractile response to phorbol dibutyrate in the presence or absence of calcium, or the expression of HSP70. In conclusion, endothelial dysfunction plays an important role in the augmentation of aortic contractility, after brief exposure to cobalt chloride.

Heat-shock response is associated with enhanced contractility of vascular smooth muscle in isolated rat aorta.

Stress proteins have been implicated in pathological cardiovascular conditions. We hypothesized that a heat-shock response modulates contractility of vascular smooth muscles. Rat aortic ring preparations were mounted in organ baths, exposed to 42 degrees C for 45 min, and subjected to contractions. Expression of HSP70 and phosphorylation of myosin light chain were examined with immunoblots. Heat shock enhanced contractile response to KCl in parallel with HSP70 expression in rat aortic rings from 8 h but not 1 h after the end of heat shock. Heat shock also augmented vascular contractility to phenylephrine whether endothelium was intact or denuded. Treatment of heat shock-preconditioned aortic rings with Bay K8644, a calcium channel activator, but not treatment with phorbol dibutyrate (1 micromol/l), a protein kinase C activator, enhanced contractions of the rings as compared with those of the control. The levels of phosphorylation of myosin light chains after administration of phenylephrine in heat shock-preconditioned tissues were statistically significantly higher than those in control tissues. Pretreatment with wortmannin (300 nmol/l), an inhibitor of myosin light chain kinase, decreased both contractility and phosphorylation of myosin light chains in parallel. However, heat-shock response did not affect relaxation responses to either acetylcholine in endothelium-intact aortic rings or sodium nitroprusside in endothelium-denuded rings. These results suggest that the heat-shock response is associated with enhanced vascular smooth muscle contractility through a modulation of thick-filament regulation.

Implication of lymph node metastasis detected on 18F-FDG PET/CT for surgical planning in patients with peripheral intrahepatic cholangiocarcinoma.

OBJECTIVES: Intrahepatic cholangiocarcinoma (ICC) is the second most common primary hepatic malignancy after hepatocellular carcinoma. ICC can be divided into 2 types according to their location: peripheral and hilar types. Intense F-FDG uptake on PET was reported in peripheral ICC. However, the usefulness of PET/CT in detecting tumors and predicting prognosis in peripheral ICC has not been fully evaluated. In this study, we evaluated the clinical role of F-FDG PET/CT to predict the recurrence after the curative resection in patients with surgically indicated peripheral ICC. METHODS: Eighteen patients with ICC underwent preoperative CT and F-FDG PET/CT scans. SUVmax of tumor, tumor to normal liver SUV ratio (TNR), lymph node status evaluated by F-FDG PET/CT, tumor and lymph node size measured by CT, vascular invasion confirmed by pathology, and satellite nodules found on CT were compared between 1-year recurrence group and recurrence-free group by chi-square test. RESULTS: Of total 23 measurable lymph nodes, 4 nodes were positive and other 19 nodes were negative or equivocal on CT. Among those 23 nodes, 9 nodes were positive and other 14 nodes were negative on F-FDG PET/CT. The sensitivity and specificity of CT were 20.0% and 86.4%, and those of F-FDG PET/CT were 80.0% and 92.3%. In the comparison between 1-year recurrent and nonrecurrent groups, lymph node metastasis detected on F-FDG PET/CT had statistically positive correlation with the 1-year recurrence after surgical resection (P = 0.02). Other factors showed no statistically significant difference between the groups. CONCLUSION: We found that lymph node metastasis detected on F-FDG PET/CT correlated positively with 1-year recurrence after surgical resection in patients with peripheral ICC.

Peripheral bone involvement of intravascular large B-cell lymphoma on 99mTc-MDP bone scan and 18F-FDG PET/CT.

A 64-year-old man was admitted for evaluation of recently developed anemia and bone pain. The bone scan showed diffusely active lesions in the peripheral bones, symmetrically. Interestingly, 18F-FDG PET/CT revealed the hypermetabolic changes in the peripheral bones as well as the internal organs. Biopsy of bone marrow confirmed the diagnosis of intravascular B-cell lymphoma. After the 3 cycles of R-CHOP chemotherapy, 18F-FDG PET/CT showed improvement of the previous hypermetabolic lesions, suggesting good response. Intravascular B-cell lymphoma is a rare and aggressive variant of diffuse large cell lymphoma characterized by proliferation of malignant cells within the vascular lumina.

Development of a real-time PCR probe for quantification of the heterotrophic dinoflagellate Cryptoperidiniopsis brodyi (Dinophyceae) in environmental samples.

A TaqMan format real-time PCR probe was developed against the internal transcribed spacer 2 ribosomal DNA region for the specific detection and quantification of Cryptoperidiniopsis brodyi in environmental samples. The assay specificity was confirmed by testing against related dinoflagellates and verified by sequencing PCR amplicons from natural water samples. Phylogenetic analysis of the sequenced environmental samples also showed that this assay is specific to C. brodyi. The C. brodyi-specific assay was used in conjunction with Pfiesteria piscicida- and Pfiesteria shumwayae-specific real-time PCR assays to investigate the temporal variations of C. brodyi, P. piscicida, and P. shumwayae abundance in the Derwent estuary, Tasmania. The 18-month field survey from November 2004 to April 2006 revealed that C. brodyi occurred in all seasons at very low densities, mostly below 25 cells liter(-1), with higher abundance (maximum, 112 cells liter(-1)) in April and May. P. piscicida was detected only once, in May 2005 at 60 cells liter(-1). P. shumwayae was not detected during the survey.